Effect of citrulline on muscle protein turnover in an in vitro model of muscle catabolism.

OBJECTIVE: Muscle net catabolism, as seen after severe trauma or sepsis or in postoperative situations, is mediated by hormones (e.g., cortisol) and proinflammatory cytokines (e.g., tumor necrosis factor alpha [TNF-α]). Specific amino acids may be able to limit this muscle mass loss. Citrulline (CIT) stimulates muscle protein synthesis in various situations, but little data exist on hypercatabolic situations and the effects on protein breakdown are unknown. Our aim was to assess the effect of CIT on protein turnover in an in vitro model of muscle hypercatabolism. METHODS: Myotubes derived from C2C12 myoblasts were treated with 150 nM dexamethasone (DEX), 10 ng/mL TNF-α, or 0.006% ethanol (as control [CON]) for 24 h. Subsequently, myotubes were incubated with or without 5 mM CIT for 6 h. Muscle protein synthesis rate was evaluated by the surface sensing of translation method and by l-[3,5-3H]tyrosine (Tyr) incorporation. The muscle protein breakdown rate was evaluated from Tyr release into culture medium. CIT action was analyzed by non-parametric Kruskal-Wallis and Mann-Whitney tests. RESULTS: CIT treatment significantly increased protein synthesis rates compared with the DEX or TNF-α group (surface sensing of translation method; DEX + CIT versus DEX; P = 0.03 and TNF-α+CIT versus TNF-α; P = 0.05) and significantly decreased protein breakdown rate in the CON and DEX groups (CON + CIT versus CON; P = 0.05 and DEX + CIT versus DEX; P = 0.05). CONCLUSIONS: CIT treatment regulated muscle protein turnover in an in vitro model of muscle net catabolism. Exploring the underlying mechanisms would also be of interest.

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.

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.