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

Mol Endocrinol. 2008 May;22(5):1105-12. doi: 10.1210/me.2007-0448. Epub 2008 Feb 14.

Abstract

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 alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from AMPK alpha2 knockout (KO), AMPK gamma3 KO, and respective wild-type (WT) littermates (C57BL/6) were incubated in the presence of 5-aminoimidazole-4-carboxamide-1-beta-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 (Ser235/236), and 4E-BP1 (Thr37/46) (P < 0.01) in WT, AMPK alpha2 KO, and AMPK gamma3 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 alpha2 or gamma3 AMPK KO mice, indicating functional alpha2 and gamma3 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 (Ser235/236), and 4E-BP1 (Thr37/46). In conclusion, functional alpha2 and gamma3 AMPK subunits are required for AICAR-induced inhibitory effects on mTOR signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases
  • Animals
  • Female
  • Immunoblotting
  • Insulin / pharmacology
  • Mice
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / metabolism*
  • Muscle, Skeletal / metabolism*
  • Phosphorylation / drug effects
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Signal Transduction / genetics
  • Signal Transduction / physiology*
  • TOR Serine-Threonine Kinases

Substances

  • Insulin
  • Multienzyme Complexes
  • Protein Subunits
  • Protein Kinases
  • TOR Serine-Threonine Kinases
  • mTOR protein, mouse
  • Prkag3 protein, mouse
  • Protein-Serine-Threonine Kinases
  • AMP-Activated Protein Kinases