Increasing leucine concentration stimulates mechanistic target of rapamycin signaling and cell growth in C2C12 skeletal muscle cells

Nutr Res. 2014 Nov;34(11):1000-7. doi: 10.1016/j.nutres.2014.09.011. Epub 2014 Oct 2.


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.

Keywords: Amino acids; Anabolic signaling; C2C12; Protein synthesis; Skeletal muscle; mTOR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Animals
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Cell Cycle Proteins
  • Cell Line
  • Dose-Response Relationship, Drug
  • Eukaryotic Initiation Factors
  • Leucine / blood*
  • Leucine / pharmacology*
  • Mice
  • Muscle Fibers, Skeletal / cytology
  • Muscle Fibers, Skeletal / drug effects*
  • Muscle Fibers, Skeletal / metabolism
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein Biosynthesis / drug effects
  • Ribosomal Protein S6 / genetics
  • Ribosomal Protein S6 / metabolism
  • Ribosomal Protein S6 Kinases, 70-kDa / genetics
  • Ribosomal Protein S6 Kinases, 70-kDa / metabolism
  • Signal Transduction*
  • TOR Serine-Threonine Kinases / genetics
  • TOR Serine-Threonine Kinases / metabolism*


  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • Cell Cycle Proteins
  • Eif4ebp1 protein, mouse
  • Eukaryotic Initiation Factors
  • Phosphoproteins
  • Ribosomal Protein S6
  • proline-rich Akt substrate, 40 kDa protein, mouse
  • ribosomal protein S6, mouse
  • mTOR protein, mouse
  • Ribosomal Protein S6 Kinases, 70-kDa
  • TOR Serine-Threonine Kinases
  • Leucine