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Role of Adenosine 5'-Monophosphate-Activated Protein Kinase Subunits in Skeletal Muscle Mammalian Target of Rapamycin Signaling

Department of Molecular Medicine and Surgery (A.S.D., Y.C.L., J.R.Z.), Section of Integrative Physiology, Karolinska Institutet, S-171 77 Stockholm, Sweden; Section of Human Physiology (J.T.T., J.F.P.W.), Department of Exercise and Sport Sciences, Copenhagen Muscle Research Centre, University of Copenhagen, DK-2100 Copenhagen, Denmark; Institute Cochin (B.V.), Université Paris Descartes, Centre National de la Recherche Scientifique (Unité Mixte de Recherche 8104), and Institut National de la Santé et de la Recherche Médicale (B.V.), Unité 567, 75016 Paris, France

Address all correspondence and requests for reprints to: Juleen R Zierath, Department of Molecular Medicine and Surgery, Section for Integrative Physiology, Karolinska Institutet, von Eulers väg 4, 4th Floor, S-171 77 Stockholm, Sweden. E-mail: Juleen.Zierath{at}ki.se.

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 2 and regulatory 3 subunits. Extensor digitorum longus muscle from AMPK 2 knockout (KO), AMPK 3 KO, and respective wild-type (WT) littermates (C57BL/6) were incubated in the presence of 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR), insulin, or AICAR plus insulin. Phosphorylation of AMPK, Akt, and mTOR-associated signaling proteins were assessed. Insulin increased Akt Ser⁴⁷³ phosphorylation (P < 0.01), irrespective of genotype or presence of AICAR. AICAR increased phosphorylation of AMPK Thr¹⁷² (P < 0.01) in WT but not KO mice. Insulin stimulation increased phosphorylation of S6K1 (Thr³⁸⁹), ribosomal protein S6 (Ser^235/236), and 4E-BP1 (Thr^37/46) (P < 0.01) in WT, AMPK 2 KO, and AMPK 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 2 or 3 AMPK KO mice, indicating functional 2 and 3 subunits of AMPK are required for the reduction in mTOR signaling. AICAR alone was without effect on basal phosphorylation of S6K1 (Thr³⁸⁹), ribosomal protein S6 (Ser^235/236), and 4E-BP1 (Thr^37/46). In conclusion, functional 2 and 3 AMPK subunits are required for AICAR-induced inhibitory effects on mTOR signaling.