Streptozotocin | Diabetes Mellitus

Table of Contents
Introduction

Materials And Methods

Care and management of animals

Induction of experimental diabet...

Determination of Body Weight and...

Biochemical estimations

Assay for triglycerides

Assay for Total Cholesterol

Assay for HDLC

Low density lipoprotein - choles...

Statistical Analysis

Results

Changes in weight

Changes in the blood glucose lev...

Discussion

Abstract
This study assessed the changes in serum lipid profiles of experimentally-induced diabetic Wistar
rats with view to elucidate the effects STZ induced diabetes on the serum levels of cholesterol
and triglycerides of Wistar rats. Twenty adult Wistar rats were randomly assigned into two
groups (A and B) of ten rats each. Group A was the control, while Group B was the STZ treated
group. The body weight, blood glucose level and serum lipid profiles were monitored in all the
animals for four weeks before the commencement of the experiment and throughout the
experimental period. Diabetes mellitus was experimentally induced in groups B rats by daily
intra-peritoneal administration of multiple doses of 40mg/kg streptozotocin dissolved in 0.1M
sodium citrate buffer for 5 consecutive days. The control group was given equivalent volume of
citrate buffer. The animals were monitored for four weeks after streptozotocin administration.
The data obtained were analyzed using descriptive and inferential statistics. The result revealed a
significant (P < 0.05) increase in the serum level of total cholesterol, triglyceride, low-density
lipoprotein cholesterol, and very low-density lipoprotein cholesterol of diabetic rats when
compared with the control rats while a significant decrease in the high-density lipoprotein
cholesterol was obtained. The study revealed that induction of diabetes in rats using STZ result
in development of hyperlipidemia in these rats.

Introduction
Diabetes mellitus is a chronic metabolic disorder characterized by a high blood glucose
concentration caused by insulin deficiency, often combined with insulin resistance. Diabetes
mellitus is a major cause of disability and hospitalization and it results in significant financial
burden 1 . By the year 2010, the total number of people worldwide with diabetes mellitus is
projected to reach 239 million. Region with greatest interest are Asia and Africa, where diabetes
rates could rise to 2-3 folds than the present 2 .

Diabetes mellitus is associated with an increased risk of thrombotic, atherosclerotic and

cardiovascular disease. About 70- 80% of deaths in diabetic patients are due to vascular disease
3
. Hyperglycemia, the primary clinical manifestation of diabetes, is thought to contribute to
diabetic complications by altering vascular cellular metabolism, vascular matrix molecules and
circulating lipoproteins 3 . Hyperglycemia increases diacylglycerol levels and activates protein
kinase C activity in the aorta of streptozotocin - induced diabetic rats 4 and dogs 5 . Thickening of
the basement membranes in renal glomeruli and peripheral capillaries has been observed in STZ-
induced diabetic rats 6 and hyperlipidemia is a feature of drug induced diabetes in rats 7 and
rabbits 8,9 .

Streptozotocin (STZ) has long been used as a drug of choice to induce diabetics type II in
various animal models. This well-established model is characterized by insulin deficiency
associated with insulin resistance 10 . It was reported that a single intravenous injection of STZ
could cause increased plasma glucose levels, decrease in body weight and 17% mortality in rats
10
.

Hyperlipidemia is a metabolic complication of both clinical and experimental diabetes 11 . Low-

density lipoprotein in diabetic patients leads to abnormal metabolism and is associated with
increase in very low-density lipoprotein (VLDL) secretion and impaired VLDL catabolism.
Ultimately this leads to atherosclerotic plaque 12 . A number of known factors for coronary artery
disease such as hypertension, obesity and dyslipidemia are more common in diabetics than in the
general population. The World Health Organization (WHO) predicts that the number of cases
worldwide for diabetes, as at now is 171 million and it will touch 366 million or more by the
year 2030 13 . Patients with DM are more likely to develop microvascular and macrovascular
complications than the non diabetic population 14 . Dyslipidemia is a frequent complication of
DM and is characterized by low levels of high-density lipoprotein-cholesterol (HDL-C) and high
levels of low density lipoprotein cholesterol (LDLC) and triglyceride (TG). The specific aim of
this study is to characterize the progression of STZ-induced diabetes on the lipid profiles of
Wistar rats

Materials And Methods

Care and management of animals
Twenty healthy adult male Wistar rats (Rattus norvegicus), weighing between 150 and 250g were
used for the experiment. The rats were bred in the animal holding of department of Anatomy and
Cell Biology Obafemi Awolowo University Ile Ife; these animals were kept in individual cages
under natural light and dark cycles at room temperature. They were maintained on standard rat
pellet (Ladokun feeds, Ibadan, Nigeria) and water given ad libitum. The animals were randomly
assigned into two groups A and B of ten rats each. Group A was the control, while group B was
the experimentally-induced diabetic group of rats. There was a pre-experimental period of four
weeks during which the body weight, blood glucose level and serum lipid profiles were
monitored in the animals before the commencement of the experiment. The animal care
conformed to the Guide for the care and use of Laboratory Animals published by the US
National Institutes of Health (NIH Publication No 85-2 3, 1996)

Induction of experimental diabetics

Diabetes mellitus was induced in group B animals by intraperitoneal administration of multiple
low doses of streptozotocin (Sigma, St. Louis, USA) (40mg/kg body weight) dissolved in freshly
prepared 0.1M sodium citrate buffer pH 6.3 for five consecutive days while the animals in group
A were given equivalent volume of the citrate buffer intraperitoneally. The rats were fasted
overnight before STZ administration.

Determination of Body Weight and Blood Glucose Level

The body weights of the animals were measured using a top loader weighing balance. Blood
sample was obtained from the tail vein of the animals and their fasting blood glucose level was
determined in mmol/L using a digital glucometer (Accu-chek Advantage, Roche Diagnostic,
Germany). The animals were fasted for a period of 16 hours before their blood glucose level was
measured.
Biochemical estimations
The serum levels of triglyceride (TGL), total cholesterol (TC) and high-density lipoprotein-
cholesterol (HDLC) were determined spectrophotometrically, using enzymatic colorimetric assay
kits (Randox, Northern Ireland) while low-density lipoprotein cholesterol (LDLC), very low-
density lipoprotein cholesterol (VLDLC) and antiartherogenic index (AAI) were calculated.
Animals were fasted for 12-16 hours before blood samples were obtained. About two milliliters
of blood was collected from the tail vein of each rat into an ice-cold centrifuge tubes. The blood
samples were centrifuged in a Denley BS400 centrifuge (England) at 5000 R.P.M for 5-minutes.
The supernatant (serum) collected was assayed for the serum levels of TGL, TC and HDL-C
using the Randox Biochemical kits while LDL-C and VLDL-C were calculated.

Assay for triglycerides

The serum level of TGL was determined by the method of Treitz 15 . 1000 l of the reagent was
added to 10l each of the sample and standard. This was incubated for 10 minutes at 20-25 0 C
and the absorbance of the sample (A sample) and standard (A standard) was measured against the
reagent blank within 30 minutes.

Assay for Total Cholesterol

The serum level of TC was determined after enzymatic hydrolysis and oxidation of the sample as
described by Richmond 16 and Roeschlau et al.,17 . 1000 l of the reagent was added to 10l each
of the sample and standard. This was incubated for 10 minutes at 20-25 C and the absorbance
of the sample (A sample) and standard (A standard) was measured against the reagent blank within 30
minutes.

Assay for HDLC

Low-density lipoproteins (LDL and VLDL) and chylomicron fractions in the sample were
precipitated quantitatively by the addition of phosphotungstic acid in the presence of magnesium
ions. The mixture was allowed to stand for 10 minutes at room temperature centrifuged for 10
minutes at 4000rpm. The supernatant represented the HDLC fraction. The cholesterol
concentration in the HDL fraction, which remains in the supernatant, was determined.

Low density lipoprotein - cholesterol

The concentration of LDL cholesterol was calculated mmol/L using Friedewalds equation 18 as
stated below.

Very low density lipoprotein - cholesterol

The concentration of VLDL cholesterol was calculated mmol/L using Friedewalds equation 18 as
stated below.

Antiatherogenic Index (AAI)

The antiatherogenic index was calculated according to the method of Guido and Joseph 19 . AAI
was calculated from total cholesterol and HDL cholesterol using the formula below. The values
were expressed as a percentage

Statistical Analysis
The data were analysed using descriptive and inferential statistics. All values are presented as
mean standard error of mean (SEM) for ten rats in each of the two group of rats. The
significance of difference in the means of all parameters reported for the two groups of animals
was determined using paired sample student t test and a p value of < 0.05 (two tailed) was
considered as significant

Results
Changes in weight
Prior to STZ administration, there was no significant difference in the average weights of the
control and diabetic group of rats. By the end of the first week after diabetes mellitus was
experimentally induced, the weights of diabetic rats were significantly reduced despite the
increase in food and fluid intake in these animals. This weight loss continued for four week after
STZ administration (fig.1). At the end of the experimental period, there was a significant (p <
0.05) decrease in the body weights of diabetic rats (171.28 5.143) when compared to the
control (204.28 8.307) (table 1).

Fig. 1 Weekly Changes in the Body Weights of Control and Diabetic Rats

TABLE 1: Changes in the Body Weight and Blood Glucose Level of Control and Diabetic
Groups of Rats after STZ Administration

Values are given as mean SEM for ten rats in each group.

a, b within column signifies that means with different letters differs significantly at P < 0.05 (two
tailed T-test) while means with the same letters does not differ significantly at P <0.05 (two tailed
T-test)

Changes in the blood glucose level

Prior to STZ administration, the fasting blood glucose level did not differ significantly (p < 0.05)
between the control and diabetic groups of rats. The blood glucose level gradually increased
during the five days period of STZ administration. One week after administration of STZ, the
blood glucose level was significantly (p < 0.05) higher in groups B rats. The blood glucose level
of these rats remains elevated over a period of four weeks (fig. 2). Control rats treated with
citrate buffer maintains a normal blood glucose level throughout the period of experiment. At the
end of the experiment, there was a significant difference (p < 0.05) in the blood glucose level of
groups A and B rats (table 1, fig. 2)
Fig. 2 Weekly Changes in the Blood Glucose Level of Control and Diabetic Rats

Tables 2 and 3 illustrate the effects of streptozotocin on the levels of total cholesterol,
triglycerides, HDLC, LDLC, VLDLC and AAI in the serum of experimentally induced diabetic
rats. The levels of total cholesterol, triglycerides LDL-C and VLDL-C were significantly (p <
0.05) increased in diabetic rats whereas the level of HDL-C and the percentage of AAI (ratio of
HDL to total cholesterol) were significantly (p < 0.05) reduced in these rats when compared to
the control normal rats.

Table 2: Effects of STZ on the Serum Total Cholesterol, Triglycerides and High Density
Lipoprotein Cholesterol (HDLC) of Experimentally-Induced Diabetic Rats

Table 3: Effects of STZ on the Serum Low Density Lipoprotein Cholesterol (LDLC) Very Low
Density Lipoprotein Cholesterol (VLDLC) and Antiartherogenic index (AAI) of Experimentally-
Induced Diabetic Rats

Discussion
When rats are injected with streptozotocin, they provide an animal model of insulin-dependent
diabetes mellitus. In this model, severe hyperglycaemia appears throughout the period of
induction with a partial deficiency in insulin 20 . In the present study, the streptozotocin induced
diabetic rats showed significantly higher levels of fasting blood glucose levels and lower body
weight compared to normal control rats. This was consistent with early reports 21,22 . Diabetes
mellitus is a complex metabolic disease caused by impairment of insulin signaling, pathways,
and the defect usually results from pancreatic -cell deficiency and/or a deficiency of insulin 23
This disease causes many chronic complications such as vascular disease, retinopathy,
neuropathy, kidney disease and heart disease. Cardiovascular disease is one of the major causes
of death in diabetic patients. Diabetes mellitus is associated with profound alteration in the serum
lipid and lipoprotein profile with an increased risk in coronary heart disease 24 . Hyperlipidemia
is a recognized complication of Diabetes mellitus characterized by elevated levels of cholesterol,
triglycerides and phospholipids; and changes in lipoprotein composition 25

The present study supports the results of other investigators 26 who proved significant increase in
total cholesterol, triglyceride, LDL-Cholesterol, VLDL-Cholesterol and significant decrease of
HDL-Cholesterol in STZ-induced diabetic rats. Mathe 27 reported that hypercholesterolemia in
STZ-induced diabetic rat's results from increased intestinal absorption and synthesis of
cholesterol. The low levels of HDL-Cholesterol in diabetic rats negatively modulate endothelial
function through a lack of oxidation inhibition and a concomitant over expression of adhesive
molecules (vascular cell adhesive molecules, VCAM, and inter cellular adhesion molecules
ICAM) 28 . Natarajan and Nadler 29 suggested that monocyte adhesion to endothelial cells as well
as excessive proliferation and migration of vascular smooth muscle cells (VSMC) are key events
in the development of atherosclerosis in diabetes 29 . These processes are mainly mediated by
growth factors, inflammatory cytokines, chemokines and related factors released by various cells
in the vessel wall 29 . The mechanism of action of these factors are however not clear. These
growth factors and cytokines acting on VSMC and endothelial cells can activate phospholipases
with the release of lipids such arachidonic and lipoleic acids 29 . These lipids can be further
metabolized by several pathways including the lipoxygenase (LO) pathway. These oxidative
pathways may lead to the formation of free radicals and lipid peroxides. LO products have been
shown to be associated with oxidant stress that may lead to atherosclerosis, hypertension and
related diabetic complications 29 . There is increasing evidence that lipid peroxidation plays an
important role in the premature development of atherosclerosis 30,31 . Abnormally high levels of
free radicals, lipid peroxidation and simultaneous decline in antioxidant defense mechanism can
lead to damage of cellular organelles and enzymes. Elevated levels of lipid peroxidation in
circulation of diabetic rats are one of the characteristic features of chronic diabetes 32 .

In conclusion, our findings suggested that administration of multiple low doses of streptozotocin
had a potential hyperglycemic activity in rats. In addition to increasing serum glucose level, it
also increases serum levels of total cholesterol, triglycerides, and LDL cholesterol which are
characteristic features of hyperlipidemia.

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This article was last modified on Mon, 19 Oct 09 18:10:08 -0500