Adipogenesis is a tightly regulated cellular differentiation process and the understanding of mechanism and manipulation of adipogenesis might contribute to a reduction in the burden of obesity and diabetes.Multiple transcription factors and signaling pathways are involved in the regulation of adipogenesis.Here we report that in preadipocytes,AMP-activated protein kinase activator,5-aminoimidazole-4-carboxamide ribonucleoside(AICAR)can activate AMPK to increase GSK3β phosphorylation,leading to inactivation of GSK3β.The AICAR stimulated GSK3β phosphorylation is mediated by increased AKT activity by AICAR.By contrast,with adipocyte differentiation,AICAR effect on GSK3β phosphorylation is blocked while AKT still regulates GSK3 activity via phosphorylating it,suggesting distinct underlying mechanism of regulating GSK3 activity in preadipocytes and adipocytes,respectively.GSK3 activity is also found to be implicated in adipogenesis process as specific GSK3 inhibitor blocked adipogenesis as indicated by decreased lipid droplet accumulation in 3T3-L1 cells and decreased expression of master adipogenic factors.Notably,GSK3 activity is demonstrated to regulate insulin action and glucose metabolism in matured adipocytes.Different GSK3 inhibitors show distinct effect on insulin action and glucose metabolism.LiCl stimulation can improve both basal glucose uptake and insulin induced glucose uptake.However,SB216763 only increases basal glucose uptake.AICAR stimulation only influences basal glucose uptake but not insulin stimulated glucose uptake.Taken together,these data indicate that AICAR effect on GSK3 activity is mediated by AICAR stimulated AKT activity.GSK3 activity negatively regulates adipocyte differentiation,glucose metabolism and insulin action.