Studies On The Effects And Mechanisms Of Beganatide On Experimental Diabetes

Type 2 diabetes mellitus (T2DM) s a progressive disease characterized by worsening of multiple abnormalities and loss of glycaemic control over time. A worldwide survey reported that diabetes mellitus is affecting nearly 10% of the population every year. T2DM constitutes 90% to 95% of all cases of diabetes. The disease course is primarily characterised by worsening of insulin resistance and a decline inβcell function. T2DM leads to microvascular and macrovascular complications, such as retinopathy, nephropathy, and atherosclerosis, including cardiovascular disease, stroke, and peripheral vascular disease. The treatment of diabetes mellitus in clinical practice has been confined to use of oral hypoglycemic agents and insulin. Although they lower glucose, do not show this progressive loss ofβcell function and mass, and their use is also associated with hypoglycaemia and weight gain. Thus, the need for additional glucose-lowerying therapies that can haltβcell deterioration without contributing to weight gain continues.GLP-1 is a naturally occurring 30-amino acid peptide synthesised in intestinal endocrine L cells. GLP-1 mediates glucose homeostasis through stimulation of glucose-dependent insulin secretion, biosynthesis of insulin and inhibition of glucagon secretion. These effects have potential clinical value in type 2 diabetes. However, because native GLP-1 is rapidly degraded to its inactive form by dipeptidyl peptidase-IV, it has a short half-life in vivo. Exenatide is a 39-amino acid peptide originally derived from the venom of the Gila monster lizard, and has a longer half-life than human GLP-1, twice-daily with diabetes. Beganatide belongs in a class of drugs called Exenatide because these drugs mimic the structure of Exenatide. Beganatide behaves like a long-acting version of the exenatide-that is, it acts as a long-acting GLP-1 agonist.In the present study, we utilized improvement solid phase synthesis to synthesize Beganatide, then we used the dexamethasone(Dex) induction IR mice model, the alloxan induction diabetes mice model, induced T2MD rats by STZ and high glucose and lipid feeding, and rat insulin-producing RINm5F cells to assesse Beganatide biological activity. The study is carried by following several parts:In the first part, Beganatide was designed and prepared with Fmoc-solid phase peptide synthetic strategy, purified by RP-HPLC and identified with Q-TOF-MS and Fourier infrared spectroscopic analysis. The IR and MS results indicated that the synthetic substance was the target compound.In the second part, Dex-induced IR mice and alloxan-induced diabetes mellitus mice were used in order to investigate the effects of Beganatid on the levels of blood glucose, insulin, glucose tolerance and TC, TG, FFA, MDA, SOD contents in serum. Pancreas samples were stained with Aldehyde Fuchsin and examined. Rusults, Beganatide ameliorated the glucose tolerance and reduced level of insulin in serum in IR mice(p<0.01). Meanwhile, Beganatide could reduced TC, TG, FFA level in IR mice(p<0.05). Treatment with Beganatide markedly decreased FPG (p<0.05) and accelerated the exudation of insulin in diabetic mice(p<0.05); Beganatide significantly blocked the increase of MDA production and increased SOD activity in serum as compared with the diabetic group (p<0.05). Histopathological showed that Beganatide restored the damage of pancreas tissues in mice with diabetes mellitus.In the third part, the rats with T2DM were induced by intraperitoneal injections low-dose ST(25mg·kg-1, twice)and high sucrose-fat diet. All rats were followed up to assess the body weight and plasma glucose each week. Eight weeks later,the oral glueose tolerance test (OGTT) was performed in all animals,the changes of FINS, HbA1c, TG, TC, FFA, HDL concentrations were measured. The morphological change in panereatic was examined by HE and AF staining of paraffin sections. The expression of insulin in pancreatic island was detected by immunohistochemistry. Results, after 8 weeks of Beganatide treatments reduced plasma glucose concentrations and HbA1c, increased plasma insulin level(p<0.05), and reduced body weight more than T2DM group with high sucrose-fat diet. Furthermore, Beganatide improved glucose tolerance by attenuating insulin response to oral glucose loading. Meanwhile, Beganatide in diabetic rats promoted a decrease in serum TG, TC and FFA contents, with a increase in serum HDL level(p<0.05). Beganatide also attenuated pathologic alterations in pancreatic islet. Insulin expressions in the pancreatic islet were significantly increased in Beganatide group than in T2DM group.In the fourth part, Beganatide was studied on both growth and function of RINm5F cells, an insulin-producing cell line derived from rat insulinoma tissue. we first investigated the effect of Beganatide on RINm5F cellular mulitiplication capacity by the MTT colorimetric assay. We further used the ELISA kit to investigate the effects of Beganatide on secretion of insulin in RINm5F cells and stimulating insulin secretion under different concentration glucose condition(4.5mM and 20mM). Then, for inducing RINm5F cell apoptosis, we pretreated RINm5F cells with Glu(12.5mM) and Pal(0.1mM) for 48 hour before experiment, we studied the cellular death apoptosis by MTT assay. At the last, Expression of Bcl-2, Bax, Sirt1 were determined by RT-PCR and Real-time PCR. The protein expression of Sirt1 was measured by Western-blot. Base on our previous experiments, preincubation with Beganatide dose-dependent increased the viability of RINm5F cells. Glucose-stimulated insulin output was significantly increased in the Beganatide groups. The addition of 5mM to 20mM glucose, markedly potentiated insulin release. Beganatide inhibited of apoptosis and increased cell survival in RINm5F cells with apoptosis by Pal-Glu. The results demonstrated that, treatment of the RINm5F cells with Beganatide increased the expression of Bcl-2 by about 1.57 fold relative to the apoptosis group. While decrease the expression of Bax by about 1 fold relative to the apoptosis group. Furthermore, the over-expression of Sirt1 in the RINm5F cells exposed to Beganatide increased to 1.8 fold that of the apoptosis. Subsequent studies demonstrated that Beganatide could increase the level of Sirt1 under normal condition, and Beganatide can reserve the Sirt1 protein level under apoptosis state which the blank group the Sirt1 protein level has been decreased drastically.The results of the present study, we arrived at the following concludes:1)We can synthesize Beganatide by improvement the solid-phase synthesis, the structure had been proved consistent with the theoretical design.2)Beganatide may process a significantly improved the insulin resistance, and reduced serum lipid level in IR mice.3)Beganatide can protective effect onβcell damage and the mechanism may be related with hypoglycemic effect and reduction of oxidative stress in alloxan-induced diabetic mice.4)Beganatide can significantly reduce blood glucose lever, control body weight, reduce lipid levels effectively and minimizing the injury of pancreatic islet in the T2DM rats.5)Beganatide plays a role in multiplication and inhibiting apoptosis on RINm5F cell, it also improved the insulin secretion. Beganatide can protect islet cells, this may be related to increased expression of Bcl-2 and decreased expression of Bax.6)The changes of Sirt1 expression take an important action in apoptosis of isletβcell, regulation of Sirt1 expression may be a general mechanism of Beganatide in antiapoptosis in this way.