Role of adenosine 5′-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

AMP-activated protein kinase ( AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin ( mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 ( S6K1) and eukaryotic initiation factor 4E-binding protein 1 ( 4E-BP1). AMPK activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha 2 and regulatory gamma 3 subunits. Extensor digitorum longus muscle from AMPK alpha 2 knockout ( KO), AMPK gamma 3 KO, and respective wild-type ( WT) litter-mates ( C57BL/ 6) were incubated in the presence of 5-aminoimidazole-4-carboxamide-1-beta-D-ribo-nucleoside ( AICAR), insulin, or AICAR plus insulin. Phosphorylation of AMPK, Akt, and mTOR-associated signaling proteins were assessed. Insulin increased Akt Ser(473) phosphorylation ( P < 0.01), irrespective of genotype or presence of AICAR. AICAR increased phosphorylation of AMPK Thr(172) ( P < 0.01) in WT but not KO mice. Insulin stimulation increased phosphorylation of S6K1 ( Thr(389)), ribosomal protein S6 ( Ser(235/ 236)), and 4E- BP1 ( Thr(37/ 46)) ( P < 0.01) in WT, AMPK alpha 2 KO, and AMPK alpha 3 KO mice. However, in WT mice, preincubation with AICAR completely inhibited insulin-induced phosphorylation of mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued in extensor digitorum longus muscle from either alpha 2 or gamma 3 AMPK KO mice, indicating functional alpha 2 and gamma 3 subunits of AMPK are required for the reduction in mTOR signaling. AICAR alone was without effect on basal phosphorylation of S6K1 (Thr(389)), ribosomal protein S6 ( Ser(235/ 236)), and 4-EBP1 ( Thr(37/ 46)). In conclusion, functional alpha 2 and gamma 3 AMPK subunits are required for AICAR-induced inhibitory effects on mTOR signaling.