(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 Ͻ 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 Ͻ 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. eukaryotic initiation factor 4E-binding protein 1; mitogen-activated protein kinase; phosphatidylinositol 3-kinase; muscle; myotube; amino acids; protein synthesis; mammalian target of rapamycin CITRULLINE (CIT) is a nonprotein amino acid. It takes its name from the watermelon (citrullus vulgaris). CIT is known mainly as an intermediary of ureagenesis in periportal hepatocytes (24) and at the whole body level is produced almost exclusively by enterocytes (4). Intestinal CIT production is controlled largely by dietary protein supply (37). CIT has also emerged as an important regulator of nitrogen homeostasis in both humans and animals, as reviewed recently (6). For example, in pioneering work, we used a model of aged malnourished rats to demonstrate that CIT-enriched diet stimulated muscle protein synthesis (MPS) (ϩ80%), and led to a net muscle protein gain (ϩ20%) (10, 33). Interestingly, this metabolic effect was accompanied by muscle histological change and an increase in maximum strength as well as increased traction in treated animals (11). Similar results were obtained in healthy aged rats, in which CIT increased muscle protein content (28). This was also found in other situations such as fasting or caloric restriction in adults rats (22,38). Finally, our experimental work showing the ability of CIT to modulate MPS was confirmed in humans; in a crossover trial, Jourdan et al. (20) showed that CIT administration to healthy volunteers fed a hypoprotein diet increased MPS compared with a nonessential amino acid mixture. Thus all these studies confirm the ability of CIT to modulate MPS in vivo, but no work has yet determined the underlying mechanisms of CIT action (an increase in nitrogen...

Exercise and citrulline (CIT) are both regulators of muscle protein metabolism. However, the combination of both has been under-studied yet may have synergistic effects on muscle metabolism and performance. Three-month-old healthy male rats were randomly assigned to be fed for 4 weeks with either a citrulline-enriched diet (1 g·kg·day) () or an isonitrogenous standard diet (by addition of nonessential amino acid) () and trained (running on treadmill 5 days·week) () or not. Maximal endurance activity and body composition were assessed, and muscle protein metabolism (protein synthesis, proteomic approach) and energy metabolism [energy expenditure, mitochondrial metabolism] were explored. Body composition was affected by exercise but not by CIT supplementation. Endurance training was associated with a higher maximal endurance capacity than sedentary groups (<0.001), and running time was 14% higher in the group than the group (139±4 min versus 122±6 min, <0.05). Both endurance training and CIT supplementation alone increased muscle protein synthesis (by +27% and +33%, respectively, versus ,<0.05) with an additive effect (+48% versus ,<0.05). Mitochondrial metabolism was modulated by exercise but not directly by CIT supplementation. However, the proteomic approach demonstrated that CIT supplementation was able to affect energy metabolism, probably due to activation of pathways generating acetyl-CoA. CIT supplementation and endurance training in healthy male rats modulates both muscle protein and energy metabolisms, with synergic effects on an array of parameters, including performance and protein synthesis.

Background Animal studies and clinical data support the interest of citrulline as a promising therapeutic for sarcopenia. Citrulline is known to stimulate muscle protein synthesis, but how it affects energy metabolism to support the highly energy‐dependent protein synthesis machinery is poorly understood. Methods Here, we used myotubes derived from primary culture of mouse myoblasts to study the effect of citrulline on both energy metabolism and protein synthesis under different limiting conditions. Results When serum/amino acid deficiency or energy stress (mild uncoupling) were applied, citrulline stimulated muscle protein synthesis by +22% and +11%, respectively. Importantly, this increase was not associated with enhanced energy status (ATP/ADP ratio) or mitochondrial respiration. We further analysed the share of mitochondrial respiration and thus of generated ATP allocated to different metabolic pathways by using specific inhibitors. Our results indicate that addition of citrulline allocated an increased share of mitochondrially generated ATP to the protein synthesis machinery under conditions of both serum/amino acid deficiency (+28%) and energy stress (+21%). This reallocation was not because of reduced ATP supply to DNA synthesis or activities of sodium and calcium cycling ion pumps. Conclusions Under certain stress conditions, citrulline increases muscle protein synthesis by specifically reallocating mitochondrial fuel to the protein synthesis machinery. Because ATP/ADP ratios and thus Gibbs free energy of ATP hydrolysis remained globally constant, this reallocation may be linked to decreased activation energies of one or several ATP (and GTP)‐consuming reactions involved in muscle protein synthesis.

Besides their main contribution as substrates for protein synthesis, amino acids as signaling molecules could exert some regulatory functions on protein synthesis and/or proteolysis that have been emphasized in a number of recent studies. Several publications have highlighted supplemental roles of those amino acids in protein metabolism as well as in immunity, heat shock response, or apoptosis processes. In this way, via their regulatory properties, selected amino acids (such as leucine, glutamine, arginine, citrulline, or methionine) directly influence the proteome. In this review, we are proposing an overview of the regulation of the proteome by amino acids in mammals.

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Among a plethora of dietary supplements, amino acids are very popular with athletes for several reasons (e.g., to prevent nutritional deficiency, improve muscle function, and decrease muscle damages) whose purpose is to improve performance. However, it is difficult to get a clear idea of which amino acids have real ergogenic impact. Here, we review and analyze the clinical studies evaluating specific amino acids (glutamine, arginine, leucine, etc.) in athletes. Only english-language clinical studies evaluating a specific effect of one amino acid were considered. Despite promising results, many studies have methodological limits or specific flaws that do not allow definitive conclusions. To date, only chronic β-alanine supplementation demonstrated an ergogenic effect in athletes. Much research is still needed to gain evidence-based data before any other specific amino acid can be recommended for use in athletes.

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