Effect of branched-chain amino acid ratio on the proliferation, differentiation, and expression levels of key regulators involved in protein metabolism of myocytes

Nutrition. 2017 Apr:36:8-16. doi: 10.1016/j.nut.2016.10.016. Epub 2016 Nov 22.


Objectives: Branched-chain amino acids (BCAAs), including leucine (Leu), isoleucine (Ile), and valine (Val), are key regulators of protein synthesis in muscle. The aim of this study was to investigate the effect of different BCAA ratios (Leu:Ile:Val) on the proliferation, differentiation, and expression levels of the regulators related to protein metabolism of C2 C12 myocytes.

Methods: Studies were conducted in C2C12 myocytes exposed to different BCAA ratios (Leu: Ile: Val = 0, 1:0.25:0.25, 1:1:1).

Results: The ratio of 1:0.25:0.25 increased cell viability and promoted cell cycle progression from G0/G1 phase to S phase, which was an indicator of proliferation enhancement (P < 0.05). Moreover, this optimal ratio (1:0.25:0.25) promoted the differentiation of myocytes into myotubes by upregulating myogenin and interleukin-15 gene expression, and differently regulated the expression of L-type amino acid transporter 1 and 4 and system ASC amino acid transporters 2. Furthermore, the ratio stimulated mTOR expression at the mRNA and phosphorylated protein levels, as well as ribosomal protein S6 kinase and regulatory-associated protein of mTOR (raptor). In contrast, the optimal ratio decreased the amount of ubiquitin ligase muscle-specific RING finger 1 and muscle atrophy F-box during proliferation and differentiation (P < 0.05). No change was observed in the expression of key genes related to energy metabolism except for uncoupling protein 3 (P > 0.05).

Conclusions: The results suggested that appropriate BCAA ratios could enhance proliferation and differentiation of the C2 C12 myocytes, also mediate the key regulators related to protein metabolism including the mTORC1 pathway. A proper utilization of balanced BCAA ratio in food would be beneficial to human and animal nutrition.

Keywords: Branched-chain amino acid ratio; Myocyte; Proliferation and differentiation; Protein metabolism; mTORC1.

MeSH terms

  • Amino Acids, Branched-Chain / pharmacology*
  • Animals
  • Cell Differentiation / drug effects*
  • Cell Line, Tumor
  • Cell Proliferation / drug effects*
  • Cell Survival / drug effects
  • Down-Regulation
  • Mechanistic Target of Rapamycin Complex 1
  • Mice
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism
  • Muscle Cells / drug effects*
  • Muscle Cells / metabolism
  • Muscle Fibers, Skeletal / cytology
  • Muscle Fibers, Skeletal / drug effects
  • Muscle Fibers, Skeletal / metabolism
  • Muscle Proteins / genetics
  • Muscle Proteins / metabolism
  • Protein Biosynthesis*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • SKP Cullin F-Box Protein Ligases / genetics
  • SKP Cullin F-Box Protein Ligases / metabolism
  • TOR Serine-Threonine Kinases / genetics
  • TOR Serine-Threonine Kinases / metabolism
  • Tripartite Motif Proteins / genetics
  • Tripartite Motif Proteins / metabolism
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism
  • Uncoupling Protein 3 / genetics
  • Uncoupling Protein 3 / metabolism
  • Up-Regulation


  • Amino Acids, Branched-Chain
  • Multiprotein Complexes
  • Muscle Proteins
  • RNA, Messenger
  • Tripartite Motif Proteins
  • Ucp3 protein, mouse
  • Uncoupling Protein 3
  • Fbxo32 protein, mouse
  • SKP Cullin F-Box Protein Ligases
  • Trim63 protein, mouse
  • Ubiquitin-Protein Ligases
  • Mechanistic Target of Rapamycin Complex 1
  • TOR Serine-Threonine Kinases