A Novel Cell Model Targeted On GPR40 Receptor For Discovery Of New Anti-diabetics & Ameliorating Effects Of TSP On Insulin Resistance Induced By High Fat Diet In C57BL/6J Mice

The incidence rate of obesity, insulin resistance, and diabetes mellitus worldwide is currently at epidemic proportions, which are associated with diet modification and shortage of exercise and physical activity and have been widely recognized as a major health problem affecting many people’s life. Owing to the side effects of marketed oral anti-diabetic drugs and the increasing incidence of failure with oral hypoglycemic agents in clinics, more attention must be paid to searching new safety and effective oral anti-diabetic drugs. The GPR40 (G-protein-coupled receptor 40) has been shown to be a physiologically relevant receptor for medium/long-chain fatty acids. It belongs to a family A G-protein-coupled receptor highly expressed in theβ-cell. Accumulating evidence suggests that GPR40 mediates the majority of the effects of FFA on glucose-stimulated insulin secretion. The oral administration of GPR40 agonists could promote glucose-dependent insulin secretion and significantly reduce blood glucose in mice. So, activation of GPR40 represents a new therapeutic approach to treatment of type 2 diabetes. The agonists of GPR40 are in development all over the world. The aim of this project is to establish a GPR40 signaling pathway targeted cell model, for screening the new class of GPR40 receptor agonists as anti-diabetic candidates.Insulin resistance is now considered as a fundamental aspect of etiology of type 2 diabetes and other pathologic defects such as obesity, cardiovascular diseases and atherosclerosis. Exploring the molecular basis of insulin resistance and searching for potent insulin sensitizer has been the focus of research worldwild. Considering the multifaceted effects of insulin resistance in various tissues, aiming at multi-targets will be a more promising strategy for the prevention or treatment of insulin resistance. Therefore, traditional Chinese medicine recipe represents new approach in the search for alternative anti-insulin-resistance drugs. Tangshunping (TSP) is composed of three effective fractions of Chinese herbal drugs from Jinqijiangtang tablet, which is an oral anti-diabetic traditional Chinese medicine recipe. Ameliorating effects of TSP on insulin resistance induced by high fat diet in C57BL/6J mice are described in the dissertation. First, the hGPR40, hElkl (active domain) and Gal4 (DNA binding domain) were amplified by polymerase chain reaction (PCR). After special restrictive endonuclease action and ligation with proper plasmid, a set of recombined plasmids, Peak13-CD5L-GPR40, pCMV-hElkl-Gal4-tag5A, Peakl2-6xGal4-luci were constructed. Secondly, transient transfect these recombined plasmids into HEK293 cell, and then detects the responsibility and specificity of transfected cell to GPR40 agonists. It indicates that this cell model can response to GW9508 (an artificial synthetic GPR40 agonist) or palmitic acid (an endogenous GPR40 ligand) in a concentration-dependent manner. This novel cell model has been used in screening GPR40 agonists. Further work was going to optimize the cell model for high-throughput drug screening.Part II Ameliorating effects of TSP on insulin resistance induced by high fat diet in C57BL/6J miceAfter feeding a high fat diet (HFD) for 3-4 months, C57BL/6J mice could develop insulin resistance with obesity, dyslipidemia, increased fasting blood glucose level, hyperinsulinemia, accompanied by glucose intolerance and insulin resistance. It has been shown that the metabolic disturbances induced by high fat feeding were similar between animal models and humans. We therefore investigated the effect of TSP on insulin resistance in HF-C57 mice. The results showed TSP could suppress body weight gain, serum cholesterol, serum NO, HOMA-IR (the Homeostasis Model Assessment ratio of insulin resistance) index, while improve impaired glucose and insulin tolerance, reduce serum glucose and insulin. Moreover, TSP increased glucose infusion rate (GIR) and activated liver glycogen syntheses under a high glucose loading in hyperglycemic clamp test. These results suggest that TSP can ameliorate insulin resistance by regulating abnormal glucose and lipid metabolism, reducing serum NO, and increasing insulin sensitivity. Activating AMPKa maybe is responsible for its ameliorating effect on insulin resistance.