title: Increasing leucine concentration stimulates mechanistic target of rapamycin signaling and cell growth in C2C12 skeletal muscle cells.
abstract: Leucine is a key amino acid for initiating translation in muscle cells, but the dose-dependent effects of leucine on intracellular signaling are poorly characterized. This study examined the effect that increasing doses of leucine would have on changes in mechanistic target of rapamycin (mTOR)-mediated signaling, rates of protein synthesis, and cell size in C2C12 cells. We hypothesized that a leucine "threshold" exists, which represents the minimum stimulus required to initiate mTOR signaling in muscle cells. Acute exposure to 1.5, 3.2, 5.0, and 16.1 mM leucine increased phosphorylation of mTOR(Ser2448) (~1.4-fold; P < .04), 4E-BP1 (Thr37/46) (~1.9-fold; P < .001), and rpS6(Ser235/6) (~2.3-fold; P < .001). However, only p70S6k(Thr389) exhibited a dose-dependent response to leucine with all treatments higher than control (~4-fold; P < .001) and at least 5 mM higher than the 1.5-mM concentration (1.2-fold; P < .02). Rates of protein synthesis were not altered by any treatment. Seven days of exposure to 0.5, 1.5, 5.0, and 16.5 mM leucine resulted in an increase in cell size in at least 5 mM treatments (~1.6-fold, P < .001 vs control). Our findings indicate that even at low leucine concentrations, phosphorylation of proteins regulating translation initiation signaling is enhanced. The phosphorylation of p70S6k(Thr389) follows a leucine dose-response relationship, although this was not reflected by the acute protein synthetic response. Nevertheless, under the conditions of the present study, it appears that leucine concentrations of at least 5 mM are necessary to enhance cell growth.
authors.affiliation: Exercise and Nutrition Research Group, School of Medical Sciences, RMIT University, Melbourne, Victoria, Australia.
authors.forename: José L
authors.initials: JL
authors.lastname: Areta
authors.affiliation: Exercise and Nutrition Research Group, School of Medical Sciences, RMIT University, Melbourne, Victoria, Australia; Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, United Kingdom. Electronic address: john.hawley@acu.edu.au.
authors.forename: John A
authors.initials: JA
authors.lastname: Hawley
authors.affiliation: Molecular Pharmacology for Diabetes Group, Health Innovations Research Institute and School of Health Sciences, RMIT University, Melbourne, Victoria, Australia.
authors.forename: Ji-Ming
authors.initials: JM
authors.lastname: Ye
authors.affiliation: Molecular Pharmacology for Diabetes Group, Health Innovations Research Institute and School of Health Sciences, RMIT University, Melbourne, Victoria, Australia.
authors.forename: M H Stanley
authors.initials: MH
authors.lastname: Chan
authors.affiliation: School of Exercise and Nutrition Sciences and Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia.
authors.forename: Vernon G
authors.initials: VG
authors.lastname: Coffey
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D039642:Eukaryotic Initiation Factors; D007930:Leucine / Q000097:blood* / Q000494:pharmacology*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects; D038601:Ribosomal Protein S6 / Q000235:genetics / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C499527:Eif4ebp1 protein, mouse; D039642:Eukaryotic Initiation Factors; D010750:Phosphoproteins; D038601:Ribosomal Protein S6; C508191:proline-rich Akt substrate, 40 kDa protein, mouse; C448579:ribosomal protein S6, mouse; C546843:mTOR protein, mouse; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D007930:Leucine
keywords: Amino acids; Anabolic signaling; C2C12; Protein synthesis; Skeletal muscle; mTOR