Effect Of Zinc On Glucose Metabolism And Its Mechanism In HepG2 Cells With Insulin Resistance

ObjectiveTo investigate the affect of trace element zinc on glucose metabolism and the mechanism in hepatocyte with insulin resistance state,to provide the experimental evidence of zinc on the mechanism of regulating glucose metabolism.MethodsInsulin resistance model was established in human hepatoma cell line HepG2 cells with different concentrations of insulin(10-7,5 × 1 0-7,1 06,5 × 10-6,10-5,5×10-5mol/L)treatment in 24 hours and the established model was evaluated.Consumptions of glucose were tested by glucose oxidase reaction,glycogen contents were detected by glycogen kit after insulin and zinc action,to investigate the effect of zinc in normal cells and insulin resistance cells on glucose metabolism.Insulin resistant HepG2 cells were divided into 6 groups:they were normal control,normal control with insulin,insulin resistance model,insulin resistance model with insulin,insulin resistance model with zinc(100?mol/L or 200?mol/L),insulin resistance model with insulin and zinc(100?pmol/L or 200p.mol/L).The mRNA expression of PEPCK,G-6-Pase,GCK were detected by real-time PCR.The protein expression of Akt/PKB,GSK,FoxO1 were tested by western blot.ResultsThe glucose consumptions of cells decreased after different concentrations(10-7,5×10-7,10-6,5×10-6,10-5,5×10-5 mol/L)of 24h insulin action while glucose consumptions was the least with 10-6Mol/L insulin(P<0.05).Therefore concentration of 10-6mol/L was chosen to build resistance model.Insulin stimulation increased the consumption of glucose significantly in normal cells and the model cells but to a lesser extent,indicating decreased sensitivity to insulin stimulation in model cells.Low concentrations of zinc(20?50?mol/L)increased glucose consumption slightly but the differences were not statistically significant(P>0.05).Higher concentrations(200?mol/L)increased glucose consumption in both normal and model cells in the absence of insulin(P<0.05).Zinc concentration of 20?mol/L group didn't increase the consumption of glucose in insulin stimulation groups.Concentration of 50,100,200?mol/L groups enhanced the effective of insulin,and 100,200?mol/L groups acted stronger than 50?mol/L group in normal cells,the differences were statistically significant(P<0.05).Compared with non insulin stimulated groups,glycogen contents increased in insulin stimulated groups(P<0.05).Without insulin stimulation,compared with normal cells,glycogen content in model cells and Zn50?mol/L groups did not change significantly(P>0.05),glycogen content in 100?mol/L and 200?mol/L zinc treatment groups increased,the differences were statistically significant(P<0.05).With insulin stimulation,glycogen contents in model cells decreased compared with the normal group(P<0.05),glycogen contents in 50?mol/L and 100?mol/L zinc treated groups did not change(P>0.05),200?mol/L zinc treatment made glycogen content increased,the difference was statistically significant(P<0.05).Insulin stimulation increased GCK mRNA expression(P<0.05),but there were no significant differences between zinc groups and model cells(P>0.05),PEPCK and G-6-Pase mRNA expression in insulin resistant cells increased while zinc made PEPCK and G-6-Pase mRNA expression decreased(P>0.05).Zinc didn't affect protein expressions of unphosphorylated Akt,GSK,FoxO1(P>0.05).Compared with normal cells,Akt phosphorylation level in resistanse model was significantly reduced,(P<0.05).Protein expressions of pAkt increased in 100?mol/L,200?mol/L zinc treatment groups,pAtk/Atk also increased,(P<0.05).Compared with non insulin stimulated groups,insulin stimulation enhanced pGSK expression(P<0.05).Expressions of pGSK in 100?mol/L and 200?mol/L zinc treatment groups increased compared with model group,pGSK/GSK ratio also increased(P<0.05).Compared with normal cells,expression of pFoxO1 in resistance cells decreased and increased in 100?mol/L,200?mol/L dose groups(P<0.05).pFoxO1/FoxO1 ratio increased in zinc 200?mol/L group,the differences were statistically significant(P<0.05).ConclusionsWe used a high concentration of insulin(10?mol/L)to build HepG2 insulin resistance model cells with 24 hours treatment..Zinc can promote glycogen synthesis in singal treatment,and have a Synergistic effect with insulin,which enhanced insulin action and improved glucose metabolism in resistant cells?Zinc may regulate glucose metabolism in following mechanisms:zinc can inhibit gluconeogenesis process together with insulin,by affecting mRNA level of key enzyme glucose metabolism and phosphorylation level in PI3K pathway.