Participation of AMPK in the Control of Skeletal Muscle Mass



Fig. 12.1
Accepted metabolic effects of AMPK in skeletal muscle. AMPK is activated by several metabolic stresses such as energy depletion, myokines, adipokines, oxidative stress, and Ca2+ release from sarcoplasmic reticulum. Subsequently, activated AMPK induces diverse metabolic adaptations in skeletal muscle and thereby contributes to promote health



According to these varied and central roles of AMPK in muscle metabolism, we hypothesized that AMPK regulates muscle mass. The association between AMPK and muscle mass regulation was first suggested in 2002 by Bolster et al. [7], who showed novel evidence that AMPK modulates protein synthesis in rat skeletal muscle. Moreover, Mu et al. [102] showed that the soleus and extensor digitorum longus muscles of muscle-specific kinase-dead AMPKα2 transgenic mice tended to be larger than those of their wild-type littermates. Several subsequent studies have demonstrated crucial regulatory roles of AMPK in the control of skeletal muscle mass. In this chapter, we provide an overview of the role of AMPK in the regulation of skeletal muscle mass.



12.2 AMPK: Subunit Structure and Activation Mechanism


AMPK comprises a catalytic α-subunit and the regulatory subunits β and γ [45] in a total of 12 possible heterotrimeric combinations of two α-, two β-, and three γ-subunits [46]. In the skeletal muscle, the predominant heterotrimeric complexes include α1/β2/γ1, α2/β2/γ1, and α2/β2/γ3 [170]. In these complexes, the α-subunit has a catalytic domain that contains the activating phosphorylation site (Thr172) at the N-terminus, an auto-inhibitory domain, and a conserved C-terminal domain that interacts with β- and γ-subunits [17, 60, 61, 112, 169]. The two distinct α isoforms have different localization patterns in mammalian cells, and whereas α1 is expressed widely, α2 is dominant in the skeletal muscle, the heart, and the liver [142]. The regulatory β-subunit contains a C-terminal region that interacts with α- and γ-subunits and a central region that binds glycogen [87]. Moreover, the regulatory γ-subunit contains a number of repeating domains that are involved in the activation of the AMPK complex, including Bateman domains that bind adenine nucleotides (AMP, ADP, or ATP) [46].

Muscle AMPK classically acts as a signaling molecule and monitor of cellular energy levels and is sensitive to AMP/ATP ratios, creatine/creatine phosphate ratios, and AMP levels [45]. Binding of AMP to Bateman domains of the γ-subunit of AMPK causes allosteric activation of AMPK and phosphorylation of the Thr172 residue of the α-subunit, which increases AMPK activity by tenfold. In contrast, phosphorylation of Thr172 by upstream kinases including liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) increases the AMPK activity by 100-fold [48]. Although the LKB1 complex is constitutively active and is not activated directly by AMP, the binding of AMP to AMPK leads to a structural change that facilitates phosphorylation of AMPK by the LKB1 complex [47, 130]. On the other hand, CaMKK can activate AMPK in response to increased intracellular Ca2+ levels independently of energy status [49, 56, 171].


12.3 AMPK and Skeletal Muscle Hypertrophy


Skeletal muscle hypertrophy is defined as a gain of muscle mass due to increases in sizes rather than numbers of pre-existing muscle fibers and occurs in response to increased loading such as resistance training [111], mechanical stretching [37], heat stress [40], and anabolic hormonal stimulation [93].

Although numerous AMPK transgenic and knockout (KO) animals have been produced, few studies show direct roles of AMPK under hypertrophic conditions (Table 12.1). Functional overload is commonly used in studies of muscle hypertrophy, and AMPKα1 activity is stimulated in these models [99]. Moreover, diminished AMPKα1 activity during functional overload reportedly accelerated muscle hypertrophy [99]. Functional overload-induced hypertrophy in plantaris muscles has also been shown to be diminished in aged (30 month) rats [149] and in the soleus muscles of obese rats [66]. Besides, both of these rat models exhibited hyper-phosphorylation of AMPKα Thr172 during overload, with a significant negative relationship (r = −0.82) between AMPK phosphorylation status and percent hypertrophy [66, 149]. In addition, hypertrophic consequences of constitutive activation of Akt, which is induced by infection with NH(2)-terminal myristoylation signal-attached Akt (MyrAkt), were reportedly elevated in the tibialis anterior muscles of AMPKα1-deficient mice in vivo [99] and in AMPKα1/α2-deficient muscle cells in vitro [73]. In our previous in vitro study [23], the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) inhibited myotube hypertrophy in skeletal muscle cells, and this response was not induced in AMPKα1/α2 knockdown cells. Taken together, these data indicate that AMPK is a negative regulator of muscle mass under hypertrophic conditions.


Table 12.1
Muscle mass in genetic animal models of AMPK
















































































































































Animal model

Type of muscle

Treatment

Change

References

Normal condition

AMPKα2 DN

EDL, SOL


Mass↑

[102]

AMPKγ1R70Q



Mass→

[3]

AMPKα1−/−

PLA


Mass↓

[99]

AMPKα1−/−α2−/−

SOL


Mass↑, CSA↑

[73]

AMPKγ3R225Q

GAS


Mass→

[176]

AMPKβ1−/−β2−/−

SOL, EDL


CSA→

[109]

AMPKα1−/−

TA


Mass↓, CSA↓

[30]

AMPKα2−/−

TA


CSA↑

AMPKα2DN

QC


Mass→, CSA→

[110]

AMPKβ1−/−β2−/−

TA


CSA↓

[148]

LKB1−/−

GAS


Mass↓

[139]

TA, EDL


Mass→

AMPKα2−/−

TA, GAS, SOL, EDL


Mass↓

[13]

AMPKα1DN

SOL, EDL, GAS/PLA


Mass→, CSA→

[22]

AMPKγ3R225Q

PLA


Mass↑

[125]

AMPKγ3−/−

PLA


Mass→

Hypertrophic condition

LKB1−/−

PLA

Overload

Mass→

[89]

AMPKα1−/−

TA

MyrAkt, transfection

CSA↑

[99]

AMPKα1−/−

PLA

Overload

Mass↑

[99]

AMPKγ3R225Q

PLA

Overload

Mass→

[125]

Atrophic condition

AMPKα1 DN

SOL

Hindlimb suspension

Mass↑, CSA↑

[22]


↑ increase or preventing decrease; ↓ decrease; → no change

EDL extensor digitorum longus, SOL soleus, PLA plantaris, TA tibialis anterior, GAS gastrocnemius, QC quadriceps, CSA muscle fiber cross-sectional area, DN dominant negative


12.4 AMPK and Skeletal Muscle Atrophy


Skeletal muscle atrophy is defined as a decrease in muscle mass due to disuse [108], aging [44, 78], malnutrition [54], and disease such as diabetes [15], cancer cachexia [153], sepsis [67], chronic renal failure [164], and chronic obstructive pulmonary disease (COPD) [4]. In particular, disuse muscle atrophy is a serious health care issue during aging and is closely related to the bedridden state.

Observational studies have demonstrated associations between AMPK and disuse muscle atrophy. In human studies, 7 [126] or 24 days [8] of bed rest in young men did not affect the phosphorylation status of AMPKα Thr172 in vastus lateralis muscles. In addition, 14 days of immobilization in young adult men and women had no effect on the phosphorylation status of AMPKα Thr172 or the expression of AMPKα2 and AMPKβ2 subunits in vastus lateralis muscles [26]. In contrast, 14 days of immobilization in young and older men led to increased expression of AMPKα1, AMPKβ1, and AMPKγ3 subunits, but did not affect the other subunits or the phosphorylation status of AMPKα Thr172 [161].

In animal studies, 14 days of hindlimb unloading in young male rats decreased the AMPKα expression and the phosphorylation status of AMPKα Thr172 in soleus muscles [43]. Moreover, 28 days of hindlimb unloading in young male mice reduced the phosphorylation status of AMPKα Thr172 in gastrocnemius muscles [80]. In contrast, long-duration (4–13 weeks) hindlimb unloading in male rats enhanced the phosphorylation status of AMPKα Thr172 in soleus muscles [52, 174]. Moreover, short-duration (3 days) hindlimb unloading in male mice increased the phosphorylation status of ACC Ser79 [11], which is an endogenous indicator of AMPK activity [20, 114].

Although the dynamics of AMPK activity following muscle disuse remains controversial, we demonstrated direct evidence of AMPK-mediated progress of skeletal muscle atrophy in transgenic mice (AMPK-DN) expressing a dominant negative mutant of AMPKα1 in the skeletal muscle [22], in which predominant reduction of AMPKα2 activity rather than AMPKα1 activity has been observed [32, 65, 91, 145]. In the experiments, 14 days of hindlimb unloading led to a 30% decrease in soleus muscle mass and muscle fiber CSA in wild-type littermate mice, but only a 15% decrease in AMPK-DN mice [22], indicating that the deficiency of skeletal muscle AMPK activity (mainly AMPKα2) hinders the progress of skeletal muscle atrophy during disuse.

Taken together, these data suggest that AMPK is required for proper adaptation of muscle mass during disuse-induced skeletal muscle atrophy. Skeletal muscle atrophy due to disuse, including bed rest or immobilization, occurs especially during the early phase (∼10 days) [5, 96, 162]. Hence, because AMPK activation is enhanced during the early phase (∼3 days) of muscle disuse [11] and returns to basal or lower levels in the following phase [26, 43, 126, 161], AMPK may regulate muscle atrophy during the early phase of disuse. However, further evidence is required to clarify the precise roles of AMPK in the regulation of skeletal muscle atrophy.


12.5 Molecular Mechanisms of AMPK-Mediated Regulation of Muscle Mass


AMPK is a master regulator of metabolism and multiple signaling molecules are implicated in AMPK-mediated regulation of muscle mass (Fig. 12.2).

A372420_1_En_12_Fig2_HTML.gif


Fig. 12.2
Molecular mechanisms of AMPK-mediated regulation of muscle mass. AMPK downregulates mTOR signaling and inhibits protein synthesis. In contrast, AMPK stimulates protein degradation systems including ubiquitin–proteasome and autophagy. These regulations are mediated by multiple signaling molecules such as myostatin, HSP72, FoxOs, NFkB, MuRF1, Atrogin-1/MAFbx, and Ulk1. The arrow indicates positive regulation whereas the blocked line indicates negative regulation


12.5.1 Protein Synthesis Pathway


The mammalian target of rapamycin (mTOR) plays crucial roles in the regulation of protein synthesis [6] as a serine/threonine protein kinase of phosphoinositol-3-kinase (PI3K)-related kinase family. Specifically, mTOR interacts with several proteins to form the distinct mTOR complexes 1 (mTORC1) and 2 (mTORC2), which are known to differ in subunit composition, sensitivity to rapamycin, and cell signaling activity [74]. It is accepted that mTORC1 signaling is more closely related to the control of protein synthesis than mTORC2 and is negatively regulated by the tumor suppressor tuberous sclerosis complex 1/2 (TSC 1/2) [146]. Increased activity of mTORC1 stimulates protein translation and synthesis following phosphorylation of key direct downstream effectors, including the 70-kDa ribosomal protein S6 kinase (S6 K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) [33, 74]. S6 K1 activation also enhances translation elongation via signals that eventually lead to decreased phosphorylation of eukaryotic elongation factor 2 (eEF2) [163]. Moreover, AMPK has been shown to interact with mTOR signaling, and activation by AICAR injection inhibits mTOR signaling and protein synthesis in rat skeletal muscle [7]. AMPK phosphorylates and activates TSC2 [59] to inhibit mTORC1 and S6 K1 activity [58] and directly phosphorylates the mTOR binding partner raptor, leading to binding of 14-3-3 to raptor and allosteric inhibition of mTORC1 [42]. Thus, mTORC1 is a potent intermediate of AMPK-mediated muscle mass regulation.

In a previous study, impairment of muscle hypertrophy during functional overload in aged rats was accompanied by reduced activation of mTOR/S6 K1/eEF2 and mTOR/4E-BP1 signaling, and these responses were negatively correlated with the phosphorylation status of AMPKα Thr172 [150]. Furthermore, Viollet and colleagues showed that S6 K1 and 4E-BP1 signaling are promoted in AMPKα1 KO mice during functional overload-induced muscle hypertrophy [99]. In addition, increased cell size in AMPKα1/α2-deficient myotubes was prevented by treatments with the mTOR inhibitor rapamycin [73]. Hence, crosstalk between AMPK and mTOR/S6 K1 signaling is likely important for muscle mass control under conditions of hypertrophy.

In contrast with these studies, we found little association of mTOR signaling with skeletal muscle atrophy [22]. Specifically, protein synthesis and phosphorylation status of S6K1 Thr389 were decreased during disuse-induced soleus muscle atrophy. However, suppression of AMPK activity did not rescue these responses [22], indicating that AMPK/mTOR/S6K1 signaling may not be a crucial axis in skeletal muscle atrophy.

Distinct roles of α isoforms (α1 and α2) of AMPK may influence the regulation of muscle mass via mTOR/S6K1 signaling. Accordingly, Viollet and colleagues indicated that AMPKα1-mTOR/S6K1 interactions are important in skeletal muscle hypertrophy. However, another study showed that both skeletal muscle hypertrophy and activation of mTOR/S6K1 signaling remained unchanged in response to functional overload in LKB1 KO mice [89], in which AMPKα2 but not AMPKα1 activity is completely abolished. Consistent with these findings, Viollet and colleagues showed that MyrAkt-induced hypertrophy was similar in muscle fibers lacking AMPKα2 and in control fibers [98]. These results suggest that AMPKα1 is more closely associated with mTOR/S6 K1 activation and skeletal muscle hypertrophy than AMPKα2.

In contrast with these studies, we demonstrated attenuation of unloading-induced skeletal muscle atrophy in AMPK-DN mice, in which AMPKα2 activity was almost completely inhibited but AMPKα1 activity was reduced by only 20% [22]. AMPK is activated in response to energy-depleting stresses [50], and AMPKα2 is more sensitive to energy depletion than AMPKα1 [131, 143]. Mitochondrial dysfunction-mediated impairment of ATP production has been suggested to activate AMPK under conditions of disuse and thereby contributes to muscle atrophy through protein degradation rather than protein synthesis [123]. Taken together, these observations indicate that AMPKα2 regulates skeletal muscle atrophy via mechanisms that are independent of protein synthesis pathways. These findings imply that there are isoform-dependent regulation of muscle mass, although multiple roles of AMPK α, β, and γ isoforms in muscle mass regulation remain unresolved.


12.5.2 Ubiquitin–Proteasome System


The ATP-dependent ubiquitin–proteasome system is the primary protein degradation pathway and intrinsically induces degradation of myofibrillar proteins in skeletal muscle [104]. The ubiquitin ligase enzyme E3 binds its protein substrate and catalyzes the movement of ubiquitin from the ubiquitin-conjugating enzyme (E2) to the substrate. This rate-limiting step of the ubiquitination influences subsequent proteasome-dependent degradation. The human genome contains more than 650 ubiquitin ligases [75], and important skeletal muscle-specific ubiquitin E3 ligases include muscle-specific RING finger protein 1 (MuRF1) and atrogin-1/muscle atrophy F-box (MAFbx). MuRF1 and atrogin-1/MAFbx are both primarily expressed in skeletal muscle [5]. Whereas MuRF1 ubiquitinates several myofibrillar proteins, such as myosin heavy chains [16] and actin [122], atrogin-1/MAFbx targets growth-related proteins such as MyoD [152] and eIF3f [18]. Moreover, MuRF1 and atrogin-1/MAFbx mRNA levels are rapidly upregulated in numerous models of muscle atrophy and play accepted roles in the initiation of atrophy [28].

In the past decade, AMPK has been shown to interact with several E3 ubiquitin ligases. In skeletal muscle, MuRF1 and atrogin-1/MAFbx are potent targets of AMPK-mediated ubiquitination, and MuRF1 and atrogin-1/MAFbx mRNAs were upregulated following treatment with the AMPK activators AICAR [23, 68, 107, 155] and metformin [68] in skeletal muscle cells in vitro. Our recent in vivo studies demonstrated that suppression of AMPK activity attenuates increases in ubiquitination and MuRF1 mRNA expression, thus inhibiting the progress of disuse-induced muscle atrophy in mice soleus muscles [22]. Other studies have also suggested associations between AMPK and E3 ligases during muscle atrophy. Specifically, TNF receptor adaptor protein 6 (TRAF6) was associated with the dimeric ubiquitin-conjugating enzyme 13/ubiquitin-conjugating enzyme variant 1A and promoted the formation of Lys63-linked poly-ubiquitin chains rather than conventional Lys48-linked poly-ubiquitin chains, which are target proteins for degradation [71, 121]. Muscle-specific TRAF6 KO mice were resistant to muscle atrophy following denervation, cancer, or starvation, and TRAF6-mediated ubiquitination was shown to be mediated partly by activation of AMPK [116, 117]. Thus, various relationships between AMPK and the ubiquitin–proteasome system are important in the regulation of skeletal muscle mass.


12.5.3 Autophagy


Autophagy is an important cell proteolytic system that controls protein turnover in skeletal muscle [85] and is regulated by multiple proteins. Among these, Unc-51-like kinase 1 (Ulk1, also known as Atg1) is considered an important serine/threonine protein kinase during the initial stage of autophagosome formation [168]. In subsequent stages, microtubule-associated protein light chain 3 (LC3) is converted to the active form of LC3 (named LC3II) through lipidation and then participates in the formation and elongation of autophagosomes. The presence of LC3 in autophagosomes and the conversion of LC3 to the lower migrating form LC3II have often been used as indicators of autophagic activity [63]. In the final step of autophagy, autophagosomes fuse with lysosomes and their cargoes are degraded [94]. The ubiquitin-binding protein p62, which binds to LC3, is preferentially degraded during autophagy [113], and thus breakdown of p62 is generally used as a marker of autophagy flux [95].

Recently, AICAR-induced AMPK activation was reported to stimulate autophagosome formation in skeletal muscle cells [133]. Accordingly, interactions of AMPK, mTOR, and Ulk1 appear to play crucial roles in the modulation of autophagy [1, 76]. Specifically, mTOR directly binds and negatively regulates Ulk1 activity by phosphorylating the protein at Ser637 and Ser757 [35, 140]. In contrast, AMPK binds and phosphorylates Ulk1 at Ser317, Ser467, Ser555, Thr575, Ser637, and Ser777 and thereby promotes Ulk1 activity and disrupts the Ulk1–mTOR interaction [1]. AMPK also indirectly induces autophagy by inhibiting the mTORC1 complex [35, 76].

Roles of AMPK-mediated autophagy in muscle mass regulation have been reported, and in vitro treatments with the synthetic glucocorticoid dexamethasone have been shown to increase the ratios of LC3II/LC3I dependently of AMPK in skeletal muscle cells [159]. In addition, chronic dexamethasone treatment led to muscle atrophy and AMPK activation in mice, and the muscle atrophy was attenuated by blocking AMPK activation [81]. These results indicate associations of AMPK-mediated autophagy and dexamethasone-induced muscle atrophy.

In a previous study, we demonstrated that increased LC3II/LC3I ratios in mouse soleus muscles during unloading-induced muscle atrophy were attenuated following suppression of AMPK activity [22]. However, unloading was previously shown to result in p62 protein accumulation in atrophic soleus muscles, indicating impairment of autophagic flux [95]. Accordingly, p62 protein expression was increased by unloading in mouse soleus [22], tibialis anterior, and gastrocnemius muscles [80], and no changes in LC3 expression or decreases in other autophagy-related proteins (Atg7 and Beclin-1) were reported after unloading. Taken together, these data warrant further research to precisely determine the roles of AMPK in autophagy activation during disuse-induced muscle atrophy.

AMPK-mediated autophagy has been shown to play critical roles in the regulation of skeletal muscle mass during fasting and/or aging [10]. These investigators showed that myofiber CSA in tibialis anterior muscles was reduced by fasting in wild-type mice but not in muscle-specific AMPK-KO mice and that LC3II/LC3I ratios and phosphorylation states of Ulk1 Ser555 after fasting were higher in wild-type than muscle-specific AMPK-KO mice. In addition, AMPK deficiency resulted in increased activation of mTOR and inhibitory phosphorylation of Ulk1 Ser757. These findings suggest that induction of muscle atrophy during fasting follows AMPK-mediated activation of autophagy. In addition, they also showed that AMPK deficiency led to greater aging-associated impairments of muscle quality, with increased central nuclei, large round fibers, small angular fibers, and necrotic fibers, and decreased muscle force production from that in wild-type mice. Besides, low muscle quality was reportedly accompanied by p62 accumulation, implying that aging-induced myopathy follows impairments of AMPK-mediated autophagic activity. Collectively, these findings indicate that both activation and inactivation of AMPK-autophagy systems result in muscle loss, and hence activation of autophagy systems by AMPK might be essential for muscle mass homeostasis.


12.5.4 Forkhead Box O (FoxOs)


The FoxOs family of transcription factors has been associated with ubiquitin–proteasome and autophagy systems [132, 144]. Among these, FoxO1, FoxO3 (also known as FoxO3a), FoxO4, and FoxO6 have been identified in skeletal muscle, and FoxO1 and FoxO3a are key factors in muscle homeostasis. Akt inhibits FoxO3a by phosphorylating residues Thr32, Ser253, and Ser315 [9], and phosphorylation of FoxO3a at Ser253 results in exclusion from the nucleus and inhibition of transcription [9]. In contrast, AMPK phosphorylates FoxO3a at six regulatory sites (Thr179, Ser399, Ser413, Ser355, Ser588, and Ser626) and promotes FoxO3a transcriptional activity without affecting subcellular localization [41].

Previous studies suggest that AMPK-mediated upregulation and/or nuclear translocation of FoxO1 and FoxO3a contributes to activation of the ubiquitin–proteasome system in skeletal muscle cells [107, 133, 155]. Furthermore, phosphorylation of FoxO3a Ser588 by AMPK leads to increased expression of autophagy-related proteins such as beclin and LC3II [133]. In our experiments, decreased phosphorylation of FoxO3a Ser253 in soleus muscles was accompanied by Akt inactivation during disuse-induced muscle atrophy, but suppression of AMPK activity maintained phosphorylation of FoxO3a Ser253 at the basal level despite Akt remained inactivated [22]. These data indicate that AMPK affects the phosphorylation status of FoxO3a Ser253 through an Akt-independent mechanism during muscle atrophy. Thus, although the mechanisms behind the relationship between AMPK and FoxOs activities in muscle mass regulation have not been characterized, AMPK appears to directly or indirectly regulate FoxOs expression and/or nuclear translocation and thereby stimulates ubiquitin–proteasome and/or autophagy systems.


12.5.5 Nuclear Factor κB (NF-κB)


The transcription factor NF-κB is sequestered in the cytoplasm by a family of inhibitory proteins known as IκBα [101]. In this mechanism, the IκB kinase complex phosphorylates IκBα, resulting in its degradation, and thus facilitates nuclear translocation and activation of NF-κB. NF-κB activation has been detected under physiological and pathological atrophic conditions, including unloading, denervation, aging, cancer, sepsis, and diabetes, and pharmacological or genetic inhibition of NF-κB can protect from skeletal muscle atrophy [79, 165]. Moreover, NF-κB signaling was reportedly more important than FoxOs signaling in disuse muscle atrophy, since NF-κB sites, but not FoxOs sites, for transcription of MuRF1 during hindlimb unloading [172]. In our study, the expression of IκBα tended to decrease with muscle atrophy in mice and was higher in AMPK-DN mice than in wild-type mice after hindlimb unloading [22]. Hence, AMPK may regulate NF-κB signaling via the expression of IκBα during disuse-induced muscle atrophy. However, no other reports show associations of AMPK with NF-κB signaling during muscle mass regulation, warranting further research to clarify the precise roles of NF-κB signaling in AMPK-mediated muscle mass regulation.


12.5.6 Heat Shock Proteins (HSPs)


HSPs are stress-induced molecular chaperones that play crucial roles in maintaining correct the folding and intracellular transport of proteins. Accordingly, as regulators of cell signaling, HSPs have various reported cytoprotective functions [69, 82, 115]. Multiple HSPs have been identified in skeletal muscle, including HSP27, HSP47, HSP60, HSP70, HSP90, and HSP110. Among these, inducible HSP70 (so-called HSP72) is the most widely studied in skeletal muscle, and profound impacts on muscle adaptation have been shown [82]. HSP72 expression is increased in skeletal muscle following exercise [97] and muscle injury [138], and during muscle regrowth and regeneration [135, 138], but is decreased by muscle disuse [22, 137]. Accordingly, overexpression of HSP72 in skeletal muscle prevented immobilization-induced atrophy in rats [137] and improved structural and functional recovery from atrophy of mouse muscles [92]. Moreover, inhibition of FoxOs and NF-κB signaling is considered a potent mechanism by which HSPs regulate skeletal muscle mass [21, 70, 136, 137]. Therefore, HSP72 may be a positive regulator of skeletal muscle mass that downregulates protein degradation pathways.

In our studies, AMPK was implicated in the regulation of HSP72 expression during skeletal muscle hypertrophy and atrophy. Specifically, AICAR-induced AMPK activation decreased HSP72 protein expression in skeletal muscle cells and was not induced following suppression of AMPKα1/α2 [23]. This was the first report to show that AMPK modulates HSP72 expression in skeletal muscle. Furthermore, we recently demonstrated that metformin-induced AMPK activation also downregulated HSP72 mRNA and protein expression in skeletal muscle cells [24], suggesting transcriptional regulation of HSP72 by AMPK. In addition, in vivo experiments showed higher HSP72 expression in intact muscle from AMPK-DN mice than from wild-type mice, and decreased HSP72 expression during unloading-induced muscle atrophy was attenuated in AMPK-DN mice [22]. Taken together, these data suggest an inverse relationship between AMPK and HSP72 in skeletal muscle and indicate that AMPK-mediated regulation of muscle mass may be mediated by HSP72.

The transcription of HSPs is mediated by HSF1 binding to the corresponding regulatory elements [82]. Hence, if AMPK affects the HSF1-mediated transcription of HSPs, several HSPs may be induced by AMPK activation. However, our experiments showed that activation of AMPK by AICAR treatment affected HSP72, but not HSP25 or constitutively expressed HSC70 [23]. Furthermore, HSF1 expression was downregulated following disuse-induced muscle atrophy independently of AMPK activity [22]. These observations indicate that AMPK modulates the expression of HSPs through HSF1-independent mechanisms.


12.5.7 Myostatin


Myostatin is known as growth differentiation factor 8 (GDF-8) and is a member of the transforming growth factor-β (TGFβ) superfamily, which has emerged as central to the regulation of skeletal muscle mass [90]. Myostatin binding to activin type IIB receptor (ActRIIB) leads to the recruitment of activin-like kinase-4 (ALK-4) or ALK-5, and phosphorylation and activation of Smad2 and Smad3, which form complexes with Smad4 [124, 128]. Subsequent nuclear translocation of the Smad2/Smad3/Smad4 complex promotes the transcription of genes involved in proliferation and differentiation and the protein metabolism in skeletal muscle [72, 134, 147]. Activation of Smad2 and Smad3 also impairs Akt activity, leading to inhibition of mTOR-dependent protein synthesis pathways [158] and stimulation of FoxOs-dependent protein degradation pathways [88]. Consequently, myostatin acts as a negative regulator of muscle mass.

Myostatin was shown to be associated with AMPK in skeletal muscle during diabetic muscle atrophy, with concomitant AMPK Thr172 phosphorylation and increased myostatin expression [55]. Accordingly, some studies have shown that AMPK activation by AICAR stimulates myostatin expression in skeletal muscle cells [19, 77]. These findings suggest that AMPK stimulates myostatin expression and thereby negatively regulates skeletal muscle mass. This hypothesis is supported by our unpublished findings showing that increases in myostatin mRNA expression in atrophic soleus muscles are abolished by suppression of AMPK. We also observed lower plasma myostatin levels in AMPK-DN mice than in wild-type mice (unpublished data). Hence, AMPK may regulate muscle mass by modulating skeletal muscle and/or whole-body myostatin expression. In addition, treatment of skeletal muscle cells with recombinant myostatin reportedly stimulated AMPK by depleting energy stores [14], suggesting that a feed-forward loop between AMPK and myostatin contributes to the control of muscle mass.


12.6 AMPK and Myogenesis


Myogenesis is defined as the formation of muscular tissue and includes growth, differentiation, and repair of muscles. Myogenic regulatory factors (MRFs) include basic helix-loop-helix (bHLH) transcription factors that are essential for determinations of muscle lineage. Among these, myogenic factor 5 (Myf5), myogenic differentiation (MyoD), myogenin, and MRF4 are characterized by the capacity to convert various cell lines into myocytes and to activate muscle-specific gene expression [141]. Myf5 is the first MRF to be expressed during embryonic development and integrates multiple developmental signals to initiate myogenesis [29]. MyoD is also an important transcriptional regulator of myogenesis and is considered a master switch gene for muscle formation [120]. Myogenin and MRF4 are involved in later stages of myogenesis and are directly associated with differentiation processes that lead to myotube formation and maturation [2, 84].

As shown in Table 12.1, AMPK transgenic and KO mice exhibit higher or lower muscle mass and muscle fiber CSA under normal growth conditions, further indicating that AMPK is involved in myogenesis. Indeed, several studies have provided evidence that AMPK mediates myogenic processes by regulating MRFs, especially myogenin. Accordingly, low myogenin, but not MyoD and MRF4, levels were observed in primary myoblasts from AMPKα1 KO mice, which were incapable of forming myotubes [31]. Furthermore, AICAR-induced activation of AMPK in skeletal muscle cells increased myogenin expression and promoted myotube formation [31]. Epigenetic histone and DNA modifications are considered important for cell differentiation and tissue development [119, 129], and histone deacetylase 5 (HDAC5), which belongs to the class IIa HDAC family, was reported to act as a conserved transcriptional repressor of myogenin [83]. In agreement, AMPK-mediated myogenin expression and myogenesis have been shown to be mediated by HDAC5 expression in skeletal muscle cells of the tibialis anterior [30]. Taken together, these studies suggest that AMPK positively regulates myogenesis through a HDAC5–myogenin mechanism.

Previous studies have demonstrated that AMPK is an important player during muscle development. Specifically, deletion of LKB1, a crucial upstream kinase of AMPK, led to defects in muscle development and growth through AMPK-mediated myoblast proliferation and differentiation [139, 151]. It has also been demonstrated that impairments of AMPK activity in fetal muscles of obese sheep were associated with decreased densities of muscle fibers per area [178]. In another study, metformin-induced AMPK activation attenuated developmental impairments of skeletal muscle in the offspring of obese mice [154]. In addition to muscle development, the importance of AMPK in muscle regeneration from injury has been reported. The postinjury regeneration process induced by cardiotoxin in tibialis anterior muscle followed the growth of new muscle fibers, which were characterized by central locations of nuclei, but deficiencies of AMPKα1 or LKB1 retarded these regeneration processes [100, 139].

In contrast, AMPK was shown to act as a negative regulator of myogenesis. Glucose restriction-mediated inhibition of myoblast differentiation was shown to be AMPK dependent [34]. Moreover, treatment with high doses of AICAR (0.5–1.0 mM), which were sufficient to fully activate AMPK, inhibited myoblast fusion to myotubes and decreased levels of myogenin [34, 139] and MyoD [166]. However, low doses of AICAR (0.125 mM), which induces moderate AMPK activation, were shown to promote myogenesis [31]. Taken together, these data suggest that whereas AMPK is basally required for myogenesis, excessive stresses such as glucose restriction or high doses of pharmacological stimulation that induce robust AMPK activation may inhibit myogenesis through undetermined mechanisms.

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Oct 1, 2017 | Posted by in MUSCULOSKELETAL MEDICINE | Comments Off on Participation of AMPK in the Control of Skeletal Muscle Mass

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