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Autophagy regulation in human skeletal muscle during exercise

In skeletal muscle, endurance training is a stress that induces substantial metabolic adaptations such as improvement of cell oxidative capacity. A single exercise bout may cause alterations in intracellular systems activity and damage to cell components. Chronic exercise can affect the basal activity of cellular pathways and modulate the number of cell constituents, and improve both their functioning and turnover (Sanchez et al. 2014). In recent years, several studies have been conducted in identifying molecular pathways underlying skeletal muscle protein turnover in response to exercise. Among them, the role of the autophagy system has been increasingly considered but data in humans are limited. Autophagy is a highly regulated system critical to cell metabolism and organelle turnover. The importance of autophagy in skeletal muscle homeostasis was for a long time much neglected. However, it is now widely acknowledged that autophagy is essential for muscle homeostasis in response to cellular stress such as physical exercise or hypoxia. Importantly, the energy sensor adenosine monophosphate-activated protein kinase (AMPK) has been shown to be involved in both transcriptional regulation of autophagy by regulating forkhead box class ‘other’ O (FOXO) transcription factor activity, and in post-translational regulation of this pathway through Unc-51-like kinase 1 (ULK1) phosphorylation (Sanchez et al. 2014). AMPK has also been suggested as a major regulator of exercise-induced autophagy in rodent skeletal muscle. In addition, mechanistic/mammalian target of rapamycin complex 1 (MTORC1), a major complex involved in protein translation and sensitive to nutrient availability and growth factors, is now presented as a key regulator of autophagy in skeletal muscle since it negatively regulates autophagy initiation through phosphorylation of ULK1. MTOR phosphorylates ULK1 at Ser-757 preventing autophagosome formation. The majority of studies performed in mice found that a single bout of exercise induces an increase in several autophagy flux markers, including increases in the microtubule-associated protein 1 light chain 3-II to I (LC3-II/LC3-I) ratio, LC3-II protein content and a decrease of p62/sequestosome 1 (SQSTM1) protein levels. In animal models, autophagy seems important to prevent mitochondria alteration and exacerbated oxidative stress during severe acute exercise. In addition, autophagy was shown to have a major role in the improvement of endurance capacity during endurance training protocols. However, studies in human skeletal muscle are more controversial and need further work. While autophagy flux markers are increased after strenuous exercises such as ultra-endurance running (Jamart et al. 2012), other reports suggest the contrary, for example the data from Møller et al. (2015) found that even if autophagy signalling is initiated (i.e. ULK1 activation) in human skeletal muscle after 60 min of exercise, lipidation of LC3 may decrease. Thus, discrepancies may exist concerning the modulation of skeletal muscle autophagy between rodents and humans. In addition, data concerning the regulation of skeletal muscle autophagy in response to chronic exercise are missing in humans. In a recent study published in The Journal of Physiology, Fritzen and coworkers (Fritzen et al. 2016) investigated the modulation of autophagy markers in human skeletal muscle during both acute and chronic exercise. For this purpose, seven moderately trained subjects performed a single 60 min bout of one-legged knee extensor exercise at 80% of their peak work load with two 5 min intervals at 100%. The other leg was used as a rested control. Muscle biopsies were obtained from vastus lateralis muscles of both legs before, immediately after, and 4 h after the end of exercise. The authors also used a rodent model in which rats were subjected to a total of 4 h swimming exercise divided into eight bouts of 30 min with 5 min of rest between each session. By using immunoblotting analysis, the authors first demonstrated that acute exercise induces a decrease in LC3-II protein expression and failed to modulate p62/SQSTM1 protein content in human skeletal muscle. Moreover, a decrease in LC3-II/LC3-I ratio was found immediately after exercise and at 4 h of recovery, suggesting that acute exercise decreases the autophagosome content by local contraction-induced mechanisms. However, in rat skeletal muscle, the authors found an increase of LC3-II/LC3-I ratio, in agreement with other studies performed in animals (Pagano et al. 2014). Thus, these results seem to indicate that a species difference exists in exercise regulation of autophagy. To test the central role of AMPK in the modulation of autophagy during exercise, the authors correlated the modulations of AMPK activity with the changes in LC3-II/LC3-I ratio in the human samples. Interestingly, they found no association between the regulation of LC3 and the activities of the AMPK subunits, but ULK1 phosphorylation at Ser-555 (an AMPK phosphorylation site) was correlated with the activity of the AMPK α1β2γ1 complexes. The authors completed these experiments in mouse skeletal muscle and with cultures of human myotubes in order to test the effects of AICAR, an exercise mimetic that activates AMPK, on the modulation of autophagy. Consistent with the previous results, AICAR treatment tends to increase LC3-I protein content and ULK1 phosphorylation (Ser-555), but without any changes in autophagosome content markers (i.e. LC3 lipidation) in human cultured myotubes. Interestingly, in mouse skeletal muscle, AICAR treatment leads to an increase in ULK1 phosphorylation at Ser-555 and ULK1 protein content, but no change in LC3 lipidation was found. These results strongly suggest that AMPK activation alone and AICAR treatment may lead to an increase in the autophagic transcriptional programme and activation of the ULK1 axis that is not sufficient to induce autophagosome formation in human skeletal muscle. Nonetheless, these findings do not rule out that AMPK induced regulation of ULK1 during exercise is a contributing component in order to normally regulate autophagy. In addition, the authors investigated the role of insulin in the regulation of autophagy during the recovery of exercise. Twelve healthy athletes performed a similar exercise protocol to that previously described, then the participants were subjected to