Complications-Hypoglycemia Ada PDF

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Guided Audio Tour poster ADA-Funded Research
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COMPLICATIONSHYPOGLYCEMIA
Guided Audio Tour: Understanding Hypoglycemia in Diabetes (Posters 378-P
to 385-P), see page 15.
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378-P
History of Severe Hypoglycemia and Score on Clarke Questionnaire
is Associated With Blunted Counterregulatory Response to Experi-
mental Hypoglycemia in Patients With Type 1 Diabetes
AMIR MOHEET, ANJALI KUMAR, LISA CHOW, LYNN E. EBERLY, ELIZABETH R.
SEAQUIST, Minneapolis, MN
Clarke questionnaire is widely used to identify patients with type 1 dia-
betes (T1DM) who have impaired hypoglycemia awareness (IHA). It consists
of 8 questions about hypoglycemia(HG) symptoms and past episodes of HG;
scores of 4 or more imply IHA. While the accuracy of CQ has been exam-
ined using patient reported HG and HG associated symptom scores, the
relationship between HG-induced counterregulatory (CR) responses, which
are responsible for the generation of many HG associated symptoms, and
CQ scores has not been examined. In this study we tested the hypothesis
that there is a signicant relationship between high CQ scores and reduced
CR response to HG.18 subjects (8F/10M, 42 12 yrs, BMI 26.7 3.9 kg/m
2
,
mean SD) with T1DM and IHA (dened as score >4 on CQ) were examined.
They underwent a 2 hour hyperinsulinemic (2.0 mU/kg/min) clamp with a
glucose target of 50 mg/dl. During HG, mean glucose was maintained at 48
5 mg/dl. Blood was collected at baseline and every 10 minutes during HG for
measurement of CR hormones. During HG, epinephrine (Epi) ranged within
15-584 pg/dl and norepinephrine (Nepi) 38.3-528.5 pg/dl.Using Spearmans
correlation, a signicant inverse relationship was found between CQ score
and mean Epi (rs = -0.52, p =0.03) and mean Nepi (rs = -0.42, p = 0.08) dur-
ing HG. Using Wilcoxon Two-Sample tests, a signicant inverse relationship
was found for a history of severe HG within the last year (question 4 on CQ)
with mean Epi (p = 0.009) and with mean Nepi (p = 0.003) responses, but not
with other CR hormones. History of moderate HG (question 3 on CQ) was not
found to be associated with CR responses. These results demonstrate that
history of severe HG and high total score on CQ is signicantly related to re-
duced CR response to HG in patients with T1DM. Therefore, such responses
on the CQ may indicate those patients with the most profound IHA, which
can be of value in both the research and the clinical setting.
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379-P
Antecedent Hypoglycemia does not Impair the Glycemia-Increasing
Effect and Counterregulatory Responses of a 10-Second Sprint in
People With Type 1 Diabetes
PAUL A. FOURNIER, RAYMOND J. DAVEY, NIRU PARAMALINGAM, ELIZABETH A.
DAVIS, TIMOTHY W. JONES, Perth, Australia
A 10-second sprint can not only increase blood glucose concentrations
when plasma insulin are at basal levels, but also prevent blood glucose lev-
els from falling post-exercise if performed after moderate intensity exer-
cise in people with type 1 diabetes mellitus (T1D), thus providing a means
of reducing the risk of exercise-mediated hypoglycemia. Since an episode
of hypoglycemia attenuates the counterregulatory responses to a sub-
sequent bout of moderate-intensity exercise, the aim of this study was to
investigate whether antecedent hypoglycemia inhibits the counterregula-
tory responses and glycemia-increasing effect of a 10-second sprint. Eight
individuals with T1D (6M, 2F; age 21.32.7 years; BMI of 25.95.1 kg.m
-2
;
HbA1c of 7.71.0%; meanSD) were subjected in the morning of different
days to either a one-hour episode of hypoglycemia or euglycemia by infus-
ing insulin at a constant rate of 80 mU.m
-2
.min
-1
and adjusting the infusion
rate of a 20% dextrose solution. Immediately after,all participants were ex-
posed to a three-hour insulin-only euglycemic clamp before they performed
a 10-second sprint on a cycle ergometer under basal insulin conditions.
During the morning clamps, blood glucose levels were signicantly differ-
ent between the hypoglycemic (2.80.3 mmol/L) and euglycemic (5.40.5
mmol/L; p<0.05) conditions. In response to the afternoon sprint, blood glu-
cose increased signicantly, reaching similar maximal levels at 45 minutes
post-sprint (6.51.1 and 6.50.7 mmol/L in the hypoglycemia and euglycemia
treatments, respectively). Sprinting resulted in a signicant post-exercise
increase in plasma catecholamines, cortisol and growth hormone levels in
both treatments (p<0.05). In conclusion, antecedent hypoglycemia does not
affect the glycemia-increasing effect of a 10-second sprint in individuals
with T1D, thus supporting further the benets of sprinting in hypoglycemia
prevention.
Supported by: National Health and Medical Research Council of Australia
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380-P
Prediction of Severe Inpatient Hypoglycemia Using Blood Glucose
Trends
RANEE R. LLEVA, SANDI-JO GALATI, KARRIE C. HENDRICKSON, JANIS E. BOZZO,
ZHENQIU LIN, SILVIO E. INZUCCHI, New Haven, CT, New York, NY
Inpatient hypoglycemia (hypo) is associated with higher mortality. An
AACE/ADA consensus on hospital glucose management suggests reassess-
ment of anti-hyperglycemic regimens with blood glucose (BG) <100mg/dL.
The value of point-of-care (POC) BG monitoring in predicting inpatient hypo is
not known.We examined POC BGs in adult DM patients (pts) on general med-
ical-surgical wards at our hospital over 1 year. Of the 480 pts with at least
1 BG<50mg/dL (severe hypo), 368 experienced their event after hospital
day 2, had at least 2 BGs recorded during those 48 hours, and were treated
with insulin/sulfonylureas. DM controls (2379) were on similar therapies and
had no documented BG<50mg/dL during their stays. Pts were matched for
age, length of stay, service, and number of BG tests; 211 pairs were identi-
ed. All BGs 48 hrs prior to the hypo event in the 211 cases, as well as a
randomly selected 48-hr period of BGs in controls, were categorized into ve
glycemic windows: <60, <70, <80, <90, & <100mg/dL. Chi-square tests were
then used to compare the 2 groups.The proportion with BG<100mg/dL was
higher in cases vs. controls. The relative frequencies became increasingly
disparate as antecedent BGs fell <90mg/dL; values <60mg/dL occurred >10
times more frequently in cases vs. controls.We conclude that inpatients with
severe hypo frequently have BGs in the low-normal/mild-moderately hypo
range in the 48 hours preceding their events. BG <100mg/dL should serve
as a marker to consider adjustment of the anti-hyperglycemic regimen, sup-
porting the AACE/ADA recommendations. The need to adjust therapy seems
more compelling the further the BG falls <100mg/dL.
Percent of pts with low-normal/mildly hypo BGs preceding severe hypo event
Antecedent BGs (mg/dL)
<100 <90 <80 <70 <60
Severe hypo 60.2% 46.0% 33.7% 22.8% 11.4%
No severe hypo 50.2% 25.1% 16.1% 6.2% 1.0%
P-value 0.0398 <0.0001 <0.0001 <0.0001 <0.0001
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381-P
Hypoglycemia Alert DogsInnovative Assistance for People With
Type 1 Diabetes
DANA S. HARDIN, DUSTIN HILLMAN, JENNIFER CATTET, Indianapolis, IN, West
Lafayette, IN
Hypoglycemia (hypo) is the most common side effect of insulin therapy in
people with type 1 diabetes (T1D). Despite education directed to prevent and
treat it, hypo continues to cause disruption in lives of people with T1D. For
the past 10 years, the Indiana Canine Assistance Network (ICAN) has trained
dogs for mobility assistance for people with disabilities. This abstract
chronicles results of training our rst hypo alert dog. A 2-year-old mobility
assistance Labrador/Golden Retriever dog was trained to recognize hypo.
Perspiration samples were collected from patients during hypo (BG <65 mg/
dL) and normoglycemia. Samples were blinded in containers placed on a Lazy
Susan apparatus. The dog was rewarded for appropriate recognition of the
hypo samples and trained to alert by nudging the trainers arm. After the dog
was introduced to the potential owner, the owners perspiration samples
were used to complete training before placement. A questionnaire consist-
ing of validated and nonvalidated components was used to assess frequency
and severity of hypo as well as emotional responses at 2 weeks pre- and 6
months post-dog placement.Figure 1 displays 2 of the questionnaire results.
The correlation between alerts and hypo (by self-monitored blood glucose)
was greater than 98% by 3 months post-dog placement.We demonstrated
clear impact on hypo in a patient with T1D. These preliminary qualitative
(n=1) ndings support our continued training (10 additional dogs are currently
in training) and monitoring. Additional studies are planned to try to elucidate
the specic biomarkers scented by the dogs.
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Supported by: Eli Lilly and Company
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382-P
Effects of Antecedent GABA A Receptor Activation on Counterregu-
latory Responses to Subsequent Exercise in Type 1 Diabetes
MAKA S. HEDRINGTON, MAIA MIKELADZE, NINO G. JOY, CHERYL YOUNG,
DONNA B. TATE, LINDSAY PULLIAM, LISA M. YOUNK, IAN C. DAVIS, CHELSEA
YOUNK, STEPHEN N. DAVIS, Baltimore, MD
The effects of the Benzodiazepine - GABA(A) receptor agonist alprazolam
(Alp) on physiologic responses during next day exercise in type 1 diabetes
(T1DM) is unknown. To test the hypothesis that Alp would blunt counter-
regulatory responses during subsequent exercise, 30 non-obese (16M / 14F)
T1DM (HBA1c 7.50.3) were studied during separate 2 day protocols. Day 1
consisted of morning and afternoon 2 hr euglycemic (eugly) or hypoglycemic
(hypo) clamps with or without 1 mg Alp given 30 min before each clamp. Day
2 consisted of 90 min euglycemic cycling exercise at 50% VO2 max. Tritiated
glucose was used to measure glucose kinetics. Despite equivalent day 2
insulin (131 U/ml) and glucose levels (951.6 mg/dl), plasma epinephrine,
norepinephrine, glucagon, cortisol and growth hormone responses (nal 15
minutes) were reduced similarly following either Alp or hypo as compared
to eugly control. Lipolysis (glycerol, NEFA) and endogenous glucose produc-
tion (EGP) were also reduced during exercise similarly following Alp or hypo
(Table 1). In summary, selective activation of GABA(A) receptors with Alp
can reduce key neuroendocrine, autonomic nervous system and metabolic
counterregulatory responses to subsequent exercise in patients with T1DM.
We conclude that activation of GABA (A) receptors can result in widespread
counterregulatory failure similar to antecedent hypo during subsequent ex-
ercise in individuals with T1DM.
Table 1 Mean SEM *p<0.05 compared to eugly control
Day 1eugly+Alp Day 1eugly Day 1hypo
AEpinephrine (pg/mL) 609* 11218 5810*
ANorepinephrine (pg/mL) 32163* 67098 41648*
AGlucagon (pg/mL) 42* 92 41*
AGrowth Hormone (ng/mL) 52* 91 31*
AGlycerol (mol/L) 6112* 10617 639*
ANEFA (mmol/L) 8356* 24059 7138*
EGP (mg/kg/min) 2.50.4* 3.90.5 3.10.2
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383-P
Recurrent Hypoglycemia Reduces Adrenal Epinephrine Release
Evoked by Splanchnic Nerve Stimulation
BRANLY O. ORBAN, VANESSA H. ROUTH, BARRY E. LEVIN, JOSHUA R. BERLIN,
Newark, NJ, East Orange, NJ
Tight glycemic control with intensive insulin therapy is used to prevent
diabetic complications due to hyperglycemia. However, intensive insulin
therapy can lead to insulin induced hypoglycemia (IIH). Recurrent hypogly-
cemia (RH) blunts the bodys counterregulatory response (CRR) to subse-
quent hypoglycemia leading to decreased epinephrine (Epi) release and an
impaired ability to restore euglycemia. Most studies of RH havefocused on
the contribution of the brain to reduced Epi release, but whether RH affects
adrenal medullary function has not been investigated.In this study, IIH (565
mg/dL) was produced (1 U/kg, sc) in 8-wk old male Sprague Dawley rats
daily for 3 days (RH). Control rats (Con) received saline injections. In one ex-
periment, all animals received insulin on day 4 and plasma glucose, Epi, and
corticosterone (Cort) were monitored. The time course of glucose decline
was identical, but the elevations in plasma Epi at 60 and 90 min and CORT
at 90 and 120 min after insulin injection were signicantly smaller in RH
rats (P<0.05). Thus, RH blunted the CRR in this protocol.In a second experi-
ment, on day 4, rats were anesthetized, and an electrode was placed on the
splanchnic nerve branch innervating the left adrenal gland. The left renal
vein was catheterized and the outow of this gland was isolated from the
systemic circulation to determine Epi release in response to nerve electrical
stimulation. Before and during stimulation, mean arterial pressure and heart
rate in Con and RH groups were similar. Epi release produced by sustained (5
min) electrical stimulation at 30Hz was less in the RH animals (P<0.05). Epi
release produced by short stimulations (30 sec) over a range of frequencies
(1Hz to 100Hz) tended to be less in the RH group. Total Epi content, measured
in right adrenal glands, was similar in Con and RH groups. These results
suggest that RHimpairs intrinsic adrenal medullary Epi release in addition to
blunting central stimulation of sympathetic outow.
Supported by: NIH (5F31DK086681-02)
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384-P
To What Extent Does Exercise Reduce Sympathoadrenal and Symp-
tomatic Response to Subsequent Hypoglycemia in Humans?
NADIA KHOURY, W. TODD CADE, AMY C. HAUCH, TAMARA HERSHEY, ANA MA-
RIA ARBELAEZ, St. Louis, MO
Episodes of hypoglycemia following play and exercise in patients with
type I diabetes are frequent clinical complaints. Exercise (~50% V0
2 max
x
90 min x 2) has been reported to reduce sympathoadrenal and symptomatic
responses to hypoglycemia the following day in nondiabetic adults (Am J
Physiol 280: E908, 2001) and adults with type 1 diabetes (Diabetes 53:1798,
2004). However, we found that higher intensity exercise at ~70% V0
2 max
x
90 min x 2 did not reduce the plasma epinephrine or symptomatic responses
to hypoglycemia at 55mg/dL or 45 mg/dL the following day in healthy young
nondiabetic adults (n=9). In this study, we observed that an intravenous glu-
cose infusion of an average of ~10 mgkg
-1
min
-1
during the second bout of
exercise was required to maintain plasma glucose concentrations at 90 mg/
dL. Thus, to test the hypothesis that this glucose infusion prevented attenua-
tion of the responses to hypoglycemia on Day 2, we repeated the study with-
out glucose infusion during exercise (n=8). Although mean plasma glucose
concentrations declined to a nadir of 46 + 2 mg/dL during the second bout
of exercise (rising to an average of 69 + 2 mg/dL), the plasma epinephrine
and symptomatic responses to hypoglycemia were not reduced the follow-
ing day. Thus, in 17 studies, by repeated measures ANOVA, there was no
signicant difference in plasma epinephrine and symptomatic responses to
hypoglycemia following antecedent exercise or rest.
Day 1 Day 2
Condition Glucose
(mg/dL + SE)
Epinephrine
(pg/mL + SE)
Symptom
Score
Rest 91 + 1 35 + 7 2 + 1
Exercise 90 + 1 72 + 22 3 + 1
Rest 56 + 1 233 + 32 8 + 2
Exercise 56 + 1 230 + 41 8 + 2
Rest 47 + 1 539 + 66 17 + 3
Exercise 47 + 1 477 + 62 15 + 2
These data indicate that in healthy individuals any effect of exercise to
reduce sympathoadrenal and symptomatic responses to subsequent hypo-
glycemia is small.
Supported by: Childrens Discov Inst 1KL2RR024994-01 and WU Mallinckrodt Inst
Radiol Pilot
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385-P
Brain Glycogen Is Important for Motor Learning during Hypoglycemia
STACI A. WEAVER, SHANNON N. SHARPE, BARTHOLOMEW A. PEDERSON,
Muncie, IN
Hypoglycemia is the major obstacle preventing the benets associated
with intensive insulin therapy in individuals with diabetes. An acute effect of
hypoglycemia is compromised cognitive function, including impaired mem-
ory. Brain glycogen, the only signicant energy store in the brain, is utilized
during hypoglycemia and has been hypothesized to be important for learning
under normal conditions. The objective of this study was to utilize geneti-
cally engineered mice (MGSKO/GSL30) with decient brain glycogen stores
to determine the importance of brain glycogen for learning under hypogly-
cemic conditions. Our hypothesis was that if brain glycogen is important for
learning during hypoglycemia, mice decient in brain glycogen would exhibit
impaired learning during hypoglycemia. Wild type and MGSKO/GSL30 mice
were subjected to a rotarod motor learning protocol for 4 trials over a course
of 8 days. Latency to fall was the measurement of learning. For trial 1 (day
1), mice were trained in the fed state. For trial 2 (day 2) and trial 3 (day 6),
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mice were fasted and injected with either vehicle or with insulin, to induce
hypoglycemia (blood glucose ~3.5 mM). In trials 1, 2 and 3, fed and vehicle in-
jected wild type and MGSKO/GSL30 mice performed similarly to each other.
During trials 2 and 3, hypoglycemia impaired performance similarly in both
genotypes. For trial 4 (day 8), mice were tested in the fed state. Wild type
mice that had been hypoglycemic during trials 2 and 3 had similar latency in
trial 4 as compared to wild type mice that were previously fed or vehicle-
injected. However, MGSKO/GSL30 mice that had been hypoglycemic during
trials 2 and 3 had decreased latency in trial 4 as compared to both wild type
mice subjected to the same conditions and MGSKO/GSL30 mice not previ-
ously hypoglycemic. These data suggest that brain glycogen is not required
for motor learning in normoglycemic conditions but is important for motor
learning during hypoglycemia.
Supported by: DK078370
Guided Audio Tour: Stop Hypoglycemia (Posters 386-P to 393-P), see page
15.
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386-P
Effect of Antecedent Exercise on the Glycemia-Increasing Effect
and Counterregulatory Responses of a Short Sprint
PAUL A. FOURNIER, TARA D. JUSTICE, GRETA L. HAMMER, RAYMOND J. DAVEY,
NIRU PARAMALINGAM, KYM J. GUELFI, ELIZABETH A. DAVIS, TIMOTHY W.
JONES, Perth, Australia
Performing a short sprint under basal insulin conditions in individuals with
type 1 diabetes mellitus (T1D) not only increases blood glucose levels, but
also prevents glycemia from falling post-exercise if performed immediately
after moderate-intensity exercise, thus reducing the risk of exercise-mediat-
ed hypoglycemia. Since moderate-intensity exercise can blunt the counter-
regulatory hormone responses to an identical bout of exercise performed
hours later in the day, the aim of this study was to investigate whether ante-
cedent exercise can inhibit the counterregulatory responses and glycemia-
increasing effect of sprinting in non-diabetic individuals. Eight non-diabetic
males were subjected to a familiarization session and two trials (adminis-
tered following a counterbalanced design) during which they either rested
(CON) or cycled at 65% VO
2
peak for 60 min (EX) before being infused with
[6,6-
2
H]glucose. Three hours later, they performed a 30-sec maximal sprint on
a cycle ergometer. In response to sprinting, blood glucose increased signi-
cantly and reached similar peak levels at 10 min of recovery in EX and CON,
but fell more rapidly afterward in EX. This rise in glycemia may be explained
by the higher glucose rate of appearance (Ra) compared with glucose rate of
disappearance (Rd; p < 0.05). Plasma insulin levels peaked in both trials after
30 min of recovery, but were lower in EX at 45 min (p = 0.042) of recovery.
Growth hormone levels increased during recovery, but to a lesser extent in
EX (p<0.05). The post-exercise rise in cortisol and norepinephrine levels did
not differ between trials. In conclusion, antecedent exercise does not at-
tenuate the glycemia-increasing effect of a sprint performed hours later, but
accelerates the subsequent decline in blood glucose levels. It remains to be
seen whether antecedent exercise affects the glycaemia-increasing effect
of sprinting in individuals with T1D.
Supported by: National Health and Medical Research Council of Australia
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387-P
Prospectively Planned Meta-Analysis Comparing Hypoglycemia
Rates of Insulin Degludec With Those of Insulin Glargine
ROBERT E. RATNER, STEPHEN GOUGH, CHANTAL MATHIEU, STEFANO DEL
PRATO, BRUCE W. BODE, HENRIETTE MERSEBACH, LARS ENDAHL, BERNARD
ZINMAN, Hyattsville, MD, Oxford, United Kingdom, Leuven, Belgium, Pisa, Italy,
Atlanta, GA, Sborg, Denmark, Toronto, ON, Canada
Insulin degludec (IDeg), a new basal insulin that forms soluble multi-
hexamers after s.c. injection, has an ultra-long and stable glucose-lowering
effect, with low day-to-day variability. The objective of this pre-specied
analysis was to compare the rate of hypoglycemia with IDeg vs. insulin
glargine (IGlar) across trials. Hypoglycemia was dened as rates of self-
reported conrmed hypoglycemia (PG<56 mg/dL or severe hypoglycemia
requiring assistance) and nocturnal conrmed hypoglycemia (00:01-05:59
incl.). Rates were analyzed with a negative binomial regression model on
patient level data. All open-labeled, randomized, treat-to-target phase 3a
trials of 26 or 52 weeks were included in which once-daily IDeg (n=2899)
was compared to IGlar (n=1431) in T1DM (2 trials) and T2DM (5 trials). IDeg
resulted in statistically signicantly lower rates of overall and nocturnal hy-
poglycemia compared to IGlar in T2DM, and for nocturnal hypoglycemia in
T1DM (Table). Signicant benets of IDeg were also evident for the pooled
population (T1DM+T2DM). Analyses of the maintenance period (stable gly-
cemia and insulin dose [obtained from 16 weeks onward]), demonstrated
more pronounced benets of IDeg. Additional heterogeneity analysis re-
vealed signicantly less risk of severe hypoglycemia with IDeg vs. IGlar in
insulin-nave T2DM (rate ratio 0.14 [0.03; 0.70]
95%CI
). In conclusion, this meta-
analysis demonstrates that treatment with IDeg provides important clini-
cal advantages with signicantly lower rates of both overall and nocturnal
hypoglycemia than IGlar at similar levels of A1c.
Table 1: Meta-analysis of conrmed hypoglycemia
Hypoglycemiarate ratio
(IDeg/IGlar)
Nocturnal hypoglycemiarate
ratio
(IDeg/IGlar)
Total
period
Maintenance
period
b
Total
period
Maintenance
period
b
T2DM 0.83
[0.74; 0.94]*
0.75
[0.66; 0.87]*
0.68
[0.57; 0.82]*
0.62
[0.49; 0.78]*
T2DM-insulin-nave 0.83
[0.70; 0.98]*
0.72
[0.58; 0.88]*
0.64
[0.48; 0.86]*
0.51
[0.36; 0.72]*
T1DM 1.10
[0.96; 1.26]
1.02
0.88; 1.19]
0.83
[0.69; 1.00]*
0.75
[0.60; 0.94]*
T1DM+T2DM
a
0.91
[0.83; 0.99]*
0.84
[0.75; 0.93]*
0.74
[0.65; 0.85]*
0.68
[0.58; 0.80]*
a
The pooled analysis was the primary endpoint;
b
From 16 weeks onwards;
[ ] = 95% condence intervals; * p<0.05
Supported by: Novo Nordisk A/S
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388-P
Type of Insulin and Age are Predictors of Hospitalization due to Se-
vere Hypoglycemia: The EpiHypo Study
SARI MKIMATTILA, PASI KORHONEN, JARI HAUKKA, FABIAN HOTI, PIA PA-
JUNEN, TERO SAUKKONEN, Espoo, Finland
Incidence and recurrence of severe hypoglycemic (SH) events among patients
with diabetes mellitus (DM) was evaluated in a retrospective nation-wide reg-
ister-based linkage study in Finland. SH was dened as a hospitalization or a
secondary health care visit due to DM with severe hypoglycemia (ICD E10.00 or
E11.00). Total population (n=140,035) comprised patients who purchased insulin
during 2000-2009 and were followed-up for SH events until end of year 2009 or
death. The present analysis comprised those 77,046 patients who had not used
insulin glargin (IGla), insulin detemir (IDet) or NPH insulin (NPH) before year 2000.
Stratied incidence rates with 95% CIs were calculated. Hazard ratios (HR) were
estimated by Coxs proportional hazards model. 9,716 SH events were identied.
Type of DM (type 1 or 2) was not associated with risk of SH. Compared to IGla,
risk of SH was lower during use of IDet (HR 0.76, CI 0.67-0.87), and higher during
use of NPH (HR 1.19, CI 1.11-1.28) (Fig. 1 upper panel). Female gender predicted
lower risk (HR 0.93, CI 0.88-0.98), and increasing age predicted higher risk of
SH (Fig.1 lower panel). Risk of SH recurrence was lower during IDet (HR 0.60,
CI 0.52-0.69), and higher during NPH (HR 1.58, CI 1.46-1.71) compared to IGla.In
conclusion, our data shows that increasing age and type of long-acting insulin are
predictors of hospitalization due to SH. Risk of hospitalization due to SH could
potentially be modied by selection of long-acting insulin.
Figure 1: Occurrence of the rst hospitalization or secondary care visit due to SH
by type of insulin (upper panel) and by age group (lower panel).
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389-P
Hypoglycemia in Diabetes Requiring Emergency Services Interven-
tion: Patient Characteristics and Mortality
ALICE C. HUSKINSON, MATTHEW J. BOTTOMLEY, LINDA CLAPHAM, CATHRYN
JAMES, SAAD H. ALZAHRANI, RHODRI KING, PETER J. GRANT, RAMZI A. AJ-
JAN, Leeds, United Kingdom
Hypoglycemia is a common complication of diabetes treatment and can
be life-threatening. The aim of this work was to characterize individuals with
diabetes sustaining severe hypoglycemia requiring emergency services inter-
vention, covering a population of approximately 28,000 diabetes subjects over
a period of 5.5 years.A total of 1312 episodes were recorded in 858 subjects,
with 55% occurring in individuals with type 1 diabetes (T1DM), 39% in type
2 diabetes (T2DM) and a minority unclassied. Two third of T1DM subjects
were males, whereas gender distribution was equal amongst T2DM individu-
als. Mean age (range) of T1DM subjects was49 yrs (8-95), whereas T2DM
subjects were older at 73 yrs (32-97). Of the T2DM subjects, 27% were not
on insulin treatment with the majority receiving a sulphonylurea (90%). Mean
capillary blood glucose (SEM) at arrival of emergency services was lower
in T1DM compared with T2DM subjects (1.710.03 mmol/L and 1.960.04
mmol/L, respectively; p<0.001). In T2DM individuals, the severity of hypogly-
cemia in insulin users and non-insulin users was similar at 1.930.05 mmol/L
and 1.980.06 mmol/L, respectively (p=0.51). HbA
1c
was higher in T1DM than
T2DM subjects (661 mmol/mol and 601 mmol/mol, respectively; p<0.001),
and insulin users in T2DM had signicantly higher HbA1c compared with those
not on this therapy. Around one third of patients self-reported hypoglycemic
unawareness. Mortality within one year of the severe hypoglycemic event
was 17.1% in the whole population, which is comparable to the one year mor-
tality of 15.6% following myocardial infarction in diabetes subjects in our area.
This observational study demonstrates that severe hypoglycemia is a common
complication in both T1DM and T2DM, and frequently occurs in non-insulin
treated individuals. We show high mortality in the rst year following severe
hypoglycemia, although it is unclear at present whether low blood glucose
directly contributes to death in these individuals.
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Spontaneous Versus Insulin Induced Hypoglycemia and Mortality in
Hospitalized Patients
RAJESH GARG, APOORVA TRIVEDI, SHELLEY HURWITZ, ALEXANDER TURCHIN,
Boston, MA
Hypoglycemia is a risk factor for increased mortality in hospitalized pa-
tients. However, most studies suggest it to be a marker rather than a cause
of increased mortality. Therefore, insulin induced hypoglycemia may have
lower mortality than spontaneous hypoglycemia. We investigated the re-
lationship between hypoglycemia and mortality and the impact of insulin
treatment on this relationship. Data were obtained from electronic medical
records of patients admitted between 4/1/2008 and 11/30/2010. Patients
with one or more blood glucose values s50 mg/dl from the point-of-care
glucose testing were considered hypoglycemic. Patients treated with insulin
were assumed to have insulin induced hypoglycemia. No patient was re-
ceiving any non-insulin anti-diabetic agents. Age, gender and race matched
patients with blood glucose values >70 mg/dl were included as the non-
hypoglycemic control group. Thus, we had four groups of patients: 1) non-
insulin treated hypoglycemia (NIH) (N=135); 2) insulin treated hypoglycemia
(IH) (N=961); 3) non-insulin treated control (NIC) (N=1058) and 4) insulin
treated control (IC) (N=736). Insulin treated patients were older: age 58 15,
60 15, 58 16 and 63 14 years in NIH, IH, NIC and IC groups respectively.
Death during hospitalization was 35 %, 20%, 1% and 5 % in the four groups
respectively. Death rate was signicantly higher in hypoglycemic compared
with control groups and in NIH compared with IH (p <0.0001) but lower in NIC
compared with IC group (P <0.0001). After controlling for age, gender and
Charlson comorbidity score all pair-wise comparisons for death rate were
still signicant (p<0.001). On multivariable logistic regression analysis, odd
ratio of death in IH relative to NIH was 0.48 [95% CI: (0.33, 0.72), p<0.001].
These data suggest a higher mortality rate among patients who develop
hypoglycemia, spontaneous as well insulin induced, although patients with
insulin induced hypoglycemia have lower mortality than those with sponta-
neous hypoglycemia.
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391-P
The Impact of Insulin Type on Severe Hypoglycemia Events Requir-
ing Inpatient and Emergency Department Care in Patients With Type
2 Diabetes
MATTHEW SOLOMON, SANDEEP VIJAN, FELICIA FORMA, RYAN CONRAD, NICK
SUMMERS, DARIUS LAKDAWALLA, Stanford, CA, Ann Arbor, MI, Bridgewater, NJ,
Santa Monica, CA, Los Angeles, CA
Although clinical trials suggest that basal analogues have a lower risk of
hypoglycemia than other forms of insulin, conclusive data are lacking. We
evaluated the risk from different insulin types on severe hypoglycemia (SHG)
events requiring inpatient (IP) or emergency department (ED) care in patients
with Type 2 diabetes (DM).Using a large database of privately-insured work-
ing-age US adults from 1998-2009, we conducted a retrospective cohort study
of DM patients newly started on insulin after at least 6 months of treatment
with oral anti-diabetic medications (OADs). We measured SHG events after
insulin initiation occurring in the IP or ED setting. Patients were classied into
an insulin group based on the most frequently used insulin type prior to the
SHG event. Multivariate Cox models assessed the association between insu-
lin type and the risk of subsequent SHG events, controlling for demographics,
comorbidities, and the number and type of concomitantly prescribed OADs.The
cohort included 8,626 patients (age 53.5 yrs; 55% female) with DM followed
for an average of 4.0 yrs after insulin initiation. Of these, 161 (1.9%) had a
SHG event at an average of 3.1 yrs after insulin initiation. Patients with SHG
events were slightly older (56.1 yrs vs 53.4 yrs), used a similar number of OADs
(1.1 vs 1.2), and were more likely to have multiple comorbidities compared to
those without SHG events. In multivariate Cox models, premixed insulin (HR
2.07; p<0.01), isophane insulin (NPH) (HR 2.01; p<0.01), and rapid acting insulin
(HR 2.78; p< 0.01) had a higher risk of SHG events compared to glargine, and
detemir showed a non-signicant trend toward more SHG events (HR 1.20;
p=0.73). These results were unchanged in sensitivity analyses of more inclu-
sive denitions of SHG events.Among patients with DM who initiate insulin
therapy, we found glargine users had lower rates of SHG events requiring IP or
ED care compared to users of other insulin formulations.
392-P
WITHDRAWN
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COMPLICATIONSMACROVASCULARATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES
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Higher Levels of Cytokines and Glycemic Variability are Associated
With Hypoglycemia and Increased Mortality in Medical Intensive
Care Unit Patients
FRANCISCO PASQUEL, CHRISTOPHER NEWTON, FARNOOSH FARROKHI, SAU-
METH CARDONA, DAWN SMILEY, LIMIN PENG, GUILLERMO UMPIERREZ,
Atlanta, GA
The prognostic importance of increased levels of inammatory cytokines
on glycemic variability and outcome in critically ill patients with hypergly-
cemia is not known. Accordingly, we determined the association between
inammatory cytokines (IL6, TNF-o), oxidative stress markers (DCF, TBA),
glycemic variability (determined by standard deviation of BG measures), hy-
poglycemic events and mortality in 150 medical ICU patients (age: 5812 yr;
male: 54%; known diabetes: 54%, [mean SD]) treated with continuous insu-
lin infusion (CII). The mean BG before CII was 18960 mg/dL and decreased
to a mean BG of 12123 mg/dL during CII. The overall mortality was 24%.
Non-survivors had higher levels of baseline IL-6 and TNF-o compared to
survivors (both, p=0.01). We found mortality to be strongly associated with
log IL6 values (died vs. lived: 3.91.3 vs. 3.11.6 log pg/mL, p=0.01) and log
TNF-o (3.01.0 vs 2.60.6 log pg/mL, p=0.01), but not with admission or hos-
pital BG concentrations (P=NS). Glycemic variability correlated with plasma
IL-6 (p=0.001) and TNF-o (p=0.028) levels but not with oxidative stress mark-
ers (p=NS). Patients with hypoglycemia (<70mg/dL) during CII had a higher
average log TNF-o levels during CII (2.90.8 vs 2.70.6 log pg/mL; p=0.029),
and a longer length of stay (2318 days vs 1725 days, p=0.005) compared
to patients without hypoglycemia. Patients with more hypoglycemic events
(0 vs 1-2 vs >2) had higher average log TNF-o during CII levels (2.70.6 vs
2.90.95 vs 2.90.4 log pg/mL; p=0.036). In addition, non-survivors with
hypoglycemia had higher glycemic variability than survivors with hypoglyce-
mia (24.48 vs 31.112 mg/dL; p=0.023). In summary, higher plasma levels of
inammatory cytokines (IL6 and TNF-o) and glycemic variability were posi-
tively correlated with increased risk of hypoglycemic events and hospital
mortality in critically ill patients with hyperglycemia in the ICU.
Supported by: sano-aventis
COMPLICATIONSMACROVASCULAR
ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND
HUMAN DIABETES
Guided Audio Tour: Diabetes and the Macrovascular TreePathology,
Function, and Assessment (Posters 394-P to 401-P), see page 17.
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394-P
The Detection of Ischemia in Asymptomatic Diabetics (DIAD) Study
Risk Score
DEBORAH A. CHYUN, CHARLES M. CLELAND, LAWRENCE H. YOUNG, SILVIO E.
INZUCCHI, JANICE E. DAVEY, FRANS J. WACKERS, FOR THE DIAD STUDY INVES-
TIGATORS, New York, NY, New Haven, CT
Coronary heart disease (CHD) is the leading cause of death in type 2
diabetes (T2DM). Current risk score calculators typically do not include
diabetes-specic prognostic factors and do not perform well in this popula-
tion. Validated diabetes-specic risk calculators are needed to estimate CHD
risk in this population. We analyzed data from the Detection of Ischemia
in Asymptomatic Diabetics (DIAD) study in order to identify factors associ-
ated with cardiac events (cardiac death, acute coronary syndromes, heart
failure, and revascularization) and to develop and validate a risk score for
older adults with T2DM. The mean age of the 1,119 subjects (54% male)
was 60.76.6 years, with mean T2DM duration of 8.57 years, mean HbA1c,
7.11.5% and 23% using insulin. During 5 years of follow-up, there were 94
(8.4%) cardiac events. Cox proportional hazards regression revealed the fol-
lowing independent predictors of events: black ethnicity (HR 0.37, p=.01);
T2DM duration (HR 1.07 per year, p<.0001); HbA1c (HR 1.26 per 1% increase,
p=.0008); insulin use (HR 0.51; p=.01); extremity numbness (HR 1.87, p=.003);
the lowest quartile of Valsalva ratio (VR) (HR 1.58, p=.04); highest quartile
of pulse pressure (HR 2.04, p=.001); family history of CHD (HR 1.61, p=.05);
waist:hip ratio (HR 1.04 per each .01 increase; p=.003); no regular physi-
cal activity (HR 1.53, p=.05); and minor ST-T-wave abnormalities on ECG (HR
1.74, p=.03). A risk score equation was developed based on comparison to
a low risk subject; equations were developed with and without VR and ECG
data as these are often not routinely available. Internal validation of the
model, using 250 bootstrap resamples, revealed adequate predictive ability
of the models (Somers D rank correlation and concordance index). These
results suggest that incorporation of diabetes-related factors is important
in cardiac risk stratication in older patients with T2DM. Accurate CHD risk
prediction in the T2DM population may enhance care delivery and improve
outcomes.
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Updated UKPDS Risk Engine that Estimates Primary and Secondary
Cardiovascular Disease Risk in People With Recently-Diagnosed or
Established Type 2 Diabetes
RUTH L. COLEMAN, RICHARD J. STEVENS, RURY R. HOLMAN, Oxford, United
Kingdom
The UKPDS Risk Engine is a diabetes-specic risk calculator that uses the
20-year UK prospective diabetes study (UKPDS) trial data to estimate future
coronary heart disease (CHD) and stroke risk. Recognized limitations are
that it does not estimate total cardiovascular disease (CVD) risk, can only be
used in individuals with newly-diagnosed type 2 diabetes and does not take
account of albuminuria.We have produced a new UKPDS Risk Engine that
incorporates the 10-year UKPDS post-trial monitoring data. It utilizes over
40,000 patient-years of data with 1,115 cardiovascular disease (CVD) events,
dened as: CVD death, non-fatal myocardial infarction (MI), non-fatal stroke
or ischaemic heart disease. Patients were entered into the model at ran-
dom intervals after diagnosis of diabetes so that risks could be estimated
in people with varying degrees of diabetes duration, and with a history of
previous MI or stroke where these had occurred prior to model entry.New
statistically-independent risk factors, in addition to those in the old model
(age, gender, ethnicity, smoking status, HbA1c, systolic blood pressure, total-
to-HDL cholesterol ratio and atrial brillation), were: previous MI or stroke,
macroalbuminuria, microalbuminuria, duration of diagnosed diabetes and
body mass index, with hazard ratios of 3.06, 1.56, 1.32, 1.05 and 1.03 respec-
tively in the CVD equations. CHD risk estimates generated by the new model
were more precise than the old model, providing 22% narrower 95% Con-
dence Intervals.The updated UKPDS Risk Engine provides a more precise and
more generalizable diabetes-specic risk calculator that estimates the risk
of CVD and CVD death, in addition to CHD and stroke. It can estimate risks
for primary or secondary CV events, and in people who have established or
newly-diagnosed diabetes.
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Acute Hyperglycemia Reduces Cerebral Vasomotor Reactivity in
Insulin-Resistant Subjects
PAOLA PALAZZO, ILARIA GIORDANI, ILARIA MALANDRUCCO, FABIANA PIC-
CONI, FRANCESCO PASSARELLI, FABRIZIO VERNIERI, RICCARDO ALTAVILLA,
SIMONA FRONTONI, Rome, Italy
Cerebral Vasomotor Reactivity (CVR), a reliable method to measure
cerebral hemodynamics, is reduced in type 2 diabetic patients, thus con-
tributing to cerebrovascular morbility and mortality. This study aimed to
investigate the impact of acute hyperglycemia on cerebral reactivity, in a
condition of insulin-resistance, before the onset of overt hyperglycemia.We,
therefore, studied seventeen patients with metabolic syndrome (MS) and 3
age-matched controls. Metabolic syndrome was dened according to the
International Diabetes Federation criteria. All subjects underwent a 2-hour
hyperglycaemic clamp (HC), at a blood glucose level of 13.9 mmol/l. In order
to avoid the possible confounding effect of hyperinsulinemia, endogenous
insulin secretion was inhibited by octreotide. At baseline, 60 and 120 min
of the HC, and 2 hours after the end, CVR was evaluated, by means of bi-
lateral Transcranial Doppler measurement of middle cerebral artery mean
ow velocity, and expressed as percentage change during inhalation of
a mixture of air and 7% CO
2
.In MS patients, but not in controls, CVR was
signicantly reduced after 1 hour and 2 hours of stable hyperglycemia vs.
baseline (50.23% vs. 62.38%, p=0.002 and 54.27% vs. 62.38%, p=0.015 re-
spectively). Two hours after the end of HC, CVR value returned to baseline.
The nding that acute hyperglycemia worsened CVR only in patients with
metabolic syndrome, but not in controls, suggests that insulin resistance per
se plays a role in CVR response to acute hyperglycemia, before the onset of
diabetes.This observation has many clinical implications, particularly on the
possible prognostic meaning of the stress-induced hyperglycemia, in terms
of cerebrovascular disease in patients with metabolic syndrome, but with-
out type 2 diabetes.
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Post-Operative Glycemic Control and Survival 1 Year after Cardiac
Surgery: Ten Year Experience With Intensive Glycemic Manage-
ment at the Pittsburgh VA
R.H. RAO, PETER L. PERREIAH, CANDACE A. CUNNINGHAM, Pittsburgh, PA
Intensive glycemic management was implemented in 2111 patients under-
going cardiac surgery (924 with diabetes) at our hospital, initially to a 90-150
mg/dl target blood glucose [BG] with a formula-based algorithm, tightened
further to 80-120 mg/dl after 2005, using an adaptive basal-bolus Glycemic
Expert system for Nurse-Implemented Euglycemia (GENIE). We analyzed the
impact of postoperative glycemic exposure (Time-Weighted Excess Glucose
[TWEG], dened as the integrated area under the curve of BG >120 mg/dl
over time) and severe hypoglycemia (BG<40mg/dl) on mortality 1 year after
surgery.One year mortality showed a clear separation, akin to a threshold
effect, between patients with TWEG ~4000 mg.dl-1.hr (equivalent to main-
taining BG ~140 mg/dl for 200h) and those with lower levels of glycemic
exposure (Odds Ratio, OR, 4.5 [95% CI 3.2- 6.5] p<0.001), there being no mor-
tality differences between various subgroups with TWEG <4000. Increased
1 year mortality was also associated with the occurrence of 1 or more severe
hypoglycemic events (OR 12.04 [CI 6.5-22.3] p<0.001) but, when glycemic ex-
posure was taken into account, the effect of hypoglycemia on mortality was
seen only in association with TWEG ~4000 (OR 8.2 [CI 3.8-18] p<0.001), not
with TWEG <4000 (OR 3.6 [CI 0.8-17] p NS). Finally, patients without a prior
diagnosis of diabetes at the time of surgery had a signicantly higher 1 year
mortality than those with a prior diagnosis of diabetes (OR 1.6 [CI 1.1-2.3]
p<0.01).Our results show that decreased long-term survival after cardiac
surgery is associated with (a) relatively modest levels of glycemic exposure,
and (b) the occurrence of severe hypoglycemia, but only in the context of
higher glycemic exposure. On the other hand, (c) survival is better in patients
with a prior diagnosis of diabetes compared to those who develop postop-
erative (stress) hyperglycemia without a prior diagnosis of diabetes.
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398-P
Sexual Dysfunction as a Marker of CVD in Males With 50 or More
Years of Type 1 Diabetes
SARA J. TUREK, STEPHANIE M. HASTINGS, JENNIFER K. SUN, GEORGE L. KING,
HILLARY A. KEENAN, Boston, MA
Increasing rates of diabetes and associated cardiovascular (CV) compli-
cations have a substantial adverse effect on quality of life and nancial
burden on the healthcare system. There is increasing evidence that sexual
dysfunction may be a predictor of increased cardiometabolic risk in aging
men. The goal of this analysis was to investigate the association of sexual
dysfunction with clinical markers of vascular disease among males using
data from the 50 Year Medalist Study who have documented 50+ years of
type 1 diabetes. Males (48.8%, n=316/658) in this group had mean age of
71.3 (8.41) y, mean diabetes duration of 59.1 (7.22) y, mean BMI of 26.3
(3.74) kg/m
2
and mean HbA1c of 7.04% (0.89). Prevalence of sexual dys-
function was characterized by a yes response to the question: Have you
ever had sexual problems?(69.8%). Risk factors associated with sexual
dysfunction included elevated HbA1c (7.1 0.9% v. 6.8 0.8%, p<0.001),
BMI (26.7 3.9 v. 25.2 3.1 kg/m
2
, p=0.02), higher total cholesterol (159.9
31.8 v. 150.1 30.6 mg/dL, 0.02), and lower HDL (54.9 16.1 v. 61.7 17.4
mg/dL, p<0.01). Age and duration (71.4 8.1 v. 71.2 9.1 y, p=0.7; 59.3 7.8
v. 59.0 7.0 y, p=1.0, respectively) were not associated with dysfunction.
In this group, sexual dysfunction was not signicantly associated with any
microvascular complication (p>0.05); however, dysfunction was associated
with CVD OR: 1.7 (95% CI:1.02, 2.7) independent of age (CV OR adjusted for
age 1.7 (1.02, 2.8)) and BMI (CV OR adjusted for BMI 2.3 (1.1, 4.6)). Congruent
with its association with CV, levels of inammatory markers including IL-6
(median 0.1 [Q2 0.06, Q3 0.3] v. 0.09 [0.04, 0.19] pg/mL, p=0.03) and PAI-1
(204.1 [ 106.4, 246.5] v. 160.0 [99.0, 203.5], pg/mL p=0.08) were also higher in
those reporting dysfunction. These ndings indicate that sexual dysfunction
may be a marker for CV in those with extreme duration diabetes. This nding
is of clinical importance as sexual dysfunction is a more overt sign of CV risk
than clinical markers of the disease.
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399-P
Effects of Insulin and Combined Hyperglycemia and Elevated FFA
on Endothelial Function, Adhesion Molecules and Platelet Aggrega-
tion in Healthy Man
NINO G. JOY, CHERYL YOUNG, DONNA B. TATE, MAKA S. HEDRINGTON, MAIA
MIKELADZE, IAN C. DAVIS, LINDSAY PULLIAM, LISA M. YOUNK, CHELSEA
YOUNK, STEPHEN N. DAVIS, Baltimore, MD
The acute vascular effects of combined hyperglycemia and elevated FFA
in the presence of clamped hyperinsulinemia are largely unknown. This study
tested the hypothesis that, hyperinsulinemia could rescue endothelial func-
tion; reduce platelet aggregation and pro atherogenic mechanisms during
moderate hyperglycemia and hyperlipidemia in healthy humans. Eighteen
individuals (9M/9F), age 402 years, BMI 28.31 kg/m2 were studied during
4 separate randomized protocols. The pancreatic clamp was combined with
a 20 % Intralipid infusion and 4 hour glucose clamps consisting of either;
1) euinsulinemic/ euglycemia (901mg/dl), 2) euinsulinemic /hyperglycemia
(2002mg/dl), 3) hyperinsulinemic /euglycemia or 4) hyperinsulinemic/hyper-
glycemia. Two D doppler ultrasound was used to determine ow mediated di-
lation (FMD) of the brachial artery.Results mean SEM from the nal 30 min
of clamps are shown in table 1.In Summary, in the presence of moderately
increased hyperglycemia and FFA; insulin can signicantly reduce P-Selectin,
ICAM, VCAM levels and improve Nitric Oxide mediated endothelial function.
Additionally insulin can reverse the effects of FFA to impair endothelial func-
tion and elevate P-Selectin and adhesion molecules. We conclude that acute
hyperinsulinemia can reverse the deleterious vascular effects of combined
FFA and hyperglycemia or FFA alone on endothelial function, platelet aggre-
gation and pro atherogenic mechanisms in healthy man.
Table 1: *p<0.05 to euins/hypergly; p<0.05 to euins/eugly
Euins/
eugly
Hyperins/
eugly
Euins/
hypergly
Hyperins/
hypergly
AP-selectin pg/mL 436 -45* 557 155*
AFMD % change -1.91.3 -0.11.9* -4.81.8 -1.22*
AVCAM ng/mL -222 -3142* 11030 -128-46*
AICAM ng/mL 86 -166* 14.8 -9-5*
FFA mol/L 993157 837157 866225 940195
Insulin U/mL 256 239 11319 10520
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An Alternative Pathway for Hypobrinolysis in Type 2 Diabetes: The
Role of Complement C3
K. HESS, SAAD ALZAHRANI, JACKIE PRICE, MARK STRACHAN, NATALIE OX-
LEY, FLADIA PHOENIX, NIKOLAUS MARX, VERENA SCHROEDER, RHODRI KING,
RAMZI AJJAN, Aachen, Germany, Leeds, United Kingdom, Edinburgh, United King-
dom, United Kingdom, Bern, Switzerland
Plasminogen activator inhibitor (PAI)-1 is regarded as the main antibrin-
olytic protein in diabetes but recent work indicates that complement C3,
an inammatory protein, directly modulates brinolysisin type 1 diabetes
(T1DM). Therefore, we investigated the role of complement C3 in brinolysis
in a large cohort of T2DM subjects.Fibrin clot lysis was determined in 875
patients enrolled on the Edinburgh type 2 diabetes study using a turbidime-
tric assay. Plasma levels of complement C3, C-reactive protein (CRP), PAI-1
andbrinogen were analysed by ELISA.Clot lysis time showed a highly sig-
nicant correlation with C3 and PAI-1 plasma levels (r=0.25, p<0.0001 and
r=0.15, p<0.0001;respectively). In contrast, a relatively weak correlation was
detected with CRP (r=0.08, p=0.02) and brinogen (r=0.08, p=0.01). Plasma
levels of C3, CRP, brinogen or PAI-1 did not differ in the presence of previ-
ous history of myocardial infarction or cerebrovascular disease. Plasma lev-
els of all four proteins correlated with body mass index, but only brinogen
showed an interaction with age and duration of diabetes. Ina regression
model involving these proteins, C3 was a predictor of lysis time [predictive
value for 0.1 mg/ml change in plasma levels 14.4 (95% CI 7.9, 21.0) p<0.001],
as was PAI-1 (predictive value for 0.1 ng/ml change in plasma levels 8.2 (4.2,
12.1; p<0.001) with a smaller effect shown for 0.1 mg/ml change in brinogen
[5.3 (0.01-10.5); p=0.049] and none detected for CRP [-2.5 (-9.7, 4.8); p=0.50].
No correlation was demonstrated between C3 and PAI-1 plasma levels in-
dicating the two proteins affect different pathways in brin clot lysis. In
multivariable analysis, drug therapies failed to predict C3 plasma levels,
although a trend was observed with antiplatelet treatment in men. C3 is at
least as strong as PAI-1 at predicting brin clot lysis in subjects with T2DM.
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Supported by: NIHR and the BHF
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Assessment of Asymptomatic Coronary Artery Diseases in Chinese
Adults With Different Glycaemic Status
GUANG NING, YU XU, YUFANG BI, MIN XU, MIAN LI, TIANGE WANG, YU LIU,
JIELI LU, YUHONG CHEN, WEIQING WANG, Shanghai, China
To evaluate the risks of subclinical coronary artery disease (CAD) in early
diabetes and prediabetes, the non-invasive dual-source computed tomogra-
phy coronary artery angiography (DSCT-CA) was used to visualize the coro-
nary artery lumen, identifying the location, severity, and composition of ath-
erosclerotic plaques. A total of 401 age- and sex-matched participants with
normal glucose regulation (NGR), prediabetes or diabetes diagnosed less
than 5 years were randomly selected from a community-dwelling population
aged 40-60 years and asymptomatic of CAD to undergo a comprehensive
examination including a detailed questionnaire, anthropometric measure-
ments, biochemical evaluations and a DSCT-CA examination. Obstructive
CAD was dened as more than 50% narrowing of vessel lumen. Coronary
artery characteristics of the study population according to glucose meta-
bolic status are shown in table 1. Multivariate logistic regression analysis
revealed few independent risk factors for obstructive CAD. Women were
strongly protected from having obstructive CAD (OR [95% condence inter-
val (CI)]: 0.33 (0.14-0.81), P = 0.016). Prediabetes was not associated with an
increased risk of obstructive CAD, whereas diabetes was associated with
a signicant 1.34-fold elevated risk (OR (95% CI): 2.34 (1.01-5.43), P =0.047)
compared with NGR after adjusted for other conventional cardiometabolic
risk factors. In conclusion, using DSCT-CA to anatomically assess subclini-
cal coronary atherosclerosis in a real world Chinese population, we found a
markedly increased risk of obstructive CAD in early diabetes.
Table 1. Coronary artery characteristics of study population by glucose meta-
bolic status
Characteristics
of coronary
arteries
NGR
(n=135)
Prediabetes
(n=132)
Diabetes
(n=134)
Age-
and sex-
adjusted
P values
(Prediabetes
vs. NGR)
Age-
and sex-
adjusted
P values
(Diabetes
vs. NGR)
Age-
and sex-
adjusted
P values
(Diabetes vs.
Prediabetes)
Age (years) 52.3 4.4 52.6 4.2 52.8 4.4 0.53 0.34 0.75
Male n (%) 56 (41.5) 67 (50.8) 67 (50.0) 0.13 0.16 0.90
Family history
of CAD n (%)
22 (16.3) 20 (15.2) 26 (19.4) 0.90 0.44 0.36
Current smoker
n (%)
40 (29.6) 43 (32.6) 46 (34.3) 0.47 0.74 0.61
Coronary cal-
cium score > 0
n (%)
15 (11.1) 15 (11.4) 30 (22.4) 0.98 0.024 0.021
Any coronary
plaques n (%)
58 (43.0) 77 (58.3) 74 (55.2) 0.026 0.067 0.58
Obstructive
CAD n (%)
10 (7.4) 10 (7.6) 22 (16.4) 0.90 0.042 0.027
Guided Audio Tour: Diabetes and Cardiovascular DiseaseFrom Coro-
nary Calcication to Cardiovascular Disease Mortality (Posters 402-P to 407-
P), see page 17.
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The Association of Vascular Atherosclerotic Plaque and Cardiovas-
cular Mortality in the Diabetes Heart Study
SUBHASHISH AGARWAL, AMANDA J. COX, DAVID M. HERRINGTON, NEAL
JORGENSEN, JIANZHAO XU, J.J. CARR, BARRY I. FREEDMAN, DONALD W.
BOWDEN, Novi, MI, Winston-Salem, NC, Seattle, WA
Coronary artery calcium (CAC), carotid artery calcium (CAAC), and abdomi-
nal aorta calcium (AAC) are noninvasive measures of atherosclerosis that
predict mortality. In a prior study we demonstrated that CAC is a powerful
predictor of all-cause mortality (odds of death six fold higher) and cardiovas-
cular (CVD) mortality (odds of death eleven fold higher) in type 2 diabetes
in CAC > 1000 compared to CAC < 10 beyond traditional risk factors. Here
we compared the predictive value of atherosclerotic plaque across multiple
vascular beds for CVD mortality. 854 participants, ages 39-86, with com-
plete data on diabetes, imaging and covariates in the Diabetes Heart Study
(DHS) were followed for an average of 7.4 years. Atherosclerosis imaging
was performed utilizing fast-gated helical CT scan at baseline to obtain mea-
sures of coronary, carotid, and abdominal aorta Agatston calcium scores.
Seven-year risk estimates for CVD mortality were obtained using logistic
regression models adjusted for age, gender, smoking, systolic blood pres-
sure, antihypertensive medication use, total and high-density lipoprotein
cholesterol, and race/ethnicity including log transformed (CAC+1), (CAAC+1),
and (AAC+1).8% (68/854) of participants died during follow-up. CAC was as-
sociated more strongly than either carotid or abdominal calcium with mortal-
ity. After adjusting for confounders, and each other (CAC, CAAC, and AAC),
the odds ratio of CVD mortality increased 1.67 fold (95% CI, 1.06-2.72) for
each 1-standard deviation (SD) increment of log transformed CAC score, 1.50
fold (95% CI, 0.99-2.32) for each 1-SD increment of log CAAC, and 1.21 fold
(95% CI, 0.67-2.30) for each 1-SD increment of log AAC. A receiver operating
characteristic curve analysis also suggested that CAC score was a better
predictor of mortality than was CAAC and AAC, with AUCs of 0.78, 0.77, and
0.77, respectively.In this study CAC score is a better predictor of CVD mortal-
ity than carotid or abdominal aorta vascular beds in diabetes.
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403-P
Patients With Type 2 Diabetes Demonstrate Reduced Cardiac Ener-
getics and Increased LV Torsion Compared to Healthy Age Matched
Controls
MICHAEL KUEHL, MARTIN J. STEVENS, MUHAMMAD AZAM, ROGER BEADLE,
Birmingham, United Kingdom
T2DM induced heart failure can occur in the absence of coronary artery
disease and might be related to the altered cardiac substrate metabolism.
Magnetic resonance spectroscopy (MRS) allows the measurement of car-
diac energetics (CE) non-invasively. Our aim was to determine CE and cardiac
function in a cohort of otherwise healthy patients with longstanding T2DM
and to compare the ndings to sex and age matched healthy controls (HC).A
cross-sectional study was conducted in 27 subjects with T2DM free of
known coronary heart disease attending the outpatient clinic of the Birming-
ham Heartlands Hospital, UK and compared to 17 HC. Phosphocreatine/-
ATP ratios as marker of CE were measured with a Philips Achieva 3T MRI/
MRS scanner (Philips Healthcare, Best, the Netherlands). Speckle-tracking
echocardiography was performed using a GE Vivid 7 ultrasound (GE Vingmed
Ultrasound, Horten, Norway).27 subjects (age, 53 12 years, diabetes dura-
tion 11 2 years, female, 42%, HbA1c, 8.4 1.1%, BMI 30.6 4.5%) and 17
control age and sex matched subjects (age, 48 11, female 50%, BMI 24.1
3.7%) have been recruited so far. The phosphocreatine/-ATP ratio were
reduced in patients with T2DM compared to HC (1.07 0.37 vs 1.54 0.53,
p<0.01 respectively). In concert, left ventricular (LV) torsion was increased in
patients with T2DM compared to HC (2.4 0.76 vs 1.4 0.7/cm, p=<0.001).
There was however no signicant correlation between torsion and cardiac
energetics.We demonstrate in patients with T2DM free of coronary artery
disease that cardiac energetics are reduced and LV torsion is increased.
These ndings are consistent with our previous results in patients with
T1DM, suggesting that impaired cardiac energetics and increased LV torsion
are early complications of otherwise healthy asymptomatic diabetes and
reect the consequences of metabolic dysfunction. The role of these early
decits in the development of cardiac failure warrants further investigation.
Supported by: Eli Lilly
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404-P
Impact of Heart Failure on Hypoglycemic Complications in Patients
With Type 2 DiabetesAn Analysis of the DiaRegis Registry
DIETHELM TSCHPE, PETER BRAMLAGE, CHRISTIANE BINZ, MICHAEL KREKLER,
KRISTIAN LBNER, EVELIN DEEG, ANSELM K. GITT, Bad Oeynhausen, Germany,
Mahlow, Germany, Mnchen, Germany, Wedel, Germany, Ludwigshafen, Germany
Heart failure and type-2 diabetes is a high risk disease constellation.
We hypothesized that hypoglycaemia might be more frequent in patients
with diabetes and heart failure than in those without heart failure and may
worsen prognosis. Analyses were based on DiaRegis which is a prospective
registry in Germany including 3810 patients with type-2 diabetes receiving
antidiabetic treatment, oral mono- or oral dual combination therapy. For
3,746 patients data on the presence of heart failure were available. Patients
with heart failure (n=370; 9.9%) had a considerable cardiovascular disease
burden with coronary artery disease (OR 7.02; 95%CI 5.59-8.82), peripheral
arterial disease (3.55; 2.56-4.91) and blindness (12.29; 2.74-55.12) showing
the highest increase over patients without heart failure. Hypoglycaemia
within 12 months prior to baseline was almost twice as frequent in diabetic
patients with heart failure (17.8%) than in those without (10.0%; OR 1.96;
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95%CI 1.47-2.61). Associations were signicant for the incidence of symp-
tomatic hypoglycaemia with (OR 4.05; 95%CI 1.91-8.58) or without help (OR
1.79; 95% CI 1.27-2.53) as well as for those with need of medical assistance
(OR 7.93; 95%CI 2.65-23.73). 61 of 325 patients with heart failure at baseline
(18.8%) had any episode of hypoglycaemia during the 12 months follow-up.
Myocardial infarction (1.6 vs. 0%), stroke/TIA (OR 4.59; 95%CI 1.29-16.39)
and depression (OR 2.98; 95%CI 1.33-6.70) were more frequent in patient
with heart failure and incident hypoglycaemia than in those without. We
conclude that hypoglycaemia was almost twice as frequent in diabetic pa-
tients with heart failure than in those without and was associated with a
worse prognosis. The ndings suggest that particular attention should be
given to an appropriate treatment strategy in patients with type-2 diabetes
and overt heart failure to improve patient outcomes.
Supported by: Bristol-Myers Squibb/AstraZeneca
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405-P
Prevalence and Incidence of Peripheral Artery Disease is Higher in
Indian Women
JAYASHEEL O. ESHCOL, RAJENDRA PRADEEPA, SIVASANKARAN SUBHASHINI,
SARAVANAN JEBARANI, RANJIT M. ANJANA, RANJIT I. UNNIKRISHNAN,
VISWANATHAN MOHAN, Iowa City, IA, Chennai, India
Peripheral artery disease (PAD) is a common complication of diabetes
associated with increased cardiovascular morbidity but the disease is not
well characterized in India. The available research shows a lower prevalence
than the west, but studies are few and none have assessed the incidence
and progression of PAD. We sought to determine the factors associated with
the incidence and progression of PAD in the Asian Indian Type 2 diabetic
population.We retrospectively examined all patients who had an ankle bra-
chial index (ABI) measurement at a diabetes centre in 2001. The baseline
clinical and biochemical characteristics and all follow up ABI measurements
were abstracted from the electronic medical record. PAD was dened as an
ABI <0.9 and incidence was identied as the rst occurrence of an ABI <0.9.
A change in ABI >0.15 was dened as signicant progression. Risk factors for
prevalent PAD and for progression were analysed by logistic regression and
by time-to-event analysis for incident PAD.Two thousand ve hundred and
twelve patients were followed for an average of 7 years. 7.6% of the study
population had PAD in 2001, (11.8% in women and 5.1% in men, adjusted OR
2.7 [CI 1.7-4.2]). Prevalent PAD was associated with high mortality, adjusted
HR 3.3[1.4-7.7]. There were 280 new cases of PAD identied resulting in a
crude incidence rate of 17 per 1000 patient years. Females had a 2-fold in-
creased rate of PAD, adjusted HR 1.94 [CI 1.4-2.7]. Other adjusted variables
that predicted incident PAD were increasing age, duration of diabetes and
LDL cholesterol. A drop in ABI > 0.15 was seen in 5.5% of patients and this
was associated with increasing age, hypertension and LDL.Among Asian In-
dian type 2 diabetic patients seen at a tertiary diabetic centre in southern
India there is a striking female predominance in incident and prevalent PAD.
Age and duration of diabetes are the strongest predictors of both incident
and prevalent PAD. This study suggests that in the Indian diabetic popula-
tion, women are at higher risk for PAD.
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The Association Between Ideal Cardiovascular Health and Coronary
Artery Calcication in Adults With and Without Type 1 Diabetes
AMY ALMAN, DAVID M. MAAHS, MARIAN REWERS, JANET K. SNELL-BER-
GEON, Aurora, CO
In 2010, the American Heart Association identied national 7 goals for
ideal cardiovascular health (ICH). The purpose of this study is to examine the
prevalence of attaining ICH goals in a population of adults with and without
type 1 diabetes (T1D) and to examine the association of ICH with coronary
artery calcication (CAC). Since the goal of a fasting plasma glucose <100
mg/dL is not relevant in T1D, this analysis focused on the remaining 6 metrics
(see Table).The CACTI study has followed adults with (n=652) and without
T1D (n=764) for 6.10.5 years. At baseline, diet, smoking, and physical ac-
tivity were assessed, and BMI, cholesterol, and blood pressure were mea-
sured. Complete ICH data were available on 1177 subjects, and 856 had CAC
progression data from the follow-up exam. Descriptive analyses and mul-
tivariate logistic regression were performed to examine the prevalence of
ICH and its association with CAC presence and progression.The table shows
the prevalence of the ICH metrics. Adults with T1D were more likely to meet
cholesterol goals than non-diabetics. . A greater number of ICH goals met
was associated with a signicant reduction in the odds of any CAC at base-
line (OR=0.71 (0.62, 0.82)) and the progression of CAC (OR=0.82 (0.70, 0.96)),
adjusted for age, sex, diabetes, hemoglobin A1c, triglycerides, and baseline
CAC score.In conclusion, the prevalence of ICH metrics is similar in T1D
adults and non-diabetics, and is low in both groups for physical activity and
diet. An increasing number of ICH metrics was associated with signicantly
reduced odds of CAC presence and progression.
Prevalence of Ideal Cardiovascular Health Metrics by Diabetes Status
ICH Metric Type 1 diabetes
(n=546)
Non-diabetics
(n=631)
p-value
Never smoking 364 (66.7) 435 (68.9 0.41
BMI < 25 kg/m2 238 (43.6) 294 (46.6) 0.30
Physical activity* 54 (9.9) 51 (8.1) 0.28
Healthy diet** 8 (1.5) 13 (2.1) 0.44
Total cholesterol <200 mg/dl 425 (77.8) 284 (45.0) <.0001
Blood pressure <120/80 mm Hg 254 (46.5) 284 (45.0) 0.60
Data are presented as N(%). * at least 150 min/week moderate intensity or
at least 75 min/week vigorous intensity. ** 4-5 of the following: at least 4.5
cups of fruit or vegetables/day, two 3.5 oz servings of sh/week, at least 3 oz
servings of ber-rich whole grains/day
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Comparison of Hospital Admissions in Patients With DM and Non-
DM Admitted With Acute Coronary Syndrome
VINCENT WOO, AMANDA HAK, Winnipeg, MB, Canada
The objective of this study was to compare cardiac risk factors, treatment,
and outcome between DM and non-DM patients (pts) admitted to a tertiary
care centre with acute coronary syndrome (ACS). Data was collected from
charts of patients who were admitted to the Health Sciences Centre in Win-
nipeg, Canada with ACS between August 2007 and July 2009.The charts of
190 DM pts, of which 96% had DM2, and 263 non-DM pts were examined.
Of all the pts included in the study, at the time of admission, 64% were male
and the average age was 66 13 years. In terms of cardiac risk factors,
non-DM pts were more likely to smoke (37.3%) than DM pts (19.6%); but DM
pts were more likely to be obese (BMI > 30; 39% vs. 28%), aboriginal (13.2%
vs. 3%) or hypertensive (89% vs. 62%) than non-DM pts. DM pts tended to
have lower levels of total cholesterol (4.11mmol/L vs. 4.41mmol/L) and LDL
(2.12mmol/L vs. 2.47mmol/L), but higher levels of triglycerides (1.59mmol/L
vs. 1.29mmol/L) and serum creatinine (157.8mol/L vs. 97.3mol/L) than non-
DM pts. DM pts tended to have lower peak levels of creatine kinase (621.3U/L
vs. 1009.1U/L) and troponin T (1.81ng/mL vs. 2.87ng/mL); but tended to have
lower ejection fractions (43% vs. 49%). As for treatment in hospital, DM pts
were as likely to receive thrombolytics or CABG as non-DM pts. Although not
signicant, DM pts were slightly less likely to have an angiogram or stents
(p-values 0.08 and 0.06, respectively). DM pts were more likely to be dis-
charged on a calcium channel blocker (32% vs. 19%) or a diuretic (37% vs.
17%); but less likely to be discharged on clopidogrel (44% vs. 60%). Finally,
9.5% of DM pts and 6.1% of non-DM pts died because of ACS (p = 0.18). All
differences are statistically signicant at o = 0.05 unless otherwise noted.
Risk factors for ACS such as smoking and high total cholesterol were more
prevalent among non-DM pts than DM pts; however, obesity and hyperten-
sion were more common among DM pts. In conclusion there continues to be
important differences between DM and non-DM pts.
Guided Audio Tour: Cardiovascular Disease in Patients with Diabetes
From Characterization to Mechanisms (Posters 408-P to 413-P), see page 13.
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Associations between Circulating Leukocyte Subtype Counts and
Carotid Intima-Media Thickness in Japanese Subjects With Type
2 Diabetes
TAKESHI MATSUMURA, KAYO TAKETA, HIROYUKI MOTOSHIMA, TAKAFUMI
SENOKUCHI, NORIO ISHII, HIROYUKI KINOSHITA, KAZUKI FUKUDA, TAKESHI
NISHIKAWA, EIICHI ARAKI, Kumamoto, Japan
The increase in leukocyte count is an independent risk factor for car-
diovascular events. However, the association between leukocyte subtype
counts with carotid atherosclerotic regression in patients with diabetes is
unknown. Therefore, we investigated the correlation between leukocyte
subtype counts and atherosclerotic markers in subjects with type 2 diabe-
tes. Overall, 360 outpatients with type 2 diabetes were enrolled and blood
samples were measured. The intima-media thickness of the common carotid
artery (CCA-IMT) was measured by high-resolution B-mode ultrasonogra-
phy. Brachial ankle pulse wave velocity (baPWV) was measured using an
ABI-form device. CCA-IMT was positively correlated with age, systolic blood
pressure (SBP), baPWV and duration of diabetes (r = 0.279, P < 0.001; r = 0.113,
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P = 0.032; r = 0.269, P < 0.001; r = 0.147, P < 0.005, respectively), and nega-
tively correlated with body mass index and diastolic blood pressure (DBP) (r =
-0.158, P = 0.003; r = -0.112, P = 0.034, respectively). baPWV was positively
correlated with age, SBP, DBP, CCA-IMT, urinary albumin and duration of
diabetes. Although white blood cell, monocyte, neutrophil, lymphocyte and
eosinophil counts were all positively correlated with CCA-IMT (r = 0.301, P <
0.001; r = 0.698, P < 0.001; r = 0.265, P < 0.001; r = 0.135, P = 0.01; r = 0.115, P =
0.03, respectively), only the monocyte count was correlated with baPWV.
Multiple regression analyses showed that the monocyte count, age and
baPWV explained 95.8% of the total variance in CCA-IMT, with the mono-
cyte count alone explained 69.7% of the variance in CCA-IMT. This indicates
that the monocyte count is a strong independent predictor of CCA-IMT. In
comparison, age, SBP and CCA-IMT explained 85.5% of the total variance
in baPWV. In conclusion, leukocyte counts, particularly the monocyte count,
may be convenient and valuable markers for detecting the progression of
macrovascular complications in patients with type 2 diabetes.
409-P
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Specicity of Gray Matter Aging in Middle-Aged Patients With Type
1 Diabetes
TIMOTHY M. HUGHES, Pittsburgh, PA
Adults with Type 1 diabetes (T1D) develop cognitive and structural brain
abnormalities similar to those observed in older adults without diabetes.
Initial studies indicate that gray matter volume (GMv) loss in T1D patients
is focal rather than global. However, the spatial distribution of GMv loss in
middle-aged T1D patients is not known. We apply MRI with high spatial reso-
lution to compare GMv of middle-aged T1D adults with age, gender matched
controls. We hypothesize that between-group differences are stronger in
regions known to be affected by aging (frontal lobes, hippocampus, putamen
and thalamus) than in other regions.GMv using 3 Tesla MRI was measured in
33 patients with T1D and 33 age, gender matched non-diabetic controls aged
30-50 years. Between-group differences in GMv were quantied by general
linear models adjusted for intracranial volume, blood pressure, total choles-
terol levels, smoking status, years of education and waist circumference.
A Bonferroni threshold (p<0.004) was applied to account for multiple com-
parisons (n=20) and minimize type-1 error.Between-group GMv differences
met Bonferroni thresholds in areas affected by aging, with smaller GMV
seen in the frontal lobes (11% smaller, p<0.001), thalamus (28% smaller,
p0.15) or hippocampus (5% larger, p=0.02). Between-group GMv differences
were also signicant in areas not related to the aging process, including the
paracentral lobule (17% smaller, p<0.001) and postcentral gyri (11% smaller,
p=0.002). GMv of these regions was not signicantly correlated with dura-
tion of T1D, insulin dose and estimated number of months with elevated A1c
at p<0.05.Compared to matched controls, T1D patients had smaller GMv in
areas affected in the aging process, including the frontal lobes, thalamus
and putamen, but not in the temporal lobes or hippocampus. T1D-related
factors did not correlate with GMv in these regions. Potential mechanisms
underlying lower GMv seen in T1D, such as microvascular disease, need to
be further explored.
Supported by: R01 Grants from NIDDK (DK089028, DK034818) and NIMH
(HL089850)
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411-P
Plasma Leptin is Associated With Vascular Endothelial Function,
but Not With Smooth Muscle Function in Type 2 Diabetic Patients
TOMOAKI MORIOKA, MASANORI EMOTO, YUKO YAMAZAKI, NAOYA KAWANO,
HIROMI URATA, SHOKO TSUCHIKURA, KOKA MOTOYAMA, KATSUHITO MORI,
SHINYA FUKUMOTO, TETSUO SHOJI, MASAAKI INABA, Osaka, Japan
Leptin plays major roles in the pathogenesis of atherosclerosis. How-
ever, the association of leptin with vascular endothelial or smooth muscle
function, that is impaired in the early stage of atherosclerosis, is yet to be
determined in human subjects with type 2 diabetes. Therefore, we investi-
gated the association of plasma leptin levels with endothelium-dependent,
ow-mediated dilatation (FMD) and endothelium-independent, nitroglyc-
erin-induced dilatation (NMD) of the brachial artery in 156 type 2 diabetic
patients (age, 63 10 (SD) years, BMI, 24.4 4.7 kg/m
2
). The mean values
of FMD, NMD and plasma leptin in those subjects were 6.6 3.7 %, 14.1
6.4% and 4.9 4.8 ng/ml, with ranges 0.7 - 18.9, 0.5 - 28.9, and 0.2 - 29.5,
respectively. In simple regression analyses, FMD was negatively correlated
with age (r = -0.266, p = 0.001), systolic blood pressure (SBP, r = -0.211, p =
0.008), and positively with plasma leptin (r = 0.232, p = 0.004), while NMD
was negatively correlated with age (r = -0.414, p < 0.001), SBP (r = -0.348,
p < 0.001), but not with plasma leptin (r = -0.099, p = 0.269). In multiple
regression analyses including age, sex, diabetes duration, BMI, SBP, serum
creatinine, smoking habit, glycated hemoglobin, lipid prole, and plasma lep-
tin, signicant independent contributors to FMD were age ( = -0.100, p =
0.010), BMI ( = -0.281, p = 0.004), SBP ( = -0.042, p = 0.010), and plasma
leptin ( = 0.363, p < 0.001), whereas those to NMD were age ( = -0.368, p <
0.001), BMI ( = -0.440, p = 0.012), SBP ( = -0.097, p = 0.003), but not plasma
leptin ( = 0.016, p = 0.928). In conclusion, plasma leptin level is signicantly
associated with vascular endothelial function, but not with smooth muscle
function, independently of obesity and other cardiovascular risk factors in
type 2 diabetes. This study suggests that the vasodilatory effect of leptin is
able to be demonstrated in type 2 diabetes through vascular endothelium-
dependent mechanisms.
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412-P
MT Preservation of Akt2 Function by Inhibition of Akt Negative Reg-
ulators Prevents Diabetic Inhibition of Cardiac Insulin Signaling
YI TAN, YUEHUI WANG, XIAOKUN LI, LU CAI, Louisville, KY, Wenzhou, China,
Changchun, China
Cardiac insulin resistance is a key pathogenic factor for diabetic cardio-
myopathy, but its mechanism remains largely unclear. Here we reported that
diabetes, induced by streptozocin, signicantly inhibited Akt phosphoryla-
tion at both sites of Ser473 and Thr308 from 2 wks to 2 months, but not
5 months. In cardiac-specic metallothionein-transgenic (MT-TG) diabetic
mice, both phosphorylation sites of Akt were preserved normally at all times
of diabetes. Analysis of Akt isoforms revealed that Akt2, but not Akt1, ex-
pression and phosphorylation were signicantly decreased as the progres-
sion of diabetes in wide-type (WT) mice, but not MT-TG mice. Diabetes also
increased Akt negative regulators, PTEN phosphorylation and TRB3 expres-
sion, in WT but not MT-TG mice, at 5 months of diabetes, suggesting that MT
preservation of Akt2 expression and function may be due to its suppression
of diabetic up-regulation of Akt negative regulators. For mechanistic study,
cardiac H9c2 cells with and without forced MT overexpression (MT-H9c2)
were treated with tert-butyl hydroperoxide (tBHP, an organic oxidant). tBHP
did not affect basal Akt phosphorylation, but signicantly inhibited insulin-
stimulated total Akt phosphorylation and increased PTEN phosphorylation
and TRB3 expression only in H9c2 cells. Furthermore, tBHP reduced Akt2
phosphorylation in the basal and insulin-stimulating conditions, which were
signicantly, but not completely, attenuated in MT-H9c2 cells. Silencing
TRB3 expression with its SiRNA completely blocked tBHP-induced inhibi-
tion of insulin-stimulated Akt2 phosphorylation. These results suggest that
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oxidative stress-attenuated cardiac Akt, especially Akt2, function via up-
regulating Akt negative regulators plays a critical role in diabetic inhibition
of insulin signaling in the heart. MT preservation of Akt2 function by inhibi-
tion of Akt negative regulators prevents diabetic inhibition of cardiac insulin
signaling.
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413-P
Amelioration of Diabetic Cardiomyopathy By TAT-KMediated En-
hanced Delivery of Metallothionein and SOD
YONGSOO PARK, JAETAEK KIM, YOUNGHYO LIM, LEEJIN PARK, HYUNOK KIM,
SANGMO HONG, Seoul, Republic of Korea
Diabetic cardiomyopathy (DCMP) might be resulted from oxidative stress
in heart tissues and antioxidant treatment may improve underlying condi-
tions. To circumvent their limited access into the biologic membrane, we
made Tat-MT and Tat-SOD constructs applying cell penetrating peptide
technologies, delivered to primary cultured neonatal cardiomyocytes and
studied cell viability in different injury conditions of high glucose, hypoxia
and AGE treatment. The protective effect of Tat-MT and Tat-SOD in com-
bination against the development of DCMP was also studied in OLETF rats.
Tat-MT and Tat-SOD were successfully delivered to cardiomyocytes, and the
intracellular activities of MT and SOD increased in line with the amount of
protein delivered. These agents inhibited three different oxidative injuries as
well as angiotensin II mediated direct injuries, and changes in the expression
of the downstream signaling molecules in caridiomyocytes. A single intrap-
eritoneal injection of Tat-MT and Tat-SOD resulted in the delivery of these
antioxidants to the heart. Continuous treatment decreased expression of
ROS and downstream signaling molecules in heart tissues, and delayed the
clinical development of DCMP. Sixteen weeks after treatment, Tat-MT and
Tat-SOD combination treatment group showed complete recovery of diastol-
ic dysfunction compared with Tat-GFP group. Perivascular and/or interstitial
brosis were signicantly decreased in antioxidant treatment group. TGF-
immunoreactivity in the interstitial and perivascular tissue was decreased
in antioxidant treatment group. The ameliorative effects of these agents
against the development of DCMP in vivo might be related to the inhibition
of ROS itself and downstream signaling pathway. We conclude that effec-
tive delivery of a combination antioxidant treatment may prevent and treat
the pathophysiology in patients with DCMP.
Supported by: Basic Science Research Program through the National Research
Foundation
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415-P
HbA1c Reduction and Risk of Cardiovascular Diseases in Type 2 Dia-
betes: An Observational Study from the Swedish NDR
KATARINA EEG-OLOFSSON, BJRN ELIASSON, BJRN ZETHELIUS, ANN-MARIE
SVENSSON, SOFFIA GUDBJRNSDOTTIR, JAN CEDERHOLM, Gteborg, Sweden,
Uppsala, Sweden
The relationship between glycemic control and cardiovascular risk in type
2 diabetes (T2D) remains unclear. The aim of this observational study was to
assess the association between improved glycemic control during follow-up
and risk for coronary heart disease (CHD), cardiovascular disease (CVD) and
total mortality in patients with T2D from the Swedish National Diabetes
Register, baseline HbA1c 7-8.9%, aged 30-75 years, with no previous CVD,
followed-up from 2004 to 2009 (mean 5.7 years).Two groups of patients
were created based on the median for change in HbA1c during follow-up:
8923 patients (mean baseline age 62.0 years, diabetes duration 8.3 years)
with HbA1c decreasing 0.1% or more from baseline to follow-up (mean base-
line HbA1c 7.80.5% and nal 7.00.6%), and 9112 patients (mean baseline
age 61.3 years, duration 9.2 years) with stable or increasing HbA1c 0.1% or
more (mean baseline HbA1c 7.70.5% and nal 8.30.9%).Absolute risk of
rst-incident fatal/nonfatal CVD was 15.1 events per 1000 person-years in
patients with decreasing HbA1c and 26.1 in patients with stable/increasing
HbA1c. Adjusted hazard ratios at Cox regression analysis for rst-incident
fatal/nonfatal CHD, fatal/nonfatal CVD and total mortality, with decreasing
HbA1c compared to stable/increasing HbA1c, were 0.61 (95% CI 0.54-0.69),
0.63 (0.57-0.70), and 0.55 (0.49-0.63), respectively, all p<0.001. Adjustment
was made for a propensity score with stratication by quintiles, including
covariates age, sex, diabetes duration, baseline HbA1c and traditional CVD
risk factors and treatments, as well as changes in these risk factors and
treatments during the study period. Differences in included covariates be-
tween the two groups were balanced when adjusted with the propensity
score.HbA1c reduction of mean 0.8% from mean 7.8% to 7.0% (to the treat-
ment target) was associated with substantially lower risk of CHD, CVD and
total mortality, compared to stable/increasing HbA1c of 7.7 to 8.3.
416-P
The Association of Insulin Resistance and Carotid Intima Media
Thickness (IMT) With Thigh and Calf Circumference in Patients
With Type 2 Diabetes Mellitus
JONG SUK PARK, MIN KYUNG KIM, JIYOON HA, YOUNGMI LEE, EUN JIN KWON,
JIWOON KIM, SHIN AE KANG, CHUL WOO AHN, KYUNG RAE KIM, Seoul, Re-
public of Korea
The relationship between body composition parameters such as thigh
and calf circumference and insulin resistance or atherosclerosis in type
2 diabetes is poorly understood. The aim of this study was to investigate
the relationship between insulin resistance, atherosclerosis, and thigh and
calf circumference in type 2 diabetic patients.A total of 4,427 subjects with
type 2 diabetes were enrolled in this study. Insulin sensitivity was assessed
according the rate constant for plasma glucose disappearance (Kitt) deter-
mined via the short insulin tolerance test. Biochemical and anthropometric
proles were measured according to a standardized protocol. Visceral fat
thickness and carotid intima media thickness (IMT) were measured by ultra-
sonography.Insulin sensitivity index was signicantly correlated with weight
adjusted thigh and calf circumference (r=0.244, r=0.211 in men, r=0.279,
r=0.258 in women, P<0.01). Thigh circumference was inversely associated
with IMT in men and women (r= -0.102, r= -0.170, P<0.05) and calf circum-
ference was negatively correlated with IMT in women (r= -0.173, P<0.01).
Multiple regression analysis revealed that thigh circumference was inde-
pendently correlated with insulin sensitivity index (Kitt) (= 0.128 in men, =
0.141 in women, P<0.05) and IMT after adjusting for age (= -0.096 in men,
= -0.156 in women, P<0.05).Calf and thigh circumference were correlated
with insulin resistance and IMT, and thigh circumference was independently
associated with insulin resistance and IMT in type 2 diabetic patients. These
results suggest that thigh circumference may be a new anthropometric
marker of insulin resistance and carotid atherosclerosis.
WITHDRAWN
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417-P
Genetic Variability in Serine/Threonine Kinase 11 (STK11) is Asso-
ciated With the Risk of Coronary Artery Disease in Type 2 Diabetes
in a Chinese Han Population
XIAOWEI MA, GE BAI, LINJUAN HUANG, JIANWEI ZHANG, RUIFEN DENG, SHAN
DING, NAN GU, XIAOHUI GUO, Beijing, China
Recent studies indicated that the LKB1 is a key regulator of the AMP-
activated protein kinase (AMPK), which plays a crucial role in protectingcar-
diac muscle from damage during ischemia. This study aimed to investigate
whether genetic variations in the STK 11 gene encoding LKB1 affect the
risk of coronary artery disease (CAD) in Chinese type 2 diabetics. 5 hap-
lotype-tagging single nucleotide polymorphisms (tag-SNPs) were selected
from CHB HapMap database ( Phase II, R#27), and total 288 CAD-positive
cases and 159 CAD-negative controls with type 2 diabetes were genotyped
for the 5 tag-SNPs by PCR-RFLP assay. The frequencies of allele A at SNP
rs12979689 were 34.9% in the CAD group and 29.4% in the control group,
respectively. The carriers of minor allele A at rs12977689 had a higher risk
of CAD compared to the homozygotes of CC (OR =1.572, 95%CI 1.039-2.376,
p = 0.035), and the difference was still signicant after adjustment for the
other known CAD risk factors (OR =1.184 , 95%CI=1.036-1.353, p = 0.013)
(Tab.1). The male carriers of major allele C at another SNP rs6510599 had
a higher risk of CAD compared to the female homozygotes of TT (adjusted
OR=5.075, 95%CI 1. 405-18.329, p = 0.013). Our ndings suggest that ge-
netic variation(s) in STK 11 may be associated with CAD risk in type 2 diabe-
tes in the Chinese population.
Association between CAD and SNP12977689 at STK 11 locus
genetype CAD+
n(%)
CAD-
n(%)
OR
(95%CI)
p OR
(95%CI)
p
AA/AC 161(61.5) 71(50.4) 1.572
(1.039-2.376)
0.035 1.184
(1.036-1.353)
0.013
CC 101(38.5) 70(49.6) 1 1
Supported by: National 973 Project (2006CB503903, 2006CB503908)
418-P
Effect of Liraglutide on Carotid Intima-Media Thickness in Patients
With Type-2 Diabetes: A 4-Month Prospective Study
MANFREDI RIZZO, ANGELO MARIA PATTI, VITTORIA DI BARTOLO, ROSARIA VIN-
CENZA GIGLIO, GIUSEPPE MONTALTO, ALI A. RIZVI, Palermo, Italy, Columbia, SC
There is currently high interest in the non-glycemic effects of incretin-
based therapies, specically glucagon-like peptide 1 (GLP-1) analogues, such
as those on cardiovascular risk markers. Liraglutide has been available in
the Italian market for the past one year, where it has been approved to be
prescribed in combination with oral hypoglycemic agents. Liraglutide has
several non-glycemic properties, but its effect on carotid intima-media
thickness (IMT), a recognized marker of subclinical atherosclerosis, is still
unknown. We evaluated in our Italian center the effect of liraglutide on
carotid IMT, as assessed by B-mode real-time ultrasound, in a prospective
study of 33 patients with type 2 diabetes (58% males, age: 599 years),
when added to metformin at a xed dose of 1500/daily. Patients were newly
diagnosed or previously treated subjects with oral hypoglycemic agents.
The dose of liraglutide was 0.6mg/daily for the rst 2 weeks, followed by a
dose of 1.2mg/daily. Statistical analysis was done using paired t-test and the
Spearman correlation method. At baseline patients weighted 829 Kg, with
average fasting glucose 9.51.4 mmol/L and HbA1c 8.30.6%. The following
mean changes were recorded after 4 months of therapy: weight decreased
by 3.1 kg, fasting glucose by 2.3mmol/l, and HbA1c by 1.8% (p<0.0001 for
all). Carotid IMT decreased from 1.550.45 mm to 1.360.31 mm (p=0.0003).
Changes in carotid IMT did not correlate with changes in body weight,
fasting glucose, or HbA1c. In conclusion, liraglutide has desirable actions
on carotid IMT, a surrogate marker of subclinical atherosclerosis, in type 2
diabetes after only 4 months of therapy. This effect seems to be achieved
by mechanisms independent of its effect on glucose metabolism. Further
studies are needed to explore the nonglycemic athero-metabolic benets of
liraglutide and other agents of the GLP-1 analogues class.
419-P
Increased Incorporation of Antiplasmin into the Fibrin Network: A
Possible Mechanism Behind Impaired Fibrinolysis in Patients With
Type 1 Diabetes
ANNA GREN, GUN JRNESKOG, PER-ERIC LINS, GRACIELA ELGUE, HKAN
WALLEN, BJRN WIMAN, Stockholm, Sweden
Patients with type 1 diabetes have a tighter brin network, which has
been associated with impaired brinolysis. Antiplasmin is an important -
brinolysis inhibitor. We thus investigated the incorporation of antiplasmin
into brin network in patients with type 1 diabetes.237 age and sex matched
patients (107 women) with type 1 diabetes, mean age 4413 and diabetes
duration 2314 years were investigated. 78 healthy subjects (44 women)
with mean age 4810 years served as controls. We measured the incorpora-
tion of antiplasmin into a brin clot (by an ELISA method), plasma levels of
plasminogen activator inhibitor type 1 (PAI-1), tissue plasminogen activator
(tPA)- PAI-1 complex, antiplasmin, plasmin-antiplasmin (PAP) complex, D-
dimer and the glycemic control by HbA1c (Mono S; reference level <5.2%).
More antiplasmin was incorporated into the brin network in patients (1.66
0.31 g/mL) vs controls (1.35 0.18 g/mL) (p<0.01) and no gender differ-
ences were found. PAI-1 activity was lower in patients than in controls,
median 2.2 (IQR 1.0-5.4 IU/mL) vs 4.3 (IQR 2.0-9.1 IU/mL) (p<0.01), while tPA-
PAI-complex, PAP-complex and D-dimer were not signicantly different. In
females, plasma levels of antiplasmin were lower in patients than in controls
(8111.4 vs 8916 g/mL; p<0.01). In patients, no gender differences were
found in levels of PAI-1 activity and PAP, while women had higher levels
of antiplasmin, D-dimer, lower tPA-PAI-complex (p<0.01, for all) and higher
HbA1c (7.21.5%) than men (6.81.2%) (p<0.02).In conclusion, more antiplas-
min is incorporated into brin network in patients with type 1 diabetes, sug-
gesting a more stable brin. This could be one of the mechanisms adding to
the risk for cardiovascular disease or to the cause of diabetes angiopathy.
The reasons for the reduced levels of PAI-1 in the whole diabetes group and,
in addition, the lower plasma levels of antiplasmin in women with type 1
diabetes are unclear.
420-P
The Role of SET7/9 and LSD1 on Macrovascular Complications in
Patients With Type 2 Diabetes
YONG XU, GUO CHEN, CHEN-LIN GAO, Luzhou, Sichuan, China
Covalent modication of the histone represent a important mechanism
by which cells control the structure and function of chromatin. Methylation
on different histones has distinct functions. However, the effect of histone
methylation on the diabetic nephropathy is still unknown. To explore the
role of histone methyltransferase SET7/9 and Lysine-specic histone dem-
ethylase 1(LSD1) on the diabetic macrovascular diseases, we selected 10
healthy volunteers randomly as Normal control group (NC group), 10 type
2 diabetes patients without macrovascular complication group (DM group)
and 10 type 2 diabetes patients with macrovascular complications group (DV
group). Peripheral blood sample was drew from each research object. West-
ern blot and RT-PCR were used to detect the protein and mRNA expression
of SET7/9,LSD1 and inammation factor, nuclear factor kappa B (NF-kB). The
results showed that, compared with NC group, the expression of SET7/9 and
NF-kB was increased in DM group (P<0.05) and further increased in DV group
(P<0.05). Compared with NC group ,the expression of LSD1 was decreased
in DM group and further decreased in DV group (P<0.05,respectly). In con-
clusions, imbalance of histone methyltransferase SET7/9 and demethylase
LSD1 may be the important epigenetic mechanism of the occurrence and
development of type 2 diabetic macrovascular complications.
Supported by: NNSF of China
421-P
MYH9 Gene Polymorphisms are Associated With the Cerebral Blood
Flow in Type 2 Diabetes Patients
CHAO LING, CHUNYOU CAI, BAOCHENG CHANG, FENGJIANG WEI, PING YU,
WENTAO SHI, LIMING CHEN, WEI-DONG LI, Tianjin, China
Previous studies have shown that intensive glucose control does not af-
fect the incidence of stroke in patients with type 2 diabetes mellitus (T2D).
In order to decipher genetic components of diabetic cerebrovascular dis-
eases, we carried out a quantitative trait association study in T2D patients
among candidate gene polymorphisms and transcranial Doppler sonography
(TCD) measurements.We studied 337 T2D individuals with diabetes. Cere-
bral blood ow velocities were evaluated by TCD, and 54 phenotypes were
documented, including the peak, minimum, and mean blood ow velocities
of the bilateral cerebral arteries, vertebral artery, and the basilar artery.
Outliers (> 4 SD) were deleted from the data set, and all phenotypes were
adjusted by age within sex. We have genotyped 52 single-nucleotide poly-
morphisms (SNPs) from 31 candidate genes. Quantitative allelic associations
and haplotype analyses were performed with the PLINK program.Two SNPs
of gene MYH9 (rs875726, rs735853) were associated with the peak veloc-
ity of the right cerebral middle artery (P=0.0044, and 0.0019, respectively).
SNPs rs875726, rs2009930, and rs375246 were associated with the mean
velocity of the right-anterior and posterior cerebral artery (P=0.0030, 0.0037,
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and 0.0087, respectively). Haplotype analysis results showed that multiple
haplotypes of MYH9 were associated with the TCD phenotypes.Our analysis
indicates that polymorphisms of MYH9 are associated with blood ow veloc-
ity of cerebral arteries in T2D patients. MYH9 polymorphisms might account
for genetic susceptibility to cerebrovascular disease in diabetes patients.
Supported by: Chinese National Natural Science Foundation Grant 81070576
(W.D.L.)
422-P
Associations of Coronary Artery Calcication and Possible M Value
With Incidence of Coronary Heart Disease in Japanese Patients
With Type 2 Diabetes
TAKAYOSHI SASAI, NORIKO TAKEBE, MITSUTAKA ONO, HARUHITO TANEICHI,
JO SATOH, Morioka, Japan
The Coronary artery calcication score (CACS) is a known predictor of
coronary heart disease (CHD) in Western countries. In this study, we ob-
served a relationship of CACS with incidence of CHD and cerebrovascular
disease (CVD) in Japanese patients with type 2 diabetes.Subjects were 589
patients with type 2 diabetes. CACS was measured by multi-detector com-
puted tomography. The patients were divided into three groups according to
the CACS (Agatston unit [AU]); no CAC (0 AU), mild-moderate CAC (1-400),
and extensive CAC (> 400). The period to onset of CHD or CVD from the
time of the CACS measurement was retrospectively calculated and analyzed
by the Kaplan-Meier method and Cox proportional hazards model.Kaplan-
Meier analysis indicated that incidence (per 1,000 person-years) of CHD was
0, 7.6, and 11.6 in the no CAC, the mild-moderate CAC, and the extensive
CAC, respectively (Log-rank test: P = 0.045), whereas that of CVD was 0, 7.6,
and 11.6 (Log-rank test: P = 0.055). By the Cox proportional hazards model,
adjusting for multiple covariates, the CACS is an independent predictor for
CHD (hazard ratio [HR] = 1.002 per 1 AU; 95% condence interval [CI], 1.000 -
1.003, P < 0.05), whereas no independent predictor was obtained for CVD. In
addition, the M value, a marker of blood glucose variability, was a marginally
signicant predictor for CHD (HR, 1.017; 95% CI, 0.999 - 1.035; p = 0.059).
CACS is a signicant predictor for CHD in Japanese patients with type 2
diabetes. The M value may also be a predictor.
423-P
Left Ventricle Hypertrophy With Preserved Systolic Dysfunction, a
Common Feature of Cardiomyopathy in Type 2 Diabetic Patients
Additional Role of Ischemia and Cardiac Autonomic Neuropathy
MINH TUAN NGUYEN, ISABELLE PHAM, EMMANUEL COSSON, ISABELA BANU,
SABRINA CHIHEB, PAUL VALENSI, Bondy, France
Diabetic cardiomyopathy is dened by structural or functional myocardial
alterations related to diabetes after exclusion of hypertension and coronary
disease. Aim: to estimate in asymptomatic type 2 diabetic patients the prev-
alence of left ventricle hypertrophy (LVH) and systolic dysfunction in those
free of silent myocardial ischemia (SMI) and hypertension and examine the
role of SMI, silent coronary stenoses (CS) and cardiac autonomic neuropathy
(CAN) in myocardial alterations.We included 656 patients, 59.78.7 years,
HbA1c 8.72.1%, with diabetes for 148 years and other risk factors but no
coronary or heart failure history. They were screened for LVH and systolic
dysfunction (hypokinesia, ejection fraction) using transthoracic echocar-
diography, SMI (abnormal stress scintigraphy and/or echocardiography), CS
(angiography performed in SMI+ patients) and CAN (~1 abnormal test among
deep breathing, lying-to-standing, Valsalva).SMI and CS were detected in
206 and 71 patients, respectively, and CAN in 199 of 282 patients tested. LVH
prevalence was high (26%) among patients free of SMI and hypertension, did
not differ according to SMI and/or CAN, but was higher in the patients with
CS (44% vs 32% in the others; p<0.05). Hypokinesia affected only 3 of the
109 patients free of SMI and hypertension. Hypokinesia was more prevalent
in the patients with both SMI and CAN than in those free of SMI and CAN
(SMI- CAN-/SMI+ CAN-/SMI- CAN+/SMI+ CAN+: 2/0/8/17%, respectively;
p<0.05) with a lower ejection fraction (706/696/688/6512%; p<0.01). In
multivariate analysis, SMI+ CAN+ status was associated with hypokinesia
(OR=4.1[1.4-12.2]), independently of hypertension.In conclusion, in high risk
type 2 diabetic patients, asymptomatic cardiomyopathy is mostly character-
ized by LVH with preserved systolic function; systolic dysfunction must lead
to suspect the combined role of SMI and CAN.
424-P
Adipose Triglyceride Lipase and Comparative Gene Identica-
tion-58 are Downregulated in the Hearts of Diabetic Fatty db/db
Mice: A Possible Animal Model for Diabetes-Related Triglyceride
Deposit Cardiomyovasculopathy
TOMOAKI INOUE, KUNIHISA KOBAYASHI, TOYOSHI INOGUCHI, NORIYUKI SONO-
DA, EIICHI HIRATA, HISASHI YOKOMIZO, YOHEI MINAMI, RYOICHI TAKAYANAGI,
Fukuoka, Japan
Adipose triglyceride lipase (ATGL) was recently identied as a rate limiting
triglyceride (TG) lipase and ATGL activity is stimulated by comparative gene
identication-58 (CGI-58). Mutations in the human ATGL or CGI-58 gene are
associated with neutral lipid storage diseases characterized by excessive
accumulation of TG in multiple tissues. The cardiac phenotype, known as
triglyceride deposit cardiomyovasculopathy (TGCV), shows massive TG ac-
cumulation in both coronary atherosclerotic lesions and the myocardium.
Recent reports show that myocardial TG content is signicantly higher in
patients with prediabetes or diabetes and is associated with impaired left
ventricular diastolic function. Therefore, we investigated roles of ATGL and
CGI-58 in the development of myocardial steatosis in the diabetic state or
possibility for diabetes-related TGCV.Triglyceride contents in the hearts of
diabetic fatty db/db mice are higher than in those of db/+ control mice. His-
tological examinations assessed with oil red O staining showed marked lipid
deposition within the hearts of db/db mice. Next, we determined expres-
sion of genes and proteins that affect triglyceride metabolism and found
that ATGL and CGI-58 expression were decreased in the hearts of db/db
mice. Also, we found increased expression of genes that affect triglycer-
ide synthesis (SREBP1c, monoacylglycerol acyltransferases and diacylglyc-
erol acyltransferases). In addition, we analyzed the expression levels of key
modulators of apoptosis and PKC activity. It was found that Bcl-2 levels were
lower and phosphorylation of PKC was increased in diabetic hearts. These
results suggest that reduced ATGL and CGI-58 expression with increased TG
synthesis may induce myocardial steatosis and cardiac apoptosis, leading to
cardiomyopathy in the diabetic state.
425-P
Serum FGF21 Concentration is Associated With Cardiometabolic
Risk Factors and Pericardial Fat Accumulation, Independent of Obe-
sity, But Not With Current Coronary Artery Disease
YENNA LEE, EUN SHIL HONG, HAK C. JANG, SUNG HEE CHOI, EUN KY KIM,
MIN KYEONG KIM, SOO LIM, JUNG HEE KIM, SEON MEE KANG, Seoungnam-Si,
Gyeonggi-Do, Republic of Korea, Seoul, Republic of Korea
Fibroblast growth factor 21 (FGF21) is an emerging metabolic regulator
associated with glucose and lipid metabolism. These associations have
been demonstrated in humans, but have been largely confounded by the
effects of obesity. We investigated the association between serum FGF21
concentrations and glucose and lipid metabolism, coronary artery disease,
and pericardial fat deposition in subjects strictly matched for obesity pa-
rameters.We enrolled 189 patients who had undergone cardiac multide-
tector coronary computed tomography. We measured various metabolic
parameters and serum FGF21 levels within body mass index (BMI)-matched
groups. Correlations and linear regressions were analyzed between serum
FGF21 levels, pericardial fat volumes, and various cardiometabolic param-
eters. Serum FGF21 levels were compared in patients with and without
diabetes, metabolic syndrome, and coronary artery disease.Serum FGF21
levels were signicantly higher in BMI-matched patients with metabolic
syndrome (107.2 83.6 vs. 82.1 67.4 ng/L without metabolic syndrome,
P < 0.05) but not those with diabetes (84.32 56.39 vs. 96.30 98.91 ng/L
without diabetes, P = 0.300) or coronary artery disease (89.6 65.8 vs. 84.2
83.1 ng/L without coronary artery disease, P = 0.633). Serum FGF21 levels
correlated positively with triglycerides, low-density lipoprotein-cholesterol,
insulin, HOMA-IR, and pericardial fat volume. They showed an independent
association with pericardial fat volume ( = 0.108 0.052, P < 0.05).Serum
FGF21 concentrations are signicantly associated with lipid proles, insulin
resistance, pericardial fat volume, and metabolic syndrome, independent of
obesity, but not with overt coronary artery stenosis or diabetes.
Supported by: National Project for Personalized Genomic Medicine (PGM21),
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426-P
Progression of Vascular Calcication is Increased With Statin Use
in the Veterans Affairs Diabetes Trial (VADT)
ARAMESH SAREMI, GIDEON BAHN, TOM MORITZ, PETER D. REAVEN, Phoenix,
AZ, Hines, IL
The effect of statins on progression of vascular calcication in type 2
diabetes (T2DM) remains uncertain. As part of the RACED sub-study of the
VADT, the association between statin use and progression of coronary and
abdominal aortic artery calcium (CAC and AAC) was determined in 197 T2DM
patients. Progression (average follow-up of 4.6 yrs) was estimated as the
difference between square root transformation of the follow-up and base-
line volumetric scores (mm
3
). Information regarding concomitant medication
was updated at each visit (14-28 visits).Participants who reported statin use
s 50% of visits (n= 36) were compared with those who reported statin use
> 50% (n=161). Except for a higher prevalence of CVD events history in those
with statin use > 50% (41% vs. 19% P<0.01), there were no signicant differ-
ences between the two groups at baseline. Despite improved lipids at the
end of the study, CAC progression was signicantly (P <0.001) accelerated
in those with more frequent statin use (Figure). Adjustments for age, dura-
tion of diabetes, hypertension, CVD events, baseline CAC, race/ethnicity,
treatment assignment (intensive vs. standard), time on study and change in
BMI, TC/HDL, systolic, diastolic blood pressure, did not change the results.
Furthermore, after adjustment for all of the above covariates, every 10%
increase in frequency statin use (continuous variable), was associated with
a 0.33 0.01 mm
3
increase in CAC progression (P =0.04). Exclusion of those
with prior or new CVD events did not change results. In conclusion, statin
use is associated with accelerated CAC progression in T2DM patients.
Supported by: Carl T. Hayden Medical Research Foundation
427-P
Hyperglycemic Induced Myocardial Mitochondrial DNA (mtDNA)
Damage is Mediated through Dysfunctional Mitochondrial Topoi-
somerases
STEVEN HICKS, BRIAN PITEO, JAMIE MATHEW, DIMTRI LAURENT, MARIA MIT-
RY, NAZAR LABINSKYY, JOHN G. EDWARDS, Valhalla, NY
Mitochondrial dysfunction has a signicant role in the development and
complications of diabetic cardiomyopathy. Less clear are the mechanisms
that lead to failure since the mitochondria have defense mechanisms to
manage diabetic-induced oxidant stress. We have reported previously in the
H9c2 cell line that chronically elevated glucose induced mtDNA damage was
linked to dysfunctional mitochondrial topoisomerase activity. The present
study extends those observations to include both neonatal cardiomyocytes
as well as the type 2 diabetic Goto-Kakizaki (GK) rat. Diabetes signicantly
decreased cytochrome oxidase activity in GK LV compared to Wistar LV
and decrease in ATP production in cardiomyocytes. Mitochondrial dysfunc-
tion was associated with signicantly increased mtDNA damage without a
change in mitochondrial copy number. TTGE analysis of the Cytochrome Oxi-
dase Subunit 3 identied several different nucleotide substitutions in the GK
LV that altered the primary protein sequence; none were observed in Wistar
LV. In isolated mitochondria, diabetes signicantly increased mitochon-
drial dependent DNA cleavage. Immunoprecipitation with a mitochondrial
topoisomerase I antibody partially blocked mitochondrial dependent DNA
cleavage indicating a functional role for this enzyme. Incubation of mito-
chondrial extracts with 50 M H
2
O
2
increased mitochondrial topoisomerase
dependent DNA cleavage, implicating ROS as a modier of topoisomerase
function. Separate from a direct impact of oxidative stress on mtDNA, ROS-
induced alteration of mitochondrial topoisomerase function propagated
mtDNA damage. These ndings indicate a signicant role for mitochondrial
topoisomerase function in the development and complications of diabetic
cardiomyopathy.
Supported by: NIH (HD065551, HL43023)
428-P
Preserved Cardiac Structure, Function and Sympathetic Innerva-
tion in Subjects With Type 1 Diabetes of Extreme Duration
OMAR ASGHAR, CHRISTOPHER MILLER, PARTHIBAN ARUMUGAM, MATTHIAS
SCHMITT, IAN ARMSTRONG, RAYAZ A. MALIK, Manchester, United Kingdom
The cardiac phenotype in Medalists (individuals with type 1 diabetes
(T1DM) ~50 years) has not been previously described. We characterized
cardiac structure, sympathetic innervation and autonomic function in this
subgroup.12 Caucasian subjects (628 years, M:F 4:8) with T1DM of long
duration (48.95 years) and no clinical history, ECG features or symptoms
of cardiac disease were studied. Ischemia was excluded using vasodilator
stress CMR. LV structure, volumes, function and myocardial deformation
(standard CMR sequences) and diffuse brosis (T1 mapping) were assessed.
Cardiac sympathetic innervation and autonomic function were assessed
with I-123 MIBG scintigraphy and cardiovascular reex tests, respectively.
Mean 15 year metabolic prole indicated suboptimal glycemic control
(HbA1c 8.51.0%), favorable lipid prole (total cholesterol 4.70.3, HDL
1.80.5, LDL 2.20.5, triglyceride 1.10.4 mmol/l) and mean eGFR of 7616.6
ml/Kg/min. Mean BP was 146/7318/10 mmHg. LV mass (83.820.2 g), mass
index (LVMI) (44.57.9g/m2), stroke volume (94.318.9 ml) and ejection frac-
tion (70.96.7%) were normal. Global circumferential LV strain (-20.73 vs
-20.61.0%, p=NS) and diffuse brosis (0.310.04 vs 0.270.01%, p=NS) did
not differ signicantly from controls. Sympathetic innervation (late HMR)
(1.60.2, normal >1.6) and cardiac autonomic function (LFA/RFA (1.291.57),
E:I ratio (1.10.1) and 30:15 ratio (1.10.1) were normal and LF/HF (1.41.6)
and valsalva ratio (VR) (1.10.1) were only mildly reduced.Patients who have
survived ~ 50 yrs of Type 1 diabetes (Medalists) exhibit normal cardiac struc-
ture and function without brosis and sympathetic denervation with minimal
autonomic dysfunction despite suboptimal glycemic and BP control, but fa-
vorable lipid prole. These results extend previous limited data on medalists
and microvascular complications to include cardiac disease, supporting the
need for further studies.
Supported by: NIHR
429-P
Comparative Effectiveness of Percutaneous Stenting Versus By-
pass Surgery Among Adult Type 2 Diabetes Patients With Periph-
eral Arterial Disease: A Nationwide Cohort Study
CHIA-HSUIN CHANG, JOU-WEI LIN, LI-CHIU WU, MEI-SHU LAI, LEE-MING CH-
UANG, Taipei, Taiwan
Background: The long-term comparative effectiveness of percutaneouss-
tenting and bypass surgery for diabetic patients with peripheral arterydis-
ease remained unclear.Methods: A retrospective cohort study using the Tai-
wan National Health Insurance claims database was conducted to identify
adult patients with type 2 diabetes mellitus and who received peripheral ar-
tery bypass surgery (n=5,652) or stenting (n=659) during 2000-2007. Primary
outcome was lower extremity amputation, further classied as above-knee
(thigh) orbelow-knee (leg, ankle, and foot). Patients were followed from the
date of index hospitalization to the earliest of outcome occurrence, death, or
December 31 2008. A propensity-score matched cohort was created for com-
parison. We estimated adjusted hazard ratios and 95% condence intervals
(CIs) with Cox proportional hazards models controlling for age, gender, and
covariates that were imbalanced after propensity score matching.Results:
The incidence rate of lower limb amputation per 1,000 person-years was
94.5 for bypass surgery recipients (186 cases) and 57.8 for stent recipients
(49 cases) during an average follow-up of 2 years. A signicant decrease in
risk was found for patients receiving percutaneous stenting (adjusted haz-
ard ratio 0.67; 95% condence interval: 0.48-0.92). Risk was signicantly
reduced for below-knee amputation, but not for above-knee. In contrast, a
signicantly increased risk for stenting as compared with bypass surgery
was observed for patients with chronic renal failure (adjusted hazard ratio
1.97; 95% condence interval: 1.00-3.91) for future amputation.Conclusions:
Percutaneous stenting was associated with a signicantly lowered risk of
lower limb amputation for diabetic patients withperipheral artery disease.
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However, bypass surgery may provide better outcome for patients with
chronic renal failure.
430-P
Diabetics Show Better Collaterals toward the Culprit Vessel at the
Time of Their First Acute Coronary Event
DARIO PITOCCO, FRANCESCO ZACCARDI, GIAMPAOLO NICCOLI, ANDREA LEO,
NICOLA COSENTINO, DOMENICO DAMARIO, CARLO TRANI, ITALO PORTO,
FRANCESCO BURZOTTA, FILIPPO CREA, GIOVANNI GHIRLANDA, Rome, Italy
Diabetes is associated with a more extensive and severe coronary ar-
tery disease (CAD); however similar age of presentation of non-diabetics
has been described despite a more extensive CAD at the time of a rst
acute coronary syndrome (ACS), thus suggesting that protective mecha-
nisms may act in diabetics. We evaluated angiographic CAD severity and
quality of the collateral circulation toward the culprit vessel according to
the diabetic status in consecutive patients at their rst ACS. One-hundred
and sixty-seven consecutive patients undergoing coronary angiography be-
cause of non-ST-elevation (NSTE)-ACS, as their rst clinical manifestation of
CAD, and showing obstructive atherosclerosis were prospectively included.
CAD severity was graded according to Bogatys stenosis score and extent
index, while collateral lling was graded according to Rentrop classication
toward culprit vessel showing a TIMI ows2. Forty-seven patients were
diabetics. Diabetics and non-diabetics were similar for age (6912vs6614,
p=0.11). Multivessel disease rate was higher in diabetics compared to non-
diabetics (68%vs42%, p=0.003). At multivariate analysis both stenosis score
and extent index were predicted by diabetes (p=0.001), stenosis score was
predicted by male sex (p=0.03) and extent index by lack of statins (p=0.05).
Good collaterals (Rentrop 2-3) were observed in 28 over 83 culprit vessel
with a TIMI ows2. At multivariate analysis, good collaterals were predicted
by diabetes (OR 2.33, 95% CI 1.45-6.54, p=0.002) and inversely by extent
index (OR 0.87, 95% CI 0.51-0.96, p=0.03). In conclusion, diabetics show at
their very rst ACS a more extensive and severe coronary atherosclerosis as
compared to non diabetics, despite a similar age of presentation. However,
angiographically visible collaterals toward the culprit vessel are better de-
veloped in diabetics, suggesting that collaterals may delay onset of a rst
ACS acting as a protective mechanisms.
431-P
Effects of Exenatide vs. Metformin on Endothelial Function in Obese
Patients With Pre-Diabetes
AARON S. KELLY, RICHARD M. BERGENSTAL, J.M. GONZALEZ-CAMPOY, HAR-
OLD KATZ, ALAN J. BANK, Minneapolis, MN, St. Louis Park, MN, Eagan, MN, St.
Paul, MN
The effect of glucagon like peptide-1 (GLP-1) receptor agonist treatment
on endothelial function in humans has not been described. Therefore, we
conducted a study comparing the acute and chronic effects of the GLP-1
receptor agonist exenatide vs. metformin on endothelial function in patients
with obesity and pre-diabetes. We performed a randomized, open-label,
clinical trial in 50 individuals (mean age 58.5 10.0; 38 females) with abdom-
inal obesity and either impaired fasting glucose, elevated HbA1c, or impaired
glucose tolerance (IGT) who were randomized (1:1) to receive 3-months of ex-
enatide or metformin. Microvascular endothelial function (primary endpoint),
assessed by digital reactive hyperemia (reactive hyperemic index: RHI),
C-reactive protein (CRP), circulating oxidized LDL (oxLDL), and vascular cell
adhesion molecule-1 (VCAM-1) were measured at baseline and 3-months.
Seven subjects with IGT participated in a sub-study comparing the effects of
acute administration of exenatide and metformin on postprandial endothe-
lial function. The groups were similar at baseline for all measured variables.
There were no differences for the change in RHI (A exenatide: 0.01 0.68
vs. A metformin: -0.17 0.72, P = 0.348), CRP (A exenatide: -0.4 2.2 mg/L
vs. A metformin: -0.4 2.2 mg/L, P = 0.987), oxLDL (A exenatide: -0.1 41.5
U/L vs. A metformin: -16.5 30.4 U/L, P = 0.123), or VCAM-1 (A exenatide:
10.4 83.2 ng/mL vs. A metformin: -15.3 101.3 ng/mL, P = 0.336) between
exenatide and metformin treatment. Triglycerides were reduced more with
exenatide compared to metformin (A exenatide: -25.5 45.7 mg/dL vs. A
metformin: -2.9 22.8 mg/dL, P = 0.032). In the sub-study, there was no
difference in postprandial RHI between exenatide and metformin. Three
months of exenatide therapy had similar effects on microvascular endothe-
lial function, markers of inammation, oxidative stress, and vascular activa-
tion, as metformin, in patients with obesity and pre-diabetes.
Supported by: Amylin and Eli Lilly
432-P
Treatment of Obstructive Coronary Artery Disease With the Reso-
lute Zotarolimus-Eluting Stent in Patients With Diabetes Mellitus
SCOTT W. LEE, JORGE BELARDI, MARTIN B. LEON, LAURA MAURI, IAN T. MER-
EDITH, FRANZ-JOSEF NEUMANN, SHIGERU SAITO, PATRICK W. SERRUYS, PETR
WIDIMSKY, STEPHAN WINDECKER, ALAN YEUNG, FRANCINE R. KAUFMAN,
SIGMUND SILBER, RESOLUTE CLINICAL INVESTIGATORS, Northridge, CA, Bue-
nos Aires, Argentina, New York, NY, Boston, MA, Clayton, Australia, Bad Krozingen,
Germany, Kamakura, Japan, Rotterdam, The Netherlands, Prague, Czech Republic,
Bern, Switzerland, Palo Alto, CA, Munich, Germany
Current guidelines support the use of drug-eluting stents (DES) for treat-
ment of coronary artery disease in patients with diabetes, but no specic
DES is yet indicated for use in the US in this high-risk population. We evalu-
ated 1-year outcomes in patients with diabetes treated with the Resolute
stent. A prespecied analysis cohort to obtain the indication for use of the
Resolute DES in patients with diabetes in the US included 878 patients with
Type 2 diabetes and 1903 patients without diabetes from 5 RESOLUTE trials.
Patients were treated for 1 or 2 lesions in separate vessels. Safety outcomes
include the composite of cardiac death or myocardial infarction related to
the treated vessel (TVMI), and stent thrombosis. Efcacy is reported as the
rate of target lesion revascularization. Target lesion failure, a composite
endpoint including cardiac death, TVMI, and target lesion revasculariza-
tion, represents stent-related outcomes. Of 878 patients with diabetes, 250
were taking insulin. At baseline, the use of insulin did not result in signicant
differences between groups except that more women were taking insulin
(43.6% vs 29.6%, p<0.001) and had hypertension (91.6% vs 86.0%, p=0.023).
Major outcomes at 1 year are in the Table. There were no statistically sig-
nicant differences in the composite endpoint between patients not taking
insulin, and patients without diabetes (5.0% vs 4.9%, p=NS). The Resolute
DES was shown to be as safe and effective in non-insulin requiring patients
with diabetes as in patients without diabetes. Both groups had similar car-
diovascular outcomes at 1 year.
Cardiac Outcomes with the Resolute DES
Outcome % (n) Patients
Without
Diabetes
(n=1903)
Patients With
Diabetes Not
Taking Insulin
(n=628)
Patients With
Diabetes
Taking Insulin
(n=250)
Cardiac death or target vessel MI 3.2 (59) 2.6 (16) 6.1 (15)
Target lesion revascularization 2.0 (38) 2.6 (16) 5.3 (13)
Target lesion failure 4.9 (92) 5.0 (31) 10.6 (26)
Stent thrombosis 0.3 (6) 0.2 (1) 0.8 (2)
Supported by: Medtronic, Inc.
433-P
Blood Pressure Trajectories before Death in Patients With Type 2
Diabetes
SANJOY PAUL, KERENAFTALI KLEIN, GIJO THOMAS, KAMLESH KHUNTI, Bris-
bane, Australia, Leicester, United Kingdom
The aim of this study is to examine the trajectory of blood pressure (BP)
over two yrs before death or censoring and its association with mortality
in patients (pts) with T2DM.A cohort of 19966 pts with minimum 3 yrs of
diabetes duration (DD) was selected from the UK General Practice Research
Database (1990-2008), who had four consecutive 6-monthly BP measures
over 2 yrs immediately before death or censoring. In the cohort, 46% were
female, mean (SD) age of 68 (11) years and 14% had at least one cardiovas-
cular event (CVE) before diagnosis of diabetes. During 6.5 years of median
duration of follow-up, 7% pts died, of whom 83% were aged ~ 68 yrs, 53%
and 20% pts had CVE respectively in dead and alive groups.During 2 yrs of
follow-up, pts systolic and diastolic BP trajectories were signicantly higher
by 5 and 2 mmHg respectively in those who died, compared to those who
were alive (Fig 1). These estimates were adjusted for the effects of age,
DD, sex, CVE, glucose-lowering and anti-hypertensive medication usages,
baseline weight and HbA1c.Compared to pts with systolic BP level 125 - 130
mmHg, pts with BP < 110 and > 140 mmHg had signicantly higher mortality
risk by 240% (adjusted odds ratio : 2.4, 95% CI: 1.9, 3.1) and 210% (adjusted
odds ratio: 2.1, 95% CI: 1.8, 2.5) respectively. Mortality risk was not higher
in patients with BP within 130 - 140 mmHg (Fig 1).The population level analy-
ses of BP trajectories leading up to death are revealing, and provide clear
message in terms of the requirement for intensive BP control and related
management policies at primary care level. Our ndings also establish the
J-shaped relationship between systolic BP and mortality.
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Genetic Variability in AMPK1 Gene Is Associated With Risk of
Coronary Artery Disease in Chinese Type 2 Diabetics
XIAOWEI MA, JIANWEI ZHANG, RUIFEN DENG, SHAN DING, XIAODAN MA,
NAN GU, GE BAI, JIANPING LI, XIAOHUI GUO, Beijing, China
The increasing evidence suggested that AMP-activated protein kinase
(AMPK) played a critical physiological role in the cardiovascular system. To
assess the possible association between the genetic variability in AMPKo1
gene and the risk of cardiovascular disease in type 2 diabetics, we selected
5 tag-SNPs (rs3805489, rs249428, rs133671707, rs837103 and rs3805486)
at AMPKo1 locus according to CHB database from HapMap R#27 (r
2
<0.8
and MAF~0.05) and genotyped 400 unrelated Han subjects with type 2
diabetes, 255 individuals with coronary artery diseases(CAD+) as cases
and 154 controls (CAD-) by using PCR-RFLP assay. We found that minor al-
lele C at rs3805489 was protective from CAD in type 2 diabetics compared
to allele A (OR=0.688, 95%CI 0.491-0.963, p=0.035) . Interestingly, when
we stratied the data by smoking status, the results showed that smoking
status had a synergistic effect with rs3805489 on CAD risk (p = 0.036, for
interaction). The carriers of genotype AA at rs3805489 had a higher risk of
CAD compared to non-carriers in the subjects who ever smoked (OR=3.069,
95%CI =1.529-6.161, p = 0.002; OR=2.615,95%CI =1.255-5.453, p = 0.010
after adjusted for other known CAD risk factors. The smokers with genotype
AA at the SNP had 3 times risk of CAD as high as the non-smokers with
genotype AC or CC (OR=3.312, CI=1.533-7.155, p=0.002) (Fig.1). Our ndings
suggest that genetic variability at AMPKo1 locus may be associated, and
interact with smoking on the risk of CAD in type 2 diabetes in the Chinese
Han population.
Supported by: National 973 Project (2006CB503903, 2006CB503908)
435-P
Slower Psychomotor Speed Predicts Faster Gray Matter Volume
Loss Within the Prefrontal Cortex Over a Three Year Follow-Up
Time
CATERINA ROSANO, CHRISTOPHER RYAN, JUDITH SAXTON, RACHEL MILLER,
TIMOTHY HUGHES, ELSA S. STROTMEYER, TREVOR J. ORCHARD, Pittsburgh, PA
Adults with T1D often show psychomotor speed slowing. Smaller left dor-
solateral prefrontal cortex (ldPFC) is associated with slower psychomotor
speed in older adults independent of other chronic conditions, including dia-
betes. We conducted this study to identify potential determinants of rate of
change in ldPFC over a 3-year period of time.High-resolution brain magnetic
resonance images (MRI, 3-Tesla) and Digit Symbol Substitution Test (DSST)
scores were obtained concurrently in 2008 from 9 participants (50+ 5.3 yrs
old, 2 men) in the Epidemiology of Diabetes Complications Study, an ongo-
ing prospective childhood onset (1950-1980) cohort followed biennially since
1986-88. MRI were repeated in 2011 and changes in gray matter volumes of
total brain and of ldPFC were computed. Age (or disease duration), cumula-
tive HbA1C since diagnosis, insulin dose, retinopathy, urinary albumin/crea-
tinine ratio and number of hypoglycemic episodes were obtained concurrent
with the 1
st
MRI.After adjusting for total brain volume change, slower DSST
was signicantly associated with greater percent gray matter volume loss
in the ldPFC (standardized beta = 4.8%, p=0.03) and explained 32% of the
variance (F change: 7.954, p=0.03). Associations between other diabetes-
related measures and dLPFC volume changes were all not signicant (p>0.2
for all). Slower DSST was not signicantly associated with total brain gray
matter volume loss (standardized beta = 0.4%, p=0.9).This is the rst report
of an association between a common manifestation of cognitive dysfunction
in T1D middle-aged adults and longitudinal rate of focal volumetric reduc-
tions within the frontal lobe. ldPFC may be a brain region particularly vul-
nerable to atrophy in T1D because of its non-anastomosing vascularization.
Future studies should examine potential applications of DSST as an indicator
of focal accelerated brain aging in T1D.
Supported by: R01 DK034818-25 and R01 DK089028-01
436-P
The Association Between G174C Polymorphism on Interleukin-6
Gene With Macrovascular Complications in Subjects With Type 2
Diabetes Mellitus
STAVROULA PAPAOIKONOMOU, NIKOLAOS TENTOLOURIS, DIMITRIS TOUSOU-
LIS, DIMITRIS PAPADOGIANNIS, ANTIGONI MILIOU, GEORGE HATZIS, NIKOLAOS
PAPAGEORGIOU, KOSTAS MAKRILAKIS, STAVROS LIATIS, CHRISTODOULOS
STEFANADIS, Athens, Greece
Low grade inammation is involved in the pathogenesis of atherosclero-
sis. Genes encoding for inammatory cytokines such as iterleukin-6 (IL-6)
are candidates for predisposing to the risk of atherosclerosis. Subjects with
type 2 diabetes mellitus (T2DM) are vulnerable to the development of mac-
rovascular disease. The aim of this cross-sectional study was to examine
for association between Guanine/Cytosine (G>C) polymorphism at position
-174 of the IL6 gene (IL6G174C polymorphism) with the prevalence of macro-
vascular complications in subjects with T2DM. A total of 431 subjects with
T2DM (mean age 66.5 9.96 years, male n=218, female n=213) were exam-
ined. IL6G174C polymorphism was detected using polymerase chain reaction
and appropriate restriction enzyme. A detailed history and examination was
performed for the diagnosis of macrovascular complications. The genotype
distribution was 49.1% GG, 26.8% GC and 24.1% CC, with no signicant gen-
der difference. The prevalence of coronary artery disease (26.7%; 23.6%;
24.2%), peripheral arterial disease (37.6%, 35.5%, 36.4%), stroke (14.9%;
12.7%, 13.1%) or any macrovascular disease (55.9%; 42.7%; 48.5%) was not
signicantly different (P>0.05) among participants with GG, GC or CC geno-
type, respectively. However, participants carrying the C allele (GC and CC)
had signicantly lower prevalence of any macrovascular complication than
GG homozygotes (45.5% vs 55.9% respectively, p=0.034). In addition, this
association remained signicant by multivariate analysis after adjustment
for gender, age, duration of diabetes, body mass index, smoking, hyperten-
sion, lipids, HbA1c, and glomerular ltration rate, (GC+CC vs GG: odds ra-
tio= 0.55, 95% condence intervals 0.35-0.87, p= 0.012). In conclusion, in
subjects with T2DM expression of the C allele by IL6G174C polymorphism
protects from the development of macrovascular disease.
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Resistin, Major Cardiovascular Events and All-Cause Mortality in
Patients With Type 2 Diabetes
CLAUDIA MENZAGHI, SIMONETTA BACCI, LUCIA SALVEMINI, CHRISTINE POW-
ERS, GIUSEPPE PALLADINO, ANTONELLA MARUCCI, CONCETTA DE BONIS,
DAVIDE MANGIACOTTI, GRAZIA FINI, SABRINA PRUDENTE, SALVATORE DE
COSMO, ALESSANDRO DORIA, VINCENZO TRISCHITTA, San Giovanni Rotondo,
Italy, Boston, MA, Rome, Italy
High serum resistin has been associated with increased risk of cardio-
vascular disease (CVD) in the general population. Only sparse and conict-
ing results have been reported in patients with type 2 diabetes (T2D) of
Asian ancestry. Our aim was to study the role of serum resistin on major
cardiovascular events and all-cause mortality in European patients with
T2D.To this purpose two prospective studies were carried out. 1) The Gar-
gano Heart Study-GHS-prospective design for major cardiovascular events
(i.e. cardiovascular death, non fatal myocardial infarction-MI-and non fatal
stroke), of 350 patients with T2D and CAD, (follow-up=1,108 person-year). 2)
The Gargano Mortality Study (GMS) for all-cause mortality, of 1,028 patients
(follow-up=5,659 person-year). Serum resistin levels were available in all the
GHS-prospective patients and in 783 participants from the GMS. The corre-
lation between serum and mRNA resistin levels in white blood cells (WBC)
from 68 patients belonging to the GMS has been also evaluated.Among the
GHS-prospective participants, 32 cardiovascular deaths, 3 non fatal MIs and
8 non fatal strokes occurred (annual incidence rate 0.04). Serum resistin pre-
dicted major cardiovascular events with age, sex and smoking habit adjusted
HR per SD increment of 1.35 (95% CI: 1.14-1.59).Among the GMS participants
150 deaths occurred (annual incidence rate 0.027). Serum resistin predicted
all-cause mortality with adjusted HR per SD increment of 1.17 (95% CI: 1.07-
1.27). These associations were unaffected by further adjustment for BMI,
diabetes duration and current therapy. WBC RETN mRNA and serum resistin
were positively correlated (R=0.317, p=0.005).High serum resistin (very likely
a consequence of increased resistin mRNA expression) is a strong, indepen-
dent risk factor for CVD and all-cause mortality in T2D. This points to resistin
as a novel biomarker in improving predictability of CVD and premature death
in such patients.
Supported by: EFSD/Pzer
438-P
A Genome-Wide Association Analysis of Coronary Calcication in
the Diabetes Heart Study
AMANDA J. COX, MAGGIE C. NG, JIANZHAO XU, J.J. CARR, BARRY I. FREED-
MAN, CARL D. LANGEFELD, DONALD W. BOWDEN, Winston-Salem, NC
Studies of genetic risk factors for cardiovascular disease (CVD) in individu-
als with type 2 diabetes mellitus (T2DM) may reveal biological pathways
which explain the increased risk for macrovascular complications in T2DM.
The aim of the current study was to examine association signals for sub-
clinical CVD in analyses restricted to individuals with T2DM.A genome-wide
association study (GWAS) for coronary artery calcied plaque (CAC) was
performed in the family-based Diabetes Heart Study (DHS). Genotyping was
completed using the Affymetrix 5.0 Array. Association analysis was per-
formed using a variance components analysis implemented in SOLAR v6.3.4
for all n=1181 European American DHS participants (ALL) and then restricting
to the n=989 T2DM affected DHS participants only (T2D). Poorly performing
SNPs (CR<0.95; Hardy-Weinberg p-value<1x10
-6
; MAF<0.01) were excluded
from further analyses. SNPs with association p-values <1x10
-4
in either ALL
or T2D were selected and association signals compared between the two
analyses.Overall GWAS ndings for ALL included n=43 SNPs with associa-
tion p-values <1x10
-4
with the top hit on Chr6q15 (rs1144159; p=1.18x10
-7
;
MAP3K7) and a lack of association across the previously well-documented
9p21 region. When comparing association signals between ALL and T2D,
n=65 SNPs met the threshold of p<1x10
-4
in either ALL or T2D with more than
half of the selected SNPs (n=35) showing stronger association signals in the
T2D analysis. This included n=7 SNPs from the region 6q15 encompassing
the genes CGA and ZNF292. Finally, of those SNPs with stronger associa-
tion signals in the T2D analysis, 23/35 SNPs failed to meet the threshold of
p<1x10
-4
in ALL.These data suggest there is potential for novel GWAS signals
for coronary calcication in T2DM. A meta-analysis of vascular calcication
signals from T2DM cohorts should further elucidate these relationships and
identify variants contributing to risk for vascular calcication in T2DM.
439-P
Predictors of Coronary Artery Disease in Type 1 Diabetes (T1D) Dif-
fer by Level of Glycemic Control
RACHEL G. MILLER, TREVOR J. ORCHARD, Pittsburgh, PA
Though glycemia has not been a consistent predictor of coronary artery
disease (CAD) in T1D, white blood cell count (WBC), hemoglobin (Hgb), and
erythropoietin (EPO), have recently emerged as potential risk factors. To ex-
plore whether these factors may partially explain why individuals with good
glycemic control remain at high risk for CAD, we examined risk factors for
CAD incidence by level of HbA1c.Data are from 520 individuals in the Epide-
miology of Diabetes Complications (EDC) study of childhood-onset (<17 years
old) T1D without baseline CAD, but with measured endogenous EPO level
(baseline mean age 27.7, T1D duration 19.6 yrs, 50.2% female). Incident CAD
(n=130, 25%) was dened as a conrmed event (CAD death, myocardial in-
farction, revascularization/blockage~50%, ischemic ECG, or EDC physician-
diagnosed angina) occurring prior to the 18-yr exam. The EDC cohort was
divided into 2 groups based on baseline median HbA1c <8.5% and ~8.5%.
Separate proportional hazards models were t by HbA1c level. Hazard ratios
(HR) are per standard deviation.There was no difference in CAD incidence
between glycemic groups (~25% in both). However, higher ln(EPO) (HR=4.5,
95% CI 1.5, 13.4) and WBC (1.6, 95% CI 1.3, 12.1) were univariately only as-
sociated with CAD incidence in those with HbA1c <8.5%, while higher -
brinogen (1.4, 95% CI 1.2, 1.7) was associated with CAD incidence in HbA1c
~8.5%. Standard lipid and blood pressure measures also predicted CAD in
both groups. After covariate adjustment, beyond age, only triglycerides re-
mained a signicant predictor of CAD in those with HbA1c ~8.5%, while in
HbA1c <8.5%, ln(EPO) (2.8, 95% CI 1.0, 7.8), red blood cell count (RBC) (0.6,
95% CI 0.4, 0.8), WBC (1.4, 95% CI 1.1, 1.9), LDL-c, DBP, and BP medication
use also predicted CAD.These data suggest risk factor effects differ by gly-
cemic status and emphasize that in the setting of lower HbA1c in T1D, EPO,
RBC, WBC, lipids, and blood pressure become particularly important factors
to monitor and potentially treat.
440-P
Macrovascular Complications in Patients With Type 2 Diabetes are
Associated With Abnormal HDL Contents of Oxidized Fatty Acids
and Haptoglobin
CECILIA MORGANTINI, BEATRICE BOLDRINI, ELENA VENTURI, DAVID MERI-
WETHER, YUEN YIN LEE, SIMONA BALDI, ANDREA NATALI, SRINIVASA T. RED-
DY, Pisa, Italy, Los Angeles, CA
We have previously shown that in type 2 diabetes (T2D) the anti-inam-
matory and anti-oxidant HDL functions are impaired. Recent studies sug-
gest that HDL dysfunction correlates with and may be caused by the content
of oxidized fatty acids derived from arachidonic (HETEs) and linoleic acid
(HODEs), as well as haptoglobin. In this study, we examined whether HDLs
from T2D patients contain elevated levels of oxidized fatty acids and hapto-
globin and whether these changes are more severe in T2D with macrovas-
cular complications (MVC).HDL contents of HETEs and HODEs were deter-
mined by LC-MS/MS and haptoglobin levels were measured by ELISA in 20
non diabetics (ND), in 25 T2D without (DMVC[-]) and 25 with MVC (DMVC[+]).
HDL function was evaluated by a cell-free assay using dichlorouorescein
diacetate.T2D patients and ND were similar with respect to major cardiovas-
cular risk factors. HDL content of HETEs, HODEs, and haptoglobin progres-
sively increased from ND to DMVC[+] patients.
ND DMVC[-] DMVC[+] p (ANOVA)
5-HETE (ng/mgHDL-Chol) 19.712.6 42.122.6 76.274.2 <0.05
9-HODE(ng/mgHDL-Chol) 4.12.4 6.84.5 13.314.4 <0.05
13-HODE (ng/mgHDL-Chol) 4.42.1 8.24.9 15.114.8 <0.05
Haptoglobin(ng/ml) 18791084 20751648 32922391 <0.05
HDL anti-oxidant index was signicantly impaired only in 5 DMVC[+] patients
when compared to ND (1.140.64 vs 1.910.93, p<0.05).A relative HDL enrich-
ment in oxidized fatty acids from arachidonic and linoleic acids and in hapto-
globin is observed in T2D patients, particularly in diabetic patients with MVC.
A loss of HDL antioxidant function is evident in patients with severe vascular
disease. Novel therapeutic agents such as apoA-I mimetic peptides, which
bind oxidized fatty acids and reduce atherosclerosis in mouse models of diabe-
tes might be an important tool in the prevention of vascular events in T2D.
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Predicting Cardiovascular Outcomes by Intima-Medial Thickness
(IMT)
SANGMO HONG, MIN-YOUNG CHUN, JEONG TAEK WOO, KWAN WOO LEE,
MUN-SUK NAM, SEI HYUN BAIK, YOUNG SEOL KIM, YONGSOO PARK, Seoul,
Republic of Korea, Suwon, Republic of Korea, Incheon, Republic of Korea
Type 2 diabetes (T2D) is a major cause of cardiovascular disease. Carotid
intima-media thickness (IMT) or presence of plaques in common carotid ar-
tery has been shown to have a strong relationship with cardiovascular ac-
cidents (CVA). However, only few studies have evaluated the usefulness of
IMT or presence of plaques in predicting future development of CVA in Asian
T2D patients. Using a total of 1489 patients with T2D (M:F=839:650, mean
age: 55.1 yr at baseline) recruited from the Korea National Diabetes Program
(KNDP), a prospective natural history study of T2D, we examined the inu-
ence of the progression of IMT on predicting the development of CVA in
a Korean T2D cohort. B-mode carotid ultrasonographic measurements (the
mean IMT, the maximum IMT and the presence of plaques) were performed
and repeated annually for a mean of 3.1 years of follow-up. Development of
CVAs was evaluated prospectively for 3.1 years, and anthropometrics and
biochemical parameters were also measured annually. During the follow-
up, 74 patients (5%) developed CVAs and the annual progression of mean
IMT was 0.0580 0.1423mm. Newly development of CVAs was associated
with abnormal mean IMT (> 0.08 cm) [OR (95% condence interval); 1.29
(1.01-1.66)] and the presence of plaques [OR (95% condence interval); 1.34
(1.06-1.68)] at baseline. After adjusting for anthropometrics and biochemical
parameters with logistic regression, the presence of plaques were clearly
associated with the development of CVAs [OR (95% CI); 1.65 (1.02-1.79)], but
the association with abnormal mean IMT decreased due to interaction with
age (=0.09; p<0.001), duration of diabetes (=0.04; p=0.02), and HDL cho-
lesterol (=-0.02; p=0.06). Although the presence of plaques and elevation
of IMT were all found to be associated with cardiovascular outcomes, only
the presence of plaques predicted cardiovascular outcomes independent
from conventional cardiovascular risk factors in Asian T2D patients during
short-term follow-up.
443-P
Stress and Response in Association With Coronary Artery Disease
(CAD) in Type 1 Diabetes
TINA COSTACOU, YUEFANG CHANG, GERALD L. SCHAFER, RHOBERT W. EVANS,
TREVOR J. ORCHARD, Pittsburgh, PA
We aimed to assess whether the degree to which an individual is able
to respond to oxidative stress modies the risk associated with oxidative
stress and the CAD development. To do this, we evaluated whether CAD
incidence was related to plasma levels of vitamin E (o- and -tocopherol) and
creatinine adjusted urinary 15-isoprostane F
2t
(IsoP) measured at three time
points during the 20 year follow up of the Pittsburgh Epidemiology of Diabe-
tes Complications (EDC) study of childhood onset type 1 diabetes (n=658,
baseline mean age 28 and duration 19 years). EDC participants with three
samples available during follow up (n=429) were selected for study. Gener-
ally, analyses conducted at each time point showed no consistent associa-
tion between serum vitamin E and urinary IsoP levels and CAD, adjusting for
diabetes duration. In multivariable mixed model analyses, however, allowing
for univariately signicant risk factors, o-tocopherol was inversely (=-0.03,
p=0.08) and IsoP directly (=0.02, p=0.03) associated with CAD; no asso-
ciation was seen for -tocopherol (=0.006, p=0.57). Moreover, the ratio of
o-tocopherol to IsoP (used as a measure of response to stress) was inversely
related to CAD incidence (=-0.02, p=0.003). All models adjusted for univari-
ately signicant variables (body mass index, lipid levels, albumin excretion
rate (log) and white blood cell count). These data provide some support for
the hypothesis that a greater capability (o-tocopherol) to respond to oxida-
tive stress (IsoP), relates to CAD incidence.
Supported by: NIDDK (DK082900-01A1 and DK34818)
444-P
WITHDRAWN
WITHDRAWN
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Survival in Diabetic Foot Ulcer Patients After Hyperbaric Oxygen
Therapy
MAGNUS LNDAHL, CHRISTER HAMMARLUND, PER KATZMAN, Lund, Sweden,
Helsingborg, Sweden
Increasing evidence support the use of Hyperbaric Oxygen Therapy (HBO)
in diabetic foot ulcer healing. However, concerns for long-term risks of HBO
treatment have been raised. The aim of the present study was to record and
discuss three-year survival data following a randomised, double-blinded,
placebo-controlled HBO study in diabetic patients.94 patients were ran-
domly assigned to forty 90 minutes long treatment sessions with either HBO
(n=49) or hyperbaric air (HBA) (n=45) at 2.5 atmospheres absolute pressure.
Nonparametric tests were used for comparison, Kaplan-Meier curves for
life-table analysis and Cox proportional hazards models to estimate hazard
ratios (HR) and 95% CIs.At inclusion patients were 68.5 (48.3-82.1)(median
and 10
th
and 90
th
percentiles) years old and 67% had type 2 diabetes. One
fth of the patients were females. There were no differences in basal char-
acteristics between groups.HR for three-year survival for those in the HBO
group was 1.78 (95% CI 0.80-3.97, p=0.15).The per-protocol analysis evalu-
ated survival in patients receiving more than 35 treatment sessions (HBO
n=38, HBA n=37). The HR for three-year survival in this population was 3.04
(95% CI 1.08-8.5, p=0.026). In a third analysis we compared survival be-
tween the per-protocol HBO group and those patients < 36 HBO sessions
(HBA and drop-outs). HR for three-year survival in this analysis was 3.17
(95% CI 1.19-8.45 p=0.021) in favour of HBO treatment.In terms of long-term
effects HBO does not seem to increase mortality. Contrarily, our study indi-
cates an increased survival in our selected group of diabetic patients with
chronic foot ulcers.
Supported by: Thelma Zoegas Foundation, Swedish Diabetes Foundation, R and
D Skane County
446-P
Plasma Levels of Circulating miR-191 are Altered in Diabetes As-
sociated Impaired Wound Healing
SEEMA DANGWAL, BERND STRATMANN, KATRIN SCHWARZ, JOHAN LOREN-
ZEN, CLAUS J. SCHOLZ, THOMAS THUM, DIETHELM TSCHPE, Hannover, Ger-
many, Bad Oeyn hausen, Germany, Wrzburg, Germany
MicroRNAs (miRNAs), master regulators of gene expression, are stably
present in blood. Here we investigated the levels of circulating plasma miR-
NAs in diabetic patients with or without impaired wound healing.MiRNAs
proling using affymetrix array was performed on pooled RNA from the
plasma of 36 diabetic patients with or without impaired wound healing. Re-
sults were validated by quantitative real-time PCR in patients with diabetes
associated impaired wound healing (n=17) and age- and body mass index-
matched disease controls i.e. diabetic patients (n=12) presenting no wounds
as well as age-matched healthy controls (n=20).Array data revealed a dif-
ferential regulation of 41 miRNAs in diabetic patients with impaired wound
healing vs. disease controls. PCR validation conrmed signicantly lower
level of miR 191 (ps0.05) in disease control vs. healthy control which was
reverted in diabetics with impaired wound healing, whereas another 2 miR-
NAs (miR 126 and miR 200b) showed a similar trend. Elevated plasma level
of miR 191 was associated with manifestation of myocardial infarction (MI)
and coronary artery disease whereas miR 126 with MI, and miR 200b with
peripheral artery disease. Diabetics with impaired wound healing showed
higher levels of cholesterol, liver transaminase and BNP vs. disease controls
(all ps0.05).Lower plasma levels of miR 191 in diabetics with no wounds
are reversed in diabetic subjects with impaired wound healing reecting a
complex pathology of diabetic wound healing complications. Hence miR 191
may serve as novel diabetic biomarker for the patient subset with no wound
healing impairment only.
Correlation analysis of differentially regulated circulating plasma miRNA
miRNA Variables in patients without
impaired wound healing
(correlation coefcient, p value, n=12)
Variables in patients with
impaired wound healing
(correlation coefcient, p value, n=17)
miR-191 C-peptide (r=0.643, p=0.02) Triglyceride (r=-0.522, p=0.03);
C-reactive protein (r=0.500, p=0.04)
miR-126 CRP (r=0.664, p=0.05);
Body weight (r=0.664, p=0.018)
LDL (r=-0.503, p=0.04);
Lipoprotein(a) (r=-0.514, p=0.04)
miR-200b Cholesterol (r=-0.587, p=0.05);
GFR (r=0.572, p=0.05)
Creatinin (r=0.619, p=0.008)
447-P
NT-proBNP: A Potential Cardiorenal Biomarker in the Management
of Diabetes
RIEKO KOMI, IKUE NAKADAIRA, JUN SUZUKI, AIKO OHOKA, ASAMI TANAKA,
MASAHIRO TAKAHASHI, KUMIKO HAMANO, Kamakura, Japan, Kawasaki, Japan
Individuals with diabetes and CKD are at particularly high risk for CVD and
ESRD. We recently reported that NT-proBNP could be a marker of silent myo-
cardial ischemia in type 2 diabetes. CVD accelerates CKD progression and
in turn, CKD itself amplies CVD risk, known as cardiorenal syndrome.In the
present study, we tested whether NT-proBNP could be a biomarker of both
CKD and CVD in type 2 diabetes.We consecutively recruited 109 patients
(mean age 61.8, HbA1c9.1, Cr0.76 mg/dl) with CKD stage1 and 2. Serum NT-
proBNP measurement (Roche) and ultrasonographic echocardiogram were
performed and subjects were followed up to 5 years. Primary endpoints were
dened as 1) renal:30% decrease of eGFR and 2) CVD and all-cause mortality.
NT-proBNP was well correlated with left ventricular mass index and eGFR.1)
Fourteen patients reached the renal endpoint and had signicantly higher
baseline NT-proBNP (169 vs. 52 pg/ml, p<0.05). Patients were stratied into
2 groups by NT-proBNP concentrations above and below the optimal cut-off
points calculated by ROC analysis. Kaplan Meier analysis demonstrated that
signicantly higher proportions of subjects with NT-proBNP above 120pg/ml
had progressive decline of eGFR (Log rank test: p=0.0054).2) Fourteen pa-
tients had new incidences of CVD or death. Mean levels of NT-proBNP were
142 and 44 pg/ml for those with and without new events. Subjects with NT-
proBNP level above 153pg/ml had signicantly higher risks of CVD and death
than those below 153pg/ml (HR 3.68, adjusted for sex and age, p=0.038).
Elevations of NT-proBNP predicted not only CVD event or death as well as
CKD progression in type 2 diabetes. As CKD is a major risk factor for CVD
in diabetes, NT-proBNP might be one of key molecules linking kidney with
heart. It is possible to stratify diabetic patients by simple measurement of
NT-proBNP, which is reproducible and inexpensive, and enables CVD preven-
tion and CKD prevention as well. Optimal cut-off value must be elucidated
by further investigation.
448-P
Chemerin is Associated With the Metabolic Syndrome But is Not
Linked to Angiographically Determined Coronary Artery Disease
CHRISTOPH H. SAELY, AXEL MUENDLEIN, ALEXANDER VONBANK, PHILIPP REIN,
KATHRIN GEIGER, HEINZ DREXEL, Feldkirch, Austria, Triesen, Liechtenstein, Phila-
delphia, PA
The novel adipocytokine chemerin has been suggested to be linked to in-
sulin resistance and to the metabolic syndrome (MetS). Its association with
coronary artery disease (CAD) is unclear. We hypothesized that chemerin
is associated with both angiographically determined CAD and with the
MetS. We measured serum chemerin in 498 patients undergoing coronary
angiography for the evaluation of established or suspected stable CAD; the
MetS was dened according to NCEP-ATPIII criteria; signicant CAD was
diagnosed when coronary stenoses ~50% were present.Chemerin was
higher in MetS patients (n=150) than in subjects without the MetS (18477
vs. 15062 ng/ml; p<0.001). It did not differ signicantly between patients
with signicant CAD (n=250) and those without signicant CAD (p=0.327).
When both, MetS and CAD status were considered, chemerin was higher
in MetS patients both among those who had signicant CAD (18280 vs.
15260 ng/ml; p=0.002) and among those who did not have signicant CAD
(18773 vs. 14863 ng/ml; p<0.001); it did not differ signicantly between
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patients with signicant CAD and subjects without signicant CAD among
MetS patients (p=0.248) nor among subjects without MetS (p=0.263). Analy-
sis of covariance (ANCOVA) showed that from the NCEP-ATPIII metabolic
syndrome traits large waist circumference as well as elevated trigylcerides
were independent predictors of elevated serum chemerin (F=12.5; p<0.001
and F=8.5; p=0.004).We conclude that chemerin is signicantly associated
with the MetS but not with angiographically determined CAD. The overall
association of chemerin with the MetS is carried by its association with
visceral obesity and elevated triglycerides.
Supported by: Research Foundation of the Austrian National Bank
449-P
Coronary Artery Disease as a Risk Factor for Developing Type 2 Dia-
betes Mellitus
CHRISTOPH H. SAELY, ALEXANDER VONBANK, PHILIPP REIN, HEINZ DREXEL,
Feldkirch, Austria, Triesen, Liechtenstein
Diabetes mellitus is a major risk factor for coronary artery disease (CAD);
whether conversely CAD confers an increased risk for diabetes has not been
studied so far.We prospectively recorded incident diabetes over 7.5 years
in 506 consecutive non-diabetic Caucasian patients undergoing coronary
angiography for the evaluation of stable CAD, covering 3795 patient years.
During follow-up, diabetes was newly diagnosed in 107 patients, i.e. in
21.1% of the study population or in 2.8% per year. Patients with signicant
CAD (n=293) when compared to subjects who did not have signicant CAD
at the baseline angiography were at a 33% (p = 0.027) increased diabetes
risk. However, the relationship between CAD and incident diabetes was at-
tenuated and no longer statistically signicant after adjustment for potential
confounders including metabolic syndrome (MetS) status. The MetS as diag-
nosed according to the current consensus denition in turn was strongly pre-
dictive of diabetes, in particular when the more selective NCEP-ATP-III waist
cut-off values were applied for its diagnosis (OR = 2.91 [1.83-4.64]; p <0.001).
We conclude that albeit apparently not causally related to diabetes inci-
dence, the presence of CAD indicates a strongly increased risk for incident
diabetes. Repeated diabetes screening of coronary patients and targeted
programs to prevent diabetes in these high-risk patients are warranted.
Supported by: Jubilumsfonds der sterreichischen National Bank
450-P
Progression of Diabetic Retinopathy is Associated With Left Ven-
tricular Diastolic Dysfunction in Patients With Type 2 Diabetes
SOICHI KURIOKA, Osaka, Japan
Diabetic retinopathy (DR) is anindependent predictor of left ventricular
diastolic dysfunction (LVDD) inpatients with diabetes mellitus. However, it
is unclear if progression of DR isassociated with LVDD, which is recognized
to result in subsequent heart failure.The subjects in this study were 120 con-
secutive patients with type 2 diabetesmellitus (T2DM) [72 men (60%); age
6610 years old (meanSD); diabetic duration119 years; HbA1c 8.01.8%].
Subjects with overt heart failure or NYHA class>1, history of coronary artery
disease, severe valvulopathy or chronicatrial brillation were excluded from
the study. All patients underwentclinical evaluation, laboratory tests, and
echocardiographic examination. Dopplerechocardiographic indices including
peak early diastolic mitral annularvelocity (E) and early diastolic myocar-
dial velocity (E) were obtained in eachpatient. The patients were divided
into three groups according to DR stage: nodiabetic retinopathy (n=80),
simple retinopathy (n=20), and preproliferative orproliferative retinopathy
(n=20). No patients showed systolic impairment oeft ventricular function
(LVEF >50%), whereas LVDD (E/E >8) was detectedin 105 cases (87.5%).
E/E was correlated with age (r=0.303, p=0.001), sex(r=0.208, p=0.021), DR
stage (r=0.241, p=0.007), systolic blood pressure(r=0.199, p=0.031), and se-
rum creatinine level (r=0.265, p=0.003). In multipleregression analysis, age
(=0.322,p<0.0001) and DR stage (=0.266, p=0.002)were independently
correlated with E/E. In this study, almost all asymptomaticpatients with
T2DM had LVDD. Progression of DR is associated with LVDD and thismay at
least in part explain the increased incidence of heart failure inpatients with
diabetes mellitus and DR.
451-P
Adiponectin Receptors and Adiponectin Action in Circulating Hu-
man CD14+ Monocytes in Patients With Diabetes and Cardiovascu-
lar Disease
LING TIAN, DENNIS STEVERSON, WEI ZHANG, YUCHANG FU, W. TIMOTHY
GARVEY, Birmingham, AL
We have previously shown that adiponectin regulates insulin action and
foam cell formation via adipoR1 and R2 in cultured cells. To investigate a role
of the adiponectin-adipoR1/2 axis in humans with cardiometabolic disease,
we studied adipoR1/2 expression in circulating CD14+ monocytes obtained
from insulin sensitive normoglycemic subjects (IS, n=12) vs. T2DM patients
with coronary artery disease (DM&CVD, n=13).Flow cytometry found no sig-
nicant differences in CD14, CD16, CD45 and CD11c expression in circulating
mononuclear cells between groups. CD14+ monocytes were then cultured for
8 days followed by adiponectin and oxLDL treatment. Adiponectin increased
AdipoR1 and R2 mRNA expression in both IS (p<10E-5, p<0.03, respectively)
and DM&CVD (p<10E-5, p<0.001), however, there were no signicant inter-
group differences. Further analyses revealed gender differences that male
IS have a trend for a greater adiponectin-mediated adipoR1 increase than
male DM&CVD (p=0.13), while the response was signicantly less in female
IS vs. female DM&CVD (p=0.01). There were no group differences in cellular
cholesterol levels and secretions of MCP-1, IL-6 and IL-1Ra. We did observe
that cholesterol accumulation was correlated with adipoR2 levels in total
subjects (p=0.0398). Importantly, as we previously reported that adiponec-
tin-adipoR1/2 signals via APPL1 in THP-1 macrophages, we found that APPL1
mRNA levels were signicantly higher in the IS vs. DM&CVD (p=0.024).In
conclusion: 1) circulating monocytes do not exhibit an adiponectin-resistant,
pro-inammatory, or lipid accumulating status in DM&CVD; 2) there are
gender differences in adiponectins ability to modulate adipoR1 expression;
3) APPL1 expression is greater in IS. Hence, monocytes may become more
pro-inammatory only after entering adipose tissue or the vascular wall in
cardiometabolic disease, and this may be related to an intrinsic down-regu-
lation of APPL1 expression.
Supported by: DK038746 DK083562 P60-DK079626 Merit Review Program of VA
452-P
Oxidative Stress Injury in Type 2 Diabetics Undergoing Coronary
Artery Bypass Surgery
ASHVINI MENON, EBRAHIM MULLA, S. HUGHES, J. MASCARO, MARTIN STE-
VENS, ROBERT BONSER, Birmingham, United Kingdom
Poly (ADP-ribose) polymerase (PARP) is a nuclear enzyme that repairs
damaged DNA strands. Increased oxidative stress in T2DM excessively ac-
tivates PARP and depletes its substrate resulting in energy deciency and
cell death. This may relate to a reduced ability of the diabetic myocardium
to cope with ischaemia-reperfusion injury. Pre-operative PARP status has
not been characterised in humans. We therefore evaluated myocardial
PARP activation and plasma protein carbonyls (marker of oxidative injury) in
T2DM and non-T2DM patients undergoing CABG.Right atrial biopsies were
obtained at initial venous cannulation. Samples were xed in 10% formal
saline and 4m parafn sections prepared. Sections were incubated with
mouse monoclonal anti-PAR (poly-ADP-ribose) antibody. Images were cap-
tured using a Ziess Axioshop-plus microscope and analysed using Axiovision
4.4 programme. The percentage of PAR stained nuclei was determined by
image analysis using a Scion image programme. Plasma protein carbonyl
was measured using the Biocell PC Test kit (Biocell, Auckland, New Zea-
land) at 0,6,12,24,48 and 72 hours following CABG.Twenty non-T2DM and 28
T2DM right atrial biopsies have been analysed so far. The median age (78
vs. 73 years), extent of coronary artery disease and median ejection fraction
(>50% in both groups) were not different. A signicantly higher percentage
of PAR positive nuclei was detected in the T2DM group (71 [65.2-76.9] %)
compared to the non-T2DM subjects (45.2 [38.6-51.8] %), p<0.001. In both
groups, protein carbonyl values varied signicantly over time (p=0.02). Pro-
tein carbonyl values at 12, 24, 48 hours post CABG were signicantly higher
in T2DM (p<0.05).Cardiac PARP activation and oxidative stress markers are
increased in T2DM. This may promote increased susceptibility to ischaemia-
reperfusion injury and may contribute to worse post-CABG outcomes in
T2DM. Ongoing studies are in progress to evaluate this relationship.
453-P
Effects of Atorvastatin on Endothelial Nitric Oxide Release, Blood
Pressure and Nitroxidative Stress Levels in Hypertensive Rats With
Diabetes
R. PRESTON MASON, ROBERT F. JACOB, J. JOSE CORBALAN, ALEKSANDER
CISZEWSKI, TADEUSZ MALINSKI, Boston, MA, Beverly, MA, Athens, OH
Clinical trials have shown that patients with diabetes benet from treat-
ment with the HMG-CoA reductase inhibitor, atorvastatin, despite having
normal LDL levels. Most patients with diabetes also have hypertension,
both of which are cardiovascular risk factors associated with endothelial
cell (EC) dysfunction and reduced nitric oxide (NO) release. In this study, we
tested the hypothesis that atorvastatin reverses EC dysfunction in diabetic,
spontaneously hypertensive (SH) rats by enhancing endothelial NO synthase
(eNOS) activity. Male SH rats with normal LDL levels were treated with
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streptozotocin (65 mg/kg/BW) to induce diabetes, followed by treatment
with atorvastatin at 20 mg/kg/day (or vehicle for control animals). After 5
weeks, glomerular ECs were isolated from renal tissue and assayed for NO
and peroxynitrite (ONOO
) release using amperometric nanosensors. Study

animals were also examined for changes in fasting glucose and mean blood
pressure (BP) levels. As an additional control, we performed similar analy-
ses in non-diabetic SH rats. Atorvastatin was found to increase NO release
in diabetic SH rats by 98% (63 7 nM to 125 16 nM) while decreasing
ONOO
-
release by 38% (180 12 nM to 112 16 nM) as compared to vehicle
alone (p<0.001). The NO/ONOO
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increased more than three-fold with atorvastatin treatment. Diabetic SH
rats also had elevated fasting glucose (355 38 mg/dL) and mean BP (172
15 mmHg) levels. Atorvastatin reduced mean BP by 21% (to 136 18 mmHg,
p<0.001) but had no signicant effect on fasting glucose levels. Atorvastatin
also increased the NO/ONOO
ratio in non-diabetic SH rats by more than

two-fold (p<0.001) and reduced mean BP by 11%. These results suggest that
atorvastatin treatment reverses endothelial dysfunction and reduces blood
pressure in diabetic SH rats by enhancing eNOS coupling and NO release
independently of glucose control.
454-P
The Inuences of High Glucose and Hypoxia on Hcdc14A, Cyclin Ex-
pressions, Cell Proliferation and Apoptosis
HOUGUANG ZHOU, LING LIU, YU ZHANG, YANYAN HUANG, ZHUANGLI GUO,
RENMING HU, QIANG DONG, Shanghai, China, Nanjing, China, Qingdao, China
Long-term hyperglycemia in T2DM increased glycated hemoglobin, re-
duced red blood cell oxygen-carrying capacity, raised blood viscosity, and
caused microvascular perfusion defects and chronic hypoxia in tissue cells.
But the mechanism of vascular injury and complication induced by high glu-
cose and hypoxia(HGH) in T2DM is still not completely clear. The purpose of
this study was to observe the inuences of HGH on human cell division cycle
protein 14a(Hcdc14A), the related cycle protein expressions, cell prolifera-
tion and apoptosis in human brain vascular endothelial cells(HBVEC). Using
different conditions of high glucose and hypoxia stimulated HBVEC. RT-PCR
and Western-blot(WB) detected Hcdc14A mRNA and protein expression
respectively. CyclinB, cyclinD3, cyclinE and P53 protein expressions were
tested with WB. XTT and CCK8 methods were used for the determination
of the rate of cell proliferation and survival, ow cytometry and caspase3
activity for apoptosis, ow cytometry for cell cycle, uorescent probe for
cytoskeletal protein F-actin spatial structure, and electron microscope for
utrastructure. HGH could downregulate Hcdc14A mRNA and protein expres-
sions, reduced cyclinB, cyclinD3 and cyclinE protein expressions, while up-
regulated P53 protein expression. HGH reduced the cell proliferation and
survival rates, increased the apoptosis rate and the caspase-3 activity,
signicantly lowered the cells in S phase and G2-M phase, inuenced cy-
toskeletal protein F-actin space structure. Electron microscope observation
also revealed that apoptosis cells increased. In conclusion, HGH could obvi-
ously downregulate the Hcdc14A in HBVEC, inuence related cyclin expres-
sions, block cell cycle, reduce cell proliferation, induce apoptosis, and affect
cytoskeletal protein F-actin spatial structure and cell ultrastructure, which
representing some key factors in the development of micro- and macrovas-
cular dysfunction in T2DM.
Supported by: The National Natural Science Foundation of China (81170322)
455-P
Inhibition of an Olfactory Receptor Mediates Cell Death in Human
Arterial Endothelial Cells
JENIE Y. HWANG, CHANG HEE JUNG, WOO JE LEE, JOONG-YEOL PARK, Seoul,
Republic of Korea
Olfactory receptors (ORs) are the largest gene family in human genome.
Although they are expected to be expressed specically in olfactory tissue,
about 10% of ORs have ectopic expression. However, the function of ec-
topic ORs is not well explored except their chemotactic function of sperm.
The present study explores the expression of one of the representative OR,
OR1D2, in a human aortic endothelial cells (HAEC) and assesses the potential
functional implication of such ectopic expression.HAECs(BioWhittaker) were
cultured in endothelial groth medium-2 supplemented with specic growth
factors. Using RT-PCR, we demonstrated the existence of OR1D2 in HAEC.
Also, we identied that well-known odorant bourgeonal, rosiglitazone and
alpha-lipoic acid(aLA) elicit activation of OR1D2 in endothelium. By using
siRNA, expression of OR1D2 was down-regulated and apoptosis of human
endothelial cells was seen in dose dependent manner. Exposure to siRNA
resulted in the activation of members of the MAPK family and inhibition of
cell proliferation. Our data give support to the hypothesis that OR signaling
may mediate cell proliferation in HAEC , thus specic receptor ligands might
be potential candidates for endothelial dysfunction.
456-P
The Estrogen-Related Receptor g (ERRg) Is Involved in the Develop-
ment of Vascular Calcication
JI-HYUN KIM, YOUNG-KEUN CHOI, CHAE-MYEONG HA, EON-JU JEON, HYUN-
AE SEO, JI-YUN JEONG, YEON KYUNG CHOI, KWI-HYUN BAE, SANG-JUN LEE,
KEUN-GYU PARK, JUNG-GUK KIM, IN-KYU LEE, Daegu, Republic of Korea
Vascular calcication, which refers to ectopic mineralization in vascular
cells occurs frequently in many diseases such as chronic kidney disease,
atherosclerosis, and diabetes. Vascular calcication is vastly correlated with
high risk of cardiovascular morbidity and mortality, due to arterial stiffness
and impaired vascular compliance. Estrogen-related receptor (ERR), a
member of orphan nuclear receptor superfamily is shown to be related to
bone formation. However, the role of ERR in vascular calcication has not
yet been investigated. This study was undertaken to examine the role of
ERR in vascular calcication.ERR expression was upregulated during in-
organic phosphate (Pi)-induced calcication of vascular smooth muscle cells
(VSMCs), along with increased expression of bone morphogenic protein-2
(BMP2), osteocalcin and alkaline phosphatase. Transient overexpression of
ERR stimulated promoter activity of osteogenic genes including BMP2 and
osteocalcin. Adenovirus-mediated overexpression of ERR also augmented
osteogenic gene expression and induced BMP2 expression and phosphory-
lation of Smad1,5,8, consequently, exacerbates Pi-induced calcication of
VSMCs. Moreover, inhibition of ERR by a selective inverse agonist attenu-
ated both Pi-induced osteogenic gene expression and calcium deposition in
cultured VSMCs. Finally, ex vivo aortic tissue culture showed that the ERR
inverse agonist signicantly reduced Pi-induced calcication in mouse aorta.
These results demonstrate that ERR stimulates vascular calcication by up-
regulating osteogenic genes. Therefore, our ndings suggest that inhibition
of ERR may be a potential therapeutic strategy for prevention of vascular
calcication.
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Comparison of Doppler (D) and Photoplethysmography (PPG) in
Measuring the Ankle-Brachial Index (ABI) in People With Diabetes
LIVIU DANESCU, CHERYL ROE, LEWIS JOHNSON, Syracuse, NY
American Diabetes Association guidelines recommend measuring ABI in
people >50 yrs old with diabetes. It is infrequently done in ofce practice,
in part, because the D technique is time consuming and difcult to master.
Previous studies have compared use of PPG and D but not specically in dia-
betes. We report results from our diabetes clinic comparing the PPG and D
methods for measuring ABI. Consecutive patients with diabetes (n=103) had
ABI measured by both methods. In 3 individuals we were unable to obtain
PPG readings. Mean age was 60 12.8 yrs, 57% were female, 79% had type
2 diabetes and the average duration of diabetes was 17 12 yrs. Of 200 limbs
evaluated, 17 (8.5%) had non-compressible vessels (NC). A comparison of
the 2 methods on the remaining 183 limbs yielded a correlation coefcient of
0.864 (Figure). Mean ABI was 1.11 0.14 for D and 1.12 0.14 for PPG. The dif-
ference between the means was non-signicant. ABI results were classied
as discordant if D and PPG varied by more than 0.15 (reported variability of
D), placing them in different diagnostic categories [low (<0.9), normal (0.9-
1.3) and high (>1.3) / NC groups]. There were only 2 (1%) discordant results.
In conclusion, an excellent correlation was found when comparing PPG with
the gold standard D technique in the measurement of the ABI in people with
diabetes. PPG requires less training and takes less time to perform making it
highly suitable for use in the ofce setting.
458-P
Reinfarction and Mortality Rate in Patients With Coronary Artery
Disease and the Relation to Glucose Tolerance
JEANETTE KUHL, GUN JRNESKOG, MALIN WEMMINGER, MATTIAS BENGTS-
SON, MAJID KALANI, Stockholm, Sweden
We investigated the importance of glucose tolerance for long term car-
diac prognosis in patients with coronary artery disease (CAD).1151 consecu-
tive patients, 836 men and 315 women, aged 32-80 years, admitted to the
coronary care unit at Danderyd University Hospital, Stockholm, for acute
coronary syndrome (ACS) from year 2006 to 2008. At discharge, the patients
were categorized according to an oral glucose tolerance test (OGTT) as
having normal (NGT; n=303 (26%)) or impaired (IGT; n=311 (27%)) glucose
tolerance, or diabetes (n=537 (47%)). In 2010, after a mean follow-up time
of 3 years, all patients were invited to answer a questionnaire and to give
fasting venous blood samples for analysis of metabolic control. Information
about reinfarction and mortality were obtained from the Swedish Coronary
Angiography and Angioplasty Registry and from the Swedish National Reg-
istration.At the follow-up, 36 patients (12%) in the NGT group had had rein-
farction compared to 50 patients (16%) in the IGT and 123 patients (23%) in
the diabetes group. Mortality was 7.1% (82 patients): 3 patients with NGT
(1.0%), 8 with IGT (2.6%), and 71 patients with diabetes (13.2%). 548 (48%)
patients (136 women, 412 men) accepted the invitation for a follow-up. At
the follow-up, a signicant increase in fP-glucose was seen in all groups as
compared to baseline values (p<0.05) despite intensied antidiabetic and
risk factor medications since discharge from the hospital. In contrast, lipo-
protein prole had improved in all groups as compared to baseline (p<0.001).
Patients with diabetes showed a signicant increase in serum creatinine
(93.04.0 mol/L vs 97.24.2 mol/L; p<0.05), while no signicant changes
were seen in patients with NGT or IGT. In conclusion, a majority of patients
admitted for ACS have disturbed glucose metabolism including diabetes.
Long term cardiac prognosis is poor in patients with CAD and dysglycaemia
despite intensied antidiabetic and risk factor medications.
459-P
The Metabolic Syndrome Signicantly Affects the Association be-
tween Resting Heart Rate and All Cause as Well as Cardiovascular
Mortality
ALEXANDER VONBANK, FABIAN SCHMID, PHILIPP REIN, CHRISTOPH H. SAELY,
HEINZ DREXEL, Feldkirch, Austria, Triesen, Liechtenstein, Philadelphia, PA
Epidemiological studies suggest that the resting heart rate (RHR) is an
independent predictor of cardiovascular and all cause mortality. However,
the power of the RHR to predict cardiovascular events in patients with the
metabolic syndrome (MetS) is not known.We prospectively investigated the
relationship between RHR and cardiovascular events in 756 consecutive pa-
tients undergoing coronary angiography for the evaluation of coronary artery
disease (CAD) over a follow-up period of 7.1 0.1 years. The MetS was de-
ned according to NCEP-ATPIII criteria.In the total study population, both all
cause and cardiovascular mortality were increased with an increasing RHR
(standardised adjusted HRs 1.03 [1.01-1.04]; p = 0.001 and 1.15 [1.03-1.47];
p = 0.001, respectively). From our patients, 357 (47.2%) had the MetS and
399 did not have the MetS. Among patients without the MetS, a higher
baseline RHR indicated a signicantly higher risk of total mortality (HR = 1.14
[111 - 1.16], p = 0.001) and cardiovascular mortality (HR = 1.13 [1.12- 1.16], p =
0.001) after multivariate adjustment. However, the RHR did not signicantly
affect total mortality (p = 0.120) or cardiovascular mortality (p = 0.244) in
patients with the MetS. Interaction terms RHRxMetS were signicant for
both total and cardiovascular mortality (p = 0.027 and p = 0.037, respec-
tively), indicating that the respective risks conferred by a high RHR were
signicantly higher in patients without the MetS than in patients with MetS.
We conclude that among angiographically characterized coronary patients,
the metabolic syndrome status signicantly affects the association of the
RHR with total and cardiovascular mortality: RHR is a strong predictor of
both total and cardiovascular mortality among subjects without the MetS,
but not among MetS patients.
460-P
One in Three Patients Referred to The Type 2 Diabetes Clinic, Steno
Diabetes Center Suffers from Obstructive Sleep Apnea
HEIDI STORGAARD, THOMAS P. ALMDAL, MICHAEL LAUB, LISE TARNOW,
Gentofte, Denmark, Copenhagen, Denmark
Several studies suggest that obstructive sleep apnoea (OSA) is associ-
ated with cardiovascular disease (CVD), and CVD is common among patients
(pts) with type 2 diabetes (T2DM). We therefore aimed to investigate the
prevalence of OSA among T2DM pts referred to Steno Diabetes Center.All
pts referred to SDC T2DM clinic September 2010-June 2011 were offered
to participate in a three-stage screening program for the diagnosis of OSA;
1.The Berlin questionnaire. A high likelihood of OSA-score qualied for;
2. An outpatient ApneaLink
monitoring. Pts with a positive test (apnoea-

hypopnoea index (AHI) ~ 5/hour) were referred to; 3. Diagnostic polygraphy
(with Embletta
) in a sleep clinic.A total of 180 pts (men, 61%; age 59.7

10.5 years, BMI, 31.8 6.7 kg/m2, T2DM duration 8.2 6.3 years, HbA1c,
7.3 1.2%) participated. Amongst these 104 (61%) had a high likelihood of
OSA-score according to the Berlin questionnaire, and 77 pts (43%) were af-
ter ApneaLink
study referred to the sleep clinic. So far, polygraphy has been

performed in 49 pts, and 44 have been diagnosed with OSA - corresponding
to an estimated prevalence of 34 % of the entire population (n=180).Ap-
neaLink
positive (n = 77) pts had higher BMI (34.3 7.4 versus 30.3 5.7 kg/
m2, p <0,001 ), higher HbA1c (7.6 1.3 versus 7.2 1.1%, p = 0.008) and lower
HDL-chol (1.1 0.3 versus 1.3 0.4 mmol / l, p = 0.003) as compared with
those without symptoms (n = 77) or signs of OSA on ApneaLink
(n = 26).
The groups were comparable with respect to: blood pressure, albuminuria,
p-creat, retinopathy, LDL chol, treatment with OAD, RAS blockade and sta-
tins. In a multiple linear regression analysis, AHI increased signicantly with
higher age, increased BMI and declining HDL-chol. Sex, HbA1c and insulin
dose per kg bodyweight were not related to AHI.These preliminary results
suggest that more than 30 % of T2DM pts referred to a hospital outpatient
clinic have OSA - this may play a role in the high cardiovascular mortality
seen in these pts.
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Serum Cystatin C Shows a Greater Association With Peripheral Ar-
tery Disease than Albuminuria in Type 2 Diabetes With Normal or
Mild Impairment of Renal Function
JANG-YEL SHIN, MI YOUNG LEE, CHOON HEE CHUNG, Wonju-Si, Gangwon-Do,
Republic of Korea
Some studies have shown a link between chronic kidney disease and
peripheral artery disease (PAD) in general population. Cystatin C has been
suggested to be a sensitive marker for early detection of renal impairment
in type 2 diabetes. We examined the association of serum cystatin C with
PAD in type 2 diabetes with normal or mild impairment of renal function.
We enrolled 272 type 2 diabetic patients [age 57.79.7 years; male 58.5%;
duration of diabetes 6.36.0 years; HbA1c 7.71.8%]. Patients were excluded
if they had estimated glomerular ltration rate (eGFR) < 60 mL/min per 1.73
m
2
, 24 hour urine albumin (24h-uAlb) ~ 300 mg/day, serum creatinine (Cr) >
1.3 mg/dL, or ankle-brachial index (ABI) > 1.4. eGFR was calculated using
the Modication of Diet in Renal Disease equation. PAD was dened as an
ABI s 0.9.Median values (inter-quartile range) of cystatin C, eGFR, and 24h-
uAlb were 0.92 (0.80, 1.08) mg/L, 93.7 (82.1, 109.2) mL/min per 1.73 m
2
, and
18.3 (10.1, 36.0) mg/day. Patients with PAD were 7.4%. Comparing to those
without PAD, patients with PAD had more likelihood to be hypertensive and
current smoker, higher values of 24h-uAlb, cystatin C, and serum Cr, and
lower value of eGFR. In partial correlation after adjusted for age and gender,
cystatin C showed positive correlations with waist circumference, smoking,
24h-uAlb, and serum Cr, but was negatively correlated with eGFR. Also, after
adjusted for age and gender, PAD was positively correlated with smoking,
cystatin C, and 24h-uAlb. Odds ratios (ORs) for PAD after adjusted for age,
gender, and smoking, were 1.81 (95% CI, 1.10-2.99) for cystatin C and 1.61
(95% CI, 1.01-2.56) for 24h-uAlb per 1 SD increase. After further adjustment
for 24h-uAlb or cystatin C, only the OR for cystatin C remained signicant.Our
nding suggests that serum cystatin C shows a greater positive association
with PAD than albuminuria in type 2 diabetes with normal or mild impairment
of renal function.
462-P
Prevention by Sulforaphane of Diabetic Cardiomyopathy is Associ-
ated With Nrf2 Up-Regulation and Activation
YANG BAI, YANG ZHENG, QIANG CHEN, XIAO MIAO, CHI ZHANG, WENPENG CUI,
YI TAN, LU CAI, Louisville, KY, Changchun, China, Wenzhou, China
This study was to investigate whether sulforaphane (SFN) as one of NF-E2-
related factor 2 (Nrf2) activator can protect diabetic cardiomyopathy. Type 1
diabetes was induced in 8-week-old male FVB mice by multiple intraperitoneal
injections of low-dose streptozotocin (STZ). Five days after the last injection of
STZ, mice with hyperglycemia (blood glucose levels ~ 250 mg/dl) were dened
as diabetic. Diabetic and age-matched control mice were subcutaneously
given SFN at 0.5 mg/kg for ve days of each week for 3 months. At the end
of the experiments, cardiac function was assessed using M-mode echocar-
diography and cardiac pathological changes, brosis, inammation and oxi-
dative damage were assessed by western blotting and immunohistochemical
staining. SFN was found to signicantly prevent diabetes-induced cardiac
dysfunction, shown by increased left ventricular posterior wall and decreased
left ventricular ejection fraction. SFN also signicantly prevented diabetes-
induced structural disarrangements, examined by hematoxylin-eosin staining,
and cardiac remodeling, detected by brotic makers of transforming growth
factor (TGF)-1 expression and collagen deposition (Sirius-red staining materi-
als). These pathological changes were accompanied by signicant increases
in oxidative damage, measured by protein nitration (3-nitrotyrosine), lipid per-
oxidation (4-hydroxynonenal), inammation (plasminogen activator inhibitor-
1,PAI-1) in diabetic heats, but not in SFN-treated diabetic hearts. SFN induced
signicant increases in Nrf2 expression, measured by western blotting and
function, reected by increase Nrf2 downstream gene NAD(P)H: quinone oxi-
doreductase1 (NQO1) expression. These results suggest that diabetes-induced
cardiac structure disarrangement and remodeling as well as oxidative dam-
age can be signicantly prevented by SFN probably via up-regulation of Nrf2
expression and function.
463-P
464-P
Coronary Response to an Increased Oxygen Demand is Altered in
Asymptomatic Type 2 Diabetic Patients With Coronary Artery Dis-
ease: A New Non-Invasive Approach
EMMANUEL COSSON, MINH TUAN NGUYEN, ISABELLE PHAM, ALAIN NITEN-
BERG, PAUL VALENSI, Bondy, France
Cold pressure test (CPT) induces an increase of double product (ADP:
blood pressure x heart rate after/before, %) and a dilation of epicardial
coronary arteries. The aim of the study was to investigate if the attenu-
ated dilation during CPT previously shown by coronary angiography in type
2 diabetic patients (T2Ds) without coronary artery disease (CAD) was also
observed with a non invasive method (NCT00685984).The ow velocity was
measured in the left anterior descending artery (aCV) by trans-thoracic echo-
doppler (3-7MHz) before and after CPT in 118 T2Ds, 30 overweighed or obese
non diabetic subjects (OS), and 25 control subjects s 40 years. T2Ds were
screened for silent myocardial ischemia (SMI: abnormal stress myocardial
scintigraphy and/or echocardiography), and in case of SMI for CAD (coro-
nary angiography).Thirty-ve T2Ds had SMI, 15 of them CAD. aCV during
CPT could be measured in 56% T2Ds, 37% OS and 64% controls. At rest DP
(p<0.0001) and aCV (p<0.01) were higher in T2Ds than in OS and controls, and
aCV correlated with DP (p<0.05). During CPT, ADP (4334/3627/3219%
increase) and change in aCV (AaCV: 3026/2134/1710% increase) did not
differ signicantly in the three groups. AaCV and ADP correlated in con-
trols (r=0.58, p<0.05), OS (r=0.78, p<0.01) and T2Ds without CAD (r=0.56,
p<0.0001) but not in T2Ds with CAD. SMI status was associated in T2Ds
with AaCV (SMI- 2122, SMI+CAD- 3628 and SMI+CAD+ 3926%, p=0.03)
despite similar ADP; and with gender, BMI, nephropathy, triglycerides and
haptoglobin (p=0.02 to 0.05). In multivariate analysis, SMI was only associ-
ated with AaCV (per 10% increase OR: 1.3[1.1-1.5], p<0.05). To conclude, (i)
non invasive study of the coronary reactivity is feasible by trans-thoracic
echography-doppler with a lower feasibility in OS, (ii) the coronary response
to CPT is increased but unadapted in T2Ds with silent CAD, probably through
defects in coronary dilation or microcirculation.
Supported by: Socit Francophone de Diabtologie
465-P
Alterations in Monocyte Surface Markers in Diabetes Complications
DANQING MIN, BELINDA BROOKS, JENCIA WONG, ROBERT SALOMON, WEN-
SHENG BAO, BRIAN HARRISBERG, STEPHEN M. TWIGG, DENNIS K. YUE, SUSAN
V. MCLENNAN, Sydney, Australia
The monocyte and macrophage cell lineage is central to the inamma-
tory response and alterations in this response are associated with diabe-
tes and its complications. Monocytes express many cell surface markers
indicative of their inammatory and activation status. Whether these mark-
ers are affected by diabetes and its complications is not known and was
investigated in this study. Blood was obtained from 22 non-diabetic controls
and 43 diabetic patients with duration of diabetes >10 years, including 25
without and 18 with diabetic complications. In each subject the percentage
of monocytes (CD45
+
CD14
+
) expressing pro- or anti-inammatory markers
(CD16 and CD163 respectively), various activation markers and chemokine
receptors were determined by ow cytometry. Clinical data were collected
and selected plasma cytokines and chemokines were also measured. Dia-
betes increased monocyte number (P<0.05) but did not alter the expression
of cell surface markers related to activation status or the expression of
chemokine receptors. However, the percentage of both pro-inammatory
CD16
+
and anti-inammatory CD163
+
monocytes were decreased in the
group with diabetic complications compared to those without (1.9 and 16.2
fold respectively, each P<0.05). The plasma pro-inammatory cytokines in-
cluding interleukin-6, interleukin-8, tumour necrosis factor-o and interferon-
WITHDRAWN
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gamma also showed the same pattern of decrease in those with complica-
tions. These results suggest that monocyte phenotype is altered by diabetic
complications status. Both anti- and pro-inammatory surface markers are
decreased by the presence of complications but a higher anti-inammatory
prole is associated with no diabetic complications. These changes may be
causally related and if conrmed in subsequent longitudinal series, could
potentially be used to predict susceptibility to diabetic complications. How
these ndings affect monocyte and macrophage function also warrants fur-
ther investigation.
Supported by: Endocrinology and Diabetes Research Foundation, The University
of Sydney
466-P
A Novel Diagnostic Procedure of Asymptomatic Coronary Artery
Disease in Diabetic Patients using Carotid-Aall Intima-Media
Thickness as a Surrogate Marker and Coronary Computed Tomog-
raphy Angiography
YUICHIRO YOSHIKAWA, HIROKI KAGAYA, TAKAHIRO KAGEYAMA, HIROMI
MAEDA, SHIGEKI IMAMURA, KAZUO MISUMI, KEMPEI MATSUOKA, AIZAN HI-
RAI, Togane, Japan, Matsudo, Japan, Tokyo, Japan
Diabetes is associated with a marked increase in the risk of coronary
artery disease (CAD). Also, diabetic patients without previous myocardial
infarction have as high a risk of myocardial infarction as non-diabetic pa-
tients with previous myocardial infarction. It is well known that patients
with diabetes have often asymptomatic CAD. Carotid-wall intima-media
thickness (IMT) is a surrogate marker of atherosclerosis associated with
cardiovascular risk factors and with cardiovascular outcomes. The present
study was performed in order to establish a diagnostic procedure for as-
ymptomatic CAD in diabetic patients using the maximum IMT (maxIMT) of
carotid artery as a surrogate marker followed by coronary computed tomog-
raphy angiography (CCTA), a new noninvasive diagnostic test for CAD. In the
present investigation, 317 diabetic patients and 224 non-diabetic patients
with lifestyle-related diseases (hypertension, dyslipidemia) without any epi-
sode of chest pain and having maxIMT over 1.5mm were studied. CCTA using
a 256 channel MDCT scanner was performed for all of them. Then coronary
angiography (CAG) was performed for the patients with positive ndings in
CCTA. In the present study, 51.4% of diabetic patients and 25.4% of non-
diabetic patients have coronary lesions with stenosis more than 25% in the
CAG (p <0.0001). Also, 36.3% of diabetic patients and 13.8% of nondiabetic
patients have coronary lesions with stenosis more than 75% in the CAG (p
<0.0001). False-positive rate of CCTA was 9.1% in diabetic patients, 17.4% in
non-diabetic patients (p <0.0001), respectively. Among various parameters,
age, HDL-C, eGFR and maxIMT have correlation with the degree of coro-
nary lesion. Thus, maxIMT of carotid artery over 1.5mm is a useful surrogate
marker of asymptomatic CAD in diabetic patients. The present diagnostic
procedure requires further studies of subsequent outcomes.
467-P
Effects of GLP-1 Receptor Agonist on Ca2+ Handling of Coronary
Smooth Muscle Cells from Metabolic Syndrome Ossabaw Swine
With Coronary Artery Disease
MIKAELA L. MCKENNEY, MOUHAMAD ALLOOSH, KYLE SCHULTZ, NICKI BELL,
NAGA CHALASANI, MIKE STUREK, Indianapolis, IN
Chronic effects of the glucagon-like peptide (GLP-1) receptor agonist
AC3174 were studied in Ossabaw swine with metabolic syndrome (MetS).
MetS was induced by hypercaloric atherogenic diet for 6 months. Subcuta-
neous injections of placebo or AC3174 were given twice daily for 6 months
after induction of MetS. Consistent with clinical effects, AC3174 attenuated
food consumption from 961% to 822% of allotted amount (p<0.05) and
weight gain decreased from 116.22.4 kg to 99.55.8 kg, respectively. Both
groups were glucose intolerant, but placebo pigs had greater intolerance
after the 6 month treatment as determined by intravenous glucose tolerance
tests. AC3174 stimulated a trend towards an increase in insulin secretion
with peak insulin levels at 12325 vs 6712 U/mL (p=0.07). MetS increases
coronary artery disease (CAD) and dysfunction of intracellular Ca
2+
handling
by coronary smooth muscle (CSM). CSM cells were isolated enzymatically
from the coronary arteries and Ca
2+
was digitally imaged with the uores-
cent Ca
2+
indicator fura-2. Ca
2+
levels were measured by a uorescence ra-
tio of 360/380nm. There was no difference in basal Ca
2+
levels. In normal
physiological salt solution we released sarcoplasmic reticulum (SR) stores
with caffeine, which binds to ryanodine receptors in the SR membrane.
AC3174 cells showed a larger peak increase in Ca
2+
in response to caffeine,
AF360/380=0.260.02, compared to AF360/380=0.160.01. A sustained
Ca
2+
signal was present during recovery to basal levels in the treated cells,
implying there is greater Ca
2+
inux after chronic AC3174 treatment. The sus-
tained uorescence ratio from the treated cells was 0.070.01 compared to
a lesser signal of 0.040.003. It appears chronic AC3174 treatment elicits
greater Ca
2+
release and inux in MetS. Our interpretation is that AC3174
attenuates the decline in CSM Ca
2+
handling associated with more severe
CAD and dedifferentiation of CSM.
Supported by: NIH (HL062552), Amylin Pharmaceuticals, Inc.
468-P
MACE Associated With Pioglitazone: A Meta-Analysis of Random-
ized, Controlled Trials
ALFONSO PEREZ, ERIC SONG, ANTHONY EDMONDS, Deereld, IL
The benet of pioglitazone (PIO) with respect to major adverse cardiovas-
cular (CV) events (MACE) has been shown in previous studies. This analy-
sis evaluated risk of MACE (primary; composite of CV death and nonfatal
myocardial infarction or stroke) and serious CHF (secondary) in patients
with type 2 diabetes mellitus (T2DM) receiving PIO (n=12,506) vs placebo
or active control (n=10,212) in all 36 randomized controlled trials in the PIO
clinical database. Treatment durations were 4 to 42 months. Events were
identied by searching preferred terms of adverse events identied through
standard safety monitoring; in 4 studies, pre-specied central adjudication
was performed. The primary endpoint was time from rst dose to rst event;
hazard ratios (HRs) and 95% CIs for events on PIO vs comparator (COMP)
were calculated using a Cox proportional-hazards model stratied by study
with treatment as the independent variable. Kaplan-Meier estimates were
also produced. The primary analysis showed a statistically signicant reduc-
tion in risk of MACE with PIO vs COMP (HR, 0.82; Table), and Kaplan-Meier
plots showed shorter time to rst event with COMP. Secondary analyses
as well as sensitivity analyses (also in the Table) showed similar results.
The analysis also conrmed the known CHF risk of pioglitazone (HR for CHF
causing hospitalization, 1.41; 95% CI, 1.15-1.74), with no corresponding in-
crease in mortality. This analysis supports the CV safety of PIO with respect
to standard MACE endpoints; PIO consistently showed favorable CV out-
comes compared with placebo and active control in T2DM, independent of
background CV risk.
MACE in Pioglitazone Trials: Comparison between Pioglitazone and Active or
Placebo Control
Analysis Study Set N HR (95% CI) for MACE
(PIO vs COMP)
Primary analysis All controlled studies 22,718 0.82 (0.71, 0.95)
Secondary analyses Double-blind
controlled studies
22,131 0.82 (0.71, 0.95)
Controlled studies
excluding PROactive
17,480 0.82 (0.62, 1.09)
PROactive study 5238 0.82 (0.70, 0.97)
Sensitivity analyses
(all controlled studies)
Excluding events >30
days after last dose
22,718 0.83 (0.72, 0.97)
Excluding studies
with no MACE
20,569 0.82 (0.71, 0.95)
Supported by: Takeda Global Research and Development Center, Inc.
469-P
P53 Silenced Endothelial Progenitor Stem Cells (EPC) Improve Col-
lateral Circulation Post Femoral Artery Occlusion in Diabetic Mice
SABYASACHI SEN, MARY YOUNG, CYRIL CHOU, BROOKE BENTLEY, JOSEPH
JERRY, Springeld, MA, Amherst, MA
Literature shows that EPCs contribute to increased collateral vessel for-
mation following vaso-occlusion. However diabetes reduces EPC number and
reduces collateral vessel formation. We cultured human EPCs and exposed
them to 5.5 mM (equivalent to 99mg%) and 20mM (equivalent to 360mg%,
HG) glucose, which resulted in signicant EPC death within 48hrs. We noted
HG exposure was associated with up-regulation of P53 and its downstream
genes such as P21, PUMA and Caspase-3. We hypothesized that EPC reduc-
tion in hyperglycemia is secondary to up-regulation of pro-apoptotic gene,
P53. We obtained mouse peripheral blood derived EPCs from P53 null mice
and observed that p53 KO EPCs are more resistant to cell death in HG. P53
null EPCs evolved into mature mouse EC (MEC) and retained endothelial
properties such as cobblestone appearance and tube-formation on matrigel.
Next, we used Lenti and Adenovirus mediated si-RNA methods to silence
P53 (long and short term respectively) in human EPCs and P53 silenced EPc
showed better survival in HG. We developed femoral artery occlusion model
to mimic peripheral vascular disease in diabetic animals. We used strepto-
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zotocin induced type 1 diabetic or Leptin resistant type 2 diabetic mouse
model. We delivered saline, P53 WT and P53 null EPCs intra-muscularly
around the femoral occlusion in these mouse models (n=10 in each group)
and counted CD31+ve number of capillaries in a 20x microscopic eld. High-
est number was noted in the group that received P53null EPCs ( 26.34.2,
49.67.4 and 95.79.7 respectively). Summary: Transplantation of P53 null
EPCs in hyperglycemic mice with femoral occlusion demonstrate better col-
lateral vessel formation post femoral artery occlusion setting compared to
WT-EPC transplanted group. Conclusion: This nding indicates towards pos-
sible therapeutic benet in transplanting short-term P53 silenced human EPC
to prevent peripheral vascular disease in patients with diabetes.
Supported by: Collaborative Biomedical Research Fund
470-P
Effects of Amaryl on Aquaporin-1 Expression in Diabetic Rats
YADONG SUN, HAIYING WANG, Changchun, China
Role of inammation in the pathogenesis of diabetic cardiomyopathy is
concerned recently. Inammatory reaction is increased by obesity resulting
from excessive intake of nutrient in the early stage of type 2 diabetes. In-
ammatory cytokines in conjunction with other harming factors of diabetes,
by causing oxidative stress and cell death, have a bad effect on the cardio-
vascular system generally and locally. There is an urgent need to develop a
hypoglycemic agent with protective effects on cardiovascular system. Ama-
ryl, an agent of glimepiride, has excellent glycemic controls, and decrease
insulin resistance, even in nearly diagnosed type 2 diabetic patients.Studies
have shown that inammatory cytokines such as tumor necrosis factor-o and
IFN- have increased the expressing of aquaporin (AQP). AQPs are a series of
intrinsic homologous membrane proteins related to transporting water, and
it has been conrmed that a variety of AQPs are expressed in myocardial
tissue, including AQP1.After establishment of type 2 diabetes model induced
by combined high sugar and high fat diet with low-dose streptozotocin (STZ)
intraperitoneal injection, the 25 diabetes rats were randomly divided into
three groups: low dose Amaryl (2mg/kg, i.g., once daily), high dose Amaryl
(4mg/kg, i.g. once daily), model group (an equal volume of normal saline, i.g.,
once daily). After 8 weeks, levels of serum cardiac enzyms and HbA1c were
assayed, and the expression of AQP1 in myocardial tissue was evaluated by
immuno-histochemistry and Western bolt individually. Compared with the
model group, the levels of cardiac enzymes and HbA1c were signicant de-
creased in treatment groups (P<0.01), the expression of AQP1 proteins was
increased in low dose group (P<0.05) and signicantly increased in high dose
group (P<0.01).As relation above, we concluded that Amaryl attenuates the
occurrence and development of diabetic cardiomyopathy, and its mechanism
may be related to stabling the expression of AQP1 in myocardial tissue.
471-P
Type 2 Diabetes and the Progression of Visualized Coronary Athero-
sclerosis to Clinical Cardiovascular Events
HEINZ DREXEL, ALEXANDER VONBANK, PHILIPP REIN, KURT HUBER, CHRIS-
TOPH H. SAELY, Feldkirch, Austria, Triesen, Liechtenstein, Vienna, Austria
We aimed at prospectively evaluating to what extent pre-existing coro-
nary artery disease (CAD) accounts for the increased long-term vascular
event risk of patients with type 2 diabetes (T2DM).Vascular events were
recorded over 8 years in 750 consecutive patients whose baseline CAD
state was veried angiographically.The baseline prevalence of CAD (87.8%
vs. 80.4%; p=0.029) and of signicant coronary stenoses ~50% (69.5% vs.
58.4%; p=0.010) as well as the extent of CAD, dened as the number of
signicant coronary stenoses (1.71.6 vs. 1.41.5; p=0.014) were higher in
patients with T2DM (n=164) than in non-diabetic subjects. During follow-
up, T2DM and CAD proved to be mutually independent predictors of vas-
cular events: T2DM predicted vascular events (n=257) independently from
the presence and extent of baseline CAD (hazard ratio (HR) 1.36 [1.03-1.81];
p=0.032); conversely, the presence and extent of baseline CAD predicted
vascular events independently from T2DM (HRs 3.29 [1.93-5.64]; p<0.001
and 1.37 [1.23-1.53]; p<0.001, respectively). However, the overall risk in-
crease conferred by T2DM was driven by an extremely high 53.3% event
rate of patients with both T2DM and signicant CAD at baseline; individuals
with T2DM but without signicant baseline CAD showed a signicantly low-
er event rate (22.0%; p<0.001).We conclude that T2DM and angiographically
visualized coronary atherosclerosis are mutually independent predictors of
vascular events. However, the overall risk increase conferred by T2DM is
driven by accelerated progression of pre-existing atherosclerosis to clini-
cal cardiovascular events, whereas vascular risk is much lower in diabetic
patients without pre-existing signicant CAD.
Supported by: Jubilumsfonds der sterreichischen National Bank
472-P
Cardiovascular Dysfunction in Newly Diagnosed Pre-Diabetic
Subjects
JUNPING CHEN, DONGXU FU, MINGYUAN WU, JULIE A. STONER, TIMOTHY J.
LYONS, Oklahoma City, OK
Cardiovascular dysfunction (CVD) is responsible for a majority of morbidity
and mortality in diabetes, and is thought to commence in the pre-diabetic
stage. We hypothesized that early cardiovascular dysfunction is present
in pre-diabetes and is associated with inammation and accumulation of
advanced glycation end products (AGEs). In a cross-sectional study, 131 sub-
jects were classied by 2-hour 75-g oral glucose tolerance tests into pre-
diabetic (n=73, 12M/61F) and normal glucose tolerant (NGT, n=58, 8M/50F)
groups. Cardiovascular function was assessed non-invasively by the pulse
wave analysis (PWA). Fasting serum CRP, TNF-alpha, IL-1-alpha, adiponec-
tin, leptin, VCAM-1, and ICAM-1 were analyzed by ELISA assays. Skin AGE
content was determined non-invasively by an investigational device (Scout,
Veralight, Inc.) which scans skin AGE-related autouorescence to provide a
SCOUT score. Means were compared between groups using a two sample
t-test, correlations were quantied using Pearsons correlation coefcient
for unadjusted analyses, and partial correlation coefcients were calculated
for adjusted analyses. Compared to NGT, newly diagnosed pre-diabetic sub-
jects exhibited signicantly lower large and small artery elasticity indexes
(P=0.002 and 0.03), and higher pulse pressure (p=0.004) and total vascular
impedance (p=0.01). In addition, they had signicantly higher SCOUT scores
(p=0.006); higher serum CRP (p=0.01) and TNF-alpha (p=0.01), and lower adi-
ponectin (p=0.01) and IL-1alpha (p=0.06). SCOUT score and serum levels of
CRP, leptin, and adiponectin were signicantly correlated with certain pa-
rameters from PWA with and without adjustment for age, BMI, and gender.
Newly diagnosed pre-diabetic subjects exhibited abnormal cardiovascular
function and pro-inammatory status. The associations of AGEs and the
tested pro-inammatory markers with cardiovascular dysfunction suggest
these factors may be implicated in the early pathogenesis of the disease.
Supported by: Talley Research Award and OCAST Award
473-P
Metabolic Predictors of Ischemic Heart Disease and Cerebrovascu-
lar Attack in Elderly Diabetic Individuals: The Roles of HDL-Choles-
terol and the LDL-C/HDL-C Ratio
TOSHIO HAYASHI, HIDEKI NOMURA, KOICHIRO INA, HIROSHI WATANABE,
KOUTARO YOKOTE, KOUTARO YOKOTE, TAKASHI OHRUI, JAPAN CDM GROUP,
Nagoya, Japan, Hamamatsu, Japan, Chiba, Japan, Sendai, Japan
Objectives: Lipids, especially LDL-cholesterol (LDL-C) , are risk factors for
ischemic heart disease (IHD) in middle aged diabetic individuals, however it
is not wellknown what predicts IHD and cerebrovascular attack (CVA) in older
patients.Research Design and Methods: This study was designed as a pro-
spective cohort study (Japan Cholesterol and Diabetes Mellitus Study) with
4,014 type2 diabetic patients (1,936 women; 67.49.5y.o., n=1261<65,1731
from 65 to 74 and 1016>75y.o.; disease duration 9.68.0yrs.). The levels of lip-
ids, glucose, and other factors like blood pressure were investigated relative to
IHD or CVA by stepwise and multiple logistic regression. Results: 153IHD and
104CVA (7.8 and 5.7/ thousand people year) occurred over 5.5-year. The risk
factors for IHD and CVA in elderly are different from those in younger. Namely,
lower HDL-cholesterol (HDL-C) was related to IHD in patients>=75 y.o. (odds
ratio (OR):0.953, P<0.02), compared to higher hemoglobinA1C (HbA1C) and LDL-
C in subjects<65 y.o. (p<0.05) and LDL-C/HDL-C in patients<74 y.o. (n=2992,
P=0.006). HDL-C was also related to CVAs in patients >=75y.o. (OR;0.852,
p=0.021). Kaplan-Meier estimator curves showed that IHD was signicantly
frequent in patients <65 y.o. in highest quartile for L/H ratio while in patients
>=75 y.o. in lowest quartile for HDL. CVA was frequent in patient>=75 y.o. in
lowest quartiles for HDL-C compared with other groups.Conclusion:IHD and
CVA in elderly diabetic patients are preditcted by HDL-C. While, LDL-C and
HbA1C are risk for IHD in non-elderly, and LDL-C/HDL-C may represent both
effects of LDL-C and HDL-C. These differences in risk by age are important for
an individualized strategy to prevent atherosclerotic diseases.
Supported by: Japanese Ministry of Health, Welfare and Labor
474-P
Serum Cystatin C is not Associated With Arterial Stiffness in Type 2
Diabetes Patients With Normal Renal Function
HYEONGKYU PARK, SEOKCHUN YEOM, YEOJOO KIM, DONGWON BYUN, KYOIL
SUH, MYUNGHI YOO, Seoul, Republic of Korea
Cardiovascular disease (CVD) is the main cause of mortality in patients
with type 2 diabetes (T2DM). Several studies showed that increased ar-
terial stiffness is an independent risk factor for CVD. Pulse wave velocity
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(PWV) is known as a marker for large vessel stiffness. Recent studies show
that serum cystatin C, a marker of renal function, is associated with PWV
and may predict future cardiovascular events, even in subjects with normal
renal function. However, there have been few studies, especially in Asian
populations, for the relationship between cystatin C and arterial stiffness in
patients with T2DM. In this study, we investigated the relationship between
serum cystatin C and aortic PWV in T2DM patients with normal renal func-
tion. Patients with urinary albumin/creatinine ratio(ACR) higher than 300 ug
albumin/mg creatinine, estimated glomerular ltration rate less than 60 ml/
min or systemic infection were excluded. A total of 124 patients(64 M/60 F,
age 60 1 yr, ACR 35 5 ug/mg) were included. Doppler-derived aortic PWV,
serum cystatin C, leptin, adiponectin, and resistin were measured. Cystatin
C is signicantly related to age(r=0.48, P < 0.001), leptin(r=0.24, P <0.05),
HDL cholesterol (r= -0.20, P < 0.05), and lipoprotein(a) (r=0.21, P < 0.05). Aor-
tic PWV is signicantly associated with age (r=0.28, P < 0.01), leptin(r=0.21,
P <0.05), and cystatin C(r=0.21, P < 0.05). In multiple regression analysis,
however, there is no signicant association between aortic PWV and serum
cystatin C levels. In summary, serum cystatin C is not associated with arte-
rial stiffness in T2DM patients with normal renal function.
475-P
Reduced Vasodilation and Enhanced Vasoconstriction of Renal Ar-
teries in Insulin Resistance Rats Induced by High-Sucrose Diet
YU GAO, GUANGYAO SONG, HUIJUAN MA, Chengde, China, Shijiazhuang, China
The aim of this study was to investigate the effects of long-term high
sucrose diet on relaxation and contraction of the renal arteries in insulin re-
sistance (IR) rat. Wistar rats were fed by chow diet(control)and high-sucrose
diet (HS) (n=14 in each group) respectively. IR was graded by glucose infu-
sion rate (GIR) using hyperinsulinemic euglycemic clamp technique. Blood
pressure, body weight (BW), plasma triglycemia (TG), free fatty acid (FFA),
insulin (INS), fasting blood glucose (FBG), endothelin-1 (ET-1) and nitric oxide
metabolite (NO2-/NO3-) were determined at 12 and 24 weeks. Arterial con-
tractility was evaluated by concentration-response curves to 10nM-100uM
noradrenaline. High-sucrose diet caused moderate rise in blood pressure at
12 and 24 weeks (P<0.01), accompanied by increase in plasma lipids and de-
crease of whole body insulin sensitivity. A reduction in endothelium-depen-
dent vasorelaxation of renal arteries in HS rats was observed at 12 and 24
weeks (P<0.01), while the contraction responses of renal arteries responded
to cumulative dose of noradrenaline was increased. No signicant differ-
ence on endothelial-independent max vasorelaxation to sodium nitroprus-
side between control and HS groups was observed. This study suggests that
high-sucrose diet can lead to dyslipidemia, increased IR and hypertension re-
sulted from decreased endothelial-dependent vasorelaxation and increased
cumulative dose contraction.
Table 1. Body weight and biochemistry in control and HS rats at 12
th
and 24
th
week after high-sucrose diet.
Time Group BW FBG TG INS FFA NO
2
-/ NO
3
- ET-1
(g) (mmol/L) (mmol/L) (mU/L) (mmol/lL) (umol/L) (pg/mL)
12w Normal
chow diet
401.0022.40 5.280.40 0.740.23 20.672.07 0.760. 07 68.334.58 212.4316.58
High-sucrose 408.3311.69 6.530.29
A
0.820.20 23.151.61 0.970.13* 48.265.61
A
245.2837.87
A
24w Normal
chow diet
450.0060.39 5.260.34 0.950.19 20.811.79 0.780.07 64.7915.46 231.8620.33
High-sucrose 480.7119.88
A
6.900.17
A
1.380.30
A
36.334.76
A
1.170.16
A
39.335.94
A
274.7924.53
A
*
p <0.05 ,
A
p <0.01 vs. control.
476-P
Aortic Stiffness is Associated With White Matter Integrity in Type
1 Diabetes Mellitus
LINDA D. VAN SCHINKEL, NATHANJA TJEERDEMA, JEROEN VAN DER GROND,
ALBERT DE ROOS, JOHANNES W. SMIT, Leiden, The Netherlands
Diabetes mellitus type 1 (T1DM) is associated with white matter injury.
Aortic stiffness may contribute to white matter damage in those patients.
Diffusion tensor imaging (DTI) is a MRI marker of early white matter disease.
Our purpose was to assess the association between aortic pulse wave ve-
locity (PWV), as a marker of aortic stiffness, and white matter brain integrity
in patients with T1DM, using MRI techniques.Forty-two T1DM patients (23
men, mean age 4412 years, mean DM duration 2413 years) and 17 age
and gender matched healthy controls were included. Aortic PWV was as-
sessed with a 1.5 Tesla MRI. Brain DTI measurements were performed on
a 3 Tesla MRI. Fractional anisotropy (FA) and apparent diffusion coefcient
(ADC) were calculated for white and gray matter integrity. Multivariable lin-
ear regression analyses including cardiovascular risk factors as covariates
were used for statisticsWhite matter FA was increased and white matter
ADC was decreased in T1DM compared to the control group (p=0.019 for FA
resp. p=0.046 for ADC). No differences in gray matter FA or ADC between
T1DM and controls were observed. Multivariable linear regression analyses
revealed aortic PWV as an independent predictor for white matter integrity
(Beta=-0.920 p=0.001 for FA resp. Beta=0.662 p=0.030 for ADC) in T1DM
patients. This effect was independent of age, gender, MAP, BMI, smoking,
duration of diabetes and HbA1c levels. Aortic PWV did not predict gray mat-
ter integrity.In conclusion: aortic stiffness is an independent predictor of
white matter integrity in patients with DM1.
Supported by: The Netherlands Heart Foundation (Project UL 2009-4548)
477-P
Cardiovascular Event Rates in Adults With Type 2 Diabetes Melli-
tus: SHIELD 5-Year Perspective
SANDRA J. LEWIS, KATHLEEN M. FOX, ELISE HARDY, SUSAN GRANDY, SHIELD
STUDY GROUP, Portland, OR, Monkton, MD, Wilmington, DE
Macrovascular complications of type 2 diabetes mellitus (T2DM) include
myocardial infarction (MI), stroke, and coronary revascularization. This study
ascertained the self-reported incidence rates of cardiovascular disease
(CVD) events over 5 years among adults with and without T2DM. Respon-
dents to the US Study to Help Improve Early evaluation and management
of risk factors Leading to Diabetes (SHIELD) surveys reported at baseline
(2004) whether they had ever been told by a healthcare professional they
had a heart attack, stroke, heart bypass surgery, angioplasty, or stents. In
the subsequent 5 years, any new CVD events (incident event[s] reported
subsequent to baseline) reported by T2DM respondents and those without
diabetes were captured.There were 2,305 T2DM respondents and 7,207 re-
spondents without diabetes who reported no prior history of CVD events
at baseline. A greater proportion of T2DM respondents had at least 1 inci-
dent CVD event over 5 years, compared with respondents without diabetes
(16.7% vs. 10.3%, p <0.0001). In addition, 32.8% of T2DM respondents and
31.4% of respondents without diabetes had multiple CVD events (2 or more)
over 5 years (p = 0.69). Among T2DM respondents with a prior history of CVD
events (n = 898), 47.7% reported a new CVD event (different from baseline
event) over the subsequent 5 years. For those without diabetes but with a
prior history of CVD events (n = 1,744), 45.6% reported a new CVD event over
5 years (p = 0.33 in comparison with T2DM group). For those with a prior his-
tory of CVD events, 23.6% of T2DM respondents and 21.8% of respondents
without diabetes had multiple CVD events over 5 years (p = 0.51). There is
a high prevalence of self-reported CVD events in respondents with T2DM.
For T2DM respondents without a prior CVD history, there was a signicantly
higher incidence rate (62% relative increase), compared with those without
diabetes and no prior history of CVD events. This study is consistent with
previous reports suggesting that T2DM is a CVD risk equivalent.
478-P
Blood Glucose Improvement Ameliorates Nocturnal Oxygen De-
saturations in Type 2 Diabetic Patients
ALBERT LECUBE, ANDREEA CIUDIN, GABRIEL SAMPOL, SILVIA VALLADARES,
JORDI MESA, CRISTINA HERNNDEZ, RAFAEL SIM, Barcelona, Spain
There is growing evidence suggesting an association between type 2
diabetes and sleep apnea syndrome. However, there are no studies to deter-
mine whether blood glucose control could prevent nocturnal arterial oxygen
desaturation. For this purpose we designed a case-control between 30 type
2 diabetic patients and 10 non-diabetic subjects closely matched by age,
gender, and BMI who where admitted to our Diabetes Unit. Nocturnal oxim-
etry was assessed with a pulse oximeter (Model 3100
; Nonin Medical Inc,

Plymouth, MN USA) at baseline and 5-days after improving blood glucose
control. A desaturation event was considered when the haemoglobin satura-
tion level (SaO2) fell bellow 3%, 4%, and 6% from baseline saturation. The
total number of desaturations was divided by the hours in bed and oxygen
desaturation index per hour (ODI) was obtained. Type 2 diabetic patients
showed a higher percentage of ODI-3%, ODI-4%, and ODI-6% in comparison
with non-diabetic subjects at baseline. Signicant positive correlations be-
tween fasting plasma glucose and HbA1C with pulse oximeter parameters
were detected. In addition, multiple linear regression analyses showed that
HbA1c was independently associated with ODI-3%, 4%, and 6% at baseline.
Finally, a signicant reduction in HbA1c (10.2 1.9 at baseline vs. 9.9 1.9%
at discharge, p<0.001) and fructosamine (320.1 73.6 vs. 304.3 59.6 mg/
dl, p = 0.005) were associated with a signicant reduction in ODI-3% (39.8
27.5 at baseline vs. 32.5 27.5 events/hour at discharge, p<0.001), ODI-4%
(28.5 23.7 vs. 23.5 24.2 events/h, p<0.001), and ODI-6% (15.1 16.8 vs.
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COMPLICATIONSMACROVASCULARCELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES
12.9 17.3 events/h, p=0.009) in diabetic patients. However, no changes
in ODI events were observed in non-diabetic patients after 5-days of fol-
low-up. Therefore, glycemic control is related to nocturnal arterial oxygen
desaturation in type 2 diabetes. Our results suggest that improvement in
nocturnal arterial oxygen desaturation can be added to the benecial ef-
fects of metabolic optimization.
479-P
Impact of CVD History and GXT Abnormalities on Weight Loss and
Fitness Gain
JEFFREY M. CURTIS, TINA KILLEAN, GEORGE A. BRAY, LAWRENCE J. CHESKIN,
EDWARD S. HORTON, KAREN C. JOHNSON, JOHN M. JAKICIC, F. XAVIER PI-
SUNYER, JUDITH G. REGENSTEINER, PAUL M. RIBISL, LYNNE WAGENKNECHT,
MARK A. ESPELAND, Phoenix, AZ, Shiprock, NM, Baton Rouge, LA, Baltimore, MD,
Boston, MA, Memphis, TN, Pittsburgh, PA, New York, NY, Aurora, CO, Winston-
Salem, NC
Whether a history of cardiovascular disease (CVD) or an abnormal graded
exercise test (GXT) inuences the capacity to lose weight or improve tness
is unknown.Look AHEAD is a multicenter randomized trial of a behavioral
weight loss intervention (ILI) in overweight or obese adults with type 2 dia-
betes (T2D), aged 45-76 years. Participants underwent a maximal GXT at
baseline and submaximal GXTs one and four years later. This analysis in-
cludes only ILI participants with follow-up data (n=2514, 97.8% of ILI group).
At study entry 356 participants reported a history of CVD events (including
myocardial infarction, stroke, coronary bypass, etc.). Baseline maximal GXTs
were deemed abnormal by angina, ST segment changes, ventricular arrhyth-
mia (VA), poor heart rate recovery (HRR), or peak workload < 5 METs; 491 had
abnormal baseline GXTs. We compared those with and without a history of
CVD or abnormal baseline GXT with regard to weight loss and tness gains
over 4 years of Look AHEAD intervention.Those without a history of CVD
events (p=0.02), lost more weight (6.7%) than those with such history (6.0%).
Age- and sex-adjusted weight losses, were similar for those with (6.8%) and
without (6.5%) abnormal baseline GXT. However, by specic GXT abnormal-
ity, those with VA (n=6, 12.4 vs. 6.6%, p=0.008) or angina (n=14, 10.3 vs.
6.6%, p=0.01) during exercise, or poor HRR (n=72, 7.9 vs. 6.5%, p=0.03) lost
more weight than those without.A history of a CVD event (0.43 vs 0.61 METs,
p=0.006) or low peak workload (n=247, 0.25 vs. 0.62 METs, p=0.01) was as-
sociated with lower tness gains. However, age-, sex-, and baseline tness-
adjusted tness gains were similar for those with and without abnormal
GXT (0.46 vs. 0.61 METs, p=0.89), and better for those with than without VA
(1.47 vs. 0.58 METs, p=0.04).A history of CVD events or low peak workload
on maximal GXT appear to hamper tness gains, but VA is associated with
better tness gains. A history of CVD may hinder weight loss, whereas ab-
normalities on the GXT (VA, angina, or abnormal HRR) do not.
480-P
Associations of Fetuin-A Levels With Insulin Resistance and Vascu-
lar Complications in Type 2 Diabetic Patients
CHAN-HEE JUNG, BO-YEON KIM, CHUL-HEE KIM, SUNG-KOO KANG, JI-OH
MOK, Bucheon, Republic of Korea
Contradictory results have been reported regarding the role of fetuin-A in
macroangiopathies. Moreover, there is few data available for associations
of fetuin-A with microangiopathies in T2DM. Therefore, we examined the
relationship between fetuin-A and macro- and microangiopathies including
cardiac autonomic neuropathy(CAN) in Korean T2DM patients. A total of 185
T2DM patients (119 males and 53 females) were recruited and evaluated for
diabetic nephropathy, retinopathy, peripheral neuropathy and CAN. Serum
fetuin-A levels were assessed by ELISA and the insulin resistance status
was assessed by the HOMA-IR. Atherosclerotic burden was assessed by
ankle-brachial index(ABI) and brachial-ankle pulse wave velocity(baPWV).
The mean levels of serum fetuin-A were 525 162 ug/ml and not differ-
ent between males and females. Serum fetuin-A levels showed signicant
positive correlations with HOMA-IR (r=0.196, p=0.022), baPWV (r=0.165,
p=0.036), systolic BP (r=0.165, p=0.036), total cholesterol (r=0.164, p=0.032),
triglycerides (r=0.215, p=0.006) and signicant negative correlations with
ABI (r=-0.183, p=0.019). Serum fetuin-A levels were also negatively corre-
lated with serum adiponectin (r=-0.187, p=0.027) and positively correlated
with serum TNF-alpha (r=0.204, p=0.016). The mean levels of HOMA-IR were
borderline signicantly increased progressively across fetuin-A tertiles (p
for trend=0.06). The mean levels of serum fetuin-A were not signicantly
different according to the presence of each microvascular complications.
This present study showed that levels of serum fetuin-A are signicantly as-
sociated with IR and arterial stiffness while there are no associations with
microangiopathies in T2DM patients. Future prospective studies with larger
numbers of patients are required to establish a direct relationship between
serum fetuin-A levels and the development or severity of diabetic vascular
complications.
481-P
COMPLICATIONSMACROVASCULAR
CELLULAR MECHANISMS OF ATHEROGENESIS
IN DIABETES
Guided Audio Tour: Diabetes and the Arterial WallFocus on the Endo-
thelium (Posters 482-P to 489-P), see page 13.
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Targeted Imaging of Myocardial Angiogenesis in Type-1 Diabetes
With Novel PET Radiopharmaceutical Demonstrates Impairment of
Alpha-v Integrin Activation
MATTHEW R. SCHUELKE, IWONA T. DOBRUCKI, SUZANNE LAPI, LAWRENCE W.
DOBRUCKI, Urbana, IL, St. Louis, MO
We have previously assessed temporal changes in peripheral angiogen-
esis in mice subjected to type-1 diabetes (DM). Peripheral angiogenesis in
DM was signicantly reduced as assessed by microSPECT-CT imaging using
Tc-99m labeled agent targeted at ov integrin. We hypothesize that a novel
PET radiopharmaceutical based on cRGD peptide structure can assess the
differences in chronic myocardial angiogenesis associated with DM.Surgical
ligation of LAD was performed on male non-DM (n=3) and DM Lewis rats
at 8 weeks after streptozotocin treatment (n=4, 50 mg/kg i.p., bolus). DM
rats demonstrated signicant weight loss and fasting glycemia (>400 mg/
dL). At two weeks post-ligation, Cu64-cRGDyK (~400uCi) was injected and
60min later in vivo contrast microCT followed by microPET imaging were
performed. Blood was collected for measurement of glucose and plasma
insulin (assessed with ELISA kit). After imaging, rats were euthanized and
organs collected for biodistribution studies. Hearts were excised, cleaned
with ice-cold saline and imaged postmortem with a hybrid microPET-CT
scanner. After imaging, hearts were cut into 2-mm short axis slices and
anterior, septal, posterior, and lateral segments for gamma well counting
(GWC) of Cu64 activity.In DM rats, glucose levels were chronically elevated,
and insulin levels signicantly reduced (p<0.05). Non-invasive microPET-CT
imaging with Cu64-cRGDyK and GWC analysis demonstrated a signicant
(p<0.05) impairment of ov integrin activation in all myocardial segments of
DM rats.A novel Cu64-cRGDyK agent targeted at activated ov integrin pro-
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vided favorable distribution for PET-CT imaging of myocardial angiogenesis
which appears to be impaired in the onset of uncontrolled DM. This targeted
ov integrin imaging strategy may allow for monitoring of therapeutic inter-
ventions directed at stimulation angiogenesis early post-MI.
Supported by: AHA (10SDG4180043)
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483-P
Ablation of PDK1 in Vascular Endothelial Cells in Diabetic Condition
Deteriorates Vascularity Only in Skeletal Muscles But Not in Liver
KAZUHITO TAWARAMOTO, MITSURU HASHIRAMOTO, TOMOHIKO KIMURA,
HIDENORI HIRUKAWA, MASASHI SHIMODA, WATARU OGAWA, MASATO KA-
SUGA, KOHEI KAKU, Okayama, Japan, Hyogo, Japan, Tokyo, Japan
We previously reported that the vascular endothelial cell-specic PDK1
knockout (KO) mice with diabetes deteriorated systemic insulin sensitivity
due to lowered vascularization in the skeletal muscle. In contrast, the he-
patic insulin sensitivity was not deteriorated, whereby we hypothesized that
vascularization in the liver is intact in KO mice compared to the control. KO
and control mice (Con) at 6 weeks of age were injected intraperitoneally
with streptozotocin (STZ), 150g/kgBW, or vehicle (Veh). As results, vascu-
larization in gastrocnemius muscle (Gastro) and the liver was not different
between Veh-treated Con and KO mice (Liver; Veh-Con 0.5280.145m
2
vs.
Veh-KO 0.8040.141m
2
, n.s., Gastro; Veh-Con 0.4160.049m
2
vs. Veh-KO
0.4610.045m
2
, n.s.). In STZ-injected group, however, vascularization in
Gastro was expectedly lowered in KO (Veh-KO 0.4610.045m
2
vs. STZ-KO
0.3230.035m
2
, p<0.05), but not in Con (Veh-Con 0.4160.049m
2
vs. STZ-
Con 0.4530.045 m
2
, n.s.). On the other hand, vascularization in the liver
was not signicantly different between STZ-Con and STZ-KO. Protein ex-
pression of vascular cell adhesion molecule 1 (VCAM1), located in PI3K signal
and regulated by PDK1, was signicantly up-regulated by 2.4 times higher
in Gastro of STZ-KO than in that of Veh-KO, but in the liver, no signicant
difference was observed between two groups. Vascular endothelial growth
factors (VEGF), VEGF receptor (VEGFR), and hypoxia induced factor 1 (HIF1)
gene expressions in both Gastro and liver were not different between STZ-
KO and Veh-KO. The present results indicated that ablation of PDK1 in vascu-
lar endothelial cells in diabetic state induces the lower vascularization and
VCAM1-upregulation in skeletal muscle but not in liver, and the underlying
mechanism may be totally different from the reported angiogenic factors,
including VEGF and HIF1.
&
484-P
Glucagon-Like-Peptide-1 (GLP-1) Activates AMPK and Diminishes
Inammation in Human Aortic Endothelial Cells
NADIA M. KRASNER, JOSE M. CACICEDO, YASUO IDO, NEIL B. RUDERMAN,
Boston, MA
GLP-1 therapy is prescribed for type 2 diabetes treatment due to its ef-
fects on insulin sensitivity and secretion, glucagon secretion, and food in-
take, resulting in improved glycemic control. Recent epidemiological studies
also suggest that it may have cardio- and cerebrovascular benets; however,
the mechanisms responsible for them are not known. AMP- activated pro-
tein kinase (AMPK) has been implicated as a therapeutic target for the pre-
vention of cardiovascular disease by virtue of its effects on lipid metabolism,
inammation and other atherogenic events in vascular cells. In the present
study, we examined whether GLP-1 activates AMPK and affects inamma-
tion in human aortic endothelial cells (HAECs). We found that GLP-1 (0.3nM)
transiently activates AMPK in HAECs, with phosphorylation of its substrate
acetyl-CoA carboxylase (ACC), peaking between 5-15min, and activation
of CREB by 20min. GLP-1 also signicantly decreased the inammatory re-
sponse (indicated by increased expression of adhesion factors; VCAM-1,
ICAM and TNFa) caused by hyperglycemia (HG, 25mM), lipopolysaccharide
(LPS, 1ug/mL) or TNFo (10ug/mL). Treatment of HAECs with LPS or TNFo
also increased monocyte adhesion and this too was diminished by GLP-1.
RT-PCR revealed that HAECs also express dipeptidyl peptidase-4 (DPP4),
the primary enzyme responsible for GLP-1 degradation. Incubation with HG
increased HAEC DPP4 activity, as well as GLP-1 degradation, potentially ex-
plaining the reduced level of GLP-1 in diabetic patients. The results suggest
that the benecial effects of GLP-1 on atherosclerotic risk could be medi-
ated by AMPK activation and the resulting decrease in inammation. Further
studies are needed to determine if the sustained effect of GLP-1 is attribut-
able to AMPK, and if so, whether it works by acute effects on metabolism
and/or the regulators of transcriptional activation.
&
485-P
Circulating Microparticles From db/db Diabetic Mice Induce Dys-
function and Apoptosis to Cerebral Endothelial Cells
JI CHEN, CHENG ZHANG, PENG LIU, SHUZHEN CHEN, ZHEN YAO, YANFANG
CHEN, Dayton, OH, Sanya, China
Type-2 diabetes is a well known risk factor for cerebral vascular diseases.
Our previous study showed that circulating microparticles (MPs) of db/db
diabetic mice are high in number and cause dysfunction to endothelial pro-
genitor cells. This study was designed to investigate whether circulating
MPs from db/db mice have direct effects on cerebrovascular endothelial cell
(cECs). Adult (9-12 weeks) male db/db and their age-matched control (db/+)
mice were used for the study. Circulating MPs were isolated from db/db
(dMPs) and db/+ mice (cMPs). Primary cECs were cultured from db/+ mice.
The 3-5 passages of cECs were used for pre-incubation with dMPs, cMPs (5
x 10
5
/ml) or vehicle (culture medium) for 48 hrs. The tube formation ability of
cECs was determined using a commercial assay kit. Cell apoptosis was ana-
lyzed by ow cytometric method. The expression levels of endothelial nitric
oxygen synthase (eNOS) and zonula occludens protein-1 (ZO-1) were mea-
sured by western blot. Caspase-3 activity was determined by uorogenic
substrate assay. Results showed: 1). Pre-incubation with dMPs decreased
the ability of tube formation (15.8 0.6, 29.6 2.2 and 32.6 3.2 tubes/eld,
n=5/group, P<0.01, dMPs vs. cMPs or vehicle) and increased the rate of cell
apoptosis (20.6 1.2%, 6.2 0.4% and 5.6 0.2%, n=5/group, P<0.01, dMPs
vs. cMPs or vehicle) in cECs; 2) Pre-incubation with dMPs down-regulated
eNOS and ZO-1 ( eNOS: 0.58 0.08, 1.09 0.08 and 1.00 0.05; ZO-1: 0.62
0.08, 1.08 0.07 and 1.02 0.06 fold of control, n=4/group, P<0.01, dMPs
vs. cMPs or vehicle) and up-regulated caspase-3 activity (1.1 0.1 nmol, 0.5
0.04 nmol and 0.4 0.02 nmol, n=4/group, P<0.01, dMPs vs. cMPs or vehicle)
in cECs. In conclusion, our data suggest that circulating MPs could be one of
the mechanisms which mediate cEC dysfunction and/or injury in diabetes.
&
486-P
Derivation of Induced Pluripotent Stem Cells from Patients With
Greater Than 50 Years Duration of Type 1 Diabetes
SHWETA BHATT, ADRIAN K. TEO, SUSANA SILVA, CHONG WEE LIEW, DAN
KAWAMORI, REBECCA WINDMUELLER, HILLARY KEENAN, GEORGE L. KING,
AMY J. WAGERS, ROHIT N. KULKARNI, Boston, MA
Type1 diabetes (T1D) is an autoimmune disorder characterized by the loss
of insulin-producing -cells. At Joslin, a cohort of patients who survived ~50
years of T1D (medalists), with or without complications presented a unique
opportunity to study mechanisms underlying the disease. The impetus for
the study came from observation that serum from several medalists tested
positive for C-peptide, and autopsied pancreases from 9 patients showed
insulin+ -cells, suggesting unique mechanism(s) protecting these -cells
and preventing some Medalists from developing complications. To inves-
tigate pathways protective towards the -cells and underlying mechanisms
for diabetic complications we obtained skin broblasts from medalists or
age-matched controls and subjected them to in vitro reprogramming using
excisable polycystronic lentivirus expressing OCT4, SOX2, KLF4 and c-MYC
to induce pluripotent stem cells (iPSCs). Although reprogramming was nor-
mal in both groups, evidenced by positive staining for early (Alkaline phos-
phatase), intermediate (OCT4, NANOG, SOX2, SSEA4) and late (Tra1-60 and
1-81) markers of pluripotency, we observed signicantly compromised ef-
ciency in establishing iPSCs from medalists, both with (0.002%) and with-
out (0.004%) complications, as compared to age-matched controls (0.02%)
[p<0.001; n=3-5]. Intriguingly, medalist iPSCs also exhibited striking morpho-
logical differences and reduced proliferation rates (20% of controls, n=5).
Further, iPSCs derived from medalists with complications demonstrated
impaired spontaneous differentiation potential (10%), unlike the iPSCs from
controls (90%) or from medalists without complications (70%) [p<0.001].
These data suggest that unique cellular mechanisms regulate the growth
and differentiation of iPSCs from medalists, alterations in which, through
genetic manipulation or in vitro disease modeling, may enable the discovery
of novel therapeutics to reduce the burden of T1D.
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487-P
Hyperglycemia Exacerbates Endothelial Cell Injury during Hypoxia/
Reoxygenation via p300-Mediated Persistent Epigenetic Regula-
tion of Egr-1 Gene
YAQIAN DUAN, BO ZHOU, YUHANG LIU, CHAO DU, HONG SU, Chongqing, China
Hyperglycemia is an independent risk factor of short- and long-term mor-
tality in acute myocardial infarction (AMI) patients with and without diabe-
tes. However, the mechanisms are not fully understood. We investigated
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whether p300, a transcriptional coactivator with histone acetyl transferase
(HAT) activity, mediates glucose-induced upregulation of transcription factor
early growth response gene (Egr)-1 and subsequent increase of vasoactive
factors during hypoxia/reoxygenation (H/R) in human umbilical vein endothe-
lial cells (HUVECs). A H/R injury model was established by exposing HUVECs
to 5h of oxygen and glucose deprivation, followed by 24h of reoxygenation
in different glucose concentrations. Hyperglycemia (25mM glucose) led to
up-regulated p300 expression and increased Egr-1 levels in H/R injured HU-
VECs. ChIP assay of the Egr-1 promoter in H/R injured HUVECs showed that
hyperglycemia promoted p300 binding and acetylation of histones H3 and
H4. Treatment with the p300 activity inhibitor, garcinol, or shRNA targeting
p300 caused obvious decreases in p300-Egr-1 promoter binding and Ac-H3
and Ac-H4 levels, ultimately resulting in down-regulation of Egr-1 expres-
sion. p300 inhibition also prevented the hyperglycemia-induced increase of
two Egr-1-activated genes: intercellular adhesion molecule 1 (ICAM-1) and
tissue factor (TF). Furthermore, during 24h of exposure to hyperglycemia, ac-
tive recruitment of p300 to the Egr-1 promoter was observed, accompanied
by increasing histone acetylation. Remarkably, these epigenetic changes
persisted during subsequent incubation at 5.5mM glucose for three days,
and were prevented by p300 inhibition.Our data suggest that p300 plays an
important role in hyperglycemia-induced endothelial cell injury during H/R.
Epigenetic histone modications may be a key mechanism that promotes the
progression of vascular damage in AMI patients, even after normoglycemia
is achieved.
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488-P
Endothelial Overexpression of PKC2 Accelerates the Develop-
ment of Atherosclerosis
QIAN LI, KYOUNGMIN PARK, CHENZHONG LI, CHRISTIAN RASK-MADSEN, AKI-
RA MIMA, GEORGE L. KING, Boston, MA
Endothelial dysfunction due to loss of insulin action on the endothelium
clearly can contribute to the risk of atherosclerosis. At the molecular level,
we have reported that hyperglycemia and elevated fatty acid can activate
several protein kinase c (PKC) isoforms and selectively inhibit insulins ac-
tivation of endothelial nitric oxide synthase (eNOS) via phophatidylinositol
3-kinase (PI3K)/Akt pathway. This study demonstrated the effect of PKC2
activation in the endothelial cells can increase endothelial dysfunction and
atherosclerosis in ApoE null mice. PKC2 isoform was overexpressed spe-
cically using endothelial promoter VE-Cadherin and crossed into ApoE null
mice (PKCEApoE). Being on high fat diet, PKCBEApoE null mice did not dif-
fer from ApoE control in weight, systemic insulin sensitivity, glucose toler-
ance, plasma lipid or blood pressure. However, the size and complexity of
the atherosclerotic lesions in the aorta increased by 70% as measured by en
face fat staining and plaque content of smooth muscle cells and extracellular
matrix. Insulin specic action on the endothelial cells and femoral artery
from the PKCEApoE null mice was impaired by 40% with respect to PI3K/
Akt/eNOS phsophorylation (activation) selectively, since the activation of
MAPKkinase was not effected. We have reported that angiotensin II (Ang
II) can activate PKC and phosphorylate Thr 86 of P85/PI3K to inhibit insulin
phosphorylation of eNOS. In PKCBEApoE null mice, the same phophorylai-
ton site on P85/PI3K was increased in endothelial cells. Infusion of Ang II
increased ET-1 expression in the aorta of PKCEApoE by 1.7 fold more than
ApoE null control. Thus, we propose that the acceleration of atherosclerosis
in diabetes and insulin resistance is due to loss of endothelial insulin action
caused by PKC activation and subsequent enhancement of angiotensins
atheogenic actions in the vessel wall.
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489-P
Endogenous Secretory Receptor for Advanced Glycation Endprod-
ucts (esRAGE) and AGEs-to-esRAGE Ratio as Biomarkers of Athero-
sclerosis Risk in Type 2 Diabetes
ZDENKA TURK, SPOMENKA LJUBI
C, JOZO BORAS, Zagreb, Croatia

Endogenous secretory receptor for advanced glycation endproducts (es-
RAGE) an isoform of soluble RAGE, acts as decoy for AGEs. We hypothesized
ratio of AGE-to-esRAGE could be link to atherosclerosis in diabetes. To
examine this issue, we compared serum levels of esRAGE and methylgly-
oxal-adduct as well as the AGE-to-esRAGE ratio between type 2 diabetic
patients with and without established macrovascular disease.Methylglyox-
al-adducts, AGEs and esRAGE were measured in type 2 diabetes (n=130) and
healthy controls (n=30). A history of macrovascular events (cardiovascular
disease, cerebral vasculopathy or peripheral vascular disease) was recorded
in 50 patients.esRAGE levels were lower in type 2 diabetic patients com-
pared with control subjects (0.3300.197 vs. 0.4580.074 ng/ml, p=0.003). In
diabetic population esRAGE correlated positively with MG-adducts, HbA1c,
BMI, and triglyceride, and inversely with CRP, LDL and homocystein. Mul-
tiple regression analysis was performed to determine which parameters
best predicted esRAGE level. Finally, urinary MG-adduct excretion (p<0.001),
AGEs (p<0.001), and AGE/RAGE (p<0.001, inversely) remained to be indepen-
dently associated circulatory esRAGE. Multiple stepwise regression model
was used to evaluate the relationship between macrovascular disease as a
dependent, and metabolic and AGE-parameters as independent variables.
Analysis pointed to LDL (=0.40 P<0.001), HbA1c (=0.37 P<0.001), AGE/es-
RAGE (=0.31 P=0.007) and homocysteine (=0.25 P=0.013) as signicant
independent contributors to diabetic macrovascular disease.High AGE/es-
RAGE ratio and esRAGE down-regulation observed in type 2 diabetes were
found to be independent predictors of macrovascular disease. A decreased
level of esRAGE may therefore be a biomarker of atherosclerosis acting as
either a decoy receptor or another mechanism.
490-P
491-P
Serum- and Glucocorticoid-Inducible Kinase-1 Increases Telom-
erase Activity and Delays the Onset of Endothelial Cell Senes-
cence
KATIA BASELLO, FRANCESCA FERRELLI, MARCO FELICE LOMBARDO, DONA-
TELLA PASTORE, BARBARA CAPUANI, ANDREA COPPOLA, ROBERTO ARRIGA,
SARA CARATELLI, FRANCESCA PACIFICI, GIULIA DONADEL, PAOLO SBRACCIA,
MASSIMO FEDERICI, DAVIDE LAURO, Rome, Italy
In Type 2 Diabetes (T2D) precocious endothelial cell senescence increases
the risk of developing atherosclerosis; telomeres shortening is a molecular
clock of ageing. Telomerase, a RNA-directed DNA polymerase, extends te-
lomeres length of eukaryotic chromosomes and human telomerase reverse
transcriptase (hTERT) is the catalytic subunit of the telomerase. In this study,
it has been examined whether Serum- and Glucocorticoid-inducible Kinase
(SGK)-1 can regulate cellular senescence in human umbilical vein endothelial
cells (HUVECs). SGK-1 is a serine threonine kinase that promotes cell survival
and reduces the intracellular levels of ROS. HUVECs have been infected with
SGK-1wt (full length), SGK-1A60 (deleted of the Nh2- 60aa) or the empty vec-
tor using a retroviral system. Telomerase activity, detected with TRAP as-
say, was found increased in HUVECs infected with SGK-1wt (p<0.001), after
eighth populations doubling level (PDL). Protein levels and phosphorylation
of hTERT and SGK-1 have been measured using Western Blot. Furthermore,
specic inhibition of SGK-1 with GSK 650394 completely prevented hTERT
phosphorylation at Ser
824
, with or without insulin stimulation. SGK-1 was
differently localized after infection of HUVECs with SGK-1A60 or SGK-1wt
constructs in cytoplasmic or nuclear compartments, respectively. Moreover,
the senescent status has been determined with SA--galactosidase in situ
staining, with a signicant decrease of senescent cells (p< 0.05) in HUVECs
infected with SGK-1wt after 14 PDL. These results demonstrate that over-
expression of SGK-1 delays the onset of senescence with a telomerase-
dependent mechanism. In conclusion, SGK-1 increases telomerase activity
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and reduces the beta-galactosidase activity. SGK-1 may have an anti-aging
effect that need further investigation and can be a new molecular target for
anti-aging therapy.
Supported by: Rome Foundation, Ministry of University 2009, Di Mario Foundation
492-P
Rho-Kinase Mediates ER Stress-Induced Endothelial Dysfunction
DAIJI KAWANAMI, KEIICHIRO MATOBA, RINA OKADA, MASAMI TSUKAMOTO,
JUN KINOSHITA, TOMOKO ITO, SHO ISHIZAWA, YASUSHI KANAZAWA, TAMOT-
SU YOKOTA, KAZUNORI UTSUNOMIYA, Tokyo, Japan
The process of atherosclerosis is affected by interactions among numer-
ous biological pathways including endoplasmic reticulum (ER) stress. C/EBP
homologous protein (CHOP), which induces cellular apoptosis in response
to ER stress, has been reported to promote atherosclerosis. Rho-kinase, an
effector of small G protein Rho, has been implicated in the pathogenesis of
atherosclerosis. However, its role in ER-stress mediated cellular response
remains unknown. In this study, we investigated the role of Rho-kinase in ER
stress-mediated endothelial dysfunction. Tunicamycin, an ER-stress inducer,
increased Rho-kinase activity in human umbilical vein endothelial cells. Tu-
nicamycin also increased CHOP mRNA expression. Interestingly, fasudil, a
specic Rho-kinase inhibitor, inhibited this induction. From a mechanistic
standpoint, we found that fasudil attenuated eukaryotic initiation factor
(eIF)2o phosphorylation and subsequent induction of activating transcrip-
tion factor (ATF)4 that are key factors to induce CHOP. It is shown that eIF2o
activates not only ATF4 but also NF-kB signaling pathway. Parthenolide, a
specic NF-kB inhibitor, attenuated tunicamycin-mediated vascular cellular
adhesion molecule 1 (VCAM-1) expression, suggesting that VCAM-1 induc-
tion by tunicamycin is NF-kB-dependent. We next used a variety of MAPK in-
hibitors to elucidate signaling pathway that is involved in VCAM-1 induction.
SB203580, a specic p38MAPK inhibitor, attenuated VCAM-1 induction.
Furthermore, fasudil inhibited tunicamycin-mediated p38MAPK phosphory-
lation, indicating that Rho-kinase mediates VCAM-1 induction via p38MAPK
signaling pathway under ER stress. These ndings demonstrate that Rho-
kinase plays a critical role in tunicamycin-mediated endothelial dysfunction
by activating eIF2o and subsequent ATF4 and NF-kB/p38MAPK-dependent
signaling pathways. We conclude that Rho-kinase is an attractive target
against atheroscelrosis by inhibiting ER stress-induced endothelial dysfunc-
tion.
493-P
Role of p66Shc in Cytokine-Mediated Leukocyte Recruitment by
Endothelial Cells
MAURA R. ORLANDO, LUIGI LAVIOLA, MARIA A. INCALZA, ANNA LEONARDINI,
ANGELO CIGNARELLI, FEDERICA TORTOSA, ROSSELLA LABARBUTA, MARI-
ANGELA MELCHIORRE, SABINA MARTEMUCCI, SEBASTIO PERRINI, ANNALISA
NATALICCHIO, FRANCESCO GIORGINO, Bari, Italy
Endothelial cells actively participate in vascular inammatory events by
regulating leukocyte recruitment via the expression of adhesion molecules.
The protein p66Shc has been proposed as a mediator of vascular dysfunc-
tion and a sensor of oxidative stress, and is upregulated in diabetes. The aim
of this work was to investigate the role of p66Shc in regulating leukocyte
recruitment by human umbilical vein endothelial cells (HUVEC). Exposure
of HUVEC to 50 ng/ml TNF-alpha resulted in increased phosphorylation of
p66Shc on Ser36 and intracellular ROS levels, and promoted leukocyte trans-
migration through the HUVEC monolayer (p<0.05). TNF-alpha also induced
a marked increase in the expression of the adhesion molecule E-Selectin
(p<0.05). Adenovirus-mediated overexpression of p66Shc in HUVEC resulted
in enhanced p66Shc phosphorylation on Ser36, increased ROS and E-Selec-
tin levels, and marked augmentation of leukocyte transmigration (p<0.05),
which appeared to be further increased when cells were exposed to TNF-
alpha. Conversely, overexpression of a phosphorylation-defective p66Shc
protein, in which Ser36 was replaced by Ala, did not augment ROS levels or
E-Selectin expression beyond those found in wild-type cells, and minimally
inuenced leukocyte transmigration. Moreover, silencing of p66Shc with
two different siRNAs markedly reduced E-Selectin expression, both under
basal conditions and following TNF-alpha exposure (p<0.05), and this was
associated with decreased leukocyte transmigration (p<0.05). In conclusion,
p66Shc acts as a novel signaling intermediate in the TNF-alpha pathway
regulating E-Selectin expression and the adhesive properties of endothelial
cells, and its action requires phosphorylation of p66Shc on Ser36.
494-P
Suppressive Effect of Acute Hyperinsulinemia on Serum Soluble
Forms of Adhesion Molecules in Young Healthy Subjects
MONIKA KARCZEWSKA-KUPCZEWSKA, AGNIESZKA ADAMSKA, AGNIESZKA
NIKOLAJUK, MAGDALENA ZIELINSKA, NATALIA MATULEWICZ, MARIA GOR-
SKA, IRINA KOWALSKA, MAREK STRACZKOWSKI, Bialystok, Poland, Olsztyn,
Poland
Markers of endothelial dysfunction, including soluble E-selectin (sE-
selectin), soluble intercellular adhesion molecule 1 (sICAM-1) and soluble
vascular cell adhesion molecule 1 (sVCAM-1) are positively related to insu-
lin resistance and cardiovascular disease. Atherosclerosis is increased in
hyperinsulinemic states, but the direct effect of insulin on atherogenesis
remain unclear. The aim of the present study was to investigate the effect
of acute hyperinsulinemia on serum sE-selectin, sICAM-1 and sVCAM-1
concentrations in young healthy population. We studied 67 healthy male
subjects (mean age, 23.332.54; mean body mass index, 25.543.79 kg/m2).
Serum sE-selectin, sICAM-1 and sVCAM-1 concentrations were measured
before and after 2 hour euglycemic hyperinsulinemic clamp. In 20 subjects,
clamp was prolonged to 6 hours and at the end additional measurements of
serum sE-selectin, sICAM-1 and sVCAM-1 were taken. Hyperinsulinemia de-
creased serum sE-selectin, this effect was present in both 2 and 6 hours of
the clamp (both p<0.001). The suppressive effect of hyperinsulinemia on se-
rum sICAM-1 was also observed in 2 and 6 hours of the clamp (both p<0.001).
We also observed decrease in serum sVCAM-1 after 2-hour and 6-hour acute
infusion of insulin (both p<0.001). Baseline sE-selectin was positively related
to BMI (r=0.37, p=0.02), waist circumference (r=0.42, p<0.001) and triglycer-
ides (r=0.32, p=0.008) and negatively to insulin sensitivity (r=-0.29, p=0.017).
Our data show that acute hyperinsulinemia decreased soluble forms of ad-
hesion molecules, suggesting the antiatherogenic effect of insulin in healthy
subjects.
Supported by: Grant UDA-POIG.01.03.01-00-128/08; from the Program Innovative
Economy 2007
495-P
Willow Bark Extract is a Robust Natural Indirect Antioxidant
through the NRF2 Activation
ATSUSHI ISHIKADO, YOKO SONO, MOTONOBU MATSUMOTO, AYA OKUNO,
STACEY ROBIDA-STUBBS, GEORGE L. KING, T. KEITH BLACKWELL, TAKETOSHI
MAKINO, Osaka, Japan, Boston, MA
NRF2 is a redox-sensitive master regulatory transcriptional factor that
plays an important role in vascular protection. NRF2 activation may prevent
some diabetic vascular complications such as nephropathy. In this study,
we have discovered that willow bark extract (WBE), which is listed in Eu-
ropean Pharmacopoeia, induces a robust phase II response as indicated
by NRF2 activation in human umbilical vein endothelial cells (HUVECs) and
Caenorhabditis elegans (C. elegans). WBE (25-400 g/ml) dose-dependently
increased NRF2 target genes HO-1 (200 g/ml; 20.7 fold), GCLM (3.6 fold),
GCS-1 (1.5 fold) or p62 (2.8 fold) mRNA and their protein expressions, and
also increased intracellular total glutathione level. WBE (200 g/ml) in-
creased the intranuclear expression (3.2 fold) and DNA binding (2.8 fold) of
NRF2. WBE also increased luciferase activity (200 g/ml; 14 fold) assessed
by ARE (antioxidant response element) reporter plasmid dose-dependently.
The siRNA of NRF2 signicantly reduced the WBE-induced HO-1, GCLM or
p62 mRNA and their protein expressions in HUVECs. Pretreatment with
WBE (100 g/ml) prevented the tert-butyl hydroperoxide (tBHP)-induced cell
toxicity, while the effect of WBE was canceled by the siRNA of NRF2. WBE-
induced mRNA expressions of NRF2 downstream molecules were partially
reduced by a specic inhibitor of p38, SB203580, but not those of PI3K, PKC
and MAPK. We also evaluated in vivo the effects of WBE on NRF2 activation
and antioxidative stress activity using C. elegans. WBE (10 mg/ml) signi-
cantly increased GFP expression in C. elegans using a gcs-1 transgene fused
with GFP and pretreatment of WBE signicantly extended the longevity of
C. elegans in case of providing the oxidative stress with tBHP. These re-
sults provided the rst evidence that WBE increased antioxidative activity
through the activation of NRF2 and may contain substances that can protect
against the development of diabetic vascular complications and the other
oxidative stress-induced diseases.
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Insulin Pensitizer Therapy Improves Atherothrombotic and Inam-
matory Biomarkers
ROZALINA GRUBINA, BRIAN IRVING, MATTIAS SOOP, MANIVANNAN SRINI-
VASAN, LAURA TATPATI, SUMIT BHAGRA, LISA CHOW, AUDREY WEYMILLER,
RICKEY CARTER, K. SREEKUMARAN NAIR, Rochester, MN
Insulin resistance, a dening feature of diabetes, metabolic syndrome,
critical illness, and other chronic diseases, is associated with disproportion-
ately high rates of atherothrombotic complications such as cardiovascular
disease (CVD). Several pro-thrombotic and pro-inammatory biomarkers
have been identied as risk factors for CVD and mortality. In a randomized
double-blinded placebo controlled study, we demonstrated that targeted
pharmacologic improvement of insulin sensitivity (S
I
) signicantly lowers
these risk factors. Individuals with impaired fasting glucose or diabetes
who never received anti-hyperglycemic therapy (n = 28), were randomized
to receive placebo or combination of metformin and pioglitazone (M/P) for
3 months; 25 participants completed the study. S
I
, measured by the hyper-
insulinemic-euglycemic clamp, improved signicantly with metformin/pio-
glitazone (n = 12), while fasting glucose, insulin, and HbA
1c
decreased with
treatment (all p< 0.001). Although BMI increased slightly with treatment
(32.43 5.53 kg/m
2
to 32.89 5.49 kg/m
2
), there were no signicant changes
in fat free mass or percent body fat. There was also no signicant change in
resting heart rate and blood pressure. After adjustment for interim changes
in fasting glucose, HbA
1c
, and BMI, M/P resulted in signicant improvements
in key CVD risk factors compared to placebo (n = 13): 10% increase in HDL
cholesterol (p = 0.03), 5.5% decrease in non-HDL cholesterol (p = 0.03), 12%
decrease in triglycerides (p = 0.02), 42% decrease in plasminogen-activator
inhibitor-1 (p = 0.001), 45% decrease in CRP (p = 0.03), 9% decrease in TNF-o
(p = 0.008), and 150% increase in adiponectin (p = 0.001). Fibrinogen did not
change with treatment. Interventions to improve insulin sensitivity may thus
be considered as a therapeutic options to reduce the burden of CVD in insulin
resistant states. ClinicalTrials.gov number NCT00443755.
Supported by: NIH RO1-DK41973-A23, KL2-RR024151, CTSA UL1-RR24150
497-P
Endothelial Cells Protection by G-CSF Against FFAs-Induced Oxida-
tive Stress and Apoptotic Cell Death was Mediated by NF-B Path-
way and Cell Cycle Regulatory Protein Hcdc14a
HOUGUANG ZHOU, LING LIU, YU ZHANG, YANYAN HUANG, ZHUANGLI GUO,
RENMING HU, QIANG DONG, Shanghai, China, Nanjing, China, Qingdao, China
Plasma free fatty acid (FFA) concentrations increase in state of metabolic
syndrome and impair endothelial function. The damage of vascular endothe-
lial cells is the key event in the pathogenesis of atherosclerosis. The aim of
this study was to elucidate whether FFA inuence human cell division cycle
protein 14a(Hcdc14a), oxidative stress, proliferation and apoptosis in human
brain vascular endothelial cells(HBVEC), and to clarify whether granulocyte
colony stimulating factor(G-CSF) possess ECs protection, and identify its
possible signaling pathway. After culturing HBVEC with FFA (50-200 mol/l,
24-72 h), we examined FFAs actions on Hcdc14a, cell cycle distribution, as-
sociated cell cycle protein expressions, oxidative stress and apoptosis in
HBVEC. We investigated whether NF-kB mediated FFA-induced injury and
apoptosis and G-CSF inuenced the above effects. After exposure to FFA,
Hcdc14a gene and protein expressions in HBVEC were downregulated, P53
protein expression was upregulated, and cyclinD3 downregulated. There
were low proliferation, high apoptosis, obviously increased ROS and de-
creased NO, eNOS and MMP in HBVEC. FFA also reduced signicantly gene/
protein expressions of NF-kBs inhibitor, IkBo. G-CSF(100nM) obviously pre-
vented above alterations. In conclusion, high concentration FFA damaged
HBVEC through downregulating Hcdc14a, inducing cell cycle arrest, initiat-
ing oxidative stress and apoptosis, and activating NF-kB pathway, which
representing key factors in the development of micro- and macrovascular
dysfunction. G-CSF could signicantly prevent these effects of FFA on HB-
VEC and possess ECs protection.
Supported by: The National Natural Science Foundation of China (81170322)
498-P
Differential Role of miRNA-9 in Fibronectin Production in Organs
Affected by Chronic Diabetic Complications
BIAO FENG, SHALI CHEN, SUBRATA CHAKRABARTI, London, ON, Canada
MicroRNA alterations play important roles in several biological processes.
Mir-9 shows widespread biological effects including tumorigenesis and cel-
lular differentiation including dedifferentiation of broblasts. Furthermore,
it targets bronectin (FN), an important extracellular matrix (ECM) protein,
which is known to be upregulated in chronic diabetic complications and
plays important roles in cell adhesion, migration, growth and differentia-
tion. In this study, we investigated the role of microRNA-9, a FN targeting
microRNA, in the endothelial cells and in the organs of diabetic animals.PCR
array was used to examine microRNA (miR) expression in endothelial cells
(HUVEC) exposed to glucose. miR-9 expression was validated by TaqManTM
PCR. Retinal, renal and cardiac tissues from the streptozotocin (STZ)-induced
diabetic rats after one month were examined for FN mRNA and protein lev-
els. Luciferase assay was used to determine the interaction between FN and
miR-9.PCR array showed glucose-induced alterations of 125 miRNAs (out
of 381) in HUVECS exposed to 25mM glucose (HG) compared to 5mM glu-
cose. Fifty-one miRNAs were upregulated and 74 were downregulated. HG
decreased miR-9 expression and increased FN mRNA and protein levels. Lu-
ciferase assay showed the binding of FN 3-UTR with miR-9 but not with the
mutated FN 3-UTR. Diabetic animals showed hyperglycemia, reduced body
weight and albuminuria. Interestingly, miR-9 expression was decreased in
the hearts of the diabetic animals, but was increased in the retinas and kid-
neys. However, FN mRNA expression was increased in the retinas, hearts
and kidneys.These studies indicate a novel glucose-induced molecular
mechanism of increased ECM protein production in which miR-9 regulates
FN expressions in diabetes. However, such effects may vary among vari-
ous organs. Identifying such mechanisms may lead to potential RNA based
treatment for diabetic complications.
Supported by: Canadian Diabetes Association and Heart and Stroke Foundation
of Ontario
499-P
Toll-Like Receptor 2 Mediates Impairment of Vascular Actions of
Insulin in Response to High Fat Diet
HYUN-JU JANG, SIMONE D. RIDGEWAY, PRATIBHA VASHISTA, DANIEL HWANG,
MICHAEL J. QUON, JEONG-A KIM, Birmingham, AL, Davis, CA, Baltimore, MD
Obesity is a chronic pro-inammatory state that promotes insulin resis-
tance in liver, adipose tissue, and skeletal muscle as well as impairing insulin
action in vascular endothelium that contributes to endothelial dysfunction.
Vascular insulin resistance promotes atherosclerosis, in part, by facilitat-
ing macrophage inltration and inammatory responses. Toll-like receptors
(TLRs) that initiate innate immune signaling are implicated in high fat diet
(HFD)-induced impairment of energy metabolism as well as cardiovascular
complications. TLR4 helps to mediate vascular insulin resistance through
fatty acid stimulated pro-inammatory responses including increased pro-
duction of cytokines and activation of JNK and NF-kB. However, the role of
TLR2 in impairment of vascular actions of insulin in response to HFD is un-
known. Therefore, we examined the specic role of TLR2 in HFD or saturated
fatty acid (SFA)-mediated impairment of vascular actions of insulin. siRNA
knock-down of TLR2 in primary endothelial cells reduced both SFA-stimulat-
ed production of pro-inammatory cytokines and splicing of X box protein
(XBP)-1. Moreover siRNA knock-down of XBP-1 or inositol requiring enzyme
1o (IRE-1o)reduced production of TNF-o from endothelial cells. Thus, SFA
stimulates pro-inammatory and unfolding protein responses to SFA require
TLR2. Moreover, impairment of insulin-stimulated phosphorylation of eNOS
and production of NO in response to SFA was opposed by siRNA knock-down
of TLR2. Mesenteric arteries isolated from TLR2 knock-out mice were pro-
tected HFD-induced impairment of insulin-stimulated vasorelaxation when
compared with arteries isolated from wild type mice on HFD. We conclude
that TLR2 in vascular endothelium helps mediate HFD-induced pro-inam-
matory and unfolding protein response that may contribute importantly
to impairment of vascular actions of insulin, endothelial dysfunction, and
atherogenesis.
Supported by: NIH
500-P
Diabetes Induces Lung Endoplasmic Reticulum Stress and Cell
Death in an Angiotensin II Type 1 Receptor-Dependent Manner
JUNLING YANG, FENGLIAN ZHAO, PENG CHEN, LU CAI, Changchun, China, Lou-
isville, KY
Diabetes damages multiple organs to cause life-threatening complica-
tions. Our previous studies showed that diabetes can induce pulmonary
brosis, in which angiotensin II (Ang II) played a critical role, but how Ang II
initiates lung brosis remains unclear. The present study was to investigate
whether diabetes induces endoplasmic reticulum stress (ERS) and associ-
ated cell death, which has been recognized as an initiator of tissue brosis,
and whether Ang II involves in this pathogenic effect. FVB mice were induced
to type 1 diabetes with multiple low-doses of streptozotocin (STZ, 50 mg/
kg daily for 5 days) and hyperglycemic mice at 7 days after the last injection
of STZ was dened as diabetic. Three months after diabetes onset, lungs
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were collected from these diabetic mice and their age-matched controls.
ERS-associated proteins were examined at both protein and mRNA levels
with Western blotting and real-time PCR assays. Apoptotic cell death was
examined with TUNEL assay and Western blotting assays. Results showed
signicant increases in protein and mRNA expressions of ERS-related mol-
ecules, including PERK, ATF6, and IRE1, in diabetic lungs compared to con-
trol lungs. There was also a signicant increase in ERS-related cell death,
measured by CHOP, JUNK, and caspase-12, in diabetic lungs. Ang II type 1
(AT1) receptor blocker (Losartan) can signicantly prevent diabetes-induced
lung ERS and associate cell death, as we observed for its inhibition of dia-
betic lung brosis in previous study. These results suggested that diabetes
induced ERS and associated cell death via Ang II/AT1-dependent pathway,
which may be the major cause of diabetes-induced lung brosis.
Supported by: Natural Science Foundation of Jilin Province in China
501-P
High Glucose Effects on eNOS and SIRT1 Expression and Chromatin
Acetylation in Endothelial Cells
ALESSANDRO DE MARCO, GLORIA FORMOSO, NATALIA DI PIETRO, ASSUNTA
PANDOLFI, VINCENZO DE LAURENZI, MASSIMO FEDERICI, AGOSTINO CONSOLI,
Chieti Scalo, Italy, Rome, Italy
The bad metabolic legacy exposing to increased vascular risk diabetic
individuals who experienced poor glycemic control in the early stages of the
disease might be mediated by hyperglycemia related epigenetic modica-
tions in the vascular cells.The NAD+ -dependent protein deacetylase SIRT1
regulates gene expression programs in response to cellular metabolic status
by modulating chromatin function via several epigenetic mechanisms: a) di-
rect histone deacetylation, b) nuclear enzymes recruitment into chromatin
or c) transcription factors and coregulators deacetylation. Since increased
eNOS expression and activity have been described among the hypergly-
cemia related endothelial cells modications, we set to investigate eNOS,
SIRT1 and chromatin modications (Histones H3 and H4 Lysine acetylation)
in HUVEC and HAEC cultured in Normal Glucose (NG, 5 mmol/L) and High
Glucose (HG, 25 mmol/L) conditions for 48 hours.HG increased both SIRT1
and eNOS protein levels. Thus, to investigate whether SIRT1 might affecte
NOS expression, HUVEC and HAEC were transfected so to over-express or
to down-regulate SIRT1 expression. SIRT1 overexpression resulted in in-
creased eNOS protein levels under both NG or HG conditions, while silencing
SIRT1 prevented the increase in eNOS levels observed in HG exposed cells.
Furthermore, by Chromatin Immunoprecipitation analysis we observed that
HG exposure in HAEC resulted in changes in the acetylation levels of Histone
H3 at the eNOS promoter. As compared to NG exposed HAEC, acetylation
was less pronounced in the enhancer region (-4178 to -4596 bp) and greater
in upstream promoter region (-891 to -794 bp).Taken together, these data
indicate that, in endothelial cells, SIRT1 is involved in modulating eNOS ex-
pression in response to metabolic stress such as hyperglycemia, potentially
by epigenetic mechanisms.
Supported by: EFSD, sano-aventis
502-P
Antithrombotic Function of Apelin is Preserved During Obesity
BRUNO M. FEVE, FRDRIC ADAM, JOS LOPEZ, CAMILLE VATIER, SABRINA
TURPIN, ADELINE MUSCAT, CLAUDE LALOU, ISABELLE CASTAN, PHILIPPE VA-
LET, ABDEL-MAJID KHATIB, RGIS BOBE, GRALDINE SIEGFRIED, Paris, France,
Le Kremlin-Bictre, France, Toulouse, France
Antithrombotic function of apelin is preserved during obesityObesity
and type 2 diabetes are associated with an increased thrombotic risk that
is partly related to a disturbed metabolic and adipokine pattern. Apelin is
an adipokine with pleiotropic functions on cardiovascular system, and on
uid and energy homeostasis. Apelin acts through its cognate seven-trans-
membrane domain receptor APJ, and is overexpressed in adipose tissue and
plasma from obese individuals. We identied a new biological and potent
antithrombotic function of apelin in vitro and in vivo. Apelin strongly inhib-
its thrombin- or collagen-induced platelet aggregation, while it does not
interfere with ADP-induced aggregation or ADP secretion. Likewise, apelin
prevents activation of ERK1/2, P38 MAPK, and Akt signaling pathways by
thrombin or collagen. Accordingly, intravenous injection of apelin increas-
es tail bleeding time and delays chemically-induced vessel occlusion and
thrombus stabilization. The use of a mutant form of apelin that is resistant to
proteolytic cleavage demonstrates that the peptide maturation is required
for its antithrombotic properties. Finally, APJ is overexpressed in platelets
from genetic or nutritional murine models of obesity or from obsese humans,
and the anti-aggregant effect of apelin is preserved under this condition.
Collectively, these ndings show a new biological function of apelin that
could be of potential interest to reduce the increased risk of thrombosis in
patients with obesity, type 2 diabetes, or metabolic syndrome.
Supported by: INSERM, Universities Paris 6, Paris 7, Paris 11, and UPS of Tou-
louse
503-P
Evaluation of Dysfunctional High-Density Lipoprotein in Patients
HIROSHI MURAKAMI, KOKI MATSUMURA, MAKI YAMASHITA, KOTA MATSUKI,
JUTARO TANABE, HIROSHI MURAKAMI, JUN MATSUI, NAOKI TAMASAWA,
TOSHIHIRO SUDA, Hirosaki, Japan
Study Aim: We simultaneously evaluated several functions of HDL in pa-
tients with type 2 diabetes, including the cholesterol efux, anti-oxidative
and anti-inammatory activities.Methods: (1) Subjects (n=30; 16 male and
14 female) were patients with type 2 diabetes admitted to our hospital for
diabetic control and education. Clinical characteristics were as follows (data
are average values): age, 55 y; HbA1c (JDS), 9.3%; total cholesterol (TC),
180 mg/dl; triglyceride (TG), 132 mg/dl; and HDL-C, 40.7 mg/dl. (2) The HDL
fraction of human plasma was separated by PAGE (Lipophor
; Jokoh, Tokyo,
Japan) and the sharp HDL band was applied to protein recovery equipment
(Phoreto-Yield
; Jokor) with a cut-off MW of 15,000. The HDL fraction was

then recovered with elution buffer. (3) Cholesterol efux capacity (Efux)
was determined as reported previously. Values were corrected against
those with non-specic BSA. (4) In HDL fraction, paraoxonase 1 (PON1) ac-
tivity (maker of anti-oxidation capacity), and levels of serum amyloid A (SAA)
(marker of inammatory capacity) and carboxymethy lysine (CML) (marker of
ApoA-I glucoxidation) were determined.Results: (1) HbA1c levels were nega-
tively correlated with PON1 activity in the HDL fraction (r=-0.32, P<0.10).
However, neither SAA nor CML showed any correlations with HbA1c. (2)
Efux (%) ranged from 19.1 to 28.9%, showing a positive correlation with
PON1 activity (r=0.38, p<0.05), and a weak negative correlation with SAA
levels (r=-0.32, p<0.10). No relationship was seen between Efux and CML
levels.Conclusion: We established a method that is able to rapidly separate
the HDL fraction from plasma in patients with type 2 diabetes. We then
conrmed that HDL-mediated Efux is reduced by decreases in PON1 activity
and increases in SAA levels in the HDL fraction. These ndings indicate that
diabetic status drives HDL remodeling, leading to dysfunctional HDL, and
this may explain the pathogenesis of atherosclerosis in type 2 diabetes.
504-P
Vascular Smooth Muscle Cell Proliferation by Intermittent Hypoxia
is Independent on the Glucose-Induced Insulin Secretion
YOJI KYOTANI, HIROYO OTA, ASAKO ITAYA-HIRONAKA, AKIYO YAMAUCHI,
SUMIYO SAKURAMOTO-TSUCHIDA, JING ZHAO, HIROSHI KIMURA, MASAYUKI
UNO, SHIN TAKASAWA, MASANORI YOSHIZUMI, Kashihara, Japan
Sleep apnea syndrome (SAS), characterized by intermittent hypoxia (IH)
during sleep, is a risk factor of cardiovascular diseases as well as type 2 dia-
betes. The proliferation of vascular smooth muscle cells (VSMC) is a pivotal
factor in cardiovascular diseases. Recently, we found that IH attenuates glu-
cose-induced insulin secretion. Therefore, there are two possibilities to in-
duce VSMC proliferation by IH: One is IH directly induces VSMC proliferation
and the other is IH induces VSMC proliferation via induction of diabetes. In
this study, we investigated direct effects of IH on cultured rat aortic smooth
muscle cell (RASMC) proliferation. WST-8 assay revealed that IH induced
the RASMC proliferation (P<0.0001), but neither normoxia nor sustained hy-
poxia did. On the other hand, the decrease of apoptosis was not observed in
IH-treated RASMC by TUNEL assay. In order to see what induces the RASMC
proliferation, we measured of several mRNAs for growth factor receptors
which are concerned with VSMC proliferation using real-time RT-PCR and
found that the mRNA level of erbB2 receptor, a member of epidermal growth
factor (EGF) receptor, was increased by IH, but not by sustained hypoxia. We
next investigated the changes of mRNAs for ligands of EGF family recep-
tor and found that epiregulin, amphiregulin and neuregulin-1 mRNAs were
increased by IH. We also conrmed that IH specically increased the level of
epiregulin in the conditioned medium and phosphorylation of erbB2 receptor
of RASMC at the specic residue that mediates intracellular signal for cell
proliferation. These results indicate that VSMC proliferation by IH is inde-
pendent on the attenuation of glucose-induced insulin secretion by IH, and
suggest that the EGF family-erbB2 receptor signal system is involved in the
IH-induced VSMC proliferation.
Supported by: Grants-in-Aid from the MEXT, Japan
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Decreased Expression and Activity of Kruppel Like Factor-2 in
Monocytes Under Hyperglycemia
XINPU CHEN, SRIDEVI DEVARAJ, Houston, TX
Diabetes is now a worldwide epidemic and contributes to increased vas-
cular complications. Kruppel-like factors (KLFs) are a subclass of the zinc-
nger family of transcription factors. KLF2 inhibits proinammatory activa-
tion of monocytes. Furthermore, KLF2 expression in circulating monocytes is
reduced in patients with chronic inammatory conditions such as coronary
artery disease. Diabetes is a proinammatory state and we and others have
shown increased monocyte activation in diabetic conditions in vitro and in
vivo. However, there is a paucity of data on KLF-2 exression and activity
in the diabetic milieu and its regulation of monocyte inammation. Thus,
the aim of this study was to examine the expression and activity of KLF-2
in hyperglycemia and examine its modulation of monocytic inammation.
THP-1 cells were incubated with mannitol, low glucose (5.5mM glucose) or
high glucose (15mM glucose) for 48 hours. KLF-2 expression was assessed
by western blotting, mRNA and DNA binding was assessed by EMSA. KLF-
2 protein expression, mRNA as well as DNA binding activity was signi-
cantly inhibited in hyperglycemia compared to either low glucose or man-
nitol, which was used as hyperosmolar control. This was associated with
signicantly increased activity of NF-kappaB and p47 phox activation and
resulted in increased release of cytokines IL-1 and IL-6 from cells. We have
shown previously that Resveratrol, a avonoid, found in grapes and red
wine, signicanly reduces inammation and monocyte activation under high
glucose conditions. Addition of resveratrol (100uM) to cells treated with high
glucose signicantly reversed HG-suppressed KLF-2 expression and activ-
ity and resulted in decreased monocyte inammation. Thus, KLF-2 apears
to be a negative regulator of monocytic activity in the diabetic milieu and
strategies to upregulated KLF-2 may be effective in decreasing inammation
associated with diabetes and cardiovascular disease.
506-P
Diabetes Causes Persistent Insulin Resistance and Impairment of
Angiogenic Function of Mesenchymal Stem Cells (MSC) by Protein
Kinase C (PKC) Activation
KOJI MIZUTANI, IN-KYUNG JEONG, AKIRA MIMA, QIAN LI, KYOUNGMIN PARK,
CHRISTIAN RASK-MADSEN, DAVID J. MOONEY, GEORGE L. KING, Boston, MA,
Cambridge, MA
Infusion of bone marrow derived MSC can improve perfusion to isch-
emic hindlimbs possibly by increases in angiogenesis which is abnormal in
diabetes. To test the hypothesis that the dysfunction of MSC in diabetes
is due to loss of insulin action, we implanted MSC isolated from control or
STZ induced diabetic mice with or without insulin incorporated into poly-
mer scaffolds for local delivery into the ischemic hindlimb of nondiabetic
mice. One week after implantation, VEGF mRNA expression in ischemic
thigh muscle was signicantly increased by 2-fold in control MSC, insulin
scaffold, and combination groups vs diabetic MSC with and without insulin.
After 4 weeks, insulin alone, control MSC, insulin with control and diabetic
MSC groups showed signicantly higher perfusion than scaffold alone. How-
ever, MSC alone or MSC and insulin had signicantly better perfusion than
diabetic MSC or diabetic MSC with insulin scaffold groups, respectively. In
MSC cultured for 2-3 months from control and diabetic mice, insulin induced
tyrosine phosphorylation of its receptors (IR-) and IRS1 was decreased in
diabetic vs control MSC by 71% and 64%, respectively, which were trans-
lated into comparable decreases of Akt activation (-51%, p<0.05) although
activation of p-Erk1/2 was not affected. mRNA and protein levels of VEGF
were reduced in diabetic MSC vs control, (p<0.05). Interestingly, PKC activity
and PKC2 protein expression in the membrane were persistently increased
in diabetic vs control MSC. Addition of PKC inhibitor (GFX) or PKC-specic
inhibitor (RBX) partially reversed the inhibitory effect of diabetes on insulins
activation of p-Akt by 44% and 25%, and enhanced mRNA expression VEGF
by 2.1 and 1.5-fold. Thus, diabetes can cause persistent insulin resistance in
MSC via PKC2 activation, which could be a therapeutic target to improve
wound healing in diabetic patients.
507-P
Pro-Inammatory Actions of TNF- in Vascular Endothelial Cells
are Reduced by Treatment With the Green Tea Polyphenol EGCG via
p38 MAPK-, STAT3-, and COX-2-Dependent Mechanisms
HUI CHEN, JI-WON LEE, YUNHUA LI, MARIA DE LA LUZ SIERRA, MICHAEL J.
QUON, Bethesda, MD, Baltimore, MD
Pro-inammatory cytokines including TNF-o contribute to the pathophys-
iology of diseases impacted by vascular endothelial dysfunction including
diabetes. The green tea polyphenol epigallocatechin gallate (EGCG) exerts
multiple benecial metabolic and vascular actions. Indeed, EGCG treatment
of SHR rats reduces hypertension, and ameliorates endothelial dysfunction
and insulin resistance. Additional biological actions of EGCG remain undis-
covered. In the present study, we investigated anti-inammatory actions of
EGCG in vascular endothelial cells. TNF-o treatment of human aortic en-
dothelial cells (HAEC) stimulated phosphorylation of STAT3 and p38 MAPK
(upstream of STAT3) as well as STAT3 transcriptional activity that were all
inhibited by EGCG co-treatment (10 M) or inhibition of p38 MAPK (using
siRNA knockdown or SB203580 (p38 MAPK inhibitor)). TNF-o treatment
also increased COX-2 expression in HAEC that was inhibited by co-treatment
with EGCG. These anti-inammatory actions of EGCG were abrogated by
siRNA knockdown of STAT3. Likewise, TNF-o treatment of HAEC stimulated
secretion of PGE
2
(COX-2 product) that was inhibited by EGCG co-treatment
or siRNA knockdown of STAT3. Further, TNF-o treatment stimulated IL-6 se-
cretion from HAEC that was inhibited by co-treatment with EGCG or siRNA
knockdown of COX-2. Finally, IL-6 treatment of HAEC stimulated secretion
of TNF-o that was inhibitable by co-treatment with EGCG. Thus, TNF-o and
IL-6 synergistically promote pro-inammatory actions through feed-forward
mechanisms that are dampened by actions of EGCG to inhibit STAT3 and
COX-2 function. Our ndings dene novel molecular mechanisms for anti-
inammatory actions of EGCG in vascular endothelium that may be relevant
for developing effective therapies targeting diabetes and its cardiovascular
complications.
Supported by: Intramural Research Program, NCCAM, NIH
508-P
Intermittent High Glucose Reduces Androgen Receptor Expression
of HUVECs via PI3K/Akt/mTOR Signaling Pathway
LIN LI, XUEYAO YIN, FANG WU, JIAQIANG ZHOU, HONG LI, Hangzhou, China
Glucose uctuation plays a more signicant role in the pathogenesis of
vascular diabetic complications than constant high glucose. Androgen recep-
tor (AR) is important in coronary atherosclerosis, and decreasing AR expres-
sion correlate with more extensive atherosclerosis. But the regulation of AR
gene expression in the uctuated glucose remains poorly understood, and
the signal transduction pathways correlate with AR has not been dened
well in the cardiovascular system. This study focuses on the effect of in-
termittent high glucose concentrations on AR expression and the PI3K/Akt/
mTOR signaling pathway in human umbilical vein endothelial cells (HUVECs).
HUVECs were incubated in media containing different glucose concentra-
tions: 5.5mmol/l, 30mmol/l, or alternating 5.5 and 30mmol/l glucose twice a
day. AR expression in HUVECs was downregulated in constant high glucose
condition than that in normal concentration condition. Furthermore, AR was
reduced more in HUVECs exposed to intermittent rather than constant high
glucose concentration. Constant and intermittent high glucose concentra-
tion could induced activation of the PI3K/Akt/mTOR pathway. When we
blocked or knocked down PI3K/Akt/mTOR signaling pathway, by LY 294002,
rapamycin, Akt siRNA and mTOR siRNA, led to increasing levels of AR mRNA
and protein associated with reduced phosphorylation of Akt or mTOR. These
results suggest that intermittent high glucose has more prominent effect in
reducing AR expression in HUVECs than constant high glucose. The patho-
genesis of this effect at least partly related to the enhanced activation of
PI3K/Akt/mTOR signaling pathway.
Supported by: S&T Major Project of Zhejiang (2009C03010-4) National Key R&D
(2009BAI80B01)
509-P
Early Intensive Insulin Treatment Stimulated Production of Nitric
Oxide via Activation of the AMPK/Akt/eNOS Pathway in Aorta of
Diabetic Rats
DONGHONG FANG, ZHIMIN HUANG, HONGYU GUAN, HAI LI, HAIPENG XIAO,
YANBING LI, Guangzhou, China
In this study, we compared the effects of early versus late intensive insu-
lin therapy on aortic endothelial function and explored the potential causal
mechanisms.High-fat diet and low-dose-STZ induced diabetic rats were ran-
domly divided to diabetes group (DM group, no insulin treatment, n=8), early
intensive insulin therapy group (EI group, n=9) and late insulin therapy group
(LI group, n=7). Non-diabetic SD rats served as controls (NC group, n=8). As
soon as diabetes was conrmed, NPH (6-10 U per day) was used in the EI
group to maintain near-normal glycemic control for the entire 16 weeks. In
the LI group, near-normal glycemic control was induced after 8 weeks of
poor glycemic control. Nitric oxide (NO) productions in blood serum at base-
line were similar among the four groups. After completing the treatment,
the DM group was markedly declined to 156.6231.49mol/L (p<0.05). The
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EI group was higher than the DM group (227.9910.39, p<0.05), while the LI
group was not different from the DM group (147.9525.27, p=0.54). Western
blot revealed that as compared with the NC groups, p-eNOS (active)/ total
eNOS was decreased in the DM groups. In the EI group, p-eNOS/t-eNOS was
maintained at a near normal level, whereas in the LI group was similar to the
DM group. The activities of AMPK, ACC and Akt proteins showed similar
trends (Fig. 1).Our results suggested that the decreased production of NO
in diabetic rats was counteracted by early, but not late insulin therapy. The
aortic endothelial protective function of early intensive insulin treatment
was mediated by AMPK/Akt/eNOS Pathway.
510-P
Effect of In Vivo Hyperglycemia on Granulocyte and Monocyte Acti-
vation Markers in Type II Diabetes
PETER HORVATH, STACY R. OLIVER, SHLOMIT RADOM-AIZIK, FRANK P. ZALDI-
VAR, PIETRO R. GALASSETTI, Irvine, CA
Atherosclerosis and cardiovascular complications of Type II Diabetes
(T2DM) are mediated by chronic inammatory processes, including exagger-
ated activation of immune cells such as granulocytes (Gc) and Monocytes
(Mc). Activation of these cells can be measured by levels of expression of
cell surface markers (CD11b, CD66b etc), adhesion molecules which facili-
tate anchoring to the endothelium and onset/progression of vascular dam-
age. While pro-inammatory effects of hyperglycemia on immune cells are
established in vitro, Gc and Mc activation during in vivo hyperglycemia,
when interacting systemic factors may drastically affect inammatory re-
sponses, remains unexplored. We therefore performed a 4-h hyperglycemic
clamp (240 mg /dl, simulating common post-prandial hyperglycemia) in 7
T2DM subjects, sampling at 0, 30, 90, 120, 180, 240 min. Mean uorescence
intensity (MFI) of CD11b, CD14, CD16, CD62L, and CD66b was measured by
ow cytometry. During the rst 2 h of hyperglycemia CD11b and CD66b ex-
pression in Gc increased signicantly. In Mc, the % of CD14+CD16+ cells
(abundant in atherosclerotic tissue), and CD14 expression in this cell sub-
type, also increased. Our data indicates that in T2DM in vivo hyperglycemia,
of magnitude/duration similar to that typically observed postprandially even
in relatively well controlled patients, markedly increases Gc and Mc adhe-
sion potential and activation state. This effect may be a signicant factor
in the postprandial component of long-term cardiovascular risk, and further
underscores the necessity of avoiding hyperglycemic peaks of even moder-
ate elevation.
Supported by: NIH (P01HD048721 and UL1RR031985)
511-P
Tribble 3 is Induced in Macrophages During Foam Cell Formation
and Facilitates Lipid Accumulation in a TLR4 Manner
DENNIS STEVERSON, LING TIAN, TIMOTHY GARVEY, Birmingham, AL
The tribbles homolog-3 gene (TRIB3) has beenreported to be upregulated
in human atherosclerotic plaque, however, the roleof TRIB3 in foam cell
formation has not been studied. Therefore, we transformed THP-1 cells,
ahuman monocytic cell line, into macrophage foam cells via treatment with
oxidizedlow-density lipoprotein (oxLDL), and found that TRIB3 was also in-
duced. In THP-1 cell with stable overexpression ofTRIB3, cholesterol accu-
mulation upon oxLDL treatment was increased by 1.2 foldincrease (p<.01)
indicating a potential role for TRIB3 in facilitating foamcell formation. oxLDL
and other lipidsinteract with Toll-like receptor 4 (TLR4) on the macrophage
cell surface, andTLR4 has been implicated in the cellularuptake of lipid
and formation of atherogenic plaques. To examine whether TRIB3 induc-
tion wasdependent of TLR4, peritoneal macrophages from wild type (WT)
and TLR4-/- micewere treated with 100ug/ml of lipopolysaccharide (LPS), a
strong activator ofTLR4. After 24 h, WT macrophagesexhibited a 10.1 fold
increase in TRIB3 expression over that in untreatedcontrols (p<.01). Further,
co-incubatedof WT cells with both 100ug/ml oxLDL and 100ug/ml of LPS led
to an augmentationof TRIB3 expression up to 17.4 fold (p<.01). However in
macrophages from TLR4 -/- mice, theTRIB3 response was markedly reduced
under these conditions; specically, theinduction of TRIB3 was signicantly
decreased by 5.5 fold following treatment withLPS, and decreased by 5.7
fold following treatment with LPS + oxLDL, whencompared with responses
in the WT cells.In conclusion: we have shown for the rst time that: 1) TRIB3
isinduced during foam cell formation and can mediate an acceleration in lipi-
daccumulation, 2) The induction of TRIB3 in response to oxLDL and LPS is
inlarge part dependent upon TLR4. Thus,TRIB3 is mechanistically implicated
in foam cell formation and potentiallyrepresents a novel target for preven-
tion of atherosclerosis.
512-P
MD-2 Plays an Important Role in High Glucose-Enhanced, IFN
Receptor/TLR4-Mediated MMP-1 Expression in U937 Mononuclear
Cells
ZHONGYANG LU, YANCHUN LI, DEVADOSS J. SAMUVEL, JUNFEI JIN, XIAOMING
ZHANG, MARIA F. LOPES-VIRELLA, YAN HUANG, Charleston, SC
Although it is known that interferon gamma (IFN) receptor and toll-like
receptor (TLR)4 play important roles in diabetic complications, the molecular
mechanisms involved in the interaction between IFN receptor and TLR4
under hyperglycemic condition have not been well understood. In this study,
we showed that while either IFN or lipopolysaccharide (LPS) stimulated
matrix metalloproteinase (MMP)-1, a key proteinase involved in athero-
sclerosis, by 2.7- and 16.5-fold (0.54 and 3.3 vs 0.2 ng/ml), respectively, in
U937 mononuclear cells, priming of U937 cells with IFN augmented LPS-
stimulated MMP-1 expression by 76.5-fold (15.3 vs 0.2 ng/ml). We further
demonstrated that high glucose markedly enhanced the priming effect of
IFN on LPS-stimulated MMP-1 expression by 984-fold (196.8 vs 0.2 ng/ml).
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COMPLICATIONSNEPHROPATHY
To explore the underlying mechanisms, we focused on TLR4, CD14 and MD-2
expression since they are all essential for TLR4 signaling. We found that
while IFN stimulated MD-2 expression by 3.2-fold and high glucose alone
had no effect, treatment of U937 cells with IFN and high glucose simultane-
ously increased MD-2 expression by 5.6-fold. In contrast, high glucose and
IFN had no effect on TLR4 and CD14 expression, suggesting that MD-2 may
be specically involved in high glucose-enhanced, IFN receptor/TLR4-me-
diated MMP-1 expression. To conrm the critical role of MD-2 in the relation
of MMP-1 expression, we showed that neutralizing MD-2 antibody or MD-2
siRNA signicantly reduced IFN receptor/TLR4-mediated MMP-1 expres-
sion under high glucose condition. Surprisingly, results showed that neutral-
izing MD-2 antibody or siRNA also signicantly reduced MMP-1 expression
induced by IFN alone, suggesting that MD-2 is involved in not only TLR4
signaling, but also IFN receptor signaling. Finally, we demonstrated that
IFN and high glucose upregulated MD-2 expression via mitogen-activated
protein kinase (MAPK) pathways and AP-1 transcription factor.
Supported by: Merit Review Grant from Department of Veterans Affairs and NIH
(RO1 DE016353)
513-P
Angiopoietin-Like 4 is Elevated in Type 2 Diabetes But is Not Asso-
ciated With Angiographically Determined Coronary Artery Disease
HEINZ DREXEL, KATHRIN GEIGER, ALEXANDER VONBANK, PHILIPP REIN, AXEL
MUENDLEIN, CHRISTOPH H. SAELY, Feldkirch, Austria, Philadelphia, PA, Triesen,
Liechtenstein
Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor), a protein
inhibitor of lipoprotein lipase, is synthesized and secreted during fasting
in adipose tissue and the liver. Its associations with metabolic syndrome
traits are uncertain, and it is not known whether it is associated with type
2 diabetes (T2DM) or coronary artery disease (CAD).We therefore measured
serum ANGPTL4 in 493 patients undergoing coronary angiography for the
evaluation of established or suspected stable CAD; signicant CAD was
diagnosed when coronary stenoses ~50% were present.ANGPTL4 was
signicantly positively correlated with age (r=0.177; p <0.001) and fasting
glucose (r=0.112; p=0.013) but was not correlated with waist circumference,
triglycerides, HDL cholesterol, systolic blood pressure or diastolic blood
pressure. ANGPTL4 was signicantly higher in patients with T2DM (n=115)
than in non-diabetic subjects (2832 vs. 2538; p=0.032); however, it was
not signicantly different between patients with signicant CAD (n=246) and
individuals without signicant CAD (p=0.112).We conclude that ANGPTL4 is
positively correlated with fasting glucose and elevated in T2DM but is not
signicantly associated with angiographically determined CAD.
Guided Audio Tour: Experimental Models of Diabetic Kidney Disease
(Posters 514-P to 521-P), see page 15.
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514-P
The Role of Thioredoxin Interacting Protein (TXNIP) in the Develop-
ment of Diabetic Nephropathy
ANU SHAH, LING XIA, HOWARD GOLDBERG, KEN W. LEE, CATHARINE WHITE-
SIDE, SUSAN E. QUAGGIN, I. GEORGE FANTUS, Toronto, ON, Canada
Thioredoxin-interacting protein (TXNIP) is an endogenous inhibitor of
thioredoxin (Trx), a ubiquitous thiol oxidoreductase that regulates cellu-
lar redox status. TXNIP is upregulated by high glucose (HG) and promotes
oxidative stress. We have found that Hcb-19 mice (lack TXNIP) are partially
protected from streptozotocin-induced diabetes and diabetic nephropathy
(DN). Since HG and reactive oxygen species (ROS) are key mediators of the
microvascular complications of diabetes, we investigated the potential role
of TXNIP in the pathogenesis of DN by utilizing primary mouse mesangial
cells (MC) culture from the 2 strains and then exposing MC to HG. C3H MC
exposed to HG (25 mM) for 3h showed a signicant increase in total cell ROS
determined by DCF (2.29 fold, p<0.001) as well as MitoSox (mitochondrial
ROS, 3.54 fold, p<0.001) uorescence, while Hcb-19 MC had no response.
Trx activity was decreased by HG only in C3H MC (i.e. 69% decrease). Of
interest lucigenin based NADPH oxidase assay revealed activation by HG
only in C3H (2.210.28 fold, p<0.05) but not in Hcb19 MC. Recently, the
mitochondria-localized NADPH oxidase isoform, Nox4 has been implicated
in HG-mediated ROS generation. Nox4 protein level was increased by HG in
C3H total cell lysates (1.70.19 fold, p<0.05) and isolated mitochondrial frac-
tions (2.140.26 fold, p<0.05), but not in Hcb19 MC. To verify our ndings, we
transfected Hcb19 MC with TXNIP adenovirus and C3H with siRNA against
TXNIP, and then repeated the aforementioned experiments. Overexpression
of TXNIP in Hcb19 MC in normal glucose (5.5 mM) augmented cellular ROS
formation, NADPH oxidase activity, NOX4 levels, and collagen IV (brosis
marker) expression. In contrast, TXNIP siRNA blocked all these effects of
HG in C3H MC. These data indicate that TXNIP is a critical component of
the HG-ROS signaling pathway, apparently required for the induction of the
mitochondrial NADPH oxidase isoform Nox4. Thus, TXNIP may be a promis-
ing target to prevent the complications of diabetes.
Supported by: NSERC-CGS M, Ontario Graduate Scholarship, and BBDC Gradu-
ate Awards
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High Glucose Induces Platelet Derived Growth Factor C (PDGF-C)
Expression in Glomerular Mesangial Cells via a Glucose Respon-
sive Transcription Tactor, Carbohydrate Response Element Binding
Protein (ChREBP)
HIROYA KITSUNAI, YUICHI MAKINO, HIDEMITSU SAKAGAMI, KATSUTOSHI MI-
ZUMOTO, TSUYOSHI YANAGIMACHI, YASUTAKA TAKEDA, YUKIHIRO FUJITA,
ATSUKO ABIKO, YUMI TAKIYAMA, MASAKAZU HANEDA, Asahikawa, Japan
High glucose evokes a variety of gene expression in mesangial cells
(MC) that alter cellular functions responsible for the development of dia-
betic glomerulopathy. We have recently shown that, in MC exposed to high
glucose, a glucose responsive transcription factor carbohydrate response
element binding protein (ChREBP) activates HIF-1o and downstream gene
expression leading to an extracellular matrix expansion in diabetic glomeruli.
Besides such observation, however, a role of ChREBP in gene regulation in
MC under diabetic circumstances has been scarcely documented. To provide
more insight into glucose-responsive gene regulation via ChREBP in MC, we
performed genome wide analysis by chromatin immunoprecipitation with
anti-ChREBP antibody followed by DNA microarray assays using cultured hu-
man MC and identied platelet derived growth factor C (PDGF-C) as a direct
target of ChREBP. Consistently, sequence analysis found the ChREBP binding
site at 1.6 kbp upstream of transcriptional start site for human PDGF-C gene.
In quantitative analyses, high glucose enhanced PDGF-C mRNA expression
in both human and mouse cultured MC. Similarly, PDGF-C protein levels in
MC increased in a glucose concentration-dependent manner. PDGF-C is
known for its mitogenic and brogenic action in various disease condtions
such as cardiac and liver brosis, renal interstitial brosis in non-diabetic
chronic kidney diaseases. In support of these issues, knockdown of PDGF-C
in mouse MC abrogated high glucose-mediated increase in mRNA levels of
brogenic genes including Collagen 4a-1 and Collagen 6a-1. Taken together,
high glucose-mediated induction of PDGF-C via ChREBP could contribute to
the development of the mitogenic and brotic lesion in diabetic nephropathy
and might provide a novel platform for therapeutic intervention of the com-
plications in diabetic patients.
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Leptin-Dependent Effects of CP-900691, a Highly Selective and Po-
tent PPAR- Agonist on Diabetic Nephropathy in the BTBR ob/ob
Mouse
TOMASZ WIETECHA, KELLY L. HUDKINS, EDWARD J. FOX, TRI Q. NGUYEN, LAW-
RENCE A. LOEB, CHARLES E. ALPERS, BARDIA ASKARI, Seattle, WA
We have recently characterized a new model of type II diabetic neph-
ropathy (DN), the BTBR ob/ob leptin-decient mouse. Current therapies for
DN (control of hyperglycemia, blood pressure and inhibition of the renin-
angiotensin system) while effective, are not completely efcacious. Studies
suggest that peroxisome proliferator-activated receptor (PPAR) agonists are
promising agents in the treatment of DN. Piperidine-based PPAR-o agonists
are novel agents that improve lipid, glycemic, and inammatory indicators in
mammalian models of diabetes and obesity. The purpose of this study was
to determine whether CP-900691 (CP), a member of this class of agents,
could prevent the development of DN in BTBR ob/ob mice. 4 week-old female
BTBR ob/ob mice and wild-type controls received either reglar chow or one
containing CP (3mg/kg/day) for 16 weeks. CP treatment reduced body weight
in BTBR (28.9 2 vs 19.7 1.5 g, untreated vs treated, respectively, p<0.001)
and BTBR ob/ob (57.8 5.2 vs 51 6 g, untreated vs treated, respectively,
p<0.001). Treatment also reduced fasting blood glucose in BTBR ob/ob (373
48.1 vs 182 10.9 mg/dL, untreated vs treated repectively, p<0.001), plasma trig-
lycerides (124 11.9 vs 47 9.2 mg/dL, untreated vs treated respectively, p<0.01),
free fatty acids (1.01 0.2 vs 0.43 0.06, untreated vs treated, p<0.01) and
interleukin 6 (35.5 14.8 vs 8.4 3.3 pg/ml, untreated vs treated, p<0.01).
CP improved insulin resistance (P<0.001) but not plasma insulin levels in the
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BTBR ob/ob. Despite these positive effects, CP did not prevent the develop-
ment of DN in the BTBR ob/ob, having no effect on glomerular hypertrophy,
mesangial matrix expansion and renal macrophage accumulation. CP also
had differential effects on the activation of fatty acid oxidation genes in the
liver and kidney. These results suggest that normal plasma leptin levels are
integral for PPAR-o agonist efcacy in the treatment of the renal complica-
tions of type II diabetes.
Supported by: MMPC MICROMouse Program (U24-DK76126)
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CCR2 Inhibition Improves Renal Function in Diabetic BKS db/db
Mice
TIMOTHY J. SULLIVAN, ZHENHUA MIAO, BIN N. ZHAO, ROBERT D. BERAHOVICH,
JAY P. POWERS, LINDA ERTL, JUAN C. JAEN, THOMAS J. SCHALL, Mountain
View, CA
Diabetic Nephropathy (DN) is a major complication of uncontrolled diabe-
tes. The chemokine receptor CCR2 has been implicated in the renal recruit-
ment of blood monocytes in response to hypertension and hyperglycemia.
We have assessed the therapeutic benet of CCR2 antagonism in a model
of diabetic nephropathy.CCX872 was dosed daily to extremely diabetic and
proteinuric 28-week old male db/db mice for 13 weeks. Assessment of 24-hr
excretion of albumin (UAER) and creatinine was performed every 3 weeks. At
the end of study, GFR was measured via inulin-FITC clearance. Renal histol-
ogy included assessment of inammatory cell inltration, glomerular size,
podocyte density, and extent of mesangial matrix.At study initiation, mice
were extremely diabetic (fasting plasma glucose 500-600 mg/dL) and pro-
teinuric (UAER 700-800 g/day). Treatment with a CCR2 antagonist signi-
cantly improved multiple renal parameters in obese, diabetic mice, including
albuminuria and serum markers of renal function. CCX872 treatment also
improved histologic measures of renal health including leukocyte inltra-
tion and podocyte density. There were no treatment-related effects on body
weight throughout the study and the improvements in renal parameters
were seen independently of any glycemic improvements. Profound improve-
ments of albuminuria, renal function and renal inammation/brosis were
seen following pharmacological intervention with a small-molecule CCR2
antagonist in a mouse model of severe diabetic nephropathy. These results
support the clinical investigation of CCR2 antagonists, such as CCX140-B, for
the treatment of diabetic nephropathy.
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Rho-Kinase Inhibition Prevents the Progression of Diabetic Neph-
ropathy via Downregulation of Hypoxia-Inducible Factor 1
KEIICHIRO MATOBA, DAIJI KAWANAMI, RINA OKADA, MASAMI TSUKAMOTO,
JUN KINOSHITA, TOMOKO ITO, SHO ISHIZAWA, YASUSHI KANAZAWA, TA-
MOTSU YOKOTA, NORIYUKI MURAI, SENYA MATSUFUJI, JUNKO TAKAHASHI-
FUJIGASAKI, KAZUNORI UTSUNOMIYA, Tokyo, Japan
The small GTPase Rho and its effector, Rho-kinase (Rho-associated coiled-
coil containing protein kinase, ROCK), are implicated in the pathogenesis of
diabetic nephropathy. Recent studies have shown that hypoxia-inducible fac-
tor 1o (HIF-1o) is a potent inducer of renal brosis under diabetic conditions.
However, the interactions of Rho-kinase and HIF-1o in the development of
renal dysfunction have not been dened. In the present study, we assessed
whether Rho-kinase blockade attenuates HIF-1o expression and subsequent
brotic response using type 2 diabetic mice and cultured mesangial cells
(MES-13). Eight-week-old db/db mice were administered Rho-kinase inhibi-
tor fasudil (100 mg/kg/day) for 24 weeks through the drinking water. Urinary
albumin excretion, mesangial matrix expansion and the expression of brotic
mediators were attenuated by Rho-kinase inhibition. From a mechanistic
standpoint, we observed that HIF-1o accumulation and the expression of its
target genes that contribute to diabetic glomerulosclerosis were also atten-
uated by fasudil in the renal cortex. Under hypoxic conditions (1% oxygen),
Rho/Rho-kinase signaling was activated in mesangial cells. Rho-kinase in-
hibitor decreased the levels of HIF-1o protein and its target genes compared
with untreated cells, whereas HIF-1o mRNA levels were not altered. Fur-
thermore, proteasome inhibitor MG132 prevented the effect of Rho-kinase
blockade on HIF-1o expression. These observations were also conrmed by
hypoxia mimetic deferoxamine. Our data indicated that the Rho/Rho-kinase
pathway might be involved in the prevention of proteasome-dependent HIF-
1o degradation under low oxygen conditions. The present study shows an
unrecognized mechanism for the renoprotective properties of Rho-kinase
inhibition and suggests that Rho-kinase could be regarded as a potential
therapeutic target for the treatment of diabetic nephropathy.
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Hyperglycemia-Induced SHP-1 Expression Cause Insulin Resis-
tance in Podocytes
NICOLAS DRAPEAU, FARAH LIZOTTE, PEDRO M. GERALDES, Sherbrooke, QC,
Canada
Renal podocytes apoptosis induced by hyperglycemia and insulin re-
sistance is an early event leading to diabetic nephropathy. A recent study
reported that insulin is critical for podocyte survival. However, how hyper-
glycemia induces insulin resistance in podocytes is not fully understood.
Recent studies demonstrated that SHP-1, a protein tyrosine phosphatase,
is elevated in renal cortex of type 1 diabetic mice (Akita). The aim of this
study is to evaluate the role of SHP-1 actions on hyperglycemia-induced
insulin resistance leading to podocyte dysfunction. We exposed cultured
podocytes to 5.6 mmol/L (NG) or 25 mmol/L (HG) of glucose concentration up
to 3-4 days with or without insulin (10-50 nmol/L). SHP-1 mRNA and protein
levels were evaluated by qPCR, and western blot. Effects of HG and SHP-1
on insulin signaling pathway (Akt and ERK) were assessed using adenovirus
of SHP-1 dominant negative and wild-type. Glomerular ltration rate (GFR),
urine albumin/creatinine ratio (uACR), foot process effacement, mRNA and
protein expression of SHP-1 and insulin signaling pathway were performed
on 9 months non-diabetic (NDM) and diabetic Akita (C57BL/6J-Ins2
Akita
)
mice. Podocytes exposed to HG expressed high mRNA and protein levels
of SHP-1. Moreover, HG concentrations decreased insulin-induced Akt and
ERK phosphorylation by 33% and 77%, respectively. Overexpression of
SHP-1 dominant negative in podocytes prevented HG effects and restored
insulin actions. Renal pathology was conrmed in Akita mice by elevated
GFR (53%), uACR (4-fold) and foot process effacement as compared to NDM
mice. Insulin injection increased Akt and ERK phosphorylation in renal cortex
of NDM mice but were unchanged in renal Akita mice. This renal insulin
resistance correlates with elevated SHP-1 mRNA (glomeruli) and protein
(cortex) levels of Akita compared to NDM mice. In conclusion, our results
showed that hyperglycemia and diabetes increase SHP-1 expression causing
insulin resistance, podocyte loss and diabetic nephropathy.
Supported by: Kidney Foundation of Canada, JDRF
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The Effects of Diabetes and Angiotensin on the Loss of Glucagon-
Like Peptide-1s Protective Actions in Diabetic Endothelial Glom-
erulopathy
AKIRA MIMA, QIAN LI, KOJI MIZUTANI, KYOUNGMIN PARK, CHRISTIAN RASK-
MADSEN, GEORGE L. KING, Boston, MA
The nding of Glucagon-like peptide-1 (GLP-1) receptors on renal glomeruli
suggests that they may have protective actions in glomerular functions. We
studied the expression and signaling of GLP-1R on mice glomerular endothe-
lial cells and made the novel nding that GLP-1 can activate protein kinase A
to phosphorylate c-Raf at serine259 to inhibit angiotensin II (Ang II) induced
phosphorylation of serine338 of c-Raf and ERK1/2 and increased the expres-
sion of PAI-1, NF-kB, TNF-o, IL-6 and CD68. When rat glomerular endothelial
cells were exposed to phorbol 12-myristate13-acetate (PMA; 100nM), PKC
is activated and decreased GLP-1R expression by 464% and its actions. Dia-
betic mice also exhibited decreased GLP-1R expression by 359% and loss
of GLP-1s protective action and increase Ang IIs actions on phosphor-c-Raf
(Ser338), phosphor-Erk1/2 and inammatory cytokine productions (PAI-1; by
1.3 0.1-fold,NF-kB; by 1.4 0.3-fold, TNF-o; by 1.4 0.2-fold, IL-6; by 1.5
0.5-fold, CD68; 6.1 2.8-fold). Mice overexpressing PKC2 species to the
endothelial cells by VE-cadherin (EC-PKC2Tg) exhibited decreased GLP-1R
expression by 4519% and increased phosphor-c-Raf(Ser338) leading to en-
hanced effects of Ang II. Diabetic EC-PKC2Tg mice exhibited greater loss
of endothelial GLP-1R expression by 513% and exendin-4 protective actions
and exhibited more albuminuria (by 1.5 0.5-fold) and mesangial expansion
(by 1.4 0.4-fold) than diabetic controls.Thus, GLP-1 has renal protective
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actions on the glomerular endothelial cells which are mediated by inhibit-
ing Ang II action at phosphor-c-Raf(Ser338) via phosphor-c-Raf(Ser259). This
action of GLP-1 is diminished in diabetes by PKC activation via downregula-
tion of GLP-1R and the enhancement of Ang II active in diabetes.
Supported by: DERC (P30DK036836)
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Hyperuricemia Induce Renal Injury by Down-Regulating CD2-
Associated Protein
WEI REN, JIANJIN GUO, DONGMING SU, WEIPING JIA, Shanghai, China, Nan-
jing, China
The increasing incidence of the link between hyperuricemia and the
metabolic syndrome has been noted. Mild hyperuricemia has been shown
to induce hypertension, cardiovascular disease, and renal brosis. The CD2
associated protein (CD2AP) is characterized as an adaptor protein that impli-
cated in podocyte homeostasis, signal transduction, dynamic actin remodel-
ing and also membrane trafcking. CD2AP knockout mice exhibit defects in
podocyte foot processes, accompanied by mesangial cell hyperplasia and
extracellular matrix deposition, suggesting that CD2AP plays an essential
role in cell-to-cell union. In this study, we examined the effect of hyperuri-
cemia on the expression of CD2AP in renal injury. Hyperuricemia has been
developed in rat models by blocking uricase enzyme with oxonic acid and
low-salt diet. Serum creatinine (Cr), uric acid (UA) and urinary album in excre-
tion rate (UAER) were measured. The concentration of CD2AP in renal cortex
was analyzed by immunohistochemistry and Western blotting. All statistical
analyses were performed using SPSS 13.0. Compared with the control group,
the plasma uric acid level (0.9 0.1 vs. 2.2 0.5 mg/dL, P < 0.01), Cr (0.45
0.1 vs. 1.5 0.5 mg/dL, P < 0.01) and UAER(0.44 0.1 vs. 1.12 0.4 mg/24h,
P < 0.05) were markedly increased in hyperuricemia groups, and UARE was
positively correlated with plasma UA level (r = 0.72; P < 0.01). The expression
of CD2AP in kidneys was decreased signicantly in hyperuricemia groups (P
< 0.01). The plasma UA level plays an important role in the development of
nephropathy.The mechanisms maybe related to uric acid level decreased the
expression of CD2AP resulting in the dysfunction of podocyte.
Supported by: National Basic Research Program of China (2011CB504000)
Guided Audio Tour: Clinical Studies of Diabetic Kidney Disease (Posters
522-P to 529-P), see page 15.
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522-P
Reduced White Blood Cell Telomere Length in Type 1 Diabetes (T1D)
Patients, is Consistent With Accelerated Aging, but Lacks Relation-
ship With Vascular Complications
ANDRZEJ S. JANUSZEWSKI, SURYA SUTANTO, CONNIE KARSCHIMKUS, DAVID
N. ONEAL, SUSAN V. MCLENNAN, STEPHEN M. TWIGG, ANTHONY KEECH, ALI-
CIA J. JENKINS, Melbourne, Australia, Sydney, Australia, Footscray, Australia
Telomeres regulate cell ageing and death. Shorter telomeres are associated
with vascular risk factors, inammation and oxidative stress, and are associ-
ated with and predictive of vascular disease. T1D is commonly regarded as
a condition of accelerated aging.We measured telomere length in T1D and
non-diabetic (CON) subjects, to determine its association with age, renal
and vascular status, inammation and glycemia. T1D subjects; n=144, aged
(meanSD) 3814 yrs; 83F / 61M 54 with microvascular complications (CX+).
118 CON subjects: 3614 yrs, 64F / 54M were studied. telomere length was
measured in whole blood using quantitative PCR and expressed as T/S ratio in
arbitrary units (AU).Age- and gender-adjusted mean T/S in CON vs. T1DM was
1.650.04 vs. 1.510.04 AU respectively; p=0.01. T/S was similar in T1DCX[-]
1.450.06 AU vs. T1DCX[+]1.520.05 AU respectively; p=NS. T/S correlated
with age: CON (r=-0.21; p=0.02); T1D (r=-0.33; p=0.00005) and T1D duration (r=-
0.21; p=0.01). For each decade T/S was less in CON by 0.07 AU and by 0.11 AU
in T1D; p=0.01.In CON T/S correlated with CRP (r=-0.23; p-0.02), small artery
elasticity (r=0.27; p=0.004), systemic vascular resistance (r=-0.22; p=0.02) and
GFR (r=0.27; p=0.004). In T1D T/S was not correlated signicantly with HbA1c,
renal or vascular function, lipids or CRP. In multiple regression analysis of the
combined group T1D (p=0.02), age (p<0.0001) and CRP (p=0.04) were signi-
cant determinants of T/S.Age-related telomere shortening was greater in T1D
than in non-diabetic subjects suggesting accelerated aging. Telomere length
correlated with T1D duration, but did not differ by complication status nor cor-
relate with vascular health or risk factors. In non-diabetic subjects telomere
length correlated with inammation, renal function and vascular health but
not with glycemia or lipids. Age, diabetes and CRP levels were independent
determinants of telomere length.
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523-P
Patterns of Urinary Albumin Excretion Rate (AER) in Normoalbu-
minuric Normotensive (NANT) Patients (pts) With Type 1 Diabetes
(T1D)
AMEER A. KHOWAJA, MICHAEL MAUER, M. LUIZA CARAMORI, Minneapolis, MN
We previously evaluated the patterns of AER in normoalbuminuric (NA)
T1D pts in the diabetic nephropathy (DN) Natural History Study (NHS). AER
levels in these pts followed one of 4 patterns (Table). We analyzed the
data from the Renin Angiotensin System Study (RASS) to explore the re-
lationships between RAS blockers and patterns of AER in NANT T1D pts.
RASS was a multicenter, randomized, double blinded, placebo controlled
trial comparing enalapril and losartan to placebo on progression of DN
over 5 years. AER was measured at baseline, quarterly and 2 months af-
ter drug washout. Patterns were classied per NHS. AER at exit was cat-
egorized as < or ~ 20 g/min. Chi-square and analysis of variance (ANOVA)
were used for analysis. There were no statistically signicant differences
in the distribution of AER patterns among groups (Table). There was also
no group difference in the proportion of pts with AER < or ~ 20 g/min at
exit (p=0.1). At exit, AER values were higher in the losartan [4.80 (0.90-
264.30) g/min] than in the placebo [4.30 (0.40-23.90); p=0.007] group.
There was no within group differences in AER levels at exit and washout.
AER levels increased from baseline to washout among pts randomized to
placebo (p=0.01) or losartan (p= 0.003), while they did not change among
pts randomized to enalapril. However, there were no group differences in
AER values at washout (p=0.139). Our results did not show a treatment ef-
fect on AER patterns over 5 years in NANT T1D. Pts randomized to losartan
showed the greatest increase in AER levels from randomization till exit.
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524-P
Insulin Dose is Associated With Susceptibility to Overt Nephropa-
thy in Type 1 Diabetes
TREVOR J. ORCHARD, TINA COSTACOU, DEMETRIUS ELLIS, Pittsburgh, PA
Kidney disease increases mortality risk in type 1 diabetes, but in its ab-
sence mortality risk is similar to that of the general population. We assessed
the association between modiable risk factors and susceptibility to overt
nephropathy (ON). We used 18 year follow up data from the Pittsburgh EDC
study, a prospective investigation of childhood onset type 1 diabetes (n=658,
baseline mean age, 28 and duration, 19 years). ON (albumin excretion rate
>200 g/min in multiple timed urine specimen) susceptibility was dened
as developing ON within 20 years of diabetes onset and at least four years
before developing either of the other two advanced microvascular complica-
tions (proliferative retinopathy or conrmed distal symmetric polyneuropa-
thy). ON resistance was dened as being ON free after 20 years of diabetes
or developing ON within 20 years of diabetes duration but at least four years
after developing both other microvascular complications. Thus, although
susceptible to microvascular disease in general, these groupings assured
specic susceptibility or resistance to ON. Out of 419 that could be so cat-
egorized, 26 (6.2%) were ON susceptible and 393 ON resistant. Of the 393
resistant, 259 were assessed at similar diabetes duration (<20 yrs) to sus-
ceptible individuals. Compared to resistant, ON susceptible individuals were
more likely to be female (p=0.05), with higher insulin dose per body weight,
systolic and diastolic blood pressure (all p-values <0.05). In multivariable lo-
gistic regression models, female gender (OR=3.74, 95% CI: 1.33-10.49), insu-
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lin dose per body weight (OR=24.92, 95% CI: 3.62-171.56) and systolic blood
pressure (OR=1.07, 95% CI: 1.03-1.11) were independently associated with
ON susceptibility. The association with insulin dose merits further study as it
is unlikely to solely reect insulin resistance or glycemic control, for neither
a previously validated equation to estimate glucose disposal rate (eGDR,
p=0.36) nor HbA1c (p=0.93) differed by susceptibility group.
Supported by: NIH (DK34818)
&
525-P
Development of Risk Prediction Models for Chronic Kidney Disease
in Type 2 Diabetes Using Genetic and Clinical Variables
GUOZHI JIANG, ERIC S. LAU, YING WANG, ANDREA O. LUK, CLAUDIA H. TAM,
JANICE S. HO, VINCENT K. LAM, HEUNG MAN LEE, MAGGIE C. NG, XIAODAN
FAN, WING YEE SO, JULIANA C. CHAN, RONALD C. MA, Hong Kong, China
This hypothesis-generating study examined whether T2D or obesity-
related genes can predict development of chronic kidney disease (CKD).
Thirty-six single nucleotide polymorphisms (SNPs) associated with T2D,
hyperglycaemia or obesity, identied through GWAS, were genotyped in
5371 T2D patients without CKD at baseline from the Hong Kong Diabetes
Registry, of which 1287 (24.0%) developed CKD during a median follow-up
period of 8 (IQR 4.8-11.0) years. A feature selection method based on Akaike
Information Criterion (AIC) was used to select an informative variable sub-
set, and Cox proportional hazards regression was used to develop models
and detect association effects. Predictive performance was evaluated and
comparisons using C statistics and time-dependent area under the curve
(AUC) of the ROC curves.Top eighteen variables were selected as predictors
of CKD in T2D, including ACR, age, HbA1c, presence of neuropathy, as well
as 6 genetic variants. The AUC was 0.80, with tendency to decrease with
increasing follow-up time (from 0.87 to 0.80 between 0 and 10 years). The
c statistics based on variable subsets were 0.857(inter-quartile range [IQR]:
0.853-0.861) and 0.853(IQR: 0.846-0.858) on training and test data sets re-
spectively. Removing genetic variants from the variable subsets resulted in
lowering of the c statistics to 0.854(IQR: 0.851-0.858) and 0.851(IQR: 0.845-
0.855) correspondingly. Three SNPs were independently associated with
CKD after adjusting for top clinical variables, with respective hazard ratios
(95% condence interval [CI]): 0.85 (0.78-0.94, P=0.001), 1.19 (1.05-1.35,
P=0.005) and 1.17 (1.03-1.32, P=0.015). Analysis of joint effect of these 3
SNPs showed additive effect with increasing number of risk alleles (Ptrend =
4.610-5). By using the feature selection method based on AIC, we success-
fully identied an informative variable subset from our dataset containing
genetic and clinical variables.
Supported by: Innovation and Technology Fund (ITS/487/09FP)
&
526-P
Prevalence of Stage 5 Chronic Kidney Disease by Cause in a Nation-
wide Study of People With Diabetes
INEZ BRADY, ON BEHALF OF THE SCOTTISH RENAL REGISTRY AND THE SCOTTISH
DIABETES RESEARCH NETWORK, EPIDEMIOLOGY GROUP, Dundee, United
Kingdom
Our aims were to examine the prevalence of stage 5 chronic kidney disease
(CKD5) and use of renal replacement therapy (RRT) in the Scottish population
with diabetes, to examine the primary renal diagnosis (PRD) among those
on RRT and to test whether survival following RRT varies by PRD.We linked
records from the national database of all those registered with diabetes in
2006 -2008 (SCI-DC) to the Scottish Renal Registry that captures all RRT
recipients in Scotland. CKD5 by end 2008 was dened as 2 recent MDRD
derived eGFR readings s15/ml/min/1.73m
2
or being in receipt of RRT. Logistic
and Cox regression were used for analyses.The point prevalence of CKD5 in
2008 was 1.6% of 19,847 people with type 1 diabetes (T1D) compared with
0.6% of 168,176 people with type 2 diabetes (T2D) (Odds ratio [OR] = 3.98,
p<0.0001) with the difference explained by duration (OR on adjustment for
duration = 0.95). 83% of CKD5 T1DM patients and 61% of T2D CKD5 patients
were receiving RRT. Diabetic nephropathy (DN) was the PRD assigned by
the renal physician in 88% of those with T1D and 56% with T2D (p<0.0001).
Other common PRDs in T2D were multisystem diseases (16%), interstitial
nephropathies (7%) and primary glomerulonephritis (5%). In those with T2D
the following were associated with a PRD of DN; longer duration of diabetes
(OR = 1.08, p=0.0001), higher HbA
1C
(OR = 1.31, p=0.01), absence of cardio-
vascular disease pre-RRT (OR = 0.50, p=0.02) and presence of retinopathy
pre-RRT (OR= 4.82, p=0.005). Three years from start of RRT 50% (CI 45%
- 57%) of those with DN were alive with survival rates being similar for pri-
mary glomerulonephritis 54% (CI 36% - 81%), interstitial nephropathies 41%
(CI 27% - 62%) and multisystem diseases 39% (CI 29% - 53%).This provides
contemporary data on the prevalence of CKD5 in diabetes and illustrates the
heterogeneity of causes of CKD among those with T2D. However mortality
rates after starting RRT were not signicantly different in diabetic vs. non-
diabetic kidney disease among those with T2D in this study.
Supported by: Scottish Government and Wellcome Trust Scottish Health Infor-
matics Programme
&
527-P
Renal Structural Changes in Patients With T2DM, Impaired Renal
Function and Albuminuria
ELIF I. EKINCI, ALISON SKENE, KAREY CHEONG, DAVID POWER, SIANNA PA-
NAGIOTOPOULOS, KAREN MCNEIL, SOPHIE CLARKE, SCOTT BAKER, PAULA
FIORETTO, GEORGE JERUMS, RICHARD MACISAAC, Melbourne, Australia, Van-
couver, BC, Canada, Padua, Italy
The structural basis of nonalbuminuric renal insufciency in T2DM re-
mains to be elucidated. We compared renal biopsy ndings in outpatients
with T2DM, eGFR<60ml/min/1.73m
2
and normo-(N), micro-() or macro-(M)
albuminuria attending a tertiary hospital. A minimum of 6 glomeruli was ex-
amined by light microscopy (LM) which included immuno-uorescence/his-
tochemistry. In subjects with N or , eGFR was supplemented by iGFR (DTPA).
All biopsies were assessed by light (LM) and electron (EM) microscopy and
the ndings categorised according to Fioretto classication
1
- Category 1
(C1): Normal/near normal, Category 2 (C2): Typical diabetic nephropathy (DN)
& Category 3 (C3): Atypical (disproportionately severe interstitial/tubular/
vascular damage with no/mild diabetic glomerular changes). There was a
predominance of typical DN (C2) in all stages of albuminuria. Subjects with
M all had C2. In subjects with N, 7/8 had varying degrees of arteriosclero-
sis. Histological evidence of DN, manifesting as either glomerulopathy (C2)
or as interstitial/vascular damage (C3) is still likely to be found in patients
with T2DM and non albuminuric renal insufciency (table). This suggests
that age, BP and vascular disease contribute to changes in renal structure
and function. In subjects the present results differ from previous data
1
in
34 subjects with T2DM, albuminuria and GFR of 101 ml/min/1.73m
2
. In that
study, the biopsies were classied as 35% C1, 30% C2 and 35% C3. This
raises the possibility that GFR in subjects in C1 and C3 does not decline as
fast as in C2, but studies of GFR trajectory would be needed to conrm this.
Fioretto Classication (1. P Fioretto et al.,
Diabetologia 39, 1569 (1996)
N
(n = 8)
(n = 6)
M
(n = 17)
C1 1 (12.5%) 0 (0%) 0 (0%)
C2 4 (50%) 5 (83%) 17 (100%)
C3 3 (37.5%) 1 (17%) 0 (0%)
Supported by: NHMRC
&
528-P
IGF-II Associated Genes May Prevent Progression of Diabetic
Nephropathy
RAM P. NARAYANAN, SIMON G. ANDERSON, BO FU, ANTHONY PAYTON, JOHN
P. NEW, ADRIAN H. HEALD, WILLIAM E. OLLIER, JOHN M. GIBSON, Salford,
United Kingdom, Manchester, United Kingdom
IGF-II is expressed in the kidney with increased expression in some re-
nal neoplasms. Interactions of IGF-II in diabetic nephropathy are unknown
despite its structural and functional similarities with insulin. We studied
single nucleotide polymorphisms (SNPs) of the IGF2 gene, its closely coupled
regulatory gene H19 and the IGF-II receptor gene IGF2R with respect to lon-
gitudinal trends in glomerular ltration rate (eGFR)528 Caucasian individu-
als with type 2 diabetes were studied. Phenotypic data were derived from
linked primary care and hospital records. Genotyping of HapMap selected
haplotype tagging SNPs was performed on Sequenom iPlex . Quality assur-
ance using SVS7 (Golden Helix) was performed. Mixed effects regression
was used to analyse longitudinal trends over 8 years in eGFR in STATA 10SE
corrected gene-wise for multiple testing.Eight IGF2, eleven H19 and twelve
IGF2R SNPs were selected . 7 SNPs were highly associated with longitu-
dinal trends in eGFR adjusted for gender, age, diabetes duration and ACE
inhibitor use. eGFR was calculated from serum creatinine using the Modi-
cation of Diet in Renal Disease (MDRD) formula.Minor alleles of three IGF2
SNPs were associated with preserved eGFR - rs734351 ( 0.53,[95% CI 0.35
to 0.71], p<0.001); rs10770098( 0.33,[95% CI 0.13 to 0.53], p=0.001) and
rs1003483 ( 0.32,[95% CI 0.12 to 0.52], p=0.001). Also associated with good
renal function were rs6578973 ( 0.29, [95% CI 0.09 to 0.49], p=0.003) near
the H19 gene as well as the IGF2R SNP rs609207 (=0.28, [95% CI 0.10 to
0.46], p=0.002).Minor alleles of two SNPs favoured a decline in eGFR- IGF2
SNP rs2000993 ( -0.49, [95% CI -0.66 to -0.31], p<0.001), and IGF2R SNP
rs761389 ( -0.57, [95% CI -0.85 to -0.28], p<0.001).All associations were
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sustained after Bonferroni correction.These novel associations of IGF2 and
related genes with longitudinal renal function suggest a protective role for
IGF-II in preventing progression of diabetic nephropathy with implications
for cellular action in the kidney.
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529-P
Relationship Between Abdominal Obesity and Diabetic Nephropa-
thy According to the Distribution of Fat Tissue in Korean Type 2 Dia-
betic Patients
MIN YOUNG CHUNG, JIN OOK CHUNG, DONG HYEOK CHO, DONG JIN CHUNG,
Gwangju, Republic of Korea
Waist circumference correlates to both visceral and subcutaneous fat
tissue. Recently, abdominal obesity assessed by waist circumference has
been implicated in the pathogenesis of diabetic nephropathy. However, the
impacts of visceral and subcutaneous fat tissue on cardiometabolic disease
have been suggested to be different from each other. In addition, the im-
pact of perirenal fat tissue on diabetic nephropathy has not been studied.
The aim of this study was to evaluate the difference between visceral, sub-
cutaneous, and perirenal fat tissue in relation to diabetic nephropathy in
Korean type 2 diabetic patients. Two hundred and twenty type 2 diabetic
patients (M: 113, F: 107; mean age: 60.9 11.6 years) were recruited. All
subjects were assessed for the metabolic proles, urinary albumin excre-
tion rate (UAE) and estimated glomerular ltration rate (eGFR). The visceral,
subcutaneous and perirenal fat tissue were measured by ultrasonography.
In the univariate correlation analysis, visceral fat tissue was positively as-
sociated with UAE. Also, there was a signicant positive association be-
tween subcutaneous fat tissue and eGFR. However, perirenal fat tissue was
not signicantly associated with UAE or eGFR. In a multivariate analysis,
increased visceral fat tissue was an independent positive determinant for
macroalbuminuria (>300 g/mg creatinine) after adjustment of age, gender,
diabetes duration, glycated hemoglobin, systolic blood pressure, and use of
angiotensin-converting enzyme inhibitor and angiotensin-receptor blocker,
while increased subcutaneous fat tissue showed an independent inverse as-
sociation for reduced eGFR (< 60 mL/min/1.73 m
2
) in multivariate analysis.
This study suggests that abdominal fat tissue is associated with the differ-
ent natural course of diabetic nephropathy according to body fat distribution
in Korean type 2 diabetic patients.
530-P
Obstructive Sleep Apnea is Independently Associated With Dia-
betic Nephropathy in Patients With Type 2 Diabetes
ABD A. TAHRANI, ASAD ALI, SAFIA BEGUM, KIRAN DUBB, SHANAZ MUGHAL,
BIJU JOSE, MILAN PIYA, ANTHONY BARNETT, MARTIN STEVENS, Birmingham,
United Kingdom, Coventry, United Kingdom
Diabetic nephropathy (DN) causes signicant morbidity and mortality.
Understanding DN pathogenesis is essential to develop new therapies. Ob-
structive sleep apnea (OSA) is prevalent in patients with type 2 diabetes
(T2D). Since OSA and DN may share common oxidative stress and inamma-
tory mechanisms, we hypothesized that OSA is associated with DN.Adults
with T2D were recruited randomly from the diabetes clinic of a UK-based
hospital. Patients with known respiratory disorders (including OSA) and
end-stage renal disease were excluded. DN was dened by eGFR of <60 ml/
min/1.73m2 or the presence of albuminuria after exclusion of other causes.
OSA was assessed using home-based portable multi-channel respiratory
monitoring (Alice PDX, Philips Respironics, USA). OSA diagnosed when the
apnoea-hypopnea index was ~5 events/hour (OSA+).T2D patients (n=234)
were included, 64.5% were OSA+ and 39.7% and 36.2% had DN and albu-
minuria respectively. OSA+ patients were older, more obese and had higher
BP compared to those without OSA. DN (29.7% vs. 55.9%, p<0.001) and
albuminuria (24.3% vs. 43.2%, p=0.007) prevalence were higher in OSA+ pa-
tients. OSA+ patients had lower eGFR (92.9 25.2 vs. 82.4 26.4, p=0.006).
OSA+ remained independently associated with DN (OR 2.251, 95%CI 1.069-
4.741, p=0.033) after adjusting for age, gender, ethnicity, BP, HbA1c, total
cholesterol, triglycerides, HDL, diabetes duration, smoking, alcohol, obesity,
and the use of anti-platelet, anti-diabetes, lipid-lowering and anti-hyper-
tensive treatments. In addition, nadir nocturnal oxygen saturations were an
independent predictor of DN (OR 0.957, 95% CI 0.920-0.995, p=0.027) when
it replaced OSA in the model.In conclusion, we describe a novel association
between OSA and DN and nocturnal hypoxia in patients with T2D. Ongoing
studies are exploring the mechanisms involved. The ability of OSA treatment
to impact DN warrants determination.
Supported by: NIHR (UK)
531-P
Triglycerides, NMR Lipoprotein Subclasses and Albuminuria in Men
With Type 2 Diabetes: A Veterans Affairs Diabetes Trial Sub-Study
ALICIA JENKINS, DERRICK G. KAUFMAN, JULIE STONER, RICK L. KLEIN, MARIA
LOPES-VIRELLA, TIMOTHY J. LYONS, VA DIABETES TRIAL INVESTIGATORS, Okla-
homa City, OK, Hines, IL, Charleston, SC
In diabetes, nephropathy can induce dyslipidemia which in turn may accel-
erate renal decline. Nuclear magnetic resonance (NMR) lipoproteinanalysis
can provide detail regarding the lipoprotein species implicated. NMR (Lipo-
Science Inc, Raleigh, NC) was performed on a sub-set of 331 VADT men (age
60 (9) y (mean (SD)): diabetes duration 11 (7) y; BMI 32.6 (5.1) kg/m
2
; HbA1c 7.7
(1.5)%), of whom 91% had hypertension, and 88% were on lipid drugs. Fast-
ing samples were obtained within 6 months of the 2-year post-randomiza-
tion visit. In cross-sectional analysis, using linear regression of the associa-
tion between (natural log-transformed) urine albumin:creatinine ratio (ACR)
(dependent variable) and (standardized natural log-transformed) lipoprotein
measures, adjusted for total LDL particle concentration but unadjusted for
other covariates, fasting triglyceride was the only conventional lipid asso-
ciated with ACR regression coefcient =0.23, p=0.036). Triglycerides re-
mained associated with ACR when adjusted for age, diabetes duration, race,
VADT randomization status (hence HbA1c), hypertension, lipid drugs, BMI
and WHR (=0.22, p=0.049). NMR lipoproteins that were inversely related
to ACR in adjusted cross-sectional analyses were: LDL particle size; particle
concentrations of large LDL and total and medium HDL concentrations (all
p<0.05). In longitudinal follow-up (1-5y, mean 2.3y), triglycerides were as-
sociated with average ACR over time (=0.34, p=0.019, fully adjusted), and
the following NMR lipoprotein subclasses were inversely associated with
average ACR over time: LDL particle size and medium HDL concentration
(both p<0.05, fully adjusted). No lipoprotein measure predicted decline in
ACR over time. In conclusion, in cross-sectional and longitudinal analyses,
triglycerides, and NMR measures of LDL and HDL were associated with ACR
during the VADT intervention period, but none predicted change in ACR over
an average of 2.3 y.
532-P
Estimated Glomerular Filtration Rate and Albuminuria: True Predic-
tors of Cardiovascular Events in Obese Patients With Type 2 Dia-
betes?
MICHAEL RESL, GREISA VILA, RICHARD PACHER, ANTON LUGER, MARTIN HL-
SMANN, STEPHANIE NEUHOLD, HELMUT BRATH, RUDOLF PRAGER, MARTIN
CLODI, Vienna, Austria
The widely used MDRD formula underestimates kidney function in obese
diabetic patients. Therefore, we aimed to evaluate the impact of this cal-
culation bias on the predictive value of eGFR for cardiovascular events in a
typical cohort of diabetic patients.In general 988 patients were analysed.
Cox Regression models including the variables HbA1c, age, duration of dia-
betes, eGFR and urinary albumin to creatinine ratio (UACR) were run. At rst
the whole collective was analysed, in a second step the cohort was split into
4 different groups according to BMI and eGFR (Group 1, 475 Pts : eGFR > 60
ml/min; BMI < 30 kg/m
2
, Group 2, 274 Pts : eGFR > 60 ml/min; BMI >30 kg/m
2
,
Group 3 110 Pts, : eGFR < 60 ml/min; BMI > 30 kg/m
2
and Group 4, 129 Pts. :
eGFR < 60 ml/min; BMI < 30 kg/m
2
). The endpoint was dened as unplanned
cardiovascular hospitalization.Patients (571 male, 417 female) were 61 22
years of age, mean duration of diabetes was 14.3 12.3 years. After a me-
dian follow up of 29 months 95 (9.6%) patients reached the dened endpoint.
The rst model, including all patients showed that UACR (HR 1.001, p <0.001)
and eGFR (HR 0.957, p<0.001) were signicant predictors of the composite
endpoint. In obese patients eGFR completely lost its predictive value for
cardiovascular events.Noteworthy the prevalence of normoalbuminuria in
patients with an eGFR below 60 ml/min was 59.4%. In obese patients eGFR
is not predictive for cardiovascular events.
533-P
Associations of Serum Pigment Epithelium Derived Factor Levels
With Renal Dysfunction, Body Habitus and Dyslipidemia in Type 2
Diabetes Patients
ALICIA JENKINS, DONG-XU FU, JULIE STONER, DERRICK KAUFMAN, SARAH X.
ZHANG, RICK KLEIN, MARIA LOPES-VIRELLA, JIAN-XING MA, TIMOTHY J. LY-
ONS, VA DIABETES TRIAL INVESTIGATORS, Oklahoma City, OK, Hines, IL, Charles-
ton, SC
The serpin Pigment Epithelium Derived Factor (PEDF) has anti-angiogenic,
anti-inammatory, and anti-oxidant effects, and in cell culture and animal
models, is protective against microvascular damage. We determined if se-
rum PEDF in Type 2 diabetes patients was related to vascular risk factors
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and renal function. Serum PEDF was quantied by ELISA in a sub-study of
254 male Veterans Affairs Diabetes Trial (VADT) subjects, sampled within 6
months of the 2-year post-randomization visit (age: 60 (9) y (mean (SD); diabe-
tes duration: 11 (7) years; BMI: 32.6 (4.8) kg/m
2
; HbA1c: 7.7 (1.4)%), of whom
88% were hypertensive and 85% were taking lipid-lowering drugs. Univari-
ate, cross-sectional associationsbetween PEDF and risk factors were quan-
tied by Spearmans rank correlation coefcients. Mixed model regression
analyses were used to evaluate longitudinal associations between PEDF and
renal function (dependent variable), with and without adjustment for age,
ethnicity,diabetes duration, hypertension, body habitus, VADT randomiza-
tion, and lipid drugs. Cross-sectional study: Among 17 variables tested, PEDF
(ng/ml) correlated with body habitus (BMI: p=0.26, p<0.0001; waist-to-hip
ratio: p=0.20, p=0.0018), renal function (serum creatinine: p=0.29, p<0.0001;
estimated GFR (mL/min/1.73 m
2
): p=-0.27, p<0.0001), and lipids (fasting
triglycerides (mg/dl): p=0.35, p<0.0001; HDL-cholesterol(mg/dl): p=-0.33,
p<0.0001). PEDF correlated inversely with CrCl (Cockcroft-Gault) and GFR
(regression coefcient =-2.13, p<0.0001 and =-1.95, p<0.0001), but not
with log-transformed urine ACR (p=0.11)after adjustment for confounders.
Longitudinal study: PEDF was not associated with longitudinal renal function
changes. In conclusion, serum PEDF in VADT Type 2 diabetes patients was
associated with adiposity, dyslipidemia and with renal dysfunction, but was
not associated with subsequent changes in renal function.
534-P
Obesity Modies the Association between Leptin Levels and Kidney
Function Decline in Patients With Type 2 Diabetes
KO HANAI, TETSUYA BABAZONO, YASUKO UCHIGATA, Tokyo, Japan
We have recently demonstrated that both low and high serum leptin levels
are risk factors for kidney function decline in patients with type 2 diabetes
(T2D). Obesity has been shown to be a modier of the association between
leptin levels and cardiovascular events. We tested the hypothesis that
obesity modies the relationship between serum leptin levels and kidney
function decline in patients with T2D.This was a longitudinal observational
cohort study of 411 Japanese adult patients with T2D (mean [ SD] age: 58
13 years, men: 61.1%). Patients were classied into 2 groups by sex-specic
median of serum leptin levels. Obesity was dened as body mass index ~ 25
kg/m
2
. Outcome measurement was the annual rate of decline in estimated
glomerular ltration rate (eGFR).During the median follow-up period of 4.8
years (range: 2.0 - 8.2), the mean rate of change in eGFR was -1.5 3.7 mL/
min/1.73 m
2
/year. A signicant interaction between serum leptin levels (low
or high) and obesity (present or absent) on the rate of change in eGFR was
detected (P interaction =0.01). In the non-obese group, the adjusted rate of
change in eGFR (mean SE) in patients with low leptin was steeper than that
in patients with high leptin (-1.9 0.3 and -0.6 0.5 mL/min/1.73 m
2
/year,
p =0.04, ANCOVA). In the obese group, the adjusted rate of change in eGFR
in patients with high leptin was not signicantly different with that in pa-
tients with low leptin (-1.5 0.3 and -0.8 0.5 mL/min/1.73 m
2
/year, p =0.25).
When leptin levels were treated as a continuous variable, logarithmically
transformed leptin levels were positively associated with the rate of change
in eGFR, independent of other variables in non-obese patients (standard-
ized estimate = 0.22, p <0.01). In contrast, in obese patients, leptin levels
were negatively associated with the rate of change in eGFR (standardized
estimate = -0.24, p =0.03).The effects of leptin levels on the kidney function
decline may depend on the presence of obesity in patients with T2D.
535-P
The Prevalence of Albuminuria According to Status of Autoimmu-
nity and Insulin Sensitivity among Youth With Diabetes
AMY K. MOTTL, ABIGAIL LAUER, RALPH B. DAGOSTINO, JR., MICHAEL MAUER,
DAVID MAAHS, LAWRENCE M. DOLAN, LISA K. GILLIAM, JEAN M. LAWRENCE,
MARYAM AFKARIAN, KRISTI REYNOLDS, SANTICA M. MARCOVINA, GIUSEPPI-
NA IMPERATORE, DANA DABELEA, ELIZABETH MAYER-DAVIS, FOR THE SEARCH
FOR DIABETES IN YOUTH STUDY GROUP, Chapel Hill, NC, Winston-Salem, NC,
Minneapolis, MN, Aurora, CO, Cincinnati, OH, Seattle, WA, Pasadena, CA, Atlanta,
GA
It is unknown how autoimmunity and insulin resistance affect the natural
history of diabetic kidney disease. To address this issue, we evaluated the
effect of these characteristics on the risk of albuminuria in children with dia-
betes.The SEARCH study is an observational study of children with type 1 or
2 diabetes who are followed longitudinally. Diabetes autoantibodies (DAA),
insulin sensitivity (using a validated equation based on euglycemic hyper-
insulinemic clamp data) and urinary albumin:creatinine ratio (UACR) from a
single, random urine specimen were measured. Data from the most recent
visit were used for all analyses. Participants were grouped into four etiologic
subclasses of diabetes: 1) DAA+/insulin sensitive (IS) (n=1833); 2) DAA+/in-
sulin resistant (IR) (n=1462); 3)DAA-/IS (n=682) and 4) DAA-/IR (n=978). Mul-
tivariate regression analyses adjusted for race/ethnicity, sociodemographics
and diabetes duration, to assess the relationship between these subclasses
of diabetes and albuminuria (UACR ~ 30ug/mg). Interaction with race/eth-
nicity, mean arterial pressure (MAP), hemoglobin A1C and body mass index
was explored.Participants included in the analysis (n=4,955) had a mean age
of 13.7 yrs (SD=4.4) and mean diabetes duration 49 mos (SD=39). Prevalence
of albuminuria was 8.2% DAA+/IS, 9.8% DAA+/IR, 7% DAA-/IS and 17%
DAA-/IR group. With DAA+/IS as the reference group, the adjusted odds
ratio (OR) of albuminuria was signicant only in the DAA-/IR group (OR=1.6;
95% CI 1.2, 2.1). Only MAP modied this association, wherein the higher
the MAP, the greater the relative difference in prevalence of albuminuria in
the DAA-/IR vs DAA+/IS groups (interaction term p-value=0.0003).In youth
with diabetes of relatively short duration, , there is a signicantly increased
prevalence of albuminuria in those with IR and DAA- but not in those with
IR and DAA+, compared to those with DAA and IS. This distinction may be
mediated by differences in MAP.
Supported by: The CDC with support from the NIDDK
536-P
Estimating Early Glomerular Filtration Rate (GFR) Decline in Dia-
betes by the Chronic Kidney Disease-Epidemiology Collaboration
(CKD-EPI) Equation
RICHARD J. MACISAAC, KAREY CHEONG, ELIF EKINCI, SHILPA VERMA, EROSHA
PREMARATNE, ZHONG X. LU, KEN A. SIKARIS, GEORGE JERUMS, Melbourne,
Australia, Heidelberg, Australia, Collingwood, Australia
Evaluating GFR using the modication of diet in renal disease (MDRD)
equation underestimates reference GFR levels >60 ml/min/1.73m
2
, whereas
an estimated GFR (eGFR) derived from the CKD-EPI equation is reported to be
less biased. However, the accuracy of the CKD-EPI equation in people with
diabetes remains to be dened. We therefore assessed the performance of
an eGFR calculated from the CKD-EPI or MDRD equations compared with
reference GFR measurements in subjects with diabetes. The reference GFR
was measured using
99m
Tc-DTPA plasma clearance (iGFR) and creatinine was
measured by an enzymatic method. In a cross-sectional study, bias (eGFR -
iGFR) was compared for the CKD-EPI and MDRD equations for subjects with
iGFR measurements > 60 ml/min/1.73m
2
. In a longitudinal study of subjects
with an early decline in GFR i.e., initial iGFR >90 ml/min/1.73m
2
and AiGFR
~3.3 ml/min/1.73 m
2
per year, AiGFR (based on initial and nal values) was
compared with AeGFR by the CKD-EPI and MDRD equations over a mean of
9 years. In the cross-sectional study, iGFR for the whole group was 80 2.2
ml/min/1.73m
2
(n=199, 75% type 2 DM). For subjects with an iGFR >90 ml/
min/1.73m
2
(iGFR: 112 2.0, n=76), both equations signicantly underesti-
mated iGFR to a similar extent: bias for CKD-EPI -12 1.4 ml/min/1.73m
2
(p <
0.001) and for MDRD -11 2.1 ml/min/1.73m
2
(p < 0.001). By contrast, for sub-
jects with an iGFR between 60 and 90 ml/min/1.73m
2
(iGFR 77 1.2, n=59),
there was a non-signicant trend for both equations to overestimate iGFR.
In the longitudinal study (n=30, 66% type 1 DM), initial and nal iGFR values
were 102.8 6 and 54.6 6.0 ml/min/1.73m
2
, respectively. Mean AGFR (ml/
min/1.73m
2
per year) was 6.2 by iGFR compared with 3.1 by MDRD and 3.3 by
CKD-EPI (both p < 0.05 vs iGFR). In conclusion, both the CKD-EPI and MDRD
equations underestimate reference GFR values >90 ml/min/1.73m
2
as well as
an early decline in GFR to a similar extent.
537-P
Microvascular Disease in Type 1 Diabetes (T1D): The Predictive
Power of A1c Variability (A1cV)
STEPHEN A. DELURGIO, PRIYA S. RAMAN, DAVID D. WILLIAMS, MARK A. CLE-
MENTS, Kansas City, MO
A1cV has been shown to correlate with increased risk of microvascular
disease (MD) for those with T1D; whether A1cV is useful in predicting MD
is unknown. This study explores the utility of the standard deviation (SD) of
A1cs in predicting level II nephropathy among T1D patients using Cox Propor-
tional Hazard models. The DCCT study cohort (n=1439, 333 events) was split:
2/3 (n=965, 222 events) for model development and 1/3 (n=474,111 events)
to test models in risk predictions. All models (M, S, B, F) include age, sex,
T1D duration, and treatment group as covariates. Model M included only the
Mean (X
2
=141, p<0.000); S, the SD alone (X
2
=120, p<=0.000); B, Both mean
and SD (X
2
=147, p<0.000). F is Model B tted to the Full cohort and serves
as a benchmark for accurate predictions (X
2
=201, p<=0.000) for the 474 pa-
tients withheld when developing models M, S, and B. Using SPSS 19, risk
predictions with Model B were more accurate than with M. Correlations of
the risks of F with those predicted with M, S, and B were r
FM
=.952, r
FS
=.913,
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and r
FB
=.986, (p< 0.0000001 for all). Statistical difference tests of rs yield
t-values of 8.59 for r
FB
-r
FM
, 14.30 for r
FB
-r
FS
, and 9.59 for r
FM
-r
FS
(p<0.00001
for all), indicating signicant differences between r
FB
, r
FM
, and r
FS
. Kendalls
W tests conrmed that there are differences in the 474 predicted risks of all
models at p<0.000001. Using both the mean and SD as predictors of level
II nephropathy better discriminates between those with medium and low
hazards compared to using only the mean (hazard curves, Figure 1). Further
analyses using retinopathy data will be presented.
538-P
High Glucose Induces ROS Production by NADPH Oxidases via a
Src-Dependent Mechanism
KEN W. LEE, LING XIA, HOWARD GOLDBERG, ANU SHAH, CATHARINE WHITE-
SIDE, I. GEORGE FANTUS, Toronto, ON, Canada
The pathogenesis of diabetic nephropathy (DN), a leading cause of end
stage renal disease, remains incompletely understood. The increased gen-
eration of reactive oxygen species (ROS) and consequent oxidative stress
is a mediator of the pathologic changes caused by chronic exposure to high
glucose (HG). Src has been reported to be both upstream and downstream of
ROS. To determine its role in DN, cultured primary rat mesangial cells (MCs)
were exposed to either normal glucose (NG 5mmol/L) or HG (25 mmol/L)
and total cell ROS measured using 2,7-dichlorouorescein (DCF) and dihy-
droethidium (DHE), uorescence. NADPH oxidase activity was assayed with
lucigenin. Src activity was blocked by the inhibitors, Dasatinib (DS) and AZD,
as well as by transfection with Src-specic siRNA. After 3 h, HG signicantly
induced cell ROS and NADPH oxidase activity that were markedly decreased
by DS, AZD, and Src siRNA. In contrast, mitoSOX loaded cells showed that
HG-induced mitochondria-specic ROS were unaffected. Thus, we examined
the role of Nox2 and Nox4. Nox2-specic siRNA abrogated ROS production
in short-term HG (1-3 h), while Nox4-specic siRNA blocked ROS production
only in long-term HG (24-48 h). Consistent with these results, HG induced
Nox4 protein showed a time-dependent signicant increase at 24-48 h.
Treatment with DS, AZD, and Src-specic siRNA blocked Nox4 induction. Ex-
posure to hydrogen peroxide in the setting of NG and in the presence of Src
inhibitors, augmented Nox4 protein, while the antioxidant Tempol blocked
HG induction of Nox4. These data indicate that a Src-dependent rapid ac-
tivation of Nox2 by HG generates a ROS signal to upregulate Nox4, that is
responsible for long term increased ROS which mediates the pathological
effects of chronic hyperglycemia.
539-P
Prognostic Value of Resting Heart Rate on Renal Outcomes in Type
2 Diabetic Patients: A Competing Risk Analysis in a Prospective
Cohort
SAMY HADJADJ, AURELIE MIOT, STEPHANIE RAGOT, WALA HAMMI, PIERRE-
JEAN SAULNIER, PHILIPPE SOSNER, XAVIER PIGUEL, RICHARD MARECHAUD,
RONAN ROUSSEL, Poitiers, France
Epidemiological studies and clinical trials suggest that resting heart rate
(RHR) was an independent predictor of cardiovascular (CV) risk and all-cause
mortality. However no data evaluated the relationship between RHR and re-
nal complications in type 2 diabetes (T2D) patients.Method: We performed
a single-centre, prospective analysis in 1088 T2D patients. RHR was deter-
mined by ECG. The study outcome was time to a renal composite criterion
(renal replacement therapy or chronic doubling of baseline serum creatinine)
adjusted for all-cause death as a competing event.Results: During median
follow-up of 4.2 years, 62 patients (6%) experienced the study outcome. At
baseline, a history of CV and of renal disease was noted in 336 (31%) and
367 (34%) patients, respectively. An interaction was found between RHR
and history of CV (but not renal) disease at baseline for the relation between
RHR and study outcome (p
interaction
=0.03). In those patients with CV disease
at baseline (CVD+), those developing a renal outcome had a higher RHR than
non-affected patients: 77 13 versus 66 12 bpm (p <0.0001), while no such
effect was noticed in those patients non-CVD+.In CVD+, RHR was associ-
ated with renal risk when adjusted for all-cause death as a competing event
(p <0.0001). In multivariate analysis in CVD+, predictors of the renal outcome
were RHR (SHR=1.04; p=0.001) and renal disease at baseline. In non-CVD+,
no relation was found between RHR and the incidence of CV and/or renal
events.Conclusion: In the real-life setting, RHR constitutes an easy, quick,
efcient and cost-effective factor that would permit identication of dia-
betic patients with an increased risk for severe renal complications.
540-P
Impact of High Glucose on the Histone Ubiquitylation in Rat Glom-
erular Mesangial Cells
YONG XU, CHENLIN GAO, GUO CHEN, Luzhou, Sichuan, China
The modication of histones by ubiquitylation is a prominent epigenetic
mark that features in a variety of chromatin-based events such as histone
methylation, gene silencing, and repair of DNA damage.The bulk of histone
ubiquitylation occurs on chromatin by the addition of a single ubiquitin mol-
ecule via an isopeptide linkage to a specic lysine residue of histones H2A
and H2B. Ubiquitylation on different histones has distinct functions. Howev-
er, he effect of histone ubiquitylation on the diabetic nephropathy is unclear.
Recently, we cultured HBZY-1 rat glomerular mesangial cells(GMCs) and
divided them into 6 groups : normal glucose group( 5.6mmol/L),high glucose
group(10,20,30 mmol/L),mannitol group was used as high osmosis control
and proteasome inhibitor group (30mmol/Lglucose pretreated with MG132
as a proteasome specic inhibitor).The ubiquitylation of histones was mea-
sured by Western blot and indirect immunuorescence laser scanning con-
focal microscope respectively. The results showed that the ubiquitylation
of H2A in GMCs in normal group was weak, whereas the ubiquitylation of
H2A in GMCs in high glucose group was stronger than that in normal group
(P<0.05) in a concentration -dependent manner. After pretreated with pro-
teasome inhibitor MG132, the ubiquitylation of H2A in GMCs was decreased
obviously compared with 20,30mmol/L glucose group (all P<0.05).But for the
ubiquitylation of H2B,we found the opposite changes in high glucose groups
(P<0.05).These data support the hypothesis that high glucose affects the
ubiquitylation of histone in GMCs and imply that the ubiquitylation of his-
tone may be take part in the pathogenesis of diabetic nephropathy.
541-P
The 9p21 Coronary Artery Disease Locus and Kidney Dysfunction in
Patients With Type 2 Diabetes Mellitus
SALVATORE DE COSMO, SABRINA PRUDENTE, OLGA LAMACCHIA, HETAL SHAH,
CHRISITINE MENDONCA, DANIELA LUCCHESI, LAURA PUCCI, LUANA MERCURI,
DIEGO BAILETTI, GIUSEPPE PENNO, MAURO CIGNARELLI, ALESSANDRO DORIA,
VINCENZO TRISCHITTA, San Giovanni Rotondo, Foggia, Italy, Boston, MA, Pisa,
Italy, Rome, Italy
Cardiovascular disease and kidney dysfunction share a common patho-
genic soil, raising the hypothesis that they also share, at least in part, a
common genetic background. We investigated whether rs2383206 at the
chromosome 9p21 locus, a single nucleotide polymorphism (SNP) which is
strongly associated with coronary artery disease (CAD) both in the general
population and in diabetic patients, is also associated with low estimated
glomerular ltration rate (eGFR) or increased urinary albumin excretion (UAE)
in patients with type 2 diabetes mellitus (T2DM). Four independent samples
(three from center-south Italy, one from Boston, MA) of White patients with
T2DM, for a total of 3,167 individuals, were studied. Low eGFR (<60 ml/
min/1.73 m
2
) was calculated from serum creatinine according to the MDRD
Study equation. Increased UAE was dened as an albumin-creatinine ratio
(ACR) > 2.5 in men or > 3.5 mg/mmol in women. All study participants were
typed for SNP rs2383206 by means of TaqMan assay. No association was
found between rs2383206 and low eGFR or increased UAE in each individual
sample. Similarly, in the pooled analysis, no association was found between
s2383206 and low eGFR (594 cases and 2,573 controls; additive OR=1.07,
95% CI=0.94-1.22, p=0.31) or increased UAE (1,009 cases and 2,121 controls;
additive OR=1.00, 95% CI=0.90-1.12, p=0.95). No interaction was observed
between rs2383206 and HbA1c (i.e., below or above the median value of the
pooled sample=7.7%) in modulating eGFR or UAE (p for SNP-by-A1c inter-
action=0.42 and 0.90, respectively).In conclusion, variability at the chromo-
some 9p21 locus is unlikely to play a role in modulating the risk of developing
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kidney dysfunction in patients with T2DM. Whether other genetic markers
are shared between CAD and kidney dysfunction in diabetic patients will
have to be investigated by means of further collaborative studies.
542-P
Adolescent Type 1 Diabetes Cardio-Renal Intervention Trial: Screen-
ing Results
M. LOREDANA MARCOVECCHIO, ON BEHALF OF THE ADDIT STUDY GROUP, Cam-
bridge, United Kingdom
To describe baseline markers of cardiovascular and renal disease in the
AdDIT screening population.Adolescents with type 1 diabetes (T1D) are be-
ing screened with 2 sets of 3 consecutive early morning urines for measure-
ment of albumin-creatinine ratio (ACR). Tertiles of ACR are calculated using
an algorithm adjusting for age, gender and duration: the upper tertile identi-
es the high-risk, the lower and middle tertiles the low-risk group. Blood
samples are being collected for centralized measurement of cardiovascular
and renal markers and standardized cardiovascular assessments are being
performed.2515 adolescents (54% males, mean (SD) age:13.11.7yr; median
[IQR] T1D duration 4.8 [2.4-7.7]yr) have been assigned to ACR tertiles: 919
(36.5%) upper, 829 (33%) middle,767 (30.5%) lower. ACR progressively in-
creases from the lower to the upper tertile (0.51 [0.42-0.62], 0.70 [0.58-0.85],
1.26 [0.91-1.82] mg/mmol, p<0.001); mean HbA1c is 8.41.4%, with small
differences across ACR tertiles (8.31.3%, 8.41.3%, 8.51.6%, p=0.03).
Renal markers (creatinine and symmetric dimethylarginine), available from
171 high- and 76 low-risk participants, are signicantly lower in the high-risk
group (48.5 [42.9-57.9] vs 53.9 [46.2-62.7] mol/l, p=0.002; 398.5255.53 vs
424.4360.37 nmol/l, p=0.001, respectively) indicating hyperltration. The
prevalence of abnormal lipid proles is higher in the high-risk group (total
cholesterol ~5.2mmol/l: 19.9 vs 13.2%) but this is not signicant. The cardio-
vascular assessments (n 270 high-risk, n 121 low-risk) indicate a higher prev-
alence of high diastolic blood pressure (20.2 vs 8.3%, p=0.003), and greater
pulse wave velocity (5.00 [4.45-5.50] vs 4.70 [4.40-5.00] m/s, p=0.001) in
the high-risk group, indicating increased arterial stiffness.These preliminary
data indicate early abnormalities in renal and cardiovascular function in ado-
lescents with T1D and ACR in the upper part of the normal range, suggesting
a higher risk of developing complications.
Supported by: JDRF, Diabetes UK, British Heart Foundation
543-P
Podocyte Excretion in the Urine of Patients With Diabetic Neph-
ropathy as a Marker of Podocyte Injury
MASAO TOYODA, EITARO TANAKA, NAOYUKI YAMAMOTO, MASAAKI MI-
YAUCHI, MORITSUGU KIMURA, TOMOYA UMEZONO, MITSUNORI YAGAME,
MASANORI HARA, DAISUKE SUZUKI, MASAFUMI FUKAGAWA, Isehara, Japan,
Niigata, Japan
According to various reports, detection of podocytes in the urine indicates
severe injury to these cells in the glomeruli. Therefore, investigation of uri-
nary podocyte excretion in relation to the development and progression of
diabetic nephropathy (DN) is thought to be important. Cilnidipine is an oral
N/L-type calcium channel blocker that has been reported to inhibit sympa-
thetic activity and has a stronger renoprotective effect than L-type calcium
channel blockers.To investigate the relationship between the stage of DN
and the urinary excretion of podocytes, the number of podocytes in the urine
wvas measured in patients with type 2 diabetes. Furthermore, we studied
the inuence of N/L-type calcium channel blocker treatment on albuminuria
and podocyturia.One hundred patients with type 2 diabetes, ranging from
normoalbuminuria to end-stage renal failure, were enrolled in this study. We
evaluated the correlations between the number of urinary podocytes and
clinical parameters. Thirteen patients with podocyturia and urinary albumin
excretion (UAE) >300 mg/g Cr despite treatment with angiotensin-converting
enzyme inhibitor (ACE-I) or angiotensin II receptor blocker (ARB) were given
additional treatment with the N/L-type calcium channel blocker cilnidipine.A
signicant increase in the number of urinary podocytes was seen along with
the progression of albuminuria, and there was a signicant positive correla-
tion between the number of podocytes excreted in the urine and UAE. Treat-
ment with cilnidipine for 3 months did not reduce the mean UAE. However,
there was a signicant reduction in the number of urinary podocytes com-
pared with the baseline value.Measurement of the urinary podocyte count
might be useful for detecting podocyte injury and for assessing the reno-
protective effect of treatment. Our results also suggest that cilnidipine may
have an additional podocyte protective effect in patients with DN who are
already receiving recommended renoprotective treatment.
544-P
The Expression and Role of Notch Signal Related Molecules in Me-
sangial Cells Induced by High Glucose
YONG XU, LIU LI, GAO CHENLIN, Luzhou, Sichuan, China
The Notch signaling pathway is a widespread and highly conserved signal-
ing pathways. Recent studies have conrmed a member of the Notch family
involved in podocyte injury,tubular differentiation and renal brosis. Gamma
secretase is the key proteases of cut Notch receptor and form reactive Notch
intracellular dormin (NICD), so it is also the core link of regulating Notch sig-
naling pathways . This study observe the changes of Notch signal related
protein molecules (Notch1, Jagged1, Hes1) and brosis molecules(Fibronectin
and TGFbeta) in the rat glomerular mesangial cells (GMC) stimulated by high
glucose or gamma - secretion inhibitors (DAPT) and explore the mechanism
of Notch signaling pathway in diabetic nephropathy. We divided cultured
rat GMC into 4 groups:the normal control group (5.5mmol/L glucose), high
glucose group (30mmol/L glucose),high glucose + DAPT group and mannitol
group (high osmosis control). Western-blot,RT- PCR techniques and immu-
nouorescence confocal microscopy were used to detect the expression of
Notch1, Jagged1, Hes1, Fibronectin (FN), TGFbeta protein and mRNA expres-
sion.We found the protein and mRNA expression of Notch1, Jagged1, Hes1 in
normal cells were weak. After high glucose treated, the protein expression
was enhanced signicantly ( P < 0.01 ) . Pretreated DAPT to high glucose, the
protein and mRNA expression of Notch1, Jagged1, Hes1 were decreased (P
< 0.01 ). The expression of FN and TGF beta were shown the same changes.
Our study indicate that high glucose is involved in the development of dia-
betic glomerular brosis via Notch pathway activation.
Supported by: NNSF of China
545-P
Glycemia Inuences Cystatin C in Youth With Diabetes: SEARCH for
Diabetes in Youth Study
ROOPA KANAKATTI SHANKAR, DAVID MAAHS, LAWRENCE M. DOLAN, AN-
DREA ANDERSON, GIUSEPPINA IMPERATORE, DANA DABELEA, KRISTI REYN-
OLDS, IRL HIRSCH, ELIZABETH MAYER-DAVIS, SANTICA M. MARCOVINA,
RALPH B. DAGOSTINO, JR., MICHAEL MAUER, AMY K. MOTTL, Cincinnati, OH,
Denver, CO, Winston-Salem, NC, Atlanta, GA, Pasadena, CA, Seattle, WA, Chapel
Hill, NC, Minneapolis, MN
There has been increasing interest in serum cystatin C as a potential
marker of glomerular ltration rate (GFR) in children. The distribution and
factors inuencing cystatin C levels in pediatric patients with diabetes are
largely unknown.The SEARCH for Diabetes in Youth Study is a large, mul-
ticenter cohort study of youth with type 1 and 2 diabetes identied soon
after diagnosis and followed longitudinally. Cystatin C was measured in 952
participants (825 with Type 1 and 127 with Type 2 diabetes) with a mean
duration of diabetes of 31 +/- 9.6 months. We used step-wise regression
models to explore the inuence of diabetes duration, fasting plasma glu-
cose, glycated hemoglobin (A1c), systolic and diastolic blood pressure (BP)
and body mass index (BMI), adjusting for known cystatin C determinants
such as age, gender, and race/ethnicity. Given the small sample size of type
2 diabetes and differences in comorbidities by diabetes type, analyses were
run separately for Type 1 and Type 2 diabetes.In youth with type 1 diabetes,
adjusted cystatin C levels were negatively associated with glucose con-
centration (p<0.001, = -0.00023) and A1c (p<0.001, = -0.01126). In youth
with type 2 diabetes, adjusted cystatin C was negatively associated with
glucose concentration (p<0.001, = -0.00069) but not signicantly with A1c,
and it was positively associated with BMI Z-score (p=0.005, = 0.05224).
We found no statistically signicant association with diastolic or systolic BP.
Hyperglycemia is associated with lower cystatin C levels in both Type 1 and
Type 2 diabetes of short duration, likely reecting acute hyperltration. The
effects of acute vs. chronic hyperglycemia on cystatin C may differ by type of
diabetes and needs to be further explored. In children with Type 2 diabetes
who have a greater prevalence of obesity, studies exploring the relationship
of cystatin C to BMI are also needed.
546-P
Sleep-Disordered Breathing as a Modiable Risk Factor for Micro-
vascular Complications in Japanese Type 2 Diabetes Mellitus
SHINYA FURUKAWA, TERUKI MIYAKE, SHINYA YAMAMOTO, TETSUJI NIIYA,
TERUHISA UEDA, TERU KUMAGI, TAKENORI SAKAI, HIROAKI MIYAOKA,
SUSUMU SAKURAI, ISAO SAITO, BUNZO MATSUURA, TAKESHI TANIGAWA,
MORIKAZU ONJI, Toon, Japan, Matsuyama, Japan, Yawatahama, Japan
Background: The associations between sleep-disordered breathing (SDB)
and type 2 diabetes mellitus (T2DM) have been reported in cross-sectional
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study. Although SDB and cardiovascular disease are closely related, the as-
sociation between SDB and microvascular complications remains unclear.
We hypothesized SDB may be a potential and treatable risk factor for mi-
crovascular complications. Aim: To estimate the prevalence of SDB among
Japanese T2DM and to examine the association between SDB and micro-
vascular complications. Method: We conducted a cross-sectional study of
690 Japanese T2DM aged from 20 to 85 years old. All participants com-
pleted self-administered questionnaires on the duration of diabetes, ethanol
intake per day, numbers of cigarettes smoked per day, use of medications
(antihypertension and antihyperlipidemia) and the score on the Epworth
Sleepiness Scale. Urinary albumin-creatinine ratio (UACR) was calculated
using rst morning urine sample, and microalbuminuria was dened by UACR
of 30 mg/gcr or greater. A pulse-oximeter was used to assess SDB.Results:
The prevalence of SDB, dened by 3% oxygen desaturation index (ODI) of 5
events/hr or more, was 45% among Japanese T2DM. There were positive
associations between 3% ODI and body mass index (BMI), hypertension, hy-
perlipidemia and microalbuminuria (P<0.0005 in all). Multivariate analysis
identied BMI (P=0.0001), age (P=0.002), hyperlipidemia (P=0.028), hyper-
tension (P=0.030) and microalbuminuria (P=0.032) as associated factors for
SDB. Finally, hypertension (P=0.0001) and SDB (P=0.0115) are independently
associated with microalbuminuria after adjusting age, sex, BMI and dura-
tion of T2DM. Conclusion: We identied the prevalence of SDB in Japanese
T2DM, and SDB was independently associated with microalbuminuria. SDB
may be a potential and treatable risk factor to prevent microvascular com-
plications in T2DM.
Supported by: Grants-in-Aid for Young Scientist (B) (No. 217090583)
547-P
Zinc Deciency Exacerbates Diabetes-Induced Renal Oxidative
Damage, Inammation and Fibrosis via Down-Regulation of Nrf2
Expression
WENPENG CUI, BING LI, YI TAN, YANG BAI, QIANG CHEN, XIAO MIAO, LINING
MIAO, LU CAI, Louisville, KY, ChangChun, China, Jilin, China
Zinc (Zn) deciency often occurs in patients with diabetes; therefore, here
the effect of Zn deciency on diabetic renal damage was investigated. Type
1 diabetes was induced in FVB mice with multiple low doses of strepto-
zotocin. Once hyperglycemia was established, diabetic and age-matched
control mice were treated with and without Zn chelator, N, N, N, N-tetrakis
(2-pyridylemethyl) ethylenediamine (TPEN) at 5 mg/kg daily for 4 months.
Renal oxidative damage, inammation and brosis were examined by his-
topathological observation, Naphthol AS-D Chloroacetate esterase assay,
immunouorescent staining, and Western blotting assay. Mechanistic study
was done with HK-11 renal tubular cells in vitro. Chronic TPEN treatment sig-
nicantly decreased renal Zn levels in both diabetic and control mice. Com-
pared to groups of diabetes and TPEN alone, Diabetes/TPEN group showed
a signicant decrease in nuclear factor-erythroid 2-related factor 2 (Nrf2)
expression along with signicant increases in renal oxidative damage (pro-
tein nitration and lipid peroxidation), inammation [inltrated inammatory
cells and inammatory mediators (PAI-1 and ICAM-1)], and brosis (collagen
accumulation in glomerulus mesangial area and up-regulation of probrotic
mediator CTGF). Treatment of HK 11 cells with TPEN signicantly removed
intracellular Zn with a signicant increase in CTGF expression, which de-
creased Nrf2 expression and nuclear distribution and also prevented sul-
foraphane-induced Nrf2 expression and function. These PTEN effects could
be signicantly attenuated by Zn supplementation with signicant increase
in Nrf2 expression and function. These results indicated that Zn is required
in part for Nrf2 expression and function; Zn deciency signicantly enhanced
diabetes-induced renal oxidative damage, inammation and structural re-
modeling, via down-regulation of Nrf2 expression and function.
548-P
Interaction of Angiotensinogen and Atrial Natriuretic Peptide Un-
der Hyperglycemia in the Kidney
SHYI J. SHIN, CHAO S. LO, CHAO H. CHEN, KUN D. LIN, Kaohsiung, Taiwan
Hyperglycemia can activate intrarenal angiotensinogen(ANG) and atrial
natriuretic peptide (ANP) synthesis. We recently reported that renal ANP
can attenuate high glucose-activated TGF-1, collagen I and NF-kB ex-
pression through auto/paracrine action in renal tubular cells (J Cell Physiol
219:776-86, 2009). We aimed to investigate the interaction between ANG
and ANP in the kidneys under hyperglycemia. In this study, we found high
glucose signicantly increased ANG mRNA and ANGII immunoreactivity at
12, 24, and 24 hr and ANP mRNA and ANP immunoreactivity at 24 and 48 hr
in NRK-52E cells. The increase of ANP mRNA and immunoreactivity stimu-
lated by high glucose was attenuated by the addition of losartan (10
-6
M Los)
and PD123319 (10
-6
M PD) in cultured NRK-52E cells and by the administra-
tion of angiotensin converting enzyme inhibitor in STZ-induced diabetic rats.
ANP mRNA and immunoreactivity increased 1.75 and 2.45-fold at 1 and 2 hr
by adding 10
-6
M ANGII in NRK-52E cells. Urine ANP excretion from bilateral
ureter was signicantly increased at 1 and 2 hr after perfusion of 500ng/
kg/hr angiotensin II through left renal artery of normal rats, but no change
during perfusion with 0.9% NaCl, 3% NaCl and 50 % glucose. In NRK-52E
cells transfected with ANG siRNA, high glucose-stimulated increase of ANP
mNRA and immunoreactivity was signicantly decreased as compared to
control siRNA. Similarly, the transfection of ANP siRNA aggravated the
stimulation of ANG, TGF-1 mRNA and immunoreactivity by high glucose in
NRK-52E cells. In conclusion, high glucose-activated ANG can increase ANP
synthesis that then attenuates ANG, and TGF-1 synthesis.
549-P
Association between TGF-B1, IGF-1 and HLA and Diabetic Neph-
ropathy in T1DM Pediatric Patients from Brazil
KARLA S. SOUZA, MARCELA A. URURAHY, YONARA M. OLIVEIRA, MELINA B.
LOUREIRO, HEGLAYNE P. SILVA, FRANCISCO P. FREIRE-NETO, GUSTAVO H. OL-
IVEIRA, FABRICIO M. SANTOS, RICARDO F. ARRAIS, ROSARIO D. HIRATA, MARIO
H. HIRATA, EDUARDO A. DONADI, MARIA G. ALMEIDA, ADRIANA A. REZENDE,
Natal, Brazil, So Paulo, Brazil, Ribeiro Preto, Brazil
Diabetic nephropathy (DN) is the leading cause of renal failure. TGF-B1
and IGF-1 are crucial mediators in the pathogenesis of DN and likewise HLA
region has a possible inuence. Thus, the objective of present study was
to investigate the association between TGF-B1 (rs1800469), IGF-1 (rs35767)
and HLA (HLA-DRB1,-DQA1, and -DQB1) polymorphisms with DN in type 1
diabetic children and adolescents assisted at a pediatric hospital (HOSPED/
UFRN) in Natal-RN/Brazil. This study included 101 type 1 diabetic patients
(T1DM group) and 106 normoglycemic subjects (NG group) aged between 6
and 20 years. Metabolic control was evaluated by glucose and glycated he-
moglobin. Albumin to creatinine ratio (ACR) was calculated. Polymorphisms
was determined by allelic discrimination technique in real time PCR using
TaqMan
pre-designed SNP assays from Applied Biosystems. TGF-B1 and

IGF-1 mRNA expression were also evaluated by real time PCR. HLA gene
alleles were examined by the polymerase chain reaction (PCR-SSO reverse).
Glucose and glycated hemoglobin were signicantly increased in T1DM
group compared to NG, indicating a poor metabolic control of these patients.
Enhanced ACR was observed in T1DM group when compared to NG, suggest-
ing an initial stage renal injury. Seven T1DM patients had microalbuminuria
and presented the following alleles: DRB1*04:05 (p=0,009; OR=0,009) and
DQA1*03:02 (p=0,009; OR=0,009) and genotypes: DRB1*03:01 DRB1*0405
(p=0,041; OR=0,070) and DQA1*03:02 (p=0,041; OR=0,070), but no signicant
association was seen. TGF-B1 (p=0.015) and IGF-1 (p=0.002) polymorphisms
were signicantly associated with ACR in those patients, which may have
generated a signicant increase in TGF-B1 (p=0.001) and IGF-1 (p=0.029)
mRNA expression in T1DM patients when compared to NG, which can lead
to renal damage. These results suggest that genes TGF-B1 and IGF-1 can
inuence the genetic susceptibility to microalbuminuria in patients with
T1DM.
Supported by: CNPq/Brazil
550-P
Aging, Kidney Function, and Treatment for Type 2 Diabetes: The
RIACE Study
ANNA SOLINI, GIUSEPPE PENNO, GIACOMO ZOPPINI, CECILIA FONDELLI, GIAM-
PAOLO ZERBINI, EMANUELA ORSI, ROBERTO TREVISAN, MONICA VEDOVATO,
MARCO G. BARONI, MAURO CIGNARELLI, FRANCESCO GIORGINO, RAFFAELLA
BUZZETTI, ELE FERRANNINI, GIUSEPPE PUGLIESE, Pisa, Italy, Verona, Italy, Siena,
Italy, Milan, Italy, Bergamo, Italy, Padova, Italy, Cagliari, Italy, Foggia, Italy, Bari, Italy,
Rome, Italy
Use of some glucose-lowering agents in type 2 diabetes raises concern in
the elderly or in pts with renal impairment. Few studies have described the
prevalence of these treatments in real-life situations. We used data from
the Renal Insufciency And Cardiovascular Events (RIACE) Italian Multi-
center Study. The cohort consists of 15,773 type 2 diabetes pts attending 19
diabetes clinics in 2007-2008. Pts were divided into age quartiles (quartile
median [IQR]: 53 [8], 63 [4], 69 [3] and 77 [6] yrs); treatment was coded as
no pharmacological therapy (Diet), metformin (Met), sulphonylureas (SU),
glinides (Gli), thiazolidinediones (TZD), or insulin (Ins). Glomerular ltration
rate (eGFR) was estimated by the CKD-EPI equation, identifying four classes
of eGFR: 1 (>90); 2 (60-89); 3 (30-59); 4 (<30) ml/min/1.73m
2
. Across age
quartiles, BMI declined (from 29.95.8 to 27.84.5 kg/m
2
, p<0.0001), while
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pulse blood pressure increased (from 5313 to 6317 mmHg, p<0.0001).
HbA
1c
, LDL-cholesterol, and triglycerides decreased slightly if signicantly
(all p<0.01) across age groups, while HDL-cholesterol increased (from 4713
to 5114 mg/dl, p<0.0001). eGFR declined progressively (from 9618 to 6719
ml/min/1.73m
2
, p<0.0001) - at a time-linear rate of 1.1 ml/min/1.73m
2
equally
in men and women. Likewise, micro- and macro-albuminuria increased with
age quartile (from 18.3 to 27.0% and from 3.9 to 6%, p<0.0001). Between the
lowest and highest age quartile, diet declined from 17 to 11%, Met and TZD
fell (from 59 to 47% and from 5 to 2%), while SU and Gli rose from 26 to 41%
and from 8 to 11% (all p<0.0001): Ins use was stable (24 to 26%). In CKD-EPI 4
class (n=304, age 749 yrs, eGFR 236 ml/min/1.73m
2
, 73% micro- or macro-
albuminuric), Gli and SU usage was 16 and 18%, respectively, and Met was
14%. Even in CKD-EPI 3 (n=2411, age=739 yrs, eGFR=498 ml/min/1.73m
2
),
Met and/or SU was 76%. In real-life situation, use of agents that are not
recommended in older pts with severe renal impairment is still prevalent.
Supported by: Research Foundation of the Italian Society of Diabetology
551-P
Taurine Ameliorates the Progression of Diabetic Nephropathy in
Type 2 Diabetic Rat Model
EUN SOO LEE, YEON SIK CHOI, MI YOUNG LEE, EUN YOUNG LEE, CHOON HEE
CHUNG, WonJu, Republic of Korea, Soonchunhyang, Republic of Korea
Diabetic nephropathy is a major cause of end stage renal disease in the
diabetic complication. Recent studies suggested that high glucose increased
reactive oxygen species which lead to tissue damage through the expression
of inammatory factors.Taurine is a 2-aminoethanesulfonic acid which is
found in the most animal tissues. According to previous studies, taurine have
several functions such as osmoregulation, bile acid conjugation, cell mem-
brane stabilization, cholesterol excretion and cell volume regulation. And it
prevents hyperglycemia induced insulin resistance through the reduction of
oxidative stress.Thus, we investigated whether taurine may reduced blood
glucose, oxidative stress and inammation or not.Experimental animals were
divided into four groups; normal group (LETO, n=10), DM group (OLETF, n=10),
taurine treated group (OLETF, n=10) and losartan treated group (OLETF, n=10).
We treated them for 20 weeks from 26 to 45 weeks of age, and measured
blood glucose, urine ACR, MCP-1 levels, GBM thickness, slit pore numbers
and glomerular volume.As a result, body weight was decreased in DM group
compared with control group, which was recovered similar to that of control
group in taurine and losartan treated groups. And 24hrs urinary albumin and
ACR were signicantly reduced in taurine treated group compared with DM
group. Moreover, we found taurine decreased vascular endothelial growth
factor (VEGF) protein and mRNA as well as, monocyte chemotacticprotein-1
(MCP-1) mRNA expression in the kidney tissue. It also decreased glomerular
basement membrane thickness and glomerular volume, and increased open
slit pore numbers between podocyte and foot processes. But there were no
signicant difference in IPGTT data between taurine treated and DM groups.
In conclusion, these results suggest that taurine ameliorates the progression
of diabetic nephropathy. We need more studies to search the mechanisms
why taurrine protect the renal injury in type 2 diabetic rat model.
552-P
Proteinuria and Chronic Kidney Disease Predict 10-year Cancer-
Related and All-Cause Mortalities in Type 2 Diabetic Patients
TSE-YA YU, HUNG-YUAN LI, YI-DER JIANG, TIEN-JYUN CHANG, JUNG-NAN
WEI, LEE-MING CHUANG, Tainan, Taiwan, Taipei, Taiwan
Proteinuria has been found to be associated with cancer mortality in gen-
eral population. However, this relationship remains unknown in type 2 dia-
betes. We conducted a cohort study to determine if proteinuria, associated
with diabetic nephropathy, predicts cancer-related mortality in subjects
with type 2 diabetes. Between July 1996 and June 2003, we enrolled 646
subjects with type 2 diabetes at the National Taiwan University Hospital. A
spot urine sample was collected to determine the presence of proteinuria at
enrollment. The vital status of all subjects was ascertained by linking their
data with computerized death certicates in Taiwan. The medium follow-up
period was 10.4 years. Subjects with proteinuria had a hazard ratio (HR) of
2.83 (95% CI 1.87-4.29) for all-cause mortality and 2.02 (95% CI 1.03-4.00)
for cancer-related mortality when adjusted for age, gender, smoking, his-
tory of cardiovascular disease, obesity, hypertension, use of angiotensin-
converting enzyme inhibitors or angiotensin II receptor blockers, hemoglobin
A1c, diabetes duration, total cholesterol, use of statins, abnormal ankle-bra-
chial index, and estimated glomerular ltration rate. The presence of protei-
nuria signicantly improved the predictive ability of cancer-related mortality
(increase in concordance statistics = 0.03 and increase in area under the
ROC curve = 0.02). Similarly, the presence of chronic kidney disease (CKD)
was associated with increased all-cause and cancer-related mortality (HR
2.12 [95% CI 1.46-3.09], and 1.94 [95% CI 1.04-3.51], respectively). In conclu-
sion, proteinuria and CKD can predict 10-year all-cause mortality and cancer-
related mortality independently in individuals with type 2 diabetes, over and
above the established risk factors associated with type 2 diabetes.
553-P
Low-Grade Albuminuria and the Role of Urine Storage in Type 2
Diabetes
NADAN GREGORIC, DRAZENKA P. BARLOVIC, JELKA ZALETEL, Ljubljana, Slovenia
Microalbuminuria is associated with an increased risk for development
of kidney and vascular disease in type 2 diabetes. Screening for microalbu-
minuria is therefore an important tool in preventive care. Albuminuria deter-
mined from overnight urine collections is one of the most direct measures of
urinary albumin excretion. However, one of its drawbacks is the requirement
of urine storage at low temperatures. Therefore, the aim of our study was
to assess whether a more convenient storage of urine samples at ambient
temperature affects the reliability of results.In patients with type 2 diabetes
included in DEMAND (delapril and manidipine for nephroprotection in dia-
betes) study albuminuria was determined by radioimmunoassay with series
of three consecutive timed overnight samples on two different occasions.
The samples were stored at ambient temperatures. We calculated a differ-
ence in albuminuria measurement between the fresh sample and samples
stored for 1 or 2 days, respectively. A paired-sample T-test was used to test
for a statistical signicance between the two groups.From 68 patients with
type 2 diabetes (age 67+/-8,5 years; glycated hemoglobin 8,4+/-1,3 %; se-
rum creatinine 76+/-13,3 mol/L; blood pressure 147/81+/-15/8 mmHg; body
mass index 30,5+/-5,1 kg/m2; mean+/-SD) 15 were microalbuminuric and 53
normoalbuminuric. The absolute differences in albuminuria among timed
overnight urine samples that were stored for 1 or 2 days compared to fresh
sample on two different occasions were not statistically signicant (4,3+/-
7,4 vs. 4,8+/-7,8 g/min and 5,5+/-8,4 vs. 4,3+/-5,8 g/min, respectively, all
p values >0,05).Urine storage at ambient temperature for 2 days does not
affect reliability of albuminuria values in patients with type 2 diabetes and
low-grade albuminuria.
554-P
Predicting Decline of Renal Function among People With Stage 3
Chronic Kidney Disease and Type 2 Diabetes
HELEN C. LOOKER, HARSHAL DESHMUKH, ANDREW D. MORRIS, COLIN N. PALM-
ER, HELEN M. COLHOUN, Dundee, United Kingdom, Kircaldy, United Kingdom
The power of trials of drugs for improving renal function in diabetes rely
on progression rates in entrants who are typically at stage 3 of chronic kid-
ney disease (CKD). Further stratication for rapid progressors among such
entrants would help to improve trial design. Therefore we have used longi-
tudinal estimated glomerular ltration rate (eGFR) data from a contemporary
cohort of people with type 2 diabetes (T2D) and stage 3 CKD to identify risk
factors which predict a reduction of ~50% of baseline eGFR within a 5 year
period.Patients were identied from the GoDARTS study which recruited
6500 people with T2D in Tayside, Scotland, between 1 October 1997 and
1 July 2006. EGFR and covariate data were obtained by data linkage to the
national clinical diabetes database (SCI-DC) which contains data on ~99% of
people with diagnosed diabetes in Scotland. We selected those with stage 3
CKD on enrolment into GoDARTS (eGFR = 30-59 mls/min/1.73m
2
) and at least
one subsequent eGFR measure. Cases were dened as people who under-
went a 50% reduction in eGFR during follow-up and controls as people with
a reduction of eGFR <10% over a maximum follow up of 5 yearsOf the 1997
people with a stage 3 CKD at baseline 266 were cases and 265 controls.
Median time to a 50% drop in eGFR was 1.9 years (95% Condence Interval
(CI) 1.1-2.8), and median follow up for the controls was 3.3 years (95% CI 2.8-
4.0).In a multivariate logistic regression model the following baseline factors
were signicantly associated with being a case after adjustment for base-
line eGFR: diabetes duration (OR per 5 years = 1.30, 95% CI 1.14-1.48), and
systolic blood pressure (OR per 10 mmHg= 1.15, 95% CI 1.05-1.26). Lipids and
HbA1c were not associated with a drop in eGFR ~50%.Stratication with
risk factors for renal progression could improve clinical trial power; the role
of the predictors reported here now needs validation in additional datasets.
Supported by: SUMMIT Consortium Funded by the IMI-JU
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555-P
Clinicopathological Risk Factors for the Prognosis of Diabetic
Nephro pathy in Japanese Patients With Type 2 Diabetes
NOSE CHIKAKO, MIHO SHIMIZU, KENGO FURUICHI, TADASHI TOYAMA, AKINORI
HARA, KIYOKI KITAGAWA, YASHUNORI IWATA, SHUICHI KANEKO, TAKASHI
WADA, Kanazawa, Japan
We evaluated the predictive impact of albuminuria, glomerular ltration
rate (GFR), and renal pathological factors for the prognosis of Japanese pa-
tients with type 2 diabetes.Two hundred and sixty Japanese patients with
histologically proven diabetic nephropathy (164 males and 96 females) were
examined. Patients were classied by urinary albumin excretion and esti-
mated GFR (eGFR) at the time of renal biopsy. Renal events were assessed
by requirement for dialysis or 50% reduction of eGFR. Predictors of renal
events, cardiovascular events and mortality were examined by the Cox pro-
portional hazards regression.A mean follow-up period was 7.9 years. At the
time of renal biopsy, the number of cases with normoalbuminuria (<30mg/
day) or urinary protein (-) or (), microalbuminuria (30-299mg/day) or urinary
protein (+), and macroalbuminuria (~300mg/day) or urinary protein~(2+)
were 47, 50 and 163, respectively. The proportion of subjects with low eGFR
(<60ml/min/1.73m
2
) was 36% in patients with normoalbuminuria, 42% in pa-
tients with microalbuminuria, and 71% in patients with macroalbuminuria.
During the observational periods, 116 patients experienced renal events, 62
patients experienced cardiovascular events, and 45 patients experienced
death. Regarding of renal events, cardiovascular events, and mortality, mac-
roalbuminuria was the main predictor. Patients with macroalbuminuria and
low eGFR had a 16.3-fold higher risk of renal events, a 5.7-fold higher risk
of cardiovascular events and a 13.8-fold higher risk of death than that of
normoalbuminuric patients with preserved eGFR (~60ml/min/1.73m
2
). Patho-
logical risk factors for renal events were atherosclerosis, nodular lesions,
exudative lesions, mesangiolysis, diffuse lesions, and interstitial brosis.
These results suggest that macroalbuminuria was the main predictor of re-
nal events, cardiovascular events, and mortality of diabetic nephropathy in
Japanese patients with type 2 diabetes.
556-P
Phycocyanin and Phycocyanobilin from Spirulina Platensis Amelio-
rate Diabetic Nephropathy via Attenuating Oxidative Stress
JING ZHENG, TOYOSHI INOGUCHI, YASUTAKA MAEDA, MARK MACARTY, SHUJI
SASAKI, MASAKAZU FUJII, NORIKO IKEDA, KUNIHISA KOBAYASHI, NORIYUKI
SONODA, RYOICHI TAKAYANAGI, Fukuoka, Japan, Encinitas, CA
In recent years, bilirubin has been paid attention to as an endogenous
strong antioxidant. We and other investigators have reported that bilirubin
and its precursor biliverdin may have benecial effects on diabetic vascular
complications, including nephropathy, via its antioxidant effects. Here, we
investigated whether phycocyanin derived from Spirulina platensis, a blue-
green algae, and its chromophore phycocyanobilin, which has a chemical
structure similar to that of biliverdin, protect against oxidative stress and
renal dysfunction in db/db mice, a rodent model for type 2 diabetes. Oral
administration of phycocyanin (300 mg/kg) for 10 weeks protected against
albuminuria and renal mesangial expansion in db/db mice, and normalized
tumor growth factor- and bronectin expression. In parallel, phycocyanin
normalized oxidative stress makers such as urinary levels of 8-hydroxy-2-
deoxyguanosine and 8-iso-prostaglandin F
2o
, and renal dihydroethidium
(DHE) staining and normalized the expression of NAD(P)H oxidase compo-
nents (Nox4, p22phox and p47phox). Oral administration of phycocyanobilin
(15 mg/kg) for 2 weeks also showed similar antioxidant effects. In cultured
renal mesangial cells, phycocyanobilin as well as bilirubin and biliverdin in-
hibited NADPH oxidase-dependent superoxide production in a dose depen-
dency evaluated by the Lucigenin assay. Phycocyanobilin also inhibited an-
giotensin II-induced intracellular reactive oxygen species in mesangila cells
evaluated by DHE staining. In conclusion, oral administration of phycocyanin
and phycocyanobilin may offer a novel and feasible therapeutic approach for
preventing diabetic nephropathy.
557-P
Differential Effect of Vitamin C Deciency on the Pathogenesis of
Diabetic Nephropathy
HIROSHI OKADA, TAKAFUMI SENMARU, YOSHITAKA KONDO, AKIHITO ISHIGA-
MI, HIROSHI OBAYASHI, MAI ASANO, MASAHIRO YAMAZAKI, MICHIAKI FUKUI,
GOJI HASEGAWA, NAOTO NAKAMURA, Kyoto, Japan, Tokyo, Japan
Vitamin C (VC)can be attributed to its biological functions as a cofactor for
a number ofenzymes and as an antioxidant. It can alsoparticipate in oxida-
tive protein folding in the endoplasmic reticulum (ER). Inthis study, we inves-
tigated the effect of VC deciency on the pathogenesis of diabetic nephrop-
athy using senescence marker protein-30 (SMP30)/glucolactonaseknockout
mouse, the VC-decient animal model. Diabetes was inducedusing strepto-
zocin in male SMP30 Y/- mice (KO) and wild-type, SMP30 Y/+, mice (WT) at 8
weeks of age. Diabetic (DM) or control (C) mice were divided into 2 groups;
a VC-supplemented (VC (+)) group and a VC-decient (VC (-)) group. After 12
weeks of diabetes, the renal histology and expression (qRT-PCR) of markers
of ERstress, oxidative stress, inammation and brosis were estimated.It
was noteworthy that cortical brosis area (Sirius red stain, x1005 elds/N)
was markedly increased only in KO-DM-VC(-) (200003.232707.2mm
2
,
P<0.0001). There were no differences among the other groups including
KO-C-VC(-)(45276.49933.2~7477012901.7mm
2
). On the other hand, in
%mesangium/glomerular area (PAS stain, 10 glomeruli/N),there were no sig-
nicant differences among the groups. MCP-1 expression wassignicantly
increased in KO-DM-VC(-) and KO-DM-VC(+) compared to the othergroups
(P<0.0001). The expression of p67phox, PPAR, and TGF-were increased
in DM groups of both strains, and PPAR expression in KO-DM-VC(-)was
signicantly lower than KO-DM-VC(+) (P<0.03). These results suggest that
thereis a distinct difference in the pathogenesis between glomerular lesion
andinterstitial brosis (tubulointerstitial lesion) of diabetic nephropathy.
Themechanisms induced by VC deciency are likely to be complex includin-
goxidative stress, inactivation of some enzymes, and mitochondrialinjury.
This study points to potential new therapeutic approaches for advanced-
stage of diabetic nephropathy.
558-P
IL-1B and TNF-A Genes may be Associated With Microalbuminuria
Onset in T1DM Pediatric Patients from Brazil
MARCELA A. URURAHY, KARLA S. SOUZA, YONARA M. OLIVEIRA, MELINA B.
LOUREIRO, HEGLAYNE P. SILVA, FRANCISCO P. FREIRE-NETO, JOO F. BEZERRA,
RAUL H. BORTOLIN, SONIA Q. DOI, RICARDO F. ARRAIS, ROSARIO D. HIRATA,
MARIA G. ALMEIDA, MARIO H. HIRATA, ADRIANA A. REZENDE, Germantown,
MD, Natal, Brazil, So Paulo, Brazil
Accumulating evidence indicates that immunologic and inammatory
mechanisms play a signicant role in development and progression of dia-
betic nephropathy. In this way, polymorphisms in pro-inammatory cytok-
ines genes may be associated with renal injury in diabetic patients. Thus,
the objective of present study was to investigate the association between
IL-1B (rs1143634 and rs16944) and TNF-A (rs1800629) polymorphisms with
microalbuminuria in type 1 diabetic children and adolescents assisted at a
pediatric hospital (HOSPED/UFRN) in Natal-RN/Brazil. This study included
130 type 1 diabetic patients (T1DM group) and 132 normoglycemic subjects
(NG group) aged between 6 and 20 years. Metabolic control was evalu-
ated by glucose and glycated hemoglobin. Albumin to creatinine ratio (ACR)
was calculated. Polymorphisms was determined by allelic discrimination
technique in real time PCR using TaqMan
pre-designed SNP assays from

Applied Biosystems. IL-1B and TNF-A mRNA RNA expression were also
evaluated by real time PCR. Glucose and glycated hemoglobin were signi-
cantly increased in diabetic individuals compared to NG, indicating a poor
metabolic control of these patients. Enhanced ACR was observed in T1DM
group when compared to NG, suggesting an initial stage renal injury. IL-1B
(rs16944) and TNF-A (rs1800629) polymorphisms were signicantly associ-
ated with ACR in those patients (p=0.0022 and p=0.0092, respectively). IL-1B
rs1143643 polymorphism was signicantly associated with glycated hemo-
globin values (p=0.0208), showing that this polymorphism is contributing
to a poor glycemic control, which would induce an inammatory process.
This could be evidenced by the signicantly higher IL-1B mRNA expression
in T1DM patients when compared to NG (p=0.001) and could lead to renal
injury. These results suggest a possible association of IL-1B (rs1143634 and
rs16944) and TNF-A (rs1800629) polymorphisms with microalbuminuria on-
set in T1DM patients.
Supported by: CNPq, CAPES
559-P
Urinary Type IV Collagen is an Important Predictor for the Incidence
of Microalbuminuria in Young Patients With Type 1 Diabetes
MIWA MORITA, YASUKO UCHIGATA, Shimane, Japan, Tokyo, Japan
Urinary type IV collagen is the main component of glomerular basement
membrane and mesangial matrix. Recently, we have demonstrated that el-
evated levels of urinary type IV collagen are associated with kidney function
decline in patients with type 1 diabetes (T1D). There were some reports by
cross-sectional design that an increase in urinary type IV collagen occurs
before an incidence of microalbuminuria. However, there were no longitudi-
nal reports. Therefore, we aimed to clarify whether urinary type IV collagen
is a predicator for the incidence of microalbuminuria in patients with T1D.
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A longitudinal observational cohort study was conducted, which included
normoalbuminuric patients diagnosed with T1D before the age of 30 years,
who were < 40 years old at the start of observation. Patients with < 1 year
follow-up period or < 18 years old were excluded. Overall, 225 patients were
enrolled (mean [SD) age: 255 years, men: 32.9%). Microalbuminuria was
dened as 30 s urinary albumin-creatinine ratio (ACR) < 300 mg/g. The
primary endpoint was the incidence of microalbuminuria. During the mean
follow-up period of 8.33.5 years, 14 patients reached the endpoint. In the
univariate Cox regression analysis, elevated logarithmically-transformed
urinary type IV collagen-creatinine ratio (T4C) was identied as a prognostic
factor for the endpoint (p < 0.001). In the multivariate analysis using conven-
tional risk factors as independent variables, including HbA1c, blood pressure
and logarithmically-transformed ACR, elevated T4C was also identied as a
prognostic factor for the endpoint (p = 0.022). When patients were classied
into 2 groups by median of T4C, patients with high T4C levels had a higher
risk for the endpoint than those with low T4C in both the univariate and
multivariate Cox regression analysis (p = 0.008 and 0.017). In conclusion,
urinary type IV collagen may be an important predictor for the incidence of
microalbuminuria in young patients with T1D.
560-P
Mannan-Binding Lectin and Activation of Complement in Model of
Type 1 Diabetes
JAKOB A. STERGAARD, METTE BJERRE, FREDERIK DAGNAES-HANSEN, TRO-
ELS K. HANSEN, STEFFEN THIEL, ALLAN FLYVBJERG, Aarhus, Denmark
Blood levels of mannan-binding lectin (MBL) are increased in type 1
diabetes patients. High MBL levels are associated with the development
of diabetic nephropathy in humans. In animals, a direct effect of MBL on
diabetic kidney changes is observed. We hypothesized that MBL level and
detrimental complement activation increase as a consequence of diabetes.
To evaluate the effects of diabetes in mice, we measured MBL before and 6
weeks after multiple low-dose streptozotocin-induced diabetes. Liver MBL
expression by RT-PCR was used to explain changes in circulating MBL levels.
MBL elimination was estimated by half-life of intravenously injected recom-
binant human MBL. Complement activation was measured by immunohis-
tochemistry in glomeruli and by C5a concentration in plasma.Mice have two
MBLs, MBL-A and MBL-C. Diabetes led to a 3.6-fold increase in MBL-C con-
centration, P<0.001 (95% condence interval (CI95%: 2.9;4.5) compared with
0% in controls, P=0.93 (CI95% -7;9%). MBL-C changes differed signicantly
in the two groups, i.e., effect-modication (P<0.001). A minor difference in
change in MBL-A was initially observed between diabetic and control ani-
mals (P = 0.02). However, in separate analyses, MBL-A did not change in
diabetic animals (P = 0.12) or in controls (P = 0.12).In accordance with the ob-
served changes in circulating MBL levels, liver expression of MBL-C mRNA
was increased in diabetes by 28% (CI95%: 8;53%), P = 0.006. MBL-A ex-
pression did not differ between diabetic and control animals.The half-life of
recombinant human MBL was signicantly prolonged in mice with diabetes
compared with controls, 14.3 hours (CI95%: 11.3;19.4 hours) vs. 11.8 hours
(CI95%: 11.0;12.9 hours), respectively (P = 0.017). Complement activation in
plasma and glomeruli did not differ between groups.We conclude that our
ndings support previous clinical observations and furthermore show that
MBL increases as a direct result of diabetes. This change may be explained
by alternations both in MBL production and turnover.
561-P
Mechanism of Cell Matrix Accumulation in Diabetes: Tuberin Phos-
phorylation/Deciency Enhances Cell Matrix Protein Accumula-
tion
SAMY L. HABIB, MUKESH YADAV, ANTHONY VALENTE, San Antonio, TX
Tubular cells are a primary target of hyperglycemia and chronic exposure
to elevated blood glucose levels contribute to the tubulointerstitial changes
seen in overt diabetic nephropathy. The mechanisms by which hyperglyce-
mia contributes to matrix expansion and brosis are not known. Deciency
in tuberin resulted in 27% increased in kidney mass compared to wild type
rats. Increase the basal levels of phospho-tuberin as well as deciency in tu-
berin is associated with increased bronectin expression in kidney cortex of
TSC2+/- rats compared to wild type rats as well as in TSC2+/- renal primary
proximal tubular cells compared to TSC2+/+ cells isolated from rats. Phos-
phorylation of tuberin is associated with increased bronectin expression
in kidney cortex of diabetic rats compared to control rats. Insulin treatment
prevented these changes in diabetic animals. Pre-treatment RPTE cells with
Akt inhibitor or rapamycin signicantly reduced HG-induced bronectin ac-
cumulation. Introduction of TSC2 cDNA into tuberin-decient cells abolished
bronectin protein expression while downregulation of tuberin by siRNA
resulted in signicant decrease of bronectin in cells treated with HG. In
addition, rapamycin treatment signicantly decreased bronectin accumula-
tion in the kidney cortex of tuberin-decient mice (TSC2+/-). Transcriptional
activity of bronectin was measured in PRPT cells exposed and pretreated
with actinomycin D before exposed to high glucose. Treatment cells with
actinomycin D signicantly decreased HG-induced increased in mRNA of -
bronectin expression. In addition pretreated the cells with rapamycin prior
exposed to HG signicantly decreased the promoter activity of bronectin.
These indicate that tuberin is a major molecule that regulates kidney hyper-
trophy and cell matrix proteins by modulating protein synthesis.
Supported by: AHA and Merit Review Award (S.L.H.)
562-P
Inammatory Stress Increase Human Mesangial Foam Cell Forma-
tion by Increasing SCAP Glycosylation in Golgi
YANG YUAN, ZI L. SUN, ZHONG X. RUAN, NanJing, China, London, United Kingdom
Diabetic nephropathy caused by inammatory stress is associated with
lipid accumulation in glomeruli. This study was designed to investigate
whether interleukin-1(IL-1) increase lipid accumulation in human mesan-
gial cells (HMCs) via LDL receptor pathway by increasing SREBP cleavage-
activating protein (SCAP) transcription and its glycosylation in Golgi.HMCs
were treated with IL-1. Intracellular cholesterol content was assessed by
Oil Red O staining and cholesterol enzymatic assay. Expression of mRNA and
protein of molecules controlling cholesterol homeostasis in the treated cells
was examined by real-time quantitative PCR and western blotting, respec-
tively. Golgi enzymes activity was determined using enzyme-based method.
SCAP translocation was detected by confocal microscopy.IL-1 increased
cholesterol accumulation in HMCs. Exposure to IL-1 increased expression
and abnormal translocation of SCAP from ER to Golgi even in the presence
of a high concentration of LDL. The increased SCAP translocation carried
more transcription factor SREBP-2 to the Golgi for activation by cleavage
which enhanced gene transcription of HMGCoA reductase and LDL recep-
tor. Furthermore, IL-1 enhanced SCAP glycosylation by upregulating Golgi
mannosidase activity. This prolonged the half-life and enhanced recycling
of SCAP between the ER and the Golgi. The effects of IL-1 were blocked
by inhibitors of Golgi mannosidases.Inammatory stress increased lipid
synthesis and uptake, thereby causing foam cell formation via increasing
transcription and protein glycosylation of SCAP by Golgi enzymes in HMCs.
These data imply that Golgi enzymes inhibitors might have a potential renal
protective role in prevention of mesangial foam cell formation.
Supported by: sKidney Research UK (RP372008), Natural Science Foundation
(BK2011601)
563-P
Renoprotective Advantage of an L/N-Type Calcium Channel Blocker
Compared With L-Type Calcium Channel Blockers in Type 2 Diabetic
Patients With Early-Stage Nephropathy
SHINYA FUKUMOTO, EIJI ISHIMURA, KOJI TAKEDA, YOHEI MIMA, KOKA MO-
TOYAMA, TOMOAKI MORIOKA, KATSUHITO MORI, TETSUO SHOJI, MASANORI
EMOTO, YOSHIKI NISHIZAWA, MASAAKI INABA, CLEARED STUDY INVESTIGA-
TORS, Osaka, Japan
We evaluated the antiproteinuric advantage of cilnidipine, an N/L-type
calcium channel blocker (CCB), compared with L-type CCBs in early-stage
diabetic nephropathy. The study was a multicenter, non-randomized cross-
over trial pre-registered as a CLEARED study (UMIN ID: 000000835). Par-
ticipants were 90 type 2 diabetic patients exhibiting either normo- or mi-
croalbuminuria, and undergoing CCB treatment for ~6 months prior to study
entry. The CCB at the time of entry was continued for the rst 6 months
(Treatment period 1). Treatment was subsequently switched from cilnidipine
to an L-type CCB, or vice versa, for the second 6-month observation period
(Treatment period 2). During period 1, the L-type CCB group (n=69) showed
a signicant increase of urinary albumin excretion (UAE) over time (53.27.2
mg/gCr (meanSEM) 96.318.4 mg/gCr, p<0.01), while the cilnidipine
group (n=21) showed no signicant elevation. During period 2, switching of
the treatment from the L-type CCB to cilnidipine resulted in signicant reduc-
tion of the UAE (96.318.4 mg/gCr 59.210.3 mg/gCr, p<0.01), whereas
switching from cilnidipine to the L-type CCB resulted in no signicant change
in the UAE. Multivariate logistic regression analysis showed that cilnidipine
was a signicant factor reducing the risk of UAE-increase (OR: 0.324 (95%CI,
0.107-0.983) for period 1, OR: 0.246 (95%CI, 0.074-0.823) for period 2) inde-
pendent of age, sex, blood pressure, RAS inhibitor, and HbA1c. This study
demonstrated that, in patients with early-stage diabetic nephropathy, the
antiproteinuric effect of cilnidipine, but not the L-type CCBs, was sustained
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COMPLICATIONSNEUROPATHY
even in patients treated for a long time. In addition, the renoprotection can
be anticipated after switching from an L-type CCB to cilnidipine, but not vice
versa.
Guided Audio Tour: New Ideas on Clinical Diagnosis and Mechanisms of
Neuropathy (Posters 564-P to 571-P), see page 13.
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Peripheral Neuropathy in the SEARCH for Diabetes in Youth Cohort:
A Pilot Study
EVA L. FELDMAN, CATHERINE L. MARTIN, RONNY A. BELL, ABIGAIL LAUER, JAS-
MIN DIVERS, DANA DABELEA, DAVID J. PETTITT, SHARON SAYDAH, BARBARA
LINDER, CATHERINE PIHOKER, DEBRA A. STANDIFORD, BEATRIZ L. RODRIGUEZ,
RODICA POP-BUSUI, FOR THE SEARCH FOR DIABETES IN YOUTH GROUP, Ann Ar-
bor, MI, Winston-Salem, NC, Aurora, CO, Santa Barbara, CA, Atlanta, GA, Bethesda,
MD, Seattle, WA, Cincinnati, OH, Honolulu, HI
Estimates of the prevalence of diabetic peripheral neuropathy (DPN) in
adult populations range from 30 to 70%. Data on DPN in children, adoles-
cents and young adults, however, are limited. In a cross-sectional pilot study,
DPN was assessed using the Michigan Neuropathy Screening Instrument
(MNSI), a validated DPN screening tool, in a subset of SEARCH for Diabetes
in Youth Study participants The MNSI includes a 15-item, self-administered
questionnaire and a structured examination for foot abnormalities, distal
vibration perception and ankle reexes. An MNSI questionnaire (MNSIQ)
score of > 7 is positive for DPN; an MNSI exam (MNSIE) score of > 2 is
positive for DPN.The MNSIQ and MNSIE were completed in 413 and 418
SEARCH participants respectively; 53% were female; 83% had type 1 diabe-
tes (T1D). For youth with T1D, mean age was 15.6 years (standard deviation
[SD] 4.4 years] with duration of 6.2 years (SD=0.9); for youth with Type 2
diabetes (T2D), mean age was 21.6 years of age (SD=3.2), with duration of
7.6 years (SD=1.8). Mean A1C was similar for T1D and T2D in a subset of
subjects (n=305) with A1C measured within 3 months: [8.8% (SD=1.9) vs. 8.7
(SD=3.1), p=0.34].Two SEARCH subjects (<1%) met the MNSIQ categorical
threshold for DPN. Mean MNSIQ score was 1.1 (SD=1.5) and was higher for
T2D vs. T1D [1.8 (SD=1.6) vs. 1.0 (SD=1.4), p<0.001]. A cumulative logit model
showed positive association between A1C and MNSIQ scores (p=0.04).
Mean MNSIE score was 0.8 1.2 and was higher for T2D (1.5[SD=1.5]) vs.
T1D [0.7(SD=1.1), p<0.001]. DPN prevalence by MNSIE (e.g., score > 2) was
11.3% (T2D 26.0% vs. T1D 8.2%; p<0.001) and was associated with older
age (p < 0.001).DPN prevalence measured by MNSIE among T2D SEARCH
participants approached rates reported in adult diabetes populations. Our
ndings suggest that children, adolescents and young adults with diabetes
are not only at risk for DPN, but that many already show measurable signs of
DPN based on the MNSIE.
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565-P
Early Detection of Nerve Fiber Loss by In Vivo Corneal Confocal Mi-
croscopy in Recently Diagnosed Type 2 Diabetic Patients
DAN ZIEGLER, ANDREY ZHIVOV, STEPHAN ALLGEIER, KARSTEN WINTER, NIKO-
LAOS PAPANAS, IRIS ZIEGLER, SABINE PESCHEL, BERND KOEHLER, OLIVER
STACHS, RUDOLF F. GUTHOFF, MICHAEL RODEN, GDC STUDY GROUP, Dsseldorf,
Germany, Rostock, Germany, Karlsruhe, Germany, Leipzig, Germany
Corneal confocal laser scanning microscopy (CCM) has emerged as a non-
invasive technique to quantify the subbasal nerve plexus (SNP), but it is un-
clear whether it allows for detection of small nerve ber pathology at early
stages of type 2 diabetes. We assessed parameters of CCM, corneal sensa-
tion, and peripheral nerve function in 50 patients with recently diagnosed
type 2 diabetes (age: 58.58.5 [meanSD] years, diabetes duration: 2.51.8
years, BMI: 32.15.1 kg/m, HbA1c: 6.60.9%) and 28 healthy subjects of
similar age. Intraepidermal nerve ber density (IENFD) in 3-mm punch biop-
sies from the distal leg was quantied in a subset of 29 subjects with and
24 without diabetes. Image data acquired from automatic CCM focus scans
were processed with digital image processing algorithms to eliminate mo-
tion artifacts and reconstruct images of the SNP devoid of anterior corneal
mosaic deformations. Nerve bers were segmented and analyzed by a newly
developed software. Corneal nerve ber density (CNFD) was reduced in pa-
tients with diabetes (0.0220.007 vs. control: 0.0280.006 m/m; P=0.001)
as was nerve branch density (328163 vs. 417170/mm; P=0.026), number of
connecting points (37.113.5 vs. 50.219.2/mm; P=0.003), while a trend to-
wards reduction was noted for nerve ber thickness (2.510.39 vs. 2.670.37
m; P=0.065). In the entire population, CNFD and number of connecting points
correlated with IENFD (both r=0.39; p=0.004). CNFD was reduced below the
5
th
percentile in 28% of the diabetic patients. Similar numbers of patients
showed concomitantly abnormal CNFD and normal IENFD and vice versa. No
difference between patients and controls was noted for corneal sensation.
In conclusion, CCM detects early nerve ber loss in recently diagnosed type
2 diabetes, but not necessarily in the same patients as skin biopsy, suggest-
ing a patchy pattern of small ber neuropathy which is detectable in the
cornea even before the lower limbs are affected.
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The Cingulum Bundle is Altered by Type 2 Diabetes
WOUTER S. HOOGENBOOM, VERONICA L. FLORES, NICOLAS R. BOLO, DONALD
C. SIMONSON, ALAN M. JACOBSON, MARTHA E. SHENTON, GAIL MUSEN, Bos-
ton, MA, Mineola, NY
The cingulum bundle (CB) is a major white matter tract that connects
the frontal and parietal lobes with parahippocampal and adjacent tempo-
ral regions, and is involved in memory, executive function, and emotion. It
also plays a role in connecting the default mode network (DMN) and the
frontal and temporal regions that support memory -- a cognitive process
often impaired in T2DM. In addition, altered connectivity in the DMN has
been identied in T2DM and this may be an early biomarker for Alzheimers
disease (AD). We used diffusion tensor imaging (DTI) to measure diffusion
in the CB of 13 T2DM (age = 576 yrs, BMI = 305 kg/m2, HbA1c = 7.51.9 %,
FPG = 13551 mg/dl, IQ = 11412) and 17 control subjects (age = 547 yrs,
BMI = 284 kg/m2, HbA1c = 5.60.4 %, FPG = 836 mg/dl, IQ = 10414). After
regions-of-interest (ROI) were placed on a color-by-orientation map for each
individual, we calculated DTI-derived diffusion values from the resulting ROI
seed-based tractography to assess overall white matter health [fractional
anisotropy (FA, trace)], as well as pathology related to axonal integrity (axial
diffusion) and axonal myelination (radial diffusion). People with T2DM had
lower FA (-5.5% in left CB: T2DM 34516 vs controls 36538; and -8.4% in
right CB: T2DM 31528 vs controls 34427), lower axial diffusion (-1.3% in
left CB: T2DM 114835 vs controls 116347; and -3.1% in right CB: T2DM
110939 vs controls 114453), and higher radial diffusion (+3.1% in left CB:
T2DM 66732 vs controls 64750; and +2.3% in right CB: T2DM 67943 vs
controls 66447), statistically signicant for FA and axial diffusion in the
right CB (p<0.05, IQ-corrected). Also, axonal integrity was correlated with
performance on delayed-recall and letter-uency in controls, but not in
T2DM. Our ndings suggest that deciencies in axonal health underlie al-
tered diffusivity in the CB in cognitively intact people with T2DM, which may
provide a neuroimaging biomarker for AD.
Supported by: National Institute of Aging (5R01AG34165-2)
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567-P
Measurement of Gastric Emptying in Diabetic Patients With Ra-
diopaque Markers: Correlation With Scintigraphy and Upper GI
Symptoms
EVA A. OLAUSSON, HAKAN GRUNDIN, MATS ISAKSSON, CHRISTINA BROCK,
PER-OVE STOTZER, HASSE ABRAHAMSSON, STIG ATTVALL, MAGNUS SIMRN,
Gothenburg, Sweden, Aalborg, Denmark
Upper gastrointestinal (GI) symptoms and delayed gastric emptying are
common in insulin treated diabetes mellitus (DM). Scintigraphy, the current
gold standard to measure gastric emptying, is expensive and not widely
available. Emptying of radiopaque markers (ROM) from the stomach using
uoroscopy is an easy and inexpensive method, available in all hospitals.In
this study we validated ROM emptying in insulin treated DM and assess the
correlation with gastric scintigraphy and upper GI symptoms.On the same
day we measured gastric emptying of a standard meal using scintigraphy
(omelet labeled with 15 MBq
99
mTc, and 150ml water) and emptying of twen-
ty spheric ROM (density 1.27 g/cm3 and 4 mm diameter) using uoroscopy in
diabetic subjects. To investigate upper GI symptoms the subjects completed
the validated questionnaire, Patient Assessment of Upper Gastrointestinal
Symptom Severity Index, PAGI-SYM.We included 115 patients with insulin
treated DM age 5212 (mean SD) (range 25-69) years; 59 females. The
mean duration of diabetes was 2614 (range 2-63) years. 86 (75%) patients
had delayed gastric emptying rate with the scintigraphy, whereas 29 pa-
tients had delayed emptying of ROMs. Of these 29 subjects, 28 also had
delayed scintigraphic emptying, whereas 58 of the patients with a normal
ROM test had delayed gastric emptying with scintigraphy, yelding a sen-
sitivity of the ROM test 33 %, whereas the specicity 97%. The strongest
correlations between gastric emptying and upper GI symptoms were found
for scintigraphic emptying and nausea/vomiting (r=0.30; p<0.001) and post-
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prandial fullness/early satiety (r=0.34; p<0.0001).In summary, the sensitivity
of the ROM emptying test had a high specicity but rather low sensitivity.
We found a strong correlation between scintigraphic emptying and nausea/
vomiting and postprandial fullness/early satiety.
Supported by: Health Care in the West Region of Sweden
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568-P
RAGE Deciency Improves Post-Injury Sciatic Nerve Regeneration
in Type 1 Diabetic Mice
JUDYTA K. JURANEK, MATTHEW S. GEDDIS, FEI SONG ZOU, YU SHAN, JOSE
GARCIA, XIAOPING (JENNIFER) SHEN, BIN CHENG, ANDREAS POLLREISZ, PRA-
TIK KOTHARY, ROSA ROSARIO, LOUIS WEIMER, THOMAS H. BRANNAGAN, ANN
MARIE SCHMIDT, New York, NY
Diabetes is of one the most prevalent metabolic, non-communicable dis-
orders of today, becoming the main public health challenge worldwide. One
of the major complications, affecting 30 - 50 % of all diabetic patients, is a
peripheral diabetic neuropathy (PDN). Although molecular mechanisms un-
derlying the pathogenesis of the disease are yet not fully understood, it has
been shown that one of the possible contributors to the disease might be
RAGE (Receptor for Advanced End-Glycation products). Studies showed that
in normoglycemia peripheral RAGE contributes to effective axonal regenera-
tion prompted by acute crush injury while in diabetes, intact peripheral nerve
studies showed that RAGE signaling is a deteriorating factor. In the present
study we hypothesized that in diabetes, RAGE contributes to progressive
neuropathy and that upon superimposed acute injury, RAGE-dependent
neuronal dysfunction would worsen, thereby attenuating neurite outgrowth
and further suppressing effective axonal regeneration and re-innervation
of target tissues. To validate the hypothesis we have set non-diabetic and
diabetic (streptozotocin injected) groups of transgenic RAGE decient (RKO)
and wild type (WT) C57 BL6 mice, performed sciatic nerve injury two months
after diabetes induction, 21 days post surgery performed conduction veloc-
ity tests and collected nerve tissue for morphological, immunohistochemical
and mRNA analysis. Results depicted on gure 1. The ndings of our study
provide evidence that RAGE suppresses effective axonal regeneration in
diabetic peripheral nerve after acute injury.
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569-P
GLP-1 Analogs Represent a Novel Treatment Strategy for Diabetic
Encephalopathy
MASAHIRO OKOUCHI, NAOTSUKA OKAYAMA, GOJI SHIGEKI, TADASHI IWAMA,
TETSUYA KATSUNO, KAORU ASADA, KOHEI HATTORI, AKITOMO GOTO, YASU-
TAKA KAMIYA, TSUNEO OHNO, TAKASHI JOH, Inazawa, Japan, Nagoya, Japan
Diabetic encephalopathy, characterized by cognitive impairment in pa-
tients with diabetes, involves increased neuronal apoptosis and impaired
adult neurogenesis in the hippocampal regions, followed by hippocampal
atrophy. Recent evidence indicates that GLP-1 analogs cross the blood-
brain barrier and increase neural stem/progenitor cell proliferation in the
hippocampal regions. Therefore, we rst compared hippocampal atrophy on
MRI between 400 type 2 diabetic patients and 400 control subjects, and
determined the important factors affecting hippocampal atrophy. In vitro, rat
pheochromocytoma cells used commonly as neuronal cell models were ex-
posed to 1 mM methylglyoxal (MG) with or without 3.3 g/ml GLP-1, and the
intercellular signaling of GLP-1 protection against neuronal apoptosis were
determined. Additionally, by using immunohistochemistry method, we inves-
tigated whether the number of progenitor cells or young neurons in the hip-
pocampal regions was inuenced by treatment with GLP-1. Diabetic patients
had more hippocampal atrophy than control subjects. Poor glycemic control,
and marked glycemic uctuation were associated with hippocampal atrophy,
while the use of GLP-1 analogs was negatively associated with hippocampal
atrophy. In vitro, GLP-1 induced phosphorylation of Akt, mTOR, p70S6K, the
upregulation of GCL, the inhibition of caspase-9 and -3 activation, and pro-
tected against MG-induced neuronal apoptosis. We also found an increase
in progenitor cells or young neurons in the hippocampal regions after GLP-1
analogs treatment. These results suggest that poorly glycemic control or
marked glycemic uctuation were closely associated with hippocampal atro-
phy in diabetic patients. Additionally, GLP-1 analogs treatment may prevent
hippocampal atrophy through protecting neuronal apoptosis and increasing
neural stem/progenitor cell proliferation in the hippocampal regions, and
thus represent a promising treatment modality for diabetic encephalopathy.
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570-P
Effects of Hyperglycemia on the Expression of Neprilysin (NEP) and
Substance P Receptor (NK1R), and the Activity of MMP2 and MMP9
in Adult Dermal Fibroblasts
ALLISANDRA MOWLES, GENER AUGUSTIN, FRANK W. LOGERFO, LEENA PRAD-
HAN-NABZDYK, Boston, MA
Impairment in wound healing is a major complication of diabetes. Fibro-
blasts play an important role in the wound healing process, especially in the
remodeling phase. Previously we have shown that hyperglycemia impairs
Adult Dermal Fibroblast (ADF) migration and proliferation while Substance
P (SP) mitigates these effects. In vitro ADF cultured in DMEM were treated
with D-glucose (5mM, 10mM or 30mM) and co-treated with SP (100nM) for
24 hours or 5 days. NEP and NK1R protein expression was assessed by west-
ern blot. MMP2 and MMP9 activity was assessed via zymography. Data are
expressed as mean fold changeSEM compared to 5mM glucose (N=3-6).
Compared to 5mM glucose, 30mM glucose signicantly increased NEP pro-
tein expression at 5 days (1.00 vs. 2.081.27). There were no differences in
NK1R protein expression between different treatment groups; however, a
trend towards up-regulation is observed for ADF treated with 10mM glucose
when compared with other treatments. Compared to 5mM glucose, 30mM
glucose signicantly decreased MMP2 (1.00 vs. 0.640.036) and MMP9
(1.00 vs. 0.6020.079) activity at 5 days and this was not restored by SP co-
treatment. In conclusion, these data suggest that hyperglycemia increases
the expression of NEP, the enzyme that breaks down SP whereas it does not
alter the expression of SP receptor NK1R in AFB. Hyperglycemia also alters
the activity of MMP2 and MMP9, which cannot be restored by addition of
SP suggesting that the previously observed increase in AFB migration and
proliferation upon co-treatment with SP is most likely not through MMP2
or MMP9.
Supported by: NIH (5R01NS066205-02)
&
571-P
Inhibition of Chymase Protects Against Brain Oxidative Stress and
Cognitive Dysfunction in Diabetic db/db Mice
EIICHI HIRATA, NORIYUKI SONODA, YOHEI MINAMI, TAKAHIRO KATO, YOSHI-
HIRO SEKI, AKIRA MONJI, RYOICHI TAKAYANAGI, TOYOSHI INOGUCHI, Fukuoka,
Japan
Accumulating evidence suggests that the tissue renin-angiotensin system
may be involved in the progression of diabetic complications. Chymase is a
potent local angiotensin II-forming enzyme. We have reported that a causal
role of chymase in diabetic nephropathy and the therapeutic effectiveness
of chymase inhibition (Am J Physiol 2010), and that NAD(P)H oxidase activa-
tion , consequently elevation of oxidative stress and inammatory cascade
might be causative pathogenic mechanisms in diabetes-associated cognitive
impairment in middle-aged diabetic rodent model (ADA 2011). Therefore, we
speculated that chymase-dependent ANG II production may play an impor-
tant role in the enhancement of oxidative stress in diabetic brain, thus con-
tributing to the development of diabetes-associated cognitive impairment.
In the present study, we report the therapeutic effectiveness of chymase-
specic inhibitior(TEI-F00806) for cognitive function and its association with
oxidative stress using diabetic db/db mouse model.Overproduction of ROS
assessed by dihydroethidium staining and elevation of lipid peroxidation
products in 20-week aged db/db mice brain were partially reduced by TEI-
F00806. Furthermore, Upregulation of NAD(P)H oxidase components includ-
ing Nox 2 and pro-inammatory cytokines in db/db mice was supressed by
TEI-F00806, whereas downregulation of anti-inammatory cytokines was
corrected by TEI-F00806. However,there was no deposition of amyloid beta
in either db/db and db/+ or the chymase inhibitor administrated db/db mice
brain. In correlation with these ndings, impairment of working memory in
db/db mice assessed by radial arm water maze test was partially restored
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by TEI-F00806.This study showed for the rst time that chymase inhibition
may protect against oxidative stress, and cognitive dysfunction in diabetes.
These ndings suggest that chymase offers a new therapeutic target for
diabetes-associated cognitive impairment.
572-P
Intermittent High Glucose, More than Constant High Glucose, En-
hances Apoptosis Related to Oxidative Stress in Schwann Cells
LIAN Q. SUN, YING Y. CHEN, XUE WANG, BING XUE, XIAO J. LI, JU M. LU, Beijing,
China
To explore the effect of intermittent high glucose (IHG) on Schwann cells
(SCs) apoptosis induced by oxidative stress, Rat SCs were primarily cultured
for 2 days in media containing different glucose concentrations: 5.6mM,
50mM, or 8h alternating 5.6 mM or 50 mM glucose and osmotic controls.
Apoptosis was studied by Annexin-V and TUNEL analysis. Intracellular ROS
generation and mitochondrial transmembrane potential were detected by
ow cytometry analysis. The concentration of 8-OHdG was detected as
oxidative stress marker. Real-time PCR was performed to analyze the ex-
pression levels of Bax and BcL-2. Western blot were performed to analyze
the expression levels of some important transcription factors. Our data indi-
cated that IHG induced high levels of ROS production and mitochondrial dys-
function, which triggered high frequency of SCs apoptosis. IHG up-regulated
Bax expression and release of cytochrome c and AIF nuclear translocation,
but down-regulated the expression of BcL-2. In addition, treatment with IHG
induced activation of caspase-3 and -9 and mitigated the cleavage of PARP.
More importantly, the cytotoxic effect of IHG was signicantly more potent
than that of constant high glucose (HG) and the cytotoxicy is not related
to the changes of osmolarity. Our results suggested that IHG induced SCs
apoptosis in both caspase-dependent and caspase-independent pathways
by activating the mitochondrial pathway related to oxidative stress. These
ndings suggest that variability in glycemic control could be more deleteri-
ous to SCs than a constant high concentration of glucose.
Supported by: Postdoctoral Science Foundation of China (No. 20090461435)
573-P
Cardiovascular Autonomic Neuropathy in the SEARCH for Diabetes
in Youth Cohort: A Pilot Study
RODICA POP-BUSUI, CATHERINE L. MARTIN, RONNY A. BELL, ABIGAIL LAUER,
JASMIN DIVERS, DANA DABELEA, DAVID J. PETTITT, SHARON SAYDAH, BAR-
BARA LINDER, CATHERINE PIHOKER, DEBRA A. STANDIFORD, BEATRIZ L. RO-
DRIGUEZ, EVA L. FELDMAN, FOR THE SEARCH FOR DIABETES IN YOUTH STUDY
GROUP, Ann Arbor, MI, Winston-Salem, NC, Aurora, CO, Santa Barbara, CA, Atlanta,
GA, Bethesda, MD, Seattle, WA, Cincinnati, OH, Honolulu, HI
Data regarding cardiovascular autonomic neuropathy (CAN) in young
people with diabetes are limited. Several measures of CAN [resting heart
rate (HR), R-R variability at rest and during deep breathing] were assessed
in a standardized manner (ANSAR system, ANSAR Inc. PA) in a pilot study
that included 222 subjects from the SEARCH for Diabetes in Youth study (a
large U.S. population-based study in people diagnosed with diabetes be-
fore age 20). Analyses used the Wilcoxon two-sample test for continuous
outcomes and Chi-square test for categorical outcomes.The mean age of
the pilot study cohort was 17.4 4.9 years; 78% with type 1 diabetes (T1D)
(age 16.1 4.4 years, duration 6.2 0.9 years, 53% female), and 22% with
type 2 diabetes (T2D) (age 22.1 3.1 years, duration 8.0 1.9 years, 59%
female). Resting HR and ratio of sympathetic/parasympathetic activity were
higher among T2D vs. T1D participants (77.1 vs. 72.0, p=0.004 and 1.6 vs.
1.1, p=0.003). E/I ratio, a measure of R-R variability during deep breathing,
was lower in T2D participants (1.3 vs. 1.4, p=0.001). Higher resting HR was
negatively associated with age (p=0.003) and with higher recent A1C value,
which was obtained within 3 months of CAN testing on a subset of 146 sub-
jects (p = 0.023). Lower E/I ratios were negatively associated with older age
(p = 0.029) and diabetes type (p = 0.028).In this cross-sectional pilot study
of young people with diabetes measures of CAN were associated with older
age and with diabetes type. The lack of normal values for the CAN measures
in an age-matched healthy population limits our ability to dene the preva-
lence of CAN among SEARCH participants. These data suggest vulnerability
in this cohort of young people with diabetes for CAN-associated compli-
cations, placing them at higher cardiovascular risk. Studies are ongoing to
better dene the prevalence, risks and outcomes associated with CAN in
SEARCH participants.
574-P
Lower Heart Rate Variability is Associated With Increased Arterial
Stiffness in Youth With Type 1 Diabetes: The SEARCH CVD Study
MAMTA JAISWAL, Aurora, CO
Lower heart rate variability is associated with increased arterial stiffness
in youth with type 1 diabetes: the SEARCH CVD study.Lower heart vari-
ability (HRV), a marker of subclinical cardiac autonomic neuropathy (CAN),
and increased arterial stiffness (AS), a marker of subclinical cardiovas-
cular disease, have been shown to develop and progress silently in youth
with type 1 diabetes (T1D), which might predispose them prematurely to
cardiovascular events. The SEARCH CVD study assessed the associations
between measures of HRV and measures of AS in youth with and without
T1D. Participants were 402 youth with T1D (mean age 19 years, duration 9.8
years, 87% non-Hispanic white (NHW) and 206 youth without T1D (mean age
19 years, 75% NHW). SphygmoCor device was used to measure HRV using
SDNN (Standard deviation of NN interval), HF (high frequency) and LF (low
frequency) power variables, AS measured by pulse wave velocity (PWV), and
augmentation index adjusted to a heart rate of 75/min (AIx75). Brachial dis-
tensibility (BrachD), was measured on a Dynapulse instrument. Multivariate
linear regression analyses explored the associations between markers of
reduced HRV (lower SDNN, lower HF and higher LF) and increased AS (higher
PWV and AIx75 and lower BrachD), adjusting for age, sex and race/ethnic-
ity, separately among youth with and without T1D. Among youth with T1D,
lower SDNN was associated with higher PVW in the carotid-femoral seg-
ment (p=0.0009), higher AIx75 (p =0.01) and lower BrachD (p= 0.03). Lower
HF and higher LF were also associated with lower BrachD among youth with
T1D (p=0.005 for each). No signicant associations were observed among
youth without T1D. The results were similar after additional adjustment for
lipid levels (LDL,HDL, triglycerides), systolic and diastolic blood pressure and
BMI. Our results suggest a parallel evolution of subclinical CAN and AS in
youth with T1D and point toward a common mediator, likely represented by
hyperglycemia.
575-P
Improved Diagnostic Accuracy of the LDIare in Diagnosing Clini-
cal Neuropathy Using Age-Related Centile Charts
PRASHANTH VAS, SANJEEV SHARMA, GERRY RAYMAN, Ipswich, United King-
dom
The Laser Doppler Imaging are (LDIare) is a non-invasive test of small
bre function assessed on the dorsum of the foot involving skin heating and
measurement of the axon-mediated are area using a laser Doppler imager.
We have previously used ROC analysis to determine sensitivity and specici-
ty in detecting neuropathy [based on the Neuropathy Disability Score(NDS)].
However, having now established normative reference ranges we believe
these will further improve the diagnostic accuracy. This study therefore
compares the sensitivities and specicities derived by these two methods.
The normative centiles were determined by assessing the LDIare in 94
(m=45;f=49) healthy controls (HC) between the ages of (20-80 years). LDI-
ares were also determined in 66 diabetes individuals with (n=31, NDS>3)
and without clinical neuropathy (n=35, NDS<3). Standard ROC analysis was
used to generate the sensitivity and specicity. Additionally, sensitivity and
specicity based on the normative centiles, were determined by dening as
abnormal any LDIare areas below the 5
th
centile for ageAmong HC, there
was an age dependant decrease in small bre function (r=-0.42, p<0.0001).
There was no signicant correlation with gender, height, and weight. The
ROC analysis suggested an optimal threshold cut off of <3.7 c m
2
. This gave
a sensitivity of 76%, specicity of 84%, positive predictive value (PPV) of
77%, and negative predictive value (NPV) of 87%. Using 5th centile cut-offs,
the sensitivity was similar at 77% but there was improved specicity (90%),
PPV (80%) and NPV (91.1%).Although both analytic methods demonstrate
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excellent sensitivity and specicity for the LDIare method to detect clini-
cal neuropathy, the application of centile derived normative values further
enhances the diagnostic accuracy. We would recommend the use of centile
charts in future studies of small bre function.
576-P
Interplay of Sorbitol Pathway of Glucose Metabolism, 12/15-Lipox-
ygenase, and Mitogen-Activated Protein Kinases in the Pathogen-
esis of Diabetic Peripheral Neuropathy
ROMAN STAVNIICHUK, HANNA SHEVALYE, HIROKO HIROOKA, JERRY L. NA-
DLER, IRINA G. OBROSOVA, Baton Rouge, LA, Mie, Japan, Norfolk, VA
The interactions among multiple pathogenetic mechanisms of diabetic pe-
ripheral neuropathy largely remain unexplored. Increased activity of aldose
reductase (AR), the rst enzyme of the sorbitol pathway, leads to accumula-
tion of cytosolic Ca
++
, essentially required for 12/15-lipoxygenase (12/15-LO)
activation. The latter, in turn, causes oxidative-nitrosative stress, an impor-
tant trigger of MAPK phosphorylation. We therefore evaluated the interplay
of AR, 12/15-LO, and MAPKs in diabetic peripheral neuropathy. In study 1,
male control and streptozotocin (STZ)-diabetic mice were maintained with or
without the AR inhibitor darestat, 16 mg kg
-1
d
-1
, for 12 wks. In study 2, male
control and STZ-diabetic wild-type (C57Bl6/J) and 12/15-LO-decient mice
were used. Fidarestat treatment did not affect diabetes-induced increase in
glucose concentrations, but normalized sorbitol and fructose concentrations
(enzymatic spectrouorometric assays) as well as 12(S) hydroxyeicosatet-
raenoic concentration (ELISA), a measure of 12/15-LO activity, in the sciatic
nerve and spinal cord. 12/15-LO expression in these two tissues (Western
blot analysis) as well as dorsal root ganglia (immunohistochemistry) was
similarly elevated in untreated and darestat-treated diabetic mice. 12/15-
LO gene deciency prevented diabetes-associated p38 MAPK and ERK, but
not SAPK/JNK, activation in the sciatic nerve (Western blot analysis) and all
three MAPK activation in the dorsal root ganglia (immunohistochemistry).
In contrast, spinal cord p38 MAPK, ERK, and SAPK/JNK were similarly acti-
vated in diabetic wild-type and 12/15-LO
-/-
mice. These ndings identify the
nature and tissue specicity of interactions among three major mechanisms
of diabetic peripheral neuropathy, and suggest that combination treatments,
rather than monotherapies, can sometimes be an optimal choice for its man-
agement.
Supported by: NIH (DK074517, DK077141, DK081147)
577-P
Insulin Treatments Ameliorate Tau Hyperphosphorylation in a Rat
Model of Type 2 Diabetes
YANG YAN, YUAN GANG, Wuhan, China
Brain insulin could regulate brain glucose metabolism, and accelerate the
memory and cognition.The lack of brain insulin is one of the mechanisms of
Alzheimers disease (AD), the most common form of dementia.It has been
proved that type 2 diabetes (T2D), the characteristics of which is insulin
resistance/hyperinsulinmia, has the potiential to increase the risk of AD.
However, the underlying mechanism is obscured. We evaluted the effects
of abnormal brain insulin on pathophysiological changes in the brain with
streptozocin (STZ) induced T2D rat model with a high protein, high glucose
and high fat diet. Our data showed that brain insulin in T2D rats were signi-
cantly decreased compared with control group(CTL)(1.20.2 vs 2.90.4,uIU/
ml, p<0.01). Hyperphosphorylated tau protein, which represented of AD-like
changes was presented in the brains of T2D rats. And glycogen synthase
kinase-3 (GSK-3) was activated in T2D brain also. Furthermore we found
that administration of insulin in abdomen decreased the level of blood glu-
cose, but could not change the hyperphosphorylation of tau or insulin de-
cency in brain. In additional, intranasal insulin treatment could reduce AD-
like changes in brain, and light the activities of GSK-3. Taken together, our
present data indicated that insulin is deency in type 2 diabetic brain, which
is a key factor involved Alzheimers disease, and intranasal insulin treatment
could reverse the AD-like changes in T2D.
Supported by: National Natural Science Foundation of China (81100582)
578-P
Autonomic Nervous System and Cystatin C Assessment in Type 1
Diabetes Mellitus
TRIANTAFILLOS DIDANGELOS, EFSTRATIOS MORALIDIS, FOTIOS ILIADIS, ANES-
TIS ZANTIDIS, CHARALAMPOS MARGARITIDIS, ANNA GOTZAMANI-PSARRAK-
OU, APOSTOLOS HATZITOLIOS, Thessaloniki, Greece
Aim: This study investigates if there is an association between Cystatin-C
measurement, as index of renal function, and autonomic function assess-
ment in patients with type 1 diabetes mellitus (T1DM).Patients-methods:
Forty nine patients (21 female), aged 3610 years (range 19-62), with a dura-
tion of T1DM 196 years (range 7-31) and receiving only insulin were enrolled
prospectively. Participants were evaluated for autonomic dysfunction with
Autonomic Function Tests (AFT) [mean circular resultant (MCR),Valsalva
maneuver (Vals), postural index (PI), orthostatic hypotension (OH)] assess-
ment and cardiac
123
I metaiodobenzylguanidine (MIBG) imaging [with the
ratio of the heart to upper mediastinum count density (H/M) at 4 hours
post-injection calculated].Within one month patients underwent glomerular
ltration rate measurement with
51
Cr-EDTA (expressed in ml/min/1.73m
2
),
Cystatin-C (CC, mg/l) and Serum Creatinine (SC, mg/dl) measurements as
indices of renal function.Results: The values of examined variables were as
follows [mean1SD (range)]: GFR 9817 (61-137), MCR 4029 (5-108), Vals
1.550.30 (1.11-2.29), PI 1.330.17 (1.03-1.81), OH 59 (0-30), CC 0.780.14
(0.49-1.06), SC 0.740.16 (0.51-1.12) and H/M 1.650.20 (1.30-2.34).GFR cor-
related signicantly with age (r=-0.377, p=0.011), duration of diabetes (r=-
0.514, p=0.000), Vals (r=0.377, p=0.010), OH (r=-0.448, p=0.002), number of
abnormal AFT (r=-0.366, p=0.012), CC (r=-0.362, p= 0.017) and SC (r=0.465,
p=0.001). MCR, PI and the H/M showed no signicant correlation with GFR.
CC showed signicant correlation with GFR and SC but not with AFT and
MIBG.Conclusions: In present study in T1DM patients CC is associated with
GFR and serum creatinine but not with AFT (which predominantly address
the parasympathetic system) and MIBG measurements (which reect sym-
pathetic dysfunction).
579-P
Severity of Retinopathy, Nephropathy and Peripheral Neuropathy
Correlates With Autonomic Dysfunction in Type 1 Diabetes: The
DAN-Study
JESPER FLEISCHER, SIMON L. CICHOSZ, KLAUS S. JENSEN, POUL ERIK JAKOB-
SEN, KNUD YDERSTRAEDE, ELISABETH GULICHSEN, PERNILLE HOEYEM, ESBEN
LAUGESEN, TROELS K. HANSEN, HANS NYGAARD, EBBE ELDRUP, HANS HENRIK
LERVANG, LISE TARNOW, NIELS EJSKJAER, Aarhus, Denmark, Copenhagen, Den-
mark, Aalborg, Denmark, Odense, Denmark, Herlev, Denmark, Gentofte, Denmark
OBJECTIVE: Clinical correlates and predictors of diabetic cardiovascular
autonomic neuropathy (CAN) include the presence of retinopathy, nephropa-
thy and peripheral neuropathy. The objective of this study was to elucidate
the association of tests for subclinical CAN with the presence of these com-
plications.RESEARCH DESIGN AND METHODS: We compared a selected co-
hort consisting of 290 type 1 diabetes patients from the DAN-study (Danish
multi-center study focusing on CAN) and 89 sex- and age-matched healthy
volunteers. The presence of CAN was quantied by measuring heart rate
variability (HRV) in time and frequency domains during cardiovascular re-
ex tests (valsalva ratio [VT], response to standing [RT] and deep breathing
[E:I]) and during 5 minutes of supine resting. In order to describe possible
associations between severity of complications and measures of autonomic
dysfunction, multivariate analysis was performed.RESULTS: In all tested pa-
rameters, HRV analysis revealed signicant differences (P<0.05) between
the control and the type 1 diabetes group. After adjusting for diabetes
duration, sex, age, pulse pressure and heart rate, autonomic dysfunction
remained signicantly correlated with severity of retinopathy, nephropathy
and peripheral neuropathy in individuals with type 1 diabetes patients. For
retinopathy, subgroup analysis showed that only spectral analysis of active
tests (response to standing and deep breathing) were signicant different
from all stages of P<0.05). CONCLUSIONS: Our results support the use of
a combination of different measures and tests for early detection of auto-
nomic dysfunction. This study corroborates that the severity of retinopathy,
nephropathy and peripheral neuropathy correlates with autonomic dysfunc-
tion.
580-P
Correlation between Small Fibre Neuropathy Assessed by the
LDIare Technique and the SUDOSCAN
in Type-1 Diabetes
SANJEEV SHARMA, PRASHANTH R. VAS, GERRY RAYMAN, Ipswich, United
Kingdom
Assessing small bre neuropathy in a busy clinical setting is a challenge.
The SUDOSCAN
(Impeto Medical, France), a new device for quickly assess-

ing sudomotor function may have such applicability. This study assesses the
SUDOSCAN
in patients with type-1 diabetes (T1DM), comparing its results

with those of the Laser Doppler Imaging (LDI) are technique -a non-invasive
method shown to be sensitive and specic for assessing C-bre function.39
subjects with T1DM [mean age 42.3 (SD=11.3)], 23 with (MV+) and 16 with-
out (MV-) microangiopathy, and 42 healthy controls [HC, mean age 47.1
(SD=15.9)] were studied. The LDIare involves heating dorsal foot skin and
measuring the axon-mediated are area with a laser Doppler imager. The
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EZSCAN
measures sudomotor function by assessing electrochemical skin

conductance (ESC) utilizing reverse iontophoresis and chronoamperometry
in the hands, feet and forehead.LDIares were reduced in T1DM compared
to HC (6.5 3.5 v 9.6 1.9 cm
2
; p<0.001). ESC in the feet was also reduced
in T1DM (70.3 1 9.6 v 77.4 7.7 S; p=0.029). Furthermore, MV+ compared
with MV- subjects had lower LDIares (4.3 2.5 v 9.8 1.98; p<0.0001) as
well as ESC in the feet (64.4 23.5 v 77.1 8.9; p=0.046). In contrast, neither
LDIare nor ESC responses differed between in MV- and HC groups in keep-
ing with our previous observation that C-bre function is not impaired in the
absence of microvascular disease. Across all subjects the methods correlat-
ed (r=0.2; p=0.02), as well as within the T1DM group (r=0.364; p=0.03). There
was no correlation within HC (r=-0.21; p=0.2).In conclusion, SUDOSCAN
results correlate with those of the LDIare technique; an established marker

of small bre function. Furthermore, as with the LDIare technique, abnor-
mal small bre function was found in MV+ but not in MV- patients. Because
it is simple and quick to perform, the SUDOSCAN
may serve as a useful tool

in assessing small bre neuropathy in the busy clinic setting.
581-P
Suppression of Rho/Rho-Kinase Pathway in Diabetic Rats Provides
a Therapeutic Effect on Diabetic Neuropathy
YASUSHI KANAZAWA, JUNKO TAKAHASHI-FUJIGASAKI, NAOKO TAKA-
BAYASHI, KUMIKO ISHIBASHI, SHO ISHIZAWA, KEIICHIRO MATOBA, DAIJI
KAWANAMI, TAMOTSU YOKOTA, KAZUNORI UTSUNOMIYA, Tokyo, Japan
Rho-kinase is a serine-threonine kinase, a downstream effector of a small
GTPase protein Rho. Rho/Rho-kinase pathway participates in various bio-
logical events, such as cell adhesion. We previously found that excessive
enhancement of Rho and Rho-kinase activity related to pathogenesis of
nerve decits of the diabetic rats. In the animal models, when a selective
Rho-kinase inhibitor, fasudil, is administrated from onset of diabetes, fasudil
inhibits development of both motor nerve conduction velocity (MNCV) and
sensory decit. In this study, we aim to examine whether fasudil could be
effective, when it is started after development of the diabetic nerve de-
cits.Sprague-Dawley rats were rendered diabetic with streptozotocin, and
divided in the 3 groups, normal control, diabetic, and diabetic rats treated
with fasudil. Fasudil administration started after 4 weeks of induction of
diabetes, and the rats were maintained totally for 8 weeks. To evaluate
the sensory decit, the latency of the hindpaw withdrawal evoked by ther-
mal stimulation was determined weekly. After 8 weeks, MNCV of sciatic
nerve was examined, and RhoA and Rho-kinase activities were estimated
biochemically using resected sciatic nerves.RhoA and Rho-kinase activities
were enhanced signicantly in the diabetic rats and were attenuated by ad-
ministration of fasudil. Both MNCV and sensory decits of the diabetic rats
were improved after the 4 weeks administration of fasudil. In the previous
study, we found that enhanced Rho-kinase activity signicantly altered Rho-
kinase related adhesion structures in Schwann cells, and the alteration was
restored by fasudil. This might be one of the underlining mechanisms for the
effectiveness of fasudil. This study showed that fasudil was effective, even
when it was started after development of the nerve decits. This also indi-
cates that disturbance of the Rho-kinase related adhesion could be restored
by fasudil, if it is once induced by diabetic condition.
582-P
Glycemic State has Real-time Impact on Beading Size and Fre-
quency while Requiring Several Years to Inuence Nerve Fibers by
Corneal Confocal Microscopy (CCM)
FUKASHI ISHIBASHI, Hiroshima, Japan
Hyperglycemia induces swelling and then, degeneration of mitochondria
(Mt) by over-produced reactive oxygen species, initiating neuron death.
Beading in CCM represents Mt. It remains to be settled what happens rst
upon hyperglycemia or glycemic control among corneal nerve (CN) struc-
tures_ber density (CNFD) and length (CNFL), branch density (CNBD) and
length (CNBL), tortuosity grade (TG), and beading frequency (BF) and size
(BS)_as identied by CCM. BS was assessable by enlarging and smoothing
of beads using S-Spline Max algorithm. We claried impact of prior hyperg-
lycemia on BS and BF in 38 type 1 diabetic patients (T1DM, HbA1c;7.30.1%,
follow-up period;11.51.2yrs) compared with 38 age-matched controls,
and impact of strict glycemic control (HbA1c;10.30.46.90.1% during
11.80.4 M) on BS and BF in 32 type 2 diabetic patients (T2DM). Compared
with controls, T1DM had lower BF (22.40.73 vs 30.40.14/0.1 mm, p<0.001),
larger BS (10.70.12 vs 9.70.15 m
2
, p<0.001), lower skewness of BS distri-
bution (0.300.032 vs 0.610.052, p<0.001), reduced CNFD (p<0.001), CNFL
(p<0.04), and increased TG (p<0.001). HbA1c at CCM, 1-3 M, or mean HbA1c 1
yr before CCM was an independent predictor for BF (p=0.0442-0.005) and BS
(p=0.049-0.004), whereas CNFD (p=0.02-0.003) and CNFL (p=0.042-0.002)
inversely correlated with mean annual HbA1c 7-10 yrs before CCM. Glycemic
control in T2DM increased BF (18.60.5120.70.34, p<0.001) and CNBD
(p<0.01) and decreased BS (9.60.138.40.09, p<0.001) and TG (p<0.001)
without inuencing CNFD and CNFL. BS decreased correlated (p=0.012-
0.031) with HbA1c 1-3 M before last CCM, and BF increased inversely cor-
related (p=0.012-0.035) with HbA1c 8-10 M before last CCM. In conclusion,
change in glycemic state was quickly reected in BS and BF and preceded
changes in CNFD and CNFL, suggesting that changes in bead (Mt) play a
pivotal role in subsequent changes in nerve bers.
583-P
SUDOSCAN: A Simple, Rapid, and Objective Method of Diagnosing
Diabetic Autonomic Neuropathy
SOLOMON TESFAYE, TOM CASH, ADITHYA SANKAR, JOSIE REEVES, JENNI-
FER DAVIES, GANESH RAO, IAIN WILKINSON, DINESH SELVARAJAH, Shefeld,
United Kingdom
Diabetic cardiac autonomic neuropathy (CAN) is a serious complication
of diabetes and carries up to a ve-fold increased risk of mortality. Current
methods of detecting CAN are based on a battery of cardiovascular reex
tests (CARTS) are impractical for widespread use in busy clinical practice.
SUDOSCAN is a new and rapid method of assessing sudomotor function by
measuring electrochemical skin conductance (ESC) through reverse ionto-
phoresis. The aim of the present study was to evaluate if SUDOSCAN can
reliably diagnose CAN.Methods: 41 subjects with type 1 diabetes and 13
healthy volunteers (HV) underwent detailed assessments including clini-
cal, neurophysiological (NISLL+7) and 5 CARTs (OBrien protocol). Based on
CARTs subjects were divided into CAN [3 abnormal CART results], Subclini-
cal CAN [1-2 abnormal results] and No-CAN [0 abnormal results]. All subjects
underwent the SUDOSCAN test. Participants placed their hands and feet on
electrodes, and an incremental low direct current (<4V) was applied to the
anode for 2min. ESC (Siemens) as a measure of sudomotor function was
obtained from the voltage and current and an autonomic risk score was cal-
culated.Results: The results show that SUDOSCAN can detect established
CAN having corrected for age and BMI.Conclusion: SUDOSCAN a simple,
rapid (<5min) and objective method of screening that doesnt require special
patient preparation or medical personnel training may serve as screening
test for CAN. It could help adhere to ADAs recommendation of annual AFT
for all diabetic patients which is currently not fullled as current tests are
cumbersome and time consuming.
HV No
CAN
Subclinical
CAN
CAN p-value p-value
adjusted
Age 4118 4313 5516 5014 0.068
BMI (Kg/M2) 23.93.7 28.45.9 29.65.7 30.36.0 0.018
Systolic BP 13218 14011 14014 13917 0.503 0.409
ESC hands (S) 6215 5912 6218 6115 0.893 0.886
ESC feet (S) 786 766 789 6026 0.003 0.004
ESC feet:hands ratio 1.30.3 1.40.3 1.30.3 1.00.4 0.045 0.044
Autonomic risk score (%) 2214 3015 3718 3913 0.022 <0.001
Supported by: Impeto Medical
584-P
The Functions of Glucose-Responsive ATP-Sensitive K (K
ATP
) Chan-
nels in Peripheral Nervous Systems
TATSUHITO HIMENO, HIDEKI KAMIYA, YUSUKE SEINO, KEIKO NARUSE, TETSUJI
OKAWA, JIRO KATO, MASAKI KONDO, ATSUSHI FUJIYA, EITA UENISHI, SHIN
TSUNEKAWA, YOJI HAMADA, YUTAKA OISO, SUSUMU SEINO, JIRO NAKA-
MURA, Nagoya, Japan, Nagakute, Japan, Kobe, Japan
Background:K
ATP
channels in pancreatic -cells are metabolic sensors
that determine glucose-responsive membrane excitability in the regulation
of insulin secretion. The K
ATP
channel is an octameric protein consisting of
two subunits: the pore-forming inward rectier K
+
channel member Kir6.1 or
Kir6.2, and the sulfonylurea (SU) receptor SUR1 or SUR2. In central nervous
systems, glucose responsive neurons share the same molecular composi-
tion Kir6.2 and SUR1 with -cells. Here we investigated functions of K
ATP
channels and potential physiological effects of SU agents in peripheral ner-
vous systems (PNS).Research design and methods:The expressions of Kir6.1,
Kir6.2 and SURs in PNS were determined with RT-PCR, immunohistochem-
istry (IHC) and western blotting (WB). Kir6.2-/- mice (KO) and wild type mice
(WT) were used in this study. In 4- and 24-week old mice, current percep-
tion thresholds (CPTs), thermal plantar test (TPT), and motor and sensory
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nerve conduction velocities (MNCVs and SNCVs, respectively) were evalu-
ated. Dorsal root ganglion (DRG) neurons derived from WT were cultured
with or without glibenclamide (GB) or tolbutamide (TB) for 24hs, and were
immunostained with an anti-neurolament M antibody to evaluate neurite
outgrowth.Results:RT-PCR, IHC and WB revealed the expression of Kir6.2 in
PNS. The expression of SUR2 in PNS was conrmed with RT-PCR. CPTs in KO
were signicantly increased compared with those in WT, indicating hypoal-
gesia. In TPT, paw withdrawal latencies in KO were delayed compared with
those in WT (WT: 6.1 1.3 s, KO: 11.1 5.3). SNCVs in KO were signicantly
delayed (WT: 34.8 9.2 m/s, KO: 25.0 2.6). The neurite outgrowth of DRG
neurons was remarkably reduced in the presence of GB (303.6 68.2 m) or
TB (403.1 46.9) compared with that in the absence of these compounds
(698.2 91.1).Conclusions:These results suggest that K
ATP
channels could
have important physiological roles in the functions of PNS.
585-P
Diabetic Autonomic Neuropathy and Cutaneous Circulation in Sub-
jects With Type 2 Diabetes Mellitus
IOANNA ELEFTHERIADOU, PINELOPI GRIGOROPOULOU, STAVROS LIATIS, ALEX-
ANDER KOKKINOS, VASILIKI ARGIANA, DESPOINA PERREA, NICHOLAS KATSIL-
AMBROS, NICHOLAS TENTOLOURIS, Athens, Greece
Transcutaneous oxygen tension (TcPO2) maps the oxygen supply available
for the skin tissue and is an indicator of the microvascular function. Patients
with type 2 diabetes (T2DM) have lower TcPO2 values in comparison with
subjects without diabetes. Previous data showed that diabetic individuals
with peripheral arterial disease (PAD) and peripheral neuropathy (PN) have
lower TcPO2 values in comparison with patients without these complica-
tions. In the present study we examined the effect of cardiac autonomic
neuropathy (CAN) on cutaneous circulation assessed by determination
of TcPO2 in patients with T2DM. A total of 100 patients with T2DM were
recruited (mean age 66.58.7 years, duration of diabetes 14.010.8 years).
TcPO2 was measured on the dorsum of the feet between the rst and second
metatarsal heads. Diagnosis of CAN was based on the battery of the four
autonomic function tests. Diagnosis of PAD was based on the absence of
triphasic waveform in the posterior tibial artery, while of PN on neuropa-
thy disability score and vibration perception threshold. A total of 20 sub-
jects had CAN. Subjects with CAN had more often PAD and PN and lower
TcPO2 levels in comparison with those without CAN (43.615.0 vs. 49.111.1
mmHg, p=0.033). Univariate regression analysis showed that diabetes dura-
tion, HbA1c, presence of PAD, presence of PN and presence and severity of
CAN were signicantly and negatively associated with TcPO2. Multivariate
analysis demonstrated that after adjustment for gender, age, duration of
diabetes and HbA1c, severity of CAN and presence of PAD was signicantly
and negatively associated with TcPO2. The same analysis showed that after
controlling for gender, age, diabetes duration and HbA1c, severity of CAN
and presence of PN were signicantly associated with lower TcPO2 levels.
In conclusion, presence and severity of CAN is associated with reduction in
cutaneous perfusion irrespective of the presence of PAD or PN.
586-P
The Effect of Aliskiren on Cardiovascular Autonomic Function
RAELENE E. MASER, M. JAMES LENHARD, PAUL KOLM, Newark, DE
The renin-angiotensin-aldosterone system (RAAS) and autonomic nervous
system (ANS) regulate the cardiovascular system. Dysfunction of the ANS
results in increased risk of mortality. Autonomic neuropathy is a multifacto-
rial pathogenic process (e.g., metabolic insult; neurovascular insufciency).
Pharmacological agents directed at components of the pathogenic process
have shown varied results. Aliskiren, a direct renin inhibitor, may offer an ad-
vantage over other mediators of the RAAS as renin catalyzes the rate-limiting
step of the RAAS. We investigated the effect of aliskiren in a double-blind,
placebo-controlled study. Nerve function was assessed by reex tests (e.g.,
RR-variation during deep breathing (assesses parasympathetic function)) and
power spectral analysis (PSA). RR-variation was measured by vector analysis
(i.e., mean circular resultant (MCR) and expiration/inspiration (E/I) ratio). Sub-
jects (n=60, 55% men, 48% type 2, aged 5112 (meanSD) yrs, duration1612
yrs) were randomly assigned to 300mg/day aliskiren (n=30) or placebo (n=30)
for 6-wks. Measures of ANS were analyzed by linear mixed effect models
comparing change in outcome from baseline to follow-up for aliskiren vs. pla-
cebo. There was a signicant interaction (aliskiren x visit) for MCR (p<0.003)
and E/I ratio (p<0.003) indicating improvement in MCR and E/I ratio for those
on aliskiren. Table 1 shows the meanSD for MCR and E/I ratio at baseline and
follow-up for aliskiren vs. placebo. Measures of PSA, however, did not differ
for baseline vs. follow-up in either group. Those on aliskiren did show improve-
ment in some measures of paraysmpathetic nerve function.
Table 1. Tests of RR-variation During Deep Breathing for Aliskiren vs. Placebo
Baseline Follow-up p-value
MCR for those on Aliskiren 41.819.7 50.826.1 <0.001
MCR for those on Placebo 38.223.6 37.524.1 NS
E/I ratio for those on Aliskiren 1.220.12 1.280.15 =0.001
E/I ratio for those on Placebo 1.210.14 1.200.14 NS
Supported by: Delaware INBRE, NIH Grant 2 P20 RR016472-11 from the NCRR
587-P
Cardiovascular Autonomic Neuropathy in Type 1 and Type 2 Diabe-
tes and Associations With Albuminuria, Retinopathy, Peripheral
Neuropathy, Pulse Pressure and Obesity: The DAN-Study
JESPER FLEISCHER, KNUD YDERSTRAEDE, ELISABETH GULICHSEN, POUL ERIK
JAKOBSEN, HANS HENRIK LERVANG, EBBE ELDRUP, HANS NYGAARD, LISE TAR-
NOW, NIELS EJSKJAER, Aarhus, Denmark, Odense, Denmark, Gentofte, Denmark,
Aalborg, Denmark, Herlev, Denmark
OBJECTIVE: To identify the presence of subclinical cardiovascular au-
tonomic neuropathy (CAN) in a cohort of individuals with diabetes in four
outpatient clinics in Denmark and to evaluate the association between CAN
and other risk factors.RESEARCH DESIGN AND METHODS: The DAN-Study
is a Danish multi-center study focusing on diabetic autonomic neuropathy.
Over a period of twelve months, 382 type 1 and 271 type 2 individuals with
diabetes were tested for CAN. Patients were randomly recruited and test-
ed during normal visits to outpatient clinics at four Danish hospitals. The
presence of CAN was quantied by measuring cardiovascular reex tests
(valsalva, response to standing and deep breathing) and analysis of heart
rate variability in time and frequency domain. In order to describe possible
associations, multivariate analysis with CAN as the dependent variable was
performed.RESULTS: Besides heart rate above 80 (ps0.001) and age above
60 years (p<0.02) in both type 1 and type 2 patients multiple ordinal logistic
regression analysis revealed that in Type 1 diabetes patients CAN was as-
sociated with microalbuminuria (p=0.015), macroalbuminuria (p=0.005), and
proliferative retinopathy (p=0.021). Among Type 2 diabetes patients CAN
was associated with high pulse pressure (p<0.001) and BMI above 35 kg/m2
(p=0.025).CONCLUSIONS: In this cross-sectional observational study CAN
is associated with proliferative retinopathy, micro- and macroalbuminuria
in type 1 diabetes patients, whereas in Type 2 diabetes patients CAN is as-
sociated with pulse pressure and obesity.
588-P
Skin Intrinsic Fluorescence is Associated With Nerve Fiber Morpho-
logy and Conduction Velocity in Persons With Diabetic Neuropathy
BAQIYYAH CONWAY, MICHAEL E. MAY, ILIZA MYERS, JOHN D. MAYNARD, KAY
ARTIBEE, WILLIAM J. BLOT, AMANDA PELTIER, Nashville, TN, Albuquerque, NM
Advanced glycation end products (AGEs) are thought to play a role in dia-
betic neuropathy (DN) and are observed in both myelinated and unmyelinated
diabetic nerve bers. Certain dermal collagen AGEs uoresce and thus skin
intrinsic uorescence (SIF) can be quantied and act as a novel marker of colla-
gen AGEs. We hypothesized that alterations in intrapapillary dermal myelinat-
ed nerve endings (IME) and sensory mechanoreceptos (Meissner corpuscles,
MCs) may correlate with AGEs as indicated by SIF. We compared SIF with
morphology data from skin biopsies in individuals with DN, (6 type 1 and 8 type
2 diabetes) and healthy controls (HC, n=9). We also compared SIF with nerve
conduction studies (NCS) in DN cases, HC, and 5 non-diabetic individuals with
idiopathic neuropathy (neuropathy controls, NC). Immunohistochemistry was
performed on 2 and 3 mm skin punches from the lateral index nger and distal
leg, respectively. Spearman correlations were used to compare SIF with age
adjusted skin morphology data and age and height adjusted NCS data. Mean
age and diabetes duration was 54.0 and 20.3 yrs, respectively, in those with
DN, while mean age were 53.8 and 58.8 in HC and NC, respectively. Average
MC density was 10.9 8.0 MCs/ mm
2
and 18.4 8.0 MCs/ mm
2
in HC (p=0.07).
Intraepidermal nerve ber density (IENFD) in the distal leg was 4.7 4.8 bers/
mm in DN and 12.0 4.7 in HC (p=0.001). SIF was higher in DN compared to HC
(p=0.03) but not compared to NC (p=0.31). SIF was inversely associated with
MC density (r= -0.93, p=0.0009) and IME density (r= -0.93, p=0.0007) in DN
only. SIF was not associated with IENFD in DN cases or healthy controls. SIF
showed a strong inverse relationship with peroneal motor amplitude (r=-0.74,
p=0.009) in DN only. In conclusion, noninvasively measured SIF suggests an
association between myelinated ber and mechanoreceptor loss and elevated
AGEs, but not unmyelinated ber loss.
Supported by: NINDS K23 NS056009, Vanderbilt CTSA Grant 1 UL1 RR024975
from NCRR/NIH
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Retinoic Acid Increases Nerve Growth Factor and Prevents Neu-
ropathy in OLETF Rats
JONG SUK PARK, MIN KYUNG KIM, JIYOON HA, YOUNGMI LEE, EUN JIN KWON,
JIWOON KIM, SHIN AE KANG, CHUL WOO AHN, KYUNG RAE KIM, Seoul, Re-
public of Korea
Retinoic acid is known to increase the endogenous expression of nerve
growth factor (NGF) which regulates neuronal differentiation and regenera-
tion. The aim of this study was to investigate the effects of all trans retinoic
acid (ATRA) on diabetic neuropathy in Otsuka Long-Evans Tokushima Fatty
(OLETF) rats, an animal model of type 2 diabetes.20 OLETF rats were re-
ceived with an oral dose of 10 mg/kg/day ATRA, and 20 OLETF rats and 10
Long-Evans Tokushima Otsuka (LETO) rats were received with the cellulose
as a vehicle once a day for 16 weeks. At the end of the treatment, glucose
levels and contents of NGF in serum and sciatic nerve were measured. Neu-
rometer test was conducted to examine improvements of the current stimu-
lus thresholds. The neurite growth from dorsal root ganglion (DRG) cells ex-
posed to NGF were assessed and mRNA of NGF and NGF receptors p75
NGFR
and trkA in response to ATRA concentration in DRG cells were measured by
RT-PCR and real-time PCR.Fasting glucose levels were signicantly higher
in the ATRA-non-treated OLETF rats (178.5 38.3 mg/dL) than in the ATRA-
treated OLETF rats (151.0 25.1 mg/dL) (P<0.01) or in the LETO rats (105.4
14.4 mg/dL) (P<0.01). Contents of NGF in LETO rats were 1423.50 + 503.21
pg/mL in serum and 243.2 41.9 pg/g in nerve; in ATRA-non-treated OLETF
rats the values were 683.71 169.89 pg/mL in serum and 150.0 28.8 pg/g
in nerve; and in ATRA-treated OLETF rats, 1855.17 667.34 pg/mL in serum
and 377.9 61.5 pg/g in nerve (P<0.05). A signicant decrease of threshold
at 2000, 250 Hz was observed in ATRA-treated OLETF rats than in ATRA-non-
treated OLETF rats. Neurite outgrowth and length in DRG cells increased in
NGF and ATRA dose dependent. But ATRA did not increase the expression of
the NGFR, p75
NGFR
and trkA mRNA levels.Our results have shown that ATRA
treatment increases serum and nerve contents of NGF in OLETF rats and
inuence nerve cell regeneration by promoting the synthesis of the NGF and
suggest that ATRA has a potential therapeutic role for diabetic neuropathy.
590-P
Evaluation of Diabetic Neuropathy Using Pupillary Light Reex Test
in Patients With Diabetes Mellitus
TAKESHI KUROSE, SHUUICHI FUJIWARA, KOIN WATANABE, TAKANORI HYO,
DAISUKE YABE, YUTAKA SEINO, Osaka, Japan
Since cardiovascular autonomic neuropathy has been postulated as a risk
of sudden death and affects the mortality of diabetic patients, reliable and
easy bedside test for evaluation of diabetic neuropathy is needed. In this
study we examined the efcacy of pupillary light reex test in the evaluation
of diabetic neuropathy. A total of 282 (189 male, 93 female) patients were
recruited in this study. The pupillary light reex test was performed using
a portable infrared video-pupillography system (Iriscorder Dual C10641
;
Hamamatsu Photonics). The measured parameters are as follows: D1, ini-
tial diameter before light stimulus (mm); D2, minimum diameter after light
(mm); CR, constriction ratio [CR=(D1 - D2)/D1]; VC, the maximum velocity of
constriction (mm/s). Mean age was 62, mean duration of diabetes was 11.2
years, and mean HbA1c level was 8.6 %. CR was signicantly associated
with coefcient of variation of R-R interval of 100 heart beat at rest (CV
R-R) (p<0.01), however, CR showed no association with the frequency of
orthostatic hypotension. On the other hand, VC was signicantly associ-
ated with both of CV R-R and orthostatic hypotension. Accordingly VC may
represent the autonomic neuropathy more precisely than CR. We have also
evaluated vibration perception threshold using C-64 tuning folk as a marker
of somatic nerve dysfunction. VC showed signicant correlation with vibra-
tion perception threshold (p<0.01). From the regression analysis of vibration
sensation and VC, VC which corresponds to the lower limit of normal sensa-
tion of vibration was 3.52. The frequency of abnormal Achilles tendon reex
in groups of less than 3.6 VC was more than the group of over or equal to
3.6 VC (p<0.01). Since the somatic nerve dysfunction appears earlier than
autonomic neuropathy, cut-off point of 3.6 VC may represent the early stage
of autonomic neuropathy. These results suggest that VC may be a good and
easy test for the detection of early stage of diabetic neuropathy.
591-P
The Functions of Gastric Inhibitory Polypeptide Signals in Periph-
eral Nervous Systems in Mice
TETSUJI OKAWA, HIDEKI KAMIYA, TATSUHITO HIMENO, NORIO HARADA, JIRO
KATO, KEIKO NARUSE, ATSUSHI FUJIYA, YUSUKE SEINO, AYAKO FUKAMI, SHIN
TSUNEKAWA, YOSHITAKA HAYASHI, YOJI HAMADA, YUICHIRO YAMADA, NO-
BUYA INAGAKI, YUTAKA SEINO, YUTAKA OISO, JIRO NAKAMURA, Nagoya, Ja-
pan, Nagakute, Japan, Kyoto, Japan, Akita, Japan, Osaka, Japan
The gastric inhibitory polypeptide (GIP) is one of the incretin hormones
secreted from the intestine on ingestion of glucose or nutrients to stimu-
late insulin secretion. GIP exerts its action by binding to a specic receptor
(GIPR) that belongs to the G-protein coupled receptor families. In addition to
insulinotropic effects, GIP has been reported to play several roles in various
tissues and organs. Here we investigated the effect of GIP signals on periph-
eral nerve functions.The presence of GIPR in dorsal root ganglions (DRGs)
was evaluated by immunohistochemistry (IH), westen blotting (WB) and RT-
PCR. DRG neurons were isolated from wild type (WT) and GIPR knockout
mice (KO), and were cultured in the presence or absence of a human recombi-
nant GIP. Then the neurite outgrowth of DRG neurons was determined. In the
animal model experiments, 5-month old KO and age-matched WT were used.
Peripheral nerve functions were evaluated by current perception thresholds
(CPTs), thermal plantar test (TPT), and motor and sensory nerve conduction
velocity (MNCV and SNCV, respectively). Sciatic nerve blood ow (SNBF)
and plantar skin blood ow (PSBF) were also evaluated.The expression of
GIPR in DRG neurons was conrmed by IH, WB and RT-PCR. GIP signicantly
promoted the neurite outgrowth of DRG neurons derived from WT in a dose
dependent manner. On the other hand, the neurite outgrowth of DRG neu-
rons derived from KO was signicantly reduced compared with that from
WT. KO showed hypoalgesia (TPT; WT: 9.10.9 s, KO: 15.82.1, p < 0.0001),
and delayed MNCV and SNCV (MNCV; WT: 47.71.5 m/s, KO: 36.49.2, p <
0.0001, SNCV; WT: 46.21.4, KO: 29.05.2, p < 0.0001). There were no differ-
ences in SNBF or PSBF between WT and KO.Our ndings indicate that GIP
signals would play important roles in maintaining peripheral nerve functions,
which might be mediated through their direct actions on DRG neurons and
axons.
592-P
Cardiovascular Autonomic Neuropathy is Associated With Arte-
rial Stiffness and Carotid Intima-Media Thickness in Patients With
Type 2 Diabetes
SU JIN OH, WON CHUL HA, HYUN SHIK SON, TAE SEO SOHN, Uijeongbu, Re-
public of Korea
Cardiovascular autonomic neuropathy (CAN) represents a signicant
cause of morbidity and mortality in patients with diabetes and is associ-
ated with a high risk of cardiac arrhythmia and sudden death. The aim of
this study is to evaluate the association between CAN and arterial stiffness
and carotid intima-media thickness (CIMT) in patients with type 2 diabe-
tes (T2DM).CAN, pulse wave velocity (PWV), ankle-brachial index (ABI), and
CIMT were evaluated in a cross-sectional sample of 268 patients (126 men
and 146 women) with T2DM. CAN was assessed by Ewings method which
employs ve non-invasive tests of autonomic function.Among 268 patients,
CAN was found in 48 patients (24%). Compared to the patients without CAN,
those with CAN were signicantly older and had signicantly longer duration
of diabetes, lower GFR, higher hs-CRP, mean CIMT, PWV, and ABI. The CAN
scores correlated positively with mean CIMT and PWV (P<0.05). Using multi-
variate regression analysis, CAN score independently predicted mean CIMT
in patients with T2DM after adjustment for age.In conclusion, CAN was as-
sociated increased arterial stiffness and CIMT in patients with T2DM. CAN
may inuence the development of cardiovascular disease through arterial
stiffness and CIMT in T2DM.
593-P
Role of Cholesterol in Neuronal Cell Function in Diabetes
KENJI FUKUI, C. RONALD KAHN, Boston, MA
Diabetes is associated with neurologic and cerebral complications, includ-
ing increased decline in cognitive function with the age, higher prevalence
of depression and increased risk of Alzheimer disease. Recently we have
shown that this is due, at least in part, to a reduction of SREBP-2 activity
leading to reduced brain cholesterol synthesis and reduced synapse forma-
tion. To determine the effect of reduced cholesterol content on neuronal
cells, we have mimicked the effect of diabetes to reduce cholesterol in GT1-7
neuronal cells by 3 approaches: treatment with the cholesterol-depleting
agent methyl-beta-cyclodextran (MBCD), treatment with the HMG-CoA
reductase inhibitor and cholesterol lowering drug simvastatin; and stable
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knockdown of the regulator of cholesterol synthesis, SREBP2. Each of these
treatment reduced cholesterol content by 20-40%. The resultant choles-
terol depleted neuronal cells exhibited impaired proliferation (69.75.0%
of control, p<0.01) and a parallel decrease in DNA synthesis (63.45.7% of
control, p<0.01). Cells treated with MBCD or symvastatin also showed de-
creased insulin and IGF-1 stimulated phosphorylation of AKT and ERK, with
no change in expression of these proteins or the insulin receptor. NGF and
BDNF induced phosphorylation of AKT and ERK was also suppressed in cho-
lesterol depleted cells. When cholesterol was chronically depleted by stable
knockdown of SREBP2, insulin, IGF-1 and NGF stimulated phosphorylation
of phosphorylation of AKT was decreased to the same extent as acute cho-
lesterol depletion with MBCD or simvastatin, but ERK phosphorylation was
increased. Cholesterol depleted cells also showed a tendency to reduced
secretion of GnRH following stimulation of cAMP. These result suggest that
reduction of cholesterol in neurons by multiple methods, including reduced
SREBP-2 as in diabetes and treatment with statins, impairs neuronal cell
proliferation and function and alters insulin signaling. These effects could
contribute to the CNS dysfunction observed in diabetes.
594-P
Sudomotor Function for Screening of Peripheral Neuropathy
RAMAKRISHNAN SANTOSH, RAJITHA K. AFSAR HUSAIN, ABHILASH PILLAI,
JEAN PAUL DESLYPERE, Hyderabad, India, Red Hill Forum, Singapore
Sensorimotor dysfunction is mainly considered for the diagnosis of periph-
eral neuropathy (PN). Autonomic small C-Fibers innervating sweat glands
are thin, long and unmyelinated and thus can be damaged very early in the
diabetes process. Sweat dysfunction leading to abnormal skin conditions
including dryness and ssures could increase diabetic foot risk. A consen-
sus statement of the ADA has suggested that sudomotor function should
be included in diagnostic tests for the detection of neuropathies in diabetes
but lack of simple, and quick methods have restricted the widespread of
such examination. The aim of this study was to evaluate SUDOSCAN
a new
quick, non-invasive, quantitative method to measure sudomotor dysfunction
as a screening tool for diabetic peripheral neuropathy.68 patients with type
2 diabetes (age 48 12 years, BMI 27 5 kg/m
2
) were measured for vibration
perception threshold (VPT) using a biothesiometer with ~ 15 V as cut-off
value for diagnosis of PN. Retinopathy status and serum creatinin level were
also assessed as indicator of microvascular lesions. Sudomotor dysfunction
was assessed by measuring electrochemical sweat conductance (ESC). Pa-
tients place their hands and feet (area with highest sweat gland density)
on large stainless-steel plates where a low voltage (<4V) is applied. ESC
results from chloride movements and electrochemical reaction in response
to electric stimulation.Based on biothesiometer examination 19 patients had
peripheral neuropathy. Using biothesiometer as reference, measurement
of sweat dysfunction had 95% sensitivity, 95% negative and 37% positive
predictive value for diagnosis of PN. Among the false positives 17 (out of
31) had diabetic retinopathy and/or high serum creatinin level indicating mi-
crovascular lesions.Sweating status using SUDOSCAN
a simple, quick and

quantitative method may be used as a screening tool of diabetic peripheral
neuropathy. Relevance of the method could be explained by earlier damages
in small C-ber when compared to large bers.
595-P
Reproducibility of a Rapid Point-of-Care Sural Nerve Conduction
Test
XUAN KONG, BONNIEJEAN BOETTCHER, KENNETH SNOW, SHAI GOZANI,
Waltham, MA
Nerve conduction studies (NCS) are the most accurate and reproduc-
ible test for diabetic peripheral neuropathy (DPN). They are used in clini-
cal settings and as an outcome measure in neurotherapeutic clinical trials.
However, NCS have not been widely adopted in the clinical management of
diabetes due to expense, limited availability, and complexity. A 1-minute,
low cost, sural nerve conduction test is now available as a point-of-care
(POC) biomarker of DPN. In order to be clinically useful, this test must have
high reproducibility so small changes in nerve function are reliably identied.
The objective of this study was to quantify the reproducibility of POC sural
nerve conduction.Seventeen subjects (15 male, age 27-64) without periph-
eral neuropathy were evaluated in two sessions 7-14 days apart. In each
session, sural conduction velocity (CV) and amplitude (Amp) were measured
twice in each leg. The device was removed and repositioned with every
test. The device corrects CV for skin temperature below 30
o
C. The primary
outcome measure was variance component analysis and the correspond-
ing coefcient of variation (CoV).A total of 136 tests were conducted. CV
was 55.83.5 m/s and Amp was 14.34.2 V. Between-session CoV was 3.6%
and 8.8% for CV and Amp, respectively. Within-session CoV was 2.4% (CV)
and 8.2% (Amp). The session-test CoV, which quanties the total variation
between sessions, was 4.3% (CV) and 12.0% (Amp). Log-transforming the
Amp reduced its CoV to 5.4%. Averaging the left and right limbs reduced the
session-test CoV to 3.4% (CV), 8.5% (Amp), and 3.8% (log(Amp)).A 1-minute
POC sural nerve conduction test demonstrated high reproducibility in a non-
neuropathic cohort. Reproducibility can be improved by log transforming
the amplitude and combining bilateral data. These results are comparable
to benchmark studies in which measurements were performed manually in
controlled multi-center clinical trials.
596-P
Correlation of Prolonged QTc Interval With Multi-Cardiovascular
Risk Factors in Patients With Type 2 Diabetes Mellitus
ZHANGRONG XU, HUI REN, AIHONG WANG, YUZHEN WANG, Beijing, China
Objective: To investigate the relationship between the prolonged QTc
interval and the multi-cardiovascular risk factors in patients with T2DM.
Methods: Medical data of 3940 T2DM patients were analyzed. After pa-
tients who may have cause of abnormal QTc were excluded, a total of 3426
T2DM patients who had underwent diabetic complication assessment and
ECG recording were enrolled. They were divided into normal QTc interval
group(group A, QTc <0.44 s, n = 2157) and prolonged QTc interval group(group
B, QTc~0.44s, n = 1269). The multi-cardiovascular risk factors were com-
pared between the two groups and further analyzed.Results: Compared
with group A, the patients in group B were older[(55.611.0) vs (54.610.9)
yrs], had longer diabetes duration, more females (51.5% vs 41.5%), short-
er body height[ (1.640.08) vs (1.650.08) m], bigger BMI [(26.23.6) vs
(25.83.2) kg/m
2
], bigger WC[(89.79.6) vs (88.29.5) cm], bigger hip circum-
ference[(95.47.3) vs (94.46.6)cm], higher WHR [(0.940.07) vs (0.930.08)],
higher BP[(134.820.3) /(76.910.2) vs (130.119.1) /(74.59.6) mmHg], higher
heart rate[(82.910.2) vs (70.710.1)beats/min], higher HbA1c [(8.02.0)% vs
(7.71.9)%], higher post-meal glucose[(13.75.1) vs (12.84.8) mmol/L], higher
fasting insulin[(9.48.6) vs (8.27.5)IU/ml], higher TC[(5.11.1) vs (5.01.1)
mmol/L], higher TG [(2.42.6) vs (2.22.5) mmol/L], and higher Alb/Cr [15(8,
36) vs 12 (7, 27) mg/g, median(P25, P75)]. All these indices were signicantly
different between the two groups(P<0.05). Logistic regression analysis
showed that factors including age, sex, BMI, waist circumference, WHR,
heart rate and Cr, were signicantly associated with prolonged QTc interval.
Conclusions: More than one third of T2DM patients present with prolonged
QTc interval and more severe muslti-cardiovascular risk factors.
597-P
Upregulation of S100B Protein in Astrocytes Cultured Under High
Glucose Condition
KEVIN K. YUE, MAN TAK CHU, Hong Kong, China
Diabetes mellitus (DM) is characterized by hyperglycemia and diabetic
complications. Recently it has been reported that there is an increase risk
of developing neurodegernative diseases like Alzheimer disease (AD) in
diabetic patients. Activation of astrocytes has been shown to be involved
in neuronal dysfunction, and S100B is a calcium binding protein that is pri-
marily expressed in astrocytes within central nervous system. In addition,
modulation of astrocytic activities and increase of S100B content were
found in different brain regions upon onset of neurodegenerative diseases.
Therefore, the correlation between DM and neuronal dysfunction was inves-
tigated, and more specically the levels of S100B protein and glial brillary
acidic protein (GFAP), a marker of astroctye activation, were determined in
astrocytes cultured under high glucose condition.Primary astrocyte cultures
and D1TNC1 type 1 astrocyte cells were treated with high glucose levels
(12 mM, 25 mM and 50 mM) for different time periods, with normal glucose
level (5.5 mM) as control. GFAP S100B and GFAP mRNA and protein levels
were then determined by real time PCR and immunoblotting.Astrocytic cul-
tures were shown to have a signicant increase of 20% in GFAP protein
expression after challenged with hyperglycemia stress for 3 and 4 days but
not in earlier time points. Similar results were obtained from primary astro-
cytes. Up-regulation of S100B mRNA levels were found in both astrocyte
cultures, with an increase of 23% and 27% in D1TNC1 type 1 astrocytes
and primary astrocytes respectively. From western analysis, the increase of
S100B protein in these cells were 30% and 33% respectively. Furthermore,
up-regulation of S100B was found to coincide with the changes in GFAP
expression.These results therefore imply that high glucose stress leads to
up-regulation of S100B protein and activation of astrocyte, and may play a
role in neuronal dysfunction resulting in diabetic cerebral vasculopathy and
other neurodegenerative diseases.
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599-P
Intraepidermal Nerve Fiber Density Decreases in Advance of Large
Fiber Neuropathy of Diabetic Patient
AIKO ARIMURA, TAKAHISA DEGUCHI, HIROSHI TAKASHIMA, YOSHIHIKO NISH-
IO, Kagoshima, Japan
Diabetic polyneuropathy (DP) consists of small and large ber neuropathy.
The large ber neuropathy can be assessed with nerve conduction study
(NCS) and the ndings of NCS have been used for the diagnosis or staging of
the DP. The small ber neuropathy, however, has not been fully investigated
in relation to the staging of the DP.Using intra-epidermal nerve ber density
(IENFD) and CVR-R as quantitative markers for the small ber neuropathy,
we compared them with the ndings of NCS.A total 44 patients with dia-
betes were subjected to analyses. All patients underwent full neurological
examination, skin biopsy to evaluate the IENFD, NCS, and measurement of
CVR-R.The 44 diabetic patients with DP were classied into stage I - V ac-
cording to the criteria by the Diabetic Neuropathy Study Group in Japan.
No signicant differences in age, BMI, A1C, or duration of diabetes were
found among each stage of the patients. Findings of NCS such as motor
and sensory nerve conduction velocity, their action potential, and F-wave
latency were impaired as the stage progressed. Similarly, IENFD and CVR-R
decreased signicantly with progressing the stage. However, the patients
at the early stages such as stageIand II showed signicant decreases in the
IENFD (40% and 60%, respectively). Similarly, 10% of patients at stage I and
20% of those at stage II showed signicant decreases in the CVR-R.Diabetic
small neuropathy assessed with IENFD and CVR-R developed in accordance
with the progression of the stage of DP. However, signicant proportion of
the patients with normal NCS showed decreases in IENFD and CVR-R sug-
gesting that small ber neuropathy may develop in advance of the large ber
neuropathy.
600-P
Prevalence of Autonomic Dysfunction and its Risk Factors in Pa-
tients With Impaired Glucose Tolerance (IGT)
RIDDHI PATIRA, SHARAD SHARMA, DEEPAK VYAS, MAHESH GUPTA, NAVNEET
AGARWAL, Gwalior, India, Agra, India
Autonomic dysfunction is well known in diabetic population. There is
evidence that autonomic neuropathy may start even earlier in IGT(dened
by American Diabetes Association as two-hour plasma glucose value dur-
ing a 75 g oral glucose tolerance test between 140 and 199 mg/dL), lead-
ing to increased incidence of cardiovascular complications. Variables like
Hypertension, BMI>25, poor glycemic control and smoking may also act as
addendum to autonomic neuropathy in patient with IGT. Our aim was to as-
sess prevalence of autonomic dysfunction and variables like Hypertension
and BMI in patients with IGT. We randomly selected 72 patients with IGT
from diabetic screening program in Gwalior, India. Autonomic neuropathy
test was based on criteria by Ewing et al (blood pressure response to stand-
ing, Valsalva test, 30th:15th beat ratio and deep breathe test). 32 (44.4%)
were found to have denite parasympathetic damage i.e. minimum two
parasympathetic function test results abnormal. Deep breathe test which
is predictor of parasympathetic abnormality, was present in highest num-
ber of patients 60 (83.33%) suggesting that this test may be able to detect
autonomic dysfunction earlier than other tests. However, this can be only
conrmed with further studies where diagnosis of autonomic dysfunction
can be made before performing this test. We also found that prevalence
of autonomic neuropathy in IGT patents with BMI>25 was 27/51(52.94%).
Similarly, prevalence of autonomic neuropathy in hypertensive IGT pateints
was 21/36 (58.33%). We conclude that autonomic dysfunction occurs more
widely in IGT than was hitherto suspected and the autonomic neuropathy is
more prevalent in IGT patients with BMI>25 and Hypertension.
601-P
Hyperglycemia Alters the Apoptotic Pathway via Heat Shock Pro-
tein, and Insulin-Like Growth Factor Binding Protein Alterations in
the Mammalian Amygdala
JESSICA R. HAYDEN, EDWARD P. SAENZ, REJEANA M. STEPHENS, CARLOS A.
GARCIA, Kingsville, TX
Investigators have reported hyperglycemia alters the functioning of the
central nervous system (CNS). Abnormalities including cognitive impair-
ments are well documented in diabetics. Cognitive decits may be tied to
decreases in brain volume. The amygdala, an important brain region that
regulates emotional responses and memory, contains receptors for leutiniz-
ing hormone, vasopressin and somatostatin may be impacted by hypergly-
cemia. Early events, prior to clinically signicant CNS changes, may include
alterations in apoptotic protein levels. The purpose of the current work is
to test the hypothesis that hyperglycemia induces apoptotic pathways in
the cells of the mammalian amygdala by altering the concentrations of heat
shock proteins (Hsp) and both insulin-like growth factor proteins (IGF) and
insulin-like growth factor binding proteins (IGFBP) of streptozotocin (STZ)-
induced diabetic Wistar rats. Age and sex-matched rats were separated into
two groups (n=3), control and diabetic (STZ: 60 mg/kg dissolved in 0.5ml
0.05M citrate buffer, pH 4.2). Three months after the onset of diabetes,
both groups were sacriced and brains dissected. The amygdala was col-
lected for biochemical analysis. The content of Hsp-27, Hsp-60, Hsp-70, IGFI,
IGF II, and IGFBP1-IGFBP6 was measured by an apoptosis antibody array in
amygdala tissue homogenates. The levels of Hsp-27 (43%), Hsp-60 (48%)
and Hsp-70 (36%) decreased in the diabetic amygdala along with decreases
in IGFI (38%), IGFII (42%) and all of the IGFBPs tested. The results suggest
an alteration of apoptotic pathways in the amygdala are stimulated by hy-
perglycemia. Cellular survival mechanisms mediated by HSPs, IGFs, and
IGFBPs were down regulated pointing to an increase in cell death due to
apoptosis. This study provides novel therapeutic molecular pathways that
could be targets for future drug intervention for diabetics that experience
cognitive decits.
Supported by: HRSA H9CRH22885 and a University Research Award
WITHDRAWN
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Sympathovagal Imbalance and Abnormal Cerebrovascular Reserve
in Impaired Glucose Tolerance (IGT) and Type-2 Diabetes
DINESH SELVARAJAH, TIM HUGHES, ELAINE CACHIA, SOLOMON TESFAYE, IAIN
WILKINSON, Shefeld, United Kingdom
Type 2 DM imposes a 2-3 fold risk of stroke and poorer outcomes. The abil-
ity to alter blood ow in response to haemodynamic stress helps maintain
adequate brain perfusion. Impaired vascular reactivity is associated with
increased stroke risk and poor recovery. This study examines the relation-
ship between cardio-vagal autonomic function and cerebrovascular hae-
modynamic abnormalities in diabetes and prediabetes.Method: Each group
[T2DM with no history of stroke, IGT and healthy volunteers (HV)] underwent
standard tests of cardiovascular autonomic function (OBriens protocol)
and baroreex sensitivity. Quantitative measures of internal carotid artery
ow (ICA-F ml/s) and reactivity (ICA-R) were determined using MR Phase
Contrast Angiography. MR data was acquired before and 30min after IV ad-
ministration of 1g acetazolamide (ACZ), a potent intracerebral vasodilator.
ICA-R = percentage change in ICA-F.Results: There were no signicant group
differences in autonomic tests. T2DM subjects had signicantly lower ICA-F
[pre-ACZ 4.3(1.0) ANOVA p=0.01; post-ACZ 6.1(1.5), ANOVA p=0.002] com-
pared with IGT [ICA-F pre-ACZ 4.9(0.9); post-ACZ 6.7(1.0)] and HV [ICA-F pre-
ACZ 4.3(1.0); post-ACZ 6.9(1.1)].Mean ICA-R was signicantly lower in both
T2DM [43.1(18.3)] and IGT [40.1(19.8)] compared with HV [58.6(15.2), ANOVA
p=0.01].There was signicant positive correlation between ICA-R and LF:HF
ratio (p=0.45, p=0.04) and relative proportion of LF (p=0.46, p=0.03) and
negative correlation with proportion HF (p=-0.46, p=0.03).Conclusions: This
study provides new insights into the pathophysiology of cerebrovascular
disease.We have demonstrated signicantly lower ICA-R in IGT and asymp-
tomatic T2DM subjects. Moreover, abnormal sympatho-vagal balance was
strongly associated with ICA-R reduction. Future work should examine if ab-
normal sympatho-vagal balance results in worse outcomes and if targeted
risk factor modications can ameliorate this.
603-P
The Role of Acupuncture in the Management of Painful Diabetic
Neuropathy (PDN): A Randomized Clinical Trial
ADAM GARROW, MEI XING, JOANNE VERE, BARBARA VERRALL, LIFEN WANG,
EDWARD JUDE, Salford, United Kingdom, Ashton-under-Lyne, United Kingdom,
Manchester, United Kingdom
In this single-blind placebo controlled Randomised Controlled Trial we
examined the role of acupuncture as an additional treatment in theman-
agement of Painful Diabetic Neuropathy (PDN). A total of 59 primary and
secondary care patients with Type 1 or Type 2 diabetes and a clinical di-
agnosis of PDN were recruited. 28/59 (47%) were randomly allocated to
received a 10-week course either of real (53%) or sham (47%) acupuncture.
5 standardised acupuncture points on the lowerlimb of each leg used in the
study: Liv-3, Kid-3, Sp-6, Sp-10, St-36. The Principal Outcome Measure was
the Leeds Assessment of Neuropathic Symptoms and Signs (LANSS) scale.
Secondary outcomes were lower-limb pain intensity (VAS); Sleep Problem
Scale (SPS); Measure Yourself Medical Outcome Prole (MYMOP); SF36 and
resting Blood Pressure (BP). Over the 10-week treatment period, patients
receiving active acupuncture showed statistically signicant improvements
in VAS -14 (-26,-3.5): MYMOP -0.89 (-1.4,-0.3) SPS -2.5 (-4.3,-0.82) and Rest-
ing Diastolic Blood Pressure -5.2 (-10.4,-0.14). In contrast, there was little
change in those receiving sham acupuncture. A signicant moderate treat-
ment effect in favour of active acupuncture was detected in MYMOP scores
-0.66 (-0.96 to -0.35). Non-signicant improvements were also seen for the
LANSS Pain Scale -0.37 (-2.2 to 1.4) Resting Diastolic BP -0.50 (-3.0 to 1.99)
and the Sleep Problem Scale -0.51 (-2.2 to 1.4). Acupuncture treatment was
well tolerated with no appreciable side effects. Further randomised trials
are needed to conrm the clinical and cost-effectiveness of acupuncture in
the treatment of Painful Diabetic Neuropathy.
Supported by: UK NIHR Research for Patient Benet Scheme
604-P
Impact of the Glyco-Metabolic Control on Erectile Dysfunction in
Patients With Type 2 Diabetes Mellitus
GIUSEPPE DEROSA, CARMINE TINELLI, ANGELA DANGELO, ELENA FOGARI,
ALDO BONAVENTURA, PAMELA MAFFIOLI, Pavia, Italy
The aim of this study was to evaluate the impact of the glyco-metabolic
control on erectile dysfunction (ED) in patients with type 2 diabetes mel-
litus. We evaluated 88 males affected by type 2 diabetes mellitus, with a
mean age of 62.789.26 years. We administered patients the IIEF (Interna-
tional Index of Erectile Function) questionnaire to assess erectile function,
organ function, sexual desire, and satisfaction level during and after the
sexual intercourse. We also administered SAS (self-rating anxiety scale) and
SDS (self-rating depression scale) questionnaires to evaluate anxiety and
depression. We also evaluated some parameters such as BMI, abdominal
circumference, glycated hemoglobin (HbA
1c
), fasting plasma glucose (FPG),
fasting plasma insulin (FPI), HOMA index, lipid prole, testosterone, free tes-
tosterone, dihydrotestosterone, and sex hormone binding globulin (SHBG).
The IIEF questionnaire showed that in the examined sample there were 50
patients (56.8%) affected by ED, and 38 patients (43.2%) without ED. Com-
paring the two groups, 57.9% of patients without ED, and 70.0% of patients
with ED were smokers, and the difference between the two groups was
signicant (p<0.05). Furthermore, 23.7% of patients withoutED, and 38.0%
of patients with ED had a history of chronic ischemic heartdisease (p<0.05
between the two groups). Patients with ED were older, and, surprisingly, had
lower levels of HbA
1c
. Furthermore, patients with EDhad higher levels of FPI,
and lower levels of testosterone and dihydrotestosterone.In conclusion our
study showed that the prevalence of ED in Italian males with type 2 diabetes
mellitus with mean age of 62 years, is about 56% and it is linked to higher
levels of FPI, but lower levels of HbA
1c
, and lower levels of free testosterone
and dihydrotestosterone.
605-P
Executive Function and Diabetic Peripheral Neuropathy
JASON L. RUCKER, STEPHEN D. JERNIGAN, JOAN M. MCDOWD, PATRICIA M.
KLUDING, Kansas City, KS, Kansas City, MO
There is growing evidence that cognitive impairments in executive function
(EF) contribute to the functional decits associated with diabetes mellitus
(DM). We examined EF in adults with DM and diabetic peripheral neuropathy
(DPN) as compared to age-matched control subjects. Twenty subjects with
DM and DPN (DM; 8 female; 58.4 6.2 years) and 20 control subjects (CN; 14
female; 54.9 6.1 years) completed the study. Subjects were administered
Becks Depression Inventory (BDI) and the Mini Mental Status Examination
(MMSE). EF was assessed via measures of attention (digit span), working
memory (reverse digit span), shifting (Trial Making Test), verbal organiza-
tion (letter and category uency), perceptual organization (Rey-Osterrieth
Complex Figure; ROCF), and dual task performance (Timed Up and Go with
mental subtraction; cTUG). Severity of DPN was quantied via tibial and per-
oneal nerve conduction testing. The CN group had greater overall cognition
(MMSE; p=0.001), lower BMI (p=0.02), and less depression (BDI; p<0.001).
The DM group performed worse on letter uency (p=0.003), category u-
ency (p=0.001), and the ROCF (p<0.001). The DM group also required more
time to complete the TUG (p<0.001) and cTUG (p<0.001); however cognitive
and motor dual task costs were not signicantly different. Reduced peroneal
nerve amplitude was signicantly associated with lower MMSE scores
(r=0.51) and greater cognitive dual task costs (r=0.51), while depression was
signicantly associated with lower MMSE score (r=-0.46) and a longer TUG
(r=0.54) and cTUG time (r=0.52). Obesity (BMI) and glycemic control (HbA1c)
were not signicantly correlated with any cognitive or functional measure.
Our data indicate that adults with DM and DPN exhibit verbal and perceptual
organization decits. As problems with such executive functions have been
linked to impairments in many aspects of functional ability, future research
should examine how these and other cognitive factors, such as depression,
impact function in this population.
606-P
Gastrointestinal Symptoms in Patients With Insulin Treated Diabe-
tes Mellitus and the Correlation With Mental and Physical Health
EVA A. OLAUSSON, JAN BRUN, GISELA RINGSTROM, CHRISTINA BROCK, KLAS
BUNNER, PETER FORS, MAGNUS SIMRN, STIG ATTVALL, Gothenburg, Sweden,
Aalborg, Denmark, Kungalv, Sweden, Alingsas, Sweden
In this study we characterized the spectrum of GI symptoms in patients
with insulin treated DM and how these correlate with physical and mental
health.We invited 801 patientswith insulin treated DM attending two diabe-
tes outpatient clinics to participate. They were asked to complete a ques-
tionnaire to rate the severity of twenty GI symptoms during the previous
two weeks (Patient Assessment of Upper Gastrointestinal Symptom Sever-
ity Index (PAGI-SYM). A proportion of the subjects also completed question-
naires to assess quality of life (QOL) (SF-36), and anxiety and depression
(HAD).475 patients returned completed questionnaires (response rate 59%)
(age 4915 (meanSD) years; 266 males). In the non-responder group a larger
male predominance was observed (65 vs. 56%; p=0.009), but there was no
age difference between the groups. 75 (16%) reported no GI symptoms at all
and the majority of patients reported very mild or mild symptoms. Using at
least moderate symptom severity as a cut-off, the most prevalent GI symp-
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COMPLICATIONSOCULAR
toms were stomach fullness (29.4%), visible abdominal distension (20.5%),
postprandial fullness (17.3%), bloating (17.3%) and loss of appetite (10.8%),
whereas nausea and vomiting of at least moderate severity was reported
by 8.6% and 4% of the patients, respectively.The GI symptom severity was
associated with impaired physical and mental QOL, as well as with anxi-
ety and depression. Stronger correlations with the severity of GI symptoms
were seen for anxiety than for depression, and for physical vs. mental QOL.
In summary, GI symptoms are common in DM, and fullness, bloating and
distention are particularly common. are the most common GIS in these DM
patients, but the vast majority of DM patients with GIS report mild symptom
severity. GI symptoms were associated with impaired physical and mental
health and paying attention to and treating these symptoms seems to be
clinically important.
COMPLICATIONSOCULAR
Guided Audio Tour: Diabetic RetinopathyBench to Bedside (Posters 607-
P to 614-P), see page 13.
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Design, Characterization, and Structure-Activity Relationships of
Substituted 2-Amino-4-Phenyl-Thiophenes as Novel Atypical Pro-
tein Kinase C Inhibitors for Diabetic Macular Edema
PAUL M. TITCHENELL, JEAN-FRANOIS PONS, DAVID A. ANTONETTI, Hershey,
PA, Abingdon, United Kingdom, Ann Arbor, MI
Diabetic retinopathy remains a leading cause of blindness worldwide.
Growth factors such as vascular endothelial growth factor (VEGF) and in-
ammatory cytokines such as tumor necrosis factor (TNF) are increased in
the diabetic eye and contribute to the loss of the blood-retinal barrier and
subsequent macular edema associated with vision loss. Although anti-VEGF
therapy improves visual outcome for some patients, there remain a subset
of individuals that fail to respond. Additionally, current anti-VEGF therapies
require repeat intra-ocular injections. Our laboratory has recently demon-
strated that atypical protein kinase C (aPKC) isoforms are required for both
TNF and VEGF induced endothelial permeability. Further, a series of small
molecules inhibitors of aPKC isoforms that are effective at preventing VEGF
and TNF induced retinal permeability were identied from a Chembridge
library screen. Here, we describe a detailed drug discovery and medicinal
chemistry optimization approach to further characterize and dene these
novel small molecule inhibitors of aPKC isoforms on a phenyl-thiophene plat-
form. Structure-activity relationships (SAR) were dened by the synthesis
and screening of over 200 phenyl-thiophene analogues for their ability to
inhibit aPKC in vitro. From this analysis a detailed SAR model was built and
a pharmacophore elucidated that confers inhibitor activity. Lead compounds
with favorable physicochemical parameters were further tested for their
ability to prevent TNF and VEGF induced permeability in primary culture.
Lead compounds were shown to be specic, potent, and exhibit high efcacy
for their ability to prevent TNF and VEGF induced permeability. Importantly,
2-amino-4-phenyl-thiophene derivatives may be developed to preserve the
blood-retinal barrier and ameliorate the macular edema associated with
retinal diseases such as diabetic retinopathy.
Supported by: JDRF (D.A.A.), NIH EY012021 (D.A.A.), FFS
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The Role of VLDLR in Down-Regulation of Wnt/-Catenin Signaling
and Diabetic Retinopathy
KYUNGWON LEE, JIAN-XING MA, Oklahoma City, OK
Diabetic retinopathy (DR) is a severe ocular complication of diabetes,
involving progressive retinal inammation, vascular leakage and neovascu-
larization (NV). Recent studies revealed that Wnt/-catenin signaling is im-
plicated in the pathogenesis of DR. Retinal levels of -catenin and Wnt co-
receptor low-density lipoprotein-related receptor 5/6 (LRP5/6) are increased
in humans with DR and in DR animal models. Very low-density lipoprotein
receptor knockout (vldlr
-/-
) mice show up-regulated Wnt/-catenin signaling
with increased levels of -catenin and LRP6 in the retina. Moreover, absence
of vldlr exhibits retinal NV and retinal inammation, pathological changes
similar to DR, suggesting that VLDLR may play inhibitory roles for DR via
suppression of Wnt/-catenin signaling.In the present study, we dem-
onstrated that VLDLR and LRP6 formed heterodimerization (VLDLR:LRP6)
through ectodomains using co-immunoprecipitation assay. The soluble
VLDLR ectodomain sufciently attenuated Wnt3A-induced phosphoryla-
tion of LRP6 and TCF/-catenin transcriptional activity in retinal pigment
epithelial cells. In addition, VLDLR ectodomain accelerated turn-over rate
of LRP6, a possible mechanism by which the VLDLR:LRP6 heterodimeriza-
tion inhibits Wnt/-catenin signaling.A monoclonal antibody (mAb) specic
for the VLDLR ectodomain that blocks VLDLR:LRP6 heterodimerization, en-
hanced Wnt/-catenin signaling via activation of LRP6. Intravitreal injection
of the anti-VLDLR mAb stabilized -catenin and increased the expression of
Wnt target genes including VEGF and TNF-o in the retina. Taken together,
these observations demonstrated that the VLDLR:LRP6 heterodimerization
represents a mechanism for VLDLR to inhibit Wnt/-catenin signaling. These
ndings suggest that VLDLR ectodomain has therapeutic potential for the
treatment of DR.
Supported by: NIH (EY018659, EY012231, EY019309, P20RR024215)
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Decient XBP1 Signaling in Endothelial Cells Exacerbates Leuko-
stasis and Tight Junction Damage in Diabetic Retinopathy
SARAH X. ZHANG, JINGMING LI, CHEN CHEN, GUANGJUN JING, JOSHUA J.
WANG, Oklahoma City, OK
Endoplasmic reticulum (ER) stress has been widely implicated in chronic
inammatory diseases, such as diabetes and its complications. X-box bind-
ing protein 1 (XBP1) is a central coordinator of cellular responses during ER
stress. The objective of this study is to investigat the role of XBP1 in en-
dothelial inammation and vascular dysfunction associated with diabetic
retinopathy (DR). Endothelium-specic XBP1 decient (XBP1EC-/-) mice were
generated by using the Cre-loxp system. Diabetes was induced by intrap-
eritoneal injection of streptozotocin (STZ). Expression of inammatory cy-
tokines and tight junction proteins, leukostasis and vascular permeability
were evaluated in diabetic XBP1EC-/- and wild type (XBP1EC+/+) mice. As
expected, retinal expression of intracellular adherent molecule-1 (ICAM-1)
and vascular adherent molecule-1 (VCAM-1) was signicantly increased,
in parallel with enhanced leukocytes adhesion to retinal vasculature in
diabetic XBP1EC+/+ mice. When compared to XBP1EC+/+ mice, diabetic XB-
P1EC-/- mice showed much higher levels of adhesion molecules and greater
increase in leukostasis in the retina. In addition, retinal expression of ZO-1,
a major tight junction protein, was much lower in XBP1EC-/- mice, indicat-
ing impaired blood-retinal barrier. Furthermore, XBP1EC-/- mice developed
more severe retinal vascular leakage compared with XBP1EC+/+ mice. Con-
sistent with the in vivo results, endothelial cells isolated from XBP1EC-/-
mice expressed much higher level of ICAM-1, but lower level of ZO-1 after
incubation with pro-inammatory cytokine TNF-o. This suggests that loss of
XBP1 sensitizes endothelial cells to inammation and tight junction damage.
Taken together, our results indicate that XBP1 plays a critical role in regulat-
ing immune homeostasis and barrier function in endothelial cells. Impaired
XBP1 signaling may contribute to vascular inammation and retinal edema
in diabetic retinopathy.
Supported by: NIH EY019949, AHAF, OCAST
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610-P
Kaplan-Meier Analysis of Time to First Signicant Vision Improve-
ment in Two Phase III Trials of Ranibizumab for Diabetic Macular
Edema (DME)
CARL D. REGILLO, SUNIR J. GARG, JASON HSU, LINDA YAU, YAN ZHENG, J. JILL
HOPKINS, JASON S. EHRLICH, LISA TUOMI, Philadelphia, PA, San Francisco, CA
DME is a serious vision-threatening complication of diabetic retinopathy
(DR). Vascular endothelial growth factor A (VEGF-A) promotes vascular per-
meability and is upregulated in DR. Ranibizumab is a monoclonal antibody
fragment (Fab) that binds to and inhibits VEGF-A. The phase 3 trials, RIDE and
RISE, enrolled 759 patients with DME randomized 1:1:1 to monthly intravitreal
ranibizumab (0.5mg or 0.3mg) or sham. Macular laser was available for all arms
starting at month 3. Using the Kaplan-Meier (KM) method, we estimated time
to rst signicant improvement in best corrected visual acuity (BCVA) dened
as a gain of ~15 letters from baseline on the Early Treatment Diabetic Retin-
opathy Study (ETDRS) chart or Snellen equivalent of 20/40 or better. Vision
worse than 20/40 has been shown to adversely affect patient self-care and
independent function. Comparing the ranibizumab and sham KM curves, a
signicantly shorter time to rst improvement in BCVA of 20/40 or better was
seen in ranibizumab-treated patients (P < 0.001) within the 24-month controlled
treatment period (Table). More than half of the ranibizumab-treated patients
rst achieved 20/40 vision or better within 2 months of treatment compared
to >6 months in sham patients. Over half of the ranibizumab-treated patients
who ever gained ~15 letters during the study did so after at least ve months
of treatment, indicating that signicant BCVA improvement could be achieved
with ongoing therapy even if the initial response was suboptimal.
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Median number of days (95% CI)
to rst visit with outcome & cumulative
% of patients who ever achieved
the outcome
a
by Month 24
Ranibizumab
Sham (n=257) 0.3 mg (n=250) 0.5 mg (n=252)
Gain of ~15 ETDRS letters >720 (NE) 337 (272-426)
b
331 (244-427)
b
cumulative % with outcome 37% 69% 65%
Snellen equivalent of 20/40 or better
c
217 (119-329) 71 (59-120)
b
58 (35-91)
b
cumulative % with outcome 67% 81% 83%
NE = not estimable because less than half of the patients reached the endpoint
in the rst 24-months.
a
Outcome not necessarily maintained at month 24.
b
P <
0.001, obtained from a log-rank test between the treatment arm vs. sham arm.
c
Proportion of patients with Snellen equivalent of 20/40 or better at baseline:
19.5%, 16.4% and 19.4% in the sham, 0.3 mg and 0.5 mg groups respectively.
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Novel Lipid Mediator Maresin Isomer Harnesses Mesenchymal
Stem Cells to Ameliorate Diabetes Mellitus and Diabetic Retinopa-
thy in db/db Mice
SONG HONG, HAIBIN TIAN, STEPHANIE R. GROSS, YAN LU, JOSE A. GALINDO,
QUANSHENG WANG, BRUCE A. BUNNELL, CHRISTIAN T. SHELINE, New Orleans, LA
Stem cell-based therapies provide promise to cure diabetic mellitus and
complications. Lipid mediators derived from docosahexaenoic acid are po-
tently anti-inammatory and tissue-protective. Herein, we tested the hypoth-
esis that Maresin isomer (7S-MaR1; 7S,14R-dihydroxy-4Z,8E,10Z,12Z,16Z,19Z-
docosahexaenoic acid), can promote stem cell functions in amelioration of
diabetic mellitus and retinopathy. GFP-labeled bone marrow mesenchymal
stem cells (MSCs), derived from C57BL/6J mice, were treated with 7S-MaR1
or control medium and then delivered into 16-week old diabetic db/db mice
via tail vein every 5 days for 35 days. Compared to control or untreated MSC
groups, the treatment with 7S-MaR1-treated-MSCs to db/db mice reduced
blood glucose level (522.7 46.3 vs 611.2 14.0 (control) or 632.7 23.1 (un-
treated) mg/dl at day 35 of treatment, n = 6, p < 0.05 ); improved glucose tol-
erance, increased blood insulin level (1.3 0.1 vs 0.7 0.1, 0.6 0.1 ng/ml, n =
6, p < 0.05); increased insulin+ -cell ratio (77.7 1.8% vs 72.8 1.4%, 68.6
43.2%, n = 60, p < 0.05) and decreased glucogan+ o-cell ratio (22.4 1.3%
vs 27.0 1.3%, 28.8 2.4%, n = 60, p < 0.05) in islets; promoted vascular-
ity, and reduced macrophage inltration in pancreas. 7S-MaR1 augmented
MSC functions of promoting MIN6 -cell viability and insulin secretion in
vitro. Additionally 7S-MaR1 promoted MSC paracrine functions of increasing
the generation of hepatocyte growth factor and vascular endothelial growth
factor. Moreover, 7S-MaR1 enhanced MSC functions to ameliorate diabetic
damage on retinal endothelial cells, pericytes, and ganglion cells at early
stage of diabetic retinopathy of db/db mice by increasing their density in ret-
ina and decreased apoptotic cells in retina compared to control or untreated
MSC groups. This work provided novel lead to develop better modality based
on 7S-MaR1-prompted-MSCs for human diabetic mellitus and retinopathy.
Supported by: NIH Grant 1-R01-DK087800 (S.H.)
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A Simple Algorithm to Estimate Time to Development of Sight-
Threatening Retinopathy
IRENE M. STRATTON, AMANDA I. ADLER, STEPHEN J. ALDINGTON, DAVID TAY-
LOR, MARK HISTED, PETER H. SCANLON, Gloucester, United Kingdom, Cambridge,
United Kingdom
The American Diabetes Association and UK National Health Service rec-
ommend annual screening for diabetic retinopathy (DR) to refer sight threat-
ening retinopathy (STDR) to ophthalmology clinics. Using longitudinal data
from Gloucester Retinal Screening Service we developed a model based
on retinal photographs to inform screening frequency. From 2005, 14,554
patients with non-referable DR (no DR or microaneurysms (MA) only) at 2
consecutive annual screenings were grouped on presence of MA in neither,
1 or both eyes at each screening and followed thereafter for a median 3
years (Table 1). Of 7,246 with no MAs at both screenings, 120 progressed to
STDR, 0.7% by 1 year. Of 1,778 with MAs in neither eye at rst and 1 eye at
second screening, 80 progressed to STDR, 1.9% by 1 year, hazard ratio (HR)
2.9 (95% CI 2.2 to 3.8) compared to those with no MA. Of 1,159 with MA in
both eyes at both screenings 299 progressed to STDR, 11% by 1 year, HR
18.2 (14.7 to 22.5) compared with no MA (Fig 1). Those in higher risk groups
were likely to progress to serious DR, and those with little or no DR unlikely
to progress to STDR in the one year screening interval. Results from 2 an-
nual screenings enable progression risk estimation which informs screening
frequency decisions even without other clinical data.
Characteristics of DR categories and progression to sight threatening refer-
able retinopathy
First Screen R0
Both eyes
R1
one eye
R1
Both eyes
R0 or R1 in
either eye
R0
Both eyes
R1
one eye
R1
Both eyes
Second
Screen
R0
Both eyes
R0
Both eyes
R0
Both eyes
R1
one eye
R1
Both eyes
R1
Both eyes
R1
Both eyes
N 7246 1266 343 3031 853 656 1159
Age (years) 64.5 (13.0) 66.3(12.2) 66.8(12.6) 65.5(12.7) 68.0 (12.2) 64.1(14.1) 63.0 (14.6)
proportion
male (%)
55.6% 59.4% 53.9% 55.9% 53.6% 55.0% 58.9%
Progressed to
referable DR
120 30 12 152 82 108 299
Hazard ratio 1.0 1.5
(1.0 to 2.3)
2.6
(1.4 to 4.7)
3.3
(2.6 to 4.2)
6.0
(4.5 to 7.9)
10.0
(7.7 to 13.0)
18.2
(14.7 to 22.5)
Expected pro-
portion with
DR 2 years
after second
screening
1% 2% 3% 4% 7% 12% 21%
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Puerarin Inhibits the Retinal Pericyte Apoptosis Induced by Ad-
vanced Glycation End Products In Vitro and In Vivo by Inhibiting
NADPH Oxidase-Related Oxidative Stress
JUNGHYUN KIM, CHAN-SIK KIM, EUNJIN SOHN, YUN MI LEE, KYUHYUNG JO,
JIN SOOK KIM, Daejeon, Republic of Korea
Retinal pericyte loss is one of the histopathological hallmarks of early
diabetic retinopathy. Puerarin (4-7-dihydroxy-8-beta-d-glucosylisoavone),
which is an isoavone-C-glucoside, causes various pharmacological ef-
fects that include anti-hyperglycemic and anti-inammatory activities. In
the present study, we determined the efcacy and possible mechanism of
puerarin on the advanced glycation end product (AGE)-modied bovine se-
rum albumin (BSA)-induced apoptosis of cultured bovine retinal pericytes
and rat retinal pericytes in intravitreally AGE-modied rat serum albumin
(RSA)-injected eyes. Puerarin signicantly inhibited pericyte apoptosis, the
generation of reactive oxygen species (ROS) and NADPH oxidase activity by
inhibiting the phosphorylation of p47phox and Rac1 which were induced by
the AGE-BSA treatment. The puerarin treatment markedly suppressed the
activation of nuclear factor-kappaB (NF-kB). In addition, the in vivo apoptosis
of the retinal pericyte of rats that was stimulated by the intravitreal injec-
tion of AGE-RSA was evidently attenuated by the puerarin treatment. These
results demonstrate that puerarin may exert inhibitory effects on AGE-
induced pericyte apoptosis by interfering with the NADPH oxidase-related
ROS pathways and blocking NF-kB activation, thereby ameliorating retinal
microvascular dysfunction.
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Two New Loci Associated With Onset and Progression of Diabetic
Retinopathy Identied by a Genome-Wide Association Study
HETAL S. SHAH, JAN SKUPIEN, JASON NOBLE, AHMED Z. SOLIMAN, PAOLO A.
SILVA, GABRIEL D. POZNIK, ADAM M. SMILES, LLOYD P. AIELLO, JENNIFER K.
SUN, ANDRZEJ S. KROLEWSKI, ALESSANDRO DORIA, Boston, MA
Diabetic retinopathy (DR) is a leading cause of vision loss in developed
countries. Although susceptibility to proliferative DR (PDR) is inuenced by
heritable factors, the genes involved are still unclear. The aim of this study
was to identify genetic variants associated with DR onset and progression
in subjects with type 1 diabetes (T1D). Using a time-to-event approach, we
conducted a genome-wide association study of time from diabetes onset
to four DR severity levels (mild, moderate, and severe non-proliferative DR,
and PDR), as documented in the medical records of 872 White T1D subjects:
352 with normoalbuminuria, 311 with microalbuminuria, and 209 with protei-
nuria. A total of 2.4 M single nucleotide polymorphisms (SNPs), genotyped
(Illumina 300K or Affymetrix 500K chip) or imputed (MACH 1.0), formed our
GWAS array. Data were analyzed by means of a Cox proportional hazards
model stratied by kidney status, applying a robust variance estimator to
account for the correlated times to consecutive DR stages and for the clus-
tering of events between the two eyes. Using a joint 2 d.f. test of genetic
marginal association and gene-environment interaction, with glycemic con-
trol (HbA1c below or above the median, 8.3%) as the interactor, two SNPs
reached genome-wide signicance: rs7668242 on chromosome 4 (p=1.2 x 10
-
8
) and rs4755904 on chromosome 11 (p=4.2 x 10
-8
). Both genetic effects were
exclusively observed in the high HbA1c stratum, with hazard ratios (HRs) of
2.0 and 2.6, respectively, as compared to HRs of 0.97 and 0.85 in the low
HbA1c stratum. Rs7668242 is located in the neural retina-expressed gene
GP6MA, and is also near WDR17, a candidate gene for retinal disease. Three
other loci approached genome-wide signicance on chromosomes 3, 10 and
15. In conclusion, we have identied two new loci potentially contributing to
DR onset and progression. Studies are underway to further replicate these
ndings and dissect the mechanisms underlying these genetic effects.
615-P
Rod Degeneration Accelerates Cone Photoreceptor Death in Dia-
betes
YUN-ZHENG LE, KE SHI, Oklahoma City, OK
Diabetic retinopathy (DR) is traditionally considered as a microvascular
complication in diabetic retina. However, emerging evidences suggest that
the loss of retinal neuron function and the death of retinal neurons are
involved in DR. To determine the relationship between rods and cones in
diabetes, we utilized a rod-specic Bcl-x knockout (KO) mice that had been
shown to have impaired stress-induced survival previously and determined
whether rod death affected cone survival in diabetic conditional Bcl-x KO
mice. In our study, diabetes was induced with streptozotocin. Retinal func-
tion was measured with electroretinography (ERG) and retinal morphol-
ogy was assessed with hematoxylin & eosin (H&E) stained sections. Cone
density was evaluated with immunohistochemistry. Thirty two weeks after
inducing diabetes, rod-specic Bcl-x KO mice demonstrated accelerated
loss of outer nuclear layer (ONL) thickness and rod photoreceptor function,
compared with that of wild-type animals. Interestingly, cone photoreceptor
function, as measured by photopic ERG, was signicantly reduced, compared
with wild-type controls. Immunohistochemical analysis of S-opsin and M-
opsin, markers of cone photoreceptors, showed that cone density was also
signicantly reduced in diabetic rod-specic Bcl-x KO mice, compared with
wild-type animals. Our data showed that BCL-x
L
, a PI3K-AKT survival path-
way downstream target, is involved in rod photoreceptor protection under
diabetic condition. Our results also suggest that cone photoreceptors are
vulnerable to diabetes-induced rod photoreceptor death. We are currently
investigating the underlying mechanism of this observation.
Supported by: R01EY20900, P20RR024215, OCAST Contract HR09-058
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Vitamin D Is Not Associated With Diabetic Retinopathy in Patients
With Type 1 Diabetes and Type 2 Diabetes
JOHANNA M. BRIX, ASTRID DOSSENBACH-GLANINGER, SIMON BRUNNER,
EVA KRZIZEK, GERIT H. SCHERNTHANER, GUNTRAM SCHERNTHANER, Vienna,
Austria
Diabetic Retinopathy (DR) is the leading cause for blindness in the West-
ern world. Hypothesis suggests that Vitamin D deciency may be associated
with diabetic microvascular disease, but a study in patients carefully clas-
sied for stages of DR is lacking. Thus, we investigated Vitamin D levels in
patients with type 1 diabetes (T1D) and type 2 diabetes mellitus (T2D) well
dened for different stages of DR and compared it to healthy controls (CO).
Vitamin D levels were determined in 90 T1D and 135 T2D patients, both with
and without DR and in 25 CO. Mean age was 4112 in T1D, 6210 in T2D and
613 years in CO. Patients were classied for DR: 0 - no DR, 1 - mild nonpro-
liferative DR, 2- moderate-severe nonproliferative DR, 3 - mild proliferative
DR and 4 - high proliferative DR. Patients were staged according to a simpli-
ed Early Treatment of Diabetic Retinopathy Study classication. Vitamin D
was measured as 25-Hydroxy (OH) Vitamin D (LC-MS-MS reference method).
Patients with T2D had signicantly lower Vitamin D levels than CO (12.97.5
ng/ml vs 20.57.8 ng/ml; p<0.001). Patients with T1D had signicantly higher
Vitamin D levels (27.711.2 ng/ml vs 12.97.5 ng/ml; p<0.001) than patients
with T2D, but they were signicantly younger (p<0.001). Vitamin D levels did
not signicantly differ in patients with or without DR and were not related
to the stages of DR either in T1D or in T2D (Table 1).In summary, patients
with T2D and different stages of DR have signicantly lower 25-OH Vitamin
D levels compared to healthy controls. However, no association was found
between Vitamin D deciency and DR in patients with either T1D or T2D.
Table 1
Diabetic Retinopathy Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 p-value
Type 1 diabetes 28.19.9 29.610.4 19.518.1 27.410.8 22.917.3 0.54
Type 2 diabetes 13.88.3 15.18.1 9.56.1 13.14.7 10.94.6 0.34
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Increased Hemoglobin AGE Levels in Type 1 Diabetes Patients and
Correlation With Vascular Inammation
ANDRZEJ S. JANUSZEWSKI, DANIEL CALANDRO, DAVID N. ONEAL, CONNIE S.
KARSCHIMKUS, ALICIA J. JENKINS, Melbourne, Australia, Footscray, Australia
Hyperglycemia, inammation and Advanced Glycation End-Products
(AGEs) may promote diabetes complications. Glycemia is reected by HbA1c,
but clinically useful AGE measures are lacking. Given their lifespan and ac-
cessibility Red Blood Cell (RBC) related AGEs are of interest. We quantied
hemoglobin (Hb) AGEs by a previously published assay and by our novel as-
say of globin autouorescence (AF) in a cross-sectional study of 147 (18-73
year old Type 1 diabetic (T1DM) patients (including 54 with and 93 without
complications) and 60 age and gender matched healthy non-diabetic con-
trols. Renal failure subjects were excluded. MeanSD diabetes duration
was 2113 years. HbA1c in T1DM and controls was 7.91.4% and 5.00.3%
respectively (p<0.00001).Relative to controls Hb-AGEs were increased in
T1D 0.190.09 vs. 0.140.03 AU, p=0.0003; as was Globin AF 2.370.16 vs.
2.190.09, p<0.00001. There were no signicant differences in either AGE
related to T1D complication status nor use of insulin pumps vs. multiple
daily insulin injections. Hb-AGEs and globin AF correlated in T1DM (r=0.41;
p<0.00001) but not in controls (r=0.05; p=NS). Only Globin-AF correlated with
vascular health (mean BP r=0.39; p=0.002 and Small Artery Elasticity r=-
0.30; p=0.02 in controls, but not in T1D patients. In T1D Globin AF correlated
with measures of vascular inammation (ELISA, RnD, Minneapolis, MN):
se-Selectin r=0.26; p=0.002; sICAM r=0.23; p=0.007 and sVCAM-1 r=0.19;
p=0.03. Neither AGE level correlated with age, HbA1c or renal dysfunction
in T1D.Relative to controls, T1D is associated with signicantly higher levels
in two Hb related AGEs, which correlate with measures of vascular inam-
mation, but not with vascular complications. These simple, low-cost repro-
ducible assays may facilitate monitoring of anti-AGE therapies and vascular
inammation. Longitudinal studies are merited.
619-P
Cases of Vision-Threatening Diabetic Retinopathy Avoided Using
Ranibizumab for Visually-Impairing Diabetic Macular Edema in the
United States
ROHIT VARMA, NEIL M. BRESSLER, QUAN DOAN, PAUL LEE, IVAN SUER, MARK
DANESE, CHANTAL M. DOLAN, ADAM TURPCU, ABRAHAM LEE, JASON S. EH-
RLICH, SHOSHANA COLMAN, Los Angeles, CA, Baltimore, MD, Westlake Village,
CA, Durham, NC, Tampa, FL, San Francisco, CA
The number of non-Hispanic White and Hispanic persons aged 45 years
and older in the US with diabetic retinopathy and center-involved diabetic
macular edema (DR with DME) who would avoid vision-threatening DR over
a 2-year period following treatment with ranibizumab 0.5 mg was estimated
using a Monte Carlo simulation model. The outcome was dened as cases
that did not have severe non-proliferative DR (NPDR) or proliferative DR
(PDR) at baseline and worsened to severe NPDR or PDR (i.e., vision threaten-
ing DR) at 2 years. Incident cases of DR with DME were estimated based
on: the US population size in 2011, the prevalence of diagnosed diabetes,
and the 1-year incidence rate for DR with DME with vision impairment. All
insured patients (private and Medicare/Medicaid) were included. Baseline
DR severity in the population was assumed to be similar to baseline DR dis-
tribution of patients enrolled in the RISE and RIDE clinical trials. DR changes
with and without ranibizumab were based on the RISE and RIDE data and
were conditioned upon patients baseline DR status.The model predicted
that 15,293 incident cases of visually-impairing DR with DME were eligible
for ranibizumab treatment. Without ranibizumab, 1,263 (95% condence
interval, 312 to 3,069) new cases of vision-threatening DR were expected
over 2 years. Ranibizumab treatment reduced the estimated new cases of
vision-threatening DR by 83% (81% to 84%) to 220 (55 to 537) cases, with
1,043 (257 to 2,542) cases avoided. The results suggest that monthly ranibi-
zumab could substantially reduce worsening to vision-threatening DR within
2 years after treatment of non-Hispanic white and Hispanic patients. This
benet may be an underestimation since the model only included incident
cases and not prevalent cases of visually-impairing DR with DME.
620-P
Interactive Effect of Aldose Reductase Z-4 Microsatellite Polymor-
phism and Glycaemic Control on Cataract Development in Type 2
Diabetes
YING WANG, ANDREA O. LUK, CALVIN C. PANG, MAGGIE C. NG, VINCENT K.
LAM, RICHARD CHOY, SHAO C. LEE, WING YEE SO, DENNIS S. LAM, RONALD C.
MA, JULIANA C. CHAN, Hong Kong, China
Aldose reductase (ALR2) of the polyol pathway is a key enzyme implicated
in formation of cataracts in diabetes. We examined the interactive effect
of the z-4 microsatellite polymorphism of ALR2 gene and glycemic control
on risk of cataract in a cross-sectional cohort of Chinese type 2 diabetic
patients. The (CA)n microsatellite polymorphism of ALR2 was determined
using polymerase chain reaction followed by capillary gel electrophoresis.
Cataract was dened by presence of lens opacity on direct ophthalmoscopy
or history of cataract surgery. A non-linear curve approach was used to iden-
tify the threshold of glycated hemoglobin (HbA1c) at which the odds ratio
(OR) for cataract started to increase. The association of z-4 allele with cata-
ract, above and below this threshold, was assessed using multiple logistic
regression analysis.Of the 5,823 patients examined, 28.1% had cataracts.
Those with cataracts were older, had longer duration of diabetes and worse
metabolic control. After adjusting for conventional risk factors and using
non-z-4 carriers with HbA1c<8.0 % as referent group (n=3173), the OR (95%
condence intervals) for cataract was highest in z-4 carriers with HbA1c~8.0
% [1.43(1.05-1.96), n=244], compared to non-z -4 carriers with HbA1c~8.0
[1.27(1.10-1.47), n=1836] and z-4 carriers with HbA1c<8.0% [1.01(0.77-1.29),
n=420, Ptrend<0.001). This interaction remained signicant after additional
adjustments for drug use (Ptrend=0.002) and renal function (Ptrend=0.01). In
type 2 diabetic patients with suboptimal glycemic control, z-4 microsatellite
polymorphism of ALR2 gene was independently associated with increased
susceptibility to cataracts.
Supported by: Innovation and Technology Fund (ITS/487/09FP), and the CUHK
Focused Investment
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Impaired Flow-Mediated Vasodilatation and Diabetic Retinopathy
in Patients With Type 2 Diabetes Mellitus
SEUNG-HYUN KO, JUNG-MIN LEE, HYUK-SANG KWON, SUNG-DAE MOON,
YONG-MOON PARK, YU-BAE AHN, Suwon, Gyeonggi-Do, Republic of Korea,
Seoul, Republic of Korea
Endothelial dysfunction plays an important role in the development of
atherosclerosis and vascular complications in type 2 diabetic patients. We
investigated the relationship between the endothelial-dependent vasodila-
tion and diabetic retinopathy (DR) in patients with type 2 diabetes.167 pa-
tients with type 2 diabetes (90 men and 77 women) were enrolled. Patients
were excluded if they had a history of cardiovascular disease, acute infec-
tions, aged more than 75 years, uncontrolled diabetes (HbA1c > 10.0%), or
uncontrolled hypertension (> 160/ > 100 mmHg). We assessed endothelial
dysfunction by ow-mediated vasodilation (FMD) method of the brachial ar-
tery using high-resolution ultrasound, and changes in vasodilation (%FMD)
were expressed as percent change over baseline values. The presence of DR
was also assessed.The mean age was 54.1 8.6 years. The mean diabetic
duration and HbA1c level were 7.3 6.0 years and 7.5 1.0%, respectively.
The median value of FMD was 3.85% (0-7.89). Twenty-nine patients (17.4%)
had DR, and the %FMD was signicantly lower in patients with DR than
without DR (0.65% (-0.33-4.20) vs. 4.87% (0.60-8.58), P < 0.001). Groups
were divided according to FMD tertiles (T1 s1.35, T2=1.35-5.95, T3 > 5.95%).
The prevalence of DR decreased across increasing tertiles of %FMD. After
adjusting for patients age, sex, diabetes duration, use of insulin, antihyper-
tensive, anti-platelet, or lipid lowering medications, systolic blood pressure,
fasting and 2-hour plasma glucose, HbA1c, and urinary albumin excretion,
participants with a reduced %FMD were more likely to have DR. (Odds ratio
11.37 [95% condence interval (CI) 2.14 - 60.31]), comparing the lowest and
highest tertiles of %FMD).Endothelial dysfunction was an independent pre-
dictor of DR, and this was most visible when the endothelial dysfunction was
the most severe. Our study provides insights into the possible mechanism of
endothelial dysfunctions inuence on the development of DR.
623-P
Current Prevalence and Correlates of Retinopathy in Patients With
Type 2 Diabetes
GIUSEPPE PUGLIESE, ANNA SOLINI, ENZO BONORA, EMANUELA ORSI, CECILIA
FONDELLI, GIANPAOLO ZERBINI, ROBERTO TREVISAN, MONICA VEDOVATO,
GABRIELLA GRUDEN, FRANCO CAVALOT, MAURO CIGNARELLI, LUIGI LAVIOLA,
SUSANNA MORANO, ANTONIO NICOLUCCI, GIUSEPPE PENNO, THE RENAL IN-
SUFFICIENCY AND CARDIOVASCULAR EVENTS (RIACE) STUDY GROUP, Rome,
Italy, Pisa, Italy, Verona, Italy, Milan, Italy, Siena, Italy, Bergamo, Italy, Padua, Italy,
Turin, Italy, Foggia, Italy, Bari, Italy, Santa Maria Imbaro, Italy
The natural history of diabetic complications, including diabetic retinopa-
thy (DR), is changing due to improved diabetes care. This study was aimed
at assessing current prevalence of DR and its association with risk factors
and complications, particularly chronic kidney disease (CKD), in subjects
with type 2 diabetes mellitus (T2DM). Patients with T2DM from the Renal
Insufciency And Cardiovascular Events (RIACE) Italian Multicenter Study
(n=15,773), visiting consecutively 19 hospital-based Diabetes Clinics in years
2007-2008, were examined. DR was assessed by fundoscopy. CKD was
dened based on albuminuria and glomerular ltration rate, as estimated
from serum creatinine. Major acute cardiovascular disease (CVD) events
were adjudicated based on hospital discharge records. Any retinopathy
was observed in 22.2% (3,497) of patients; 12.4% had non-advanced DR,
whereas 9.8% had advanced DR (non-proliferative 4.2%, proliferative 4.2%,
maculopathy 1.3%, blindness 0.1%). Non-advanced and advanced DR were
independently associated with HbA
1c
, diabetes duration and treatment (par-
ticularly with insulin) hypertension, any previous CVD, albuminuria and the
albuminuric CKD phenotypes and, inversely, with age and age at diabetes
diagnosis. Maculopathy alone was also associated with female sex, but not
with HbA
1c
, hypertension and age. Triglycerides were independently associ-
ated with presence versus absence of CKD in subjects with advanced retin-
opathy. Other correlates differed according to the CKD phenotype, with male
sex, hypertension, and previous CVD associated with the albuminuric forms,
female sex with the nonalbuminuric phenotype, and age with reduced GFR,
independent of albuminuria. Data from this large cohort show that retin-
opathy is present in less than one fourth of subjects, with a relatively high
prevalence of advanced forms, and correlates with surrogate indexes of
chronic hyperglycaemia, hypertension, cardiovascular events, and albuminu-
ria in subjects with T2DM.
Supported by: Italian Society of Diabetology (Fo.Ri.SID)
Guided Audio Tour: Diabetic Dyslipidemia (Posters 624-P to 631-P), see page
17.
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624-P
Enteral Glucose Enhances Chylomicron Production in Healthy
Humans
CHANGTING XIAO, SATYA DASH, LINDA SZETO, GARY F. LEWIS, Toronto, ON,
Canada
Overproduction of VLDL by the liver is an important contributor to diabetic
dyslipidemia. We and others have recently shown that intestinal lipopro-
tein (chylomicron) production is also increased in insulin resistance and is
similarly regulated by several nutrient and hormonal factors that control
hepatic lipoprotein production. High carbohydrate diets and hyperglycemia
are associated with hypertriglyceridemia, believed to be mainly due to VLDL
hypersecretion, whereas chylomicron secretion is not known to be modied
by the addition of glucose to ingested fat. Here we examined the effects of
lipid, with or without added glucose, delivered directly into the duodenum,
on hepatic and intestinal lipoprotein metabolism in humans. Seven normolip-
idemic, normoglycemic men underwent 2 separate studies each, 4-6 weeks
apart, in random order. During each study the subjects received a constant
infusion of lipid (20% Intralipid, 60ml/h) plus either normal saline (60ml/h)
(NS study) or glucose (25% dextrose, 60ml/h) (G study) through a nasoduo-
denal tube for 15 hours. TG-rich lipoprotein (TRL) apoB-100 and B-48 kinetics
were assessed with a primed, constant infusion of deuterated leucine under
pancreatic clamps. Compared with lipid infusion alone, intraduodenal co-
infusion of glucose increased plasma and TRL TG concentrations by 41% and
50%, respectively. TRL apoB-48 concentrations (NS=1.4+/-0.5, G=3.3+/-0.9
mg/L, P=0.04) and production rates (NS=0.04/-0.02, G=0.25+/-0.10 mg/kg/
day, P=0.004) were markedly increased with glucose co-infusion. TRL apoB-
100 concentrations (NS=37.8+/-7.7, G=47.5+/-6.3 mg/L, P=0.19) or production
rates (NS=7.8+/-2.6, G=10.1+/-2.2 mg/kg/day, P=0.21) were not signicantly
affected by treatments. These results show that enteral glucose enhances
chylomicron particle production and may have important implications for di-
etary recommendations especially for individuals predisposed to hypertrig-
lyceridemia, such as those with insulin resistance and type 2 diabetes.
Supported by: Canadian Institutes of Health Research
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625-P
APOA5 Genotype Modulates 2-Year Changes in Lipid Prole in Re-
sponse to Weight-Loss Diet Intervention: The Pounds Lost Trial
XIAOMIN ZHANG, QIBIN QI, FRANK B. HU, FRANK M. SACKS, LU QI, Boston, MA
Apolipoprotein A5 gene (APOA5) is a major gene involved in lipid metabo-
lism and modulated by dietary factors. A new variant rs964184 in APOA5 was
recently identied to be associated with lipids in genome-wide association
studies (GWAS). We aimed to examine whether APOA5 variant might modify
changes of lipid levels in response to 2-year weight-loss diet intervention
in a randomized trial. We genotyped APOA5 rs964184 in 734 overweight or
obese adults who were randomly assigned to one of four diets differing in
the percentages of energy derived from fat, protein and carbohydrate for 2
years. We evaluated the change of fasting serum concentrations of total
cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density li-
poprotein cholesterol (HDL-C), and triglyceride (TG) from baseline to 2 years
of follow-up. After 2-year dietary intervention, we found signicant interac-
tion between the APOA5 rs964184 polymorphism and dietary fat intake (low
vs high) modulating changes in TC, LDL-C and HDL-C concentrations (P for
interactions=0.010, 0.007 and 0.021, respectively). In low-fat intake group
(20% of energy derived from fat), carriers of the risk allele (G allele) exhibited
a greater reduction in TC and LDL-C than the non-carriers (Padd=0.040 and
0.042); the genotype effects were not observed in high-fat group (40% of
energy derived from fat). Analyses in the white participants showed similar
results. Our data suggest that a long-term low dietary fat intake may im-
prove lipid prole in the APOA5 rs964184 risk allele carriers.
Supported by: HL071981, RR-02635
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Plasma Angiopoietin-Like Protein 4 is Increased in Patients With
Type 2 Diabetes and the Metabolic Syndrome in the Presence of
Low Grade Inammation
NATHANJA TJEERDEMA, JACQUELINE JONKER, SANDER KERSTEN, PATRICK C.
RENSEN, JOHANNES W. SMIT, Leiden, The Netherlands, Wageningen, The Neth-
erlands
Angiopoietin-like 4 protein (Angptl4) is an adipocytokine that plays a role
in lipid metabolism by inhibition of lipoprotein lipase and has been asso-
ciated with dyslipidemia. Experimental studies suggest that Angptl4 also
plays a role in inammation, but it is unknown how Angptl4 levels are inu-
enced by inammation.Therefore the objective of this study was to assess
plasma Angptl4 in T2DM, in subjects with MetS in the presence or absence
of low grade inammation, and in healthy controls (Controls).A total of 268
male subjects (T2DM n=67, MetS with low grade inammation, n=67, MetS
without low grade inammation n=67 and Controls n=67) were included
in this study. All subjects were matched for age and waist circumference.
The IDF-criteria for the MetS were used and low grade inammation was
dened as serum CRP ~1.8 mg/L. As Angptl4 levels are not normally distrib-
uted, comparisons between groups were performed with Wilcoxon signed
rank tests adjusted for the number of comparisons.In all patients combined,
mean(SD) age was 56.8(5.8) years and mean waist circumference was
105(9) cm. Angptl4 [median(interquartile range)] was increased in subjects
with T2DM [6.4(5.2-7.8) ng/mL, P<0.001] and in subjects with MetS with low
grade inammation [6.8(5.3-8.1) ng/mL, P<0.001] compared to healthy con-
trols [4.0(2.8-5.6) ng/mL]. This elevation was independent of obesity as sub-
jects were matched for waist circumference. Patients with the MetS with-
out inammation did not show elevated levels of Angptl4 [3.8(2.8-5.2) ng/
mL, P=0.751].In conclusion, increased plasma Angptl4 levels are observed in
subjects with T2DM and MetS with, but not without, low grade inamma-
tion. We hypothesize that systemic inammation plays an important role in
the association between Angptl4 in MetS and T2DM. Further studies into
the causal relationships are required.
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627-P
Relationship Between Lipoprotein Kinetics and Lipoplysacharide
Distribution Among Lipoproteins in Normal Subjects and Patients
BRUNO VERGS, CHRISTELLE VACHOU, LAURENCE DUVILLARD, EMMANUEL
FLORENTIN, JEAN-MICHEL PETIT, MARIE-CLAUDE BRINDISI, REMY BURCELIN,
Dijon, France, Toulouse, France
Lipopolysacharides (LPS) are components of gram-negative bacterial
membrane that are associated in plasma with lipoproteins. LPS binding with
lipoproteins may promote their catabolism and then reduce their pro-inam-
matory effects. However, data on LPS distribution among lipoproteins are
scarce and the relationship between LPS distribution and lipoprotein metab-
olism remains unknown. This prompted us to perform an in vivo lipoprotein ki-
netic study in 31 individuals (16 with type 2 diabetes [T2DM], 15 controls) and
to analyze the relationship between lipoprotein kinetics and LPS distribution
among VLDL, LDL and HDL.Plasma LPS levels were not different between
T2DM patients and controls but LPS distribution was different between the
2 groups with, in T2DM vs. controls, higher proportion of LPS-VLDL (317
vs. 2111%,p=0.001), LPS-HDL (289 vs. 2010%,p=0.03), free LPS (104
vs. 74%,p=0.04) and lower part of LPS-LDL (3013 vs. 5116%,p=0.001). In
T2DM patients, VLDL catabolism was decreased when VLDL production and
HDL catabolism were increased. In multivariate analysis, for the whole stud-
ied population: LPS-VLDL was associated with LPS-HDL (p<0.0001) but not
with apoB kinetics; LPS-LDL was associated with LPS-VLDL (p=0.003) and
VLDL catabolism (p=0.008) but not with LPS-HDL; LPS-HDL was associated
with free LPS (p<0.0001) and LPS-VLDL (p=0.041) but not with HDL kinetics;
free LPS was only associated with LPS-HDL.In conclusion, our data indicate
that an important LPS transfer occur in vivo between free LPS and HDL as
between HDL and VLDL whereas LPS-LDL is mainly derived from VLDL ca-
tabolism and may represent a catabolic pathway for LPS. T2DM patients
show a signicant decrease of LPS bound to LDL that may represent an im-
paired LPS catabolic pathway. Further studies are needed to see whether
these modications of LPS distribution among lipoproteins in T2DM patients
may increase intra-vascular pro-inammatory state.
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628-P
Hyperadiponectinemia is Strongly Linked to Elevated HDL-Choles-
terol (HDL-C) in Type 1 Diabetes (T1D)
ROSSANA M. CALDERON, ANGELA SZETO, THAMER ALESSA, ARMANDO MEN-
DEZ, RONALD B. GOLDBERG, Miami, FL
Although HDL-C levels are often increased in T1D, the basis for this nd-
ing and its clinical signicance are not well understood. Adiponectin (APN)
levels have been shown to correlate strongly with HDL-C, suggesting a
mechanistic relationship between the two. Since APN levels are increased
in T1D, we investigated whether elevated APN is related to increased HDL-C
in T1D. We studied 114 T1D subjects attending our Diabetes Clinic (diabetes
duration 25.81.4 yrs [meanSEM], HbA1c 7.80.4%) and 64 controls (CON).
T1D were slightly older (42.31.4 vs 371.4yrs; p<0.05) and had a higher BMI
(26.30.4 vs 24.50.4 Kg/m
2
; p<0.01) than CON. Both HDL-C (642 vs 522
mg/dL; p<0.005) and APN (13.96.5 vs 10.84.0 g/ml; p<0.001) were higher
in T1D than CON, and women (W) had higher HDL-C, apo A-I and APN than
men (M) in both groups (Table). 44% of T1D W and 50% of M had HDL-C >90
th
percentile of the CON group values (W>76; M>54 mg/dL).
T1 Diabetes Controls
Men (n=53) Women (n=61) Men (n=31) Women (n=33)
BMI (kg/m
2
) 27.2 0.5
b
25.6 0.6 25.1 0.4 24 0.8
Triglyceride (mg/dL) 97 7.4 88 6.6 125 14.5 91 8.1
a
HDL-C (mg/dL) 56 2
b
71 2
a,b
45 2 59 2
a
apo A-I (mg/dL) 153 3.4
b
183 4.3
a,b
137 2.8 162 4.4
a
APN (g/mL) 10.2 0.6 16.9 0.9
a,b
9.6 0.6 11.5 0.6
a
p<0.05, M vs W within Group;
b
p<0.05, T1D vs CON between genders.
APN was correlated with HDL-C and apo A-I in both T1D (r=+0.56, p<0.001
and r=+0.42, p<0.001, respectively) and CON (r=+0.65, p<0.001 and r=+0.41,
p<0.001). In step-wise regression analysis APN remained associated with
HDL-C in both T1D W and M after adjusting for age, diabetes duration, HbA1c,
serum creatinine, CRP, BMI, and triglyceride (r
2
M=0.08, p<0.05; W=0.20,
p<0.001). Our results demonstrate that APN levels are strongly linked to HDL-C
values in T1D, supporting the notion that the hyperadiponectinemia of T1D is
in part responsible for the frequent nding of elevated HDL-C in this condition.
The implications of these ndings and its impact on cardiovascular risk remain
to be determined.
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629-P
Effects of AMR101 on Lipid and Inammatory Parameters in Patients
With Diabetes Mellitus-2 and Residual Elevated Triglycerides (200-
500 mg/dL) on Statin Therapy at LDL-C Goal: The ANCHOR Study
ELIOT A. BRINTON, CHRISTIE M. BALLANTYNE, HAROLD E. BAYS, JOHN J.
KASTELEIN, RENE A. BRAECKMAN, PARESH N. SONI, Salt Lake City, UT, Houston,
TX, Louisville, KY, Amsterdam, The Netherlands, Bedminster, NJ
AMR101 is a novel omega-3 fatty acid agent containing ~96% pure icosa-
pent ethyl, the ethyl ester of eicosapentaenoic acid. The phase 3 ANCHOR
study evaluated the efcacy and safety of AMR101 in patients with residual
high fasting TG levels (200-500 mg/dL) despite optimized LDL-C (40-100 mg/
dL) on statins. Of 702 patients randomized to AMR101 4 g/d, 2 g/d, or place-
bo for 12 weeks, 514 (73%) had diabetes mellitus-2. Intent-to-treat analysis
of the effects of AMR101 on median placebo-adjusted percent change from
baseline in endpoint parameters was performed in 3 subgroups: total (all
subjects with diabetes), well-controlled diabetes, and less-controlled dia-
betes (</> median HbA1c; see Table for 4 g/d results). Baseline parameters
were similar among all groups except hsCRP, which was higher in the total
and less-controlled diabetes groups. AMR101 signicantly reduced TG, non-
HDL-C, apo B, and RLP-C in all diabetes groups, LDL-C in the total diabetes
group, and hsCRP in the total and less-controlled diabetes groups (Table).
Interestingly, decreases in hsCRP and apo B appeared to be far greater in pa-
tients with less-controlled diabetes. Importantly, there were no signicant
increases in FPG, HbA1c, insulin, or HOMA-IR in any group. In conclusion, in
patients with diabetes and mixed dyslipidemia, AMR101 4 g/d signicantly
improved lipid and lipid-related parameters without worsening glycemic
control, with possibly greater effects among those with less-controlled
diabetes.
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Median Placebo-adjusted Percent Change in Lipid and Inammatory Parame-
ters From Baseline to Study End in Patients With Diabetes Treated With
AMR101 4 g/day
% Change in: Well-controlled
Diabetes*, P
Less-controlled
Diabetes**, P
Total Diabetes,
P
TG (n=78, 87, 165) -21.0<0.0001 -24.8<0.0001 -23.2<0.0001
LDL-C (n=78, 87, 165) -6.60.0831 -5.70.1304 -6.30.0227
Non-HDL-C (n=78, 87, 165) -11.30.0019 -18.0<0.0001 -14.4<0.0001
hsCRP (n=75, 85, 160) -4.00.7372 -34.60.0002 -21.50.0028
RLP-C (n=28, 32, 60) -26.70.0468 -21.30.0364 -25.00.0024
Apo B (n=75, 85, 160) -6.10.0170 -12.8<0.0001 -9.5<0.0001
P-values are from Wilcoxon rank-sum test.*HbA1c < median of 6.8%. **HbA1c
~ median of 6.8%.Abstract abbreviations: Apo B=apolipoprotein B; FPG=fasting
plasma glucose; HbA1c=hemoglobin A1c; non-HDL-C=non-high-density lipopro-
tein cholesterol; HOMA-IR=homeostasis model index insulin resistance;
hsCRP=high-sensitivity C-reactive protein; LDL-C=low-density lipoprotein cho-
lesterol; RLP-C=remnant-like particle cholesterol; TG=triglyceride.
Supported by: Amarin, Inc.
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630-P
Estimation of Plasma Free Fatty Acids through Integrated Analysis
of Exhaled Breath in Obese and T2DM Adults
STACY R. OLIVER, MATTHEW K. CARLSON, ROBERT NEWCOMB, SIMONE MEIN-
ARDI, DONALD R. BLAKE, PIETRO R. GALASSETTI, Irvine, CA
Volatile organic compounds (VOCs) represent ideal, non-invasive markers
of endogenous biochemical processes; however, clinically usable metabolic
tests from VOCs has proven challenging. We previously estimated plasma
glucose, insulin, and free fatty acids (FFAs) by integrating exhaled VOCs. If
developed, a breath-based lipid test would greatly improve the diagnosis
and management of obesity and diabetes (OW, T2DM).Therefore we stud-
ied 10 T2DM (5M, 443yrs) and 5 OW subjects (5F, 415yrs) during 4 hours
of controlled glycemic and insulinemic uctuations (hyperglycemia of ~220
mg/dL; euglycemic-hyperinsulinemia). Plasma, room air and exhaled gases
were sampled at 12 different time points.Concentrations of ~100 VOCs were
determined by gas chromatography and matched with plasma FFAs. Multi-
linear models to reconstruct plasma FFAs concentrations for each subject
were generated by least squares regression on several subsets of exhaled
VOCs. The gas clusters yielding the strongest correlations with plasma FFAs
were acetone, CH3I, CH2Br2, n-butane in OW (r = 0.927); and n-butane, m\p-
xylene, i-pentane, ethylbenzene, MeONO2 in T2DM (r = 0.943). Additional
gas combinations were identied allowing broad range applicability across
both groups.Our data show the feasibility of accurately estimating plasma
FFAs in OW and T2DM subjects during glucose and insulin uctuations via in-
tegrated analysis of multiple exhaled gas proles. Our observations indicate
the potential of this methodology for non-invasive diagnosis and monitoring
of metabolic variables relevant to obesity and diabetes.
Supported by: NIH (1UL1RR031985, K24 DK085223)
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631-P
Activation of GPR119 Reduces the Appearance of Labeled Choles-
terol in an Oral Fat Tolerance Test
KATHLEEN K. BROWN, MELANIE K. SHADOAN, DALLAS K. CROOM, ANDREW
J. CARPENTER, DEREK J. NUNEZ, ANDREW A. YOUNG, PAUL L. FELDMAN, Re-
search Triangle Park, NC
Agents which lower cholesterol in circulation by impeding intestinal ab-
sorption offer an alternative to statins for reduction in cardiovascular risk.
GPR119, identied as a sensor of fatty acid derivatives, is present in the
gastrointestinal tract, but effects on lipid homeostasis have not previously
been described. To track the appearance and accumulation of chylomicrons
in circulation, fasted male rats were gavaged with radiolabeled cholesterol
in olive oil. Triton was injected intravenously to block LPL-mediated clearance
and thus enable tracking of cholesterol appearance over the next 5 hours.
Effects are reported as counts, relative to those at corresponding time points
in vehicle-treated rats (=100% in Fig). Ezetimibe used as a positive control,
decreased tracer appearance to 19 3.4 % of control by 4 hrs. Each in a struc-
turally diverse set of GPR119 agonists tested (30 mg/kg p.o. at t=-2 hr) slowed
appearance of labeled cholesterol, suggesting the effect was GPR119-specif-
ic. The relative decreases for all agonists compared to vehicle control were 50
8.1, 51 11.3, 55 9.6, 6315.4 and 5810.2 (meanSEM, n=5-8/group) for
GSK263, GSK706, Arena, Prosidion and MBX respectively. These previously
unreported effects of GPR119 agonists are predicted to have therapeutic ben-
et in the setting of dyslipidemia, metabolic syndrome and T2DM.
632-P
A Role for GPR119 in Lipid and Lipoprotein Metabolism
KATHLEEN K. BROWN, MANDY L. BERGQUIST, MELANIE K. SHADOAN, ANDREW
J. CARPENTER, ANDREW A. YOUNG, PAUL L. FELDMAN, DEREK J. NUNEZ, ANI-
TA VAN DEN HOEK, HANS M. PRINCEN, Research Triangle Park, NC, Leiden, The
Netherlands
Activation of GPR119 on islet o and -cells and enteroendocrine cells in
the gastrointestinal (GI) tract coordinates insulin secretion and the release of
gut peptides to promote the disposal of meal-derived nutrients. At present,
the signicance of its role as a fatty acid sensor in the GI tract is unclear. Fe-
male APOE3Leiden.hCETP transgenic mice, fed a western diet, were used to
investigate the effects of acute (single dose) or chronic (5 weeks) treatment
with a potent and selective GPR119 agonist, on lipid homeostasis. For each
study, groups (n=8/group), matched for total cholesterol (TC), triglycerides
(TG) and age, were treated with either vehicle (V) or GSK2041706 (GSK706)
at 10 or 30 mg/kg/d. To measure acute effects on TG appearance, overnight
fasted mice received a single gavage of V or GSK706. Mice received 200
l of olive oil 2hr later (with [
3
H]-triolein and [
14
C]-oleic acid) immediately
after iv injection of Triton (T) to block TG hydrolysis. Blood was collected at
0, 1, 2, 3 and 4h to measure plasma radioactivity and TG. To assess VLDL
production, overnight fasted mice received a single gavage of V or GSK706,
were anesthetized 1.5hr later and injected iv with 0.1 mL PBS containing 100
CiTran
35
S to measure de novo ApoB synthesis. Mice received an iv injec-
tion of T 30 min later, and samples were collected at 0, 15, 30, 60 and 90 min
to measure plasma TG. After 90 min, mice were sacriced and blood was
collected for isolation of VLDL. There were no acute treatment related ef-
fects on TG absorption, hepatic VLDL-TG secretion or de novo ApoB synthe-
sis. However, there were changes in lipid and lipoprotein parameters with
chronic dosing manifested by decreased FFA (24% and 30%, p<0.001, @ 10
and 30 mg/kg) and decreased serum TC and TG (36% and 37%, p<0.05, @
30mg/kg) compared to V. TC in the VLDL/LDL peak on FPLC was decreased by
27% and 39% (p<0.05, @ 10 and 30 mg/kg). There were no effects on CETP
mass or activity or on post-heparin plasma lipolytic activity. This suggests a
therapeutic role for GPR119 in regulation of plasma TC and TG.
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Fasting and Postprandial Overproduction of Apolipoprotein B48-
Containing Lipoproteins in Juvenile Zucker Fatty Rats: Impact of a
Cinnamon Extract on Enterocytes
BOLIN QIN, RICHARD A. ANDERSON, Beltsville, MD, Spring Hill, TN
Emerging evidence suggests that the overproduction of intestinal apoli-
poprotein (apo) B48 is associated with obesity and insulin resistance. We
investigated the metabolism of apoB48-containing lipoproteins in male juve-
nile Zucker lean (Zl) and fatty (Zf) rats. At 5 weeks of age, Zf rats had higher
body weight and epididymal white adipose mass compared to Zl rats, and
elevated area under the curve of glucose response to an oral glucose chal-
lenge. After an overnight fast, rats were subjected to an oral olive oil loading
test. The fasting and postprandial triglyceride (TG) levels and TG-rich lipo-
proteins (apoB48) in Zf rats were signicantly higher compared with Zl rats.
The small intestinal mRNA expression of insulin signaling genes, including
insulin receptor (Ir), and insulin receptor substrates-1&2 (Irs1 and 2 ) were
lower. The gene expression related to lipogenic signaling and cholesterol
metabolism including sterol regulatory element binding protein (Srebp)-1c,
microsomal triglyceride transfer protein (Mtp) and scavenger receptor type
B class I (Scrbi ) were higher; and the mRNA levels of ATP-binding cassette
transporters G (Abcg )5, ATP-binding cassette transporters A (Abca1), Nie-
mann-pick C1-like (Npc1l )1 and peroxisome proliferator-activated receptor
(Ppar)g were down regulated in Zf rats compared to the Zl rats. An aqueous
cinnamon extract (CE, 10g and 100g/mL) reversed the expression of mul-
tiple dysregulated genes in the primary enterocytes of Zf rats and inhibited
the oversecretion of apoB48 into the media. In summary, obesity-induced
systemic insulin resistance is associated with compromised signaling path-
ways related to apoB48 production in the small intestinal tissue of juvenile
Zf rats and CE is benecial in the control of intestinal apoB48 metabolism.
Supported by: USDA/ARS CRADA No.58-3K95-7-1184 with Integrity Nutraceuti-
cals, Inc.
634-P
Crosstalk between Salivary Gland Chronic Inammation and the En-
docrine Function of Adipocytes in Insulin Resistance
YOSUKE SHIKAMA, QUINKAI LI, YUKIKO BANDO, NANAKO AKI, CHISATO KO-
SUGI, MAKOTO FUNAKI, Tokushima, Japan
In insulin resistance, free fatty acids (FFAs) in plasma are increased due
to adipocyte dysfunction, which inhibits insulin action in the muscle and
liver and contributes to a development of type 2 diabetes (T2D). Emerging
evidence suggests that, among FFAs, saturated fatty acids (SFAs) are po-
tent activators of toll-like receptors (TLRs), which initiate a proinammatory
response in these organs, such as activation of mitogen-activated protein
kinase (MAPK) or nuclear factor-kB (NF-kB). Epithelial cells in salivary glands
also express TLRs. Interestingly, it has been reported that sjgren syndrome
(SS), in which salivary gland epithelial cells exhibit chronic inammation due
to autoimmunity and secrete interleukin (IL)-6, is frequently accompanied
by T2D or dyslipidemia.In this study, we investigated the effect of SFAs on
a proinammatory response in salivary gland epithelial cells and whether
or not adipocyte function could regulate it. Here we report that treatment
with SFAs, but not with unsaturated fatty acids, activated p38 MAPK and
NF-kB, which led to an increased secretion of IL-6 in a human salivary gland
cell line, HSY cells. HSY cells expressed both AdipoR1 and AdipoR2 recep-
tors, and PPAR1. SFA-induced secretion of IL-6 from HSY cells was inhibited
by globular adiponectin or simvastatin, but not by full-length adiponectin.
These results suggest a putative crosstalk between chronic inammation of
salivary glands and the endocrine function of adipocytes, which affect other
organs by adipocytokine secretion in insulin resistance.
635-P
Differential Effects on Lipid Proles between Alogliptin and Sita-
gliptin in Newly Diagnosed, Drug Nave Subjects With T2DM
EIJI KUTOH, YASUHIRO UKAI, Tokyo, Japan
Different DPP-4 inhibitors show signicant structural differences. As an
initial step towards nding any differences in their pharmacological actions,
effects on glycemic and other parameters were compared between sita-
gliptin (sita) and alogliptin (alo). Newly diagnosed, drug naive subjects with
T2DM were assigned to either 25-50mg/day sita (n=21) or 12.5-25mg/day
alo (n=22) monotherapy. At 3 months, the levels of these parameters were
compared with those at baseline. This study is a head-to-head comparison.
At baseline, the levels of the measured parameters were similar between
these two groups. At 3 months, similar glucose lowering efcacies were
observed in these groups (HbA1c: from 10.46 to 8.32%, p<0.001 for sita and
from 10.4 to 8.63%, p<0.002 for alo). In contrast to this, distinct regulatory
patters were observed with lipid proles. Briey, in the alo group, signi-
cant reductions of non-HDL-C (-9.8%, p<0.005) and LDL-C (-9.2%, p<0.05)
levels were observed. Changes of (A)HbA1c levels were weakly correlated
with those of (A)non-HDL-C (R=0.291) or ALDL-C (R=0.327). By contrast, non-
HDL-C and LDL-C levels had little, if any, changes in the sita group. Other
parameters including TG, HDL-C, FFA and body weight had no change in both
groups. Besides these lipid parameters, HOMA-B increased in both groups
(+56.3% for alo and +89.7% for sita) with signicant inter-group differences
(p<0.05). Both drugs are well tolerated and no clinically signicant adverse
events were observed. In conclusion, this study suggests that: 1) Both sita-
gliptin and alogliptin are effective and safe as an initial therapy for T2DM.
2) Alogliptin, but not sitagliptin, can ameliorate atherogenic lipid proles.
Further, the atherogenic lipids may be associated with the glycemic effect
during alogliptin treatment. 3) Although similar glucose lowering efcacies
were observed, sitagliptin had a higher degree of enhancing beta-cell func-
tion than alogliptin.
636-P
Cardiovascular Risk Management in Diabetes Patients
DOO MAN KIM, SHIN GON KIM, WON YOUNG LEE, CHONG HWA KIM, CHUL
SIK KIM, DONG HYEOK CHO, GYU JANG WON, YOUNG JOO KIM, Seoul, Repub-
lic of Korea, Gyeonggi-do, Republic of Korea, Gwangju, Republic of Korea, Daegu,
Republic of Korea
Current guidelines on cardiovascular (CV) disease prevention recommend
targeted management after assessment of CV risks using many of available
method even if the patient is asymptomatic. This study was performed to
explore how CV high risk is differently detected between 2 distinct meth-
ods: non-invasive test (NIT) and risk calculation and how the awareness of
CV high risk impacts physician and patient behavior for risk management in
diabetes patient.A prospective, observational study was carried out in 22
hospitals in Korea. 622 type 2 diabetes patients aged ~40 years were as-
sessed by Carotid Ultrasound (CUS) and United Kingdom Prospective Diabe-
tes Study (UKPDS) risk engine. CV high risk from CUS was dened as carotid
intima-media thickness ~ 1mm or plaque presence. Before and 6months af-
ter the test, patients completed a questionnaire on health-related behaviors
and physicians collected data on treatment patterns via chart review.Among
622 (mean age: 60.029.50 years), 271 (43.5%) and 66 (10.6%) patients were
stratied as CV high risk from CUS and UKPDS, respectively. Approximately
40% of patients at moderate and low risk from UKPDS were determined
as high risk from CUS. The awareness of high risk from CUS altered physi-
cians treatment patterns (P=0.021) for managing major CV risk factors: blood
pressure (BP) and lipid. Along with CUS, risk levels by UKPDS also impacted
physicians behavior: more changes of treatment pattern in high than in low
risk level for BP (12.0 vs 15.6 %) and lipid (13.8 vs 21.6 %). Patients noted
increased health-related behaviors: smoking cessation and dietary changes
(P<0.005, respectively) in 6 month follow up than before CUS and bigger in
high risk.This study identied NIT could detect more CV high risk patients
than risk calculation. However, awareness of CV high risk itself has a posi-
tive impact on physician and patient behavior, regardless of assessment
methods. Therefore, assessing CV risks using varied methods with the pa-
tient could be better for risk management in diabetes patients.
Supported by: Pzer Pharmaceuticals Korea Ltd.
637-P
A Multi-Centric, Prospective, Randomized, Double Blind Study to
Evaluate the Safety and Efcacy of 2mg and 4mg of ZYH1 Compared
to Placebo in Hypertriglyceridemia With Type II Diabetes Not Con-
trolled With Atorvastatin Therapy
RAJENDRAKUMAR H. JANI, DHIRAJ GAMBHIRE, GUNJAN JARIWALA, Mumbai,
India
ZYH1 is a new, predominantly PPAR alpha agonist and developed by Zy-
dus Cadila. It demonstrated favorable anti-dyslipidemic and safety prole in
phase 1 and 2 studies. In this randomized, double blind, placebo control, ICH
GCP compliant phase 3 study, either 2mg ZYH1, 4mg ZYH1 or placebo was
given orally once daily (OD) for 12 weeks to subjects with diabetic dyslipi-
demia (DD). All the subjects were on Atorvastatin (Ato) 10 mg OD for mini-
mum of 4 weeks before enrolment and had Triglyceride (TG) 200-500 mg/
dl. ZYH1 2mg + Ato 10mg, ZYH1 4mg + Ato 10mg and placebo + Ato 10mg
reduced TG by 44.9%, 47.2% and 25% respectively. Other lipid markers and
fasting plasma glucose were also corrected favorably in ZYH1 arms (Table).
The frequency of mild to moderate adverse events was comparable in ZYH1
2 mg (11%), ZYH1 (16%) and Placebo (9.8%). Gastritis was frequent in ZYH1
(4%). ZYH1 plus Ato was better than Ato alone for the control of DD.
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FOOT CARELOWER EXTREMITIES
Table: Per protocol analysis ZYH1 2mg, ZYH1 4mg and Placebo in addition to
Ato 10mg in DD (*P < 0.05)
Laboratory Parameter Visit ZYH1 2 mg(N=80)
Mean SD (%
change)
ZYH1 4 mg(N=78)
Mean SD (%
change)
Placebo(N=83)
Mean SD (%
change)
Triglyceride
(mg/dL)
Baseline 273.8 79.18 284.9 87.80 284.7 80.15
Week
12
149.7 70.07
(-44.9*)
142.1 67.26
(-47.2*)
204.0 97.13
(-25)
HDL-Cholesterol
(mg/dL)
Baseline 36.2 7.87 38.9 11.28 38.6 12.32
Week
12
40.0 11.48
(9.9*)
39.9 8.80
(8.1*)
37.0 7.88
(0.5)
Apolipoproteins A1 (mg/
dL)
Baseline 134.3 33.77 135.1 29.31 138.7 28.40
Week
12
155.6 47.39
(22.4*)
142.4 48.57
(9.2)
145.1 49.32
(9.2)
LDL-Cholesterol-Direct
(mg/dL)
Baseline 131.5 30.67 140.2 29.38 140.2 33.50
Week
12
97.0 29.93
(-26.2)
94.0 27.60
(-30.7*)
103.3 30.53
(-22.8)
Apolipoproteins B
(mg/dL)
Baseline 97.6 22.27 102.2 21.40 103.4 23.39
Week
12
71.0 22.64
(-26.6)
67.3 19.29
(-32.1*)
77.0 18.79
(-22.4)
Total Cholesterol
(mg/dL)
Baseline 198.6 38.30 209.9 36.89 208.8 39.35
Week
12
155.3 36.10
(-22.1)
151.9 33.55
(-25.8*)
168.4 35.23
(-17.4*)
Non-HDL Cholesterol
(mg/dL)
Baseline 162.4 37.47 171.0 37.26 170.2 41.51
Week
12
115.3 35.91
(-28.8*)
112.0 33.52
(-32.4*)
131.4 36.49
(-20)
Fasting Plasma Glucose
(mg/dL)
Baseline 177.0 66.29 175.1 64.01 180.2 68.47
Week
12
150.9 75.28
(-10.5*)
147.8 60.75
(-10.5*)
181.6 94.38
(7.4)
Guided Audio Tour: Walking on the Neuropathic Diabetic Foot (Posters 638-
P to 645-P), see page 17.
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638-P
Early Detection of Peripheral Neuropathy Using Virtual Obstacle
Crossing
BIJAN NAJAFI, GURTEJ GREWAL, RASHAD SAYEED, STEVE YASCHECK, YASER
KHAN, ROBERT A. MENZIES, TALAL K. TALAL, Chicago, IL, Tucson, AZ, Doha, Qa-
tar
This is a pilot study observing the effect of virtual reality technique on the
early detection of diabetic peripheral neuropathy (DPN). We predicted that
DPN patients would show a misjudgment of foot position during obstacle
crossing, an altered reaction time, and an impaired balance.An innovative
virtual obstacle crossing (VOC) paradigm using wearable sensors was de-
veloped in attempts to detect lower extremity nerve damage due to DPN.
Sixty-eight participants including diabetes with no, moderate and severe
neuropathy and aged matched healthy controls were recruited. Severity of
neuropathy was quantied using vibratory perception threshold (VPT) values
.The ability of perception of lower extremity was quantied by measuring
the rate of obstacle crossing success (OCS), reaction time (T
R
), and foot
position while crossing a series of virtual obstacles with heights ranging
from 5% to 20% of the subjects leg length. Additionally, balance was as-
sessed pre, during and post VOC trials.No signicant difference was found
between groups for age and BMI (p>0.05). Results suggest VOC test allows
separating between groups. The rate of OCS was signicantly reduced with
increasing neuropathy severity (p<0.05). Results also suggest a signicant
correlation between T
R
and VPT values (r=0.5, p<10
-5
). Finally, results sug-
gest a signicant deterioration in balance due to diabetes, irrespective of
neuropathy severity (p<0.05).The results proposed the benet of virtual ob-
stacle crossing as an objective method for detecting peripheral neuropathy
at early stage. This is based on the reasoning that lower extremity proprio-
ception decreases with increasing nerve damage. The increased reaction
time, decreased OCS, and increased sway of the DPN patients in this study
suggests their decreased proprioception, and, therefore, increased periph-
eral nerve damage. Further studies should be addressed to compare VOC
with other standard methods to conrm whether VOC can detect DPN earlier
than current methods.
Supported by: QNRF (NPRP08-499-3-109) and NIH (T35DK074390)
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639-P
Naltrexone Targets Angiogenesis Pathways to Accelerate Wound
Closure
PATRICIA J. MCLAUGHLIN, JESSICA A. IMMONEN, IAN S. ZAGON, Hershey, PA
Delayed wound closure is a major complication of diabetes. These studies
investigated an underlying biological axis, the opioid growth factor (OGF) -
opioid growth factor receptor (OGFr) that has been shown to modulate epi-
thelial cell replication. Blockade of opioid receptors from endogenous opioid
peptides such as OGF (chemical name, [Met5]-enkephalin) which inhibit cell
replication, by topical application of opioid antagonists such as naltrex-
one (NTX), reversed delays in closure of 6 mm diameter full-thickness skin
wounds in streptozotocin-induced type 1 diabetic (T1D) rats. Application 3
times daily of NTX decreased wound size over a 9 day period by more than
50% relative to residual wound area in T1D rats receiving vehicle. Histo-
pathological evaluation of skin samples from T1D rats treated with NTX or
vehicle, as well as normal tissue, has indicated that markers of angiogenesis
were signicantly repressed in T1D rats, but were markedly elevated in T1D
rats receiving NTX, relative to normal levels. Granulation tissue was evalu-
ated periodically between 3-10 days following wounding using antibodies
specic for broblast growth factor (FGF), vascular endothelial growth factor
(VEGF), and alpha smooth muscle actin (o-SMA). FGF, a marker for angiogen-
esis initiation, was reduced 64% in T1D rats on day 3, but elevated to normal
levels in T1D rats receiving NTX. The number of VEGF- positive vessels was
signicantly increased more than 2-fold in T1D rats receiving NTX relative
to T1D-vehicle rats on days 3, 5, and 8. Moreover, the number of o-SMA
positive cells in T1D skin was markedly (p<0.05) elevated in tissue topically
treated with NTX on days 3, 5, 8, and 10 relative to T1D-vehicle specimens.
These data suggest that blockade of the OGF-OGFr axis with opioid antago-
nists alters angiogenesis, an underlying physiological pathway associated
with wound healing in diabetes, and provides additional support for the use
of NTX as a non-toxic, effective, topical therapy for wound closure.
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H2S Improves Wound Healing via Restoring EPCs Functions in Type
2 Diabetic Mice
FANG LIU, DANDAN CHEN, JIEMEI WANG, FUWANG LI, MINGWEI ZHANG,
WEIPING JIA, ALEX F. CHEN, Shanghai, China, Pittsburgh, PA
Angiogenesis is crucial to wound healing in diabetes. Hydrogen suldes
(H
2
S) pro-angiogenic effects have been recently reported. This study inves-
tigated the effectiveness and possible mechanism of H
2
S donor in improving
wound healing in db/db diabetic mice.A 6mm dorsal wound was made on
male db/db and their matched control mice. The db/db mice were treated
with sodium hydrosulde (NaHS), 4-hydro- xythiobenzamide group (HTB), or
saline; The control mice were treated with DL-PAG or saline for 18 days.
Wound size was recorded every other day; skin tissue in wound area and the
plasma were harvested, and endothelial progenitor cells (EPCs) were cul-
tured from bone marrow cells. Plasma H
2
S concentrations were measured.
EPC tube formation and adhesive function after in vivo H
2
S therapy and in
vitro treatment with NaHS, PAG, or si-CSE were determined. The expres-
sion of CSE, CBS,VEGF, and angiopoitien-1 (Ang-1) in skin tissue and EPCs
were detected by Western blotting.The plasma H
2
S levels of db/db mice was
signicantly lower than controls (P<0.01), and signicantly increased in the
NaHS and HTB groups compared to db/db controls(both P<0.01). The wounds
in NaHS and HTB groups healed signicantly faster than db/db group (both
P<0.01) from day 4 to 16. PAG delayed the wound closure rate in the db/+
mice(P<0.05). The tube lengths of EPCs and the adhesive EPC numbers in-
creased in NaHS and HTB groups, but decreased in PAG group. The tube
formation and adhesion of EPCs from db/+ mice reduced signicantly after
in vitro PAG intervention (both P<0.01) or CSE silencing (both P<0.01). Con-
trarily, the tube length of EPCs increased signicantly in EPCs modied by
NaHS than in db/db EPCs (all P<0.01). The expression of Ang-1 and VEGF
in wound skin tissue and in EPCs were up-regulated in the NaHS and HTB
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celerates wound healing in diabetic mice; its effects are related to restore
the EPC angiogenic function.
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Fluctuating Sensorimotor Responses in Routine Foot Screening As-
sessmentsResults from the West of Ireland Diabetes Foot Study
ADAM GARROW, LORNA HURLEY, LAURA KELLY, LIAM GLYNN, CAROLINE
MCINTOSH, SEAN DINNEEN, Salford, United Kingdom, Galway, Ireland
Sensorimotor testing is an established part of diabetic foot screening. This
analysis examined the variability of common screening tests over time.563
primary care patients attended a standardised foot ulcer risk assessment
including light touch (10g monolament); sharp/blunt; temperature; vibration
perception (VPT) and ankle reex response. 377/563 (67%) patients attended
a follow-up assessment 19 (sd 2.1) months after the baseline visit. All as-
sessments were carried out by health care professionals trained in senso-
rimotor screening. The analysis examined changes in response in each of the
clinical tests between baseline and follow-up. Kappa (k) statistics show the
level of agreement between the two time points over and above what would
be expected by chance. K values <0.2 indicate Poor; 0.2-0.4 Fair & 0.4-0.6
Moderate agreement. The proportion of patients with normal sensation at
baseline and also at 19 months was sharp/blunt 87.3%; monolament 82.3%;
VPT 80.3%; temperature 72.5%; and ankle reex 69.8%. In contrast, a large
proportion of patients with sensorimotor dysfunction at baseline were re-
corded as having normal sensation at follow-up: sharp/blunt 43.9%; mono-
lament 41.8%; VPT 43.9%; temperature 36.9%; and ankle reex 49.6%. The
large variability was reected in Kappa values which suggested only fair to
moderate levels of agreement between the baseline and follow-up assess-
ments. (sharp/blunt 0.42; monolament 0.50; VPT 0.43; temperature 0.36 &
ankle reex 0.28).Whilst instrument measurement error and inter-observer
variability may partly explain differences between baseline and follow-up
measures, the observed improvements in sensorimotor function were un-
expected and perhaps counter-intuitive. The results may reect uctuations
in peripheral nerve function in patients with partial rather than complete
sensory loss. Caution is required when interpreting the results of clinical
sensorimotor tests in diabetes.
Supported by: Diabetes Federation of Ireland, The Health Research Board
642-P
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25-Hydroxyvitamiv D [25(OH)D] Levels and Diabetic Foot Ulcer: Is
There any Relationship?
MOHAMMAD ZUBAIR, JAMAL AHMAD, ABIDA MALIK, Aligarh, India
In recent years, there has been an effort to understand possible roles
of 25(OH)D, including its role in the immune system particularly on T cell-
medicated immunity, pancreatic insulin secretion and insulin action. 25(OH)
D stimulates the cell differentiation and reduces cell proliferation, which is
essential for cell growth and wound healing. However, data on the associa-
tion between low level of plasma 25(OH) D and diabetic foot syndrome are
scarce. Circulating plasma 25(OH) D levels were measured in diabetic pa-
tients with ulcer (n=162) and without ulcer (n=162) in a case control study. Of
these patients, 85.1% had type 2 diabetes. Subjects with diabetic foot ulcer
showed lower median plasma level of 25(OH)D [6.3(4.2-11.1) vs. 28.0(21.4-
37.0)]ng/ml after adjusting the age and BMI. Regardless of the low levels
of 25(OH) D in cases and controls, it was associated with neuropathy, sex
(female), duration of ulcer healing, and smoking status and independent of
confounding factors, including BMI (kg/m
2
), A1c (%), hypertension, neph-
ropathy, foot ulcer, retinopathy, CAD, PAD, HDL-C (mg/dl) and LDL-C (mg/dl).
The factors which predict the risk of developing ulcer independent of 25(OH)
D status were BMI (>25kg/m
2
) [OR 20.18; RR 1.45], A1c (>6.9%) [OR 4.37;
RR 1.77], HDL-C(<40mg/dl) [OR 1.16; RR 1.07], LDL-C (>100mg/dl) [OR 1.07;
RR 1.03], triglycerides(>200mg/dl) [OR 1.40; RR 1.19], neuropathy [OR 6.88;
RR 3.12], retinopathy [OR 3.34; RR 1.91], hypertension [OR 1.64; RR 1.28],
nephropathy [OR 3.12; RR 1.87] & smoking [OR 4.53; RR 2.99] using odds and
risk ratios. In conclusion, it is not clear whether the suppression of delayed
wound healing seen during 25(OH)D deciency is due to the secondary ef-
fect or is a direct action of vitamin D on certain components of the immune
system. Long-term randomized trials are needed to see the impact of vitamin
D supplementation on the outcome of diabetic foot patients
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644-P
Serum Magnesium Levels Are Signicantly Correlated With the Se-
verity of Inammation in Patients With Diabetic Foot
SALIM OZENC, SIRZAT YESILKAYA, MUSTAFA CAKAR, EROL ARSLAN, SEREF
DEMIRBAS, BAYRAM KO, KENAN SAGLAM, Ankara, Turkey
Hypomagnesemia is not an uncommon nding in patients with uncon-
trolled diabetes mellitus. It is associated with abnormal platelet action
and the development of neuropathy, both of which are risk factors for the
evolution of ulcers of the feet. A lately suggested data from animal studies
shows that during chronic hypomagnesemia, loss of neutral endopeptidase
activity contributes to neutrophil activation. In another, high CRP levels were
found associted with lower magnesium levels in healthy groups. We aimed
to investigate the correlation between serum magnesium levels and indica-
tors of inammation in diabetic foot patients.Our study was made on the
total of 88 patients; 57 diabetic foot patients and 31 controls. Median age
of patient and control groups was 64 and 51, respectively. 25.8 % (n = 7) of
controls and 17.5 % (n=10) of patient group were female. Wagner grade of
the patients foot ulcers were grade 2 on 18 patients and grade 1, 3 and 4
on each 13 patients. Serum magnesium levels of diabetic foot patients (me-
dian:1,92 mmol/l) were signicantly lower than controls (median:2.1 mmol/l)
(p<0.05). Sedimentation levels of diabetic foot ulcer patients (median: 38
mm/hr) were signicantly higher than controls (median: 14 mm/hr)(p<0.05).
Serum CRP levels of diabetic foot patients (median: 18.0 mg/l) were signi-
cantly higher than controls (median: 13.8 mg/l)(p<0.05). On Pearson correla-
tion analysis, there was a signicant negative correlation between serum
magnesium levels and serum CRP levels of the patient group (p<0.05).Serum
magnesium depletion is present and shows a relationship with develop-
ment of ulcers in subjects with type 2 diabetes. It has lately been proposed
that severe hypomagnesemia is associated with low grade inammation in
metabolic syndrome and cardiovascular inammation. In the literature, to
our knowledge, our study is the rst to say that serum magnesium levels are
signicantly associated with serum CRP levels in diabetic foot patients.
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645-P
Psychological Stress and Diabetic Foot Ulcer Healing: Preliminary
Findings
LORETTA VILEIKYTE, BIING JIUN-SHEN, LAURA CAMPBELL, ANDREW J. BOUL-
TON, MATTHEW J. HARDMAN, Manchester, United Kingdom, Athens, OH
Research indicates that psychological stress (PS)-induced immunomodu-
lation delays acute wound repair. Here we explored the potential role of PS
in the more complex diabetic foot ulcer (DFU)-healing paradigm. Ninety three
type 2 DM patients (84% male; mean age 57yrs) with plantar neuropathic
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DFU (University of Texas Classication: 69% grade 1A; 11% 1B; 16% 2A; and
4% 2B) completed baseline self-report measures of generalized (Perceived
Stress Scale, PSS; Hospital Anxiety and Depression Scale, HADS; and State-
Trait Anger Expression Inventory, STAXI) and DFU-specic PS (NeuroQoL-In-
terpersonal Burden (NeuroQoL-IP) and Patient Interpretation of Neuropathy
(PIN) Scales: PIN-Amputation Worry and PIN-Anger at Docs). DFU-specic
biomarkers (IL6, IL1-beta, MMP2 and MMP9) were determined via quanti-
cation of immunohistochemical tissue localization and normalized biopsy
gene expression. Systemic biomarkers (IL6 and IL1-beta) were measured
from patient serum via ELISA. Bivariate analyses revealed multiple mea-
sures of increased generalized and DFU-specic PS were associated with:
a) decreased local IL1-beta at baseline: HADS-Depression (r=-.27; p<.001)
and NeuroQoL-IP (r=-.38; p<.01); b) decreased MMP9: HADS-Depression
(r=-.29; p<.05); PIN-Worry (r=-.34; p<.05) and PIN-Anger (r=-.37; p<.01); c) in-
creased MMP2: PIN-Worry (r=.32; p<.05) and PIN-Anger (r=.31; p<.05). STAXI
was associated with higher levels of baseline systemic IL6 (r=.32; p<.01).
Intriguingly, greater than 80% DFU area reduction at 6 weeks was less likely
in patients reporting more severe PIN-Worry (r=-.36; p<.01) and PIN-Anger
(r=-.30; p<.01) and associated with higher baseline levels of systemic IL6
(r=-.29; p<.05) and local MMP2 (r=-.32; p<.05). These preliminary data iden-
tify potential psychological stress-induced biomarkers linking stress to DFU
chronicity.
Supported by: 5-R01-DK07-1066-05
646-P
Cathepsin D, Adiponectin, TNF-, IL-6 and hsCRP Plasma Levels in
Subjects With Diabetic Foot and Possible Correlation With Clinical
Variables: A Multicentric Study
JAMAL AHMAD, MOHAMMAD ZUBAIR, ABIDA MALIK, MOHAMMAD A. SID-
DIQUI, SUBHASH K. WANGNOO, Aligarh, India, New Delhi, India
Diabetic foot, characterized by a pronounced inammatory reaction, de-
creased collagen content and biosynthesis and accelerated degradation are
crucial in wound healing. Cathepsin D has been implicated in cell growth,
apoptosis and its inhibitor to reverse the inhibition of collagen biosynthesis
in wounded rat skin with diabetes. To date, the increased proteolytic activity
of cathepsin D in diabetic foot has not been evaluated and the pathogenic
signicance of this inammation has received little attention. Circulating
levels of cathepsin D, acute-phase reactant and cytokines were measured in
diabetic foot ulcer (DFU) (n=211) and without ulcer (n=208). Of the patients,
89.73% had type 2 diabetes. Subjects with DFU showed higher median
plasma level of Cathepsin D [556.3(312.6-587.3) vs 306.3(92.6-337.3)] RFC/
ml, TNF-o [96.6(79.9-121.5) vs 8.4(7.1-9.20)] ng/ml, IL-6 [32.2(8.52-48.4) vs
4.9(4.5-5.6)] ng/ml, hsCRP [12.6(11.2-14.1) vs 3.90(3.50-4.60)] mg/ml and
lower median plasma levels of adiponectin [8.50(7.10-9.5) vs 13.3(12.1-14.2)]
ng/ml. A positive correlation was found between grades of ulcer, BMI, A1c
and retinopathy for cathepsin D, for adiponectin, between grades of ulcer,
BMI, retinopathy, nephropathy & smoking, for IL-6, between grades of ulcer,
BMI, nephropathy, CAD & smoking, for hsCRP, grades of ulcer, BMI, LDL-C,
nephropathy & smoking while total cholesterol, nephropathy, PAD, smoking
and neuropathy for TNF-o. Diabetic subjects with various grades of DFU
showed a higher plasma cathepsin D, IL-6, hsCRP, TNF-o and lower adi-
ponectin levels in comparison with diabetes without foot ulcer independent
of the concomitant infections. It would be interesting to nd out whether an
activation of immune system precedes the development of foot ulcer and
whether cathepsin D inhibitor & anti-inammatory therapies will be effec-
tive in improving the outcome in such patients
647-P
All Negative Pressure Dressings Are Not Equal: A Comparison of
Negative Pressure Therapies in Porcine Wound Healing Model
NICHOLAS J. TANNOUS, DEBORAH NOBLE, EDWARD ROY, JIYING HUANG,
GEORGETTE ONI, IMELDA DELGADO, GAURAV THAKRAL, LAWRENCE LAVERY,
KATHRYN DAVIS, Dallas, TX
Negative pressure dressings are now a common modality in both inpa-
tient and ofce based wound care. There are, however, several available
options with varying economic implications. Variations in dressing type and
suction pressure may have effects on the wound both on macroscopic and
microscopic levels. This study looks at the outcome on wound healing in a
porcine model when comparing the lower pressure Convatech negative pres-
sure dressing to the higher pressure KCI system.Five animals were included
in this IACUC approved study. For each animal, three circular full thickness
wounds, 5 cm in diameter, were surgically created down to fascia on their
dorsum. One wound was covered using the Engenex
Bio-dome
technol-
ogy from Convatec, which consists of negative pressure wound therapy at
75 mm Hg. The second was covered using V.A.C.
therapy from KCI at a

negative pressure of 125 mm Hg. The third was a control wound that was
covered with a thin layer of Medline Skin Integrity
Hydrogel, followed by a
non-occlusive gauze dressing. The area of each wound was recorded twice
weekly over a 21 day period.KCI wounds were signicantly smaller on day 3
than the control (p= 0.036) and Convatech (p=0.048). By day 10 there were
no signicant differences between control wounds and negative pressure
dressings (p= 0.7455). By day 21 control wounds were signicantly small-
er than wounds treated with negative pressure systems (p= 0.0434). KCI
wounds were smaller than Convatech wounds by day 21 but not signicantly
so (p=0.23).This study demonstrates that there are signicant differences
between negative pressure systems. This difference may be attributable
to the suction pressure. After 10 days the positive effect of these dressings
appears to be reduced. This has implications for clinical practice and further
studies are warranted to investigate the optimum timing and pressure for
negative pressure wound therapy.
648-P
Obstructive Sleep Apnea is Associated With Oxidative Stress and
Microvascular and Endothelial Dysfunction in Patients With Type
2 Diabetes
ABD A. TAHRANI, KIRAN DUBB, ASAD ALI, MANJIT SINGH, ANTHONY H. BARNETT,
MARTIN J. STEVENS, Birmingham, United Kingdom, Coventry, United Kingdom
We aimed to assess the impact of OSA on oxidative stress (OS) and mi-
crovascular function in adults with T2D. Patients were recruited randomly
from the diabetes clinic of a UK hospital. OSA (apnoea hypopnea index (AHI)
~5 events/h (OSA+))was assessed using home based multi channel device
(Alice PDX, Philips Respironics, USA). Microvascular skin ow was assessed
using laser speckle contrast imaging (Moor Instruments Ltd, UK) at the mid
thigh level. OS was assessed by measuring serum 3-nitrotyrosine (NT) (nM)
and plasma lipid peroxide (LP) (M/ml).Patients (n=102) were included (73
OSA+). NT (23.5 (16.7-36.1) vs. 15.5 (11.5-24.3), p=0.007) and LP (18.4 (8.3-
37.4) vs. 7.9 (0.8-22.8), p=0.01) levels were higher in OSA+ patients and cor-
related with OSA severity (Table 1).After adjustment for a wide range of
variables, OSA remained an independent predictor of NT (B=0.16, p=0.01)
and LP (B=1.09, p=0.03) levels, OSA+ patients had lower blood ow and im-
paired endothelium responses (Table 2).OSA increases OS and is associated
with impaired microvascular function which might contribute to the devel-
opment of microvascular complications. Trials examining the impact of OSA
treatment on these parameters are needed.
Table 1: The correlation between oxidative stress measures and OSA. ODI:
Oxygen desaturation Index
AHI ODI Time spent
with oxygen
saturations < 80%
Nadir
nocturnal oxygen
saturations
Serum nitrotyrosine r=0.38,
p<0.001
r=0.36,
p<0.001
r=0.25,
p=0.01
r=-0.23,
p=0.02
Plasma lipid peroxide r= 0.19,
p=0.06
r=0.22,
p=0.03
r=0.25,
p=0.02
r= -0.24,
p=0.02
Table 2: Data presented as median (IQR). P values adjusted for age, BMI and
diabetes duration
OSA-
(n=24)
OSA+
(n=47)
P value-
unadjusted
P value-
adjusted
Baseline ux 35.60
(26.75-42.37)
30.80
(15.70-20.70)
< 0.001 < 0.001
Heating induced ux 168.90
(133.20-209.05)
154.60
(129.30-192.80)
0.405 0.726
Acetylcholine
induced ux
130.95
(99.07-177.52)
99.30
(68.90-120.20)
0.01 0.029
Na nitroprusside
induced ux
146.50
(117.05-204.62)
103.00
(62.30-135.10)
0.001 < 0.001
Baseline conductance
(measured as ux/
Mean arterial BP)
0.40
(0.28-0.48)
0.20
(0.16-0.31)
< 0.001 < 0.001
Acetylcholine
conductance
1.43
(1.09-1.83)
1.07
(0.75-1.29)
0.002 0.023
nitroprusside
conductance
1.61
(1.15-2.14)
1.16
(0.62-1.41)
0.001 < 0.001
Supported by: NIHR (UK)
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649-P
Study of Heat Shock Protein Hsp47 Expression in Chronic Wound of
Controlled and Uncontrolled Type 2 DiabetesMellitus
DEEPA POKHARIA, HEMANT KUMAR, MANISH MISHRA, AWADHESH K. ARYA,
RICHIK TRIPATHI, RAKESH K. SINGH, MOHAN KUMAR, KAMLAKAR TRIPATHI,
Varanasi, India, Kingstown, Saint Vincent and the Grenadines
HSP47, a collagen binding stress proteinplays a vital role in procollagen
processing during collagen synthesis. This studyreveals the possible ef-
fect of type 2 diabetes mellitus (T2DM) on Hsp47 expression in broblast
cells. Forthis 50 T2DM subjects having chronic wound and 20 non diabetic
subjects havingwound had been registered. They were divided into three
groups 20 subjects werenon diabetic having wound (G1), 25 subjects were
controlled diabetic having wound (G2)(HbA1c %< 7.0, FBS<7.7mmol/l) and
25 subjects were uncontrolled diabetic havingwound (G3) (HbA1c %> 7.0,
FBS>7.7mmol/l). They were evaluated forglycemic control (FBS, PPBS, HbA1c
%) and expression of Hsp47 at wound area ofthe subjects. Biochemical tests
were analyzed by Syncron CX5 autoanalyzer. Expression ofHsp47 were as-
sayed by immunohistochemistry method and the intensity of immunoreac-
tivity of Hsp47 was evaluatedaccording to a scale of zero no expression,
1faint,2 moderate and 3 strong expression. Group G3 shows the poor expres-
sionof Hsp47 (score 1+), Hsp47 expressed moderately in group G2 score 2+
and groupG1scored 3+ for Hsp47. Hsp47 expressed strongly in dermal bro-
blastsof the granulation tissue of G1. G2 express Hsp47 moderately while
G3 express minimally.Our studies indicate that Hsp47 mediates aberrant col-
lagen homeostasis inuncontrolled T2DM wound; this may be due to dysfunc-
tion of broblast cells. Suchimpairments could contribute to delay wound
healing. Several pharmacologicaland genetic approaches may be considered
to address Hsp47 directed therapies fortreating non healing wound.
Supported by: ICMR-SRF
650-P
HbA1c and Long-Term Mortality in Patients With Diabetic Foot Ulcer
MAGNUS LNDAHL, KATARINA FAGHER, ANDERS NILSSON, Lund, Sweden, An-
gelholm, Sweden
Recommendations for glycemic control in patients with cardiovascular
disease have been debated. The aim of this study was to evaluate the im-
pact of HbA1c on long-term mortality in the high-risk group of patients with
diabetic foot ulcers. 8-year mortality was evaluated in a consecutive and
prospectively registered group of type 2 diabetes patients, younger than 80
years, visiting a multidisciplinary diabetic foot clinic with an ulcer that did
not heal within a period of 4 weeks treatment. Of the 223 eligible patients
nine were excluded due to lack of HbA1c and the remaining 214 patients
(37.9% females, age 69.1 (63-76) years) grouped according to their baseline
HbA1c (<7.5% (58mmol/mol) (n=81, group 1), 7.5-8.9% (n=70, group 2) and
9.0%- (n=63, group 3)). There were no differences between groups in sex
distribution or prevalence of myocardial infarction, heart failure, hyperten-
sion, dyslipidemia, end-stage renal disease or peripheral vascular disease,
but patients in group 3 were younger than those in group 1. Eight-year mor-
tality is given in gure 1 and age adjusted HR for mortality are given in table
1. Despite similar baseline cardiovascular co-morbidities, HbA1c below 7.5%
was associated with increased mortality in this study population of patients
with diabetic foot ulcer.
Table 1
HR (95% CI)
HbA1c <7.5% vs
HbA1c 7.5-8.9
p-value HR (95% CI)
HbA1c <7.5% vs
HbA1c 9.0%-
p-value HR (95% CI)
HbA1c <7.5% vs
HbA1c 7.5%-
p-value
2-year mortality 2.20(1.09-4.46) 0.03 1.83(0.89-3.70) 0.10 1.91(1.08-3.39) 0.026
4-year mortality 1.84(1.12-4.46) 0.02 1.96(1.14-3.40) 0.02 1.83(1.19-2.80) 0.006
6-year mortality 1.46(0.95-2.25) 0.08 1.64(1.02-2.63) 0.04 1.49(1.02-2.16) 0.04
8-year mortality 1.43(0.96-2.13) 0.08 1.51(0.98-2.32) 0.06 1.42(1.01-2.02) 0.047
Supported by: Thelma Zogas Foundation, Swedish Diabetes Foundation, R&D
County of Skane
651-P
Use of Biomarkers of Oxidative Stress as Indicators of Risk Factor
for Diabetic Foot
CAIO J. TEIXEIRA, ANA CARLA P. OLIVEIRA, TALITHA F. STEFANELLO, MRCIA
A. CARRARA, ANACHARIS B. S-NAKANISHI, JURANDIR F. COMAR, CARLOS A.
SANTOS, ROBERTO B. BAZOTTE, MRCIA R. BATISTA, Maring, Brazil
We investigated the role of oxidative stress markers as indicators of risk
of injury of the lower extremities in patients with type 2 diabetes mellitus.
In 29 patients were assessed the following parameters: 1. Anthropometric
measures for calculating the Body Mass Index (BMI); 2. Glycated hemoglo-
bin (HbA1c); 3. Blood markers of oxidative status (total antioxidant capacity
- TAC, reduced protein thiols and levels of carbonylated proteins), and 4.
Test of sensation in the feet (National Hansens Disease Program - U.S.).
Patients were divided into two groups according to the index of sensation:
group 1 (low risk - 18 patients) and group 2 (high risk of injury - 11 patients).
The metabolic evaluations were performed at two moments in each group,
baseline and later (110 days). The results were: 1. The group 1 had a mean
age 56.98.7 years and the mean duration of disease of 8.86.9 years, while
group 2 had a mean age of 63.47.6 years and the mean of disease duration
of 18.810.4 years (P<.001); 2. According to BMI, the group 2 was framed as
obese (BMI=32.46.7 kg/m) and insulin users, while group 1 was classied
as overweight (BMI=29.544.3 kg/m) and users of oral antidiabetics; 3. The
group 1 presented an increase of HbA1c (%) during the study period (from 5.8
to 7.4, P=.0002), while group 2 showed only a trend to increase (from 8.1 to
8.9, P=.1748); 4. The TAC (mg/plasma albumin) in group 1 ranged from 21.9
to 17.3 (P<.0001) and group 2 from 20.8 to 16.7 (P=.0020). For thiols groups
(mg/ jmol albumin), in group 1 ranged from 11.4 to 10.3 (P=.0013) and group
2 from 10.2 to 10 (P=.3652). Concerning the levels of protein carbonyls (mg/
jmol albumin), in group 1 ranged from 4.4 to 7.3, (P<.0001) and group 2 from
4.7 to 7.5 (P=.0029). The results suggest that the evaluation of the plasma
levels of carbonylated proteins, TAC and reduced protein thiols could furnish
additional information to prevent the risk of diabetic amputation considering
that the alteration of these biomarkers was associated with loss of sensitiv-
ity and foot ulcerations.
652-P
Initial Vascular Response to CLEAR Cleat Cycling in Patients at Risk
for DFU
RYAN T. CREWS, STEVEN R. SMITH, SHANNON B. LIU, Chicago, IL
Exercise has been linked to improved wound healing. Potential mecha-
nisms for this improvement are increases in skin blood ow and skin oxy-
gen tension. While diabetic foot ulcers (DFU) are extremely difcult to heal,
typical wound locations prohibit most types of exercise. The CLEAR Cleat
was designed to allow DFU patients to safely participate in aerobic exercise
by minimizing physical stress to the wound (ofoading).This study sought
to determine whether CLEAR Cleat cycling increased blood ow to the
forefoot.Ten subjects with diabetic peripheral neuropathy, with or without
addition DFU risk factors, were recruited to participate (aged 518 y; BMI
326). Individuals with active DFU were excluded. Each subject completed
two 5-minute stationary cycling sessions at the same self-selected cadence
and resistance level. Standard athletic shoes and pedals were used during
one session, and the CLEAR Cleat was used with one foot during the other
A166
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DIABETES EDUCATION
session. Perfusion levels were examined at the hallux by surface laser Dop-
pler probe (tpu - tissue perfusion units) and the forefoot by plantar surface
temperature (infrared thermography) both pre and post each cycling bout.
The cycling signicantly increased microcirculation at the hallux during both
cycling conditions (pre 3.152.9 vs. post 6.86.3 tpu). Mean forefoot temper-
ature did not differ between groups or pre (28.2.7C) vs. post (28.2.8C)
exercise. With the exception of mild leg cramping and muscle tightness in
two subjects, no adverse events occurred. The increased microcirculation in
these high risk subjects indicates that the cycling may improve blood ow
to forefoot DFU and therefore aid healing. The lack of change in the gross
assessment of surface temperature may be due to the limited duration of
cycling or the relatively low exertion level by the subjects. Future studies
utilizing subjects with active DFU should look at the use of the cleat in a rou-
tine exercise regimen. They may determine if the thermal response changes
over multiple cleat sessions.
Supported by: NIH (T35DK074390)
653-P
Intravenous Antimicrobial Therapy of Diabetic Foot Ulcers: An Ef-
fective Outpatient Program in Tanzania
ZULFIQARALI G. ABBAS, JANET LUTALE, LENNOX K. ARCHIBALD, Dar es Salaam,
United Republic of Tanzania, Gainesville, FL
Diabetic foot ulcer infection cause substantial morbidity and mortality,
prolonged hospital stays, and increased hospital costs in Tanzania. Thus,
we initiated an outpatient program for (IV) antimicrobial therapy in two out-
patient clinics in Dar es Salaam, Tanzania. During February 2005 -December
2011 (study period), diabetes patients with infected foot ulcers were evalu-
ated then started on IV antimicrobial therapy administered twice daily for
10-15 days depending on severity. Treatment included various combinations
of second and third-generation cephalosporins, penicillins, macrolides, qui-
nolones, and anti-anaerobic agents. Logistic regression analysis was carried
out and adjusted odds ratio (AOR) and 95% condence intervals (CI) calcu-
lated. Of 909 patients treated during the study period, 595 (65%) were male;
median age was 55 years; 900 patients received an extended-spectrum
third-generation cephalosporin and complete ulcer healing was attained in
760 (84%). Independent factors associated with complete healing were ulcer
area <1,000 mm
2
(AOR: 2.1, CI: 1.4-3.1) or concomitant receipt of an anti-
anaerobic agent (AOR: 4.5, CI: 3.0-6.8). Independent factors associated with
delayed ulcer healing included macrovascular disease (AOR: 2.1, CI: 1.4-3.2);
hypertension (AOR: 1.6, CI: 1.04-2.4); and addition of amoxicillin/clavulanic
acid (AOR: 2.7, CI: 1.5-4.7) or quinolones (AOR: 5.1, CI: 3.3-7.9) to the thera-
peutic regimen. In conclusion, we established the feasibility of affordable,
outpatient IV antimicrobial therapy for diabetic foot ulcer infection in Tan-
zania. Best outcomes were documented for patients who received a single
extended-spectrum third-generation cephalosporin with an anti-anaerobic
agent. Augmentation of this regimen with penicillins, macrolides, or quino-
lones, did not enhance ulcer healing rates. These ndings potentially have
cost saving implications for the management of infected diabetic foot ulcers
in Tanzania.
654-P
Daily Foot Care Adherence and Patient Education
JINSUP SONG, GARY FOSTER, GUENTHER BODEN, DANA TANGO, REAGAN
KANE, ALEXANDRA HANLON, JAMES FURMATO, Philadelphia, PA
While studies have shown that diabetic education can improve foot care,
many diabetic patients are experiencing non-traumatic lower limb amputa-
tion. This study examines if a novel diabetic foot care education using per-
sonal plantar pressures improves adherence to daily foot care. Study partici-
pants (N=96) were at-risk type 2 DM patients (63.5% male; mean age 53.9
years; diabetic foot risk score: 36.5% 1; 47.9% 2A; 8.3% 2B; 2.1% 3A; 5.2%
3B). Study participants were randomly assigned to the control (45) or the in-
tervention group (51). All participants received standard foot care brochures,
but only the subjects in the intervention group were shown their barefoot
plantar pressures and received an explanation as to why they need to in-
spect their feet daily and wear protective shoes. All subjects received rou-
tine foot care at baseline, 1, 3, 6, 9, and 12 months. Adherence to daily foot
care behaviors was assessed using Footcare Behavior Questionnaire. While
the subjects in the intervention group had greater adherence to daily foot
inspection than the control group throughout the study period, a signicant
difference was noted only at 3 month evaluation. No signicant difference
was noted in barefoot indoor walking. Additional studies and independent
measures of foot care are needed to evaluate if the proposed diabetic foot
education based on personal plantar pressures yields improved adherence
and clinical outcomes.
Table 1. Percentages of adherence by item, group, and visit. Item responses
were compared by group according to the percent of responses that im-
proved from baseline using Fishers Exact tests.
During the
past week
Control Intervention
Visit Adherence
(%)
N Adherence
(%)
N p value
examined
feet
baseline 64.4 45 70.6 51
daily or month 1 76.7 43 87.5 48 0.3376
twice per
day
month 3 71.1 38 88.9 45 0.0422
month 6 70.6 34 83.3 42 0.3715
month 9 65.5 29 83.8 37 0.1507
month 12 77.8 18 86.4 22 0.3047
never walk baseline 46.7 45 49.0 51
barefoot
indoor
month 1 53.5 43 66.7 48 0.3048
month 3 52.6 38 71.1 45 0.1293
month 6 58.8 34 66.7 42 0.4219
month 9 69.0 29 81.1 37 0.1304
month 12 61.1 18 63.6 22 0.4117
655-P
Implications of Plantar Loading in Gait on Microvascular Function
in Diabetes and Pre-Diabetes
JAMES A. FURMATO, JINSUP SONG, DANA N. TANGO, REAGAN KANE, Phila-
delphia, PA
The relationship of walking to neuropathic foot ulcer (DNU) genesis is un-
known. We hypothesize soft tissue property changes around the sub-papillary
venous plexus delays onset of postocclusive hyperemic response (POHR) in in-
dividuals with a history of DNU compared to healthy subjects. If similar chang-
es are found in obese individuals (where pre-diabetes is prevalent), subclinical
glycation may occur before it is recognized and addressed by a physician. We
measured POHR to loads applied under the hallux in weightbearing subjects.
Nine diabetic individuals with a history of DNU and 9 non-diabetic, healthy
controls enrolled in a pilot study. The ndings were compared to 18 nondiabet-
ic subjects in a weight loss study. We titrated each subject for the compres-
sion needed to blanch and applied a 90s continuous load and 90 cycles pulsed
load then followed each with 180s of refractory time. Laser Doppler signals
were analyzed to nd the latency time between the end of occlusion and the
initiation of POHR (TL) for the three groups. The mean TL for pulsed compres-
sion was statistically greater in the diabetic group than in the control group
(100.024.7 vs 68.71-22.2 s; p=0.010). The TL for continuous compression
was higher in the diabetic individuals as well (0.0610.02 vs 0.0470.020s;
p=0.17). The obesity subjects showed mean pulsed TL=78.535.5s, longer than
healthy controls, and continuous compression TL=0.0600.031, closer to that
of diabetic subjects suggesting deviation from normal. Prolonged TL in walking
may lead to hypoxia in sensate individuals and DNUs in diabetic individuals.
We suggest temporal sequencing of gait and a physiological test be used to
indicate changes at the tissue and cellular level to mark progression of glyca-
tion in diabetic and pre-diabetic feet.
DIABETES EDUCATION
Guided Audio Tour: Innovative Approaches that Foster Self-Management
(Posters 656-P to 663-P), see page 17.
&
656-P
Specialty Trained Certied Diabetes Educator (CDE) With Endocri-
nologist Oversight Providing Metabolic Management (MM) With
Diabetes Self-Management Training (DSMT) to Increase Rural
Health Access in the Adult Patient With Uncontrolled Type 2 Diabe-
tes Mellitus Improves HgA1c and Patient Satisfaction
MARY A. JOHNSON, JOHN W. KENNEDY, H.L. KIRCHNER, JEFFERY M. BARRETT,
Danville, PA
The increase in uncontrolled Type 2 DM, combined with a shortage of
endocrinologists, led a rural health system in Pennsylvania to pilot the use

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A99

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) release using amperometric nanosensors. Study

ratio, an indicator of normal EC function,

ratio in non-diabetic SH rats by more than

monitoring. Pts with a positive test (apnoea-

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study referred to the sleep clinic. So far, polygraphy has been

; Nonin Medical Inc,

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; Jokor) with a cut-off MW of 15,000. The HDL fraction was

pre-designed SNP assays from Applied Biosystems. TGF-B1 and

pre-designed SNP assays from

(Impeto Medical, France), a new device for quickly assess-

in patients with type-1 diabetes (T1DM), comparing its results

measures sudomotor function by assessing electrochemical skin

results correlate with those of the LDIare technique; an established marker

may serve as a useful tool

a simple, quick and

therapy from KCI at a

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