A pharmacological dimension to personalised phosphoproteomics of human skeletal muscle

Recent studies by our group suggest that the kinase mTORC1 (the mammalian target of rapamycin) may regulate changes to insulin sensitivity in skeletal muscle following exercise. To investigate this directly in humans we extended an approach recently developed by our group ("personalised phosphoproteomics") to include a pharmaco-dimension through the addition of treatment with the mTORC1 inhibitor Rapamycin. We recruited 13 healthy human subjects and administered a single dose of Rapamycin using a double blinded placebo crossover study design. Subjects performed one-legged exercise for 2 hours, and 4 hours post-exercise a 2 hour hyperinsulinemic-euglycemic clamp was performed. Muscle biopsies from m. vastus lateralis were obtained immediately prior to the clamp in both the rested and exercised leg, and at the conclusion of the clamp. Frequent blood samples from catheterised femoral and arterial veins were additionally taken, concomitantly with blood flow measurements, enabling the direct measure of glucose utilization by the muscle bed. Our global phosphoproteomics analysis quantified over 60,000 Class I phosphopeptides in human skeletal muscle tissue, and through comprehensive mass spectrometry-based analyses we identified and quantified phosphosites within the mTOR pathway across eight different conditions in 13 subjects (104 biopsies in total). As we observed previously, skeletal muscle phosphoproteomes exhibited high inter-subject variation, however our paired experimental design enabled us to uncover numerous novel phosphorylation events modulated by mTORC1 and its downstream effectors. Ongoing investigation into these phosphosites and their potential kinases are shedding light on the complex interplay between mTOR, insulin and exercise at an individual and group level.