Clinical Science (1995)
88, 301-306 (Printed in Great Britain)
30I
Development of decreased insulin-induced glucose
transport in skeletal muscle of glucoseintolerant hybrids
of diabetic GK rats
Lorraine A. NOLTE, Samy M. ABDEL-HALIM*, Iva K. MARTIN, Amel GUENIFI*,
Juleen R. ZIERATH, Claes-Goran OSTENSON* and Harriet WALLBERG-HENRIKSSON
Department of Clinical Physiology and *Department of Endocrinology, Karolinska Hospital,
Karolinska Institute, Stockholm, Sweden
(Received
15 July17 October 1994; accepted 21 November 1994)
1. The effect of glucose intolerance on insulinstimulated glucose transport in isolated skeletal
muscles was investigated in male F, hybrids of
spontaneously diabetic GK (Goto-Kakizaki) and
control Wistar rats at 1 and 2 months of age.
2. Hybrid rats are characterized by markedly
impaired glucose-induced insulin secretion. The area
under the blood glucose curve was significantly higher
following an intraperitoneal glucose injection (2 g/kg)
in hybrid rats in both age groups than in the control
rats (P<O.OOl). In 2-month-old hybrid rats the incremental area under the insulin curve during the intraperitoneal glucose tolerance test was not different
from that of control rats. Serum cholesterol, triacylglycerol or plasma free fatty acid levels did not differ
between the groups. Fasting and post-prandial plasma
glucose concentrations were elevated in 2-month-old
hybrid rats compared with control rats (54%,
P < 0.05, and 27%, P < 0.05, respectively), but were
not differerent in 1-month-old rats. Plasma insulin
did not differ between the hybrid and control rats in
the fasting or post-prandial state at either age
studied.
3. The insulin dose-response curves for 3-0methylglucose transport did not differ between
1-month-old hybrid and control rats for either the
soleus or epitrochlearis muscle. The insulin doseresponse curve for the epitrochlearis, but not for the
soleus, muscle from 2-month-old hybrid rats was
shifted to the right compared with the curve from the
control animals (P<0.05).
4. In conclusion, the hybrid rat is a non-obese, nonhyperinsulinaemic animal model, which at a young
age is characterized by impaired insulin secretion and
moderate glucose intolerance. In this glucoseintolerant rat model, mild peripheral insulin resistance gradually develops, as reflected by the
decreased insulin-induced glucose transport in the
fast-twitch epitrochlearis muscle. It is suggested that
the elevated blood glucose per se may have contri-
buted to the slight decrease in peripheral insulin
action.
-
INTRODUCTION
The GK (Goto-Kakizaki) rat is a spontaneously
(non-obese, non-ketotic) type 2 diabetic rat model
[l]. The diabetic state of the GK rat was produced
by selective inbreeding over several generations of
normal Wistar rats which demonstrated the highest
blood glucose levels during an oral glucose tolerance test [l]. GK rats display glucose intolerance at
a very young age [2]. In the isolated perfused
pancreas and pancreatic islets of adult GK rats,
glucose-stimulated insulin release is markedly
impaired [3-51. By breeding a normal Wistar female
with a diabetic GK male rat, an F, hybrid rat has
been developed [2, 61 which, like the GK rat,
demonstrates glucose intolerance at a very young
age [2]. Hybrid GK-Wistar rats display glucose
tolerance as well as glucose-induced insulin responses which are intermediate between GK and
control rat levels [2].
Impaired insulin secretion and/or peripheral insulin resistance are characteristic features associated
with type 2 diabetes mellitus [7-91. During glucose
infusion, skeletal muscle is considered the most
important in uiuo site for the uptake of glucose, and
thus the primary site of peripheral insulin resistance
[lo, 111. The glycaemic state has been shown to
autoregulate glucose uptake in skeletal muscle and
adipocytes, by the mass action effect of glucose
[12-14]. Additionally, hyperglycaemia per se has
been suggested to be a contributing factor for the
development of peripheral resistance in skeletal
muscles of diabetic patients [141.
The present investigation was designed to examine
the effect of glucose intolerance on peripheral
insulin resistance in isolated rat skeletal muscles.
For this purpose glucose-intolerant rats, F, hybrids
Key words: GK rats, glucose intolerance, insulin resistance, Wmethylglucose transport, skeletal muscle.
Abbreviations: ANOVA, analysis of variance; BSA, bovine serum albumin; KHB, Krebs-Henseleit buffer.
Correspondence: Dr Harriet Wallberg-Henriksson. Department of Clinical Physiology, Karolinska Hospital, Box 60500, 5171 76 Stockholm, Sweden.
302
L. A. Nolte et al.
of the spontaneously diabetic GK rat, with the
primary lesion in the pancreas, were studied at 1 or
2 months of age in order to follow a potential
alteration on skeletal muscle glucose transport
owing to the long-lasting period of impaired glucose
tolerance.
METHODs
Animals and muscle preparation
Male F, hybrid GK-Wistar rats were obtained
from the Department of Endocrinology (Karolinska
Hospital, Stockholm, Sweden) and control, nondiabetic Wistar rats were obtained from B&K
Universal (Sollentuna, Sweden). All rats were maintained on a 12h-12h light-dark cycle, 07.00 to
19.00 hours, and received a diet of standard rat
chow and water ad libitum. Rats were studied after
an overnight fast at either 1 month [weight range=
66-134g: hybrid (n=32) 104+3g and control
(n=35) 95)2g] or 2 months [weight range=181251 g: hybrid (n=37) 204f3g and control (n=40)
201 k 3 g] of age. An intraperitoneal glucose tolerance test was performed 1 week before the in uitro
skeletal muscle experiments. All rats were anaesthetized intraperitoneally (5 mg/100 g body weight of
pentobarbital sodium) and thereafter the muscles
were dissected out. The intact epitrochlearis or the
split soleus muscle was used for the in uitro incubation procedure. The soleus muscles were divided
into strips according to the protocol described by
Henriksen et al. [15]. The mean size of the extracellular space for both the soleus and epitrochlearis
muscles of the 1- and 2-month-old animals was
0.31 fO.01 and 0.28 fO.O1 ml/g wet weight respectively. The mean values did not differ with increasing concentrations of insulin or between the two
types of rats studied.
Glucose tolerance test
All rats were fasted overnight and a fasting blood
sample was obtained by a tail vein incision at 09.00
hours on the morning of the test. Thereafter, the
rats were injected intraperitoneally with 2 g of
glucose/kg body weight. Blood samples were further
taken at 15, 30, 60, 90, and 120min after the glucose
injection. Blood glucose was measured by a glucose
oxidase method using reagent strips (BM-test
Glycaemia 1-44; Boehringer-Mannheim, Mannheim,
Germany), read for absorbance in a reflectance
meter (Reflolux S, Boehringer-Mannheim).
In another set of experiments, 2-month-old hybrid
and control rats were used to determine the insulin
response throughout the intraperitoneal glucose
tolerance test. The glucose tolerance tests were
performed exactly as described above plus additional blood (250 pl) was obtained at 0, 5, 15, 30, 60,
and 120min for analysis of serum insulin levels.
Serum immunoreactive insulin was assayed by the
Pharmacia Insulin RIA 100 method (Kabi Pharmacia Diagnostics, Uppsala, Sweden) using human
insulin standards. The lower limit of sensitivity for
this method was 18 pmol/l.
Muscle incubation
After dissection, intact epitrochlearis and split
soleus muscles were preincubated (1 h) in sealed
glass flasks containing 2 ml of Krebs-Henseleit
buffer [161 supplemented with 5 mmol/l Hepes
(KHB), 0.1% radioimmunoassay-grade bovine serum
albumin (BSA), 8 mmol/l glucose, 32 mmol/l mannito1 and in the absence or presence of increasing
concentrations of human insulin. Glycogen levels in
isolated rat muscles remain unaltered during 14h
incubation [17, 181.
Following preincubation, muscles were rinsed
(10min) in a glucose-free KHB medium containing
0.1% BSA and 40mmol/l mannitol with the same
concentration of insulin as in the preceding step. All
flasks were continuously gassed with 95% 02-5%
CO, in a shaking waterbath maintained at 30°C.
Measurement of 3-0-methylglucose transport
The glucose transport rate was measured using
the non-metabolizable glucose analogue 3-0methylglucose as previously described by WallbergHenriksson et al. [19]. Briefly, after the rinse step
the muscles were transferred to a flask containing
l m l of KHB supplemented with 0.1% BSA,
8 mmol/l 3-0-[3H]methylglucose (437 pCi/mmol)
and 32 mmol/l ['4C]mannitol (8 pCi/mmol), and the
same concentration of insulin as in the preceding
two incubation steps. All muscles were incubated for
10min at 30°C; afterwards muscles were quickly
blotted on ice-cold filter paper, excess tissue and
tendons were trimmed away and the muscles were
freeze-clamped with tongs cooled to the temperature
of liquid nitrogen (within 20 s). Muscle samples were
stored at -80°C and later processed for 3-0methylglucose transport [191.
Blood sampling
Following muscle dissection, blood was drawn
(approximately 2-3 ml) from the descending aorta.
One millilitre of blood was placed in a tube with no
additive for analysis of serum cholesterol and triglyceride levels. The second millilitre of blood was
placed in a tube containing EDTA and centrifuged
at 4°C for 15min. Plasma samples were stored at
- 20°C for subsequent analysis of fasting insulin
and glucose levels. Plasma glucose was analysed by
the glucose dehydrogenase method (Merck kit
12194, Darmstadt, Germany). Immunoreactive insulin was assayed by the Pharmacia Insulin RIA 100
method (Kabi Pharmacia Diagnostics, Uppsala,
Sweden) using human insulin standards. The lower
limit of sensitivity of this method was 18pmol/l
insulin.
Effect of glucose intolerance on glucose transport
Table 1. Fasting plasma insulin, glucose, free fatty acid and serum
cholesterol and triacylglycerol levels in I- and Z-monthold hybrid
and control rats. Values are expressed as means f SEM for 7-15 samples
per group. *P<O.OS versus control in the same weight category.
One monthold rats
Measurement
Control
Insulin (pmol/l)
Glucose (mmol/l)
Free fatty acids (mmol/l)
Triacylglycerol (mmol/l)
Cholesterol (mmol/l)
Control
43f3
37k4
3.0 f0.4
0.8 0.I
1.8kO.l
3.6 f 0.3
108k12
101k7
5.4f0.7
8.3 f I .O*
0.3 I f0.04 0.35k0.05
0.8kO.l
0.8fO.l
1.5kO.O
1.6f0.1
0.8 k0.1
1.9k0.1
Table 2. Postprandial (approximately I h) plasma insulin and glucose levels in I- and 2-month-old hybrid and control rats. Values are
expressed as meansf SEM for 6-7 samples per group. * P i O . O S versus
control in the same weight category.
Twemonth-old rats
Hybrid
Hybrid
In a separate set of experiments fasting plasma
samples were drawn to determine free fatty acid
levels. All rats were anaesthetized intraperitoneally
( 5 mg/100 mg body weight of pentobarbital sodium)
and blood samples were drawn (approximately 2 ml)
from the descending aorta. The blood was immediately placed in a tube containing sodium heparin
and centrifuged at 4°C for 15 min. Plasma samples
were stored at -80°C until later analysis of free
fatty acid levels using the Farb-Test Colorimetric
method (Boehringer-Mannheim kit 1383175).
In another set of experiments post-prandial
(approximately 1 h) blood samples were taken at
08.00 hours following a night of normal feeding.
Rats were anaesthetized intraperitoneally (5 mg/
lOOg body weight of pentobarbital sodium) and
decapitated. Blood samples were collected in
EDTA-containing tubes, centrifuged at 4°C for
15min and stored at -20°C for later analysis of
post-prandial insulin and glucose levels.
Chemicals
All chemicals were obtained from Sigma (St
Louis, MO, U.S.A.). The insulin (Actrapid) was a
product of Novo Nordisk (Copenhagen, Denmark).
All radioactive products were obtained from New
England Nuclear (Boston, MA, U.S.A.).
303
One-month-old rats
Twemonth-old rats
Measurement
Control
Hybrid
Control
Hybrid
Insulin (pmol/l)
Glucose (mmol/l)
109+22
10.5f 1.3
86f13
10.3f0.3
121f6
9.0 f0.7
I I.4f 0.8*
143k26
fasting and post-prandial glucose levels did not
differ between 1-month-old hybrid and control rats,
the 2-month-old hybrid rats demonstrated higher
fasting and post-prandial plasma glucose levels than
control animals (P<O.O5, Tables 1 and 2). Fasting
serum cholesterol and triacylglycerol levels were not
different between control and hybrid rats in the two
age categories studied (Table 1). The fasting plasma
free fatty acid levels of 2-month-old hybrid rats were
similar to levels in control rats (Table 1).
Glucose tolerance test
Hybrid rats demonstrated moderately impaired
glucose tolerance to an intraperitoneally glucose
load of 2 g glucose/kg body weight in both age
groups studied (Fig. 1). The area under the glucose
curve for the 1-month-old hybrid rats was 162% of
that of the controls (P<O.OOl), and the area under
the glucose curve for the 2-month-old hybrid rats
was 270% of that of the control curve (P<O.OOl).
However, the incremental area under the insulin
curve for the 2-month-old hybrid animals was not
different from that of the control rats (1962f 1338
and 2406f1416pmol 2 h - l l - I for hybrid and
control rats respectively). The insulinogenic index of
the control rats was slightly higher than that of the
hybrid rats, although this difference was not significant (1.19k0.85 and 1.83f1.10 for hybrid and
control respectively).
Statistical analysis
Values are reported as the meansfSEM. For
multiple comparisons a two-way analysis of variance (ANOVA) was used to evaluate statistical
significance. When the ANOVA indicated significant
differences, a Newman-Keul’s test was used for post
hoc analysis. Integrated glucose or insulin responses
were calculated as the areas under the curves. A
Student’s unpaired t-test was used for assessing
statistical differences when two groups of data were
compared.
RESULTS
Blood indices
Insulin levels in the fasting or post-prandial state
did not differ in 1- or 2-month-old hybrid compared
with control rats (Tables 1 and 2). Although the
Skeletal muscle insulin doseresponse
Intact epitrochlearis and split soleus muscles were
preincubated (1 h) in increasing concentrations of
insulin before measurement of 3-0-methylglucose
transport. The insulin dose-response curves were
not different in 1-month-old hybrid rats compared
with control rats in either muscle studied (Fig. 2).
At 2 months of age no difference in the doseresponse curves was detected in the soleus muscle,
however a significant rightward shift in the doseresponse curve occurred in the epitrochlearis muscle
of the hybrid animals (Fig. 3). At 1200 and
2400 pmol/l of insulin the rate of 3-0-methylglucose
transport in the epitrochlearis muscle was significantly decreased ( P <0.09, however at 6000 pmol/l
of insulin there was no difference between the
groups (Fig. 3).
L. A. Nolte et al.
304
T
0’
0
0’
15
30
60
Time (min)
0.0 -11-
I
I20
1
0
15
30
60
Time (min)
I20
0
300
600
6000
Insulin (pmol/l)
0.0
1
0
300
600
1
7
6OOo
Insulin (pmol/l)
Fig. I. Effect of an intraperitoneal glucose load on blood glucose
levels in I- (a) and 2-month-old (6)hybrid GK-Wistar ( 0 )and
rats. Blood samples were obtained from a tail vein in
control Wistar (0)
overnight-fasted rats before (time 0) and after (time 15, 30, 60, 90, 120min),
an intraperitoneal injection of glucose (2glkg). Blood glucose was assessed
as described in Methods. Values are expressed as meansf SEM for 6-15
animals per group.
Fig. 2. Insulin dose-response curves in epitrochlearis (a) and soleus
( b ) muscles from I-monthold hybrid GK-Wistar ( 0 )and control
Wistar (0)
rats. Isolated epitrochlearis and split soleus muscles were
incubated (I h) in KHB containing 8mmol/l glucose and 32mmol/l mannitol
and in the absence or presence of increasing concentrations of insulin.
Thereafter, the rate of 3-0-methylglucose transport was assessed as
described in Methods. Results are meansf SEM for 5-9 muscles per group.
DISCUSS10N
This study presents a glucose-intolerant rat
model, the hybrid GK-Wistar, which progressively
develops mild skeletal muscle insulin resistance. In
2-month-old hybrid rats, a mild decrease in muscle
insulin action was observed in the fast-twitch, highly
glycolytic epitrochlearis muscle. There was not evidence of decreased muscle insulin action in the
1-month-old hybrid rats. These observations were
based on measurement of the transmembrane transport of glucose, which is generally accepted to be
rate-limiting for overall glucose use under normal
physiological conditions [181. However, it cannot be
excluded that a defect in insulin action, for example
decreased glycogen synthesis activity, may prevail in
the presence of normal glucose transport activity
c201.
Hybrid rats demonstrate a primary defect, for
their glucose interolance, at the level of the pancreatic beta-cell [3, 21, 221. The glucose-stimulated insu-
lin response is markedly impaired in the perfused
pancreas as well as in isolated pancreatic islets of
2-month-old hybrid rats [2, 231. As early as 1 week,
hybrid animals demonstrate a defect in islet insulin
secretion (C.-G. Ostenson and S.M. Abdel-Halim,
unpublished work).
Despite the early pancreatic secretory impairment,
changes at the muscle cellular levels were not
evident until the fasting blood glucose concentration
was no longer maintained within normal limits.
These results are in agreement with human studies
in which the degree of peripheral insulin resistance
is highly correlated to the fasting glucose concentration [24]. The results suggest that the elevated
blood glucose levels per se may have contributed to
the development of peripheral insulin resistance in
these hybrid rats. However, the insulin resistance
developed in the hybrid rats was much less pronounced than the skeletal muscle insulin resistance
demonstrated in other animal models of diabetes
Effect of glucose intolerance on glucose transport
T
,
0.00 J
,
I
I
0 600 12W
2400
Insulin (pmol/l)
1
T
(b'
0.25
0.00
6000
305
The precise mechanism responsible for decreased
insulin sensitivity in the epitrochlearis muscle of the
2-month-old animals is not known. However, this
finding might be attributed to a receptor defect
since a rightward shift in the dose-response curve
reflects decreased hormone sensitivity [28]. A postreceptor defect cannot completely be ruled out since
at 2400 pmol/l insulin, muscle insulin responsiveness
was decreased in the hybrid rats [28]. The decreased skeletal muscle insulin action of the
2-month-old hybrid rats is similar to the findings of
studies in isolated human adipocytes from glucoseintolerant subjects, in which decreased insulin sensitivity, but no apparent change in responsiveness, is
present [29]. Hyperinsulinaemia results in an insulin
receptor down-regulation and decreased insulin
sensitivity in human adipocytes [30]. The incremental area under the insulin curve of the hybrid rats
was not different from the curve of the control rats.
Therefore, it appears that the hybrid rats are not
hyperinsulinaemic and this condition cannot
account for the changes in cellular muscle insulin
resistance in this model.
Speculatively, hyperglycaemia per se may have
decreased muscle sensitivity to insulin in these
hybrid rats. The effect of glucose on peripheral
insulin-mediated glucose uptake is a phenomenon
which has been studied extensively. Hyperglycaemia
leads to the development of insulin resistance in
partially pancreatomized rats [31]. In addition, a
decrease in skeletal muscle insulin response has been
observed in normal rats infused with elevated levels
of glucose [32, 331. Hypothetically, hyperglycaemia
may decrease glucose transport by increasing the
internalization of the specific glucose transporter
proteins [12].
In conclusion, the hybrid GK-Wistar rat appears
to be an appropriate animal model for studying
non-obese, glucose-intolerant patients with mild to
moderate hyperglycaemia and normal insulin levels.
In this model, hyperglycaemia, due to defective
beta-cell secretory function, precedes the development of mild insulin resistance in fast-twitch glycolytic skeletal muscle.
1
0 600 I200
2400
6000
Insulin (pmol/l)
Fig. 3. Insulin doseresponse curves in epitrochlearis ( a ) and soleus
(b) muscles from 2-monthold hybrid GK-Wistar ( 0 )and control
Wistar (0)
rats. Isolated epitrochlearis and split soleus muscles were
incubated (I h) in KHB containing 8mmol/l glucose and 32mmol/l mannitol
and in the absence o r presence of increasing concentrations of insulin.
Thereafter, the rate of 3-0-methylglucose transport was assessed as
described in Methods. Results are means+SEM for 4-17 muscles per group.
*P<0.05 versus control (twc-way ANOVA).
[18] or in isolated human skeletal muscle obtained
from type 2 diabetic patients [25]. Thus, it appears
that factors other than fasting hyperglycaemia are
necessary for the development of pronounced peripheral insulin resistance.
The insulin-induced glucose transport rate was
significantly decreased in the fast-twitch, glycolytic
epitrochlearis muscle, but not in the slow-twitch,
oxidative soleus muscle. Similar results were
obtained in a recent study [26], in which the uptake
of 2-deoxyglucose in 8-week-old female G K rats
during a hyperinsulinaemic-euglycaemic clamp
(plasma insulin levels approximately 2400 pmol/l)
was decreased in the epitrochlearis but not in the
soleus muscle. The present investigation in hybrid
rats and the findings of Bisbis et al. [26] support
the notion that insulin resistance preferentially develops in muscles rich in glycotic fibres rather than
in oxidative muscles [27].
ACKNOWLEDGMENTS
This study was supported by grants from the
Swedish Medical Research Council ( 5 117, 10627 and
00034), The Bank of Sweden Tercentenary Foundation, The Swedish Diabetes Association, The
Nordisk Insulin Foundation, The Thurlings Foundation, The Wibergs Research Foundation, Gustav
V's Research Foundation, and from Novo-Nordisk
A/S.
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