Autophagic cellular responses to physical exercise in skeletal muscle.

Sports Med DOI 10.1007/s40279-013-0140-z

REVIEW ARTICLE

Autophagic Cellular Responses to Physical Exercise in Skeletal Muscle Bjorn T. Tam • Parco M. Siu

Ó Springer International Publishing Switzerland 2014

Abstract Autophagy is an evolutionarily conserved biological process that functions to recycle protein aggregate and malfunctioned organelles. The activation of autophagy can be stimulated by a number of ways including infection, caloric restriction, and physical exercise. In addition to cellular metabolism and cell survival/death machinery, autophagy plays an important role in the maintenance of cellular homeostasis in skeletal muscle especially during physical exercise in which energy demand can be extremely high. By degrading macromolecules and subcellular organelles through the fusion of autophagosomes and lysosomes, useful materials such as amino acids can be released and re-used to sustain normal metabolism in cells. Autophagy is suggested to be involved in glucose and lipid metabolism and is proposed to be a critical physiological process in the regulation of intracellular metabolism. The effects of physical exercise on autophagy have been investigated. Although physical exercise has been demonstrated to be an autophagic inducer, cellular autophagic responses to exercise in skeletal muscle appear to be varied in different exercise protocols and disease models. It is also not known whether the exercise-induced beneficial health consequences involve the favorable modulation of cellular autophagy. Furthermore, the cellular mechanisms of exercise-induced autophagy still remain largely unclear. In this review article, we discuss the general principle of autophagy, cellular signaling of autophagy, autophagic responses to acute and chronic aerobic exercise, and the potential cross-talks among autophagy, mitochondrial

B. T. Tam P. M. Siu (&) Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China e-mail: [email protected]

biogenesis, and ubiquitination. This article aims to stimulate further studies in exercise and autophagy.

1 Introduction Skeletal muscle is an organ with remarkable adaptive capability in the body [1, 2]. Conventionally, the main function of skeletal muscle is limited to the control of movement and posture. Recently, skeletal muscle was found to be related to various health problems such as obesity and type 2 diabetes mellitus [2]. Accumulating findings suggest that skeletal muscle plays a central role in orchestrating the metabolism and functions of different organs. Lack of physical activity owing to different reasons, such as microgravity [3], cancer cachexia [4, 5], and aging [6, 7], may lead to muscle atrophy and other consequences that are detrimental to human health. Different interventions using physical exercise are shown to be useful to counteract the negative consequences brought by muscle atrophy [8–10]. However, how metabolic and molecular remodeling of skeletal muscle mediates the benefits of exercise remains unclear. Malfunction of autophagy may cause different diseases such as Parkinson disease, amyotrophic lateral sclerosis, and Alzheimer disease [11]. Moreover, understanding the molecular pathways that rule muscle growth and death is indispensable to improve human health. Autophagy, a catabolic machinery of muscle cells, is undoubtedly an important mechanism in human development and exercise adaptation as the cytosolic rearrangement after exercise might require the mediation of autophagy. Autophagy is a lysosomal pathway involving the degradation of protein aggregates and subcellular organelles in cells. The degraded products can be recycled to yield energy to support cell

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metabolism. There are three different kinds of autophagy, namely microautophagy, chaperone-mediated autophagy, and macroautophagy. In this review, we discuss the cellular responses of macroautophagy, which is primarily responsible for the elimination of macromolecules and subcellular organelles [12], to physical exercise in skeletal muscle. In myofiber atrophy, the rate of protein breakdown operates over the rate of protein synthesis. In eukaryotic cells, there are two proteolytic systems involved in protein degradation: the ubiquitin–proteasome system and the autophagy lysosomal system. For the autophagy lysosomal system, a double membrane named phagophore is formed to engulf the target proteins and/or organelles. An autophagic vacuole named autophagosome is formed after sequestering the target materials. Then, the autophagosome fuses with the lysosome to create an autolysosome and the autophagosome is degraded by lysosomal enzymes [13]. Muscle mass has been demonstrated to be affected by the ubiquitin–proteasome system and the autophagy lysosomal system. The over-activation of the systems has been suggested to result in muscle atrophy. In the following paragraphs, we focus on the molecular mechanisms of the autophagy lysosomal system and provide evidence showing the regulation of autophagy through physical exercise.

target of the therapeutic strategy. In a cell culture study, H2O2-treated senescent fibroblasts have been shown to be protected from apoptosis by the activation of autophagy [21]. In contrast, the dysregulation of autophagy has been shown to contribute to myopathy. A recent study [22] indicated that defective autophagy contributes to skeletal muscle cell death, which leads to collagen VI muscular dystrophies. Intriguingly, re-activation of autophagy by different means was demonstrated to restore myofiber survival and relieve the dystrophic muscle phenotype of the collagen VI-null mice. The Forkhead box protein transcription factors (FoxO) have been shown to be upregulated through the expression of inhibitor of pyruvate dehydrogenase complex leading to 50 AMP-activated protein kinase (AMPK) activation. This reinforces the cytoprotective role of autophagy in cells as AMPK inhibits the mammalian target of rapamycin (mTOR) [23]. mTOR is extensively manipulated to induce or to suppress autophagy. Suppression of mTOR relaxes the inhibition of autophagy, which recycles the protein aggregates. However, a study has shown that the use of mTOR inhibitors (e.g., rapamycin) might lead to adverse effects on inclusion body myopathy [24]. Therefore, it is noted that the activation of autophagy induced by the inhibition of mTOR may not necessarily deliver beneficial effects to skeletal muscle.

2 Physiological Function of Autophagy Autophagy has physiological functions in maintaining cellular homeostasis especially during energy-deficit situations. Mice lacking the autophagy-related protein (Atg) Atg5 at birth have been shown to die within 1 day of delivery [14]. This result has proved that the conversion of intracellular proteins to amino acids is important in the restoration of energy homeostasis during early development. Besides maintenance of homeostasis, autophagy at the basal level in different cells can assure the intracellular quality of cells. Ubiquitinated proteins and other malfunctioned protein aggregates accumulate in cells [15]. Tissue-specific knockout models have shown the importance of autophagy in different tissues. Liver-specific Atg7-/- mice have been demonstrated to experience hepatomegaly and hepatic failure [16]. Neural cell-specific Atg5 and Atg7 knockout mice were shown to develop neurodegeneration and progressive motor deficits [15]. In fact, many neurodegenerative diseases including Alzheimer disease, Parkinson disease, and Huntington disease are related to aberrant autophagy. Intriguingly, the symptoms of these diseases were shown to be relieved or prevented by activating autophagy [17–20]. Therefore, it is very likely that the accumulation of long-lived proteins contributes to the pathology of the neurodegenerative disorders. Reactivation of autophagy might be proposed as a potential

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3 Molecular Mechanism of Autophagy Autophagy is highly inducible. Physical exercise [25, 26], nutrient starvation [27], and withdrawal of growth factor have been shown to induce autophagy [28]. In the above stresses to muscle cells, autophagy is crucial in recycling of macromolecules to provide nutrients and energy to muscle cells. A number of Atg proteins have been identified to control the major steps of autophagy. To date, 31 Atg genes have been identified [29, 30]. Autophagy consists of several essential processes namely initiation, nucleation, elongation, fusion, and degradation. These processes are tightly regulated to locate and gather the targeted organelles and long-lived protein to autophagosomes, which will then be fused with lysosomes for breakdown. The contents inside the autophagosomes will ultimately be degraded by the hydrolytic enzymes provided by the lysosomes [31]. 3.1 Initiation The formation of an autophagosome starts from a phagophore, although the origin of the phagophore remains unclear. The formation of the phagophore should be built up on a platform called a phagophore assembly site [32]. The origin of the phagophore has been proposed to be from

Exercise-Induced Autophagy in Skeletal Muscle

plasma membrane or organelles possessing a lipid membrane [33–36]. Both endoplasmic reticulum [33] and mitochondria [35] are suspected to be the source of the phagophore. In summary, no conclusion on the exact origin of the phagophore has been reached as yet. mTOR is a central switch for the activation of autophagy (Fig. 1). Autophagy has been demonstrated to be induced by the inhibition of mTOR because of pharmacological induction [37], nutrient deprivation [27], and exercise training [25]. A stable complex has been shown to form by mTOR, serine/threonine-protein kinase (Ulk1), FAK-family interacting protein (FIP200), Atg13, and Atg101 under a nutrient-rich environment. Conversely, under a nutrient-deficient situation, mTOR detaches from the complex. The remaining multi-protein complex localizes to the phagophore [38, 39]. 3.2 Nucleation and Elongation Nucleation of the phagophore requires a complex comprising of phosphoinositide 3-kinase (class III PI3K complex), Beclin-1, vacuolar protein sorting (Vps) 15, ambra1, Vps34, and Atg14/ultraviolet irradiation resistance-associated tumor suppressor gene (UVRAG). Immunoprecipitation analysis has shown that the class III PI3K complex contains either Atg14 or UVRAG [40]. The complex containing Atg14 has been found during the early autophagosome formation and the complex containing UVRAG was found during endosome maturation [41]. Both B-cell leukemia/lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-XL) are able to bind to Beclin1. This binding inhibits autophagy because Bcl-2 affects the interaction between the Vps34 and Beclin-1 [42]. Phosphorylation of Bcl-2 or Beclin-1 has been shown to result in the dissociation of Bcl-2 from Beclin-1 [25]. It has been demonstrated that C-Jun N-terminal kinase 1 (JNK1) can phosphorylate Bcl-2 [43, 44]. Therefore, JNK1 can also trigger autophagy by releasing Beclin-1 from the binding of Bcl-2. In addition, Beclin-1 has been shown to be phosphorylated by the death-inducing kinase (DAPkinase) [45]. The unbound Beclin-1 is proposed to be a key autophagic protein governing autophagy by recruiting Atg proteins for autophagosome formation (Fig. 1) [46]. The elongation of an autophagosome involves different Atgs. Briefly, Atg5 covalently binds to Atg12 through the catalysis of Atg7 and Atg10. The Atg5–Atg12 complex then interacts with Atg16 to form the Atg12–Atg5–Atg16 complex, which later mediates the conjugation of phosphatidylethanolamine (PE) to the microtubule-associated protein 1 light chain 3-I (LC3-I) to form LC3-II, which binds to the autophagosome membrane (Fig. 1). There are several steps involved in the formation of LC3-II from LC3. The LC3 has an amino acid tail. Atg4 removes the

C-terminal arginine of LC3; then, the glycine residue left on the LC3 is activated by Atg7, which then transfers the LC3-I (the activated form of LC3) to Atg3. Finally, the PE binds to the LC3-I through the mediation of Atg3 and the Atg12–Atg5–Atg16 complex to form LC3-II [29, 47]. The amount of LC3-II has been shown to correlate with the extent of autophagosome formation [48]. 3.3 Maturation and Degradation After elongation, the autophagosome fuses with the lysosome, an acidic and enzyme-rich organelle, for the degradation of proteins inside the autophagosome. During maturation, the small GTP-binding protein Ras-related protein (Rab7) and lysosomal membrane protein 1 (LAMP1) and 2 (LAMP-2) are required to facilitate maturation and degradation [49, 50]. Amino acids are exported to the cytosol after the degradation process through the lysosomal amino acid transporter 1 and 2 [51, 52]. During exercise, skeletal muscle is damaged mechanically and metabolic homeostasis is disturbed. Under such a stressful condition, skeletal muscle can remove damaged proteins and organelles by autophagy to release useful materials to the nutrient pool inside the muscle cells (Fig. 1). Therefore, this series of molecular mechanisms has been proposed to play an important role in maintaining homeostasis of skeletal muscle during physical exercise [15, 20, 53].

4 Role of FoxO Transcription Factors in Autophagy in Skeletal Muscle 4.1 FoxO-Mediated Autophagy in Skeletal Muscle It has been reported that activation of FoxO3 is essential for fiber atrophy and the expression of atrogin-1 during muscle denervation and fasting [54]. Besides activation of the ubiquitin–proteasome system, the autophagy lysosomal system has been shown to mediate the process of muscle proteolysis. FoxO3 induces the expression of different Atg genes such as LC3-II, gamma-aminobutyric acid receptorassociated protein-like 1 (GABARAPL1), Vps34, Ulk2, Atg12, Atg4, and Beclin-1 in denervated and fasted adult mice [55]. Recent works have shown that post-translational modifications of FoxOs are important in the coordination of muscle mass. The activity of FoxOs can possibly be altered by different mechanisms such as phosphorylation, protein-protein interaction, and acetylation. Phosphorylation of FoxO3 by AMPK or serine/threonine-specific protein kinase (Akt) may activate or inhibit the transcriptional activity of FoxO3. Phosphorylation of FoxO3 by Akt causes inhibition of transcriptional activity whereas

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Fig. 1 The key molecular signaling pathways and cellular events in exercise-induced autophagy. (1) Exercise activates AMPK as the AMPto-ATP ratio increases. Moreover, the skeletal muscle contractions during exercise stimulate Akt. A downstream target of AMPK and Akt is mTOR, which can be inhibited by AMPK and activated by Akt simultaneously. mTOR is regarded as a central switch that balances the input signals from AMPK and Akt. In exercise-induced autophagy, mTOR may exert an inhibitory effect on Ulk1/2, which leads to the induction of autophagy. Ulk1/2 can also phosphorylate Beclin-1, which leads to the detachment of Beclin-1 from Bcl-2. In addition, exercise increases the activity of JNK, which phosphorylates Bcl-2. Phosphorylated Bcl-2 can also detach from Beclin-1. Unbound Beclin-1 induces autophagy. (2) Exercise leads to phosphorylation of FoxO, which is mediated by AMPK. After phosphorylation, the transcriptional activity of FoxO will be enhanced. FoxO is an important transcription factor of different autophagy-related genes such as Bnip3, LC3, and Atgs. (3) During autophagy induction, the formation of autophagosome starts from a phagophore, which is suspected to develop from membranes of mitochondria and endoplasmic reticulum. Nucleation of phagophores requires a complex comprised of Beclin-1. Elongation of autophagosomes involves different Atg proteins. Atg5 covalently binds to Atg12 through the catalysis of Atg7 and Atg10. The Atg5–Atg12 complex

will then interact with Atg16 non-covalently to form the Atg12–Atg5– Atg16 complex, which mediates conjugation of PE to the LC3-I (which is formed by cleaving LC3 at its C-terminus by Atg4) to form LC3-II that binds to autophagosome membranes. Autophagosomes sequester malfunctioned organelles and long-lived proteins. Finally, autophagosomes fuse with lysosomes, which degrade the contents inside autophagosomes. (4) Activation of AMPK through exercise triggers deacetylation of PGC-1a, which is a transcription coactivator. The deacetylated PGC-1a stimulates mitochondrial biogenesis and angiogenesis, which enhance fiber-type switching. By the mediation of AMPK, autophagy and mitochondrial biogenesis may be activated simultaneously. Any mismatch between them may have detrimental effects to cells. AMP Adenosine monophosphate, AMPK 50 AMPactivated protein kinase, Atg Autophagy-related proteins, ATP Adenosine triphosphate, Akt Serine/threonine-specific protein kinase, Bcl-2 B-cell leukemia/lymphoma-2, Bnip3 Bcl-2/adenovirus E1B interaction protein 3, FoxO Forkhead box protein, JNK c-Jun N-terminal kinases, LC3 Microtubule-associated protein 1 light chain 3, mTOR Mammalian target of rapamycin, PE Phosphatidylethanolamine, PGC-1a Peroxisome proliferator-activated receptor gamma coactivator 1-alpha, PPARc Peroxisome proliferator-activated receptor gamma, Ulk1/2 Unc-51-like kinase 1/2

phosphorylation of FoxO3 by AMPK on Ser588 leads to the upregulation of transcriptional activity (Fig. 1) [56]. For protein-protein interaction, no interactions have been detected between FoxO3a and Beclin-1, Ulk1, Atg13,

Atg5, LC3, or GABARAPL1. These results have indicated that FoxO3a acts only as the transcription factor [56]. It has been postulated that FoxO3 interacts with the histone acetyl-transferase p300, and the acetylation of FoxO3

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causes cytosolic degradation via the proteasome system [57]. Although the effect of the acetylation of FoxO3 on autophagy remains unclear, it is possible that the degradation of FoxO3 suppresses autophagy as FoxO3 is known to induce autophagy in skeletal muscle [58, 59]. Simultaneously, p300 has been shown to be responsible for the acetylation of different Atg proteins. The knockdown of p300 has been used to experimentally activate autophagy [60]. Therefore, p300 can regulate both the FoxO3 and Atg proteins by acetylation. Fine regulation of FoxO family is important in regulating autophagy. It is noted that the relationship between exercise and FoxO expression remains unclear. Additional studies are needed to explore how exercise regulates FoxO expression and post-translational modifications of transcription factors and kinases in muscle autophagy. 4.2 Links Between the Ubiquitin Proteasome System and the Autophagy Lysosomal System A link has been evidently proposed between the ubiquitin proteasome system and the autophagy lysosomal system [56, 57, 61]. Recently, deficiency of MuRF1 has been demonstrated to cause elevation of expression of Muscle Atrophy F-box (MAFbx) and reduction of expression of Beclin-1 in skeletal muscle after denervation, suggesting that deficiency of MuRF1 may downregulate the denervation-associated activation of autophagic signaling [62]. As FoxO3 has been shown to regulate both the ubiquitin proteasome system and the autophagy lysosomal system, the change in expression of FoxO3 can unsurprisingly affect the two systems simultaneously [63]. The molecular mechanisms underlying the FoxO3-mediated connection between the ubiquitin–proteasome system and the autophagy lysosomal system remain largely unclear. More studies are required to gain a clear picture of the link and interactions between autophagy and the proteasome system in skeletal muscle. Under some conditions such as ultra-endurance exercise [64] and muscle atrophy [65], the ubiquitin proteasome system and the autophagy lysosomal system have been shown to be activated simultaneously. Failure of the activation of autophagy has been found in Atg5-/- or Atg7-/mice, leading to the aggregation of ubiquitinated proteins [66, 67]. It has been speculated that the ubiquitinated proteins could be degraded via autophagy as significant accumulation of ubiquitin is uncommonly seen in mice with basal autophagy [12]. As the ubiquitin proteasome system and the autophagy lysosomal system have been shown to be responsive to physical exercise, it is possible that exercise can be taken as an intervention used to preserve muscle mass possibly through the mediation of these two cellular systems [67]. In the next section, we discuss the effects of physical exercise on skeletal muscle autophagy.

5 Effects of Physical Exercise on Skeletal Muscle Autophagy Different responses of autophagy in skeletal muscle have been reported following different kinds of physical exercise, namely single bout exercise exposure and habitual/ regular chronic exercise training. In this part, we discuss different autophagic responses to a single bout of aerobic exercise and chronic aerobic exercise training. The findings of the effects of acute and chronic exercise on muscle autophagy are summarized and presented in Tables 1 and 2, respectively. 5.1 Acute Effect of Single Bout of Aerobic Exercise LC3-II and Atg12–Atg5 conjugation have been demonstrated to be increased after ultra-endurance exercise [68]. Both LC3-II and Atg12-Atg5 conjugation are suggested to be related to damage of muscle tissue. They are also regarded as two important protein complexes in regulating the size of autophagosomes [13, 69] and imposing curvature on the crescent phagophore [45]. Moreover, it is important to keep replenishing the LC3 protein so as to maintain autophagy because LC3 is continuously consumed during autophagy [59]. During the situation of energy deficit such as ultra-endurance exercise, muscle protein is degraded into amino acids to provide energy to muscle cells to sustain the demand of energy metabolism. Inverse correlation between Akt activity and autophagy induction has been demonstrated in wild-type muscle after 1 h of treadmill exercise [70]. The decrease in the phosphorylation state of Akt has been suspected to cause the increase in the LC3-II/LC3-I ratio. The dephosphorylation of Akt has been postulated to be associated with the dephosphorylation of FoxO3 [63]. Dephosphorylated FoxO3 can freely translocate to the nucleus for the transcription of other genes. Upregulation of LC3 has been shown to be regulated by FoxO3 [59]. Therefore, it is reasonable to suspect that ultra-endurance exercise might stimulate autophagy via dephosphorylation of FoxO3. An ultra-endurance running study has shown that the phosphorylation state of FoxO3 is decreased by about 50 % and the expression of LC3-II is increased by more than fivefold after exercise [68]. The effect of dephosphorylation of FoxO3 is not only limited to the regulation of autophagy but extended to the effects on the ubiquitin proteasome system. MuRF1 protein expression has been shown to be increased after ultra-endurance exercise [68]. The alterations of the ubiquitin proteasome system and the autophagy lysosomal system have been suggested to be regulated by the FoxO3–Bnip3 pathway in C2C12 myotubes [55, 59]. This may imply a link between the ubiquitin proteasome system and the autophagy lysosomal system during skeletal

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B. T. Tam, P. M. Siu Table 1 Autophagic response to short-term exercise Animal type

Exercise intervention

Major findings on autophagic responses

References

Mice

Wild-type mice performing 80 min (*900 m) of running or maximal exercise (soleus, tibialis anterior and extensor digitorum longus)

:Number of autophagosomes (*48 % in soleus; 93 % in tibialis anterior; 83 % in extensor digitorum longus)

[25]

BCL2AAA mice performing 80 min (*900 m of running) or maximal exercise (tibialis anterior and extensor digitorum longus)

:Conversion of LC3-I to LC3-II :Degradation of p62 ;Bcl-2–Beclin-1 complex ;Plasma insulin (*90 %) after performing maximal exercise ;Blood glucose (*51 %) after performing maximal exercise :Number of autophagosomes (*54 % in tibialis anterior; *33 % in extensor digitorum longus) ;Conversion of LC3-I to LC3-II ;Degradation of p62 ;Dissociation of the Bcl-2–Beclin-1 complex ;Plasma insulin (*53 %) after maximal exercise

Mice

Mice exercising on a treadmill for 50 min at a speed of 12.3 m/min with a slope of 5° (gastrocnemius)

;LC3-II ([90 % at 3, 6, and 12 h compared with the control)

[79]

;Beclin-1 ([90 % immediately after exercise; *80 % at 3 h; *62 % at 6 h; *52 % at 12 h) ;Atg7 (*50 % immediately after exercise; *53 % at 3 h; *80 % at 6 h; *70 % at 12 h) ;Atg12-5 (*30 % immediately after exercise; *23 % at 3 h; *50 % at 6 h; *81 % at 12 h) ;Lamp2a (no significant change after exercise; *67 % at 3 h; *76 % at 6 h; *84 % at 12 h)

Mice

WT mice running for 1 h, speed increasing from 10 to 40 cm/s in the training hour

:LC3-II/LC3-I (*580 %, compared with the wild-type sedentary group)

Col6a-/- mice running for 1 h, speed increasing from 10 to 40 cm/s in the training hour

;P-Akt (*50 %, compared with the wild-type sedentary group)

[70]

;LC3-II/LC3-I (*89 %), compared with the wild-type exercise group) :P-Akt (*205 %, compared with the wild-type exercise group)

Humans

Male subjects running at their freely chosen speed for 24 h without sleep (vastus lateralis)

:LC3-II (554 %)

[68]

:Atg12-5 (36 %) No significant change for Atg7, Atg3, and Beclin-1 ;Phosphorylation state of Akt (*74 %) ;Phosphorylation state of FoxO3a (*49 %) ;Phosphorylation state of mTOR at Ser2448 (*32 %) ;Phosphorylation state of 4E-BP1 (*34 %) :Phosphorylation state of AMPK (*247 %)

Humans

Male runners running a 200-km race in, on average, 28 h 3 min (vastus lateralis)

:Atg 4b mRNA (*49 %)

[64]

:Atg 12 mRNA (*57 %) :Gabarapl1 mRNA (*286 %) :LC3 mRNA (*103 %) :Cathepsin L mRNA (*123 %) :Bnip3 and Bnipl mRNA (*113 %)

4E-BP1 4E-binding protein 1, Akt serine/threonine-specific protein kinase, AMPK 50 AMP-activated protein kinase, Atg autophagy-related proteins, Atg12-5 Atg 12-5 complex, Bcl-2 B-cell leukemia/lymphoma-2, BCL2AAA mice knock-in mutation mice without exercise-induced phosphorylation, Bnip3 Bcl-2/adenovirus E1B interaction protein 3, Bnipl Bcl-2/adenovirus E1B interaction protein 3-like, FoxO3a forkhead box protein 3a, Gabarapl 1 gamma-aminobutyric acid receptor associated protein-like 1, Lamp2a lysosomal membrane protein 2a, LC3-I microtubule-associated protein 1 light chain-I, LC3-II microtubule-associated protein 1 light chain-II, mTOR mammalian target of rapamycin, p62 sequestosome, : increased, ; decreased

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Exercise-Induced Autophagy in Skeletal Muscle Table 2 Autophagic response to long-term exercise Animal type



References

Mice

Functional overload: mice were subjected to bilateral functional overload of plantaris muscle by surgically removing their soleus and one-half of the gastrocnemius muscles (plantaris)

:Akt Ser-473 phosphorylation (*100 % on seventh day of FO)

[114]

Myostatin null mice swimming at 35–37 °C for 60 min/day, five times a week, for 5 weeks (digitorum longus, gastrocnemius, rectus femoris)

:LC3 mRNA (*93 % on 2nd day after training)

Myostatin null mice running in unidirectional running wheel for 2–2.5 km/day

:Bnip3 mRNA (*48 % on 2nd day after training) :CathepsinL mRNA (*107 % on 2nd day after training)

Wild-type mice running (or swimming) according to the above protocols

:Beclin-1 mRNA (*112 % on 2nd day after training)

Mice

Mice

Wild-type mice running voluntarily for 3 months Col6a-/- mice running voluntarily for 3 months

Mice

:p70s6k Thr-389 phosphorylation (*185 % on seventh day of FO)

[90]

:Bnip3 mRNA (*15 % on 2nd day after training) ;p62 mRNA (*20 % on 2nd day after training) :CathepsinL mRNA (*27 % on 2nd day after training)

:ATF4 mRNA (*84 % on 2nd day after training) No significant change in the following mRNA: LC3, Bnip3, p62, CathepsinL, ATF4, and Beclin-1 No significant change in the LC3-II/LC3-I ratio

[70]

;LC3-II/LC3-I ratio (*56 %, compared with the wildtype running group) :p-Akt (*460 %)

4-Week voluntary running

Two muscles were analyzed

Protein expression in two different types of muscle would be analyzed

Plantaris (mixed fiber type)

[92]

:LC3-II (*92 %) :LC3-II/LC3-I (*36 %) ;p62 (*18 %) :LC3 (*77 %) :Atg6 (*45 %) :Bnip3 (*47 %) Soleus (oxidative fiber type) :LC3-II (*83 %) :p62 (*51 %) :LC3 (*103 %) :Atg7 (*67 %) :Atg6 (*50 %)

Mice

Wild-type mice running on a treadmill for 40 min at 16.3 m/min on a slope of 5, 5 days/week for 8 weeks

For young mice (2 months old)

[93]

;Beclin-1 in EDL (*39 %) ;Beclin-1 in gastrocnemius (*50 %) ;Atg7 in EDL (*18 %) For old mice (10 months old) :LC3-II in EDL (*62 %) :LC3-II in gastrocnemius (*26 %) :Beclin-1 in EDL (*60 %) :Atg7 in EDL (*32 %) :MuRF-1 in EDL (*80 %) :MuRF-1 in gastrocnemius (*65 %)

Rats

Following the initial adaptation phase, rats exercising for 60 min/day at 30 m/min for 5 days (approximately equal to 70 % of maximum oxygen consumption) (soleus)

No significant change was found in Atg12 mRNA, Atg12 protein, Atg12–Atg5 protein, Atg4 mRNA, Atg4 protein, Atg7 protein, LC3 mRNA, LC3-II/ LC3I, Cathepsin B mRNA, Cathepsin D mRNA, Cathepsin L mRNA, and Cathepsin L protein

[84]

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B. T. Tam, P. M. Siu Table 2 continued Animal type



References

Rats

Rats at 24 months of age with 8 % life-long calorie restriction performing voluntary exercise (peak running activity: 2,500 m/day; average: 1,145 m/day) (plantaris)

No significant change (compared with rats with 8 % life-long calorie restriction only) was found in Beclin1, LC3-II/I ratio, and LC3 mRNA

[27]

:Atg7 (*42 %) :Atg9 (*43 %) :Lamp2 mRNA (*129 %)

Rats

Rats running at 20 m/min and a grade of 5° for 1 h per day, 6 days per week for 8 weeks (soleus)

:Atg7 (*80 %)

[87]

:Beclin-1 (*197 %) :LC3 (*124 %) ;PGC-1a (*31 %) :p53 (*145 %) :p21 (*67 %) :MnSOD (*88 %) :FoxO3mRNA (*115 %)

Rats

Diabetic rats swimming for 1 h per day, 5 days per week for 4 weeks (digitorum longus)

;LC3 (*55 %) compared with the non-exercise group

[91]

Humans

Following the initial adaptation phase, participants (overweight women) walking at an intensity level of 13 on the Borg Perceived Exertion scale and performing strength training at a level of 15–16 for 6 months (vastus lateralis)

:Atg7 mRNA (threefold)

[89]

:LC3 mRNA (threefold) :Lamp2 mRNA (threefold, p = 0.07) :FoxO3A mRNA (eightfold, p = 0.057)

Akt serine/threonine-specific protein kinase, ATF4 activating transcription factor 4, Atg autophagy-related proteins, Bnip3 Bcl-2/adenovirus E1B interaction protein 3, Bnipl Bcl-2/adenovirus E1B interaction protein 3-like, EDL extensor digitorum longus, FoxO3 forkhead box protein 3, FoxO3a forkhead box protein 3a, Gabarapl 1 gamma-aminobutyric acid receptor associated protein-like 1, Lamp2 lysosomal membrane protein 2, LC3 microtubule-associated protein 1 light chain 3, LC3-I microtubule-associated protein 1 light chain-I, LC3-II microtubule-associated protein 1 light chain-II, MnSOD manganese superoxide dismutase, MuRF-1 muscle-specific RING finger protein-1, P21 cyclin-dependent kinase inhibitor 1, P53 tumor protein 53, p62 sequestosome, p70s6k p70S6 kinase, PGC-1a peroxisome proliferator-activated receptor gamma coactivator 1-alpha, : increased, ; decreased

muscle degradation after performing a long duration of endurance exercise. Similarly, another study has demonstrated that ultra-endurance exercise increases the gene expression of Atg4b, Atg12, cathepsin L, GABARAP1, LC3, Bnip3, and Bnip3l [64]. GABARAP1 and LC3 are homologs of Atg8, which are important in the formation of the autophagosome [71]. Generally, cathepsin L is regarded as a typical marker of muscle protein breakdown [72]. The increased expression of cathepsin L may indicate the high rates of protein turnover. Bnip3 and Bnip3l have been shown to induce autophagy by disrupting the Bcl-2–Beclin1 complex and by antagonizing the activity of Bcl-2 [73]. Although the gene expressions of the autophagic factors have been shown to be increased after ultra-endurance exercise, the protein expressions of the key autophagyrelated factors have not been examined. It is worth noting that variations might exist for the correlation of mRNAprotein expressions [74]. Even though findings related to the expression of autophagic factors at mRNA level are valuable for understanding the mechanisms of exercise-

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induced autophagy, the mRNA data might not be exactly predictive of protein alteration [74, 75] and, more importantly, direct evidence of activation of autophagy. In addition, it is worth noting that the intensity and duration of exercise and the food and water intake were unable to be controlled in the situation of this ultra-endurance exercise study. Athletes were permitted to eat and drink ad libitum during the experimental period. If some athletes chose to eat less, caloric deficiency or restriction can be a factor influencing autophagy [64]. Acute treadmill exercise has been shown to induce autophagy in skeletal muscle [25]. Key autophagic proteins including LC3-II and p62 are found to be significantly altered after exercise. The change in autophagic proteins is consistent with the observation of increased autophagosomes after exercise. More importantly, disruption of the Bcl-2–Beclin-1 complex is demonstrated to be the key step in autophagy-mediated exercise adaptation. Bcl-2–Beclin-1 complexes are found to be largely disrupted 30 min after exercise. Subsequently, genetic mutant Bcl-2AAA mice


(mice lacking three conserved phosphorylation residues for impairing exercise-induced phosphorylation), Beclin-1?/mice, and Atg16L1HM mice (both mice exhibit deficiency of exercise-induced autophagy in skeletal muscle) are used to further demonstrate the importance of autophagy on glucose metabolism [25]. The mutant mice have been shown to have impaired redistribution of glucose transporters 4 (GLUT4) in muscle in response to exercise. This impairment has been shown to be associated with decreased exercise-induced AMPK phosphorylation, which is known to be important in the regulation of localization of GLUT4 and glucose metabolism in skeletal muscle [76]. In the same study, exercise training has been shown to improve the glucose tolerance in mice fed with a high-fat diet but not in Bcl-2AAA mice (mice that are incapable of inducible autophagy in skeletal muscle). This suggests that activation of autophagy induced by physical exercise is critical to mediate the exercise-induced improvement of glucose metabolism in diet-induced obesity [25]. Nonetheless, deficiency of autophagy in skeletal muscle has also been shown to increase beta oxidation and protect animals from high-fat diet-induced insulin resistance [77]. These conflicting results have demonstrated that both activation and inhibition of autophagy might improve glucose homeostasis. While additional efforts are required to reveal the exact role of autophagy in glucose and energy metabolism in muscle, the use of different transgenic mice may account for the different results obtained. Transgenic mouse models have been widely used to study the roles and functions of genes. However, a global gene knockout approach [25] may give results different from those obtained from a study adopting a tissue-specific knockout approach [77] as secondary/indirect effects might have arisen from other organs that contribute to the observed phenotypes [78]. Recently, a single bout of exercise was shown to decrease the expression of some autophagic proteins including LC3-II, Beclin-1, Atg7, and LAMP-2 during the recovery period [79]. These findings demonstrated that a single bout of treadmill exercise might attenuate the autophagic signaling in skeletal muscle. Notably, the autophagic response observed in this study is contrary to the findings reported by He et al. [25]. In accompaniment with the decreased expression of autophagic proteins, MuRF1 protein expression is significantly increased during the recovery period [79]. It has been argued that the protein degradation regulated by MuRF1 might serve as a compensatory mechanism for the reduction of autophagy. Furthermore, both the duration and intensity of exercise applied might be insufficient to trigger autophagy. There are some possible explanations for the contradictions observed among different studies. First, differences in energy consumption may determine the activation of

autophagy. The ratio of AMP-to-ATP has been considered an important upstream signal to activate AMPK [80, 81]. The increase in the ratio of AMP-to-ATP increases the activity of AMPK. The enhanced activity of AMPK may activate autophagy through the mTOR-dependent pathway [23]. Therefore, differences in energy consumption leading to a different ratio of AMP-to-ATP may cause different autophagic responses. Second, pre-exercise muscle glycogen concentration may result in different autophagic responses. The pre-exercise muscle glycogen concentrations have been found to be inversely correlated to the degree of activation of AMPK [82, 83]. Last but not least, there are a number of intrinsic and extrinsic factors that may affect the results of studies such as the muscle type being studied, time of sacrifice and tissue collection, diet, and age.

5.2 Chronic Effect of Long-Term Aerobic Exercise Aside from the single bout exercise, long-term chronic exercise training has also been shown to result in different autophagic responses in skeletal muscle. The results of long-term exercise training are found to be diversified as summarized in Table 2. No significant changes in protein expressions of Beclin1, Atg12, Atg4, Atg7, LC3, Atg12–Atg5 complex, and cathepsin L have been demonstrated in mice after performing 5 consecutive days of treadmill exercise for 60 min/day at 30 m/min [84]. In contrast to the aforementioned acute exercise studies that harvested the muscle tissues within 3 h after the single bout of exercise, the muscle tissues were collected 24 h after the last bout of exercise and, therefore, basal autophagy rather than acute autophagic response was evaluated in this chronic exercise study. Exercise has been shown to protect muscle against doxorubicin-induced autophagic changes in skeletal muscle. Doxorubicin is an anti-tumor agent used in cancer treatment but doxorubicin is known to have toxicity that causes significant muscle wasting associated with elevation of autophagy [85]. The LC3-II/LC3-I ratio has been shown to be significantly increased after doxorubicin therapy [84]. Intriguingly, endurance exercise training has been found to restore elevated levels of the expression of key autophagic genes such as Beclin-1, Atg12, Atg4 and Atg7, and the LC3-II/LC3-I ratio in the skeletal muscle of doxorubicintreated mice [84]. It is speculated that endurance exercise training upregulates major antioxidant enzymes such as superoxide dismutase and glutathione peroxidase in muscles [86]. Thus, muscles are protected against the oxidative stress induced by doxorubicin. In this connection, endurance exercise training might inhibit the doxorubicininduced activation of autophagy.

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Caloric restriction has been found to attenuate aging [27]. A mouse study has attempted to examine the additional benefit gained from performing life-long daily voluntary wheel running together with 8 % caloric restriction in diet [27]. No significant difference has been found in protein expressions of Beclin-1, LC3-I, LC3-II, Atg7, and Atg9 in muscles of rats with 8 % caloric restriction when compared with muscles of rats with 8 % caloric restriction plus life-long exercise [27]. Life-long exercise is found not to confer additional effects on autophagy. In contrast, Atg7, Beclin-1, FoxO3, and LC3 have been found to be upregulated in muscle of rats after 8 weeks of training comprising running on a motorized treadmill at 20 m/min and a grade 5° for an hour per day, 6 days per week [87]. In the same study, it has been concluded that exhaustive exercise activates autophagy, leading to muscle atrophy that impairs the endurance capacity of skeletal muscle [87]. Conversely, different types of exercise have been widely reported to bring various health benefits to humans [88]. Even in this study, however, exhaustive exercise led to over-activation of autophagy and had detrimental effects on skeletal muscle. The combination of dietary restriction and exercise has been hypothesized to activate autophagy and the ubiquitin proteasome pathway and reduce inflammation and apoptosis [89]. Obese older adults who participated in a 6-month weight loss program combined with moderate intensity exercise have showed significant changes in autophagy markers including LC3, Atg7, and LAMP-2 at the mRNA level [89]. However, it is worth noting that, in this study, the changes in gene expression were not solely due to the effects of exercise. It would be interesting if the measurements of the mRNA and protein expressions can be conducted in an exercise-only group, so that the effects of exercise can be interpreted. Most of the studies did not consider the sustainability of the activation of autophagy in response to exercise. In most of the chronic exercise training studies, animals were sacrificed at least 24 h after completing the last bout of exercise to study the basal level of autophagy [87, 90–92]. The basal autophagy flux and autophagy protein expression have been reported to be increased after 4 weeks of voluntary running [92]. Some key markers important in examining autophagic flux including the LC3-II/LC3-I ratio, LC3-II, and p62 have been found to be upregulated [92]. Proteins important in initiating and regulating autophagy such as Beclin-1, Bnip3, and Atg7 have also been found to be upregulated [92]. Different types of muscles have been shown to have different autophagic responses even when exposed to the same exercise stimulus [92]. Basal autophagy flux and autophagy protein expression have been shown to be increased in parallel with mitochondrial biogenesis in plantaris with mixed fiber

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types. However, no significant increase in basal autophagy flux and mitochondrial biogenesis was observed in oxidative soleus muscle after a 4 weeks of voluntary running. A transgenic animal model has been developed to investigate whether basal autophagy is associated with contractile activity or the mitochondrial contents of muscle [92]. Mice with muscle-specific over-expression of PGC-1 (characterized with an increase in mitochondrial content but unchanged contractile activity) have been used. Intriguingly, the increased mitochondrial content has been demonstrated to be associated with elevated basal autophagy in skeletal muscle. It is suggested that mitochondrial biogenesis and mitophagy (a specific autophagic elimination of mitochondria) should be in equilibrium to prevent abnormal accumulation or excessive degradation of mitochondria. However, basal autophagy has been shown to be attenuated with aging and the attenuation of autophagy might reduce the efficiency of the degradation of unfolded proteins and malfunctioned organelles [93]. In this regard, aerobic exercise training has been shown to enhance basal autophagy in aged mice. LC3-II, Beclin-1, and Atg7 have been demonstrated to be upregulated following 8 weeks of aerobic exercise training [93]. Nonetheless, the expressions of Beclin-1 and Atg7 in extensor digitorum longus were found to be downregulated in young trained mice [93]. This is contradictory to one of the previously mentioned studies that showed that both autophagic flux and autophagy proteins are upregulated after 4 weeks of exercise training [92]. Data concerning fiber-type shifting and mitochondrial contents may help to explain these discrepancies. However, no such data are available to further explain the contradictory findings and further investigations are required to fully understand how exercise regulates autophagy in regard to fiber-type transformation and mitochondrial biogenesis. Exercise produces free radicals and reactive oxygen species (ROS) in skeletal muscle [94–96]. ROS has been suggested to play an important role in both physiological and pathological adaptations in skeletal muscle. ROS has recently been identified to regulate autophagy, which recycles excess protein and damaged organelles [97]. Oxidative stress may stimulate mitophagy, which has been shown to be mediated by p38 mitogen-activated protein kinase [98, 99]. The reduction of mitochondrial contents may affect cell functions and susceptibility to cell death. Therefore, it is important to maintain muscle integrity by enhancing mitochondrial biogenesis to maintain normal cellular function. PGC-1a has been shown to stimulate mitochondrial biogenesis and accelerate the turnover of intra-cellular mitochondria [100]. The recycled mitochondria are very likely to be replaced by the regenerated mitochondria. Exercise has been proposed to induce autophagy [25, 64, 68, 70, 79, 92] and mitochondrial


biogenesis [101–105]. The dynamic equilibrium between these two intracellular events should be important but the interactions between autophagy and mitochondrial biogenesis remain largely undefined and require further investigation. Myostatin is a differentiation factor inhibiting muscle growth. The upregulation of myostatin leads to muscle atrophy, which is commonly seen in patients with cancer, chronic obstructive pulmonary disease, and a very low physical activity level [106–110]. It has been demonstrated that myostatin increases the mRNA expressions of Atg4b and Ulk-2, the protein expression of LC3-II, and the number of autophagosomes in C2C12 murine muscle cells. Activation of autophagy has been accompanied by activation of proteasomal degradation contributing to muscle atrophy [111]. Conversely, decreased expression of myostatin may cause inactivation of autophagy. Myostatin null (MSTN-/-) mice exhibit abnormal muscle hypertrophy without the increase in muscular strength [112]. This may be explained by the unfavorable accumulation of sarcoplasmic reticulum, which negatively affects muscle contractility [112]. Exercise has been shown to be useful to restore the force-generating ability in MSTN-/- mice to the level of wild-type mice. Surprisingly, significant increases in the mRNA levels of Bnip3 (in both swim training and wheel running), LC3 (in swim training), cathepsin L, ATF4, and Beclin-1 (in wheel running) have been shown to be accompanied by enhanced oxidative phenotypes such as an increase in the number of succinate dehydrogenase (SDH)positive fibers and an increase in capillary density. Basal autophagy has also been found to be activated in skeletal muscle with an enhanced oxidative phenotype in some previously discussed studies [89, 92]. In collagen VI null (Col6a1-/-) mice, myofiber degeneration, spontaneous apoptosis, and decreased muscle strength together with impaired autophagic flux have been reported in skeletal muscles that are deficient of Col6a1 [70]. Unlike the autophagic response to exercise observed in MSTN-/mice, both acute and prolonged exercises were found to exacerbate the dystrophic phenotype of Col6a1-/-. This observation seems to conflict with the previously mentioned studies related to exercise-induced autophagy, as activation of autophagy after exercise was not found in muscle of Col6a1-/- mice. The LC3-II/LC3-I ratio has been reported to be reduced after long-term voluntary exercise in Col6a1-/- mice [70]. Moreover, autophagy flux has not been shown to be increased by contractile activity [70]. Of note, basal autophagy was found to be associated with mitochondrial content in skeletal muscle [92]. The improved oxidative phenotype has led to upregulation of basal autophagy. In Col6a1-/- mice, tibialis anterior muscle was shown with abnormalities of SDH staining, implying the presence of dysfunctional mitochondria and

altered sarcoplasmic reticulum. In addition, autophagy flux was found to be impaired. Further investigation is required to dissect the relationship between PGC-1a and the enhancement of basal autophagy. The effect of metabolic adaptation (e.g., mitochondrial biogenesis and angiogenesis) and contractile adaptation on basal autophagy should be one of the focuses in the area of exercise-induced autophagy. Diabetes can reduce skeletal muscle mass by inducing muscle atrophy [113]. A recent study has revealed that rats with induced diabetes show a significant weight loss. Histological analysis indicates that muscle fiber diameter was significantly reduced in diabetic mice [91]. LC3 expression was quantified to reveal the underlying mechanism beneath muscle atrophy in diabetic muscle. In diabetic mice, the LC3 expression was found to be significantly increased. Compared with the control diabetic rats, the body mass of diabetic rats with exercise (swimming 1 h/day and 5 days/week for 4 weeks) was increased by about 30 % whereas LC3 expression was reduced by 75 % [91]. The reduction in autophagy appears to conflict with the current thought that autophagy could be induced by exercise. However, LC3 expression was not reduced to a level lower than that of the healthy control group. LC3 expression only returned to the basal level after exercise. The restoration of LC3 expression might actually contribute to reverse skeletal muscle wasting in diabetic mice. Similar results can be found in another study that showed that the phosphorylation of Akt and p70S6K1 are significantly increased in gastrocnemius muscle of mice subjected to bilateral functional overload [114]. Although the actual LC3 expression was not measured, the signaling pathway has implications on the autophagic response. Akt and p70S6K1 act as upstream signaling factors for autophagy while Akt negatively regulates autophagy [55, 59]. Phosphorylation of Akt may lead to the inactivation of autophagy through the PI3K–Akt–mTOR-mediated pathway. In the diabetic study, exercise-inhibited autophagy seems to protect the diabetic rats from high blood glucose and muscle wasting [91]. This is contradictory to a previous study that demonstrated that exercise-induced autophagy improves impaired glucose tolerance in diet-induced obesity [25]. There are some possible explanations for the discrepancies between these two studies. First, muscle tissues were collected at different time points. Autophagy level varies with time and cellular nutrient state, therefore, protein expression is likely to be different at different times and nutrient states [115]. It has been demonstrated in cardiac muscle that LC3-II expression increases 1 h after exercise and returns to basal level 3 h after exercise [26]. A similar study should be conducted in skeletal muscle to depict the pattern of autophagic activation and de-activation in skeletal muscle. Second, swimming and wheel

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running have been demonstrated to alter gene expression differently in skeletal muscle [90]. Different exercises may affect autophagy in skeletal muscle differently. How different types/forms of exercise affect autophagy should be investigated in the future as different types/forms of exercise at different intensities and frequencies affect contractile activity and the cellular nutrient states of skeletal muscle differently. Both contractile activity and cellular nutrient states have been demonstrated to stimulate Akt [116–118] and AMPK [119–121] signaling, which regulate autophagy via the mTOR-dependent pathway [56, 63, 122]. A previous study has discussed the association of GLUT4 localization with activation of autophagy [25]. GLUT4 expression is reduced in skeletal muscle of type 1 and type 2 diabetic patients [123, 124]. The traditional diabetic drug metformin pharmacologically activates AMPK [125, 126] and, consequently, stimulates glucose uptake by skeletal muscle and inhibits liver gluconeogenesis [126, 127]. Interestingly, exercise can also stimulate the phosphorylation of AMPK [128, 129], which regulates autophagy and GLUT4 localization. Hence, uptake of glucose by skeletal muscle can be enhanced by exercise. Notably, the AMP/ATP ratio is continuously altered during exercise. This alteration can be sensed by AMPK [121], which can activate autophagy. However, a recent study has shown that exercise alone cannot reduce the level of serum glucose and improve insulin resistance in mice fed with a high-fat diet through the activation of AMPK [130]. After 6 weeks of exercise training, the phosphorylation state of AMPK and the LC3-II/LC3-I ratio remained unchanged. It is possible that AMPK was not activated by the exercise protocol in a mildly energy-deprived state [130]. It is believed that further understanding of the role of autophagy in exercise adaptation would help to hasten the development of novel effective exercise-based therapies to combat metabolic diseases such as diabetes mellitus.

time points for sacrificing animals, transgenic animal models, disease models, and exercise types/forms/protocols may affect autophagic response to physical exercise. Although the roles of different autophagic signaling molecules have been studied extensively, how exercise modulates autophagy in skeletal muscle remains a mystery. Some unanswered questions remain to be solved. Muscular contraction and metabolic adaptation seem to be associated with the alteration of autophagy in skeletal muscle during exercise. However, how the effect of exercise is mediated with the autophagic signaling pathway is not known. The relationship between mitochondrial biogenesis and autophagy needs to be comprehensively studied as any imbalance between these two vital intracellular processes may cause detrimental effects to skeletal muscle cells. Furthermore, the term ‘‘exercise-induced autophagy’’ has been generally used to describe the activation of autophagy following physical exercise. However, transient upregulation of autophagy and chronic elevation of basal autophagy following exercise intervention are not usually comprehensively distinguished in the literature. Future studies are needed to further investigate how these two adaptations (i.e., transient upregulation of autophagy and chronic elevation of basal autophagy following exercise) contribute to the resulting metabolic effects of exercise. Importantly, the LC3-II/LC3-I ratio, quantification of LC3-II expression and p62 degradation, and identification of autophagosomes are suggested as means for unambiguously monitoring cellular autophagic activity [131]. Experimental findings collected and integrated from multiple aspects/levels (i.e., mRNA level, protein level, qualitative measurement, and actual function of the skeletal muscle) are desirable when deciding whether autophagy is induced after exercise intervention. In summary, the efforts and findings in the past years have considerably improved our knowledge in skeletal muscle autophagy in response to the stimulus of physical exercise.

6 Conclusion and Perspective

Acknowledgments During the written process of the present article, the research work of PM Siu was supported by the General Research Fund (PolyU 5632/10M) from the Hong Kong Research Grants Council, Hong Kong Jockey Club Charities Trust, and the Hong Kong Polytechnic University Research Fund. The authors have no potential conflicts of interest that are directly relevant to the content of this review.

Autophagy is important in maintaining the cellular homeostasis of skeletal muscle fiber. Exercise appears to be an effective intervention to activate autophagy. However, our understanding of the effects of autophagy on skeletal muscle adaptation is very limited. Autophagy has been considered to be an important mechanism to recycle longlived proteins and malfunctioned subcellular organelles [25]. Disruption of autophagy would seriously destroy the homeostasis of skeletal muscle cells. However, new findings suggested that inhibition of autophagy may confer protection against high-fat diet-induced obesity and insulin resistance [77]. Furthermore, differences in signaling pathways, metabolic characteristics of skeletal muscles,

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Physical exercise increases autophagic signaling through ULK1 in human skeletal muscle.

Altered cellular distribution of hexokinase in skeletal muscle after exercise.

Skeletal muscle metaboreceptor exercise responses are attenuated in heart failure.

Autophagic Adaptations to Long-term Habitual Exercise in Cardiac Muscle.

Skeletal muscle aging: influence of oxidative stress and physical exercise.

Lifelong physical exercise delays age-associated skeletal muscle decline.

Dietary nitrate reduces skeletal muscle oxygenation response to physical exercise: a quantitative muscle functional MRI study.

Regular physical exercise improves cardiac autonomic and muscle vasodilatory responses to isometric exercise in healthy elderly.

Resistance exercise training and circulatory responses to feeding and skeletal muscle protein anabolism in older men.

Acute and chronic responses of skeletal muscle to endurance and sprint exercise. A review.

Simplified data access on human skeletal muscle transcriptome responses to differentiated exercise.

Cardiac, skeletal muscle and serum irisin responses to with or without water exercise in young and old male rats: cardiac muscle produces more irisin than skeletal muscle.

Cellular mechanisms of fatigue in skeletal muscle.

Cellular players in skeletal muscle regeneration.

Road to exercise mimetics: targeting nuclear receptors in skeletal muscle.

Autophagic signaling and proteolytic enzyme activity in cardiac and skeletal muscle of spontaneously hypertensive rats following chronic aerobic exercise.

Exhaustive physical exercise and acid hydrolase activity in mouse skeletal muscle. A histochemical study.

The pleiotropic effect of physical exercise on mitochondrial dynamics in aging skeletal muscle.

Human skeletal muscle mRNAResponse to a single hypoxic exercise bout.

Ibuprofen treatment blunts early translational signaling responses in human skeletal muscle following resistance exercise.

Exercise Promotes Healthy Aging of Skeletal Muscle.

Skeletal muscle hypertrophy after aerobic exercise training.

Inflammatory Mechanisms Associated with Skeletal Muscle Sequelae after Stroke: Role of Physical Exercise.

Autonomic responses to exercise: cortical and subcortical responses during post-exercise ischaemia and muscle pain.