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

Abstract

(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...

Citrulline stimulates muscle protein synthesis, by reallocating ATP consumption to muscle protein synthesis

“…However, the mechanism by which CIT provides the necessary energy for this process is not fully understood. Interestingly, most of the studies showing a positive effect of CIT on muscle protein synthesis observed this under conditions of protein/energy deficiency . We observed a similar pattern here, with CIT‐stimulated protein synthesis exclusively detected after amino acid/serum deprivation or mild uncoupling, when ATP/ADP ratios were decreased, but not in standard conditions ( Figure ).…”

“…Interestingly, most of the studies showing a positive effect of CIT on muscle protein synthesis observed this under conditions of protein/energy deficiency. 1,3,31 We observed a similar pattern here, with CIT-stimulated protein synthesis exclusively detected after amino acid/serum deprivation or mild uncoupling, when ATP/ADP ratios were decreased, but not in standard conditions (Figure 1). CIT stimulated protein synthesis while ATP/ADP ratios, respiration, and AMPK activation remained unchanged.…”

“…Nutrients such as amino acids not only serve for protein synthesis but also have important regulatory properties. Among them, CIT is well known to stimulate muscle protein synthesis via an activation of the mTORC1 pathway . However, the mechanism by which CIT provides the necessary energy for this process is not fully understood.…”

“…Among them, CIT is well known to stimulate muscle protein synthesis via an activation of the mTORC1 pathway. 1,3,29,30 However, the mechanism by which CIT provides the necessary energy for this process is not fully understood. Interestingly, most of the studies showing a positive effect of CIT on muscle protein synthesis observed this under conditions of protein/energy deficiency.…”

“…Since then, several studies confirmed the positive effect of CIT on muscle protein synthesis, especially in the malnourished state. 2 Using a myotube in vitro model, Le Plénier et al 3 confirmed a direct positive effect of CIT on protein synthesis in muscle during amino acid/serum deficiency. They could link this effect to a stimulation of the mTORC1 pathway, a key activator of protein synthesis.…”