The Effect Of Caveolin-3 On Glucose Metabolism In C2C12 And H9C2 Cells And Effect Of (-)-epicatechin Against H9C2 Cell Death Induced By High Glucose And High Lipid

Part Ⅰ The Effect of Down-Regulation of Caveolin-3 on Glucose Metabolism in Skeletal Muscle C2C12 Cells and Myocardial H9C2 CellsObjective Caveolin-3(CAV3)is a muscle specific protein,which plays an important role in maintaining muscle health and glucose homeostasis.Previous work of the team found that the increased expression of CAV3 in skeletal muscle cells promoted the uptake and utilization of glucose without insulin stimulation,suggesting that CAV3 may participate non-insulin-mediated glucose uptake.Therefore,in this study,we will transfect CAV3 siRNA to down-regulate CAV3 expression and explore the effect of CAV3 on insulin and non-insulin-mediated glucose metabolism.Methods CAV3 siRNA was transfected into skeletal muscle C2C12 cells and myocardial H9C2 cells,and CAV3 protein was down-regulated.The cells were cultured in high glucose medium with or without insulin(where in C2C12 cells use culture medium without fetal bovine serum and H9C2 cells use culture medium containing fetal bovine serum).The glucose assay kit detects cellular glucose uptake,the glycogen synthesis kit detects cellular glycogen synthesis,the Western blotting detects changes in key proteins in glucose uptake and glycogen synthesis.Results1.In the presence of insulin,down-regulation of CAV3 protein expression can significantly reduce glucose uptake within 48h of skeletal muscle C2C12 cells and myocardial H9C2 cells(p<0.01).In terms of signaling molecules that regulate glucose uptake,the down-regulation of CAV3 protein expression significantly reduced the expression of glucose transporter 4(p<0.01)and phosphorylation of protein kinase B(p<0.05)in skeletal muscle C2C12 cells.2.In the absence of insulin,down-regulation of CAV3 protein expression can significantly reduce glucose uptake within 48h of skeletal muscle C2C12 cells and myocardial H9C2 cells(p<0.05).Signal molecules that regulate glucose uptake and glycogen synthesis,the down-regulation of CAV3 protein expression significantly reduced the glucose transporter 4(p<0.01)protein in skeletal muscle C2C12 cells,and significantly reduced expression of glucose transporter 4(p<0.05)and phosphorylated of glycogen synthesis kinase 3β(p<0.01)in myocardial H9C2 cells.Conclusion Down-regulation of CAV3 protein expression reduced glucose uptake and glycogen synthesis in skeletal muscle C2C12 cells and myocardial H9C2 cells with or without insulin,suggesting that CAV3 can not only enhance insulin-mediated glucose uptake by promoting glucose transporter 4 related pathways and can enhance non-insulin-mediated glucose uptake.Part Ⅱ Effect of(-)-Epicatechin Against H9C2 Cell Death Induced by High Glucose and High LipidObjective(-)-Epicatechin(EC)is a flavonoid that has important effects on Antioxidant anti-apoptosis,Glucose homeostasis,and insulin sensitivity.The purpose of this study is to observe the effect of EC against high glucose and high lipid induced H9C2 cell death.Methods H9C2 cells were divided into normal Control group,high glucose and high lipid(HGHL)group,DMSO+HGHL group and EC+HGHL group.The latter groups were treated with DMSO and EC was pretreated for 2h and then incubated with high glucose and high lipid for 24h.Cell viability was detected by CCK-8 method,cell morphology was observed by inverted microscope,cell death and nuclear changes were observed by Hoechst 33258 staining,lactate dehydrogenase(LDH)and superoxide dismutase(SOD)activities,reduced glutathione(GSH)content were determined by chemical colorimetry.Results1.Compared with the Control group,the cell survival rate of the HGHL group and the DMSO+HGHL group was significantly reduced,the cell morphology was abnormal,the number of vacuolated cells was increased,the nuclear were fragmented,the content of reduced GSH and the activity of SOD were significantly reduced(p<0.01),LDH activity increased significantly(p<0.05).2.Compared with the HGHL group,the cell survival rate of the EC+HGHL group was significantly improved(p<0.05),the cell morphology tended to normal,the number of vacuolated cells was reduced,the nuclear structure was complete,and the content of reduced GSH and SOD activity were significantly increased(p<0.05),LDH activity was significantly reduced(p<0.01).Conclusion EC increases the ability of anti-oxidation and scavenging free radicals to maintain the integrity of cell membranes and nucleus,improve cell survival rate,and resist H9C2 cell death induced by high glucose and high lipid.