Regulation of Protein Synthesis by Leucine Starvation Involves Distinct Mechanisms in Mouse C2C12 Myoblasts and Myotubes

Abstract

Leucine modulates protein translation in higher eukaryotes by affecting phosphorylation and the function of proteins that regulate the initiation and/or elongation steps. These include the initiation factor 4E binding protein 1 (4E-BP1), initiation factor 4E (eIF4E), initiation factor 2 (eIF2alpha), ribosomal S6 kinases (S6K1/2), and elongation factor 2 (eEF2). The alteration of protein translation by leucine starvation was studied during myogenic differentiation using the mouse C2C12 cell line as well as the role of rapamycin-sensitive mTOR (mammalian target of rapamycin) in the signaling of leucine in myotubes. A time course study showed that 1 h of leucine starvation decreased protein synthesis and S6K1 phosphorylation in myoblasts, whereas 3-5 h of starvation were necessary to induce such an alteration in myotubes. Although S6K1 phosphorylation was reduced in leucine-deprived myotubes, S6K2 and S6 phosphorylation were not affected. In contrast, rapamycin decreased the phosphorylation of S6K2 and S6 in myotubes. It is therefore likely that under the conditions present, the rapamycin-sensitive mTOR was not affected by leucine starvation. S6K1 dephosphorylation may thus be mTOR independent, and the functional mTOR/S6K2 pathway may maintain S6 phosphorylation. An increased phosphorylation of eEF2 in myoblasts and myotubes indicated that global protein synthesis was reduced via a decrease in translation elongation. An increased association between 4E-BP1 and eIF4E, and increased phosphorylation of eIF2alpha also contributed to decreasing protein synthesis in leucine-starved myoblasts. In contrast, in leucine-starved myotubes, there were no change in the 4E-BP1-eIF4E association or eIF2alpha phosphorylation, suggesting that these factors were not rate limiting for decreasing protein synthesis in leucine-deprived myotubes.

Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs

“…It has been reported that leucine deprivation in myoblast and myotubes increased (78) and leucine administration in cell culture (44,66), rats (87,88), and humans (29,41) decreased the phosphorylation of eEF2, a key component of the elongation process. In the current study, phosphorylation of eEF2 did not differ between the CON ϩ LEU and the CON ϩ ALA groups.…”

Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro

“…Second, ingestion of small quantities of leucine rich essential amino acids activate the downstream mTORC1 target p70S6k and MPS in a comparable manner to 20–25 g of whey protein, and to a greater extent than a bolus of leucine‐deficient essential amino acids (Bukhari et al, 2015; Churchward‐Venne et al, 2012), and removal of leucine from an essential amino acid supplement following muscle loading attenuates mTORC1 signaling (Moberg et al, 2014). Finally, in C2C12 myotubes in vitro, leucine exhibits the most potent stimulation of mTORC1 signaling compared to all other amino acids (Atherton, Smith, Etheridge, Rankin, & Rennie, 2010), and its deprivation impairs protein synthesis and phosphorylation of p70S6k (Talvas, Obled, Fafournoux, & Mordier, 2006). …”

“…In vitro cultures of skeletal muscle provide a controlled and isolated environment in which to understand cellular and molecular adaptation, and have improved our understanding of the importance of amino acids, and in particular leucine, for skeletal muscle growth (Areta, Hawley, Ye, Chan, & Coffey, 2014; Atherton, Smith et al, 2010; Talvas et al, 2006). However, a limitation of conventional in vitro methods is the inability of the rigid 2‐dimensional substrate to support muscle contraction, and as such only acute experiments are typically possible.…”

Content of selected amino acids in the gastrocnemius muscle during experimental hypothyroidism in rats

“…Leucine is an essential amino acid with a branching structure and a twin nature: it exhibits both anabolic and catabolic activities. It stimulates oxidative metabolism, especially that of 5′adenosine monophosphate-activated protein kinase (AMPK), which is the main regulator of cellular energy management (27). In the case of leucine supplementation in Ay mice, increased oxygen consumption was observed, suggesting increased energy expenditure through selective stimulation in the expression of genes important in the metabolism of fatty acids and in the biogenesis of mitochondria in the skeletal muscles (10).…”

“…It is also notable as a stimulator of muscular metabolism, boosting cellular synthesis of proteins and participating in mRNA translation. In vitro tests performed on mouse C2C12 cellular lines demonstrated that subjecting them to leucine starvation lowers protein synthesis in myoblasts (27). This amino acid also plays an important role as a repair factor.…”