446 C. McFarlane et al.
Shyu, K. E., Lu, M. J., Wang, B. W., Sun, H. Y., Chang, H., (2006) Myostatin expression in
ventricular myocardium in a rat model of volume-overload heart failure. European Journal of
Clinical Investigation, 36, 713–719.
Siciliano, G., Tessa, A., Renna, M., Manca, M. L., Mancuso, M., Murri, L. (1999). Epidemiology
of dystrophinopathies in North-West Tuscany: A molecular genetics-based revisitation.
Clinical Genetics, 56, 51–58.
Siriett, V., Platt, L., Salerno, M. S., Ling, N., Kambadur, R., Sharma, M. (2006). Prolonged
absence of myostatin reduces sarcopenia. Journal of Cellular Physiology, 209, 866–873.
Siriett, V., Salerno, M. S., Berry, C., Nicholas, G., Bower, R., Kambadur, R., Sharma, M. (2007).
Antagonism of myostatin enhances muscle regeneration during sarcopenia. Molecular
Therapy, 15, 1463–1470.
Sironi, M., Cagliani, R., Comi, G. P., Pozzoli, U., Bardoni, A., Giorda, R., Bresolin, N. (2003).
Trans-acting factors may cause dystrophin splicing misregulation in BMD skeletal muscles.
FEBS Letters, 537, 30–34.
Skipper, M. (2006). A lean feat – microRNAs and muscle mass. Nature Reviews. Genetics, 7,
491.
Snow, M. H. (1977). The effects of aging on satellite cells in skeletal muscles of mice and rats.
Cell and Tissue Research, 185, 399–408.
Souchelnytskyi, S., Tamaki, K., Engstrom, U., Wernstedt, C., Ten Dijke, P., Heldin, C. H. (1997).
Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with
Smad4 and is required for transforming growth factor-beta signaling. The Journal of Biological
Chemistry, 272, 28107–28115.
Steelman, C. A., Recknor, J. C., Nettleton, D., Reecy, J. M. (2006). Transcriptional profiling of
myostatin-knockout mice implicates Wnt signaling in postnatal skeletal muscle growth and
hypertrophy. The FASEB Journal, 20, 580–582.
Szabo, G., Dallmann, G., Muller, G., Patthy, L., Soller, M., Varga, L. (1998). A deletion in the
myostatin gene causes the compact (Cmpt) hypermuscular mutation in mice. Mammalian
Genome, 9, 671–672.
Thies, R. S., Chen, T., Davies, M. V., Tomkinson, K. N., Pearson, A. A., Shakey, Q. A., Wolfman,
N. M. (2001). GDF-8 propeptide binds to GDF-8 and antagonizes biological activity by inhib-

iting GDF-8 receptor binding. Growth Factors, 18, 251–259.
Thomas, M., Langley, B., Berry, C., Sharma, M., Kirk, S., Bass, J., Kambadur, R. (2000).
Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast prolifera-
tion. The Journal of Biological Chemistry, 275, 40235–40243.
Tidball, J. G. (1995). Inflammatory cell response to acute muscle injury. Medicine and Science in
Sports and Exercise, 27, 1022–1032.
Trendelenburg, A. U., Meyer, A., Rohner, D., Boyle, J., Hatakeyama, S., Glass, D. J. (2009).
Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and
myotube size. American Journal of Physiology. Cell Physiology, 296, C1258–C1270.
Tseng, B. S., Zhao, P., Pattison, J. S., Gordon, S. E., Granchelli, J. A., Madsen, R. W., Folk, L. C.,
Hoffman, E. P., Booth, F. W. (2002). Regenerated mdx mouse skeletal muscle shows differen-
tial mRNA expression. Journal of Applied Physiology, 93, 537–545.
Tsuchida, K., Arai, K. Y., Kuramoto, Y., Yamakawa, N., Hasegawa, Y., Sugino, H. (2000).
Identification and characterization of a novel follistatin-like protein as a binding protein for the
TGF-beta family. The Journal of Biological Chemistry, 275, 40788–40796.
Tsuchida, K., Matsuzaki, T., Yamakawa, N., Liu, Z., Sugino, H. (2001). Intracellular and extracel-
lular control of activin function by novel regulatory molecules. Molecular and Cellular
Endocrinology, 180, 25–31.
Tsuchiya, S., Okuno, Y., Tsujimoto, G. (2006). MicroRNA: Biogenetic and functional mecha-
nisms and involvements in cell differentiation and cancer. Journal of Pharmacological
Sciences, 101, 267–270.
Wang, H., Zhang, Q., Zhu, D. (2003). hsgt interacts with the N-terminal region of myostatin.
Biochemical and Biophysical Research Communications, 311, 877–883.
447Role of Myostatin in Skeletal Muscle Growth and Development
Watt, D. J., Morgan, J. E., Clifford, M. A., Partridge, T. A. (1987). The movement of muscle
precursor cells between adjacent regenerating muscles in the mouse. Anatomy and Embryology
(Berlin), 175, 527–536.
Watt, D. J., Karasinski, J., Moss, J., England, M. A. (1994). Migration of muscle cells. Nature,
368, 406–407.
Wehling, M., Cai, B., Tidball, J. G. (2000). Modulation of myostatin expression during modified

muscle use. The FASEB Journal, 14, 103–110.
Welle, S., Bhatt, K., Shah, B., Thornton, C. (2002). Insulin-like growth factor-1 and myostatin
mRNA expression in muscle: Comparison between 62–77 and 21–31 yr old men. Experimental
Gerontology, 37, 833–839.
Whittemore, L. A., Song, K., Li, X., Aghajanian, J., Davies, M., Girgenrath, S., Hill, J. J., Jalenak, M.,
Kelley, P., Knight, A., Maylor, R., O’hara, D., Pearson, A., Quazi, A., Ryerson, S., Tan, X. Y.,
Tomkinson, K. N., Veldman, G. M., Widom, A., Wright, J. F., Wudyka, S., Zhao, L.,
Wolfman, N. M. (2003). Inhibition of myostatin in adult mice increases skeletal muscle mass
and strength. Biochemical and Biophysical Research Communications, 300, 965–971.
Wolfman, N. M., Mcpherron, A. C., Pappano, W. N., Davies, M. V., Song, K., Tomkinson, K. N.,
Wright, J. F., Zhao, L., Sebald, S. M., Greenspan, D. S., Lee, S. J. (2003). Activation of latent
myostatin by the BMP-1/tolloid family of metalloproteinases. Proceedings of the National
Academy of Sciences of the United States of America, 100, 15842–15846.
Xiong, Y., Hannon, G. J., Zhang, H., Casso, D., Kobayashi, R., Beach, D. (1993). p21 is a univer-
sal inhibitor of cyclin kinases. Nature, 366, 701–704.
Yablonka-Reuveni, Z., Seger, R., Rivera, A. J. (1999). Fibroblast growth factor promotes recruit-
ment of skeletal muscle satellite cells in young and old rats. The Journal of Histochemistry and
Cytochemistry, 47, 23–42.
Yang, J., Ratovitski, T., Brady, J. P., Solomon, M. B., Wells, K. D., Wall, R. J. (2001). Expression
of myostatin pro domain results in muscular transgenic mice. Molecular Reproduction and
Development, 60, 351–361.
Yang, W., Chen, Y., Zhang, Y., Wang, X., Yang, N., Zhu, D. (2006). Extracellular signal-regulated
kinase 1/2 mitogen-activated protein kinase pathway is involved in myostatin-regulated dif-
ferentiation repression. Cancer Research, 66, 1320–1326.
Yarasheski, K. E., Bhasin, S., Sinha-Hikim, I., Pak-Loduca, J., Gonzalez-Cadavid, N. F. (2002).
Serum myostatin-immunoreactive protein is increased in 60–92 year old women and men with
muscle wasting. The Journal of Nutrition, Health & Aging, 6, 343–348.
Zachwieja, J. J., Smith, S. R., Sinha-Hikim, I., Gonzalez-Cadavid, N., Bhasin, S. (1999). Plasma
myostatin-immunoreactive protein is increased after prolonged bed rest with low-dose T3
administration. Journal of Gravitational Physiology, 6, 11–15.

Zammit, P. S., Golding, J. P., Nagata, Y., Hudon, V., Partridge, T. A., Beauchamp, J. R. (2004).
Muscle satellite cells adopt divergent fates: A mechanism for self-renewal? The Journal of Cell
Biology, 166, 347–357.
Zhang, Y., Musci, T., Derynck, R. (1997). The tumor suppressor Smad4/DPC 4 as a central media-
tor of Smad function. Current Biology, 7, 270–276.
Zhou, G. Q., Xie, H. Q., Zhang, S. Z., Yang, Z. M. (2006). Current understanding of dystrophin-
related muscular dystrophy and therapeutic challenges ahead. Chinese Medical Journal
(England), 119, 1381–1391.
Zhu, X., Hadhazy, M., Wehling, M., Tidball, J. G., Mcnally, E. M. (2000). Dominant negative
myostatin produces hypertrophy without hyperplasia in muscle. FEBS Letters, 474, 71–75.
Zhu, X., Topouzis, S., Liang, L. F., Stotish, R. L. (2004). Myostatin signaling through Smad2,
Smad3 and Smad4 is regulated by the inhibitory Smad7 by a negative feedback mechanism.
Cytokine, 26, 262–272.
Zimmers, T. A., Davies, M. V., Koniaris, L. G., Haynes, P., Esquela, A. F., Tomkinson, K. N.,
Mcpherron, A. C., Wolfman, N. M., Lee, S. J. (2002). Induction of cachexia in mice by sys-
temically administered myostatin. Science, 296, 1486–1488.

449
G.S. Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness,
DOI 10.1007/978-90-481-9713-2_19, © Springer Science+Business Media B.V. 2011
Abstract While the importance of b-adrenergic signalling in the heart has been
well documented for more than half a century and continues to receive significant
attention, it is only more recently that we have begun to understand the importance
of this signalling pathway in skeletal muscle. There is considerable evidence
regarding the stimulation of the b-adrenergic system with b-adrenoceptor agonists
(b-agonists) in animals and humans. Although traditionally used for the treatment
of bronchospasm, it became apparent that some b-agonists, such as clenbuterol, had
the ability to increase skeletal muscle mass and decrease body fat (Ricks et al. 1984;
Beerman et al. 1987). These so-called “repartitioning effects” proved desirable for
those working in the livestock industry trying to improve feed efficiency and meat

quality (Sillence 2004). Not surprisingly, b
2
-agonists were soon being used by those
engaged in competitive bodybuilding and by other athletes, especially those engaged
in strength- and power-related sports (Lynch 2002; Lynch and Ryall 2008).
As a consequence of their muscle anabolic actions, the effects of b-agonist
administration on skeletal muscle have been examined in a number of animal
models (and in humans) with the hope of discovering therapeutic applications,
particularly for muscle wasting conditions including sarcopenia (age-related mus-
cle wasting and associated weakness), cancer cachexia, sepsis, and other forms of
metabolic stress, denervation, disuse, inactivity, unloading or microgravity, burns,
HIV-acquired immunodeficiency syndrome (AIDS), chronic kidney or heart
failure, chronic obstructive pulmonary disease, muscular dystrophies, and other
neuromuscular disorders. For many of these conditions, the anabolic properties of
b-agonists have the potential to attenuate (or potentially reverse) the muscle
Role of b-Adrenergic Signalling in Skeletal
Muscle Wasting: Implications for Sarcopenia
James G. Ryall and Gordon S. Lynch
G.S. Lynch (*)
Department of Physiology, Basic and Clinical Myology Laboratory,
The University of Melbourne, Victoria, Australia
e-mail:
J.G. Ryall
The Laboratory of Muscle Stem Cells and Gene Regulation,
National Institute of Arthritis, Musculoskeletal and Skin Diseases,
National Institutes of Health (NIH), Bethesda, MD, USA
e-mail:
450 J.G. Ryall and G.S. Lynch
wasting, muscle fibre atrophy, and associated muscle weakness. b-agonists also
have clinical significance for enhancing muscle repair and restoring muscle function

after injury or following reconstructive surgery.
In addition to having anabolic effects on skeletal muscle, b-agonists have also
been associated with some undesirable side effects, including increased heart rate
(tachycardia) and muscle tremor, which have so far limited their therapeutic potential.
In this chapter we describe the physiological significance of b-adrenergic signalling
in skeletal muscle and discuss the therapeutic potential of b-adrenergic stimulation
for age-related muscle wasting and weakness. We describe the effects of current
b-agonists on skeletal muscle and identify novel research strategies to minimize the
unwanted side-effects associated with systemic b-adrenergic stimulation.
Keywords β-adrenoceptor agonist • β-adrenergic signalling • cardiac muscle •
fibre type • G-protein couple receptor • heart • muscle hypertrophy • muscle wasting
• skeletal muscle
1 Overview of b-Adrenergic Signalling
Before discussing the therapeutic potential of b-adrenergic stimulation for sarcopenia,
it is important to characterize the role of this important signalling pathway in
normal healthy skeletal muscle.
b-adrenoceptors belong to the guanine nucleotide-binding G-protein coupled
receptor (GPCR) family (Fredriksson et al. 2003), and are activated endogenously via
adrenaline (epinephrine) and/or noradrenaline (norepinephrine). One of the defining
features of the GPCR superfamily is that all of the receptors couple to heterotrimeric
guanine-nucleotide-binding regulatory proteins (G-proteins). These molecules
received their name from the typical three subunit composition (designated ‘abg’).
All GPCRs (including b-adrenoceptors) have a conserved seven transmembrane
a-helical structure forming three extracellular loops; including an amino-terminus
and three intracellular loops, including a carboxy-terminus (Johnson 2006; Morris
and Malbon 1999). The third-fifth intramembranous regions are believed to be
important in ligand binding, while the third intracellular loop of the GPCR has a
central role in G-protein coupling (Johnson 2006).
The G-proteins are located in the cytoplasmic space and act intracellularly,
interacting with an intracellular loop of the GPCR (Fig. 1). The G-protein bg sub-

units (Gbg) form a tightly interacting dimer which is bound to the intracellular
plasma membrane via an isoprenyl moiety located on the C-terminus of the g sub-
unit, whereas the G-protein a subunit (Ga), in its inactive state, remains attached
to the Gbg dimer (Bockaert and Pin 1999). Activation of the GPCR causes a pro-
found change in the conformation of the intracellular loops and uncovers a previ-
ously masked G-protein binding site (Filipek et al. 2004; Klco et al. 2005; Meng
and Bourne 2001). Specifically, the third intracellular loop of the GPCR is
involved in G-protein binding (Kobilka et al. 1988). Upon binding of a ligand to
451
Role of b-Adrenergic Signalling in Skeletal Muscle Wasting: Implications for Sarcopenia
the GPCR, guanosine diphosphate (GDP) is released from the Ga subunit, and
subsequent guanosine triphosphate (GTP) binding occurs, which activates the Ga
subunit and exposes effector-interaction sites in the Gbg dimer (Bockaert and Pin
1999; Gilman 1995; Hampoelz and Knoblich 2004; Rodbell et al. 1971).
The Ga-subunits can be divided into four main families, based on their primary
sequence: Ga
s
, Ga
i/o
, Ga
q/11
and Ga
12
, which regulate the activity of many different
second messenger systems (Lohse 1999; Wilkie et al. 1992). b-adrenoceptors
couple predominantly with Ga
s
and Ga
i
isoforms to initiate downstream effector

pathways including adenylyl cyclase (AC), transmembrane protein kinases, and
phospholipases (Dascal 2001; Wenzel-Seifert and Seifert 2000).
Three subtypes of b-adrenoceptors have been identified and cloned; b
1
-, b
2
- and
b
3
-adrenoceptors (Dixon et al. 1986; Emorine et al. 1989; Frielle et al. 1987), each
with a 65–70% homology in their amino acid composition (Kobilka et al. 1987).
Skeletal muscle contains a significant proportion of b-adrenoceptors, mostly of the
b
2
-subtype, but also include approximately 7–10% b
1
-adrenoceptors (Kim et al. 1991;
Williams et al. 1984) and a smaller population of a-adrenoceptors, usually in higher
P-GSK3β
βγ α
Non-Canonical β-AR signalling
Canonical β-AR signalling
Extracellular
Intracellular
PIP
3
PI3
PKA
PIP
2

PDK1/2
cAMP
b-adrenoceptor
P-Akt
P-TSC2
Rheb
mTORC1
P-FoxO1/3
G
G
Fig. 1 b-adrenergic signalling in skeletal muscle. Traditionally, the stimulated b-adrenoceptor
has been thought to couple with the stimulatory Ga subunit (Ga
s
) of the heterotrimeric G-protein
(Gabg) and adenylate cyclase (AC), resulting in conversion of ATP to cAMP and the activation of
protein kinase A (PKA). Stimulation of this pathway has been linked to the inhibition of proteolytic
pathways and possibly to protein synthesis. In the non-canonical signalling pathway b-adrenoceptors
signal via the G-protein Gbg subunits to promote phosphorylation of phosphatidylinositol-4,5-
bisphosphate (i.e. PIP
2
becomes PIP
3
) by phosphatidylinositol 3-kinase (PI3-K), leading to Akt
activation. These events trigger the downstream activators, glycogen synthase kinase 3b (GSK3b),
tuberous sclerosis complex 2 (TSC2, an activator of mammalian target of rapamycin complex-1,
mTORC1) and the forkhead box O (FoxO) family of transcription factors. Thus, b-adrenoceptor
stimulation can influence protein synthesis and degradation by several mechanisms
452 J.G. Ryall and G.S. Lynch
proportions in slow-twitch muscles (Rattigan et al. 1986). Slow-twitch muscles like
the soleus have a greater density of b-adrenoceptors than fast-twitch muscles, such as

the extensor digitorum longus (EDL) (Martin et al. 1989; Ryall et al. 2002, 2004).
Although the functional significance of this difference in b- adrenoceptor density is
not yet understood fully, the response to b-agonist administration appears to be
greater in fast-, than in slow-twitch skeletal muscles (Ryall et al. 2002, 2006).
The Ga
s
-AC-cyclic AMP (cAMP) is the most well characterized of the b
2
-
adrenoceptor signalling pathways and is generally thought to be, at least partially,
responsible for the b
2
-adrenoceptor mediated hypertrophy in skeletal muscle
(Hinkle et al. 2002; Navegantes et al. 2000). The production of cAMP results in the
activation of numerous downstream signalling pathways, including the well-
described protein kinase A (PKA) signalling pathways.
Following cAMP activation, PKA is thought to phosphorylate and regulate the
activity of numerous proteins. In addition, PKA is capable of diffusing passively
into the nucleus, where it can regulate the expression of many target genes via
direct phosphorylation of the cAMP response element (CRE) binding protein
(CREB), or via a modulator that acts on second generation target genes (Carlezon
et al. 2005; Mayr and Montminy 2001).
The CRE binding protein is a nuclear transcription factor that is expressed ubiq-
uitously and has been implicated in many processes, including cell proliferation,
differentiation, adaptation, and survival (Mayr and Montminy 2001). CREB forms
a homodimer and binds to a conserved CRE-region on DNA. Nuclear entry of
PKA, phosphorylates CREB at a single serine residue site (Ser
133
) (Hagiwara et al.
1993). Phosphorylation of Ser

133
promotes transcription at the CRE-region through
recruitment of the transcriptional co-activators CREB-binding protein (CBP) and
p300, which mediate transcriptional activity through their association with RNA
Polymerase II (Goodman and Smolik 2000; Mayr and Montminy 2001). CREB-
phosphorylation promotes activation of genes containing a CRE-region, of which
there are >4,000 in the human genome (Pourquié 2005; Zhang et al. 2005). Finally,
CRE-gene activation is terminated by dephosphorylation of CREB, a process regu-
lated by the serine/threonine phosphatases PP-1 and PP-2A (Hagiwara et al. 1992;
Wadzinski et al. 1993).
One target for b-adrenoceptor mediated CRE activation in skeletal muscle is the
promoter region of the orphan nuclear receptor, NOR-1 (NR4A3) (Ohkura et al.
1998; Pearen et al. 2006). b
2
-adrenoceptor activation is associated with an increased
expression of NOR-1 and the related orphan nuclear receptor nur-77 (NR4A1)
(Maxwell et al. 2005; Pearen et al. 2006). Interestingly, Pearen and colleagues
(2006) found that siRNA mediated inhibition of NOR-1 expression was associated
with a dramatic increase (>65 fold) in the levels of myostatin mRNA in C2C12
cells. Myostatin is a member of the transforming growth factor-b superfamily and
a potent negative regulator of muscle mass (McPherron et al. 1997). Thus, b-adre-
noceptor activation, through increased NOR-1 expression, may inhibit myostatin
expression and hence promote skeletal muscle growth.
The transcriptional adapters, CBP and p300, promote skeletal muscle myogenesis
via the coactivation of a number of myogenic basic helix-loop-helix (bHLH) pro-
453
Role of b-Adrenergic Signalling in Skeletal Muscle Wasting: Implications for Sarcopenia
teins (Eckner et al. 1996; McKinsey et al. 2002; Sartorelli et al. 1997). The family
of myogenic bHLH proteins, including MyoD, myogenin, myf5 and MRF4, activate
muscle gene transcription via pairing with the ubiquitously expressed E-box consen-

sus sequence in the control regions of muscle-specific genes (McKinsey et al. 2002;
Molkentin and Olson 1996). Sartorelli and colleagues (1997) found that p300 and
CBP may positively influence myogenesis by acting as a ‘bridge’ between the
myogenic bHLH and the myocyte enhancer factor 2 (MEF2) family of proteins.
In addition to transcriptional coactivation, CBP and p300 have intrinsic histone
acetyltransferase (HAT) activity (Goodman and Smolik 2000; Roth et al. 2003;
Thompson et al. 2004). Histone acetyltransferases are believed to play an important
role in transcription, since they catalyze the transfer of acetyl groups from acetyl-
coenzyme A to the e-amino group of lysine side chains of specific proteins, includ-
ing several transcriptional regulatory proteins (Yang 2004). Therefore, the
b-adrenoceptor mediated actions of CBP and p300 could increase the accessibility
of docking sites for transcriptional proteins and regulators (Ogryzko et al. 1996;
Thompson et al. 2004).
Chen and colleagues (2005) identified an unexpected role for PKA/CREB sig-
nalling during myogenesis, proposing that myogenic gene expression of Pax3,
MyoD, and Myf5 is dependent on AC/cAMP mediated phosphorylation of PKA
and subsequent activation of CREB. The authors demonstrated the importance of
CREB in the developing myotome, since CREB
−/−
mice did not express Pax3,
MyoD, or Myf5 and myotome formation was defective (Chen et al. 2005). It
remains to be determined whether b-adrenoceptor mediated activation of PKA/
CREB signalling has a similar response during myogenesis.
Berdeaux and colleagues (2007) demonstrated a novel role of CREB in mediat-
ing the activity of MEF2. They showed that b-adrenergic stimulated CREB modu-
lated the phosphorylation status of the class II histone deacetylase HDAC5 in
mouse skeletal muscle, by increasing the expression of salt inducible kinase 1
(SIK1). Activated SIK1 phosphorylated HDAC5, resulting in its nuclear exclusion
and subsequent activation of the MEF2 myogenic program (Berdeaux et al. 2007).
These exciting results demonstrated the complexity of the downstream activators

of the b-adrenergic signalling pathway and highlighted the previously unappreci-
ated role of this pathway in skeletal muscle.
In addition to the well-described Ga
s
-cAMP signalling pathways, studies have
implicated the Gbg subunits in various cell signalling processes, which may
also play important roles in b-adrenoceptor signalling in skeletal muscle (Crespo
et al. 1994; Dascal 2001; Diversé-Pierluissi et al. 2000; Ford et al. 1998; Mirshahi
et al. 2002). Specifically, in vitro cell culture experiments have revealed that the
Ga
i
linked Gbg subunits activate the phosphoinositol 3-kinase (PI3K)-AKT
signalling pathway (Lopez-Ilasaca et al. 1997; Murga et al. 1998, 2000; Schmidt
et al. 2001).
The PI3K-AKT signalling pathway has been implicated in protein synthesis,
gene transcription, cell proliferation, and cell survival (Bodine et al. 2001b;
Glass 2003, 2005; Kline et al. 2007; Pallafacchina et al. 2002; Rommel et al.
2001). Although there are three distinct isoforms of AKT, the predominant
454 J.G. Ryall and G.S. Lynch
skeletal muscle isoform is AKT1 (Nader 2005). Activation of PI3K phosphorylates
the membrane bound PIP
2
, creating a lipid-binding site on the cell membrane for
both AKT1 and 3¢-phoshphoinositide-dependent protein kinase 1 (PDK). PDK
then phosphorylates AKT1 at the membrane (Nicholson and Anderson 2002). Akt
activation, in turn, results in the phosphorylation of numerous downstream activa-
tors, including glycogen synthase kinase 3b (GSK3b), tuberous sclerosis complex
2 (TSC2, leading to the subsequent activation of mammalian target of rapamycin
complex1, mTORC1) (Garami et al. 2003; Latres et al. 2005) and members of the
forkhead box O (FOXO) family of transcription factors (Sandri et al. 2004; Stitt

et al. 2004).
Kline and colleagues (2007) found that stimulation of the b-adrenoceptor signal-
ling pathway resulted in AKT phosphorylation and subsequent activation of
mTORC1. Initiation of mTORC signalling phosphorylates and subsequently acti-
vates p70
s6
kinase (p70
S6K
), while concomitantly inactivating 4EBP-1 (also termed
PHAS-1). p70
S6K
mediates the phosphorylation of the 40S ribosomal S6 protein,
resulting in the upregulation of mRNA translation encoding for ribosomal proteins
and elongation factors (Jefferies et al. 1997). Inactivation of 4EBP-1 removes its
inhibitory action on the protein initiation factor eukaryotic initiation factor 4E (eIF-
4E) (Lai et al. 2004; Nave et al. 1999). These findings supported those of Sneddon
and colleagues (2001) who reported an increased phosphorylation of 4E-BP1 and
p70
S6K
in rat plantaris muscle after 3 days of clenbuterol treatment.
GSK-3b is reported to be a negative regulator of protein translation and gene
expression in cardiac (Hardt and Sadoshima 2002) and skeletal muscle (Childs
et al. 2003; Bossola et al. 2008). Following b-adrenoceptor stimulation, GSK3b
is phosphorylated and subsequently inactivated by AKT1 (Yamamoto et al.
2007), resulting in the expression of a dominant negative form of GSK3b. Since
GSK3b normally acts to inhibit the translation initiation factor eIF2B, blockade
of GSK3b by AKT1 might promote protein synthesis (Bodine et al. 2001b;
Rommel et al. 2001).
AKT1 signalling is not only involved in the signalling pathways responsible
for muscle hypertrophy, but it has been implicated in the inhibition of signalling

pathways responsible for “muscle atrophy”. AKT1 inactivation of FOXO leads to
nuclear exclusion and inhibition of the forkhead transcriptional program. The
DNA displacement and subsequent nuclear exclusion of FOXO requires the
involvement of 14-3-3 proteins, which bind to FOXO following AKT1-mediated
phosphorylation (Tran et al. 2003). 14-3-3 proteins are among a family of
chaperone proteins that interact with specific phosphorylated protein ligands
(Tran et al. 2003).
Activation of the forkhead transcriptional program is necessary for induction of both
muscle RING finger 1 (muRF1) and muscle atrophy F-box (MAFbx, also called
atrogin-1) (Sandri et al. 2004; Stitt et al. 2004). Both muRF1 and MAFbx encode ubiq-
uitin ligases which conjugate ubiquitin to protein substrates, and are upregulated in
numerous models of muscle atrophy (Bodine et al. 2001a; Tintignac et al. 2005). Thus,
by phosphorylating and inactivating FOXO, AKT1 blocks the induction of FOXO-
mediated atrophy signalling via muRF1 and MAFbx. b-Adrenoceptor activation
455
Role of b-Adrenergic Signalling in Skeletal Muscle Wasting: Implications for Sarcopenia
reduces the expression of muRF1 and MAFbx in skeletal muscle from denervated and
hindlimb-suspended rats, an effect possibly mediated via AKT1-initiated inhibition of
the forkhead transcriptional program (Kline et al. 2007).
It is interesting to note that while FOXO1 regulates the expression of both
MAFbx and muRF1 (Stitt et al. 2004), FOXO3a appears only to activate the MAFbx
promoter (Sandri et al. 2004). In addition, while measurable levels of FOXO4 have
been identified in skeletal muscle (Furuyama et al. 2002), very little is known about
its role in skeletal muscle atrophy. Furuyama and colleagues (2002) characterized
the expression pattern of FOXO1, FOXO3a and FOXO4 with ageing and caloric
restriction in rats. FOXO4 mRNA expression increased from 3 to 12 months and
then decreased from 12 to 26 months. A similar pattern was observed for FOXO3a
expression (Furuyama et al. 2002). Interestingly, FOXO1 mRNA expression
remained unchanged. In contrast, caloric restriction resulted in an increase in the
expression levels of both FOXO4 and FOXO1, but not FOXO3a (Furuyama et al.

2002). These results indicate the complexity of the forkhead transcriptional program
in the regulation of skeletal muscle atrophy (Kandarian and Jackman 2006).
Several studies have identified a role for FOXO1 in binding to the promoter
region of 4EBP-1 which resulted in increased mRNA and protein expression (Léger
et al. 2006; Wu et al. 2008). Associated with the increase in 4EBP-1 was a reduction
in mTORC activation and p70
S6K
. Thus, in addition to previously reported roles in
atrophic signalling pathways, FOXO1 also plays an active role in inhibiting protein
synthesis (Yang et al. 2008).
A number of researchers have identified genes that are activated by b-adreno-
ceptor stimulation, but the mechanism for their activation remains unclear. For
example McDaneld and colleagues (2004) examined differential gene expression in
skeletal muscle after b-agonist administration to evaluate the role of genes thought
responsible for muscle growth. Decreased mRNA abundance following b-adreno-
ceptor stimulation was confirmed for DD143 identified as ASB15, a bovine gene
encoding an ankyrin repeat and a suppressor of cytokine signalling (SOCS) box
protein, in both cattle and rats (McDaneld et al. 2004, 2006; Spangenburg 2005).
The authors reported that ASB15 was a member of an emerging gene family
involved in a variety of cellular processes including cellular proliferation and dif-
ferentiation (McDaneld et al. 2004).
Similarly, Spurlock and colleagues (Spurlock et al. 2006) examined gene
expression changes in mouse skeletal muscle 24 hours and 10 days after b-adreno-
ceptor stimulation and identified genes involved in processes important to skeletal
muscle growth, including regulators of transcription and translation, mediators of
cell-signalling pathways, and genes involved in polyamine metabolism. They
reported changes in mRNA abundance of multiple genes associated with myogenic
differentiation relevant to the effect of b-adrenoceptor stimulation on the prolifera-
tion, differentiation, and/or recruitment of satellite cells into muscle fibres to pro-
mote muscle hypertrophy. Similarly, they showed an upregulation of translational

initiators responsible for increasing protein synthesis (Spurlock et al. 2006).
More recently, Pearen and colleagues (2009) profiled skeletal muscle gene
expression in mouse tibialis anterior muscles at 1 and 4 h after systemic administration