The actions of exogenous leucine on mTOR signalling and amino acid transporters in human myotubes

Human myotubes were treated with leucine, insulin or co-treated with leucine and insulin for 30 min, 3 h or 24 h. Activation of mTOR signalling kinases were examined, together with putative nutrient sensor human vacuolar protein sorting 34 (hVps34) and gene expression of selected amino acid transporters. Phosphorylation of mTOR and p70S6K was transiently increased following leucine exposure, independently to insulin. hVps34 protein expression was also significantly increased. However, genes encoding amino acid transporters were differentially regulated by insulin and not leucine.mTOR signalling is transiently activated by leucine within human myotubes independently of insulin stimulation. While this occurred in the absence of changes in gene expression of amino acid transporters, protein expression of hVps34 increased.Amino acids are essential for the regulation of cell growth and proliferation [1,2] in two ways; by providing the substrate required for polypeptide biosynthesis, and by modulating signalling pathways responsible for protein synthesis [3-6]. Various cell models have examined the anabolic potential of the branched chain amino acid (BCAA), leucine, to stimulate skeletal muscle growth via mammalian target of rapamycin (mTOR) signalling [7,3,10]. Phosphorylation of mTOR complex 1, a rapamycin-sensitive kinase, is vital for downstream activation of phosphokinases required for translational initiation. Stimulation of mTOR by nutrients or insulin activates p70 ribosomal S6 kinase (p70S6K), a key mediator of the protein synthesis cascade [11]. Active p70S6K subsequently leads to phosphorylation of its downstream target, ribosomal protein S6 kinase (S6). This results in the translation of messenger RNA (mRNA) which encode for ribosomes and transcription factors, an essential process leading to increased cellular capacity to undergo protein synthesis [12]. Moreover, mTOR-catalysed stimulation of eukaryotic initiation factor 4E-binding protein (4E-BP1) results in