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

Abstract

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.

1. 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 β₂-adrenergic receptors (β₂-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 contains a significant proportion of β-ARs. The β₂ subtype is the most abundant, while ~7–10% of ARs are the β₁ subtype [1, 2]. Furthermore, β₂-AR is more dense in slow-twitch muscles than in fast-twitch muscles [3, 4]. However, the magnitude of anabolic responses to β₂-adrenergic agonists is greater in fast-twitch muscles than in slow-twitch muscles [5–8].

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 β₂- and β₃-ARs in skeletal muscle are also capable of coupling to an inhibitory guanine nucleotide-binding protein, Gα_i [9]. β₂-AR activates the Gα_s/adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP)/cAMP-dependent protein kinase A (PKA) signaling pathway. The signaling pathway is at least in part responsible for the anabolic response of skeletal muscle to β₂-AR stimulation. Further, in addition to the well-documented inhibition of AC activity [10], β₂-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 NH₂ terminus and 3 intracellular loops that include a COOH terminus [12]. β₂-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 β₂-AR responsiveness in various conditions.

The adaptive responses of β₂-AR expression to anabolic and catabolic conditions in skeletal muscles are shown in Figure 1. Understanding the correlation between changes in muscle mass and β₂-AR expression in several anabolic or catabolic conditions present scientific evidence to eradicate sports doping and identify novel approaches for attenuating muscle atrophy concomitant with disuse and various diseases. This paper will discuss the effects of (1) pharmacological β₂-AR stimulation (sports doping), (2) muscle hypertrophy (exercise training), and (3) muscle atrophy (catabolic conditions and hormones) on β₂-AR expression in skeletal muscles.

Figure 1.

Changes in β₂-AR expression in hypertrophied and atrophied skeletal muscles. (a)  β₂-AR stimulation using anabolic drugs downregulates β₂-AR expression in hypertrophied fast-twitch muscles but not in slow-twitch muscles [4, 7, 8, 14–17]. (b) Exercise training such as endurance training upregulates β₂-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 β₂-AR expression. The differential effects of types of exercise training on physiological responses such as β₂-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 β₂-AR expression in nonatrophied slow-twitch muscles but not fast-twitch muscles [16, 20, 21]. (d) Muscle disuse downregulates β₂-AR expression in atrophied slow-twitch muscle, whereas no changes or upregulation of receptor expression are observed in fast-twitch muscles [14, 22]. Up arrow (open arrow): upregulation of β₂-AR expression; down arrow (filled arrow): downregulation of β₂-AR expression; lateral arrow (shade arrow): no change.

2. Pharmacological Stimulation of β₂-AR

2.1. Muscle Hypertrophy and β₂-AR

A β₂-adrenergic agonist, clenbuterol [1-(4-amino-3,5-dichlorobenzyl)-2-(tert-butylamino) ethanol], is used as a nonsteroidal anabolic drug for sports doping. According to the recent World Anti-Doping Agency (WADA) documents, clenbuterol was the seventh most commonly used anabolic agent in 2009 (67 cases; 2.0% of all anabolic agents used).

Numerous studies have shown that the administration of β₂-adrenergic agonists induces muscle hypertrophy in many species [23–25]. Experiments using mice lacking β₁-AR, β₂-AR, or both demonstrate that β₂-adrenergic agonist-induced functions such as muscle hypertrophy are mediated by β₂-AR [26]. β₂-Adrenergic agonists promote muscle growth by increasing the rate of protein synthesis and/or decreasing protein degradation [23–25]. Furthermore, β₂-adrenergic agonists induce slow-to-fast [myosin heavy chain (MHC)I/β → MHCIIa → MHCIId/x → MHCIIb] transformation of muscle fibers.

The β₂-AR signaling pathway involves the agonist-dependent activation of Gα_s, which in turn activates AC, resulting in increased cAMP production. Cyclic AMP-activated PKA initiates the transcription of many target genes via the phosphorylation of cAMP-response-element-(CRE-) binding protein (CREB) or adaptor proteins such as CREB-binding protein (CBP) and p300, subsequently promoting protein synthesis [23]. While β₂-AR-mediated signaling was traditionally believed to involve selective coupling to Gα_s, recent studies revealed that β₂-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 β₂-adrenergic agonist-induced hypertrophy in skeletal muscles [23].

In addition to promoting protein synthesis, the hypertrophic response of skeletal muscles following β₂-adrenergic agonist administration is associated with decreased protein degradation. β₂-Adrenergic agonists attenuate protein degradation predominantly via Ca²⁺-dependent proteolysis and the ATP/ubiquitin-dependent pathway [27–31]. However, there is little knowledge regarding the preventive effects of β₂-adrenergic agonists on the proteolysis system compared with the protein synthesis system.

The hypertrophic responses to β₂-adrenergic agonists are observed much frequently in fast-twitch muscle than in slow-twitch muscle. Our group previously demonstrated that clenbuterol administration (1.0 mg·kg⁻¹·day⁻¹) to rats for 10 days increases the mass of fast-twitch (extensor digitorum longus: EDL) muscle without altering in slow-twitch (soleus) muscle [7, 8]; other groups also observed the same tendency [5, 6, 32–35]. However, the mechanisms of the fiber-type-dependent effects of β₂-adrenergic agonists on muscle hypertrophy remain unclear.

Pearen et al. [36, 37] and Kawasaki et al. [38] identified that β₂-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 β₂-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 slow-twitch 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, β₂-AR-mediated signaling pathways tend to promote muscle hypertrophy to a greater extent in fast-twitch muscle than in slow-twitch muscle.

2.2. Posttranslational Regulation of β₂-AR

As shown in Table 1, some reports focus on the responses of β₂-AR expression to β₂-AR stimulation in skeletal muscles [4, 7, 8, 14–17]. This is because β₂-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].

Table 1.

Responses of β₂-AR expression in skeletal muscle to anabolic and catabolic conditions.

Conditions	Species	β2-AR		Other findings	References
		Protein	mRNA
β2-AR stimulation
Fenoterol	Rat	↓ (FT)	n.d.		[4]
(1.4 mg · kg−1 · day−1, 4 weeks)		→(ST)
Clenbuterol	Rat	n.d.	↓ (FT)	β1-AR mRNA ↓ (LV)	[7]
(1.0 mg · kg−1 · day−1, 10 days)			→ (ST)	β2-AR mRNA ↓ (LV)
Clenbuterol	Rat	n.d.	↓ (FT)	GR mRNA ↓ (FT)	[8]
(1.0 mg · kg−1 · day−1, 10 days)			→ (ST)	HuR mRNA ↓ (FT)
				AUF1 mRNA ↓ (FT)
				hnRNP A1 mRNA ↓ (FT)
Fenoterol	Rat	→ (FT, ST)	↓ (FT, ST)	Gαs content→(FT, ST)	[14]
(1.4 mg · kg−1 · day−1, 2–7 days)				AC activity→ (FT, ST)
Clenbuterol	Rat	↓ (FT+ST)	n.d.		[15]
(2.0 mg · kg−1 · day−1, 18 days)
Clenbuterol	Rat	↓ (FT)	n.d.	β2-AR affinity→ (FT)	[16]
(4.0 mg · kg−1 of feed, 10 days)
Clenbuterol	Rat	↓ (FT+ST)	n.d.		[17]
(0.2 mg · kg−1 · day−1, 7 days)
Clenbuterol (50 μM)	Mouse (ex vivo)	Phosphorylated	n.d.	cAMP concentration ↑	[41]
Formoterol (100 μM)		β2-AR ↑ (ST),		(FT, ST)
Salbutamol (500 μM)		→(FT)
Endurance training
Treadmill (12 weeks)	Rat	↓ (FT)	n.d.	β2-AR afflnity →	[18]
				AC activty ↓
				Gαs content ↓
Treadmill (18 weeks)	Rat	→ (FT)	n.d.	AC activity ↑ (FT, ST)	[19]
		↑ (ST)		β2-AR density→(acute)
Catabolic conditions
Dexamethasone	Rat	→ (FT, ST)	→ (FT)	GR mRNA ↓ (FT, ST)	[20]
(1.0 mg · kg−1 · day−1, 10 days)			↓ (ST)	CREB mRNA ↓ (ST)
				AUF1 mRNA ↑(FT)
Dexamethasone	Rat	n.d.	→ (FT)	GR mRNA ↓ (FT, ST)	[21]
(1.0 mg · kg−1 · day−1, 10 days)			↓ (ST)	β1-AR mRNA ↑ (LV)
Dexamethasone	Rat	→ (FT)	n.d.	β2-AR affinity→(FT)	[16]
(0.2 mg · kg−1 · day−1, 10 days)
Casted-immobilization	Rat	→ (FT, ST)	→ (FT)	GR mRNA ↓ (ST)	[22]
(10 days)			↓ (ST)	GR protein ↓ (ST)
Aging	Rat	→ (FT, ST)	n.d.		[4]
Injury	Rat	↑ (FT)	↑ (FT)	Gαs content ↑ (FT), ↓ (ST)	[14]
(bupivacaine injection)		↓ (ST)	↓ (ST)	AC activity ↑ (FT, ST)

FT, fast-twitch muscle; ST, slow-twitch muscle; LV, left ventricle muscle. Up arrow, increase; down arrow, decrease; lateral arrow, no change. n.d., no data.

The desensitization of β₂-AR is associated with receptor phosphorylation. McCormick et al. [41] demonstrate that fast-twitch fibers mainly express nonphosphorylated β₂-AR, whereas slow-twitch fibers predominantly express phosphorylated β₂-AR. Furthermore, treating muscle fibers with β₂-adrenergic agonists (e.g., clenbuterol, formoterol, and salbutamol) increases the phosphorylation of β₂-AR in slow-twitch 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 β₂-adrenergic agonist administration [42]. Thus, there is a negative correlation between the level of phosphorylated β₂-AR and receptor kinase. Therefore, further investigation is needed to reveal the detailed mechanism of β₂-AR phosphorylation.

Following β₂-AR phosphorylation, the receptor is internalized into the cytosol. The internalized β₂-AR is then degraded or dephosphorylated and subsequently recycled to the membrane [13, 43–45]. Prolonged administration of β₂-adrenergic agonists leads to the downregulation of β₂-AR density in skeletal muscles [15–17]. These posttranslational regulations are advantageous for maintaining the rate of muscle protein synthesis and/or degradation.

2.3. Short-Term and Chronic Transcriptional Regulation of β₂-AR

β ₂-AR synthesis, including transcription and subsequent translation, is required to restore transmembrane receptor density. The process of β₂-AR synthesis can be separated into 2 pathways: (1) the positive autoregulation of β₂-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 β₂-AR gene via interaction between hormones and the nuclear receptor complex and response elements on the β₂-AR promoter region [48]. In particular, the transcription of the β₂-AR gene and the subsequent mRNA expression via cAMP-mediated CRE activation increased in response to short-term β₂-adrenergic agonist exposure [46, 47]. Moreover, treatment with glucocorticoids or thyroid hormone transactivates the β₂-AR gene both in vitro and in vivo [48–51].

Our previous reports demonstrate that clenbuterol administration (1.0 mg·kg⁻¹·day⁻¹) for 10 days to rats decreases β₂-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 β₂-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 β₂-AR expression [16, 50, 51]. These findings corroborate our results that there is a positive correlation between the expression levels of β₂-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 β₂-AR mRNA expression in the EDL and soleus muscles. Thus, in contrast to the transactivation of the β₂-AR gene and increase in the mRNA level in response to short-term agonist exposure, chronic β₂-adrenergic stimulation inhibits β₂-AR synthesis in skeletal muscles.

2.4. Posttranscriptional Regulation of β₂-AR

In addition to post-translational and transcriptional regulation, several groups focus on the posttranscriptional regulation of β₂-AR mRNA. β₂-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) [52–55]. These factors play a role in the regulation of β₂-AR mRNA stability [52–55]. Our study demonstrates that clenbuterol-induced stimulation of β₂-AR decreases the mRNA expressions of these factors in the EDL but not in the soleus muscle [8], suggesting that the posttranscriptional process of β₂-AR synthesis requires the stability of its mRNA to be regulated.

3. Exercise Training and β₂-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 β₂-AR in skeletal muscles differ with the type of exercise training.

3.1. Strength Exercise Training and β₂-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 transporter1 mRNA expression is completely offset by strength training [64]. Thus, there are no synergetic effects of a combination of strength training and β₂-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 β₂-AR stimulation is insufficient because the experimental models of strength-trained animals are not fully established.

3.2. Endurance Exercise Training and β₂-AR

In contrast to strength training, β₂-AR stimulation affects endurance-training-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 Ca²⁺ sensitivity in fast-twitch fibers. These results suggest that endurance-training-heightened muscle aerobic capacity is attenuated by β₂-AR stimulation-induced muscle fiber-type 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 β₂-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 β₂-AR activation. Furthermore, the Ca²⁺-signaling [70] and p38 MAPK pathways [71], which is downstream of β₂-AR, are activated in skeletal muscles in response to exercise, which regulates PGC-1α expression. Since PGC-1α promotes mitochondrial biogenesis [72], the exercise-induced activation of β₂-AR may in part enhance aerobic capacity by increasing PGC-1α expression. Thus, β₂-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 β₂-AR Expression to Exercise Training

As mentioned above, the functional roles of β₂-AR during exercise training are physiologically important in skeletal muscles. Therefore, changes in the expression and sensitivity of β₂-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 β₂-AR expression to exercise training in skeletal muscles. However, many studies demonstrate the effects of exercise training on β₂-AR expression in several tissues 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 β₁-, β₂-, and β₃-AR densities in the rat left ventricle. In adipocytes, the exercise-induced trafficking of β₂-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 β₂-AR mRNA in macrophages and highlight the significance of β₂-AR in the exercise training-induced improvement of macrophages' innate immune function. Thus, changes in β₂-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 β₂-AR expression and signaling in slow-twitch muscle contributes to the adaptation of metabolic and anabolic capacities during exercise.

4. Muscle Atrophy and β₂-AR

4.1. Preventive Roles of β₂-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 β₂-adrenergic agonists provide therapeutic potential for attenuating or reversing muscle wasting, muscle fiber atrophy, and muscle weakness. These β₂-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 β₂-AR as a target of pharmacological stimulation. Since 2000, many groups have focused on the preventive effects of β₂-adrenergic agonist on disuse-induced muscle atrophy [4, 34, 35, 79].

Yimlamai et al. [35] demonstrate that clenbuterol attenuates the hindlimb unweighting-induced atrophy and reduces ubiquitin conjugates only in fast-twitch plantaris and tibialis anterior muscles but not in the slow-twitch soleus muscle; this suggests that clenbuterol alleviates hindlimb unweighting-induced atrophy, particularly, in fast-twitch muscles at least in part through a muscle-specific inhibition of the ubiquitin-proteasome pathway. However, Stevens et al. [34] report that clenbuterol treatment accelerates hindlimb unweighting-induced slow-to-fast (MHCI/β → MHCIIa → MHCIId/x → MHCIIb) transformation in the soleus muscle.β₂-Adrenergic agonist also reverses muscle wasting and weakness in several conditions such as aging [4], muscular dystrophy [29], denervation [80], cancer cachexia [28], and myotoxic injury [81].

4.2. Preventive Roles of β₂-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 ubiquitin-proteasome pathway [82, 83]. Sepsis also elevates plasma glucocorticoids and adrenocorticotropic hormone (ACTH) levels [84]. Therefore, several studies focus on the counteractive effects of β₂-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, β₂-AR stimulation plays an inhibitory role in muscle atrophy and weakness induced by catabolic diseases, mechanical unloading, catabolic hormones, and pharmacological agents.

4.3. Response of β₂-AR Expression to Catabolic Hormones

Although the effectiveness of β₂-AR stimulation on muscle atrophy is well documented, catabolic condition-induced changes in the expression of β₂-AR in skeletal muscles are not fully understood. Understanding the responses of β₂-AR expression to muscle atrophy is required to establish treatments for muscle atrophy.

Table 1 shows the catabolic-condition-induced changes in β₂-AR expression in skeletal muscles. Our group investigated whether catabolic hormones or agents alter β₂-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 β₂-AR mRNA in the soleus muscle without altering that in the EDL muscle, although the expression of β₂-AR protein in the EDL and soleus muscles is not altered [20, 21]. Dexamethasone also does not alter β₂-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 β₂-AR gene in the human hepatoma cell line (HepG2) [48], subsequently leading to the upregulation of β₂-AR levels in DDT₁ 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 β₂-AR gene [46, 47], in the soleus and EDL muscles, respectively [20]. These findings suggest that the dexamethasone-induced decrease in the expression of β₂-AR mRNA in the slow-twitch soleus muscle is associated with transcriptional regulations.

4.4. Response of β₂-AR Expression to Muscle Disuse

The effects of physiological and pathological catabolic-condition-induced muscle atrophy on β₂-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 β₂-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 β₂-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 aging-induced muscle wasting is observed in the EDL and soleus muscles, although there are no age-associated changes in β₂-AR density in these muscles. Furthermore, in the regeneration process from muscle injury induced by bupivacaine injection, β₂-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 β₂-AR expression are different from and/or dependent on the conditions, especially in fast-twitch muscles, whereas decreasing tendencies are observed in slow-twitch muscles.

Both pharmacological and mechanical studies indicate that the preventive effects of β₂-AR stimulation on muscle atrophy and weakness are limited by decreased β₂-AR synthesis and subsequently decreased density. In order to use β₂-adrenergic agonists as a therapeutic agent for muscle wasting, further studies are necessary to obtain detailed evidence regarding the responses of β₂-AR expression and function to muscle atrophy.

5. Conclusions

In this paper, we discussed adaptive responses of β₂-AR expression in skeletal muscles to β₂-adrenergic agonist treatment, exercise training, muscle disuse, and glucocorticoid treatment. This paper also outlined the functional roles of β₂-AR in skeletal muscles. Skeletal muscle partly requires β₂-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.