Physiological, Morphological and molecular biological studies of the effect of Glucagon-like peptide-1 and exenatide in the diabetic rat pancreas

Abstract

Diabetes mellitus (DM) is a major health problem currently affecting over 225 million people worldwide. It is often described as a major metabolic disorder, which can result
in numerous long-term complications including retinopathy, nephropathy, neuropathy and cardiomyopathy. DM is due to a deficiency of insulin or insulin resistance. Of the
225 million diabetic patients, around 5-10% suffer from type 1 DM (T1DM) and the remaining 90-95% suffer from type 2 diabetes (T2DM). T1DM is due to insulin deficiency whereas T2DM is due to either a reduction in insulin secretion or insulin resistance. Patients with both T1DM and T2DM normally require insulin and hypoglycaemic drugs, respectively. Changes in life style habits, regular exercise and healthy diets can also help to control blood glucose in T2DM patients. Mediators that can help to increase the health of pancreatic islets to synthesize and secrete insulin will be of tremendous benefit to diabetic patients. This study investigated the beneficial effects of incretins, substances such as glucagon-like peptide-1 (GLP-1) and its synthetic agonist, exenatide on the diabetic rat pancreas compared to healthy, agematched
controls. These incretins exert their beneficial effects by repairing the pancreatic islets. Thus, increasing pancreatic beta (β) cell mass and in turn it will help
to synthesize and secrete insulin into the circulation. The rationale of this study was to find out how these two incretins can improve insulin secretion both in vivo and in vitro employing the rat model of T1DM following injection with streptozotocin (STZ). The project employed six groups of rats, with three groups serving as age-matched, healthy
controls and the other three groups rendered diabetic. One set of rats from each group was untreated while the rats from the other four groups were given either GLP-1(50
nmol/kg body weight) or exenatide (1 μg/kg body weight) over 10 weeks. The project measured body weight, levels of blood glucose and insulin. The plasma levels of liver
and kidney markers were also determined. The in vitro study measured insulin secretion from pancreatic fragments, the distribution of insulin- and glucagon-positive cells in
pancreatic islets, granules, co-localization of different peptides in the islets, biochemical, and molecular biological changes, which may occur in the pancreas
during the experimental period.

For the in vivo study, the results have shown mild gain in body weight and no change in blood glucose levels in both treated and untreated age-matched normal control rats.
Furthermore, the results show a significant reduction in blood glucose levels in diabetic rats treated with either GLP-1 or exenatide, but the beneficial effect was more
pronounced following GLP-1 treatment. The results also show no changes in glucose handling between normal treated or normal untreated rats following blood glucose tolerance test (GTT). However, in diabetic rats, the results show that the GTT reveals a better glucose tolerance in these diabetic animals treated with either GLP-1 or exenatide
but the effect was more significant with GLP-1 compared with untreated diabetic rats. The present study shows that diabetic rats secreted significantly less insulin in the blood than normal healthy rats and a significant increase in serum insulin was detected in both normal and diabetic rats treated with either GLP-1 or exenatide compared to untreated controls. The results also show significant reductions in the liver enzymes, aspartate transferase and alanine transferase in the diabetic rats. A similar beneficial effect on kidney function was obtained owing to a small reduction in blood urea nitrogen, serum
creatinine and serum uric acids in both normal and diabetic rats treated with either GLP-1 or exenatide. In the lipid profile study, the results show a mild reduction in
serum cholesterol and a marked reduction in serum triglyceride in both normal and diabetic rats treated with either GLP-1 or exenatide.

The results from the in vitro study show that either GLP-1 or exenatide can evoke marked dose-dependent release (secretion) of insulin from pancreatic tissue fragments
of normal and diabetic rats, indicating that there is a clear role for either GLP-1 or exenatide in inducing insulin secretion.

In this study, an attempt was also made to investigate both the number and distribution of endocrine cells in the control and diabetic rat pancreas using immnohistochemistry.
The results show a significant increase in the number of cells containing either insulin or GLP-1 in both normal and diabetic treated rats. However, in the case of exenatide,
catalase and glutathione reductase-positive cells were only significantly increased in diabetic rats, but the increase was not significant in normal rats treated with either GLP-
1 or exenatide. These results show that the significant increase in number of catalase and glutathione-positive cells in diabetic rats treated with either GLP-1 or exenatide reveal the beneficial antioxidant effect of both GLP-1 and exenatide in treatment of
oxidative stress, which usually occurs in DM. On the other hand, there was a significant decrease in glucagon-positive cells in both normal and diabetic rats treated with either
GLP-1 or exenatide.

The immunohistochemical and immunofluorescent studies also revealed that insulinpositive cells were distributed both in the central and peripheral portions of the islets of
Langerhans in normal pancreas. In contrast, glucagon-positive cells were located in the peripheral part of the islets of Langerhans. After the onset of diabetes, the number of insulin-positive cells was reduced significantly. In contrast, the number of glucagonpositive cells increased significantly with abnormal pattern of distribution compared to normal pancreas. The pattern of distribution of both GLP-1 and exenatide has indicated co-localization not only with insulin, but also with glucagon. Furthermore, catalase and glutathione reductase-positive cells were distributed homogenously all over the islet of Langerhans with no specific co-localization with specific type of endocrine cell.

In the gene expression study, the results show significant increases in the levels of mRNA of pancreatic duodenal hoeobox-1, heat shock protein-70, glutathione peroxidase, insulin receptor and glucagon like peptide-1 receptor in both normal and diabetic rats treated with either GLP-1 or exenatide. However, the increase was not significant in mRNA gene expression of either insulin receptor or glucagon like peptide-1 receptor in normal rats treated with GLP-1. On the other hand, the gene expression results show that glucagon mRNA level was significantly decreased in both
normal and diabetic rats treated with either GLP-1 or exenatide.

In conclusion, the results of this study have clearly demonstrated that both GLP-1 and exenatide have marked beneficial effects on pancreatic islet cells, especially β- and α- cells, which produce insulin and glucagon, respectively. The two incretins seem to repair the diabetic pancreas, which in turn secretes more insulin and less glucagon.