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Am J Physiol Endocrinol Metab

280: E23±E30, 2001.

Gluconeogenesis in moderately and severely hyperglycemic patients


with type 2 diabetes mellitus
1 1 2
GUENTHER BODEN, XINHUA CHEN, AND T. PETER STEIN
1
Division of Endocrinology/Diabetes/Metabolism and the General Clinical Research Center,
2
Temple University Hospital, Philadelphia, Pennsylvania 19140; and the Department of
Surgery, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine,
Stratford, New Jersey 08084
Received 20 March 2000; accepted in ®nal form 6 September 2000

Boden, Guenther, Xinhua Chen, and T. Peter Stein. gluconeogenesis (GNG) (7, 8, 26, 28, 32, 41). The valid-ity of
Gluconeogenesis in moderately and severely hyperglycemic patients this widely accepted concept, however, can be questioned.
with type 2 diabetes mellitus. Am J Physiol Endo-crinol Metab 280: First, the notion that postabsorptive EGP is uniformly elevated
E23±E30, 2001.ÐWe tested the generally accepted concept that in hyperglycemic patients has recently come under attack (1,
increased gluconeogenesis (GNG) and endogenous glucose
17). Second, the data that were the basis for the thesis that
production (EGP) are the main reasons for postabsorptive
hyperglycemia in patients with type 2 diabetes mellitus (T2DM). patients with high fasting plasma glucose (FPG) had increased
GNG was measured with the 2H2O method by use of both the C5-to- rates of GNG were obtained with methods that, for several
C2 ratio (C5/C2, with gas chromatography-mass spectrometry) and reasons, did not allow quantitative measurements of GNG.
the C5-to-2H2O ratio (C5/2H2O, with isotope ratio mass Usually only one (or at most two) GNG precursor was used.
To determine total GNG would have re-quired extrapolation
spectrometry), and EGP was measured with 3-[ 3H]glucose in 27
patients with T2DM [13 with fasting plasma glucose (FPG) .10 mM
from the conversion to GNG from one to all precursors. This
and 14 with FPG ,10 mM] and in 7 weight- and age-matched is dif®cult, because it has been shown that infusion of some
nondiabetic controls. The results showed 1) that GNG could be precursors (for in-stance glycerol) decreased the fractional
determined accurately with 2H2O by using either C5/C2 or C5/2H2O; conversion of other precursors (for instance amino acids) (16,
2) that whereas after an overnight fast of 16 h, GNG was higher in 38). Another, more serious problem was that the hepatic GNG
the entire group of patients with T2DM than in controls (6.4 vs. 5.0 precursor speci®c activity was unknown, because the label
mmolz kg21 z min21 or 60.4 vs. 51.4% of EGP, P , 0.02), GNG was was diluted in the oxaloacetate pool, which is shared by GNG
within normal limits (less than the mean 6 2 SD of controls or , and the tricarboxylic acid cycle (19). This usually resulted in a
65.3%) in 11/14 (79%) patients with mild to moderate hyperglycemia systematic underestimation of GNG (19). Recently, new
(FPG ,10 mM) and in 5/13 (38%) of patients with severe methods have become available that for the ®rst time have
hyperglycemia (FPG 10±20 mM); 3) that elevated GNG in T2DM allowed quantita-tive in vivo determination of GNG in human
was asso-ciated with a 43% decrease in prehepatic insulin secretion, subjects (13, 20, 22, 23, 33).
i.e., with hepatic insulin de®ciency; and 4) that FPG corre-lated
signi®cantly with glucose clearance (insulin resistance) (r 5 0.70)
and with GNG (r 5 0.50) or EGP (r 5 0.45). We conclude 1) that 2
peripheral insulin resistance is at least as important as GNG (and In the current study, we used the H2O technique, which
EGP) as a cause of postabsorptive hyperglycemia in T2DM and 2) was recently developed and validated by Landau and
that GNG and EGP in T2DM are increased under conditions of colleagues (22, 23). Major advantages of this method are that
signi®cant hepatic insulin de®ciency and thus probably represent a it determines GNG from all precursors (including glycerol)
late event in the course of T2DM. and that it avoids the problems, related to unknown precursor
speci®c activity in the liver, that have plagued all previous
endogenous glucose production; insulin resistance; prehe-patic isotopic methods. We used this new method to examine the
insulin secretion; deuterated water method hypothesis that GNG was elevated in patients with type 2
diabetes (T2DM) and was responsible for their postabsorptive
hyperglycemia. To this end, we measured GNG in 27 patients
HYPERGLYCEMIA AFTER AN OVERNIGHT FAST is
a major hall- with T2DM fasted for 16 h who were subdi-vided into two
mark and an important diagnostic criterion of diabe-tes. groups, those who had either mild to moderate (,10 mM) or
Postabsorptive hyperglycemia of .7.8 mM in dia-betic patients severe (.10 mM) postabsorp-tive hyperglycemia.
is generally believed to be due to increased endogenous
glucose production (EGP), which in turn is believed to be
caused by increased rates of
The costs of publication of this article were defrayed in part by the payment
Address for correspondence: G. Boden, Temple Univ. Hospital, 3401 North of page charges. The article must therefore be hereby marked ``advertisement''
Broad St., Philadelphia, PA 19140 (E-mail: bodengh @tuhs.temple.edu). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Copyright © 2001 American Physiological Society. All rights reserved.
E24 GLUCONEOGENESIS IN TYPE 2 DIABETES

Table 1. Study subjects


T2DM
All patients Glucose .10 mM Glucose ,10 mM Controls

Gender (male/female) 11/16 7/6 4/10 4/3


Plasma glucose, mM* 10.66 0.8 14.26 0.8 7.26 0.4 5.560.2
(range) (4.7±19.3) (10.2±19.3) (4.7±9.9) (4.9±6.1)
Age, yr 596 2 556 3 626 3 5863
Wt, kg 84.46 4.3 93.26 7.0 76.26 4.5 80.765.6
Ht, cm 164.16 2.0 167.46 2.2 1616 3.0 173.162.5
Fat, % 36.26 1.9 37.46 3.0 35.16 2.3 28.662.5
BMI, kg/m2 31.06 1.4 32.96 2.5 29.26 1.4 26.861.3
Duration of T2DM, yr 14.56 1.8 17.06 2.3 11.86 2.8
(range) (1±28) (8±28) (1±20)
Values are means 6 SE. * After an overnight fast (16 h). Statistical analysis: glucose, P _ 0.001, type 2 diabetes mellitus (T2DM) all patients vs. all other
groups; P _ 0.05, T2DM (,10 mM) vs. controls; age, P _ 0.01 controls vs. T2DM .10 mM and ,10 mM; Ht, P _ 0.02 T2DM (all patients) vs. controls; Fat %, P _
0.005 controls vs. T2DM (.10 mM); P _ 0.03, T2DM (all patients) vs. controls; body mass index (BMI), P _ 0.05 controls vs. T2DM (.10 mM); P _ 0.04, T2DM
(all patients) vs. controls.

METHODS In a separate study, designed to determine 2H enrichment in


plasma water, the fast was extended to 24 h in 14 diabetic patients (7
Subjects
with plasma glucose of .10 mM and 7 with plasma glucose of ,10
Twenty-seven patients with T2DM and seven age- and weight- mM) and in the 7 age- and weight-matched controls. Blood samples
matched nondiabetic controls participated in this study. Thirteen of were collected at 16, 20, and 24 h for measurement of 2H enrichment
the diabetic patients had high (mean 14.2, range 10.2±19.3 mM) and of plasma water (Table 2).
14 had either normal or moder-ately elevated (mean 7.2, range
4.7±9.9 mM) postabsorptive glucose levels. All patients in this group, Analytical Procedures
however, had ele-vated glucose levels (.11.11 mM) during a 2-h oral
glucose tolerance test. Some of their characteristics are given in Table Determination of GNG. This method depends on the incor-
1. All patients had been treated with oral hypoglycemic agents poration of 2H from 2H2O into glucose. After 2H2O adminis-tration,
2
(sulfonylureas or biguanides or both), and some had received, in H enrichment at the glucose carbon 5 (C5) divided by 2H enrichment
addition, small doses of NPH insulin (5±20 units) at bedtime. These of plasma water or at the glucose carbon 2 (C2) equals the fractional
medications were withheld starting 3 days before the studies in all but contribution of GNG to EGP. This is so because the conversion of
three patients (one discontinued medications 2 days before, the other every molecule of pyruvate to glucose involves addition of an H from
two, one day before the studies). The patients' body weights were body water to C2 of the intermediate phosphoenolpyruvate. This
stable for _2 mo, and their diets contained a minimum of 250 g/day carbon becomes C5 of glucose (22, 23).
of carbo-hydrates for _2 days before the studies. Informed written
During conversion of glycerol to glucose, one H from body water
consent was obtained from all of the subjects after explana-tion of the is added to C2 of glyceraldehyde 3-phosphate during isomerization
nature, purpose, and potential risks of the study. The study protocol
with dihydroxyacetone 3-phosphate. That car-bon also becomes C5
was approved by the Institutional Review Board of Temple of glucose. Thus enrichment in C5 of glucose re¯ects glucose
University Hospital.
production from pyruvate and glyc-erol, i.e., from all GNG
precursors (22, 23). Therefore, the ratio of 2H labeling at C5 of
Experimental Design glucose to plasma water is a measure of GNG relative to EGP.
2
The subjects were admitted to the Temple University Hos-pital H enrichment on C2 re¯ects glucose production from GNG and
General Clinical Research Center on the day before the studies. At 6 from glycogenolysis (GL). This is so because one H from body water
PM, the subjects ingested a meal containing 14 kcal/kg of body is added to C2 of glucose 6-phosphate when fructose 6-phosphate is
weight. The meal was composed of 53% carbohydrate, 15% protein, converted to glucose 6-phosphate during GNG. Furthermore, glucose
and 32% fat. They then fasted for 16 h but were allowed water ad 6-phosphate, which is also formed as an intermediate during GL,
libitum. At 11 PM, a baseline blood sample was obtained. The equilibrates ex-tensively with fructose 6-phosphate, resulting in the
subjects drank 2.5 g of 2H2O (99.9% 2H; Isotec, Miamisburg, OH) ex-change of the H bound to C2 of the glucose 6-phosphate with that
in body water. There is, however, no labeling at C5
per kg of body water at 11 PM and again 4 h later at 3 AM. Body
water was assumed to be 50% of body weight in women and 60% of
body weight in men. Additional water ingested during the fast was
2
en-riched to 0.5% with 2H2O to prevent dilution of the isotopic steady Table 2. H enrichment in plasma water
state. The studies began at 8 AM the following day with the subjects 16 h 20 h 24 h
reclining in bed. A short polyethylene catheter was inserted into an
antecubital vein for infusion of isotopes. Another catheter was placed T2DM .10 mM
into a contralateral forearm vein for blood sampling. This arm was (n _ 7) 0.5026 0.019 0.5056 0.013 0.5056 0.013
T2DM ,10 mM
wrapped with a heating blanket (;70°C) to arterialize venous blood.
(n _ 7) 0.4736 0.031 0.5006 0.037 0.4896 0.038
Blood samples were drawn after 16 h of fasting for determination of Controls (n _ 7) 0.4366 0.011 0.4386 0.009 0.4366 0.007
glucose turnover, GNG, substrates, and hormones.
Values are means 6 SE.

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Copyright © 2001 American Physiological Society. All rights reserved.
GLUCONEOGENESIS IN TYPE 2 DIABETES E25
2
Table 3. Comparison of C5/ H2O and C5/C2 (at 16 (Diagnostic Products, Los Angeles, CA) and epinephrine with a 3H
h) radioenzymatic assay (Amersham, Piscataway, NJ). Plasma free fatty
acid (FFA) concentration was determined with a kit from Wako
T2DM (Richmond, VA). Plasma glycerol, lactate, alanine, glutamine,
.10 mM ,10 mM Controls glutamate, b-hydroxybutyrate (b-OHB), and acetoacetate (AcAc)
(n _ 13) (n _ 14) (n _ 7) were determined enzymat-ically.
C5 0.2916 0.019 0.2466 0.016 0.2276 0.013
C2 0.4446 0.028 0.4346 0.033 0.4546 0.030 Statistical Analysis
C5/2H2O 0.6226 0.042 0.5326 0.032 0.5206 0.025
C5/C2 0.6396 0.043 0.5606 0.033 0.5076 0.033 All data are expressed as means 6 SE. Statistical analysis was
performed using the SAS program (SAS Institute, Cary, NC).
Values are means 6 SE. C5, 2H enrichment on carbon 5 of glucose; C2, 2H ANOVA with repeated measures was used to determine the
enrichment of carbon 2 of glucose; 2H2O, 2H enrichment in plasma water. differences in GNG, GL, and EGP across time points. Pairwise
C5/2H2O and C5/C2, ratios of C5 to 2H2O and to C2, respectively. comparison to each time point was then performed if overall
comparison was statistically signi®cant. Correla-tions between GNG
and EGP or glucose were determined by least squares regression
analysis.
during GL. Therefore, the ratio of 2H labeling at C5 to C2 of glucose
is a measure of GNG relative to EGP (22, 23). RESULTS
2
H enrichment of C5 and C2 was determined by gas chro-
matography-mass spectrometry (Hewlett-Packard 5989 MS, HP 5890 Effects of a 16-h Fast
GC) as previously described (5). GNG (mmolz kg21 z min21) was Postabsorptive (16-h) plasma glucose concentrations were
calculated by multiplying the ratio of C5 to 2H2O (C5/2H2O) or that 10.6 6 0.8 mM in patients with T2DM [n 5 27, 14.2 6 0.8
of C5 to C2 (C5/C2) with EGP. GL was calculated as the difference (range 10.2±19.3) mM and 7.2 6 0.4 (range 4.7±9.9) mM,
between EGP and GNG. Thus GL is that part of glycogenolysis that respectively, in patients with FPG .10 mM (n 5 13) and ,10
becomes glucose. mM (n 5 14)] and were 5.5 6 0.2 (range 4.9±6.1) mM in
Enrichment of 2H in plasma water was determined in all subjects controls (n 5 7) (Fig. 1).
with an isotope ratio mass spectrometer (PDZ Eu-ropa, London, UK)
by use of an ABCA-G module and a standard curve with known GNG
enrichments ranging from 0.25 to 1.0%. Rates of GNG were higher in patients with T2DM than in
21
In the current study, GNG was determined with both C5/ 2H2O and controls (60.4 6 2.4% of EGP or 6.35 6 0.25 mmolz kg z
21
C5/C2. Both methods gave similar results (Ta-ble 3). We are, min vs. 51.4 6 2.6% or 5.0 6 0.3 mmolz
therefore, presenting pooled data obtained with both methods.
Glucose turnover. Glucose turnover was determined with 3-
[3H]glucose. 3-[3H]glucose was infused intravenously for 2.5 h
starting with a bolus adjusted proportionally to the degree of
hyperglycemia (40 mCi 3 mM glucose/5.5) (15), followed by
continuous infusion of 0.4 mCi/min. Glucose was isolated from
blood for determination of 3-[3H]glucose speci®c activity as
previously described (36). Rates of total body glucose appearance
(GRa) and disappearance (GRd) were cal-culated using Steele's
equation for non-steady-state condi-tions (37). The rate of EGP was
equal to GRa, because no glucose was infused during these studies.
Glucose clearance rates were calculated as G Rd/FPG. C-peptide
kinetics. Approximately 1 wk before the studies,
a 50-nmol intravenous bolus of biosynthetic human C-pep-tide (Eli
Lilly, Indianapolis, IN) was administered to each subject after an
overnight fast. Plasma C-peptide concentra-tions were then
measured, and C-peptide kinetic parameters were calculated at
frequent intervals for 3 h as described by Polonsky et al. (30).

Insulin secretory rates. The C-peptide kinetic parameters were


used to calculate prehepatic insulin secretion rates (ISR) for each
time interval between successive blood sam-ples by deconvolution of
peripheral C-peptide concentrations, according to Polonsky et al. (30)
and Eaton et al. (11). Fig. 1. Plasma glucose concentrations and rates of gluconeogenesis (GNG)
Substrate and hormone analyses. Plasma glucose was mea-sured and endogenous glucose production (EGP) in 14 patients with type 2 diabetes
with a glucose analyzer (YSI, Yellow Springs, OH). C-peptide was mellitus (T2DM) with fasting plasma glucose (FPG) ,10 mM, 13 patients with
determined by RIA (Linco, St. Charles, MO). Insulin was determined T2DM with FPG .10 mM, and 7 nondi-abetic controls. Values are means 6
after deproteinization by RIA by use of an antiserum with minimal SE. Broken horizontal lines in GNG and EGP panels represent mean 1 2 SD
of controls (64.3%). Statistical analysis: GNG, .10 mM vs. ,10 mM, P , 0.03; .
(,0.2%) cross-reactivity with proinsulin (Linco). Human growth
10 mM vs. controls, P , 0.02. EGP, .10 mM vs. ,10 mM, P , 0.01; .10 mM vs.
hormone and glucagon were determined by RIA. Cortisol was controls, P , 0.05.
measured with a kit

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Copyright © 2001 American Physiological Society. All rights reserved.
E26 GLUCONEOGENESIS IN TYPE 2 DIABETES

21 21 21 21
kg z min , P , 0.02). GNG was also higher in the (4.1 6 0.3 mmolz kg z min ) in the .10 mM glucose group,
21 21
.10 mM glucose group than in the ,10 mM glucose group 43.7 6 3.1% (3.8 6 0.4 mmolz kg z min ) in the ,10 mM
21 21 21
(64.8 6 3.3% or 8.5 6 1.1 mmolz kg z min vs. glucose group, and 48.6 6 2.6% (4.8 6 0.5 mmolz kg z
21 21 21
56 6 3.1% or 4.8 6 0.6 mmolz kg z min , P , 0.03) and in min ) in the controls. GL in all T2DM and in the .10 mM
the controls. The difference between the T2DM with ,10 mM glucose group was signi®cantly lower than GL in controls (P ,
glucose and the control group was not statistically signi®cant. 0.03).
There was a trend for GNG to rise with higher FPG, which EGP
was re¯ected in a signif-icant but modest correlation between
FPG and GNG (r 5 0.50, P , 0.005; Fig. 2). Rates of EGP were higher in the .10 mM glucose group
than in the ,10 mM glucose group (12.6 6 1.2 vs. 8.6 6 0.8
Postabsorptive GNG levels (at 16 h) were similar in the 7 21 21
mmolz kg z min , P , 0.01) and in the controls (9.8 6 0.6
elderly controls (this study) and in 12 younger (36 6 3 yr) and 21 21
mmolz kg z min , P , 0.05). The differences between all
leaner (body mass index 24.7 6 1.3) nondiabetic subjects (51.4 21 21
T2DM (10.5 6 0.8 mmolz kg z min ) and controls and
6 2.6 vs. 54.2 6 3.2%, non-signi®cant), suggesting that our between the ,10 mM glucose and control group were not
normal range was representative of that in a larger nondiabetic statistically signi®cant.
popula-tion. FPG correlated signi®cantly but modestly with EGP (r 5
0.46, P , 0.01; Fig. 2).
GL
Glucose Clearance
Rates of GL were 39.6 6 2.4% of EGP (3.9 6 0.3 mmolz
21 21 Glucose clearance was used to estimate insulin sen-sitivity.
kg z min ) in patients with T2DM, 35.2 6 3.3% Rates of glucose clearance were 1.12 6 0.10, 0.90 6 0.08, 1.33
21 21
6 0.15, and 1.79 6 0.10 mlz kg z min in all T2DM, the .10
mM and ,10 mM groups, and controls, respectively. All of
these differences were highly signi®cant (P , 0.01). Glucose
clearance corre-lated closely with FPG (r 5 0.70, P , 0.001;
Fig. 2).

Hormones and Substrates


All T2DM had signi®cantly higher plasma insulin, growth
hormones, cortisol, FFA, and ketone body lev-els than controls
(Table 4). The .10 mM glucose group had signi®cantly higher
plasma insulin and ketone body levels than the ,10 mM
glucose group and sig-ni®cantly higher plasma insulin,
glucagon, FFA, ke-tone bodies, and GNG precursor levels
than the con-trols.

Elevated vs. Normal GNG


To gain insight into factors responsible for the in-creased
rates of GNG, we compared the 11 patients (8 from the .10
mM, 3 from the ,10 mM group) who had GNG .65.3% (i.e.,
higher than the mean 1 2 SD of controls) with the remaining
16 T2DM patients and with the 7 controls who had normal
GNG. Compared with the 16 patients with normal GNG, the
11 patients with high GNG had signi®cantly lower prehepatic
ISR (101 6 18 vs. 178 6 32 pmol/min, P , 0.05) but the same
21
glucose clearance (1.13 6 0.12 vs. 1.14 6 0.15 mlz kg z
21
min ; Fig. 3). They also had higher plasma b-OHB (323 6 68
vs. 173 6 32 mmol/l, P , 0.005) and total ketone body levels
(393 6 71 vs. 228 6 46 mmol/l, P , 0.05) but comparable
plasma AcAc (69 6 10 vs. 55 6 15 mmol) and FFA levels (638
6 37 vs. 596 6 46 mM) (Fig. 4). On the other hand, there were
no signif-icant differences in plasma glucagon (68 6 6 vs. 67 6
Fig. 2. Correlation between FPG at 16 h of fasting and rates of GNG ( top),
EGP (middle), and glucose clearance (bottom) in 13 patients with T2DM and
5 pg/ml) and in plasma epinephrine levels (18 6 3 vs. 20 6 3
FPG .10 mM, 14 patients with T2DM and FPG ,10 mM, and 7 nondiabetic pg/ml). These results indicated that high GNG in patients with
controls. The correlation FPG vs. glucose clearance (G Rd/FPG) is valid, T2DM was associated with hepatic insulin de®ciency.
because FPG2 vs. GRd remains signif-icantly correlated (r 5 0.51, P , 0.002).

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Copyright © 2001 American Physiological Society. All rights reserved.
GLUCONEOGENESIS IN TYPE 2 DIABETES E27

Table 4. Hormones and substrates (at 16 h)


T2DM
All patients Glucose .10 mM Glucose ,10 mM Controls
(n _ 27) (n _ 13) (n _ 14) (n _ 7)
Insulin, pmol/l 87612² 1306 20* P _ 0.003 51.6 9 406 7
Glucagon, pg/ml 7064 776 7* 636 6 P _ 0.01 606 8
Epinephrine, pg/ml 1962 196 3 206 3 176 3
hGH, ng/ml 0.3660.05² 0.406 0.05 0.346 0.05 P _ 0.01 1.126 0.20
Cortisol, nmol/l 313625³ 2616 17 3616 39 2256 31
GNG precursors, mmol/l 2,1356123 2,1226 132* 2,1486 208 1,8326 114
FFA, mmol/l 609630² 6386 42* 5826 42 P _ 0.02 3626 68
Total ketones, mmol/l 294641² 3856 72* P _ 0.04 2106 31 P _ 0.05 1406 23
Values are means 6 SE. hGH, human growth hormone; GNG, gluconeogenesis; FFA, free fatty acids. *P _ 0.01, T2DM (.10 mM) vs.
controls; ²P _ 0.01, T2DM (all patients) vs. controls; ³P _ 0.05, T2DM (all patients) vs. controls.

DISCUSSION GNG. The method has been extensively validated in normal


subjects (5, 23) and has been used in pregnant women (18)
Can GNG be Accurately Quantitated in T2DM with and in patients with cirrhosis of the liver (29). So far, it has not
2
H2O? been used in diabetic patients. In this study, GNG was
To assess its role in the pathogenesis of elevated FPG in determined in patients with T2DM and in nondiabetic controls
patients with T2DM, we measured GNG with the recently by use of C5/C2 and C5/H2O. C5/C2 does not require steady-
2 2
developed H2O method (22, 23). This method determines state condi-tions, only suf®cient H enrichment to measure C5
2 2
GNG from all precursors (includ-ing glycerol) and avoids the and C2. C5/ H2O does need complete mixing of H and body
problems related to un-known precursor speci®c activity in 2
water. This is obtained ;1 h after the last H2O inges-tion (23).
the liver that have previously prevented quantitative 2
determination of Not surprisingly, therefore, we found that H enrichment in
plasma water did not change between 16 and 24 h, indicating
that a steady state existed at 16 h (Table 2). Con®rming a
2
previous report from our labo-ratory, H enrichment on C2 of
glucose and in plasma water was the same in nondiabetic
2
subjects (5). In diabetic patients, H enrichment of plasma
water was slightly but not signi®cantly higher than that on C2,

Fig. 3. Rates of GNG, glucose clearance, insulin secretion (ISR), and plasma
glucagon levels in 16 patients with T2DM with normal GNG, in 11 patients Fig. 4. Plasma concentrations of glucose, ketone bodies (acetoacetate plus b-
with T2DM with high GNG (higher than mean 6 2 SD of controls), and in 7 hydroxybutyrate), and free fatty acids (FFA) in 16 patients with T2DM and
nondiabetic controls. Statistical analysis: *P , 0.05, **P , 0.001 vs. controls; normal GNG, 11 patients with T2DM and high GNG, and 7 normal controls.
1P , 0.05, 11P , 0.001 vs. T2DM with normal GNG. Statistical analysis: *P , 0.05, **P , 0.005 vs. controls; 1P , 0.05 vs. T2DM
with FPG ,10 mM.

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Copyright © 2001 American Physiological Society. All rights reserved.
E28 GLUCONEOGENESIS IN TYPE 2 DIABETES

resulting in GNG rates that were 3±5% higher when C5/C2 was linear over time. It appears more likely, however, that
was used instead of C5/H2O (Table 2). Both methods can liver glycogen decreases dose dependently, i.e., at a faster rate
therefore be used to quantitate GNG in patients with T2DM. during the early hours of the fast than later (14). Assuming a
linear decrease would therefore result in underestimation of
For this study, C5/C2 as well as C5/H2O was obtained for all
the glycogen content and GL and overestimation of GNG
data points, and the re-sults were pooled. during the initial 15 h of fasting. In another study, Tayek and
13
Katz (39), using mass isotopomer [U- C]glucose analysis,
GNG in T2DM
reported GNG rates of 48.8 6 5.7% of EGP in nine patients
We found that patients with T2DM, as a group, had higher with T2DM (FPG 11.8 6 1.3 mM) and 46.6 6 4.0% in eight
rates of GNG than controls (60.4 6 2.4 vs. 51.4 6 2.6%, P , controls (P , 0.05).
0.03). In one of the two other studies in which GNG was When we studied 27 patients with T2DM, it became
13
quantitatively determined [with C nuclear magnetic apparent that there was considerable variation in their rates of
resonance spectroscopy (NMR)], Magnusson et al. (24) GNG. For instance, GNG was elevated infre-quently in
reported rates of GNG of 88 6 2% of EGP in seven patients patients with FPG ,10 mM; in fact, only 1 of 14 (7%) had
with T2DM (FPG 14.6 6 1.2 mM) and of 70 6 6% in ®ve GNG exceeding the mean 1 3 SD of controls, whereas 3 of 14
nondiabetic controls. Although both studies agreed that GNG (21%) exceeded the mean 1 2 SD. Elevated GNG was more
was elevated in T2DM, the GNG values in the Magnusson common in patients with more severe hyperglycemia (FPG .10
study were higher than those in our study. The differences, mM). Here 5 of 13 (38%) exceeded the mean 1 3 SD and 8 of
however, were more apparent than real when the dif-ferences 13 (61.5%) exceeded the mean 1 2 SD of controls. This also
in methodology are considered. Magnuson et al. determined meant, however, that GNG remained within normal limits in
mean rates of GL and obtained GNG by subtraction of GL more than one-third (5 of 13) of even severely hyper-glycemic
from EGP. There are several reasons why this method could patients (FPG 10.2±19.3 mM). One patient in this group had
underestimate GL and there-fore overestimate GNG. 1) The discontinued his Glipizide medication only 1 day before being
NMR spectroscopy method does not detect renal GL. This studied. It is, therefore, possi-ble that this patient's GNG
may be incon-sequential in nondiabetic subjects, whose (49.5%) might have been slightly higher had the drug been
kidneys con-tain only trivial amounts of glycogen (2). Patients discontinued 2 days earlier. Thus, whereas there was a trend
with T2DM, however, accumulate glycogen in their kidneys for GNG to rise with rising FPG levels, the correlation
and are able to produce glucose by GL (2). This alone could between GNG and FPG was modest (r 5 0.50, P , 0.005).
1
account for much of the difference between the two studies.
2) In the Magnusson study, liver glycogen concentrations were GNG and Insulin
much lower in diabetic patients than in controls (131 vs. 282
mmol/l), possibly due to differences in prestudy food intakes. To learn why GNG was high in some diabetic pa-tients but
Because GL de-pends on glycogen levels (40), this could have not in others, we compared the 11 patients with elevated GNG
been a reason for low GL and high GNG rates in the diabetic (. mean 1 2 SD of controls) with the remaining 16 patients
patients. 3) Magnuson et al. determined average rates of GL who had normal GNG (Fig. 3). This comparison revealed that
from changes in hepatic glycogen concentrations based on the patients with elevated GNG had similar plasma levels of
NMR measurements obtained between 4 and 22.5 h after the the major GNG-promoting hormones and substrates, including
last meal. Therefore, to provide accu-rate rates of GL, the ®rst glucagon, epinephrine, cortisol, FFA, and GNG precur-sors
NMR measurement needed to coincide with the peak of (lactate, alanine, glutamate, glutamine, and glyc-erol; Table 5).
postmeal hepatic glycogen accumulation. If hepatic glycogen In addition, they were as insulin resis-tant (as judged by their
increased after the ®rst NMR measurement (4 h after the glucose clearance) as the patients with normal GNG.
meal), which may occur in diabetic patients who not Noteworthy, however, their livers were exposed to ;40% less
infrequently have delayed absorption, GL would have been insulin (as
under-estimated and GNG overestimated. 4) Liver volume (to
calculate glycogen content from glycogen concentra-tion) was
determined once (14.5 h after the last meal). It was assumed Table 5. Plasma GNG precursor levels in subjects with
that the decrease in liver volume [observed previously to be normal and high rates of GNG
23% over 67 h of fasting (33)]
Normal GNG
Controls T2DM High GNG T2DM
(n _ 7) (n _ 16) (n _ 11)
1
For instance, if the kidneys contributed 25% of total EGP, as has Lactate 8456 74 1,148 6 156 1,138 6 148
been reported by Meyer et al. (25), and if 50% of this renal EGP was Alanine 2896 21 412 6 43 355 6 29
from GL (a reasonable assumption), then 1.39 mmolz kg21 z min21 of a Glutamate 896 9 79 6 10 91 6 24
total EGP of 11.1 mmolz kg21 z min21 in the Magnusson study (24) Glutamine 5416 36 478 6 36 380 6 52*
would have been renal GL. Hepatic plus renal GL would have been Glycerol 1166 18 91 6 10 84 6 11
2.7 mmolz kg21 z min21, i.e., 24% of EGP, and GNG would have been Total precursors 1,8326 114 2,208 6 191 2,048 6 158
76%, the same as the 75% that we found in seven patients with
comparable FPG after 24 h of fasting (this study). Values are means 6 SE. *P _ 0.05 vs. controls.

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Copyright © 2001 American Physiological Society. All rights reserved.
GLUCONEOGENESIS IN TYPE 2 DIABETES E29

judged by a ;40% reduction in prehepatic ISR). This suggested It needs to be pointed out, however, that the patients in the .
that hepatic insulin de®ciency was a major cause for the 10 mM glucose group had not only grossly elevated plasma
increased GNG in these patients, because insulin is known to glucose but also abnormally high se-rum insulin levels, both
suppress GNG (34), and insulin de®ciency is known to of which should have de-pressed EGP (31, 35). The fact that in
increase gene transcription of at least three of the four key over 50% of these patients EGP was within normal limits
GNG enzymes (phosphoenol-pyruvate carboxykinase, instead of reduced indicated that these patients had hepatic as
glucose-6-phosphatase, and fructose 1,6-biphosphatase) (12). well as peripheral insulin resistance.

Insulin is also a powerful suppressor of ketone body In summary, our data showed 1) that the deuterated water
formation (27). Presence of insulin de®ciency in these method (either C5/C2 or C5/H 2O) can be used to accurately
patients was further supported by the ®nding that their plasma measure GNG in patients with T2DM; 2) that GNG was
b-OHB levels were higher, whereas their FFA levels (i.e., the infrequently (;20%) elevated in pa-tients with FPG ,10 mM
ketone body precursors) were sim-ilar compared with those of but was commonly elevated (;60%) in patients with FPG .10
the diabetic patients with normal GNG. mM; 3) that hepatic insulin de®ciency seemed to be a major
reason for increased GNG; and 4) that peripheral insulin resis-
tance together with inappropriate glucose production caused
GNG and FPG the development of fasting hyperglycemia. On the basis of
Whether EGP (or GNG) is elevated in mildly to moderately these ®ndings, we conclude that the widely held concept that
hyperglycemic patients with T2DM has remained FPG of .7.8 mM is mainly due to increased EGP or GNG
controversial. Older studies have frequently reported greatly overemphasizes the impor-tance of EGP/GNG and
increased rates of EGP, which has led to the conclusion that underemphasizes the impor-tance of insulin resistance.
elevated levels of FPG in T2DM were primarily the result of
increased EGP (3, 4, 10, 21). More recently, however, it has We thank the nurses of the General Clinical Research Center for help with
become clear that in most of these studies, EGP was the studies and for excellent patient care, Karen Kresge and Maria Mozzoli for
overestimated in pro-portion to the degree of the patient's outstanding technical assistance, and Con-stance Harris Crews for typing the
manuscript.
hyperglycemia. The main reason for this was incomplete This work was supported by National Institutes of Health Grants R01-AG-
equilibration of the labeled and nonlabeled glucose in the 07988, R01-AA-10221 (to G. Boden), R01-AG-14098 (to T. P. Stein), and
expanded glucose pool. In studies in which an isotopic steady RR-349 (to the General Clinical Research Center).
state was achieved, EGP was usually found to be ele-vated
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