L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS-Dependent Mechanism

PLoS One. 2015 Oct 29;10(10):e0141572. doi: 10.1371/journal.pone.0141572. eCollection 2015.


Dietary L-citrulline is thought to modulate muscle protein turnover by increasing L-arginine availability. To date, the direct effects of increased L-citrulline concentrations in muscle have been completely neglected. Therefore, we determined the role of L-citrulline in regulating cell size during catabolic conditions by depriving mature C2C12 myotubes of growth factors (serum free; SF) or growth factors and nutrients (HEPES buffered saline; HBS). Cells were treated with L-citrulline or equimolar concentrations of L-arginine (positive control) or L-alanine (negative control) and changes in cell size and protein turnover were assessed. In myotubes incubated in HBS or SF media, L-citrulline improved rates of protein synthesis (HBS: +63%, SF: +37%) and myotube diameter (HBS: +18%, SF: +29%). L-citrulline treatment substantially increased iNOS mRNA expression (SF: 350%, HBS: 750%). The general NOS inhibitor L-NAME and the iNOS specific inhibitor aminoguanidine prevented these effects in both models. Depriving myotubes in SF media of L-arginine or L-leucine, exacerbated wasting which was not attenuated by L-citrulline. The increased iNOS mRNA expression was temporally associated with increases in mRNA of the endogenous antioxidants SOD1, SOD3 and catalase. Furthermore, L-citrulline prevented inflammation (LPS) and oxidative stress (H2O2) induced muscle cell wasting. In conclusion, we demonstrate a novel direct protective effect of L-citrulline on skeletal muscle cell size independent of L-arginine that is mediated through induction of the inducible NOS (iNOS) isoform. This discovery of a nutritional modulator of iNOS mRNA expression in skeletal muscle cells could have substantial implications for the treatment of muscle wasting conditions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antioxidants / pharmacology*
  • Catalase / genetics
  • Catalase / metabolism
  • Cell Line
  • Citrulline / pharmacology*
  • Mice
  • Muscle Fibers, Skeletal / drug effects
  • Muscle Fibers, Skeletal / metabolism*
  • Nitric Oxide Synthase Type II / genetics
  • Nitric Oxide Synthase Type II / metabolism*
  • Oxidative Stress
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Superoxide Dismutase / genetics
  • Superoxide Dismutase / metabolism
  • Superoxide Dismutase-1


  • Antioxidants
  • RNA, Messenger
  • Citrulline
  • Catalase
  • Nitric Oxide Synthase Type II
  • Sod1 protein, mouse
  • Sod3 protein, mouse
  • Superoxide Dismutase
  • Superoxide Dismutase-1

Grants and funding

This work was funded by a grant from the Ajinomoto Amino Acid Research Program (3ARP: 09/07). D.J. Ham & M.K. Caldow were funded by an ESPEN postdoctoral fellowship.