Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies

Am J Physiol Endocrinol Metab. 2017 Jan 1;312(1):E27-E36. doi: 10.1152/ajpendo.00203.2016. Epub 2016 Nov 8.


Citrulline (CIT) is an endogenous amino acid produced by the intestine. Recent literature has consistently shown CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both interorgan and intraorgan trafficking and metabolism, we combined three approaches: in vivo, ex vivo, and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated (P < 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, P < 0.05), and the CIT effect was abolished in the presence of wortmannin, rapamycin, and PD-98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD-98059 inhibited the CIT effect on S6K1, whereas only LY-294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control.

Keywords: amino acids; eukaryotic initiation factor 4E-binding protein 1; mammalian target of rapamycin; mitogen-activated protein kinase; muscle; myotube; phosphatidylinositol 3-kinase; protein synthesis.

MeSH terms

  • Androstadienes / pharmacology
  • Animals
  • Carrier Proteins / drug effects*
  • Carrier Proteins / metabolism
  • Chromones / pharmacology
  • Citrulline / metabolism
  • Citrulline / pharmacology*
  • Enzyme Inhibitors / pharmacology
  • Flavonoids / pharmacology
  • In Vitro Techniques
  • Intracellular Signaling Peptides and Proteins
  • Male
  • Malnutrition / metabolism*
  • Mechanistic Target of Rapamycin Complex 1
  • Mitogen-Activated Protein Kinases / drug effects*
  • Mitogen-Activated Protein Kinases / metabolism
  • Morpholines / pharmacology
  • Multiprotein Complexes / drug effects
  • Multiprotein Complexes / metabolism
  • Muscle Fibers, Skeletal / drug effects*
  • Muscle Fibers, Skeletal / metabolism
  • Muscle Proteins / biosynthesis
  • Muscle Proteins / drug effects*
  • Muscle, Skeletal / drug effects*
  • Muscle, Skeletal / metabolism
  • Phosphatidylinositol 3-Kinases / drug effects*
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphoproteins / drug effects*
  • Phosphoproteins / metabolism
  • Protein Kinase Inhibitors / pharmacology
  • Proto-Oncogene Proteins c-akt / drug effects
  • Proto-Oncogene Proteins c-akt / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Signal Transduction
  • Sirolimus / pharmacology
  • TOR Serine-Threonine Kinases / drug effects
  • TOR Serine-Threonine Kinases / metabolism
  • Wortmannin


  • Androstadienes
  • Carrier Proteins
  • Chromones
  • Eif4ebp1 protein, rat
  • Enzyme Inhibitors
  • Flavonoids
  • Intracellular Signaling Peptides and Proteins
  • Morpholines
  • Multiprotein Complexes
  • Muscle Proteins
  • Phosphoproteins
  • Protein Kinase Inhibitors
  • Citrulline
  • 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
  • TOR Serine-Threonine Kinases
  • Mechanistic Target of Rapamycin Complex 1
  • Proto-Oncogene Proteins c-akt
  • Mitogen-Activated Protein Kinases
  • 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one
  • Sirolimus
  • Wortmannin