Muscle Plasticity and β2-Adrenergic Receptors: Adaptive Responses of β2-Adrenergic Receptor Expression to Muscle Hypertrophy and Atrophy

We discuss the functional roles of β2-adrenergic receptors in skeletal muscle hypertrophy and atrophy as well as the adaptive responses of β2-adrenergic receptor expression to anabolic and catabolic conditions. β2-Adrenergic receptor stimulation using anabolic drugs increases muscle mass by promoting muscle protein synthesis and/or attenuating protein degradation. These effects are prevented by the downregulation of the receptor. Endurance training improves oxidative performance partly by increasing β2-adrenergic receptor density in exercise-recruited slow-twitch muscles. However, excessive stimulation of β2-adrenergic receptors negates their beneficial effects. Although the preventive effects of β2-adrenergic receptor stimulation on atrophy induced by muscle disuse and catabolic hormones or drugs are observed, these catabolic conditions decrease β2-adrenergic receptor expression in slow-twitch muscles. These findings present evidence against the use of β2-adrenergic agonists in therapy for muscle wasting and weakness. Thus, β2-adrenergic receptors in the skeletal muscles play an important physiological role in the regulation of protein and energy balance.


Introduction
The skeletal muscle is the most abundant tissue in the human body comprising 40-50% of body mass. Skeletal muscle protein undergoes rapid turnover, which is regulated by the balance between the rates of protein synthesis and degradation. Physical activity (exercise training) and anabolic hormones and drugs (sports doping) increase muscle protein content. However, sarcopenia and muscle disuse (due to unloading, microgravity, or inactivity) and diseases decrease muscle protein content. The rate of protein synthesis is at least in part mediated by β 2 -adrenergic receptors (β 2 -ARs) in skeletal muscles in both anabolic and catabolic conditions. ARs belong to the guanine nucleotide-binding G-protein-coupled receptor (GPCR) family. Skeletal muscle con-tains a significant proportion of β-ARs. The β 2 subtype is the most abundant, while ∼7-10% of ARs are the β 1 subtype [1,2]. Furthermore, β 2 -AR is more dense in slow-twitch muscles than in fast-twitch muscles [3,4]. However, the magnitude of anabolic responses to β 2 -adrenergic agonists is greater in fast-twitch muscles than in slow-twitch muscles [5][6][7][8].
The family of β-ARs was originally believed to signal predominantly via coupling with a stimulatory guanine nucleotide-binding protein, Gα s ; however, recent studies revealed that both β 2 -and β 3 -ARs in skeletal muscle are also capable of coupling to an inhibitory guanine nucleotide-binding protein, Gα i [9]. β 2 -AR activates the Gα s /adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP)/cAMP-dependent protein kinase A (PKA) signaling Fast-twitch muscle (exercise nonrecruited) Fast-twitch muscle (nonatrophied) Figure 1: Changes in β 2 -AR expression in hypertrophied and atrophied skeletal muscles. (a) β 2 -AR stimulation using anabolic drugs downregulates β 2 -AR expression in hypertrophied fast-twitch muscles but not in slow-twitch muscles [4,7,8,[14][15][16][17]. (b) Exercise training such as endurance training upregulates β 2 -AR expression in exercise-recruited slow-twitch muscles, whereas no changes or downregulations are observed in fast-twitch muscles [18,19], although muscle mass is not altered. However, although exercise training such as isometric strength training induces muscle hypertrophy, there is no insight regarding the effects of such exercise on β 2 -AR expression. The differential effects of types of exercise training on physiological responses such as β 2 -AR expression and muscle hypertrophy should be clarified in more detailed and are currently being investigated by our group. (c) Catabolic hormones or drugs such as glucocorticoids downregulate β 2 -AR expression in nonatrophied slow-twitch muscles but not fast-twitch muscles [16,20,21]. (d) Muscle disuse downregulates β 2 -AR expression in atrophied slow-twitch muscle, whereas no changes or upregulation of receptor expression are observed in fast-twitch muscles [14,22]. pathway. The signaling pathway is at least in part responsible for the anabolic response of skeletal muscle to β 2 -AR stimulation. Further, in addition to the well-documented inhibition of AC activity [10], β 2 -AR coupling to Gα i activates Gα s -independent pathways [11]. β 2 -AR has 7 transmembrane α helices forming 3 extracellular loops, including an NH 2 terminus and 3 intracellular loops that include a COOH terminus [12]. β 2 -AR contains phosphorylation sites in the third intracellular loop and proximal cytoplasmic tail. Phosphorylation of these sites triggers the agonist-promoted desensitization, internalization, and degradation of the receptor [13]. These regulatory mechanisms contribute to maintaining agonist-induced β 2 -AR responsiveness in various conditions. The adaptive responses of β 2 -AR expression to anabolic and catabolic conditions in skeletal muscles are shown in Figure 1. Understanding the correlation between changes in muscle mass and β 2 -AR expression in several anabolic or catabolic conditions present scientific evidence to eradicate sports doping and identify novel approaches for attenuating Journal of Biomedicine and Biotechnology 3 muscle atrophy concomitant with disuse and various diseases. This paper will discuss the effects of (1) pharmacological β 2 -AR stimulation (sports doping), (2) muscle hypertrophy (exercise training), and (3) muscle atrophy (catabolic conditions and hormones) on β 2 -AR expression in skeletal muscles. Numerous studies have shown that the administration of β 2 -adrenergic agonists induces muscle hypertrophy in many species [23][24][25]. Experiments using mice lacking β 1 -AR, β 2 -AR, or both demonstrate that β 2 -adrenergic agonistinduced functions such as muscle hypertrophy are mediated by β 2 -AR [26]. β 2 -Adrenergic agonists promote muscle growth by increasing the rate of protein synthesis and/or decreasing protein degradation [23][24][25]. Furthermore, β 2 -adrenergic agonists induce slow-to-fast [myosin heavy chain (MHC)I/β → MHCIIa → MHCIId/x → MHCIIb] transformation of muscle fibers.

Pharmacological
The β 2 -AR signaling pathway involves the agonistdependent activation of Gα s , which in turn activates AC, resulting in increased cAMP production. Cyclic AMPactivated PKA initiates the transcription of many target genes via the phosphorylation of cAMP-response-element-(CRE-) binding protein (CREB) or adaptor proteins such as CREBbinding protein (CBP) and p300, subsequently promoting protein synthesis [23]. While β 2 -AR-mediated signaling was traditionally believed to involve selective coupling to Gα s , recent studies revealed that β 2 -AR exhibits dual coupling to both Gα s and Gα i in skeletal muscles [9,23]. In addition to Gα s , Gα i -linked Gβγ subunits play an active role in various cell signaling processes such as the phosphoinositol 3 kinase (PI3 K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70S6 K and PI3 K/Akt/forkhead box-O (FOXO) pathways. These signaling pathways play important roles in β 2 -adrenergic agonist-induced hypertrophy in skeletal muscles [23].
In addition to promoting protein synthesis, the hypertrophic response of skeletal muscles following β 2 -adrenergic agonist administration is associated with decreased protein degradation. β 2 -Adrenergic agonists attenuate protein degradation predominantly via Ca 2+ -dependent proteolysis and the ATP/ubiquitin-dependent pathway [27][28][29][30][31]. However, there is little knowledge regarding the preventive effects of β 2 -adrenergic agonists on the proteolysis system compared with the protein synthesis system.
Pearen et al. [36,37] and Kawasaki et al. [38] identified that β 2 -AR activation increases the expression of the orphan nuclear receptor, NOR-1 (NR4A3), a negative regulatory factor of myostatin (a member of the transforming growth factor-β superfamily and a potent negative regulator of muscle mass), in fast-twitch muscles without altering that in slow-twitch muscles. Furthermore, Shi et al. [32] demonstrate the possibility that β 2 -adrenergic agonist-induced fiber-type-dependent hypertrophy is in part due to the extracellular signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) pathway. Moreover, the pharmacological inhibition of the PI3 K/Akt/mTOR signaling pathway revealed that the attenuation of the anabolic response to clenbuterol is greater in fast-twitch muscles than in slowtwitch muscles [30]. In addition to the protein synthesis system, Yimlamai et al. [35] found that clenbuterol inhibits ubiquitination more strongly in fast-twitch muscles than in slow-twitch muscles. Thus, β 2 -AR-mediated signaling pathways tend to promote muscle hypertrophy to a greater extent in fast-twitch muscle than in slow-twitch muscle. Table 1, some reports focus on the responses of β 2 -AR expression to β 2 -AR stimulation in skeletal muscles [4,7,8,[14][15][16][17]. This is because β 2 -AR functions such as muscle hypertrophy are maintained via receptor density, including synthesis and downregulation as well as receptor sensitivity, which includes receptor sensitization, desensitization, phosphorylation, and internalization [13,39,40].

Posttranslational Regulation of β 2 -AR. As shown in
The desensitization of β 2 -AR is associated with receptor phosphorylation. McCormick et al. [41] demonstrate that fast-twitch fibers mainly express nonphosphorylated β 2 -AR, whereas slow-twitch fibers predominantly express phosphorylated β 2 -AR. Furthermore, treating muscle fibers with β 2adrenergic agonists (e.g., clenbuterol, formoterol, and salbutamol) increases the phosphorylation of β 2 -AR in slowtwitch fibers but not in fast-twitch fibers [41]. On the other hand, the receptor phosphorylation occurs via the actions of protein kinases (such as PKA) and/or GPCR kinase (GRK). Rat skeletal muscles contain predominantly GRK2 and GRK5; GRK protein is expressed more in fast-twitch muscles than in slow-twitch muscles. These expression levels in each type of muscle fiber are not altered by β 2 -adrenergic agonist administration [42]. Thus, there is a negative correlation between the level of phosphorylated β 2 -AR and receptor kinase. Therefore, further investigation is needed to reveal the detailed mechanism of β 2 -AR phosphorylation.

Conditions
Species (1.0 mg · kg −1 · day −1 , 10 days) regulations are advantageous for maintaining the rate of muscle protein synthesis and/or degradation.

Short-Term and Chronic Transcriptional
Regulation of β 2 -AR. β 2 -AR synthesis, including transcription and subsequent translation, is required to restore transmembrane receptor density. The process of β 2 -AR synthesis can be separated into 2 pathways: (1) the positive autoregulation of β 2 -AR gene transcription via receptor-mediated elevation of cAMP concentration followed by the phosphorylation and activation of CREB [46,47] and (2) the transactivation of the β 2 -AR gene via interaction between hormones and the nuclear receptor complex and response elements on the β 2 -AR promoter region [48]. In particular, the transcription of the β 2 -AR gene and the subsequent mRNA expression via cAMP-mediated CRE activation increased in response to short-term β 2 -adrenergic agonist exposure [46,47]. Moreover, treatment with glucocorticoids or thyroid hormone transactivates the β 2 -AR gene both in vitro and in vivo [48][49][50][51].
Our previous reports demonstrate that clenbuterol administration (1.0 mg·kg −1 ·day −1 ) for 10 days to rats Journal of Biomedicine and Biotechnology 5 decreases β 2 -AR mRNA expression in the fast-twitch EDL muscle without altering that in the slow-twitch soleus muscle [7,8]. Furthermore, the mRNA expression of glucocorticoid receptors (GRs) was also decreased with clenbuterol treatment in the EDL muscle but not in the soleus muscle [8]. Glucocorticoids and the GR complex activate the transcription of the β 2 -AR gene via interaction with glucocorticoid response elements (GREs), consensus cis-acting DNA sequences (i.e., AGA ACA nnn TGT TCT) on its promoter regions [48], thus upregulating β 2 -AR expression [16,50,51]. These findings corroborate our results that there is a positive correlation between the expression levels of β 2 -AR and GR in skeletal muscles. Beitzel et al. [14] also report that administrating the β-adrenergic agonist, fenoterol (1.4 mg·kg −1 ·day −1 , i.p.), for 5 days decreases β 2 -AR mRNA expression in the EDL and soleus muscles. Thus, in contrast to the transactivation of the β 2 -AR gene and increase in the mRNA level in response to short-term agonist exposure, chronic β 2 -adrenergic stimulation inhibits β 2 -AR synthesis in skeletal muscles.

Posttranscriptional Regulation of β 2 -AR.
In addition to post-translational and transcriptional regulation, several groups focus on the posttranscriptional regulation of β 2 -AR mRNA. β 2 -AR mRNA contains an AU-rich element (ARE) within the 3 -untranslated region (3 -UTR) that can be recognized by several mRNA-binding proteins, including Hu antigen R (HuR), AU-rich element binding/degradation factor1 (AUF1), and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) [52][53][54][55]. These factors play a role in the regulation of β 2 -AR mRNA stability [52][53][54][55]. Our study demonstrates that clenbuterol-induced stimulation of β 2 -AR decreases the mRNA expressions of these factors in the EDL but not in the soleus muscle [8], suggesting that the posttranscriptional process of β 2 -AR synthesis requires the stability of its mRNA to be regulated.

Exercise Training and β 2 -AR
Strength-resistance training increases muscle mass [56], fiber cross-sectional area [57], protein and RNA contents [58], and the capacity to generate force [59]. In contrast to strength training, endurance training is characterized by increased mitochondrial mass [60], increased oxidative enzymes [61], decreased glycolytic enzymes [62], increased slow contractile and regulatory proteins [62], and decreased fast fiber area [63]. These findings suggest that the functional roles of β 2 -AR in skeletal muscles differ with the type of exercise training.

Strength Exercise Training and β 2 -AR.
Mounier et al. [64] investigated the changes in the weight of the EDL muscle induced by clenbuterol administration, strength training, and a combination of both. They found that the effects of strength training and clenbuterol on muscle hypertrophy were not additive in fast-twitch muscles. Their report also demonstrates that the strength-training-induced enhancement of lactate dehydrogenase-specific activity is completely inhibited by clenbuterol administration, while the clenbuterol-induced decrease in monocarboxylate trans-porter1 mRNA expression is completely offset by strength training [64]. Thus, there are no synergetic effects of a combination of strength training and β 2 -AR stimulation on muscle mass. Furthermore, strength training counteracts molecular modifications such as glycolytic control induced by chronic clenbuterol administration in fast-twitch muscles to some extent. However, our evidence regarding the synergistic effects of strength training and β 2 -AR stimulation is insufficient because the experimental models of strengthtrained animals are not fully established.

Endurance Exercise
Training and β 2 -AR. In contrast to strength training, β 2 -AR stimulation affects endurancetraining-induced modulations such as contractile activity [65], muscle fiber-type shift [65], metabolic enzyme activity [66], and insulin resistance [67,68]. Lynch et al. [65] demonstrated that low-intensity endurance training prevents clenbuterol-induced slow-to-fast (type I fiber → type II fiber) fiber-type transformation in the EDL and soleus muscles, and thereby offsets the clenbuterol-induced decrease in Ca 2+ sensitivity in fast-twitch fibers. These results suggest that endurance-training-heightened muscle aerobic capacity is attenuated by β 2 -AR stimulation-induced muscle fibertype transformations. Furthermore, pharmacological β-AR blockage diminishes the endurance-training-induced increase in citrate synthase activity in the fast-twitch plantaris muscle [66]. Moreover, clenbuterol administration prevents the endurance-training-induced improvement in insulin-stimulated glucose uptake and attenuates the increase in citrate synthase activity in the skeletal muscles of obese Zucker rats [67,68]. These findings demonstrate that the endurance-training-induced increase in aerobic metabolism in skeletal muscles requires moderate but not excessive stimulation of β 2 -AR.
Recently, Miura et al. [69] demonstrated that an increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA in response to exercise is mediated by β 2 -AR activation. Furthermore, the Ca 2+ -signaling [70] and p38 MAPK pathways [71], which is downstream of β 2 -AR, are activated in skeletal muscles in response to exercise, which regulates PGC-1α expression. Since PGC-1α promotes mitochondrial biogenesis [72], the exerciseinduced activation of β 2 -AR may in part enhance aerobic capacity by increasing PGC-1α expression. Thus, β 2 -AR stimulation is essential for enhancing the effects of exercise training on muscle functions such as fiber-type shift as well as oxidative and anaerobic metabolism.

3.3.
Response of β 2 -AR Expression to Exercise Training. As mentioned above, the functional roles of β 2 -AR during exercise training are physiologically important in skeletal muscles. Therefore, changes in the expression and sensitivity of β 2 -AR should be important for the metabolic, anabolic, and catabolic adaptations of skeletal muscles during exercise training. Nevertheless, there is little information on the response of β 2 -AR expression to exercise training in skeletal muscles. However, many studies demonstrate the effects of exercise training on β 2 -AR expression in several tissues 6 Journal of Biomedicine and Biotechnology and cell types such as myocardia [73,74], adipocytes [75], and macrophages [76]. Barbier et al. [73] demonstrated that exercise training induces changes in the distribution of β 1 -, β 2 -, and β 3 -AR densities in the rat left ventricle. In adipocytes, the exercise-induced trafficking of β 2 -AR into the cell membrane from the cytosol is coupled with adipocytes' function to increase intracellular cAMP production [75]. Kizaki et al. [76] also found a reduction in the expression of β 2 -AR mRNA in macrophages and highlight the significance of β 2 -AR in the exercise training-induced improvement of macrophages' innate immune function. Thus, changes in β 2 -AR expression play a role in physiological adaptations to exercise training in several tissues.
A few studies also report the effects of exercise training on β-AR in skeletal muscles [18,19,77,78] (Table 1). Nieto et al. [18] demonstrate that β-AR density and Gα s content in the fast-twitch gastrocnemius muscle are significantly lower in endurance-exercised rats than in controls. They also reveal that exercise reduces receptor-and nonreceptor-mediated (i.e., pharmacological stimulation of AC by forskolin) AC activity in muscles [18]. However, Buckenmeyer et al. [19] report that endurance training increases β-AR density in slow-twitch muscles that are primarily recruited during endurance training, whereas β-AR density is not altered in fast-twitch muscles. Their report also demonstrates that receptor-mediated AC activity in slow-twitch muscles is increased by endurance training, and nonreceptor-mediated AC activity is increased by training in both fast-and slow-twitch muscles [19]. In contrast to chronic endurance training, the effects of acute exercise on β-AR density and AC activity in each type of muscle were not observed [19]. Therefore, endurance-exercise-training-induced changes in β 2 -AR expression and signaling in slow-twitch muscle contributes to the adaptation of metabolic and anabolic capacities during exercise.

Preventive Roles of β 2 -AR in Disuse-Induced Muscle
Atrophy. Muscle wasting and weakness are common in physiological and pathological conditions, including aging, cancer cachexia, sepsis, other forms of catabolic stress, denervation, disuse (e.g., unloading, inactivity, and microgravity), burns, human immunodeficiency virus-(HIV)acquired immunodeficiency syndrome (AIDS), chronic kidney or heart failure, chronic obstructive pulmonary disease (COPD), and muscular dystrophies. For many of these conditions, the anabolic properties of β 2 -adrenergic agonists provide therapeutic potential for attenuating or reversing muscle wasting, muscle fiber atrophy, and muscle weakness. These β 2 -adrenergic agonists also have important clinical significance for enhancing muscle repair and restoring muscle function after muscle atrophy.
In particular, muscle disuse, which is mainly reflected by increased myofibrillar protein breakdown, causes a progressive decrease in muscle strength associated with a decreased cross-sectional area of muscle fibers. Therefore, preventing disuse-induced muscle atrophy is a problem requiring urgent attention and highlights β 2 -AR as a target of pharmacological stimulation. Since 2000, many groups have focused on the preventive effects of β 2 -adrenergic agonist on disuse-induced muscle atrophy [4,34,35,79].

Preventive Roles of β 2 -AR in Catabolic Hormone-Induced
Muscle Atrophy. Prolonged muscle disuse and/or unloading increases the secretion of glucocorticoids, which promotes the catabolism of muscle proteins via the ubiquitinproteasome pathway [82,83]. Sepsis also elevates plasma glucocorticoids and adrenocorticotropic hormone (ACTH) levels [84]. Therefore, several studies focus on the counteractive effects of β 2 -AR stimulation on glucocorticoid-induced muscle atrophy [16,85]. Huang et al. [16] report that clenbuterol almost prevents the decrease in the weight of gastrocnemius/plantaris muscle bundles induced by dexamethasone, a synthetic glucocorticoid. Pellegrino et al. [85] demonstrate that concurrent treatment of clenbuterol with dexamethasone minimizes MHC-transformation-induced by clenbuterol (slow-to-fast) or dexamethasone (fast-to-slow) alone. Thus, β 2 -AR stimulation plays an inhibitory role in muscle atrophy and weakness induced by catabolic diseases, mechanical unloading, catabolic hormones, and pharmacological agents.

Response of β 2 -AR Expression to Catabolic Hormones.
Although the effectiveness of β 2 -AR stimulation on muscle atrophy is well documented, catabolic condition-induced changes in the expression of β 2 -AR in skeletal muscles are not fully understood. Understanding the responses of β 2 -AR expression to muscle atrophy is required to establish treatments for muscle atrophy. Table 1 shows the catabolic-condition-induced changes in β 2 -AR expression in skeletal muscles. Our group investigated whether catabolic hormones or agents alter β 2 -AR expression in skeletal muscles [20,21]. Dexamethasone administration (1.0 mg·kg −1 ·day −1 ) to rats for 10 days decreases the expression of β 2 -AR mRNA in the soleus muscle without altering that in the EDL muscle, although the expression of β 2 -AR protein in the EDL and soleus muscles is not altered [20,21]. Dexamethasone also does not alter β 2 -AR density in gastrocnemius/plantaris muscle bundles [16]. These phenomena are specifically observed in skeletal muscles; meanwhile, glucocorticoids and the GR complex activate the transcription of β 2 -AR gene in the human hepatoma cell line (HepG2) [48], subsequently leading to the upregulation of β 2 -AR levels in DDT 1 MF-2 smooth muscle cells [50] and lung tissue [16,51]. Furthermore, dexamethasone decreases the expression of GR mRNA in the soleus muscle [20,21]. Dexamethasone also decreases and increases the expression of CREB mRNA, a transcription factor of the β 2 -AR gene [46,47], in the soleus and EDL muscles, respectively [20]. These findings suggest that the dexamethasone-induced decrease in the expression of β 2 -AR mRNA in the slow-twitch soleus muscle is associated with transcriptional regulations.

4.4.
Response of β 2 -AR Expression to Muscle Disuse. The effects of physiological and pathological catabolic-conditioninduced muscle atrophy on β 2 -AR expression have also been studied (Table 1) [4,14,22]. Our recent investigation demonstrates that casted immobilization (knee and foot arthrodesis) for 10 days markedly induced atrophy in the soleus muscle, whereas it decreased the expression of β 2 -AR mRNA [22]. Decreased GR mRNA and protein expression was also detected in the soleus muscle [22]. These results suggest that casted immobilization decreases the expression of β 2 -AR mRNA in slow-twitch muscles via the downregulation of GR levels and subsequent glucocorticoid signals. On the other hand, Ryall et al. [4] demonstrate that aginginduced muscle wasting is observed in the EDL and soleus muscles, although there are no age-associated changes in β 2 -AR density in these muscles. Furthermore, in the regeneration process from muscle injury induced by bupivacaine injection, β 2 -AR density and mRNA expression as well as Gα s content are decreased in the soleus but increased in the EDL muscle [14]. Thus, the effects of catabolic conditions such as disuse, aging, and injury on β 2 -AR expression are different from and/or dependent on the conditions, especially in fasttwitch muscles, whereas decreasing tendencies are observed in slow-twitch muscles.
Both pharmacological and mechanical studies indicate that the preventive effects of β 2 -AR stimulation on muscle atrophy and weakness are limited by decreased β 2 -AR synthesis and subsequently decreased density. In order to use β 2adrenergic agonists as a therapeutic agent for muscle wasting, further studies are necessary to obtain detailed evidence regarding the responses of β 2 -AR expression and function to muscle atrophy.

Conclusions
In this paper, we discussed adaptive responses of β 2 -AR expression in skeletal muscles to β 2 -adrenergic agonist treatment, exercise training, muscle disuse, and glucocorticoid treatment. This paper also outlined the functional roles of β 2 -AR in skeletal muscles. Skeletal muscle partly requires β 2 -AR activation for hypertrophy, regeneration, and atrophy prevention; however, its functions and responsiveness must be adaptively regulated by the receptor itself via downregulation, synthesis, and desensitization. New insight in the form of scientific evidence is needed to eradicate sports doping and to identify new therapeutic targets for attenuating muscle atrophy induced by physiological and pathological conditions.