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Jing Ai, Ning
Wang, Mei Yang, Yong-Chun Zhang, Bao-Feng Yang,
Department of Pharmacology, Harbin
Medical University, Bio-Pharmaceutical Key Laboratory of
Heilongjiang Province, Harbin 150086, Heilongjiang Province, China
Zhi-Min Du, State Base for Drug Clinical Trial, Harbin
Medical University, Harbin 150086, Heilongjiang Province, China
Supported by the Key Found of the Technological Office of
Heilongjiang Province, No. 20010101001-00, the National Natural
Science Foundation of China, No. 30371647, Foundation of Educational
Office of Heilongjiang Province, No. 10531094
Co-correspondents: Jing Ai
Correspondence to: Professor Bao-Feng Yang, Department of
Pharmacology, Harbin Medical University, Harbin 150086, Heilongjiang
Province, China. yangbf@ems.hrbmu.edu.cn
Telephone: +86-451-86671354
Fax: +86-451-86669482
Received: 2004-06-28
Accepted: 2004-08-05
Abstract
Aim: To
establish a simplified and reliable animal model of insulin
resistance with low cost in Wistar rats.
Methods: Wistar
rats were treated with a high fat emulsion by ig for 10 d. Changes
of the diets, drinking and body weight were monitored every day and
insulin resistance was evaluated by hyperinsulinemic-euglycemic
clamp techniques and short insulin tolerance test using capillary
blood glucose. Morphologic changes of liver, fat, skeletal muscles,
and pancreatic islets were assessed under light microscope. mRNA
expressions of GLUT2 and a-glucosidase in small intestine
epithelium, GLUT4 in skeletal muscles and Kir6.2 in beta cell of
islets were determined by in situ hybridization.
Results: KITT
was smaller in treated animals (4.5±0.9) than in
untreated control Wistar rats (6.8±1.5), and so
was glucose injection rate. Both adipocyte hypertrophy and large
pancreatic islets were seen in high fat fed rats, but no changes of
skeletal muscles and livers were observed. mRNA levels of GLUT2, a-glucosidase
in small intestinal epithelium and Kir6.2 mRNA in beta cells of
islets increased, whereas that of GLUT4 in skeletal muscles
decreased in high fat fed group compared with normal control group.
Conclusion: An
insulin resistance animal model in Wistar rats is established by ig
special fat emulsion.
ã 2005
The WJG Press and Elsevier Inc. All rights reserved.
Key words: Wistar rat; Insulin resistance; Model
Ai J, Wang N, Yang M, Du ZM, Zhang YC, Yang BF. Development of
Wistar rat model of insulin resistance. World J Gastroenterol 2005; 11(24): 3675-3679
http://www.wjgnet.com/1007-9327/11/3675.asp
INTRODUCTION
Epidemiological studies suggest that insulin resistance is not only
an independent risk factor that induces type 2 diabetic mellitus,
but also the common cause of hypertension, coronary heart disease,
and cerebral vessel disease, thus the key to cure and prevent heart
and cerebral vessel disease. It is of paramount importance to
establish an insulin resistance animal model, in order to have a
better understanding of the pathological process of insulin
resistance and to develop therapeutic drugs. Several insulin
resistance animal models are available, including hereditary ob/ob
mice and SD or Wistar rat models developed by either injecting
low-dose dexamethasone (2 mg/d)
into abdominal cavity or feeding food rich in fructose and sucrose[1-7].
The major disadvantages of all these animal models are the long
experimental cycles (4-30 wk) and the less relevant pathologic
status of insulin resistance induced by a sole factor as opposed to
the natural multi-factoral process. The present study was to
establish an insulin resistance animal model using Wistar rats with
more clinically relevant pathophysiological characteristics of
insulin resistance based on glucose utility of the body and
alterations of various cellular and molecular events related to
insulin resistance.
MATERIALS AND METHODS
Animals and reagents
Male Wistar rats weighing 180-220 g were obtained from Department of
Animals, Harbin Medical University. Insulin was purchased from the
First Biochemical Drug Company of Shanghai. The kit for in situ
hybridization of small-intestine GLUT2, a-glucosidase,
Kir6.2 in islet beta cells and GLUT4 in skeletal muscles was
purchased from BOSD Biotech, Wuhan, China.
Preparation of fat emulsion
A constant volume of 100 mL fat emulsion containing 20 g lard, 1 g
thyreostat, 5 g cholesterol, 1 g sodium glutamate, 5 g sucrose and 5
g saccharose, 20 mL Tween 80, 30 mL propylene glycol was prepared by
adding distilled water and stored at 4 ℃.
Animal treatment
Twenty-four Wistar rats were randomly divided into normal control
group and high fat emulsion group, 12/group. Rats in normal control
group received common water, rats in high fat emulsion group
received fat emulsion for 10 d.
Insulin resistance sensitivity assay by short insulin
tolerance test using capillary blood glucose[8]
Rats were weighed and placed into mouse cage after fasting
overnight. Blood sugar in rats was detected six times after ip
insulin (0.05 U/kg) using a blood sugar detector. Abscissa indicates
time and ordinate expresses nature logarithm of blood sugar.
Regression coefficient (r) or slope was determined by linear
regression and KITT
was calculated by multiplying r by 100. K value
indicates insulin sensibility with smaller K values for lower
sensibilities.
Hyperinsulinemic-euglycemic clamp experiment
As described previously[9],
food was withdrawn 12 h before the
experiment. The rats were then
anesthetized by ip amobarbital sodium (25 mg/kg) after they had been
weighed. Rats were cannulated in the jugular vein for infusion of
glucose and insulin (dual cannula) and in the carotid artery for
sampling. All cannulae were tunneled subcutaneously, and encased in
silastic tubing (0.08 cm) sutured to the skin. After infusion of
glucose (10%) and insulin (1 IU/mL) from dual cannula (constant
velocity), blood sugar was measured. To keep the blood sugar in a
relatively steady state, the rate of glucose infusion was
continuously adjusted. Glucose injection rate (GIR) was measured
under homeostasis six times during the experiment.
Morphological alterations of rats with insulin resistance
After rats were fasted for 3 h, perirenal fat, gastrocnemius muscle,
liver, and pancreas were dissected. The tissues were then fixed with
4% paraformaldehyde, embedded in paraffin wax, sectioned, and
stained with eosin by sappan wood. Morphological alterations were
examined under a light microscope. The procedure for mRNA levels was
described previously. The positive expression of a-glucosidase
and Kir6.2 showed brown staining signals in villi of small intestine
and in pancreatic islet b-cells.
GLUT2 mRNA was expressed in the incisurae between two villi of small
intestine and in cytoplasm of skeleton muscle. Expression level was
assayed by the degree of color.
Statistical analysis
Data were analyzed by Student's
t-test.
The results were expressed as mean±SD.
RESULTS
Changes of diet, drinking, body weight, and visceral fat in
Wistar rats fed with fat emulsion
Compared with normal control group, the drinking, body weight, and
visceral fat increased markedly 10 d after ig fat emulsion (Table 1,
P<0.05). In contrast, no changes were observed in diet
group.
Table 1 Changes of
diet, drinking, body weight, and visceral fat in Wistar rats fed
with fat emulsion (mean±SD,
n = 12)
| Groups |
n |
BW
(g) |
Diet
(g) |
Drink
(mL) |
Visceral
fat (g) |
| NC |
12 |
202.9±22.7 |
77.3±6.5 |
152.0±20.0 |
9.2±4.1 |
| FD |
12 |
229.4±23.4a |
81.5±7.4 |
247.0±26.5b |
16.3±5.4a |
aP<0.05,
bP<0.01
vs control. BW: body weight; NC: control group; FD: fat
emulsion group.
Insulin resistance sensitivity assay by short insulin
tolerance test using capillary blood glucose and
hyperinsulinemic-euglycemic clamp experiment in Wistar rats
The results of short insulin tolerance test using capillary
blood glucose revealed that KITT
decreased to 4.50.9 in rats treated with fat emulsion (ig, 10 d) (P<0.05,
n = 12). Our hyperinsulinemic-euglycemic clamp test indicated
that the GIR for keeping homeostasis of blood sugar in rats of high
fat emulsion group was decreased in fat treated group (Figure 1A).
Figure 1
(PDF) Results of insulin resistance sensitivity assay in
hyperinsulinemic-euglycemic clamp test (A) and changes of
glucose induced by insulin injection (B). NC: control diet
group; FD: fat emulsion diet group; GIR: glucose injection rate. aP<0.05,
bP<0.01
vs NC.
Morphological alterations of skeletal muscle, liver, fat, and
pancreas islets in rats with insulin resistance
No morphological changes were observed in skeletal muscle and liver,
but in larger adipocytes and pancreas islets when the tissues were
stained with HE in high fat emulsion group compared to control group
(Figure 2).
Figure 2 Morphological
observation of fat and pancreas islets stained HE in rats with
insulin resistance (60×). A:
Fat cells in control group; B:
fat cells in high fat emulsion group; C:
pancreas islets in control group; D:
pancreas islets in high fat emulsion group.
mRNA expression assay of GLUT2 and a-glucosidase in small intestinal
epithelium, Kir6.2 mRNA in beta cells of islets and GLUT4 in
skeletal muscles of rats with insulin resistance
Both GLUT2 and a-glucosidase mRNAs in small intestinal
epithelium were increased in fat emulsion group compared to control
group (Figures 3A-D). The number of cells expressing a-glucosidase
mRNA in high fat emulsion group was 506 and 405 in control group (P<0.05,
n = 12). GLUT2 mRNA was diffused in the incisurae between two
villi of small intestine. In the sight of 100 cm2,
the expression area of GLUT2 mRNA in high fat emulsion group was
increased to 3.8±0.6 cm2
from 3.0±0.4 cm2
in control group (P<0.05, n = 12). In cytoplasm of
skeleton muscle of fat emulsion group, the expression of GLUT4 mRNA
was lower, as indicated by the brighter brown color, than in control
group (Figures 3E and F). Kir6.2 mRNA in b-cells showed a tendency
to increase in fat emulsion group because its positive expression
density did not change but the volume increased compared to control
group.
Figure
3 mRNA expression
assay of GLUT2 and a-glucosidase
in small intestinal epithelium and GLUT4 in skeletal muscle in rats
with insulin resistance (30×). A:
GLUT2 in control group; B:
GLUT2 in high fat emulsion group; C:
a-glucosidase
in control group; D:
a-glucosidase
in high fat emulsion group; E: GLUT4 in control group; F:
GLUT4 in high fat emulsion group.
DISCUSSION
In the present study, we developed a new formula of fat emulsion
based on the insulin-resistant animal models established by other
laboratories. The formula for preparing fat emulsion is to combine
high fat diets with fructose and sucrose. Moreover, the fat emulsion
was administered to rats by ig but not by food feeding so as to
control the daily fat intake. The possible imbalance of fat intake
due to decreased appetite as a result of high fat diets was avoided.
Increased drinking accompanying an increase in body weight and
visceral fat was observed though the rats were administered high fat
emulsion for 10 d. This is typical of the clinical phenotype.
In order to clarify if the insulin-resistant
animal model was established in our study, we detected the dynamic
characteristics of blood sugar after insulin injection by short
insulin tolerance test using capillary blood glucose. The result
demonstrated that the KITT
value decreased markedly after ig fat emulsion for 10 d compared to
control group, indicating that the rats are insensitive to exogenous
insulin, i.e. insulin resistance.
Insulin resistance refers to the insensitivity of
tissues (such as skeletal muscle, liver, kidney, and adipose tissue)
to insulin action, i.e. the weaker glucose utilization of body after
insulin action that results in hyperglycemia. The classical test
used to evaluate insulin resistance is hyperinsulinemic-euglycemic
clamp experiment[10,11].
We determined the insulin resistance using
hyperinsulinemic-euglycemic clamp technique that is known to be the
most reliable method for verifying whether or not insulin resistance
is achieved in rats after feeding fat emulsion. According to
negative feedback mechanism, if we want to keep blood sugar in the
basal level by changing injection rate of exogenous glucose, we
should regulate the injection rate of exogenous insulin. The
exogenous GIR is equal to the glucose utilization rate of peripheral
organs because the endogenous glucose production (from liver) could
be completely inhibited when the plasma insulin level is excessively
high. Hence, GIR can be considered as an index for evaluating the
action of insulin on peripheral organs.
Our data showed that the GIR was indeed decreased
in rats treated with high fat emulsion, indicating that insulin
resistance is achieved in fat emulsion group.
Diabetes mellitus is a pathological process
affecting the whole body system. Skeletal muscle, fat, and liver are
considered as the insulin-sensitive tissues[12-16].
Alterations of the functional status of these tissues may result in
insulin resistance of the body. The main manifestation is the
dysfunction of glucose absorption and utilization, metabolism
disturbance of glucose in liver cells and lipo-metabolism
disturbance in adipose cells[15,17].
Several papers have reported increases in adipose cell volume and
number accompanied with redistribution of these cells over the body
in insulin-resistant animals and humans. Nevertheless, it is more
important that adipose cell volume increases when insulin resistance
occurs[14].
Our experiments demonstrated that insulin resistance could be
induced in rats after being treated with high fat emulsion by ig.
However, we still do not know if morphological changes occur as a
result of dysfunction. We therefore carried out histopathological
assay by HE staining of insulin-sensitive tissues, such as fat,
skeletal muscle, and liver. We found no morphological changes in
both skeletal muscle and liver, but adipose cell volume was markedly
enlarged in rats of fat emulsion group. In high fat emulsion group,
pancreatic islets enlarged but no changes in the other part of
pancreas were seen. The morphological changes in both fat cells and
pancreatic islets in high fat emulsion group confirmed the success
of our insulin-resistant animal model.
Glucose is an important substance that keeps the
balance of energy metabolism and life. Glucose is a polarity
molecule that cannot pass the lipid bilayer of cell membrane by free
diffusion. Small intestine and renal tubule can absorb glucose and
other tissues must intake glucose by facilitating diffusion with
glucose transporter proteins in cell membrane. Among the various
glucose transporter proteins, GLUT2 and GLUT4 have been more
intensively studied.
GLUT2 is an important glucose transporter protein
which distributes in hepatocytes, pancreatic islet b-cells,
small intestine, and kidney[18-20].
People pay close attention to GLUT2 because it is related to
pathogenesis of diabetes mellitus. GLUT2 expression in pancreatic
islet b-cells
decreases in BB rats and Zuker obesity rats with autoimmune diabetes
mellitus. Some studies reported that GLUT2 mRNA and protein
expressions are enhanced in the liver of diabetes mellitus rats
induced by streptozotocin, but others found no changes. Studies on
expression of GLUT2 in epithelial cells found that GLUT2 protein
expression in sarcolemma of small intestine increases in both STZ-induced
diabetes mellitus rats and high sugar feeding rats, but no changes
occurred in hyperglycemia rats caused by glucose filling, while
glucose transporter activity increases in these conditions[21-23].
Until now, study on mRNA expression of GLUT2 in small intestine of
insulin-resistant rats has not been reported.
GLUT4 is an insulin reaction protein transporter
which mainly distributes in adipose cells and striated muscles[24].
On the one hand, GLUT4 is the richest protein transporter in both
adipose cells and striated muscles. On the other hand, GLUT4
expression level is coincident with glucose utilization ability of
various types of adipose cells. Almost all the glucose absorbed by
muscles gets converted into glycogen in high insulin condition.
Glucose transport of muscular tissues is very important for the
glucose utility of body because glucose transport is the
rate-limiting step of glucose utility of muscle tissue. To date,
studies have evidenced that not only the protein expression of GLUT4
decreases in insulin-resistant condition, but also the translocation
of GLUT4 from cytoplasm to cell membrane is defected[25].
Based on the above reasons, we measured mRNA
expression of GLUT2 in small intestinal epithelial cells and GLUT4
in gastrocnemius muscle in our insulin-resistant rat model. On the
one hand, we detected mRNA expression of GLUT2 in small intestinal
epithelial cells. On the other hand, we further verified at molecule
level that we successfully established the insulin-resistant animal
model. Our study demonstrated that mRNA expression of GLUT2 in small
intestinal epithelial cells increased in high fat emulsion group
compared with normal control group. In contrast, GLUT4 mRNA
expression decreased. The results reveal that the insulin-resistant
animal model is successfully established.
In order to ensure that sugar can be absorbed in
small intestine, polysaccharide must be divided into monosaccharide
by a-glucose glycosidase of small intestine. In our experiments,
mRNA expression of a-glucose glycosidase in small intestine
increased in fat emulsion group. The mRNA expression of b-cell
Kir6.2 in pancreatic islets did not change in fat emulsion group,
but the total expression level increased because of the enlargement
of pancreatic islet volume.
In conclusion, an insulin-resistant rat model is
established by ig special fat emulsion.
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