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

José L. Areta, John A. Hawley, Ji Ming Ye, M. H Stanley Chan, Vernon G. Coffey

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19 Citations (Scopus)

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 mTORSer2448 (~1.4-fold; P < .04), 4E-BP1 Thr37/46 (~1.9-fold; P < .001), and rpS6Ser235/6 (~2.3-fold; P < .001). However, only p70S6kThr389 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 p70S6kThr389 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.

Original languageEnglish
Pages (from-to)1000-1007
Number of pages8
JournalNutrition Research
Volume34
Issue number11
DOIs
Publication statusPublished - 2014
Externally publishedYes

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Cell growth
Sirolimus
Leucine
myocytes
Muscle Cells
leucine
Muscle
skeletal muscle
cell growth
Skeletal Muscle
Cells
Growth
Phosphorylation
Cell Size
translation (genetics)
phosphorylation
Proteins
Translational Peptide Chain Initiation
dosage
protein synthesis

Cite this

Areta, José L. ; Hawley, John A. ; Ye, Ji Ming ; Chan, M. H Stanley ; Coffey, Vernon G. / Increasing leucine concentration stimulates mechanistic target of rapamycin signaling and cell growth in C2C12 skeletal muscle cells. In: Nutrition Research. 2014 ; Vol. 34, No. 11. pp. 1000-1007.
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Increasing leucine concentration stimulates mechanistic target of rapamycin signaling and cell growth in C2C12 skeletal muscle cells. / Areta, José L.; Hawley, John A.; Ye, Ji Ming; Chan, M. H Stanley; Coffey, Vernon G.

In: Nutrition Research, Vol. 34, No. 11, 2014, p. 1000-1007.

Research output: Contribution to journalArticleResearchpeer-review

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AU - Areta, José L.

AU - Hawley, John A.

AU - Ye, Ji Ming

AU - Chan, M. H Stanley

AU - Coffey, Vernon G.

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AB - 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 mTORSer2448 (~1.4-fold; P < .04), 4E-BP1 Thr37/46 (~1.9-fold; P < .001), and rpS6Ser235/6 (~2.3-fold; P < .001). However, only p70S6kThr389 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 p70S6kThr389 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.

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