MUSCULAR SYSTEM - ANATOMY, FUNCTIONS AND INJURIES

BASIC BIOLOGY
AND CURRENT UNDERSTANDING
OF SKELETAL MUSCLE

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MUSCULAR SYSTEM - ANATOMY, FUNCTIONS AND INJURIES

BASIC BIOLOGY
AND CURRENT UNDERSTANDING
OF SKELETAL MUSCLE

KUNIHIRO SAKUMA
EDITOR

New York

Copyright 2013 by Nova Science Publishers, Inc.

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Contents
Preface

vii

Chapter 1

Stem Cell Hierarchies in Muscle Satellite Cells

Yusuke Ono

Chapter 2

Cellular Mechanisms Regulating Protein Metabolism

in Skeletal Muscle Cells: Age-Associated Alteration
of Intracellular Signaling Governing Protein Synthesis
and Degradation
Mitsunori Miyazaki

Chapter 3

Chapter 4

Chapter 5

21

Biological Actions of Insulin-Like Growth Factor-I (IGF-I)

Isoforms and IGF Binding Proteins in Skeletal Muscle
Akihiko Yamaguchi, Kunihiro Sakuma and Isao Morita

53

An Overview of the Therapeutic Strategies for

Preventing Sarcopenia
Kunihiro Sakuma and Akihiko Yamaguchi

87

Oxidative Stress-Induced Signal Transduction

in Skeletal Muscle
Wataru Aoi

123

Chapter 6

The Functional Role of Heat Shock Proteins in Skeletal Muscle

Tomonori Ogata

147

Chapter 7

Mechanical Stress and Myofibrillar Structure

Fuminori Kawano

171

Chapter 8

AMPK: Molecular Mechanisms of Metabolic

Adaptations in Skeletal Muscle
Masataka Suwa

205

A Role for Mitochondria as a Potential Regulator

of Myogenesis
Akira Wagatsuma

251

Chapter 9

vi
Chapter 10
Index

Contents
Skeletal Muscular Adaptation and Local Steroidogenesis
Katsuji Aizawa

289
303

Preface
Skeletal muscle is a highly plastic tissue that constitutes approximately thirty percent of
total body mass and adapts rapidly to changing functional demands. Skeletal muscle is not
only the generator of force production, but also plays a crucial role in whole body metabolism
and energy consumption. In this book, leading experts in the area of exercise biochemistry
and molecular biology in skeletal muscle provide an up-to-date view of the molecular basis of
various adaptations of skeletal muscle, with emphasis on new biological concepts (muscle
stem cells, muscle steroidogenesis, etc). This book deals with the recent intriguing role of
heat shock protein (HSP), AMPK and reactive oxygen species (ROS) for muscle morphology,
function and metabolism. Discussed also is the molecular mechanism for protein metabolism
and therapeutic application for sarcopenia. The deeper understanding of the signal
transduction and modification in skeletal muscle will develop new therapeutic strategies for
preventing physical disability and increased risk of morbidity/mortality due to the loss of
muscle mass.
Chapter 1 - Satellite cells are muscle stem cells located between the basal lamina and
plasma lemma of myofibres and play important roles in adult skeletal muscle repair and
regeneration as well as postnatal muscle growth. Recent findings indicate that satellite cells
exist as a genetically and functionally heterogeneous population among muscles, which is not
only based on fibre types, but also embryonic origins. Satellite cells are also a heterogeneous
population within a single myofibre. Importantly, only a small population of satellite cells
appears to possess stemness that generates robust myogenic progeny and maintains the
satellite cell pool throughout life. Recent studies have discovered the cellular and molecular
characteristics of satellite cell heterogeneity including Pax7+Myf5- satellite stem cells,
immortal DNA strand-retaining cells, satellite-side population cells, highly Pax7-expressing
cells and slow-dividing cells. In this chapter, the authors discuss emerging findings of satellite
cell heterogeneity and stem cell hierarchies in satellite cell populations in adult muscle.
Further understanding of the hierarchical regulation of satellite cell populations is an
important issue to develop cell-based therapies of muscular dystrophies and age-related
sarcopenia.
Chapter 2 - Skeletal muscle is the largest organ in the human body, making up more than
30-40% of total body weight in healthy adults. The wasting of skeletal muscle mass, which is
induced by unloading, malnutrition, aging or several kinds of diseases, leads to the loss of
functional human performance, long-term health issues and a low quality of life. It has been

viii

Kunihiro Sakuma

generally accepted that the net balance between protein synthesis and degradation is a critical
determinant of the regulation of skeletal muscle mass.
Recent studies indicate that the skeletal muscle protein metabolism in response to the
environmental cues (i.e. physical activity and nutrients) is blunted in the elderly, which then
leads to the age-associated gradual loss of muscle mass. The aim of the present chapter is to
summarize and discuss, 1) recent progress in the understanding of cellular mechanisms in
anabolic (protein synthesis) and catabolic (protein degradation) signaling pathways that
govern the regulation of skeletal muscle mass, 2) altered capacity in the protein metabolism
of aged skeletal muscle in response to exercise and nutrients. A better understanding of the
anabolic and catabolic processes which regulate skeletal muscle mass is critical for the
development of more effective therapeutic interventions to prevent the loss of muscle with
aging and disease.
Chapter 3 - Insulin-like growth factor-I (IGF-I) is an important growth factor mediating
cell proliferation, differentiation and cell survival in skeletal muscles. In humans, there are
three types of IGF-I isoforms derived from the differential E domain, called IGF-I Ea, Eb and
Ec. IGF-I Ec is also called mechano growth factor (MGF), because of the marked
upregulation in exercised and damaged muscles. In rodents, IGF-I isoforms are composed of
two types of IGF-I, IGF-I Ea and MGF. IGF-I isoforms serve as the IGF-I precursor peptides.
IGF-I isoforms have multiple transcriptional initiation sites and derived from some alternative
splicing. After post-translation modification, the IGF-I precursor peptides are grown into a
mature IGF-I. IGF-I isoforms are produced by various tissues, including liver, cartilage and
skeletal muscle, and act through endocrine and autocrine/paracrine pathways. E peptides
derived from the IGF-I isoforms are likely to have different growth-promoting effects on
skeletal muscles.
IGF binding protein (IGFBP) family is composed of six different members, which are
IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6. IGFBPs are characteristic
of high affinity for IGFs binding, whereas IGFBP related proteins with the low affinity
binding are also found and distinguished from IGFBPs. Most of the circulating IGF-I exists in
a large tripartite complex with IGFBP-3 and the acid labile subunit (ALS). IGF-I also exists
in binary or ternary complexes with another member of the IGFBP family. IGF-I is removed
from the complexes, and free IGF-I acts on muscle growth via the IGF-I receptors. IGFBPs
have stimulating and inhibitory effects depending on the IGF-I as well as IGF-independent
actions. The same IGFBP can act to promote or suppress IGF actions in association with
posttranslational modification, such as proteolytic cleavage, dephosphorylation. Gene
expressions and their functions of IGF-I isoforms and IGFBPs in skeletal muscle are
discussed in the present review.
Chapter 4 - The world's elderly population is expanding rapidly, and we are now faced
with the significant challenge of maintaining or improving physical activity, independence,
and quality of life in the elderly. Sarcopenia, the age-related loss of skeletal muscle mass, is
characterized by a deterioration of muscle quantity and quality leading to a gradual slowing of
movement, a decline in strength and power, increased risk of fall-related injury, and often,
frailty. Since sarcopenia is largely attributed to various molecular mediators affecting fiber
size, mitochondrial homeostasis, and apoptosis, the mechanisms responsible for these
deleterious changes present numerous therapeutic targets for drug discovery. Resistance
training combined with amino acid-containing supplements is often utilized to prevent agerelated muscle wasting and weakness. In this chapter, the authors summarize recent

Preface

ix

therapeutic strategies using supplemental, pharmacological, and hormonal approach for

counteracting sarcopenia. Treatment with ghrelin seems to be an interesting approach for
preventing sarcopenia in the near future. EPA and ursolic acid seem to be effective as
therapeutic aqgents, because they attenuate the degenerative symptoms of muscular dystrophy
and cachexic muscle. The activation of peroxisome proliferator-activated receptor
coactivator 1 (PGC-1) in skeletal muscle by exercise and/or unknown supplementation
would be an intriguing approach to attenuating sarcopenia. In contrast, muscle loss with age
may not be influenced positively by treatment with a proteasome inhibitor or antioxidant.
Chapter 5 - Reactive oxygen species (ROS) are produced via several sources in the
skeletal muscle. In particular, the mitochondrial electron transport chain in the muscle cells
could be a major source of ROS, with an elevation of oxygen consumption. Physical exercise,
a sedentary lifestyle, and the aging process can all generate oxidative stress. Such oxidative
stress can cause transcriptional and post-translational regulation of key proteins and affect
their functionality. Evidence has suggested that some muscle proteins (i.e., myofiber proteins,
metabolic signaling proteins, and inflammatory factors) can be the targets of oxidative stress.
Continuous or excess elevation of ROS in muscle tissues results in inflammation, loss of
muscle mass, and metabolic dysfunction. In contrast, growing evidence has suggested that
moderate and transient elevations in ROS contribute to the metabolic improvement and
maintenance of muscle mass via modulating related proteins, which mediate health benefits
induced by moderate exercise. The oxidative proteins may be also useful as potential
biomarkers to examine the oxidative stress levels, antioxidant compounds, and their possible
benefits or dysfunction.
Chapter 6 - Heat shock proteins (HSPs) are known as highly conserved prosurvival
molecules. HSPs have been demonstrated to contribute to improving several pathological
alterations in skeletal muscle. It has been reported that several HSPs attenuate contractioninduced and age-related muscle damage in overexpression models. Activation of the ubiquitin
proteasome pathway induces skeletal muscle atrophy during immobilization, but
overexpression of HSPs can block the activity. In muscular dystrophy, enhanced HSP72
preserves muscle function and slows progression of muscle atrophy. Furthermore, the
enhancements of HSP72 in skeletal muscle potentially contribute to preventing obesity- and
hyperlipidemia-induced insulin resistance through regulation of inflammatory factors, thereby
preventing diabetes. This review focuses on recent progress in the understanding of HSP
functions in skeletal muscle.
Chapter 7 - Myofibrils are the contractile components of the skeletal muscle fibers.
However, the myofibril does not simply work as a contractile apparatus, but also plays a
crucial role in the sensing of mechanical stress and capture of the signaling molecules. The
giant sarcomeric protein, titin, spans from the Z-disc to the M-band, and acts as a mechanical
sensor. Muscle-specific RING finger 1, E3 ubiquitin ligase, and calpain-3 bind to titin and
contribute to the stress response of myofibrils following the structural change of titin. Lack of
these proteins causes a severe myopathic disease. The Z-disc mediates the important
signalings related to the muscle characteristics via various proteins, including calcineurin, the
protein phosphatase that activates the slow-twitch muscle-specific gene transcription in a
Ca2+ concentration-dependent manner, and the muscle LIM protein, the transcriptional coregulator of the basic helix-loop-helix responsible consequence such as MyoD and myogenin.
Furthermore, some differences are found in the core structure of sarcomere, including a
thicker Z-disc, a longer thin actin filament, and longer titin, in slow-twitch compared to fast-

Kunihiro Sakuma

twitch muscle fibers. These properties potentially affect the mechanical strength and the
signal transduction. The adaptive transformation is postulated on the structural organization
as well as its components, in accordance with the level and the pattern of the muscle activity.
Chapter 8 - Skeletal muscle possesses a great degree of metabolic plasticity, which can be
controlled by exercise, cytokines, pharmaceuticals and nutrients. There are large variations in
the human skeletal muscle metabolic capacity, and they are linked with the prevalence of
chronic metabolic disorders such as obesity, type II diabetes mellitus, hypertension and
dyslipidemia, as well as exercise endurance.
The metabolic profiles of skeletal muscle have been investigated for several decades. 5AMP-activated protein kinase (AMPK) is one of the most important metabolic regulators in
skeletal muscle. AMPK is also activated by energy deprivation (i.e., an increasing AMP:ATP
ratio) and served as a cellular energy sensor. AMPK is activated by muscle contraction (e.g.,
exercise), drugs used to treat type 2 diabetes mellitus, cytokines, nutrients, reactive oxygen
species and other chemically synthesized activators.
Activated AMPK promotes the catabolic pathways that generate ATP (e.g., glucose
uptake, fatty acid uptake and oxidation, and mitochondrial respiration). AMPK promotes the
translocation of glucose transporter 4 (GLUT4) from the cytoplasm to the cell membrane and
the subsequent glucose uptake into a cell independent of the insulin system. AMPK also
accelerates the mitochondrial fatty acid uptake by inhibiting the acetyl-coenzyme A
carboxylase activity. AMPK phosphorylates several downstream transcription factors and
coactivators that regulate energy catabolism. AMPK activation results in the upregulation and
the direct phosphorylation of the peroxisome proliferator-activated receptor coactivator-1
(PGC-1), which is an important regulator for mitochondrial biogenesis. In addition, AMPK
indirectly deacetylates PGC-1 via SIRT1. These characteristics make AMPK a valuable
therapeutic target for chronic metabolic diseases associated with insulin resistance.
Chapter 9 - Mitochondria serve a critical function in the maintenance of cellular energy
supplies, calcium homeostasis, and cell death. Aside from these major roles, several lines of
evidence suggest that mitochondria are involved in cell cycle control, cell growth and
differentiation in a wide variety of cell types including myogenic cells.
When the myoblasts differentiate into myotubes, the abundance, morphology, and
functional properties of mitochondria is being dynamically altered. Mitochondrial mass,
mitochondrial DNA content, mitochondrial enzyme activities, and mitochondrial respiration
are markedly increased during myogenesis.
This strongly suggests that myogenesis is accompanied by increased mitochondrial
biogenesis and that the metabolic shift from glycolysis to oxidative phosphorylation as the
major energy source occurs during myogenesis. Intriguingly, when myoblasts are exposed to
mitochondrial genetic and metabolic stress, they fail to differentiate into multinucleated
myotubes, suggesting that mitochondria may play a certain role in myogenesis.
The process is relatively well understood phenomenologically, but the underlying
molecular mechanisms have been surprisingly slow to emerge. Understanding how
mitochondria are involved in myogenesis will provide a valuable insight into the underlying
mechanisms that regulate the maintenance of cellular homeostasis. Here the authors will
summarize the current knowledge regarding the role of mitochondria as a potential regulator
of myogenesis.
Chapter 10 - The plasticity of skeletal muscle facilitates adaptation to various stimuli.
Exercise training induces skeletal muscle adaptation such as muscle strength and hypertrophy,

Preface

xi

while inactivity leads to muscle atrophy such as sarcopenia. Sex steroid hormones (androgens
and estrogens) often mediate muscle plasticity. Indeed, these hormones induce various effects
including growth, strength, metabolism, and antioxidant levels as well as muscle atrophy.
Though sex steroid hormones play an important role in skeletal muscular homeostasis, the
role of the endocrine system in muscle plasticity is unknown.
Sex steroid hormones are produced by various peripheral target tissues including the
kidney, liver, and brain in addition to endocrine organs such as the testis or ovary. Sex steroid
hormones are synthesized from cholesterol by steroidogenic enzymes, such as cholesterol
side-chain cleavage (P450scc), cytochrome P450 enzyme 17-hydroxylase (P450c17), 3hydroxysteroid dehydrogenase (HSD), and 17-HSD, with testosterone being irreversibly
converted to estrogen by aromatase cytochrome P450 (P450arom). Testosterone is also
converted into its bioactive metabolite dihydrotestosterone (DHT) by 5-reductase.
The functional importance of sex steroid hormones derived from extragonadal tissues has
been gaining support in recent years. For instance, steroidogenic enzymes expressed in
skeletal muscle have been reported to locally synthesize sex steroid hormones from
circulating dehydroepiandrosterone (DHEA) or testosterone in response to exercise. Thus,
local steroidogenesis in skeletal muscle may play an important role in the plasticity of skeletal
muscle. This review focuses on the steroidogenesis of skeletal muscle and discusses the
physiological significance of the sex steroid hormone network of circulation and skeletal
muscle.

In: Basic Biology and Current Understanding of Skeletal Muscle ISBN: 978-1-62808-367-5
Editor: Kunihiro Sakuma
2013 Nova Science Publishers, Inc.

Chapter 1

Stem Cell Hierarchies in

Muscle Satellite Cells
Yusuke Ono
Department of Stem Cell Biology, Nagasaki University
Graduate School of Biomedical Sciences, Nagasaki, Japan

Abstract
Satellite cells are muscle stem cells located between the basal lamina and plasma
lemma of myofibres and play important roles in adult skeletal muscle repair and
regeneration as well as postnatal muscle growth. Recent findings indicate that satellite
cells exist as a genetically and functionally heterogeneous population among muscles,
which is not only based on fibre types, but also embryonic origins. Satellite cells are also
a heterogeneous population within a single myofibre. Importantly, only a small
population of satellite cells appears to possess stemness that generates robust myogenic
progeny and maintains the satellite cell pool throughout life. Recent studies have
discovered the cellular and molecular characteristics of satellite cell heterogeneity
including Pax7+Myf5- satellite stem cells, immortal DNA strand-retaining cells, satelliteside population cells, highly Pax7-expressing cells and slow-dividing cells. In this
chapter, we discuss emerging findings of satellite cell heterogeneity and stem cell
hierarchies in satellite cell populations in adult muscle. Further understanding of the
hierarchical regulation of satellite cell populations is an important issue to develop cellbased therapies of muscular dystrophies and age-related sarcopenia.

Keywords: Skeletal muscle, myoblasts, heterogeneity, stemness

Corresponding author: Yusuke Ono, Ph.D. Department of Stem Cell Biology, Nagasaki University Graduate
School of Biomedical Science, 1-12-4 Sakamoto, Nagasaki 852-8523 Japan. Tel: +81 (0)95 819 7099; Fax:
+81 (0)95 819 7100.E-mail: yusuke-ono@nagasaki-u.ac.jp.

Yusuke Ono

1. Introduction
Satellite cells are resident muscle stem cells located between the basal lamina and
plasmalemma of myofibres, which were discovered by electron microscopy analysis over 50
years ago [1-3]. Satellite cells play important roles in providing myonuclei for postnatal
muscle growth as well as muscle maintenance, hypertrophy, repair and regeneration in adults
[4, 5]. Satellite cells generate muscle progenitors, termed myoblasts, which extensively
proliferate before fusing with existing myofibres to contribute toward myonuclei, or fuse with
each other to form myotubes that eventually become mature myofibres. Satellite cells also
self-renew to maintain the stem cell pool for future needs [6, 7].
In healthy adult muscle, satellite cells are mitotically quiescent and express the pairedbox transcription factor Pax7. Satellite cells are activated in response to stimulation such as
muscle injury. Activated satellite cells up-regulate MyoD expression and undergo mitosis.
Following cell proliferation, the majority of satellite cells down-regulates Pax7, maintains
MyoD, expresses myogenin and undergoes myogenic differentiation to produce myosin
heavy chain (MyHC)+ new myonuclei. A minority population down-regulates MyoD,
maintains Pax7 and returns to a quiescent state to self-renew [8-10], as observed in mouse
myoblast cell lines [11]. Interestingly, the total number of satellite cells in adult muscle
remains relatively constant following repeated muscle injury and regeneration throughout life,
indicating that self-renewal of satellite cells is carefully regulated. Indeed, failure of satellite
cell functions leads to impaired recovery following muscle damage and severe muscular
dystrophic phenotypes [12-18]. In addition to Pax7 [19], satellite cells can be also identified
by the expression of CD34 [20], caveolin-1 [21], M-cadherin [22], calcitonin receptors [23],
1-integrin [24], c-Met [25], syndecan-3/4 [26], Myf5/ -gal [20], NCAM [27], VCAM-1 [28]
and CXCR4 [29] (Figure 1).

2. Stem Cell Potential of Satellite Cells

Transplantation of freshly isolated satellite cells has been shown to significantly generate
new muscle and re-populates satellite cells in host muscle [6, 7, 30]. Collins et al.
transplanted freshly isolated single myofibres containing satellite cells into irradiated mdxnude mice to investigate the potential of pure and small populations of anatomically defined
satellite cells [6]. Approximately seven satellite cells within a single myofibre can generate
more than 100 new myofibres that contain thousands of myonuclei as well as re-populate
satellite cells in host muscle. This study indicated that satellite cells possess potent myogenic
and self-renewal abilities for adult muscle regeneration in vivo. Surprisingly, Sacco et al.
showed that engraftment of only a single muscle stem cell gives rise to extensive proliferative
cells and newly formed myofibres in regenerating muscle, as determined by real-time
quantitative and kinetic analyses of mice [31]. Mdx mice with deficient telomerase activity,
which leads to shortened telomeres, exhibit failure of long-term satellite cell proliferation, and
thus display a severe muscular dystrophic phenotype [12].
Notably, muscle-wasting severity in mdx mice lacking telomerase activity parallels a
decrease in satellite cell regenerative capacity, and muscle-wasting severity is significantly
rescued by transplantation of wild-type satellite cells.

Stem Cells Hierarchies in Muscle Satellite Cells

Figure 1. Muscle satellite cells and cell fate choice. (A) Freshly isolated extensor digitorum longus
(EDL) myofibres with their associated satellite cells were immediately fixed and stained for caveolin1(red)+ and Pax7 (green) + satellite cells. (B) Pax7+ quiescent satellite cells become activated to coexpress both Pax7 and MyoD (Pax7+MyoD+). The vast majority of activated satellite cells then
undergoes proliferation before down-regulating Pax7, maintaining MyoD, and differentiating (Pax7MyoD+). The minority of satellite cells maintains Pax7 but lose MyoD to self-renew and become
quiescent. (C) Freshly isolated satellite cells were cultured in differentiation medium for 5 days after
culture in growth medium for 7 days. Mononucleited proliferating cells in growth condition and
multinucleited myotubes in differentiation condition were observed in culture. (D) MyHC+ myotubes
shown in (C) were visualised by immunocytochemistry.

These data revealed that pathological phenotypes of Duchenne muscular dystrophy

(DMD) are caused by cell autonomous failure of satellite cell function, and satellite cells
possess an indispensable role in maintaining the damage-repair cycle of DMD [12]. There is
much evidence showing that aged muscle exhibits reduced muscle function. Interestingly,
transplantation of single myofibres containing satellite cells together with muscle injury
stimulus markedly prevents age-related muscle wasting and improves muscle force generation
throughout life by altering the aged environment of host muscle [32].
On the other hand, non-satellite cell populations contribute to muscle regeneration, and
thus, if satellite cells are the only source for muscle regeneration remains unclear. Recent
studies have addressed whether satellite cells are actually necessary for muscle regeneration
in vivo and whether there is any significant contribution by non-satellite cell populations to
muscle regeneration following acute muscle injury.
Conditional depletion of satellite cells in mice expressing the human diphtheria toxin
receptor (DTA) under the control of the mouse Pax7 locus or inducible ablation of Pax7+
cells by crossing Pax7CreERT2 mice with R26RDTA mice has been performed [16, 17, 33].
Satellite cell depletion results in complete loss of regenerated muscle. Indeed, these studies
clearly provide genetic and direct evidence of the indispensability of satellite cells for muscle
regeneration in adults.

Yusuke Ono

3. Satellite Cell Heterogeneity

3.1. Evidence of Satellite Cell Heterogeneity among Muscles
During embryonic development in vertebrates, skeletal muscles in the trunk and limbs are
derived from myogenic stem cells that migrate from the somite, whereas satellite cells in most
head muscles originate from non-somite cells [34, 35]. It has been reported that there are
distinct genetic networks that specify pre-myogenic progenitors between head and trunk/limb
muscles during development [34]. Pax3 is not expressed in head muscle progenitors, and
mutations of Pax3 gene affects only limb myogenesis, but not head myogenesis [36]. In
contrast, double-knockout of MyoR and Tcf21 in mice results in only a lack of facial muscles
including the masseter during development [37]. Bicoid-related homeodomain transcription
factor Pitx2 and T-box transcription factor Tbx1 are involved in the specification of
branchiomeric muscle progenitor cells [38, 39]. Recent findings also suggest that either Myf5
or Mrf4 is required to initiate extraocular myogenesis [40]. Interestingly, Wnt signalling is a
positive regulator of myogenesis in trunk mesoderm, although Wnt blocks myogenic
differentiation of progenitors in the head [41]. Recently, it has been shown that satellite cells
in adult muscle retain developmental identities [40, 42, 43]. Gene expression profiles reveal
that Pax3 and Lbx1 mRNA are virtually undetectable in eye and masseter-derived satellite
cells, but are detected in limb-derived cells [40, 43]. In contrast, Tcf21 is robustly expressed
in head satellite cells, but not in limb cells [40, 42, 43].
Trunk muscles function in posture and locomotion, whereas head muscles mainly control
facial expression, eye movement and feeding activity. Because certain muscular dystrophies
severely affect particular muscles, but not others [44], the various functions and different
regulatory networks between trunk/limb and head muscles presumably link to muscle-specific
phenotypes in muscular dystrophies [45]. Thus, the heterogeneity of satellite cells among
muscles may be related to myopathic phenotypes, even though transplanted satellite cells can
generate new myofibres in distinct originated muscles [40, 42, 43, 46]. Previous findings
suggest that masseter muscles damaged by myotoxin injection regenerate poorly compared
with that of limb muscles in adults [47]. This phenomenon can be explained by recent
findings showing that masseter muscles contain a significantly lower number of satellite cells
per myofibre, compared with that in limb muscles [43]. In addition, masseter-derived satellite
cells tend to maintain a longer proliferative state, presumably to generate an appropriate
number of progenitors, thus, myogenic differentiation proceeds over a longer period
compared with that in limb muscles [43]. Interestingly, it has been reported that extraocular
muscles are not affected morphologically or functionally in DMD and age-related sarcopenia,
whereas limb muscles are severally affected [48-50]. The mechanisms of this effect remain
unclear, but hypotheses have proposed that extraocular muscle, with a distinct origin from
that of limb muscles, contains more satellite cells per myofibre, which possess higher
proliferation and differentiation abilities as well as resistance to apoptosis and oxidative stress
compared with those in limb muscles [51]. The mechanism of heterogeneous dysfunction of
satellite cells among muscles, which contributes to muscular dystrophic pathology, is still
unclear. It would be necessary to understand the relationship between developmental
programs in different muscles and their satellite cell function to provide a mechanistic insight
into the muscle myopathies of specific susceptible muscles.

Stem Cells Hierarchies in Muscle Satellite Cells

Old age is associated with a significant decline in the mass, strength and regenerative
capacity of skeletal muscles [52]. While the myogenic potential of aged satellite cells is not
compromised [53, 54], their self-renewal appears to fail [55]. This observation may explain
the decline in satellite cell numbers with age [54, 56]. However, it is unlikely that the agerelated decrease in satellite cell numbers occurs in all muscles throughout the body. For
example, age-related decline in rat satellite cell numbers occurs in tibialis anterior muscle,
extensor digitorum longus (EDL) and soleus [56, 57], whereas no significant reduction is
observed in levator ani muscle [58]. Additionally, although the number of Pax7+ satellite cells
per EDL fibre significantly decreases with age, conversely, the number of satellite cells per
masseter fibre almost doubles in aged mice, compared with that in young mice [43]. This
finding may explain that both the structure and function of masseter muscles are preserved
with age, compared with those of limb muscles [59]. These observations, therefore, indicate
that age-dependent changes in satellite cell number and function may not be a universal
property of muscle aging, suggesting distinct alterations in muscle function with age.

3.2. Evidence of Satellite Cell Heterogeneity within Fibres

Accumulating evidence indicates that satellite cells are a functionally heterogeneous
population not only among muscles but also within a single myofibre [5, 60]. Satellite cells
can be potent myogenic progenitors. Engraftment of a single EDL myofibre can not only
generate substantial amounts of muscle, but also repopulate host muscle with donor-derived
new satellite cells [6]. However, there is great variation in the amounts of regenerative fibres
and satellite cells generated by individual grafted myofibres, and only a small population of
satellite cells possess stem cell properties [6].
Whether all tissue stem cells are equal or some particular cells retain more stem cell-like
potential than others is a central question in stem cell biology. In cell biology experiments,
studies usually provide average data from bulk cells. Thus, the behaviour, function and
importance of small cell populations may be masked by the majority [61]. Single cell studies
may achieve precise characterisation of cells with functional heterogeneity. Individual
evaluation of satellite cells by clonal analysis is, therefore, necessary to determine whether all
satellite cells retain stem cell function. Clonal analysis demonstrated that proliferation
abilities vary widely among satellite cell clones in muscle [55, 62, 63]. The analysis of a
range of satellite cell behaviours on a per myofibre basis showed that there is a large range
in proliferative potential among satellite cell clones including those from the same myofibre
[43]. It is interesting to note that the range form the most to least proliferative clone has been
demonstrated as over 100-fold within the same myofibres after 10 days in culture of masseterderived satellite cells [43]. Individual satellite cell-derived clones show a strong positive
correlation between the number of cells with the Pax7+MyoD self-renewal phenotype and
the colony size [43], indicating that self-renewal correlates with proliferation. These findings
revealed that satellite cells within a muscle exist as a functionally heterogeneous population,
with some possessing more stem cell properties including high proliferation associated with
high self-renewal. Grafting a single muscle stem cell resulted in extensive proliferation and
new muscle fibres in vivo [31]. Importantly, only a small percentage of single cells exhibited
extensive expansion after transplantation, implying again that a sub-population of satellite
cells has stem cell properties.

Yusuke Ono

In vitro studies demonstrated that an undifferentiated non-fused subpopulation of

myoblasts self-renews and becomes quiescent-like during myogenic progression [9, 11, 64].
However, whether all satellite cells possess self-renewal remained unclear. Clonal analysis
showed that some satellite cells (~20%) from EDL in mice are solely composed of
Pax7MyoD+ cells committed to myogenic differentiation after 10 days of culture, even under
a growth condition [43]. This observation suggests that not all satellite cells can self-renew
and some cells exist as a population committed to myogenic differentiation.
As we discuss below, satellite cell self-renewal appears to fail in aged muscle. However,
clonal analysis of older satellite cells also revealed that, although there is a significant
decrease in the maximum and mean number of population doublings, which is consistent with
previous studies [56, 65], a strong positive correlation between the number of Pax7+MyoD
self-renewing cells and colony size in older muscle is observed, as is the case with young
muscle [43]. Importantly, a population of satellite cells with stem cell characteristics is also
preserved even in aged muscle [43]. These results support a previous observation that older
satellite cells grafted into young muscle remain capable of generating as much new muscle
and satellite cells as grafting cells from young mice [54].

3.3. Molecular Characteristics of Satellite Cell Heterogeneity within Fibres

In stem cell fate choice, the balance between self-renewal and differentiation is important
to maintain stem cell pools and tissue homeostasis. Asymmetric cell division allows a stem
cell to generate a daughter cell that self-renews and another that undergoes differentiation and
provides a mechanism for protecting against DNA mutation involved in cell division of tissue
stem cells from damage and loss throughout life [66]. Functional heterogeneity among
satellite cells raises the question of whether satellite cells within a myofibre actually have
distinct molecular characteristics. Using a multiplex single-cell RT-PCR assay, Cornelison
and Wold reported that individual satellite cells within myofibres have different gene
expression profiles [67]. It has been also shown that the Myf5 locus is active in approximately
90% of quiescent satellite cells in EDL in adults [20]. Kuang et al provided genetic evidence
of the satellite cell heterogeneity [68]. Using Myf5-Cre/Rosa26-YFP mice, ~90% of Pax7+
satellite cells within myofibres in EDL demonstrate a myogenic history by expressing Myf5
at some point, and the remaining 10% of Pax7+ cells do not express Myf5 at any point during
development, postnatal growth and adult homeostasis. Pax7+Myf5- satellite cells were named
satellite stem cells with stem cell-like characteristics, which asymmetrically produce a selfrenewable Pax7+Myf5 stem cell and a Pax7+Myf5+ daughter cell committed to transitamplifying progeny that undergo limited symmetric proliferation to generate myonuclei [68]
[but see [69]].
Further study elucidated the molecular mechanism in satellite stem cells, showing that the
Wnt receptor Fzd7 is highly expressed only in Pax7+Myf5- satellite stem cells, but not in
committed Pax7+Myf5+ satellite cells. Wnt7a secreted from regenerating fibres only permits
Pax7+Myf5- satellite stem cells to undergo symmetric cell division to contribute toward
efficient muscle regeneration [70].
Recently, a subset of resident satellite cells possessing both side-population [SP] and
satellite cell characteristics, which express the SP marker ABCG2 as well as satellite cell
markers Pax7 and Syndecan-4, has been identified and named satellite-SP cells. [71].

Stem Cells Hierarchies in Muscle Satellite Cells

Although the majority of SP cells exist outside the satellite cell position, satellite-SP cells
are localized in the satellite cell position and spontaneously fuse to form myotubes and
produce myogenic progeny [71]. When transplanted into regenerating muscle, satellite-SP
cells generate satellite cell progeny more efficiently.
In 1975, the immortal strand hypothesis has been proposed as a protective mechanism
against genomic mutations during DNA replication in stem cells [72]. According to this
hypothesis, a stem cell produces a committed cell with newly synthesized DNA and a
daughter cell that retains older DNA strands by asymmetric cell division. This phenomenon
has been also observed for satellite cells [73, 74]. Recently, using transgenic Tg:Pax7-nGFP
mice, Rocheteau et al showed that a highly Pax7-expressing (Pax7-nGFPhigh) cell population
has a lower metabolic status and delayed first cell division compared with those of a lower
Pax7-expressing (Pax7-nGFPlow) cell population [75]. Pax7-nGFPhigh cells can self-renew and
generate Pax7-nGFPlow cells after serial transplantations. Interestingly, they found that the
majority of Pax7-nGFPhigh cells asymmetrically segregate older DNA strands to renewing
cells during cell division; thus, quiescent Pax7-nGFPhigh cells represent a reversible dormant
stem cell state during muscle repair and regeneration, whereas Pax7-nGFPlow cells perform
random DNA segregation [75].

3.4. Slow-Dividing Satellite Cell Populations

In adults, tissue stem cells retain the ability to proliferate, differentiate and self-renew
throughout life to maintain tissue homeostasis and repair injuries. It remains unclear how
tissue stem cells carefully coordinate a precise balance between proliferation, differentiation
and self-renewal, and how stem cells retain long-term self-renewal. Most tissue stem cells are
maintained in a quiescent state, and the majority divide extensively in response to tissue
injury. Recent studies have provided evidence of some cell populations that proliferate
extremely slowly with more stem cell properties in mouse skin [76, 77], intestine [78], male
germ line [79] and among cardiac stem cells [80]. Hematopoietic stem cell division frequency
inversely correlates with long-term self-renewal [81, 82]. Dormant hematopoietic stem cells
that slowly divide possess a long-term repopulation potential and produce fast-dividing
committed cells that repopulate over a short term [82]. These fast-dividing committed cells
give rise to multipotent progenitors that generate nearly a billion circulating blood cells per
day. Importantly, slow-dividing cells provide a slow-dividing self-renewable daughter and
transit-amplifying committed progeny that contributes to tissue homeostasis and repair [83].
In the postnatal growing muscle of rats, approximately 80% of the satellite cell
population is a highly proliferative population that is readily labelled by BrdU pulsing,
whereas the remaining cells divide slowly [84]. Fast- and slow-dividing cell populations
among activated satellite cells in adult muscles have also been observed [63, 64, 73, 74]. It
has been thought that the slow-dividing minority population reverts to quiescent selfrenewable cells and the fast-dividing majority population undergoes limited cell division as
transiently amplifying cells before myogenesis. However, there was no direct evidence
regarding whether these two populations have such distinct roles during myogenic
progression.
Recently, the relationship between proliferation behaviour and satellite cell function was
investigated [85].

Yusuke Ono

Figure 2. A slow-dividing cell population present in activated satellite cells. To monitor cell-cycle
frequency, freshly isolated satellite cells were labelled with a fluorescent membrane dye PKH26
(Sigma). PKH26-labelled cells were maintained in growth medium for 4 days. Typical images
immediately (Day 0) or at 4 days (Day 4) after PKH26 staining. The vast majority of activated satellite
cells are PKH26low fast-dividing cells, whereas PKH26high slow-dividing cells are observed as a
minority population [85].

To assess the frequency of cell division, satellite cells isolated from mouse EDL muscle
were labelled with the fluorescent lipophilic dye PKH26 and stimulated to proliferate under a
growth culture condition for several days. The vast majority of activated satellite cells are
PKH26low retaining fast-dividing cells that generate a greater number of both multinucleated
myotubes and self-renewing cells, compared with those of PKH26high retaining slow-dividing
cells as the minority population (Figure 2). However, cells derived from the slow-dividing
cell population efficiently form secondary myogenic colonies after passaging, whereas those
derived from the fast-dividing cell population rapidly commit to myogenic differentiation
after a few rounds of cell division. Thus, the fast-dividing cell population is exhausted after
several passages, but the slow-dividing cell population retains long-term self-renewal ability
[85]. A single satellite cell clonal assay demonstrated that large colonies contain numerous
self-renewable and differentiated progeny, which suggests cells that generate large colonies
possess stem cell characteristics [43]. Importantly, only cells derived from large colonies can
generate highly proliferative progeny with long-term self-renewal, unlike those from small
colonies (our preliminarily observation). Indeed, the satellite cell population that generates
large colonies probably contains slow-dividing cells. Moreover, in vivo transplantation
analysis demonstrated that slow-dividing cells extensively produce regenerative myofibres in
muscles with cardiotoxin-induced injury. Following transplantation, the effect of secondary
cardiotoxin injection to stimulate muscle regeneration was also examined.
Interestingly, after the second cardiotoxin injection, more newly regenerated myofibres
were observed only in slow-dividing cell-transplanted muscle, but not in fast-dividing cell-

Stem Cells Hierarchies in Muscle Satellite Cells

transplanted muscle, compared with levels after the first cardiotoxin injection. These findings
indicate that self-renewing cells derived from the slow-dividing cell population undergo a
second round of proliferation within injured muscle after the second muscle injury, and slowdividing cells, therefore, retain stem cell-like properties that extensively contribute to
regeneration in host muscle by repeatedly producing myogenic progeny. A previous study by
Beauchamp et al showed that the majority of immortal myoblasts quickly die after
transplantation into irradiated dystrophic muscles, but a minority, which are slow-dividing in
vitro, efficiently survive and are more successfully engrafted [86], Therefore, the in vivo
regenerating environment appears to directly and preferentially favour engraftment of slowdividing cells.

3.5. Molecular Characteristics of Slow-Dividing Cells

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor- family
and initiate signalling via binding to transmembrane type 1 and 2 BMP receptors (BMPRs).
Upon BMP stimulation, BMPRs phosphorylate R-Smads (Smad1, Smad5 and Smad8).
Phosphorylated Smad1/5/8 then translocates into the nucleus to regulate the expression of
target genes such as Id1 gene, a helix-loop-helix protein. BMP signalling can be suppressed
by secreted antagonists such as Noggin and Chordin, which bind BMPs with high affinity to
interfere with interactions between BMPs and receptors [87]. Although BMPs are known to
be crucial for bone and cartilage formation and repair, BMPs from the lateral plate mesoderm
also inhibit myogenic differentiation of the dermomyotome to regulate the onset of
myogenesis during development [88, 89]. In adult muscle, recent studies showed that satellite
cells start to express BMPR-1A together with pSmad1/5/8 and Id1 in the nucleus after
activation [90, 91]. Disrupting interactions between BMPs and their receptors by treatment
with the BMP antagonist Noggin or soluble BMPR-1A fragments induces precocious
myogenic differentiation in myoblasts. Similarly, genetic disruption of endogenous BMP
signalling by siRNA mediated knockdown of BMPR-1A or pharmacological inhibition of
Smad1/5/8 phosphorylation with dorsomorphin also causes premature myogenic
differentiation through down-regulation of Id1 protein expression. Interestingly, the level of
endogenous Noggin expression increases in satellite cell progeny committed to myogenic
differentiation, and Noggin knockdown by siRNA promotes proliferation and disrupts
myoblast fusion into multinucleated myotubes, indicating that Noggin produced from
differentiating cells antagonises BMPs to facilitate myogenic progression. Crucially,
inhibition of BMP signalling pathway during muscle regeneration results in smaller
regenerated myofibres with excess collagen deposition.
These findings suggest that the BMP-Smad-Id axis is required for population expansion
to generate appropriate numbers of myogenic progenitors prior to myogenic differentiation,
thus acting as a potent regulator of routine satellite cell functions to balance between
proliferation and differentiation during muscle regeneration [90]. In support of these
observations, ectopic activation of BMP signalling during foetal muscle growth in chicks
increases muscle progenitors, resulting in increase of myofibres. Conversely, interfering with
BMP signalling reduces muscle progenitors leading to smaller myofibres, suggesting that
BMP signalling is essential for regulating satellite cell numbers at the postnatal muscle
growth stage [92].

10

Yusuke Ono

Figure 3. Role of BMP signalling during muscle regeneration in satellite cells. During muscle
regeneration, quiescent Pax7+ satellite cells are activated to co-express Pax7 and MyoD. Satellite cellderived myoblasts proliferate extensively before many cells then downregulate Pax7 and differentiate to
either repair injured muscle fibres or fuse together to generate new myofibres. Other satellite cells
maintain Pax7 expression but lose MyoD, and self-renew to maintain a stem cell pool. Normal BMP
signalling through Smad1/5/8 phosphorylation and Id1 induction is required during muscle regeneration
to allow the satellite cell-derived myoblast population to expand, by preventing precocious myogenic
differentiation. Noggin, a negative regulator for BMP signalling, is up-regulated to antagonise the BMP
signal to facilitate the myogenic differentiation programme [90].

Together, BMP signalling is clearly a part of the programme that regulates satellite cell
numbers and functions from development through to growth and regeneration of muscle
(Figure 3).
It is important to note that some slow-dividing cells tend to differentiate and fuse to form
myotubes at an early time point in culture [85]. This observation indicates that slow-dividing
cells are still a heterogeneous population that may correspond to committed myogenic
progenitors that immediately undergo myogenic differentiation with minimal or no cell
division in vivo [93] and in vitro [43]. As mentioned above, Id1 as a critical target of BMP
signalling plays an important role to maintain the undifferentiated state of activated satellite
cells [90, 91, 94]. Interestingly, Id1 is expressed by all activated satellite cells, but the
expression level varies within the slow-dividing cell population. Because slow-dividing cells
with low levels of Id1 protein tend to undergo myogenic differentiation immediately, longterm self-renewable cells in the slow-dividing cell population are restricted to an
undifferentiated population that expresses high levels of Id1 protein [85]. Thus, slow-dividing
cells with a highly BMP signalling (Id1high) in the activated satellite cells are a minority cell
population that retains long-term self-renewal throughout life, while committed progenitors
that express low levels of Id1 protein may proliferate minimally before differentiation into
muscle.
Speculatively, the small population of satellite cells capable of robust re-population of
satellite cells and efficient muscle regeneration, which is observed in previous studies [6, 31,
32, 68], may also contain cells that generate slow-dividing cells.

Stem Cells Hierarchies in Muscle Satellite Cells

11

The cell cycle frequency of satellite-SP cells that are capable of efficiently generating
satellite cell progeny is much lower compared with that of other satellite cells in vitro [71]. In
addition, cell cycle entry for the first cell division by the Pax7high cell population is much
slower than that of the Pax7low cell population [75]. Future studies should be valuable for
defining whether slow-dividing cells correspond to other stem cell-like populations found
previously, such as Pax7+Myf5+ cells, older DNA retaining cells, satellite-SP cells and
Pax7high cells (Figure 4).

3.6. Slow-Dividing Cells and Aging

A hallmark of age-related muscle weakness called sarcopenia is characterised by a
significant decline in muscle mass, strength, and regenerative ability. While the myogenic
potential of aged satellite cells is not compromised, the self-renewal ability may fail [56, 95].
This finding explains the decline in satellite cell numbers with age [54, 56]. Tissue-specific
niche provides a microenvironment for stem cells to regulate tissue maintenance, repair and
regeneration.
Satellite cells are localised between the plasma membrane and the basal lamina of
myofibres, where the function of satellite cells is modified by cell-adhesion molecules such as
cadherins [22] and regulated by the basal lamina via integrins [96].

Figure 4. A possible model for stem cell hierarchies of satellite cells. Satellite cells within a fibre exist
as a functionally heterogeneous population [43]. There seems to be hierarchical regulation of satellite
cell populations in adult muscle. In activated satellite cells, there are at least two populations; fastdividing cells as a majority and slow-dividing cells as a minority [85]. Slow-dividing cells that express
high levels of Id1 protein retain long-term self-renewal ability and so may be at the top of the satellite
cell hierarchy. As a transit amplifying cells, fast-dividing cells that highly express Id1 protein give rise
to a greater number of multinucleated myotubes and self-renewing cells. Self-renewed cells derived
from the fast-dividing cell population are committed cells that express low levels of Id1 protein and
rapidly undergo myogenic differentiation after a few rounds of cell division without producing any selfrenewing cells when reactivated, whereas cells derived from the slow-dividing cell population that
highly express Id1 protein are capable of generating both slow- and fast-dividing cell populations [85].
It is unclear whether the slow-dividing cells are corresponding to the other stem cell-like populations,
such as Pax7+Myf5- cells [68], Pax7high cells [17], SP-satellite cells [71] and older DNA-retaining cells
[17, 73, 74].

12

Yusuke Ono

Satellite cells in their niche are also regulated by growth factors and cytokines secreted
from myofibres or interstitial cells [97]. Indeed, the satellite cell niche is a critical factor in
the maintenance of stem cell function.Whether or not slow-dividing cell population in
satellite cells is maintained throughout life or affected by aging remained unclear. Recently,
Chakkalakal et al. have shown that the function of slow-dividing cells is partially disrupted
during aging. Using the transgenic mice expressing a histone2B-GFP reporter driven by a
tetracycline-inducible transactivator, they monitored proliferative state of satellite cells
throughout life on the basis of label retention in vivo [98].The study showed that the rate of
satellite cell cycling differed between adult and aged histone2B-GFP mice and found that
aged satellite cells spend less time in a quiescent state compared with adult satellite cells and
aged satellite cells lose robust self-renewal potential after cell cycle entry, suggesting that
aged satellite cells lose their ability to retain a quiescent state. Slow-dividing cell population
in aged muscles displayed reduced cell growth, decreased expression of Pax7 compared with
young slow-dividing cells.However, transplanted slow-dividing cells from aged muscle into
pre-injured adult muscles can give rise to produce more numbers of both myonuclei and
Pax7+satellite cells compared with fast-dividing cells, indicating that aged slow-dividing cells
still retain stemness.

Conclusion
More than 50 years after the discovery of satellite cells, new insights during this decade
have significantly advanced our knowledge of the molecular mechanisms underlying satellite
cell fate choice. Recent studies also indicate that resident satellite cells are directly or
indirectly affected in some types of muscular dystrophy such as DMD, Emery-Dreifuss,
oculopharyngeal and facioscapulohumeral [45]. How distinct genetic networks in muscle
progenitors during the development of different muscles affect satellite cell properties in
adults, and how satellite cells are directly or indirectly compromised by genetic mutations in
specific muscular dystrophies will be emerging research areas in satellite cell biology. In this
chapter, we discussed recent findings of functionally heterogeneous and stem cell-like
subpopulations among satellite cells in adult muscle.
Considering current research progress on satellite cell heterogeneity, we may need to
revise satellite cell criteria, while discovery of new markers to define the satellite cell
population that retains more stem cell-like properties will be important to advance satellite
cell research [99].
There appears to be hierarchical regulation of satellite cell populations in adult muscle.
Slow-dividing cells avoid the risk of DNA mutation by repetitive replication and may be at
the top of the satellite cell hierarchy.
Future in vitro and in vivo studies will be valuable to define stem cell-like populations,
including slow-dividing cells, to better understand the molecular mechanisms of muscle
homeostasis and efficient repeated regeneration of adult muscle throughout life to extend the
window of opportunity for satellite cell-based therapies of muscular dystrophies and
sarcopenia.

Stem Cells Hierarchies in Muscle Satellite Cells

13

Acknowledgments
This work was supported by The Nakatomi Foundation, JSPS KAKENHI and Special
Coordination Funds for Promoting Science and Technology from JST.

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In: Basic Biology and Current Understanding of Skeletal Muscle ISBN: 978-1-62808-367-5
Editor: Kunihiro Sakuma
2013 Nova Science Publishers, Inc.

Chapter 2

Cellular Mechanisms Regulating

Protein Metabolism in Skeletal Muscle
Cells: Age-Associated Alteration
of Intracellular Signaling Governing
Protein Synthesis and Degradation
Mitsunori Miyazaki
Department of Physical Therapy, School of Rehabilitation Sciences
Health Sciences University of Hokkaido
Tobetsu-cho, Ishikari-gun, Hokkaido, Japan

Abstract
Skeletal muscle is the largest organ in the human body, making up more than 3040% of total body weight in healthy adults. The wasting of skeletal muscle mass, which
is induced by unloading, malnutrition, aging or several kinds of diseases, leads to the loss
of functional human performance, long-term health issues and a low quality of life. It has
been generally accepted that the net balance between protein synthesis and degradation is
a critical determinant of the regulation of skeletal muscle mass.
Recent studies indicate that the skeletal muscle protein metabolism in response to the
environmental cues (i.e. physical activity and nutrients) is blunted in the elderly, which
then leads to the age-associated gradual loss of muscle mass. The aim of the present
chapter is to summarize and discuss, 1) recent progress in the understanding of cellular
mechanisms in anabolic (protein synthesis) and catabolic (protein degradation) signaling
pathways that govern the regulation of skeletal muscle mass, 2) altered capacity in the
protein metabolism of aged skeletal muscle in response to exercise and nutrients. A better
understanding of the anabolic and catabolic processes which regulate skeletal muscle
mass is critical for the development of more effective therapeutic interventions to prevent
the loss of muscle with aging and disease.

Keywords: Sarcopenia, protein synthesis, protein degradation, mammalian target of

rapamycin, Atrogin1, muscle ring finger 1

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Mitsunori Miyazaki

Introduction
Skeletal muscle is the largest organ in the human body, making up more than 30-40% of
total body weight in healthy adults [1]. The maintenance of skeletal muscle mass is critical for
long-term health and quality of life, because a decrease in muscle mass during aging is highly
associated with functional impairment and disability, which then leads to the loss of
independence and increased risk of morbidity and mortality [2, 3]. Sarcopenia is the term
widely used to describe the age-associated and progressive loss of skeletal muscle mass and
strength. This degenerative loss of skeletal muscle occurs at a rate of 1-2% per each year after
the age of 50 yr. [4-6], and this is accelerated with advancing of age such that lean muscle
mass declines to ~50% when reaching an age of 75-80 yr. compared to the healthy young
adults [7-10]. Particularly, the reduced muscle mass largely reflects a loss of myofibrillar
proteins [11]. Despite the significance of skeletal muscle loss and weakness as inevitable
concomitants with aging, the potential mechanisms underlying the development of sarcopenia
are only partially understood.
The etiology of sarcopenia is very complex and characterized by the combination of
multiple factors including individual muscle fiber atrophy, degenerative loss of fiber number
or preferential loss of type II (fast, glycolytic) muscle fiber (summarized in Table 1). In
addition, situations are more complicated because of changes in elderly such as sedentary life
style, malnutrition or susceptibility to diseases, all of which can accelerate/influence the
potential loss of muscle mass.
Table 1. The potential etiologies leading to age-associated muscle wasting
Mitochondrial dysfunction
Increased oxidative stresses
DNA damage
Increased myonuclei apoptosis
Increased level of pro-inflammatory cytokines
Decreased or altered responsiveness of sex/anabolic hormones
Compromised protein metabolism (reduced protein synthesis / increased protein degradation)
Decreased number of muscle precursor cells
Loss of alpha-motor neuron input
Degenerative loss of muscle fiber number
Individual muscle fiber atrophy
Infiltration with fat and connective tissues
Preferential loss of type II fiber (fast / glycolytic)
Vitamin D deficiency
Increased susceptibility to chronic diseases
Inadequate nutritional intake
Decreased level of physical activity, Sedentary life style

Significant progress has been made over the past decade to identify some of the potential
contributors to the development of age-associated muscle loss. Skeletal muscle mass is
largely determined by the net balance between protein synthesis and protein degradation. In
the catabolic conditions such as aging, disuse or inflammatory diseases, the rate of protein

Cellular Mechanisms Regulating Protein Metabolism in Skeletal Muscle Cells

23

degradation in skeletal muscle exceeds relative to the protein synthesis rates such that there is
a net negative balance in cellular protein contents leading to atrophy of individual muscle
fibers and decreased muscle mass.
Recently, the impaired balance of protein metabolism is indicated as an important factor
that contributes to age-associated muscle loss. Here in this chapter, we will highlight the
advances that have been made over the past few years in our understanding of the cellular and
physiological mechanisms regulating the protein metabolism in the development and
treatment of age-associated muscle loss.
Particular emphasis has been placed on 1) cellular mechanisms regulating mammalian
target of rapamycin (mTOR, also called as mechanistic target of rapamycin)-dependent
signaling and protein synthesis, 2) potential mechanisms govern the protein degradation
through ubiquitin-proteasome system, 3) age-associated alteration of protein metabolism in
skeletal muscle.

1. Translational Control of Muscle Protein

Synthesis mTOR - A Central Regulatory Hub
for Protein Synthesis
Skeletal muscle proteins turnover regularly such that 1-2% of proteins in skeletal muscle
are metabolized daily [12]. The turnover of skeletal muscle proteins involves the ongoing
processes of protein synthesis and breakdown respectively. To date, numerous studies have
shown that protein kinase mTOR plays a crucial role in regulating the rate of protein
synthesis in skeletal muscle cell [13-16]. mTOR is a serine/threonine kinase of the
phosphatidylinositol kinase-related kinase family that is highly conserved from yeast to
mammals [17]. In skeletal muscle, mTOR is found in two distinct multi-protein complexes:
mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Essential components of
mTORC1 are mTOR, regulatory-associated protein of mTOR (Raptor), G protein -subunitlike (Gl/also known as mLST8) and proline-rich Akt substrate 40 (PRAS40), whereas
mTORC2 is comprised of at least six different proteins, some of which overlap with
mTORC1: mTOR, rapamycin-insensitive companion of mTOR (Rictor), mammalian stressactivated-protein-kinase-interacting protein 1 (mSIN1), protein observed with Rictor-1
(Protor), Gl and Deptor [18-24]. mTORC1 and mTORC2 have been respectively
characterized as the rapamycin-sensitive and rapamycin-insensitive protein complexes. The
crucial role of mTOR (particularly mTORC1) in mediating protein synthesis and subsequent
muscle growth/hypertrophy is supported by genetic and pharmacological studies. Musclespecific knockout of mTOR or raptor causes reduced postnatal growth and severe myopathy
[25-27]. A specific inhibitor of mTOR, rapamycin, acts especially on mTORC1 although
mTORC2 is also affected during long-term treatment [28]. Rapamycin administration
prevents skeletal muscle growth and hypertrophy in vivo, including muscle growth during
regeneration [29], compensatory muscle hypertrophy induced by synergist ablation [13] and
muscle re-growth during reloading of unloaded muscles [30].
As a rapamycin-sensitive complex, mTORC1 plays a central role in transmitting
information from extracellular anabolic signals including growth factors, nutrients and
cellular stresses into pathways that mediate several aspects of the cell growth machinery

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Mitsunori Miyazaki

(Figure 1). Particularly, mTORC1 promotes protein translation through phosphorylating

eukaryotic initiation factor 4E-binding protein 1 (4EBP1) and p70 ribosomal S6 kinase 1
(S6K1), the most well characterized downstream targets of mTORC1 [31].

Figure 1. Simplified schema depicting signaling networks regulating mTORC1 activity and protein
synthesis in skeletal muscle. Insulin or IGF-1 activates class I PI3K, which then leads to the
phosphorylation and activation of Akt. Akt directly phosphorylates TSC2 on multiple residues and
inhibits its GAP activity, thereby allowing Rheb to accumulate in its active GTP-bound form and
leading to activation of mTORC1 and its downstream effectors S6K1 and 4EBP1. Activated Akt also
phosphorylates PRAS40, thereby releasing its inhibitory function toward mTORC1. Activation of
MEK/ERK pathway leads to TSC2 phosphorylation at S664 may contributes to mTORC1 activation. A
heterodimeric complex of the Rag proteins plays a fundamental role in amino acid-induced regulation
of mTORC1 signaling. Rag GTPases are heterodimers of either RagA or RagB with either RagC or
RagD. In the presence of amino acids, the Rag GTPases are converted to the active conformation, in
which RagA/B is loaded with GTP and RagC/D is loaded with GDP. The active form of the Rag
heterodimer physically interacts and relocalizes mTORC1 to a perinuclear membrane-bound
compartment that contains the mTORC1 activator Rheb. REDD1 and REDD2 function by inhibiting
the interaction of TSC2 with the scaffold protein 14-3-3, thereby promoting assembly of the
TSC1/TSC2 complex and subsequent inhibition of Rheb, which then lead to mTORC1 inhibition.
Under energy-deprived conditions (increased AMP to ATP ratio), activated AMPK phosphorylates
TSC2 and enhances its inhibitory function leading to decreased mTORC1 activity. AMPK also directly
phosphorylate the Raptor and inhibit mTORC1 activity.

Following the phosphorylation by mTORC1, a translational repressor 4EBP1 dissociates

it from an inhibitory complex with the translation initiation factor eukaryotic initiation factor
(eIF) 4E, allowing for the complex formation of eIF4A/4G/4E thereby promoting translation
initiation of mRNAs with 7-methyl guanine cap [32-34]. In parallel, mTORC1 also

Cellular Mechanisms Regulating Protein Metabolism in Skeletal Muscle Cells

25

phosphorylates and activates S6K1, which subsequently phosphorylates several downstream

substrates such as ribosomal protein S6 (rpS6) and eukaryotic elongation factor 2 (eEF2)
kinase. Phosphorylation of rpS6 has been reported to enhance cell size and cell proliferation
[35] by an unknown mechanism although the role of rpS6 has been heavily debated [36].
Phosphorylation of eEF2K at Ser366 by S6K1 relieves its inhibitory regulation toward eEF2,
thereby enhancing protein translation elongation [37, 38]. More comprehensive reviews on
protein translation regulation can be found elsewhere [34, 36, 39-42].

Regulatory Mechanisms of mTORC1 Activation in Skeletal Muscle Insulin/IGF-1-Dependent Pathway

Currently, the most well characterized mechanism regulating mTORC1 activity in
skeletal muscle is the insulin like growth factor-1 (IGF-1)/insulin-dependent pathway [13,
43]. Stimulation of muscle cells with insulin or IGF-1 lead to activation of class I
phosphoinositide 3-kinase (PI3K), which catalyzes the synthesis of the lipid
phosphatidylinositol 3,4,5-trisphosphate (PIP3). Subsequent to class I PI3K activation,
generation of PIP3 at the plasma membrane results in the recruitment of the phosphoinositidedependent protein kinase 1 (PDK1) and Akt, which then leads to the phosphorylation and
activation of Akt [44, 45]. Akt controls mTORC1-dependent signaling through regulation of
tuberous sclerosis complex (TSC) 1/TSC2, a heterodimeric protein complex which functions
as a negative regulator of mTORC1 [27, 46-50]. Within the TSC1/TSC2 protein complex,
TSC2 functions as a GTPase activating protein (GAP) for a small G protein Ras homolog
enriched in brain (Rheb), an mTORC1 activator. Akt directly phosphorylates TSC2 on
multiple residues (at least two sites, Ser939 and Thr1462) and negatively regulates its GAP
activity, thereby allows Rheb to accumulate in its active GTP-bound form, which then leading
to activation of mTORC1 and its downstream effectors 4EBP1 and S6K1 [51-54]. Activated
Akt also phosphorylates PRAS40, releasing PRAS40 from the mTORC1 complex and
enhances its binding to the cellular anchor protein 14-3-3 [55, 56]. Conversely, in the absence
of growth factors, PRAS40 is hypo-phosphorylated and remains bound to mTORC1 and
thereby inhibits binding of other mTOR substrates, such as S6K1 and translational repressor
4EBP1, which then leads to suppression of protein translation initiation [56].
Mitogen-Activated Protein Kinase Pathway
Another important signaling involved in regulating protein translation initiation through
mTORC1 is the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated
kinase (ERK)-dependent pathway. MEK/ERK pathway contributes to mTORC1 activation
most likely through direct phosphorylation of TSC2 [57-59] or indirect regulation through
p90 ribosomal protein S6 kinase (RSK) [60, 61]. Phosphorylation of TSC2 at both S540 and
S664 sites have been shown to be directly phosphorylated by ERK and contribute to enhanced
mTORC1 activity, likely through inhibiting TSC2 GAP activity thereby allowing Rheb to
accumulate in its active GTP-bound form [57-59]. We have recently reported that mechanical
overload-induced activation of mTORC1 signaling in skeletal muscle is mediated
independently of PI3K/Akt pathway and possibly through TSC2 phosphorylation at S664 via
MEK/ERK regulation [62]. In addition, it has also been indicated that RSK, which is
downstream effector of ERK, affects mTORC1 activity through phosphorylating TSC2 at

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Mitsunori Miyazaki

S1798 site or raptor, a component of mTORC1 [60, 61]. MEK/ERK signaling can also
enhance protein synthesis independent of the mTORC1 pathway through RSK regulation
toward rpS6 [63, 64] or MAP kinase-interacting kinase 1 (MNK1) signaling to eIF4E [16, 65,
66].
Nutrient-Dependent Pathway
In addition to growth factors, availability of amino acids, particularly leucine, has also
been shown to play an important role in the regulation of mTORC1 signaling and protein
synthesis rate [21, 67-70]. Unlike the growth factor-dependent pathway, it has been suggested
that amino acids activate mTORC1 pathway through independent of a canonical class I
PI3K/Akt signaling [70-72]. Currently the precise mechanisms regulating amino aciddependent activation of mTORC1 signaling in skeletal muscle are largely unknown, however,
some potential candidates have been implicated. A class III PI3K, human vacuolar proteinsorting-associated protein 34 (Vps34) has been suggested as a mediator of amino acidinduced activation of mTORC1 signaling, as siRNA knockdown of Vps34 blocks amino acidinduced S6K1 activation but has no effect on Akt activation [71, 73, 74]. A heterodimeric
complex of the Rag proteins, subfamily of Ras small GTPases, has also been implicated as an
effecter of amino acid availability upstream of mTORC1 [75, 76]. In the presence of amino
acids, the Rag GTPases are converted to the active conformation, in which RagA or RagB is
loaded with GTP and RagC or RagD is loaded with GDP. The active form of Rag
heterodimer physically binds to mTORC1 and promotes its translocation to a membranebound compartment that contains the mTORC1 activator Rheb through regulation of the
Ragulator protein complex (trimeric protein complex encoded by the MAPKSP1, ROBLD3
and c11orf59 genes) [75-77].
Cellular Stresses and mTORC1 Inhibition
It has been indicated that mTORC1-dependent signaling is negatively regulated by the
cellular stresses such as hypoxia or energy deprivation, which then leads to the diminished
rate of protein synthesis. Recently, stress response genes designated as regulated in
development and DNA damage responses 1 (REDD1) and REDD2 have been suggested as
important molecules that negatively regulate mTORC1 signaling in skeletal muscle [78-84].
Both REDD1 and REDD2 are indicated as inhibitor of mTORC1 signaling and cell growth in
response to several cellular stresses such as hypoxia, energy stress, or glucocorticoid
treatment [85-88]. REDD1/REDD2 functions by inhibiting the interaction of TSC2 with the
scaffold protein 14-3-3, thereby promoting assembly of the TSC1/TSC2 complex and
subsequent inhibition of Rheb, which then lead to mTORC1 inhibition [83, 89]. Interestingly,
gene expression profiles have indicated the skeletal muscle-enriched expression of REDD2
vs. the ubiquitous expression of REDD1 both in mouse and human tissues [83, 90]. It is of
great interest in REDD2 function in skeletal muscle because gene expression of REDD2 has
been shown to be significantly sensitive in response to the mechanical loading state of
skeletal muscle: REDD2 mRNA expression was down-regulated approximately 50% in both
young and old human skeletal muscle in response to an anabolic stimulus [90] and by 90%
following mechanical overload of the mouse plantaris muscle by synergist ablation (Miyazaki
et al., unpublished observation), whereas increased expression of REDD2 in response to
unloading, a model of muscle atrophy that is associated with diminished mTORC1 activity
[91-93].

Cellular Mechanisms Regulating Protein Metabolism in Skeletal Muscle Cells

27

Energy Status and mTOR Activity

In addition to the availability of growth factors and amino acids, the rate of protein
synthesis is regulated by the cellular energy status both in muscle and non-muscle cells [94,
95]. It is currently suggested that mTORC1 functions as a sensor of cellular energy state
through input from the AMP-activated protein kinase (AMPK) pathway [53]. AMPK is a
heterotrimeric serine/threonine kinase complex comprised of a catalytic and regulatory
and subunits. Recently, a number of upstream kinases have been identified, including the
tumor suppressor LKB1 (STK11) kinase, Ca2+/calmodulin-dependent protein kinase kinases
CaMKK and CaMKK, and TGF-activated kinase-1 (TAK1) [96-101]. AMPK is well
known as a sensor of cellular energy status, which is regulated by changes in the cellular
levels of AMP-to-ATP ratio [102]. When cellular energy is decreased, causing an increase in
the AMP-to-ATP ratio, AMP binds to the -subunits which then leads to the allosteric
activation of AMPK as well as to enhanced phosphorylation of Thr172 on the subunit by
inhibiting its dephosphorylation by protein phosphatases [103-105].
The mTORC1-dependent signaling contains multiple potential sites for regulatory
integration with AMPK. Inoki et al. [53] have reported that, under low cellular energy
conditions, activated AMPK phosphorylates TSC2 on Thr1227 and Ser1345 residues (these
residues correspond to Thr1271 and Ser1387, respectively, in human TSC2) and enhances its
inhibitory function leading to decreased mTORC1 activity. This research group has also
reported that AMPK-dependent phosphorylation of TSC2 on Ser1387 primes TSC2 for
further phosphorylation by GSK-3 on multiple residues. This series of phosphorylation steps
leads to subsequent inhibition of mTORC1 activity under energy-deprived conditions [106].
In addition, a recent study has indicated that AMPK can directly phosphorylate the Raptor
(component of mTORC1 protein complex) on two well-conserved serine residues
(Ser722/Ser792), and Raptor phosphorylation is required for the inhibition of mTORC1
activity through cellular energy stress-induced AMPK activation [107].
Consistent with these observations in non-muscle cells, a strong negative correlation has
been reported between increased phosphorylation of AMPK and reduced muscle hypertrophy
in overloaded muscles in rats [108]. In human skeletal muscle, decreased mTORC1 activation
is coincident with maximal activation of AMPK during resistance exercise [109].
Furthermore, activation of AMPK, by treatment with 5-aminoimidazole-4-carboxamide-1-bD-ribofuranoside (AICAR), results in decreased protein synthesis and a repression of
mTORC1-mediated signaling in skeletal muscle in both in vitro and in vivo experimental
models [110-114]. In contrast, mechanical overload-induced muscle hypertrophy is
accelerated in mice lacking AMPK1, which is accompanied by hyperactivation of mTORC1
signaling [95]. These studies indicate that AMPK is a potentially important negative regulator
of mTORC1 signaling and activation of AMPK in skeletal muscle will contribute to
diminished protein synthesis and hypertrophy.

2. Molecular Mechanisms Regulating Protein

Degradation in Skeletal Muscle
Skeletal muscle contains some distinct proteolytic systems such as the lysosomal system,
the caspase system, the calpain system, and the proteasome system. Although evidence has

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Mitsunori Miyazaki

suggested that lysosomal cathepsins and cytosolic calcium-activated calpains contribute to

acceleration of muscle proteolysis during the catabolic conditions [115-120], the primary
regulator responsible for the degradation of contractile proteins in skeletal muscle is the ATPubiquitinproteasome dependent system [121, 122].
The muscle-specific E3 ubiquitin ligases, muscle atrophy F-box (MAFbx or also called as
Atrogin1) and muscle ring finger 1 (MuRF1) are originally identified as atrogenes, a set of
atrophy-related transcripts whose expression are highly induced or suppressed following
various types of muscle atrophy in rodents [123, 124].

Figure 2. Schematic representation of the molecular mechanisms in the regulation of Atrogin1/MAFbx

and MuRF1 under catabolic conditions. The forkhead families of transcriptional factors, FOXOs have
been indicated as transcriptional activator of muscle-specific E3-ubiquitin ligases Atrogin1 and
MuRF1. Insulin/IGF-1-dependent activation of Akt controls gene expression of Atrogin1 and MuRF1
through modulating protein phosphorylation of FOXO1/FOXO3. The transcription factor NFB also
plays a role in mediating the process of skeletal muscle catabolism through regulation of MuRF1
expression.

In addition to Atrogin1 and MuRF1, another RING-type ubiquitin ligase Cbl-b has also
been suggested as the primary ubiquitin ligase responsible for microgravity-induced skeletal
muscle atrophy [125, 126]. Another E3 ubiquitin ligase found to play a critical role in skeletal
muscle atrophy is tripartite motifcontaining protein 32 (Trim32), which mediates the
ubiquitination of thin filament (actin, tropomyosin, troponins) and Z-band (-actinin)
components and promotes their degradation [127]. Tumor necrosis factor receptor-associated
factor 6 (TRAF6) is also suggested as another E3 ubiquitin ligase, which intercedes
starvation-induced skeletal muscle atrophy [128].

Cellular Mechanisms Regulating Protein Metabolism in Skeletal Muscle Cells

29

Currently, expression of these atrogenes, particularly Atrogin1 and MuRF1 expression

have been indicated as molecular markers of muscle wasting, since these two genes are
specifically up-regulated during multiple models of skeletal muscle atrophy including fasting,
cachexia, diabetes, microgravity, immobilization and nutrient deprivation [123, 124, 129131].
The crucial role of Atrogin1 and MuRF1 expression in muscle atrophy was also
confirmed through knockout mice studies where an absence of Atrogin1 or MuRF1
demonstrates significant sparing of muscle mass following denervation [123].

Transcriptional Regulation of Atrogin1 and MuRF1 Expression

The forkhead families of transcriptional factors, FOXO have been identified as key
regulator of the skeletal muscle atrophy process through modulating transcriptional regulation
of many atrogenes including Atrogin1 and MuRF1 (Figure 2) [132, 133]. Expression levels
of FOXO family are enhanced during the pro-catabolic condition in skeletal muscle [130,
134]. Overexpression of FOXO3 acts on the Atrogin1 promoter to cause Atrogin1
transcription and FOXO3 expression is sufficient to induce significant atrophy in skeletal
muscle cell [119, 132]. FOXO1 has been shown to be required for Atrogin1 and MuRF1
expression, but its overexpression is not sufficient to induce atrophy in skeletal muscle [121,
132, 133]. Atrogin1 expression is also regulated through FOXO4 in response to the proinflammatory cytokine tumor necrosis factor alpha (TNF-) [135]. Akt controls these
forkhead family members. In addition to its role as an activator of protein synthesis, PI3K/Akt
signaling pathway can dominantly suppress the atrophy-associated increases in Atrogin1 and
MuRF1 expression through modulating protein phosphorylation of FOXO1/FOXO3. Akt
phosphorylates FOXOs on multiple sites, promoting their nuclear export into the cytoplasm
where FOXO proteins are retained through association with the 14-3-3 proteins and thus
inhibiting their transcriptional function [133, 136]. The transcription factor nuclear factor
kappa B (NFB) also plays a role in mediating the process of skeletal muscle catabolism
[137-139]. Activation of NFB through muscle-specific transgenic expression of activated
IB kinase causes profound decrease in muscle mass and this is induced through increased
MuRF1 expression [140]. Recently, myogenin has also been suggested as promoter of
skeletal muscle atrophy upon denervation or spinal muscular atrophy by directly activating
the expression of Atrogin1 and MuRF1 [141, 142]. Upregulation of myogenin in response to
denervation is controlled by a transcriptional pathway in which histone deacetylases 4 and 5
are initially induced and, in turn, repress the expression of Dach2, a negative regulator of
myogenin [141].
Targeted Substrates of Atrogin1 and MuRF1
While Atrogin1 and MuRF1 are known as E3 ubiquitin ligases, the specific protein
targets for polyubiquitination and subsequent proteasome degradation during skeletal muscle
atrophy are still under intense debate. It has been indicated that Atrogin1 and MuRF1
possibly regulate the degradation of key proteins involved in skeletal muscle growth,
differentiation and formation of contractile machinery. Member of myogenic regulatory
factors, MyoD and myogenin have been indicated as substrates of Atrogin1 [143-145]. Since

30

Mitsunori Miyazaki

myogenic regulatory factors are essential for muscle cell differentiation and formation of
myofiber, Atrogin1-dependent degradation of MyoD and myogenin likely contribute to the
loss of myofibrillar proteins such as myosin heavy chain [144, 145].
The regulatory subunit of the eIF3 (eukaryotic Initiation Factor 3) complex; eIF3f, has
been also identified as a major target of Atrogin1 for ubiquitination and degradation by the
proteasome during skeletal muscle atrophy [146, 147]. Ectopic expression of Atrogin1 in
skeletal muscle cells induces atrophy and degradation of eIF3f, whereas blockade of Atrogin1
prevents eIF3f degradation undergoing atrophy [147]. Interestingly, the eIF3f has also been
indicated as a scaffold protein that interconnects mTORC1 and downstream S6K1 to
coordinate a promotion of protein synthesis, and genetic activation of eIF3f is sufficient to
induce skeletal muscle hypertrophy and blockade of atrophy [146-148]. The central role of
eIF3f in both pathways regulating protein synthesis and degradation is attractive as a
therapeutic target.
In contrast, it appears that MuRF1 may directly targets contractile and/or myofibrillar
proteins for degradation. MuRF1 associates with titin at the M band of the sarcomere and
potentially contributes titin-dependent signaling [149-151]. A yeast two-hybrid screening of
skeletal muscle cDNA libraries with MuRF1 baits has identified eight myofibrillar proteins as
potential binding partner of MuRF1: titin, nebulin, the nebulin-related protein NRAP,
troponin-I, troponin-T, myosin light chain 2, myotilin and T-cap [152]. Within these potential
candidates, troponin-I has been confirmed as the direct substrate of MuRF1 for
polyubiquitination in cardiac muscle [153]. It has also been indicated that protein components
of thick filaments including myosin heavy chain proteins, myosin-binding protein C, myosin
light chain-1 and myosin light chain-1 are directly degraded through MuRF1-dependent
polyubiquitination [154, 155].

3. Basal Protein Metabolism in Skeletal Muscle

Between Young and Elderly
Initial studies on protein metabolism during age-associated loss of muscle mass have
hypothesized that gradual muscle wasting in elderly is caused by a decrease in basal rate of
protein synthesis, increased basal rate of protein degradation or a combination of these two
processes resulting in a negative net protein balance. While some early studies observed
substantially lower basal protein synthesis in skeletal muscle in elderly compared to the
young adults [156-161], more recent studies could not reproduce those findings and generally
show little or no differences in basal muscle protein synthesis between young and elderly
[162-167]. Similarly, although there are some reports showing the higher rate of basal protein
degradation in elderly as compared with the younger individuals [168], many studies have
confirmed that basal protein degradation in skeletal muscle is consistent to remain essentially
unchanged with advancing of age [157, 158, 160, 162, 167]. The reasons for these apparent
discrepancies are still unclear, but it is likely that differences in the health status, physical
activity levels and/or nutrient status may be attributed. Therefore, although it is still not
conclusive, there has been a general agreement in the current research field that basal net
balance of muscle protein metabolism (both in protein synthesis and protein degradation) is
likely not compromised with advancing of age in healthy individuals.

Cellular Mechanisms Regulating Protein Metabolism in Skeletal Muscle Cells

31

Age-Associated Anabolic Resistance in Skeletal Muscle

Although basal rates of protein synthesis are similar between age groups, skeletal muscle
in elderly individuals may have anabolic resistance, impairment in their ability to properly
respond to anabolic stimuli including resistance exercise, nutritional intake or growth factor
such as insulin compared to the young subjects [169]. It has been suggested that this blunted
response of muscle protein synthesis following anabolic stimuli is likely a key factor in
gradual loss of skeletal muscle mass with advancing of age.
Resistance Exercise
High-intensity resistance exercise is well established as a potent stimulus to enhance
muscle protein synthesis which then promoting skeletal muscle hypertrophy [157, 170-175].
Skeletal muscles of elderly individual are also capable of increases in protein synthesis and
subsequent muscle hypertrophy following resistance exercise, however, the enhancement of
muscle protein synthesis in response to those anabolic stimuli in elderly subjects is likely
blunted compared to the young individuals. It was reported that 16 weeks of resistance
exercise training at a relatively high intensity/volume induce increased muscle strength and
significant muscle fiber hypertrophy both in young and elderly, but the growth response was
significantly lower in elderly individuals compared to young [173]. This blunted adaptation to
resistance exercise in elderly may be caused by an inability of the exercise bout to accelerate
muscle protein synthesis. A single bout of resistance exercise accelerates protein synthesis
rates in skeletal muscle within a few hours (2-4 hrs.), and this increased protein synthesis
rates persists for up to 24-48 hrs. in young subjects [8, 172, 176, 177]. In contrast, recent
studies have shown that initial increase in protein synthesis rates following a single bout of
resistance is blunted in elderly [170, 171, 175, 178, 179]. Therefore, it has been suggested
that a reduced hypertrophic response in elderly people is likely caused by inadequate response
of muscle protein synthesis following acute bout of resistance exercise, thereby leading to a
blunted accrual of muscle proteins over time after repeated bouts of exercise.
The increase in muscle protein synthesis appears to be mediated primarily through
changes in protein translation. In particular, mTORC1-dependent signaling pathway has been
indicated as a major regulator of muscle protein synthesis and subsequent overall muscle fiber
size by controlling translation initiation and elongation [180]. A seminal study by Baar and
Esser [181] has demonstrated that acute mechanical loading of the skeletal muscle resulted in
the phosphorylation of S6K1, which is the direct downstream effector of mTORC1 signaling,
and the magnitude of the increased S6K1 phosphorylation was highly correlated with the
concomitant hypertrophic response of the muscle in rodents. The association between
contraction-induced increase in protein synthesis and activation of mTORC1-dependent
signaling in skeletal muscle has been further supported by the observation that rapamycin,
mTORC1-specific inhibitor, completely blocked the mechanical load-induced activation of
mTORC1 and subsequent hypertrophic responses both in rodents and human (increase in
muscle protein synthesis and muscle fiber hypertrophy) [13-16, 62, 182]. Age-associated
defect of muscle protein synthesis following acute bout of resistance exercise is likely
explained by the inadequate response of mTORC1 signaling in elderly. There have been
several studies that report age-associated decline in activation state of mTORC1-dependent
signaling following resistance exercise [90, 170, 173, 178, 179]. Kumar et al. have reported
an age-associated differential response to resistance exercise in elderly subjects that show

32

Mitsunori Miyazaki

blunted phosphorylation of two key downstream targets of mTORC1, S6K1 and 4EBP1 at 1
hr. after exercise [170]. Recently, Fry et al. have also confirmed that age-associated decline in
protein synthesis rates and blunted responses of mTORC1 signaling persist for at least 24 hrs.
following resistance exercise in elderly subjects [179]. These data clearly suggest that proper
control of mTORC1-dependent signaling in response to anabolic stimuli is critical key factor
for developing more effective therapeutic interventions to prevent the loss of muscle with
aging.

Amino Acids Supplementation

Availability of amino acids or proteins is well known to be a key nutrient factor for the
stimulation of muscle protein synthesis [183, 184]. Among the amino acids, the essential
amino acids (EAAs) are primarily responsible for the regulation of muscle protein synthesis
[185]. Furthermore, the branched-chain amino acid, particularly leucine, is recognized to have
a potential role in the regulation of muscle protein synthesis [186-188]. There have been
several reports that ingestion or infusion of amino acids potentially stimulates muscle protein
synthesis both in young and elderly subjects [167, 185, 189]. However, evidence has
indicated that there is an anabolic resistance in response to the availability of amino acids in
elderly compared to young subjects. Volpi et al. have reported that muscle protein anabolism
is blunted in healthy elderly subjects during the intake of amino acids due to an impaired
response of muscle protein synthesis [167]. Some follow-up studies have also confirmed the
anabolic resistance of skeletal muscles in elderly to a physiological dose of amino acids [163166]. This blunted response to amino acids availability in aged skeletal muscle is likely
associated with the dysregulation of mTORC1-dependent signaling [164, 190]. Eventually,
the use of high-protein diets alone to increase muscle mass and strength in elderly has been
mostly ineffective [191, 192].
As mentioned above, resistance exercise is well established as an excellent enhancer of
muscle protein synthesis both in young and elderly subjects. However, the aged skeletal
muscle potentially shows an impaired ability to respond to anabolic stimuli. To improve this
anabolic resistance in elderly subjects, nutritional supplementation combined with resistance
exercise program has been evaluated over the last couple of years. It has been well
documented in young subjects that muscle protein synthesis is enhanced when EAAs or
protein are ingested following resistance exercise [193-195]. The addition of carbohydrates to
EAAs solution augments insulin secretion in humans [167, 195, 196]. Protein synthesis rates
and mTORC1-dependent signaling are further synergistically enhanced by the ingestion of
leucine-enriched EAAs plus carbohydrates following a single bout of resistance exercise
[197, 198]. This additive effects of protein anabolic response to combined program of
resistance exercise and ingestion of carbohydrate with EAAs are likely achieved by the full
activation of each independent signal inputs toward mTORC1 regulation, including growth
factor (insulin)-dependent pathway, amino acids-dependent pathway and mechanical stressdependent pathway in skeletal muscle cells. Therefore, it is currently considered that
resistance exercise in combination with proper nutrients supplementation (EAAs and
carbohydrates) is an only proven and effective strategy to counteract to the age-associated
muscle wasting.

Cellular Mechanisms Regulating Protein Metabolism in Skeletal Muscle Cells

33

4. Muscle Protein Degradation during Aging and

Chronic Diseases - Inflammation and Cachexia
In several chronic diseases with high prevalence in elderly population such as cancer,
chronic heart failure or chronic obstructive pulmonary disease, skeletal muscle mass is lost
very rapidly and this pathological condition instigated by primary diseases is referred to as
cachexia. Acute loss of skeletal muscle mass is largely caused by the acceleration of
ubiquitin/proteasome-dependent muscle protein degradation. Although there is still no widely
accepted definition, growing evidence suggests that systemic effects of increased proinflammatory cytokines may play an important role in the pathology of cachexia. The
inflammatory cytokines that have been implicated in cachectic muscle wasting are including
TNF-, interleukin-1 beta (IL-1), interleukin-6 (IL-6), interferon-gamma (IFN-), or TNFlike weak promoter of apoptosis (TWEAK) [199-202]. In patients exhibiting inflammation
diseases, elevated levels of circulating pro-inflammatory cytokines is a hallmark of cachexia,
and directly contribute to a negative balance of protein metabolism [200]. There have been
several studies which showing that pro-inflammatory cytokines, particularly TNF- can
enhance protein degradation in skeletal muscle through modulating the ubiquitin-proteasomedependent system [135, 203, 204]. It has been suggested that expression of muscle specific
E3-ubiquitin ligase Atrogin-1 is upregulated by TNF- via a p38 MAPK-dependent
mechanism [138]. Expression of another muscle specific E3-ubiquitin ligase MuRF1 is also
enhanced by pro-inflammatory cytokines through modulating NFB or FOXO4 signaling
pathway [135, 205, 206]. Importantly, increased level of pro-inflammatory cytokines and
expression of E3-ubiquitin ligases correlates with protein degradation and skeletal muscle
wasting in patients with cachexic conditions [207, 208]. These data have suggested that
ubiquitin/proteasome-dependent protein degradation may play a central role in rapid loss of
muscle mass during inflammatory diseases.

Chronic Low - Grade Inflammation and Sarcopenia

Number of epidemiological studies has indicated that levels of local or systemic
inflammatory cytokines, such as IL-6 or TNF-, are consistently increased with advancing of
age, even in apparently healthy individuals or in the absence of acute infection/diseases [209213]. This condition of chronic, low-grade inflammation in elderly population has been
termed as inflamm-aging, and implicated as a possible contributor to age-associated muscle
loss [214]. In the Health, Aging, and Body Composition (Health ABC) Study that is
community population-based American cohort study, higher plasma concentrations of IL-6
and TNF- were generally associated with lower muscle mass and lower muscle strength in
well-functioning older population [215]. Follow-up study also indicated that TNF- and its
soluble receptors were highly associated with 5-year decline in muscle mass and strength
[216]. Similarly, it was found in the cohort of Framingham Heart Study that increased level of
inflammatory cytokines in elderly subjects, particularly IL-6, was a significant predictor of
sarcopenia [217, 218]. In a cohort of Italian older subjects (InCHIANTI Study), higher levels
of IL-6 or C reactive protein (CRP) were associated with the poor physical performance and
muscle strength [209, 219, 220]. Based on these epidemiological data, chronic low-grade

34

Mitsunori Miyazaki

inflammation in elderly population has been implicated as a possible contributor responsible

for the age-associated muscle loss [3, 6, 214, 221]. Whereas there is a clear association
between inflammation status and gradual muscle loss in elderly, however, the practical effects
of low-grade chronic inflammation on muscle catabolism (particularly muscle protein
degradation) is still under debate. Importantly, while significant muscle wasting occurs with
advancing of age, loss of muscle mass during aging in non-frail healthy individual is not as
severe and acute as that seen in frail subjects with chronic diseases. In addition, increased
levels of inflammatory cytokines in healthy elderly are much smaller than that seen during
disease conditions. Indeed, several studies have found no changes in expression level of
atrogenes, either Atrogin1 or MuRF1, in skeletal muscles from elderly subjects [222-225].
Furthermore, it has also been confirmed that basal protein degradation in skeletal muscle is
consistently unchanged with advancing of age [157, 158, 160, 162, 167]. Thus, although it
still remains controversial, the ubiquitin-proteasome dependent protein degradation is likely
not playing a fundamental role in age-associated gradual muscle loss in non-frail healthy
individuals.

Conclusion
Significant progresses have been made over a past decade to identify some of the
potential mechanisms contributing to the development of sarcopenia. In elderly individuals,
impaired balance of protein metabolism has been indicated as a critical factor which
contributing age-associated muscle loss. Although basal rates of protein synthesis and protein
degradation are essentially unchanged with advancing of age, skeletal muscle in elderly
individuals may have an impaired ability to properly respond to anabolic stimuli. This
imbalance of protein metabolism results in a minor and daily muscle protein loss, which leads
to the long-term gradual muscle wasting in the elderly. At this time, only proven and effective
intervention to counteract to this age-associated anabolic resistance and the subsequent
muscle loss is the resistance exercise in combination with adequate nutrients supplementation.
However, considering the multifactorial nature of muscle loss and weakness in elderly
populations such as potential disease status or sedentary life style, more comprehensive
interventions will be necessary. It is quite possible that precise understanding of cellular
mechanisms that govern protein metabolism in skeletal muscle will develop new therapeutic
strategies for preventing physical disability and increased risk of morbidity/mortality due to
the loss of muscle mass with aging.

Acknowledgments
This work was supported by the JSPS KAKENHI Grant Number 24800056 and the
research grant provided from The Uehara Memorial Foundation.

Cellular Mechanisms Regulating Protein Metabolism in Skeletal Muscle Cells

35

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In: Basic Biology and Current Understanding of Skeletal Muscle ISBN: 978-1-62808-367-5
Editor: Kunihiro Sakuma
2013 Nova Science Publishers, Inc.

Chapter 3

Biological Actions of Insulin-Like

Growth Factor-I (IGF-I) Isoforms and
IGF Binding Proteins in Skeletal Muscle
Akihiko Yamaguchi1, Kunihiro Sakuma2 and Isao Morita3
1

School of Rehabilitation Sciences, Health Sciences University of Hokkaido,

Ishikari-Tobetsu, Hokkaido, Japan
2
Health Science Center, Toyohashi University of Technology, Toyohashi, Aichi, Japan
3
School of Nursing and Social Services, Health Sciences University of Hokkaido,
Ishikari-Tobetsu, Hokkaido, Japan

Abstract
Insulin-like growth factor-I (IGF-I) is an important growth factor mediating cell
proliferation, differentiation and cell survival in skeletal muscles. In humans, there are
three types of IGF-I isoforms derived from the differential E domain, called IGF-I Ea, Eb
and Ec. IGF-I Ec is also called mechano growth factor (MGF), because of the marked
upregulation in exercised and damaged muscles. In rodents, IGF-I isoforms are composed
of two types of IGF-I, IGF-I Ea and MGF. IGF-I isoforms serve as the IGF-I precursor
peptides. IGF-I isoforms have multiple transcriptional initiation sites and derived from
some alternative splicing. After post-translation modification, the IGF-I precursor
peptides are grown into a mature IGF-I. IGF-I isoforms are produced by various tissues,
including liver, cartilage and skeletal muscle, and act through endocrine and
autocrine/paracrine pathways. E peptides derived from the IGF-I isoforms are likely to
have different growth-promoting effects on skeletal muscles.
IGF binding protein (IGFBP) family is composed of six different members, which
are IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6. IGFBPs are
characteristic of high affinity for IGFs binding, whereas IGFBP related proteins with the
low affinity binding are also found and distinguished from IGFBPs. Most of the
circulating IGF-I exists in a large tripartite complex with IGFBP-3 and the acid labile
subunit (ALS). IGF-I also exists in binary or ternary complexes with another member of
the IGFBP family. IGF-I is removed from the complexes, and free IGF-I acts on muscle
growth via the IGF-I receptors. IGFBPs have stimulating and inhibitory effects

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Akihiko Yamaguchi, Kunihiro Sakuma and Isao Morita

depending on the IGF-I as well as IGF-independent actions. The same IGFBP can act to
promote or suppress IGF actions in association with posttranslational modification, such
as proteolytic cleavage, dephosphorylation. Gene expressions and their functions of IGF-I
isoforms and IGFBPs in skeletal muscle are discussed in the present review.

Introduction
Skeletal muscle results from morphological and biochemical changes in response to
different environments and stimuli. Skeletal muscle is also influenced by hormonal
conditions, such as growth hormone (GH) and testosterone, and mechanical loading, such as
exercise, and results from the changes of muscle mass and protein synthesis. Insulin-like
growth factor-I (IGF-I) is one of the most important growth-promoting factors mediating
numerous gene expressions in the skeletal muscles. Originally, somatic growth is thought to
be controlled by pituitary GH and its action was mediated by circulating IGF-I, which is
mostly derived from the liver. It has been well-established as GH/IGF-I axis. The GH/IGF-I
axis is important for normal growth, and the disruption leads to the retardation of body and
muscle growth. Most of the circulating IGF-I exists in a large tripartite complex with IGF
binding protein (IGFBP)-3 and the acid labile subunit (ALS) [1]. IGF-I also exists in binary
or ternary complexes with another member of the IGFBP family [1]. IGF-I is removed from
the complexes, and the free IGF-I exerts various biological functions via the IGF-I receptors.
The large complex of IGF-I in circulation cannot cross the vascular endothelium unless this
complex is broken down and therefore acts to regulate the endocrine actions of IGF-I. In
contrast, the remaining IGF/IGFBP complex is easily cross the vascular endothelium and is
thought to be locally bioavailable to the target tissues [1]. Thus, it is thought that IGFBPs
function as carrier proteins for circulating IGFs and regulate IGF turnover, transport, and
tissue distribution [2].
IGF-I gene expression in the liver is tightly regulated by GH, whereas IGF-I expression
in skeletal muscle is regulated by mechanical stimuli in addition to GH [3]. Enhanced IGF-I
expression in skeletal muscle induces various growth-promoting actions, such as cell
proliferation and differentiation [4, 5]. Local IGF-I expression in response to mechanical
overloading is thought to be a most important factor for regulating muscle hypertrophy. The
multiplicity of growth-promoting actions of IGF-I is related to the diversity of intracellular
signaling, and proceeds on to the differential cascades [4, 5]. The multiplicity is also related
to a variety of IGF-I isoforms and IGFBPs expressed in the skeletal muscle. Several studies
have shown that IGF-I isoforms are expressed at the point of differential timing in response to
exercise and injury [6, 7]. IGFBPs are also expressed individually in response to various
stimuli [8, 9, 10]. The differential expressions and distinct roles of IGF-I isoforms and
IGFBPs in skeletal muscle would be discussed in the present review.

IGF-I Isoforms
Human insulin-like growth factor-I (IGF-I) gene originally called somatomedin C is
located within a region of the long arm of chromosome 12 [11]. It contains two promoters and
six exons, and yields multiple and heterogeneous mRNA transcripts. The multiple mRNA

Biological Actions of Insulin-Like Growth Factor-I (IGF-I) Isoforms

55

transcripts result from multiple transcriptional initiation sites, alternative splicing and
different polyadenylation signals [12]. The IGF-I mRNA transcripts receive post-translation
modification and grow a mature IGF-I of 70-amino acid peptide.
IGF-I gene contains two promoters initiating at alternate 5' exons, exon 1 or 2. Promoter
1 initiates at exon 1 and produces IGF-I transcripts called class 1. Promoter 2 initiating at
exon 2 results from class 2 IGF-I transcripts. The transcripts of class 2 are predominantly
expressed in the liver, and thought to be highly responsive to a GH [13]. In contrast,
transcripts of class 1 are widely expressed in all tissues. Each promoter initiating at exon 1
and 2 is spliced to the common exon 3 and 4, and also spliced to exon 5 and/or 6 according to
various combinations (Figure 1).
Exon 3 and 4 of IGF-I gene are common to all transcripts and encode to the mature IGF-I
peptide. First 16-amino acids common to all E peptide of IGF-I are encoded by exon 4. The
remainder of E peptide is encoded by exon 5 and/or 6. Alternative splicing of IGF-I gene at
exon 5 and 6 yields different IGF-I isoforms. The isoforms, which contain exon 5, are
classified as IGF-I Eb and those of exon 6 are classified as IGF-I Ea. Transcripts of IGF-I Eb
are thought to be abundant in the liver, whereas transcripts of IGF-I Ea are predominantly
expressed in extra-hepatic tissues [14]. In the human skeletal muscle, as well as other tissues,
a third alternative splicing variant is also produced. The transcript is named IGF-I Ec, which
is alternatively called mechano-growth factors (MGF), and contains both exon 5 and 6 [15].
As IGF-I Ec transcript contains only the first 49 base pairs from exon 5, it leads to a reading
frame shift of carboxyl terminal ends during the translation process. Thus, IGF-I Ea, IGF-I Eb
and IGF-I Ec mRNA transcripts are composed of exons 3-4-6, exons 3-4-5 and exons 3-4-5
(49 bp insert)-6, respectively (Figure 1). IGF-I Eb isoform in humans has been identified in
the liver [18] and the skeletal muscle [19], however, its role still remains unclear.

Figure 1. IGF-I isoforms in humans. IGF-I gene consists of six exons including two promoters. The
isoforms of mRNA and precursor peptides are produce by the combination of alternate promoters and
different E domains. A mature IGF-I peptide is composed of B, C, A, and D domains, which are
derived from exon 3 and 4. There are three different E peptides in humans, called Ea, Eb and Ec (MGF)
peptides. The Ea, Eb and Ec peptides in humans are derived from exons 4-6, exons 4-5 and exons 4-56, respectively. The translated parts into proteins are described as the gray and black boxes. Numbers
described in parenthesis represent the numbers of amino acid in proteins.

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Akihiko Yamaguchi, Kunihiro Sakuma and Isao Morita

IGF-I mRNA transcripts encode several IGF-I precursor peptides. The IGF-I precursor
peptides differ in the length of the amino-terminal (N-terminal) ends and the structure of
carboxyl-terminal (C-terminal) ends, called the E-domain. There are two types of IGF-I
precursor peptides derived from different promoters, called class 1 IGF-I and class 2 IGF-I.
There are three types of IGF-I precursor proteins derived from differential E domains, called
IGF-I Ea, IGF-I Eb and IGF-I Ec in humans. Taken together, the number of IGF-I mRNAs
and precursor peptides are expected to consist of six types of the isoforms. To date, four types
of splicing variants have been identified (Figure 1). The E-domains in the different IGF-I
precursor peptides share only 50% similarity [20, 21].
A mature IGF-I polypeptide is composed of a B-, C-, A- and D-domains. The E-domain
of mature IGF-I is trimmed off the precursor peptides through post-translational modification
by proteolytic cleavage. The mature IGF-I has a 70-amino acid long single chain polypeptide
and a 7,649 Da of the molecular weight [22]. The identical mature IGF-I peptide results from
all precursor peptides via post-translational modification (Figure 1).
The transcriptional and post-transcriptional regulation, alternative splicing, and posttranslational modification of IGF-I gene have given rise to several IGF-I precursor peptides,
and mature IGF-I and E peptide. The different E-peptides are thought to be removed from the
precursor IGF-I intracellularly [23]. Several studies have shown that these peptides have their
multiple biological roles [24, 25]. The different E-peptides have been shown to modulate IGF
actions and exert IGF-independent bioactivities [25, 26, 27].

IGF Binding Proteins

IGF-I in body fluid is normally bound to a variety of binding protein, called IGF binding
proteins (IGFBPs). IGFBPs are composed of six different members, which are IGFBP-1,
IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6 [2, 28]. Many kinds of tissues express
more than one IGFBP [29, 30, 31]. Muscle cells are known to produce and secrete several
IGFBPs [10, 29, 32].
IGFBPs circulate in the free form or in the complexes with IGFs. The free and binary
complexes are thought to exist in the vascular and extracellular compartment, whereas the
ternary complexes essentially confine in the vascular compartment. IGFBPs are thought to
function as carrier proteins for circulating IGFs and regulate IGF turnover, transport, and
tissue distribution [2].
It is characteristic of IGFBPs that the binding for IGF-I is equal or has higher affinities
than those of IGF receptors. IGFBPs are composed of N-terminal domain, C-terminal
domain, and the central linker domain (L-domain) separated by the N-terminal and Cterminal domains. The N-terminal and C-terminal domains are cysteine rich, and share high
similarity in their amino acid sequences. The L-domain is little similarity among IGFBPs.
The N-terminal domains of IGFBPs contain 80-93 amino acid residues after the signal
peptides, and share approximately 58% similarity. Ten to 12 of the 16-20 cysteines found in
IGFBPs are located in N-terminal domain (Figure 2). These cysteines in IGFBPs are
significantly conserved. Within the N-terminal domain, a local motif of GCGCCxxC is highly
conserved among IGFBPs, with the exception in IGFBP-6. GCGCCxxC motif can be found
in several other related proteins, however, the function remains unclear at present [33].

Biological Actions of Insulin-Like Growth Factor-I (IGF-I) Isoforms

57

Figure 2. The structure of IGFBPs and biological actions of their domains. IGFBPs are composed of Nterminal domain, L-domain and C-terminal domain. The N-terminal and C-terminal domains are
cysteine rich, and sharing high similarity in their amino acid sequences. The L-domain is poorly
conserved and contains a number of proteolysis, glycosylation and phosphorylation sites. The Cterminal domain contains heparin-binding motifs, which are associated with the binding to cell surfaces
and the extracellular matrix, nuclear localization sequence, integrin binding sequence as well as IGF
binding site. IGF-independent actions are also associated with the C-terminal domain. Arrowhead
indicates cysteine residue.

The L-domains of IGFBPs contain 55-94 amino acid residues, and share less than 15%
similarity. It is thought that this region structurally acts as a hinge between the N-terminal and
C-terminal domains. The L-domain contains a number of proteolysis, glycosylation and
phosphorylation sites. All IGFBPs can be cleaved by specific protease. The IGF binding
activities in IGFBPs are reduced by the proteolytic cleavages. IGFBP-3 and IGFBP-4 are Nglycosylated, and IGFBP-5 and IGFBP-6 are O-glycosylated [34]. The glycosylation of
IGFBPs influences the resistance to proteolysis of IGFBPs [35], and makes the molecule less
susceptible to proteolysis [36]. Potential phosphorylation sites are found in IGFBPs, only
phosphoisoforms of IGFBP-1, IGFBP-3 and IGFBP-5 have been reported so far [37].
Phosphorylation of IGFBP-1 has been shown to increase IGF binding by at least 5-fold [38].
Phosphorylated IGFBP-1 inhibits IGF-I actions, whereas the dephosphorylated IGFBP-1
appears to enhance IGF-I-induced DNA synthesis [39, 40]. Thus, the post-translational
modification in the L-domain would alter the property of IGFBPs to modulate IGF
bioactivity.
The C-terminal domains of IGFBPs, like the N-terminal domains, are highly conserved,
and share approximately 34% similarity. The C-terminal domain contains six cysteines of the
total 16-20 cysteines in IGFBPs (Figure 2). The amino acid residues including the last 5
cysteines in the C-terminal domain share 37% similarity with the thyroglobulin-type-I domain
[41]. The thyroglobulin-type-I domain is composed of about 65 amino acid residues, found in
a number of proteins with various physiological functions in different organisms [41]. In
IGFBPs, the C-terminal domain is involved in the bindings to IGFs, cell surfaces,
extracellular matrix (ECM) and other ligands. The C-terminal domains of IGFBP-3, IGFBP-5
and IGFBP-6 contain heparin-binding motifs, which are associated with the binding to cell

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Akihiko Yamaguchi, Kunihiro Sakuma and Isao Morita

surfaces and ECM [42, 43, 44, 45]. The C-terminal domains of IGFBP-3 and IGFBP-5 also
have a functional nuclear localization sequence (NLS) and demonstrate localization in the
nucleus [46]. The C-terminal domains of IGFBP-1 and IGFBP-2 have an arginie-glycineglutamate (RGD) motif, which are involved in binding to integrins, located on the cell surface
[47]. The C-terminal domains of IGFBP-3 and IGFBP-5 have a nuclear localization sequence
(NLS) [48].

IGF Binding of IGFBPs

IGFBPs are encoded by four exons, with the exception of IGFBP-3 including an
untranslated exon 5. Exon 1 encodes for the N-terminal domain, 5'-untranslated region and a
few amino acid residues of L-domain in IGFBPs. The Exon 2 exclusively encodes for the Ldomain. The C-terminal domain in IGFBPs is encoded within exon 3 and exon 4.
It is thought that IGF affinity is related to the N-terminal domain in IGFBPs. Limited
proteolysis of human IGFBP-3 with serine protease prostate specific antigen [49, 50] or with
plasmin [51] caused a small fragment corresponding to N-terminal domain and part of the Ldomain, and the IGF affinities of the fragments were weak or not detectable. Proteolytic
IGFBP-4 fragment corresponding to N-terminal domain and a small portion of the L-domain
also had lower IGF-I and IGF-II affinity in comparison with intact IGFBP-4 [52].
In the N-terminal domain, it is thought that the portion corresponding to the last 4
cysteines are important for interaction with IGFs. In a human IGFBP-4 mutation study, Qin et
al. [53] have reported that the deletion of Leu72-Ser91 in the N-terminal domain is
undetectable for IGF-II binding by Western ligand blot analysis. In addition, they have shown
that a structure disruption generated by the point mutation from His74 to Pro74 reduces IGF-II
affinity by 50-fold as compared with the full length IGFBP-4 [53]. In a point mutation study
using bovine IGFBP-2, it has been reported that the substitution of Tyr60 to Ala60 or Phe60 in
the N-terminal domain, which is the residue corresponding to Tyr65 of human IGFBP-2,
reduces the affinities for IGF-I and for IGF-II [54, 55]. Mutations of the adjacent residues did
not reduce the affinity. From these results, they have asserted that Tyr65 in human IGFBP-2 is
probably one of the contact points of IGFs. By NMR studies of a recombinant IGFBP-5
fragment, Kalus et al. [56] have shown that Tyr50, corresponding to Tyr65 of human IGFBP-2
interacts with IGF-II as well as the residues of Val49, Pro62 and Lys68-Leu74. Imai et al. [57]
have confirmed by a mutagenesis study that the residues corresponding to 68-70 and 73-74 in
IGFBP-5 and the homologous residues in IGFBP-3 are important for IGF binding.
It is thought that the C-terminal domain in IGFBPs is also related to IGF affinity.
Removals of most or all of C-terminal domain in human IGFBP-3 [58], IGFBP-4 [53], or
IGFBP-5 [59] disrupt IGF binding. In the C-terminal domain of human IGFBP-1, Brinkman
et al. [60] showed that the deletion of the 20 residues and the substitution of Cys226 to Tyr226
abolished IGF binding. Bramani et al [61] have reported that specific mutagenesis of Gly203
or Gln209 in rat IGFBP5 reduces IGF-I binding affinity by 8-fold and 6-fold, respectively.
Allan et al. [62] have shown that cumulative mutagenesis of the basic amino acids in the 201218 region of IGFBP-5 lead to a progressive loss of IGF-I binding. In IGFBP-4, Qin et al.
[53] have reported that the residues of Cys205-Val214 are important for IGF binding. In
IGFBP-2, fragments containing residues of 148-270, 169-289 or 181-289 in the C-terminal

Biological Actions of Insulin-Like Growth Factor-I (IGF-I) Isoforms

59

domain have been shown to be related to IGF binding [63, 64]. Forbes et al. [65] has reported
that Lys222-Asn236 in bovine IGFBP-2, which is corresponding to those of 224-238 of human
IGFBP-2, was important for IGF binding, and speculated that these residues must lie in close
proximity to the N-terminal domain to allow both domains to interact with IGFs. Kibbey et al
[66] have confirmed that upstream and downstream regions contained the residues of 247-250
in the C-terminal domain of human IGFBP-2 participate in IGF-I binging. Thus, it is thought
that the C-terminal domain of IGFBP-2 plays a key role in IGF binding and inhibition of IGF
binding to the IGF-IR [67].
Some studies have been reported that both the N-terminal and C-terminal domains need
for high affinity of IGF binding in IGFBP-2 [68] and IGFBP-3 [69]. By using biosensor
analysis of IGFBP-2 fragments, Carrick et al. [68] have reported that the N-terminal domain
has a fast association component, and the C-terminal domain contributes to the stability of
IGF/IGFBPs complex, and that these domains must be combined to form one high affinity
binding site [68]. In IGFBP-3, Payet et al. [69] have shown that each fragment of N-terminal
or C-terminal domains has a low affinity binding site, but co-incubating the fragments of
these domains can form high affinity binding site for IGFs. In addition, the ternary complexes
containing the N-terminal and C-terminal fragments and IGF appear to show some binding to
ALS, but not able to bind with ALS in the binary complexes between N-terminal or Cterminal fragments and IGF [69]. Headey et al [70] have reported that the presence of both
the N-terminal and C-terminal domains in IGFBP-6 is insufficient to hold high affinity IGF
binding using the coincubation technique. They have discussed that linkage of the N-terminal
and C-terminal domains, such as an intact IGFBP, is necessary for high affinity IGF binding.
The L-domain of IGFBPs does not directly bind IGFs, but appears to modulate the IGF
binding by post-translational modification. It has been reported that phosphorylation in the Ldomain changes the property of IGFBPs to modulated IGF binding [71]. Its contribution to
IGF binding is likely related to its ability to promote a tertiary structure, which permits
optimal relationships between the N-terminal and C-terminal domains. In a chimeras study
constructed between IGFBP-3 and IGFBP-2, Hashimoto et al. [72] have shown that IGF-II
binding of IGFBP-3 does not change, when the C-terminal domain of IGFBP-3 is exchanged
for the C-terminal domain of IGFBP-2. In contrast, replacement of IGFBP-3 L-domain to the
IGFBP-2 L-domain reduces the relative affinity of the resultant chimera for IGF-II by 37%. It
is possible that the different L-domain of IGFBPs influences the properties of IGF binding of
specific IGFBP.

IGFBP Related Proteins

Cysteine rich protein groups with structural and functional similarities to the IGFBPs
exist. These protein groups are called IGFBP-rPs. IGFBP-rPs are known to be composed of
nine members, IGFBP-rP1 to IGFBP-rP9. The IGFBP-rPs have the N-terminal domain with
40-57 % similarities to those of IGFBPs, and scarcely have amino acid residues common to
IGFBPs in the other domains. In addition, IGFBP-rPs appear to have at least 100-fold lower
affinity for IGFs as compared with IGFBPs. IGFBP-rPs are distinguished from IGFBP family
by the weakness of the structural and functional similarities, and considered to be the IGFBP

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Akihiko Yamaguchi, Kunihiro Sakuma and Isao Morita

superfamily [33]. Consequently, the IGFBP superfamily can be classified into IGFBP family
proteins with high affinities for IGFs and IGFBP-rPs with low affinities.
IGFBP-rP1 is firstly named Mac 25 [73], and provisionally IGFBP-7 [74]. This protein is
similar to tumor adhesion factor (TAF) [75] or prostacyclin stimulating factor (PSF) [76].
Structurally, IGFBP-rP1 consists of the N-terminal domain, Kazal-type serine proteinase
inhibitor and immunoglobulin-like domain. The region of similarity to the IGFBPs is
confined to the N-terminal domain. The IGFBP-rP1 appears to have multiple roles including
the ability to bind IGFs and insulin. It has been reported that Mac mRNA expression is higher
in dividing mouse myoblasts than in undivided, undifferentiated myotubes [77]. It suggests
that IGFBP-rP1 may play a role in differentiation of muscles. IGFBP-rP1 appears to
specifically accumulate in new blood vessels of various human cancer tissues and in cultured
vascular endothelial cells [78], suggesting that IGFBP-rP1 may also be involved in the
formation of new capillary vessels. IGFBP-rP1 is also capable of stimulating and enhancing
IGF and insulin mediated fibroblast cell growth [79]. Thus, IGFBP-rP1 has diverse biological
roles.
CCN family proteins are a group of cysteine rich proteins, which is coined by Bork [80].
The nomenclature of CCN family is based on the initial letters of main members: Cystein-rich
61 (Cyr61, CCN1), Connective tissue growth factor (CTGF, CCN2), and Nephroblastoma
Overexpressed gene (Nov, CCN3). CCN family proteins are shown to contain a N-terminal
domain of similarity to those of IGFBPs, and consist of six members: CTGF, Nov, Cyr61,
WISP-2, WISP-1 and WISP-3. These proteins are alternatively named IGFBP-rP2, IGFBPrP3, IGFBP-rP4 IGFBP-rP7, IGFBP-rP8 and IGFBP-rP9, respectively. WISP-1, WISP-2 and
WISP-3 are regulated by Wnt-1, which is a glycosylated signaling protein critical in the
developmental process [81, 82]. Each protein of the CCN family is composed of four
domains: the N-terminal domain similar to IGFBPs, the Von Willebrand factor type C repeat
(VWC), the thrombospondin type I repeat, and the C-terminal domain with cysteine knot and
heparin binding sites. IGFBP-rP7 only has the first three conserved protein domains: the Nterminal domain, the VWC and the thrombospondin type I repeat, and lacks the last Cterminal domain unlike the other CCN family proteins. In general, it is thought that the
members of the CCN family have 1,000-fold lower affinity for IGF-I as compared with
IGFBP family [83].
It is now thought that CCN family proteins are not growth factors but matricellular
proteins that modify signaling of other molecules, in particular those associated with ECM
[84]. CCN family proteins appear to be involved to mitosis, adhesion, apoptosis, extracellular
matrix production, growth arrest and migration of multiple cell types. [84]. CCN family
proteins are induced by growth factors and cytokines such as transforming growth factor
(TGF)- and endothelin 1 and cellular stress such as hypoxia, and integrate communication
between ECM and the cell surface [85]. It has been reported that IGFBP-rP2 is selectively
upregulated by TGF- in fibroblast cells, which is a potent stimulator of fibroblast cell
proliferation [86]. Some of the biological effects of TGF- on fibroblast and endothelial cells
appear to be mediated by the upregulated IGFBP-rP2 [87, 88, 89, 90]. It has been known that
IGFBP-rP4 is associated with the ECM and cell surfaces, probably through its heparin
binding sites [91, 92]. IGFBP-rP4 promotes the adhesion of fibroblasts and epithelial cells,
enhances chemotaxis of fibroblasts, and enhances growth factor-stimulated DNA synthesis in
fibroblast and endothelial cells [92, 93]. It is thought that the interactions between integrins or

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61

heparin sulfate proteoglycans and CCN family proteins are important for the adhesive and
mitogenic functions [85, 94, 95].
IGFBP-rP5 is structurally composed of three domains: a N-terminal domain similar to
those of the IGFBPs, Kazal-type serine proteinase inhibitor and immunoglobulin-like domain,
and a large domain having similarity with bacterial serine protease of HtrA class [96].
IGFBP-rP5 is known to be serine protease specifically cleaving IGFBP-5 [83]. IGFBP-rP6 is
alternatively named an endothelial cell-specific molecule-1 (ESM-1) and endocan. Expression
of this molecule appears to be restricted to human lung tissue. IGFBP-rP6 is composed of two
potential protein domains: a N-terminal domain similar to those of the IGFBPs and a Cterminal domain that does not share the similarity with any known proteins. It has been
reported that IGFBP-rP6 is upregulated by cytokines, TNF, interleukin 1 [97], and growth
factors such as vascular endothelial growth factor-A (VEGF-A) [98].
The IGFBP-rPs are regulated by a variety of growth factors and cytokines, and involved
in various biological functions independent of IGF actions. As IGFBP-rPs have at 100-fold
lower affinity for IGFs as compared with IGFBPs, the modulation for IGF action may be
weak from the properties with the low affinity binding. The biological function of IGFBP-rPs
for IGF actions is obscure at present. The details of IGFBP superfamily and CCN family
proteins are discussed in other reviews [33, 84, 85].

Circulating and Local IGF-I

IGF-I is produced by various tissues, including the liver, cartilage and skeletal muscle. It
has been thought for a long time that GH controls the production and release of IGF-I in these
tissues, and regulates muscle growth via metabolic and anabolic actions of IGF. To date, it
has been widely known as GH/IGF-I axis. The GH/IGF-I axis is important for normal growth,
and the disruption leads to the retardation of body and muscle growth [99].
Mature IGF-I is mostly circulating in blood, which is mainly derived from the liver, but
also from skeletal muscle and adipose tissue [100]. The circulating IGF-I is thought to be a
marker of GH action in the liver, and act in an endocrine manner [101]. Serum IGF-I levels
are largely dependent on nutritional and GH status, and its levels are reduced in underfed and
hypophysectomized rats. The underfed and hypophysectomized rats showed a 50% and 80%
decrease in the serum IGF-I levels after 4 weeks, respectively, and also reduced the weights
of hind limb and jaw muscles [99]. Rats which suffered a polyclonal antiserum of GH had a
decrease of 80-90% in serum IGF-I levels, and substantially reduced muscle weights of hind
limbs [102]. Yaker et al. [103] have reported that 85-90% decreases of circulating IGF-I
levels and retardation of post-natal growth were caused by a double IGF-I gene inactivation
model of LID/ALSKO, which is a liver-specific IGF-I deficient gene and the ALS deleted
gene. Yaker et al. [103] assert that post-natal growth to some extent would be influenced by
endocrine IGF-I. Thus, it has been reported that circulating IGF-I has an important
physiological role in fetal and post-natal growth and development of this tissue [104].
Growth-promoting action of GH is likely to be primarily mediated by activation of IGF-I.
GH administration on IGF-I null mice failed to stimulate their growth, but the injection on
wild-type mice accelerated growth [105]. Transgenic MKR mice, which over-express a
dominant negative IGF-IR specifically in the skeletal muscle, showed significant lower levels

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Akihiko Yamaguchi, Kunihiro Sakuma and Isao Morita

of muscle mass at 3 weeks of age [106]. Although the MKR mice were treated with
recombinant human GH for 4weeks, MKR mice failed to exhibit the GH-induced increases of
muscle fibers and myogenic regulatory factors observed in wild-type mice. The proliferation
of satellite cells in MKR mice also failed to exhibit the effects of GH administration [106].
Thus, growth-promoting action of GH is mainly mediated by IGF-I action via IGF-IR.
Local IGF-I production in the skeletal muscle is regulated by GH-dependent and GHindependent stimuli and is thought to act in autocrine or paracrine manners. Several
investigators have reported that hypophysectomy induces a decreased expression of IGF-I
mRNA in the skeletal muscle [99, 107], and that GH treatment in hypophysectomized rats
cause the increases of IGF-I mRNA expression in skeletal muscle [108]. In contrast,
compensatory overload due to synergic ablation increases the expression of IGF-I mRNA in
hypophysectomized muscle as well as in the controls [108, 109]. IGF-I is composed of two
different isoforms, called IGF-I Ea and MGF, in rodent skeletal muscles. Lowe et al. [110]
have reported that the expressions of IGF-I Ea and MGF mRNAs in the kidney, lung, and
heart are coordinately increased by the administration of GH. Iida et al. [111] have shown that
exogenous GH administration to GH-deficient mice increased the expressions of IGF-I Ea
and MGF mRNAs in the skeletal muscle. Administration of recombinant human GH to
hypophysectomized rats caused a marked increase in both MGF and IGF-I Ea muscle mRNA
levels [112]. In addition, Yamaguchi et al. [113] have also shown that both IGF-I Ea and
MGF mRNAs exhibit an increased expression following compensatory overload independent
of the GH status. Thus, local IGF-I expressions are thought to be modulated through both
GH-dependent and GH-independent manners.
Different expression patterns of the IGF-I isoforms have been shown in overloaded or
damaged muscles. Owino et al. [17] have reported that MGF mRNA expression, but not IGFI Ea mRNA, is specifically stimulated by mechanical overloading. Cheema et al. [114] have
also reported that using an in vitro culture system, the distinct expression responses between
MGF and IGF Ea mRNAs are induced by mechanical signals, static load, ramp stretch or
cyclical stretches. In humans, Hameed et al. [16] have shown that a single bout of highresistance exercise in young subjects results in a significant increase in MGF mRNA, but not
in IGF-I Ea mRNA. Thus, MGF appear to be more susceptible to the mechanical stimuli than
IGF Ea.
Muscle hypertrophy is caused by mechanical overloading, but circulating IGF-I hardly
changes in spite of the increased expression of IGF-I in skeletal muscle [115]. Exogenous
administration of GH induces the increase of serum IGF-I, but does not stimulate muscle
hypertrophy in the absence of mechanical loading [116]. In the liver IGF-I deficient mouse
model, a severe deficiency of serum IGF-I did not prevent muscle hypertrophy in response to
resistance exercise [117]. Mechanical overloading to hypophysectomized muscles also
induced the increases of muscle fiber areas and IGF-I expression [115]. In addition, a greater
hypertrophied portion in a muscle following mechanical overloading has exhibited a larger
increase of muscle IGF-I expression [109]. Thus, exercise-induced localized muscle
hypertrophy is thought to be associated with locally produced IGF-I in the skeletal muscle.
In humans, GH levels start to increase 10 to 20 min after the onset of exercise. The GH
levels reach their peak at the end of the exercise, and remain elevated for up to 2h after
exercise [118, 119, 120]. The serum GH levels are increased according to the intensity of
exercise [121]. GH secretion correlates positively with duration of exercise, when the
intensity is maintained at a constant state [122]. Total IGF-I, IGFBP-3 and ALS in serum

Biological Actions of Insulin-Like Growth Factor-I (IGF-I) Isoforms

63

increase slightly during exercise, and free IGF-I does not change during and after exercise
[123]. The physiological meaning of these changes is unknown. It has been known that IGF-I
stimulates glucose transport as potently as insulin in addition to involving of skeletal muscle
remodeling. IGF-I deficiency results in an impaired insulin action in skeletal muscle [124].
Circulating IGF-I as well as locally produced IGF-I would also have an impact on these
metabolic effects.

Modulation of IGFs Action by IGFBPs

IGFs exert mitogenic activities by interacting with IGF receptors, which are located on
the cell surface. Two types of IGF receptors are known at present. Type-I IGF receptor (IGFIR) has been shown mainly to mediate the biological effects of IGF-I and IGF-II, including
cell growth, migration, and survival [125]. The IGF-IR is composed of two subunits and
two subunits. The subunit contains a cysteine rich ligand-binding site, and the subunit
has tyrosine kinase activity. The IGF-IR is homologous to the insulin receptor. Type-II IGF
receptor (IGF-IIR) exclusively binds to IGF-II. The IGF-IIR has been shown to interact with
G protein pathway, and serves as a receptor for mannose-6-phosphate-cotaining ligands
[126]. Recently it has been reported that IGF-II binding to the IGF-IIR activates ERK1/2
mitogen-activated protein (MAP) kinase cascade through a mechanism involving sphingosine
kinase (SK)-dependent transactivation of G protein-coupled sphingosine-1-phosphate (S1P)
receptors [127]. However, the role of the IGF-IIR in mediating IGF action is less defined.
The bioactivities of IGFs are modulated by the affinity of IGFBPs for IGFs as well as the
interaction between IGFs and the receptors. IGFBPs bind IGFs with affinities that are equal to
or greater than those of the IGF-IR, and function as carrier proteins of IGFs in the circulation.
Most IGF-I in circulation forms a large complex of 150 kDa, consisting of IGF, IGFBPs and
ALS. IGF-I cannot cross the vascular endothelium unless this complex is broken down. In
contrast, the remaining IGF/IGFBP complex forms a small complex of 40-50 kDa, and easily
cross the vascular endothelium [1]. Proteolysis of the 150 kDa and 50 kDa complexes by
protease releases bioavailable IGF-I in the circulation and local fluid, resulting that the
releasing IGF-I easily interact with IGF-IR. Dephosphorylation of IGFBPs and interaction of
IGFBPs with cellular surfaces and ECM reduce IGF-I binding affinity. The reduced affinity
of IGFBPs would be easy to interact IGF-I with IGF-IR. It is thought that these changes are
related to an inhibitory or stimulatory effect for the IGF actions [34].
Each IGFBP has distinct structural and biochemical properties. IGFBP-1 and IGFBP-2
have an RGD motif in the C-terminal domain. The RGD sequence mediates binding to
integrins, which is associated with cell motility. IGFBP-3 and IGFBP-5 form a ternary
complex with IGFs and ALS, and have a NLS in their sequences. IGFBP-3, IGFBP-5 and
IGFBP-6 have a heparin-binding motif, which is associated with the binding to cell surfaces
and ECM [42, 43, 44, 45]. IGFBP-3 and IGFBP-4 are N-glycosylated, and IGFBP-5 and
IGFBP-6 are O-glycosylated [34]. IGFBP-6 binds IGF-II with an affinity of 100--fold higher
than those for IGF-I, and predominantly inhibits IGF-II action [36, 128]. IGFBP-4 and
IGFBP-6 consistently inhibit IGF actions, whereas IGFBP-1, IGFBP-2, GFBP-3, and IGFBP5 inhibit or potentiate IGF actions, depending on various conditions such as culture
conditions, cell type and IGFBP dose [129, 130, 131, 132].

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Akihiko Yamaguchi, Kunihiro Sakuma and Isao Morita

Figure 3. Local IGF/IGFBPs complex, IGFBPs and IGF signaling in skeletal muscle. IGF/IGFBPs
complex is prompted to bind IGF-IR after post-translational modification of IGFBPs, including
proteolytic cleavage, de-phosphorylation, ECM binding, and interaction to cell surface. IGFBPs also
have cellular bioactivity through IGF-independent manner. IGF-I signaling processes three different
cascades, called MAP kinase, PI3-K/AKT, and calcineurin signaling pathways. IGF-I signaling in
skeletal muscle modulates the proliferation and differentiation as well as protein synthesis and
degradation through these different pathways, and result from muscle hypertrophy.

Either inhibitory or stimulatory effect of IGFBPs for IGF actions is influenced by the
specific cleavage of IGFBP protease (Figure 3). Parker et al. [133] have shown that a
calcium-dependent serine protease activated by IGFs specifically cleaves IGFBP-4 into
fragments with a low affinity for IGF-I, and that the IGFBP-4 fragments decreases the
inhibitory effects for IGF-I actions in comparison with intact IGFBP-4. In addition, a
protease-resistant mutation of IGFBP-4 has been shown to inhibit DNA synthesis, cell
migration, and muscle growth in response to IGFs [134, 135]. Proteolyzed IGFBP-5 could not
modulate IGF growth stimulation in cultured fibroblasts [136]. Protease resistant mutation of
IGFBP-5 inhibits IGF-I stimulated DNS and protein synthesis and migration of porcine
smooth muscle cells [137]. Proteolyzed IGFBP-3 fragments have also been shown to lose the
binding affinity for IGFs and diminish the inhibitory effects of IGFBP-3 [51, 138].
The modulation of IGFBPs for IGF actions is also dependent upon ECM association and
interactions with cell surface proteins (Figure 3). IGFBP-5 is known to contain ECM binding
sites, which are located on amino acid residues of Arg201-Arg218 [45]. IGFBP-5 interacts

Biological Actions of Insulin-Like Growth Factor-I (IGF-I) Isoforms

65

specifically with thrombospondin-1 and osteopontin, which are ECM proteins [139]. ECMbound IGFBP-5 has a several-fold decrease of IGF-I affinity [136]. Binding to ECM of
IGFBP-5 results from the increased interaction between IGF-I and IGF-IR, and potentiates
IGF actions. Disruption of the ECM binding sites in IGFBP-5 abolishes ECM binding, and
the potentiated IGF actions in smooth muscle [140].
As the affinity of IGFBP-1 for IGF-I is higher than that of IGF-I for the IGF-IR, IGFBP1 reduces free IGF-I levels and inhibits a signaling through IGF-IR. IGFBP-1 is secreted as a
phosphoprotein, and contains three serine phosphorylation sites in humans. The affinity of
phosphorylated IGFBP-1 for IGF-I is several-fold higher than that of a non-phosphorylated
one [71]. Some studies have shown that dephosphorylated IGFBP-1 enhances IGF-I-induced
DNA synthesis, but phosphorylated IGFBP-1 inhibits IGF-I actions [39, 40]. Phosphorylation
of IGFBP-3 has also been shown to reduce IGF binding [141, 142]. The phosphorylation of
IGFBP-3 inhibits cell surface interaction and proteolytic cleavage [142]. Thus, it is thought
that phosphorylation of IGFBPs is associated with the modulation of IGF binding (Figure 3).
Intriguingly, in rats, the phosphorylation of IGFBP-1 has been reported not to change the
affinity for IGF-I [143].

IGF-Independent Actions of IGFBPs

IGFBPs itself appear to have cellular bioactivity, apart from modulation of IGF actions.
The C-domains of IGFBP-1 and IGFBP-2 contain RGD motifs, which bind to 51 integrin.
Jones et al. [144] have reported that Chinese hamster ovary cells transfected with IGFBP-1
causes an increase in cell migration, and that the cell migration mediated through 51
integrin binding of IGFBP-1. The action has been shown to be IGF-I independent, as the cells
do not produce IGFs and exogenous IGFs administration could not increase the cell
migration.
Similarly, several studies have demonstrated that IGFBP-2 can act in an IGF-independent
manner, at least in part by an interaction with 51 integrin [145, 146] (Figure 3).
In mouse fibroblast cell line with a disrupted IGF-IR gene, Valentinis et al. [147] have
reported that the growth inhibition by IGFBP-3 does not involve IGF binding or the signaling
via IGF-IR. A 16 kDa IGFBP-3 fragment, which is devoid of affinity for IGFs, has been
shown to inhibit the mitogenic effects resulting from IGF-IR activation [148, 149]. Gill et al.
[150] have shown that IGFBP-3 enhances ceramide analogue-induced apoptosis in the IGFunresponsive breast cancer cell line, Hs578T.
In mutant osteoblasts that produce neither of the two IGFs, Miyakoshi et al. [151] have
shown that IGFBP-5 increases cell proliferation and alkaline phosphatase. In addition, they
have reported that local injection of IGFBP-5 to the parietal bone of IGF-I knockout mice
leads to the increases of bone formation markers comparable to those seen in wild-type mice.
In mouse myoblasts transfected by wild-type or non-IGF binding IGFBP-5 cDNAs, both
myoblasts increased cell survival and decreased apoptosis [152]. In overexpressing mice of
wild-type IGFBP-5 or mutant IGFBP-5 with negligible IGF binding affinity, IGFindependent actions of IGFBP-5 have been demonstrated in the liver, brain and skeletal
muscle during development [153]. Thus, several studies have demonstrated that IGFBPs have
cellular bioactivity through an IGF-independent manner.

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Intracellular Signaling in Association

with IGF-I and IGFBPs
IGF-I is a unique growth factor, as the signaling induces both proliferation and
differentiation via the IGF-IR. IGF-I peptide regulates muscle mass by several mechanisms,
stimulating proliferation, inducing differentiation, inhibiting protein degradation, and
stimulating protein synthesis [4, 5]. IGF-I induces phosphorylation of the receptor, and
subsequently processes three different cascades, myoblast proliferation through MAP kinase
signaling, phosphatidylinositol 3-kinase (PI3-K) and AKT (PI3-K/AKT) activation, and
calcineurin signaling pathways [5] (Figure 3).
First, IGF-I acts via the MAP kinase pathway. Inhibition of MAP kinase, but not PI3-K
or mammalian target of Rapamycin (mTOR), inhibits IGF-I-stimulated proliferation of rat
L6A1 myoblasts [154]. MAP kinase pathway activates cell cycle markers, such as cyclin D,
cdk4, c-fos, c-jun, and induces the proliferation of skeletal muscle. Secondly, IGF-I acts via
the PI3-K/AKT pathway, which appears to be critical for the regulation of growth promoting
action. Pharmacological inhibition of this pathway prevents up-regulation of muscle
hypertrophy [155]. PI3-K/AKT pathway activates the protein synthesis through the activation
of mTOR and the inhibition of glycogen synthase kinase 3 (GSK3) [156]. PI3-K/AKT
pathway inhibits the increases of non-phospholylated FOXO, a key transcription factor,
which is responsible for muscle degradation. [5]. PI3-K/AKT pathway also activates
expression of terminal muscle differentiation markers, such as p21, MyoD, and myogenin
[157]. Also AKT regulates apoptotic and anti-apoptotic proteins of the Bcl-2 family, and
enhances cell survival. The third pathway is that IGF-I acts via calcineurin-dependent
signaling. The inhibition of calcineurin completely blocked the growth of IGF-I-treated
myotubes in vitro [158]. Calcineurin activates the expression of GATA-binding protein-2 and
nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), which
are transcription factors [159]. Calcineurin is also associated with the activation of MyoD and
MEF2, and induces muscle differentiation [160, 161]. Thus, IGF-I signaling in the skeletal
muscle is thought to modulate the proliferation and differentiation as well as protein synthesis
and degradation through various kinds of pathways, and result from muscle hypertrophy
(Figure 3).
Several studies have demonstrated that IGFBP-1 has cellular bioactivity through an IGFindependent manner. RGD motifs of C-terminal domain of IGFBP-1 can associate with cell
surface integrins, mediating IGF-independent effects. Bioactivity of IGFBP-1 on cell
migration occurs via binding of its RGD motifs to 51 integrin, leading to activation of focal
adhesion kinase (FAK) and stimulation of MAP kinase pathway [162, 163]. FAK also
regulates the activities of AKT and GSK-3 [164]. (Figure 3)
IGF-independent action of IGFBP-2 has been shown to be associated with
dephosphorylation of FAK and p42/44 MAP kinase [145]. Phosphatase and tensin homolog
deleted on chromosome 10 (PTEN), which is a negative regulator of PI3-K and MAP kinase,
has been shown to be inhibited by binding of IGFBP-2 to 51 integrin in breast cancer cells
[165]. Several studies have shown evidence for the nuclear localization of IGFBP-5 [166] and
putative receptors of IGFBP-3 [167, 168]. In one study using mutant IGFBP-5 with negligible
IGF binding affinity, IGF-independent actions of IGFBP -5 have been shown to be associated
with AKT and p38 MAP kinase activations [153].

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IGF-I isoforms and Skeletal Muscle Growth

Satellite cells, normally quiescent myogenic precursor cells, play an important role in the
hypertrophic and regenerative growth of skeletal muscles [169]. Mechanical overloading
activates satellite cells and stimulates the expression of myogenic markers [170, 171, 172]. In
humans, skeletal muscles of power lifting athletes contain a higher number of satellite cells in
comparison with un-trained subjects [173]. Strength training for 90 days induces the increase
of satellite cell numbers [174]. In contrast, inactivation of satellite cells by using gamma
irradiation prevents muscle hypertrophy following mechanical overloading [175, 176].
Satellite cells are located between the basal lamina and the sarcolenma of a muscle fiber,
and anatomically can be discriminated from myonuclei. These cells fulfill the basic condition
of stem cells that can give rise to differentiated cell types, and that can maintain themselves
by self-renewal [177]. In response to various stimuli, such as overload or injury, satellite cells
are activated to enter the cell cycle, and then the activated satellite cells are proliferated, and
differentiated into myofibers. The activated satellite cells also fuse into pre-existing
myofibers, and are served as a new myonuclei. Most of muscle hypertrophies after
mechanical overloading and the repair after muscle damages are caused through a series of
these stages [169].
Activated satellite cells express myogenic regulatory factors and several cell cycle
markers. When quiescent satellite cells are activated to enter the cell cycle, the activated cells
express myf5 and MyoD. myf5 and MyoD are co-expressed during the proliferation phase
[178]. In addition, proliferating cell nuclear antigen (PCNA), a marker for cell proliferation,
is expressed in proliferating satellite cells [178]. Expression of myogenin initiated in the
activated satellite cells continues through fusion and differentiation [178]. Differentiating
satellite cells also express p21, one of the cyclin-dependent kinase inhibitors, which mediates
withdrawal from the cell cycle [179, 180].
The concept of myonuclear domain has been proposed because of a correlation between
the muscle fiber areas and myonuclei on power lifting athletes [173, 181]. Kadi et al. [181]
have proposed that the incorporation of satellite cells into preexisting fibers to maintain a
constant nuclear to cytoplasmic ratio is a fundamental mechanism for muscle fiber growth.
Petrella et al. [182] have shown that 16 weeks of strength training causes the differential
muscle hypertrophies among young and older men and women, and that young men with
superior hypertrophy had increased myonuclear numbers. Petrella et al. [183] have also
demonstrated that the subjects who showed robust muscle hypertrophy in response to 16 wk
of resistance training have a large pool of muscle satellite cells, and that the subjects exhibit
the greater increases of satellite pool and myonuclei after the training. Expansion of the
myonuclear domain may drive additional new nuclei into preexisting fibers to produce
adequate gene expression for muscle growth.
In vitro studies have demonstrated that IGF-I increases cell proliferation and induces a
differentiation process of satellite cells [184, 185]. In addition, IGF-I expression in the
skeletal muscle has been shown to extend the replicative life span of satellite cells [186].
Jacquemin et al. [187] have shown that IGF-I-induced hypertrophy can be triggered in the
absence of proliferation by recruiting reserved mononuclear cells. IGF-I acts to increase the
diameter of myotubes, and suppress protein degradation, increase amino acid uptake and
stimulate protein synthesis in muscle cells [156, 188, 189]. Barton-Davis et al. [190] have

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Akihiko Yamaguchi, Kunihiro Sakuma and Isao Morita

shown that IGF-I-induced muscle hypertrophy is only partially repressed by gamma

irradiation, which inactivates muscle satellite cells. The actions of IGF-I would not only
target satellite cells, but also target differentiated muscle cells.
In humans, a number of investigators have reported that the expressions of muscle IGF-I
isoforms are regulated differentially after a single bout of exercise [16, 191, 192, 193].
Psilander et al. [194] have shown that IGF-I Ea mRNA expression in the skeletal muscle is
down-regulated after a single bout of exercise. Hameed et al. [16] have found that MGF
mRNA in the skeletal muscle is up-regulated 2.5h after a single bout of resistance exercise,
but not in the case of IGF-I Ea. Bamman et al. [195] have also shown that MGF mRNA, but
not IGF-I Ea, in the skeletal muscle is up-regulated 24 h after a single bout of resistance
exercise. In contrast, several studies have reported that muscle IGF-I Ea mRNA expression
increases after a single bout of resistance exercise [7, 192, 193]. McKay et al. [7] have
showed that muscle MGF mRNA expression increased 24 h after acute resistance exercise,
and that the IGF-I Ea and Eb mRNA expression increased 72 h after the exercise.
Each IGF-I isoform in rodents has been shown to be differentially expressed in exercising
and damaged muscles. Owino et al. [17] have shown that MGF mRNA expression, but not
IGF-I Ea mRNA, is specifically stimulated by mechanical overloading. Hill and Goldspink
[6] have shown that the timing of IGF-I mRNA expression in response to mechanical and
pharmacological damages is different between IGF-I isoforms, and that the expression of
MGF mRNA precedes those of IGF-I Ea expression. In one study using an in vitro culture
system which involved mechanical signals, the expression of IGF-I Ea was upregulated by a
single ramp stretch in myoblasts and myotubes, but reduced by repeated cyclical stretches. In
contrast, MGF did not show constitutive expression in static culture, but was upregulated by
the ramp and repeated cyclic stretches [114]. Muscle IGF-I Ea and MGF expressions were
differentially regulated in response to recombinant GH administration in elderly subjects [19].
In rodents, muscle MGF mRNA in a GH-deficient state was increased by GH administration
more rapidly than IGF-I Ea, but not observed in a GH-sufficient state [111]. Taken together,
MGF and IGF-I Ea would have a different sensibility to various stimuli.
In humans, mRNA expression of myogenic regulatory factors is upregulated after a
single bout of exercise, and the relationships between myogenic regulatory factors and IGF-I
isoforms have been examined [192, 193, 195]. Bamman et al. [195] have reported that
myogenin mRNA is increased in relation to MGF and IGF-I Ea expression after a single bout
of exercise and after 16 wk of resistance training, but MyoD is not related to those
expressions. Haddad and Adams [192] have shown that MGF and IGF-I Ea mRNA
expression after a single and a double bout of exercise is associated with the increase of
myogenin mRNA. Recently, McKay et al. [7] have demonstrated that MGF mRNA is
expressed earlier than those of IGF-I Ea and IGF-I Eb after exercise-induced muscle damage,
and that the temporal expression of MGF is correlated with the increases of MyoD and
Myof5. Additionally, McKay et al. [7] have shown that the expression of IGF-I Ea and IGF-I
Eb are correlated with increased expressions of myogenin and myf6 [7]. In rodent muscles,
MGF mRNA expression following mechanical and pharmacological damages has been shown
to rapidly express and then decline within a few days, and precede that of MyoD [6]. In
contrast, the expression of IGF-I Ea mRNA following the damages is slowly upregulated and
later than that of MyoD [6]. Thus, it is thought that MGF would serve as an initial pulse
responsible for satellite cell activation, and that IGF-I Ea would serve as a main regulator for
acceleration of protein synthesis after muscle damages.

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In in vivo gene transfer and transgenic approaches, overexpression of IGF-I Ea has been
reported to promote the increase of muscle mass and strength in adult mice [196]. In the
combination of overexpression of IGF-I Ea and resistance training in rats, the hypertrophic
effects of IGF-I Ea have been demonstrated [104]. E peptides of IGF-I appear to have
biological activities. Pfeffer et al. [27] demonstrated that IGF-I Ea and Eb did not modulate
IGF-I secretion, but that they increased cell entry of IGF-I compared with the mature IGF-I
alone. Overexpressions of IGF-I Ea and Eb promote muscle hypertrophy, but the
overexpression of mature IGF-I fails to increase muscle mass [197]. When several
recombinant Ea peptides derived from rainbow trout are synthesized and administrated to
three types of cell lines, all Ea peptides have been shown to exert the mitogenic activities
[26]. The Eb peptide also has growth-promoting effects in human bronchial epithelial cells
[198]. Thus, it is thought that E peptides retain the function of growth-promoting factors.
In C2/C12 cell cultures treated with IGF-I Ea and MGF, Yang & Goldspink [25] have
demonstrated that IGF-I Ea-treated cells initiates the fusion of myoblasts to form myotubes,
and that MGF-treated cells show evidence of proliferation, but remains in the mononucleated
state. Mills et al. [199] have also reported that synthesized E peptide of MGF induced the
proliferation of human myogenic precursor cells and inhibit the differentiation. To compare
the actions of IGF-I isoforms, Barton [24] performed viral mediated delivery of IGF-I Ea and
MGF into the skeletal muscle of young and adult mice. The injection of IGF-I Ea in young
and adult mice produced the increases of muscle IGF-I protein and induced the increases of
muscle mass. In contrast, MGF injection increased the IGF-I protein and muscle mass in
young mice, but did not in adult mice. Shavlakadze et al. [200] have reported that the overexpression of IGF-I Ea in transgenic mice do not stimulate the early regeneration after whole
muscle grafts, suggesting that another IGF-I such as MGF may play a role in the early stages
of skeletal muscle regeneration. Taken together, IGF-I Ea and MGF would play differential
roles on muscle growth. IGF-IR on muscle fibers would be required for the hypertrophy,
regardless of which isoform is expressed. Barton et al. [197] have shown that overexpressions
of IGF-I Ea and Eb result in the increase of muscle mass in wild mice, but could not cause
hypertrophy after injection into the muscles of MKR mice, which lack functional IGF-IR. In
contrast, there are some evidences that the signaling of the E peptides is not mediated by the
IGF-IR [25, 197, 198, 199, 201]. Siegfried et al. [198] have reported that a monoclonal
antibody antagonist to the IGF-IR could not suppress the proliferating response induced by
IGF-I Eb. An antibody against IGF-IR abolished the proliferating and migrating effects of
IGF-I on myoblasts, but did not influence the mitogenic activities in E peptide of MGF [25,
199, 201]. Barton et al. [197] have shown that MGF, but not IGF-I Ea, acts through an IGFIR-independent pathway to cause increased MMP-13, a member of collagenase sub-family
and a potent degrading enzyme of the muscle extracellular matrix. Thus, the different actions
of E peptides may be modulated by the distinct receptor regulation. It remains unclear
whether specific receptors and intracellular signaling for each E peptide exists, or not.

IGFBPs and Skeletal Muscle Growth

The IGFBPs in skeletal muscle are expressed individually in response to various stimuli
[8, 9, 10]. IGFBP-4 mRNA expression is increased in overloading rodent skeletal muscle, but

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Akihiko Yamaguchi, Kunihiro Sakuma and Isao Morita

not in IGFBP-5 mRNA [8]. In contrast, IGFBP-5 mRNA expression is increased in unloaded
muscle but not in IGFBP-4 mRNA [8]. Awede et al. [202] have shown that IGFBP-4 and
IGFBP-5 mRNA is increased by clenbutenol-induced muscle hypertrophy. Denervation
results in upregulation of IGFBP-4 and IGFBP-5 transcripts, but there was no change in
IGFBP-6 [203]. In rat regenerating muscles, all members of the IGFBPs appear to be
upregulated after muscle damage [9, 10]. Jennische and Hall [9] have reported that IGFBP-3,
IGFBP-4, IGFBP-5 and IGFBP-6 mRNAs were upregulated in the regenerating skeletal
muscle. Yamaguchi et al. [10] have shown that all members of IGFBPs are upregulated with
different timings during the regenerating process after muscle damage. In humans, prolonged
exercise induced a marked increase of serum IGFBP-1 level, but did not change IGFBP-3
[204, 205]. Dall et al. [206] have reported that serum IGFBP-1 and IGFBP-6 levels increase
after submaximal rowing exercise, whereas IGFBP-2 and IGFBP-4 do not change after the
exercise. Schwarz et al. [207] have shown that serum IGFBP-3 is increased after low and high
intensity exercises.
It has been reported that overexpressions of IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-5
in transgenic mice lead to various growth retardations [208, 209]. In contrast, IGFBP-2 null
mice exhibit normal body weight gains during pre-natal and post-natal growth [210, 211].
IGFBP-5 deficient mice have also shown indistinguishable changes of body weights in
comparison with wild-type mice from birth through postnatal 42 days [212]. In IGFBP-2 null
mice, mRNA expression of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6
were significantly increased as compared with the control [211]. In IGFBP-5 deficient mice,
IGFBP-3 was upregulated according to the loss of IGFBP-5 [212]. Thus, null mutation of an
IGFBP induces the elevated expression of the other members. It is thought that the
expressions of IGFBPs in various conditions would be regulated in harmony with the other
members of the IGFBP family.
In skeletal muscle, it has been well known that IGFBP-4 has an inhibitory effect [213,
214]. In contrast, IGFBP-5 has either an inhibitory or stimulatory effect for IGF actions [215,
216]. In C2C12 myoblast culture system, IGFBP-5 expression is increased during myoblast
differentiation [203]. In the cultured myoblast, Knockdown of IGFBP-5 decreases the
expression of myogenin and impairs the myoblast differentiation [217]. In the IGFBP-5
overexpressing mice, whole body growth inhibition and retarded muscle development are
observed [209]. Further, IGFBP-5 has been shown to inhibit muscle differentiation by
blocking IGF actions in cultured myoblasts [152, 218]. Thus, the same IGFBP could act to
potentiate or inhibit IGF actions depending on various conditions such as culture conditions,
cell type and IGFBP dose [129, 130, 131, 132].
Bach et al. [219] have reported that IGF-II-induced proliferation and differentiation are
inhibited by IGFBP-6 in L6A1 rat myoblasts, and that the inhibition levels depend upon the
affinities of IGFBP-6. All IGFBPs can be cleaved by specific protease. The IGF binding
activities in IGFBPs are reduced by the cleavage. Dephosphorylation of IGFBPs and
interaction of IGFBPs with cellular surface and ECM also reduce IGF-I binding affinity. The
reduced affinity of IGFBPs would be easy to interact IGF-I with IGF-IR. It is thought that
these changes result from an inhibitory or stimulatory effect for the IGF actions [34]. In
humans, both increased [207] and unchanged [206] IGFBP-3 proteolytic activities have been
reported after exercise. Interestingly, Rosendal et al. [220] have reported that prolonged
physical training increases IGFBP-3 proteolysis in previously untrained individuals, but not
in trained individuals, indicating an association with the level of training in the individuals.

Biological Actions of Insulin-Like Growth Factor-I (IGF-I) Isoforms

71

Yamaguchi et al. [10] have showed that the expressions of specific IGFBPs are
associated with those of specific myogenic markers in regenerating muscles in vivo. The time
course expression of IGFBP-3 and IGFBP-2 mRNA was significantly correlated with those of
MyoD and PCNA. In contrast, the expression of IGFBP-1 and IGFBP-5 mRNAs was
significantly correlated with those of myogenin and p21. These suggest that IGFBP-2 and
IGFBP-3 are associated with the proliferating process in regenerating muscles and that
IGFBP-1 and IGFBP-5 are related to the differentiation process but not to proliferation. James
et al. [32] have reported that IGFBP-5 is expressed during myoblast differentiation in a
culture system. Ren et al. [217] have shown that knockdown of IGFBP-5 inhibits myogenic
differentiation in vitro. Despite the significant sequence homology among the six IGFBPs,
each IGFBP exhibit distinct structural and biochemical properties [48]. For example, IGFBP1 and IGFBP-2 have RGD sequences, which are associated with binding to integrins. IGFBP3 and GFBP-5 have a NLS in their sequence, and IGFBP-3, IGFBP-5 and IGFBP-6 have a
heparin-binding motif. These distinct characteristics of IGFBPs may support the various
biological effects of IGF-I.
It has been known that IGFBP-6 binds IGF-II with an affinity 100--fold higher than those
for IGF-I, and predominantly inhibits IGF-II action [36, 128]. Yamaguchi et al. [10] have
shown that the expression of IGFBP-2, IGFBP-3 and IGFBP-6 in IGFBP family is only
correlated with the expression of MGF in regenerating muscles. However, the relationships
between IGFBPs and IGF-I isoforms are still unknown.

Conclusion
Insulin-like growth factor-I (IGF-I) plays important roles for various developmental
processes in skeletal muscles. IGF-I is known as an inter-mediator regulated by GH. IGF-I in
skeletal muscle is regulated in a GH-independent manner as well as a GH-dependent manner.
IGF-I locally produced in skeletal muscle is thought to play an important role for muscle
growth in response to mechanical stimuli. Mechanical overloading increases IGF-I expression
in the skeletal muscle via a GH-independent manner. IGFBPs have a characteristically high
affinity for IGFs binding, and form binary or ternary complexes with IGFs. In the case when
IGF-I is removed from the complexes, then free IGF-I acts on muscle growth via the IGF-I
receptors. IGFBPs in skeletal muscle are expressed individually in response to various
stimuli.
IGFBPs have stimulating and inhibitory effects depending on the IGF-I as well as IGFindependent actions. IGFBPs can act to promote or suppress IGF actions in association with
the posttranslational modification, such as proteolytic cleavage and dephosphorylation. There
are three different cascades of IGF-I intracellular signaling, which are MAP kinase, PI3K/AKT, and calcineurin signaling pathways. Several IGF-I isoforms are produced by multiple
transcriptional initiation sites and alternative splicing, and developed a mature IGF-I after
post-translation modification. Each IGF-I isoform appears to play distinct roles for the
growth-promoting effects. Proliferation and differentiation in the skeletal muscle are thought
to be associated with the multiplicity of the IGF isoforms, the diversity and various
posttranslational modifications of IGFBPs, and the multiple and complicated intracellular
signaling.

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Akihiko Yamaguchi, Kunihiro Sakuma and Isao Morita

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In: Basic Biology and Current Understanding of Skeletal Muscle ISBN: 978-1-62808-367-5
Editor: Kunihiro Sakuma
2013 Nova Science Publishers, Inc.

Chapter 4

An Overview of the Therapeutic

Strategies for Preventing Sarcopenia
Kunihiro Sakuma1, and Akihiko Yamaguchi2
1

Research Center for Physical Fitness, Sports and Health,

Toyohashi University of Technology, Toyohashi, Japan
2
School of Dentistry, Health Sciences University of Hokkaido,
Kanazawa, Ishikari-Tobetsu, Hokkaido, Japan

Abstract
The world's elderly population is expanding rapidly, and we are now faced with the
significant challenge of maintaining or improving physical activity, independence, and
quality of life in the elderly. Sarcopenia, the age-related loss of skeletal muscle mass, is
characterized by a deterioration of muscle quantity and quality leading to a gradual
slowing of movement, a decline in strength and power, increased risk of fall-related
injury, and often, frailty. Since sarcopenia is largely attributed to various molecular
mediators affecting fiber size, mitochondrial homeostasis, and apoptosis, the mechanisms
responsible for these deleterious changes present numerous therapeutic targets for drug
discovery. Resistance training combined with amino acid-containing supplements is often
utilized to prevent age-related muscle wasting and weakness. In this chapter, we
summarize recent therapeutic strategies using supplemental, pharmacological, and
hormonal approach for counteracting sarcopenia. Treatment with ghrelin seems to be an
interesting approach for preventing sarcopenia in the near future. EPA and ursolic acid
seem to be effective as therapeutic aqgents, because they attenuate the degenerative
symptoms of muscular dystrophy and cachexic muscle. The activation of peroxisome
proliferator-activated receptor coactivator 1 (PGC-1) in skeletal muscle by exercise
and/or unknown supplementation would be an intriguing approach to attenuating
sarcopenia. In contrast, muscle loss with age may not be influenced positively by
treatment with a proteasome inhibitor or antioxidant.

Corresponding author: Kunihiro Sakuma, Ph.D., Research Center for Physical Fitness, Sports and Health,
Toyohashi University of Technology, 1-1 Hibarigaoka, Tenpaku-cho, Toyohashi, 441-8580, Japan. E-mail:
ksakuma@las.tut.ac.jp; Tel: 81-532-44-6630; Fax: 81-532-44-6947.

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Keywords: Sarcopenia, exercise, myostatin, ursolic acid, PGC-1, skeletal muscle

Abbreviations
ACE
ActRIIB
ALK
BCAA
CR
CrM
DHEA
DMD
eIF
EPA
FOXO
GH
IGF-I
IKK
IL
mTOR
MuRF1
NF-B
PGC-1
PI3-K
p70S6K
RDA
Rheb
ROS
TNF-
TSC

angiotensin-converting enzyme
activin receptor IIB
activin receptor-like kinase
branched chain amino acid
calorie restriction
creatine monohydrate
dehydroepiandrosterone
Duchenne muscular dystrophy
eukaryotic initiation factor
eicosapentaenoic acid
forkhead box O
growth hormone
insulin-like growth factor-I
inhibitor of B kinase
interleukin
mammalian target of rapamycin
muscle ring-finger protein 1
nuclear factor-kappaB
peroxisome proliferator-activated receptor coactivator 1
phosphatidylinositol 3-kinase
p70 ribosomal protein S6 kinase
recommended dietary allowance
Ras homolog enriched in brain
reactive oxidative species
tumor necrosis factor-
tuberous sclerosis complex

1. Introduction
Skeletal muscle contractions power human body movements and are essential for
maintaining stability. Skeletal muscle tissue accounts for almost half of the human body mass
and, in addition to its power-generating role, is a crucial factor in maintaining homeostasis.
Given its central role in human mobility and metabolic function, any deterioration in the
contractile, material, and metabolic properties of skeletal muscle has an extremely important
effect on human health. Aging is associated with a progressive decline of muscle mass,
quality, and strength, a condition known as sarcopenia [1]. Although this term is applied
clinically to denote loss of muscle mass, it is often used to describe both a set of cellular
processes (denervation, mitochondrial dysfunction, inflammatory and hormonal changes) and
a set of outcomes, such as decreased muscle strength, decreased mobility and function [2],

An Overview of the Therapeutic Strategies for Preventing Sarcopenia

89

increased fatigue, a greater risk of falls [3], and reduced energy needs [4]. In most countries,
there has been a rapid and continuing increase in life expectancy. By the year 2030, 20% of
the adult US population will be older than 65 years [5]. In the 27 member states of the EU,
the percentage of people aged 65 years and older will rise from 17.1 in 2008 to 25.4 in 2035
and to 30 in 2060 [6], therefore, age-related losses in skeletal muscle mass and function
present an extremely important current and future public health issue.
Lean muscle mass generally contributes up to ~50% of total body weight in young adults
but declines with aging to be 25% at 75-80 years old [7, 8]. Loss of muscle mass is typically
offset by gains in fat mass. Loss of muscle mass is most notable in the lower limb muscle
groups, with the cross-sectional area of the vastus lateralis being reduced by as much as 40%
between the age of 20 and 80 years old [9]. On a muscle fiber level, sarcopenia is
characterized by specific type II muscle fiber atrophy, fiber necrosis, and fiber-type grouping
[10-12]. In elderly men, Verdijk et al. [12] showed a reduction in type II muscle fiber satellite
cell content with aging. Although several investigators support such an age-related decrease
in the number of satellite cells [12-15], some reports [16, 17] indicate no such change. In
contrast, most studies point to an age-dependent reduction in muscle regenerative capacity
due to reduced satellite cell proliferation and differentiation.
Another morphologic aspect of sarcopenia is fat accumulation. In the US, it was
estimated that the prevalence of obesity in elderly Americans, aged 60 years and older, would
increase from 23.6% in 1990 to 37.4% in 2010 [18]. Obesity is associated with increased
morbidity and mortality, and there is unchallenged evidence that obesity increases the risk of
hypertension, dislipidemia, type 2 diabetes mellitus, and several cancers [19-21]. Although
the methods used to classify sarcopenic obesity vary [22-25], sarcopenic obesity has been
attributed as a major cause of disability in old age. Indeed, Bouchard et al. [24] demonstrated
that obesity contributed more to lower physical capacity than sarcopenia. Fat distribution
changes with age such that there is an increase in visceral fat, which is more marked in
women than in men. Also, fat is increasingly deposited in skeletal muscle [26, 27] and in the
liver. Higher visceral fat is a main determinant of impaired glucose tolerance in the elderly.
Increased intramuscular and intrahepatic fat contributes to impaired insulin action through
locally released adipokines and fat free fatty acids. Broadwin et al. [23] have illustrated that
an increased percentage of fat mass was associated with greater functional disability in older
adults.
Several possible mechanisms of age-related muscle atrophy have been described;
however, the precise contribution of each is unknown. Age-related muscle loss is a result of
reductions in the size and number of muscle fibers [28], possibly due to a multi-factoral
process that involves physical activity, nutritional intake, oxidative stress, and hormonal
changes [3, 29]. The specific contribution of each of these factors is unknown but there is
emerging evidence that the disruption of several positive regulators (Akt and serum response
factor) of muscle hypertrophy with age is an important feature in the progression of
sarcopenia [30-32]. In contrast, many investigators have failed to demonstrate an age-related
enhancement in levels of common negative regulators [Atrogin-1, myostatin, and calpain] in
senescent mammalian muscles [31, 32].
Resistance training combined with amino acid-containing supplements is an effective
candidate to prevent age-related muscle wasting and weakness [31-33]. In particular,
sarcopenia has been most attenuated by treatment with many essential amino acids plus high
leucine [31-33]. In addition, many researchers have focused on inhibiting myostatin to treat

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Kunihiro Sakuma and Akihiko Yamaguchi

various muscle disorders such as muscular dystrophy, cachexia, and sarcopenia [34, 35].
Furthermore, more recent studies have indicated the possible application of new supplements
to prevent muscle atrophy [36, 37]. This chapter aims to address strategies using exercise, and
hormonal, pharmacological, and supplemental approaches for inhibiting muscle wasting, in
particular sarcopenia. Figure 1 indicates a summary of therapeutic approach for sarcopenia.

2. Exercise
2.1. Resistance Training
One resistance exercise bout can, within 1 hour, increase muscle protein synthesis [38],
which can last up to 72 hour after exercise [39]. Resistance training has shown the most
promise among interventions aimed to decrease the effects of sarcopenia, as it enhances
strength, power, and mobility function and induces varing degrees of skeleteal muscle
hypertrophy [40-42]. For example, 12 weeks of whole-body resistance training resulted in an
increase in type II muscle fiber area in men aged 64-86 year [43] and 65-72 year [44, 45]. A
2-year longituidinal trial of resistance training found increases in leg press (32%) and military
press (90%) 1 repetition maximum and kneee extensor muscle cross-sectional area (9%) in
60-80 year old men and women [46]. The functional benefits of resistance training have been
evaluated in a large-scale trial of 72- to 98-year olds and frail nursing home residents, with
resistance training increasing muscle strength (113%), gait velocity (12%), stair-climbing
power (28%), and spontaneous physical activity [41]. In addition, 6 weeks of training for
elderly persons (68.45.4 years) improved their physical activity profile (6-min walk, 30second chair stand, chair sit and reach and back scratch) as well as muscle strength.

Figure 1. A summary of therapeutic approaches for sarcopenia.

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91

In the elderly, resistance training induces the muscle expression of insulin-like growth
factor-I (IGF-I) [47], myogenic regulatory factors [48], and interleukin (IL)-6 [49], which
contribute to muscular hypertrophy by regulating the activation, proliferation, and
differentiation of satellite cells. In addition, resistance training decreases oxidative DNA
damage and improves the electron transport chain function [50]. One bout of resistance
exercise for elders can enhance the rate of synthesis of muscle protein [51, 52]. However,
several studies using humans and rodents indicated a lower degree of activation in mitogenactivated protein kinase and Akt-mammalian target of rapamycin (mTOR) pathways after
muscle contraction or mechanical overload than occurs in young adults [51, 53, 54]. More
recently, Mathew et al. [55] indicated that one bout of resistance exercise elicited a similar
extent of activation in translational signaling [Akt, p70 ribosomal protein S6 kinase
(p70S6K), ribosomal protein S6, and 4E-binding protein 1] between young and old subjects.
In contrast, physical activity can affect muscle inflammation. Recent evidence shows that
chronic resistance physical training contributes to the control of locally-derived inflammation
through adaptations to repeated and acute increases in proinflammatory mRNA within muscle
[56]. Several studies [47, 57] have shown that excess intensive strength training for the
elderly impairs the effective gain of muscle strength and mass particularly in women.
Therefore, careful attention should be paid when determining the amount and frequency of
resistance training for the elderly.

2.2. Endurance Training

A previous study has shown that endurance exercise effectively stimulates mitochondrial
biogenesis and increases muscle oxidative capacity [58]. Chow et al. [59] investigated distinct
mechanisms underlying exercise-induced improvements in mitochondrial function in a
murine model of endurance training. Eight weeks of treadmill training (80% of peak O2
uptake, 5 days/week) augumented mitochondrial function, as reflected by increased
mitochondrial enzyme activities, maximal rate of adenosine triphosphate synthesis in isolated
mitochondria and whole body maximal O2 uptake. Exercise-induced increases in
mitochondrial function were associated with increased transcript levels of nuclear and
mitochondrial genes encoding mitochondrial proteins, mitochondrial DNA abundance, and
the expression of peroxisome proliferator-activated receptor coactivator (PGC-1) and
mitochondrial transcription factor A [59]. A key player controlling mitochondrial function is
PGC-1, a master regulator of mitochondrial biogenesis. Indeed, PGC-1 levels in skeletal
muscle decrease during aging [60]. In addition, oxygen availability for mitochondria is
decreased in sedentary older people, associated with reduced muscle capillarity, decreased
maximal blood flow, and disruption of the microvascular endothelium [61].
Very intriguingly, the expression of PGC-1, which is elicited by endurance training
[62], drives not only mitochondrial biogenesis and the establishment of oxidative myofibers,
but also vascularization [63, 64]. Indeed, a recent study demonstrated that transgenic
overexpression of PGC-1 in skeletal muscle improved sarcopenia and obesity associated
with aging in mice [65]. In skeletal muscle, PGC-1 can also prevent muscle wasting by
regulating autophagy [60] and stabilization of the neuromuscular junction program [66] in the
context of muscle atrophy during disease. The health-promoting effects of increased PGC-1
expression in skeletal muscle have been shown in different mouse models with affected

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Kunihiro Sakuma and Akihiko Yamaguchi

muscle such as Duchenne muscular dystrophy (DMD) [67], denervation-induced atrophy

[60], and mitochondrial myopathy [68]. Thus, the well-known sarcopenia-attenuating effects
of endurance training may be attributable to protection against mitochondrial disorders
(apoptosis, oxidative damage, etc) by the increased PGC-1 amount.

2.3. Combined Exercise

The American College of Sports Medicine recommends a multi-component training
exercise programme (strength, endurance, balance, flexibility) to improve and maintain
physical function in older adults [69]. Resistance exercise has been investigated as an
approach to counteract sarcopenia by stimulating protein synthesis and causing muscle
hypertrophy with increased muscle strength and with improved physical performance [70].
Endurance training improves aerobic capacity. Most of the studies had a multicomponent
program of 3 90-min sessions per week, consisting of 15 min of balance training, 15 min of
flexibility, 30 min of aerobic exercise and 30 min of high-intensity resistance training.
To study the impact of each exercise modality in more detail, Davidson et al. [71]
randomized 60- to 80-year-old obese subjects into 4 groups: a control group, a group that had
progressive resistance training, a group that performed aerobic exercise and a group that
combined progressive resistance training with aerobic exercise. After 6 months, body weight
decreased by 0.6 kg in the resistance, by 2.8 kg in the aerobic and by 2.3 kg in the combined
exercise group. Abdominal fat and visceral fat decreased and endurance capacity improved
significantly in the aerobic and combined exercise group. Skeletal muscle mass and muscle
strength increased in the resistance and combined exercise groups only. Insulin resistance
improved by 31 % in the aerobic group and by 45% in the combined exercise group, whereas
it did not change in the resistance training group. The fear of negative interference from
endurance and resistance training was not substantiated [72, 73]. Thus, the combination of
progressive resistance training and aerobic exercise is the optimal exercise strategy for
simultaneous improvement of insulin resistance and functional limitations in the elderly.
Aerobic exercise only is the second best choice, although exercise training in obese old
people is very hard to describe.

3. Supplemental Approach
3.1. Amino Acid Supplementation
Many Americans consume more than the recommended dietary allowance (RDA) of
protein; however, research shows that a significant number of elderly people do not meet the
estimated average requirement, let alone the RDA [74]; 32% to 41% of women and 22% to
38% of men aged 50 and older consume less than the RDA of protein [75]. Epidemiological
studies show that protein intake is positively associated with preservation of muscle mass. For
example, in a recent study, 38 healthy, normal-weight, sedentary women aged 57 to 75 were
recruited to determine whether a higher muscle mass index was associated with animal or
vegetal protein intake [76].

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Many reviews indicate that certain nutritional interventions such as a high protein intake
or an increased intake of essential amino acids and the branched chain amino acid (BCAA)
leucine with resistance training may help to attenuate fiber atrophy in sarcopenic muscle by
the modulation of both anabolic and catabolic pathways [77-79]. In particular, leucine can be
considered a regulatory amino acid with unique characteristics. It plays several roles in
muscle metabolism regulation, which includes translational control of protein synthesis [80]
and glucose homeostasis [81]. In addition, leucine has been demonstrated to be a nitrogen
donor for the synthesis of muscle alanine and glutamine [80]. Considering these findings, the
use of leucine as an anti-atrophic agent is biologically justified.
It has been documented that oral post-exercise amino acid supplementation had a
synergistic effect on the contraction-induced escalation of muscle protein synthesis following
an acute resistance exercise bout [82-84]. Treatment with amino acids has been shown to
induce additive hypertrophy in response to continuous resistance training [85]. Recent human
studies have shown that amino acids play a role in the phosphorylation of translational factors
called eukaryotic initiation factors, especially eukaryotic initiation factor (eIF)4F and
p70S6K, through an mTOR-mediated mechanism [84, 86] On the other hand, several studies
have not found benefits from protein supplementation [87-89]. These studies utilized a single
bout or short-term (10 days) ingestion to examine the rate of myofibrillar synthesis [89] or
protein synthesis [87]. In contrast, Godard et al. [88] tried to investigate the long-term
supplementation of several amino acids and carbohydrate with resistance training.
Unfortunately, they conducted the examination of total muscle cross-sectional areas by only
magnetic resonance imaging, and did not perform a detailed morphological analysis (crosssectional area of muscle fiber by biopsy sample). Since the evaluation of muscle crosssectional area by magnetic resonance imaging appears to be influenced by the inner amount
of adipose tissue, connective tissue, or water, it is unknown whether their protein
supplementation actually failed to elicit positive effects on the morphometry of muscle fiber.
More recently, the administration of many essential amino acids tends to achieve a
positive effect on muscle mass and protin synthesis both under normal conditions [90-92] and
with resistance training [86]. Although a positive attenuating effect on sarcopenia has been
observed in almost all studies utilizing many essential amino acids plus a high amount of
leucine, supplementation with essential amino acids not enriched with leucine may fail to
enhance muscle protein synthesis in the elderly. In addition, a higher amount of leucine
should be supplemented along with large amounts of isoleucine and valine in order to avoid
an imbalance of BCAA levels, as pointed out by Nicastro et al. [91] in their recent review.

3.2. Creatine Supplementation

Creatine monohydrate (CrM) is among the most widely used and researched ergogenic
aids [93]. At the cellular level, CrM may reduce oxidative stress, prevent motor nerve dropout [94], enhance mitochondrial function [95], and reduce neuronal apoptosis. Creatine is a
guanidine compound that is produced endogenously from arginine, glycine, and methionine
in liver, kidney, and pancreas [96]. Exogenous creatine is obtained mainly from meat (1 kg of
meat contains ~ 5 g of creatine). Phosphocreatine plays an important role in supporting
metabolism during high-intensity exercise. Impairments in phosphocreatine metabolism may
therefore hinder muscle performance and reduce muscle mass [97, 98], although it remains

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unclear whether or not creatine content is altered by the aging process, different to the marked
reduction of phosphocreatine in muscle of metabolic myopathic patients [99]. Although some
studies did not show a beneficial effect from CrM supplementation during resistance training
for elderly individuals [100, 101], many studies have reported that CrM supplementation
during resistance training increases muscle mass and muscular strength, endurance, and
power in older individuals [99, 101-104]. Rawson et al. [101] reported that 1 month of CrM
supplementation (20 g/d 10 d 4 g/d 20 d) did not enhance fat-free mass, total body
mass, or upper extremity strength gains, yet there was less leg fatigue in the CrMsupplemented group.
In addition, a 2-month resistance exercise program (67-80 years) supplemented with CrM
(20 g/d 5 d 3 g/d) did not influence training-induced increases in total body mass or
strength gain [100]. In contrast, studies of longer duration (> 4 months) reported beneficial
effects of CrM supplementation in further enhancing the strength and muscle mass gains
attained with a resistance-training program [101, 102, 105]. There are several effects of CrM
administration that may enhance resistance exercise-induced strength gains in elderly
including activation of myogenic determination factors [106], enhancement of satellite cell
activation and recruitment [107], and reduction of amino acid oxidation and protein
breakdown [108]. There are a number of important mechanistic questions that remain to be
answered, including whether the gains are maintained over a longer period (i.e., > 6 months)
post-study and if there is a true enhancement of functional capacity, what is the mechanism of
action (i.e., more contractions over time vs. activation of satellite cells [109]. CrM
supplementation without resistance training in older adults has also been shown to have
minimal benefits [101, 109].

3.3. Ursolic Acid

A water-insoluble pentacylic triterpenoid, ursolic acid is the major waxy component in
apple peels [110]. It is also found in many other edible plants. Interestingly, because it exerts
beneficial effects in animal models of diabetes and hyperlipidemia [111, 112], ursolic acid is
thought to be the active component in a variety of folkloric antidiabetic herbal medicines
[112, 113]. As predicted by conectivity maping, Kunkel et al. [114] found that ursolic acid
reduced skeletal muscle atrophy in the setting of two-distinct atrophy-inducing stresses
(fasting and muscle denervation). A major strength of the connectivity map is that it takes into
account positive and negative changes in mRNA expression that together constitute an
authentic mRNA expression signature. Thus, by querying the connective map with signatures
of muscle atrophy, Kunkel et al. [114] were, in effect, querying with the reciprocal signature
of muscle atrophy but also induced muscle hypertrophy.
Ursolic acid might increase muscle mass by inhibiting atrophy-associated skeletal musle
gene expression. Indeed, Kunkel et al. [114] found that acute ursolic acid treatment of fasted
mice reduced Atrogin-1 and MuRF1 mRNA levels in association with reduced muscle
atrophy. Similarly, chronic ursolic acid treatment of unstressed mice reduced Atrogin-1 and
MuRF1 expression and induced muscle hypertrophy. Although ursolic acid increased skeletal
muscle Akt phosphorylation in vivo, the experiments could not determine if it acted directly
on skeletal muscle, how quickly it acted, and if the effect required IGF-I or insulin, which are
always present in healthy animals, even during fasting. To address these issues, Kunkel et al.

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[114] studied serum-starved skeletal myotubes and found that ursolic acid rapidly stimulated
IGF-I receptor and insulin receptor activity, but only if IGF-I or insulin was also present.
Taken together, their data suggest that ursolic acid first enhances the capacity of pre-existing
IGF-I and insulin to activate skeletal muscle IGF-I receptors and insulin receptors,
respectively. Importantly, ursolic acid alone was not sufficient to increase phosphorylation of
the IGF-I receptor or insulin receptor. Rather, its effects also required IGF-I and insulin,
respectively. This suggests that ursolic acid either facilitates hormone-mediated receptor
autophosphorylation or inhibits receptor dephosphorylation. The latter possibility is supported
by previous in vitro data showing that ursolic acid directly inhibits protein tyrosine
phosphatase 1B [115], a tyrosine phosphatase that dephosphorylates (inactivates) the IGF-I
and insulin receptors [116]. Further research is needed to elucidate the effect of
supplementation with ursolic acid in skeletal muscle and to attenuate muscle wasting (ex.
sarcopenia).

3.4. Eicosapentaenoic Acid (EPA)

EPA is a 20-carbon omega (n)-3 polyunsaturated fatty acid with anti-inflammatory
properties, which is synthesized from ingested alpha-linolenic acid or is consumed in fish and
fish oil such as cod liver, sardine, and salmon oil [117]. There is no established Dietary
Reference Intake for n-3 fatty acids; yet, adequate intake is set at 1.6 and 1.1 g/d for men and
women, respectively. While intake in the United States occurs at levels much lower than the
proposed adequate intake and no signs of deficiency are observed, the adequate intake is
proposed to provide optimal health benefits associated with consuming omega-3
polyunsaturated fatty acids [118]. Several clinical trials have reported potential health benefits
of omega-3 polyunsaturated fatty acids in many diseases, including cardiovascular diseases
[119], epilepsy, inflammatory bowel disease, exercise-trained subjects [120], and cancerassociated cachexia [121]. In particular, the administration of omega-3 fatty acids and EPA
capsules or supplements or supplements with EPA has been shown to be associated with
weight stabilization, gains in lean body mass, and improvements in quality of life markers in
weight loosing patients with advanced pancreatic cancer. In addition, EPA has also been
shown to inhibit the proinflammatory transcription factor nuclear factor kappaB (NF-B)
[121, 122], to reduce tumor necrosis factor- (TNF-) production by macrophages [123] and
to prevent the damaging effects of TNF- during skeletal muscle differentiation in vitro
[124]. Furthermore, short-term treatment with EPA (16 day, 100 mg/Kg) attenuates the
muscle degenaration of mdx mice, a model of DMD [125]. EPA treatment decreased creatine
kinase levels and attenuated myonecrosis (decrease in Evans-blue dye-positive fibers and a
concomitant increase in peripheral nucleated fibers), and reduced the levels of TNF-.
Some evidence suggests omega-3 polyunsaturated fatty acids to also be a potentially
useful therapeutic agent for the treatment and prevention of sarcopenia. In a more recent
study [126], sixteen healthy, older adults were randomly assigned to receive either omega-3
fatty acids or corn oil for 8 week. In their study, the rate of muscle protein synthesis and the
phosphorylation of key elements of the anabolic signaling pathway were evaluated in three
different conditions. Smith et al. [126] found that omega-3 fatty acid supplementation had no
effect on the basal rate of muscle protein synthesis but augmented the hyperaminoacidemia-

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hyperinsulinemia-induced increase in the rate of muscle protein synthesis probably due to a

greater increase in muscle p70S6KThr389 phosphorylation.

3.5. Antioxidant Supplementation

Free radicals are a highly reactive chemical species with a single unpaired electron in its
outer orbit seeking to pair with another free electron [127]. In particular, reactive oxygen
species (ROS), deriving from oxidative metabolism, have higher reactivity than O2. ROS are
constantly generated in cells of aerobic organisms, in particular skeletal muscle, by the
addition of a single electron to the oxygen molecule with subsequently damage of biological
macromolecules (etc, lipids). The interaction of ROS with normal cellular structures leads to
potentially nonreversible modifications, with consequent cellular loss of function and death
[128]. ROS production has been shown to increase in skeletal muscle during aging [129,
130]. During the aging process, it is probable that increased levels of ROS lead to the
modification of mitochondrial DNA and result in increases in myonuclear apoptosis [131].

Figure 2. Supplementation with ursolic acid upregulates the amount of IGF-I and insulin and then
stimulates protein synthesis by activating Akt/mTOR/p70S6K pathway. Amino acid supplementation
enhances protein synthesis by stimulating mTOR. Akt blocks the nuclear translocation of FOXO to
inhibit the expression of Atrogin-1 and MuRF1 and the consequent protein degradation. In cachexic
muscle, supplementation with EPA downregulates the amount of TNF- and NF-B. Resistance
training also induces IGF-I expression and activates mTOR. Both resitstance and endurance exercise
inhibit TNF- expression. Endurance training increases the amount of PGC-1 through calcineurin- or
CaMK-dependent signaling. Activated PGC-1 protects several mitochondrial disorders (apoptosis,
oxidative damage, etc.) elicited by the increase in NF-B and Bax and/or the decrease in Bcl-2 in
senescent muscle.

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In the case of experimental diabetes, antioxidant supplementation seems to effectively

prevent the muscle atrophy [132, 133]. The effect on cancer cachexia are partial although
significant [134]. In contrast, the data of antioxidant supplementation for mammalian
sarcopenia is extremely limited and controversial, despite the clinical relevance and large
interest (both from a research and commercial standpoint of view). Several studies have
investigated the possibility of delaying the aging process by enhancing antioxidative capacity
[135, 136]. For example, resveratrol, a natural polyphenol found in grapes, peanuts, and
berries [137], has shown a protective effect against oxidative stress in skeletal muscle.
Although most of human studies analyze the relationship between dietary antioxidant
supplementation and physical performance or muscle strength measures, the effect is still and
largely controversial. As pointed out by a more recent review [138], there are currently no
trials verifying the effects of antioxidant supplementaion on sarcopenia (as identified by one
of several the consensus definitions provided by international groups of experts). As proposed
by Bonetto et al. [139], oxidative stress probably would behave as an additional factor that
certainly amplifies the wasting stimuli, but probably does not play a leading role in many
other cases, which did not demonstrate the effectiveness of antioxidant therapy. Very
intriguingly, a recent statement from the Society on Sarcopenia, Cachexia, and Wasting
Disease does not even mention antioxidant supplementation as a possible tool to manage
sarcopenia in older persons [140]. Figure 2 provides an overview of the molecular pathways
of muscle growth and the action point of exercise and supplemental approaches for
counteracting sarcopenia.

4. Hormonal Supplementation
4.1. Testosterone
In males, levels of testosterone decrease by 1% per year, and those of bioavailable
testosterone by 2% per year from age 30 [141, 142]. In women, testosterone levels drop
rapidly from 20 to 45 years of age [143]. Testosterone increases muscle protein synthesis
[144] and its effects on muscle are modulated by several factors including genetic
background, nutrition and exercise [145]. Numerous studies of treatment with testosterone in
the elderly have been performed over the past few years [146-148]. In 1999, Snyder et al.
[148] suggested that increasing the level of testosterone in old men to that seen in young men
increased muscle mass but did not result in functional gains in strength. Systemic reviews of
the literature [149] have concluded that testosterone supplementation attenuates several
sarcopenic symptoms including decreases in muscle mass [147, 148] and grip strength [146].
For instance, a recent study of 6 months of supraphysiological dosage of testosterone in a
randomized placebo-controlled trial reported increased leg lean body mass and leg and arm
strength [150]. Although there are significant increases in strength among elderly males given
high doses of testosterone, the potential risks may outweigh the benefits. Risks associated
with testosterone therapy in older men include sleep apnea, thrombotic complications, and the
increased risk of prostate cancer [151].
These side effects have driven the necessity for drugs that demonstrate improved
therapeutic profiles. Novel, non-steroidal compounds, called selective androgen receptor

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modulators, have shown tissue-selective activity and improved pharmacokinetic properties.

Whether these drugs are effective in treating sarcopenia has yet to be shown [152].
Dehydroepiandrosterone (DHEA) is marketed as a nutritional supplement in the US and is
available over the counter. Unlike testosterone and estrogen, DHEA is a hormone precursor
which is converted into sex hormones in specific target tissues [153]. However,
supplementation of DHEA in aged men and women resulted in an increase in bone density
and testosterone and estradiol levels, but no changes in muscle size, strength, or function
[154, 155].

4.2. Estrogen
It has been hypothesized that menopause transition and the subsequent decline in
estrogen may play a role in muscle mass loss [156]. Van Geel et al. [157] reported a positive
relationship between lean body mass and estrogen levels. Similarly, Iannuzzi-Sucich et al.
[158] observed that muscle mass is correlated significantly with plasma estrone and estradiol
levels in women. However, Baumgartner et al. [159] reported that estrogen levels were not
associated with muscle mass in women aged 65 years and older. The mechanisms by which
decrease in estrogen levels may have a negative effect on muscle mass are not well
understood but may be associated with an increase in pro-inflammatory cytokines, such as
TNF- and IL-6, which might be implicated in the apparition of sarcopenia [160].
Furthermore, estrogen could have a direct effect on muscle mass since it has been shown that
skeletal muscle has estrogen beta-receptors on the cell membrane [161]. Therefore, a direct
potential mechanistic link could exist between low estrogen levels and a decrease in protein
synthesis. Further studies are needed to investigate this hypothesis. Neverthless, before
reaching a conclusion on the contribution of estrogens to the onset of sarcopenia, it would be
important to measure urinary estrogen metabolites since a relationship between breast cancer
and urinary estrogen metabolites has been shown [162].

4.3. Growth Hormone (GH)

GH is a single-chain peptide of 191 amino acids produced and secreted mainly by the
somatotrophs of the anterior pituitary gland. GH coordinates the postnatal growth of multiple
target tissues, including skeletal muscle [163]. GH is controlled by the actions of two
hypothalamic factors, GH releasing-hormone (GHRH), which stimulates GH secretion, and
somatostatin, which inhibits GH secretion [164]. The secretion of GH is maximal at puberty
with a gradual decline during adulthood.
Indeed, circulating GH levels decline progressively after 30 years of age at a rate of ~1%
per year [165]. In aged men, daily GH secretion is 5- to 20-fold lower than that in young
adults [166]. The age-dependent decline in GH secretion is secondary to a decrease in GH
releasing hormone and to an increase in somatostatin secretion [167].
The effects of GH administration on muscle mass, strength and physical performance are
still under debate [31, 168]. In animal models, GH treatment is very effective at inhibiting
sarcopenic symptoms, particularly in combination with exercise training [169]. The effect of
GH treatment on elderly subjects is controversial. Some groups have demonstrated an

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improvement in strength after long-term administration (3-11 months) of GH [170]. In

contrast, many researchers have found that muscle strength or muscle mass did not improve
on supplementation with GH [168, 170]. One recent study reported a positive effect on
counteracting sarcopenia after the administration of both GH and testosterone [171]. Several
reasons may underlie the ineffectiveness of GH treatment in improving muscle mass and
strength in the elderly, such as a failure of exogenous GH to mimic the pulsatile pattern of
natural GH secretion, the induction of GH-related insulin resistance, or reduced GH receptor
expression in skeletal muscle [172]. It should also be considered that the majority of trials
conducted on GH supplementation have reported a high incidence of side effects, including
soft tissue edema, carpal tunnel syndrome, arthralgias, and gynecomastica, which pose
serious concerns, especially in older adults; therefore, very careful attention should be paid
when administering GH to the elderly.
There is evidence that the age-asociated decline in GH levels in combination with lower
IGF-I levels contributes to the development of sarcopenia [173]. IGF-I is perhaps the most
important mediator of muscle growth and repair [174] possibly by utilizing Akt-mTORp70S6K signaling. The administration of IGF-I to the elderly has resulted in controversial
findings on muscle strength and function [175]. The ineffectiveness may be attributable to
age-related insulin resistance to amino acid transport and protein synthesis [176] or a marked
decrease in IGF-I receptors [177] and receptor affinity for IGF-I [178] in muscle with age.
Wilkes et al. [179] demonstrated a reduced effect of insulin on protein breakdown in the legs
of older versus younger subjects, probably due to the blunted activation of Akt by insulin.
More comprehensive reviews on insulin resistance in sarcopenia can be found elsewhere
[176].

4.4. Ghrelin
Ghrelin, is a 28-amino acid peptide mainly produced by cells in the stomach, intestines,
and hypothalamus [180]. Ghrelin is a natural ligand for the GH-secretagogue receptor, which
possesses a unique fatty acid modification, an n-octanoylation, at Ser 3 [181]. Ghrelin plays a
critical role in a variety of physiological processes, including the stimulation of GH secretion
and regulation of energy homeostasis by stimulating food intake and promoting adiposity via
a GH-independent mechanism [180].
In contrast, ghrelin inhibits the production of anorectic proinflammatory cytokines,
including IL-1, IL-6 and TNF- [182]. Because of their combined anabolic effects on
skeletal muscle and appetite, ghrelin and low molecular weight agonists of the ghrelin
receptor are considered attractive candidates for the treatment of cachexia [183]. For example,
Nagaya et al. [184] gave human ghrelin (2 g/Kg twice daily intravenously) for 3 weeks to
cachexic patients with chronic obstructive pulmonary disease in an open-label study. After
ghrelin therapy, significant increases from baseline measurements were observed for body
weight, lean body mass, food intake, hand grip strength, maximal inspiratory pressure, and
Karnofsky performance score [184]. In another unblinded study, the same group
demonstrated that treatment with human ghrelin (2 g/Kg twice daily intravenously, 3 weeks)
significantly improved several parameters (e.g., Lean body mass measured by Dual-energy
X-ray Absorption and left ventricular ejection fraction) in 10 patients with chronic heart
failure [185]. In a 1-year placebo-controlled study in healthy older adults over the age of 60

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years given an oral ghrelin-mimetic (MK-677), an increase in appetite was observed [186].
The study did not show a significant increase in strength or function in the ghrelin-mimetic
treatment group, when compared to the placebo group, however a tendency was observed
[186]. As pointed out in a recent review by Nass et al., [187], the use of this compound
induces the potential deterioration of insulin sensitivity and development of diabetes mellitus
in older adults with impaired glucose tolerance.

5. Pharmacological Approach
5.1. Myostatin Inhibition
Myostatin was first discovered during screening for novel members of the transforming
growth factor- superfamily, and shown to be a potent negative regulator of muscle growth
[188, 189]. Mutations in myostatin can lead to massive hypertrophy and/or hyperplasia in
developing animals, as evidenced by knockout experiments in mice. Moreover, mouse
skeletal muscles engineered to overexpress the myostatin propeptide, the naturally occurring
myostatin inhibitor follistatin, or a dominant-negative form of activin receptor IIB (ActRIIB:
the main myostatin receptor [193]) all display similar, if not greatr, increase in size [190].
Myostatin levels increase with muscle atrophy due to unloading in mice and humans
[191, 192], and with severe muscle wasting in HIV ptients [193]. The increased levels of
myostatin are widely accepted to lead to muscle wasting [194]. Although many researchers
consider myostatin levels to increase with age, studies using sarcopenic muscles have yielded
conflicting results [53, 195, 196]. Intriguingly, Carlson et al. [195] showed enhanced levels of
Smad3 (possible myostatin-downstream regulator) but not myostatin in sarcopenic muscles of
mice. More recently, McKay et al. [197] observed more abundant myostatin-positive type IIassociated stem cells in older than younger males after muscle loading in spite of no
difference in stem cell-specific myostatin levels at baseline. Therefore, it is possible that
myostatin-dependent signaling is activated in sarcopenic mammalian muscles.
Many researchers have conducted experiments to inhibit myostatin in models of muscle
disorders such as DMD, amyotrophic lateral sclerosis, and cancer cachexia [34, 35]. In
addition, several investigators examined the effect of inhibiting myostatin to counteract
sarcopenia using animals only. A lack of myostatin caused by gene manipulation increased
the number of satellite cells, and enlarged the cross-sectional area of predominant type IIB/X
fibers in tibialis anterior muscles of mice [198]. In addition, these myostatin-null mice
showed prominent regenerative potential including accelerated fiber remodeling after an
injection of notexin [198]. Lebrasseur et al. [199] reported several positive effects of 4 weeks
of treatment with PF-354 (24 mg/Kg), a drug for myostatin inhibition, in aged mice. They
showed that PF-354-treated mice exhibited significantly greater muscle mass (by 12%), and
increased performance such as treadmill time, distance to exhaustion, and habitual activity.
Furthermore, PF-354-treated mice exhibited decreased levels of phosphorylated Smad3 and
MuRF1 in aged muscle. More recently, Murphy et al. [200] showed, by way of once weekly
injections, that a lower dose of PF-354 (10 mg/Kg) significantly increased the fiber crosssectional area (by 12%) and in situ force of tibialis anterior muscles (by 35%) of aged mice
(21-mo-old). Blocking myostatin enhances muscle protein synthesis [201] by potentially

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relieving the inhibition normally imposed on the Akt/ mTOR signaling pathway by myostatin
[202]. These lines of evidence clearly highlight the therapeutic potential of antibody-directed
inhibition of myostatin for treating sarcopenia. However careful attention would be payed for
the myostatin inhibition, as mice with null mutation of myostatin revealed to impair tendon
structure and function [203].

5.2. Angiotensin-Converting Enzyme (ACE) Inhibitors

ACE inhibitors have long been used as a treatment in primary and secondary prevention
in cardiovascular disease as well as secondary stroke prevention. It has now been suggested
that ACE inhibitors may have a beneficial effect on skeletal muscle. ACE inhibitors may
improve muscle function through improvements in endothelial function, metabolic function,
anti-inflammatory effects, and angiogenesis thereby improving skeletal muscle blood flow.
ACE inhibitors can increase mitochondrial numbers and IGF-I levels thereby helping to
counter sarcopenia [204, 205].
Observational studies have shown that the long-term use of ACE inhibitors was
associated with a lower decline in muscle strength and walking speed in older hypertensive
people and a greater lower limb lean muscle mass when compared with users of other
antihypertensive agents [206]. Several studies have shown that ACE inhibitors improved
exercise capacity in both younger and older people with heart failure [206, 207], but caused
no improvement in grip strength [208]. Few interventional studies using ACE inhibitors for
physical function have been undertaken. One study looking at functionally impaired older
people without heart failure has shown that ACE inhibitors increase 6-minute walking
distance to a degree comparable to that achieved after 6 months of exercise training [209].
However, a study comparing the effects of nifedipine with ACE inhibitors in older people
found no difference between treatments in muscle strength, walking distance, or functional
performance [210]. It is possible that frailer subjects with slower walking speeds, who have a
tendency to more cardiovascular problems, benefit more. Further evidence is required before
recommending ACE inhibitors to counter the effects of sarcopenia. However, ACE inhibitors
are associated with cardiovascular benefits and as older people frequently have underlying
cardiovascular problems, these agents are already commonly prescribed.

5.3. Proteasome Inhibitors

In a variety of conditions such as cancer, diabetes, denervation, uremia, sepsis, disuse,
and fasting, skeletal muscles undergo atrophy through the degradation of myofibrillar proteins
via the ubiquitin-proteasome pathway [211]. Recent advances assert that muscle atrophy
under these conditions shares a common mechanism in the induction of muscle-specific E3
ubiquitin ligases Atrogin-1 and MuRF1 [212, 213]. Only very indirect measurements (small
increases in mRNA levels encoding some components of the ubiquitin-proteasome pathway
[214, 215] or ubiquitin-conjugate accumulation [216]) in old muscles of rodents or humans
suggested modest activation of this pathway. The expression pattern of Atrogin-1 and/or
MuRF1 in aged muscle is highly contradictory both in rats and humans [214, 217-220]. When
even the mRNA expression of these atrogenes increased in sarcopenic muscles, the induction

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was very limited (1.5-2.5 fold) as compared with other catabolic situations (10-fold). In
addition, the major peptidase activities of the proteasome (i.e. chymotrypsin-like, trypsin-like,
and caspase-like activities) were always reduced or unchanged with aging [212, 215, 221].
Altogether, these observations clearly suggest that activation of the ubiquitin-proteasome
system contributed little to the establishment of sarcopenia in accordance with very slow
muscle mass erosion.
There are several chemical classes of compounds that inhibit proteasomal activity,
including peptide analogs of substrates with different C-terminal groups, such as aldehydes,
epoxyketones, boronic acids, and vinyl sulfones [222]. A selective boronic acid proteasome
inhibitor, Velcade (also known as PS-341 and bortezomib), directly inhibits the proteasome
complex without direct effects on ubiquitination. Velcade is orally active and is presently
approved by the Food and Drug Administration and the European Medicines Agency [223,
224]. Gazzerro et al. [225] suggested that treatment with Velcade (0.8 mg/Kg) over a 2-week
period reduced muscle degeneration and necrotic features in mdx muscle fibers, as evaluated
with Evans blue dye. In addition, they observed many myotubes and/or immature myofibers
expressing embryonic myosin heavy chain in mdx muscle after Velcade administration.
Furthermore, Gazzerro et al. [225] also demonstrated that MG-132 increased dystrophin,
alpha-sarcoglycan and beta-dystroglycan protein levels in explants from Becker muscular
dystrophy patients, whereas it increased the proteins of the dystrophin glycoprotein complex
in DMD cases. Strangely, there has been no rodent study examining the effect of these
proteasome inhibitors to prevent muscle atrophy with aging; therefore, proteasome inhibitors
do not seem to attenuate muscle wasting in cases of sarcopenia.

5.4. Vitamin D
Vitamin D has been traditionally considered a key regulator of bone metabolism, and
calcium and phosphorus homeostasis through negative feedback with the parathyroid
hormone [226]. It is also well established that vitamin D deficiency causes rickets in children
and osteomalacia and osteoporosis in adults. A large and growing body of evidence suggests
that vitamin D is not only necessary for bone tissue and calcium metabolism, but may also
represent a crucial determinant for the development of major (sub)clinical conditions and
health-related events [226, 227].
Today, approximately 1 billion, mostly elderly people, worldwide have vitamin D
deficiency. The prevalence of low vitamin D concentrations in subjects older than 65 years of
age has been estimated at approximately 50% [228, 229], but this figure is highly variable
because it is influenced by sociodemographic, clinical, therapeutic and environmental factors.
Similarly there is an age-dependent reduction in vitamin D receptor expression in skeletal
muscle [230]. Prolonged vitamin D deficiency has been associated with severe muscle
weakness, which improves with vitamin D supplementation [231]. The histological
examination of muscle tissue from subjects with osteomalacia is characterized by increased
interfibrillar space, intramuscular adipose tissue infiltrates and fibrosis [232].
A large body of evidence currently demonstrates that low vitamin D concentrations
represent an independent risk factor for falls in the elderly [233-235]. Supplementation with
vitamin D in double-blind randomized-controlled trials has been shown to increase muscle
strength and performance and reduce the risk of falling in community-living elderly and

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nursing home residents with low vitamin D levels [236-238]. In contrast, several groups
found no positive effect of vitamin D supplementation on fall event outcomes [239-241].
Cesari et al., [242] attributed these contradictory findings to the selection criteria adopted to
recruit study populations, adherence to the intervention, or the extreme heterogeneity of cutpoints defining the status of deficiency. A more comprehensive knowledge on vitamin Drelated mechanisms may provide a very useful tool preventing muscle atrophy for older
persons (sarcopenia). Figure 3 represents an overview of hormonal and pharmacological
strategies for sarcopenia.

6. The Other Candidate

6.1. Calorie Restriction (CR)
Several studies indicate the protection of age-related functional decline and loss of
muscle fibers by CR [243-245], although the mechanisms by which CR (30-40%) delays the
aging process remain to be fully elucidated. These protective effects are likely attributable to
the ability of CR to reduce the incidence of mitochondrial abnormalities (mitochondrial
proton leak), attenuate oxidative stress, and counteract the age-related increases in
proapoptotic signaling in skeletal muscle [243, 245, 246]. Noticeably, CR has been shown to
modulate the majority of the apoptotic pathways involved in age-associated skeletal muscle
loss, such as mitochondria-, cytokine/receptor-, and Ca2+/ER-stress- mediated signaling [243,
245]. For instance, CR markedly inhibits increases in several mediators of the TNF-mediated
pathway of apoptosis (TNF-, TNF-receptor 1, cleaved caspase-3 and -8) possibly by
enhancing production of a muscle-derived anabolic cytokine, IL-15, which competes with
TNF-mediated signaling [247]. In addition, the combination of CR with exercise training is
proposed to counteract the apoptosis associated with sarcopenia more effectively [248].
In contrast, CR alone or combined with life-long voluntary exercise did not modulate the
amount of several autophagy-linked molecules (Beclin-1, autophagy9, LC3) at the protein
level except autophagy7 in sarcopenic muscles of rats [249]. Therefore, the attenuating effect
on sarcopenia by CR may be independent of the autophagy signaling.
Interestingly, a study using dual-energy X-ray absorptiometry indicated an increase in skeletal
muscle among rhesus monkeys on 30% CR for 17 years [250]. More recently, McKiernan et
al. [251] showed that CR for rhesus monkeys opposed age-related reductions in the
proportion and cross-sectional area of type II fibers in a histochemical analysis of vastus
lateralis muscle biopsies. It remains to be determined whether CR is effective in counteracting
the age-related loss of muscle in human subjects and to what extent dietary intervention can
be applied in human populations. Since excessive CR (over 50%) may have a number of side
effects (e.g. weakness, loss of stamina, osteoporosis, depression, anorexia nervosa, etc.)
[252], a more mild calorie restriction should be applied in the elderly.

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Kunihiro Sakuma and Akihiko Yamaguchi

Figure 3. Myostatin signals through the ActRIIB-ALK4/5 heterodimer activate Smad2/3 with blocking
of MyoD transactivation in an autoregulatory feedback loop. Recent findings suggest that myostatinSmad pathway inhibit protein synthesis probably due to blocking the functional role of Akt. Treatment
with ACE inhibitor and testosterone upregulates the amount of IGF-I and then stimulates protein
synthesis by activating Akt/mTOR/p70S6K pathway. Testosterone also enhances protein synthesis by
stimulating mTOR. Abundant serum GH, which is induced by ghrelin, activates JAK2-STAT5
signaling to promote muscle-specific gene transcription necessary to hypertrophy. More recent finding
indicates that vitamin D enhances follistatin expression, in turn blocks the functional role of myostatin
in vitro. The direct role of vitamin D on muscle fiber remains to be elucidated.

Conclusion and Perspectives

The advances in our understanding of muscle biology that have occurred over the past
decade have led to new hopes for pharmacological treatment of muscle wasting. These
treatments will be tested in humans in the coming years and offer the possibility of treating
sarcopenia/frailty. These treatments should be developed in the setting of appropriate dietary
and exercise strategies. Currently, resistance training combined with amino acid-containing
supplements would be the best way to prevent age-related muscle wasting and weakness.
Supplementation with ursolic acid and EPA seems to be more intriguing candidates
combating sarcopenia, although systematic and fundamental research in these treatments has
not been conducted even in rodent. The well-known sarcopenia-attenuating effects by
endurance training may be attributable to the protection for mitochondrial disorders by the
increase of PGC-1 amount.

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105

Acknowledgments
This work was supported by a research Grant-in-Aid for Scientific Research C (No.
23500778) from the Ministry of Education, Culture, Sports, Science and Technology of
Japan.

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In: Basic Biology and Current Understanding of Skeletal Muscle ISBN: 978-1-62808-367-5
Editor: Kunihiro Sakuma
2013 Nova Science Publishers, Inc.

Chapter 5

Oxidative Stress-Induced Signal

Transduction in Skeletal Muscle
Wataru Aoi
Laboratory of Health Science, Graduate School of Life and
Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan

Abstract
Reactive oxygen species (ROS) are produced via several sources in the skeletal
muscle. In particular, the mitochondrial electron transport chain in the muscle cells could
be a major source of ROS, with an elevation of oxygen consumption. Physical exercise, a
sedentary lifestyle, and the aging process can all generate oxidative stress. Such oxidative
stress can cause transcriptional and post-translational regulation of key proteins and
affect their functionality. Evidence has suggested that some muscle proteins (i.e.,
myofiber proteins, metabolic signaling proteins, and inflammatory factors) can be the
targets of oxidative stress. Continuous or excess elevation of ROS in muscle tissues
results in inflammation, loss of muscle mass, and metabolic dysfunction. In contrast,
growing evidence has suggested that moderate and transient elevations in ROS contribute
to the metabolic improvement and maintenance of muscle mass via modulating related
proteins, which mediate health benefits induced by moderate exercise. The oxidative
proteins may be also useful as potential biomarkers to examine the oxidative stress levels,
antioxidant compounds, and their possible benefits or dysfunction.

Introduction
Reactive oxygen species (ROS) are generated in various conditions in the living body. A
small percent of the oxygen utilized in the mitochondria is converted to superoxide during the
electron transport chain reaction.

Corresponding autor: Wataru Aoi. Laboratory of Health Science, Graduate School of Life and Environmental
Sciences, Kyoto Prefectural University, Kyoto 606-8522, Japan. Phone: +81-75-703-5417; Fax: +81-75-7035417; E-mail: waoi@kpu.ac.jp.

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Therefore, the skeletal muscle is a major source of ROS production since it is a major
metabolic organ. It is known that oxygen consumption during aerobic exercise is elevated 10
to 20-fold in the body as a whole and over 100-fold in the skeletal muscle alone (Figure 1).
In addition, endothelium and invaded phagocytes also produces ROS in muscle tissues
via reactions by such xanthine oxidase, NADPH oxidase, and myeroperoxidase. When ROS
are generated above the protective capacity of antioxidants during physical exercise, the aging
process, and pathogenesis, they induce oxidative stress in the muscle tissues.
Cell components such as lipids, proteins, and DNA, are easy targets for ROS, and
accumulated oxidative products of these components are observed in the conditions that
generate oxidative stress. Such oxidative damage is closely associated with the pathogenesis
of life-style diseases, age-related diseases, dystrophic diseases, and exercise-induced muscle
fatigue/damage.
Interestingly, a moderate level of oxidative stress mediates the maintenance of
physiological homeostasis and additional exercise-induced beneficial adaptations.

Figure 1. Generation of reactive oxygen species in skeletal muscle. ROS are continuously generated
from various sources with various physiological and pathological conditions. Consequently, muscles
are exposed to oxidative stress and cellular components such as DNA, lipids, and proteins are oxidized.
Such oxidative stress can also cause transcriptional and post-translational regulation of key proteins and
affect their functionality.

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Growing evidence has shown that several oxidative-sensitive signaling pathways exist in
the muscle cells, and the regulation of the signaling activity is thought to be a major factor
contributing to the effect of ROS on muscular function. In particular, the post-translational
and transcriptional regulation of proteins induced by oxidative stress is closely associated
with the expression of those functions via regulating the activity of key proteins. In this
chapter, we review oxidative stress-induced signals associated with various phenotypic
functions in the skeletal muscle.

Post-Translational and Transcriptional

Regulation of Proteins Induced
by Oxidative Stress
Numerous post-translational modifications have been characterized to be the result of the
direct modification of amino acid residues or the formation of reactive intermediates via the
oxidation of other cellular components [1]. The modification of 20 different amino acids
plays an important role in the manifestation of the function of many proteins. The
modification can be subdivided into 2 general forms: reversible modifications and irreversible
modifications. Some of the lipid peroxidation products exhibit a facile reactivity with
proteins, generating a variety of intra- and intermolecular covalent adducts such as 4hydroxy-2-nonenal (4-HNE), N-(hexanoyl) lysine (HEL), and carbonylation. In addition, the
oxidation of cysteine to sulfenic, sulfinic, and sulfonic acids has been shown to occur
frequently, and these sulfenic and sulfinic acids can often be enzymatically reduced. In
addition, nitration by reactive nitrogen species, chlorination by hypochlorous acid, and
bromination by hypobromous acid of the target proteins are frequently detected
modifications.
In contrast to a reversible modification, an irreversible modification is a chemical
reaction, which is difficult to control and leads to the production of abnormal proteins.
Therefore, in many cases, since the abnormal proteins cannot be repaired, the oxidative
protein must be catabolized in the proteasome. The protein modification spreads to the
constitutive proteins and enzymatic proteins, which regulate various cellular signal
transductions.
Therefore, the inactivation of proteins in upstream signal transduction, due to oxidative
modification, can induce an abnormal cellular response. Indeed, much evidence has
demonstrated the irreversible post-translational modification of membrane transporters,
enzymes, and chaperone proteins in various cells. Such modifications can be associated with
the onset of various common diseases, including cancer, inflammation, and metabolic
disorders [2-5].
Oxidative stress also affects gene expression via the regulation of transcription factors.
Nuclear factorkappa B (NF-B) is well characterized as a representative transcription factor
that is regulated by the intracellular redox balance [6]. Under normal conditions, NF-B
heterodimers are located in the cytoplasm by binding with I-kappa B. According to the
classical descriptions of regulation, NF-B-bound I-kappa B is ubiquitinated by the
phosphorylation of upstream kinases in response to ROS, which results in NF-B moving

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from I-kappa B into nucleus. The translocated NF-B binds to the genetic region of DNA
encoding for proinflammatory cytokines, chemokines, and adhesion molecules.
Consequently, oxidative stress induces inflammation with the infiltration of phagocytes
through the expression of inflammatory mediators and NF-B activation. In addition to the
transcription factor, some transcriptional coactivators, which regulate gene expression by
interacting with transcription factors, are also shown to have redox sensitivity in the
regulation of its activity and contents.
Previously, it has been shown that several muscular proteins are also targets that can be
modified by ROS or nitrogen oxide species that are generated by physiological and
pathological conditions [7-17] (Table 1). ROS generated by high-intensity acute/chronic
exercise directly induce the oxidation of the thiol moiety on the side chain of the amino acid
cysteine residues in proteins and indirectly modify other amino acid residues of the proteins
by lipid peroxides. Such modifications could be a mechanism of delayed-onset muscle
damage and muscular fatigue [8, 10, 12, 16], as described later. In addition, oxidative protein
modifications can also be found in chronic pathological conditions (e.g., disuse atrophy and
insulin resistance) [9, 11, 13, 14] and is closely associated with these disorders via abnormal
signal transduction.

Oxidative Stress and Inflammatory Signals

Acute and chronic inflammation is developed because of the direct effect of
proinflammatory mediators on cellular signaling and can lead to oxidative stress. In addition
to muscle cells, innate immune cells, inflammatory macrophages, fibroblasts, and endothelial
cells, which release a distinct set of proinflammatory mediators, can exacerbate the generation
of ROS in muscle tissues. This can often create a vicious loop between oxidative stress and
inflammation. Oxidative stress-induced inflammation is mediated via redox-sensitive
transcription factors.
Table 1. Non-enzymatic oxidative protein modification in skeletal muscle

Exposure of cells to oxidative and proinflammatory stimuli causes the activation of a

series of upstream kinases such as mitogen-activated protein kinase (MAPK), I-kappa B
kinase, protein kinase C, phosphatidylinositol 3-kinase (PI3K), which then activate NF-B by
the phosphorylation-mediated degradation of I kappa B [18-21] (Figure 2). Activated

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127

upstream kinases may also phosphorylate p65, the active subunit of NF-B. Free, activated
NF-B, in the form of the p65-p50 heterodimer, is translocated to the nucleus. There, it binds
to the B sequences located in the promoter of the target gene.

Figure 2. Schematic of inflammation signaling cascade in response to oxidative stress in skeletal

muscle. Under normal conditions, nuclear factor-kappa B (NF-B) heterodimers (p65 and p50) are
located in the cytoplasm by binding with I-kappa B (I-B). According to the classical descriptions of
regulation, NF-B-bound I-B is ubiquitinated by the phosphorylation of upstream kinases in response
to ROS, which results in NF-B moving from I-B into nucleus. The translocated NF-B binds to the
genetic region of DNA encoding for proinflammatory cytokines, chemokines, adhesion molecules, and
ubiquitin ligases. Oxidative stress induces inflammation and proteolysis with the infiltration of
phagocytes through the expression of inflammatory mediators and NF-B activation.

Alternatively, MAPKs can activate AP-1 components, c-Jun, and c-Fos, leading to the
binding of AP-1 (c-Jun-c-Fos heterodimer) to the cyclic AMP response element (CRE)
sequences of the target promoter gene [6].
Unaccustomed and strenuous exercise causes muscle damage that presents clinically as
muscular pain and involves protein degradation and ultrastructural changes. The release of
soluble muscle enzymes, most notably creatine kinase (CK), leads to the disruption of
sarcomere architecture [22, 23] and surface membrane damage [24, 25]. Muscle damage
usually occurs after some time after exercise (rather than during or immediately after
exercise), peaks at about 2448 h, and is called delayed-onset muscle damage [26]. Muscledamaging exercise leads to phagocytic infiltration into the muscle tissue and the inflammatory
response induces delayed-onset muscle damage [27]. Previous studies have shown that
delayed-onset muscle damage is mainly induced by mechanical stress, especially eccentric
muscle contraction [28-30]. In addition, we demonstrated that delayed-onset muscle damage
induced by prolonged exercise is partly associated with inflammation through phagocyte
infiltration caused by ROS [31]. In an in vitro study that used myotube cells from rats, the
addition of hydrogen peroxide (H2O2) induced the translocation of p65 (a component of NFB) into the nucleus and, subsequently, increased the expression of cytokine-induced

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neutrophil chemoattractant-1 and monocyte chemoattractant protein-1. Prolonged acute

exercise increased the amount of nuclear p65 in rat gastrocnemius muscles. This increase
persisted 1 h after exercise, which is similar to the in vitro results. Prolonged exercise also
caused muscle damage with neutrophil invasion on the following day.
Therefore, delayed-onset muscle damage after exercise is associated with inflammation
that is secondary to phagocyte infiltration caused by the generation of ROS. The infiltration
of phagocytes into the tissues expressing these mediators results in proteolysis and
ultrastructural damage. In addition to NF-B, several studies showed that AP-1 is transiently
activated by a single bout of exercise, which can also induce an inflammatory response [32].
The activation of AP-1 may also contribute to delayed-onset muscle damage.
Metabolic disorders and cardiovascular diseases are associated with low-grade
continuous inflammation [33, 34]. When aging individuals lead a sedentary lifestyle, they
increase chronic inflammation and oxidative stress in the skeletal muscle, blood, and other
tissues. The primary sources of cytokine and ROS are not clear, but it is assumed that certain
adipokines (e.g. tumor necrosis factor- (TNF-)) which are secreted from accumulated
visceral adipose tissue can induce further production of proinflammatory cytokines and ROS
from neutrophils and macrophages. In addition, these proinflammatory cytokines induce ROS
production from mitochondria in skeletal muscle cells, which is followed by chronic NF-B
activation [35]. Growing evidence suggests that additional adipokines, including resistin, fatty
acid binding protein, and visfatin can also contribute low-grade inflammation [36-38]. In
addition, a reduction in circulating adiponectin, an adipokine with anti-inflammatory
properties, occurs with obesity and leads to low-grade inflammation in the skeletal muscle
and liver [39, 40]. Such low-grade inflammation is closely associated with insulin resistance
and muscle atrophy via disturbance in insulin signaling and the proteolytic pathway.

Oxidative Stress and Muscle Atrophy

Muscle atrophy can be due to both muscle fiber atrophy and a complete loss of muscle
fiber [41, 42] caused by several factors including the apoptosis of muscle cells [43] and
decreased differentiation of satellite cells [44], and a lack of protein caused by decreased
protein synthesis and increased protein degradation [45]. ROS affects muscle atrophy via
several pathways (Figure 3). Recently, the molecular mechanism that underlies the loss of
protein from muscles has been defined. Protein synthesis is triggered by the insulin-like
growth factor-1 signaling pathway, which activates the PI3-K/Akt pathway. The activation of
downstream targets follows (e.g. the mammalian targets of rapamycin and glycogen synthase
kinase-3). On the other hand, ROS is closely associated with protein degradation via
activation of the ubiquitin-proteasome pathway, which is one of the major causes of protein
degradation.
It has been reported that the content of polyubiquitinated proteins increases during
atrophy [46-48] and that the inhibition of proteasomes prevents protein degradation [49]. In
vitro studies have revealed that the addition of oxidants to myotubes increases protein
degradation rates, along with an increase in the ubiquitination of proteins, such as myosin,
and an increase in the expression of the major components of the ubiquitin-proteasome
pathway [50-52]. Muscle ring finger 1 (MuRF1) and muscle atrophy F-box have been
identified as the ubiquitin ligases whose activities increase during atrophy [53, 54]. NF-B

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129

can regulate the ubiquitin-proteasome proteolytic pathway through induction of MuRF1 and
proteasome expression [55-57].
Furthermore, it has been shown that the 20S proteasome can selectively degrade modified
proteins oxidatively without ubiquitination [58, 59].

Figure 3. Muscle loss signaling in response to chronic oxidative stress. Chronic oxidative stress
accelerates protein degradation via ubiquitin-proteasome- and calpain- systems, as well as an activation
of apoptotic signaling. The protein degradation and apoptosis leads to muscle atrophy with aging,
inactivity, and diseases.

These observations suggest that protein degradation could be the link between oxidative
stress and muscle atrophy. In fact, the hyperactivity of NF-B and the ubiquitin-proteasome
pathway have been identified as a major cause of aging-related muscle atrophy [60, 61]. TNF, an inflammatory cytokine, led to ROS-mediated NF-B activation, which resulted in the
reduction of total protein and a specific loss in the myosin heavy chain in C2C12 myotubes
and muscle tissues from mice [62]. Another in vivo study [63] has also shown that circulating
TNF- causes ROS-mediated NF-B activation along with contractile dysfunction in
respiratory muscles. In contrast, we found that the inhibition of NF-B reduced oxidative
stress-induced protein degradation [51]. Some antioxidants can also prevent muscle atrophy
in atrophy models.

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These observations demonstrate a close relationship between oxidative stress and protein
degradation during muscle atrophy. As another pathway, it is also possible that the
intracellular production of ROS could play a key role in the observed disturbances in calcium
homeostasis. Oxidative stress-induced decreases in Ca2+-ATPase activity in the sarcoplasmic
reticulum (SR) would reduce Ca2+ removal from the cytoplasm and promote intracellular Ca2+
accumulation, which leads to an increase in proteolysis through the activation of the calciumdependent protease calpain [64-66].
In addition to muscle fiber atrophy, a complete loss of muscle fibers also contributes to a
loss in muscle mass. A decrease in the number of muscle fiber is caused by the apoptosis,
which results from mitochondrial dysfunction and the activation of apoptotic signaling
pathways. In fact, accelerated apoptosis of muscle fibers has been documented to occur with
age [67, 68]. Apoptosis and the amount of cleaved caspase-3, an effector protease in the
apoptosis cascade, are elevated in the gastrocnemius muscles of old Fischer 344 rats
compared to that in young animal [69]. Oxidative stress induced by H2O2 leads to the
elevation of apoptotic DNA fragmentation and an increase in pro-apoptotic factors such as
Bcl2-associated X protein (Bax), caspase-3, and caspase-9 in C2C12 myotubes [70, 71]. It is
suggested that receptor signaling via TNF may contribute to the activation of caspasedependent apoptotic signaling in aged muscles. The activation of caspase-3 via caspase-8
occurs after the binding of TNF- to TNF receptor 1 and leads to nuclear DNA fragmentation
[72]. Elevations in TNF- are observed in aged muscles, suggesting that elevated oxidative
stress can lead to the apoptosis of myocytes via TNF. In addition, it was reported that
oxidative stress activates not only the caspase-dependent apoptotic pathway but also the
caspase-independent pathway via an apoptosis-inducing factor, which is released from the
mitochondria and is translocated to the nucleus where it plays a role in DNA fragmentation
[70]. Conversely, muscle apoptosis induced by menadione, a compound that instigates
oxidative stress, is attenuated along with the inhibition of lipid peroxide [73], thus supporting
the relationship between ROS and apoptosis. Such apoptosis can also cause a reduction in
fiber size [74].

Oxidative Stress and Metabolic Impairment

The basal metabolic rate and the energy metabolism of the whole body decreases with
age. A loss in skeletal muscle mass is also observed because it is the major energy-consuming
tissue in the body [75]. It has been reported that the loss of fat-free mass with age explains the
reduction in resting energy consumption [76]. In addition, the activity of enzymes involved in
aerobic metabolism in muscle mitochondria, such as cytochrome c oxidase and citrate
synthase, decreases with age [77].
Most of the ATP produced in muscular mitochondria relies on the consumption of
oxygen and, at the same time, this metabolic process generates ROS. Thus, age-related
mitochondrial dysfunction caused by oxidative stress can also lead to disturbances in energy
metabolism [78, 79]. Previously, it has been investigated that oxidative stress is strongly
associated with the clinical manifestation of insulin resistance in the skeletal muscle. Type 2
diabetes is characterized by insulin resistance in various tissues, especially in skeletal muscle
(i.e., the primary site of insulin-stimulated glucose disposal).

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Thus, oxidative stress-induced development of insulin resistance in the muscle leads to

the initiation of diabetes and the pathogenesis of late diabetic complications. Indubitably,
insulin is sensitively and inversely correlated with the levels of plasma free radicals in
diabetic patients [80, 81]. Several studies demonstrate that ROS impair insulin-mediated
glucose uptake and storage by disrupting signaling control points, such as glycogen synthase
kinase-3, Akt phosphorylation, and actin remodeling [78, 82, 83]. In muscle cells, stimulation
of oxidants such as H2O2 blocks insulin-induced glucose uptake and glucose transporter 4
(GLUT4) translocation by impairing insulin receptor activation and PI3-K/Akt signaling [84,
85]. Interestingly, oxidative products are elevated in the muscles of patients with type 2
diabetes [86, 87] and diabetic mice [88]. This finding provides support for the role of
oxidative stress in the development of glucose resistance.
Mitochondrial proteins are thought to be a major target of post-translational oxidative
modification because these proteins are close to the source of ROS production. In skeletal
muscles from Fischer 344 rats, Feng et al. [9] performed a study using quantitative
proteomics to identify mitochondrial proteins susceptible to carbonylation in an agedependent manner. In that study, 22 carbonylated proteins that included proteins associated
with fatty metabolism and the citrate cycle, thought to be associated with the development of
insulin resistance, were identified. Meany et al. [11] have also reported that a number of
mitochondrial proteins constituting the electron transport chain are carbonylated in the
muscles of aged rats compared to the muscles of young rats. In addition, we recently found
that the 3-nitro-tyrosine modification of adenylate kinase 1 (AK1), a key enzyme in the
synthesis, equilibration, and regulation of adenine nucleotides, is elevated in the aged muscles
of mice, and the modification of AK1 is involved in the impairment of glucose uptake via the
inhibition of AMP-activated protein kinase. In contrast, several antioxidants improve the
insulin sensitivity of muscle cells for glucose uptake and reduce the production of oxidative
products [83, 84, 89].
The HEL moiety is a novel adduct, formed from the reaction of linoleic acid
hydroperoxide and lysine, and is a marker of lipid peroxidation-derived protein modification
in the early stages after oxidative stress [90, 91]. Therefore, because it may be useful in
detecting rapid oxidative modification of mitochondrial proteins during acute exercise, we
hypothesized that proteins on the mitochondrial membrane are easily modified by HEL. In an
analysis of mouse gastrocnemius muscles obtained immediately after running, the
modification of carnitine palmitoyltransferase I (CPT I) by HEL was detected [7]. CPT I is
located on the mitochondrial membrane and is a rate-limiting step in fatty acyl-CoA entry into
the mitochondria in the muscle [92]. In contrast, astaxanthin, an antioxidant in the
mitochondrial membrane, limits the modification of CPT I by HEL after exercise. This
information suggests that astaxanthin traps the oxygen radicals generated by exercise.
Several studies [93, 94] have shown that the fatty acid translocase/cluster of
differentiation 36 (FAT/CD36) is associated with CPT I on the mitochondrial membrane and
increases its function. We found that the interaction between CPT I and FAT/CD36 in the
muscle during exercise was facilitated by astaxanthin [7]. Thus, the modification of CPT I by
HEL may alter the colocalization of CPT I with FAT/CD36 by changing the CPT I structure,
which could lead to the regulation of lipid metabolism during exercise. Lipolysis in the body
is important during exercise to facilitate lipid utilization in the muscle rather than to release it
from the adipose tissue. The utilization ratio of carbohydrates and lipids for energy generation
is almost equal when exercise is of low to moderate intensity.

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A possible factor influencing the utilized ratio of these energy substrates is the
colocalization of CPT I with FAT/CD36. Exercise-induced ROS may partly limit the
utilization of fatty acids by diminishing the CPT I activity caused by HEL modification.
Indeed, we and another group found that the inhibition of this modification by dietary
astaxanthin increased fat utilization during exercise and prolonged the running time to
exhaustion, when compared with mice on a normal diet [7, 95].
Therefore, HEL-modification of CPT I can partly suppress lipid metabolism during
exercise, which would affect the endurance performance and efficiency of adipose tissue
reduction with training.
Under the condition of exercise-induced muscle damage, oxidative stress can also
transiently decrease insulin-stimulated glucose uptake in skeletal muscle. In a euglycemichyperinsulinemic clamp study, Kirwan et al. first reported that, in human subjects, systemic
insulin resistance and elevated circulating CK levels persist 48 h after eccentric exercise. This
effect was not observed after concentric exercise or in the resting state [96]. We recently
reported that the insulin-stimulated uptake of 2-deoxy-[3H] glucose in damaged muscle was
significantly lower after downhill running when compared to the uptake that was found to
occur in sedentary mice [8].
Furthermore, Del Aguila et al. [97] reported that muscle-damaging exercise impairs the
insulin-stimulated activity of insulin receptor substrate-1 (IRS-1), PI3-K, and Akt in the
muscle tissues. In addition to proinflammatory cytokines such as TNF-, intereukin-1, and
interleukn-6, we observed the elevation of 4-HNE, a lipid peroxidation product that
covalently modifies the proteins on cysteine, histidine, and lysine residues, in the damaged
muscle obtained from mice on the next day after acute running [8]. Liu et al. [98] reported a
positive correlation between 4-HNE and CK activity in the blood, following strenuous
exercise in humans. Recently, insulin receptor substrate-1 (IRS-1) was detected as a 4-HNEtargeted protein in the damaged muscle [8]. IRS-1 is upstream in the PI3K/Akt-dependent
insulin-signaling pathway in muscle cells and regulates glucose uptake via GLUT4. In the
damaged muscle after strenuous exercise, insulin-stimulated glucose uptake is decreased
along with a reduction of insulin signal transduction, which suggests that 4-HNE modification
of IRS-1 is involved in the transient impairment of insulin sensitivity. In addition, Sahlin et
al. [15] showed a marked elevation in the 4-HNE modification of mitochondrial protein after
acute endurance exercise, which would also be associated with metabolic dysfunction in
damaged muscle.

Oxidative Stress and Muscle Fatigue

It is well established that ROS have important influences on force production in the
skeletal muscle. A study using spin traps and vitamin E in animals demonstrated that
scavenging ROS in muscles during exercise delays the onset of muscular fatigue [99].
Moreover, many reports have shown that the administration of the antioxidant Nacetylcysteine (NAC), which acts as a reduced thiol donor supporting glutathione resynthesis, delays muscular fatigue during a variety of submaximal exercise tasks such as
electrically stimulated fatigue of the muscle, cycling exercise, and repetitive handgrip
exercise [100-104]. In animal studies, NAC administration has also been shown to delay
fatigue in both in vitro and in situ muscle preparations [105, 106].

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133

Studies using excised muscle fiber bundles also revealed that force production during
submaximal tetanic contractions is decreased with nitric oxide (NO) donors [107, 108] and
increased with nitric oxide synthetase (NOS) inhibitors and NO scavengers [109, 110].
The influence of oxidative stress on the SR, a subcellular organelle that controls the
contractile state of the muscle by regulating the calcium concentration in the cytosol, has been
studied extensively in the skeletal muscle, and is associated with the oxidative modification of
membrane proteins [111-113]. Muscle contraction is performed by increasing intracellular
calcium concentrations, which are released from the SR via the ryanodine receptor (RyR)
calcium-release channel following active potentials during the excitation-contraction coupling
process. Afterwards, calcium is immediately taken into the SR via SR calcium-dependent
ATPase (SERCA), which relaxes the muscle. It has been known that the responsive proteins
in the SR are sensitive to redox modulation [114, 115]. The RyR appears to be in close
association with the NADP(H) oxidase(s) found in the SR, and locally generated superoxide
appears to be the major ROS capable of influencing this channel [113]. Each subunit of this
large tetrameric protein contains a small number of regulatory cysteines. ROS and NO
oxidize thiol residues on neighboring cysteines to form disulfide bonds, which induce channel
opening. Disulfide formation is reversed by reducing agents, and it provides a mechanism for
the direct redox modulation of channel activity. The SERCA, another potential target,
contains a small number of critical sulfhydryls near the SERCA active site, which has been
shown to slow the reuptake of calcium into the SR [116-118]. Exposure to elevated NO also
inhibits SERCA activity via thiol oxidation and nitration of tyrosine residues [119, 120].
Consequently, oxidation of SR proteins tends to increase cytosolic calcium levels, which
prevents of muscle relaxation. Therefore, the modification of calcium transport proteins in the
SR can causes excess or chronic muscle contraction by increasing intracellular calcium levels,
which leads to muscle fatigue.
Muscle myofilaments are also sensitive to direct redox modification [121]. Myosin heavy
chains contain several sulfhydryl residues, which are useful sites for protein labeling.
However, thiol modification generally does not dramatically alter myosin function [122]. On
the other hand, Yamada et al. [17] reported that a force reduction in the soleus muscle of
hyperthyroid rat is associated with carbonylation of the myosin heavy chain. In addition, it
has been suggested that the myosin heavy chain is easily glycosylated, which changes the
structural and functional properties of the protein [123]. However, the involvement of these
modifications on exercise-induced fatigue is unclear. In contrast, myosin light chains, actin,
and tropomyosin appear less sensitive to redox modulation [124, 125].

Oxidative Stress Induced by Moderate

Exercise and Health Benefits
Oxidative stress is associated with the development of pathogenesis and a reduction in
exercise performance. On the other hand, exercise-induced oxidative stress plays an important
role in improving metabolism adaptation through exercise. This exercise-induced effect does
not occur without oxidative stress, which is recognized as the theory of hormesis [126]
(Figure 3). Therefore, there is much debate concerning the intake of dietary antioxidants
during exercise.

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Figure 4. Beneficial effects mediated by moderate exercise-induced oxidative stress. Moderate exercise
induces transient and mild oxidative stress which plays an important role in improving beneficial
adaptations (e.g. metabolic improvement, antioxidant, and antiinflammation) through exercise.

Previously, it has been suggested that a moderate level of oxidative stress caused by lowto-moderate intensity exercise (which does not cause muscle damage) is important for signal
transduction in the cell.

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Regular exercise adaptively improves glucose and lipid metabolism, and the expression/
activity of several key proteins in the skeletal muscle is involved in the development of this
adaptation. Specifically, peroxisome proliferator-activated receptor gamma coactivator-1
alpha (PGC-1), a family of transcriptional co-activators, plays an important role as a
modulator and in improving the metabolic rate through regular exercise [127].
The activation of PGC-1 alters the metabolic phenotype through interaction with the
nuclear respiratory factor and peroxisome proliferator-activated receptor (PPAR) [127,
128], which improves lipid metabolism, elevates mitochondrial biogenesis, and facilitates a
fast-to-slow fiber type switch. Therefore, an improved understanding of the activation of the
PGC-1 protein with exercise has implications beyond better athletic performance [129, 130],
including the possibility of providing targets for the treatment of muscle weakness in the
elderly, obese, and sick (e.g., mitochondrial myopathies and diabetes) [131-133]. The
expression/activity of PGC-1 could partly be caused by ROS and the activation of other
upstream signal molecules including AMP activated protein kinase, calcineurin, CaMKs, and
p38 MAPK [134-137], which are induced by exercise. Cellular ROS levels are regulated by
the inducible defense system including antioxidant enzymes and thiol reductants. These are
predominantly regulated by the transcription factor Nrf2 (nuclear erythroid 2-related factor 2)
and its cytosolic repressor protein, Keap1 [138]. The S-thiolation of Keap1, the cytoplasmic
inhibitor of Nrf2, by ROS or electrophiles results in the dissociation of the Keap1-Nrf2
complex. Once freed from inhibition by Keap1, Nrf2 translocates to the nucleus and binds to
the antioxidant-responsive element (ARE)-containing gene. Exercise induces expression of
various antioxidant enzymes via activation of Nrf2 in human muscles, which presumably
resulted from moderate oxidative stress in response to exercise [139]. In contrast, abnormal
Nrf2/ARE signaling is found in aged skeletal muscles, and this impairs the antioxidant system
[139]. NF-B and MAPK pathways in the skeletal muscle have also been shown to enhance
the gene expression of several antioxidant enzymes, such as mitochondrial superoxide
dismutase (MnSOD) [126]. Although there is still some controversy, moderate exercise can
lead to the mild activation of NF-B and MAPK (without inflammation and with phagocyte
infiltration) via moderate oxidative stress, which contributes to the up-regulation of
antioxidant capacity. In contrast, a high-dose of dietary antioxidants combined with a dietaryexercise regimen can counteract the oxidative stress that induces beneficial effects brought
about by moderate exercise. Therefore, there is some debate about the intake of dietary
antioxidants during exercise. Gomez-Cabrera et al. [140] demonstrated, in a human doubleblind study, that oral administration of vitamin C (1 g/d) suppresses the adaptation of
endurance capacity with exercise training for 8 weeks. In this case, the suppression of PGC1 expression, a key modulator of mitochondria biogenesis, was found. Ristow et al. [141]
also reported that administration of vitamin C (1000 mg/d) and vitamin E (400 IU/d) with a 4week intervention of exercise training significantly ameliorated improvements in glucose
infusion rates (GIR) during a hyperinsulinemic, euglycemic clamp along with the downregulation of PGC-1 and Mn-SOD. Therefore, the negative effects of antioxidant vitamin
would result from its capacity to reduce the exercise-induced expression of key transcription
factors involved in nutrient metabolism and antioxidation. However, some antioxidants
accelerate energy metabolism and insulin sensitivity induced by exercise through the
elevation in the level and activity of key modulators [140-143]. This may be responsible for
the specific actions of each compound in addition to antioxidant properties. Therefore, the

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effectiveness of the compounds may differ according to gender, individual characteristics,

and mode of ingestion. In essence, optimum method of intake, the quantity and quality of the
foods to be ingested, and the timing of their intake need to be established in accordance with
the purpose of using each food or food component and understanding the physiological
changes brought on by exercise.

Conclusion
The effect of oxidative stress on cellular signal transduction has been elucidated. We
found that oxidative stress is closely associated with pathogenesis, including muscle
dystrophy, insulin resistance, and disuse muscle atrophy. In addition, phenotypic changes that
occur after acute and chronic exercise are partly mediated by oxidative stress. However, there
are many proteins modified by oxidation, which have not been yet identified. Therefore, the
effect of oxidative stress on signal transduction via the post-translational modification of
proteins remains unclear, in contrast to the phosphorylated modification of proteins. Further
studies are needed to elucidate the physiological and pathological significance of the effect of
oxidative stress on skeletal muscles.

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In: Basic Biology and Current Understanding of Skeletal Muscle ISBN: 978-1-62808-367-5
Editor: Kunihiro Sakuma
2013 Nova Science Publishers, Inc.

Chapter 6

The Functional Role of Heat

Shock Proteins in Skeletal Muscle
Tomonori Ogata
Faculty of Human Environmental Studies,
Hiroshima Shudo
University, Ozukahigashi, Asaminamiku,
Hiroshima, Japan

Abstract
Heat shock proteins (HSPs) are known as highly conserved prosurvival molecules.
HSPs have been demonstrated to contribute to improving several pathological alterations
in skeletal muscle. It has been reported that several HSPs attenuate contraction-induced
and age-related muscle damage in overexpression models. Activation of the ubiquitin
proteasome pathway induces skeletal muscle atrophy during immobilization, but
overexpression of HSPs can block the activity. In muscular dystrophy, enhanced HSP72
preserves muscle function and slows progression of muscle atrophy. Furthermore, the
enhancements of HSP72 in skeletal muscle potentially contribute to preventing obesityand hyperlipidemia-induced insulin resistance through regulation of inflammatory
factors, thereby preventing diabetes. This review focuses on recent progress in the
understanding of HSP functions in skeletal muscle.

Introduction
Metabolic disorders including hyperglycemia, hyperlipidemia, and hypertension are
attributed to individual genetic factors, aging, and lifestyle. Many people try to improve their
metabolic disorders through exercise or sports. It is well known that moderate exercise
contributes to health by preventing disease or improving the negative consequences of
conditions related to obesity, diabetes, or chronic inflammatory conditions. On the other hand,
intense exercise causes muscle fiber damage induced by excessive reactive oxygen species
(ROS), heat stress, and mechanical stretch. A multitude of physiologic stresses result in

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protein damage and misfolded protein structure, leading to cellular injury and death in
skeletal muscle [1-3].
The cell has a number of protective measures to promote survival during periods of
environmental stress. One of the most highly conserved mechanisms of cellular protection
involves the expression of a polypeptide family known as heat shock proteins (HSPs). HSPs
ubiquitously express in multiple cells and act as molecular chaperones that have
cytoprotective functions: 1) to help protein folding in various intracellular compartments, 2)
to maintain structural proteins, 3) to translocate proteins across membranes into cellular
compartments, 4) to prevent protein aggregation, and 5) to degrade unstable proteins [4-6].
Their expression is increased when cells are exposed to elevated temperatures or other
stressors inducing protein damage. In addition, HSPs are involved in many signaling
pathways and regulate cell death [7, 8], ubiquitin proteasome [9, 10], inflammation [10, 11],
and insulin sensitivity [12] in skeletal muscle, indicating they play essential roles in muscle
function and several pathologies. In fact, the ability of muscular cells to induce HSPs
following stress is significantly decreased with aging [13, 14] and pathological conditions
such as diabetes [15].
Multiple HSPs are expressed in skeletal muscle. HSPs have normally been classified
according to molecular mass. The most prominent are small HSP (A- and B- crystallin and
HSP27), HSP40, HSP60, HSP70, HSP90, and HSP110 families. HSPs are expressed either
constitutively or can be induced by stress stimulation in skeletal muscle [4, 16]. This review
focuses mainly on HSP72, which has been well characterized in skeletal muscle and which
recent evidence suggests is functionally important to skeletal muscle.

1. Expression and Localization of HSPs

in Skeletal Muscle
Molecular chaperones such as HSPs are well known to reduce cellular damage [17, 18].
HSPs have multiple functions in maintaining intracellular integrity via protection, repair, and
even control of signaling for cell death [4, 19].
HSP72, a molecular weight 72-kDa and stress-inducible isoform, is one of the most
prominent isoforms belonging to the HSP70 family, and has been well studied in mammalian
skeletal muscle. Its expression is increased by multiple stressors including thermal stress,
oxidative stress, and exercise [16, 20, 21]. In rat hindlimb studies, HSP72 expression is
upregulated immediately after thermal stress in the soleus muscle, and within 24 h in the
plantaris muscle [21]. In human skeletal muscle, both exhaustive endurance exercise by
cycling [22] and resistance exercise with maximal eccentric repetitions [23] markedly
increase HSP72 expression. On the other hand, decreased mechanical loading, such as cast
immobilization and rat tail suspension, leads to the downregulation of HSP72 with increasing
susceptibility to damage of skeletal muscle [24-26]. Aging attenuates not only HSP72
expression level with muscle weakness, but also the ability to induce HSP72 in response to
stress stimulation [13, 27, 28]. It is reported that skeletal muscle in transgenic mice that
overexpress HSP72 exhibit fewer damaged myofibers and a smaller deficit of force
generation following severe lengthening contractions induced by electrical stimulation [29].
Together, these studies suggest that the expression of molecular chaperone proteins including

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HSP72 may play important roles in protection and repair of skeletal muscle from exerciseinduced stresses.
The level of HSP72 expression is associated with the features of fiber type in skeletal
muscle. Oxidative muscle, predominantly composed of slow-twitch fibers, tends to exhibit a
higher constitutive level of HSP72 than non-oxidative muscle, predominantly composed of
fast-twitch fibers. HSP72 protein content positively correlates with type I myosin heavy chain
(MyHC) content in skeletal muscle [20, 30, 31], which is found in greater abundance in
muscle with slow twitch fibers. For example, the content of HSP72 in soleus muscle,
predominately type I MyHC, is ~2 to 5 folds greater than in plantaris and tibialis anterior
muscles, predominately type II MyHC [20]. Histochemical studies have reported that HSP72
is constitutively expressed in slow-twitch fibers, but onl`y slightly expressed in fast-twitch
fibers (Figure 1).
Slow-twitch fibers are frequently recruited in muscle contraction accompanied by
mechanical, oxidative, or heat stress, suggesting that HSP72 expression is strongly influenced
by exogenous stresses. On the other hand, endogenous factors also seem to contribute to
HSP72 expression.
During the embryonic and early neonatal periods, muscle fibers receive polyinnervation
and most muscles have similar metabolic and contractile properties [32, 33]. Exogenous stress
levels in slow-twitch and fast-twitch fibers seem to be similar in embryonic muscle, but
HSP72 already is more highly expressed in slow-twitch fibers during the embryonic period
(Figure 1). In addition, expression of HSP72 is diminished when type I MyHC content is
reduced by treatment with thyroid hormone and clenbuterol [34, 35]. These findings indicate
that the expression of HSP72 is influenced not only by exogenous stresses, but also by
endogenous factors in muscles with slow twitch fibers, suggesting that it has functions other
than cellular protection.
In response to stress stimulation, several HSPs appear to show different localized
accumulation in skeletal muscle fiber. Paulsen et al. (2007 and 2009) observed that small
HSPs (B-crystallin and HSP27) are enhanced and rapidly translocate from the cytosolic
compartment to cytoskeletal/myofibrillar structures during and/or after exercise in human
skeletal muscle.
Similarly, HSP72 was enhanced and translocated in cytoskeletal/myofibrillar structures
after exercise, but the enhancement and translocation of HSP72 required a longer time than
both small HSPs [36, 37]. Larkins et al. (2012) reported that B-crystallin and HSP27 can
tightly bind to nonmembranous myofibrillar sites at a lower temperature than HSP72 when
skinned (sarcolemma removed ) fibers were exposed to heat stress (HSP27 and B-crystallin
at ~40 vs. HSP72 at 44) [38].
These findings indicate that there are differences in the processes controlling the binding
of two small HSPs and HSP72. Thus, enhancement level and time required for translocation
of HSPs appear to differ dependent on type of HSPs and stress condition, suggesting
differential roles of each HSP according to the physiological situation.

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2. Cellular Protection Mechanisms

by HSPs in Skeletal Muscle
2.1. Enhancement of HSP72 to Exercise in Skeletal Muscle
High intensity exercise that involves repeated eccentric contraction and exhaustive
running induces skeletal muscle damage via mechanical and oxidative stresses and
subsequent activation of proteases [39, 40].

F, fibula; S, soleus; GS, superficial region of the gastrocnemius; GD, deep region of the gastrocnemius;
PS, superficial region of the plantaris; PD, deep region of the plantaris. Scale bars in the right panel
of each age indicate 50m, respectively.
Figure 1. Differential expression intensities of HSP72 in the skeletal muscle fibers. Immunohistochemical staining of plantaris muscles by anti-HSP72 and anti-type I MyHC antibodies was performed
on embryonic day 22 and postnatal day 56. The arrows indicate the same fibers in the serial sections at
each age.

It is well known that prior exercise training attenuates subsequent contraction-induced

injury with acute high-intensity exercise in skeletal muscle. For example, rats previously
trained with either downhill or uphill running show reduced myofiber injury following 90 min

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151

of downhill running as well as an attenuation of the induced increment of glucose-6phosphate dehydrogenase in muscle and creatine phosphokinase activity in plasma [41].
The acquisition of muscle tolerance to contraction-induced muscle damage through
exercise training appears to be partially associated with molecular mechanisms that include
chaperone functions of HSPs as well as neuromuscular and morphological adaptations.
Several studies have reported that prolonged exercise training increases several molecular
chaperone proteins in skeletal muscle, including HSP27, HSP72 and GRP78 [16, 42].
Interestingly, prolonged exercise training notably increases HSP72 expression compared with
a single bout of acute exercise and the enhancement persists for more than 2 weeks after the
last training session (Figure 2).
The enhancement by acute exercise, however, returns to pre-exercise levels within 2
days. This long-term enhancement of HSP72 may be one of the adaptive cellular protective
responses to repeated daily exercise in skeletal muscle.

Cont, control; PE, post-acute exercise; DT, detraining; d, days. *P<0.05 compared with control; P<0.05
compared with PE-4d; #P 0.05 compared with DT-28d. Values are means SEM. Reproduced
from Ogata et al. (2009).
Figure 2. Prolonged exercise training but not a single bout of acute exercise induces long-term
enhancement of HSP72 expression in rat skeletal muscle. Changes of HSP72 levels after acute exercise
(left) and prolonged training (right). Protein expression in plantaris muscle was analyzed by western
blotting during periods after a single bout of acute treadmill running, or treadmill running training for 8
weeks.

In addition, HSP72 protein content positively correlated with muscle weight during
detraining in that study, suggesting contribution to maintaining muscle mass after exercise
training [42].
The enhancements of HSPs in response to acute stress in eukaryotic cells are primarily
induced by transcription and translation of HSPs mRNA accompanied by the activation of
heat shock transcription factors (HSFs) [27, 43, 44]. HSF-1 is known to be activated by
several stressors such as heat, oxidative stress, and denatured proteins, and increases HSP72
mRNA via interaction with the nuclear heat shock element in the promoter region of HSP72.

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Interestingly, long-term enhancement of HSP72 protein is not accompanied by increases of

HSP72 mRNA during detraining (Figure 3).
This phenomenon leads us to hypothesize that long-term enhancement of HSP72 by
prolonged training is not accompanied by transcriptional regulation, but is regulated posttranscriptionally. The fact that HSF-1 and HSF-2 are unchanged during detraining periods
supports this hypothesis. Several previous studies have similarly reported inconsistencies
between HSP72 protein and mRNA levels [28, 45, 46]. Murlasits et al.(2006) showed that
although HSP72 mRNA expression in skeletal muscle was not changed 24 hours following
the last resistance training exercise session in either young or old rats, HSP72 content was
notably increased [28].
There are two possible reasons why the elevation of HSP72 protein content is
unaccompanied by an increase in mRNA levels during detraining periods after prolonged
training. The first is an improvement of translation efficiency from mRNA to protein after
transcription. It is thought that post-transcriptional regulation plays a role in HSP72 protein
production [47].
Under critical situations for cell survival, a damaged cell has the ability to identify and
preferentially translate HSP72 mRNA to the exclusion of other messages, indicating that
HSP72 content may be strongly influenced by post-transcriptional regulation [48, 49]. It has
been reported that microRNAs are associated with translational regulation via 3-untranslated
regions of target mRNAs in skeletal muscle [50, 51]. Xu et al. [50] demonstrated that miR-1,
a muscle-specific microRNA, reduced the level of HSP72 protein without changing the
transcription level in the H9c2 cell line, indicating that the translational efficiency of HSP72
mRNA by miR-1 was reduced. Furthermore, McCarthy and Esser [52] reported that miR-1
expression was downregulated by ~50% in skeletal muscle following seven days of functional
overload. Based on these experimental findings, we can speculate that changes in interference
by microRNAs such as miR-1 may have mediated the enhancement of HSP72 protein that
was unaccompanied by an increase in mRNA during detraining periods.

a
Figure 3. (Continued).

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153

c
Cont, control; DT, detraining; d, days. Values are means SEM. There are no statistically significant
differences among all groups. Reproduced from Ogata et al. (2009).
Figure 3. The expression of HSF-1 (A) and HSF-2 (B) proteins and HSP72 mRNA (C) during
detraining. Protein expression determined by western blotting and mRNA by real-time PCR were
analyzed in plantaris muscle during detraining after treadmill running training for 8 weeks.

A second possible reason for the disconnect between HSP72 protein and mRNA is a
decline in the degradation rate of HSP72 that prolongs the half-life. Protein degradation rate
is strongly affected by changes in cellular stress and the half-life of HSP72 will likely be as
well. The duration of the HSP72 increase caused by transient stress stimulation is short in
mammalian cells [21, 49]. Mizzen et al. (1988) indicated that the half-life of HSP72 induced
by elevated temperature using rat embryo fibroblast cells was around 48 hours [49].
In fact, HSP72 content after an acute single bout of exercise returned to control levels
within 48 hours in rat skeletal muscle. If the half-life of HSP72 is extended by prolonged
exercise training, the notable increase of HSP72 during detraining periods might result from
the accumulation of HSP72 in the muscle. Several studies suggest that exercise training
attenuates the activation of the ubiquitin-proteasome pathway [53]. Prolonged exercise
training may affect the half-life of HSP72 because degradation of HSP72 is mediated by

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Tomonori Ogata

ubiquitination [54]. However, there is no direct evidence regarding whether repeated stress
stimulation prolongs the half-life of HSP72.
Future studies are needed to determine the relation between the half-life of HSP72 and
exercise stimulation. Together, HSP72 content may be regulated not only by transcription, but
also by translational efficiency and protein half-life, depending upon changes in physiological
status within skeletal muscle.

2.2. Prevention of Cellular Damage by Heat Treatment and HSP72

Heat treatment appears to contribute to cellular protection and facilitation of cellular
remodeling after injury in skeletal muscle [24, 55]. Touchberry et al. (2012) showed that prior
heat treatment reduced creatine kinase (CK) level and mononuclear cell infiltration after
eccentric exercise (downhill running) with the increase in HSP72 in rat soleus muscle [55].
Moreover, pre-heat treated rats presented with a greater increase in total protein
concentration and neonatal MyHC expression than did non-heated rats during recovery after
eccentric exercise [55]. Selsby et al. showed that intermittent heat treatment during 1-week of
reloading after immobilization attenuated oxidative stress and improved the rate of skeletal
muscle regrowth [24]. Thus, preconditioning by heat stress seems to have a mitigating effect
on muscle fiber injury and promotes muscle remodeling (recovery from injury), suggesting
that enhancement of HSPs with hyperthermia mediates these effects.
The acquisition of muscle tolerance to stressors inducing muscle damage, such as
mechanical and oxidative stresses, is related to prevention of cell injury and facilitation of
recovery from injury (rehabilitation). There is direct evidence that HSPs contribute
significantly to provide muscle tolerance and recovery from muscle damage, supporting the
benefits of heat preconditioning [29, 56-61]. Liu et al. (2013) showed overexpression of
HSP72 in transgenic mice not only significantly lowers muscle fiber damage compared with
negative littermate control mice after exhaustive exercise, but also can delay muscle fatigue
[56]. Furthermore, Miyabara et al. (2006) observed that the number of necrotic fiber
disruptions induced by cryolesioning was significantly less in transgenic mice with HSP72
overexpression compared with wild-type mice [58]. They also reported that HSP72
overexpression improved muscle mass recuperation and increased the potential for muscle
force generation during reloading after immobilization compared with wild type mice [57].
Therefore, the inhibitory effect of cellular damage by heat treatment might be attributed to
enhanced HSP72 in skeletal muscle.
Although there is no study in genetically-modified skeletal muscle, HSP27 and Bcrystallin also may play a protective role. Koh and Escobedo (2004) demonstrated that
lengthening contraction of muscle using electrode stimulation induced phosphorylation and
translocation of HSP27 and B-crystallin to the Z-disk and membrane in mouse muscle fibers
[62]. Paulsen et al. (2009) showed in humans that eccentric exercise induced an immediate
movement of the HSP27 and B-crystallin from cytosol to cytoskeletal/ myofibrillar
structures, and a repeated bout of exercise caused an accumulation of both HSPs in the Z-disc
and area of disrupted sarcomea, indicating the possibility that small HSPs quickly stabilize
and protect the myofibrillar structure [36].

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2.3. The Impact and Contribution of HSPs on Age-Related

Muscle Weakness
Aging causes a progressive loss of muscle mass and strength, called sarcopenia,
independent of any disease process. Although lifelong preservation of skeletal muscle is
crucial to prevent elderly people from becoming bedridden, there is a normal age-related
decline in the total number of muscle fibers by ~40% between ages 20 and 80 [63]. This loss
of muscle fiber number with sarcopenia is associated with necrotic cell death and with failure
of muscle fiber regeneration, and occurs to a greater extent in fast-twitch than in slow-twitch
fibers [64, 65]. Aging is associated with an increased susceptibility to contraction-induced
muscle damage and disrupted fibers are observed more commonly in older muscle,
suggesting less protection from damage than in younger muscle [64]. The ability of the cell to
induce HSPs following stress stimulation decreases with aging. Aged skeletal muscle in rats
fails to increase HSP72 and HSP27 following muscle contractile activity, but increases do
occur in younger adult rodents [13, 14, 27].
The lack of stress response in muscles of older rodents has been proposed as a major
factor in the development of age-related functional deficits.
The constitutive overexpression of HSP72 improves even normal age-related muscle
dysfunction. Lifelong HSP72 overexpression in transgenic old mice can depress age-related
increases in lipid peroxidation, catalase activity, and protein carbonyls [59]. In addition,
pharmacological enhancement of HSP72 induced by 17-(allylamino)-17-demethoxygeldanamycin treatment facilitates successful recovery of maximum tetanic force generation in
aged-skeletal muscle at 28 days following lengthening muscle contraction [60]. This
facilitation of recovery from contraction-induced damage is similarly shown in older
transgenic mice with HSP72-overexpression [29]. Slow-twitch fibers containing
constitutively high levels of HSP72 are less susceptible to injury compared to fast-twitch
fibers with low levels of HSP72, both during aging and following lengthening-contraction
exercises. Although it is unclear how HSP72 provides protection in muscle fibers, Fu and
Tupling (2009) showed that HSP72 can directly bind sarcoplasmic reticulum Ca2+-ATPase
(SERCA)2a and that overexpression of HSP72 in HEK-293 cells not only prevents thermal
inactivation of SERCA2a but also increases maximal SERCA2a activity during heat stress
[66]. HSP72 may protect muscle fibers either through direct interaction with organelles, or
indirectly through suppression of oxidation.
Several mitochondrial HSPs, including HSP10, HSP60, and mtHSP70, are hypothesized
to be responsible for protein transport, folding, refolding, and prevention of protein
denaturation as molecular chaperones in the mitochondrial matrix [67]. Lifelong
overexpression of HSP10 in transgenic mice clearly prevented age-related decreases in
maximum force generation and fiber cross sectional area in muscle of old wild-type mice
[61]. In addition, levels of carbonylated mitochondrial proteins in transgenic mice were lower
than in wild-type mice, indicating that HSP10 may prevent age-related muscle weakness and
atrophy. It is well known that mitochondrial dysfunction is associated with progression of the
aging process [68] and that oxidative damage to mitochondrial DNA and proteins
accumulates over time due to ROS produced by the electron transport chain. Segmental
mitochondrial abnormalities containing mitochondrial DNA deletion mutations have been
observed in aged skeletal muscle [69]. Muscle fibers harboring mitochondrial mutations often

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Tomonori Ogata

display sectional atrophy, splitting, and increased steady-state levels of oxidative nucleic
damage [69, 70].
Thus, age-related mitochondrial DNA mutation is one of the leading candidates to induce
muscle fiber disruption and atrophy through ROS generation. To determine whether the
benefit from overexpression of mitochondrial HSP10 is related to the maintenance of
mitochondrial DNA and proteins in skeletal muscle, an examination of the relationship
between mitochondrial dysfunction and mitochondrial HSPs would provide major progress in
the prevention of age-related muscle atrophy, or sarcopenia.
Together, attenuated heat shock responses result from aging and cause chronic
inflammatory reactions and ROS production via organelle dysfunction, leading to age-related
muscle weakness [13]. As countermeasures, increasing HSPs can prevent or delay cellular
damage and protein degradation in aged skeletal muscle [59, 60].
These data demonstrate that the development of age-related muscle weakness and
atrophy is not inevitable. However, because specific triggers for impaired induction of HSPs
during aging remains unknown at the present time, further studies are required to elucidate
the underlying mechanisms.

3. The Prevention of Disuse-Related Muscle

Atrophy by Heat Treatment and HSPs
Disuse muscle atrophy occurs in response to prolonged bedrest, cast immobilization, or
space flight. Muscular atrophy leads to physical decline, can delay recovery from muscle
injury, and can cause elderly people to be confined to the bed. Therefore, it is important to
elucidate the mechanisms of muscle atrophy and to take preventive measures.
Prior heat stress can diminish subsequent unloading-induced skeletal muscle atrophy
[71]. Higher HSP72 levels as well as an attenuated muscle mass loss have been shown in
heat-treated animals compared with untreated animals after hindlimb unloading, suggesting a
contribution of HSP72 to muscle mass preservation [71]. In addition, prior heat stress
attenuates de novo expression of MyHC IId/x and decreasing MyHC I/ in soleus muscle
following 2 weeks of immobilization, and prevents disuse-induced fiber type transformation
from slow to fast [72]. To support this, it is reported that continuous mild heat stress induces a
fast-to-slow fiber type shift in a myocyte cell line [73]. It has been hypothesized that
increased expression of HSP72 enhances protein synthesis by maintaining the elongation
phase of protein synthesis. Furthermore, it has been hypothesized that HSP72 protects
proteins from proteolysis by refolding damaged proteins. In addition to the chaperone
functions of HSPs, it recently has been shown that HSPs could contribute to attenuation of
disuse-induced protein degradation via regulation of the ubiquitin proteasome pathway [911].
Muscle atrophy results when protein degradation exceeds protein synthesis. Preferential
degradation of muscle proteins has been observed in response to inactivity, denervation, and
fasting. Protein degradation in skeletal muscle is mainly mediated through the ubiquitin
proteasome pathway [10, 74]. E3 ubiquitin ligases, atrogin-1 (muscle atrophy F-box, MAFbx)
and muscle specific RING finger 1 (MuRF1), are upregulated in multiple muscle atrophy
models [53, 75]. MAFbx and MuRF1 knockout animals exhibit attenuated muscle atrophy

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157

following denervation [76]. These E3 ligases are known as representative marker proteins in
the ubiquitin proteasome pathway. Both MAFbx and MuRF1 are controlled directly or
indirectly by forkhead box O (FOXO) and nuclear factor-B (NF-B) [77, 78]. FOXO
signaling is able to account for ~40% of disuse muscle fiber atrophy [9]. Therefore, regulation
of FOXO and NF-B transcriptional activity plays an important role for protein degradation
via the ubiquitin proteasome pathway in muscle atrophy [9, 77].
HSP72 expression is downregulated in atrophying muscle during hindlimb
immobilization in mice [71]. On the other hand, HSP72 overexpression restrains progression
of disuse-related muscle fiber atrophy [9, 10, 79]. Senf et al. (2008) demonstrated that HSP72
overexpression by injection of an HSP72 expression plasmid abolished any increase of total
ubiquitinated proteins in muscle after seven days of immobilization [10]. In addition, they
reported that FOXO3a transcriptional activity was increased by 7-fold following seven days
of hindlimb immobilization, but was completely abolished in muscle injected with HSP72.
This interaction between HSP72 and FOXO3a is also demonstrated by the inhibition of
FOXO3a-dependent transcription of MAFbx by HSP72 [9, 10]. Similar to FOXO3a,
enhancement of NF-B transcriptional activity during muscle disuse is completely abolished
by HSP72 overexpression [10].
Pharmacological enhancement of HSP72 by 17-demethoxygeldanamycin can also
attenuate disuse-induced muscle atrophy and increasing -calpain and conjugated ubiquitin
levels [79].
Disuse-induced muscle atrophy in senescence can also be attenuated by enhancement of
HSP72 [80]. Dodd et al. (2009) reported that HSP72 overexpression by plasmid injection
prevented decreasing muscle fiber cross sectional area in soleus muscle in both young and
senescent rats following immobilization for 6 days.
In addition, HSP72 overexpression prevented the increase in NF-B and the decrease in
inhibitor of B (I-B) expression in senescent animals after immobilization [80]. Because
aging and reduced physical activity appear to have a synergistic effect on muscle wasting,
HSP72 might contribute to slower progression of age-related muscle deterioration.
HSP27 is also associated with the ubiquitin proteasome pathway during disuse-induced
muscle atrophy. Dodd et al. (2009) showed that hindlimb immobilization caused a 35%
decrease in the mean fiber cross sectional area of nontransfected muscle and an 18% decrease
in muscle transfected with an HSP27 expression plasmid, indicating attenuation of disuse
atrophy by HSP27 [11].
In this model, NF-B transcriptional activity increased ~4.5-fold following seven days of
immobilization, but HSP27 overexpression abolished this increase. In addition, HSP27
inhibited the disuse-induced increase in MuRF1 and atrogin-1 transcription. Unlike HSP72,
HSP27 had no effect on FOXO transactivation; HSP27 overexpression does not inhibit
disuse-induced FOXO-dependent reporter activity [11]. HSP27 seems to be a negative
regulator of NF-B in skeletal muscle in vivo, and inhibits MuRF1 and atrogin-1, and
attenuates skeletal muscle disuse atrophy. Therefore, HSP72 regulates the ubiquitin
proteasome pathway through both FOXO and NF-B, and HSP27 regulates it through NF-B
to attenuate disuse-related muscle atrophy.
These findings partly explain why heat stress treatment inhibits progression of disuseinduced muscle atrophy.
A common event in atrophying muscle is the loss of myonuclei or satellite cells via
apoptosis [81]. The change of physiological conditions related to muscle atrophy, such as

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hindlimb unloading, denervation, and spinal cord transection, results in a decrease in the
number of myonuclei and an increase in apoptotic myonuclei in rat skeletal muscle [82-84].
Based on the concept that the nuclear domain, which represents the amount of cytosol per one
myonucleus, is unchanged following the alteration of muscle size, a decrease in myonucleus
number by apoptosis may cause muscle atrophy.
It is indicated that HSP72 is involved in suppression of the apoptotic cell death cascade at
different levels. It can inhibit cytochrome C release from mitochondria and activation of
various caspases [85, 86]. For example, overexpression of HSP72 appears to inhibit apoptosis
by blocking caspase activation and activity in lymphoid tumor cell lines [87]. In C2C12
muscle cells, the increase of HSP27 and HSP72 by heat treatment attenuates apoptotic cell
death in response to H2O2 [88]. Because HSP72 is downregulated in atrophying muscle, the
reduced anti-apoptotic effect of HSPs may accelerate disuse-induced atrophy.
Together, disuse-related muscle atrophy is associated with protein degradation by the
ubiquitin proteasome pathway and with nuclear elimination by apoptosis induction.
Increasing HSPs presumably can attenuate muscle atrophy by regulation of signal
transduction on ubiquitin and apoptosis pathways, and by helping protein folding as a
molecular chaperone.

4. Preventive Effect of HSP72

on Muscular Dystrophies
Muscular dystrophies comprise a group of heterogeneous genetic myopathies associated
with progressive skeletal muscle wasting that causes repeated muscular cell necrosis and
regeneration. There is currently no cure for patients with muscle dystrophies. However, a
recent study indicates that HSP72 can ameliorate progressive muscular dystrophy [89].
The X-linked muscle-wasting disease Duchenne muscular dystrophy (DMD) is caused by
mutations in the gene encoding dystrophin and affects about 1 in 3,500 live male births.
Compromised intracellular Ca2+ regulation in DMD muscle triggers chronic inflammation and
repeated cycles of degeneration and regeneration of muscle fibers [90]. It has been speculated
that the dysfunction of SERCA mediating Ca2+ reuptake into the sarcoplasmic reticulum (SR)
results in a disorder of Ca2+ homeostasis and subsequent inflammatory events [91].
Goonasekera et al. (2011) showed that genetic overexpression of SERCA1 in skeletal muscle
dramatically attenuates the manifestations of muscular dystrophies such as the increase of
disrupted muscle fibers and serum CK [92]. Therefore, improvement of SERCA function has
promise in the therapy for muscle dystrophies.
Recently, it was reported that increasing the expression of intramuscular HSP72
preserves muscle strength and ameliorates the dystrophic pathology via protection of SERCA
function [89]. Gehrig et al. (2012) reported that SERCA did not properly function in two
DMD mouse models, dystrophin-deficient mdx mice and utrophin/dystrophin double
knockout mice (dko mice) [89]. They showed that muscle-specific overexpression of HSP72
in transgenic DMD mice preserved muscle strength and SERCA activity, and ameliorated
necrotic fiber disruption compared with non-transgenic DMD mice [89]. In addition,
pharmacological induction of HSP72 by BGP-15 treatment similarly prevents impaired
muscle strength and SERCA activity in both mdx and dko mice. Interestingly, overexpression

The Functional Role of Heat Shock Proteins in Skeletal Muscle

159

of HSP72 by BGP-15 treatment not only ameliorates muscle wasting, but also can prolong
lifespan in severely dystrophic dko mice. These findings provide evidence that increasing
HSP72 in skeletal muscle can become a significant therapeutic target for ameliorating DMD
via interaction with SERCA.
In skeletal muscle from DMD mice, IB kinase (IKK)/ NF-B signaling is persistently
enhanced in immune cells and in regenerating muscle fibers, contributing to chronic
inflammation [93]. Heterozygous deletion of the p65 subunit of NF-B in the mdx mice
resulted in amelioration in muscle pathogenesis and enhanced regeneration of myofibers.
Similarly, genetic deletion and pharmacological inhibition of IKK attenuated muscle
pathology and augmented skeletal muscle regeneration in the mdx mice [93]. Thus,
depression of the IKK/ NF-B-related inflammatory response seems to be one of the
therapeutic targets for DMD. On the other hand, overexpression of HSP72 by transfection in
skeletal muscle can inhibit NF-B reporter activity and progression of muscle atrophy [10]. In
addition, pharmacological activation of HSP72 can depress phosphorylation of JNK in a
mouse obesity model [12].
Together, HSP72 seems to play a role in ameliorating muscular dystrophies by stabilizing
SERCA function and attenuating inflammatory responses. The anti-apoptotic effect of HSP72
[85, 88] and its role in protein folding and refolding [17] also might ameliorate progressive
DMD. Further studies are required to confirm the biological roles of HSP72 and other HSPs
on muscular dystrophies, because there is little information at this time.

5. Role of HSPs on Insulin Sensitivity as

Important Mediators in Skeletal Muscle
Skeletal muscle takes up approximately 75 to 85% of the body's glucose load. It is the
major tissue responsible for whole-body insulin-mediated glucose disposal and contributes to
prevention of insulin resistance [94, 95]. An active lifestyle with frequent muscle contractions
contributes to prevent the onset of diabetes and is more effective than treatment with
metformin [96]. In an earlier study by Hopper et al. (1999), patients with type 2 diabetes
subjected to heat therapy using a hot tub over 3 weeks lowered their fasting plasma glucose
and glycosylated hemoglobin levels [97]. Evidence shows that heat treatment blocks the
development of insulin resistance in response to a high-fat diet [98, 99]. Gupte et al. (2009)
showed that high-fat fed rats treated weekly with heat had lower serum insulin and effective
glucose clearance after glucose injection compared with non-heat treated high-fat fed rats
[98]. They observed that heat treatment results in decreased activation of JNK and IKK-,
which are implicated in insulin resistance, and upregulation of HSP72 and HSP25. A single
bout of heat treatment also can enhance insulin-stimulated glucose disposal in aged skeletal
muscle [100]. These benefits of thermal treatment to insulin resistance have been confirmed
by studies of heat treatment-induced HSPs [12, 101].
In a human study, it was demonstrated that HSP72 mRNA in muscle was significantly
lower in type 2 diabetic patients than in healthy subjects, and HSP72 mRNA content in
muscle inversely correlated with the rate of glucose uptake and other measures of insulinstimulated carbohydrate and lipid metabolism [15]. Furthermore, obese insulin-resistant
humans have lower HSP72 protein levels compared with healthy people [12]. Although heat

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Tomonori Ogata

treatment upregulates HSP72, the increase is attenuated by a high-fat diet [12]. In addition,
the level of HSP72 enhancement by exercise training is less pronounced in diabetic animals
than in non-diabetic animals. Endurance training induces the activation and expression of
HSF-1 which is a transcriptional regulator of HSPs, in skeletal muscle from non-diabetic
animals, but not in diabetic animals [102].
It is well known that HSP72 is downregulated in aged skeletal muscle [13, 64]. However,
in a recent study using vervet monkeys, it was observed that HSP72 was not decreased with
aging when animals consumed a low-fat diet and did not have chronic insulin resistance,
which indicated that aging did not reduce HSP72 in the absence of insulin resistance [103].
Therefore, inhibition at the transcriptional level with insulin resistance may be related to
downregulation of HSP72 in skeletal muscle, but the mechanism for the reduced HSP72
expression in diabetes still remains unclear.
The interaction between HSP72 and insulin resistance has been shown in animal models
that overexpress HSP72 [12]. Chung et al. (2008) reported that transgenic mice with musclespecific overexpression of HSP72 have lower levels of fasting glucose and insulin than wildtype mice after 16 weeks on a high-fat diet. In addition, mice with HSP72 overexpression
display notably improved glucose and insulin tolerance compared with wild-type mice when
placed on a high-fat diet [12]. Upregulation of HSP72 by BGP-15 treatment also shows the
same effects as the transgenic model on insulin resistance [12]. These findings indicate that
HSP72 counteracts the high-fat diet-induced development of insulin resistance and provide
supportive evidence for the beneficial effects of heat therapy.
Obesity- and hyperlipidemia-induced insulin resistance is characterized by increased
oxidative stress and inflammation. Increased ROS and peroxidation in lipid and protein are
common features observed in aging and obesity. Oxidative stress promotes activation of
inflammatory kinases, such as JNK and IKK-, and induces insulin resistance. The effect of
HSP72 on insulin resistance may be associated with its anti-inflammatory action. Activation
of the inflammatory factors JNK and IKK is commonly observed in diabetic patients, and
can cause phosphorylation of insulin receptor substrate (IRS)-1 on serine 307, leading to
insulin resistance [104, 105]. A high-fat diet leads to increased JNK phosphorylation, but this
increase is suppressed by overexpression of HSP72 [12] and heat treatment [98], supporting
other studies that show HSP72 to be a negative regulator of JNK. HSP72 antisense
oligonucleotides block HSP72 expression in NIH 3T3 cells and concomitantly abolish the
suppressive effect of JNK activation and apoptosis [106]. Increased HSP72 by antioxidant
lipoic acid in skeletal muscle also prevents diet-induced insulin resistance through JNK
inhibition [107]. It seems that HSP72 can modulate stress-activated signaling by directly
inhibiting JNK. To support the interaction between HSP72 and JNK, phosphorylation of JNK
in response to a high-fat diet is lower in slow-twitch muscle, which has constitutively higher
HSP72 content, than in fast-twitch muscle, which has lower HSP72 content [98].
In addition to the inhibition of inflammatory kinases, HSP72 may contribute to reduced
ROS generation through the mitochondrial respiratory function and concomitantly attenuate
the development of insulin resistance [108, 109]. In human skeletal muscle, HSP72 mRNA
expression positively correlates with the activity of mitochondrial enzymes such as citrate
synthase (CS) and -hydroxyacyl-CoA dehydrogenase (-HAD) [110]. Transgenic mice that
overexpress HSP72 show higher enzyme activities of CS and -HAD in skeletal muscle than
wild-type mice [12]. HSP72 overexpression in astrocytes actually reduces the induction of

The Functional Role of Heat Shock Proteins in Skeletal Muscle

161

ROS [108]. Thus, decreasing ROS generation by HSP72 may also account for reduced insulin
resistance following heat therapy.
These findings provide strong evidence that HSP72 induction in skeletal muscle can be a
therapeutic target for insulin resistance. Because HSP72 can be easily increased by heat
treatment (e.g. sauna, hot pack, and infrared radiation) and/or exercise, it potentially has wide
clinical application for diabetic patients.
Although there is little information about the relationship between other HSPs and
diabetes, a recent study by Marker et al. (2012) indicates that HSP60 released by adipocytes
contributes to the development of insulin resistance in skeletal muscle cells [111]. They
observed that plasma HSP60 levels were higher in obese than in non-obese men, and were
positively correlated with BMI and insulin resistance. HSP60 treatment impaired insulin
signal transduction, which decreased phosphorylation of AKT and GSK3, and decreased
insulin-stimulated glucose uptake in skeletal muscle cells. In addition, treatment of HSP60
activates ERK1/2, JNK, and NF-B and increases phosphorylation of IRS-1 [111]. Therefore,
contrary to the function of HSP72, HSP60 may function as a negative regulator of insulin
signaling via an inflammatory effect, and impair insulin sensitivity in skeletal muscle,
although there is no direct evidence in vivo at this time.
Endoplasmic reticulum (ER) stress has been assumed as one of the central mechanisms of
impaired insulin sensitivity in several cell types [112, 113]. ER stress is defined as an
accumulation of unfolded proteins in the ER, and the activation of ER stress signaling
(describe as unfolded protein response) is associated with apoptotic death of -cells in the
pancreas and impaired insulin signaling [112].

Heat stress

Exercise

Inactivity

HSP72

ROS

Myofiber
damage

Ubiquitin
proteasome

Sarcopenia

Inflammation

Protein
folding

Apoptosis

Disuse
atrophy

Muscle
dystrophies

Insulin
resistance

Figure 4. Functional roles of HSP72 in skeletal muscle. The schema summarizes the functions and
contributions of HSP72 in skeletal muscle described in this chapter.

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Tomonori Ogata

On the other hand, alleviated ER stress results in restoration of systemic insulin

sensitivity and enhancement of glucose uptake in muscle [114]. It is known that GRP78 is an
abundant chaperone in ER and increasing its expression attenuates the activation of the ER
stress signal and apoptosis induction [115, 116]. In a recent study using C2C12 myotube,
however, inhibition of ER stress by GRP78 overexpression could not improve insulin signal
transduction [113]. Therefore, the relationship between ER stress and GRP78 underlying
insulin resistance in skeletal muscle is controversial.
Potential candidates of pathways mediating insulin resistance are: 1) mitochondrial
dysfunction and ROS production, 2) chronic inflammation and activation of proinflammatory factors, such as JNK and NF-B, and 3) activation of ER stress signaling.
Several HSPs appear to play roles in these pathways and positively or negatively regulate
insulin sensitivity. The fact that HSPs regulate insulin sensitivity in skeletal muscle provides
supportive evidence for prevention and improvement effects of diabetes by regular exercise.

Conclusion
In summary, several HSPs are expressed in skeletal muscle and the expression is altered
in accordance with physiological conditions. In particular, HSP72 appears to play multiple
roles in maintaining physiological function and ameliorating pathological conditions by: 1)
protecting muscle damage by several stressors including exercise, 2) ameliorating muscle
atrophies induced by aging, disuse, and pathological dystrophin deficiency, and 3)
ameliorating the development of insulin resistance in skeletal muscle (Figure 4). Accumulated
recent studies provide evidence that HSP72 is associated with the life cycle of multiple
proteins and cells through signal transduction pathways involving inflammation, the ubiquitin
proteasome, and apoptosis. Therefore, some health benefits acquired by exercise can be
accounted for in term of expression and functions of HSP72. A better understanding of the
role of HSPs in skeletal muscle promises to lead to clinical applications for multiple
physiological conditions.

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In: Basic Biology and Current Understanding of Skeletal Muscle ISBN: 978-1-62808-367-5
Editor: Kunihiro Sakuma
2013 Nova Science Publishers, Inc.

Chapter 7

Mechanical Stress and

Myofibrillar Structure
Fuminori Kawano
Department of Health and Sport Sciences, Graduate School of
Medicine, Osaka University, Toyonaka City, Osaka, Japan

Abstract
Myofibrils are the contractile components of the skeletal muscle fibers. However, the
myofibril does not simply work as a contractile apparatus, but also plays a crucial role in
the sensing of mechanical stress and capture of the signaling molecules. The giant
sarcomeric protein, titin, spans from the Z-disc to the M-band, and acts as a mechanical
sensor. Muscle-specific RING finger 1, E3 ubiquitin ligase, and calpain-3 bind to titin
and contribute to the stress response of myofibrils following the structural change of titin.
Lack of these proteins causes a severe myopathic disease. The Z-disc mediates the
important signalings related to the muscle characteristics via various proteins, including
calcineurin, the protein phosphatase that activates the slow-twitch muscle-specific gene
transcription in a Ca2+ concentration-dependent manner, and the muscle LIM protein, the
transcriptional co-regulator of the basic helix-loop-helix responsible consequence such as
MyoD and myogenin. Furthermore, some differences are found in the core structure of
sarcomere, including a thicker Z-disc, a longer thin actin filament, and longer titin, in
slow-twitch compared to fast-twitch muscle fibers. These properties potentially affect the
mechanical strength and the signal transduction. The adaptive transformation is
postulated on the structural organization as well as its components, in accordance with
the level and the pattern of the muscle activity.

Corresponding author: Department of Health and Sport Sciences, Graduate School of Medicine, Osaka University,
1-17 Machikaneyama-cho, Toyonaka City, Osaka 560-0043, Japan, E-mail: kawaco@space.hss.osaka-u.ac.jp;
Phone: +81-6-6850-6018; Fax: +81-6-6850-6030.

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Introduction
Skeletal muscle fibers undergo hypertrophy in response to enhanced mechanical stress
[1-6], a key physiological stimulus resulting from muscle activities such as exercise and
resistance training. It is well known that the level of the protein synthesis is critical for the
production of the muscle-specific proteins and contractile cytoskeletons, leading to
hypertrophy or atrophy, if the workload is altered from the sedentary level [7-9]. In addition
to the influences in the muscle mass, metabolic properties are improved by the repeated use of
muscles. In animal studies, chronic voluntary running up-regulates the mitochondrial
function, enabling it to prevent the age-related accumulation of body fat [10-12]. These facts
highlight the importance of muscle activity for the regulation of muscle characteristics.
However, it has not been fully understood how muscle fibers sense the mechanical stress
produced by the contraction and force development, and transmit the signals to the pathways
providing the muscular characteristics. To date, many studies have been carried out to
investigate the precise structure of myofibrils of skeletal muscle fibers, which form an
essential cytoskeleton generating the muscle force. In addition, it has recently been
demonstrated that myofibril does not simply function as a contractile apparatus, but it also
plays a crucial role in sensing of the mechanical stress and capture of the signaling molecules.
Skeletal muscles can be classified into mainly two types: those exhibiting a slow-twitch
contractile property with oxidative metabolism and those with a fast-twitch contractile
property with anaerobic metabolism [13-15]. In mammals, slow-twitch muscles generally
exhibit tonic activity in order to maintain the posture of an individual against the force of
gravity. In contrast, fast-twitch muscles are activated during locomotion and generate great
force. The level and the pattern of the applied mechanical stress, as well as the required
strength of myofibrils, appear to be different between slow- and fast-twitch muscles, which
may lead to the different potential to respond of the signaling pathways to the physiological
stimuli due to their unique components. This chapter reviews the current information on
myofibrillar structure, protein components and their roles in the regulation of slow- and fasttwitch skeletal muscle fibers

Basic Structure and Main Components of Myofibril

Overview
The sarcomere is the smallest unit that can induce contractile action, and is composed of
several main cytoskeletons (Figure 1). Thin and thick filaments form the main long axis of the
myofibril. Under light microscopy, the striation is shown to be made up of overlapping or
non-overlapping regions of thin and thick filaments.
In electron micrographs, I-bands lacking thick filaments appear bright, whereas the Abands form visually dark regions containing a number of cross-bridges as well as both thin
and thick filaments. M-lines (M-band), seen at the middle space between A-bands, lack the
thin filaments, and appear slightly bright because cross-bridges are not formed. The I-band is
divided by the Z-disc. The precise details in regard to the main structure of Z-discs are
presented in the sections below.

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173

Figure 1. Main structure of sarcomere. Typical differences in sarcomeres between fast- (upper) and
slow- (lower) twitch muscle fibers are shown. Note that thicker Z-disc [67], longer thin filament [31,
36], longer titin [21] and differential pattern of M-lines [61-63] are marked in the sarcomere of slowtwitch fibers.

The giant protein titin, also known as connectin, is the main source of elasticity in the
sarcomeres [16, 17]. The titin filament are inserted, along with the N-terminus, in the Z-disc
and reach all way to the center of the sarcomere, the M-band. This length corresponds to half
of a sarcomere, ~1.0 m or more depending on the stretch of the sarcomeres. Titin is
expressed in potentially millions of different isoforms generated by alternative splicing from
the transcript of a single titin gene [18, 19]. The size of the human titin isoforms sequenced so
far ranges from 625 kDa for the low-abundance novex-3 isoform up to 3,700 kDa for the
human soleus titin [20], which is the so-called N2A isoform characteristic of skeletal muscles.
In a set of ~40 different rabbit skeletal muscles, the size range of N2A isoforms was found to
be ~3,300 to 3,700 kDa [21]. The extensible segment of titin begins ~100 nm from the Z-disc
center and bridges the remaining I-band portion of the sarcomere as an elastic spring until it
enters the thick filament (A-band) portion. The PEVK domain, a part of the I-band region,
plays a key role for the elasticity of sarcomeres [17]. The static tension is not developed by
sarcomeres while the PEVK domain is kept at the slack position, even though the myofibrils
are stretched up to approximately 2.5 m of the sarcomere length [17, 22]. Beyond this
length, a linear extension of the titin PEVK domain generates an exponential increase in the
static tension. The N2A domain locates at a neighbor in the N-terminal end of the PEVK
domain in the I-band [16, 17]. This elastic property of titin is the main route of mechanical
continuity through relaxed sarcomeres and is also important for the elastic recovery of resting
sarcomere length after active shortening or passive stretch of the muscle. The tension
developed by such connection centers the A-band in the sarcomere.

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Degradation of titin and/or nebulin induces the thick filament misalignment on stretch or
activation, leading to a failure in the effective force production [23].

Thin Filament
The major component of the thin filament is F-actin. The three-component complex
consists of troponin I, troponin C and troponin T localized on the actin filament and interferes
with the mediation of muscle contraction [24, 25] (Figure 2). By shifting the position along
thin filaments in response to Ca2+ binding to troponin C, tropomyosin exposes the myosinbinding site on actin, starting the formation of cross-bridge cycling and contractile action [2630]. The length of the thin filament ranges from ~0.95 to ~1.30 m from the Z-disc in skeletal
muscles [31-33]. Slow-twitch muscle has a longer thin filament than fast-twitch muscle
(Figure 1).
Nebulin is a giant protein spanning most of the length of the thin filament with the Cterminus anchored at the Z-disc to N-terminal region, the M1M2M3 domain, directed toward
the pointed-end [34] (Figure 2). The number of its modular nebulin motifs closely correlates
with the I-band length [35]. Some recent studies in nebulin-deficient mouse models [36, 37]
and human myopathy patients [38] have shown an important contribution of nebulin to the
regulation of the thin filament length. Witt et al. [37] reported that shorter thin filaments with
an average length of ~0.8 m were noted in nebulin-deficient muscle, compared with the
length in wild type muscle, which was a constant 1.2 m. Such reduction in the thin filament
length caused the severe effects in the Ca2+ sensitivity and force production of myofibrils
[38].
Stabilization of the thin filament is also regulated by actin capping proteins (Figure 2).
CapZ is a barbed-end capping protein at the Z-disc, and organizes the correct assembly of the
filaments at the Z-disc [39]. Tropomodulin (Tmod), a pointed-end capping protein, binds
strongly to actin in the presence of tropomyosin [40]. Four isoforms of Tmod are present
within mammals (Tmod1-4). Tmod1 is expressed in terminally differentiated cells, such as
striated muscles, Tmod2 is expressed in neuronal tissues, Tmod3 is expressed ubiquitously,
and Tmod4 is restricted to skeletal muscles in mammals [41-47]. Tmod1, -3 and -4 potentially
regulate the length of the thin filaments in skeletal muscle sarcomeres. Nebulin does not
extend all the way to the pointed-end of the thin filament from the Z-disc, because the actinbinding site, the M1M2M3 domain, of nebulin is localized at a relatively fixed distance ~0.9
m from the Z-disc [31, 32]. Previous studies [31-33, 48-50] have indicated that Tmod
regulates the length of a relatively more dynamic, nebulin-free, pointed-end extension,
whereas nebulin mechanically and chemically stabilizes an ~0.9- to ~1.0-m-long core region
of the thin filament. The localization site of Tmod from the Z-disc ranges from 1.11 m in the
extensor digitorum longus muscle, to 1.22 m in the soleus muscle, to 1.26 m in the
embryonic flank, which is consistent with the lengths of the thin filaments found in rabbit and
mouse muscles [31, 36]. Furthermore, the length of the thin filament is inversely correlated
with the composition of the type IIx myosin heavy chain in human skeletal muscles, also
suggesting a shorter thin filament in fast-twitch muscles [51]. The sarcomere with a shorter
thin filament can generate the maximum force when it is lengthened to approximately 2.5 m
(the optimal sarcomere length) and thereafter the force production decreases as the sarcomere
length is stretched more [51].

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175

Figure 2. Thin filament organization. Thin filament, formed by the polymerization of actin molecules,
are capped with CapZ at the barbed-end in the Z-disc [39], and with Tmod at the pointed-end [40].
Nebulin binds to the thin filament, spans through most of the thin filament length, and stabilizes the
filament. The length of nebulin-free region observed in the pointed-end is regulated by Tmod, resulting
in the differential mechanical property (see the main text for precise details). Tropomyosin is also
known as actin binding protein, interfering the interaction of actin and myosin heavy chain. Binding of
Ca2+ to TnC triggers the formation of cross-bridges and contraction subsequently start [26-30]. Tmod,
tropomodulin; TnI, troponin I; TnC, troponin C; TnT, troponin T.

However, the optimal sarcomere length becomes slightly longer in the sarcomere with a
longer thin filament. Therefore, slow-twitch muscles enable the development of tension rather
at the stretched position.
Tmod1 null cardiac myocytes in the embryonic mouse heart completely fail to assemble
myofibrils and do not beat, resulting in the failure of cardiac looping and aborted
development [52, 53]. In contrast, Tmod1 is not necessary for the assembly of myofibrils or
for the development of the skeletal muscles, since Tmod3 and -4 compensate for the absence
of Tmod1 structurally [32]. However, the absence of Tmod1 results in depressed isometric
force production and systemic locomotor deficits in the skeletal muscles of Tmod1 null mice
[32].

Thick Filament
The thick filament is composed of myosin heavy chain and light chain. These myosins
form hexamers characterized by a two-headed structure, a flexible neck region containing the
myosin light chains and a long coiled-coil tail domain. The catalytic domain of the myosin
heavy chain contains the ATPase and actin-binding activities [54].
Myosin-binding protein C (MyBPC), an accessory protein of the thick filament, directly
interacts with both thin and thick filaments [55, 56]. To date, three isoforms have been
identified in skeletal muscles and cardiac myocytes. MyBPC1 and -2 are restricted to skeletal
muscles, whereas MyBPC3 is expressed exclusively in cardiac myocytes [57, 58]. MyBPC3
modulates the formation of cross-bridges in cardiac myocytes and directly affect the Ca2+
sensitivity in the myofibril [56]. An increase in Ca2+ sensitivity has been shown in the
MyBPC3-deficient myocardium [59]. In skeletal muscles, the mutations in the skeletal
muscle isoforms of MyBPC were also identified in patients suffering from a disorder
characterized by congenital contractures of the hands and feet [60].

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In electron micrographs of the sarcomere, the M-band, a series of several M-lines,

appears in the center of the A-bands where thick myosin filaments are positioned. Five major
M-lines (M6', M4', M1, M4 and M6) can be observed within the M-band. In general, the fasttwitch skeletal muscle fibers have a three-line pattern (M6/M6' lines missing), whereas the
slow fibers have a four-line pattern (M1 line missing) [61-63] (Figure 1). M-lines are formed
by the web structure of myomesin and M-protein. These two proteins bind to the central zone
of myosin filaments at the N-terminal domains. Myomesin interacts with the C-terminus of
titin. Titin is believed to counteract the force imbalance caused by a different profile of
activated cross-bridges, by producing the restoring force directed to the center of the
sarcomere. It is predicted that the myomesin web in the M-band supports and stabilizes the
action of the titin filaments [64].

Z-disc
The Z-disc is the essential structure for the production of tension in muscle fibers [65].
The major component of many of proteins of the Z-disc is -actinin, which plays a key role in
mediating, integrating and preserving the Z-disc [66]. The thin actin filaments, titin and
nebulin directly attach to the Z-disc. Z-discs can be observed in the longitudinal view, across
the sarcomere, of electron micrographs as dense bands with a width ranging between 30 and
50 nm in fast-twitch muscles, and 100 and 140 nm in slow-twitch and cardiac muscles [67]
(Figure 1). Z-disc widths are determined by the number of layers of -actinin, which can
range from two to six or more. Interestingly, the thinner Z-discs in fast-twitch muscles
produce the higher shortening velocities of sarcomeres. Many proteins have recently been
shown to associate with the Z-discs, and to be capable of sensitizing a mechanical stress and
subsequently mediating a signal transduction (see the sections below). The fiber type-related
differences in the thickness of Z-discs may result from the adaptation to specific activities and
stimuli.
The -actinin family comprises four members, with -actinin-1 and -4 being expressed in
non-muscle tissues and -actinin-2 and -3 being striated muscle-specific isoforms [68]. Slowtwitch skeletal muscle fibers express predominantly -actinin-2, whereas the expression of actinin-3 is restricted to fast-twitch fibers [69]. Mutant mice lacking -actinin-3 show the
compensatory replacement of missing actinin with -actinin-2, suggesting that -actinin-3 is
not a critical component for myofibril formation in fast-twitch fibers [70, 71]. In practical
terms, -actinin-3 appears to be a functionally dispensable isoform, since 16% of the world
population have a congenital deficiency of -actinin-3 [72]. In addition, it has been reported
that -actinin-3 polymorphism is closely related to the work performance in athletes [73],
although some studies have reported that the presence of -actinin-3 does not influence the
characteristics of muscle contraction [74, 75]. Nevertheless, it is the current consensus that
exercise stimulates the shift of the isoform from -actinin-3 to -2, in association with an
elevated aerobic capacity and fiber phenotype transition of fast-to-slow in fast-twitch skeletal
muscles [76, 77]. These phenomena are strongly supported by the results obtained by
MacArthur et al. [71], who demonstrated that loss of -actinin-3 triggers a shift in the muscle
metabolism toward the more efficient aerobic pathway and an increase in the intrinsic
endurance performance.

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-actinin is not only a constitutive Z-disc component but also a mediator of the particular
signaling which stimulates the slow-twitch-specific oxidative function in skeletal muscles.
Desmin, the most abundant intermediate filament protein in muscle, surrounds Z-discs
and supports their integration [78]. It is also known that expression of desmin precedes that of
other muscle-specific structural genes and some transcriptional factors, such as MyoD,
myogenin and MRF4, during muscular development [78-80]. However, it has been
demonstrated that desmin is not essential for the myogenic commitment in the early stage of
myogenesis and myofibrillogenesis, because somites and myotomes form normally and
mononucleated muscle precursor cells migrate during embryogenesis in the absence of
desmin [81]. After migration, primary and secondary myotube formation occurs in an
identical manner in desmin null and wild type mice. The Z-discs are formed normally, and the
subsequent assembly of the associated proteins is sufficient in desmin null mice [81, 82]. The
first discernable effects of the lack of desmin are seen on the muscle fiber phenotype after
birth [81]. These effects are mainly observed in the slow-twitch muscles, soleus and
diaphragm, where this modification is accompanied by morphological and biochemical
changes. These include a variability in fiber diameter, the presence of central nuclei, the
presence of crescent-shaped sarcolemmal masses, and a decrease in the amount of type II
myosin heavy chain. Despite the normal myogenesis and myofibrogenesis, skeletal muscles
of desmin null mice exhibit less strength and less fatigue resistance compared with either
heterozygous or control littermates [81]. Moreover, it is noted that muscle fibers with centralnucleation, a marker of regeneration and degeneration, are observed more frequently in
association with increases in age or mechanical loading by exercise [81, 83]. These
pathological changes are more massive in the slow-twitch skeletal muscle.
Desmin intermediate filaments connect parallel myofibrils transversely and have been
implicated in the correction for the direction of the tension transmission within the muscle
fiber. A recent study [83] has further found that muscles lacking desmin become
progressively stiffer, accumulate increased collagen, and exhibit an increased expression of
genes involved in extracellular matrix turnover. It has also been reported that cell populations
identified by the muscle progenitor marker, 7-integrin, are reduced, while cell populations
identified by the fibroadipogenic marker, Sca-1, are elevated in the skeletal muscle of desmin
null mice. In addition, the increase in macrophage infiltration is also pronounced in desmin
null mice. Most myopathies are complicated with fibrosis during an insufficient regeneration
followed by degeneration usually triggered by mechanical stress. Fibrosis is one of the
debilitating factors which interfere with muscle regeneration and the normalization of muscle
function. Desmin plays a key role in developing the integrity of myofibrils and providing
resistance against cytoskeletal ruptures.

Associated Proteins and Signal Transduction

Muscle-Specific RING Finger 1 (MuRF1) and Atrogin-1
In addition to the great importance of the activation of protein synthesis pathways during
muscular hypertrophy, proteolysis has also been reported to make an inverse contribution, by
participating in the induction of atrophy.

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The stimulation of proteolysis observed during atrophy is shown to occur at least in part
due to an activation of the ubiquitin-proteasome pathway [84]. The addition of ubiquitin to a
protein substrate acts a signal to mediate the degradation of a specific target. Three distinct
enzymatic components are required, an E1 ubiquitin-activating enzyme, an E2 ubiquitinconjugating enzyme, and an E3 ubiquitin-ligating enzyme. The E3 ubiquitin ligases are the
components which confer substrate specificity. Many different E3 enzymes have been
identified, and each molecule modulates the ubiquitination of a distinct set of substrates [85].
The E3 ubiquitin ligases, MuRF1 and atrogin-1, also known as muscle atrophy F box
protein (MAFbx), were first identified as key factors up-regulated in several atrophy models
by Bodine et al. [7]. Numerous studies [86-90] have since demonstrated the increased
expression of MuRF1 and atrogin-1 in the atrophied skeletal muscles following the
experimental models. Mice lacking MuRF1 (MuRF1/) or MAFbx (MAFbx/) appear
phenotypically normal. However, under atrophying conditions, significantly less muscle mass
is lost in either MuRF1/ or MAFbx/ animals in comparison to their control littermates
[7].
Three MuRFs (MuRF1, -2 and -3) were characterized by Centner et al. [91]. MuRF1
associates with M-lines via binding to the Ig-domains of titin (Figure 3). Overexpression of
MuRF1 results in disruption of the MuRF1-binding domain of titin, suggesting that MuRF1
plays a role in titin turnover [92]. MuRF3, which is a microtubule-associated and stabilizing
protein exclusively expressed in cardiac and skeletal muscles, localizes at Z-discs in skeletal
muscles [93]. It has also been shown that MuRF1 binds to a multitude of sarcomere proteins,
such as telethonin and myotilin, implying that MuRF1 may participate in a pathway for the
degradation of these proteins [94]. MuRF2 co-localizes with MuRF1 at the M-line and with
the microtubule-associated MuRF3 [95, 96]. However, a Z-disc localization has not been
shown yet.
Atrogin-1 interacts strongly and specifically with the Z-disc protein, -actinin-2, in
cardiac myocytes [97], although the localization and the interaction in skeletal muscles are
still unknown. Recently, MyoD [98] and calcineurin [97] have been identified as the
substrates of atrogin-1 in myoblasts and cardiac myocytes.

Calpain
Calpain is a member of the non-lysosomal Ca2+-dependent cysteine proteases, which are
involved in many cellular processes mediating the selective proteolysis of target proteins [99].
Calpain-1, -2 and -3 are predominantly expressed in skeletal muscles [100]. Calpain-1 colocalizes with -actinin at the Z-discs, and an interaction is further detected with titin at two
positions within the I-band and the lateral edge of the Z-disc [101]. This association of
calpain with sarcomeric proteins might serve a role in the signaling and the mechanical
transmission.
Calpain-3 is a skeletal muscle-specific isoform that is distinct from other calpains in
terms of its Ca2+ dependence and rapid autolysis [102]. In addition to the four universal
domains of the ubiquitous calpains, calpain-3 has three additional unique insertion sequences
called NS, IS1, and IS2 [103]. IS1 includes three autolytic sites that are involved in the
regulation of its activity [104]. The IS2 domain contains a nuclear translocation signal and a
binding site for titin [105].

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Figure 3. Titin binding proteins. MuRF1 [91] and capain-3 [108] localize at M-band by binding to titin.
MuRF1 plays a role for the titin turnover [92], whereas capain-3 shows its proteolytic activity in many
cytoskeletal proteins following autolysis [108]. MARPs bind to the N2A domain of titin in I-band,
mechanically flexible region sensitizing a force [113, 114]. In accordance with mechanical stress
applied by contraction, the I-band region of titin, including the PEVK domain, transforms and releases
MARPs, which are replaced with a competitive binding of capain-3 to this region [115]. MARPs
translocate to a nucleus and contribute to stress response-related transcription. MuRF1, muscle-specific
RING finger 1; Capn3, calpain-3; MARP, muscle ankyrin repeat protein.

Furthermore, according to the an immunoblotting analysis, calpain-3 exists primarily in

an autolyzed form [106] that consists of a small N-terminal fragment of 34 kDa and three
large C-terminal fragments of 55, 58, and 60 kDa [107].
Inactive calpain-3 is localized at the M-line and the N2A domain of titin, and is activated
by autolysis in response to an activation signal [108]. The activity of calpain-3 is directed
against all components of the cytoskeleton, potentially including constameres (integrin and
, talin, vinexin, ezrin and filament C) and M-line (M protein and myomesin) as well as Zdisc proteins. Interestingly, activated calpain-3 is translocated to constameres and the muscletendon junction, which are the potential sites of force transmission. This suggests that
calpain-3 may participate in the integrated regulation of the muscular cytoskeleton during
processes such as force generation, adaptive response to exercise, or passive stretching during
contraction.
Mutations of calpain-3 play a crucial role in the pathogenesis of limb-girdle muscular
dystrophy type 2A (LGMD2A), a disease characterized by progressive atrophy and weakness
of the proximal limb muscles [109]. Several studies on calpain-3-knockout mice have shown
that calpain-3 deficiency leads to the formation of abnormal sarcomeres, impairment of
muscle contractile capacity and loss of the muscle fibers [110, 111]. Calpain-3-dependent
proteolysis of IB controls IB turnover, and regulates NF-B-dependent transcription of
survival genes [112]. However, the elevated level of IB in both the nucleus and cytoplasm
in the muscle cells derived from LGMD2A patients leads to an accumulation of cytoplasmic
NF-B and promotes the apoptosis in the muscle cells, possibly due to the reduced expression
of survival genes [112]. In the N2A domain of titin, a binding site for the muscle ankyrin
repeat protein (MARP), which can modulate stress-dependent signal transduction, overlaps
with the binding site for calpain-3 in the I-bands [113, 114] (Figure 3).

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Ojima et al. [115] demonstrated that calpain-3 shifts its location from the M-line to the
N2A region of titin when the sarcomere is extended, suggesting that calpain-3 functions as a
sarcomere-length sensor in co-operation with titin. They also generated calpain-3-knockin
mice in which caplpain-3 is proteolytically inactive but structurally intact. The enlargement in
the proportion of central-nucleated muscle fibers was noted within the skeletal muscles of the
knockin mice, in accordance with the progression of age [116]. Exercise further promoted the
damage in the single muscle fibers. Translocation of MARP from the N2A region of titin is
thought to contribute to the adaptive response to physical stress, although the accumulation of
mutated calpain-3 in the N2A region was very slow and the level of induction of MARP was
lower than that in wild type mice. The mislocalization of calpain-3 has also been observed in
LGMD2A patients.

Cypher and Protein Kinase C (PKC)

Two distinct splicing variants of Cypher (Cypher1 and -2) were identified by Zhou et al.
[117]. Cypher is expressed exclusively in striated muscles and localized to the Z-disc. The
Cypher1 isoform contains a PDZ domain at its N-terminus, three LIM domains at its Cterminus, and nine YS/TPS/TP amino acid repeats. Of these domains, Cypher2 shares only
the PDZ domain. Consistent with their localization at the Z-disc, both Cypher isoforms can
bind to -actinin 2 through their PDZ domains (Figure 4A). Cypher-knockout mice exhibit
the normal Z-disc apparatus until birth [118]. However, at postnatal day 1, the Z-disc is
severely disorganized and disrupted after the muscle contraction is started.
Cypher is also known to bind to PKC via its LIM domains [119]. PKCs are serine/
threonine kinases that can induce a variety of cellular responses upon activation, including
transmission of growth signals, receptor desensitization, and regulation of transcription in
many other cellular functions [120]. PKCs are divided into three groups, the classical Ca2+sensitive isoforms (, I, II and ), the novel isoforms , , and , which lack the Ca2+sensitive C2 domain and thus redistribute Ca2+ independently, and the atypical PKCs ( and
/), which are neither activated by Ca2+ nor by diacylglycerol or phorbol esters. PKC , a
known mediator of cardiac hypertrophy, relocates to the Z-disc in adult cardiac myocytes
upon stimulation [121-123]. In skeletal muscle cells, a recent study has demonstrated that
PKC is rapidly translocated to membranes following pharmacological stimulation [124].
This translocation of PKC causes the activation of GLUT4 traffic, which leads to an
improvement of intracellular glucose uptake. The other potential mediator of contractionstimulated glucose uptake in skeletal muscle was PKC. It has recently been shown that the
glucose uptake of skeletal muscles from PKC-knockout mice can respond to the contraction
stimuli, indicating that PKC is not required for the contraction-stimulated glucose uptake
[125].

Actinin-Associated LIM Protein (ALP)

ALP, identified by Xia et al. [126], includes the PDZ domain, which enables ALP to bind
to the spectrin repeats of -actinin, and also contains a single LIM domain. ALP is expressed
at high levels in skeletal muscles and at lower levels in cardiac myocytes.

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Figure 4. Models of signal transduction induced by Z-disc-associated proteins. A) Localization of PKC

at Z-discs is mediated by Cypher. Translocation of PKC to membrane causes the activation of GLUT4
traffic, which lead to improve the intracellular glucose uptake [124]. B) ALP and MLP localize at Zdisc (upper). ALP stabilizes actin filament anchorage to the Z-disc [127]. MLP is the combinational
regulator of MyoD, translocates to a nucleus in response to the mechanical stress, and induces a stress
responsible program [153]. MLP also play a critical role for retaining the localization of calcineurin at
the Z-disc [151] (lower). C) NFATc is localized to Z-discs in resting skeletal muscle fibers [141].
Increased Ca2+ concentration resulted from the nerve activity promotes binding of calcineurin and
calmodulin, leading to gain the phosphatase activity of calcineurin [130]. Dephosphorylated NFATc
translocates to a nucleus and co-operates with MEF2 to transcribe slow-twitch muscle-specific genes
[133, 134, 141]. Calsarcin family, localized at Z-discs, suppresses NFATc-related transcription via
inhibition of calcineurin activity [148]. Calsarcin also binds to Cypher at Z-discs [146] (A). ALP,
actinin-associated LIM protein; MLP, muscle LIM protein; MARP, muscle ankyrin repeat protein; CnA
and CnB, calcineurin A and B, respectively; PKC, protein kinase C; CaM, calmodulin; NFATc,
nuclear factor of activated T cells c; MEF2, myocyte enhancer factor 2.

ALP enhances the ability of -actinin to crosslink actin filaments, indicating that ALP
stabilizes actin filament anchorage at Z-discs [127] (Figure 4B).
Knock-down of ALP expression affects the expression of MyoD and myogenin, resulting
in the inhibition of muscle differentiation [128]. Therefore, ALP is thought to play a crucial
role in the formation of myofibrils and transcriptional regulation in muscle differentiation.

Calcineurin and Nuclear Factor of Activated T Cells (NFAT)

Calcineurin-dependent signaling mechanisms are a very important pathway for the
induction of slow-twitch muscle-specific gene transcription. Binding of Ca2+ to a
calmodulin-calcineurin complex stimulates serine/threonine phosphatase activity of
calcineurin [129]. Low amplitude, sustained Ca2+ waves preferentially induce the calciumcalmodulin binding, which in turn is associated with the regulatory calcineurin B subunit,
thereby activating calcineurin A [130]. The members of the NFAT family are known to be
substrates of calcineurin A. Five different NFAT genes have been identified: NFATc
(NFATc1 or NFAT2), NFATp (NFATc2 or NFAT1), NFAT4 (NFATc3 or NFATx), NFAT3
(NFATc4), and NFAT5 [129, 131, 132]. Recent studies have indicated that NFATc, an NFAT
isoform present in skeletal muscles, may play a role in fast- to slow-twitch fiber
transformation [133]. Dephosphorylation of NFATc by calcineurin promotes the translocation
to the nucleus, where the NFATc associates with other transcriptional factors, such as
myocyte enhancer factor 2 (MEF2) [130], and activates the expression of slow-twitch muscle-

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specific genes such as myoglobin and troponin I slow [133, 134]. Together with the gene
transcription, activation of the calcineurin-NFATc mechanism specifically induces a muscle
fiber property switch toward a slow-twitch phenotype and oxidative metabolism, further
resulting in an enhancement of fatigue resistance. An increase in the number of slow-twitch
muscle fibers has been noted in mutant mice with forced overexpression of calcineurin [135].
On the other hand, lack of calcineurin A leads to a significant decrease in slow-twitch and
oxidative fibers. It has also been reported that slow-twitch fibers fail to respond to the
increased workload in skeletal muscle-specific calcineurin B1-null mice [136].
Motor nerve-driven tonic activity (10-15 Hz) in slow-twitch muscle fibers results in the
sustained elevation of intracellular Ca2+ concentration in a range between 100 and 300 nM,
and such sustained increase in intracellular Ca2+ has been predicted to activate calcineurin
[137]. In fast-twitch muscle fibers, the resting intracellular Ca2+ concentration is maintained
at ~50 nM. High amplitude (~1 M) Ca2+ transients induced by motor nerve activity are
predicted to be of insufficient duration to evoke the calcineurin signaling [138]. Chronic
stimulation of motor nerves innervating the fast-twitch muscle fibers results in a sustained
elevation of intracellular Ca2+ concentration and fast-to-slow fiber transformation [139, 140].
Chin et al. [133] further demonstrated that the inhibition of calcineurin activity by
administration of cyclosporin A induced a slow-to-fast fiber transformation with a
dependence on switching of the combinational transcription activity of NFATc and MEF2.
These previous findings clearly indicate the critical roles of calcineurin-NFATc mechanisms
in slow-twitch muscle-specific gene transcription.
Liu et al. [141] demonstrated the localization of NFATc in a resting state and in response
to stimulation by techniques using green fluorescent protein-fused or constitutively active
mutant NFATc. Interestingly, in resting skeletal muscle fibers, NFATc was localized at the
sarcomeric Z-discs and absent from nuclei (Figure 4C). They also simulated the activity
patterns typical of slow-twitch muscle by electrical stimulation with a continuous or trained
pulse at 10 Hz, resulting in the nuclear appearance of fluorescent foci of green fluorescent
protein-fused NFATc. However, nuclear translocation of NFATc did not occur with either
continuous 1 Hz stimulation or with the fast-twitch fiber activity pattern of short-duration
trains at 50 Hz.
Recently, Frey et al. [142] discovered a family of striated muscle-specific calcineurinbinding proteins, the calsarcins, which localize to the Z-disc of the sarcomere [143] (Figure
4C). The calsarcin family consists of 3 members. Calsarcin-1 is expressed in the adult heart
and in slow-twitch fibers of skeletal muscles. Calsarcin-2, also known as myozenin 1 [144]
or FATZ-1 [145], and calsarcin-3 are exclusively expressed in fast-twitch fibers of skeletal
muscles [142, 146]. Calsarcins also bind to -actinin, cypher, telethonin, -filament [142,
146] and myotilin [147] to localize to the Z-disc. Myozenin 1 and calsarcin-1 suppress the
NFATc-related transcription via the inhibition of calcineurin activity [148]. Thus, myozenin
1-knockout mice display a switching of fiber phenotype to the slow-twitch type as well as a
drastic improvement in the oxidative function in skeletal muscle fibers. Physical performance
as measured by the voluntary running distances is enhanced in the knockout mice. The lack of
calsarcin-1 also leads to an increase in the calcineurin activity, which in turn is associated
with an expansion of slow-twitch skeletal muscle fibers [149]. Together, these observations
strongly suggest a model in which the Z-disc-associated proteins modulate the intracellular
signal transduction due to the muscle activities.

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Muscle LIM Protein (MLP)

MLP is one of the Z-disc-associated proteins that include two LIM domains and directly
binds to -actinin [150, 151]. Two other cytoplasmic-binding partners have been identified:
telethonin [152] and calcineurin [151]. The main role of MLP in skeletal muscles is to
mediate mechanical stress via translocation to the nucleus, within which MLP is a potent
activator of MyoD [153] (Figure 4B). This nuclear activity suggests that MLP may exhibit
transcriptional activity in addition to its structural role, leading to the intriguing possibility
that MLP is a striated muscle stress sensor.
Expression of MLP mRNA has been shown to increase rapidly but transiently after
eccentric exercise [154, 155], and both mRNA and protein are increased upon the onset of
chronic low-frequency electrical stimulation [156, 157] in the fast-twitch muscle.
Transcriptions of MARP genes are also up-regulated in response to eccentric exercise [154,
155]. In MARP-knockout mice, single muscle fibers appear to be less stiff, tend to have
longer resting sarcomere lengths, and express a longer isoform of titin, indicating a role of
MARP proteins in the passive mechanical behavior of muscle [158]. MARP-knockout mice
further show the enhanced expression of MLP and MyoD in response to eccentric exercise.
Thus, the eccentric exercise-induced loss of contractile torque can be recovered normally,
possibly due to the compensatory role of MLP and MyoD. On the other hand, in MLPknockout mice, acute induction of MyoD following eccentric exercise is inhibited, although
the lack of MLP does not affect the expression of genes for the MARP family proteins [152].
The torque production lags are noted in the early stages of the recovery process after the
eccentric exercise. These results indicate an important role of MLP in the transcription and
activation of MyoD, which is necessary to promote the mechanical stress-related responses.
Binding of MLP to calcineurin is thought to be a crucial biological process for switching
both MyoD and NFATc transcription-mediated mechanisms toward "ON". Heterozygous
MLP-knockout mice show a displacement of both calcineurin and calsarcin-1 from the Zdiscs in cardiac myocytes [151]. This result displays a clear linkage between the stress sensor
MLP and calcineurin-NFATc signaling in cardiac myocytes.
Although the precise mechanisms are still unknown, a similar mechanism can be
expected in skeletal muscles. In fact, MLP is up-regulated during fast-to-slow fiber-type
transition in skeletal muscles [156, 157].
The myogenic regulatory factor family (MRF) is made up of proteins with a basic helixloop-helix (bHLH) motif, which is the basic region responsible for DNA binding. MRFs,
including MyoD, myogenin, Myf-5 and MRF4, play a key role for the regulation of
myogenesis [159]. The MRFs heterodimerize with widely expressed bHLH E proteins, such
as E12 and E47, and activate the muscle-specific gene expression by binding to E-box
consensus sequences [160-162]. Another transcriptional factor that is known to play pivotal
roles in muscle development is the MADS box MEF2 protein. MEF2 and MRFs act
synergistically to activate the myogenic program through direct interactions involving the
MADS box and bHLH domains of the respective proteins [163-165]. In addition, MRFs and
MEF2 are capable of activating the expression of each gene family member, forming an
active regulatory circuit which ensures that high levels of MEF2 and MRF gene expression
are maintained during myogenesis. MLP interacts with the bHLH of MRFs by the first LIM
motif, enhancing the DNA-binding activity of the transcriptional complex [153]. Based on the
facts that MyoD and MEF2 are necessary for stress responses [152, 158] and the slow-twitch

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muscle-specific gene transcription [130, 133, 134], it is predicted that MLP plays a crucial
role in the transcription of key genes related to the response and adaption of skeletal muscle
fibers to physiological stimuli.

Remodeling of Myofibrils
Mechanical stress on muscle fibers produced by the contraction and force development
causes myofibrillar disorganization, in which Z-disc streaming, Z-disc smearing and Z-disc
disruption occur as morphological hallmarks of delayed onset muscle soreness [166].
According to the results of previous studies using animal models simulating enhanced
mechanical stress, the desmin intermediate filaments are affected shortly after the contractile
stimulation [167-169], indicating that the morphology of desmin is a possible marker of
myofibrillar damage. For example, a focal initial loss of immunohistochemical staining for actinin, titin and nebulin is correlated with the strong expression of desmin, particularly in the
disorganized region after eccentric exercise [170]. However, an addition of new sarcomeres to
the pre-existing sarcomeres is also observed following contractile stimulation. These
observations suggest a model in which an adaptive remodeling of myofibrils is induced after
the damage. These processes lead to sarcomerogenesis and lengthening of the myofibrils
[170-173], and subsequently prevent soreness through an additional loading. A novel
contribution of two related proteins in the myofibrillar remodeling is reviewed below.

Myotilin
Myotilin, which is highly expressed in skeletal muscles and moderately expressed in the
heart [174], is localized at the Z-discs by binding to -actinin [175], actin [176], -filament
[177] and myozenin 1 [147].
Carlsson et al. [178] reported that the intensity of myotilin staining was markedly
increased in areas with myofibrillar alterations induced by eccentric exercise. Myotilin was
strongly detected in sites ranging from a single broadened Z-disc up to large lesions involving
several consecutive sarcomeres. The staining also co-existed with the areas showing increased
phalloidin staining, in which a temporary lack of -actinin, titin and nebulin was noted [170,
173]. The addition of new sarcomeres was postulated to occur either by the splitting of an
existing Z-disc or by the reintegration of -actinin and related Z-disc proteins (titin and
nebulin) in a single broadened sarcomere. They further found that the staining of myotilin
was more abundant in the Z-discs of slow- than fast-twitch skeletal muscle fibers. The
difference in the width of Z-discs may cause the alteration in the amount of myotilin captured
within the Z-discs, because the thickness of Z-discs in slow-twitch fibers is broader than that
in fast-twitch fibers [67]. It is predicted that the different fiber types will show at least some
differences in their ability to respond to the mechanical stress produced by contractile
activity.
Pathogenic mutations in the myotilin gene cause a subset of myofibrillar myopathies and
LGMD type 1A (LGMD1A) that are characterized by streaming of Z-discs and degeneration
of muscle fibers [179-181]. Typically, the skeletal muscle of LGMD1A patients exhibits

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185

multiple myopathic features, variant fiber size, fiber splitting, central nuclei, fibrosis, rimmed
autophagic vesicles and Z-disc streaming. Myotilin binds to -actinin via its N-terminus,
whereas the sites for the -filament binding and for dimerization locate at the C-terminus
[175, 177, 180]. This dimerization is necessary for the actin binding activity. Myotilin
protects the thin filament from pharmacologically induced disassembly by cross-linking the
actin filaments alone or in concert with -actinin [176]. Further, the expression of terminally
truncated myotilin fragments in differentiating muscle cells leads to myofibril disarray,
indicating an indispensable role for the full length myotilin in stabilization and anchorage of
thin filaments. In LGMD1A patients of the first-identified pedigree, one allele exhibits a
cytosine-to-thymidine mutation at position 450 of the myotilin gene, resulting in a Thr57Ile
substitution in exon 2 [180]. Since the -actinin binding activity of mutated myotilin is still
present, the mutation may interfere with the thin filament organization and lead to the failure
in the correct Z-disc integration. A recent study [182] has identified a novel mutation on exon
9 of the myotilin gene, in which the amount of myotilin monomer is increased in the patient
muscle, but the homodimer of myotilin is decreased, resulting in a clinically typical
LGMD1A pathology. Mutant myotilin in this patient further exhibits decreased binding
activity to -actinin. These observations indicate that the pathologic mutation of myotilin
loosens the complex formed by actin bundlers at the Z-discs, leading to a decrease in the
strength and the ability of the Z-discs to resist mechanical stress during muscle contraction.
In contrast to the abnormalities of myotilin function seen in LGMD1A patients, the lack
of myotilin does not influence muscle mass, fiber size or force development [66]. Myotilin is
part of a small subfamily that includes palladin and myopalladin [174]. These functionally
redundant proteins exist in all striated muscles, and their expression may be sufficient to
overcome the lack of myotilin. Myopalladin, the Z-disc protein, indirectly coordinates the
assembly of an intra-Z-disc meshwork by tethering the thin filaments via -actinin [183].
Similarly to the LGMD1A patients, the transgenic mice expressing human myotilin with a
Thr57Ile mutation also exhibit severe myopathies, Z-disc streaming, excess myofibrillar
vacuolization and plaque-like myofibrillar aggregation [184]. Surprisingly, the degree of
myofibrillar aggregation is different in various muscles.
Gastrocnemius and extensor digitorum longus muscles, composed predominantly of fasttwitch fibers, are heavily populated with the aggregation in the transgenic mice, whereas
slow-twitch muscles such as the soleus and diaphragm are completely spared of any abnormal
myopathology [184]. Furthermore, a physiological deficit in muscle stiffness, as determined
by the level of force reduction after lengthening contraction, is also noted in the fast-twitch
extensor digitorum longus muscle, while no reduction is induced in the slow-twitch soleus
muscle. These data again suggest an abundance of compensatory proteins or protective
signaling based on the thicker Z-discs of slow-twitch fibers.

25kDa Heat Shock Protein (HSP25)

Several molecules of the HSP family are known to be up- or down-regulated during
hypertrophy or atrophy of skeletal muscle fibers in rodents [4, 185-190]. Murine HSP25,
which is homologous with human HSP27, is one of the small HSPs. HSP25 is abundantly
expressed especially in slow-twitch skeletal muscles, such as the soleus and adductor longus
[187, 191]. Muscle inactivation by spinal cord isolation decreases HSP25 expression in

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association with the severe atrophy of slow-twitch soleus muscle [187, 192]. The level of
HSP25 expression, in contrast, is elevated in response to the ablation of synergists, leading to
mechanical overload and muscular hypertrophy [186]. Forced expression of HSP25 can
attenuate the disuse-induced atrophy in the slow-twitch muscle due to the inhibition of
MuRF1 and atrogin-1 transcription, as well as NF-B activation [193]. These studies clearly
indicate the importance of HSP25 in the regulation of skeletal muscle mass, although some
studies [190, 194] have documented that several HSPs are not affected by unloading.
Molecular chaperoning is a common function of HSPs. Particularly in HSP25, the
phosphorylation triggers the formation of an oligomeric complex, which is necessary for the
chaperone action and resistance against cellular stress [195]. Serine 15 and 86 (Ser15 and
Ser86, respectively) are the major phosphorylated sites of HSP25 [195-197]. In rodents, three
kinds of phosphorylated HSP25, i.e., the non-, single- (at either Ser15 or 86 site) and dual- (at
both Ser15 and 86 sites) phosphorylated forms, have been identified [186, 197-201]. The
lengthening contraction causes an immediate increase of the dual phosphorylation of HSP25
and translocation from the soluble to the insoluble fraction [200]. In humans, the eccentric
exercise stimulates the accumulation of HSP27 in the Z-discs and the disrupted sarcomeres
[202]. Non- and single-phosphorylated HSP25s are generally expressed in the cytoplasm of
normal skeletal muscles. Ser86 is thought to be a constantly phosphorylated site of HSP25
[203]. Mechanical stress applied to the skeletal muscle promotes the phosphorylation
selectively at Ser15, leading to the production of a dual-phosphorylated form and
translocation from the cytoplasm to myofibrils. Under normal conditions, HSP25 forms a
large molecular complex, likely a homo- or hetero-oligomer, with a size ranging from 600800 kDa. However, the mechanical stress stimulates the dissociation of a free single HSP25
from the complex, and an interaction directly or indirectly with desmin, not actin or actinin, in
the disorganized region of the myofibrils. Interestingly, in these lesions, desmin forms a Zdisc-like striated structure, but the interval distance is longer than in normal sarcomeres,
suggesting that the onset of sarcomerogenesis follows the disruption of myofibrillar protein
organization. Progression of phosphorylation might alter the HSP25 function toward
myofibrillar protection.

Muscle Length and Neural Control

As reviewed above, myofibrils mediate the important biological signals following
mechanical stress applied to the muscle, whereas overloading, such as eccentric exercise,
induces myofibrillar damage, which may cause a temporal loss of the signaling machinery as
well as the contractile strength. Further, loss of activity promotes the proteolysis of musclespecific cytoskeletal proteins [204-206], and may lead to the decreased capacity for the signal
transduction. Severe, unloading-associated loss of muscle mass is noted particularly in slowtwitch skeletal muscle, such as the soleus [4, 203, 204, 207-212]. Neuromuscular systems
highly regulate the relationship between force development and muscle length by the
remodeling of sarcomeres, to adapt to the given environment.

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187

Muscle Activity in Slow-Twitch Muscle

Slow-twitch antigravity muscle, such as the soleus, in most vertebrates shows tonic
neuromuscular activity for maintaining posture against the force of gravity. In addition to the
soleus muscle activity, afferent and efferent neural bursts measured at the 5th lumber
segmental level of the spinal cord [3], where the motoneurons innervating the soleus are
located, are present at 1-G in rats. The tonic activity in the soleus becomes silent if the rats are
exposed to a real microgravity environment created by the parabolic flight of a jet airplane
[213]. During the ascending phase of the airplane, the G level is increased from 1-G to
approximately 2-G. Subsequently, ~ 20 seconds of microgravity is obtained at the top of the
parabola. The level of afferent nerve activity is also decreased during microgravity, whereas
the magnitude of the change in the efferent neural activity is minor throughout the parabolic
flight. Muscle activities in fast-twitch muscles, such as the gastrocnemius and tibialis anterior,
do not change in response to exposure to microgravity. Similar responses are also seen in the
neuromuscular activities of the soleus muscle in response to acute (20 seconds) tail
suspension [3]. However, fast-twitch muscles show no response, or even an increase in the
activity of the dorsiflexor muscle, to the acute tail suspension.
These selective losses of muscle activity in the soleus during the exposure to a
microgravity environment or tail suspension are dependent on the changes in the muscle
length. In rats, the ankle joints are generally dorsiflexed up to approximately 30o during the
sedentary quadrupedal posture in a 1-G environment [2]. While assuming such a position, the
soleus muscle is stretched and the mean length of sarcomeres is greater than 3.0 m.
However, the ankle joints are plantarflexed to approximately 160o during microgravity
exposure and tail suspension, leading the sarcomere length in soleus muscle fibers to be
shortened to approximately 2.0 m. In this slack position, the force production is
minimized further, even if the muscles are neurally activated. These phenomena indicate that
unloading by the exposure to real microgravity or tail suspension inhibits both mechanical
stress and neural activity by shortening of the muscle fiber length in the soleus muscle of rats.
Plaster casting of the ankle joint at the dorsiflexed position, leading to the stretching of soleus
muscle length, can prevent the atrophy following tail suspension, indicating that a decrease in
the neuromuscular activities is the main cause for the unloading-associated atrophy in the
soleus [214].
The shortening of the muscle length is moderate in the gastrocnemius compared to the
soleus, whereas the muscle length is slightly stretched in the plantarflexor, tibialis anterior,
during tail suspension [209]. These observations also support the idea that the soleus is more
susceptible to gravitational unloading than either the fast-twitch synergist, gastrocnemius, or
its antagonist, tibialis anterior.

Adaptation of Neuromuscular Activity and Sarcomere Remodeling

Several studies [3, 4, 207, 209, 215, 216] have found that decreased soleus and afferent
neural activities are gradually recovered and reach the pre-experimental level within 2 weeks
during continuous tail suspension. The ankle joints are generally kept plantarflexed at 90
~160o during tail suspension. Passive tension development of the soleus muscle is decreased
in response to plantarflexion of the ankle joint, which has a direct influence on the muscle

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Fuminori Kawano

length, as stated above. Even though the tension development is still very low when the ankle
joint angle is 160o, it becomes capable of generating force when the ankle joint is 140o within
2 weeks of tail suspension [3].
It is clear that the number of sarcomeres is decreased in response to chronic shortening of
the muscle length and the sarcomere length at a given angle of the ankle joint is increased [3].
Therefore, the soleus muscle fibers are slightly stretched even though the ankle joints are still
plantarflexed during tail suspension. The mean sarcomere lengths at ~60o on the floor before
tail suspension and at ~90o on the 14th day during tail suspension are similar, suggesting that
the static force can be produced by the remodeling of sarcomeres even without an external
load.
Shah et al. [217] tested the roles of desmin in regulating the lengthening- or shorteninginduced remodeling of sarcomeres in the hindlimb muscles of mice. The immobilization of
ankle joints by plaster casting at the plantarflexed position caused an increase in the serial
sarcomere number of the tibialis anterior and extensor digitorum longus, due to the
lengthening of the muscles.
A consistent result was observed in the shortened soleus. Surprisingly, the muscles in
desmin null mice showed similar remodeling of sarcomeres, with an increase in the serial
sarcomere number in dorsiflexors, and inversely in the soleus. This fact indicates that desmin
is not essential for sarcomerogenesis in terms of the adaptation of muscle length in mature
skeletal muscles. However, it cannot be excluded that a defect in desmin may enhance the
myofibrillar damage and activates a regeneration/degeneration cycle [81-83, 217].

Elastic Properties
Discharges of Ia and II afferent nerve fibers in response to a given stretch of rat soleus
muscle fibers are increased after 14 days of tail suspension, suggesting that increased
connective tissues could contribute to a better transmission of passive mechanical stretch to
the muscle spindles [218]. Further, the relative proportion of type III collagen, which is more
elastic than type I, increases in response to 14 and 28 days of tail suspension in rats [219].
Passive tension of soleus fibers, atrophied after 14 days of tail suspension, increases less
steeply in response to stretching than that of normal fibers [220].
Elasticity of the I-band region of titin filaments in atrophied soleus muscle fibers is also
reduced following tail suspension, whereas that in fast-twitch extensor digitorum longus
muscle fibers is not affected [204]. Splicing variation of the titin gene results in the
production of variant proteins with different molecular sizes [18, 19]. The titin expressed in
slow-twitch muscles is larger than that in fast-twitch muscles. It is indicated that titin-borne
tension is inversely related to the molecular size of titin [21]. Titin size is also correlated with
the expression of type I myosin heavy chain or the percentage of slow-twitch fibers in the
muscle. Slow-twitch muscles usually express long titin, resulting in the low titin-based
stiffness [21, 22]. However, total passive stiffness, provided under lack of titin, is high in the
rabbit soleus, in which abundant collagen fibers in the extra-myofibrillar space are noted.
These structural features may help to protect myofibrils from the tonic mechanical stress in
slow-twitch muscles.

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189

Conclusion
Skeletal muscle fibers can adapt in various directions, e.g., hypertrophic, atrophic,
oxidative, or stress-resistive, in response to a given environment and physiological stimuli.
This adaptation results in slow- or fast-twitch characteristics, which are not only for the
differential contractile apparatus and metabolism but also for changing the capacity of
molecular responses to the muscle activity. Titin is a novel mechanical sensor that is
transformed in response to mechanical stress, and releases MARPs. Together with the
association of MuRF1 and calpain-3 with titin, these results suggest that titin plays a crucial
role in the biological process of myofibrillar kinetics. Interestingly, numerous molecules that
induce important signals are localized to the Z-disc in the sarcomere. Some of these
molecules also contribute to the myofibrillar organization, construction and repair. Again,
these structural components vary between slow- and fast-twitch muscle fibers.

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In: Basic Biology and Current Understanding of Skeletal Muscle ISBN: 978-1-62808-367-5
Editor: Kunihiro Sakuma
2013 Nova Science Publishers, Inc.

Chapter 8

AMPK: Molecular Mechanisms

of Metabolic Adaptations
in Skeletal Muscle
Masataka Suwa
Faculty of Life Design, Tohoku Institute of Technology,
Taihaku-ku, Sendai, Miyagi, Japan

Abstract
Skeletal muscle possesses a great degree of metabolic plasticity, which can be
controlled by exercise, cytokines, pharmaceuticals and nutrients. There are large
variations in the human skeletal muscle metabolic capacity, and they are linked with the
prevalence of chronic metabolic disorders such as obesity, type II diabetes mellitus,
hypertension and dyslipidemia, as well as exercise endurance.
The metabolic profiles of skeletal muscle have been investigated for several decades.
5-AMP-activated protein kinase (AMPK) is one of the most important metabolic
regulators in skeletal muscle. AMPK is also activated by energy deprivation (i.e., an
increasing AMP:ATP ratio) and served as a cellular energy sensor. AMPK is activated by
muscle contraction (e.g., exercise), drugs used to treat type 2 diabetes mellitus, cytokines,
nutrients, reactive oxygen species and other chemically synthesized activators.
Activated AMPK promotes the catabolic pathways that generate ATP (e.g., glucose
uptake, fatty acid uptake and oxidation, and mitochondrial respiration). AMPK promotes
the translocation of glucose transporter 4 (GLUT4) from the cytoplasm to the cell
membrane and the subsequent glucose uptake into a cell independent of the insulin
system. AMPK also accelerates the mitochondrial fatty acid uptake by inhibiting the
acetyl-coenzyme A carboxylase activity. AMPK phosphorylates several downstream
transcription factors and coactivators that regulate energy catabolism. AMPK activation
results in the upregulation and the direct phosphorylation of the peroxisome proliferatoractivated receptor coactivator-1 (PGC-1), which is an important regulator for
mitochondrial biogenesis. In addition, AMPK indirectly deacetylates PGC-1 via SIRT1.

Tel : +81-22-304-5599, Fax: +81-22-304-5591, E-mail: suwa-m@tohtech.ac.jp.

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Masataka Suwa
These characteristics make AMPK a valuable therapeutic target for chronic metabolic
diseases associated with insulin resistance.

Keywords: AMPK, GLUT4, mitochondria, PGC-1, skeletal muscle, type II diabetes

Introduction
One of the unique characteristics of skeletal muscle is its considerable heterogeneity in
terms of its metabolic properties. Skeletal muscle plays an important role in regulating the
whole body energy expenditure due to its great capacity for glucose and lipid metabolism.
Skeletal muscle can account for 40~50% of body weight in non-obese subjects [1] and as
much as 30% of the total resting substrate oxidation [2].
An impaired skeletal muscle metabolic capacity is considered to be associated with the
development of chronic metabolic disorders such as obesity and type 2 diabetes mellitus [3-8]
that are increasing in worldwide. The metabolic profile in skeletal muscle is also a key
determinant of physical performance [9-11].
How the metabolic properties in skeletal muscle are controlled has been investigated for
several decades. It is known there are a number of signaling molecules that regulate metabolic
adaptations in skeletal muscle, and hundreds of reports have reached the general hypothesis
that 5-AMP-activated protein kinase (AMPK) is a central energy-sensing master regulator of
cellular metabolism. AMPK was first described in 1987 as a common protein kinase
regulating fatty acid and cholesterol synthesis [12]. AMPK is present in many tissues [13]. It
regulates the carbohydrate and lipid metabolism in a cell [14] and also regulates appetite via
orexigenic neuropeptides in the hypothalamus [15], and it can control the whole body energy
metabolism.
AMPK is also considered to regulate protein synthesis and degradation [16-19], cellular
polarity [20, 21], the cell cycle [22], apoptosis [23] and autophagy [24]. AMPK has a possible
link with a variety of disorders, such as obesity, insulin resistance, metabolic syndrome,
cardiovascular diseases, stroke, dementia and cancer [14, 25]. AMPK expression is associated
with an extended life span in C. elagans [26, 27], and thus, the gene encoding it is considered
to be one of the longevity genes [28].
Skeletal muscle AMPK is activated by many stimuli including muscle contraction,
caloric restriction, hormones and cytokines, drugs, nutrients, reactive oxygen species (ROS)
and other chemically synthesized activators.
In recent years, AMPK has emerged as a critical regulator of skeletal muscle glucose
transport, fatty acid oxidation and mitochondrial biogenesis as a result of its phosphorylation
of downstream targets. AMPK is therefore a potential therapeutic target for obesity and type
II diabetes mellitus. This chapter will focus on how the AMPK regulates the skeletal muscle
energy balance and metabolic adaptations that are associated with the prevention and
therapeutics for such diseases.

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1. Evolution and Structure of AMPK

AMPK is a highly conserved heterotrimetric serine/threonine kinase and with orthologs
found in all eukaryotes, including mammals (AMPK), yeast (sucrose nonfermenting 1:
SNF1), plants (SNF1-related kinase: SnRK1), roundworms (AMP-activated kinase: AAK)
and insects (AMPK). Human AMPK consists of a catalytic - and regulatory - and subunits [29].
Figure 1 illustrates the structural elements of the AMPK subunit sequences. Two
isoforms exist for both the -subunit (1 and 2) and -subunit (1 and 2) and there are
three -subunits (1, 2, and 3) that enable the expression of 12 // heterotrimer
combinations. The -subunit contains the serine/threonine kinase domain at the N terminus,
which only has kinase activity when it has been phosphorylated by upstream kinases [30].
Three alternative splice variants exist for 1 (1.1, 1.2 and 1.3) [14]. The 1.3 isoform of
550 residues appears to be widely expressed, whereas the 1.1 isoform of 575 residues and
the 1.2 isoform of 559 residues have not been fully characterized [13, 14]. The 2 isoform of
552 residues is more restricted in its tissue distribution. It is strongly expressed in skeletal
muscle and lesser extent in the heart, brain, kidneys and liver [13]. The -subunit also
contains a subunit interacting domain that contacts the -subunit. The -subunit contains a
domain interacting with the - and -subunits, which mediates the association of the
heterotrimeric AMPK complex [31, 32]. Most -subunits also contain a glycogen binding
domain. The -subunit is composed of four repeating cystathione--synthase (CBS) domains
that can bind the AMP and induce a subsequent conformational change in the AMPK
complex, the phosphorylation of threonine 172 in the -subunit, and thus fully lead to kinase
activation [33, 34, 35]. There are three 2 isoforms (2.1, 2.2, and 2.3) and two 3 isoforms
(3.1 and 3.2), and the most common isoforms are the 2.1 and 3.1 isoforms [14].

Figure 1. The structure of AMPK. AMPK is composed of a catalytic -subunit and regulatory - and subunits. The -subunit consists of the NH2-terminal kinase domain and its auto-inhibition domain and
-subunit interacting domain. The catalytic domain can be phosphorylated by upstream AMPK kinase
on threonine 172. The -subunit consists of a glycogen binding domain and an - and -subunitsinteracting domain. The -subunit is composed of four repeating cystathione--synthase (CBS) domains
that can interact with ATP, ADP, and AMP.

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2. The AMPK Isoform Expression Patterns

in Skeletal Muscle
Most mammalian skeletal muscles are composed of slow-twitch (type I) and fast-twitch
(type II) fibers. The type II fibers are further subclassified into type IIA, IIX and IIB fibers in
rodent muscle and type IIA and IIX fibers in human muscle [36]. The type I, IIA, IIX and IIB
fibers dominantly express the myosin heavy chains 1, 2a, 2x (2x is also called 2d) and 2b,
respectively, in rodents [37, 38]. The rank order of the maximum contraction velocity in rat
skeletal muscle fibers is I < IIA < IIX < IIB [39]. The expression pattern of AMPK subunit
isoforms in skeletal muscle differs among the fiber types. The 1- and 2-subunits expression
levels are lower in the type I fiber-rich soleus muscle than in the type IIB fiber-rich white
quadriceps muscle in rats [40]. The 3-subunit of AMPK is dominantly expressed in the type
II fiber-rich gastrocnemius and extensor digitorum longus muscles in comparison to the
soleus muscle in rodents [41, 42].
The 1-subunit plays an important role in skeletal muscle growth and hypertrophy [43,
44]. The 2-subunit seems to partially regulate the metabolic and contractile properties of
skeletal muscle [45-48]. The 3-subunit controls the carbohydrate and lipid metabolism,
mitochondrial biogenesis, and endurance performance in mouse fast-twitch muscle and
human muscle [49-52]. It is therefore likely that the different characteristics among the fiber
types are at least partly attributable to the differences in the subunit expression pattern. Only
three of the 12 possible heterotrimers are present in human skeletal muscle: 1/2/1,
2/2/1 and 2/2/3 [53, 54]. The 3-containing AMPK complexes contain only 2- and
2-subunits in the skeletal muscle [41], suggesting that the 2/2/3 heterotrimer is
preferentially expressed in the fast-twitch muscle. During high-intensity exercise, only the
2/2/3 heterotrimer is activated, while the activity of 1/2/1 or 2/2/1 heterotrimers
either remains unchanged or decreases [54]. These results raise the possibility that the
expression patterns of AMPK heterotrimer species affect the characteristics and exerciseinduced adaptations of skeletal muscles.

3. Activation and Inhibition

of AMPK in Skeletal Muscle
AMPK activation is regulated by the phosphorylation of Thr172 in the catatytic -subunit
as is the case for many protein kinases. When phosphorylated, AMPKs activity can increase
by more than 100-fold [55]. The phosphorylation of AMPK is accomplished by upstream
AMPK kinases. In addition, adenosine nucleotides allosterically regulate AMPK activity.
Other physiological and chemical stimulators/inhibitors also control the AMPK
phosphorylation status and activity.

3.1. Allosteric Regulation

The ATP/AMP and ATP/ADP ratios reflect the cellular energy turnover. Because AMPK
is subjected to allosteric regulation by adenosine nucleotides, it is considered to be an energy

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sensor in cells. Increased AMP or ADP levels promote their binding to the AMPK -subunit,
which presumably results in a conformational change in the AMPK heterotrimer from the
inactive to the active conformation [56]. This allosteric regulation increases the AMPK
activity by only a few fold [56, 57]. However, the binding of AMP promotes the
phosphorylation of Thr172 in the -subunit by liver kinase B1 (LKB1) and Ca2+/calmodulindependent protein kinases (CaMKK) [58]. Once AMPK is phosphorylated, AMP can further
allosterically activate it and inhibit the dephosphorylation of Thr172 by protein phosphatases
[58, 59. 60].

3.2. AMPK Kinases

LKB1 is a well-defined upstream kinase of AMPK, and is the primary AMPK kinase in
skeletal muscle. The LKB1 kinase activity is maximally enhanced by its interaction with two
regulatory proteins termed Ste20-related adaptor (STRAD) and mouse protein 25 (MO25)
[61]. Unlike many protein kinases that are regulated by phosphorylation, the LKB1-MO25STRAD complex is a constitutively active kinase that acts through unknown,
phosphorylation-independent mechanisms [61, 62]. The ablation of LKB1 in mouse skeletal
muscle induced a decrease in the AMPK2 activity in spite of a significant decrease in the
ATP/AMP ratio [63]. In addition, the lack of LKB1 abolished the 5-aminoimidazole-4carboxamide-1--D-ribofuranoside (AICAR)- and phenformin-induced activation of
AMPK2 and phosphorylation of Thr172 in 2-subunit [63]. These results suggest that LKB1
at least partially regulates the AMPK2 activity under certain conditions.
CaMKK is another upstream AMPK kinase that can be activated in response to an
elevated intracellular Ca2+ level [64, 65]. There are two isoforms of CaMKK; CaMKK and
CaMKK [66]. The CaMKK protein is highly expressed in skeletal muscle [44, 67]. On the
other hand, CaMKK protein expression in skeletal muscle is controversial [44, 68, 69].
Forced expression of constitutively active CaMKK (caCaMKK) in mouse skeletal muscle
fibers by transfecting them with a vector encoding caCaMKK increased both the AMPK1
and -2 activities [67]. Incubation of isolated mouse soleus muscle with caffeine, which
promoted the Ca2+ release from sarcoplasmic reticulum (SR), resulted in elevated AMPK1
activity, whereas this effect was abolished by pre-incubating the muscle with either a SR Ca2+
release blocker, dantrolene, the CaMKK inhibitor, STO-609, or the calmodulin-competitive
inhibitor, KN-93 [70]. These results imply that the SR Ca2+-activated CaMKK enhances the
AMPK1 activity in skeletal muscle fibers.

3.3. AMPK Activators

3.3.1. Exercise and Muscle Contraction
Exercise and muscle contraction are the most potent physiological activator of AMPK. In
the first report in 1996 by Winder and Hardie, who demonstrated the activation of skeletal
muscle AMPK by exercise in rats, many studies concerning the relationship between AMPK
and exercise/contraction have been conducted [71]. Changes in the cellular energy stress, the
Ca2+ concentration, the production of ROS, hormones/cytokines and other signaling
molecules can promote the activation of AMPK during exercise/contraction.

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As mentioned above, AMPK acts as an energy sensor by sensing energy deprivation (i.e.
increased an AMP/ATP level). Exercise incorporates the muscle contraction which enhances
the energy consumption by up to ~ 100 fold from rested muscle [72]. When the consumption
of ATP exceeds the generation during contraction, the AMP/ATP ratio is elevated, which
triggers the activation of AMPK through allosteric regulation and phosphorylation by LKB1.
Ionized calcium is considered to be an important signaling ion in skeletal muscle [73, 74].
Increasing the intracellular Ca2+ concentration by caffeine treatment can result in AMPK
activation, which occurs at least partially via CaMKK, in isolated skeletal muscle [72, 75].
Such caffeine-induced change preferentially occurs in AMPK1 in comparison to the
AMPK2 [72, 76]. Because an increasing intracellular Ca2+ level is essential for muscle
contraction, it is possible that increasing the intracellular Ca2+ released from the SR during
muscle contraction may promote the phosphorylation of AMPK.
Superoxide anion radicals (O2-), one of the ROS, are continually produced in cells, and
the production of O2- increases substantially in contracting muscle [77, 78]. O2- is
immediately enzymatically dismutated to hydrogen peroxide (H2O2) by superoxide dismutase
[77]. In NIH-3T3 cells, H2O2 treatment phosphorylated the Thr172 in 1-subunit in a
concentration-dependent manner [79]. In addition, the contraction-induced AMPK activation
was partially blocked by the antioxidant, N-acetylcysteine, in isolated muscle [80, 81].
Collectively, these results suggest that ROS may activate AMPK during contraction [81].
H2O2-induced AMPK1 activation is not associated with increasing the AMP level or
decreasing the ATP/AMP ratio [80]. Although the mechanisms involved in AMPK activation
by ROS have been still unclear, the AMPK1 activation by acute ROS production may occur
via an adenosine nucleotides-independent mechanism.
Nitric oxide (NO) producion in a tissue is catalyzed by NO synthase (NOS). NO is
considered to be an important signaling molecule required for the metabolic adaptations in
skeletal muscle fibers [82, 83]. NOS is the primary source of NO in the contracting muscle
[84, 85]. The NOS activity and phosphorylation of neuronal NOS in skeletal muscle increased
with endurance exercise [86, 87]. The treatment of isolated skeletal muscle with NO donors
increases the AMPK1 activity, but does not affect AMPK2 [88, 89]. Therefore it is
possible that the exercise-induced activation of AMPK1 (as mentioned below) is partially
associated with the increase in NOS activity and NO production.
Mild heat stress is also associated with the AMPK activity in skeletal muscle. The
temperature of skeletal muscle reaches ~40C during exercise [90-92]. The exposure of rat
hepatocytes to heat shock increases the AMPK activity, which is considered to be a potential
protective response against the stress [93]. The AMPK phosphorylation occurring after mild
heat stress at 40C is significantly higher than that at 37C in C2C12 muscle cells [94].
Collectively, these findings indicate that it is likely that the elevated temperature of skeletal
muscle during exercise activates AMPK.
Recent studies have demonstrated that skeletal muscle expresses and secretes cytokines
(myokines) and hormones that can work in an autocrine, paracrine and endocrine fashion [95,
96]. In response to acute exercise, several myokines are released from skeletal muscle [97,
98]. Exercise and contraction induce the expression and secretion of interleukin-6 (IL-6) [99101] and brain-derived neurotrophic factor (BDNF) [102] in skeletal muscle cells. IL-6 has
been reported to be an inflammatory cytokine [103], and the plasma IL-6 concentraiton
increases by numerous inflammatory conditions and infection diseases. However, the
circulating IL-6 level increases more dramatically, up to 100-fold, in response to exercise

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[104]. IL-6 has been shown to activate AMPK [105, 106], and the AMPK activation in
skeletal muscle during exercise is diminished in IL-6 knockout mice, thus suggesting that IL6 at least partially controls the exercise-induced activation of AMPK in skeletal muscle.
BDNF is a member of the neurotrophin family, and can play an important role in
neuronal outgrowth, differentiation, synaptic connection and neuronal repair [107]. The
circulating BDNF level also increases during exercise [108]. BDNF can activate the AMPK
in skeletal muscle cells [102]. Therefore, IL-6 and BDNF are the potent candidates for
activating AMPK in skeletal muscle during exercise.
A major question has been what types of exercise(s) activate AMPK in the skeletal
muscle. The AMPK activation by endurance exercise seems to be intensity- and timedependent. Low-intensity endurance exercise (40 to 50% maximal O2 uptake) for 20 to 90
min fails to change the AMPK activity [109, 110]. On the other hand, prolonged low-intensity
endurance exercise (~45% maximal O2 uptake) until exhaustion (~3.5 h) increases the
AMPK2 activity, but not the AMPK1 activity [111]. Moderate-intensity exercise (60 to
80% maximal O2 uptake) for five to 60 min also preferentially activates the AMPK2 [54,
109, 110, 112]. It is therefore suggested that low- to moderate-intensity exercise is associated
with isoform-specific and intensity- and time-dependent increases in AMPK2 activity, but
not in AMPK1 activity. Exposure to further high-intensity exercise, such as sprint exercise,
in single or four bouts of exhausting sprint exercises for 30s, thus leads to increase in the both
the AMPK1 and 2 activities [110, 113]. Resistance exercise also increases the activity and
phosphorylation of AMPK [114-116].
3.3.2. Hypoxia
An inadequate oxygen supply (hypoxia) is considered to be another types of metabolic
stress. Hypoxia limits oxidative phosphorylation and leads to a decrease in ATP production
and the ATP/AMP ratio [117]. Hypoxia can generate ROS, and peroxynitrate is generated
from NO under hypoxic conditins [118, 119]. Under such conditions, AMPK can be rapidly
activated [119, 120]. Hypoxia promotes the activation of both AMPK1 and 2 in skeletal
muscle [47, 117, 121].
3.3.3. Secretory Factors
Beside the intracellular factors, several autocrine, paracrine and endocrine factors
including hormones and cytokines, regulate the AMPK activity [122]. Leptin, adiponectin,
and ciliary neurotrophic factor (CNTF) can all activate the skeletal muscle AMPK, as well as
IL-6 and BDNF, which were reviewed above in section 4.3.1.
Adipose tissue is currently recognized as a secretory organ, which secretes a variety of
biologically active molecules called adipocytokines. Adipocytokines play an important role
in regulating metabolism and inflammation [123]. Leptin was the first adipocytokine
identified, and it can act on central and peripheral tissues to regulate the food intake and
energy expenditure [124]. Skeletal muscle expresses leptin receptors, and the administration
of leptin to mice has been shown to promote AMPK2 activation in skeletal muscle [125].
Leptin preferentially activates the AMPK of type I muscle fibers rather than type II fibers
[125, 126]. There seems to be two ways by which leptin activates AMPK2. One is a direct
effect that occurs rapidly. Another later effect occurs through a primary action of leptin on the
hypothalamus and the subsequent secondary effects on skeletal muscle, which are mediated
by the sympathetic nervous system [125].

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Adiponectin is also an adipocytokine that regulates the peripheral metabolism [127],

protects the heart from ischemic injury [128], and controls appetite [129, 30]. Skeletal muscle
expresses two isoforms of adiponectin receptors: adipoR1 and AdipoR2 [131]. AdipoR1 was
observed to activate the AMPK by increasing the intracellular Ca2+ concentration in C2C12
skeletal muscle cell [132, 133]. On the other hand, AdipoR2 can regulate the peroxisome
proliferator-activated receptor (PPAR) signaling but not AMPK [133, 134].
CNTF is a trophic molecule of the IL-6 family that is considered to possess neurotrophic
and myotrophic effects [135, 136]. CNTF is most abundantly synthesized by the sciatic
nerves [137]. The CNTF receptor (CNTFR) is expressed in skeletal muscle as well as in the
nervous system [138], and it has been speculated that CNTF is a nerve-derived myotrophic
factor. In addition, skeletal muscle also expresses CNTF mRNA [139]. Incubation of isolated
type I fiber-rich soleus muscle of mice with CNTF results in increases in the AMPK1 and
AMPK2 activities, but this was not observed following incubation with type II fiber-rich
extensor digitorum longus muscle [140, 141]. This effect on the soleus muscle is speculated
to be due, at least in part, to the increase in AMP and corresponding decrease in the ATP
content [141]. In vivo treatment of mice with CNTF also increases the AMPK1 and
AMPK2 activities of type I and IIA fiber-rich red gastrocnemius muscle, but not type IIB
fiber-rich white gastrocnemius muscle [141]. Therefore, CNTF seems to preferentially
activate the AMPK in the type I fibers.
3.3.4. Chemical Activators
AMPK can be activated by many chemical substrates. The adenosine analogue 5aminoimidazole-4-carboximide 1--D-ribofuranoside (AICAR) was the first and is the most
widely used chemical activator of AMPK. Before recognizing the effect of AICAR on
AMPK, AICAR had been noted to reduce the myocardial ischemic injury [142-144]. In 1994,
it was reported that in vitro treatment of primary adipocyte with AICAR increased the AMPK
activity in a time- and dose-dependent manner [145]. AICAR is rapidly taken up into cells
and phosphorylated nucleotide 5-aminoimidazole-4-carboximide ribonucleoside (ZMP)
[146]. ZMP is an analogue of AMP and mimics the activating effect of AMP on AMPK
[146]. Although AICAR strongly activates AMPK, it can also activate other AMP-sensitive
enzymes [147-149], suggesting that AICAR is not a specific activator of AMPK.
A-769662 is a direct activator of AMPK that was identified by screening a chemical
library of more than 700,000 compounds using partially purified liver AMPK [150].
Although in vivo treatment of rats with A-769662 increases the concentration and metabolic
effects of AMPK in the liver, the changes in the concentrations of AMPK in the skeletal
muscle and brain are much lower than that in liver [150]. Incubation of mouse skeletal muscle
with A-769662 increased the activity of the 1-, but not 2, subunit-containing AMPK [151].
The human liver contains ~15% 1-subunit-containing AMPK [151], whereas the level is
undetectable in human muscle [151]. Therefore, A-769662 would not be an ideal AMPK
activator for skeletal muscle.
Several antidiabetic, insulin-sensitizing drugs such as the biguanides (metformin and
phenformin) and thiazolidinediones (rosiglitazone, pioglitazone and troglitazone) can activate
AMPK. Although biguanides have been used for the treatment of type II diabetes mellitus
since the 1950s, their mechanisms of action have only been understood since 2001. Zhou et
al. first indicated that metformin activates the AMPK in hepatocytes and isolated rat
epitrochlearis muscle [152]. In vivo metformin treatment also activates the AMPK in skeletal

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muscle [153, 154]. The action of metformin on the AMPK phosphorylation in skeletal muscle
is much milder than that of AICAR. A single injection of metformin takes for 5-6 h to
increase the phosphorylation of AMPK [154], whereas intraperitoneal injection of AICAR
into rats increased the AMPK phosphorylation within 30 min after the injection [155].
Collectively, these findings suggest that metformin and AICAR regulate AMPK via different
mechanisms.
The biochemical mechanisms by which metformin activates AMPK are unclear, but the
effects of metformin such as the inhibition of mitochondrial complex I of the respiratory
chain [156, 157], production of reactive nitrogen species [158] and inhibition of AMP
deaminase [159] are all considered to be potential candidates. Interestingly, the activation of
AMPK by metformin or phenformin treatment of isolated perfused rat heart is followed by an
increase in the cytosolic AMP concentration, without any change in the total AMP content or
AMP/ATP ratio [160], raising the possibility that metabolically active AMP regulates the
AMPK activity, and that the action of biguanides on AMPK occurs through such a
mechanism.
Thiazolidinediones are agonists of the transcription factor peroxisome proliferatoractivated receptor (PPAR). Thiazolidinediones can improve insulin sensitivity and reduce
the lipolysis and content of fatty acids in adipocytes through the binding and activation of
PPAR [161]. In addition, AMPK can be rapidly activated by thiazolidinediones in skeletal
muscle, with a coincident increase in the AMP level and a decrease in the ATP level [162,
163]. Long-term administration of thiazolidinediones in vivo also enhances the basal AMPK
activity in patients with type II diabetes mellitus [164, 165], and in rats [166] and mice [167].
Such treatment also increases the circulating adiponectin level and adiponectin secretion from
adipose tissue [168].
Interestingly, exposure of cultured myocytes to rosiglitazone enhances their adiponectin
production and AMPK2 phosphorylation [169], suggesting that thiazolidinediones promote
the autocrine or paracrine control of AMPK via the adiponectin secretion in skeletal muscle.
Additionally, the ablation of adiponectin abolishes the increase in AMPK activity induced by
thiazolidinediones both in vivo and in vitro [167, 169]. These results imply that AMPK
activation in the skeletal muscle by thiazolidinediones is at least partially mediated by
adiponectin signals.
Many nutraceuticals derived from foods, beverages, and herbal medicine can also
activate AMPK. It was reported that many compounds, including the polyphenol resveratrol
(present in the skin of red grapes and in red wine) [170], the flavonoid quercetin (present in
many fruits and vegetables) [171], berberine (present in genus Berberis used in Chinese
medicine) [172], compounds derived from bitter melon [173], epigallocatechin gallate
(present in green tea) [174], capsaicin (present in peppers) [175], -lipoic acid (present in the
internal organs of animals and vegetables) [176], the isoflavone genistein (present in many
plant) [177], the flavonoid tangeretin (present in the peel of citrus fruits) [178] and osmotin
(present in the skin of vegetables and fruits) [179] can activate AMPK. Resveratrol, quercetin
and berberine have all reported to increase the ADP:ATP ratio, which is accompanied by the
inhibition of mitochondrial ATP synthesis, which is associated with AMPK activation in
[180]. Osmotin is a ligand for AdipoR1 [179]. The mechanisms of action for the other
compounds are currently unknown.

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3.3.5. Caloric Restriction

Caloric restriction (CR) is the most consistent strategy for extending the lifespan in many
species, from yeast to monkeys [181]. CR induces an increase in the AMP/ATP ratio and
leads to lifespan extension in C. elegans, while the expression of an AMPK2 mutant
abolished the lifespan extension, suggesting that AMPK is required for CR-induced lifespan
extension [27]. CR and fasting can activate AMPK in the skeletal muscles of mice [182-184].
On the other hand, other studies have demonstrated that CR fails to change or decreases the
AMPK activity in skeletal muscle [185, 186]. The reasons for such incompatible results are
unclear.

3.4. AMPK Inhibitors

3.4.1. AMPK Phosphatases
Protein phosphatases can regulate the phosphorylation of AMPK. Purified protein
phosphatases 2A (PP2A) and 2C (PP2C) decrease the AMPK activity in cell-free assays
[187]. Recombinant PP2C also dephosphorylated the recombinant AMPK [188]. The affinity
of PP2C for AMPK seems to be negatively regulated by AMP [188]. Increased PP2C
expression and activity are associated with decreased AMPK activity in the skeletal muscle of
mice [189], thus suggesting that PP2C negatively regulates the AMPK activity in skeletal
muscle in vivo. PP2A is associated with ceramide-induced AMPK inhibition, as discussed
below in section in 4.4.3.
3.4.2. Chemical Inhibiton
Compound C is a synthetic AMP/ATP-competitive inhibitor of AMPK [152, 190, 191].
In skeletal muscle cells, AICAR-induced AMPK activation is abolished by Compound C
because it blocks the conversion of AICAR into ZMP [191]. On the other hand, hyperosmotic
stress- or dinitrophenol-induced AMPK activation are unaltered by Compound C [191]. In
addition, Compound C treatment does not change the contraction-induced AMPK
phosphorylation in isolated rat epitrochlearis muscle [192]. However, Compound C also
inhibits many other protein kinases [193], suggesting that it is not a specific AMPK inhibitor.
3.4.3. Nutrient Excess
High glycogen and glucose inhibit the AMPK activity. Acute glucose infusion increases
the glycogen content and decreases the AMPK activity of skeletal muscle in rats [194]. A
single bout of sprinting exercise promotes the AMPK phosphorylation in skeletal muscle,
whereas the same exercise with glucose ingestion fails to phosphorylate AMPK [195]. The
basal and AICAR-treated AMPK2 activities in skeletal muscle with a high glycogen content
using a glycogen super-compensation technique induced by a combination of swimming
exercise and a high-glycogen diet are lower than those of rats with a low glycogen content
induced by a combination of swimming exercise and a high-fat diet in rats [196]. It has been
speculated that glycogen binds to the glycogen binding domain in the -subunit that
allosterically inhibits the AMPK [197].
Lipid overload also inhibits the AMPK activity. Feeding rodents a high-fat diet impairs
the skeletal muscle AMPK activity, phosphorylation and protein expression [198, 199].

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Exposure of cultured bovine aortic endothelial cells to palmitate, a saturated free fatty acid,
decreases the AMPK phosphorylation due to an increase in the PP2A activity [200]. PP2A is
activated by ceramide [201], which is de novo synthesized from saturated free fatty acids
[202]. Additionally, a cell-permeable analog of ceramide can mimic the effects of palmitate
on AMPK phosphorylation [200]. Collectively, these data indicate that it is possible for an
excess of saturated free fatty acids to increase the intracellular ceramide concentration, which
activates the PP2A and leads to AMPK dephosphorylation.
The essential amino acid leucine is associated with AMPK activity and phosphorylation.
Treatment of C2C12 myoblasts with leucine decreased their AMPK activity and
phosphorylation [203]. Acute leucine administration to rats decreased the AMPK
phosphorylation and AMP/ATP ratio in the gastrocnemius muscle [204]. On the contrary, the
incubation of mouse extensor digitorum longus muscle with leucine also decreased the
AMPK phosphorylation and AMPK2 activity, but the concentration of adenine nucleotides
did not change [205].
3.4.4. Secretory Factors
Resistin is a newly-identified adipocytokine expressed in adipocytes and macrophages
that may play a role in obesity-induced insulin resistance [206, 207]. Chronic hyperresistinemia in rats induced by the intravenous administration of an adenovirus encoding
mouse resistin downregulated the AMPK phosphorylation in the skeletal muscle, liver and
adipose tissue [208]. Exposure of L6 muscle cells to resistin (50 nM, i.e., ~625 ng/ml, 24 h)
also decreased the AMPK phosphorylation [209]. In contrast, incubating L6 cells with a
physiological concentration of resistin (100 ng/ml, 4 h) did not affect the AMPK status [210].
Although the reason(s) for the discrepancies between these findings are still unclear, the
differences in the concentrations of resistin and the length of exposure might have led to the
inconsistent results. Tumor necrosis factor- (TNF-) is cleaved by a TNF- converting
enzyme primarily produced by macrophages [211]. TNF- is also an adipocytokine [212],
and its effects are mediated by two receptors: TNFR1 and TNFR2 [213]. TNF- can inhibit
AMPK through TNFR1 signaling in cultured skeletal muscle cells and the skeletal muscle of
mice [189, 214]. TNF- promotes the expression of PP2C, which decreases the AMPK
phosphorylation in L6 myotube [189].

4. Regulation of Glucose Metabolism by AMPK

Glucose metabolism is the one of most essential functions in most organisms. AMPK can
control the glucose metabolism in several tissues including skeletal muscle, adipose tissue
and liver. In skeletal muscle cells, AMPK is associated with glucose uptake, glycogen
synthesis and glucose phosphorylation, in cooperation with insulin signals.

4.1. Glucose Uptake

It is well known that insulin play an important role in reducing the blood glucose level by
promoting the glucose uptake of skeletal muscle (Figure 2) and adipose tissue cells, as well as

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inhibiting the hepatic glucose production and output. On the other hand, the inhibition or
ablation of insulin signaling proteins such as phosphatidylinositol-3-kinase (PI3K) [215, 216],
insulin receptor substrate 1 (IRS1) [217], or the serine/threonine-specific protein kinase Akt2
[218] did not affect the contraction-stimulated glucose uptake in skeletal muscle.
Furthermore, the effect of insulin and contraction on glucose uptake was additive [219]. It is
therefore considered that further insulin-independent mechanisms controlling glucose uptake
during contraction exist.
A great deal of evidence suggests that AMPK is an important regulator of the skeletal
muscle glucose uptake during contraction, as well as a regulator of sucrose nonfermenting
AMPK-related kinase [220] and Ca2+ [221].
The skeletal muscle glucose uptake is mainly accomplished by the translocation of
glucose transporter 4 (GLUT4) from intracellular sites into the plasma membrane. Activation
of AMPK increases the GLUT4 translocation and glucose uptake of skeletal muscle both in
vivo and in vitro [222, 223].

Figure 2. The glucose uptake induced by insulin and AMPK signals in skeletal muscle. The
autophosphorylation and activation of insulin receptor substrate 1 (IRS1) and phosphoinositide 3-kinase
(PI3K) are induced by the binding of insulin to the insulin receptor (IR). PI3K converts
phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) by
phosphorylation. PIP3 interacts with phosphoinositide-dependent kinase 1 (PDK1), which
phosphorylates and activates Akt2. Akt2 phosphorylates the Rab guanosine triphosphatases (GTPases)
tre-2/USP6, BUB2, cdc16 (TBC1) domain family proteins of member 1 (TBC1D1) and member 4
(TBC1D4), which bind to 14-3-3 protein and their activities are inhibited. AMPK is phosphorylated and
activated by liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinases (CaMKK). AMPK
is subjected to allosteric regulation by AMP and ADP. AMPK also inhibits TBC1D1 and TBC1D4, the
same as insulin signals. As a result, the level of guanosine diphosphate (GDP)-bound Rab protein
decreases, and then GLUT4 is released from intracellular sites to the plasma membrane.

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Although the mechanisms underlying how AMPK controls the GLUT4 translocation
have not been fully elucidated, it has been speculated that the tre-2/USP6, BUB2, cdc16
(TBC1) domain family proteins of member 1 (TBC1D1) and member 4 (TBC1D4, formerly
known as Akt substrate of 160 kDa), that regulate the GLUT4 trafficking by their Rab
guanosine triphosphatase (GTPase) activity [224-226], mediate the action of AMPK. Both
TBC1D1 and TBC1D4 are phosphorylated by AMPK [224, 227-229]. When phosphorylated,
TBC1Ds bind to 14-3-3 proteins and their Rab GTPase activity is inhibited. As a result, the
level of guanosine triphosphate (GTP)-bound Rab protein increases, and GLUT4 is released
from intracellular sites to the plasma membrane [224, 225, 229-231] (Figure 2).
AMPK plays a putative role in improving the insulin sensitivity of skeletal muscle. The
activation of AMPK by AICAR treatment enhances the insulin-stimulated glucose transport
to fast-twitch muscles in vivo and ex vivo [232-234]. One possible explanation for these
finding is that AMPK can activate the insulin signaling proteins, IRS1 and PI3K. AMPK
activation induces the phosphorylation of IRS1 on Ser-789 in cell free assays and in C2C12
myotubes [235]. In addition, preincubation of C2C12 myotubes with AICAR increased the
insulin-stimulated IRS1-associated PI3K activity [235]. Treatment of rats with AICAR for
five days also increased the insulin-stimulated IRS1-associated PI3K activity in isolated
skeletal muscle [236]. However, it has also been suggested that the phosphorylation of IRS1
on Ser-789 is associated with insulin resistance in genetically insulin-resistant animal models
[237]. Therefore, further studies are required to elucidate the role of AMPK in insulin
sensitivity and the phosphorylation of Ser-789 of IRS1.

4.2. Glycogen Synthesis

AMPK is associated with glycogen synthesis and breakdown. AMPK directly
phosphorylates glycogen synthase at Ser7 [238], which is known to inhibit glycogen synthase
[239]. Expose of AICAR to isolated mouse skeletal muscle also lead to the phosphorylation
of Ser7 of glycogen synthase, which decreased its activity [240]. It was therefore anticipated
that the activation of AMPK would result in a decrease in the glycogen level. However,
chronic AICAR administration to rats increased both the glycogen content [241, 155] and
glycogen synthase activity [155]. Under such a condition, the level of cytosolic glucose-6phosphate, which can allosterically activate glycogen synthase [155], increases via AMPKinduced glucose uptake and phosphorylation that may induce glycogen synthase activity.
Presumably, the increased glucose-6-phosphate overcomes the inhibitory effect of the
phosphorylation of glycogen synthase by AMPK.

4.3. Regulation of GLUT4 Expression

The GLUT4 expression level is one of the determinants of the glucose uptake capacity in
skeletal muscle and is important for the maintenance of whole body glucose homeostasis.
Overexpression of GLUT4 increases the insulin- and contraction-stimulated glucose transport
into skeletal muscle fibers [242]. The selective disruption of GLUT4 in skeletal muscle
almost completely abolished the insulin- and contraction-stimulated glucose transport,
impaired the whole body glucose tolerance and led to the development of diabetes [243, 244].

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Skeletal muscle GLUT4 expression is increased by AMPK activation both in vivo [241, 245]
and in vitro [246]. The GLUT4 gene promoter contains putative binding sites for the
transcription factors GLUT4 enhancer factor (GEF) and myocyte enhancer factor 2 (MEF2)
[247, 248]. GEF can bind to the domain I in the GLUT4 promoter, which is necessary for
GLUT4 promoter activity [248]. The MEF2 binding domain is also required for the
transcriptional regulation of GLUT4 [247, 248]. The full extent of the transcriptional activity
of the GLUT4 gene appears to depend on the interaction between GEF and MEF2A [250].
MEF2 and GEF interact with the transcriptional co-repressor, histone deacetylase 5
(HDAC5), which inhibits the GLUT4 promoter activity [251, 252]. In human skeletal muscle
cell culture, AMPK was observed to phosphorylate the HDAC5 [253]. This reduced the
association between HDAC5 and the GLUT4 promoter and nuclear content of HDAC5, and
coincidently increased the GLUT4 expression [253], suggesting that AMPK regulates the
GLUT4 expression by removing HDAC5 from the GLUT4 promoter. Furthermore, the
activation of AMPK by AICAR injection in rats induced the GEF phosphorylation, the
translocation of GEF and MEF2 into the nucleus, and the binding of these proteins to DNA,
which subsequently increased the GLUT4 mRNA expression in skeletal muscle [254].
Additionally, the removal of HDAC5 from GEF-MEF2 enables the recruitment of
transcriptional coactivators, such as peroxisome proliferator activated receptor coactivator1 (PGC-1), which is also activated by AMPK (as mentioned below), and upregulates the
GLUT4 transcription via the interaction with MEF2 [255]. Treatment of wild type mice with
AICAR for 28 consecutive days increased the GLUT4 protein expression, especially in the
white gastrocnemius and quadriceps muscles, whereas such changes are fully abolished in
PGC-1 knockout mice [256], thus suggesting that the AMPK-induced GLUT4 biogenesis
requires the PGC-1.

4.4. Regulation of Hexokinase II

Hexokinase initiates glucose metabolism by phosphorylating glucose, and maintains the
downhill glucose gradient from the capillaries to the cytosol. Four isoforms of hexokinase (IIV) exist, and hexokinase II is predominantly expressed in skeletal muscle [257]. Hexokinase
II may contribute to the insulin- and contraction-stimulated glucose uptake. Forced reduction
of hexokinase II expression by partial knockout of hexokinase II impaired the exerciseinduced glucose uptake in the type I fiber-rich soleus muscle, but not in the type II fiber-rich
gastrocnemius or superficial vastus lateralis muscles of mice [258]. Additionally, the
overexpression of hexokinase II enhanced the insulin- and exercise-induced glucose uptake
[259]. It has been speculated that glucose phosphorylation by hexokinase is the rate-limiting
step required for glucose uptake in type I fibers [260]. The activation of AMPK by AICAR
treatment in rats or isolated skeletal muscle from rats increased the hexokinase activity and
hexokinase II expression [244, 245, 261-264]. The promoter of hexokinase II gene contains a
site for cAMP-response element (CRE), which binds to the CRE binding protein (CREB) and
activating transcription factor 1 (ATF1) [265, 266]. Incubation of rat epitrochlearis muscle
with AICAR increased the phosphorylation of both CREB and ATF1, whereas the
phosphorylation of CREB was lower in the gastrocnemius and tibialis anterior muscles of
muscle-specific LKB1 knockout mice [267], thus suggesting that AMPK probably regulates
hexokinase II transcription directly via the phosphorylation of CREB and ATF1.

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5. Fatty Acid Metabolism

Fatty acid (FA) oxidation in skeletal muscle is a three-step process, which includes: 1)
FA transport across the plasma membrane, 2) FA uptake into mitochondria and 3) oxidation
processes (-oxidation, the TCA cycle and the electron transport chain) that occur in the
mitochondria. Activation of AMPK increases the skeletal muscle FA oxidation [222, 268,
269], and AMPK can regulate all three steps.

5.1. FA Transport into Skeletal Muscle Cells

FA transport across the plasma membrane is accomplished by three types of transport
proteins, including FA transporter/CD36 (FAT/CD36) [270], FA-binding protein of plasma
membrane (FABPpm) [271] and FA transport proteins (FATPs) [272, 273]. The association
of AMPK with the FA transport into cells was first demonstrated by experiments using
cardiomyocytes, in which the activation of AMPK induced the translocation of FAT/CD36
from intracellular sites to the plasma membrane, similar to the mechanism of GLUT4
recruitment [274, 275]. Concerning the skeletal muscle cells, contraction increases the cellsurface FAT/CD36 protein content with a concomitant increase of AMPK phosphorylation
and FA uptake [276, 277], whereas the resistin treatment of L6 muscle cells decreased the
cell-surface FAT/CD36 protein content and reduced the AMPK phosphorylation and FA
uptake [209]. Therefore, the role of AMPK in FAT/CD36 trafficking in skeletal muscle cells
is expected. The activation of AMPK by AICAR in vivo increased the plasma membrane
FAT/CD36 content in the skeletal muscle of rodents [278, 279], while this change did not
occur in 2 kinase-dead AMPK transgenic mice [279]. Interestingly, the AICAR-stimulated
FA uptake and oxidation in the skeletal muscle of FAT/CD36 null mice were remarkably
diminished in comparison to these in wild type mice [280]. Together, these findings suggest
that FAT/CD36 play a critical role in the AMPK-induced FA uptake and oxidation in skeletal
muscle. Although the full mechanisms regulating the FAT/CD36 trafficking in skeletal
muscle cells have been unclear, TBC1D4 and Rab GTPase mediate the insulin- and AMPKstimulated FAT/CD36 translocation, similar to GLUT4, in cardiomyocytes [275]. Figure 3
shows the AMPK-mediated regulation of FAT/CD36 translocation and FA uptake in skeletal
muscle cells.

5.2. Mitochondrial Acyl-CoA Uptake and Oxidation

The intracellular FA are converted into fatty acyl coenzyme A (FA-CoA) by FA-CoA
synthase before mitochondrial uptake. The FA-CoA transport to the mitochondrial matrix is
catalyzed by the carnitine palmitoyltransferase (CPT) enzyme complex consisting of CPT-I,
carnitine-acylcarnitine translocase (CAT) and CPT-II [281].
CPT-I is localized in the outer membrane of the mitochondria, and catalyzes the
conversion of FA-CoA to acylcarnitine [281]. The acylcarnitine can permeate the inner
membrane via the CAT system [281]. The acylcarnitine is reconverted into FA-CoA via CPTII in the mitochondrial matrix [281]. CPT-I is considered to be the rate-limiting enzyme for

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this process. CPT-I is allosterically inhibited by malonyl-CoA, the first intermediate of FA

synthesis [281]. Malonyl-CoA is carboxylated from acetyl-CoA by acetyl-CoA carboxylase
(ACC).
When the ACC activity increases, an increase in the malonyl-CoA level and the
coincident suppression of CPT-I activity occur. In skeletal muscle, although ACC is not
required for FA synthesis, because the muscle does not synthesize FA, ACC plays an
important role in controlling the CPT-I activity.

Figure 3. The role of AMPK in fatty acid (FA) metabolism in skeletal muscle cells. AMPK promotes
the translocation of FA transporter/CD36 (CD36) translocation from intracellular sites to the plasma
membrane and the subsequent FA uptake. Intracellular FAs are converted into fatty acyl coenzyme A
(FA-CoA) by FA-CoA synthase before mitochondrial uptake. FA-CoA transport to mitochondrial
intermembrane space is catalyzed by carnitine palmitoyltransferase-I (CPT-I), which converts the FACoA to acylcarnitine. The acylcarnitine can permeate the inner membrane via the carnitineacylcarnitine translocase (CAT) system. The acylcarnitine is reconverted into FA-CoA by CPT-II in the
mitochondrial matrix. CPT-I is inhibited by malonyl-CoA, which is carboxylated from acetyl-CoA by
acetyl-CoA carboxylase 2 (ACC2). When the ACC2 activity increases, an increase in the malonyl-CoA
level and a coincident decrease in CPT-I activity occur. AMPK can phosphorylate and inhibit ACC2,
which decreases the malonyl-CoA concentration and leads to an increase in CPT-I activity. Because
CPT-I is the rate-limiting enzyme for this process, the FA-CoA level in the matrix increases. The FACoA in the matrix is oxidized through -oxidation, the TCA cycle, and the electron transport chain
(ETC).

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Two isoforms of ACC have been identified (ACC1 and ACC2). The primary isoform of
ACC in skeletal muscle is ACC2, which is localized in the mitochondrial outer membrane
[282]. The activation of AMPK by AICAR increases the ACC2 phosphorylation and FA
oxidation, since it decreases the malonyl-CoA concentration in the skeletal muscle [222, 268,
269] (Figure 3).
FA-CoA in the mitochondrial matrix is oxidized through -oxidation, the TCA cycle, and
via electron transport chain processes by many oxidative enzymes. The mitochondrial
components involved in FA oxidation are increased by the chronic activation of AMPK in
skeletal muscle. Long-term AMPK activation in rats promoted an increase in mitochondrial
enzyme activity and protein expression in the skeletal muscle [261, 262, 283]. Such changes
of mitochondrial biogenesis would therefore positively contribute to enhancing the oxidative
capacity of FA.

6. Transcriptional Control of Mitochondrial

Biogenesis through PGC-1
Chronic activation of AMPK in vivo induces mitochondrial biogenesis and increases the
mitochondrial volume in skeletal muscle [261, 262, 283]. Such adaptations are speculated to
be controlled by the expression and activity of the coactivator, PGC-1 because: 1) AMPK
activation increases PGC-1 expression, 2) AMPK activates the PGC-1 via deacetylation
and phosphorylation and 3) PGC-1 regulates mitochondrial biogenesis and its oxidative
capacity by interacting with transcription factors that can control the expression of
mitochondrial components and regulators of mitochondrial biogenesis. Indeed, chronic
AICAR treatment to wild type mice increased the cytochrome C oxidase I and cytochrome C
protein expression levels, whereas the treatment to PGC-1 knockout mice did not lead to a
change in the expression levels of these proteins [256], suggesting that the AMPK-induced
increases in the expression levels of three proteins require the PGC-1.

6.1. Regulation of PGC-1 Expression

Transgenic mice overexpressing PGC-1 had increased mitochondrial biogenesis in the
skeletal muscle, with coincident increases in fatty acid oxidation, insulin sensitivity, peak
oxygen uptake, and endurance exercise performance [284-287]. PGC-1 knockout mice
showed the downregulation of mitochondrial biogenesis in skeletal muscle [288, 289]. It is
therefore speculated that the PGC-1 expression level is one of the determinants of
mitochondrial biogenesis in skeletal muscle. AMPK can regulate the PGC-1 expression in
type II fiber-dominant skeletal muscle both ex vivo and in vivo. The treatment of isolated
epitrochlearis muscle with AICAR for 18 hours increased the PGC-1 mRNA and protein
expression levels [290, 291]. The administration of AICAR to rats also increased the PGC-1
protein expression in the extensor digitorum longus muscle 24 hours after administration
[245]. Furthermore, AICAR treatment upregulated the PGC-1 mRNA level and
mitochondrial components in wild type mice, whereas such an effect was abolished in
AMPK2 knockout mice [48, 292].

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Figure 4. Transcriptional regulation of the metabolic characteristics by peroxisome proliferator

activated receptor coacivator-1 (PGC-1) in skeletal muscle cells. AMPK directly phosphorylates
the cAMP response element binding protein family (CREB-F), which can regulate the hexokinase II
gene. AMPK also induces the expression of nicotinamide phosphoribosyltransferase (NAMPT), which
provides NAD+. Then, allosteric activation of silent information regulator of transcription 1 (SIRT1)
occurs. SIRT1 deacetylates and activates PGC-1. PGC-1 is also activated via phosphorylation by
AMPK. The activated form of PGC-1 interacts with several transcription factors, which can induce the
transcription of genes related to mitochondrial biogenesis and glucose transporter 4 (GLUT4)
expression. There are positive feedback loops for PGC-1 expression, i.e., PGC-1 interacts with
transcription factors such as peroxisome proliferator-activated receptor (PPAR), nuclear respiratory
factor 1 (NRF1), estrogen-related receptor (ERR), and the interaction with CREB-F-myocyte
enhancer factor 2 (MEF2) further induces the PGC-1 expression. GEF; GLUT4 enhancer factor.

The candidates of transcriptional regulators for AMPK-induced PGC-1 expression

include transcriptional factors of the CREB family and MEF2. The mouse PGC-1 promoter
contains a CRE and MEF2 binding site [293, 294]. AMPK can phosphorylate the
transcriptional factors of CREB family such as CREB and ATF1 [265, 266]. AMPK

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phosphorylates and inhibits HDAC5 which represses the PGC-1-transcribing activity of

MEF2 [293]. Interestingly, motor nerve stimulation enhanced the PGC-1 promoter activity,
whereas mutation of the CRE and/or MEF2 binding site fully abolished it [295], suggesting
that contraction-induced PGC-1 upregulation depends on CRE and MEF2 sequence
elements. Based on these results, it is plausible that the CREB family and MEF2 play a
crucial role in the AMPK-induced PGC-1 expression. Moreover, there are positive feedback
loops involved in the PGC-1 expression. In this loop, PGC-1 interacts with and activates
the MEF2 [294], nuclear respiratory factor 1 (NRF1) [296], estrogen-related receptor
(ERR) [297] and PPAR [298], which further induce the expression of PGC-1 (Figure 4).

6.2. PGC-1 Deacetylation and Phosphorylation

AMPK can regulate the PGC-1 activity by deacetylation via silent information regulator
of transcription 1 (SIRT1). SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent
histone deacetylase, can deacetylate PGC-1, thus leading to subsequent increase in
mitochondrial biogenesis and fatty acid oxidation [299, 300]. The treatment of C2C12 muscle
with AICAR increased in the SIRT1 activity with a concomitant increase in NAD +, an
allosteric coenzyme for SIRT1 [301, 302]. The treatment also increased the expression of
nicotinamide phosphoribosyltransferase (NAMPT) expression, which provides NAD+ [301].
In addition, fasting and exercise both increased the NAMPT expression, NAD+ level and
PGC-1 deacetylation in the skeletal muscle of wild type mice, but not of AMPK 3knockout mice [303]. Collectively, these data may suggest that the AMPK-NAMPT-NAD+SIRT1 pathway regulates the deacetylation of PGC-1 in skeletal muscle (Figure 4).
Interestingly, SIRT1 can activate LKB1 [304]. In addition, the resveratrol treatment -induced
AMPK phosphorylation was abolished by the SIRT1 inhibitor, nicotinamide in L6 muscle
cells, suggesting SIRT1 mediates the AMPK phosphorylation [305]. This positive feedback
loop between AMPK and SIRT1 might further promote deacetylation of PGC-1.
AMPK can also directly phosphorylate and activate PGC-1. AMPK2 interacts with
PGC-1 and phosphorylates it at Thr177 and Ser538, which requires the PGC-1-dependent
induction of the PGC-1 promoter in cell culture [306]. Figure 4 shows the putative processes
of PGC-1 activation by AMPK in skeletal muscle cells.

6.3. Transcriptional Control by PGC-1

PGC-1 is expressed in many tissues including brown and white adipose tissue, heart,
brain, kidneys, and liver, as well as in skeletal muscle [307], and interacts with many
transcription factors and nuclear receptor targets [308]. PGC-1 may regulate the
mitochondrial biogenesis by interacting with transcription factors that promote the expression
of mitochondrial genes in skeletal muscles. The first reported transcription factor that can be
activated via the interaction with PGC-1 is NRF1 [296]. NRF1 regulates the expression of
genes encoding proteins involved in the electron transport chain proteins and mitochondrial
transcription factor A (mtTFA), which controls the transcription of mitochondrial genes [209,
310]. PGC-1 also interacts with ERR and induces ERR-mediated transcription, which
regulates the genes involved in mitochondrial biogenesis [297, 311]. PGC-1 interacts with

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and activates PPAR and the proteins cooperate to induce the expression of genes involved in
mitochondrial fatty acid uptake and -oxidation [312]. As shown in Figure 4, PGC-1 can
promote the mitochondrial biogenesis via interactions with transcription factors as well as by
altering the glucose metabolism.

7. AMPK: A Therapeutic Target

for Metabolic Disorders
Impaired metabolic functions of skeletal muscle are one of the potential predictors of
metabolic disorders such as obesity, insulin resistance and type II diabetes mellitus.
Decreased insulin-stimulated glucose transport [313], mitochondrial enzyme activity and
respiration [3, 4, 7, 314], and the accumulation of triglycerides [315] and ceramide [316-317],
and the downregulation of metabolic regulators, including PGC-1 and NRF1 [318, 319] are
observed in the skeletal muscle of such patients. As discussed in this chapter, AMPK
regulates various aspects of energy metabolism including glucose homeostasis, fatty acid
uptake and oxidation and mitochondrial biogenesis in skeletal muscle. It has been
hypothesized that the activation of AMPK in skeletal muscle improves the metabolic
disorders.
In human skeletal muscle, the AMPK protein expression and activity are maintained in
obese patients and patients with type II diabetes mellitus [320-322]. Allosteric activation by
AMP and exercise-induced activation are also identical between the subjects with and without
type II diabetes [320, 321]. On the contrary, acute exercise-induced AMPK activation in
skeletal muscle is suppressed in the patients with type II diabetes and obesity [323]. However,
the fact that this study included more female subjects in the obese and diabetic groups than in
the lean group might mask the effects of exercise. The treatment of obese diabetic patients
with AICAR significantly increased the AMPK activity, glucose uptake, and fatty acid
oxidation in their skeletal muscle [321, 324, 325]. In addition, AICAR treatment improved
the diabetic symptoms in rodents, such as by decreasing the blood glucose level and
enhancing glucose tolerance [326, 327]. These results suggest that stimulating AMPK has
therapeutic value because it can compensate for and improve repressed insulin signaling.
Indeed, several pharmaceutical agents used to treat for type II diabetes are the AMPK
activators, as mentioned above. In the future, further AMPK-activating strategies can be
developed to prevent and treat obesity and type II diabetes.

Conclusion
AMPK is a // heterotrimeric serine/threonine kinase. The AMPK activity is controlled
by upstream kinases, and also adenosine nucleotides also allosterically regulate the activity of
AMPK. In skeletal muscle cells, AMPK is activated by many types of stimuli, such as
exercise, secretory factors, nutrients, and chemical activators. AMPK regulates energy
metabolism via key signaling molecules, thereby controlling the energy homeostasis and gene
transcription. AMPK enhances the glucose and FA uptake, FA oxidation and mitochondrial
biogenesis and respiration in skeletal muscle cells. Further investigations about the

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mechanisms underlying AMPK activation and signaling will be important for better
understanding obesity and type II diabetes mellitus, and may lead to the development of novel
preventive or therapeutic strategies for these diseases.

Acknowledgments
This work was supported by a research Grant-in-Aid for Scientific Research (C) (No.
24500796) from the Ministry of Education, Science, Culture, Sports, Science and Technology
of Japan.

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reduction of genes of oxidative metabolism in humans with insulin resistance and
diabetes: Potential role of PGC1 and NRF1. Proc. Natl. Acad. Sci. U S A, 100, 84668471.
[319] Mensink, M., Hesselink, M. K., Russell, A. P., Schaart, G., Sels, J. P. & Schrauwen, P.
(2007). Improved skeletal muscle oxidative enzyme activity and restoration of PGC-1

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and PPAR / gene expression upon rosiglitazone treatment in obese patients with type
2 diabetes mellitus. Int. J. Obes, 31, 1302-1310.
[320] Steinberg, G. R., Smith, A. C., Van Denderen, B. J., Chen, Z., Murthy, S., Campbell, D.
J., Heigenhauser, G. J., Dyck, D. J. & Kemp, B. E. (2004). AMP-activated protein
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& Goodyear, L. J. (2001). AMP-activated protein kinase (AMPK) is activated in
muscle of subjects with type 2 diabetes during exercise. Diabetes, 50, 921-927.
[322] Hjlund, K., Mustard, K. J., Staehr, P., Hardie, D. G., Beck-Nielsen, H., Richter, E. A.
& Wojtaszewski, J. F. (2004). AMPK activity and isoform protein expression are
similar in muscle of obese subjects with and without type 2 diabetes. Am. J. Physiol.
Endocrinol. Metab, 286, E239-E244.
[323] Sriwijitkamol, A., Coletta, D. K., Wajcberg, E., Balbontin, G. B., Reyna, S. M.,
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Sakamoto, K. & Musi, N. (2007). Effect of acute exercise on AMPK signaling in
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B., Cameron-Smith, D., Kemp, B. E. & Steinberg, G. R. (2005). Impaired activation of
AMP-kinase and fatty acid oxidation by globular adiponectin in cultured human
skeletal muscle of obese type 2 diabetics. J. Clin. Endocrinol. Metab, 90, 3665-3672.
[325] Koistinen, H. A., Galuska, D., Chibalin, A. V., Yang, J., Zierath, J. R., Holman, G. D.
& Wallberg-Henriksson, H. (2003). 5-amino-imidazole carboxamide riboside increases
glucose transport and cell-surface GLUT4 content in skeletal muscle from subjects with
type 2 diabetes. Diabetes, 52, 1066-1072.
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Wallberg-Henriksson, H. & Zierath, J. R. (2002). 5-Aminoimidazole-4-carboxamide
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[327] Halseth, A. E., Ensor, N. J., White, T. A., Ross, S. A. & Gulve, E. A. (2002). Acute and
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In: Basic Biology and Current Understanding of Skeletal Muscle ISBN: 978-1-62808-367-5
Editor: Kunihiro Sakuma
2013 Nova Science Publishers, Inc.

Chapter 9

A Role for Mitochondria as a Potential

Regulator of Myogenesis
Akira Wagatsuma
Graduate School of Information Science and Technology,
University of Tokyo, Bunkyo-ku, Tokyo, Japan

Abstract
Mitochondria serve a critical function in the maintenance of cellular energy supplies,
calcium homeostasis, and cell death. Aside from these major roles, several lines of
evidence suggest that mitochondria are involved in cell cycle control, cell growth and
differentiation in a wide variety of cell types including myogenic cells.
When the myoblasts differentiate into myotubes, the abundance, morphology, and
functional properties of mitochondria is being dynamically altered. Mitochondrial mass,
mitochondrial DNA content, mitochondrial enzyme activities, and mitochondrial
respiration are markedly increased during myogenesis.
This strongly suggests that myogenesis is accompanied by increased mitochondrial
biogenesis and that the metabolic shift from glycolysis to oxidative phosphorylation as
the major energy source occurs during myogenesis. Intriguingly, when myoblasts are
exposed to mitochondrial genetic and metabolic stress, they fail to differentiate into
multinucleated myotubes, suggesting that mitochondria may play a certain role in
myogenesis.
The process is relatively well understood phenomenologically, but the underlying
molecular mechanisms have been surprisingly slow to emerge. Understanding how
mitochondria are involved in myogenesis will provide a valuable insight into the
underlying mechanisms that regulate the maintenance of cellular homeostasis. Here we
will summarize the current knowledge regarding the role of mitochondria as a potential
regulator of myogenesis.

Correspondence should be addressed to Akira Wagatsuma, wagatsuma1969@yahoo.co.jp.

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1. Introduction
Mitochondria not only generate most of the energy necessary for cellular function via
oxidative phosphorylation (OXPHOS) but also are involved in calcium homeostasis, reactive
oxygen species (ROS)-redox control, multiple cellular signaling pathways, and cell death.
Alterations in mitochondrial function are involved in a range of inherited and acquired human
diseases and in the aging process [1]. Therefore, proper mitochondrial function is required for
the maintenance of both cellular and tissue integrity.
There is growing evidence that mitochondrial activity are linked to cell cycle control, cell
growth and differentiation, as have been shown in a wide variety of cell types including
adipocytes [2, 3], erythroblasts [4], erythroleukemia cells [5], dendritic cells [6], epidermal
keratinocyte [7], intestinal epithelial cells [8], mastocytoma cells [9], mesoangioblasts [10],
neurons [11-13], embryonic stem cells (ESCs) [14-18], and induced pluripotent stem cells
(iPSCs) [19], and myoblasts [20-29].
Skeletal muscle comprises approximately 40% of the normal adult male and 30% of the
female body weight [30]. The skeletal muscle possesses the unique ability to increase
metabolic rate nearly 100-fold during the transition form a basal resting state to near-maximal
contractile activity [31]. Therefore, skeletal muscle is a key metabolic tissue, which is
composed of highly specialized postmitotic, multinucleated myofibers. The molecular and
cellular processes leading to the fusion of myoblast cells to form multinucleated myofibers
have been extensively studied. Myogenesis is characterized by mononucleated myoblasts
withdrawing from the cell cycle, initiating muscle specific gene expression, and subsequently
fusing with each other and with existing myotubes to form syncytial myofibers [32]. It is
orchestrated through a series of transcriptional controls governed by the myogenic regulatory
factors (MRFs). The MRFs, a group of basic helix-loop-helix (bHLH) transcription factors
consisting of MyoD, Myf-5, myogenin, and MRF4, play important regulatory functions in the
skeletal muscle developmental program [33]. MRF4 and Myf5 act in a genetic pathway
upstream of MyoD to direct embryonic cells into the myogenic lineage [34]. Myogenin,
which acts in a genetic pathway downstream of MyoD and Myf-5 [35], directly controls the
differentiation process, including the formation of myotubes [36, 37].
After the onset of myogenic differentiation, mitochondrial enzyme activity is drastically
increased [38-40]. Similarly, muscle regeneration, which proceeds through a molecular
cascade resembling embryonic muscle development, is also accompanied by an increased
mitochondrial enzyme activity [41-43]. These findings suggest that the metabolic shift from
glycolysis to OXPHOS as the major energy source occurs during myogenesis. Intriguingly,
this metabolic shift has been also reported in ESCs [15, 16] and iPSCs [19]. iPSCs have low
mitochondrial activity, relying predominantly on glycolysis for ATP generation and maintains
a state of dedifferentiation, while differentiation is accompanied by increased mitochondrial
activity [19]. Therefore, metabolic shift may be a key event initiating cell differentiation. This
shift requires an activation of mitochondrial biogenesis through coordinated expression of
nuclear and mitochondrial genomes. Mitochondrial biogenesis is tightly controlled by
transcriptional coactivators, transcription factors, and nuclear receptors [1, 44-47]. Their
expression is induced during myogenesis [39, 40] and muscle regeneration [41, 43].
The effects of impairment of mitochondrial function and activity on myogenic cells have
been investigated using antimycin [48], azide [48-51], chloramphenicol [24, 26-29], carbonyl

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253

cyanide-m-chlorophenyl-hydrazone (CCCP) [48], carbonyl cyanide p-(trifluoromethoxy)

phenylhydrazone (FCCP) [26], ethidium bromide (EtBr) [20, 22, 23, 48], myxothiazol [27],
rhodamine 6G [52], rifampicin [20], rotenone [27], oligomycin [26, 27, 50], tetracycline [25],
valinomycin [48]. Overall, these antibiotics and chemicals negatively regulate myogenesis.
For instance, respiration-deficient myoblasts devoid of mitochondrial DNA (rho0 cells) by
EtBr fail to differentiate into multinucleated myotubes [20, 22, 23]. Rifampicin, which
inhibits mitochondrial RNA synthesis, shows reversible inhibition of myotube formation [20].
Tetracycline, an inhibitor of mitochondrial protein synthesis, blocks myoblasts fusion [25].
Chloramphenicol, an inhibitor of mitochondrial translation, restricts myogenic differentiation
[24, 26-29] and interferes with muscle regeneration [43]. Despite the data being accumulated,
little is known about the molecular mechanisms underlying the regulation of myogenesis by
mitochondria. In this review, we will summarize the current knowledge regarding the role for
mitochondria as a potential regulator of myogenesis.

2. Mutations in Mitochondrial DNA (mtDNA)

Although mitochondria have their own genetic system, more than 98% of the total protein
complement of the organelle is encoded by the nuclear genome. mtDNA may be more
susceptible to damage by ROS than nuclear DNA, because of the proximity of mtDNA to the
source of ROS, a lack of histones protecting mtDNA, and inefficient DNA repair systems [53,
54]. Consequently, the rate of mutations in mtDNA is approximately 10 times greater than
that in nuclear DNA [55]. The following sections focus on the mutations in mtDNA detected
in myofibers and satellite cells.

2.1. Genes Encoded by mtDNA

Mitochondria possess their own DNA. Each mitochondrion consists of 16,569 base pairs
which encode 37 genes encoding 13 genes that are required for OXPHOS, 22 transfer RNAs,
and 2 ribosomal RNAs [56]. Despite the fact that mtDNA comprises only 1-3% of genetic
material in animal cells, several lines of evidence suggest that its contribution to cellular
physiology could be much greater than would be expected from its size alone. For instance,
(a) it mutates at higher rates than nuclear DNA; (b) it encodes either polypeptides of the
electron transport system (ETS) or components required for their synthesis such as tRNAs
and rRNAs, and therefore, any coding mutations in mtDNA will affect the ETS as a whole;
this could affect both the assembly and function of the products of numerous nuclear genes in
ETS complexes; (c) defects in the ETS can have pleiotropic effects because they affect
cellular energetics as a whole [57].

2.2. Mutations in mtDNA of Myofibers

Abnormalities of mitochondrial ETS enzymatic activities are identified using a
histochemical method. Activities of two complexes of the ETS are commonly analyzed:

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Akira Wagatsuma

complex II (succinate dehydrogenase; SDH) and complex IV (cytochrome c oxidase; COX).

Abnormal myofibers are characterized by negative for COX and hyperreactive for SDH [58].
These fibers have been termed ragged-red fibers (RRFs), which display a granular, mottled
red appearance with the modified Gomori trichrome stain [59]. RRF was initially
characterized in human mitochondrial myopathy diseases that are neuromuscular disorders
associated with defects of the mitochondrial genome [60]. Besides, RRFs are observed in
aged human and animal skeletal muscle and their occurrence is investigated in details [61-77].
For instance, the frequency of RRFs in old humans (61 to 77 years of age) is much higher
than that in young humans (21 to 31 years of age) (0.33 versus 0.02%) [78]. In animal, the
frequency of myofibers exhibiting ETS abnormalities in old (34 years) and young adult (11
years) rhesus monkeys is 2.41 and 0.16%, respectively [64]. Intriguingly, extrapolating the
data to the entire length of the fiber, up to 60% of the fibers could be estimated to display
ETS abnormalities in an aged animal, when compared to 4% in a young adult animal [64]. It
has been pointed out that these ETS abnormalities are localized segmentally along myofibers
and are associated with fiber atrophy, resulting in contributing to age-related loss of myofiber
[65]. Aiken and coworkers investigate the mitochondrial genotype of aged myofibers
exhibiting mitochondrial enzymatic abnormalities using laser-capture microdissection
coupled with polymerase chain reaction (PCR) [65]. Large mtDNA deletion mutations (4.49.7 kb), corresponding to mtDNA genomes of 11.9-6.6 kb, were detected in ETS abnormal
myofibers but not in adjacent phenotypically normal myofibers [65]. Besides deletion
mutations, mtDNA point mutation are also detected in five tRNA genes including
tRNALeu(UUR)A3243G, tRNALysA8344G, tRNAMetT4460C, tRNAMetG4421A, and a 3-bp
deletion in the tRNALeu(UUR) in COX deficient myofibers from aged human [69]. The
tRNALysA8344G mutation is originally associated with myoclonus epilepsy and ragged-red
fibres (MERRF) [79], and the tRNALeu(UUR)A3243G mutation is responsible for mitochondrial
encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) [80].

2.3. Mutations in mtDNA of Satellite Cells

Satellite cells, which are wedged between the plasma membrane of the myober and the
basement membrane [81], contribute to muscle growth, maintenance, repair, and regeneration
[82-86]. Mitochondria from satellite cell are smaller with less developed cristae than
mitochondria observed in the adjacent myofibers [87-92]. Satellite cells have usually a lower
percentage of mutated mtDNA when compared to mature myofibers [93]. This is probably
associated with basal levels of ROS production in undifferentiated myoblats being lower than
in myotubes [94-96]. mtDNA may be particularly susceptible to oxidative damage due to its
close proximity of the inner mitochondrial membrane, where ROS are generated [53, 54].
Therefore, mtDNA is continually exposed to a high steady-state level of ROS and free
radicals in the matrix of the mitochondria, leading to oxidative modification and mutation of
mtDNA occurring with great ease [97]. Indeed, it has been shown that oxidative stress can
lead to the degradation of mtDNA, and that strand breaks and abasic sites in ROS-damaged
mtDNA of mammalian cells prevail over mutagenic base lesions [98].
Satellite cells derived from patients with MERRF [99] or MELAS [100] have high
proportions of mutant mtDNA with severe mitochondrial protein translation defects. These
findings may have some possible causes as follows: (a) replicative advantage for mutant

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255

mtDNA, as shown in mtDNA harboring large-scale deletions [101]; (b) a high population of
mutant mtDNA initially present in ovum; (c) a nonrandom sampling of the mtDNA
population in embryological development [99]. Meanwhile, a sporadic patient with chronic
progressive external ophthalmoplegia, ptosis, limb weakness, sensorineural hearing loss, and
a pigmentary retinopathy that has previously been only found in association with large-scale
deletions or the A3243G mutation in the tRNAleu(UUR) gene, has shown evidence of abnormal
proliferation of mitochondria [102]. Satellite cells from the patient's skeletal muscle contain
homoplasmic wild-type mtDNA and a low or negligible amount of mutated mtDNA [102,
103]. Therefore, activation of satellite cells in adult muscle could function in the shifting of
normal mitochondrial templates to mature myofiber and restoration of a more normal
mitochondrial genotype and biochemical function [102, 103]. For instance, muscle injury
induces restoration of a wild-type mtDNA genotype in regenerating myofibers in patients
with a Kearns-Sayre Syndrome (KSS), a sporadic disease associated with large-scale mtDNA
deletions, and in sporadic patients with tRNA point mutations [104]. Resistance training also
increases the ratio of wild-type to mutant mtDNAs, in the proportion of myofibers with
normal respiratory chain activity in patient with a phenotype similar to KSS [105]. These
findings suggest that normalization of the mtDNA genotype in mature myofibers in a patient
with mitochondrial myopathy could be brought by enhancing the incorporation of satellite
cells into the myofibers with mutated mtDNA through regeneration following injury and
resistance training.

3. Mitochondrial Biogenesis During Muscle

Regeneration and Myogenesis
Muscle regeneration is characterized by a highly synchronized process including satellite
cell activation and subsequent myoblast proliferation, differentiation, and fusion, leading to
new myofiber formation in and around the damaged myofibers. Muscle regeneration is
known to stimulate mitochondrial biogenesis. Similarly to myogenesis in vivo, myogenesis,
which is recapitulated in cell culture system, is accompanied by increased mitochondrial
biogenesis. In this section, we summarize the changes in mitochondrial biogenesis during
muscle regeneration and myogenesis.

3.1. Mitochondrial Biogenesis

Mitochondrial biogenesis (also referred to as mitochondriogenesis) is highly regulated
through a network of coactivators, transcription factors and nuclear receptors. This network
allows for broad and robust activation of the mitochondrial biogenesis program in response to
a wide range of physiological stimuli such as not only nutrient availability, contractile
activity, temperature, but also myogenesis. Mitochondrial biogenesis is characterized as a
vital process in the synthesis and degradation of the organelle [106, 107]. Therefore, this
fundamental process comprehends (1) the synthesis import, and incorporation of lipids and
proteins to the existing mitochondrial reticulum; (2) the stoichiometric assembly of
multisubunit protein complexes into a functional respiratory chain; (3) replication of the

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mtDNA; (4) selective degradation of mitochondria by autophagy (mitophagy) [46, 108, 109].
When it is not indicated, in this article, mitochondrial biogenesis will be referred to as an
increase in mitochondrial volume and changes in organelle composition per tissue or cell
[108].

3.2. Mitochondrial Biogenesis during Muscle Regeneration

Muscle regeneration partially recapitulates embryonic myogenesis [110]. Muscle injury
was induced by intramuscular injection of either bupivacaine (which induces Ca2+ release
from the sarcoplasmic reticulum (SR) and simultaneously inhibits Ca2+ reuptake into the SR,
resulting in persistently increased [Ca2+] levels and leads to myofiber death), notexin (which
involves Ca2+ overload and activation of Ca2+-dependent proteases, resulting in tissue
necrosis), or freezing (which causes uniform and complete necrosis of myofibers). Such acute
muscle injury shows a rapid loss of the activities of citrate synthase [41-43], which is often
used as marker for the mitochondria content of a tissue, after 2-3 days, when degenerative
myofibers still persist and proliferating myoblasts reside [41, 43]. The activity of citrate
synthase then is increased drastically between days 5 and 10, when myoblasts differentiate
into myotubes [41, 43]. Similarly, the rate of state-3 respiration (respiratory rate during active
phosphorylation of ADP) is also recovered [41]. In contrast, the rate of state-4 respiration
(respiratory rate after exhaustion of ADP) is comparable between control and injured muscles
[41]. Accordingly, the pattern of changes in the respiratory control ratio, a measure of the
tightness of coupling between electron transport and OXPHOS, is calculated as the ratio of
state-3/state-4 respiration [111], closely resembling the pattern obtained with citrate synthase
activity [41].
An activation of mitochondrial biogenesis occurs through the coordinated expression of
peroxisome proliferator-activated receptor (PPAR)- coactivator-1 (PGC-1) family members,
transcription factors, and nuclear receptors. Among them, PGC-1 is known to play a central
role in a regulatory network governing the transcriptional control of mitochondrial biogenesis
[44-47]. Indeed, PGC-1 is upregulated at late stages of muscle regeneration, when
mitochondrial enzyme activities had already returned to nearly control levels [41]. This
finding raises the possibility that PGC-1 may be much less likely to be involved in
mitochondrial biogenesis at early stages of muscle regeneration. In support of this hypothesis,
PGC-1 is not expressed in proliferating myoblasts [112] and in both myoblasts and
myotubes [113] and is not significantly induced during muscle regeneration [43]. These
findings lead us to hypothesize that other PGC-1 family members including PRC (PGC-1related coactivator) and PGC-1 may contribute to the mitochondrial biogenesis, at least in
part, at the early stage of muscle regeneration because they can also activate mitochondrial
biogenesis in a similar fashion as PGC-1 do. There is evidence that PRC coactivates NRF-1
[114, 115] and PGC-1 interacts with NRF-1 (nuclear respiratory factor-1) and estrogenrelated receptor [116]. A number of studies have revealed the potential roles of PRC and
PGC-1 in controlling mitochondrial biogenesis. For instance, PGC-1 expression increases
with myogenic differentiation [113]. The induction of PRC and PGC-1 is observed before a
recovery of mitochondrial enzyme activity to normal levels during muscle regeneration [43].
PRC-overexpressing myotubes show an elevated fatty acid oxidation and increased
expression of mitochondrial genes [117]. Forced expression of PGC-1 in C2C12 cells results

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257

in increased mitochondrial biogenesis and oxygen consumption [118]. Skeletal musclespecific PGC-1 transgenic mice exhibit increased mtDNA amount, mitochondrial content,
mitochondrial enzyme activity [119]. Mice lacking PGC-1 show a reduced numbers of
mitochondria, decreased respiration function, and decreased expression of mitochondrial
genes [120]. Taken together, PRC and/or PGC-1 may functionally replace PGC-1 during
muscle regeneration. However, the possibility cannot be excluded that PGC-1 may
contribute to the mitochondrial biogenesis during muscle regeneration, as has been shown in
gain-of-function and loss-of-function studies [121-125]. Other studies [39, 40] also report that
PGC-1 expression increases in vitro culture system using C2C12 myoblasts. Accordingly,
further studies are required to elucidate the role of PGC-1 in mitochondrial biogenesis
during muscle regeneration.

3.3. Mitochondrial Biogenesis during Myogenesis

There is increasing evidence that mtDNA content directly reflects on metabolic state of
cells [126-128]. Indeed, the amount of mtDNA is dynamically changed when myoblasts
differentiate into myotubes [39, 40, 129, 130]. In the early study, the mtDNA content of
differentiating myotubes increases approximately 4-fold relative to that of myoblasts by
Southern hybridization [129] and subsequently, a quantitative PCR analysis reveals that the
mtDNA copy number is 4-5-fold higher in differentiated myotubes compared to myoblasts
[39, 130]. This increase in the amount of mtDNA is accompanied with upregulation of PGC1, NRF-1, and TFAM (mitochondrial transcription factor A) concomitant with induction of
genes encoding mitochondrial proteins [39, 40]. This finding suggests that mitochondrial
biogenesis during myogenesis is regulated at the transcriptional level. PGC-1 does not have
any known DNA binding domains and can only participate in protein-protein interactions
with transcription factors and transcriptional machinery at the site of transcription initiation
[131]. PGC-1 stimulates the expression of NRF-1 and NRF-2 (also referred to as GAbinding protein; GABP) [112], which are involved in the transcriptional control of nuclear
and mitochondrial genes involved in OXPHOS, electron transport (complex I-V), heme
biosynthesis, protein import/assembly, ion channels, shuttles, and translation [1]. In addition,
PGC-1 binds to and coactivates the transcriptional function of NRF-1 on the promoter for
TFAM, a direct regulator of mitochondrial DNA replication/transcription [112]. Therefore,
upregulation of these genes contribute to increase the template availability for transcription
and translation of key mitochondrial proteins necessary for myogenesis.
Consistent with the in vivo findings, the activity of mitochondrial enzymes including
citrate synthase, isocitrate dehydrogenase, 3-hydroxyacyl CoA dehydrogenase, cytochrome c
reductase, succinate dehydrogenase, and COX is drastically increased during myogenesis [38,
40]. The content of respiratory chain complexes is higher in myotubes than in myoblasts [39,
96]. The content of respiratory chain complexes including complex I, II, III, and V is
approximately 8-fold higher in fully differentiated myotubes compared to myoblasts [39].
Despite complex IV activity being increased in differentiated myotubes, the content remains
unchanged [96]. This may be explained, at least in part, by the observation that a subunit
switching from liver isoforms of subunits VIa and VIIa to heart-specific isoforms occurs
during myogenesis [132]. This switching might be responsible for enhanced activity of
complex VI. Metabolic rate dynamically changes during myogenesis [49, 133]. In

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proliferating myoblasts, ATP production is provided by anaerobic glycolysis (40%) and

OXPHOS (60%), whereas in differentiated myotubes, ATP production relies on
mitochondrial respiration as their major source of metabolic energy (approximately 87%)
[133]. These findings suggest that myogenesis is accompanied by increased mitochondrial
biogenesis and that the metabolic shift from glycolysis to OXPHOS as the major energy
source occurs during myogenesis.
In the light of energy metabolism, satellite cells seem to be a heterogeneous population. It
has been reported that satellite cells are characterized by a difference in proliferative potential
and can be divided into two main subpopulations as follows: low proliferative clones (LPC)
and high proliferative clones (HPC) [134]. The percentage of LPC or HPC is 75% or 25%,
respectively [134]. These subpopulations differ in their metabolic state. LPC show a more
oxidative phenotype characterized by a lower mitochondrial membrane potential (m),
higher rate of mitochondrial generation of ATP and lower rate of ROS production, compared
to HPC [134]. Intriguingly, when cultured in myogenic medium, LPC are found to undergo
normal myogenic differentiation while HPC spontaneously differentiate into adipocytes
[134]. These findings lead us hypothesize that their metabolic state of satellite cells may
reflect on their developmental potential.

4. Possible Role of Mitochondria

in Muscle Regeneration
Recent studies have provided evidence that mitochondria may potentially regulate muscle
regeneration [43, 135]. Muscle regeneration is impaired when mitochondrial translation is
inhibited with chloramphenicol [43], which is an antibacterial antibiotic that was first shown
to inhibit mitochondrial protein synthesis selectively in yeast [136-138]. Chloramphenicol
inhibits protein synthesis in mitochondria but not in mammalian cytoplasmic ribosomal
systems [139]. Chloramphenicol reversibly binds to the 50S subunit of the 70S ribosome and
blocks prokaryotic protein translation primarily by inhibiting peptidyl-transferase and
blocking elongation [140]. Consequently, chloramphenicol inhibits the proper assembly of 4
out of 5 respiratory chain complexes within mitochondria and therefore affects mitochondrial
biogenesis in mammalian cells. Mice were intramuscularly injected with chloramphenicol day
3, 5, and 7 after the initial freeze injury and the muscle specimens were histochemically
analyzed at day 10. Mice exhibited the poor muscle regeneration with small myofibers and
increased connective tissues [43]. Overall, this finding support in vitro data that
chloramphenicol attenuates myogenic cell proliferation and differentiation [24, 26-29].
Therefore, in vivo data, when combined with the previous data in vitro, suggests a role of
mitochondria for sustaining muscle regeneration. However, the molecular mechanisms
remain unknown although chloramphenicol has been shown to downregulate myogenin in an
avian QM7 myoblast [26, 28] and mouse C2C12 myoblast [29].
Muscle regeneration is effectively accelerated using a method for complex mediated
delivery to intracellular mitochondria [135]. The method is based on the mitochondriotropism
of a multisubunit RNA import complex (RIC) [141]. Muscle injury was induced by piercing
repeatedly with a 26-gauge hypodermic needle at an angle of ~ 45 to the longitudinal axis of
the fiber, resulting in ~ 3000 myofibers being damaged at each insertion [135]. When a

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259

combination of polycistronic RNAs encoding the guanine-rich heavy-strand (H-strand) of the

mitochondrial genome is administrated to injured muscle, it rejuvenates mitochondrial mRNA
levels, organellar translation, respiratory capacity, intramuscular ATP levels with reduced
intracellular reactive oxygen species levels [135]. This strategy increases proliferative and
differentiative potential of satellite cells concomitant with upregulation of MRFs including
Myf5, MyoD, myogenin, and MRF4, resulting in promoting muscle regeneration with the
recovery of muscle contractility [135]. One of the most intriguing aspects is that Numb,
which has been generally considered to be a negative regulator of Notch-signaling [142], is
significantly induced in myoblasts by RIC-mediated transfection strategy. MyoD-positive
mononucleated cells express Numb and they are attached to or migrating on damaged
myofibers at the injury site [135]. This finding may provide new insight into the possible
mechanism regulating muscle regeneration through enhancing mitochondrial activity. Numb
segregates asymmetrically in dividing adult mouse muscle satellite cells [143, 144].
Attenuation of Notch signaling by Numb overexpression leads to the commitment of
progenitor cells to the myoblast cell fate with increased expression of Myf5 and desmin
[143]. Therefore, RIC-induced Numb may play a certain role in muscle regeneration by
modulating Notch-signaling. However, recent evidence suggests that although forced
expression of Numb in myogenic progenitors does not abrogate canonical Notch-signaling, it
can stimulate the self-renewal of myogenic progenitors [145]. Therefore, the precise role of
Numb in muscle regeneration remains to be elucidated. Furthermore, it remains unknown
whether mitochondrial activity directly or indirectly modulates Notch signaling at the present
time.

5. Mitochondrial Activity is Required for

Sustaining Normal Myogenesis in vitro
EtBr is frequently used to produce respiratory chain-deficient myogenic cells [20, 22, 23,
48]. EtBr is a well-known inhibitor of mtDNA replication [146] and transcription [147]. In
addition, transmitochondrial cytoplasmic hybrid (cybrid) technology allows for respiratory
chain-deficient myogenic cells harboring mtDNA with large-scale deletions to investigate
their intrinsic capacity for myogenesis. These studies provide evidence that normal
myogenesis requires mitochondrial activity. The following sections focus on the myogenesis
in respiratory chain-deficient myoblast. Furthermore, we discuss the relationship between
myogenesis and ATP synthesis via OXPHOS.

5.1. Myogenesis in Respiratory Chain-Deficient Myoblast

Myotube formation can occur even in respiratory chain-deficient myoblasts derived from
MERRF patients [99]. Unfortunately, it remains unclear to what extent myotube formation
progress for lack of quantitative data [99]. Brunk et al. report that myoblast fusion is
completely suppressed when myoblasts are forced to differentiation in the presence of EtBr
[20]. The inhibitory effect is fully reversible [20]. Herzberg et al. developed the respirationdeficient myoblasts contained only ~10% mtDNA by short-term exposure to EtBr to

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investigate their fusion capacity [22]. The fusion capacity of mtDNA-depleted myoblasts falls
to one-third of control myoblasts when initiating cell differentiation in the absence of EtBr
[22]. Subsequently, Sobreira et al. investigate the extent to which myoblasts fuse into
multinucleated myotubes in respiration-deficient myogenic cybrids produced by
transmitochondrial cybrid technology [52]. They produced viable myoblasts harboring
exclusively mtDNA with large-scale deletions by treating wild-type myoblasts with
rhodamine 6G and fusing them with cytoplasts homoplasmic for two different mutated
mtDNAs. Three cell lines were used as mitochondrial donors: 143B.TK-, 206/FLP 32.39, and
206/CW 420-115. The wild-type 143B.TK- cell line contains normally respiring mitochondria
[148]. Rhodamine 6G-treated myoblasts fused with 143B.TK- cytoplasts were used as
controls. The transmitochondrial cell line 206/FLP 32.39 was obtained by fusing mtDNA less
143B206 rho0 cells with enucleated fibroblasts from KSS patient K11 [149] harboring a 1,902
bp mtDNA deletion encompassing the region between nucleotides 7846 and 9748 of mtDNA.
The transmitochondrial cell line 206/CW 420-115 was obtained by fusing mtDNA less
143B206 rho0 cells with cytoplasts from KSS patient K13 [149] harboring a 5,790 bp deletion
situated between nucleotides 10155 and 15945 of mtDNA. It has previously been
demonstrated that the threshold for normal respiratory chain function in cultured cells
harboring mtDNA with deletions is relatively high, requiring the presence of at least 15%
wild-type mtDNA [101, 150]. The residual levels of wild-type mtDNA are less than 0.005%
and 0.025% of total mtDNA, respectively for the 206/FLP 32.39 and 206/CW 420-115
myogenic cybrids, which are insufficient to sustain normal respiratory chain activity, as
shown in very low levels of their COX activity when compared to control myogenic cybrid
[52]. The cell proliferation capacity of these myogenic cybrids nearly equals to that of control
myogenic cybrid [52], suggesting that the introduction of intact mitochondria is sufficient to
overcome the growth arrest of respiratory-deficient cells and the absence of ATP generated
by OXPHOS does not impair myoblast proliferation. The myotubes derived from myogenic
cybrids harboring mtDNAs with deletions have fewer nuclei (less than 4 nuclei) than that
from control myogenic cybrid [52]. The rate of myoblast fusion is reduced, ranging from 19%
to 50% and from 13% to 28% of the control myogenic cybrid, respectively for the 206/FLP
32.39 and 206/CW 420-115 myogenic cybrids [52]. These finding suggest that myogenic
differentiation might progress to some extent depending exclusively on inefficient energy
production via glycolysis, instead of relying primarily on more efficient energy production
through OXPHOS. However, one can argue that respiratory chain activity is required for
sustaining normal myogenesis. Importantly, it should be noted that myoblast fusion is
suppressed to a greater extent than the expression of sarcomeric myosin heavy chain in
myogenic cybrids containing mutated mtDNAs, [52] suggesting that mitochondrial activity
might either directly or indirectly modulate myoblast fusion.
Myoblast fusion is a highly regulated process that is a typical and essential feature of
myogenesis [151-153]. It occurs through the sequential events including cell elongation, cell
migration, cell-cell recognition/adhesion, and membrane fusion [154]. Recent studies have
identified cell surface, extracellular, and intracellular molecules in myoblast fusion in
mammals [152, 153, 155, 156]. For instance, integrins comprise a large family of /
heterodimeric cell surface glycoproteins involved in cell-cell and cell-matrix interactions. The
integrin 1A, and 1D subunits and its partners 1, 3, 4, 5, 6, 7A, 7B, and v are expressed
in vertebrate skeletal muscle in a developmentally regulated fashion [157, 158]. Integrin 3
and 91 are observed at membrane cell surface and at close cell-cell contact of fusing

A Role for Mitochondria as a Potential Regulator of Myogenesis

261

myoblasts [159, 160]. The function of 3 integrin and 9 integrin being attenuated using
specific antibodies, myoblast fusion is suppressed in vitro [159, 160]. The 1 integrindeficient myoblasts from transgenic mice have an impaired ability to undergo cell fusion
[161]. The 1 integrin-null mice exhibit the accumulation of unfused myoblasts in skeletal
muscle [161]. Intriguingly, mtDNA-depleted myoblasts fail to form multinucleated myotubes
and show markedly reduced levels of 51 integrins around the plasma membrane [162]. 51
integrins also have been reported to regulate myogenesis [163, 164]. When myoblasts began
to fuse, 5 integrin is distributed in small adhesion plaques along the lateral edges of the
postmitotic myoblasts and early myotubes and subsequently 51 integrins is localized to
parallel streaks underneath their entire substrate surface of mature myotubes [163]. When
myoblasts are treated with a monoclonal antibody of enhancing the binding of 51 integrins
for its ligand fibronectin, it interfers with cell migration and normal myoblast fusion [164]. In
view of the suggested role of integrins in myogenesis, mitochondrial genetic stress may
suppress myoblast fusion by dysregulating integrins-mediated cell adhesion and migration.
Myoferlin, which is identified as a protein highly homologous to dysferlin [165], is highly
expressed in myoblasts undergoing fusion, and is expressed at the site of myoblasts fusing to
myotubes [166]. Myoferlin-deficient myoblasts undergo initial fusion events, but they form
large myotubes less efficiently in vitro and myoferlin null muscle lacks large diameter
myofibers [166]. Nuclear factor of activated T cells (NFAT) is a family of transcription
factors in regulating early gene transcription in response to T cell receptor-mediated signals in
lymphocytes [167]. Despite their name, the member of NFAT isoforms (NFATc1-4) is
expressed in myogenic cells and differentially translocate at different stages of muscle
development [168]. The NFATc2 isoform is activated only in newly formed or nascent
myotubes [168] where it plays a crucial role in the myotube growth [169]. NFATc2 regulates
myoblast fusion through the cytokine, interleukin-4 (IL-4), which is secreted by newly
formed myotubes, interacts with IL-4 receptor present on surrounding myoblasts, promoting
their fusion to pre-existing myotubes [170], probably through enhancing the migration of
myoblasts [171]. Prostaglandin F2 also recruits the additional myoblast fusion with preexisting myotubes via an NFATc2-dependent pathway [170]. The fusion events of myoblasts
occur in two well-characterized phases. In the first phase, mononucleated myoblasts fuse with
one another to form a nascent myotube (myoblast/myoblast fusion events). During the second
phase, additional myoblasts fuse with pre-existing myotubes to form a large and mature
myotube (myoblast/myotube fusion events) [152]. Therefore, these findings suggest that
NFATc2 signaling is involved in regulating myoblast/myotube fusion events. Actin
regulatory molecules also play key roles in myoblast fusion. Defects in neural WiskottAldrich syndrome protein (NWASP), which is important regulators of the actin cytoskeleton
and WASP-interacting protein (WIP) leads to impaired myoblast fusion [173]. Until now, the
relationship between the expression of these molecules involved in regulating cell fusion and
mitochondrial activity during myogenesis is largely unknown.

5.2. Mitochondrial Activity Regulates Myogenesis Independently of Their

Implication in ATP Synthesis
An early study [Brunk et al., 1976] showed that inhibition of mitochondrial protein
synthesis by chloramphenicol had no effect on myoblast fusion within the first 40 hours after

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Akira Wagatsuma

the initiation of differentiation. This finding is inconsistent with other studies [24, 26-29]. It is
possible that lab-to-lab variability in experimental conditions including such as cell type
(primary or immortalized cells), cell density, culture time, and drug exposure times may have
contributed to this discrepancy.
Korohoda et al. [24] have reported that chloramphenicol inhibits the fusion of myoblasts
isolated from chick embryo skeletal muscle. This is among the first study to show the
inhibitory effect of chloramphenicol on myogenesis. They show that tryptose phosphate broth
and nucleosides can restore the cell capacity to proliferate but not to fuse and differentiate in
the presence of chloramphenicol [24].
Subsequently, it has been demonstrated that mitochondrial activity is an important
regulator of myogenesis in quail myoblasts of the QM7 cell line and mouse myoblasts of the
C2C12 cell line [26, 28, 29]. Chloramphenicol-treated myoblasts proliferate at a slower rate
than control myoblasts without inducing any alteration of cell viability [26]. However, a
strong reduction in ATP levels is observed in chloramphenicol-treated myoblasts during cell
proliferation [26].
This finding raises the possibility that ATP depletion observed in chloramphenicoltreated myoblasts during the early period of cell culture could be responsible for the inhibition
of myogenic differentiation. This possibility seems to be excluded for the following the
reasons: (1) glycolysis fully compensates for mitochondrial impairment just before and during
terminal differentiation, as shown in a marked accumulation of L-lactate in the culture
medium [25, 26]; (2) myogenic differentiation is repressed especially when myoblasts are
exposed to chloramphenicol at the onset of terminal differentiation [26]. These findings
indicate that mitochondrial activity regulates myogenesis independently of their implication
in ATP synthesis [26].

6. Molecular Mechanisms of Myogenesis

Modulated by Mitochondrial Activity
To elucidate the roles of mitochondria in myogenesis, the drugs which can damage
mitochondria by inhibiting respiratory chain complex, mtDNA replication and transcription
or protein translation are frequently used. Overall, they have negative effects on myogenesis.
The results are showed in Table 1.
However, the molecular mechanisms have not been explicitly explored. The following
sections focus on several molecules reported to suppress myogenesis when exposure to
mitochondrial genetic and metabolic stress.

6.1. Myogenin
Cabellos research group has investigated the relationship mitochondrial activity and
myogenesis using chloramphenicol [26, 28, 29]. Chloramphenicol treatment inhibits
myogenesis and downregulates the expression of myogenin but not other MRFs, Myf5 and
MyoD [26, 28]. Conversely, chloramphenicol removal rejuvenates the capacity for
differentiation into myotubes and leads to the restoration of myogenin expression [26].

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A Role for Mitochondria as a Potential Regulator of Myogenesis

Table 1. Effects of mitochondrial genetic and metabolic stress on myogenesis
Cell type
Satellite cells
(Rat)
Satellite cells
(Human)
Satellite cells
(Human)
Satellite cells
(Rat)
Satellite cells
(Rat)
Satellite cells
(Chick)

Reagents

Target

Results

EtBr

mtDNA

Inhibition of myoblast fusion

EtBr

mtDNA

Rhodamine 6G

mtDNA

Protein
translation
Protein
Rifampicin
translation
Protein
Chloramphenicol
translation
Chloramphenicol

C2C12 (Mouse) Tetracyclin

Protein
translation

QM7 (Quail)

Chloramphenicol

Protein
translation

C2C12 (Mouse) Chloramphenicol

Protein
translation

QM7 (Quail)

Chloramphenicol

Protein
translation

C2C12 (Mouse) Chloramphenicol

Protein
translation

Skeletal muscle
Protein
Chloramphenicol
(Mouse)
translation
QM7 (Quail)

Oligomycin

QM7 (Quail)

FCCP

C2C12 (Mouse) Azide

C2C12 (Mouse) Oligomycin
C2C12 (Mouse) Rotenone
C2C12 (Mouse) Myxothiazol

ATP synthase

Reduced rate of cell proliferation Inhibition of

myoblast fusion
Transmitochondrial myoblasts were produced
by fusing rhodamine 6G treated myoblasts with
cytoplasts harboring wild-type or mutated
mtDNAs.
Decreased myoblast fusion and differentiation
No difference in the extent of myoblast fusion
between control and treated cultures
Inhibition of myoblast fusion
Inhibition of myoblast fusion
Decreased myotube formation and
downregulation of CK and TnI No changes in
the expression of MyoD and myogenin
Reduced rate of cell proliferation Decreased
myoblast fusion and differentiation
Downregulation of connectin and AChR
Downregulation of myogenin but not CMD1
(MyoD) and Myf5
Inhibition of myoblast fusion Downregulation
of myogenin
Decreased myoblast fusion and differentiation
Downregulation of myogenin, AChR, and
sMyHC Chloramphenicol abrogates the
downregulation of c-Myc normally occurring at
the induction of cell differentiation
Decreased myoblast fusion and differentiation
Downregulation of myogenin,TnT, and sMyHC
Chloramphenicol decreases expression of
calcineurin
Skeletal muscles forced to regenerate in the
presence of chloramphenicol are of poor repair
with small myofibers and increased connective
tissues in the damaged myofibers
Decreased myoblast fusion and differentiation

Oxidative
Decreased myoblast fusion and differentiation
phosphorylation
No difference in the extent of myoblast fusion
Cytochrome c
and expression of sMyHC between control and
oxidase
treated cultures
Inhibition of myoblast fusion
ATP synthase
Downregulation of myogenin
NADH
Inhibition of myoblast fusion
dehydrogenase Downregulation of myogenin
Cytochrome bc 1 Inhibition of myoblast fusion
complex
Downregulation of myogenin

References
Brunk et al.,
1976
Herzberg et
al., 1993
Sobreira et
al., 1999
Brunk et al.,
1976
Brunk et al.,
1976
Korohoda et
al., 1993
Hamai et al.,
1997

Rochard et
al., 2000
Pawlikowska
et al., 2006
Seyer et al.,
2006

Seyer et al.,
2011

Wagatsuma
et al., 2011
Rochard et
al., 2000
Rochard et
al., 2000
Yun et al.,
2005
Pawlikowska
et al., 2006
Pawlikowska
et al., 2006
Pawlikowska
et al., 2006

EtBr, ethidium bromide; CK, creatine kinase; TnI, troponin I; troponin T, TnT; AChR, acetylcholine
receptor subunit; sMyHC, sarcomeric myosin heavy chain; FCCP, carbonyl cyanide
p-(trifluoromethoxy) phenylhydrazone.

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Akira Wagatsuma

The functions of myogenin have been well investigated. When transfecting mesenchymal
cell line C3H10T1/2 with myogenin, it can convert them to myoblasts [174]. Mice lacking
myogenin exhibit a normal number of myoblasts but die immediately after birth because of an
absence of myofibers [36, 37]. They differ from mice lacking either Myf5 or MyoD, which
have normal skeletal muscle [175, 176]. Therefore, myogenin has a crucial role during
myogenesis [36, 37] and both Myf5 and MyoD cannot compensate for its deficit [35].
Intriguingly, similar downregulation of myogenin is commonly observed in FCCP,
myxothiazol [27], rotenone [27], and oligomycin [26, 27], which affect mitochondria at
different levels. These findings suggest that myogenin is a specific target of mitochondrial
activity. How is myogenin expression regulated by mitochondrial activity? The measurement
of half-life of myogenin mRNA after transcription inhibition with actinomycin D reveals that
chloramphenicol have no effect on myogenin mRNA stability [26], suggesting that
mitochondrial activity could regulate myogenin expression at the transcriptional level.
Unfortunately, the molecular mechanisms regulating the expression and activity of myogenin
by mitochondrial activity remain to be elucidated, although they are not only transcriptionally
regulated but also by mRNA stability, translation efficiency, protein stability, and protein
phosphorylation [177]. For instance, myogenin is a short-lived protein [178] and can be
ubiquitinated and degraded through the von Hippel-Lindau protein (pVHL)-E3 ubiquitin
ligase pathway [179]. Myogenin protein can be stabilized by interacting with EGLN3 (EGL
nine homolog 3), which encodes an intracellular prolyl hydroxylase that is involved in the
cellular response to oxygen availability by hydroxylating hypoxia inducible factor 1 (HIF1) protein [180, 181]. EGLN3 can prevent myogenin protein from pVHL-mediated
unbiquitination and degradation, leading to stabilization of myogenin protein that promotes
myogenesis [179]. Myogenin activity can be suppressed by calcium/calmodulin-dependent
protein kinase II-mediated phosphorylation [182]. Considering the role of myogenin in
myogenesis, it is expected that forced expression of myogenin can rescue cell differentiation
in chloramphenicol-treated myoblasts. However, overexpression of myogenin fails to restore
their differentiation potential in the presence of chloramphenicol [26]. This indicates that
mitochondrial activity could regulate myogenesis by decaying ability of MRFs via other
regulators and/or negative regulators of myogenesis.

6.2. c-Myc Oncogene

The same group identified c-Myc (cellular myelocytomatosis oncogene) gene, which
could be a target gene regulated by mitochondrial activity [28]. c-Myc is a proto-oncogene
encoding a transcription factor [183], which plays a role in myogenesis [184-190].
Impairment of mitochondrial activity by chloramphenicol allows to abrogate the
downregulation of c-Myc normally occurring at the induction of differentiation in control
cells [28]. Overexpression of c-Myc mimics the influence of inhibition of mitochondrial
activity on myogenic differentiation [28]. A triiodothyronine-dependent mitochondrial
transcription factor (p43) overexpression can stimulate mitochondrial activity, downregulate
c-Myc expression, and promote myogenic differentiation [28]. These findings suggest the
possibility that c-Myc could be a primary target of mitochondrial activity. Indeed, the
endogenous c-Myc is downregulated within the first 24 h after switching to a differentiation
medium [186]. Ectopic expression of c-Myc in quail myoblasts leads to form myotubes and

A Role for Mitochondria as a Potential Regulator of Myogenesis

265

downregulates MyoD, myogenin, and Myf5 expression [189]. Cotransfection of c-Myc with
MyoD and myogenin in NIH 3T3 cells inhibits myogenic differentiation [187]. While these
findings are compelling, a role of c-Myc should be carefully considered for a number of
reasons as follows. First, nuclear runoff transcription assay demonstrates that c-Myc and
skeletal muscle-specific genes could be simultaneously transcribed in both biochemically
differentiated myoblasts (no fusion) and terminally differentiated myotubes [185]. c-Myctransformed C2C12 cells retain the ability to undergo commitment and biochemical
differentiation but they are strikingly unable to fuse into multinucleated myotubes with no
change in the expression of MyoD, myogenin, and myosin heavy chain [188]. These findings
suggest that irreversible repression of c-Myc is not necessary for myogenic differentiation and
its expression is insufficient to suppress the differentiated phenotype. Secondly, c-Myc
represses p21Cip1/WAF1 expression through transcriptional activator, Miz-1 (c-Myc interacting
zinc-finger protein-1)-dependent interaction with p21Cip1/WAF1 core promoter [191]. In
addition, c-Myc interacts with Miz-1 and recruits DNA methyltransferase 3A to p21Cip1/WAF1
promoter to silence p21 transcription [192]. The expression of p21 Cip1/WAF1 is known to be a
key event triggering the withdrawal of myoblasts from the cell cycle to G0, a prerequisite to
myogenic differentiation [193]. Chloramphenicol or c-Myc overexpression decrease the
proportion of myoblasts in the G0-G1 phase whereas overexpression of p43 exerts opposite
influence [28]. These findings suggest the possibility that mitochondrial activity could
regulate myoblast cell cycle withdrawal by modulating expression of p21 Cip1/WAF1 through cMyc/Miz-1 complex. Therefore, not only c-Myc, but also Miz-1 expression levels need to be
explored in myogenic cells treated with chloramphenicol. Thirdly, Myc is a member of the
Myc/Max (Myc-associated factor X)/Mad (MAX dimerization protein) transcriptional
network comprises a group of widely expressed transcription factors [194]. For instance, cMyc/Max heterodimers transactivate its downstream genes by binding to the E-box sequence
5-CACGTG-3 in the target promoter, whereas Mad/Max heterodimers act as transcriptional
repressors at the same E-box-related DNA-binding sites [194]. Therefore, c-Myc/Max
heterodimers act by competing with Mad/Max heterodimers, resulting in controlling the
expression of their target genes. Intriguingly, a switching from Myc/Max to Mad/Max
heterodimers occurs in the process of cell differentiation. For instance, Max can interact with
Myc but not Mad in the undifferentiated U937 monocyte cell line and Mad:Max complexes
begin to accumulate rapidly 2 hr after induction of macrophage differentiation [195],
suggesting that cell differentiation is accompanied by a change in the composition of Max
heterocomplexes. The c-Myc/Max complex is readily detected in exponentially proliferating
HL60 cells but Mad is strongly induced in differentiated HL60 cells [196]. In addition, these
changes are closely associated with human telomerase reverse transcriptase (hTERT), the
catalytic subunit of telomerase/telomerase activity [196]. c-Myc induces the hTERT promoter
activity and gene expression [197] whereas Mad1 represses them [198]. Most recent
biochemical and genetic evidence suggest that the maintenance of telomere length by
telomerase is critical to sustain the proliferative ability of cells. These findings suggest that
the Myc/Max/Mad transcriptional network may constitute a molecular switch where the
abundance of Myc- versus Mad-containing heterodimers determines whether cells enter a
differentiation pathway or remain in a proliferative, undifferentiated state. Therefore, same
molecular switching may occur during myogenesis. However, this hypothesis is highly
speculative. Further studies are required to validate it in myogenic cells. Finally, a new mode
of Myc regulation has been recently reported in myogenesis. Myc protein is cleaved by a

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Akira Wagatsuma

calpain (calcium-dependent cytoplasmic cysteine proteinase) to generate a cytoplasmic form,

Myc-Nick, which retains Myc box regions but lacks nuclear localization sequence and the
bHLH/leucine zipper domains essential for heterodimerization with Max and DNA binding
activity [199]. During myogenesis, while the full-length Myc decreases, Myc-nick is
increased. Overexpression of Myc-nick in both primary and cultured myoblasts accelerates
their differentiation and increases expression of skeletal muscle-specific markers such as
desmin, tropomyosin, troponin, myosin heavy chain [199]. This finding suggests that Myc
protein can both inhibiting and facilitating myogenesis depending on the biological context.
In conclusion, important findings have been reported since early times but further studies will
be required to understand the precise role of c-Myc in myogenesis. c-Myc-mediated
regulation of myogenesis may be much more complex than one thought.

6.3. Calcineurin Signaling

To further understand the molecular mechanisms underlying the regulation of
myogenesis by mitochondrial activity, Cabellos research group [29] conducted a
comprehensive differential display analysis using total RNA from control and
chloramphenicol-treated myoblasts to search for other gene modulated by mitochondrial
activity. They identified calcineurin (also referred to as protein phosphatase 2B) [29], in
which serine/threonine protein phosphatase under the control of a eukaryotic Ca2+- and
calmodulin, plays a critical role in the coupling of Ca2+ signals to cellular responses [200].
Calcineurin is a heterodimeric enzyme consisting of a 60-kDa catalytic A subunit (calcineurin
A) and 19-kDa calcium-binding regulatory B subunit (calcineurin B). [200]. Calcineurin have
been shown to regulate myogenesis [201-206]. Generally, inhibition of calcineurin activity by
cyclosporin A or antisence calcineurin expression vectors strongly inhibits myogenesis [168,
201, 202, 207] whereas overexpression of a constitutively active form of calcineurin promote
it [29, 200]. Chloramphenicol prevents the increased expression of calcineurin A [29]
normally observed in differentiating myoblasts [29, 201]. Overexpression of mitochondrial
T3 receptor (p43), which stimulates mitochondrial activity, potentiates myogenesis
concomitant with an increased calcineurin A expression [29]. These findings suggest that
calcineurin could be a target regulated by mitochondrial activity.
How does calcineurin signaling regulate myogenesis? Calcineurin transcriptionally
regulates the myogenin expression by activating MEF2 (myocyte enhancer factor-2) and
MyoD [204]. Calcineurin dephosphorylates NFAT transcription factors, allowing NFAT to
translocate to the nucleus where it cooperates with other transcription factors to induce
transcription of target genes [167]. NFATc2/c3 synergistically cooperates with MyoD at the
myogenin promoter, which activates the expression of myogenin [206]. Calcineurin regulates
expression of the inhibitor of differentiation (Id) family of proteins, which function as
dominant negative regulators of bHLH transcriptional regulators that drive cell lineage
commitment and differentiation [208]. Id interactions with E2A proteins preclude their
heterodimerization with MyoD and thereby attenuating MyoD function [209]. Therefore,
downregulation of both Id1 and Id3 is required for in vitro myogenesis [210-212]. Indeed,
forced expression of a constitutively active form of calcineurin reduces the expression of both
Id1 and Id3 in satellite cells [204]. In addition, the skeletal muscles forced to regenerate in the
presence of cyclosporine A (calcineurin inhibitor) are of poor repair with elevated expression

A Role for Mitochondria as a Potential Regulator of Myogenesis

267

of Id1 and Id3 [204]. Taken together, mitochondrial activity may regulate myogenesis
through calcineurin-mediated myogenin and Id expression.
In contrast to the finding from myoblasts treated with chloramphenicol, calcineurin is
upregulated in mtDNA-depleted myoblasts [48, 162]. This upregulation seems to be
associated with elevated steady-state cytosolic Ca2+ level ([Ca2+]i). Myogenic cell lines
containing partially depleted mtDNA are established by chronically treated with EtBr for ~70
growth cycles, followed by single cell cloning [48]. Depletion of mtDNA leads to the
disruption of mitochondrial membrane potential and then results in elevated steady-state
cytosolic Ca2+ level ([Ca2+]i) possibly due to reduced ATP levels affecting Ca2+ efflux
through the plasma membrane [48]. Under these conditions, calcineurin A and NFATc are
strongly induced concomitant with a modest increase in calcineurin B [48]. Intriguingly, all
these changes are normalized in genetically reverted myoblasts which contain near-normal
mtDNA levels by growing mtDNA-depleted cells in normal medium, lacking EtBr, for ~30
growth cycles [48]. Therefore, calcineurin signaling may be increased in mtDNA-depleted
myoblasts. What role does calcineurin signaling play in mtDNA-depleted myoblasts? One
possibility would be that calcineurin is involved in activation of nuclear factor-B (NF-B)
signaling, which has been known to repress myogenesis [213]. In support of this hypothesis,
Alzuherri et al. [214] report that stable expression of constitutively active calcineurin in
myoblasts strongly activates NF-B. However, this finding perplexes us because previous
studies have consistently shown that calcineurin promotes myogenesis. To explain this
discrepancy, they propose a model for the dual actions of calcineurin on myogenesis:
transient activation of calcineurin promotes myogenesis, whereas prolonged activation of
calcineurin inhibits it [214]. If that's the case, chronic activation of calcineurin observed in
mtDNA-depleted myoblasts may lead to NF-B activation, resulting in interfering with
myogenesis.

6.4. Nuclear Factor-B (NF-B) Signaling

NF-B was originally described as a constitutive transcription factor in mature B-cell
lines [215]. Subsequently, it was found that NF-B was present in an inducible form in many
other cell types including myogenic cell [216, 217]. NF-B is known to play a role in
myogenesis [213]. NF-B is a heterodimeric or homodimeric complex formed from five
distinct subunits: RelA/p65 (also known as p65), RelB, c-Rel, NF-B1 (p50, which is
processed from p105), and NF-B2 (p52, which is processed from p100) [218, 219]. Only
p65, c-Rel, and RelB possess C-terminal transcriptional transactivation domains, whereas p50
and p52 lack intrinsic transactivating properties, and instead function as homodimeric
transcriptional repressors or modulators of transactivating dimer partners [211]. The activity
of NF-B is regulated by two main pathways: the canonical and non-canonical NF-B
pathways [211, 220, 221]. The canonical NF-B activation pathway applies to dimers
composed of p65, c-Rel and p50 [222]. NF-B (mostly p50/p65 heterodimer) is normally
sequestered in the cytoplasm in an inactive state through their association with IB or IB
members of the IkB family of inhibitor proteins [211, 220, 221]. When cells are stimulated by
a wide variety of different stimuli such as proinflammatory cytokines, growth factors, reactive
oxygen species, IB proteins are phosphorylated by IB kinase (IKK) complex consisting of
three subunits, IKK, IKK, and IKK [223, 224] and subsequently degraded by the 26S

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Akira Wagatsuma

proteasome, allowing NF-B dimmers to translocate into the nucleus where they regulate
target gene expression [213, 220, 221]. In the non-canonical pathway, the p52 and RelB
heterodimers are frequently activated [211, 220, 221]. IKK homodimer is activated by the
NF-B-inducing kinase. Once activated, IKK phosphorylates p100 subunit, followed by 26S
proteasome-dependent processing of p100 to p52, which can lead to the activation of
p52/RelB heterodimers that translocate to the nucleus to transactivate NF-B target genes that
may be distinct from the classical pathway [213].
NF-B DNA binding activity is decreased during myogenic differentiation [216, 217,
225-227]. Guttridge et al., investigate the relative levels and subunit composition of NFB/Rel complexes found in undifferentiated and differentiated C2C12 cell cultures [217].
They report that p65 and p50 subunits of NF-B contain abundant DNA binding activities
using an electrophoretic mobility shift assay and NF-B transcriptional activity falls by nearly
50% with initiation of myogenic differentiation [217]. Subsequently, Guttridges research
group have shown that myogenesis is enhanced in MyoD-expressing fibroblasts deficient in
p65, IKK, or IKK and in myoblasts lacking p65 or IKK [213]. Skeletal muscles from p65
or IKK mutant mice also contain higher myofiber numbers [227]. These findings suggest
that canonical NF-B signaling functions to repress myogenesis [227].
The NF-B pathway is likely to be activated in mtDNA-depleted myoblasts [162, 228].
Indeed, p50 and c-Rel is abundant in the nuclear fractions from mtDNA-depleted myoblasts
concomitant with increased NF-B DNA binding activity [162, 228]. It is, however,
debatable as to whether the levels of p65 are increased or decreased in mtDNA-depleted
myoblasts [48, 228]. Because c-Rel can form heterodimers with p50 and p65 [229], at least cRel/p50 and/or p50/p50 nuclear translocation can occur, leading to activation of NF-B
pathway in mtDNA-depleted myoblasts. Avadhanis research group focus on the role of
calcineurin in the process of NF-B activation in response to mitochondrial genetic stress.
[228]. The nuclear p65, p50, and c-Rel levels are decreased in skeletal muscles from mice
lacking calcineurin A, compared to wild-type mice [228]. When C2C12 cells are transfected
with cDNA containing calcineurin A, it enhances the levels of p65, p50, and c-Rel in the
nuclear fraction whereas FK506, (calcineurin inhibitor) decreases their levels [228]. The
DNA-bound complex supershifted with antibodies against p65, p50, and cRel is observed in
transfected myoblasts and the complex formation is decreased by treating with FK506 [228].
The NF-B DNA binding activity of transfected myoblasts is comparable to that of mtDNAdepleted myoblasts [228]. Taken together, calcineurin could contribute to activation of NF-B
signaling by modulating the levels of NF-B/Rel proteins. The same research group further
investigates the mechanism of activation of NF-B pathway in mtDNA-depleted myoblasts.
NF-B/Rel proteins (p50, c-Rel, p65) bound IB can form a ternary complex with
calcineurin, as revealed by chemical cross-linking and coimmunoprecipitation experiments.
Calcineurin can dephosphorylate IB at the C-terminal PEST (polypeptide sequences
enriched in proline, glutamate, serine, and threonine) region, which has been shown to be
important for the function of IB [230-232]. Dephosphorylation of IB by calcineurin is
inhibited by RII peptide (a specific substrate of calcineurin) in a dose-dependent manner
[228]. These findings suggest that IB is inactivated through calcineurin-mediated
dephosphorylation, thereby releasing p50/Rel complex to translocate into the nucleus, leading
to activation of target genes.
Although the levels of p50 and c-Rel are increased in mtDNA-depleted myoblasts [162,
228], we think carefully whether NF-B (p50/c-Rel) functions as a repressor of myogenesis

A Role for Mitochondria as a Potential Regulator of Myogenesis

269

because there is a report that DNA-binding activity of the NF-B subunit c-Rel cannot be
detected during myogenesis [217]. However, given that NF-B (p50/c-Rel) serves in a way
comparable to NF-B (p50/p65), they might play a role in myogenesis in response to
mitochondrial genetic stress. NF-B, which consists mostly of p50/p65, seems to interfere
with myogenesis through multiple mechanisms [227]. For instance, NF-B activates the
expression of cyclin D1 [217], which itself is capable of suppressing myogenic differentiation
[233, 234]. NF-B transcriptionally regulates cyclin D1 through direct binding of NF-B to
multiple sites in the cyclin D1 promoter [217]. Furthermore, p65 has shown to be able to
interact with cyclin D1, which contribute to cyclin D1 protein stability [235]. NF-B seems to
regulate MyoD expression through posttranscriptional gene silencing mechanism. Activation
of NF-B through p65 can destabilize MyoD mRNA levels through an mRNA cis-regulatory
motif (5-ACUACAG-3) [236, 237]. NF-B can also posttranslationally regulate MyoD.
Activation of the NF-B pathway by coexpression of IKK, IKK, or p65 modulates
myogenesis probably through its role in MyoD protein stability [238]. Tumor necrosis factor and tumor necrosis factor-like weak inducer of apoptosis activates NF-B pathway,
resulting in inhibition of myogenesis probably through destabilizing MyoD protein [238,
239]. NF-B pathway is likely to regulate myogenesis through Yin Yang1 (YY1) which is
known to be a negative regulator in myogenesis by directly repressing skeletal musclespecific genes [240-242]. NF-B regulates YY1 expression at the transcriptional levels
through direct binding of the p50/p65 heterodimer complex to the YY1 promoter and YY1
binds and inhibits myofibrillar promoters by recruiting the Polycomb group member, histone
methyltransferase (Enhancer of zente homolog 2; Ezh2) as well as histone deacethylase 1
(HDAC1) [243]. Furthermore, NF-B works in concert with YY1 to regulate microRNAs
(miRNAs). NF-B and YY1 negatively regulate the expression of miR-29b/c, which
accerilates myogenic differentiation [243]. Other miRNAs are also regulated by NF-B
through YY1 [245]. Although miR-1 and miR-133 are simultaneously induced during
myogenic differentiation, their effects are functionally antagonistic [247]. miR-1 facilitates
cell differentiation by targeting HDAC4 whereas miR-133 enhances cell proliferation by
repressing serum response factor [247]. These regulation mediated by NF-B seems to be
mostly dependent on p65 activity. Therefore, further studies may be required to determine
whether NF-B (p50/c-Rel) suppress myogenesis in response to mitochondrial genetic stress.

Conclusion
This paper provides the current knowledge about the role for mitochondria as a potential
regulator of myogenesis. Previous studies have highlighted that mitochondria play a role in
myogenesis. Several molecules have been identified as mitochondrial target. In particular,
myogenin, that is essential for myogenic differentiation, may be an important target of
mitochondrial activity because its downregulation is commonly observed in myogenic cells in
response to mitochondrial stress caused by antibiotics and chemical compounds, which affect
the organelle at different levels. In addition, NF-B may function as a suppressor of
myogenesis in mtDNA-depleted myoblasts. Guttridges research group has proposed the
model for NF-B signaling pathways during myogenesis [227]. According to them, a
temporal switch occurs between NF-B canonical and non-canonical signaling pathways

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Akira Wagatsuma

during myogenesis. In proliferating myoblasts, canonical NF-B signaling regulates gene

expression to inhibit premature myoblast differentiation. Once myogenic differentiation is
initiated, the canonical NF-B signaling is shut down and the non-canonical pathway is
activated. The non-canonical NF-B signaling occurs late in the myogenic program to
regulate mitochondrial biogenesis and myotube maintenance. This model leads us
hypothesize that the canonical NF-B pathway excessively functions in mtDNA-depleted
myoblasts, resulting in interfering with myogenesis concomitant with insufficient activation
of non-canonical NF-B pathway.
In recent years, the advantage of activating mitochondrial biogenesis for mitochondrial
disease, muscle regeneration, and maintenance of muscle mass has been understood. For
instance, activation of PPAR/PGC-1 pathway effectively improves a phenotype of
mitochondrial myopathy. When mitochondrial biogenesis is stimulated either by transgenic
expression of PGC-1 in skeletal muscle or by administration of bezafibrate, a PPAR
panagonist, these two strategies successfully delay the onset of a mitochondrial myopathy
caused by a COX deficiency [247].
As described in the previous section, mitochondrial gene therapy is effective in the
treatment of muscle injury [135]. Increased PGC-1 expression has a beneficial effect on the
development and progression of sarcopenia, which refers to the loss of skeletal muscle mass
and strength that normally occurs with ageing [248]. PGC-1 transgenic mice can preserve
not only mitochondrial function but also neuromuscular junctions, and muscle integrity
during ageing [249]. Intriguingly, elevated PGC-1 expression makes whole-body healthy
because the loss of bone mineral density and the increase of systemic chronic inflammation
are attenuated and insulin sensitivity is improved by reactivating insulin signaling in aged
mice [249]. These efforts not only promise the development of therapeutic strategies for
mitochondrial diseases and sarcopenia but also may help us to understand the role of
mitochondria in myogenesis.

Acknowledgments
This research was supported by the MEXT (The Ministry of Education, Culture, Sports,
Science and Technology) (Grant-in Aid for Scientific Research (C), 22500658), Japan.

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In: Basic Biology and Current Understanding of Skeletal Muscle ISBN: 978-1-62808-367-5
Editor: Kunihiro Sakuma
2013 Nova Science Publishers, Inc.

Chapter 10

Skeletal Muscular Adaptation

and Local Steroidogenesis
Katsuji Aizawa
Senshu University Institute of Sport, Tama-ku,Kawasaki-shi, Kanagawa, Japan

Abstract
The plasticity of skeletal muscle facilitates adaptation to various stimuli. Exercise
training induces skeletal muscle adaptation such as muscle strength and hypertrophy,
while inactivity leads to muscle atrophy such as sarcopenia. Sex steroid hormones
(androgens and estrogens) often mediate muscle plasticity. Indeed, these hormones
induce various effects including growth, strength, metabolism, and antioxidant levels as
well as muscle atrophy. Though sex steroid hormones play an important role in skeletal
muscular homeostasis, the role of the endocrine system in muscle plasticity is unknown.
Sex steroid hormones are produced by various peripheral target tissues including the
kidney, liver, and brain in addition to endocrine organs such as the testis or ovary. Sex
steroid hormones are synthesized from cholesterol by steroidogenic enzymes, such as
cholesterol side-chain cleavage (P450scc), cytochrome P450 enzyme 17-hydroxylase
(P450c17), 3-hydroxysteroid dehydrogenase (HSD), and 17-HSD, with testosterone
being irreversibly converted to estrogen by aromatase cytochrome P450 (P450arom).
Testosterone is also converted into its bioactive metabolite dihydrotestosterone (DHT) by
5-reductase.
The functional importance of sex steroid hormones derived from extragonadal
tissues has been gaining support in recent years. For instance, steroidogenic enzymes
expressed in skeletal muscle have been reported to locally synthesize sex steroid
hormones from circulating dehydroepiandrosterone (DHEA) or testosterone in response
to exercise. Thus, local steroidogenesis in skeletal muscle may play an important role in
the plasticity of skeletal muscle. This review focuses on the steroidogenesis of skeletal
muscle and discusses the physiological significance of the sex steroid hormone network
of circulation and skeletal muscle.

Address correspondence and reprint requests to: Katsuji Aizawa, Ph. D. Senshu University Institute of Sport, 2-11, Higashimita, Tama-ku, Kawasaki-shi, Kanagawa 214-8580, Japan. (TEL) +81 44 911 1024. (FAX) +81 44
911 1032. E-mail: katsuji.aizawa@gmail.com.

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1. Introduction
Skeletal muscle is plastic due in part to a balance between protein synthesis and
degradation. For example, exercise leads to muscular hypertrophy due to increased protein
synthesis, while inactivity or disuse leads to muscle atrophy due to protein catabolism. Thus,
the maintenance of skeletal muscle homeostasis is beneficial in the prevention of many
chronic diseases and can improve quality of life; however, the mechanism of skeletal muscle
plasticity is still poorly understood.
Sex steroid hormones, such as testosterone and estradiol, play important roles in
developing both strength and mass of skeletal muscle. Chronic exercise induces increases in
muscle size, strength, energy metabolism, and antioxidant capacity, as well as changes in
muscle fiber type [1, 2]. Sex steroid hormones partly contribute to these exercise-induced
muscular adaptations [3, 4, 5]. Interestingly, testosterone administration increases muscle
strength and mass, while estrogen administration has little effect on muscle development and
mass. On the other hand, loss of skeletal muscle mass with aging, known as sarcopenia, is
associated with decreased blood androgens levels. Thus, it is likely that sex steroid hormones
may be involved in the mechanism of skeletal muscular adaptation.
Sex steroid hormones are mainly secreted by the ovary, testis, and adrenal cortex and
regulate a variety of physiological processes in their target tissues, which include
reproductive tissues, bone, liver, cardiovascular tissues, brain, and skeletal muscle [6, 7].
Both classes of sex steroid hormones, androgens and estrogens, are synthesized from
cholesterol by steroidogenic enzymes, such as cholesterol side-chain cleavage (P450scc),
cytochrome P450 enzyme 17-hydroxylase (P450c17), 3-hydroxysteroid dehydrogenase
(3-HSD), 17-HSD, 5-reductase, and cytochrome P450 (P450arom) [7]. Previous reports
have shown that sex steroid hormones are secreted not only by the ovary, testis, and adrenal
gland, but also by various peripheral tissues, including bone, liver, and brain [8, 9, 10].
Recent findings indicate that skeletal muscle produces local sex steroid hormones [11] and is
activated by exercise stimulation [12]. Since sex steroid hormones participate in skeletal
muscular adaptation, local muscular steroidogenesis may have an important role in skeletal
muscle homeostasis. This review focuses on the steroidogenesis of skeletal muscle and
discusses the physiological significance of the sex steroid hormone network of circulation and
skeletal muscle.

2. Role of Sex Steroid Hormones

in Skeletal Muscle
2.1. Androgens and Estrogens
The major sex hormones, testosterone and estradiol, are produced by the testis and ovary.
In addition, dehydroepiandrosterone (DHEA) and its sulfate-bound form (DHEAS), are
mainly secreted by the adrenal gland and ovary. They are the most abundant hormones in
humans and mammals, and are important precursors of sex steroid hormones in peripheral
tissues [13, 14]. For instance, plasma DHEAS levels in human adults are 100500 times
higher than testosterone, and 1,00010,000 times higher than estradiol [14]. Thus, DHEA

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291

plays a critical physiological role for peripheral tissues in maintaining steroidogenesis as an

available precursor for conversion to testosterone and estrogen [13, 14, 15].
As a sex steroid-sensitive tissue, skeletal muscle expresses both androgen receptor (AR)
and estrogen receptor (ER) [16, 17]. Androgens and estrogens are the ligands of AR and ER,
respectively. Sex steroid hormones bind to each receptor to form complexes that bind
promoter or enhancer elements of target genes to regulate their transcription. Indeed, sex
steroid hormones can affect growth, strength, metabolism, and antioxidant levels in skeletal
muscle [3, 5, 18, 19]. For example, testosterone-induced muscle fiber hypertrophy is
associated with an increase in satellite cell number, a proportionate increase in myonuclear
number, and changes in satellite cell ultrastructure [5]. Testosterone has been reported to
increase muscle strength and protein synthesis [20]; furthermore, long-term administration of
DHEA promotes muscle strength and mass in humans [21], while sex steroid hormones
deficiencies lead to the delay of skeletal muscle development [15]. In addition, bioactive
androgen dihydrotestosterone (DHT) administration upregulates AR expression and increases
the skeletal muscle mass of castrated rats [22]. In mice, bioactive DHT promotes the
expression of genes associated with protein synthesis, cell signaling, cell proliferation, ATP
production, and muscle contraction and relaxation in skeletal muscle in vivo [23]. Regarding
the effects of estrogen on skeletal muscle, there is a striking decline in muscle strength that
can be reversed by hormonal replacement therapy (HRT) in postmenopausal women,
suggesting that female sex steroids are important modulators of muscle physiology [24]. In
fact, meta-analysis of data from female patients receiving HRT confirmed the beneficial
effects of estrogens on muscle strength [25]. However, the effects of estrogen replacement
therapy on muscle strength are still unclear; while some studies report that it increases muscle
strength [19], others indicate that it does not [26]. Estradiol also plays a role in the level of
antioxidant enzymes in skeletal muscle.
Antioxidant as well as ER- gene expression levels in the skeletal muscle of mice acutely
and chronically respond to changes in circulating estradiol; this is another indication of
estrogen-mediated mechanisms influencing contractility of skeletal muscle [27]. However,
the mechanism by which estrogen, through its action on muscle, regulates metabolism is
unclear at this time.

2.2. Sarcopenia and Sex Steroid Hormones

During aging, there is a gradual decline in serum sex steroid hormones levels [28]. A
combination of changes in testicular function, altered neuroendocrine regulation of Leydig
cells, and increased binding capacity of sex hormonebinding globulin (SHBG) results in
approximately 40% lower testosterone levels among men in their 70s compared with men in
their 20s. The marked reduction in DHEA production by the adrenal gland during aging (i.e.,
by age 75 the plasma DHEA level is ~80% lower than at age 25) results in a dramatic
decrease in androgen and estrogen levels in peripheral target tissues, which is thought to be
associated with insulin resistance, obesity, and loss of muscle mass [29]. In postmenopausal
women, the secretion of estradiol by the ovaries is decreased to levels comparable to those in
men [30], increasing the risk for brain, bone, and cardiovascular disorders as well as affecting
muscular function [31, 32]. Moreover, the dramatic reduction in hormone production after
menopause in females accelerates sarcopenia [33].

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Muscle atrophy can be caused by diverse medical conditions and results from accelerated
catabolism of muscle proteins. Ubiquitin ligases that regulate muscle proteolysis, such as
atrogin-1/MAFbx/FBXO32 [34] and Murf-1/trim63 [35], are specifically upregulated during
muscle atrophy. Overexpression of atrogin-1 leads to atrophy of cultured myotubes, whereas
atrogin-1 or Murf-1 knockout animals are resistant to muscle atrophy. Thus, the levels of
atrogin-1/Murf-1 appear to determine the extent of muscle loss.
Low testosterone levels have been associated with decreased muscle mass in humans, and
testosterone administration has been successful in improving general muscle function in
elderly men [36]. A previous study reported that testosterone deprivation induced a 31-fold
increase in testosterone-dependent fast-twitch levator ani muscle atrogin-1 mRNA and 18fold increase in Murf-1 mRNA, which were detected after 2 and 7 days of castration,
respectively [37]. Testosterone administration in the model above inhibited mRNA
expression of atrogin-1 and Murf-1 in androgen-sensitive rat skeletal muscles in vivo [37].
Testosterone also repressed MAFbx expression via association of the AR and Oct-1 with the
5 untranslated region of MAFbx just upstream of the first codon. Thus, prevention of muscle
atrophy by testosterone may be mediated by the regulation of ubiquitin ligases that control
muscle proteolysis [38].

3. Local Production of Sex Steroid Hormones

3.1. Steroidogenic Enzymes
Sex steroid hormones are produced from cholesterol by steroidogenic enzymes (Figure
1). The biosynthesis of active androgens and estrogens from DHEA is catalyzed by three
classes of enzymes [39].
Testosterone is synthesized through the metabolism of DHEA by 3-HSD and 17-HSD,
and is converted into estrogen by P450arom. The physiological balance between androgens
and estrogens is largely controlled by P450arom [40], which is encoded by a single gene. In
contrast, 12 genes encoding 17-HSDs have been identified in humans, and 10 have been
cloned so far (15, 7, 8, 1012) [41]. The 17-HSDs differ in tissue distribution, catalytic
preferences, substrate specificity, subcellular localization, and mechanisms of regulation. For
instance, rat 17-HSD type I efficiently converts androstenedione to testosterone [42].
Testosterone is converted into its bioactive metabolite DHT by 5-reductases [43, 44]. DHT
has a higher binding affinity for AR than testosterone, and is therefore a highly potent
androgen [45]. The two types of 5-reductase genes (type 1 and type 2), share approximately
50% sequence identity [46] but exhibit different tissue- and cell typespecific expression
patterns that reflect their biological roles. While the type 1 isozyme is broadly distributed, the
type 2 5-reductase are found predominantly in male sex accessory tissues such as the
epididymis, prostate, and seminal vesicles [46, 47, 48].

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293

Figure 1. A pathway showing sex steroid hormone metabolism, and associated steroidogenic enzymes.
DHEA: dehydroepiandrosterone, DHT: dihydrotestosterone, P450scc: cholesterol side-chain cleavage,
17-HSD: 17-hydroxysteroid dehydrogenase, 3-HSD: 3-hydroxysteroid dehydrogenase, P450arom:
aromatase cytochrome P450, AR: androgen receptor, ER: estrogen receptor

Numerous studies have demonstrated the expression of steroidogenic enzymes in a

variety of tissues. For instance, 3-HSD and 17-HSD are expressed in the testis, ovary,
adrenal gland, and brain tissues [10, 41, 49], whereas P450arom mRNA and activity have
been detected in the ovary, gastric mucosa, tibial growth plate, and brain [8, 50]. The
functional significance of gonadal-derived sex steroid hormones has been extensively studied;
however, the importance of local extragonadal-derived sex steroid hormones in cell
physiology and pathophysiology has only recently begun to be appreciated in nervous and
adipose tissues, and the adrenal gland [7, 30].

3.2. Steroidogenesis in Skeletal Muscle

Skeletal muscle expresses both AR and ER in various species (human, cow, mouse, and
rat). In addition, mRNA for steroidogenic enzymes, including 3-HSD, 17-HSD, and
P450arom, is detected in skeletal muscle cells of rats, both in vivo and in culture (in vitro)
[11] at levels comparable to those found in the kidney and liver. This suggests that skeletal
muscle may be an important extragonadal source of sex steroid hormones (Figure 2A, 2B).
The tissue concentrations of testosterone and estradiol in skeletal muscle were significantly
lower than in the ovary and testis. Moreover, intracellular production of testosterone and
estradiol from DHEA or testosterone in cultured muscle cells occurred in a dose-dependent
manner (Figure 3AD).
These data demonstrate the presence of key enzymes that locally synthesize testosterone
and estrogen from DHEA, namely 3-HSD, 17-HSD, and P450arom, in skeletal muscle. On
the basis of earlier reports that skeletal muscle possesses receptors for both androgen and
estrogen (ER and, the most recently isolated subtype, ER) and that, as revealed here,
skeletal muscle is capable of generating sex steroids locally, we propose the existence of a

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Katsuji Aizawa

local autocrine and/or paracrine system of testosterone and estrogen action in skeletal muscle
cells.

Figure 2. Skeletal muscle expresses steroidogenic enzymes (ref. [11]). A: mRNA expression of
steroidogenic enzymes in various rat tissues. Expression of 3-HSD, 17-HSD type I, and P450arom
mRNA was revealed by RT-PCR gels in skeletal muscle [gastrocnemius (gastro)] of the rat compared
with positive control tissues, namely ovary, testis, liver, kidney, and brain. -Actin mRNA was used as
an internal control. B: Localization of steroidogenic enzymes via protein expression from the muscle of
the rat. Representative images of immunofluorescence for P450arom in skeletal muscle (gastrocnemius)
of male rats (AD). Aromatase is stained red, and immunoreactivity is clearly seen in the cytoplasm
and cytoplasmic membrane (AD) at a magnification of 200. E: Positive staining was also observed in
blood vessels (magnification 200). F: Omission of primary antibody showed no immunoreactivity in the
skeletal muscle, indicating the specificity of the antibody (magnification 200).

Figure 3. Local steroidogenesis when DHEA and testosterone are added to cultured muscle cells (ref.
[11]). Intracellular concentration of total testosterone (A and C), and estradiol (B and D) formed after
the addition of either DHEA or testosterone (10, 100, 300, and 500 M, respectively) in cultured
muscle cells. The muscle cells treated with vehicle (0.1% ethanol) were used as a control.

Skeletal Muscular Adaptation and Local Steroidogenesis

295

In addition, muscular steroidogenesis was confirmed in perimenopausal human skeletal

muscle [51], although the influence of peripheral and systemic sex steroids on skeletal muscle
quality in pre- and postmenopausal women is different. This may be that systemically
delivered and peripherally produced sex steroids have distinct roles in the regulation of
neuromuscular characteristics during aging.

4. Exercise and Muscular Steroidogenesis

Previous studies have reported that exercise stimulation influences local steroidogenesis
in skeletal muscle [12, 52, 53]. Endurance exercise has been shown to increase intramuscular
androgen biosynthesis in rodents, providing an alternative source of androgens that may
produce intracrine and/or autocrine actions within skeletal muscle. Specifically, a single bout
of endurance treadmill running upregulates mRNA expression and protein content of 3HSD, 17-HSD, and 5-reductase type 1 isozyme within rodent skeletal muscle and increases
the intramuscular concentrations of testosterone, free testosterone, and DHT [12, 52].
Similarly, endurance training increases circulating and intramuscular concentrations of
DHEA, skeletal muscle mRNA expression of 3-HSD, and skeletal muscle mRNA and
protein expression of 5-reductase [53]. Endurance exercise training also enhances muscular
DHT concentration through 5-reductase in the skeletal muscle of rats, suggesting that local
bioactive androgen metabolism may participate in exercise traininginduced skeletal
muscular adaptation. Thus, exercise-induced alteration of steroidogenic enzymes in muscle
tissue may lead to their local conversion to testosterone, DHT and estrogens from circulating
DHEA or testosterone.
Only a single study has evaluated the intramuscular steroidogenic potential of human
skeletal muscle after exercise in healthy young eugonadal men and women [54]. The authors
reported that the intramuscular protein content of 3-HSD type 1 and 2 and 17-HSD type 3
did not change in the quadriceps musculature at 10 and 70 min after a single bout of heavy
resistance exercise (6 sets, 10 repetitions of squats) and that no alterations in intramuscular
concentrations of total testosterone were present after completion of the exercise bout [54].
However, this study did not evaluate alterations in the 5-reductase isozymes or
intramuscular DHT. This finding contrasts with previous findings in rats suggesting that a
species difference in the muscle steroidogenic response to exercise may exist.

5. Sex Steroid Hormone Network of Circulation

and Skeletal Muscle
Resistance exercise increases testosterone levels in plasma in males but not females [55,
56], although there are no reported gender differences in chronic exercise-induced adaptation
such as hypertrophy by skeletal muscle [57, 58]. This adaptation may be caused the synthesis
of sex steroid hormones from circulating DHEAs or testosterone in skeletal muscle that
would act as an autocrine and local paracrine muscle growth factor. In fact, it found that the
expression of 5-reductase type 1 and muscular bioactive androgen DHT were significantly
increased by exercise in both sexes (Figure 4A) [52]. These results strongly suggest that

296

Katsuji Aizawa

exercise enhances the local metabolism of bioactive androgens in skeletal muscle, which may
play a role in compensating for the low level of circulating testosterone in females. In
contrast, the level of muscle estradiol is increased by exercise in males, but not in females
(Figure 4B) [12]. Furthermore, it has been shown that expression of P450arom, which is
important for metabolizing androgens into estrogens [40], is increased in males but decreased
in females by a single bout of exercise [12]. Thus, 5-reductase and P450arom expression in
skeletal muscle is inversely correlated with the response to exercise in the two sexes. Since
the physiological balance between sex steroid hormones is largely controlled by aromatase or
5-reductase in exercising skeletal muscle, estrogen synthesis may increase in males while
testosterone synthesis increases in females. Thus, sex differences in the regulation of 5reductase and P450arom by exercise may in part compensate for insufficient local levels of
sex steroid hormones in skeletal muscle for each sex. Moreover, the highly effective local
synthesis of sex steroid hormones in skeletal muscle contributes to the differential capacity
for sex steroid hormone synthesis.

Figure 4. Muscular concentrations of DHT (A) and estradiol (B) in the skeletal muscle of male and
female rats with acute exercise (ref. [12, 52]).

In postmenopausal women, the secretion of estradiol by the ovaries is impaired and

decreases to levels comparable to those in men [30]. This age-induced reduction in sex steroid
hormone production increases the risk for brain, bone, and cardiovascular disorders in
postmenopausal women. A previous study demonstrated that DHEA replacement enhances
the increased muscle mass and strength induced by resistance exercise in elderly subjects
[59]. This study provides support for DHEA replacement in that it significantly potentiates
the effect of heavy resistance exercise on muscle hypertrophy and strength in elderly women
and men. A recent study also demonstrated that endurance exercise training increases serum
DHT levels (14.5%) and aerobic capacity (10.8%) among older men [60]. Taken together,
exercise stimulation influences local steroidogenesis in skeletal muscle as well as endocrine
glands such as testis, adrenal, and ovary, suggesting that sex steroid hormone synthesis in
skeletal muscle is involved in muscular adaptation in cooperation with circulating hormones
(Figure 5). In particular, local muscular steroidogenesis may contribute to the compensation
of insufficient local levels of sex steroid hormones in skeletal muscle for each sex or agerelated hormone decrease.

Skeletal Muscular Adaptation and Local Steroidogenesis

297

Figure 5. Interaction between circulating and muscular sex steroid hormones.

Conclusion
In summary, skeletal muscle can be considered an endocrine organ that produces sex
steroid hormones. We, and others, have found that exercise locally stimulates muscle
steroidogenic enzymes, thereby activating sex steroid hormone production. Furthermore, the
age-related decrease in sex steroid hormone levels is involved in the reduction of muscle
function. Thus, enhancement of muscle steroidogenesis by exercise may play an important
role in skeletal muscle homeostasis and maintenance in aging. Finally, the interaction
between circulating and muscular sex steroid hormones may contribute to skeletal muscular
adaptation.

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Index
A
Abraham, 77, 78
abuse, 80
accessibility, 144
acetylation, 247, 283
acetylcholine, 263
activation state, 31
active site, 133, 196
acute infection, 33
acute stress, 151
adaptation(s), vii, x, 31, 37, 83, 91, 108, 109, 111,
118, 121, 124, 133, 134, 135, 137, 138, 141, 145,
151, 165, 166, 176, 188, 189, 190, 194, 197, 202,
206, 208, 210, 221, 228, 232, 238, 247, 275, 278,
289, 290, 295, 296, 297, 301
adductor, 185, 298
adductor longus, 185
adenine, 131, 215, 223
adenosine, 42, 91, 208, 210, 212, 224, 240, 243
adenosine triphosphate, 91
adenovirus, 215
adenylate kinase, 131
adhesion, 11, 60, 66, 76, 77, 78, 83, 126, 127, 233,
260, 281
adipocyte, 139, 212, 238, 245, 270
adiponectin, 128, 139, 211, 212, 213, 234, 235, 237,
238, 249
adipose, 61, 93, 102, 128, 131, 132, 170, 213, 215,
223, 233, 293
adipose tissue, 61, 93, 102, 128, 131, 132, 170, 213,
215, 223, 233, 293
adiposity, 99, 169
ADP, 207, 208, 213, 216, 229, 256
adrenal gland, 290, 291, 293
adulthood, 98

adults, vii, 2, 3, 4, 6, 7, 12, 21, 22, 47, 48, 85, 89, 92,
94, 95, 99, 102, 106, 107, 108, 109, 111, 113,
116, 119, 137, 290
aerobic capacity, 92, 176, 226, 296
aerobic exercise, 35, 92, 105, 124, 142
afferent nerve, 187, 188
afferent nerve fiber, 188
age-related diseases, 124
aggregation, 148, 185
aging process, ix, 94, 96, 97, 103, 123, 124, 155, 252
agonist, 203
alanine, 93
aldehydes, 102
allele, 185
alters, 85, 112, 135, 144, 165, 194, 240
alveolar macrophage, 240
American Heart Association, 106, 109
amino, viii, 24, 26, 27, 32, 39, 40, 43, 46, 47, 48, 55,
56, 57, 58, 59, 64, 67, 74, 75, 76, 87, 88, 89, 93,
94, 98, 99, 104, 110, 111, 125, 126, 180, 190,
215, 230, 239, 249
amino acid(s), viii, 24, 26, 27, 32, 39, 40, 43, 46, 47,
48, 55, 56, 57, 58, 59, 64, 67, 74, 75, 87, 88, 89,
93, 94, 98, 99, 104, 110, 111, 125, 126, 180, 190,
215, 230
amniotic fluid, 74
amplitude, 181, 182, 198
amyotrophic lateral sclerosis (ALS), viii, 53, 54, 59,
61, 62, 63, 100, 111
anabolic steroids, 84
anabolism, 32, 46, 48
anchorage, 77, 181, 185
anchoring, 197
androgen(s), xi, 84, 97, 115, 289, 290, 291, 292, 293,
295, 296, 298, 299, 300, 301
angiogenesis, 78, 101, 109
angiotensin converting enzyme, 118
anorexia, 103

304

Index

anorexia nervosa, 103

antagonism, 240
antibiotic, 258
antibody, 69, 101, 294
antigen, 14, 67, 74, 75
antihypertensive agents, 101
antioxidant, ix, xi, 87, 97, 114, 123, 131, 132, 134,
135, 136, 137, 142, 144, 146, 160, 170, 210, 289,
290, 291, 297, 299
antisense, 77, 160
antisense oligonucleotides, 160
antisense RNA, 77
antitumor, 120
antitumor agent, 120
apoptosis, viii, 4, 22, 33, 49, 51, 60, 65, 80, 82, 83,
84, 87, 92, 96, 103, 119, 121, 128, 129, 130, 141,
157, 158, 160, 162, 163, 165, 168, 179, 196, 197,
206, 226, 233, 238, 269, 286
apoptosis pathways, 158
apoptotic mechanisms, 168
apoptotic pathways, 103
appetite, 99, 206, 212
arginine, 93
arrest, 282
arthrogryposis, 193
assessment, 299
astrocytes, 160, 169, 298
athletes, 67, 83, 84, 86, 176, 248
ATP, x, 24, 27, 28, 130, 144, 167, 205, 207, 208,
209, 210, 211, 212, 213, 214, 215, 252, 258, 259,
260, 261, 262, 263, 267, 271, 291
autocrine/paracrine, viii, 53
autolysis, 178, 179, 196
autosomal dominant, 193
avian, 15, 258

B
basal lamina, vii, 1, 2, 11, 67
basal metabolic rate, 130
base, 55, 243, 253, 254
base pair, 55, 243, 253
basement membrane, 254
beneficial effect, 94, 101, 135, 160, 270, 291
benefits, ix, 90, 93, 94, 95, 97, 101, 123, 154, 159,
162
beverages, 213
binding globulin, 291
biological activities, 69
biological fluids, 80
biological roles, 56, 60, 159, 292
biomarkers, ix, 50, 113, 123
biopsy, 93, 274

biosynthesis, 226, 257, 292, 295

births, 158
bladder cancer, 78
blood, 7, 49, 60, 61, 91, 101, 109, 128, 132, 215,
224, 249, 290, 294
blood flow, 91, 101, 109
blood vessels, 60, 294
BMI, 161
body composition, 35, 107, 111, 115, 298, 299
body fat, 172
body fluid, 56
body weight, vii, 21, 22, 35, 70, 89, 92, 99, 206, 252
bonds, 133, 144
bone, 9, 15, 19, 65, 78, 86, 98, 102, 110, 112, 115,
270, 290, 291, 296, 299
bone form, 65
bone growth, 78
bone marrow, 15, 19
bone mineral content, 112
boric acid, 115
brain, xi, 25, 36, 65, 88, 136, 192, 207, 210, 212,
223, 230, 232, 289, 290, 291, 293, 294, 296, 298
breakdown, 23, 47, 49, 94, 99, 140, 217, 280
breast cancer, 65, 66, 81, 82, 83, 98
breathing, 143
bromination, 125
bronchial epithelial cells, 69

C
C reactive protein, 33
Ca2+, ix, 27, 40, 41, 82, 103, 130, 136, 140, 143, 144,
155, 158, 171, 174, 175, 178, 180, 181, 182, 191,
198, 209, 210, 212, 216, 229, 230, 233, 234, 243,
256, 266, 267, 273, 284
Ca2+ signals, 266
cachexia, 29, 33, 49, 90, 95, 97, 99, 100, 107, 113,
115, 117, 118, 286
cadmium, 202
caffeine, 209, 210
calcineurin, ix, 64, 66, 71, 82, 96, 135, 164, 171,
178, 181, 182, 183, 195, 198, 199, 263, 266, 267,
268, 272, 283, 284, 285
calcitonin, 2
calcium, x, 28, 64, 102, 110, 130, 133, 140, 144,
165, 181, 196, 198, 210, 251, 252, 264, 266
calcium homeostasis, x, 130, 251, 252
caliber, 16
caloric restriction, 206, 238, 274
calorie, 78, 88, 103, 121, 141, 237, 238, 275
calpain-3, ix, 171, 178, 179, 180, 189
CAM, 14

Index
cancer, 17, 33, 38, 49, 60, 95, 97, 100, 101, 113, 120,
125, 136, 206, 244, 285
cancer therapy, 120, 285
candidates, 26, 30, 99, 104, 156, 162, 211, 213, 222
capillary, 60
carbohydrate(s), 32, 48, 49, 74, 93, 110, 131, 159,
206, 208, 240
carbon, 95
carboxyl, 55, 56, 76
carcinoma, 76
cardiac muscle, 30, 176, 286
cardiomyopathy, 198, 199
cardiovascular disease(s), 95, 101, 106, 108, 128,
206, 234
cardiovascular disorders, 291, 296
carpal tunnel syndrome, 99
cartilage, viii, 9, 53, 61
cascades, 15, 54, 64, 66, 71, 141
case study, 235
casein, 81
caspases, 158
casting, 187, 188
castration, 292, 299
catabolic, viii, x, 21, 22, 28, 29, 49, 50, 93, 102, 120,
205
catabolism, x, 28, 29, 34, 139, 205, 290, 292
catabolized, 125
CBS, 207
CDK inhibitor, 118
cDNA, 30, 268
cell biology, 5, 12
cell culture, 69, 74, 218, 223, 255, 262, 268
cell cycle, x, 11, 12, 13, 35, 66, 67, 77, 83, 84, 206,
251, 252, 265, 283, 286
cell death, x, 16, 82, 148, 155, 158, 166, 170, 251,
252
cell differentiation, 13, 30, 86, 113, 252, 260, 263,
264, 265, 269, 271, 281, 283, 284, 286
cell division, 6, 7, 8, 10, 11, 17
cell fate, 3, 6, 12, 15, 17, 18, 259, 280
cell fusion, 261, 283
cell line(s), 2, 65, 69, 75, 81, 152, 156, 158, 226,
260, 262, 264, 265, 266, 267, 272
cell signaling, 241, 291
cell size, 25, 37
cell surface, 57, 60, 63, 64, 65, 66, 77, 260
cellular energy, x, 27, 38, 205, 208, 209, 251
cellular homeostasis, x, 251
cellular signaling pathway, 252
ceramide, 65, 214, 215, 224, 240
cerebral cortex, 298
Chad, 111, 112
challenges, 38

305

chaperones, 148, 155, 163, 167, 170

chemical(s), 96, 102, 125, 141, 150, 170, 208, 212,
224, 253, 268, 269
chemokines, 126, 127
chemotaxis, 60
chicken, 13, 193
children, 102
chimera, 59
chinese medicine, 213
chlorination, 125
cholecalciferol, 121
cholesterol, xi, 206, 226, 289, 290, 292, 293
chromatography, 112, 137
chromium, 107
chromosome, 10, 54, 66, 193
chronic diseases, 22, 33, 34, 114, 290
chronic heart failure, 33, 99, 117
chronic hypoxia, 40
chronic illness, 115
chronic obstructive pulmonary disease, 33, 49, 99
chymotrypsin, 102
circulation, xi, 54, 63, 289, 290
city, 171
classes, 102, 290, 292
cleavage, viii, xi, 54, 56, 57, 64, 65, 70, 71, 139, 163,
283, 289, 290, 293
clinical application, 113, 138, 161, 162
clinical symptoms, 119
clinical trials, 95
clone, 5
cloning, 76, 192, 196, 230, 267
cluster analysis, 84
cluster of differentiation, 131
CNS, 234, 246
coactivators, x, 109, 126, 145, 205, 218, 248, 252,
255, 278
coding, 253, 283
codon, 292
coenzyme, x, 205, 219, 220, 223
collagen, 9, 107, 177, 188, 204
commercial, 97
communication, 60
community, 33, 49, 50, 102, 106, 115, 120
compensation, 214, 296
competition, 37, 85
complement, 139, 253
complications, 97, 131
composition, 35, 105, 174, 190, 239, 244, 246, 256,
265, 268, 272
compounds, ix, 97, 102, 123, 136, 212, 213, 269
compression, 276
conditioning, 107
congestive heart failure, 119

306

Index

conjugation, 119
connective tissue, 14, 22, 77, 78, 93, 188, 258, 263
connectivity, 94
consensus, 97, 114, 176, 183
construction, 189
consumption, 130, 145, 210
control group, 92
controlled trials, 102, 121
controversial, 34, 97, 98, 99, 106, 162, 209
cooperation, 215, 296
COPD, 117
corepressor, 283
coronary heart disease, 113
correlation, 27, 67, 191
cortex, 290
CPT, 131, 132, 136, 219, 220
creatine, 88, 93, 95, 111, 112, 127, 138, 151, 154,
263, 271, 286, 298
creatine phosphokinase, 151, 298
cross-sectional study, 105, 115
cross-validation, 35
CRP, 33
cues, viii, 21
culture, 3, 5, 6, 8, 10, 62, 63, 68, 70, 71, 81, 84, 257,
262, 283, 284, 293
culture conditions, 63, 70
culture medium, 262
cure, 158
cyanide, 253, 263
cycles, 158, 267
cycling, 12, 18, 85, 115, 132, 143, 148, 174
cyclophosphamide, 284
cyclosporine, 266, 281
cysteine, 56, 57, 60, 63, 125, 126, 132, 144, 178, 266
cytochrome, xi, 108, 130, 158, 221, 238, 254, 257,
273, 279, 289, 290, 293, 298
cytokines, x, 12, 22, 33, 60, 61, 78, 98, 99, 126, 127,
128, 132, 138, 141, 142, 205, 206, 209, 210, 211,
267
cytoplasm, x, 29, 125, 127, 130, 137, 179, 186, 205,
229, 267, 294
cytosine, 185
cytoskeleton, 36, 172, 179, 198, 202, 203, 261, 282
cytotoxicity, 166

D
daily living, 106
damages, 67, 68
deacetylation, 221, 223
decay, 113, 283, 286
defects, 15, 36, 253, 254, 277

deficiency(s), 22, 62, 63, 79, 95, 102, 103, 120, 162,
176, 179, 191, 193, 194, 196, 199, 270, 273, 278,
279, 291
deficit, 109, 145, 148, 185, 264, 286
degradation, viii, 21, 22, 23, 28, 29, 30, 33, 34, 43,
64, 66, 67, 74, 96, 101, 126, 127, 128, 129, 130,
137, 139, 140, 153, 156, 158, 165, 178, 192, 203,
206, 226, 254, 255, 264, 276, 277, 286, 290
degradation rate, 128, 153
dehydroepiandrosterone, xi, 88, 115, 289, 290, 293,
298
deltoid, 276
dementia, 206
denaturation, 155
dendritic cell, 252, 271
deoxyribonucleic acid, 81
dependent variable, 109
dephosphorylation, viii, 27, 42, 54, 66, 71, 95, 209,
215, 239, 240, 268
deposition, 9
depression, 103, 159
deprivation, x, 26, 29, 44, 169, 194, 205, 210, 292
desensitization, 180
detectable, 58
detection, 277
developmental change, 164
developmental process, 60, 71
diabetes, ix, x, 29, 94, 97, 100, 101, 108, 130, 131,
135, 137, 145, 147, 148, 159, 160, 161, 162, 169,
170, 205, 206, 212, 213, 217, 224, 225, 237, 240,
243, 248
diabetic patients, 113, 131, 138, 141, 159, 160, 161,
224
diacylglycerol, 180, 239, 240
diaphragm, 111, 137, 140, 143, 177, 185, 195
diet, 132, 142, 159, 160, 169, 214, 237, 238, 239
dimerization, 185, 265
diploid, 77
disability, vii, 22, 34, 35, 49, 89, 106
discharges, 204
discs, 172, 176, 177, 178, 181, 182, 183, 184, 185,
186
disease progression, 15
diseases, vii, x, 21, 22, 33, 95, 124, 125, 129, 206,
210, 225, 252, 254, 270, 274
disorder, 158, 175
displacement, 183
dissociation, 135, 186, 273
distribution, 38, 54, 56, 79, 86, 89, 194, 197, 199,
207, 246, 292, 300
diversity, 54, 71, 190
DNA, vii, 1, 6, 7, 11, 12, 17, 18, 22, 26, 57, 60, 64,
65, 79, 91, 108, 124, 126, 127, 130, 146, 155,

Index
156, 165, 167, 183, 218, 243, 244, 253, 257, 265,
268, 269, 274, 275, 277, 280, 282, 283, 284
DNA damage, 22, 26, 91, 146
DNA repair, 253
docosahexaenoic acid, 113
domain structure, 193
donors, 133, 210, 260
dosage, 97
dose-response relationship, 47
down-regulation, 9, 13, 135
drug discovery, viii, 87, 115
drugs, x, 97, 205, 206, 212, 236, 262
dyslipidemia, x, 205
dystrophy, ix, 3, 12, 13, 15, 16, 36, 87, 88, 90, 92,
102, 109, 136, 147, 158, 168, 179, 196, 197, 201,
281

E
ECM, 57, 60, 63, 64, 70, 75, 78
edema, 99
eicosapentaenoic acid, 88, 113
eIF4A, 24
elderly population, viii, 33, 34, 50, 87
elders, 91, 108
election, 109
electron, ix, 2, 91, 96, 123, 131, 155, 167, 172, 176,
219, 220, 221, 223, 225, 253, 256, 257, 271, 275,
276
electron microscopy, 2
ELISA, 115
elongation, 25, 31, 37, 156, 162, 258, 260
elucidation, 279
embryogenesis, 177, 273
embryology, 200
embryonic stem cells (ESCs), 252, 271
EMG, 204
emission, 226
encoding, 79, 91, 101, 126, 127, 158, 206, 209, 215,
223, 229, 248, 253, 257, 259, 264, 292
endocrine, viii, xi, 53, 54, 61, 72, 210, 211, 289, 296,
297, 300
endocrine glands, 296
endocrine system, xi, 289
endocrinology, 299
endonuclease, 276
endothelial cells, 60, 77, 78, 126, 215, 233
endothelium, 54, 63, 91, 124
endurance, x, 85, 91, 92, 94, 96, 104, 108, 109, 132,
135, 143, 144, 145, 148, 176, 194, 205, 208, 210,
211, 221, 228, 233, 248, 295, 296
energy, vii, x, 24, 26, 27, 41, 42, 44, 89, 99, 103,
105, 110, 112, 130, 131, 132, 135, 141, 205, 206,

307

209, 210, 211, 224, 225, 227, 232, 233, 246, 247,
251, 252, 258, 260, 271, 278, 279, 280, 290
energy consumption, vii, 130, 210
energy expenditure, 141, 206, 211, 225, 247
enlargement, 180
environment(s), 3, 9, 15, 54, 186, 187, 189, 203
environmental factors, 102
environmental stress, 148
enzyme(s), x, xi, 69, 88, 91, 108, 118, 119, 125, 127,
130, 131, 135, 137, 144, 160, 178, 190, 212, 215,
219, 220, 221, 224, 225, 226, 236, 244, 248, 251,
252, 256, 257, 266, 280, 289, 289, 290, 291, 292,
293, 294, 295, 297, 298, 300
enzyme inhibitors, 118, 119
EPA, ix, 87, 88, 95, 96, 104, 113
epidemic, 106
epidermis, 17
epididymis, 292
epilepsy, 95, 254, 275, 277
epithelial cells, 60, 252
erosion, 102
erythrocyte membranes, 192
ESCs, 252
estrogen, xi, 98, 222, 223, 247, 248, 256, 289, 290,
291, 292, 293, 296, 298, 299, 300
ethanol, 294
etiology, 22
eukaryotic, 24, 30, 39, 47, 88, 93, 151, 227, 266
eukaryotic cell, 151, 227
evidence, ix, x, 3, 5, 6, 7, 18, 27, 32, 33, 66, 69, 79,
85, 89, 91, 95, 99, 101, 102, 114, 123, 125, 128,
148, 154, 159, 160, 161, 162, 169, 170, 191, 194,
216, 232, 243, 251, 252, 253, 255, 256, 257, 258,
259, 265, 275
evolution, 193, 227
excitability, 168
excitation, 133
exclusion, 152
exercise performance, 118, 133, 145, 221, 246, 279
exocytosis, 282
exons, 54, 55, 58
experimental condition, 262
exposure, 140, 144, 187, 210, 213, 215, 259, 262
extensor, 3, 5, 16, 83, 90, 174, 185, 188, 208, 212,
215, 221
extensor digitorum, 3, 5, 16, 83, 174, 185, 188, 208,
212, 215, 221
extracellular matrix, 57, 60, 69, 74, 75, 77, 119, 177
extraocular muscles, 4
eye movement, 4

308

Index

F
facial expression, 4
facial muscles, 4
families, 28, 29, 148, 200, 231
family members, 18, 29, 72, 256
fasting, 29, 94, 101, 110, 156, 159, 160, 214, 223,
238, 247
fasting glucose, 160
fat, 22, 89, 92, 94, 106, 113, 130, 132, 139, 141, 159,
160, 169, 214, 228, 232, 237, 238, 239, 242, 245
fatty acids, 89, 95, 106, 112, 113, 132, 213, 215, 245
female rat, 296
fertility, 86
fiber bundles, 133
fibroblast growth factor, 82
fibroblasts, 14, 60, 64, 77, 80, 81, 82, 126, 260, 268
fibrosis, 102, 177, 185, 194
fibula, 150
filament, ix, 28, 45, 171, 173, 174, 175, 177, 179,
181, 182, 184, 185, 191, 192, 195
fish, 95
fish oil, 95
flank, 174
flexibility, 92
flight, 139, 156, 187, 203
fluid, 63
follicle, 114
food, 99, 136, 142, 194, 211, 226, 234
Food and Drug Administration, 102
food intake, 99, 211, 226, 234
force, vii, 3, 100, 121, 132, 133, 143, 146, 148, 154,
155, 166, 172, 174, 175, 176, 179, 184, 185, 186,
187, 188, 191
Ford, 240
formation, 9, 15, 24, 29, 48, 60, 75, 77, 125, 133,
136, 174, 175, 176, 177, 179, 181, 186, 196, 246,
252, 253, 255, 259, 263, 268, 278, 280, 285, 298,
299
fractures, 121
fragments, 9, 58, 59, 64, 76, 179, 185
free radicals, 131, 254
freezing, 256
fruits, 213
functional food, 136
fusion, 9, 67, 69, 194, 252, 253, 255, 259, 260, 261,
262, 263, 265, 272, 280, 281, 282, 283

G
gait, 90
gamma-tocopherol, 142

gastric mucosa, 293

gastrocnemius, 121, 128, 130, 131, 150, 187, 203,
208, 212, 215, 218, 294
GDP, 24, 26, 216
GEF, 218, 222, 243
gender differences, 295
gene expression, 6, 16, 17, 18, 26, 28, 41, 43, 44, 54,
67, 72, 77, 79, 85, 94, 108, 119, 125, 126, 135,
138, 164, 183, 194, 198, 199, 200, 237, 243, 244,
248, 249, 252, 265, 268, 270, 282, 284, 286, 291,
299
gene promoter, 72, 218, 243
gene silencing, 269, 286
gene therapy, 270
gene transfer, 69
genes, 9, 26, 29, 41, 76, 77, 82, 91, 145, 146, 177,
179, 181, 183, 184, 192, 195, 200, 206, 222, 223,
237, 248, 253, 254, 256, 257, 265, 266, 268, 269,
272, 279, 281, 286, 291, 292, 300
genetic background, 97
genetic factors, 147
genetic mutations, 12
genetic programs, 15
genetics, 168, 200, 227
genome, 254, 259, 271, 274, 286
genotype, 194, 254, 255, 277
genus, 213
germ line, 7, 17
gestation, 73
gland, 86, 293
glucocorticoid, 26, 40, 45, 195
glucocorticoid receptor, 40
glucose tolerance, 89, 100, 138, 169, 217, 224, 238
GLUT, 241, 242, 243, 244
GLUT4, x, 131, 132, 180, 181, 197, 205, 206, 216,
217, 219, 222, 228, 240, 241, 242, 243, 244, 249
glutamate, 58, 268
glutamine, 93
glutathione, 132, 143, 145
glycine, 58, 93, 111
glycogen, 66, 83, 128, 131, 207, 214, 215, 217, 227,
228, 236, 239, 242, 243
glycolysis, x, 251, 252, 258, 260, 262, 272
glycoproteins, 260
glycosylated hemoglobin, 159
glycosylation, 57, 74
gravity, 172, 187
grouping, 89
growth arrest, 60, 260
growth factor, viii, 12, 23, 25, 26, 27, 31, 32, 35, 38,
43, 50, 53, 54, 55, 60, 61, 66, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 88, 91,

309

Index
108, 111, 112, 115, 116, 118, 128, 241, 267, 295,
301
growth hormone, 54, 72, 73, 78, 79, 80, 88, 111, 116,
117
GTPases, 24, 26, 40, 216
guanine, 24, 259

H
hair, 18, 271
hair follicle, 271
half-life, 153, 154, 264
harmony, 70
health, vii, ix, 21, 22, 30, 43, 91, 95, 102, 109, 110,
116, 122, 123, 140, 145, 147, 162, 237
health status, 30
heart failure, 101, 108
heat shock protein, vii, 148, 163, 164, 165, 166, 167,
168, 169, 170, 189, 202, 203, 231
hematopoietic stem cells, 7, 18
heme, 170, 257
heme oxygenase, 170
hepatocellular carcinoma, 226
hepatocytes, 83, 210, 212
herbal medicine, 94, 213
heterogeneity, vii, 1, 4, 5, 6, 12, 13, 16, 17, 103, 206,
279
high fat, 237
hip fractures, 120
hippocampus, 168
histidine, 132
histological examination, 102
histone(s), 18, 29, 218, 223, 243, 253, 269, 283
histone deacetylase, 29, 218, 223, 243
history, 6, 17
HIV, 100, 117
homeostasis, viii, x, xi, 6, 7, 12, 18, 37, 87, 88, 93,
99, 102, 110, 124, 130, 137, 158, 170, 217, 224,
227, 246, 249, 251, 252, 278, 289, 290, 297
hormone(s), xi, 22, 79, 84, 98, 114, 115, 116, 139,
149, 206, 209, 210, 211, 240, 289, 290, 291, 292,
293, 295, 296, 297, 298, 299
hormone levels, 297
host, 2, 3, 5, 9
House, 226
HPC, 258
hTERT, 265, 283
human body, vii, 21, 22, 88
human health, 88
human milk, 75
human skin, 77
human subjects, 35, 46, 103, 110, 132
Hunter, 44, 84

hybrid, 30, 259

hybridization, 257
hydrogen, 127, 145, 210
hydrogen peroxide, 127, 145, 210
hyperactivity, 129, 246
hyperglycemia, 142, 147
hyperinsulinemia, 46, 96
hyperlipidemia, ix, 94, 147, 160
hyperlipidemia-induced insulin, ix, 147, 160
hyperplasia, 100
hypertension, x, 89, 119, 147, 205
hyperthermia, 154, 164
hypokinesia, 204
hypothalamus, 99, 206, 211, 226, 234, 240
hypothesis, 7, 16, 98, 152, 206, 233, 256, 265, 267
hypoxia, 26, 40, 41, 60, 211, 228, 233, 264, 274

I
ideal, 212
identification, 14, 42, 136, 198, 227
identity, 272, 292
IFN, 33, 49
images, 8, 294
immobilization, ix, 29, 140, 147, 148, 154, 156, 157,
188, 204
immune response, 285
immunofluorescence, 294
immunoglobulin, 60, 61, 196, 284
immunoreactivity, 294
improvements, 91, 95, 101, 108, 111, 135
in vitro, 9, 10, 11, 12, 16, 17, 27, 42, 62, 66, 68, 71,
79, 84, 85, 95, 104, 127, 132, 196, 212, 213, 216,
218, 227, 244, 257, 258, 259, 261, 266, 281, 293
in vivo, 2, 3, 5, 8, 9, 10, 12, 17, 23, 27, 35, 45, 46,
69, 71, 74, 79, 82, 85, 94, 114, 129, 141, 157,
161, 191, 196, 198, 212, 213, 214, 216, 217, 218,
219, 221, 236, 237, 238, 240, 244, 246, 255, 257,
258, 281, 284, 291, 292, 293, 300
incidence, 17, 99, 103, 169
increased workload, 182
independence, viii, 22, 87, 190
indirect effect, 236
indirect measure, 101
individual character, 136
individual characteristics, 136
individuals, 30, 31, 33, 34, 70, 94, 128, 142
inducer, 49, 166, 167, 269, 286
induction, 10, 44, 83, 99, 101, 129, 138, 156, 158,
160, 161, 162, 177, 180, 181, 183, 223, 256, 257,
263, 264
ineffectiveness, 99
infancy, 73

310

Index

infection, 210
inflammation, ix, 33, 34, 35, 50, 72, 91, 109, 123,
125, 126, 127, 128, 135, 148, 158, 159, 160, 162,
170, 211, 233, 270
inflammatory bowel disease, 95
inflammatory disease, 22, 33
inflammatory mediators, 126, 127
inflammatory responses, 159
ingestion, 32, 46, 48, 49, 93, 110, 111, 136, 214, 239
inhibitor, ix, 23, 26, 31, 36, 38, 60, 61, 81, 87, 88,
100, 102, 104, 118, 121, 135, 141, 143, 157, 209,
214, 223, 253, 259, 266, 267, 268, 271, 272
initiation, viii, 24, 25, 31, 37, 39, 45, 47, 48, 53, 55,
71, 88, 93, 110, 131, 257, 262, 268, 282, 283, 284
injections, 100
injury(s), viii, 2, 3, 7, 8, 9, 14, 19, 35, 54, 67, 87,
106, 138, 148, 150, 154, 155, 156, 165, 200, 212,
234, 255, 256, 258, 270, 273, 276
insects, 207
insertion, 178, 258
insulin resistance, ix, x, 92, 99, 106, 109, 116, 126,
128, 130, 131, 132, 136, 138, 139, 141, 142, 145,
147, 159, 160, 161, 162, 163, 169, 170, 206, 215,
217, 224, 228, 235, 237, 238, 239, 240, 241, 243,
245, 248, 291
insulin sensitivity, 100, 111, 131, 132, 135, 148, 161,
162, 169, 213, 217, 221, 238, 246, 248, 270
insulin signaling, 85, 128, 136, 161, 169, 216, 217,
224, 238, 270
insulin system, x, 205
integration, 27, 177, 185
integrin(s), 2, 11, 14, 57, 58, 60, 63, 65, 66, 71, 74,
78, 81, 83, 177, 179, 260, 281
integrity, 148, 177, 194, 252, 270
interference, 92, 152
interferon, 33
intervention, 34, 103, 135, 169
intestine, 7
intoxication, 40
intracellular calcium, 133
intramuscular injection, 256
intravenously, 99
involution, 86
ion channels, 257
iron, 113
irradiation, 67, 68, 83
ischemia, 168
isoflavone, 213
isoforms, viii, 53, 54, 55, 56, 62, 67, 68, 69, 71, 73,
82, 148, 166, 173, 174, 175, 176, 180, 190, 191,
194, 196, 207, 208, 209, 212, 218, 221, 227, 228,
229, 257, 261, 279
isolation, 13, 185, 245

isoleucine, 93
isomerization, 144
isotope, 137
isozyme(s), 197, 292, 295, 300
issues, vii, 21, 94

J
Japan, 1, 21, 53, 87, 105, 123, 147, 171, 205, 225,
251, 270, 289
joints, 187, 188
Jordan, 204

K
keratinocyte, 252
kidney(s), xi, 62, 93, 111, 112, 207, 223, 289, 293,
294
kinase activity, 36, 63, 141, 207, 209, 233, 236, 240
kinetics, 46, 112, 189, 197
Krebs cycle, 226

L
labeling, 133, 137
lactic acid, 254
L-arginine, 111
lead, 24, 25, 26, 58, 70, 96, 100, 126, 128, 130, 131,
135, 162, 172, 181, 184, 185, 186, 207, 217, 221,
225, 254, 256, 258, 267, 268, 291, 295
lean body mass, 95, 97, 98, 99, 111
legs, 99, 117
leptin, 117, 211, 234
lesions, 184, 186, 200, 254
leucine, 26, 32, 40, 42, 46, 47, 48, 49, 89, 93, 111,
112, 215, 240, 266
levator, 5, 16, 292
life cycle, 162
life expectancy, 89
ligand, 58, 63, 75, 81, 99, 213, 261
light, 30, 133, 172, 175, 258
limb weakness, 255
linoleic acid, 131
lipid metabolism, 131, 132, 135, 136, 159, 206, 208,
237
lipid peroxidation, 125, 131, 132, 138, 141, 155
lipid peroxides, 126, 143
lipids, 96, 115, 124, 131, 255
lipolysis, 213, 226, 235
liver, viii, xi, 42, 53, 54, 55, 61, 62, 65, 73, 78, 79,
89, 93, 95, 121, 128, 190, 192, 207, 209, 212,

Index
215, 216, 223, 227, 238, 239, 242, 257, 289, 290,
293, 294, 298
localization, 57, 58, 66, 72, 78, 144, 174, 178, 180,
181, 182, 197, 247, 266, 281, 292
locomotor, 175
locus, 3, 6
longevity, 206, 227
long-term health, vii, 21, 22
Louisiana, 106
low-grade inflammation, 33, 128
Luo, 165, 198, 232, 281
luteinizing hormone, 114
lymphocytes, 261
lymphoid, 78, 158
lysine, 125, 131, 132, 137, 142

M
machinery, 23, 29, 36, 37, 186, 257
Mackintosh, 241
macromolecules, 96
macrophages, 95, 126, 128, 168, 215, 240
magnetic resonance, 93
magnetic resonance imaging, 93
magnitude, 31, 187
majority, 2, 3, 5, 7, 8, 9, 11, 14, 99, 103
malnutrition, vii, 21, 22
mammalian cells, 41, 153, 248, 254, 258, 278
mammalian tissues, 237
mammals, 23, 172, 174, 207, 260, 290
man, 105, 138, 231
manipulation, 100
mapping, 76, 78, 192, 193
mass, vii, viii, ix, x, 5, 21, 22, 23, 33, 35, 47, 69, 85,
87, 88, 89, 91, 92, 94, 97, 98, 99, 105, 106, 111,
115, 117, 118, 123, 130, 137, 141, 156, 246, 251,
270, 290, 291
mass spectrometry, 137
masseter, 4, 5, 16
matrix, 77, 81, 155, 191, 219, 220, 221, 254, 260
matter, 16, 106
measurement(s), 99, 264
meat, 93
mechanical stress, ix, 32, 127, 171, 172, 176, 177,
179, 181, 183, 184, 185, 186, 187, 188, 189
mechano growth factor, viii, 53, 79, 85
mediation, 174
medical, 292
medicine, 232
MEK, 24, 25
mellitus, 100, 206, 212, 213, 224, 225
melon, 213, 238
membranes, 148, 180, 194, 245

311

menadione, 130, 141

menopause, 98, 114, 291, 299
mesangial cells, 202
mesoderm, 4, 9
messages, 152
messenger ribonucleic acid, 79
messenger RNA, 73, 165, 286
meta-analysis, 121, 291, 299
metabolic, v, 128, 130, 147, 164, 205, 224, 225, 233,
238, 247, 248, 257, 273
metabolic changes, 36
metabolic disorder(s), x, 125, 147, 205, 206, 224
metabolic dysfunction, ix, 123, 132
metabolic syndrome, 206, 232, 236
metabolites, 98
metabolized, 23, 296
metformin, 159, 169, 212, 213, 235, 236
MHC, 46, 108
microcirculation, 109
microdialysis, 46
microgravity, 28, 29, 187, 203
microRNA, 152, 165, 286
microscope, 276
microscopy, 172
migration, 60, 63, 64, 65, 66, 74, 77, 78, 81, 83, 177,
260, 281
military, 90
Ministry of Education, 105, 225, 270
mitochondria, x, 91, 103, 123, 128, 130, 131, 135,
137, 142, 158, 162, 167, 206, 219, 225, 234, 237,
246, 251, 253, 254, 255, 256, 257, 258, 260, 262,
264, 269, 270, 272, 274, 277, 279, 280
mitochondrial damage, 142
mitochondrial DNA, x, 91, 96, 108, 155, 156, 251,
253, 257, 272, 274, 275, 276, 277, 279, 280
mitochondrial electron, ix, 123, 271
mitogen, 25, 39, 63, 83, 91, 126, 137
mitosis, 2, 60
MMP, 69
models, ix, 27, 29, 91, 94, 98, 100, 129, 147, 156,
158, 160, 164, 174, 178, 184, 217
moderate exercise, ix, 123, 134, 135, 147
modifications, 71, 96, 125, 126, 133, 144
molecular biology, vii, 165, 276
molecular mass, 148
molecular mediators, viii, 87
molecular weight, 56, 99, 148
molecules, ix, 11, 26, 60, 103, 126, 127, 135, 147,
171, 172, 175, 185, 189, 206, 209, 211, 224, 260,
262, 269, 272, 280, 281
monoclonal antibody, 69, 137, 261
monocyte chemoattractant protein, 128
morbidity, vii, 22, 34, 89, 106

312

Index

morphogenesis, 15
morphology, vii, x, 184, 251
mortality, vii, 22, 34, 89, 106
Moses, 112
motif, 28, 56, 58, 63, 71, 183, 269
mRNA(s), 4, 24, 26, 39, 44, 47, 54, 55, 56, 60, 62,
68, 69, 70, 71, 72, 73, 78, 79, 84, 85, 91, 94, 101,
107, 112, 116, 119, 151, 152, 153, 159, 160, 170,
183, 196, 212, 218, 221, 226, 232, 235, 246, 259,
264, 269, 282, 283, 292, 293, 294, 295
mtDNA, 253, 254, 256, 257, 259, 261, 262, 263,
267, 268, 269, 274, 275, 277, 279, 280
multinucleated, x, 8, 9, 11, 251, 252, 253, 260, 261,
265, 272, 281
multiple factors, 22
multiple myeloma, 36
multipotent, 7
multi-protein complexes, 23
muscle contraction, x, 88, 91, 127, 133, 149, 155,
159, 174, 176, 180, 185, 205, 206, 209, 210, 291
muscle performance, 93, 299
muscle relaxation, 133
muscle stem cells, vii, 1, 2, 15, 118
muscle strength, x, 31, 33, 50, 88, 90, 91, 92, 97, 99,
101, 102, 110, 115, 116, 118, 119, 158, 235, 289,
290, 291, 298, 299
muscular dystrophy, ix, 3, 12, 13, 15, 87, 88, 90, 92,
102, 109, 147, 158, 168, 179, 196, 197, 201, 281
musculoskeletal, 107, 120
musculoskeletal system, 120
mutagenesis, 58, 75, 81, 300
mutant, 65, 66, 81, 182, 214, 254, 268, 272, 280
mutation(s), 4, 6, 7, 12, 15, 58, 64, 70, 76, 86, 101,
155, 156, 167, 175, 184, 185, 196, 201, 202, 223,
228, 229, 245, 253, 254, 255, 273, 274, 275, 276,
277
myoblasts, x, 1, 2, 6, 9, 10, 17, 60, 65, 66, 68, 69, 70,
84, 86, 140, 163, 167, 168, 178, 215, 240, 251,
252, 253, 256, 257, 259, 261, 262, 263, 264, 266,
267, 268, 269, 272, 273, 274, 282, 283, 284, 285,
286
myocardial infarction, 199
myocardial ischemia, 234
myocardium, 175
myoclonus, 254
myocyte, 84, 156, 181, 190, 218, 222, 243, 244, 266,
284
myofibres, vii, 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12
myofibril, ix, 171, 172, 175, 176, 185, 192, 196, 201
myogenesis, x, 4, 7, 9, 13, 15, 77, 82, 83, 84, 86,
106, 115, 177, 183, 199, 200, 251, 252, 253, 255,
256, 257, 259, 260, 262, 263, 264, 266, 267, 268,

269, 270, 272, 273, 274, 277, 278, 279, 280, 281,
282, 283, 284, 285, 286
myogenic progeny, vii, 1, 7, 9
myoglobin, 182
myopathy, 23, 36, 92, 109, 120, 145, 174, 191, 197,
201, 254, 255, 270, 275, 277, 279, 286
myosin, 2, 30, 45, 46, 79, 82, 102, 128, 129, 133,
139, 144, 149, 166, 174, 175, 176, 177, 188, 191,
192, 193, 195, 198, 203, 208, 226, 227, 228, 242,
260, 263, 265, 272, 283
myotubes, x, 2, 3, 7, 8, 9, 10, 11, 38, 42, 60, 66, 67,
68, 69, 95, 102, 128, 129, 130, 137, 141, 168,
195, 217, 226, 229, 231, 238, 242, 251, 252, 253,
254, 256, 257, 260, 261, 262, 264, 278, 282, 292

N
NAD, 222, 223, 247
NADH, 144, 263
National Health and Nutrition Examination Survey,
109
natural compound, 107, 112
necrosis, 28, 44, 50, 89, 113, 158, 201, 215, 239,
256, 269, 286
negative consequences, 147
negative effects, 135, 262
nerve, 93, 181, 182, 212, 223
nervous system, 212
neuroblastoma, 271
neuronal apoptosis, 93
neurons, 168, 252, 298
neuropathy, 235
neuropeptides, 206
neurotoxicity, 271
neurotrophic factors, 235
neutral, 17, 165
neutrophils, 128
nicotinamide, 222, 223
nitric oxide, 133, 143, 146, 231, 233, 277
nitric oxide synthase, 143, 231, 233, 277
nitrogen, 93, 125, 126, 213, 230, 231, 236
nonsense mutation, 194
normal aging, 121, 275
Norway, 166
NRF, 246, 256, 257, 278
Nrf2, 135, 145
nuclear genome, 253
nuclear magnetic resonance (NMR), 58, 76
nuclear receptors, 248, 252, 255, 256
nucleation, 177
nuclei, 67, 177, 182, 185, 260
nucleotides, 131, 208, 210, 215, 224, 260

Index
nucleus, 9, 58, 126, 127, 130, 135, 137, 179, 181,
183, 218, 266, 268, 279, 285
null, 61, 70, 86, 100, 175, 177, 182, 188, 192, 204,
219, 245, 246, 261
nursing, 90, 103
nursing home, 90, 103
nutraceutical, 114
nutrient(s), viii, x, 21, 23, 29, 30, 32, 34, 36, 40, 135,
205, 206, 224, 226, 255
nutrition, 97

O
obesity, ix, x, 50, 89, 91, 106, 116, 128, 139, 147,
159, 160, 163, 205, 206, 215, 224, 225, 231, 235,
240, 291
oil, 95
old age, 89, 141
oligomerization, 202
omega-3, 95, 107, 113
opportunities, 38
orbit, 96
organ(s), vii, xi, 21, 22, 72, 86, 124, 174, 211, 213,
231, 289, 297
organelle(s), 133, 155, 156, 194, 248, 253, 255, 269,
273
osteomalacia, 102
osteoporosis, 102, 103
ovaries, 291, 296
overlap, 23, 273
ovum, 255
oxidation, x, 94, 125, 126, 133, 136, 139, 140, 142,
143, 144, 155, 205, 206, 219, 220, 221, 223, 224,
225, 230, 232, 234, 237, 241, 245, 246, 247, 248,
249, 256
oxidative damage, 92, 96, 108, 124, 155, 164, 167,
254, 274, 275
oxidative stress, ix, 4, 22, 89, 93, 97, 103, 113, 114,
123, 124, 125, 126, 127, 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, 142,
144, 145, 148, 150, 151, 154, 160, 163, 165, 166,
169, 202, 230, 254, 285
oxygen, ix, 91, 96, 108, 123, 124, 130, 131, 145,
169, 211, 221, 226, 246, 257, 264, 279
oxygen consumption, ix, 123, 124, 257

P
pain, 127
pancreas, 93, 161
pancreatic cancer, 95
parallel, 24, 177, 191, 261

313

parathyroid, 102
parathyroid hormone, 102
patella, 107
pathogenesis, 38, 111, 124, 131, 133, 136, 159, 179
pathology, 4, 33, 158, 159, 185, 202
pathophysiological, 234
pathophysiological roles, 234
pathophysiology, 82, 107, 293
pathways, viii, x, 16, 23, 30, 37, 43, 53, 64, 66, 82,
91, 93, 97, 128, 135, 162, 172, 177, 190, 205,
230, 231, 237, 267, 269, 277, 284, 285
PDZ domains, 180
pedigree, 185, 201
peptidase, 102
peptide(s), viii, 53, 55, 56, 66, 69, 72, 73, 79, 85, 98,
99, 102, 117, 144, 268
percentage of fat, 89
perinatal, 39, 273, 282
peroxidation, 160, 297
peroxide, 130, 195
peroxisome, ix, x, 87, 88, 91, 135, 205, 212, 213,
218, 222, 237, 247, 248, 256, 278
peroxynitrite, 137, 140, 144, 233
phagocyte, 127, 128, 135
pharmaceutical(s), x, 205, 224
pharmacological treatment, 104
phenotype(s), 2, 3, 4, 5, 85, 122, 135, 145, 176, 177,
182, 200, 234, 244, 255, 258, 265, 270, 273, 274,
275, 282, 286
phosphate, 63, 80, 111, 151, 217, 262
phosphocreatine, 93
phosphorus, 102
physical activity, viii, 21, 22, 30, 35, 50, 87, 89, 90,
91, 157
physical exercise, 124, 145
physiological, 116, 234
physiological mechanisms, 23, 114, 300
physiology, 116, 169, 191, 253, 276, 291, 293, 298
pioglitazone, 212, 237
pituitary gland, 98
placebo, 97, 99, 113, 119
plants, 94, 207
plaque, 185
plasma membrane, 11, 25, 216, 217, 219, 220, 245,
254, 261, 267
plasmid, 157
plasticity, x, xi, 205, 230, 277, 289, 290
playing, 34
point mutation, 58, 254, 255, 275, 277
polarity, 17, 206, 226, 271
polymerase, 254
polymerase chain reaction (PCR), 6, 153, 254, 257,
294

314

Index

polymerization, 175
polymorphism, 176, 194
polypeptide(s), 56, 148, 162, 253, 268
polyunsaturated fat, 95
polyunsaturated fatty acids, 95
pools, 6
population, vii, 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16,
18, 33, 89, 176, 194, 255, 258
positive correlation, 5, 6, 132
positive feedback, 222, 223
positive relationship, 98
postnatal muscle, vii, 1, 2, 9
post-transcriptional regulation, 56, 152
potassium, 143
PRC, 256
precursor cells, 16, 22, 67, 69, 85, 177, 281
prediction models, 35
pregnancy, 300
preservation, 92, 115, 155, 156, 275
preservative, 299
prevention, 95, 101, 107, 110, 113, 121, 154, 155,
156, 159, 162, 206, 232, 290, 292
principles, 237
probe, 81
professionals, 106
progenitor cells, 4, 15, 17, 259, 280
pro-inflammatory, 22, 29, 33, 98, 162
proliferation, viii, 2, 3, 4, 5, 6, 7, 9, 17, 18, 25, 37,
50, 53, 54, 60, 62, 64, 65, 66, 67, 69, 70, 71, 73,
77, 78, 86, 89, 91, 255, 258, 260, 262, 263, 269,
271, 272, 279, 286, 291
proline, 23, 36, 38, 268
promoter, 29, 33, 55, 127, 151, 200, 218, 222, 223,
243, 244, 247, 257, 265, 266, 269, 279, 283, 291
propagation, 281
prostate cancer, 97
prostate specific antigen, 58, 75, 81
prosurvival, ix, 147
proteasome, ix, 27, 29, 30, 33, 34, 43, 51, 87, 101,
102, 107, 119, 120, 125, 128, 129, 137, 139, 140,
141, 147, 148, 153, 156, 157, 158, 162, 165, 178,
195, 203, 268
protection, 92, 103, 104, 148, 149, 154, 155, 158,
162, 163, 168, 186, 235
protective role, 154
protein components, 30, 172, 191
protein family, 200, 222
protein folding, 148, 158, 159
protein kinase C, 126, 137, 181, 197
protein kinases, 208, 209, 214, 216
protein oxidation, 137
protein structure, 81, 148
proteinase, 60, 61, 74, 266

protein-protein interactions, 257

proteoglycans, 61
proteolysis, 28, 43, 45, 46, 57, 58, 70, 86, 117, 119,
127, 128, 130, 137, 139, 140, 156, 167, 177, 178,
179, 186, 226, 292
proteolytic enzyme, 196
proteomics, 131
proto-oncogene, 264, 285
PTEN, 66, 83, 137
ptosis, 255
puberty, 98
public health, 89, 109
purification, 192

Q
quadriceps, 107, 208, 218, 295
quality of life, vii, viii, 21, 22, 87, 95, 115, 290
quercetin, 114, 213, 237

R
Rab, 216, 217, 219, 241, 245
race, 18
radiation, 83, 161
radicals, 96, 131, 210
ramp, 62, 68
reactions, 124, 156
reactive oxygen, vii, x, 96, 121, 124, 137, 140, 141,
142, 147, 165, 205, 206, 230, 231, 252, 259, 267,
271, 276
reactivity, 96, 125
reading, 55
reagents, 143
reality, 235
receptors, viii, 2, 9, 33, 53, 54, 56, 63, 66, 69, 71, 80,
86, 95, 98, 99, 116, 211, 212, 215, 234, 293, 299,
300
recognition, 260
recommendations, 107, 110
reconstruction, 191
recovery, 2, 154, 155, 156, 163, 166, 173, 183, 202,
204, 235, 256, 259
recovery process, 183
recruiting, 67, 269
red wine, 213
redistribution, 197, 245
redundancy, 193, 200
regenerate, 4, 263, 266
regeneration, vii, 1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 17, 23, 69, 72, 82, 85, 106, 139, 155, 158,

Index
159, 177, 188, 252, 253, 254, 255, 256, 258, 270,
273, 276, 277, 279
regenerative capacity, 2, 5, 89
regrowth, 154, 164
rehabilitation, 112, 114, 154
relaxation, 168, 291
relevance, 97, 232, 286
remodelling, 201
renal cell carcinoma, 279
repair, vii, 1, 2, 3, 7, 9, 10, 11, 14, 18, 67, 73, 77, 99,
148, 149, 189, 211, 254, 263, 266, 276
repetitions, 148, 295
replication, 7, 12, 255, 257, 259, 262, 282
repression, 27, 265, 282
repressor, 24, 25, 121, 135, 141, 218, 268
requirements, 105
researchers, 89, 99, 100
residues, 24, 25, 27, 56, 57, 58, 59, 64, 75, 125, 126,
132, 133, 207
resolution, 201
respiration, x, 205, 224, 236, 247, 248, 251, 253,
256, 257, 258, 259, 272, 277
respiratory rate, 256
responsiveness, 22
restoration, 16, 106, 162, 168, 248, 255, 262, 277,
279
restructuring, 271
resveratrol, 97, 114, 213, 223, 238, 248
retardation, 54, 61
reticulum, 130, 136, 137, 140, 143, 144, 155, 158,
161, 170, 209, 255, 256
retina, 111
retinoblastoma, 84
retinopathy, 255
reverse transcriptase, 265, 283
ribosomal RNA, 35, 253
ribosome, 37, 258
risk(s), vii, viii, 12, 22, 34, 87, 89, 97, 102, 120, 121,
291, 296
RNA(s), 253, 258, 259, 266, 275, 279, 280
rodents, viii, 28, 31, 53, 68, 91, 101, 155, 185, 186,
208, 214, 219, 224, 242, 295
root, 163
rosiglitazone, 212, 213, 236, 237, 249
roundworms, 207
rowing, 70
runoff, 265

S
salmon, 95
satellite cells, vii, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 62, 67, 83, 84, 89, 91,

315

94, 100, 105, 115, 128, 157, 253, 255, 258, 259,
266, 276, 279, 280
saturated fat, 240
saturated fatty acids, 240
scavengers, 133
schema, 24, 161
science, 232
sclerosis, 25, 38, 39, 88
secrete, 56
secretion, 14, 32, 62, 69, 80, 98, 99, 210, 213, 291,
296
sedentary lifestyle, ix, 123, 128
segregation, 7, 17
selectivity, 239
seminal vesicle, 292
senescence, 35, 157
sensing, ix, 39, 171, 172, 206, 210, 227, 229
sensitivity, 126, 162, 174, 175, 217, 242
sensorineural hearing loss, 255
sensors, 227
sepsis, 43, 49, 101
sequencing, 111
serine, 23, 27, 58, 60, 61, 64, 65, 76, 77, 160, 165,
169, 180, 181, 207, 216, 224, 242, 266, 268
serum, 61, 62, 70, 77, 80, 85, 89, 95, 104, 106, 114,
115, 116, 118, 158, 159, 198, 269, 278, 286, 291,
296, 301
sex, xi, 22, 80, 98, 115, 116, 289, 290, 291, 292, 293,
295, 296, 297, 298, 300, 301
sex differences, 296
sex hormones, 98, 290, 301
sex steroid, xi, 115, 116, 289, 290, 291, 293, 295,
296, 297, 298, 300, 301
sex steroid hormones, xi, 289, 290, 291, 293, 295,
296, 297, 298
shock, ix, 136, 147, 151, 156, 162, 163, 164, 165,
167, 169, 170, 202, 210
showing, 3, 4, 6, 30, 33, 95, 184, 293
side chain, 126
side effects, 97, 99, 103
signal peptide, 56
signal transduction, vii, x, 37, 125, 126, 132, 134,
136, 158, 161, 162, 168, 171, 176, 179, 181, 182,
186, 240
signaling pathway, viii, 21, 29, 31, 33, 42, 64, 66, 71,
72, 77, 82, 84, 95, 101, 118, 125, 128, 130, 132,
144, 148, 172, 190, 194, 230, 236, 238, 269, 285
signalling, 4, 9, 10, 18, 35, 36, 37, 38, 39, 41, 48,
138, 168, 199, 233, 237, 285
signals, 23, 36, 42, 49, 55, 62, 68, 79, 104, 125, 172,
180, 186, 189, 198, 213, 215, 216, 226, 227, 238,
242, 261
signs, 95, 138

316

Index

single chain, 56
siRNA, 9, 26
skeleton, 116
skin, 7, 17, 213
sleep apnea, 97
smooth muscle, 64, 65, 80, 81, 203
smooth muscle cells, 64, 80, 81
soleus, 5, 16, 41, 85, 119, 133, 148, 149, 150, 154,
156, 157, 164, 166, 167, 168, 173, 174, 177, 185,
186, 187, 188, 189, 202, 203, 204, 208, 209, 212,
218, 230, 233, 245, 246
solution, 32
somatic cell, 280
specialization, 164
species, vii, ix, x, 88, 96, 121, 123, 124, 125, 126,
137, 141, 142, 147, 165, 169, 193, 205, 206, 208,
213, 214, 230, 231, 236, 252, 259, 267, 271, 276,
293, 295
spin, 132
spinal cord, 158, 168, 185, 187, 202
spinal cord injury, 168
spindle, 204
Spring, 13, 191, 276
stability, 59, 88, 264, 269, 282, 286
stabilization, 81, 91, 95, 185, 192, 264
starch, 227
starvation, 28, 44, 115, 139
state(s), 2, 4, 7, 10, 12, 19, 26, 27, 50, 62, 68, 69, 89,
132, 133, 156, 182, 232, 252, 254, 256, 257, 258,
265, 267, 279
stem cells, vii, 1, 4, 5, 6, 7, 11, 16, 17, 18, 67, 72,
100, 139, 252, 271
steroidogenic, xi, 289, 290, 292, 293, 294, 295, 297,
298
steroids, 291, 295
stimulation, 2, 9, 32, 41, 46, 48, 64, 66, 77, 99, 131,
143, 148, 149, 153, 154, 155, 162, 178, 180, 182,
183, 184, 198, 223, 229, 239, 290, 295, 296
stimulus, 3, 26, 31, 172
stomach, 99, 117
storage, 131
strength training, 67, 91, 109, 112, 229
stress response, ix, 26, 155, 162, 171, 179, 183, 231
stressors, 148, 151, 154, 162
stretching, 179, 187, 188
stroke, 101, 120, 206, 254
structural gene, 177
structural protein, 148
structure, ix, 5, 16, 45, 56, 57, 58, 59, 74, 101, 118,
131, 154, 166, 171, 172, 173, 175, 176, 186, 191,
193, 195, 197, 207, 226, 276
style, 22, 34, 124
substitution, 58, 185, 300

substrate(s), 23, 25, 29, 30, 36, 37, 38, 102, 105, 132,
140, 160, 178, 181, 206, 212, 216, 217, 225, 241,
242, 245, 261, 268, 292, 300
sucrose, 207, 216
sulfate, 61, 115, 290
Sun, 38, 39, 49, 72, 141, 144, 169, 202, 242, 286
supplementation, ix, 32, 34, 48, 87, 93, 94, 95, 96,
97, 98, 99, 102, 103, 107, 110, 111, 112, 113,
114, 115, 121, 137, 142, 240
suppression, 25, 41, 49, 135, 155, 158, 197, 220,
239, 282
survival, viii, 36, 38, 41, 44, 53, 63, 65, 66, 72, 86,
148, 152, 179, 237
susceptibility, 22, 148, 155
Sweden, 106
sympathetic nervous system, 211
symptoms, ix, 87, 97, 98, 224
synergistic effect, 93, 157
synthesis, 23, 25, 27, 30, 31, 32, 37, 46, 47, 48, 57,
60, 64, 65, 66, 83, 91, 93, 95, 96, 104, 110, 128,
131, 132, 156, 165, 206, 213, 215, 217, 220, 253,
255, 258, 259, 262, 276, 280, 290, 291, 295, 296,
298
synthesized activators, x, 205, 206

T
T cell(s), 117, 181, 261
T cell receptor, 261
tamoxifen, 15
target, x, xi, 9, 10, 21, 23, 30, 36, 37, 38, 39, 40, 42,
43, 45, 47, 54, 66, 68, 82, 88, 91, 98, 107, 118,
125, 127, 131, 133, 136, 144, 152, 159, 161, 178,
206, 235, 240, 264, 266, 268, 269, 282, 285, 289,
290, 291, 299
techniques, 182
technology, 259, 260
telomere, 265
temperature, 149, 153, 163, 165, 210, 231, 255
tendon, 101, 107, 179
tensile strength, 194
tension, 116, 173, 175, 176, 177, 187, 188, 190, 204
testis, xi, 289, 290, 293, 294, 296
testosterone, xi, 40, 54, 79, 97, 98, 99, 104, 114, 115,
116, 289, 290, 291, 292, 293, 294, 295, 298, 300
therapeutic approaches, 90
therapeutic effects, 111
therapeutic interventions, viii, 21, 32
therapeutic targets, viii, 87, 159
therapeutics, 206
therapy, 43, 97, 99, 158, 159, 160, 161, 163, 169,
277, 291, 298, 299
thermal treatment, 159

317

Index
thiazolidinediones, 212, 213
threonine, 23, 27, 42, 180, 181, 207, 216, 224, 227,
266, 268
thyroglobulin, 57, 74
thyroid, 149
tibialis anterior, 5, 100, 119, 149, 187, 188, 203, 218
tissue, vii, 5, 6, 7, 11, 54, 56, 60, 61, 72, 73, 77, 84,
88, 98, 99, 102, 113, 127, 130, 139, 159, 196,
207, 210, 211, 246, 252, 256, 274, 279, 291, 292,
293, 295
tissue homeostasis, 6, 7
titin, ix, 30, 45, 171, 173, 174, 176, 178, 179, 180,
183, 184, 188, 189, 190, 195, 197, 203
TNF-alpha, 44, 49, 121, 189
TNF-, 88, 95, 96, 98, 99, 103, 113, 128, 132, 215,
232, 240, 241
tonic, 172, 182, 187, 188
toxicity, 243
toxin, 3
trafficking, 217, 219, 245
transcription factors, x, 44, 66, 119, 125, 126, 135,
151, 167, 198, 205, 218, 221, 222, 223, 226, 244,
252, 255, 256, 257, 261, 265, 266, 284
transcripts, 28, 54, 55, 56, 70, 85, 108, 277
transduction, 77, 136, 192
transection, 158, 202
transfection, 159, 259
transfer RNA, 253
transformation, x, 80, 156, 167, 171, 181, 182, 198,
283
transforming growth factor (TGF), 9, 27, 60, 77, 83,
100, 117
translation, viii, 24, 25, 31, 37, 39, 45, 47, 48, 53, 55,
71, 110, 151, 152, 196, 253, 254, 257, 258, 259,
262, 263, 264, 283
translocation, x, 26, 96, 127, 131, 149, 154, 166,
178, 180, 181, 182, 183, 186, 199, 203, 205, 216,
217, 218, 219, 220, 235, 240, 241, 242, 245, 268
transmission, 177, 178, 179, 180, 188
transplantation, 2, 3, 5, 8, 9, 15, 18, 85, 168
transport, ix, 54, 56, 63, 91, 99, 123, 131, 133, 155,
167, 206, 217, 219, 220, 221, 223, 224, 225, 228,
230, 231, 233, 239, 241, 242, 243, 245, 249, 253,
256, 257, 271, 275, 276
trapezius, 83
trial, 90, 97, 107, 108, 109, 113, 116, 119, 120, 121,
237, 301
triggers, 156, 158, 175, 176, 186, 210
triglycerides, 106, 224, 248
triiodothyronine, 264
trypsin, 102

tumor, 14, 27, 29, 38, 39, 40, 41, 50, 60, 77, 78, 81,
88, 95, 114, 128, 137, 140, 158, 163, 202, 240,
241, 269
tumor cells, 81
tumor necrosis factor (TNF), 29, 33, 44, 49, 88, 95,
96, 98, 99, 103, 113, 121, 128, 129, 130, 132,
140, 189, 202, 215, 232, 240, 241, 269
tumorigenesis, 279
turnover, 17, 23, 43, 54, 56, 86, 120, 177, 178, 179,
208, 277
type 2 diabetes, x, 89, 131, 138, 142, 159, 163, 169,
170, 205, 206, 225, 229, 236, 237, 248, 249
tyrosine, 63, 75, 95, 112, 131, 133, 233

U
ubiquitin, ix, 23, 28, 29, 33, 34, 43, 44, 45, 51, 101,
119, 127, 128, 129, 139, 140, 141, 147, 148, 153,
156, 157, 158, 162, 165, 167, 171, 178, 189, 194,
195, 203, 226, 264, 292, 299, 300
ubiquitin-proteasome system, 23, 102, 140
ultrastructure, 190, 291
underlying mechanisms, x, 156, 251
uniform, 256
united, 95
United States, 95
untranslated regions, 152
ursolic acid, ix, 87, 88, 94, 96, 104, 112

V
valine, 47, 93
variations, x, 205
vascular cell adhesion molecule, 14
vascularization, 91
vastus lateralis, 89, 103, 139, 166, 218
VCAM, 2
vector, 209
vegetables, 213
velocity, 90, 143, 208, 228
ventilation, 194
versatility, 18
vertebrates, 4, 187, 281
vessels, 60, 78
vision, 235
vitamin C, 135, 145
vitamin D, 102, 104, 120, 121
vitamin D deficiency, 102
vitamin E, 132, 135, 143
volleyball, 194

318

Index

W
walking, 101
water, 93, 94, 162
weakness, viii, 11, 22, 34, 59, 87, 89, 102, 103, 104,
116, 135, 148, 155, 156, 179, 191, 299
web, 176, 193
weight control, 278
weight loss, 139
weight management, 105
western blot, 151, 153
wild type, 154, 174, 177, 180, 218, 219, 221, 223,
280
Wiskott-Aldrich syndrome, 261
withdrawal, 67, 80, 265, 283, 286

Wnt signaling, 77
workload, 172
worldwide, 102, 206

Y
yeast, 23, 30, 42, 207, 214, 227, 238, 245, 258, 279,
280
yolk, 192
young adults, 22, 30, 89, 91, 98

Z
zinc, 233, 265