Distinct anabolic signalling responses to amino acids in C2C12 skeletal muscle cells

Abstract

The essential amino acids (EAA) activate anabolic signalling through mechanisms, which are unclear in detail but include increased signalling through the mammalian target of rapamycin complex 1 (mTORC1). Of all the EAA, the branched chain amino acid (BCAA) leucine has been suggested as the most potent in stimulating protein synthesis, although there have been no studies investigating the effects of each EAA on anabolic signalling pathways. We therefore undertook a systematic analysis of the effect of each EAA on mTORC1 signalling in C2C12 myotubes whereby cells were serum (4 h) and amino acid (1 h) starved before stimulation with 2 mM of each amino acid. Immunoblotting was used to detect phosphorylated forms of protein kinase B (Akt)/mTORC1 signalling enzymes. The phosphorylation of Akt was unchanged by incubation with EAA. Phosphorylation of mTOR and 4E binding protein-1 (4EBP1) were increased 1.67 +/- 0.1-fold and 2.5 +/- 0.1-fold, respectively, in response to leucine stimulation but not in response to any other EAA. The phosphorylation of ribosomal s6 kinase (p70S6K1) was increased by stimulation with all EAA with the exceptions of isoleucine and valine. However, the increase with leucine was significantly greater, 5.9 +/- 0.3-fold compared to 1.6-2.0-fold for the non-BCAA EAA. This pattern of activation was identical in ribosomal protein s6 (RPS6) with the additional effect of leucine being 3.8 +/- 0.3-fold versus 1.5-2.0-fold. Phosphorylation of eukaryotic initiation/elongation factors eIF2alpha and eEF2 were unaffected by EAA. We conclude that leucine is unique amongst the amino acids in its capacity to stimulate both mTOR and 4EBP1 phosphorylation and to enhance p70S6K1 signalling.

Conversion of leucine to β-hydroxy-β-methylbutyrate by α-keto isocaproate dioxygenase is required for a potent stimulation of protein synthesis in L6 rat myotubes

“…S6K1 was activated by L‐Leu 1.2‐fold compared with untreated cells. This result is lower than that reported by Gran and Cameron11 in primary human myotubes and observed in rodent muscle cells 6. This discrepancy could be attributed to their experimental design, in which muscle cells were amino acid starved prior the addition of L‐Leu.…”

“…L‐Leu stimulates protein synthesis in muscle cells through at least two distinct mechanisms: by stimulation of mRNA translation initiation machinery13, 35 and by activation of intracellular signalling pathways linked to mTOR 6, 36, 37. The positive effects of L‐Leu supplementation on protein synthesis have been widely studied in situations where the subjects were either in deficient or fasting conditions,8, 11, 12, 13, 14, 15 and therefore under a catabolic stimulus.…”

“…Among them, leucine is the most effective amino acid in stimulating protein synthesis through a direct activation of mTOR. Phosphorylation of mTOR drives to a 4E‐BP1 activation and an induction of S6K1 signalling 6, 10, 11, 12. Most studies on the beneficial effects of leucine enhancing protein synthesis have been carried out on leucine deficient media6, 7, 13 in in vitro studies or in models of muscle atrophy such as immobilization14 or experimental cachexia15, 16 involving a limited food intake period.…”

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.…”