Effects Of Zinc On Glucose Consumption And Mechanisms Involved In Normal And Insulin-resistant L6 Myotubes

ObjectiveTo investigate the effects of microelement zinc on glucose consumption and the expression of key target molecules,namely m TOR-S6K1 and Akt-GLUT4/GSK3β,in both normal and insulin resistance L6 myotubes,to provide a further experimental and theoratical evidence of the relationship between zinc and insulin resistance.MethodsL6 cells in logarithmic phase were cultured with DMEM supplemented with5.55mmol/L glucose containing 10% feotal serum,When cells were approximately60%-70% in confluence,they were switched to DMEM supplemented with5.55mmol/L glucose and 5% horse serum to induce differentiation into myotubes,then they were exposed to different doses of palmitic acid for 24 hours,cell viability and glucose consumption of different groups were measured to ensure the adequate dose for establishing insulin resistant model.According to the results above,insulin resistant cells were treated with different doses of zinc(0,10,20,50,100 μmol/L)in the presence or absence of insulin(100nmol/L)for 3h.,cell viability of different groups were determined to ensure the adequate doses of zinc for the subsequent experiment.Then we applied the selected doses of zinc above to test its effect on glucose consumption of normal and insulin resistant L6 myotubes in the medium by glucose oxidase method.In the following studies,the control and PA-treated L6 myotubes were exposed to different doses of zinc(0,10,20,50μmol/L)in the presence or absence of insulin(100nmol/L)for 15 min,the expression of m TOR-S6K1 and Akt-GSK3β were determined by western blotting,and lower dose of zinc(10μmol/L)was chosen for a look at the GLUT4 transmenbrane activity by immumofluorescence method.All in all,the experiments above aimed to elucidate the underlying molecular mechanism of zinc.ResultsThe concentration of 0.4mmol/L was chosen for PA to induce insulin resistance in the following studies.zinc treatment caused enhanced cell viability of insulin-resistant L6 myotubes:zinc at the concentration of 50μM and 100μM can enhance cell viability(P<0.05),50μM zinc and insulin can synergically enhance cell viability(P<0.05).zinc showed upregulated stimulation of glucose consumption in both normal and insulin resistant L6 myotubes.For normal L6 myotubes,zinc concentrations varied from 10μM to 100μM could increase glucose consumption in basal state,while for insulin resistant L6 myotubes,higher doses of zinc(from 20μM to 100μM)could increase glucose consumption in both basal and insulin stimulating state.For the key moleculars of insulin signaling pathway in normal L6 myotubes,zinc at the concentration of 20μM and 50μM could upregulate the phosphorylation of AKTSer473 and GSK3β Ser9 in basal state,while in the insulin-stimulated state,50μM zinc showed the same effect.There existed no significant influence of low dose of zinc(10μM)on the transmenbrame of GLUT4,50μM zinc were stimulatory to the expression of m TOR in insulin-stimulated states,while 20μM zinc were stimulatory to the phosphorylation of m TOR in basal states.There existed no significance influence of zin on S6K1.For insulin resistant L6 myotubes,50μM zinc was found to upregulate the the phosphorylation of AKTSer473 and GSK3β Ser9 in basal state,while in the insulin stimulating state,20μM zinc showed the same effects.There existed no significant influence of low dose of zinc(10μM)on the transmenbrame of GLUT4,50μM zinc was found to inhibite the overxpression of both m TOR and S6K1 in basal state,50μM zinc was also found to inhibite the overxpression of m TOR、p-m TOR、S6K1 and p-S6K1 in insulin stimulating state.ConclusionsZinc exhibited an inhibitory effect on palmite acid induced insulin-resistant L6 myotubes,which may be involved by activating the phosphorylation of AKT/GSK3βand decreasing the phosphorylation of m TOR/S6K1,thus improving the glucose consumption.