Roles of AMPK, CaMKII and Akt in Contraction-stimulated AS160 Phosphorylation in Isolated Rat Skeletal Muscle

Abstract

Listen

Translate article

Akt substrate of 160kD (AS160), the most distal insulin signaling protein known to be important for insulin-stimulated glucose transport, also becomes phosphorylated in response to contractile activity by skeletal muscle. AMP-activated protein kinase (AMPK) and Akt can phosphorylate AS160, but the contribution of these kinases for AS160 phosphorylation during contraction is unclear. Ca2+/calmodulin-dependent kinase (CaMK)-II has been implicated as a possible trigger for contraction-stimulated glucose transport, but its role in regulating phosphorylation of AS160 is unknown. PURPOSE: To evaluate the roles of three contraction-stimulated kinases (AMPK, CaMKII and Akt) on contraction-stimulated phosphorylation of AS160. METHODS: Isolated rat epitrochlearis muscles were incubated ± inhibitors of contraction-stimulated kinases [wortmannin (inhibitor of phosphatidylinositol 3-kinase which is upstream of Akt), Akti-1/2 (Akt inhibitor), KN93 and AIP2 (CaMKII inhibitors)] prior to and during electrical stimulation (2ms twitch, 2Hz for 20min) or resting control conditions. Phospho-specific antibodies were used to evaluate the contraction-stimulated kinases, and AS160 phosphorylation was assessed using a phospho-Akt substrate (PAS) antibody (PAS-160; PAS immunoreactivity at 160kD). RESULTS: In vitro twitch contraction increased pAMPK, pCaMKII, pGSK3 (an Akt substrate) and PAS-160. Each of the inhibitors reduced contraction-stimulated PAS-160 (wortmannin, P<0.01; Akti-1/2, P<0.01; KN93, P<0.05; AIP2, P=0.06). Wortmannin completely blocked the contraction-stimulated increase in pGSK3 and PAS-160 without altering pAMPK or pCaMKII. Akti-1/2, KN93 and AIP2 each inhibited their respective target kinase, but each also partially reduced phosphorylation of one or two of the other kinases. CONCLUSION: The data using wortmannin suggest that, in isolated rat epitrochlearis undergoing a twitch contraction protocol, phosphatidylinositol 3-kinase activation is essential for the increase in PAS-160, presumably via activation of Akt. In addition, increased pAMPK and pCaMKII are not sufficient for the contraction-induced increase in PAS-160. Supported by NIH Grants R01 AG10026 and R01 DK071771.