The Effects And Mechanism Of Glucagon-like Peptide-1 Action In Rat Skeletal Muscle-PI3K Pathway

Objective:1.Cultivate rat skeletal muscle cells in vitro.2.Study on the effects and mechanism of glucagon-like peptide-1 action in cultured rat skeletal muscle cell- the signal transduction pathway of PI3K.3.Providing theoretical basis for safety and validity of the GLP-1 related medicine to the clinical application.Method:1.Cultivate rat skeletal muscle cells in vitro,then do morphological observation through inverted microscope.2.Experiment was divided into three steps:The first step:Check whether the effect of GLP-1 and INS to survive quantity of skeletal muscle cells were cumulative through MTT.The second step:Verify signal transduction pathway of the GLP-1 on glycogen synthase of rat skeletal muscle cells- PI3K pathway through Immunohistochemistry. Experiment was divided into six groups:control group,GLP-1(1nmol/l) intervention group,GLP-1(1nmol/l) + LY294002(15μmol/l) group,INS(1nmol/l) intervention group,GLP-1(1nmol/l) + INS(1nmol/l) intervention group.The third step:Verify signal transduction pathway of the GLP-1 on glycogen synthase of rat skeletal muscle cells- PI3K pathway through Western blot examination.Experiment was divided into four groups mainly:control group,GLP-1 (1nmol/l) intervention group,GLP-1(1nmol/l) + LY294002(15μmol/l) group, LY294002(15μmol/l) intervention group.3.Measure the glycogen synthase(GS) and phospho-glycogen synthase kinase-3α/β(pGSK-3α/β) distribution and expression through immunohistochemistry.4.Measure GS and pGSK-3α/βexpression through western blot.Results:1.Rat skeletal muscle cells were well subcultured.2.Both GLP-1 and INS can induce skeletal muscle cell growth increased,the effect of GLP-1 and INS on rat skeletal muscle cell growth was cumulative.OD values of GLP-1 group,INS group,GLP-1+INS Group were significantly higher than the Ctrl group(P＜0.05),which showed that GLP-1+INS applications increase the number of cell survival,the effect of GLP-1 and INS on rat skeletal muscle cell growth was cumulative.3.Immunocytochemistry and western blot both show that compared with the GLP-1 group,expression of GS in GLP-1+PI3K inhibitor group significantly reduced (P＜0.05),which showed that the PI3K inhibitor partly blocked effect of GLP-1 increase the expression of GS in skeletal muscle cells.GLP-1 can induce the increase the GS expression in skeletal muscle cells,the increase of glycogen synthesis in rat skeletal muscle ceils induced by GLP-1 was relevant with the PI3K pathway.4.Given GLP-1 stimulation,western blot results showed that the 15min group and the 30min group showed clear expression of positive particles(pGSK-3α/β),and compared with the 15min group,the 1h group,the 2h group,the 8h group have significant differences(P＜0.05),which showed that after the GLP-1 stimulation of skeletal muscle cells,the intracellular PI3K activated,caused the inactivation of GSK3,that generate inactivated pGSK-3α/β.Conclusion:This study confirmed in vitro that GLP-1 may act directly on rat skeletal muscle cells and induce glycogen synthesis of skeletal muscle cells through increasing the activity of GS in skeletal muscle cells,Reduced the activity of GSK-3,that is,to generate inactive pGSK-3α/β,thereby increasing the non-phosphorylated GS in signaling downstream,the non-phosphorylated GS is the active form of GS.Given PI3K inhibitor,the glycogen synthase decreased significantly,show that GLP-1-induced glycogen synthesis increase partly through the PI3K pathway.