The effects of obesity on skeletal muscle regeneration

Abstract

Obesity and metabolic disorders such as type 2 diabetes mellitus are accompanied by increased lipid deposition in adipose and non-adipose tissues including liver, pancreas, heart and skeletal muscle. Recent publications report impaired regenerative capacity of skeletal muscle following injury in obese mice. Although muscle regeneration has not been thoroughly studied in obese and type 2 diabetic humans and mechanisms leading to decreased muscle regeneration in obesity remain elusive, the initial findings point to the possibility that muscle satellite cell function is compromised under conditions of lipid overload. Elevated toxic lipid metabolites and increased pro-inflammatory cytokines as well as insulin and leptin resistance that occur in obese animals may contribute to decreased regenerative capacity of skeletal muscle. In addition, obesity-associated alterations in the metabolic state of skeletal muscle fibers and satellite cells may directly impair the potential for satellite cell-mediated repair. Here we discuss recent studies that expand our understanding of how obesity negatively impacts skeletal muscle maintenance and regeneration.

Full text

REVIEW ARTICLE
published: 17 December 2013
doi: 10.3389/fphys.2013.00371
The effects of obesity on skeletal muscle regeneration
Dmitry Akhmedov and Rebecca Berdeaux
*
Department of Integrative Biology and Pharmacology and Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston,
Houston, TX, USA
Edited by:
Carlos Hermano J. Pinheiro,
Reviewed by:
Zhaoyong Hu, Baylor College of
Medicine, USA
Thomas J. Hawke, McMaster
University, Canada
James G. Ryall, The University of
Melbourne, Australia
*Correspondence:
Rebecca Berdeaux, Department of
Integrative Biology and
Pharmacology and Graduate School
of Biomedical Sciences, University
of Texas Health Science Center at
Houston, 6431 Fannin St., MSE
R366, Houston, TX 77030, USA
e-mail:
rebecca.berdeaux@uth.tmc.edu
Obesity and metabolic disorders such as type 2 diabetes mellitus are accompanied by
increased lipid deposition in adipose and non-adipose tissues including liver, pancreas,
heart and skeletal muscle. Recent publications report impaired regenerative capacit y of
skeletal muscle following injury in obese mice. Although muscle regeneration has not
been thoroughly studied in obese and type 2 diabetic humans and mechanisms leading
to decreased muscle regeneration in obesity remain elusive, the initial findings point
to the possibility that muscle satellite cell function is compromised under conditions of
lipid overload. Elevated toxic lipid metabolites and increased pro-inflammatory cytokines
as well as insulin and leptin resistance that occur in obese animals may contribute
to decreased regenerative capacity of skeletal muscle. In addition, obesity-associated
alterations in the metabolic state of skeletal muscle fibers and satellite cells may directly
impair the potential for satellite cell-mediated repair. Here we discuss recent studies that
expand our understanding of how obesity negatively impacts skeletal muscle maintenance
and regeneration.
Keywords: obesity, type 2 diabetes, lipids, skeletal muscle, muscle regeneration, satellite cells, leptin, lipotoxicity
Obesity and associated disorders are quickly reaching a global
epidemic scale. Over 500 million people worldwide are over-
weight or obese (World Health Organization, 2013). Obesity
is highly associated with development of metabolic syndrome,
type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and
cardiovascular disorders (Kahn et al., 2006; Lavie et al., 2009;
Samuel and Shulman, 2012). In obese individuals, lipids exces-
sively accumulate in adipose tissues and ectopically accumulate
in non-adipose tissues including skeletal muscle (Unger et al.,
2010). Lipids in skeletal muscle have been extensively studied in
the context of insulin sensitivity. However, lipid overload in mus-
cle appears to affect not only insulin signaling, but also muscle
maintenance and regeneration. The underlying mechanisms are
not fully understood, but recent experimental data suggest that
multiple factors such as a ccumulation of toxic lipid metabolites
and low-grade inflammation result in impaired muscle regenera-
tion under conditions of obesity. The impact of obesity on skeletal
muscle maintenance and physiology has been addressed in rodent
models of obesity, including leptin-deficient Lep
ob/ob
mice (com-
monly termed “ob/ob”), leptin receptor-deficient Lepr
db/db
mice
(termed “db/db”) and obese Zucker rats (which also have a lep-
tin receptor mutation) (Kurtz et al., 1989; Tschop and Heiman,
2001), as well as in mice and rats fed a high-fat diet. All of these
animals have increased whole body lipid content and develop
hyperglycemia and insulin resistance, a phenotype similar to type
2 diabetes (reviewed in Unger, 2003).
Here we will discuss the sources of lipids that directly affect
skeletal muscle, review studies investigating muscle regeneration
in obesity models, and discuss possible mechanisms underlying
impaired regenerative capacity of skeletal muscle in obese animals
(summarized in Figure 1).
OBESITY AND SKELETAL MUSCLE LIPID ACCUMULATION
Obesity is characterized by elevated adipose storage in subcu-
taneous and visceral adipose depots and non-adipose organs,
a phenomenon called ectopic lipid accumulation (Van Her pen
and Schrauwen-Hinderling, 2008). In addition, obese individu-
als have increased circulating fatty acids (Boden and Shulman,
2002; Mittendorfer et al., 2009) and high ectopic lipid deposi-
tion in skeletal muscle partially resulting from increased fatty
acid uptake from the circulation (Goodpaster et al., 2000b; Sinha
et al., 2002; Bonen et al., 2004;reviewedinGoodpaster and
Wolf, 2004). Lipids within skeletal muscle are comprised of two
pools: extramyocellular lipids (EMCL) localized in adipose cells
between myofibers and intramyocellular lipids (IMCL) located
within muscle cells (Sinha et al., 2002; Boesch et al., 2006). A por-
tion of EMCL comprises adipose tissue closely associated with
the muscle, referred to as intermuscular adipose tissue (IMAT)
(Goodpaster et al., 2000a). Although IMAT accumulation in
obese patients is positively correlated with insulin resistance and
reduced muscle performance (Goodpaster et al., 2000a; Hilton
et al., 2008), this adipose depot does not appear to affect mus-
cle mass (Lee et al., 2012a), and its effects on muscle regeneration
have not been addressed. IMCL are comprised of neutral lipids
triacylglycerols (TAG) and cholesterol esters, mainly localized
to lipid droplets (reviewed in Fujimoto et al., 2008; Thiele and
Spandl, 2008) as well as lipid metabolites, such as long-chain acyl
CoAs, diacylglycerols and ceramides. Elevated TAG content and
increased numbers of lipid droplets have been observed in mus-
cle biopsies from obese people (Simoneau et al., 1995; Malenfant
et al., 2001). Genetically obese mice (ob/ob and db/db) and obese
Zucker rats also have increased IMCL (Kuhlmann et al., 2003;
Unger, 2003; Fissoune et al., 2009; Ye et al., 2011). Long-chain
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Akhmedov and Berdeaux Obesity and skeletal muscle regeneration
FIGURE 1 | Major mechanisms linking obesity with impaired muscle
regeneration. Obesity is associated with insulin and leptin resistance,
elevated circulating and intramuscular fatty acids, diacylglycerols, ceramides
and pro-inflammatory cytokines. Following muscle injury, satellite cells
(depicted adjacent to muscle on left) are activated, proliferate, differentiate
and form myofibers that grow and replace damaged tissue. Impairment of
these processes underlies inefficient muscle regeneration in obese rodents.
Defective leptin signaling can contribute to decreased satellite cell
proliferation and impaired muscle hypertrophy, but the molecular
mechanisms are not known. Fatty acids, diacylglycerols (DAG) and ceramides
induce apoptosis and decrease myoblast proliferation and differentiation,
possibly via activation of myostatin and inhibition of MyoD and myogenin
expression and/or activity. Ceramides and pro-inflammatory cytokines inhibit
muscle growth in part by inhibiting the IGF-1/Akt /mTOR pathway.
acyl CoAs, diacylg lycerols and ceramides accumulate in skele-
tal muscles of obese humans, ob/ob and db/db mice and obese
Zucker rats (Turinsky et al., 1990; Hulver et al., 2003; Adams
et al., 2004; Holland et al., 2007; Magnusson et al., 2008; Lee et al.,
2013; Turner et al., 2013) and negatively affect cell signaling and
metabolism; the defects are collectively referred to as lipotoxicity
(Lelliott and Vidal-Puig, 2004; Kusminski et al., 2009). In skeletal
muscle, lipotoxic species interfere with insulin signaling and are
thought to be partly responsible for insulin resistance in obesity
(reviewed in Timmers et al., 2008; Bosma et al., 2012; Coen and
Goodpaster, 2012). However, it remains largely unknown what
other physiologic processes are impaired by these lipid metabo-
lites in skeletal muscle. In the following sections we will focus
on recent findings on how obesity, and in some cases lipids,
impair muscle progenitor cell function and muscle regeneration
and regrowth.
EFFECTS OF OBESITY ON MUSCLE PROGENITOR CELLS
Insulin resistance and mitochondrial and metabolic dysfunction
are perhaps the most prominent muscle abnormalities that neg-
atively impact whole body metabolism and physical performance
in states of obesity and type 2 diabetes. Skeletal muscle mainte-
nance depends on ongoing repair, regeneration and growth, all of
which decline during aging (reviewed in Jang et al., 2011). Obesity
rates increase with aging, which is also accompanied by reduced
regenerative capacity and muscle strength. Thus, as average life
span increases, it is of growing clinical importance to understand
whether obesity impacts muscle maintenance and regeneration
and to identify mechanisms that may be targeted for therapeutic
benefit.
Skeletal muscle regeneration after injury requires the activity
of muscle stem cells and satellite cells, which remain associated
with skeletal myofibers after development (reviewed in Wang and
Rudnicki, 2012). Muscle regeneration is commonly experimen-
tally induced by intramuscular injection of a myotoxic agent,
such as cardiotoxin, notexin or barium chloride. Freeze-induced
injur y is an alternative model of muscle injury entailing appli-
cation of steel cooled to the temperature of dry ice to the muscle
(Warren et al., 2007). In normal animals, these injuries cause local
myofiber necrosis and inflammation, followed by satellite cell
activation, proliferation, differentiation, fusion and ultimately
regrowth of myofibers to approximately the same size as the origi-
nal within about three weeks (Figure 1 and Charge and Rudnicki,
2004). Satellite cells are required for regenerative myogenesis
(Lepper et al., 2011; Gunther et al., 2013). Currently there is a
controversy regarding requirement of satellite cells for skeletal
muscle hypertrophy. Load-induced hypertrophy in humans and
rodents is accompanied by satellite cell activation, proliferation
and fusion with existing myofibers (Rosenblatt et al., 1994; Kadi
et al., 2004; Petrella et al., 2008; Bruusgaard et al., 2010). However,
genetic ablation studies in mice demonstrated that satellite cells
do not appear to be required for hypertrophy induced by mechan-
ical overload (McCarthy et al., 2011; Jackson et al., 2012; Lee
et al., 2012b). Although efficient hypertrophy in rodents does not
strictly require satellite cell fusion to myofibers, nuclear accretion
due to satellite cell fusion is thought to promote hyper trophy by
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Akhmedov and Berdeaux Obesity and skeletal muscle regeneration
supporting the grow i ng cytoplasm. In addition, muscle regen-
erative capacity declines with aging, and this is thought to be
due in part to reduced satellite cell function (reviewed in Jang
et al., 2011). Thus, although it is still not settled to what extent
this specific progenitor population is required for maintenance
of adult muscle, it is clear that identification of therapeutic tar-
gets to stimulate and maintain activity of these cells has potential
to improve metabolism and strength in aging and obese humans.
Recent data indicate that skeletal muscle regeneration is signifi-
cantly impaired in models of diabetes and obesity, possibly due to
impaired muscle progenitor cell function.
LIPOTOXICITY IN MYOBLASTS
Several groups have modeled lipid overload by incubating cul-
tured muscle cells with fatty acids or lipid metabolites. During
differentiation of L6 myoblasts, exogenous ceramides markedly
reduce expression of the myogenic transcription factor myo-
genin, likely via inhibition of phospholipase D, while inhibitors of
ceramide synthesis potentiate myogenin expression and accelerate
myotube formation (Mebarek et al., 2007). In addition, several
studies showed that increasing ceramide pools either by palmitate
loading or silencing of stearoyl-CoA desaturase 1 (SCD1), which
normally desaturates fatty acids and reduces the pool of saturated
fatty acids that are converted to ceramides, results in increased
apoptosis in differentiated L6 and C2C12 muscle cells (Tur pin
et al., 2006; Rachek et al., 2007; Peterson et al., 2008b; Henique
et al., 2010; Yuzefovych et al., 2010). These findings suggest that
the elevated fatty acids in obesity could directly harm the muscle
fibers and satellite cells.
To test the effect of intracellular free fatt y acid accumulation on
myoblast viabilit y and myogenesis, Tamilarasan, et al. used C2C12
cells stably transfected with human lipoprotein lipase (LPL),
which converts TAGs to free fatty acids and glycerol (Tamilarasan
et al., 2012). In spite of an approximately tenfold increase in
intracellular free fatty acids and TAGs, cell viability and prolif-
eration were similar to control cells. However, LPL-expressing
cells showed defective differentiation accompanied by markedly
decreased expression of MyoD, myogenin, and myosin heavy chain
as well as a reduced number of myotubes (Tamilarasan et al.,
2012). In mice, acute triglyceride infusion resulted in increased
plasma free fatty acid and diacylglycerol levels and increased
caspase-3 activity in gastrocnemius muscle (Turpin et al., 2009).
However, in the same study, ob/ob mice and mice fed high-fat diet
for 12 weeks did not show increased apoptosis, autophagy or pro-
teolysis in muscle despite elevated plasma free fatty acids, muscle
diacylglycerols and ceramides (Turpin et al., 2009). In contrast
with this result, another group observed increased caspase-3
activation in gastrocnemius muscle in mice after 16 weeks of
high-fat diet feeding (Bonnard et al., 2008), probably secondary
to elevated reactive oxygen species (ROS), oxidative stress and
mitochondrial dysfunction. Because cell viability and apoptosis
were not directly assessed in this study, it is difficult to conclude
if caspase-3 activation was accompanied by increased apoptosis
(Bonnard et al., 2008). It is possible that pro-apoptotic effects
of caspase-3 in muscle from obese animals are counteracted
by increased expression of pro-surv ival Bcl2 and transcriptional
downregulation of other pro-apoptotic genes, such as caspase8,
caspase14, Fadd, and multiple genes involved in TNF-α signal-
ing (Turpin et al., 2009). Therefore, although fatty acids and
ceramides induce apoptosis in muscle cells in vitro,itappears
that elevated lipid metabolites do not impair muscle cell viability
in vivo. In vitro studies have r aised the interesting possibility that
fatty a cids and possibly other lipid metabolites interfere with the
myogenic differentiation program, suggesting that perhaps differ-
entiation during muscle regeneration would be impaired in obese
animals.
MUSCLE REGENERATION IN OBESITY MODELS
Several recent studies have employed myotoxins and freeze injury
to evaluate muscle regeneration in obese or diabetic mice. In mice
fed high-fat diet for 8 months, Hu, et al. observed reduced tibialis
anterior (TA) muscle mass after cardiotoxin injury, associated
with smaller myofibers, larger interstitial spaces and increased
collagen deposition compared with lean mice (Hu et al., 2010).
Similarly, a short period of high-fat diet (3 weeks) in young
mice ( aged 3–6 weeks) resulted in reduced numbers of satellite
cells and impaired regeneration of TA muscle after cold-induced
injury (Woo et al., 2011). A similar effect on satellite cell num-
ber and regeneration was observed in young mice with prenatal
malnutrition, which also results in ele vated adiposity (Woo et al.,
2011). Although proliferation r ates were not directly assessed
in this study, the data collectively suggest that high adipos-
ity depresses proliferative capacity of satellite cells either due
to intrinsic metabolic properties of the muscle or satellite cells
or alterations of circulating metabolites after high-fat feeding.
However, in other studies, intermediate durations (12 weeks)
of high fat feeding did not markedly impair the size of regen-
erating fibers of extensor digitorum longus (EDL) muscle after
cardiotoxin injury (Nguyen et al., 2011). Collagen deposition
was not evaluated, but there do appear to be larger intersti-
tial spaces in histological sections of regenerating muscle from
the 12 week high-fat diet-fed animals (Nguyen et al., 2011)
consistent with the findings of Hu et al. (2010). It is notable
when comparing these studies that Hu, et al. and Woo, et al.
evaluated regeneration of TA muscle while Nguyen, et al. ana-
lyzed EDL muscle. While both muscle groups are comprised
of predominantly fast-twitch IIB/X fiber types, TA contains a
larger proportion of oxidative type IIA fibers (Bloemberg and
Quadrilatero, 2012). The choice of muscle group is an important
consideration, as slow twitch muscles contain higher numbers
of satellite cells per fiber (Gibson and Schultz, 1983). Thus,
effects of high-fat diet feeding on different functional aspects
of muscle regeneration may depend on the muscle studied
and the type of analysis performed. Ultimate conclusions will
depend on additional analyses of multiple parameters of mus-
cle regeneration in h igh-fat diet fed animals, including careful
analysis of proliferation, muscle progenitor number, as well as
resolution of inflammation, fibrosis and fiber caliber during
regrowth.
Effects of lipid overload on skeletal muscle regeneration have
specifically been assessed in transgenic mice overexpressing LPL
in skeletal muscle (Levak-Frank et al., 1995; Tamilarasan et al.,
2012). Overexpression of LPL in muscle results in an approxi-
mately eightfold increase in LPL activity, increased free fatty acid
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Akhmedov and Berdeaux Obesity and skeletal muscle regeneration
uptake and three- to fourfold increases in free fatty acid and
TAG concentrations in gastrocnemius muscle. By two months of
age, transgenic mice develop severe myopathy, which is detected
histologically as regenerating myofibers with centrally localized
nuclei, in addition to perturbed sarcomere structure, excessive
glycogen storage, increased protein degradation and apoptotic
nuclei (Levak-Frank et al., 1995; Tamilarasan et al., 2012). Ten
days after cardiotoxin injury, myofiber cross-sectional area in
LPL-transgenic mice is reduced compared to wild-type mice,
indicating that intracellular lipid accumulation impairs muscle
regeneration (Tamilarasan et al., 2012), either directly or indi-
rectly . The defect in regeneration might result from reduced
differentiation of progenitor cells, as LPL overexpression blocks
myogenic differentiation of C2C12 cells (Tamilarasan et al., 2012)
as described above. This, however, has not yet been tested. The
pronounced muscle degenerative phenotype in LPL-expressing
mice is most likely explained by lipotoxicity caused by the several-
fold increase in intracellular free fatty acid and TAG concen-
trations. In comparison, high-fat diet feeding usually results in
a 30–50% increase in intramuscular TAG in rodents (Marotta
et al., 2004; Bruce et al., 2009; Ussher et al., 2010). The ultimate
extent of lipotoxicity in skeletal muscle in vivo will therefore likely
depend on the extent of lipid infiltration.
LEPTIN SIGNALING
In genetically obese ob/ob and db/db mice, which have more severe
insulin resistance than high-fat diet-fed mice, EDL myofiber
regeneration after cardiotoxin injury is blunted (Nguyen et al.,
2011). This finding could suggest that leptin signaling is impor-
tant for skeletal muscle regeneration. In support of this model,
injury-induced satellite cell proliferation is specifically impaired
in leptin signaling-deficient mouse models, but not in the two
high-fat diet models (Hu et al., 2010; Nguyen et al., 2011).
Notably, ob/ob and db/db mice show defects of early regenera-
tion stages: decreased proliferation and reduced MyoD expression
are most evident a t day 5 post-injury (Nguyen et al., 2011). In
agreement with this result, b asal rates of satellite cell prolifera-
tion are reduced in both mice and obese rats with leptin signaling
deficiencies (Purchas et al., 1985; Peterson et al., 2008a), suggest-
ing reduced proliferative capacity. Recombinant leptin stimulates
proliferation and MyoD and myogenin expression in myoblasts
from wild-type mice, but myoblasts from mice lacking all forms of
the leptin receptor (referred to as POUND mice) show decreased
expression of MyoD and myogenin transcripts and decreased
myotube formation during differentiation ex vivo (Arounleut
et al., 2013). Moreover, administration of recombinant leptin to
ob/ob mice restores expression of the proliferation markers pro-
liferating cell nuclear antigen (PCNA) and cyclin D1, which may
account for the muscle growth-promoting effect of recombinant
leptin in leptin-deficient animals (Sainz et al., 2009). In C2C12
myoblasts, leptin also stimulates proliferation but does not appear
to promote MyoD or myogenin expression or differentiation
(Pijet et al., 2013). Although leptin clearly has stimulatory effects
on mouse myoblasts and muscle, it is not clear whether leptin
promotes myoblast proliferation in all species. Leptin receptors
are poorly abundant in porcine muscle, and recombinant lep-
tin has no effect on proliferation of primary porcine myoblasts
cultured in serum free medium or on protein accretion as these
cells differentiated (Will et al., 2012). In line with this finding,
lean and obese leptin receptor-deficient Zucker r ats exhibit com-
parable BrdU incorporation, expression of myogenic regulatory
factors, activation of pro-hypertrophic signaling pathways and
gain of muscle mass in response to overload, demonstrating that
leptin signaling per se is not required for satellite cell activation
and muscle hypertrophy, at least in rats (Peterson et al., 2008a).
In addition to the activity of satellite cells, macrophages
also contribute to regeneration of injured muscle by facilitating
removal of tissue debris (Arnold et al., 2007). Leptin stimu-
lates proliferation and activation of macrophages (Santos-Alvarez
et al., 1999; Raso et al., 2002), pointing to another possible
mechanism by which leptin resistance could impair muscle regen-
eration. Nguyen, et al. provided data supporting this hypothesis:
in injured muscle of ob/ob and db/db mice, macrophage accu-
mulation is decreased dur ing early regeneration (Nguyen et al.,
2011). In addition, these authors observed markedly decreased
angiogenesis after injury in ob/ob and db/db mice (Nguyen et al.,
2011). The data suggest that leptin could potentiate muscle regen-
eration by regulating macrophage activity and/or by stimulating
vascularization. Vascularization potentiates regrowth of regener-
ating muscle in mice (Ochoa et al., 2007; Deasy et al., 2009). It
appears that vascularization is not only important for nutrient
availability but also myofiber growth. Vascular endothelial growth
factor (VEGF), elevated during angiogenesis, promotes regenera-
tion by directly stimulating myofiber growth (Arsic et al., 2004;
Messina et al., 2007). As leptin resistance is often observed in
obese and type 2 diabetic humans (Maffei et al., 1995;reviewed
in Martin et al., 2008) it is possible that lack of leptin signaling
could contribute to poor vascularity and compromised satellite
cell function.
INFLAMMATION
In skeletal muscle, inflammation is activated after injury and is
coordinated with myogenic differentiation to achieve efficient
muscle regeneration (reviewed in Mann et al., 2011; Kharraz et al.,
2013). Immediately after muscle injury, an acute inflammatory
stage ensues characterized by infiltration of pro-inflammatory M1
macrophages that remove tissue debris. Later, a different popula-
tion of macrophages (M2) resolves inflammation. Accumulating
data show that macrophages not only mediate inflammation but
also support satellite cells during skeletal muscle regeneration.
In mice, deletion of chemokine receptor-2 (CCR-2) impairs
macrophage infiltration after muscle injury and results in inef-
ficient muscle regeneration (Warren et al., 2005). In co-culture
experiments in vitro, macrophages stimulate satellite cell prolif-
eration (Cantini et al., 1994; Massimino et al., 1997; Merly et al.,
1999). When transplanted together with satellite cells into muscle
of Dmd
mdx
mice, a mouse model of human Duchenne muscular
dystrophy, macrophages stimulate satellite cell survival and pro-
liferation (Lesault et al., 2012). This potentiation effect is likely
mediated, at least in part, by pro-inflammatory cytokines TNF-α
and IL-6, which promote myoblast proliferation and migration
in vitro (Li, 2003; Torrente et al., 2003; Wang et al., 2008; Toth
et al., 2011). However, TNF-α and another pro-inflammatory
cytokine IL-1α also prevent myogenic differentiation
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Akhmedov and Berdeaux Obesity and skeletal muscle regeneration
(Miller et al., 1988; Layne and Farmer, 1999; Langen et al., 2001;
Trendelenburg et al., 2012). During later stages of regeneration,
TGF-β and IL-10 secreted by anti-inflammatory M2 macrophages
promote myogenic differentiation (Arnold et al., 2007; Deng
et al., 2012). Thus, the interplay between macrophages and
satellite cells is precisely temporally orchestrated during skeletal
muscle regeneration.
Obesity is recognized as a state of chronic inflammation with
increased circulating pro-inflammatory cytokines TNF-α,IL-1β
and IL-6 (reviewed in Wellen and Hotamisligil, 2005; Gregor and
Hotamisligil, 2011). The effects of chronically elevated cytokines
on satellite cell maintenance, activation and proliferation are not
well understood, but it appears that chronic exposure to cytokines
has distinct effects on myoblast proliferation and differentia-
tion from acute exposure. For example, in a mouse model of
chronic inflammation in which TNF-α is constitutively expressed
in lung and becomes chronically ele vated in the circulation,
skeletal muscle becomes atrophic and myoblast proliferation and
differentiation are reduced in response to mechanical loading
(Langen et al., 2006). Similarly, chronic, local delivery of IL-6
to muscle of young rats inhibits muscle growth and stimulates
expression of cyclin-dependent kinase inhibitor p21, suggesting
decreased satellite cell proliferation, althoug h this has not been
tested (Bodell et al., 2009). It is possible that in chronic inflamma-
tion the normal coordination between macrophages and muscle
satellite cells is impaired and contributes to impaired satellite cell
function. It would be interesting to manipulate cytokine signaling
in obesity models to determine whether the chronic inflammation
that accompanies obesit y in fact does impair muscle satellite cell
proliferation and differentiation and ultimately muscle growth.
MYOSTATIN
Myostatin is a member of the TGF-β family of secreted proteins
known to prevent muscle regeneration and growth (reviewed in
Joulia-Ekaza and Cabello, 2006; Kollias and McDermott, 2008).
Interestingly, myostatin expression is increased in skeletal muscle
of extremely obese women (Hittel et al., 2009)andofob/ob and
high-fat diet-fed mice (Allen et al., 2008). In C2C12 myoblasts,
recombinant or overexpressed myostatin decreases proliferation
most likely by stimulating expression of the cyclin-dependent
kinase inhibitor p21, resulting in inhibition of C dk2 and impaired
G1 to S phase transition (Thomas et al., 2000; Taylor et al., 2001;
Joulia et al., 2003). Moreover, proliferation of satellite cells in
myostatin-null mice is mar kedly increased (McCroskery et al.,
2003). Myostatin also represses transcription of myogenic regula-
tory factors through direct activation of Smad2/3 proteins, which
repress expression of MyoD and myogenin. In addition, Smad3
represses MyoD activity through direct interaction (Liu et al.,
2001; Langley et al., 2002). Elevated myostatin in obese people
correlates with increased phosphorylation of Smad2/3 proteins
and an approximately two-fold decrease in MyoD and myogenin
transcript levels (Watts et al., 2013). Thus, increased myostatin
in obese animals may contribute to defects in regeneration and
maintenance of muscle mass (Figure 1).
The source and mechanism by which myostatin becomes
elevated in obese subjects remain obscure. Expression of the
myostatin gene is stimulated in myocytes by several pathways
including g lucocorticoid signaling (Salehian et al., 2006) possi-
bly via C/EBP-δ ( Allen et al., 2010) or repression of miR-27a/b
(Allen and Loh, 2011). Myostatin expression in muscle cells has
also been reported to be stimulated by FoxO1 and TGF-β/Smad3
(Allen and Unterman, 2007), MyoD (Spiller et al., 2002)and
a JNK/p38-mediated signaling pathway (Han et al., 2010). It
is not known which, if any, of these pathways may mediate
the increase in circulating myostatin in obese patients, but it is
tempting to speculate that elevated glucocorticoids commonly
observed in metabolic syndrome and obesity (Anagnostis et al.,
2009) could stimulate myostatin expression by promoter regu-
lation (Allen et al., 2010) and modulation of miR-27a/b (Allen
and Loh, 2011). Alternatively, insulin resistance may result in
derepression of myostatin via constitutive activation of FoxO1
(Allen and Unterman, 2007); this model would be consistent
with the observation of elevated myostatin in insulin resistant
type 2 diabetic patients and non-obese hyperinsulinemic sub-
jects (reviewed in Allen et al., 2011). Although skeletal muscle
expresses far more myostatin than other tissues, it is noteworthy
that myostatin mRNA increases by at least fifty fold in adipose
tissue (primarily adipocytes) and only twofold in skeletal mus-
cle of obese mice (Allen et al., 2008). Thus, it is possible that in
obesity a large amount of myostatin could be secreted from adi-
pose as a result of hypercortisolemia. Although myostatin is well
known for its role in regulation of muscle growth, it is not clear to
what extent myostatin contributes to impaired muscle regenera-
tion observed in rodent models of obesity. Genetic manipulations
disrupting myostatin signaling, such as expressing a dominant
negative form of the myostatin receptor in satellite cells in an
obesity model, will help to answer this question.
ADIPOGENESIS
Fibro/adipogenic progenitor (FAP) cells comprise a recently iden-
tified population of progenitors that reside in the muscle and
become activated after muscle damage in mice (Joe et al., 2010;
Heredia et al., 2013). Unlike myogenic progenitors, FAP cells
do not fuse or differentiate into myofibers. Instead, FAP cells
support myogenesis likely by enhancing proliferation and differ-
entiation of myogenic progenitors through secretion of factors
such as IL-6 (Joe e t al., 2010). The signals that regulate FAP
cell differentiation are incompletely understood. FAP cells spon-
taneously differentiate into adipocytes in vitro and when trans-
planted into skeletal muscle with fatty infiltration, but not when
transplanted into healthy skeletal muscle (Joe et al., 2010). Using
a co-culture system, Uezumi, et al. found that muscle satellite
cells inhibit adipogenic differentiation of FAP cells likely by direct
physical interaction (Uezumi et al., 2010), though the signal is
unknown. If the same regulation occurs in vivo,thenadecrease
in satellite cell number, activity or proximity to FAP cells could
result in increased adipogenic conversion of FAP cells and IMAT
accumulation. Alternatively, exciting work by Heredia, et al.
demonstrated that after skeletal muscle injury, eosinophil-derived
anti-inflammatory cytokines IL-4/IL-13 promote FAP prolifer-
ation and inhibit their differentiation to adipocytes (Heredia
et al., 2013). It is possible that under the pro-inflammatory con-
ditions of obesity, the ability of satellite cells or eosinophils to
inhibit adipogenic differentiation of FAP cells is compromised.
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Akhmedov and Berdeaux Obesity and skeletal muscle regeneration
As a result, FAP cells activated during injury could differentiate
into adipocytes, contribute to increased IMAT, and occupy areas
of the tissue once filled with skeletal myofibers. Indeed, it has
been shown that muscle side population cells from dystrophic or
injured tissue differentiate in culture to FAP cells and lose myo-
genic capacity (Penton et al., 2013). It is notable in this regard
that in patients with Duchenne muscular dystrophy, the skele-
tal muscle eventually loses capacity for ongoing regeneration and
myofibers are replaced by fatty infiltrate and collagen (Radle y
et al., 2007). It will be important for future studies to examine
the action of FAP cells in obese animals and humans.
METABOLISM
Recently it has been recognized that satellite cells exhibit different
intrinsic metabolic properties in states of quiescence, prolifera-
tion and differentiation (reviewed in Ryall, 2013). In the quiescent
state, satellite cells have low energy demands, low oxygen con-
sumption and low ATP production. In low nutrient conditions,
elevated NAD
+
levels stimulate the deacetylase SIRT1, which in
turn promotes myoblast proliferation and prevents myogenic dif-
ferentiation, in part via MyoD deacetylation (Fulco et al., 2003).
Culturing mouse myoblasts in low glucose medium similarly pre-
vents differentiation at least in part through SIRT1 activation
(Fulco et al., 2008;reviewedinRyall, 2012). It thus can be hypoth-
esized that in low energy states, limited nutrient supply and the
associated increase in SIRT1 activity would be beneficial to main-
tain a pool of muscle satellite cells. On the other hand, obesity
and nutrient overload would be expected to provide unfavor-
able conditions for maintenance of quiescent satellite cells or for
proliferation after acute injury.
Cerletti, et al. tested the corollary to this hypothesis by e val-
uating muscle satellite cell metabolism and function in mice
after short-term (12 weeks) caloric restriction. They showed
that short-term caloric restriction in mice increases both the
number and myogenic capacity of muscle-associated satellite
cells and enhances regeneration after freeze injury ( Cerletti
et al., 2012). Satellite cells isolated from calorie-restricted ani-
mals had higher mitochondrial content, enhanced oxidative
metabolism and reduced glycolytic capacity accompanied by ele-
vated SIRT1 expression. Muscle stem cells harvested from calor-
ically restricted mice also displayed improved engraftment in
dystrophin-deficient Dmd
mdx
mice that had not been previously
subjected to caloric restriction (Cerletti et al., 2012). Thus, the
altered cellular metabolic state of the satellite cells from a calorie-
restricted animal was sufficient to confer benefits on a normal
recipient. The beneficial effects of calorie restriction were not,
however, limited to the satellite cells. Tr ansplanted muscle stem
cells had much higher engraftment efficiency when transplanted
into healthy uninjured skeletal muscle of animals undergoing
calorie restriction, possibly as a result of reduced inflammation
in the muscle (Cerletti et al., 2012).
These findings strongly suggest that (1) muscle satellite cell
metabolism is profoundly altered by the systemic nutritional
environment and (2) the metabolic/ inflammatory state of the
organism, and therefore of the mature myofibers, also affects the
health or fusion capacity of satellite cells. Accumulation of SIRT1
protein in the satellite cells from calorically restricted mice could
theoretically stimulate proliferation and oxidative metabolism,
resulting in a larger satellite cell pool. In obesity, perturbations of
intrinsic satellite cell metabolism could negatively affect the pro-
liferation and activity of the satellite cell pool, but this exciting
field is still emerging.
MUSCLE REGROWTH AFTER INJURY IN OBESE ANIMALS
A common finding among the aforementioned in vivo studies of
skeletal muscle regeneration in obese animals is reduced recov-
er y of muscle mass and function after injury (Hu et al., 2010;
Nguyen et al., 2011; Tamilarasan et al., 2012). This may o ccur
secondary to reduced satellite cell function or as a result of defec-
tive hypertrophic g rowth after initial satellite cell differentiation
and fusion. In this section, we will discuss some potential mecha-
nisms underlying defective muscle regrowth after injury in obese
animals.
IGF-1/Akt SIGNALING
In normal skeletal muscle, the balance between muscle hypertro-
phy and atrophy is largely regulated by the IGF-1/Akt signaling
pathway (reviewed in Glass, 2010), which stimulates mTOR-
dependent protein synthesis and inhibits FOXO-dependent tran-
scription of muscle-specific E3 ubiquitin ligases ( Bodine et al.,
2001; Sartorelli and Fulco, 2004; Bodine, 2006). The balance
between muscle growth and atrophy is dysregulated in obesity.
In obese mice and Zucker r ats, muscle growth in response to
mechanical loading is reduced due to decreased activation of
Akt, p70S6 kinase and mTOR (Sitnick et al., 2009; Paturi et al.,
2010). Similar mechanisms might impair muscle regrowth after
injury. Indeed, in high-fat diet-fed mice, Hu, et al. found that
PIP
3
levels and PI(3)-kinase activity are reduced and expres-
sion of the lipid and protein phosphatase PTEN is increased (Hu
et al., 2010). These combined changes would result in decreased
Akt and mTOR activity and reduced hypertrophy. Pten dele-
tion in muscle is sufficient to restore Akt phosphorylation and
remarkably improves muscle growth in high-fat diet-fed mice
(Hu et al., 2010). These findings clearly demonstrate that dysreg-
ulated PI(3)-kinase/Akt pathway activity in muscle of obese mice
not only impairs insulin signaling but also interferes with muscle
growth.
On the o ther hand, Nguyen, et al. observed impaired muscle
growth after injury in obese ob/ob and db/db and but not in high-
fat diet-fed mice (Nguyen et al., 2011). Since both ob/ob and db/db
mice are deficient in leptin signaling , one interpretation is that
leptin signaling is necessary for nor mal muscle regeneration. The
authors point out that leptin could promote muscle growth by
activation of PI(3)-kinase and ERK1/2 pathways (Nguyen et al.,
2011). Consistently, administration of recombinant leptin to mice
or C2C12 myoblasts activates janus kinase 2 (JAK2), which poten-
tiates phosphorylation of insulin receptor substrates IRS1 and
IRS2, activity of PI(3)-kinase, and phosphorylation of Akt and
glycogen synthase kinase 3 (GSK3) (Kellerer et al., 1997; Kim
et al., 2000; Maroni et al., 2003, 2005). These studies suggest
the hypothesis that leptin-dependent activation of Akt is impor-
tant for regulation of muscle growth or regrowth after injury.
In further support of this model, leptin treatment of ob/ob mice
increases the mass of multiple skeletal muscle groups, including
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Akhmedov and Berdeaux Obesity and skeletal muscle regeneration
gastrocnemius, EDL and soleus, with concomitant decreased
expression of muscle-specific E3 ubiquitin ligases MAFbx and
MuRF1 in gastrocnemius muscle (Sainz et al., 2009).
The toxic lipid metabolites diacylglycerols and ceramides also
impair IGF-1/Akt signaling. In skeletal muscle and liver, diacyl-
glycerols activate PKCε and PKCθ, which phosphorylate multiple
serine residues of insulin receptor substrate-1 (IRS-1) directly or
via JNK and IKKβ ultimately leading to insulin resistance (Yu
et al., 2002; Li et al., 2004;reviewedinSamuel et al., 2010; Turban
and Hajduch, 2011). Interestingly, PKCθ deletion in dystrophic
Dmd
mdx
mice increases expression of myogenin and myosin
heavy chain and decreases necrotic areas in the muscle ( Madaro
et al., 2012). Similarly, stable PKCθ knockdown in C2C12 cells
increases expression of myogenin and myosin heavy chain and
potentiates myotube formation in vitro (Marino et al., 2013).
The other major class of toxic lipid intermediates, ceramides,
inhibits Akt by two distinct mechanisms. In C2C12 myoblasts,
3T3-L1 adipocytes and PC-12 cells, ceramides activate protein
phosphatase 2A, leading to Akt dephosphor ylation (Salinas et al.,
2000; Cazzolli et al., 2001; Chavez et al., 2003; Stratford et al.,
2004). In L6 myotubes, ceramides induce PKCζ-dependent Akt
phosphorylation on Thr34, which blocks Akt translocation to the
plasma membrane (Hajduch et al., 2001; Powell et al., 2003, 2004;
reviewed in Bikman and Summers, 2011). In addition, ceramides
impair amino acid uptake in L6 myotubes by decreasing the
amount of the membrane-associated amino acid transporter
SNAT2, with concomitant reduction of p70S6 kinase phosphory-
lation and protein synthesis (Hyde et al., 2005). All of these events
would be expected to inhibit myofiber g rowth. It is likely that
a similar mechanism contributes to impaired muscle regrowth
during regeneration in obese animals (Figure 1).
INFLAMMATION
Pro-inflammatory cy tokines TNF-α,IL-1β and IL-6 inhibit IGF-
1/Akt signaling and de-repress transcription of muscle ubiquitin
ligases Mafbx and Murf1 and potentiate skeletal muscle atrophy
(reviewed in Glass and Roubenoff, 2010). Thus, in addition to
the possible negative effects on myoblast proliferation and differ-
entiation, increased circulation of TNF-α,IL-1β and IL-6 could
counteract anabolic growth of skeletal muscle during regener-
ation in obese animals (Figure 1). For example, treatment of
human, porcine or mouse (C2C12) myoblasts with TNF-α or
IL-1β prevents IGF-1-stimulated protein synthesis (Frost et al.,
1997; Broussard et al., 2003, 2004). In rats, 16 weeks of high-fat
diet feeding results in decreased Akt and mTOR phosphoryla-
tion and increased apoptosis that correlates with upregulation of
TNF-α receptors in the muscle (Sishi et al., 2011). Interestingly,
TNF-α treatment increasesceramide synthesis in C2C12 myoblasts
and L6 myotubes, and exogenous ceramides cause atrophy of L6
myotubes (Strle et al., 2004; De Larichaudy et al., 2012). In support
of the idea that ceramides mediate effects of TNF-α on myotubes,
ceramide synthesis inhibitors block the inhibitory effect of TNF-α
on IGF-1-stimulated protein synthesis (Strle et al., 2004)and
prevent TNF-α induced atrophy (De Larichaudy et al., 2012). It is
therefore possible that in obese animals, elevated TNF-α impairs
IGF-1 signaling and muscle regrowth via ceramides and toxic lipid
intermediates, which also directly inhibit satellite cell activity.
CONCLUDING REMARKS
The influence of obesity on skeletal muscle regeneration and
maintenance is an emerging area that is poorly mechanistically
understood. So far, this topic has been primarily addressed in
studies on obese rodents. Regenerative capacity is particularly
impaired by severe obesity such as in genetically obese ob/ob
and db/db mice. Identifying factors that specifically block mus-
cle regeneration in obese animals is challenging because obesity
is accompanied by several abnormalities, including but not lim-
ited to ectopic accumulation of multiple lipid species, insulin
and leptin resistance, chronic inflammation and metabolic dis-
turbances (Figure 1). Using genetic models and pharmacological
approaches to block synthesis of specific lipid species and modu-
late production and signaling of cytokines will help to determine
which lipid species and cytokines specifically impair regeneration
in obese animals. Another challenge is determining how obesity
affects different steps during regeneration such as satellite cell
activation and proliferation, myoblast differentiation, fusion and
myofiber growth. In this regard, intriguing new studies linking
global metabolism, cellular metabolism and satellite cell capac-
ity for engraftment may facilitate identification of new molecular
mechanisms that could be targeted therapeutically. An impor-
tant open question is whether and to what extent obesity impairs
muscle regeneration in humans and whether impaired muscle
regeneration contributes to poor wound healing in type 2 dia-
betic patients (reviewed in Greenhalgh, 2003), or whether poor
vascular function itself impairs satellite cell function and skeletal
muscle regeneration in obese and type 2 diabetic people. In obese
and t ype 2 diabetic patients, exercise and low calorie diet aimed
at reducing lipid oversupply and stimulating metabolism could
be beneficial not only by improving whole body metabolism
but also perhaps by promoting anabolic growth of muscle via
improved satellite cell viability and function. Stimulation or
preservation of satellite cells could, in tur n, enable these indi-
viduals to become stronger and more active and to possibly
prevent further IMAT accumulation. In a ddition to abnormali-
ties discussed here, obese and type 2 diabetic individuals suffer
from complications, such as peripheral neuropathy, which we do
not address directly in this review (reviewed in Vincent et al.,
2011; Ylitalo et al., 2011). As innervation is required for skeletal
muscle maintenance and regeneration in rodents (d’Albis et al.,
1988; Rodrigues Ade and Schmalbruch, 1995; Billington, 1997),
it is possible that peripheral neuropathy contributes to impaired
skeletal muscle regeneration in obese and type 2 diabetic humans
and could prevent putative salutary effects of strategies to pro-
mote satellite cell function. Ultimate conclusions about the effects
of obesity on muscle regeneration await the results of the next
generation of experiments that explore signaling mechanisms and
more fully characterize muscle regeneration in obese rodents and
humans.
ACKNOWLEDGMENTS
This publication was supported by the National Institute of
Arthritis and Musculoskeletal and Skin Diseases (R01AR059847).
The content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes of
Health.
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Conflict of Interest Statement: The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Received: 01 October 2013; acce pted: 28 November 2013; published online: 17
December 2013.
Citation: Akhmedov D and Berdeaux R (2013) The effects of obesity on skeletal muscle
regeneration. Front. Physiol. 4:371. doi: 10.3389/fphys.2013.00371
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