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Introduction
Insulin resistance is a major
characteristic of various metabolic disorders, including type 2
diabetes and obesity[1]. There is strong evidence that an
important factor leading to obesity and insulin resistance is an
increase in energy intake[2]. Our previous study
demonstrated that high-fat, high-sucrose feeding led to insulin
resistance and impaired glucose tolerance in female Wistar rats[3].
In our study, female rats were given a relatively low-dose of
streptozotocin (STZ), followed by a high sucrose-fat diet containing
52% sucrose, 24% fat, and 18% protein (4.8 cal/g chow). Compared to
the control group, which did not receive either STZ or a high
sucrose-fat diet, the treatment group showed obvious obesity and
impaired glucose tolerance after 8 weeks. Fasting blood glucose
levels increased slightly or moderately, and were accompanied by
hyperinsulinemia, dyslipidemia, enhanced gluconeogenesis, and
reduced insulin tolerance. These characteristics indicated insulin
resistance and obesity with mild ¦Â-cell dysfunction, which was
further induced by the high sucrose-fat diet. A similar result is
observed in humans. Our results suggested that this model was a
successful insulin-resistant rat model and is useful in the study of
insulin resistance and insulin sensitivity.
Thiazolidinediones (TZDs), a new
class of insulin sensitizing drugs including troglitazone,
pioglitazone, and rosiglitazone, provide an effective approach for
treating type 2 diabetes. TZDs improve insulin sensitivity, impair
glucose tolerance and dyslipidemia in type 2 diabetics[4],
as well as in obese non-diabetic subjects[5]. Similar
findings have been demonstrated in a number of genetic and
non-genetic animal models of diabetes/insulin resistance[6,7].
TZDs elicit their effects through activating the peroxisome
proliferator-activator receptor (PPAR)-g, a nuclear hormone
receptor. When ligands stimulate the PPAR-¦Ã nuclear receptor, a
variety of response genes are stimulated or repressed. The TZD PPAR-¦Ã
agonists improve insulin sensitivity via multiple mechanisms.
Although the exact target genes for insulin sensitization remain
unknown, it has been demonstrated in vitro and in vivo
that treatment with TZDs affects a variety of factors involved in
lipid metabolism, insulin signal pathways, glucose phosphorylation,
and glucose transport.
In the present study, we evaluated
the effect of pioglita-zone, a PPAR-¦Ã agonist, on insulin resistance
and related abnormalities in low-dose STZ and high sucrose-fat diet
induced obese rats. Our findings demonstrated that PPAR-¦Ã agonist
treatment reduced circulating and stored lipids and improved the
insulin-resistant status in model rats.
Materials and methods
Animals
Thirty female Wistar rats,
weighing 225-250 g, were obtained from the Experimental Animal
Center, Chinese Academy of Medical Sciences, Beijing, China
[Certificate No SCXK(Jing)2000-0006], and maintained in
individual cages with a 12-h/12 h light-dark cycle. All animals were
allowed free access to water. At the beginning of the study, rats
were randomly assigned to control (n=8) and experimental
groups (n=22). Experimental groups were injected
intraperitoneally with low-dose STZ, 30 mg/kg (Sigma, St Louis, MO,
USA). After 2 weeks, a glucose tolerance test was carried out and 15
rats that had developed impaired glucose tolerance in the STZ-treated
group were selected and received a high sucrose-fat diet (containing
52% sucrose, 24% fat, 18% protein, and 4.8 cal/g chow). Rats in the
control group were fed ad libitum on a standard chow.
After 8 weeks on a high sucrose-fat
diet, weight-matched, fasting-blood-glucose-matched STZ rats were
randomly divided into two groups, a STZ group and a pioglitazone
group. STZ rats (n=8) were given vehicle solution and
pioglitazone rats (n=7) were orally administered pioglitazone
(20 mg¡¤kg-1¡¤d-1; the pioglitazone was a gift
from Prof Yu-ling LIU) as a suspension in 1% Tween 80 solution (the
high sucrose-fat diet was given to the rats during this period).
rats in each group were treated for 28 d. Glucose tolerance tests,
insulin tolerance tests and gluconeogenesis tests were carried out
over the last 14 d of the experiment. At the end of the treatment
period, all animals were decapitated after a 4-h fast. Blood samples
were collected and serum was prepared and kept at -20 ºC
for the determination of serum lipids and immunoreactive insulin.
Celiac fats were excised for weighing. Skeletal muscles and livers
were separated and frozen immediately in liquid nitrogen, and stored
at -70 ºC for further analysis of GLUT4 and IRS-1.
Biochemical analysis Serum
glucose levels were assayed using the glucose oxidase method. Serum
insulin was determined using a radioimmunoassay kit (China Institute
of Atomic Energy, Beijing, China). Free fatty acid (FFA)
concentrations were measured using the Cu2+ reagent
method. Triglyceride (TG), total cholesterol (TC), and
HDL-cholesterol (HDL-C) levels were determined using the
colorimetric method with commercial kits (Zhong sheng bei kong
Bio-Technology and Science, Beijing, China).
Intraperitoneal glucose tolerance
test (IPGTT) Baseline glucose levels were determined after a 4-h
fast, after which glucose (2 g/kg body wt) was administered
intraperitoneally to non-sedated animals. Tail vein blood was
sampled for glucose determination 30 and 120 min after glucose
admini-stration.
Insulin tolerance test
(ITT) Baseline glucose levels were determined after a 4-h fast.
Insulin (0.4 U/kg body wt) was injected subcutaneously. Blood was
sampled from the tail tip 40 and 90 min after insulin
administration, and the glucose concentration was determined.
Insulin injections and blood glucose sampling took approximately the
same amount of time per animal (ie 25 animals were injected in 12
min and blood glucose sampling of those same 25 animals also took
approximately 12 min), so that the sample times are accurate for
each animal.
Gluconeogenesis test Baseline
glucose levels were determined after an overnight fast. DL-alpha
alanine was injected intraperitoneally. Blood was sampled from the
tail and glucose levels were determined at 0 and 60 min. The percent
increase in glucose level at 60 min was calculated. Higher values
indicated higher levels of gluconeogenesis.
GLUT4 and IRS-1 protein
expression Protein extractions and immunoblots for the
determination of GLUT4 were carried out on frozen skeletal muscle
from 12 rats using a modified Klip's method[8]. skeletal
muscle (1 g) was powdered under liquid nitrogen and homogenized for
20 s in buffer (pH 7.4) containing sucrose 250 mmol/L, Tris 50
mmol/L and edetic acid 0.2 mmol/L. The homogenates were centrifuged
at 9000¡Ág for 10 min (4 ºC) and the supernatants were
reserved. The pellet was cleaned with buffer and centrifuged three
times. All three supernatants were mixed and centrifuged at 190 000¡Ág
for 60 min (4 ºC). The resulting pellet was resuspended in a small
amount of buffer (about 0.5 mL) as a total membrane fraction.
Protein concentrations of the suspensions were determined using the
Coomasine brilliant blue method[8] prior to Western blot
analysis.
Frozen tissues (100 mg liver, 100 mg
skeletal muscle) were ground into a fine powder with a mortar and
pestle and homogenized in the buffer (1% Triton X-100, sodium
pyrophosphate 100 mmol/L, Hepes 50 mmol/L, pH 7.4, NaF 100 mmol/L,
PMSF 2 mmol/L, 0.1% aprotinin, sodium edetic acid 10 mmol/L, Na3VO4
10 mmol/L) with a polytron homogenizer at 4 ºC for 6 (for
muscle) or 10 (for liver) times[9,10]. The homogenates
were centrifuged at 10 000¡Ág at 4 ºC for 60 min and the
supernatants were collected for IRS-1 analysis.
Protein extracts were resuspended in
Laemmli buffer and separated using sodium
dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10%
(for GLUT4) or 7.5% (for IRS-1) polyacrylamide gels. Proteins were
electrophoretically transferred to nitrocellulose membranes. The
nitrocellulose membranes were incubated in phosphate buffer saline
contained 0.1% Tween-20 overnight at 4 ºC to reduce non-specific
binding and blotted with GLUT4 (anti-rabbit carboxy-terminal GLUT4,
Santa Cruz Biotechnology, California, USA 1:1000) and IRS-1
(anti-rabbit carboxy-terminal IRS-1, Santa Cruz Biotechnology)
antibodies according to the manufacturer's instructions. After
incubation with peroxidase-conjugated secondary antibodies (1:2500,
Santa Cruz Biotechnology), proteins were visualized using enhanced
chemiluminescence. Band intensities were quantified by densitometry.
Statistical analysis The
values presented are expressed as mean¡ÀSD. Statistical analyses were
carried out using ANOVA. P<0.05 was considered to be
statistically significant.
Results
Effect of pioglitazone on body
weight, adipose weight, fasting blood glucose (FBG), fasting blood
insulin (FBI), and insulin sensitivity index (ISI)
STZ rats had significantly elevated
levels of glucose and insulin compared with control rats (P<0.01)
(Table 1). Reduced ISI (1/FBG¡ÁFBI) values revealed that insulin
resistance developed in STZ rats. Treatment with pioglitazone for 28
d significantly lowered fasting glucose and insulin levels, and
improved insulin sensitivity. STZ rats had significantly increased
body weight and mass of celiac fat (P<0.01). There was no
significant difference in body weight and adipose tissue weight
between STZ animals and pioglitazone-treated animals (Table 1).
Effect of pioglitazone on glucose
tolerance in STZ rats The IPGTT results revealed that rats in
the STZ group had significantly impaired glucose tolerance compared
with those in the control group. Surprisingly, the area under the
curve (AUC) indicated that no effect attributable to pioglitazone
administration on glucose tolerance was observed in the pioglitazone-treated
group, although fasting glucose was slightly but significantly
decreased (Table 2).
Effect of pioglitazone on insulin
tolerance in STZ rats The results of the ITT showed that insulin
resistance had developed significantly in STZ rats. Treatment with
pioglitazone for 19 d markedly decreased glucose levels at every
point, including AUC, indicating that insulin sensitivity was
improved. In particular, the data suggested a prolongation in
insulin action (Table 3).
Effect of pioglitazone on
gluconeogenesis in STZ rats Under conditions of insulin
resistance, gluconeogenesis from non-glucose substrates was always
elevated in STZ rats. Our results showed that pioglitazone
significantly reduced glucose production from alanine, which was
assessed by a percentage increase in blood glucose levels at 60 min
(Table 4).
Effect of pioglitazone on lipid
profiles in serum, liver, and muscle The changes in lipid
contents in these three groups (Table 5) indicated that the serum
TG, TC, and FFA levels were all elevated significantly in the
untreated STZ group, whereas HDL-C decreased. Pioglitazone treatment
decreased TG, TC, and FFA and increased HDL-C levels significantly.
Elevated liver TG, TC, FFA levels and muscle TG were observed in STZ
rats and were normalized by piogli-tazone treatment. These findings
may indicate that tissue lipid levels are closely associated with
insulin sensi-tivity. The TC/HDL-C ratio is considered to be a
predictor of cardiovascular disease (CVD). TC/HDL-C values suggested
that pioglitazone diminished the potential risk of CVD by correcting
dyslipidemia.
Effect of pioglitazone on GLUT4
content in skeletal muscle membrane membrane GLUT4 protein
content was decreased by 68.7% in skeletal muscles from untreated
STZ rats compared with control rats (Figure 1). Pioglitazone
treatment increased GLUT4 protein levels significantly.
Effect of pioglitazone on IRS-1
expression in liver and skeletal muscle statistically
significant (A, 26.7%; B, 28.6%) decreases in IRS-1 proteins were
observed in untreated STZ rats compared with control rats (Figure
2). Pioglitazone treatment for 28 d increased IRS-1 protein
expression in liver and skeletal muscle significantly.
Discussion
Insulin resistance is a
characteristic feature of type 2 diabetes and other
pathophysiological states in humans. Therefore, amelioration of
insulin sensitivity is an important therapeutic goal. Diet plays an
important role in the develop-ment of insulin resistance and type 2
diabetes. An excessive intake of fat and sugar can lead to obesity
and diabetes[11]. Previous studies have shown that high
fat (59%) feeding for 24 d leads to significant insulin resistance.
Euglycemic clamp revealed that glucose disposal rate (GDR) decreased
by 50%; however, hyperglycemia was not observed[12].
Pascoe and Storlien reported that low-dose STZ (45 mg/kg body wt)
administration to neonatal rats caused no change in glucose and
insulin levels after 8 weeks; however, hyperglycemia and insulin
resistance developed after high-fat feeding for 1 week[13].
In the present study, we injected STZ at low doses to Wistar rats to
induce light damage of islet cells, leading to glucose intolerance.
On this basis, a high sucrose-fat diet was followed to induce
obesity. Our previous study reported that glucose infusion rate (GIR)
decreased significantly in model rats compared with control rats
(15.1¡À4.8 vs 27.3¡À2.9 mg/kg per min, P<0.01)[14].
This model is similar to the genetically insulin-resistant obese
Zucker rats, which are characterized by a range of metabolic
abnormalities including severe hyperinsulinemia, dyslipidemia and
adipocyte hypertrophy; however, they possess normal blood glucose
levels[15]. The precise mechanisms responsible for this
defect remain unknown.
Major progress has been made in
understanding the mechanisms that causally underlie the metabolic
actions of TZDs. Although the exact mechanisms remain uncertain, it
has been widely reported that TZDs activate PPAR-¦Ã and stimulate
adipocyte differentiation, which might modulate the glucose
metabolism of other tissues.
In the present study, we
investigated the effects of a particular TZD, pioglitazone, on
insulin resistance induced by low-dose STZ and a high sucrose-fat
diet in Wistar rats. The model rats showed significant elevation in
fasting blood glucose and insulin levels, as well as in circulating
TG, TC, and FFA levels. Pioglitazone treatment markedly normalized
serum lipid contents and decreased lipid storage in
insulin-sensitive tissues. In our previous study, a strong
correlation was found between circulating triglycerides and in
vivo insulin resistance as assessed by the
hyperinsulinemic-euglycemic clamp. Evidence from other studies also
suggests an important role for tissue lipid levels in insulin action[16].
It was, therefore, suggested that regulating lipid metabolism might
play an important role in the insulin sensitizing effect of
pioglitazone.
Studies in vitro have shown
that a major effect of TZDs is the inhibition of gluconeogenesis in
isolated hepatocytes[17]. Our gluconeogenesis test
revealed that pioglitazone significantly inhibited the impaired
gluconeogenesis in STZ rats.
Our study also shed some light on
the potential molecular mechanisms underlying amelioration of
insulin resistance. We focused on the insulin sensitive tissues,
that is, liver and skeletal muscle. It has been reported that
glucose transport decreases in the skeletal muscle of high-fat or
high-sucrose induced insulin-resistant rats, and that GLUT4 mRNA and
protein expression also decrease[18]. In
insulin-deficient type 1 diabetic rats, GLUT4 expression also
decreased[19]. Our results are consistent with these
reports and show a 68.7% decrement in skeletal GLUT4 protein content
in STZ rats compared with control rats, although we did not measure
plasmid membrane GLUT4 content during insulin stimulation, which
reflects GLUT4 translocation. Although it is unlikely that changes
in GLUT4 content entirely explain the insulin sensitizing effects of
pioglitazone treatment because of various defects in the
intracellular insulin-signaling pathway, GLUT4 is contributing to a
certain extent.
IRS-1 is a well-described insulin
receptor substrate that, after tyrosine phosphorylation, is
associated with and activates PI3-kinase, and plays an important
role in insulin signaling[20]. Our results showed a
marked deficiency in IRS-1 protein content in untreated STZ rats,
with a significant improvement towards normal levels after
pioglitazone treatment. pioglitazone induced increases in GLUT4 and
IRS-1 protein expression, which were associated with improvements in
glucose transport and insulin signaling pathways, and this may
explain the effects of pioglitazone in improving insulin resistance
in the model animals.
In the present study, pioglitazone
treatment improved insulin sensitivity as assessed by both ISI and
ITT; however, pioglitazone had no effect on glucose intolerance in
STZ rats. Glucose intolerance pre-existed prior to high sucrose-fat
diet feeding, which means that the impaired glucose tolerance was
originally caused by STZ administration, and developed gradually
with feeding. The reasons for this remain uncertain and require
further investigation, particularly the effects on ¦Â-cell function.
In conclusion, our studies showed a
marked state of insulin resistance and obesity in STZ rats that is
associated with various defects in glucose and lipid metabolism,
including the insulin signaling-glucose transport pathway. Despite
the persistence of obesity in these animals, pioglitazone treatment
led to an apparent improvement in overall insulin sensitivity by
ameliorating dyslipidemia, hyperinsulinemia, and gluconeogenesis, as
well as affecting glucose transport and insulin signaling.
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