In humans, nutrient deprivation and extreme endurance exercise both activate autophagy. We hypothesized that cumulating fasting and cycling exercise would potentiate activation of autophagy in skeletal muscle. Well-trained athletes were divided into control (n = 8), low-intensity (LI, n = 8), and high-intensity (HI, n = 7) exercise groups and submitted to fed and fasting sessions. Muscle biopsy samples were obtained from the vastus lateralis before, at the end, and 1 h after a 2 h LI or HI bout of exercise. Phosphorylation of ULK1 Ser317 was higher after exercise (P < 0.001). In both the fed and the fasted states, LC3bII protein level and LC3bII/I were decreased after LI and HI (P < 0.05), while p62/ SQSTM1 was decreased only 1 h after HI (P < 0.05), indicating an increased autophagic flux after HI. The autophagic transcriptional program was also activated, as evidenced by the increased level of LC3b, p62/ SQSTM1, GabarapL1, and Cathepsin L mRNAs observed after HI but not after LI. The increased autophagic flux after HI exercise could be due to increased AMPactivated protein kinase a (AMPKa) activity, as both AMPKa Thr172 and ACC Ser79 had a higher phosphorylation state after HI (P < 0.001). In summary, the most effective strategy to activate autophagy in human skeletal muscle seems to rely on exercise intensity more than diet.-Schwalm, C., Jamart, C., Benoit, N., Naslain, D., Prémont, C., Prévet, J., Van Thienen, R., Deldicque, L., Francaux, M. Activation of autophagy in human skeletal muscle is dependent on exercise intensity and AMPK activation. FASEB J. 29, 3515-3526 (2015). www.fasebj.org

In this study, the coordinated activation of ubiquitin-proteasome pathway (UPP), autophagy-lysosomal pathway (ALP), and mitochondrial remodeling including mitophagy was assessed by measuring protein markers during ultra-endurance running exercise in human skeletal muscle. Eleven male, experienced ultra-endurance athletes ran for 24 h on a treadmill. Muscle biopsy samples were taken from the vastus lateralis muscle 2 h before starting and immediately after finishing exercise. Athletes ran 149.8 ± 16.3 km with an effective running time of 18 h 42 min ( ± 41 min). The phosphorylation state of Akt (-74 ± 5%; P < 0.001), FOXO3a (-49 ± 9%; P < 0.001), mTOR Ser2448 (-32 ± 14%; P = 0.028), and 4E-BP1 (-34 ± 7%; P < 0.001) was decreased, whereas AMPK phosphorylation state increased by 247 ± 170% (P = 0.042). Proteasome β2 subunit activity increased by 95 ± 44% (P = 0.028), whereas the activities associated with the β1 and β5 subunits remained unchanged. MuRF1 protein level increased by 55 ± 26% (P = 0.034), whereas MAFbx protein and ubiquitin-conjugated protein levels did not change. LC3bII increased by 554 ± 256% (P = 0.005), and the form of ATG12 conjugated to ATG5 increased by 36 ± 17% (P = 0.042). The mitochondrial fission marker phospho-DRP1 increased by 110 ± 47% (P = 0.003), whereas the fusion marker Mfn1 and the mitophagy markers Parkin and PINK1 remained unchanged. These results fit well with a coordinated regulation of ALP and UPP triggered by FOXO3 and AMPK during ultra-endurance exercise.

Activation of autophagy in skeletal muscle has been reported in response to endurance exercise and food deprivation independently. The purpose of this study was to evaluate whether autophagy was more activated when both stimuli were combined, namely when endurance exercise was performed in a fasted rather than a fed state. Mice performed a lowintensity running exercise (10 m/min for 90min) in both dietary states after which the gastrocnemius muscles were removed. LC3b-II, a marker of autophagosome presence, increased in both conditions, but the increase was higher in the fasted state. Other protein markers of autophagy, like Gabarapl1-II and Atg12 conjugated form as well as mRNA of Lc3b, Gabarapl1, and p62/Sqstm1 were increased only when exercise was performed in a fasted state. The larger activation of autophagy by exercise in a fasted state was associated with a larger decrease in plasma insulin and phosphorylation of Akt AMPK␣Thr172 , ULK1 Ser317 , and ULK1 Ser555 remained unchanged in both conditions, whereas p38Thr180/Tyr182 increased during exercise to a similar extent in the fasted and fed conditions. The marker of mitochondrial fission DRP1 Ser616 was increased by exercise independently of the nutritional status. Changes in mitophagy markers BNIP3 and Parkin suggest that mitophagy was increased during exercise in the fasted state. In conclusion, our results highlight a major implication of the insulin-Akt-mTOR pathway and its downstream targets FoxO3a and ULK1 in the larger activation of autophagy observed when exercise is performed in a fasted state compared with a fed state.LC3b; mitophagy; signaling; fission; ER stress EXERCISE DISTURBS MUSCLE CELL HOMEOSTASIS by modifying the intra-and extracellular milieu, impairing energetic status and stretching membranes. These stressors lead to muscle remodeling by regulating transcriptional and translational events aiming at coping with further exercise-induced homeostatic disturbances. These adjustments give rise to beneficial effects of exercise for health and also increase in sports performance. However, remodeling implies that protein degradation is, at least transiently, activated. Several enzymatic systems are involved in muscle protein degradation. Besides the role of calpains, caspases, and metalloproteins, the activation of the ubiquitin-proteasome pathway in skeletal muscle during endurance exercise was the subject of particular attention during the past few years (16, 18). More recently, endurance exercise has also been identified as a stimulus that induces autophagy in this tissue (13,14,16,27).Macroautophagy, here called autophagy, is a catabolic cellular process that provides cellular constituents encapsulated inside a double-membrane vesicle called an autophagosome (AP) to lysosomes, the latter taking in charge of the degradation. Autophagy can process numerous cellular constituents, including soluble proteins, protein aggregates, and mitochondria (2, 22). Identification of autophagy genes and their related proteins (Atg) in mammals highlighted ...

In the skeletal muscle, a 200-km run activates the expression of ubiquitin ligases muscle-specific RING finger 1 and muscle atrophy F-box as well as various cellular stresses, among which are ER stress, oxidative stress, and inflammation. Meanwhile, compensatory mechanisms seem also triggered: the unfolded protein response is up-regulated, and the chymotrypsin-like activity of the proteasome is repressed.

The purpose of this study was to evaluate whether ultra endurance exercise changes the mRNA levels of the autophagy-related and autophagy-regulatory genes. Eight men (44 ± 1 years, range: 38-50 years) took part in a 200-km running race. The average running time was 28 h 03 min ± 2 h 01 min (range: 22 h 15 min-35 h 04 min). A muscle sample was taken from the vastus lateralis 2 weeks prior to the race and 3 h after arrival. Gene expression was assessed by RT-qPCR. Transcript levels of autophagy-related genes were increased by 49% for ATG4b (P = 0.025), 57% for ATG12 (P = 0.013), 286% for Gabarapl1 (P = 0.008) and 103% for LC3b (P = 0.011). The lysosomal enzyme cathepsin L mRNA was upregulated by 123% (P = 0.003). Similarly, transcript levels of the autophagy-regulatory genes BNIP3 and BNIP3l were both increased by 113% (P = 0.031 and P = 0.007, respectively). Since upregulation of these genes has been related with an increased autophagic flux in various models, our results strongly suggest that autophagy is activated in response to ultra endurance exercise.

Contrary to our hypothesis and despite an increase in the mRNA level of Redd1, an inhibitor of the mTORC1 pathway, short-term acute environmental hypoxia induced a higher response of PKB and S6K1 to a meal, which may be due to increased plasma insulin concentration.

This study compared human muscles following long-term reduced neuromuscular activity to those with normal functioning regarding single fiber properties. Biopsies were obtained from the vastus lateralis of 5 individuals with chronic (Ͼ3 yr) spinal cord injury (SCI) and 10 able-bodied controls (CTRL). Chemically skinned fibers were tested for active and passive mechanical characteristics and subsequently classified according to myosin heavy chain (MHC) content. SCI individuals had smaller proportions of type I (11 Ϯ 7 vs. 34 Ϯ 5%) and IIa fibers (11 Ϯ 6 vs. 31 Ϯ 5%), whereas type IIx fibers were more frequent (40 Ϯ 13 vs. 7 Ϯ 3%) compared with CTRL subjects (P Ͻ 0.05). Cross-sectional area and peak force were similar in both groups for all fiber types. Unloaded shortening velocity of fibers from paralyzed muscles was higher in type IIa, IIa/IIx, and IIx fibers (26, 65, and 47%, respectively; P Ͻ 0.01). Consequently, absolute peak power was greater in type IIa (46%; P Ͻ 0.05) and IIa/IIx fibers (118%; P Ͻ 0.01) of the SCI group, whereas normalized peak power was higher in type IIa/IIx fibers (71%; P Ͻ 0.001). Ca 2ϩ sensitivity and passive fiber characteristics were not different between the two groups in any fiber type. Composite values (average value across all fibers analyzed within each study participant) showed similar results for cross-sectional area and peak force, whereas maximal contraction velocity and fiber power were more than 100% greater in SCI individuals. These data illustrate that contractile performance is preserved or even higher in the remaining fibers of human muscles following reduced neuromuscular activity. chemically skinned fibers; unloaded shortening velocity; fiber power; passive tension; spinal cord injury MUSCLE UNLOADING OCCURS in a variety of conditions, such as immobilization, disease, paralysis, or exposure to microgravity. The absence of normal weight-bearing activity induces a rapid decrease in muscle mass and strength, especially of antigravity muscles (1, 25). Muscle atrophy induced by unloading is associated with several structural changes, such as modifications of the myosin heavy chain (MHC) isoform expression, inducing fiber-type transitions toward a higher proportion of fast type II fibers. Experiments based on single fiber models have demonstrated great sensitivity in detecting also a certain degree of functional variability of fibers expressing the same MHC isoforms, especially when the pattern of muscle activity changes. Human studies involving 17 days of bed rest (27, 28) or spaceflight (26) revealed that maximal single fiber force (P 0 ) from the soleus muscle was decreased, mainly as a result of a decline in fiber cross-sectional area (CSA), and that maximal unloaded shortening velocity (V 0 ) was increased. Consequently, single fiber power was either maintained or depressed, depending on the study participant or fiber type (29), with fibers expressing type I MHC being generally more affected. Muscle unloading up to 4 mo in a long-term bed-rest study induced a similar ...

Our results indicate that in vivo administration of MG132 partially prevents muscle atrophy associated with disuse and highlight an unexpected regulation of MG132 proteasome inhibitor on ubiquitin-ligases.