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Impact of oxidative stress on exercising skeletal muscle

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It is well established that muscle contractions during exercise lead to elevated levels of reactive oxygen species (ROS) in skeletal muscle. These highly reactive molecules have many deleterious effects, such as a reduction of force generation and increased muscle atrophy. Since the discovery of exercise-induced oxidative stress several decades ago, evidence has accumulated that ROS produced during exercise also have positive effects by influencing cellular processes that lead to increased expression of antioxidants. These molecules are particularly elevated in regularly exercising muscle to prevent the negative effects of ROS by neutralizing the free radicals. In addition, ROS also seem to be involved in the exercise-induced adaptation of the muscle phenotype. This review provides an overview of the evidences to date on the effects of ROS in exercising muscle. These aspects include the sources of ROS, their positive and negative cellular effects, the role of antioxidants, and the present evidence on ROS-dependent adaptations of muscle cells in response to physical exercise.
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Biomolecules 2015, 5, 356-377; doi:10.3390/biom5020356
biomolecules
ISSN 2218-273X
www.mdpi.com/journal/biomolecules/
Review
Impact of Oxidative Stress on Exercising Skeletal Muscle
Peter Steinbacher * and Peter Eckl
Department of Cell Biology, University of Salzburg, A-5020 Salzburg, Austria;
E-Mail: peter.eckl@sbg.ac.at
* Author to whom correspondence should be addressed; E-Mail: peter.steinbacher@sbg.ac.at;
Tel.: +43-662-8044-5601.
Academic Editor: Michael Breitenbach
Received: 13 January 2015 / Accepted: 30 March 2015 / Published: 10 April 2015
Abstract: It is well established that muscle contractions during exercise lead to elevated
levels of reactive oxygen species (ROS) in skeletal muscle. These highly reactive molecules
have many deleterious effects, such as a reduction of force generation and increased
muscle atrophy. Since the discovery of exercise-induced oxidative stress several decades
ago, evidence has accumulated that ROS produced during exercise also have positive
effects by influencing cellular processes that lead to increased expression of antioxidants.
These molecules are particularly elevated in regularly exercising muscle to prevent the
negative effects of ROS by neutralizing the free radicals. In addition, ROS also seem to
be involved in the exercise-induced adaptation of the muscle phenotype. This review provides
an overview of the evidences to date on the effects of ROS in exercising muscle. These
aspects include the sources of ROS, their positive and negative cellular effects, the role of
antioxidants, and the present evidence on ROS-dependent adaptations of muscle cells
in response to physical exercise.
Keywords: skeletal muscle; ROS; exercise; mitochondria; force generation;
antioxidants; PGC-1
1. Introduction
Skeletal muscle is a highly specialized tissue with excellent plasticity in response to external stimuli
such as exercise and training. The repetitive muscle contractions conducted during endurance training
lead to a variety of phenotypic and physiological responses. These responses include activation of
OPEN ACCESS
Biomolecules 2015, 5 357
mitochondrial biogenesis, fiber type transformation and angiogenesis. Together, they increase the
muscle’s capacity of aerobic metabolism and its resistance to fatigue. High muscle activity also involves
a strong increase in reactive oxygen species (ROS) production. These unstable molecules and ions
contain oxygen and are extremely reactive due to an unpaired electron. Among these oxygen
intermediates are the free radicals superoxide, peroxide and the hydroxyl radicals and other highly
reactive oxidants, such as singlet oxygen and hypochlorous acid. They promote oxidation reactions
with other molecules, such as proteins, lipids and DNA and can thus be highly detrimental. However,
recent research has demonstrated that ROS also have a beneficial role in promoting the adaptive
responses of muscle to training.
More than three decades ago it was established that muscle activity leads to an increase in ROS
production and concentration of free radicals [1,2]. Since then numerous investigations in rodents
and humans have confirmed these early observations. Thus, it is generally accepted that single bouts of
aerobic or anaerobic exercise, as well as chronic exercise promote the generation of ROS (summarized
in the reviews [3,4]; and more recently e.g., [5–7]).
The great interest in this topic also stems from data that show that ROS levels are increased in
subjects with aging-related sarcopenia, cardiac reperfusion injuries or muscular diseases, i.e., muscle
dystrophies. Thus, it was assumed that exercise-induced ROS are potentially detrimental to muscle
function and lead to muscle fatigue and muscle atrophy. Hence, many investigations focused on ways
to prevent ROS production and accumulation and subsequent oxidative damage during and following
physical exercise.
2. Sources of ROS in Muscle
It has consistently been shown that muscle activity leads to a strong increase in ROS production [8].
However, there is a large debate about the sources and the extent of ROS that these sources produce.
Several potential producers of ROS have been identified in muscle cells which are likely to be activated
by different stimuli. Among these are mitochondria, nicotinamide adenine dinucleotide phosphate
(NADPH) oxidases (NOXs), phospholipase A2 (PLA2), xanthine oxidase (XO) and lipoxygenases
(Figure 1). Some of these are discussed in more detail below. In addition to these intracellular sources,
ROS has been shown to be produced from non-muscle sources. Strenuous exercise can elicit muscle
injuries, which then lead to the activation of the neutrophils and macrophages via interferon- (IFN-),
interleukin-1 (IL-1) and tumor necrosis factor (TNF) (for more detailed information see reviews [9,10]).
These immune cells excessively produce ROS (oxidative burst), which is a central component of
neutrophil defense mechanism. In addition, the exercise-induced increase of catecholamines (adrenaline,
noradrenaline, dopamine) also play a role in the generation of ROS [11], as well as ROS derived
from endothelium [12] (Figure 1).
2.1. Mitochondria
For a long period of time, mitochondria were regarded as the main producer of cellular ROS with an
estimated superoxide production rate of approximately 1%–4% of total mitochondrial O2 consumption
(see reviews [8,13,14]). More recent data demonstrate that the production of ROS in mitochondria
is by an order of magnitude smaller than originally expected and is approximately 0.15% [15].
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Mitochondria are thought to produce ROS by a leak of single electrons in the respiratory chain in the
mitochondrial inner membrane of the contracting muscle cells. Ten different sites of superoxide/H2O2
generation have been found as yet in mammalian mitochondria [16,17]. Superoxide production mainly
occurs from complexes I (NADH dehydrogenase) and III (coenzyme Q and cytochrome C oxidoreductase)
of the electron transport chain [18,19]. New findings also identify complex II (succinate dehydrogenase)
as a major source of superoxide production [16]. Using isolated mitochondria, the contribution of
each site to total H2O2 production has recently been quantified and shown to strongly depend on
the substrate being oxidized [20]. At rest, H2O2 was predominantly produced from the quinol site in
complex I (site IQ) and flavin site in complex II (site IIF), followed by sites IF and IIIQo. Under conditions
that mimic mild and intense aerobic exercise, total production is much less and the low capacity site IF
dominates [20].
Figure 1. Sources of reactive oxygen species (ROS) and endogenous antioxidants in
skeletal muscle fibers. Following exercise, ROS are produced endogenously by mitochondria,
NOXs, PLA2 and XO. In addition, exercise increases ROS production also in activated
neutrophils and macrophages, endothelia of blood vessels and by catecholamines. Regular
exercise leads to an increase of endogenous antioxidants, which are able to neutralize
free radicals. A, adrenaline; CAT, catalase; Cu, Zn-SOD, copper-zinc superoxide dismutase;
DA, dopamine; GPX, glutathione peroxidase; GSH, glutathione; IFN, interferon ;
IL-1, interleukin-1; Mn-SOD, manganese superoxide dismutase; NA, noradrenaline;
NOX, nicotinamide adenine dinucleotide phosphate oxidase; PLA2, phospholipase A2;
SR, sarcoplasmic reticulum; TNF, tumor necrosis factor; TxnRd2, thioredoxin reductase 2;
XO, xanthine oxidase.
Biomolecules 2015, 5 359
2.2. NADPH Oxidases
NADPH oxidases (NOXs) are flavoprotein enzymes that are activated by calcium, free fatty acids,
protein-protein interactions and posttranslational modifications and use NADPH as electron donors [21,22].
They are transmembrane proteins in the transverse tubules and the sarcoplasmic reticulum and transport
electrons across biological membranes to reduce oxygen to superoxide or H2O2 [21,22]. It was shown
that NOX family members contribute to cytosolic superoxide production in skeletal muscle both at rest
and during contractile activity to a larger extent than mitochondria [23–25]. ROS generated by NOXs
activates ryanodine receptors (RyR), which leads to an intracellular Ca2+ release [26–28]. More recently,
it was found that insulin induces ROS generation through NOX activation and that this ROS increase
is required for the intracellular Ca2+ rise mediated by inositol triphosphate (IP3) receptors [29].
2.3. Xanthine Oxidase
Xanthine oxidase (XO) is a cytosolic molybdoflavoenzyme that is recognized as a key enzyme in
purine catabolism in which it catalyzes the hydroxylation of hypoxanthine to xanthine and of xanthine
to uric acid [30]. In muscle, XO is present in the cytosol but also in the associated endothelial cells [8].
Upon contraction, XO activity is significantly increased and leads to increased lipid peroxidation, protein
oxidation, muscle damage and edema [31]. During intense exercise in which large amounts of ATP
are consumed, hypoxanthine and xanthine levels are rising and serve as substrates for XO to generate
ROS [32]. Interestingly, ROS generated by XO appears to be involved in the regulation of exercise-induced
mitochondrial biogenesis via peroxisome proliferator-activated receptor- coactivator-1 (PGC-1) [33].
2.4. Myostatin
Recently, it was demonstrated that myostatin, a blocker of muscle differentiation, is capable of
signaling ROS production via canonical Smad3, nuclear factor (NF)-B and TNF- in muscle cells [34].
In the absence of Smad3, myostatin induces ROS production through the activation of p38 and ERK
mitogen-activated protein kinase (MAPK) pathways mediated via TNF- , IL-6, NOX and XO [35].
2.5. Phospholipase A2
Enzymes of the phospholipase A2 (PLA2) family also contribute to intra- and extracellular ROS
increase during muscle contraction. They cleave arachidonic acid from phospholipids in the plasma
membrane, sarcoplasmic reticulum or mitochondrial membranes. Arachidonic acid is an important
lipid-signaling molecule and is a substrate for lipoxygenases for the production of ROS [36]. In addition,
the cytosolic PLA2 enzyme has been demonstrated to increase ROS by stimulating NOXs [37]. Human
muscle is known to contain approximately 15 different PLA2 isoforms that are either Ca2+-sensitive or
Ca2+-insensitive [38]. The Ca2+-independent and dependent enzymes are supposed to produce ROS
under resting and activity conditions, respectively [39].
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3. Effects of ROS on Force Generation and Muscle Atrophy
In unfatigued muscle, intracellular ROS appear to be essential for normal force generation.
Low-level ROS supplementation even increases force production [40]. A stronger increase of ROS
due to intense exercise leads to a variety of adaptations of the muscle cells. Dependent upon the ROS
concentration, duration of ROS exposure and training status of the individual, ROS can have beneficial
and detrimental effects (Figure 2). Thus, a single bout of exhaustive exercise has been shown to cause
oxidative damage in untrained persons while in trained subjects, no such effects are observed due to an
increased resistance of such persons to oxidative stress [41]. Strong increases in ROS after strenuous
exercise, aging and/or disease (e.g., chronic heart failure, COPD, cancer) can cause contractile dysfunction
and muscle atrophy, which both promote muscle weakness and fatigue [3,42].
Figure 2. Deleterious and beneficial effects of exercise-induced ROS increase. Exercise
produces ROS and whether they are beneficial or detrimental to health is dependent upon
the ROS concentration, duration of ROS exposure and training status of the individual.
A single bout of exhaustive exercise leads to strong increases of ROS, which cannot be
buffered by endogenous antioxidants, particularly in untrained individuals. This results in
severe oxidative damage, including muscle weakness and fatigue, DNA mutations, lipid
peroxidation, mitochondrial dysfunction and apoptosis/necrosis. Trained persons have a higher
level of adaptation and less health risks. ROS produced during regular exercise continuously
increase the level of adaptation by improving antioxidant capacity, mitochondrial biogenesis,
insulin sensitivity, cytoprotection and aerobic capacity of skeletal muscle.
3.1. Contractile Dysfunction
Contractile dysfunction may result from oxidative modifications of a variety of proteins in diverse
intracellular components [43,44]. However, our understanding of the processes involved is still limited
Biomolecules 2015, 5 361
and many data are equivocal. In the sarcoplasmic reticulum, the ryanodine receptor (RyR), which is
the Ca2+ release channel, was shown to be oxidized by ROS and it was hypothesized to contribute to
muscle fatigue [26–28]. However, other work demonstrated that this oxidation resulted only in increased
Ca2+-induced Ca2+ release, whereas the Ca2+ release triggered by action potentials was not affected [45,46].
From this it was inferred that ROS-mediated effects on Ca2+ release in the sarcoplasmic reticulum
are unlikely to contribute to muscle fatigue. This goes in line with recent findings that demonstrate that
the mitochondrial antioxidant SS-31 restored the decrease in sarcoplasmic reticulum Ca2+ release while
force recovery was not improved [47].
Similar uncertainty surrounds the effect of ROS on the force generating myofilaments. Initial data
have shown that brief exposure to low concentrations of H2O2 increased force by 27%, while a longer
exposure results in force decline [48]. By contrast, a short exposure of skinned fibers to 10 mM H2O2
had no effect on maximum force [45], while longer exposure to 50 mM H2O2 inhibited contractility [49].
Although the exact mechanisms are unknown, it is generally assumed that changes in force generation
are the result of changes in the myofibrillar Ca2+ sensitivity [8,44,47]. In this regard, it was most
recently suggested that troponin I, which is involved in sensing the intracellular Ca2+ levels, is a target
of ROS. Oxidized cysteine residues of troponin I can react with the antioxidant glutathione, which
helps protect the molecule from oxidative stress and make the contractile apparatus much more sensitive
to Ca2+ [50]. However, it must be mentioned that ROS may also lead to changes in the contractile
proteins. In this regard, it was demonstrated that H2O2 is able to modify the S1 fragment in the myosin
head, which then leads to a restriction of the myosin-actin dynamics in the presence of ATP [49].
3.2. Muscle Atrophy
Besides their effects on the contractile kinetics, ROS are also able to modulate various signaling
pathways, such as calcium, protein tyrosine kinases and phosphatases, serine/threonine kinases, and
phospholipases [51]. This then leads to changes in gene expression, cell function, metabolism or cell damage.
Chronic oxidative stress is associated with an increase in protein loss and muscle atrophy. High ROS
levels cause a sustained activation of NF-B and of FoxO which then activate two muscle-specific
E3 ubiquitin ligases, atrogin-1 or muscle atrophy F-box (MAFbx) and muscle RING (Really Interesting
New Gene)-finger protein 1 (MuRF-1) [52]. MAFbx and MuRF-1 then degrade various proteins,
such as titin, nebulin, troponin, myosin-binding protein C, myosin light chains 1 and 2 and myosin
heavy chain [53,54]. Recently, it was demonstrated that excessive oxidative stress also enhances
the transcription factor C/EBP homology protein (CHOP). This transcription factor also enhances
expression of MuRF1, which again results in increased protein degradation [35].
4. Antioxidants in Muscle
4.1. Enzymatic and Nonenzymatic Antioxidants
Muscle activity increases ROS but simultaneously also the body’s antioxidant defense system.
These molecules are able to neutralize free radicals by accepting the unpaired electron and thereby
inhibit the oxidation of other molecules. Depending on the oxygen consumption rate, cells constitutively
express different levels of antioxidant enzymes, including mitochondrial antioxidant manganese
Biomolecules 2015, 5 362
superoxide dismutase (Mn-SOD, SOD2), cytosolic copper-zinc superoxide dismutase (Cu, Zn-SOD, SOD1),
glutathione peroxidase (GPX) and catalase (CAT), and the nonenzymatic antioxidant glutathione
(GSH) [55] (Figure 1).
GSH is the most abundant nonprotein thiol in cells with intracellular concentrations of 1–15 mM [56].
It plays a major role in the detoxification of electrophilic xenobiotics, such as chemical carcinogens,
environmental pollutants, and the inactivation of endogenous ,-unsaturated aldehydes, quinones,
epoxides, and hydroperoxides, which are formed as secondary metabolites during oxidative stress via
members of the glutathione transferase family [57]. It also protects from oxidative stress by reducing
hydrogen peroxide and organic peroxides levels via a reaction catalyzed by GSH peroxidase thus
keeping the intracellular environment in the reduced state [55,58]. In addition, GSH is a substrate for
dehydroascorbate reductase enabling the recycling of ascorbic acid, and it is a scavenger of hydroxyl
radicals and singlet oxygen [59].
The aforementioned enzymatic reactions lead to the oxidation of GSH to glutathione disulfide
(GSSG). This molecule can inactivate a number of enzymes by reacting with protein thiols leading to
the formation of mixed disulfides (e.g., [60]). To avoid damage to intracellular constituents, GSSG is
efficiently reduced to GSH by glutathione reductase (GR) utilizing NADPH. This action of GR is
also very important during and after exercise in which a substantial amount of GSH is oxidized due to
the elevated ROS levels to keep the GSH/GSSG ratio constant thereby maintaining homeostasis.
Furthermore, exercising skeletal muscle appears to increase GSH import from plasma [61,62], and
liver can synthesize GSH de novo and supply it [58]. But also increased muscle glutathione synthetase
activities have been observed after treadmill training [63]. These exercise responses are tissue- and
fiber-specific [64].
The antioxidant enzymes SOD, CAT and GPX are the primary defense against ROS generated
during exercise and increase in response to exercise [65,66]. Recent work identified thioredoxin
reductase-2 (TxnRd2) as another key player to decrease the exercise-induced content of mitochondrial
H2O2 in skeletal muscle [67]. The same authors have shown that TxnRd2 is also able to control
mitochondrial H2O2 levels after a high-fat, high-sucrose diet in the heart but not in skeletal muscle.
Antioxidant enzyme levels vary considerably with respect to muscle fiber types, i.e., type I muscle
fibers possess higher activity of all antioxidant enzymes than the type IIA and type IIB fibers [68].
4.2. Adaptive Responses to Exercise
In general, it was found that there is an exercise-induced increase in antioxidant protein levels and
antioxidant activity. Thus, endurance training in rats leads to an increase in Mn-SOD, GPX and CAT,
while the data on Cu, Zn-SOD are somewhat less clear [69–73]. Note that from the above studies, it is
likely that upregulation of these antioxidants is muscle- and/or fiber type-specific. Many studies have
shown that even an acute bout of exercise increases SOD activity in skeletal muscle ([74–77]; for
review see [55]), and it has further been shown that Cu, Zn-SOD and Mn-SOD contents are increased.
While the Cu, Zn-SOD enzyme activity gradually returns to resting levels within three days, Mn-SOD
activity and protein content continues to increase in the post-exercise period [78]. GPX activity after
acute exercise on the other hand appears to depend on the muscle type, i.e., GPX activity was
Biomolecules 2015, 5 363
increased a day after an acute bout of treadmill running to exhaustion in rat soleus but not tibialis
muscle [78], and CAT activity appears not to be altered by acute exercise [65].
ROS generated by acute exercise can lead to increased lipid peroxidation as measured by the
formation of malondialdehyde [79]. Interestingly, this effect was only found in liver and fast skeletal
muscle in the sedentary group, whereas the endurance-trained group did not show increases in lipid
peroxidation after exercise. Lipid peroxidation generates a vast number of oxidative lipid breakdown
products for which more or less specific tests are available (for review see [59]. Some investigators
determined plasma isoprostane levels in athletes performing either a 50 km ultramarathon [80] or
exercise for 2.5 h on a treadmill [81]. Peak levels of isoprostanes were found directly post-exercise,
followed by a return to baseline within one day or one hour, respectively. Other investigators found
increased levels of pentane in the breath after exercise [1], increased levels of lipid hydroperoxides [82],
conjugated dienes [83] and oxidative DNA damage as measured by 8-hydroxydeoxyguanosine [84] or
single cell gel electrophoresis [85]. The latter investigation gave results similar to those of Alessio and
Goldfarb [79], namely a significant reduction of oxidative DNA damage in the trained compared to the
untrained group indicating that exercise training causes an adaptive response to elevated oxidative stress
by increased antioxidant enzyme activity. Powers et al. [86] studied the influence of training and observed
significantly increased SOD activity in the soleus following exercise up to 60 min/d. Conversely,
training induced significant increases in GPX activity in slow gastrocnemius only, and the magnitude
of the GPX increase was directly related to exercise duration but relatively independent of intensity.
However, CAT activity was not increased in any muscle with training. In addition, Radák et al. [87]
observed decreased DNA damage and increased DNA repair levels as well as resistance against
oxidative stress of proteins in aged rat skeletal muscle upon training. The obviously paradoxical situation
that increased exercise-induced oxidative stress causes beneficial effects is interpreted in terms of
hormesis—beneficial effects of potentially harmful agents—which apart from providing an adaptation
to the damaging agent provide also systemic beneficial effects, including improved physiological
function, decreased incidence of disease and a higher quality of life [88]. However, the beneficial
effects appear to depend upon the duration of the exercise. While a single bout of exercise is suggested
to lead to a limited adaptive response, regular exercise appears to gradually increase the level of
adaptation by the repeated activation of antioxidant genes and proteins [32]. These authors hypothesize
that it is the increased ROS level that is the important stimulus for the muscle cells to adapt to chronic
exercise. The improved capabilities to decrease ROS may then provide a better protection from ROS
during subsequent trainings but also attenuate the aging process and promote health with increased
functional capacities [32]. Therefore, exercise is very similar to the adaptive ischemic preconditioning
response [89]. Restoration of perfusion to ischemic organs results in increased ROS levels that can
lead to tissue damage, myocardial infarction and stroke. However, such deleterious effects can be
avoided by short intermittent bouts of reperfusion in which the transiently elevated ROS levels are
important mediators of a cardioprotective response [89,90].
Important mediators of the adaptive responses are the adenosine monophosphate-activated protein
kinase (AMPK), the transcription factors NF-B, together with p38 MAPK, and members of the FoxO
transcription factor family [91–93]. At low ROS levels, they promote adaptation by increasing gene
expression of antioxidant enzymes, such as Mn-SOD and Cu, Zn-SOD, CAT and GPX1 [94]. High
antioxidant capacities then diminish the deleterious effects of subsequent increases in ROS [95].
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In addition, products of radical reactions are also suggested to be the mediator of this adaptation. Of
special interest in this context are lipid peroxidation products, in particular 4-hydroxynonenal (HNE),
which has been shown to both induce DNA damage [96] but also to be involved in the regulation of
cell proliferation and growth as well as necrotic or apoptotic cell death by its marked ability to modulate
several major pathways of cell signaling and, consequently, gene expression (for review see [97]).
With respect to antioxidant gene expression it has been shown to be one of the most effective activators
of nuclear factor erythroid-derived 2-like 2 (Nrf2) [98] which on stimulation dissociates from its
cytoplasmic inhibitor Keap1, translocates to the nucleus and transactivates antioxidant-responsive
elements (ARE)-dependent genes [99]. In addition, HNE has been demonstrated to cause mitochondrial
uncoupling and thus protection from ROS specifically via the induction of the uncoupling proteins
UCP1, UCP2 and UCP3 and the adenine nucleotide translocase [100].
4.3. Exogenous Antioxidants and Exercise
Apart from the endogenous antioxidants, which are obviously regulated by exercise, exogenous
antioxidants such as vitamin C, E, and carotenoids are taken up with the food or are used as dietary
supplements. The question therefore arises whether such supplements can be considered beneficial
during exercise. To address this question, Ristow et al. [73] investigated the effects of a diet supplemented
with vitamin C and E on exercise-induced insulin sensitivity as measured by glucose infusion rates
during a hyperinsulinemic, euglycemic clamp in previously untrained and pre-trained healthy young
men. Interestingly, exercise was found to increase parameters of insulin sensitivity (including
adiponectin) only in the absence of antioxidants in both previously untrained and pretrained individuals.
This was paralleled by increased expression of ROS-sensitive transcriptional regulators of insulin
sensitivity and ROS defense capacity, peroxisome proliferator-activated receptor  (PPAR) and
PPAR coactivators PGC-1 and PGC-1 only in the absence of antioxidants. Molecular mediators of
endogenous ROS defense (Mn-SOD, Cu, Zn-SOD and GPX) were also induced by exercise, and this
effect was again blocked by antioxidant supplementation. The authors concluded that exercise-induced
oxidative stress ameliorates insulin resistance and causes an adaptive response promoting endogenous
antioxidant defense capacity and that supplementation with antioxidants may preclude these health-promoting
effects of exercise in humans. It was demonstrated that exercise causes an activation of mitogen-activated
protein kinases (MAPKs: p38, ERK 1 and ERK 2), which in turn activates nuclear factor B (NF-B)
in rat gastrocnemius muscle and consequently the expression of important enzymes associated with
defense against ROS (SOD) and adaptation to exercise—endothelial nitric oxide synthase (eNOS) and
inducible nitric oxide synthase (iNOS) [101–103]. The expression of these enzymes can be inhibited
by allopurinol, an inhibitor of XO indicating also that the prevention of ROS formation causes an
inhibition of an adaptive response. The authors therefore conclude that in all likelihood, antioxidant
supplements should not be recommended before training as they interfere with muscle cell adaptation.
Thus, physical exercise is considered a double-edged sword: when practiced strenuously it causes
oxidative stress and cell damage; in this case application of antioxidants may be helpful. But when
practiced in moderation, it increases the expression of antioxidant enzymes and thus should be
considered an antioxidant [101,103]. Supportive evidence for this assumption comes from studies on
physical overtraining. Margonis et al. [104] examined the responses of oxidative stress biomarkers to
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a resistance training protocol of progressively increased and decreased volume/intensity in male test
persons and observed significantly increased levels of urinary isoprostanes (7-fold), serum levels of
thiobarbituric acid reactive substances (TBARS), protein carbonyls, CAT, GPX, and GSSG and
significantly decreased levels of GSH, the GSH/GSSG ratio, and total antioxidant capacity in blood
serum of over-trained individuals. Similarly, Palazzetti et al. [105] investigated the effects of overloaded
training (OT) with athletes exercising for a duathlon before and after a four week OT and found that
at rest conditions, OT induced an increased plasma GPX activity and a decreased plasma total
antioxidant status, while OT resulted in higher exercise-induced variations of blood GSH/GSSG ratios,
TBARS levels and decreased total antioxidant status in exercise conditions indicating that OT could
compromise the antioxidant defense mechanisms. By comparing the oxidative stress response in
control athletes and athletes with overtraining syndrome Tanskanen et al. [106] were further able to
show that exercise to exhaustion led to an increase in oxygen radical absorbance (antioxidant) capacity
and malondialdehyde in the controls but not in the over-trained athletes. Instead, over-trained athletes
showed negative correlations between oxygen radical absorbance capacity at rest and protein carbonyls
after exhaustive exercise indicating that increased oxidative stress may play a role in the pathophysiology
of overtraining syndrome. Although these observations are not yet conclusive they indicate that
adaptation to exercise is limited and that its protective effect can be exceeded leading to oxidative
stress that cannot be dealt with by the endogenous antioxidant system. Whether it is helpful to apply
exogenous antioxidants under such conditions as suggested still has to be elucidated.
5. Training-Induced Muscular Adaptation, PGC-1 and ROS
In addition to the above-described effects of exercise on contents and activities of antioxidant
enzymes, regularly performed exercise in the form of endurance training leads to well described
adaptations of the cardiovascular and muscular system. Important responses at the intramyocellular
level include increases in size and number of mitochondria as well as such in the activities of oxidative
enzymes [107–109]. In support of the increased oxidation of fatty acids, the content of intramyocellular
lipid is also elevated [110]. Endurance exercise is also known to improve insulin sensitivity and
muscular glucose uptake [108,111]. Recent research has demonstrated that ROS also have a beneficial
role in promoting these adaptive responses of muscle to training.
5.1. Role of PGC-1 in Exercise
In rodents and humans, it has been demonstrated that peroxisome proliferator-activated receptor
gamma coactivator-1 alpha (PGC-1) is a key regulator of the exercise-induced changes of muscle
fibers towards a slow phenotype, as well as in the protection from muscle atrophy [108,112,113].
Several studies have shown that PGC-1 is upregulated after high-intensity training [114–118].
Activation of PGC-1 is likely to occur by phosphorylation of the PGC-1 protein by p38 MAPK
together with NF-B [119], both of which are known to be activated by ROS [91,92]. PGC-1 has
been demonstrated to regulate lipid and carbohydrate metabolism, and to improve the oxidative
capacity of the muscle fibers by increasing the amount and activity of mitochondria through upregulation
of nuclear respiratory factors (NRF-1, 2) and mitochondrial transcription factor A (TFAM) [120,121].
Furthermore, PGC-1 regulates genes involved in the determination of muscle fiber type.
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Overexpression of PGC-1 increases the proportion of oxidative type I fibers [122] while PGC-1
knock-out (KO) mice exhibit a shift from oxidative type I and IIA toward glycolytic type IID/X and
IIB fibers [123]. This regulatory diversity of PGC-1 is enabled by its broad binding capacity to
transcription factors in various signaling pathways. PGC-1 has multiple binding sites for the interactions
with diverse coactivators. A domain between amino acids 200 and 400 interacts with the nuclear
receptors PPAR and NRF-1 [124], which are considered as master regulators of mitochondrial
biogenesis [125]. PGC-1 binds to and activates the transcriptional function of NRF-1 on the promoter
for TFAM, a direct regulator of mitochondrial DNA replication and transcription [120]. Another domain
that predominantly binds to nuclear hormone receptors such as ERR-, PPARs, RXR, glucocorticoid
receptor, HNF4, and probably others, is an LXXLL sequence in the N-terminal region of PGC-1 [124].
This sequence is necessary for the coactivation of the nuclear receptor liver x receptor  (LXR) [126].
The transcription complex of LXR and PGC-1 then activates fatty acid synthase (FAS), a multifunctional
enzyme that catalyzes all reactions required for the de novo biosynthesis of lipid [127]. The binding
site of the nuclear receptor estrogen-related receptor  (ERR-) is also in the LXXLL region of
PGC-1 [124]. The transcription complex formed by ERR- and PGC-1 induces the expression of
vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis [128,129]. Between
amino acids 400 to 500 of the PGC-1 protein is the binding site for myocyte enhancer factor 2
(MEF2). This transcription factor is a key regulator of slow muscle identity [130]. MEF2 proteins are
activated through the calcium-regulated calcineurin signaling pathway [130,131]. When overexpressed,
MEF2C promotes the formation of slow fibers, thus enhancing running endurance in mice [132].
Genetic deletion of Mef2c has been shown to block activity-dependent (exercise-induced) fast-to-slow
fiber type transition [132]. This is in line with the proposed role of PGC-1 in such transitions.
Muscle-specific overexpression of PGC-1 has been shown to evoke a transition of glycolytic type II
in oxidative type I fibers [122]. This shift is initiated by the formation of a PGC-1/MEF2 transcription
complex, which then activates the expression of slow muscle genes [133]. Handschin et al. [123] have
shown that PGC-1 deficient mice display a significant shift from slow oxidative type I and fast
oxidative IIA toward fast glycolytic type IIX and IIB fibers, resulting in a reduced endurance capacity.
5.2. PGC-1 Regulates ROS Defense
It has been shown that oxidative stress increases the expression of PGC-1 [134]. Similarly,
depleting the endogenous antioxidant glutathione augments exercise-mediated induction of PGC-1
expression [135]. Upregulation of PGC-1 possibly involves the transcription factor Cre-binding protein
(CREB) [136]. PGC-1 then induces an increase of ROS-detoxifying enzymes, including GPX1 and
Mn-SOD [136]. Only recently, new light has been shed on the molecular mechanisms involved in
antioxidant activation. Therefore, it is highly likely that PGC-1 binds to ERR- and activates the
NAD+-dependent histone deacetylase silent information regulator 3 (SIRT3) in the mitochondrial
matrix [137]. SIRT3 is also known to regulate ROS production by directly binding and deacetylating
mitochondrial complex I and II [138,139]. Previous studies have also shown that SIRT3 is able to
deacetylate the mitochondrial enzyme Mn-SOD, thereby promoting its antioxidative activity [140–142].
Thus, it appears that PGC-1 is a powerful suppressor of ROS production mainly by upregulation of
antioxidant expression. Correspondingly, it was demonstrated that PGC-1 knock-out (KO) mice have
Biomolecules 2015, 5 367
reduced expression levels of Mn-SOD, Cu, Zn-SOD and GPX1 and are thus more sensitive to oxidative
stressors [136]. By contrast, overexpression of PGC-1 enhances antioxidant defense by upregulation
of Mn-SOD expression and a higher catalase activity [143]. Further, PGC-1 increases the expression
of uncoupling proteins 2 and 3 (UCP2, UCP3) and thereby concomitantly reduces mitochondrial ROS
production [144].
6. Conclusions
There is rapidly growing evidence that ROS have both positive and negative effects in contracting
skeletal muscle cells. The deleterious effects such as a reduction of force generation and increased
muscle atrophy appear to occur particularly after non-regular strenuous exercise, while regular training
has positive effects by influencing cellular processes that lead to increased expression of antioxidants.
These molecules then provide a better protection from ROS during subsequent trainings. However,
a diet supplemented with exogenous antioxidants such as vitamins appears to prevent health-promoting
effects of physical exercise in humans. The exercise-induced production of ROS may also be an
important signal to activate PGC-1, a key player in the adaption of muscle cells to exercise.
Author Contributions
Both authors contributed interactively to explore the background literature and to the writing
procedure. Peter Steinbacher took responsibility for preparing the final version of the manuscript.
Abbreviations
AMPK adenosine monophosphate-activated protein kinase
ARE antioxidant-responsive element
CAT catalase
CHOP C/EBP homology protein
CREB Cre-binding protein
Cu, Zn-SOD copper-zinc superoxide dismutase
eNOS endothelial nitric oxide synthase
ERR- estrogen-related receptor
FAS fatty acid synthase
GPX glutathione peroxidase
GR glutathione reductase
GSH glutathione
GSSG glutathione disulfide
HNE 4-hydroxynonenal
IFN- interferon-
IL-1 interleukin-1
iNOS inducible nitric oxide synthase
LXR nuclear receptor liver x receptor 
MAFbx muscle atrophy F-box
Biomolecules 2015, 5 368
MAPK mitogen-activated protein kinase
MEF2 myocyte enhancer factor 2
Mn-SOD manganese superoxide dismutase
MuRF-1 muscle RING-finger protein 1
NADPH nicotinamide adenine dinucleotide phosphate
NF-B nuclear factor-B
NOX nicotinamide adenine dinucleotide phosphate oxidase
NRF nuclear respiratory factor
Nrf2 nuclear factor erythroid-derived 2-like 2
OT overloaded training
PGC-1 peroxisome proliferator-activated receptor- coactivator-1
PLA2 phospholipase A2
PPAR peroxisome proliferator-activated receptor
ROS reactive oxygen species
RyR ryanodine receptor
SIRT3 silent information regulator 3
TBARS thiobarbituric acid reactive substance
TFAM mitochondrial transcription factor A
TNF tumor necrosis factor
TxnRd2, thioredoxin reductase-2
UCP uncoupling protein
VEGF vascular endothelial growth factor
XO xanthine oxidase
Conflicts of Interest
The authors declare no conflict of interest.
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... This partially decreases physiological functions and results in limited adaptive responses. However, regular exercise, due to the constant shift between exercise and rest periods, leads to the induction of antioxidant and damage repair systems resulting in protection against oxidative stress and attenuation of the aging process, leading to health promotion [11,33]. This is analogous to the hormesis phenomenon classically known from toxicology. ...
... Already 20 years ago circulating IL-6 has been shown to increase following endurance exercise [62] and muscle as its source was recognized in 2000 [63]. Today it is well established that IL-6 is secreted by contracting skeletal muscle in the absence of muscle damage and that it plays important roles in the metabolic regulation of not only muscle itself but also in other organs such as liver, adipose tissue and pancreatic ß-cells [33]. IL-6 is the reference cytokine of the IL-6 family that includes also IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), and cardiotrophin-like cytokine (CLC). ...
... IL-6 is the reference cytokine of the IL-6 family that includes also IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), and cardiotrophin-like cytokine (CLC). Members of this family signal via the ubiquitously expressed transmembrane protein gp130 (CD130) which in the case of IL-6 transmits the intracellular signal after association with the membrane-bound IL-6 receptor [33]. Although IL-6 is in principal defined as a pro-inflammatory cytokine , it has been shown to have beneficial effects not only on muscle formation but also on glucose homeostasis and lipid metabolism . ...
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Exercise training is well known to improve physical fitness and to combat chronic diseases and aging related disorders. Part of this is thought to be mediated by myokines, muscle derived secretory proteins (mainly cytokines) that elicit auto/paracrine but also endocrine effects on organs such as liver, adipose tissue, and bone. Today, several hundred potential myokines have been identified most of them not exclusive to muscle cells. Strenuous exercise is associated with increased production of free radicals and reactive oxidant species (ROS) as well as endoplasmic reticulum (ER)-stress which at an excessive level can lead to muscle damage and cell death. On the other hand, transient elevations in oxidative and ER-stress are thought to be necessary for adaptive improvements by regular exercise through a hormesis action termed mitohormesis since mitochondria are essential for the generation of energy and tightly connected to ER- and oxidative stress. Exercise induced myokines have been identified by various in vivo and in vitro approaches and accumulating evidence suggests that ROS and ER-stress linked pathways are involved in myokine induction. For example, interleukin (IL)-6, the prototypic exercise myokine is also induced by oxidative and ER-stress. Exercise induced expression of some myokines such as irisin and meteorin-like is linked to the transcription factor PGC-1α and apparently not related to ER-stress whereas typical ER-stress induced cytokines such as FGF-21 and GDF-15 are not exercise myokines under normal physiological conditions. Recent technological advances have led to the identification of numerous potential new myokines but for most of them regulation by oxidative and ER-stress still needs to be unraveled.
... However, acute exercise and subsequent recovery are marked by the disruption of homeostasis. For example, muscle damage and inflammation are increased after an exercise bout [1], with a possible increase in reactive oxygen species (ROS) in a manner dependent on the type, duration, and load of the exercise [2]. ...
... Notwithstanding, ROS at low levels, a phenomenon similar to hormesis, play an important role in exercise-induced physiological adaptation [8]. Conversely, excessive oxidative stress can result in impaired physical performance and maladaptive muscle recovery [2]. Photobiomodulation (PBM) is a low power, nonthermal delivery of photons in the visible or near infrared spectrum that stimulates, heals, regenerates, or protects tissues [9,10]. ...
... From the discovery of exercise-induced oxidative stress several decades ago, evidence has been gathered that ROS produced during exercise also have positive effects by influencing cellular processes that lead to increased expression of antioxidants. These molecules are particularly elevated in regularly exercising the muscle to prevent negative effects of ROS by neutralizing the free radicals (15). On the other hand, today herbal medicines are used along with various methods of sports activities for improving the oxidative stress markers. ...
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... NO is a double faced coin. In physiological concentrations, it is a scavenger of ROS, but in greater concentrations it forms the cytotoxic peroxinitrite radical and can contribute to fatigue (33) . ...
... The increased muscle resistance against mechanical stress, reduced activity of phospholipase A and reduced fragmentation in proteins carrying iron and zinc are others mechanisms contributing to decreasing the production of free radicals and the associated oxidative stress, as addressed in literature (10,23). ...
... For example, in specific conditions, such during exercise, il-6 demonstrates anti-inflammatory effects including an inhibitory effect on the production of tNF-alpha and the stimulation of anti-inflammatory cytokines such as il1ra and il-10. 75 on the other hand, a recent study found that sarcopenia in elderly was associated with higher il-6 and tNF-alpha levels in comparison to elder patients with conserved muscularity. 76 in order to assess the role of il-6 in cachexia, Baltgalvis et al. used a mouse model of colorectal cancer, the Apc Min/+ mouse model. ...
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... In some circumstances, such as in young or well-trained athletes, the oxidative stress can also induce an adaptive (somehow favorable) physiological response [32]. This is mainly attributable to the activation of specific redox-sensitive transcriptional pathways, which promote increased expression of endogenous antioxidants enzymes such as mitochondrial manganese-dependent superoxide dismutase (MnSOD), which finally turn into a decreased oxidative damage or an enhanced repair [33][34][35][36][37][38][39]. Several lines of evidence now attest that the antioxidant response is rapidly activated by oxidative stress during exhaustive exercise [40]. ...
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