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

• Published in: PloS one Vol. 10; no. 10; p. e0141572
• Main Authors: Ham, Daniel J., Gleeson, Benjamin G., Chee, Annabel, Baum, Dale M., Caldow, Marissa K., Lynch, Gordon S., Koopman, René
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 29.10.2015; Public Library of Science (PLoS)
• Subjects: Alanine; Amino acids; Animals; Antioxidants; Antioxidants - pharmacology; Arginine; Breakdowns; Catalase; Catalase - genetics; Catalase - metabolism; Cell Line; Cell size; Citrulline; Citrulline - pharmacology; Diet; Free radicals; Gene expression; Growth factors; Hydrogen peroxide; Inhibitors; Kinases; L-Alanine; Laboratories; Leucine; Lipopolysaccharides; Medical research; Metabolism; Mice; Muscle Fibers, Skeletal - drug effects; Muscle Fibers, Skeletal - metabolism; Muscles; Musculoskeletal system; Myotubes; NG-Nitroarginine methyl ester; Nitric oxide; Nitric Oxide Synthase Type II - genetics; Nitric Oxide Synthase Type II - metabolism; Nitric-oxide synthase; Nutrients; Nutrition research; Oxidative Stress; Physiology; Protein biosynthesis; Protein synthesis; Protein turnover; Proteins; RNA, Messenger - genetics; RNA, Messenger - metabolism; Rodents; Skeletal muscle; Superoxide dismutase; Superoxide Dismutase - genetics; Superoxide Dismutase - metabolism; Superoxide Dismutase-1; Australia; Victoria Australia
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0141572

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.