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

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 regulat-ing cell size during catabolic conditions by depriving mature C2C12 myotubes of growth fac-tors (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 treat-ment substantially increased iNOSmRNA 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, exacer-bated wasting which was not attenuated by L-citrulline. The increased iNOSmRNA expres-sion was temporally associated with increases in mRNA of the endogenous antioxidants SOD1, SOD3 and catalase. Furthermore, L-citrulline prevented inflammation (LPS) and oxi-dative 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 iNOSmRNA expression in skeletal muscle cells could have sub-stantial implications for the treatment of muscle wasting conditions