**3. AMPK and skeletal muscle autophagy**

#### **3.1. The AMP-activated protein kinase**

**2.4. The roles of autophagy in skeletal muscle**

178 Muscle Cell and Tissue

deprivation or by the absence of growth factors [108].

have regular autophagic flux in the cells, even during atrophy.

strategies attempted to control autophagy level will be promising.

Compared to other tissues like the liver or pancreas, autophagy in skeletal muscle exhibits a low protein turnover and a small size of autophagosomes. These peculiar characteristics have probably constituted a brake for the detection of autophagy in this tissue for a long time. Associated to the use of transgenic mice expressing LC3 fused with GFP, autophagy process can be now easily visualized [62]. Conversely to liver or pancreas in which autophagy is activated transiently for a few hours, in skeletal muscle, autophagy can be activated for several days [62]. As it was shown in other tissues, muscular autophagy is activated by nutrient

Although it was reported that the mRNAs coding for Atgs are present in abundance in skeletal muscle [109], the role and the regulation of basal autophagy have been poorly characterized in this tissue until recently. In order to assess the function of autophagy in skeletal muscle, Masiero and colleagues performed experiments on mice deprived of the Atg7 gene, a gene necessary to the unfolding of the autophagy program [86,110]. Importantly, mice showed obvious signs of muscular weakness and atrophy exacerbated during ageing. Mice presented an accumulation of degraded proteins and free radicals, a deterioration of the internal cellular structures, and an activation of the apoptotic program. The authors clearly defined that inhibition of basal autophagy does not protect from skeletal muscle atrophy induced by denervation or starvation, but on the contrary, contributes to its degeneration. Similar muscle alterations have been obtained in muscle-specific Atg5-/- mice [87], confirming the necessity to

In many conditions varying from fasting, denervation, inactivity, microgravity, various pathologies as cancer, AIDS, sepsis, diabetes, cardiac failure, and myopathies, autophagy is overactive and pathologic, thus leading altered metabolism and muscle loss [81,108,111]. Contribution of autophagy to muscle loss begins to be clarified with the use of different animal models and innovative techniques. Inactivation of autophagic flux by LC3 silencing partially prevents FoxO3-mediated muscle atrophy [63] and atrophy caused by the expression of mutant SOD1 G93A in skeletal muscle [112]. In another model, atrophy induced by L-type calcium channel (DHPR) inactivation is linked to the expression of autophagic genes including LC3, Vps34, BNIP3 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3), and cathepsin L (for the lysosome) [113]. During sepsis, an upregulation of autophagy was found in parallel to mitochondrial injury and decreased biogenesis, especially in locomotor muscles [114]. Reactive oxygen species overproduction by altered mitochondria is now considered as a critical signal for the promotion of skeletal muscle autophagy, thus finding an opening to practical prospects for treatment of disease [115]. Accordingly, antioxidant supplement can lead to an inhibition of skeletal muscle autophagy through a reduction of oxidative stress and an increase in antioxidant capacity [116]. Taken together, these recent results confirm that excessive activation of autophagy is a critical factor for muscle wasting determinism, and

Data concerning sarcopenia are also specific. Sarcopenia is an age-related loss of muscle mass and strength, which is associated with increased autophagy, apoptosis, and exacerbated proteolysis [117]. Elevated peroxisome proliferator-activated receptor-coactivator α (PGC-1α) expression in muscle during aging contributes to reduce the proteolytic activity associated AMPK is a serine/threonine protein kinase highly conserved through evolution. AMPK is a heterotrimeric complex composed of a catalytic subunit (AMPK-α) and two regulatory subunits (AMPK-β and AMPK-γ). Humans have seven genes encoding AMPK subunits (α1, α2, β1, β2, γ1, γ2, γ3) that can form at least 12 αβγ heterotrimers, increasing the diversity of its functions [119]. The catalytic α subunit contains the threonine phosphorylation site that upon phosphorylation leads to AMPK activation [120]. AMPK acts as a sensor of cellular energy status by regulating several intracellular systems including glucose and lipid metab‐ olisms and mitochondrial biogenesis [121]. Thus, AMPK activation leads to increased glycol‐ ysis flux [122] and fatty acid oxidation [123-126] and on the contrary, to an inhibition of glycogenogenesis [127,128] and cholesterol and fatty acid biosynthesis [129-131]. The enzyme also increases the expression of PPARα (peroxisome proliferator-activated receptor α) target genes and PGC-1 leading to mitochondrial biogenesis and enhanced oxidative metabolism of muscle cells [132,133]. AMPK is activated by a large variety of cellular signals that decrease cellular ATP levels and increase AMP in response to different kinds of stress like electricalstimulated muscle contraction, exercise, hypoxia, and heat shock or under conditions of nutrient deprivation [124,134,135]. The recognized enzymes in the regulation of AMPK under energy stress conditions are LKB1 (liver kinase B1), CaMKK (calmodulin-dependent protein kinase kinase), and TAK-1 (transforming growth factor beta-activated kinase 1).

### **3.2. Regulation of skeletal muscle autophagy by AMPK**

The role of AMPK in protein turnover has been clearly defined in recent years. AMPK has been involved in the control of protein synthesis and the repression of skeletal muscle mass by inhibiting MTOR activity [136,137] in two ways: AMPK phosphorylates TSC2 (tuberous sclerosis complex 2) at Thr-1227 and Ser-1345 and RPTOR at Ser-722 and Ser-792, leading to a reduction of MTORC1 activity. Several studies also showed that AMPK increases protein degradation through the modulation of the ubiquitin-proteasome and the autophagosomelysosome pathways [138,139]. AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribonucleo‐ side, an AMPK activator) treatment increases the expression of the E3 ligases MAFbx/ Atrogin-1 and MuRF1 in muscles cells. In addition, increase of autophagic flux by AMPK has been reported in several muscle models as C2C12 myoblasts, C2C12 myotubes, and primary myotubes [138,139]. Two major signaling pathways were characterized in AMPK-induced muscular autophagy (Fig.2).

**Figure 2.** Role of AMPK in skeletal muscle protein turnover

The first one concerns the activation of the forkhead box class O proteins (FoxO), notably involved in the regulation of protein breakdown, energy metabolism, and mitochondrial turnover [140]. FoxO factors also play an important role in exercise-induced angiogenesis by limiting it during the first days of training program [141,142]. Activation of FoxO3a by AMPK leads to an increase in several Atgs expression, including LC3-II and Gabarapl1 that act as promoters of autophagosome fabrication [139]. AMPK directly interacts with FoxO3a and phosphorylates it on Ser-588, a residue known to lead to FoxO3a activation [139,143]. The upregulation of several Atgs by FoxO factors have been described in *Drosophila* larval fat body [144], mammalian cardiomyocytes [145], hepatocytes [146], and colorectal cancer cells [147]. Regarding the regulation of FoxO3a subcellular localization in muscle cells, while long treatments (i.e., 24 h) with AMPK activators do not change FoxO3a nuclear content, an increase in the total protein level is notable after 30 min. With a short time course (30 min–6 h), the activation of AMPK by AICAR leads to a relocalization of FoxO3a into the nucleus [139]. Tong and colleagues reported that AICAR treatment causes FoxO3a nuclear relocation through a decrease in FoxO3a phosphorylation at Thr-318/321 [148]. However, Greer and colleagues have reported an increase of FoxO3a transcriptional activity without any change in the nuclear content of the factor after AMPK activation by 2-deoxyglucose (2DG) in HEK293T cells [143]. These data strongly suggest that FoxO3a relocalization into the nucleus is not necessarily required to increase its transcriptional activity. A possibility is that AMPK may also control FoxO3a protein stability.

The second pathway involves modulation of the Ulk1 complex. A multiprotein complex composed of AMPK, MTORC1, Ulk1, FIP200, and Atg13 has been identified in muscle cells (Fig.3) [139]. These data fit with the model found in other cell types showing that, under basal conditions, MTORC1 prevents autophagy by interacting with Ulk1 [72]. Under nutrient-rich conditions, phosphorylation of Ulk1 by MTORC1 represses Ulk1 kinase activity and its ability to interact with Atg13 or FIP200; thereby, it coordinates the autophagy response [65,149]. In muscle cells, activation of AMPK (by AICAR treatment) or inhibition of MTORC1 (by Torin1 treatment or amino acid privation) removes AMPK, MTOR, and RPTOR from Ulk1 [139]. These events are known to induce the Ulk1-dependent phosphorylation of Atg13 and FIP200, leading to the initiation of autophagy [70].

Proteomics screens of autophagy [150] and a co-immunoprecipitation study performed in HEK293T cells [151] showed that AMPK interacts with both Ulk1 and Ulk2. In muscle cells, Ulk1 also acts as an interacting partner of AMPK, and Ser-467 site identified by Egan and colleagues is also phosphorylated by AMPK [139]. Ulk1 phosphorylation by AMPK may participate to conformational changes and thus disrupts the interaction between Ulk1 and MTORC1, in agreement with the suppression of MTORC1 anti-autophagy activity in the Ulk1 complex [72]. Moreover, Ulk1 phosphorylation by AMPK may directly activate Ulk1 kinase activity. Indeed, in vitro studies showed that Ulk1 is highly phosphorylated and that purified Ulk1 can phosphorylate itself and requires autophosphorylation for stability [152]. In mam‐ mals, Ulk1 phosphorylation by AMPK is critical for mitochondrial homeostasis and cell survival during starvation [153]. In summary, AMPK regulates Ulk1 activity by decreasing MTORC1 activity and by phosphorylating Ulk1 [121].

**Figure 3.** The Ulk1/Atg13/FIP200/MTORC1/AMPK complex

**Figure 2.** Role of AMPK in skeletal muscle protein turnover

180 Muscle Cell and Tissue

FoxO3a protein stability.

The first one concerns the activation of the forkhead box class O proteins (FoxO), notably involved in the regulation of protein breakdown, energy metabolism, and mitochondrial turnover [140]. FoxO factors also play an important role in exercise-induced angiogenesis by limiting it during the first days of training program [141,142]. Activation of FoxO3a by AMPK leads to an increase in several Atgs expression, including LC3-II and Gabarapl1 that act as promoters of autophagosome fabrication [139]. AMPK directly interacts with FoxO3a and phosphorylates it on Ser-588, a residue known to lead to FoxO3a activation [139,143]. The upregulation of several Atgs by FoxO factors have been described in *Drosophila* larval fat body [144], mammalian cardiomyocytes [145], hepatocytes [146], and colorectal cancer cells [147]. Regarding the regulation of FoxO3a subcellular localization in muscle cells, while long treatments (i.e., 24 h) with AMPK activators do not change FoxO3a nuclear content, an increase in the total protein level is notable after 30 min. With a short time course (30 min–6 h), the activation of AMPK by AICAR leads to a relocalization of FoxO3a into the nucleus [139]. Tong and colleagues reported that AICAR treatment causes FoxO3a nuclear relocation through a decrease in FoxO3a phosphorylation at Thr-318/321 [148]. However, Greer and colleagues have reported an increase of FoxO3a transcriptional activity without any change in the nuclear content of the factor after AMPK activation by 2-deoxyglucose (2DG) in HEK293T cells [143]. These data strongly suggest that FoxO3a relocalization into the nucleus is not necessarily required to increase its transcriptional activity. A possibility is that AMPK may also control

The second pathway involves modulation of the Ulk1 complex. A multiprotein complex composed of AMPK, MTORC1, Ulk1, FIP200, and Atg13 has been identified in muscle cells (Fig.3) [139]. These data fit with the model found in other cell types showing that, under basal Time-course studies have been performed in muscle cells in order to better understand the dynamics of UlK1 complex following autophagy induction [139]. Interestingly, AMPK dissociates from Ulk1 3 h after AICAR treatment [139]. In agreement with these observations, in HeLa cells, AMPK is associated with Ulk1 only under nutrient-rich condition, and it dissociates from Ulk1 5 min after starvation [154]. Thus, in normal condition, Ulk1 is associated with AMPK; upon AICAR treatment, the complex remains stable for 3 h and then dissociates. As suggested by another group, Ulk1 dissociation from AMPK could permit to Ulk1 complex to be more active [154]. Conversely, this dissociation can constitute a negative regulatory feedback as proposed by Loffler et al. [155]. The authors showed that Ulk1 could mediate phosphorylation of AMPK on the regulatory subunits, constituting an inhibitory feedback control. Further works have to define the molecular mechanisms for these events, especially in skeletal muscle.
