Distinct Signals Regulate AS160 Phosphorylation in Response to Insulin, AICAR, and Contraction in Mouse Skeletal Muscle

Abstract

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 wortmannin 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.

General aspects of muscle glucose uptake

“…This increase has been implicated in GLUT4 translocation and glucose transport (Jessen andGoodyear 2005, Kurth-Kraczek et al 1999). In addition, calcium stimulates glucose transport in skeletal muscle by pathways, independent of contraction (Youn et al 1991).…”

“…Therefore, there are some signaling molecules related to GLUT4 translocation that are activated by both insulin and muscle contraction, and possibly the most important are the Akt substrate of 160 kDa (AS160) and TBC1D1. AS160 is a Rab GTPase-activating protein (GAP) that modulates GLUT4 trafficking in insulin-sensitive 3T3-L1 adipocytes and L6 myoblasts (Kramer et al 2006). Under basal conditions, AS160 exists primarily in an unphosphorylated state and retains GLUT4 vesicles intracellular.…”

Role of 5′AMP-activated protein kinase in skeletal muscle

“…25 Moreover, when we measured phosphorylation of Akt substrate of 160 kDa (AS160), we found it to correlate with the activation pattern of a2b2g1 but not to that of a2b2g3 AMPK. Together with the observations in mouse skeletal muscle that contraction-mediated AS160 phosphorylation is dependent on complexes of AMPK containing the a2-subunit but not the g3-subunit, 26,27 it is likely that a2b2g1 AMPK may be mediating contraction and exercise effects on AS160.…”

“…We and others have shown, using different transgenic animal models, that a2-AMPK is necessary for full AS160 phosphorylation during contractions but not during insulin stimulation in vitro. 26,27 Thus, from these data, it seems possible that AS160 is a common effector molecule for both insulin-and contraction-mediated effects on glucose uptake, and that AMPK is likely to play a key role in contraction-induced AS160 phosphorylation. Therefore, one hypothesis could be that during exercise AS160 is phosphorylated and that this phosphorylation is sustained in the hours after the initial exercise bout, so that when insulin is applied, the effect on glucose uptake is enhanced.…”

Thrifty Tbc1d1 and Tbc1d4 proteins link signalling and membrane trafficking pathways

“…In some muscle types, contraction leads to increased AMPK phosphorylation and activation, and this in turn leads to increased GLUT4 translocation. However, this may not be the case in all muscle types, and more complex patterns of regulation involving interplay between Akt phosphorylation, AMPK phosphorylation and calcium-dependent regulation take place [11,12]. Tbc1d1 with its Akt, AMPK and calmodulin-binding domains is therefore an attractive candidate molecule to act as a point of convergence between insulin action, exercise and metabolic stress pathways, but this role has yet to be established.…”