Background: Sarcopenia, (loss of muscle mass and quality with age), is a major health problem without safe and effective pharmaceuticals. The mechanistic target of the rapamycin complex 1 (mTORc1) pathway is a cell-type agnostic master regulator of metabolism, and in skeletal muscle, positively regulates proteostasis. However, studies show mTOR signaling is counterintuitively hyperactive in aged muscle, representing a potential cause of sarcopenia (e.g. via inhibition of autophagy). This study aims to comprehensively analyze mTOR-related anabolic/autophagy cell signaling via western blot comparing protein phosphorylation site activity across age (young/old) and sex (men/women).
Methods: This project was approved by the University of Nottingham Ethics Committee and complied with the Declaration of Helsinki (2015), and was registered at https://clinicaltrials.gov/(NCT02505438). This experiment includes 29 human muscle biopsy samples (fasted-state, vastus lateralis) collected from young men (22±4y, BMI:24±3, N=6), older men (69±4y, BMI: 27±2, N=8), young women (22±3y, BMI:22±2, N=7) and older women (67±4y, BMI: 26±2, N=8). Following protein extraction and standardization, mTOR-related targets were quantified, including mTOR(Ser2448), ribosomal protein S6 kinase 1 (p70S6K1 Thr389), 4E-binding protein 1 (4E-BP1 Thr37/46), ribosomal protein S6 (rps6 Ser235/236), tuberous sclerosis complex 2 (TSC2 Thr1462), adenosine monophosphate kinase (AMPK Thr172), serine-threonine protein kinase (AKT Ser473), and Forkhead family of transcription factors (FoxO families, 1a(Thr24),3a(Thr32), and 4a(Thr28)). Data were quantified via densitometry and normalized to Coomassie blue staining to account for loading error. Data were tested for normal distribution using a Shapiro-Wilk test. Comparisons between age/sex were performed using independent t-tests or non-parametric equivalent, with the alpha level of significance set at p < 0.05. Graph Pad Prism (version 9.0, La Jolla, US) was used for all analyses.
Results: mTOR phosphorylation (Ser2448) was unaffected by age, however the mTOR target S6K1 phosphorylation (Thr389) was higher in both sexes combined in old age (1.5-times) (P<0.05) than their young comparative group. In line with p70S6K1 data, phosphorylation of rps6 (Ser235/236) in both older men and both sexes combined were higher than their young counterparts (2-times; P<0.05 and 1.9-times; P<0.01, respectively). In contrast, there were no differences in 4E-BP1 (Thr37/46). AMPK phosphorylation (Thr172) in older men and women was 5.2-times (P<0.001), and 3.2-times (P<0.05) greater than their younger counterparts; and both sexes combined were 5.6-times (P<0.01) higher. Upstream Akt phosphorylation (Ser473) exhibited no difference, while TSC2 phosphorylation (Thr1462) was lower two-thirds (P<0.01) in young than older subjects (sex combined). For autophagy targets, FoxO1a and FoxO3a phosphorylation were not different (Thr24/Thr32), while FoXO4a (Thr28) phosphorylation was 5.6 (P<0.01) and 3.4-times (P<0.05) greater in men and both sexes combined in older groups, respectively.
Discussion: We find key elements of hyperactive mTOR signaling in human muscle supporting the notion of mTOR inhibition strategies e.g. rapamycin interventions, to ameliorate sarcopenia in older age. That FoxO4a phosphorylation is increased in older age, implies this target might also be important in aging autophagy dysregulation.