Ubiquitin–proteasome system markers
We next characterized the mRNA levels of E3 ubiquitin ligases transcriptionally regulated by FoxO3a (Fig. 2). We found an increase in MuRF1 at Te (+300% ± 113%) and at 3 h after exercise (+510% ± 90%; Fig. 2C) and an increase in MAFbx/atrogin-1 at 3 h after exercise (+179% ± 54%; Fig. 2D). Nonetheless, no change in Mul1 mRNA level was found (Fig. 2B). We also determined the protein expression of these three E3 ligases (Fig. 3) and found a significant increase in MuRF1 protein expression at Te (+104% ± 40%) and 3 h after exercise (+192% ± 74%) and an increase in Mul1 protein expression from 30 min to Te (Figs. 3B and A, respectively). These rises were +71% ± 23%, +118% ± 25%, +78% ± 7.9%, +123% ± 41%, and +99% ± 25% at 30 min, 60 min, 90 min, 120 min, and Te, respectively. However, we did not detect any change in MAFbx/atrogin-1 protein level (Fig. 3C). Furthermore, the ubiquitin-conjugated proteins levels were significantly increased by 52% ± 13%, 69% ± 22%, 48% ± 12%, 96% ± 19%, 125% ± 15%, and 52% ± 23% at 30 min, 90 min, 120 min, Te, 3 h, and 24 h after exercise (Fig. 3D).
Autophagy system markers
The expression of several markers of autophagy pathway is depicted in Figure 4. We found an increase in LC3B-II/LC3B-I ratio at 120 min (+131% ± 57%) and Te (+100% ± 47%), suggesting an induction of autophagy during exercise (Fig. 4A). In addition, we assessed the protein level of p62, also called sequestosome 1 (SQSTM1). p62 is a ubiquitin-binding scaffold protein that binds directly to LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy–lysosomal machinery (28). As LC3, p62 is itself degraded by lysosome. Since p62 accumulates when autophagy is inhibited, and decreased when autophagy is induced, p62 is commonly used as a marker to characterize autophagic flux (3). In accordance with LC3 analysis, p62 protein expression was significantly reduced at Te (−46% ± 11%) and a tendency was observed at 120 min (P = 0.07; Fig. 4B). Moreover, a significant increase in p62 protein expression has been found at 24 h after exercise (+65% ± 23%). Next, we assessed the phosphorylation level of Ulk1 Ser317, Ser555, and Ser757 that are known to be phosphorylated by AMPK for the first two sites and by MTOR for the last one (9,18). We reported an increase in the phosphorylation of Ser317, i.e., +110% ± 31%, +77% ± 21%, +133% ± 55%, and +132% ± 36% at 60 min, 90 min, 120 min, and Te, respectively, and an increase in Ser555 phosphorylation at 30 min (+100% ± 26%) and 60 min (+76% ± 21%) of exercise (Figs. 4C and D, respectively). A decrease in Ulk1 Ser757 phosphorylation was found at 120 min of exercise (−44% ± 26%) and at Te (−59% ± 12%), whereas an increase was obtained at 24 h after exercise (+69% ± 22%; Fig. 4E). The total form of Ulk1 was significantly increased at 90 min (+33% ± 14%), 120 min (+38% ± 21%), Te (+100% ± 39%), and at 3 h after exercise (+54% ± 40%; Fig. 4F).
Mitochondrial network remodeling markers
Next, we evaluated the modulation of major markers of mitochondrial remodeling: the phosphorylation state of DRP1 (Ser616), which is known to affect mitochondrial morphology through stimulation of mitochondrial fission, the protein levels of MFN2, a substrate of Mul1, and OPA1. The last two proteins are critical regulators of mitochondrial homeostasis because they are involved in mitochondrial fusion. Figs. 5A and B show a rise in the phosphorylation of DRP1 on Ser616 by 143% ± 63%, 178% ± 78%, 255% ± 127%, and 290% ± 108% at 60 min, 90 min, 120 min, and Te, respectively, without any change in the expression of its total form. Concerning MFN2 and OPA1, we did not find any significant variation of their protein content throughout the exercise and during recovery (Figs. 5C and D, respectively).
Protein synthesis markers
Last, we examined several major components of the Akt/MTORC1 pathway to establish whether endurance exercise may induce a decrease in protein synthesis flux (Fig. 6). We found that exercise induces hypophosphorylation of Akt Ser473 at 90 min, 120 min, and Te (−53% ± 12%, −38% ± 12%, and −52% ± 17%, respectively); MTOR Ser2448 at 90 min, 120 min, and Te (−33% ± 13%, −36% ± 5%, and −35% ± 11%, respectively); and 4E-BP1 also at 90 min, 120 min, and Te (−43% ± 13%, −59% ± 8%, and −61% ± 8%, respectively; Figs. 6A, B, and C, respectively). In addition, we assessed the Ser240/244 phosphorylation of the direct target of S6K1, rpS6, but we did not find any significant modulation, compared to preexercise condition (Fig. 6D). In sum, these results suggest that the translational machinery could be less functional, especially for the latest points of the exercise. We also assessed the phosphorylation state of the regulatory subunit of the eukaryotic initiation factor 2 (eIF2), a heterotrimeric complex that mediates the binding of tRNAmet to the ribosome in a GTP-dependent manner (Fig. 6E). The α-subunit of this factor contains the main target for phosphorylation (Ser51) and is considered as the regulatory subunit of eIF2, with its phosphorylation leading to a stabilization of the eIF2–GDP–eIF2B complex that inhibits the turnover of eIF2B (36). Thus, phosphorylation of eIF2α at Ser51 is associated with the inhibition of protein synthesis. As expected, a significant increase in the phosphorylation of this residue occurred at 60 min, 90 min, 120 min, and Te (+137% ± 29%, +101% ± 38%, +144% ± 26%, and +195% ± 25%, respectively).
Furthermore, phosphorylation of MTOR and 4E-BP1 was significantly increased at 3 h (+37% ± 11% and +93% ± 29%, respectively) and at 24 h (+32% ± 15% and +48% ± 32%, respectively) after exercise when compared to non-exercised mice.
In this study, we showed that autophagy and ubiquitin–proteasome markers, notably Mul1 protein expression, are upregulated during endurance exercise, coordinately with a downregulation of the protein synthesis pathway. While autophagy markers and phosphorylation of the mitochondrial fission marker DRP1 returned to basal level after exercise, ubiquitin–proteasome machinery markers, especially MuRF1 mRNA and protein levels, reached a peak at 3 h after exercise.
AMPK plays a major role in skeletal muscle homeostasis in response to energy stress conditions, including exercise (31). It was therefore not surprising to observe an increase in the phosphorylation state of AMPK on Thr172, which witnesses the kinase activation in response to the exercise. In our study, during the first 60 min corresponding to a low-intensity exercise, we were able to detect a fast increased AMPK phosphorylation. The low level of aerobic fitness of the sedentary mice used in the present study may contribute to explain the activation of the kinase from such a low intensity, since it is known that AMPK activation depends on the training status (26). Concerning FoxO3a, we found a decrease of Ser253 and Thr32 phosphorylation (i.e., two Akt-linked inhibiting phosphorylation) from 120 min of exercise and during the recovery period (i.e., at 3 h after exercise). This result suggests that FoxO3a is activated during exercise, especially when intensity is near to exhaustion, and also during the first hours of the recovery period. Accordingly, FoxO3a mRNA and protein levels were markedly increased at 3 h after exercise. Furthermore, among FoxO posttranslational modifications, phosphorylation of FoxO3a Ser413/588 by AMPK has been found to increase FoxO3a transcriptional activity in vitro (11,32). Thus, AMPK-mediated phosphorylation of FoxO3a could also be involved in FoxO3a activation during exercise, although we did not test this possibility because mouse-specific antibodies are not currently available.
Autophagic activity characterization represents a challenge because of the dynamics of the system. Among Atgs proteins, LC3B-II has been positively correlated with an increased number of autophagosomes and is therefore commonly used as a marker of autophagy activation (2). Nonetheless, an increase in LC3B-II expression strengthens the hypothesis of an enhanced number of autophagosomes but does not allow making clear whether this raise is the consequence of an increased autophagosome formation or a defect in their degradation through the lysosome. p62, a ubiquitin-binding scaffold protein that binds with LC3, may serve to link ubiquitinated substrates to the autophagic machinery and is itself degraded by the lysosome. Thus, modulation of p62 levels may be used as a marker to study autophagic flux (3,20). In our study, we used several markers involved in different steps of the autophagic process, LC3B-II and p62 levels, and the phosphorylation level of Ulk1 at several sites. We found that exercise increases LC3B-II and induces a drop in p62 expression near to exhaustion, suggesting an increase in autophagic flux at high intensities. These elements are in agreement with others emergent studies (12,14,19) and strengthen the idea that autophagy–lysosomal pathway is induced by exercise. Ulk1 is a key initiator of autophagy, and Ser317/555 has been recently found to be phosphorylated by AMPK in vitro during glucose starvation and autophagy activation (18). MTOR phosphorylates Ulk1 at Ser757, resulting in loss of Ulk1 kinase activity, thus preventing the initiation of autophagy. Here we showed that endurance exercise leads to an increase in the phosphorylation state of Ser317 and Ser555, suggesting that AMPK plays a role in the early step of autophagosome formation during exercise. However, these two phosphorylation sites are differentially regulated. Indeed, Ser555 is quickly and strongly phosphorylated at 30 and 60 min and thereafter go back to its basal phosphorylation level, whereas Ser317 stays phosphorylated from 60 min to Te. Although the dynamics of Ulk1 complex is not clearly established in skeletal muscle, some allosteric modifications could explain such differences. These results imply that autophagy machinery would be quickly initiated during exercise, and not only for high intensities, suggesting that autophagy may constitute a generic response to any kind of endurance activity. Moreover, Ulk1 Ser757 phosphorylation was decreased from 120 min to Te concurrently to MTOR hypophosphorylation, supporting a potential role of the inhibition of the MTOR signaling pathway in autophagy induction by exercise.
Furthermore, the present study shows for the first time a rise in the protein expression level of the E3 ligase Mul1 in response to exercise. Nonetheless, we did not detect any variation in Mul1 mRNA level, suggesting that exercise induces a stabilization of Mul1 protein. The molecular mechanisms underlying this stabilization remain to be clarified. Mitochondrial remodeling through fission and fusion is essential for the removal of damaged mitochondria. Mul1 has been shown to ubiquitinate the mitochondrial profusion protein MFN2, causing its degradation through the proteasome. Nevertheless, we did not observe a decrease in MFN2, suggesting that its degradation rates were unaltered. Thus, the rise in Mul1 expression that we observed during exercise was probably not sufficient to induce MFN2 degradation. In the same way, OPA1 protein level, another GTPase required for mitochondrial fusion, was not significantly altered during exercise. Mul1 has also been reported to stabilize the mitochondrial fission protein DRP1 in vitro, resulting in mitochondrial fragmentation (4), but these events remain to be tested in skeletal muscle. Phosphorylation of DRP1 at Ser616 stimulates mitochondrial fission and such an increase has been reported to be permissive for mitophagy (37). Here, we found that DRP1 phosphorylation state was progressively increased until exhaustion. All together, these results indicate that endurance exercise quickly promotes DRP1 activation, potentially stimulating mitochondrial fission, but not Mul1-dependent degradation of mitochondrial fusion markers contrary to what it has been reported during muscle wasting (23). Although we did not establish whether Mul1 regulates mitophagy in this study, this certainly warrants further investigations especially with a view to characterize other Mul1 partners or targets than MFN2 and to investigate the possible differential effect of acute exercise between slow and fast skeletal muscles.
Concerning protein synthesis pathway, we found that Akt, MTOR, and 4E-BP1 phosphorylation was inhibited from 90 min to Te. While protein flux synthesis has not been performed, these results suggest that protein synthesis is inhibited during endurance exercise. The significant increase in eIF2α phosphorylation observed from 60 min of exercise highlights these hypotheses. Furthermore, the slow but significant increase in MTOR phosphorylation and the subsequent rise in 4E-BP1 phosphorylation found at 3 and 24 h after exercise suggest an increase in protein translation and possibly protein synthesis during the recovery period. Endurance training is well known to promote a fast-to-slow muscle phenotype shift and mitochondrial biogenesis but not to induce muscle growth. Thus, a rise in protein synthesis in response to endurance exercise would be necessary for tissue repair and remodeling, especially for the synthesis of specific subcellular protein not involved in muscle hypertrophy as mitochondrial proteins. In contrast, force and resistance training are two major stimuli of muscle protein synthesis resulting in hypertrophy.
The physiological relevance of these events can be speculated. In our model, AMPK and/or Akt inhibition would modulate FoxO3a activity and Ulk1 axis to mobilize protein degradation as source of alternative nutrient production and energy substrates when exercise intensity/duration becomes high. In addition, autophagy system is known to be required for normal cellular function and for response to multiple types of stress to maintain skeletal muscle function. Thus, during prolonged exercise, THE autophagy–lysosomal pathway may represent a compensatory mechanism to prevent cellular loss function caused by constraints linked to metabolic stress.
In conclusion, the present study aimed to identify specific signaling events related to cellular component turnover that are modulated during endurance exercise. To the best of our knowledge, this study is the first to give a picture of the regulation of pathways implicated in protein balance and which consider mitochondrial dynamics markers at different points of endurance exercise. Our data support the idea that Akt/MTOR pathway inhibition and AMPK activation modulate protein turnover through FoxO3a and Ulk1 axes. Noticeably, the mitochondrial Mul1 ubiquitin ligase is quickly induced by endurance exercise, but this induction is not sufficient to trigger mitochondrial fusion markers alteration. Further investigations are needed to better understand the overall implications of AMPK or other metabolic sensors in the regulation of mitophagy and to also identify the precise role of Mul1 in these events. On the basis of these data, it is clearly conceivable that the involvement of the autophagic system in response to exercise must be considered not only in muscle homeostasis but also in disease. Because exercise is associated with improved quality of life and constitutes one of the best approaches to limit atrophy and metabolic disorders, these research directions are crucial in the fight against a wide spectrum of metabolic and muscle diseases.
The authors would like to thank the rodent animal facility of the laboratory Muscle Dynamics and Metabolism (RAM, Campus La Gaillarde, INRA, Montpellier) and the METAMUS platform dedicated to the functional exploration metabolism. The authors thank J. Glaviole for helpful discussion.
This project was supported by the Faculty of Sport Sciences of the University of Montpellier 1 and the Institut National de la Recherche Agronomique (INRA). No conflict of interest, financial or otherwise, is declared by the authors.
The results of the present study do not constitute endorsement by American College of Sports Medicine.
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Keywords:© 2014 American College of Sports Medicine
AMPK; PROTEIN DEGRADATION; MITOPHAGY; MITOCHONDRIA; ENDURANCE EXERCISE