The Action And Mechanism Of Glucagon-like Peptide-1 And Its Analogue On Gluconeogenesis On HepG2 Cell-the Signal Transduction Pathway Of PI3K

Objective:1. Cultivate human hepatoma cell line HepG2 in vitro.2. Study the action and its mechanism of glucagon-like peptide-1 on gluconeogenesis of HepG2 cell line-the signal transduction pathway of PI3K.3. Providing a primary theoretical basis for the clinical therapy of GLP-1 and its analogue, so as to improve glucose metabolism effectively and pointedly.Method:1. Cultivate HepG2 cell line in vitro, then do morphological observation through microscope.2. Divide the cultivated HepG2 cell line randomly into four groups, and 5 samples in every group:Control group, insulin(INS)(10nmol/L)group, GLP-1 (10nmol/L)group, and Exendin-4(10nmol/L) group. Give every group intervention for 0,4,8,16,24 hours respectively. Use western blot to measure the protein expression of PEPCK, and chose the best intervene time for the next experiment.3. Divide the cultivated HepG2 cell line randomly into nine groups:①Control group,②INS(10nmol/L)group,③GLP-1(10nmol/L)group,④Exendin-4(10nmol/L) group,⑤GLP-1(10nmol/L)+INS(10nmol/L)group,⑥Exendin-4(10nmol/L)+INS (10nmol/L) group,⑦LY294002(15μmol/L) group,⑧GLP-1(10nmol/L)+LY294002(15μmol/L) group,⑨Exendin-4(10nmol/L)+LY294002 (15μmol/L)group, and give these groups certain concentration drug intervention for 8h. Measure the PEPCK protein expression with western blot.Results:1. The HepG2 cells were well subcultured.2. Compared with the Control group, the HepG2 cells in other groups grew well, and the cells form remain intact after drug intervention for 24h.3. Measured the PEPCK protein expression quantity of different time in each group. We found in the Control group,the PEPCK protein expression on 4h was more than that on Oh,and after 16h intervention,the PEPCK protein expression curve leveled off. In the groups INS,GLP-1 and Exendin-4,the PEPCK protein expression on 8h was less than that on 4h significantly,and after 8h, the PEPCK protein expression curve leveled off.So we chose 8h as the best intervene time.4. The result of western blot indicated that INS,GLP-1,Exendin-4 can decrease the PEPCK protein expression. This effects of GLP-1,Exendin-4 and INS were cumulative. And the PI3K inhibitor LY294002 can partly block this restrain action.Conclusion:1. This study confirmed GLP-1 and its analogue Exendin-4 may act on HepG2 cells, and decrease the protein expression of PEPCK,a key enzyme of gluconeogenesis.2. To the restrain effect on PEPCK, GLP-1 and its analogue Exendin-4 can overly with INS.3. GLP-1 and its analogue decrease PEPCK protein expression, and PI3K inhibitor LY294002 can partly inhibit this effect. PEPCK is a key enzyme of gluconeogenesis, FoxOl is an important transcription factor of key enzymes in gluconeogenesis. So we think the mechanism of the restrain gluconeogenesis action of GLP-1 and its analogue may be like this:they combine with their receptors, activate PI3K/Akt pathway, then FoxOl is phosphorylated,and the p-FoxOl is removed out of cell nucleus with losing activity. Then the expression quantity of the key enzymes of gluconeogenesis are decreased.