Study Of Anti-diabetic Drugs Targeted At GLP-1 And The Cognate Receptor & The Features Of Obese MSG Mice And The Reasons For Occurring Hyperglycemia

Glucagon-like peptide-1 (GLP-1) belongs to the incretin family, which is secreted from intestinal L cells after food ingestion and absorption, and works through endocrine, nerve and substrate stimulation to enhance the biosynthesis and secretion of insulin, and restrain the secretion of glucagon. GLP-1 could also inhibit appetite, decrease glucose production, and increase the insulin sensitivity of the peripheral tissues. The most notable advantages of GLP-1 are less hypoglycemia for glucose-dependent hypoglycemic effect, and protection ofβcells from apoptosis and stimulation ofβcells proliferation and neogenesis. However, GLP-1 is quickly degraded by dipeptidyl peptidaseⅣ(DPPⅣ) as soon as secretion, with the half life only about 2 min, which makes it difficult to be used in clinic. Because GLP-1 exerts many effects via binding to its cognate receptor, scientists are striving to search GLP-1 analogs or GLP-1 receptor agonists that could resist the degradation of DPPⅣ. In the present study, we have three objectives:1) Study the DPPⅣand human plasma stability and in vivo hypoglycemic effects of BPI3006, a novel GLP-1 analog; 2) Study the anti-diabetic action and relative mechanisms of E2HSA, a novel long-term GLP-1 receptor agonist; 3) Study the features of Monosodium Glutamate (MSG)-induced mice and the possible reasons for producing hyperglycemia.PartⅠThe anti-diabetic study of novel GLP-1 analog, BPI3006BPI3006 is a novel GLP-1 analog with the second -NHCO- at N terminal replaced by a new chemical entity -CH(CF3)NH- through chemical synthesis. In vitro, BPI3006 possessed excellent DPPⅣresistance but poor human plasma stability. The report gene assay showed that BPI3006 could bind to GLP-1 receptor to stimulate the production of cAMP with the equivalent intensity with exendin-4. In vivo, acute administration of BPI3006 in normal ICR mice could significantly suppress the blood glucose variation following oral glucose loading with good dose-effect relationship, and evidently restrain the fasting blood glucose in spontaneous T2DM KKAy mice. Repeated administration of BPI3006 in KKAy mice twice daily could significantly improve the impaired oral glucose tolerance and hyperinsulinemia, and decrease the body weight gain as well as the food and water consumption. In addition, we also find other peptides, BPI3007 and BPI3008, which all displayed better hypoglycemic effect with long action through the in vivo hypoglycemic effect study.PartⅡThe anti-diabetic study of a novel long-acting GLP-1 receptor agonist, E2HSAE2HSA is a recombinant macromolecular protein obtained by combination of exendin-4 and human serum albumin. E2HSA could bind to GLP-1 receptor to stimulate the production of cAMP, and enhance the expression of insulin gene. E2HSA could not only significantly increase the NIT-1 cell viability, and promote it proliferation, but also protect it from the injury induced by Act D, phorbol ester (PMA) and water soluble cholesterol, and inhibit the apoptosis of NIT-1 cell induced by Act D and water soluble cholesterol, which might be associated with the increase of AKT and PDX-1 protein expression. In vivo, acute administration of E2HSA in normal ICR mice could significantly decrease the blood glucose variation following oral glucose loading with excellent dose-effect relationship, and the action could sustain for at least 4 days. E2HSA could also evidently decrease the food intake and body weight of normal ICR mice, and delay the movement of small intestine after a single dosing, and all the actions could last for 3 days. Repeated injection of E2HSA in alloxan-induced diabetic mice could markedly suppress the fasted and fed blood glucose, and increase the islet and blood insulin level, as well as reduce the consumption of food and water and body weight. In spite of this, repeated injection of E2HSA both in normal ICR mice and alloxan-induced diabetic mice could induce the production of antibody, with the titer of 1:1000000, which significantly influence the hypoglycemic effect of E2HSA.PartⅢStudy of the features of obese MSG mice and the reasons for occurring hyperglycemiaAs a typical model of metabolic syndrome, the main features of MSG mice include central obesity, insulin resistance and the disorder of glucose and lipid metabolism. In our study, we found that some MSG mice showed hyperglycemia, which was similar to the change of blood glucose in the insulin resistance state, whenβcell could secrete insulin in a compensatory manner to keep it in the normal level, to the decompensate state, whenβcell could not secrete more insulin to result in hyperglycemia. Therefore, in the present study we try to find out the factors that may induce hyperglycemia in MSG mice.The biochemical assay showed that the content of SOD and HDL decreased, and the content of TG increased in the pancreas and liver of MSG mice. When compared with the MSG mice, the content of TG in the pancreas and liver of hyperglycemic MSG mice significantly increases. The immunohistochemistry assay suggested that the islets were enlarged in a compensatory manner, the distribution of a cell andβcell and the content of insulin and glucagon were damaged in MSG mice, and the islets were shriveled irregularly with more glucagon and less insulin in the hyperglycemic MSG mice in comparision with MSG mice. The gene expression assay showed that the expression of many genes, such as the genes that relative to the proliferation, apoptosis, endoplasmic reticulum stress, inflammatory injury, and immune regulation ofβcells in the pancreas, the genes of inflammatory factor, immunoregulatory factor, NF-κB and JNK in the liver, and the genes of leptin, inflammatory factor and immunoregulatory factor in the adipose of MSG mice, have changed to varying degrees. When compared with the MSG mice, the decrease of MafA gene expression and the increase of CHOP, IL-6, BAX and BAK gene expression in the pancreas, and the increase of leptin, TNFa gene expression in the adipose of hyperglycemic MSG mice might explain the production of hyperglycemia. The 2 dimensional polyacrylamide gel electrophoresis results showed that the expression of many proteins changed in the liver and pancreas of MSG mice, and the differences of the hyperglycemic MSG mice from the MSG mice might represent the reasons for hyperglycemia, but needing further protein identification.