Introduction Leucine is commonly considered to be the most anabolic amino acid and its ingestion or infusion alone stimulates myofibrillar muscle protein synthesis (MyoPS)1,2, purportedly through its ability to increase mTORC1 recruitment to the lysosomal surface3. Our lab has also reported a further mechanism of mTORC1 activation in human skeletal muscle where anabolic stimuli elicit mTOR-lysosome translocation to the cell periphery concomitant with elevated mTORC1 activity. However, whether leucine ingestion alone is able to initiate either of these cellular events in human skeletal muscle and whether they are related to MyoPS is yet to be established.
Aims Therefore, the aim of this study was to investigate the impact of leucine ingestion on mTOR translocation, protein-protein interactions and localized activity in human skeletal muscle. A secondary aim was then to determine if these cellular events were associated with MyoPS rates.
Methods Eight young, healthy, recreationally active males (age – 23±3yrs, BMI – 24.2±1.9kg/m2, BF% – 22.8±3.6%) volunteered to undertake a primed continuous infusion of L-[ring-13C6]phenylalanine with repeated blood and skeletal muscle biopsy samples. All procedures were approved by the research ethics board at the University of Toronto, Canada (Protocol No. 00036752) and conformed to the Declaration of Helsinki (revised 2013). Biopsies were obtained at baseline and 30, 60 and 180 minutes after ingestion of 2g leucine and subjected to immunofluorescent microscopy analysis for mTOR colocalization/activity analysis4. MyoPS was determined by LC-MS/MS and has been previously published elsewhere5. One-factor repeated measures ANOVAs were conducted to determine differences in mTOR localization, protein-protein interactions and region-specific RPS6Ser240/244 phosphorylation, a mTORC1-mediated event, with appropriate post hoc comparisons corrected for multiple comparisons (Holm-Bonferroni). Pearson’s correlation coefficients were used to assess potential associations between these measures and MyoPS.
Results Leucine ingestion elicited increases in mTOR colocalization with WGA (sarcolemmal marker) and LAMP2 (lysosomal marker) at 30 and 60 minutes post-ingestion (18-29% & 16% respectively, p<0.025) suggesting leucine initiated mTOR translocation to the periphery and lysosomal surface respectively. p-RPS6Ser240/244 displayed increased staining intensity at all post-ingestion timepoints indicating elevated mTORC1 activity across this entire post-prandial period assessed (14-30%, p<0.03). Moreover, when assessed in a region-specific manner, peripheral (outer 5.5µm of fibre) RPS6Ser240/244 phosphorylation was elevated at all postprandial timepoints (16-33%, p<0.02) whereas central phosphorylation was only elevated at 180 minutes post-ingestion. This suggests that the primary site of mTORC1 activation following leucine ingestion is the area of the fibre to which mTOR-lysosome complexes translocate. Total fibre p-RPS6Ser240/244 staining intensity at 60 min was positively associated with MyoPS rates (r=0.74, p=0.036), however, this association became stronger when considering only peripheral RPS6Ser240/244 phosphorylation (r=0.8, p=0.016).
Conclusions Combined, these results demonstrate, for the first time, that leucine ingestion is able to stimulate mTOR translocation to the fibre periphery and lysosomal surface, two predominant mechanisms of mTORC1 activation, in human skeletal muscle. In addition, leucine-stimulated peripheral mTORC1 activity was associated with MyoPS rates, further confirming the importance of this cellular region for skeletal muscle anabolism.