Hyperactivation of Akt by insulin pretreatment is responsible for the increased AMPKSer-485 phosphorylation in HK-532 cells.
A, HK-532 cells were treated with 20 nm insulin and Akt, and ERK phosphorylation was examined. B, HK-532 cells were incubated overnight without or with 50 nm insulin (Ins) and then treated with 1 mm AICAR for 2 h. Insulin pretreatment significantly increased AICAR induced Akt phosphorylation. *, p < 0.05; #, p < 0.01 by t test. C, the cells were incubated with insulin along with 20 μm LY294002 (LY) or U0126 (U) overnight. The cells were washed and treated with 1 mm AICAR for 2 h. D, inhibiting Akt overactivation during insulin pretreatment by LY294002 prevented AMPKSer-485 hyperphosphorylation (b) and restored AICAR-induced Tau dephosphorylation. (c) but did not affect AMPKThr-172 phosphorylation (a). *, p < 0.05 compared with insulin + LY294002 + AICAR; #, p < 0.05 compared with insulin or insulin + AICAR; v, p < 0.05 compared with control.