Metabolism

Clinical and Experimental

VOL XXXVII, NO 4 APRIL 1988
PRELIMINARY REPORT
Evidence of Abnormalities of Insulin Metabolism
in Rats With Spontaneous Hypertension
Carl E. Mondon and Gerald M. Reaven
Var i ous aspec t s of i nsul i n met abol i sm wer e c ompar ed i n r at s w i t h spont aneous hyper t ensi on (SHR) and a sui t abl e c ont r ol
gr oup of si mi l ar si zed Kyot a Wi st er (WKY) r at s. The r esul t s i ndi c at ed t hat t he t ot al i nt egr at ed ser um i nsul i n r esponse t o an
or al gl uc ose c hal l enge w as si gni f i c ant l y gr eat er i n t he SHR r at s, despi t e t he f ac t t hat t he t ot al i nt egr at ed ser um gl uc ose
r esponses wer e si mi l ar i n t he t wo gr oups. Thi s i ndi r ec t evi denc e of r esi st anc e t o i nsul i n-st i mul at ed gl uc ose di sposal w as
suppor t ed by t he obser vat i on t hat t he abi l i t y of si mi l ar ser um c onc ent r at i ons of ex ogenous i nsul i n t o st i mul at e gl uc ose
di sposal w as r educ ed i n SHR r at s. I n addi t i on, t he met abol i c c l ear anc e r at e of i nsul i n w as l ow er i n t he SHR r at s. These dat a
i ndi c at e t hat abnor mal i t i es i n i nsul i n sec r et i on, ac t i on, and c at abol i sm ex i st i n r at s w i t h spont aneous hyper t ensi on.
Whet her t hi s def ec t i s uni que t o SHR r at s, or c ommon t o al l f or ms of ex per i ment al hyper t ensi on, i s an i mpor t ant i ssue.
Equal l y i mpor t ant i s t he r el at i onshi p bet ween t he obser ved c hanges i n i nsul i n met abol i sm and t he el evat ed bl ood
pr essur e.
0 1988 by Grune & Stratton, I nc.
A
LTHOUGH several recent reports have demonstrated
that serum insulin concentration is elevated in patients
with high blood pressure,le4 the implication of this finding is
not clear. One obvious question is whether or not hyperinsu-
linemia is only seen in humans with high blood pressure. The
present study was initiated in order to address this issue, and
involved assessment of insulin secretion and insulin action in
rats with spontaneous hypertension (SHR).
MATERI ALS AND METHODS
Spontaneously hypertensive rats (SHR), originally derived from
Wistar Kyoto stock from the NIH Animal Genetic Resource colony,
and the Kyota Wistar (WKY) control model for the SHR rat, were
obtained from Taconic Laboratories (Germantown, NY) at 7 and 5
weeks of age and provided Purina Laboratory Chow (No. 5012) and
water ad libitum. Systolic blood pressure readings were obtained
indirectly by tail vein occlusion one or two days prior to administra-
tion of the oral glucose tolerance test, and were significantly elevated
(P < ,001) by Students unpaired t-test in SHR as compared to
WKY rats (176 ? 4 v 117 f 4 mm Hg). We have noted and
confirmed the report by the animal supply laboratory that body
weight gain of SHRs occurs at a slower rate than that of WKY rats.
Body weight gain five to seven days before the experiment averaged
3.8 * 0.2 g/d for SHR rats and 7.8 f. 0.4 g/d for WKY rats.
Therefore, studies were performed on SHR rats that were 2 weeks
older than the WKY rats in order to control for differences in weight.
All experiments were begun midday, five hours after food withdraw-
al.
Mefabo/i:;m, Vol 37, No 4 (April). 1988: pp 303-305
Glucose Tolerance
Oral glucose tolerance tests were performed on 36 unanesthetized
rats whose body weight averaged 265 g (range 240 to 290 g).
Eighteen SHR rats were 80 + 1 I days of age and I8 WKY rats were
67 + 5 days of age. Each rat was partially sedated by wrapping the
animal in a terry cloth towel at approximately 12:45 PM. The
exposed tail was cut at the tip and an initial blood sample of
approximately 0.5 mL collected into a I .5 mL polyethylene centri-
fuge tube. The animal was unwrapped from the towel and glucose
was administered as a 6.8 g/dL solution at a dose of 170 mg12.5 mL
solution/l00 g body weight. The glucose solution was administered
by passing a polyethylene cannula (PE 200, Clay Adams, Parsippa-
ny, NJ) into the stomach. To prevent the animal from biting the
cannula, the incisor teeth were placed through quarter-inch holes
drilled into the handle ends of a closed clothespin. The jaws were
held open with release of the pin. After glucose was administered,
From the Department of Medicine, Stanford University School of
Medicine, and Geriatric Research. Education and Clinical Center.
Veterans Administration Medical Center, Palo Alto, CA.
Supported by research grants from the Sandoz Research I nsti-
tute and the Research Service of the Veterans Administration.
Address reprint requests to G.M. Reaven. MD, VA Medical
Center (GRECC 640/182B), 3801 Miranda Ave. Palo Alto, CA
94340.
D 1988 by Grune & Stratton, I nc.
0026-0495/88/3704-0001$03.00~0
303
the cannula was withdrawn and the clothespin removed. The rats
were rewrapped and maintained in the towels for additional blood
samplings at 30,60, 120 and 180 min.
Assessment of in Vivo I nsulin-Stimulated Glucose
Insulin sensitivity was evaluated using an insulin suppression test6
in rats of ages and weights as used for the oral glucose tolerance
tests. The test was begun at 1 PM following withdrawal of food at
8 AM on the day of the experiment. Blood pressure recordings on
these animals were taken on the morning of the test and averaged
169 + 4 mm Hg for nine SHR rats and 125 t 2 for nine WKY rats.
All infusions were begun after an intraperitoneal injection of sodium
thiamylal (6.0 mg/lOO g body weight) to initiate anesthesia. Subse-
quently, the right jugular was exposed and cannulated for adminis-
tration of the infusion. Rats received a continuous infusion (0.848
mL/h) of epinephrine (0.08 pg/kg/min), propranolol (1.7 pg/
kg/min), glucose (8 mg/kg/min), and insulin (2.5 mU pork
insuEn/kg/min). With this technique, the endogenous release of
insulin is inhibited by the infusion of epinephrine and propranolol,
and comparable steady state serum insulin (SSSI) levels attained
during the last hour of infusion. By comparing the steady state serum
glucose levels (SSSG) during the same period, a direct assessment of
the ability of insulin to stimulate glucose uptake can be generated.
Blood samples (0.5 mL) were drawn from the tip of the tail at 0,60,
120, 130,140,150, 160 minutes, and SSSI and SSPG concentrations
were calculated from serum values between 130 and 160 minutes.
This approach is based upon the premise that similar SSSI concen-
trations will be reached in all rats in response to the same exogenous
insulin infusion. During the course of these experiments it became
apparent that this was not the case, and that SSSI concentration
were actually higher in SHR rats. In order to compensate for this,
studies were also carried out in ten SHR rats infused at an exogenous
insulin rate of 1.5 mU/kg/min. In addition, the metabolic clearance
rate (MCR) of insulin was calculated from the following formula:
MCR (mL/min/kg) =
Insulin infusion rate (rU/min/kg)
SSSI concentration (pU/mL)
_
basal insulin concentration (pU/mL)
Serum glucose and insulin concentrations were measured as
described previously.6 Results are expressed as mean + SEM.
Differences in serum glucose and insulin responses to oral glucose
and insulin MCR were compared by Students nonpaired t-test, and
SSSI and SSSG concentration by one-way ANOVA with P < .05
set as the level of statistical significance.
RESULTS AND DISCUSSION
Serum glucose and insulin concentrations in response to
oral glucose are shown in Fig 1. Total integrated serum
glucose response areas during the 180-minute period Follow-
ing the oral glucose load were comparable in SHR (524 f 12
mg/dL) and WKY rats (509 + 10 mg/dL) (NS). In con-
trast, total integrated serum insulin response was signifi-
cantly (P < .Ol) higher in SHR (280 + 14 wU/mL) as
compared to WKY (220 f 15 pU/mL).
SSSG concentrations during the insulin suppression test
are illustrated in the left panel of Fig 2. Compared by
ANOVA, SSSG values were similar when SHR and WKY
rats were infused with insulin at a rate of 2.5 mU/kg/min,
whereas SSSI concentrations were significantly higher in the
SHR rats. These data suggest that insulin stimulation of
glucose uptake was impaired in SHR as compared to WKY
- WKY
--- SHR
L I
0 30 60 120 180
MONDON AND REAVEN
0 30 60 120 180
Time (mid
Fi g 1. Mean (+ SEMI ser um gl uc ose and i nsul i n c onc ent r a-
t i ons i n r esponse t o an or al gl uc ose c hal l enge i n SHR (O----O) and
WKY (O--+1 r at s.
rats, since SSSG levels were similar despite higher SSSI
concentrations. To pursue this issue directly, SHR rats were
also infused with a lower rate of insulin, 1.5 mU/kg/min,
and this resulted in SSSI levels that were not significantly
different from those of WKY rats infused at a rate of 2.5
mU/kg/min. However, in this instance, the SSSG concen-
trations were significantly higher in the SHR rats, Since the
SSSI concentrations were similar, higher SSG levels in SHR
rats indicate that these animals are insulin-resistent.
Since SSSI levels were higher in SHR rats when they were
infused with insulin at the same rate as WKY rats, there
appeared to be a defect in the rate of insulin removal from
serum in SHR rats. To quantify this change, the MCR of
insulin was calculated, and the results indicated that insulin
MCR was significantly reduced (P < ,001) in SHR
(31.6 -c 3.0 mL/min) as compared to WKY (47.4 + 2.6
mL/min) rats.
In summary, these results indicate that impairment in
insulin-stimulated glucose uptake, hyperinsulinemia, and a
reduced rate of insulin removal from serum all exist in SHR
rats. Thus, it appears that hyperinsulinemia is not unique to
humans with high blood pressure, and can be demonstrated
in an animal model of spontaneous hypertension. The signifi-
cance of the multiple defects in insulin metabolism in SHR
remains to be clarified, but documentation of these abnor-
malities of insulin metabolism in both hypertensive humans
and rats suggests that these issues are worthy of further
study.
Fi g 2. Mean (+ SEMI SSSG and SSSI c onc ent r at i ons i n SHR
(ml and WKY (0) r at s. The f i gur es w i t hi n eac h bar def i ne t he
i nsul i n i nf usi on r at e used i n t hat st udy 11.5 or 2.5 mU/k g/mi n).
ABNORMALITIES OF INSULIN METABOLISM 305
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