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 ₈₉ .

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

Induction of experimental diabetics

Determination of Body Weight and Blood Glucose Level

Biochemical estimations

Assay for triglycerides

The serum level of TGL was determined by the method of Treitz ₁₅ . 1000 μl of the reagent was added to 10μl 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.

Figure 1

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 μl of the reagent was added to 10μl 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.

Figure 2

Assay for HDLC

Low density lipoprotein - cholesterol

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

Figure 3

Very low density lipoprotein - cholesterol

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

Figure 4

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

Figure 5

Statistical Analysis

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).

Figure 6

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

Figure 7

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)

Figure 8

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.

Figure 9

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

Figure 10

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 ₂₁₂₂ . Diabetes mellitus is a complex metabolic disease caused by impairment of insulin signaling, pathways, and the defect usually results from pancreatic β-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 ₃₀₃₁ . 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.