The Effects Of GLP-1 To The Activity Of Glycogen Synthase And The Study With MAPK Cell Signalling Pathway In Liver Cells

Objectives1. Cultivate human liver L02 cells in vitro.2. Study on the signal transduction pathway of glucagon-like peptide -1 (GLP-1) on glycogen synthase in cultured human liver L02 cells-pathway of MAPK.3. Providing a deeper theoretical basis for the insulin-like effects in extrapancreatic tissues mediated by GLP-1, and the clinical application safety and validity of the GLP-1 related medicine.Methods1. Cultivate human liver L02 cells in vitro, then do morphological observation through inverted microscope.2. Experiment was divided into two parts: (1) PartⅠ:Check whether the effect of GLP-1 and INS to survive quantity of human liver L02 cells were cumulative through MTT. (2) PartⅡ:Verify signal transduction pathway of the GLP-1 on glycogen synthase of human liver L02 cells - MAPK pathway through Western blot examination. Experiment groups was divided into:Ctrl group, GLP-1(10nmol/l) group, GLP-1(10nmol/l)+PD (15μmol/l) group, PD group, INS(10nmol/l) group, GLP-l+INS(10nmol/l) group.3. Measure the phosphoglycogen synthase(pGS) and glycogen synthase (GS) expression through western blot.4. Determine the activity of glycogen synthase by measure the OD ratio of p-GS/GS through western blot.Results1. human liver cells were well subcultured.2. The OD values of GLP-1 group, INS group, GLP-1+INS group were significantly higher than the Ctrl group (P <0.05), and OD value of GLP-1+INS group was significantly higher than GLP-1 group and INS group.3. western blot results showed that, compared with the Ctrl group, the phospho-glycogen synthase (p-GS) expressions of the GLP-1 group and INS group decreased significantly. There are significant differences between GLP-1 group and GLP-1+PD group. Compare with the Ctrl group,GLP-1 group,INS group, the phospho-glycogen synthase (p-GS) expression of the GLP-1+ INS group decreased significantly.4. western blot results showed that, compared with the Ctrl group, the glycogen synthase (GS) expressions of the GLP-1 group and INS group increased significantly. There are significant differences between GLP-1 group and GLP-1+PD group,which showed that the MAPK inhibitor PD partly blocked the effect of GLP-1 increase the expression of GS in human liver cells. Compare with the Ctrl group,GLP-1 grop,INS group, the glycogen synthase (GS) expressions of the GLP-1+ INS group increased significantly.5. The ratio of p-GS/GS represent the activity of GS, the greater the ratio is, the lower the activity of GS. The result showed that:,compared to the Ctrl group,the activity of the GLP-1 group,INSgroup,GLP-1+INS group increased significantly (P<0.05).Compare to the GLP-1+INS group,the activity of either the GLP-1 group or the INS group is lower. (P<0.05). There is no significant difference between the PD98059 group and the Ctrl group (P>0.05). Compare to the GLP-1 group,the activity of GS in GLP-1+ PD98059 group decreased significantly. (P<0.05)Conclusion:The study showed that both GLP-1 and INS can induce human liver cell growth increased, the effect was cumulative. This study in vitro confirmed that INS and GLP-1 may act directly on human liver cells and induce the transformation from the inactived pGS to actived GS, up-regulate the expression of the glycogen synthesis, increase the activity of GS in human liver cellls to promote glycogen synthesis. MAPK inhibitor can inhibite the up-regulation effect to expression and the activity of Glycogen synthase mediated by GLP-1 in human liver cells, indicate that MAPK pathway can be important to the up-regulation with expression and activity of the Glycogen synthase mediated by GLP-1 in human liver cells.