Insulin Resistance in the Spontaneously Hypertensive Rat

Sonia Hulman, Bonita Falkner, and Y.Q. Chen

To determine experimentally if insulin resistance is associated with spontaneously occurring hypertension, insulin-stimulated
glucose metabolism was studied in an animal model of genetic hypertension. The spontaneously hypertensive rat (SHR) and its
genetic control, the Wistar-Kyoto strain (WKY) were studied with the euglycemic hyperinsulinemic clamp technique. Clamp
studies demonstrated reduced insulin-stimulated glucose uptake in SHR (P < .OOl). These data indicate that SHR is
insulin-resistant when compared with WKY. A reduction of insulin-stimulated glucose metabolism occurred in older animals of
both strains, providing evidence of an aging effect on insulin-stimulated glucose metabolism. However, the reduction of
insulin-stimulated glucose metabolism was more pronounced in the hypertensive animals. This study demonstrates the
oresence of Derioheral (skeletal muscle) insulin resistance in the SHR.
bopyright 01961 by WIB. Saunders Co&any
ECENT CLINICAL reports describe a relationship
R between insulin resistance and elevated blood pres-
sure. Experimental work has begun to describe this phenom-
enon in animal models. Young and Landsberg have demon-
strated that a sucrose-enriched diet aggravates hypertension
and causes an increase in urinary catecholamine metabolite
excretion in the spontaneously hypertensive rat (SHR). In
Sprague-Dawley rats, fructose-enriched diet induces insu-
lin resistance as measured by the insulin suppression test.
It was recently reported that young SHR have a greater
endogenous insulin response to an oral glucose challenge
compared with young Wistar-Kyoto rats (WKY). Impaired
peripheral glucose uptake in the SHR was also suggested by
the insulin suppression test.4
We undertook the following study to determine if the
SHR represents a non-obese insulin-resistant animal model
of hypertension. We used the euglycemic hyperinsulinemic
clamp technique to compare the glucose-disposal response
to exogenously infused insulin in WKY and SHR.
METHODS
Animal Preparation
Male WKY and SHR were obtained from Charles River (Wil-
mington. MA) at age 8 to 10 weeks. Animals were acclimated to the
laboratory environment, and trained for tail-cuff blood pressure
measurement (IITC, Life Sciences, Woodland Hills, CA). Animals
were weighed and had simultaneous blood pressure measurements
biweekly. The reported weight and blood pressure was that
obtained within 72 hours of cannulation and study. Animals were
studied at approximately 12 weeks of age (Table 1) after an
overnight fast. On the morning of the study, anesthesia was
induced with intraperitoneal pentobarbital, 40 mg/kg. Catheters
were placed in the external jugular vein for infusion, and the
femoral artery for sample withdrawal. The animal remained
anesthetized for the duration of the study. Hypothermia was
prevented with a heat lamp.
Euglycemic Hyperinsulinemic Clamp Study
After a sample was withdrawn for assessment of fasting glucose
and insulin concentration, hyperinsulinemia was induced with an
insulin infusion (4 mU/kg/min). Plasma glucose was measured
every 15 minutes with the glucose oxidase method (Glucostat
Model 27, YSI. Yellow Springs, OH) and hypoglycemia and its
attendant neuroendocrine counterregulatory responses was pre-
vented with a simultaneous infusion of glucose. Glucose infusion
rate was determined by the operator, because this method resulted
in smoother glucose control than the negative feedback equation
Metabolism, Vol 40. No 4 (April), 1991: pp 359-361
used in clinical studies. The glucose infusion rate required to
maintain euglycemia then becomes an index of insulin-stimulated
glucose disposal. Euglycemic hyperinsulinemia was maintained for
2 hours. The mean value of the last three samples withdrawn is
reported as clamped glucose and insulin concentration.
Laboratory Methods
Regular human insulin (Lilly, Indianapolis, IN; 100 UlmL) was
administered in normal saline solution. Glucose was administered
as 5% dextrose in water. Plasma insulin was measured with the
double-antibody radioimmunoassay technique (Amersham, Arling-
ton Heights, IL), which uses a human standard. Rat insulin and
human insulin differ by only three amino acid residues, so cross-
reactivity of the human antibody and rat antigen should be present.
To ensure that cross reactivity was occurring, radioimmunoassay of
serial dilutions of fasting SHR and WKY plasma were performed
with human antibody and compared with a human insulin standard
curve. There was parallel displacement of the rat plasma curve.
indicating cross-reactivity (data not shown), so the results are
expressed in terms of the human insulin standard. ANOVA was
performed comparing groups, followed by Scheffes test for pair-
wise comparisons. Results are expressed as mean ? SD.
RESULTS AND DISCUSSION
Two groups of rats in both SHR and WKY strains were
studied with the clamp technique: younger rats and older
rats (Table 1). Within the age groups, age and weight did
not differ between WKY and SHR, but tail cuff blood
pressure was significantly greater in SHR than WKY in
both younger and older rats. Older WKY and SHR are
significantly heavier than younger animals of the same
strain.
Results of the euglycemic clamp study in both age groups
are summarized in Table 2. Fasting plasma insulin and
glucose are the same in each rat strain and age group. In Fig
lA, the mean plasma glucose concentration in the four
different groups studied (younger WKY, older WKY,
younger SHR, older SHR) is depicted. Fasting glucose
concentration (time 0 to time 30 minutes) is followed by
From the Depanment of Pediatrics, Medical College of Penn~tva-
nia, Ph~ladeiphia, PA.
Address reprint requests to Sonia H&man, MD, Medical College of
Pennsylvania, Department of Pediatrics, 3300 Henn, Ave, Philadel-
phia, PA 19129.
Copyright 0 1991 by W. B. Saunders Company
0026-0495191t40060003.OOlO
359
360 HULMAN, FALKNER, AND CHEN
Table 1. Results: Age, Weight, and Blood Pressure in Younger and
Older Rats
Younger rats
N
Age (wk)
Weight (g)
BP (mm Hg)
Older rats
N
Age (wk)
Weight (g)
BP (mm Hg)
WKY SHR P
8 10
12.5 k 0.8 12.2 f 0.6 NS
258 -+ 11 264 rt 13 NS
137 + 11 170 r 19 <.OOl
4 4
27.3 + 2.7 27.0 k 0.3 NS
376 2 43 345 k 17 NS
140.3 + 9.8 174.0 + 19 < .OOl
40
IA
0 15
I I I I
30 45 60 75 90
Time, Minutes
glucose during hyperinsulinemia (time 30 to time 90 min-
utes). Mean plasma glucose concentration for each group is
within physiologic range, in spite of simultaneous hyperinsu-
linemia (Table 2). ANOVA comparing the four groups,
followed by Scheffes test, showed no significant differ-
ences. Figure 1B depicts the glucose infusion rate in
mg/kg/min required to maintain euglycemia, for each 15
minute interval. SHR, regardless of age, require a signifi-
cantly lower glucose infusion rate than WKY in each
15minute interval to maintain euglycemia in the face of
physiologic hyperinsulinemia (P < .OOl). The lower amount
of glucose required to maintain euglycemia during hyperin-
sulinemia in both younger and older SHR indicates that
SHR are relatively insulin-resistant when compared with
WKY.
3.75 -
325 -
-:
B
2.75 -
& 2.25 -
E
.c 1.75 -
Time, Minutes
Insulin stimulates peripheral glucose uptake (primarily
skeletal muscle). Insulin also inhibits endogenous produc-
tion of glucose, EGP (primarily hepatic).5 Smith et al6 have
shown that hepatic EGP is 90% suppressed in the conscious
chronically catheterized Sprague-Dawley rat at levels of
hyperinsulinemia comparable to that achieved in our study.
It is possible that EGP during hyperinsulinemia may have
contributed to total glucose metabolism in SHR and WKY.
The known hyperadrenergic state of the SHR could be a
plausible explanation for persistent EGP. A lack of suppres-
sion of EGP by insulin could explain the reduced require-
ment for glucose in the SHR in the face of euglycemic
hyperinsulinemia.
Fig 1. (A) Plasma glucose concentration for the four rat groups.
Times 15 and 30 are fasting glucose concentration, time 45 through SO
are glucose concentrations during hyperinsulinemia. Glucose concen-
trations are not different at any time among the four groups. (B)
Glucose infusion rate (GIR) in mg/kg/min during hyperinsulinemia for
the four rat groups. Both WKY groups require higher GIR at all times
than both SHR groups (ANOVA, P < .OOl), indicating insulin resis-
tance. f, Young WKY; 0, older WKY; *, Young SHR; x, older SHR.
EGP was not measured in this study because the animals
were sedated with pentobarbital. Pentobarbital anesthesia,
in the dose used in our study, suppresses hepatic glucose
production in the rat. Although there may have been a
Table 2. Results: Clamp Study in Younger and Older Rats
difference between SHR and WKY in persistent EGP
during hyperinsulinemia, the interpretation of EGP mea-
surement in this study would have been confounded by
pentobarbital; hence it was not performed. However, the
difference in glucose infusion rate between WKY and SHR
of both age groups is so marked, that, even in the face of
pentobarbitals effect on EGP, it can be concluded that
SHR are insulin-resistant. This study indicates insulin resis-
tance at the level of the peripheral tissue, primarily skeletal
muscle, which is consistent with the results of Mondon and
Reaven: who used the insulin suppression test.
WKY SHR P
Younger rats
Fasting glucose (mg/dL) 80.3 k 13.2 75.4 2 12.8 NS
Clamp glucose 78.1 r 10.7 78.0 + 14.4 NS
Fasting insulin ($J/mL) 41.3 +- 7.1 34.6 lr 14.1 NS
Clamped insulin 67.5 2 7.2 57.7 f 14.8 NS
Older rats
Fasting glucose (mg/dL) 79.3 k 8.0 66.8 2 6.7 NS
Clamped glucose 79.3 k 4.0 67.3 -e 4.6 NS
Fasting insulin (pU/mL) 43.7 2 8.7 54.6 -c 13.2 NS
Clamped insulin 88.8 -t 14.0 101.5 2 36.2 NS
Increased adrenergic activity also opposes the normal
insulin stimulation of peripheral glucose uptake. Chiasson
et al8 demonstrated that the addition of epinephrine to
perfusing medium of a rat hindlimb inhibited insulin-
stimulated glucose uptake. James et al added epinephrine
to the insulin infusate during a hyperinsulinemic clamp, and
demonstrated that peripheral glucose uptake was not stim-
ulated. Although plasma catecholamine levels were not
measured in this study, SHR have been reported to have
higher levels than WKY when subjected to stress. It is
possible that the enhanced catecholamine response of the
SHR may result in submaximal response to insulin, both in
the liver and in the peripheral musculature, resulting in
insulin resistance.
INSULIN RESISTANCE IN SHR 361
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