title: Rag GTPases and AMPK/TSC2/Rheb mediate the differential regulation of mTORC1 signaling in response to alcohol and leucine.
abstract: Leucine (Leu) and insulin both stimulate muscle protein synthesis, albeit at least in part via separate signaling pathways. While alcohol (EtOH) suppresses insulin-stimulated protein synthesis in cultured myocytes, its ability to disrupt Leu signaling and Rag GTPase activity has not been determined. Likewise, little is known regarding the interaction of EtOH and Leu on the AMPK/TSC2/Rheb pathway. Treatment of myocytes with EtOH (100 mM) decreased protein synthesis, whereas Leu (2 mM) increased synthesis. In combination, EtOH suppressed the anabolic effect of Leu. The effects of EtOH and Leu were associated with coordinate changes in the phosphorylation state of mTOR, raptor, and their downstream targets 4EBP1 and S6K1. As such, EtOH suppressed the ability of Leu to activate these signaling components. The Rag signaling pathway was activated by Leu but suppressed by EtOH, as evidenced by changes in the interaction of Rag proteins with mTOR and raptor. Overexpression of constitutively active (ca)RagA and caRagC increased mTORC1 activity, as determined by increased S6K1 phosphorylation. Furthermore, the caRagA-caRagC heterodimer blocked the inhibitory effect of EtOH. EtOH and Leu produced differential effects on AMPK signaling. EtOH enhanced AMPK activity, resulting in increased TSC2 (S1387) and eEF2 phosphorylation, whereas Leu had the opposite effect. EtOH also decreased the interaction of Rheb with mTOR, and this was prevented by Leu. Collectively, our results indicate that EtOH inhibits the anabolic effects that Leu has on protein synthesis and mTORC1 activity by modulating both Rag GTPase function and AMPK/TSC2/Rheb signaling.
authors.affiliation: Department of Cellular and Molecular Physiology, Penn State College of Medicine, 500 University Dr., Hershey, PA 17033, USA. lqh10@psu.edu
authors.forename: Ly Q
authors.initials: LQ
authors.lastname: Hong-Brown
authors.forename: C Randell
authors.initials: CR
authors.lastname: Brown
authors.forename: Abid A
authors.initials: AA
authors.lastname: Kazi
authors.forename: Maithili
authors.initials: M
authors.lastname: Navaratnarajah
authors.forename: Charles H
authors.initials: CH
authors.lastname: Lang
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000502:physiology*; D000818:Animals; D002478:Cells, Cultured; D000431:Ethanol / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D007930:Leucine / Q000502:physiology*; D051379:Mice; D020559:Monomeric GTP-Binding Proteins / Q000502:physiology*; D032342:Muscle Cells / Q000187:drug effects / Q000502:physiology; D009479:Neuropeptides / Q000502:physiology*; D055503:Protein Multimerization / Q000502:physiology; D000076205:Ras Homolog Enriched in Brain Protein; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000502:physiology*
chemical_list: C519509:Crtc1 protein, mouse; D009479:Neuropeptides; C430722:RRAGC protein, human; D000076205:Ras Homolog Enriched in Brain Protein; C490213:Rheb protein, mouse; C000624653:TSC2 protein, human; D014157:Transcription Factors; C000624654:Tsc2 protein, mouse; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D000431:Ethanol; D055372:AMP-Activated Protein Kinases; D020559:Monomeric GTP-Binding Proteins; D007930:Leucine