Exercise triggers skeletal muscle signalling pathways that modulate the release of circulating factors to cause systemic health benefits. Understanding the structure of this network could lead to better strategies for treating cardiometabolic diseases. We previously showed that acute exercise induces >1000 changes in protein phosphorylation in human muscle. Here we employed a strategy to deconvolute this network by analysing phosphoproteomes of myotubes treated with small molecules that target different aspects of exercise signalling.
Small molecules were selected based on their potential ability to activate various branches of the exercise network. We tested 21 compounds and selected nine treatments to measure the entire phosphoproteome, as these covered the relevant positive controls with the greatest diversity. Single-run LC-MS/MS quantified a total of 20,249 Class I phosphopeptides, of which 24.7% were regulated in at least one treatment. Comparative analysis provided a valuable resource indicating which treatments regulate specific exercise-regulated phosphosites for future kinase-substrate relationship analysis and functional investigation. In light of the important role of AMPK in exercise, we compared a panel of six AMPK activators to determine novel potential AMPK substrates and investigate differences in the mechanism of AMPK activation. Bioinformatics suggested that combining β-adrenergic and calcium agonists would yield a phosphoproteome most closely resembling exercise. Experimentally measuring these phosphoproteomes supported this and also revealed extensive interactions between the treatments. Dual stimulation promoted multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-thrombotic and fibrotic secreted protein. Secretomic analysis of L6 myotubes treated with β-adrenergic and calcium agonists revealed a significant decrease in SERPINE1 secretion and other deleterious secretory factors.
This work provides a novel approach to dissect exercise signalling, which may help to determine the mechanisms of beneficial effects of exercise. We demonstrate an underappreciated effect of exercise to reduce the circulating levels of certain factors, developing a framework for new insights into exercise benefits.