The Mechanism And Action Of Glucagon-like Peptide-1 On G6Pase Of Gluconeogenesis Of HepG2Cells

Objective:1. Cultivate human hepatic tumor cells -HepG2 cell line in vitro.2. Study the mechanism and action of glucagon-like peptide-1 (GLP-1)on glucone-ogenesis of HepG2 cells -the signal transduction pathway of phosphatidylinositol-3-kinase(PI3K).3. Providing a primary theoretical basis for the clinical therapy of GLP-1 related such as effective fall of blood glucose and improvement of insulin resistense.Method:1.Cultivate HepG2 cell line in vitro, then do morphological observation through inverted microscope.2. Divided the cultivated HepG2 cells randomly into four groups:①Control group②Insulin (10nmol/l) group,③GLP-1 (10nmol/l) group,④Exenatide(10nmol/L) group. Gave them intervention respectively in 4,8,24 hours. Used Western blot to measure the expressions of glucose-6-phosphatase(G6Pase)and choiced the best time of intervention(8 hour).3. Divided the cultivated HepG2 cells randomly into nine groups:①Control group②Insulin(10nmol/l) group,③GLP-1(10nmol/l) group,④Exenatide(10nmol/L)group,⑤LY294002(15umol/L)group,⑥GLP-1(10nmol//L)+Insulin(10nmol/L)group,⑦Exenatide(10nmol/L)+Insulin(10nmol/L)group,⑧GLP-1(10nmol/L)+LY294002(15μmol/L)group,⑨Exenatide(10nmol/L)+LY294002(15μmol/L)group.and gave them intervention respectively in 8 hours.Used Western blot to measure the expressions of G6Pase.4. Divided the cultivated HepG2 cells randomly into three groups:①Control group (5.6mmol/LGlucose),②Glucose(10mmol/L)group,③lucose (20mmol/L)group. and gave them intervention respectively in 8 hours(8h).Used Western blot to measure the expressions of G6Pase.Results:1. The HepG2 cells were well subcultured.2.The results of western blot indicated that Insulin.. GLP-1 and Exenatide can decrease the expression of G6Pase,the best effect is 8h. So, the next experiment chosed 8h as the interventional time. 3. The results of western blot indicated that Insulin,GLP-1 and Exenatide can decrease the expression of G6Pase; The effects of GLP-1,Exenatide and Insulin on the G6Pase of human hepatocyte were cumulative. The phosphatidylinositol 3 kinase inhibitor Ly294002 partly blocked the effects that GLP-1,Exenatideon the expression of G6Pase.4. The high concentrations of glucose could increase the expression of G6Pase,and increase the gluconeogenesis of human hepatocyte.Conclusion:This study confirmed in vitro that GLP-1,Exenatide may act directly on human heaPatocyte and decrease the expression of G6pase. By contraries, the highconcentra-tions of glucose could increase the expression of G6pase. The phosphatidylinositol-3-kinase(PI3K) inhibitor LY294002 could block the effects of GLP-1 on human hepatocyte. It indicate that the effect of GLP-1 and Exenatide on G6Pase of human hepatocyte was partly through the pathway of PI3K. The high concentrations of glucose could increase the expression of G6Pase,and increase the gluconeogenesis of human hepatocyte.