The aim of this study was to evaluate the anabolic effect of acute and chronic leucine and insulin stimulation in a model of human skeletal muscle. It was important to determine whether leucine could mediate mTOR-related signalling via activation of the putative regulator, hVps34, and amino acid transporters. The present work demonstrates the ability of the BCAA member, leucine, to independently stimulate mTOR signalling in an experimental model of human skeletal muscle. Although p70S6K was only transiently activated by leucine, chronic stimulation resulted in enhanced phosphorylation of the ternary kinase, eIF4G. Moreover, these data offer an explanation of the molecular mechanism by which amino acids modulate myogenic growth, via activation of the hVps34 protein. Finally, while exogenous leucine and insulin were sufficient to stimulate anabolic signalling in cultured myotubes, incubation did not alter gene expression of the amino acid transporters measured.
The use of human primary myotubes provides pertinent experimental data on amino acid-mediated anabolic signalling that occurs in human skeletal muscle tissue. Cultured muscle cells undergo differentiation to form mature myotubes [37
] thus providing a physiologically relevant model to study molecular signalling events which occur in vivo
. In agreement with data from rodent studies [17
], Chinese hamster ovary cells (CHO) [38
], pancreatic beta-cells [39
] and rat hepatocytes [40
], this study demonstrates for the first time that mTOR-related signalling kinases are responsive to a physiological dose of leucine in a human model of skeletal muscle, primary myotubes. However, while mTOR was stimulated by insulin on serine residue 2448, at all time points measured, the action of leucine was transient (1.3-fold increase) with phosphorylation diminishing after 30 min. This is consistent with Deldicque et al.
(2008) who demonstrated a 1.5-fold increase in mTOR activation within C2C12 cells, a model of murine skeletal muscle, following an acute 30 min exposure to 5 mM leucine [17
]. However, more recently it was reported that a 2 mM dose of leucine also stimulates mTOR phosphorylation on Ser2448 by a comparable magnitude [14
]. The downstream biomarker, p70S6K, followed a similar trend to mTOR phosphorylation. While leucine demonstrated transient activation of p70S6K (2.0-fold increase), insulin appeared to stimulate this kinase at all time points. Previous work has demonstrated that 5 mM leucine stimulates p70S6K activation by 10-fold [17
] and a 2 mM dose elicits a 5.9-fold increase [14
] within rodent muscle cells. Furthermore, cultured pancreatic beta-cells have been shown to respond to 3.3 mM of leucine, therefore the stimulatory effect of leucine on the phosphorylation state of p70S6K may be specific to the cell model employed where by different cell types have a varying dose-response to amino acids [39
]. Previous in vitro
experiments demonstrates the synergistic activation of p70S6K in FAO cells treated with a combination of amino acids with insulin [41
]. Results obtained from the current study did not exhibit an additive effect of leucine and insulin, despite utilising the same insulin concentration. While these experiments were conducted using leucine only, the former study used a mixture of amino acids. Furthermore, recent evidence suggests that while the activation of anabolic signalling is indeed mTOR-dependent, amino acids may signal through the Rag complex to activate mTORC1 while insulin requires the Ras homolog enriched in brain (Rheb) component to regulate anabolic signalling [42
]. Moreover, it has also been previously shown that deletion of the Ser2448 phosphorylation site on mTOR did not affect mTOR activity and was not required for the downstream phosphorylation of PHAS-1 (4EBP1) or p70S6K in human embryonic kidney cells [43
]. Therefore, it is possible that while mTOR phosphorylation in this study was transient, the downstream activation of p70S6K and eIF4G may occur through an independent signalling mechanism and requires further research.
Surprisingly, phosphorylation of eIF4G occurred following 3 h and 24 h treatment of leucine and insulin. Given that activation of eIF4G is a necessary step during formation of the translation initiation complex which is involved in cap dependent translation [13
], activation by leucine indicates that protein synthesis may be initiated without insulin.
hVps34 is postulated to be a nutrient modulator responsible for signalling to mTOR. Recently, amino acid regulation of this class III PI3 kinase has been investigated in murine myotubes [28
], HepG2 and CHO cells [29
]. During states of amino acid starvation, hVps34 demonstrates reduced function. However, this is restored with the re-addition of amino acids [27
]. Furthermore, previous reports demonstrate that endogenous expression of hVps34 is unresponsive to 1 μM insulin [29
]. Unlike these studies, human primary muscle cells demonstrated increased hVps34 protein abundance following acute and chronic stimulation of leucine with and without 100 nM insulin. The response of hVps34 to insulin in cultured myotubes may reflect the physiological need of skeletal muscle for insulin-mediated glucose uptake and for muscle anabolism [45
]. Limited studies have investigated the factors regulating hVps34 expression; however the possibility of cross-talk with insulin signalling requires further analysis.
Both system A and L solute carriers may regulate intracellular amino acid bioavailability within skeletal muscle [47
]. The activity of system A transporters has been previously demonstrated to be insulin responsive cultured adipocytes, liver and rat muscle cells [48
However, in cultured human myotubes both acute and chronic stimulation with insulin failed to increase SNAT2
gene expression. It is important to note that the aforementioned studies used supraphysiological doses of insulin, nearly 2-fold greater than the concentration used in the current study. System L transport proteins are essential for the movement of BCAA across the cell membrane of skeletal muscle cells [47
]. Leucine exposure of human cultured primary muscle cells did not exert a change in either LAT1
gene expression, despite the specificity of LAT1 for leucine [51
]. However, functional expression of LAT1 requires covalent association with the heavy chain of the CD98 antigen (CD98hc) in plasma membranes [52
]. Acute insulin exposure stimulated an increase in CD98hc mRNA expression, however longer exposure resulted in suppressed gene expression. The current study aimed to present exploratory data on the regulation of amino acid transporters by leucine. Given the physiological need for amino acids in skeletal muscle, further research is required to delineate the factors affecting both the protein expression and cellular localisation of amino acid transporters.