Insulin and contraction increase GLUT4 translocation in skeletal muscle via distinct signaling mechanisms. Akt substrate of 160 kDa (AS160) mediates insulin-stimulated GLUT4 translocation in L6 myotubes, presumably through activation of Akt. Using in vivo, in vitro, and in situ methods, insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR all increased AS160 phosphorylation in mouse skeletal muscle. Insulin-stimulated AS160 phosphorylation was fully blunted by

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... nnin in vitro and in Akt2 knockout (KO) mice in vivo. In contrast, contraction-stimulated AS160 phosphorylation was only partially decreased by wortmannin and unaffected in Akt2 KO mice, suggesting additional regulatory mechanisms. To determine if AMPK mediates AS160 signaling, we used AMPK ␣2-inactive (␣2i) transgenic mice. AICARstimulated AS160 phosphorylation was fully inhibited, whereas contraction-stimulated AS160 phosphorylation was partially reduced in the AMPK ␣2i transgenic mice. Combined AMPK ␣2 and Akt inhibition by wortmannin treatment of AMPK ␣2 transgenic mice did not fully ablate contraction-stimulated AS160 phosphorylation. Maximal insulin, together with either AICAR or contraction, increased AS160 phosphorylation in an additive manner. In conclusion, AS160 may be a point of convergence linking insulin, contraction, and AICAR signaling. While Akt and AMPK ␣2 activities are essential for AS160 phosphorylation by insulin and AICAR, respectively, neither kinase is indispensable for the entire effects of contraction on AS160 phosphorylation.