L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an <i>iNOS</i>-Dependent Mechanism

<div><p>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 <i>iNOS</i> mRNA expression (SF: 350%, HBS: 750%). The general NOS inhibitor L-NAME and the <i>iNOS</i> 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 <i>iNOS</i> mRNA expression was temporally associated with increases in mRNA of the endogenous antioxidants <i>SOD1</i>, <i>SOD3</i> and <i>catalase</i>. 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 (<i>iNOS</i>) isoform. This discovery of a nutritional modulator of <i>iNOS</i> mRNA expression in skeletal muscle cells could have substantial implications for the treatment of muscle wasting conditions.</p>