J Phys Fitness Sports Med, 2(4): 423-428 (2013)DOI: 10.7600/jpfsm.2.423

JPFSM: Review Article

How β2-adrenergic agonists induce skeletal muscle hypertrophy?Takashi Kitaura

Received: September 2, 2013 / Accepted: September 10, 2013

Abstract Some β2-adrenergic agonists (β2-agonists) can strongly induce muscular hypertrophy, and are prohibited to use as doping drugs for athletes. The pharmacological mechanism for such induced hypertrophy is not clear. These agonists affect many organs via the cAMP-PKA system. Muscular hypertrophy is most likely induced by way of protein synthesis via the IGF-1/PI3K/PKB system and negative myostatin pathway. There are some reports indicating that β2-agonists might stimulate protein synthesis via the IGF-1/PI3K/PKB system. Furthermore, it inhibits proteolysis via the ATP-dependent ubiquitin-proteasome system (UPS), autophagy-lysosome system and calcium-calpain system. In this review, some discrepancies are introduced between a basic hypertrophic mechanism and inhibited atrophic mechanism using β2-agonists. Surfaces of muscle fiber might have some various receptors, and the β2-adrenoceptor might be activated di-rectly by PI3K via Gαs to trigger skeletal muscle hypertrophy. β2-agonists might be stimulated to synthesize follistatin of the myostatin inhibitor, as well as to produce satellite cells. Furthermore, increased myostatin may work to determine the size of each muscle fiber, and an increased number of quiescent satellite cells serve as myonuclei donors for hypertrophied muscle fibers. Recently, it was reported that follistatin synthesis was regulated by microRNAs. The effect of microRNA on β2-agonists is not clear. In the cAMP/PKA/CREB pathway, β2-agonists might inhibit various proteolytic systems, resulting in an increase of structural proteins. But β2-agonists have more pharmacological functions like lipolytic action and stimulation of the central nervous system; thus, more research and analysis is needed.Keywords : β2-adrenergic agonists, muscle hypertrophy, clenbuterol, protein synthesis, protein

degradation

Introduction

Muscular hypertrophy is one of the most interesting phenomena for athletes and people with muscle-wasting disorders. There are many methods to induce muscular hypertrophy like anaerobic weight training and hormonal treatment. Some β2-adrenergic agonists (β2-agonists) like clenbuterol, fenoterol, formoterol and salbutamol are well known as bronchodilators to treat asthma, and act as decongestants or as powerful anabolic agents. Therefore, they are limited in use for doping in athletic competitions. In particular, the anabolic action is the most intrinsic property in sports. Over the past 30 years many investiga-tors have reported about skeletal and/or cardiac muscle hypertrophy with clenbuterol administration1-5). But the mechanism of the action of drugs is complicated, and still under discussion in some reviews6-9). In this review, I propose a way to clarify the problem of the mechanism. The basic idea to explain muscular hypertrophy is due to the increased structural proteins. It was caused by the increased protein synthesis and the

decreased proteolysis. The homeostatic protein content is maintained by the balance between synthesis and break-down. The grade of change is summarized into hypertro-phic grades (H1-H4), atrophic grades (A1-A4) or keeping the situation (K) with no change (Table 1). If both the increasing grade and the degradation grade are the same high level (I5 and D5), protein content shows no change (K). Therefore, we need a few analyses to determine atro-phy or hypertrophy in two different directions. But most research was done in one direction for the various reasons of time, technique, and so on. In many cases, increasing grades (I1-I5) depend on the activity of the IGF-1/PI3K/Akt signaling pathway10-12) and negative myostatin pathway for protein synthesis13-15). On the other hand, degradation grades depend on the activ-ity of the ATP dependent ubiquitin-proteosome system (UPS)16,17), autophagy-lysosome system18-20) and calcium sensitive calpain pathway21-23). They are well documented by Ito et al.24).

Increments of structural proteins

There are some reports that β2-agonists increase protein Correspondence: tkitaura@staff.kanazawa-u.ac.jp

Laboratory of Exercise Biochemistry, Division of Sports Education, Health Service Center, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan

424 JPFSM : Kitaura T

synthesis via the IGF-1/PI3K/Akt pathway in striated muscles25-29). Although β2-agonists do not bind to IGF receptors directly, clenbuterol causes a transient increase of IGF-I in cells30) and of IGF-II in rat soleus29) and mas-seter muscle31). The IGF-1/PI3K/Akt pathway might be stimulated by increased IGFs. The peptide hormone ghrelin32), which increases before meals and decreases after meals, is secreted from the stomach and the hypo-thalamus, where it stimulates the secretion of growth hormone (GH) from the anterior pituitary gland. Then GH promotes IGF-1 synthesis and muscle growth. However, the increasing mechanism of IGFs by clenbuterol remain to be resolved. Spurlock et al.33) showed that clenbuterol promotes the synthesis of IGF-1 mRNA-related muscle growth in mouse gracilis muscle. β2-agonists activate the guanine nucleotide exchange factor domain of the β2-adrenoceptor causing the exchange of GDP for GTP at the α stimulatory subunit of the guanine nucleotide-binding regulatory protein (Gαs) and the subsequent dissociation of Gαs from the tightly associated β and γ subunits (Gβγ).

It was shown by von Maltzahn et al that Gαs could di-rectly activate PI3K34). And activation of frizzled (Fzd) 7, another member of the G protein coupled receptor family, induced hypertrophy of rodent myotubes via the activation of the PI3K-Akt-mTOR pathway. Gαs-coupled receptors expressed in skeletal muscle were summarized and reported by Berdeaux and Stewart8) (Fig. 1). It may suggest that β2-adrenoceptor might be able to directly activate PI3K via Gαs to trigger skeletal muscle hyper-trophy. Skeletal muscle hypertrophy was explained by hypertrophy of individual muscle fibers and hyperplasia increasing the number of cells. The former was supported by the increased diameter of muscle fibers; the latter by the increased number of fibers. Increased mRNA and protein content of muscle structural proteins and growth factor like IGF-1, and decreased mRNA and protein con-tent of proteolytic enzymes may explain the hypertrophy of muscle. Table 1 explains high hypertrophy (e.g. H4) by the high increasing grade (e.g. I5) and low degradation grade (e.g. D1) in muscular change. Myostatin is a member of transforming growth fac-tor beta (TGF-β) and inhibits muscle differentiation and growth in myogenesis (Fig. 2). Therefore, decreased myo-statin content by varoius stimulations also will explain increased protein synthesis. However, β2-agonists surpris-ingly showed increased myostatin contents. Therefore, skeletal muscle hypertrophy by β2-agonists should be explained by increased IGF, not by increased myostatin, which may have other functions. Myostatin is expressed in satellite cells and regulates satellite cell quiescence and self-renewal35). The adult muscle cells are post-mitotic and unable to divide, they must acquire external DNA. Satellite cells retain their mitotic capacity36). Higher lev-els of myostatin have been observed in quiescent satellite cells37). It may mean that an increasing number of qui-escent satellite cells serve as myonuclei donors for hy-pertrophied muscle fibers and support preferential incre-ments of DNA content in the latter phase of hypertrophy with β-agonist Cimaterol38).

K H H H H

K H H H

K H H

K H

Deg

rada

tion

grad

e

K

Table 1. Grade of muscular change

Capital letters I and D represent increments and decrements in muscular proteins, respectively. Capital letters H and A represent hypertrophy and atrophy of muscle, respectively. Capital letter K represents no change. The additional numbers indicate the intensity of each item.

Fig. 1 GPCRs that induce myofiber hypertrophy. Four GPCRs (Fzd7, β2-AR, CRFR2, and LPA receptor) have been shown to stimulate hypertrophy in myotubes and/or myofibers. Cognate ligands are italicized. A partial view of the known signaling mediators is shown. CRFR2 stimulates myofiber growth by an uncharacterized effector pathway. β2-AR also induces fiber type transitions to fast-twitch fibers (not shown). Data are cited from Berdeaux and Stewart8).

425JPFSM : Muscle hypertrophy with β2 agonists

In the cAMP/PKA/CREB pathway, the major effector of cAMP in skeletal muscle is protein kinase A (PKA). β2-agonists bind their own receptors and increase cAMP on PKA regulatory subunits, leading to the release of PKA catalytic subunits, which then drive cAMP response ele-ment binding protein (CREB) phosphorylation on Ser133. CREB phosphorylation occurs rapidly in skeletal muscle in response to β2--agonist administration27). Many genes contain cAMP response elements, and are thus potential targets of CREB39). CREB stimulates retinoblastoma protein promoter activity, together with MyoD, to induce the differentiation of myoblasts into myotubes40). CREB interacts with MyoD on follistatin promoter, thus promot-ing follistatingene expression41) (Fig. 2). Follistatin also known as an activin-binding protein is a protein that in humans is encoded by the FSTgene and inhibits excessive muscle growth. Follistatin has been shown to induce skel-etal muscle hypertrophy, notably by inhibiting myostatin activity42,43). Follistatin-mediated skeletal muscle hyper-trophy is regulated by Smad3 and mTOR independently

of myostatin. Modulation of myostatin activity by fol-listatin could therefore contribute to the hypertrophy and anti-atrophy effects of β2-agonists. Gilson et al.44) showed that the IGF-IR/Akt/mTOR pathway plays a critical role in follistatin-induced muscle hypertrophy. There are vari-ous kinds of follistatin, which have different functions. It is said that only follistatin-344 is active in muscle tissue. Recently, it was reported that a few microRNAs (miR-NAs or miRs) regulate skeletal muscle hypertrophy45-49). For example, Clop et al.50) introduced that miR-1 and miR-206 were important in muscular hypertrophy caus-ing translational inhibition of myostatin gene. Sun Y et al.51) showed that mTOR regulated miR-1 and follistatin, i.e. the mTOR-miR-1-HDAC4-follistatin pathway. These might bring a novel explanation of muscular hypertrophy by β2-agonists.

Decrement of structural proteins

On the ubiquitin-proteosome system, two typical E3

Fig. 2 A model for Gαs-, Gαi-, and Gβγ-mediated signalling in skeletal muscle. See text for details. Dotted lines describe pathways whose molecular mechanisms and/or role in adult skeletal muscle have yet to be completely defined. Open arrows indicate a transcriptional regulation. CREB: cAMP response element binding protein; Epac: exchange protein activated by cAMP; FoxO: forkhead transcription factor; G: guanine nucleotide-binding regulatory protein; Gαi: α inhibitory subunit of G protein; Gαs: α stimulatory subunit of G protein; Gβ: β subunit of G protein. Gγ: γ subunit of G protein; GSK: glycogen synthase kinase; HDAC: histone deacetylase; IGF: insulin-like growth factor; MAFbx: muscle atrophy F-box/atrogin; MEF: myocyte enhancer factor; mTOR: mammalian target of rapamycin; MuRF: muscle RING-finger protein; PI3K: phosphoinositide 3-kinase; NR: nuclear receptor; PGC: peroxisome proliferator-activated receptor γ coactivator; PKA: cAMP-dependent protein kinase A; PKC: protein kinase C; Rap: Ras-related protein; SIK: salt induced kinase. Data are cited from Joassard et al.9).

Follistatin

Myostatin IGF-1 β2-agonists

Gβγ GαiGαs

PKA-induced Gαs

to Gαi switching

cAMP

PKA

CREB SIK1

EpacRap

HDAC Myogenin PKC

MyoD

Myf5

MEF2 MuRF1 p70S6K GSK-3β

NR4A3Follistatin PGC-1α4

PGC-1α1FoxOmTOR GSK-3β

PI3K

AKt

MAFbx/Atrogin-1 IGF-1 Myostatin

Activin type IIBreceptor

MuRF1

Protein synthesis Protein degradation

Adenylate cyclase

Muscle gene expression Protein degradation Protein synthesis

426 JPFSM : Kitaura T

ubiquitin (Ub) ligases, atrogin-1 and muscle ring-finger protein 1 (MuRF-1), are important in regulating the con-tents of MyoD and myosin, respectively, and are activated by the FOXO transcription factors (Fig. 2). Bodin et al.16) and Sandri et al.17) showed Akt-mTOR pathway protein degradation via inhibition of the ubiquitin proteasome system (UPS). Costelli et al.52) demonstrated that clen-buterol treatment suppresses the expression of ubiquitin (Ub) mRNA in rats. And Gonçalves et al.53) suggested that clenbuterol inhibited atrogin-1 expression. Recently Ijiri et al.54) reported that clenbuterol induced muscle hyper-trophy of neonatal chicks owing to decreased atrogin-1 mRNA. In the autophagy system, the breakdown of pro-teins is necessary for amino acid turnover for maintain-ing normal cellular function. Zhao et al.55) showed that inhibition of FOXO3 by activated Akt promotes skeletal muscle loss via the coordinate activation of both UPS and autophagy. Furthermore, the calcium-calpain system also plays important roles for protein turnover. Gonçalves et al.19) showed clenbuterol suppresses proteasomal and lysosomal proteolysis in rat soleus muscles. With clen-buterol, Douillard et al.56) showed the inhibition of calpain proteinase in rat skeletal muscles, and Higgins et al.21) showed the inhibited activity of calpain in lambs. Even if proteolysis is activated by β2-agonists, more activated protein synthesis will produce more structural proteins with their own fragmented amino acids and amino acids from foods. It is just like I4 and D3 representing hyper-trophy (H1) in grade of muscular change (Table 1).

Conclusion

It is true that muscular hypertrophy is induced by β2-agonists. The medical use of drugs may bring many ben-efits for many patients. The pharmacological mechanisms and effects of β2-agonists on skeletal muscle are becom-ing clearer, in part, due to new analyzing methods for analysis of signaling pathways and downstream effectors regulating protein homeostasis. However, undesirable side effects like cardiac muscle damage and hypertension from the drugs must be taken into account. More novel trials like miRNA function analysis may help to elucidate the mechanism of muscular hypertrophy and other phar-macological functions.

Acknowledgments

This work was supported by Grant-in-Aid for Scientific Re-search (No. 21500628 to T.K.) from the Ministry of Education, Science, Sports, and Culture of Japan.

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