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Redox modulation of mitochondriogenesis in exercise. Doesantioxidantsupplementation blunt thebenefits of exercise training? Mari Carmen Gomez-Cabrera, Salvador-Pascual, Helena Cabo, Ferrando, Jose Viña

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Received date: 9 January 2015 Revised date: 1 April 2015 Accepted date: 2 April 2015 Cite this article as: Mari Carmen Gomez-Cabrera, Andrea Salvador-Pascual, Helena Cabo, Beatriz Ferrando, Jose Viña, Redox modulation of mitochondriogenesis in exercise. Doesantioxidantsupplementation blunt thebenefits of exercise training?, Free Radical Biology and Medicine, http://dx. doi.org/10.1016/j.freeradbiomed.2015.04.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title:Redox

modulation

of

mitochondriogenesis

in

exercise.

Doesantioxidantsupplementation blunt thebenefits of exercise training? Authors: Mari Carmen Gomez-Cabrera, Andrea Salvador-Pascual, Helena Cabo, Beatriz Ferrando, Jose Viña. Affiliations: Department of Physiology. University of Valencia. Investigación Hospital Clínico Universitario/INCLIVA. Spain.

Running title: Redox modulation of mitochondriogenesis in exercise

Address for correspondence: Jose Viña Department of Physiology Faculty of Medicine Blasco Ibañez, 15 Valencia, Spain 46010 Tel (34) 96 386 46 54 Fax (34) 96 386 46 42 [email protected]

Key words: PGC-1α, vitamins, skeletal muscle, redox signaling

1

ABSTRACT Physical exercise increases the cellular production of reactive oxygen species (ROS) in muscle, liver, and other organs.

This is unlikely due to increased mitochondrial

production but rather to extra-mitochondrial sources such as NADPH oxidase or xanthine oxidase. We have reported a xanthine oxidase mediated increase in ROS production in many experimental models from isolated cells to humans. Originally, ROS were considered as detrimental and thus as a likely cause of cell damage associated with exhaustion. In the last decade, evidence showing that ROSact as signals has been gathered and thus the idea that antioxidant supplementation in exercise is always recommendable has proved incorrect.

In fact, we proposed that

exercise itself can be considered as an antioxidant because training increases the expression of classical antioxidant enzymes such as superoxide dismutase and glutathione peroxidase and, in general, lowering the endogenous antioxidant enzymes by administration of antioxidant supplements may not be a good strategy when training. Antioxidant enzymes are not the only ones to be activated by training. Mitochondriogenesis is an important process activated in exercise.

Many redox-

sensitive enzymes are involved in this process. Important signaling molecules like MAP Kinases, NF-κB, PGC-1α, p53, Heat Shock Factor, and others modulate muscle adaptation to exercise. Interventions aimed at modifying the production of ROS in exercise must be performed with care as they may be detrimental in that they may lower useful adaptations to exercise.

1. Historical overview of Exercise Redox Biology. Janus faced of ROS in exercise From an historical point of view free radicals have been considered damaging to tissues[1]. This idea has been entrenched in the mind of exercise physiologists for years. Figure 1 showsthe evolution of the main findings in exercise redox biology.Sixty years ago (in the 50's), electron spin resonance spectroscopy (ESR) was used to generate the first data showing that skeletal muscle contains free radicals[2]. In the 1970s, Brady et al.[3] and Dillard et al.[4]reported increased lipid peroxidation during exercise bothin rats and humans.The biological importance of this finding was unclear at the time. It was not until the early the 80's that researchers identified the first link between muscle function and free radical biology. ESR was again used to show that free radical content

2

is elevated in isolated frog limb muscles electrically stimulated to contract repetitively[5]. Figure 1

50’s

70’s

Skeletal muscle contains ROS

Exercise lipid peroxidation

Evidences against supplementation with antioxidants in exercise training

2008

80’s

Historical

ROS are generated during tetanic contractions

overview of ROS play a key role in cell signaling and adaptations to exercise

Exercise Redox Biology

1982

2000 Exercise antioxidant capacity in skeletal muscle

Vitamin E protects against ROS in muscle

90’s

Contraction-induced ROS influences muscle function and fatigue

1994

Role of GSH in regulating ROS in exercise

Antioxidant supplements in exercise

Negative effects 2010

2000

Positive effects 1990

1980

1970

1950

Shortly afterwards, a ground-breaking report was published showing a 2- to3-fold increase in free radical content of skeletal muscle studied in vivoin rats run to exhaustion[6]. These findings were associated with three aspects of fatigue that are now well-recognized: increased lipidperoxidation, decreased control of mitochondrial respiration,and decreased integrity of the sarcoplasmic reticulum[6]. The same study showed that vitamin E deficiency inflated these three changes, indicating that the exercise induced changes were sensitive to both free radical production and antioxidant buffering. Collectively, these studies stimulated interest in many laboratories to investigate whether vitamin E (and subsequently other antioxidant nutrients) could retard both tissue damage and muscle contractile dysfunction that occurs during some forms of exercise[7, 8]. These studies havecontinued to the present day. Other chainbreaking antioxidants or specific enzymatic inhibitors, such as allopurinol (a drug used 3

in clinical practice to inhibit xanthine oxidase, a free radical generating enzyme) have been recommended to athletes to prevent muscle damage during competition or in isolated sessions of exhaustive exercise [9-12] (See Figure 1).During the 90's different researchers highlighted the role of exhaustive exercise in the regulation of glutathione redox status in human and rat studies [13, 14]. We found a linear correlation betweenGSSG-to-GSH and lactate-to-pyruvate ratios. We took it to suggest that oxidative stress (evidenced by the oxidation of glutathione) is related to exhaustion (evidenced by an increase in lactate). Dietaryadministration of thiol-containing substances such as N-Acetylcysteine (NAC)or glutathione was recommended to the sport population to prevent cellular damage caused by strenuous physical exercise[15] and to inhibit muscle fatigue [16]. The idea that ROS are involved in normal muscle contraction dates back to the 90’s [17, 18]. It was Michael Reid who showed that the low levels of ROS present in skeletal muscle in basal conditions are a requirement for normal force production [19] and that antioxidant-mediated depletion of ROS from unfatigued skeletal muscle results in a depression of muscle force production[17, 18] (See Figure 1). This observation stimulated much interest in the possibility that ROS could contribute to muscle fatigue during prolonged exercise.

Antioxidants and fatigue Several research groupshave providedconvincing evidence that ROS contribute to muscle fatigue during submaximal contractions because scavenging themvia non enzymatic (allopurinol, NAC,deferoxamine, tiron and DMSO) or enzymatic mechanisms (PEG-SOD) [20, 21]reduces fatigue. This has been observed in isolated muscles[22, 23], in intact animals[24], and in humans[25].It appears, however, that antioxidant scavengers do not show effectiveness in delaying fatigue when muscle contractions are near maximum [26]. The effectiveness of dietary antioxidants (such as vitamin E) on fatigue still is a matter of debate: some research groups have shown that scavenging ROS in muscle with vitamin E during exercise delays the onset of muscle fatigue [27],but others have not [28]. NAC is a thiol containing compound that acts by minimizing the exercise-induced oxidative stress through its actions as a cysteine donor in the maintenance of glutathione homeostasis and via direct scavenging of ROS[29]. A growing number of reports 4

indicate that administration of NAC delays muscle fatigue in electrically stimulated human limb muscle [16], cycling exercise[30], and handgrip exercise [31].Experimental evidencesuggests that NAC supplementation exerts an acute ergogeniceffect minimizing oxidant interference in theactivity of key ion transporters and ion channel proteins [32]. These studies have in general, but not always [33, 34],shown an ergogenicbenefit of NAC on performance within a variety of exercisemodalities[35, 36](See Figure 2). Figure 2 Exercise ANTIOXIDANTS

T

ROS

T

ANTIOXIDANTS

Skeletal markers of Oxidative Stress

Muscle adaptations to exercise

Activation of cell signaling pathways Transcription factors Transcriptional co-activators

Mitochondrial biogenesis

Cardiac and skeletal muscle hypertrophy

mRNA levels of: Enzymes Transporters

activity of DNA repairing enzymes

Health benefits of exercise

brachial artery vasodilatation systolic and diastolic blood pressure insulin sensitivity

Performance

Skeletal markers of Muscle damage in running speed in endurance time Skeletal muscle fatigue The ingestion of high doses of antioxidant vitamins in a regular basis may hamper athletes performance

strenght recovery in VO2max

Oral antioxidant supplementation is useful before competition (or tournaments)

For years researchers have recommended supplementing the sport population with antioxidant vitamins.It is estimated that ~60% of the US population usesdietary supplements at least occasionally, and ~40% usesupplements on regular basis (10, 11). The most commonly used supplements are multivitamins, vitamin C, vitamin E, and calcium [37]. Vitamin C is one of the biggest-selling nutrients in the U.S. vitamin and mineral market, with predominantly athletes topping the buyers’ list[38].

Reactive oxygen species ascells signals in skeletal muscle in exercise Hans Selye in his classical 1937 short letter to Nature[39]described how organisms react to various external stimuli (i.e., stressors). These reactions generally follow a programmed series of events and help the organism adapt to the imposed stress. At the 5

end of the 90's and thank to the studies performed by the research groups of Ji and Powers, the antioxidant response began to be considered a common cellular reaction to exercise-induced free radicals, these being considered as stressors(See Figure 1).These two groups independently found a significant increase in the muscle antioxidant capacity after acute or chronic exercise [40-42]. The protective effect of endurance training on the ability of the muscle to resist to injuries caused by oxidation due to an increased antioxidant defence was first reported by Salminen and Vihko [43]. Based on these reports we believe that a paradigm shift [44] took place in muscle physiology in the nineties. The assumption that oxidative stress was inevitably deleterious

to

muscle

cells

and

the

unquestionable

beneficial

effects

of

antioxidants,started changing.It was established that cells respond to increased oxidative stress by adaptive changes in the expression of a variety of proteins involved in maintenance of cellular integrity. This responseincluded variations in the rate of cell growth, changes in the length of the cell cycle, and marked adaptive responses in resistance to oxidative stress including the up-regulation of antioxidant enzymes, increased activity of DNArepairing enzymes, and expression of cytoprotective proteins such as heat-shock proteins [45] (See Figure 2).It is now recognized that these adaptations canprotect skeletal muscle against further bouts of (normally)damaging contractile activity [46]. In fact, animals frequently exposed to exercise (chronic training) have shown less oxidative damage after exhaustive exercise than untrained ones. This protection is largely attributed to the up-regulationof endogenous antioxidant enzymes such as Manganese Superoxide Dismutase (MnSOD), GlutathionePeroxidase (GPx), and ϒ-Glutamylcysteine Synthase (GCS) [47]. Lester Packer and Chandan Sen first highlightedthe potential role that ROS play in modulating cell signaling processes[48] (See Figure 1).These authors showed that critical steps in the signal transductioncascade such as protein phosphorylation and binding of redox-regulated transcription factors: Nuclear Factor κB

(NF-κB) and

Activator Protein 1 (AP- 1) to the promoterregion of genes,weresensitive to oxidants and antioxidants[48]. Jackson’s groupfound that cells responses to ROS could be placed into two overlapping groups, the cellular adaptation to ROS-induced damage, and the use of ROS as intracellular messengers [45, 49].They included heat-shock factor-1 (HSF-1) as a redox-regulated transcription factor. More specifically they found that oxidation of protein thiol groups occurs rapidly in skeletal muscle following exercise, 6

suggesting a role of oxidative stress in stimulationof production of Heat Shock Proteins[50]. Previous supplementation of animals with vitamin Esuppresses these positive adaptive responses. In 2001 first in vivo evidence showing that an acute bout of exercisein rats increased NF-κB and AP-1 binding their correspondingDNA domain of the MnSOD gene, in skeletal muscle,was reported by Ji's group

[51]. Shortly afterwards we showed

thatincreased NF-κB binding was accompanied by a cascadeof events in the exercised muscle, including increased IKKα and IκBαphosphorylation, decreased IKK and IκB content in thecytosol, and increased nuclear p50 content. The magnitude of this activation was similar to that found after treatment with lipopolysaccharide. Furthermore,our data showed that this activation was almost abolished by pyrrolidine dithiocarbamatetreatment(a well-knownantioxidant inhibitor of NF-κB) prior to exercise[52]. One year later we found that the inhibition of ROS formation, with allopurinol, prevented MAP kinases (p38 and ERK1/2), NF-κB activation and therefore the subsequent up-regulation of gene expression of MnSOD, and Nitric Oxide Synthases (iNOS andeNOS) in skeletal muscle[53]. These experiments led us to propose that exercise training acts as an antioxidant because it increases the expression of endogenous antioxidant enzymes [54]. NF-κB can regulate the expression of many genes involved in inflammation such as ICAM-1,IL-2, IL-8, and NLRP3 inflammasome [55]. The inflammatory response to exercise-induced muscle damage is mediated by NF-κB signaling that not only regulates the antioxidant response but also muscle protein synthesis [56]. It has been found that the use of anti-inflammatory and analgesic drugs (Ibuprofen and acetaminophen) attenuates the rate of muscle protein synthesis normally seen 24 h after high-intensity eccentric resistance exercise [57].

However, the long-term influence of this acute

response after resistance exercise for individuals who chronically consume these drugs has not been studied. Antioxidants blunts cell signaling in skeletal muscle During the first decade of 2000 it was shown that ROS play a key role in cell signaling and that antioxidant vitamins could prevent useful cellular adaptation to exercise in skeletal muscle (See Figure 1).These data have been confirmed with other antioxidants, such as NAC, by different research groups. For instance,Xie and co-workers 7

establishedthe essential role of ROS in cardiac-induced hypertrophy, andin transcriptional regulation of several growth-related genesin cardiac myocytes [58]. Petersen and co-workers found that NAC infusion blocked skeletal muscle cell signaling pathways (JNK and NF-κB p65 phosphorylation) and attenuated gene expression of proteins involved in adaptations to exercise (MnSOD)[59]. Other signaling pathways blunted by NAC during exercise are: phosphorylation of protein kinase B (Akt), mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase (p70s6K),and p38[60]. Intravenous infusion with NAC abolishes the exercise-induced increase in Na+-K+ pump αmRNA expression inhuman muscle[61]. This pump is critical to cell survival and muscle function. Supplementation with a combination of antioxidants to humans(140 mg/l of ascorbic acid, 12 mg/l of coenzyme Q10 and 1% NAC) has also been related with significantly altered mRNA levels of exercise-sensitive genes such asHexokinase(HK-II),GLUT-4 and Sterol-Regulatory Element-Binding Factor-1c(SREBF-1c) of the carbohydrate and lipid metabolism in skeletal muscle [62] Finally, supplementation with vitamins C and E attenuates the systemic IL-6 response and blunts the increase in IL-1R and cortisol associated to exercise. IL-6 derived from skeletal muscle is involved in the systemic immunological and metabolic response to exercise[63]. So at the beginning of the 21st century the studies clearly indicate that ROSactivate importantcell signaling pathways which occur followingcertain physiological stresses in tissues, such as muscle following exercise. These findings raised the questionof the convenience of the widespread practice of supplementation with antioxidant nutrients to the sport population(Figure 1).

2.

Effects of long term supplementation with dietary antioxidants in exercise

training. Figure 2shows thattreatment with different types of antioxidantscause beneficial effects when administered before one single session of exhaustive exercise. They have been involved in the prevention of muscle damage [9-11], oxidative stress [7, 13, 53, 64], muscle fatigue [16, 22, 24], and the decrease in muscle performance associated to ROS production during one bout of exhaustive exercise[30, 32, 65]. Based on this data, a significant

number

of

athletes,

especially 8

elite

athletes,

consume

dietary

antioxidantsupplements (mainly vitamins C and E) seeking beneficial effects on performance [8].However, the antioxidant efficacy maydepend critically on the agent administered. Although some reports have concluded that vitamin C and E supplementation decreases the markers of oxidation in tissues, positive effects of dietary antioxidants against contraction-induced muscle damage and muscularfatigue are not commonly observed[29, 66]. For instance, Kaminski and Boal[67] reported that a vitamin C supplement (3g)

before eccentric exercisereducedthe intensity of calf

soreness, while Francis and Hoobler reported that muscle soreness was not reduced by vitamin E supplementation (600 IU/day) 2 days before and 2 days after a damageinducing eccentric protocol[68]. Another important aspect, apart from the type of antioxidant, is that the effects of the antioxidants can be paradoxical,depending on the dosage, or on whether the administration is chronic or acute. An acute administration of a certain type of antioxidant (such as allopurinol or NAC) before a high intensity sessionof exercise may be beneficial but its chronic administration caninterfere with cellular signaling functionsof ROS and with the improvements in performance. Finally, the training level of the individuals taking the antioxidants is another important aspect to take into account in order to understand the variable effects of antioxidants. There are several evidences supporting this assumption. For instance,Jenkins and coworkers [69] reported that highly trained subjects had significantly greater musclecatalase and superoxide dismutase activities than those subjects in a lowmoderate fit group.Robertson et al [70] examined the antioxidant status of trained runners and sedentary individuals and found that the antioxidant capacity was enhanced in the runners. Toskulkao and Glinsukon found that trained runners had higher erythrocyte enzyme activity (e.g. MnSOD, GPx, and catalase) than did untrained subjects [71]. Based on these studies, the fittest athletes who have greater antioxidant enzymeactivities and do not show an increase in oxidative stress parameters after exhaustive exercise do not seem to need antioxidant supplements during training. In reviewing the literaturewe have found that different research groupshave used a variety of supplementation strategies in subjects or animals with different training status, with variations

in

dosage,

types

of

exercise,

timing,

duration

and

type

of

supplementation[72]. This makes the comparison between studies very difficult.In any

9

case care must be taken before prescribing antioxidants to athletes and each case much be studied individually.

Ergogenic effects of long term administration of antioxidants in exercise training. In an excellent review published in 2012 by Nikolaidis and co-workers the authors performed an extensive review to evaluate whether antioxidant vitamin C and/or Esupplementation affects the favorable adaptations to exercise training. Only those studies in which supplementation was followed by more than three weeks (this time period is sufficient to allow exercise training adaptations to appear), with an appropriate control group and in which the authors measured clear biochemical and or physiological endpoints of chronic exercise were included in the analysis. Although the literature in the area of ROS and exercise has grown spectacularly in the last thirty years, studies showing ergogenic effects of dietary antioxidants are limited. As far as we know, only two studies have reported that vitamin-E-supplementation (50 IU/kg/day) inducesan ergogenic effect and promote favorable adaptations in rats trained for a long period of time (12 weeks)[73, 74]. We would like to highlight other twostudies recently published showing beneficial effects in performance after antioxidant supplementation even if they do not deal with dietary antioxidants. Slattery and coworkers foundthat9 days of oral supplementation with NAC improved cycling performance via an improved redox balance and promoted adaptive processesin well-trained athletes. Similar results have also recently been reported during repeated highintensityintermittent shuttle runs in trainedmen after a 6 day loading period with NAC [35]. However, in our opinion, the supplementation and training protocols in these two studies were not of sufficient duration tosignificantly impact training-induced physiological adaptations.Other studies have not supported the notion that long-term supplementation with dietary vitamins are ergogenic, the majority being in favor of not having an effect [75-92]or even inducing a performance impairment[93-98].

Negative effects of long term administration of antioxidants in exercise training As early as 1971, it was shown that vitamin E supplementation (400 IU/d for 6 wk) caused unfavorable effects on endurance performance in swimmers [93]. The authors literally concluded: “There is no evidence here to suggest that vitamin E has any beneficial effect on endurance performance. Indeed the evidence, if anything, suggests 10

that the vitamin has an unfavorable effect”.In 1986 Packer and co-workers found that running time to exhaustion and activities of several mitochondrial enzymes in guinea pigs were greatly reduced in animals supplemented with large doses of ascorbate compared with unsupplemented controls[94]. In 2002, it was shown that supplementation of racing greyhounds with 1g vitamin C a day for 4 weeks significantly slowed their speed [95]. Moreover, in a human study, the negative effects of ascorbic acid supplementation on the adaptive responses of endogenous antioxidant enzymes and stress proteins were demonstrated [96]. In this scenario, as stated earlier, we consider that exercise training acts as an antioxidant since the adaptation in the expression of antioxidant enzymes are dependent on the exercise-induced increase in ROS [54]. Furthermore, two independent studies have shown that supplementation with ascorbic acid[97]or a mix of antioxidants [98]does not preserve muscle function but hinders the recovery process thereby being detrimental for future performance.Malm and coworkers showed, in two consecutive studies, the deleterious effects of ubiquinone-10 supplementation on the performance of humans after a high-intensity training program [99, 100]. This was followed by a study published by Richardson and co-workersthat reported reductions in vascular function in healthy males taking an acute dose of vitamins C and E. Theyfound reduced brachial artery vasodilatation during a submaximal forearm handgrip test in the supplemented group when compared with placebo [101]. Moreover, the same research group found that antioxidant administration prevented the exercise training (6 weeks) reduced systolic and diastolic blood pressure returning old individuals to a hypertensive state and blunting training-induced improvements in flow-mediated vasodilation[102]. Reductions in exercise-induced redistribution of blood flow to skeletal muscles have been shown to reduce exercise capacity [103]. Thus, since vitamin C may reduceexercise-induced blood flow, it may decrease exercise capacity and performance[104]. Copp et al. [105]found that the combination of tempol and vitamin C, given immediately before exercise,impaired the rat’s performance due to reductions in muscle blood flow and mitochondrial oxygen utilization at rest and during contractions.Hellsten's group has recently reported thatthe training-induced improvements in blood pressure, blood cholesterol, maximal oxygen uptake and plasma lipid profile are lost when old individuals are supplemented with the antioxidant resveratrol (250 mg/day trans-resveratrol). These authors concluded that removal of ROS via resveratrol treatment may limit training-induced adaptations and 11

questioned whether, in general, the level of ROS formation in aged men indeed is detrimental

to

cardiovascular

health

as

previously

proposed[106].

Furthermore,Braakhuis et al. [107] observed that supplementationwith 1000 mg/day of vitamin C for 3 weeks slowedfemale runners during training, although no differenceswere found in a 5 km time trial or in an incrementaltreadmill test after the intervention period. In this context radicals may be seen as beneficial as they act as signals to enhance adaptations, rather than deleterious as they are when cells are exposed to high levels of these radicals. The fact that ROS play an important role in promoting adaptations in muscles represents a fundamental change in biological understanding whereby in the past it was assumed that even low dose of ROS were universally harmful. Thus, ingesting high doses of antioxidant vitamins in a regular basis in an attempt to enhance muscle performance, individuals may actually be retarding or even hampering the adaptations to exercise training [66].

3. Redox regulation of mitochondrial biogenesis in exercise training. Effect of antioxidant supplements. Mitochondrial biogenesisinvolves the orchestrated expression of the mitochondrial genome and the nuclear genes that encode mitochondrial proteins [108]. These organelles cannot be made de novo but replicate by a mechanism that recruits new proteins, which are added to pre-existing sub compartments and protein complexes. This process leads to a growth in size of the organelle that is subsequently divided by a fission process[109]. Normally, mitochondrial biogenesis is activated by changes in physiological state that require increases in the rates of ATP utilization approaching the existing capacity of the cells to produce it[110] (See Figure 3) .

12

Figure 3

Stimulus/ stimulation

Morphological adaptations

Muscle contraction Endurance training

Physical activity

Nutrient Availability

Circadian rhythms

Infectious agents

Hypoxia

Temperature

Hormones

Aging

Diseases

Growth mitochondrial size

ROS

RONS

Increase mitochondrial biogenesis

ATP

Ca2+

Byosinthetic processes Lipid

Metabolic function

Cell cycle

Heme

Proliferation

Cholesterol

Apoptosis

Nucleotide synthesis

Evidences showing that the mitochondrial size, number, and/or volume are increased within skeletal muscle in response to endurance training were found in the sixties[111]. Endurance training, employing an appropriate duration per day, frequency per week, and submaximal intensity per exercise bout, can producean increase in mitochondrial content, usuallyranging from 50 to 100% within 6 weeks [108]. The approximate 6 weeks’ time period required to achieve a new steady-state mitochondrial content in response to endurance training clearly does not reflect the early molecular events that ultimately lead to the measurable morphological changes. Indeed, changes inmitochondrial protein content can be apparentat much earlier time points. This means that a continuous exercise stimulus isrequired to maintain mitochondrial content[108]. Endurance training is associated not only with an increase in mitochondrial volume density and enzyme activity in oxidative metabolism, but with an improvement of coupling and regulatory properties of mitochondrial respiration in human skeletal muscle[108]. Although the complete pathway controlling mitochondrial biogenesis has not been elucidated, progress in identifying key players has been made in the last few years.A number of transcription factors regulate mitochondrial biogenesis. However, despite the 13

complexity of the various signalling pathways that regulate mitochondrial biogenesis, they all seem to share the PPARγ co-activator 1 (PGC-1) family of transcription factors [112].PGC-1α was initially identified as a cold-inducible co-activatorfor PPARγ in brown fat and skeletal muscle by Spiegelman's research group[112].Pioneering studies by Scarpulla and co-workers identified the transcription factors that recognize the promoters of mitochondrial OXPHOS genes, leading to the identification of nuclear respiratory factor 1 (NRF-1) and GA-binding protein (GABP), also known as NRF2[113]. The coordination between the mitochondrial and the nuclear genome is achieved by nuclear proteins, such as mtTFA[114]. MtTFA can be considered the most important mammalian transcription factor for mtDNA because it stimulates mitochondrial DNA transcription and replication (See Figure 4).

Figure 4

EXERCISE TRAINING

ROS

ANTIOXIDANTS

PGC 1 α

GPx MnSOD

NRF-1

mRNA mtDNA mtTFA mtTFA

MITOCHONDRIAL BIOGENESIS

14

The mainsignaling pathways that regulate PGC-1α expression are the Ca2+-Induced Pathways and the 5’-AMP-activated protein kinase (AMPKs) [115]. Several lines of evidence support the role ofthese molecules in mitochondrial biogenesisand function. We are going to focus on the role of RONS as signals involved in mitochondrial adaptations to exercise. The role of ROS as stimulants of mitochondrial biogenesis was first proposed by Davies and co-workers in 1982 when they demonstrated for the first time in vivo that ROS are produced in skeletal muscle after an exhaustive bout exercise. In the last sentence of the discussion section of the manuscript stated that: "It is tempting to propose that exercise induced free radicals may cause limited damage to mitochondrial membrane which, in a chronic training situation, may be the initiating stimulus to mitochondrial biogenesis" [116]. In 2006 it was shown that PGC-1α and PGC-1βwere powerfully induced by H2O2, and that these coactivators regulated a complex and multifaceted ROS defense system such as MnSOD, catalase, and GPx, as well as UCP-2 and UCP-3[117]. Furthermore,PGC-1a KO mice had a greatly increasedsensitivity to damage by oxidative stress[117]. In skeletal muscle contraction leads to an enhanced formation of ROS and an elevation in PGC-1α, UCP-3 and HKII mRNA content which is abolished in the presence of antioxidants, suggesting the role of ROS on the contraction induced increase in expression of these genes in skeletal muscle[118]. Ji and coworkersfound that an acute bout of exercise that increased ROS productionstimulated PGC-1α expression and other key proteinsin the mitochondrial biogenic pathway.Inhibition of the free radical generating enzyme xanthine oxidase withallopurinol severelyattenuated exercise activation of the PGC-1α signaling pathway, thusproviding strong evidence that mitochondrial biogenesis in skeletalmuscle is controlled at least in part by a redoxsensitive mechanism[119]. In 2008 we found thattraining-induced expression of transcription factors involved in mitochondrial biogenesis such as PGC-1, NRF-1 and mTFA and of a marker of mitochondrial content such as cytochrome c was blunted by a high dose ofvitamin C administration (500 mg/Kg in the animal study). We also found that training (6 weeks) caused an increase in two majorantioxidant enzymes (MnSOD and GPx) in skeletal muscle and that vitamin C prevented these beneficial effects. Endurance capacity is directly related to the mitochondrialcontent. This variable was seriously hampered by 15

antioxidantsupplementation in our study, whereas VO2max, which isdependent also on the cardiovascular system adaptations, was notsignificantly affected[120]. We concluded that supplementation with high doses of vitamin C lowers training efficiency because it jeopardizes mitochondrial biogenesis and questioned the common practice of taking vitamin C supplements during training (See Figure 4). One year later Ristow and colleaguesshowed that exercise training (4 weeks) increased parameters of insulin sensitivity inthe absence of antioxidants (1000 mg/day of vitamin C and 400 IU/day) in both previously untrained and pre-trained

individuals[121]. This was paralleledby

increased expression of ROS-sensitive transcriptional regulatorsof insulin sensitivity and ROS defense capacity: PPARγ, PGC-1α and PGC-1β only in the absence of antioxidants.

The antioxidant enzymes MnSOD and CuZnSOD and GPx were

alsoinduced by exercise, and this effect too was blocked by antioxidantsupplementation. In this case supplementation with antioxidants precluded the health-promoting effects of exercise in humans through the inhibition of exercise-induced ROS generation. In 2011 and in a rat study, it was found that Vitamin E (1000 IU/Kg) and α-lipoic acid (1.6 g of α-lipoic acid per kilogram offood administered) supplementation suppresses skeletal muscle mitochondrial biogenesis, regardless oftraining status.Antioxidant supplementation reducedPGC-1α mRNA, PGC-1α and COX IV protein, and citrate synthase enzyme activity in both sedentary and exercise-trainedrats (14 weeks). These findings suggest that prolonged antioxidant supplementation could potentially impair the endogenousmetabolic and redox status of skeletal muscle in sedentarypeople on top of preventing some of the beneficial adaptations to exercise training[122]. In 2014 Venditti and co-workers have found that the metabolic adaptations to training are generally prevented by antioxidant supplementation [123]. The same year, in a double-blind, randomised, controlled trial it was found that daily vitamin C (1000 mg) and E (235 mg) supplementation attenuates increases in markers of mitochondrial biogenesis following endurance training (11 weeks)[124]. Finally, the redox sensitivity of PGC-1α has been recently demonstrated both in heart and in skeletal muscle. Bouitbir and co-workers[125]showed that statins increased ROS production differently in heart and skeletal muscle and could be represented as a double edged sword: they are beneficial by playing an important role in cell signalling involved in antioxidant defence network in cardiac muscle, but could be harmful by inducing excessive oxidative stress in vulnerable skeletal muscle. It was proposed that activationof PGC-1 expression could 16

be triggered by low doses of mitochondrial ROS generation following atorvastatin treatment, in order to counteract stressor challenges and hence re-establish homeostasis. In this regard, a new concept proposesthat mitochondrial biogenesis can be triggered by low doses of ROS to counteract stressor challenges and to re-establish homeostasis. This concept is called mitochondrial hormesis or ‘mitohormesis’.It implies that different concentrations of the same agent, in this case ROS, may exert a nonlinear or J-shaped response, which has been known since the 1950s as ‘hormesis’[126]. This concept opposes the traditional view of ROS as being invariably harmful and unwanted byproducts of mitochondrial metabolism; rather, it fosters the claim that ROS are essential, health-engendering signaling molecules. However, the debate on the benefits of antioxidants supplements during training is still controversial. Data showingbeneficial effects on muscle function of this type of widespreadpractice are elusive. Different research groups have found normal adaptations to exercise training in rats [86] and humans [127] despite supplementation with dietary antioxidants during the training period. However, in our opinion, the vast majority of recent experimental evidences in the field advises against this supplementation[128]. Certainly, few, if any, studies report that supplementation with antioxidants during training is advantageous.

CONCLUDING REMARKS ROS are important metabolites in muscle. They are produced in mitochondria but also from other sources like oxidases. Excess production of ROS causes molecular and even cellular damage but ROS produced in exercise have a physiological role, i.e. they behave as signals to modulate adaptations of muscle to exercise. Paramount among these adaptations is the change in the rate of mitochondriogenesis, which is dependent of oxidants-induced activation of factors such as PGC-1α and mtTFA. Moreover, exerciseinvolves the activation of the antioxidant genes such as GPx and MnSOD. In this respect we proposed that exercise itself can be considered as an ‘antioxidant’. Nutritional antioxidants such as vitamins, especially C and E, are usually taken by the general population and even more by elite athletes in an attempt to minimize muscle damage and to serve as ergogenic aidi.e. improve performance. However, recent evidence shows that antioxidants may, by lowering the physiological concentration of 17

ROS, hamper useful adaptations to exercise. In the literature, there is controversy in that some research groups support the favourable effect of antioxidant administration, but the majority of the scientific evidence is against recommending the indiscriminate use of antioxidant supplementation for the sport population. Our opinion is that one may take antioxidant supplements in competition, when ROS formation associated with exhaustion is likely to occur, but not take them in training when adaptations are sought. These desirable adaptations may not occur if physiological ROS levels are blunted by antioxidant supplementation.

CONFLICT OF INTEREST: The authors declare that no conflict of interest exists.

ACKNOWLEDGEMENTS: This work was supported by grants SAF2013-44663-R, from the Spanish Ministry of Education and Science (MEC); ISCIII2012-RED-43-029 from the “Red Tematica de investigacion

cooperativa

en

envejecimiento

y

fragilidad”

(RETICEF);

PROMETEO2014/056 from "Conselleria d’Educació, Cultura i Esport de la Generalitat Valenciana"; RS2012-609 Intramural Grant from INCLIVA and EU Funded CM1001 and FRAILOMIC-HEALTH.2012.2.1.1-2. The study has been co-financed by FEDER funds from the European Union.

LIST OF ABBREVIATIONS Akt: Protein Kinase B AMPK: 5’-AMP-activated protein kinase AP-1: Activator Protein 1 COX IV: Cytochrome c Oxidase CuZnSOD: Copper/Zinc Superoxide Dismutase DMSO: Dimethyl sulfoxide eNOS: Endothelial Nitric Oxide Synthase ERK: Extracellular signal-Regulated Kinase ESR: Electron spin resonance spectroscopy GABP: GA-binding protein GCS: Glutamylcysteine Synthatase GPx: Glutathione Peroxidase GSH: Reduced glutathione GSSG: Oxidized Glutathione HK: Hexokinase HSF-1: Heat-Shock Factor–1 IKK: IκB Kinase 18

IL-1R: Interleukin 1 Receptor IL-6: Interleukin 6 iNOS: Inducible Nitric Oxide Synthase IU: International Units JNK: c-Jun N-terminal Kinase MAPK: Mitogen Activated Protein Kinase MnSOD: Manganese Superoxide Dismutase mtDNA: Mitoch ndrial DNA mTOR: Mammalian target of rapamycin mtTFA: Mitochondrial transcription factor A NAC: N-Acetylcysteine NF-κB: Nuclear Factor κB NRF-1: Nuclear respiratory factor 1 NRF-2: Nuclear respiratory factor 2 OXPHOS: Oxidative Phosphorylation System PEG-SOD: Polyethylene Glycol Superoxide Dismutase PGC-1α: PPARγ co-activator 1α PGC-1β: PPARγ co-activator 1β PPAR: Peroxisome proliferator–activated receptors p70s6K: p70 ribosomal S6 kinase RONS: Reactive oxygen and nitrogen species SREBF-1c: Sterol-Regulatory Element-Binding Factor-1c UCP-(1-3): Uncoupling protein (1-3)

REFERENCES [1] Sies, H.; Cadenas, E. Oxidative stress: damage to intact cells and organs. Philos Trans R Soc Lond B Biol Sci 311:617-631; 1985. [2] Commoner, B.; Townsend, J.; Pake, G. E. Free radicals in biological materials. Nature 174:689-691; 1954. [3] Brady, P. S.; Brady, L. J.; Ullrey, D. E. Selenium, vitamin E and the response to swimming stress in the rat. J Nutr 109:1103-1109; 1979. [4] Dillard, C. J.; Litov, R. E.; Savin, W. M.; Tappel, A. L. Effects of exercise, vitamin E and ozone on pulmonary function and lipid eroxidation. J. Appl. Physiol. 45:927-932; 1978. [5] Koren, A.; Schara, M.; Sentjurc, M. EPR measurements of free radicals during tetanic contractions of frog skeletal muscle. . Period Biol. 82:399-401 1980. [6] Davies, K. J.; Quintanilha, A. T.; Brooks, G. A.; Packer, L. Free radicals and tissue damage produced by exercise. Biochem Biophys Res Commun 107:1198-1205; 1982. [7] Jackson, M. J.; Jones, D. A.; Edwards, R. H. Vitamin E and skeletal muscle. Ciba Found Symp 101:224-239; 1983. [8] Quintanilha, A. T.; Packer, L. Vitamin E, physical exercise and tissue oxidative damage. Ciba Found Symp 101:56-69; 1983. [9] Gomez-Cabrera, M. C.; Martinez, A.; Santangelo, G.; Pallardo, F. V.; Sastre, J.; Vina, J. Oxidative stress in marathon runners: interest of antioxidant supplementation. Br J Nutr 96 Suppl 1:S31-33; 2006.

19

[10] Gomez-Cabrera, M. C.; Pallardo, F. V.; Sastre, J.; Vina, J.; Garcia-del-Moral, L. Allopurinol and markers of muscle damage among participants in the Tour de France. Jama 289:2503-2504; 2003. [11] Sanchis-Gomar, F.; Pareja-Galeano, H.; Gomez-Cabrera, M. C.; Candel, J.; Lippi, G.; Salvagno, G. L.; Mann, G. E.; Vina, J. Allopurinol prevents cardiac and skeletal muscle damage in professional soccer players. Scand J Med Sci Sports; 2014. [12] Vina, J.; Gimeno, A.; Sastre, J.; Desco, C.; Asensi, M.; Pallardo, F. V.; Cuesta, A.; Ferrero, J. A.; Terada, L. S.; Repine, J. E. Mechanism of free radical production in exhaustive exercise in humans and rats; role of xanthine oxidase and protection by allopurinol. IUBMB Life 49:539544; 2000. [13] Sastre, J.; Asensi, M.; Gasco, E.; Pallardo, F. V.; Ferrero, J. A.; Furukawa, T.; Vina, J. Exhaustive physical exercise causes oxidation of glutathione status in blood: prevention by antioxidant administration. Am J Physiol 263:R992-995; 1992. [14] Sen, C. K.; Atalay, M.; Hanninen, O. Exercise-induced oxidative stress: glutathione supplementation and deficiency. J Appl Physiol 77:2177-2187; 1994. [15] Sastre, J.; Asensi, M.; Gascó, E.; Pallardó, F. V.; Ferrero, J. A.; Furukawa, T.; Viña, J. Exhaustive physical exercise causes oxidation of glutathione status in blood: prevention by antioxidant administration. Am. J. Physiol. 263:R992-R995; 1992. [16] Reid, M. B.; Stoik, D. S.; Koch, S. M.; Khawli, F. A.; Lois, A. A. N-acetylcysteine inhibits muscle fatigue in humans. J.Clin.Invest. 94:2468-2474; 1994. [17] Reid, M. B.; Khawli, F. A.; Moody, M. R. Reactive oxygen in skeletal muscle. III. Contractility of unfatigued muscle. J Appl Physiol 75:1081-1087; 1993. [18] Reid, M. B.; Moody, M. R. Dimethyl sulfoxide depresses skeletal muscle contractility. J Appl Physiol 76:2186-2190; 1994. [19] Reid, M. B. Invited Review: redox modulation of skeletal muscle contraction: what we know and what we don't. J Appl Physiol 90:724-731; 2001. [20] Supinski, G.; Nethery, D.; Stofan, D.; DiMarco, A. Effect of free radical scavengers on diaphragmatic fatigue. Am J Respir Crit Care Med 155:622-629; 1997. [21] Gomez-Cabrera, M. C.; Close, G. L.; Kayani, A.; McArdle, A.; Viña, J.; Jackson, M. J. Effect of xanthine oxidase-generated extracellular superoxide on skeletal muscle force generation. American Journal of Physiology:Regulatory, Integrative and Comparative Physiology Under review; 2009. [22] Barclay, J. K.; Hansel, M. Free radicals may contribute to oxidative skeletal muscle fatigue. Can J Physiol Pharmacol 69:279-284; 1991. [23] Moopanar, T. R.; Allen, D. G. Reactive oxygen species reduce myofibrillar Ca2+ sensitivity in fatiguing mouse skeletal muscle at 37 degrees C. J Physiol 564:189-199; 2005. [24] Shindoh, A.; Dimarco, A.; Thomas, A. Effect of N-acetylcysteine on diaphragm fatigue. J. Appl. Physiol. 68:2107; 1990. [25] Reid, M. B.; Stokic, D. S.; Koch, S. M.; Khawli, F. A.; Leis, A. A. N-acetylcysteine inhibits muscle fatigue in humans. J. Clin. Invest. 94:2468-2474.; 1994. [26] Reid, M.; Haak, K.; Francheck, K. Reactive oxygen in skeletal muscle: I. Intracelular oxidant kinetics and fatigue in vitro. J. Appl. Physiol. 73:1797; 1992. [27] Novelli, G. P.; Bracciontti, G.; Falsini, S. Spin-Trappers and vitamin E prolong edurance to muscle fatigue in mice. Free Radic. Biol. Me. 8:9; 1990. [28] Coombes, J. S.; Powers, S. K.; Rowell, B.; Hamilton, K. L.; Dodd, S. L.; Shanely, R. A.; Sen, C. K.; Packer, L. Effects of vitamin E and alpha-lipoic acid on skeletal muscle contractile properties. J Appl Physiol 90:1424-1430; 2001. [29] Powers, S. K.; Jackson, M. J. Exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production. Physiol Rev 88:1243-1276; 2008.

20

[30] McKenna, M. J.; Medved, I.; Goodman, C. A.; Brown, M. J.; Bjorksten, A. R.; Murphy, K. T.; Petersen, A. C.; Sostaric, S.; Gong, X. N-acetylcysteine attenuates the decline in muscle Na+,K+-pump activity and delays fatigue during prolonged exercise in humans. J Physiol 576:279-288; 2006. [31] Matuszczak, Y.; Farid, M.; Jones, J.; Lansdowne, S.; Smith, M. A.; Taylor, A. A.; Reid, M. B. Effects of N-acetylcysteine on glutathione oxidation and fatigue during handgrip exercise. Muscle Nerve 32:633-638; 2005. [32] Medved, I.; Brown, M. J.; Bjorksten, A. R.; Murphy, K. T.; Petersen, A. C.; Sostaric, S.; Gong, X.; McKenna, M. J. N-acetylcysteine enhances muscle cysteine and glutathione availability and attenuates fatigue during prolonged exercise in endurance-trained individuals. J Appl Physiol 97:1477-1485; 2004. [33] Medved, I.; Brown, M. J.; Bjorksten, A. R.; Leppik, J. A.; Sostaric, S.; McKenna, M. J. Nacetylcysteine infusion alters blood redox status but not time to fatigue during intense exercise in humans. J Appl Physiol (1985) 94:1572-1582; 2003. [34] Edwards, D. G.; Haymann, M. A.; Roy, M. S.; Kenefick, R. W. Oral N-Acetylcysteine supplementation does not improve cycling time trial performance. Faseb J 20:A811; 2006. [35] Cobley, J. N.; McGlory, C.; Morton, J. P.; Close, G. L. N-Acetylcysteine Attenuates Fatigue Following Repeated-Bouts of Intermittent Exercise: Practical Implications for Tournament Situations. Int J Sport Nutr Exerc Metab; 2011. [36] Slattery, K. M.; Dascombe, B.; Wallace, L. K.; Bentley, D. J.; Coutts, A. J. Effect of Nacetylcysteine on cycling performance after intensified training. Med Sci Sports Exerc 46:11141123; 2014. [37] Hathcock, J. N.; Azzi, A.; Blumberg, J.; Bray, T.; Dickinson, A.; Frei, B.; Jialal, I.; Johnston, C. S.; Kelly, F. J.; Kraemer, K.; Packer, L.; Parthasarathy, S.; Sies, H.; Traber, M. G. Vitamins E and C are safe across a broad range of intakes. Am J Clin Nutr 81:736-745; 2005. [38] Sauberlich, H. E. Pharmacology of vitamin C. Annu Rev Nutr 14:371-391; 1994. [39] Selye, H. A Syndrome produced by Diverse Nocuous Agents- Nature 138:32; 1936. [40] Ji, L.; Fu, R. G. Responses of glutathione system and antioxidant enzymes to eshaustive exercise and hidroperoxide. J. Appl. Physiol. 72:549-554; 1992. [41] Ji, L. L.; Fu, R.; Mitchell, E. W. Glutathione and antioxidant enzymes in skeletal muscle: effects of fiber type and exercise intensity. J Appl Physiol 73:1854-1859; 1992. [42] Vincent, H. K.; Powers, S. K.; Demirel, H. A.; Coombes, J. S.; Naito, H. Exercise training protects against contraction-induced lipid peroxidation in the diaphragm. Eur J Appl Physiol Occup Physiol 79:268-273; 1999. [43] Salminen, A.; Vihko, V. Endurance training reduces the susceptibility of mouse skeletal muscle to lipid peroxidation in vitro. Acta Physiol Scand 117:109-113; 1983. [44] Kuhn, T. S. The Structure of Scientific Revolutions. Chicago ; London: University of Chicago Press; 1962. [45] Jackson, M. J. Free radicals in skin and muscle: damaging agents or signals for adaptation? Proc Nutr Soc 58:673-676; 1999. [46] McArdle, F.; Spiers, S.; Aldemir, H.; Vasilaki, A.; Beaver, A.; Iwanejko, L.; McArdle, A.; Jackson, M. J. Preconditioning of skeletal muscle against contraction-induced damage: the role of adaptations to oxidants in mice. J Physiol 561:233-244; 2004. [47] Brooks, S. V.; Vasilaki, A.; Larkin, L. M.; McArdle, A.; Jackson, M. J. Repeated bouts of aerobic exercise lead to reductions in skeletal muscle free radical generation and nuclear factor kappaB activation. J Physiol 586:3979-3990; 2008. [48] Sen, C. K.; Packer, L. Antioxidant and redox regulation of gene transcription. FASEB J 10:709-720; 1996. [49] Jackson, M. J.; McArdle, A.; McArdle, F. Antioxidant micronutrients and gene expression. Proc Nutr Soc 57:301-305.; 1998. 21

[50] McArdle, A.; van der Meulen, J. H.; Catapano, M.; Symons, M. C.; Faulkner, J. A.; Jackson, M. J. Free radical activity following contraction-induced injury to the extensor digitorum longus muscles of rats. Free Radic Biol Med 26:1085-1091; 1999. [51] Hollander, J.; Fiebig, R.; Gore, M.; Ookawara, T.; Ohno, H.; Ji, L. L. Superoxide dismutase gene expression is activated by a single bout of exercise in rat skeletal muscle. Pflugers Arch 442:426-434; 2001. [52] Ji, L. L.; Gomez-Cabrera, M. C.; Steinhafel, N.; Vina, J. Acute exercise activates nuclear factor (NF)-kappaB signaling pathway in rat skeletal muscle. Faseb J 18:1499-1506; 2004. [53] Gomez-Cabrera, M. C.; Borras, C.; Pallardo, F. V.; Sastre, J.; Ji, L. L.; Vina, J. Decreasing Xanthine Oxidase Mediated Oxidative Stress Prevents Useful Cellular Adaptations to Exercise in Rats. J Physiol; 2005. [54] Gomez-Cabrera, M. C.; Domenech, E.; Vina, J. Moderate exercise is an antioxidant: upregulation of antioxidant genes by training. Free Radic Biol Med 44:126-131; 2008. [55] Juliana, C.; Fernandes-Alnemri, T.; Wu, J.; Datta, P.; Solorzano, L.; Yu, J. W.; Meng, R.; Quong, A. A.; Latz, E.; Scott, C. P.; Alnemri, E. S. Anti-inflammatory compounds parthenolide and Bay 11-7082 are direct inhibitors of the inflammasome. J Biol Chem 285:9792-9802; 2010. [56] Liao, P.; Zhou, J.; Ji, L. L.; Zhang, Y. Eccentric contraction induces inflammatory responses in rat skeletal muscle: role of tumor necrosis factor-alpha. Am J Physiol Regul Integr Comp Physiol 298:R599-607; 2009. [57] Trappe, T. A.; White, F.; Lambert, C. P.; Cesar, D.; Hellerstein, M.; Evans, W. J. Effect of ibuprofen and acetaminophen on postexercise muscle protein synthesis. Am J Physiol Endocrinol Metab 282:E551-556; 2002. [58] Xie, Z.; Kometiani, P.; Liu, J.; Li, J.; Shapiro, J. I.; Askari, A. Intracellular reactive oxygen species mediate the linkage of Na+/K+-ATPase to hypertrophy and its marker genes in cardiac myocytes. J Biol Chem 274:19323-19328; 1999. [59] Petersen, A. C.; McKenna, M. J.; Medved, I.; Murphy, K. T.; Brown, M. J.; Della Gatta, P.; Cameron-Smith, D. Infusion with the antioxidant N-acetylcysteine attenuates early adaptive responses to exercise in human skeletal muscle. Acta Physiol (Oxf) 204:382-392; 2012. [60] Michailidis, Y.; Karagounis, L. G.; Terzis, G.; Jamurtas, A. Z.; Spengos, K.; Tsoukas, D.; Chatzinikolaou, A.; Mandalidis, D.; Stefanetti, R. J.; Papassotiriou, I.; Athanasopoulos, S.; Hawley, J. A.; Russell, A. P.; Fatouros, I. G. Thiol-based antioxidant supplementation alters human skeletal muscle signaling and attenuates its inflammatory response and recovery after intense eccentric exercise. Am J Clin Nutr 98:233-245; 2013. [61] Murphy, K. T.; Medved, I.; Brown, M. J.; Cameron-Smith, D.; McKenna, M. J. Antioxidant treatment with N-acetylcysteine regulates mammalian skeletal muscle Na+-K+ATPase alpha gene expression during repeated contractions. Exp Physiol 93:1239-1248; 2008. [62] Meier, P.; Renga, M.; Hoppeler, H.; Baum, O. The impact of antioxidant supplements and endurance exercise on genes of the carbohydrate and lipid metabolism in skeletal muscle of mice. Cell Biochem Funct 31:51-59; 2012. [63] Fischer, C. P.; Hiscock, N. J.; Penkowa, M.; Basu, S.; Vessby, B.; Kallner, A.; Sjoberg, L. B.; Pedersen, B. K. Supplementation with vitamins C and E inhibits the release of interleukin-6 from contracting human skeletal muscle. J Physiol 558:633-645; 2004. [64] Sen, C. K.; Atalay, M.; Hänninen, O. Exercise-induced oxidative stress: glutahione supplementation and deficiency. J. Appl. Physiol. 79 (3):675-686; 1994 [65] Leeuwenburgh, C.; Ji, L. L. Glutathione and glutathione ethyl ester supplementation of mice alter glutathione homeostasis during exercise. J Nutr 128:2420-2426; 1998. [66] McGinley, C.; Shafat, A.; Donnelly, A. E. Does antioxidant vitamin supplementation protect against muscle damage? Sports Med 39:1011-1032; 2009. [67] Kaminski, M.; Boal, R. An effect of ascorbic acid on delayed-onset muscle soreness. Pain 50:317-321; 1992. 22

[68] Francis, K. T.; Hoobler, T. Failure of vitamin E and delayed muscle soreness. Ala Med 55:15-18; 1986. [69] Jenkins, R. R. Free radical chemistry: relationship to exercise. Sports Med. 5:156-170; 1988. [70] Robertson, J. D.; Maughan, R. J.; Duthie, G. G.; Morrice, P. C. Increased blood antioxidant systems of runners in response to training load. Clin Sci (Lond) 80:611-618; 1991. [71] Clarkson, P. M.; Thompson, H. S. Antioxidants: what role do they play in physical activity and health? Am J Clin Nutr 72:637S-646S.; 2000. [72] Nikolaidis, M. G.; Kerksick, C. M.; Lamprecht, M.; McAnulty, S. R. Does vitamin C and E supplementation impair the favorable adaptations of regular exercise? Oxid Med Cell Longev 2012:707941; 2012. [73] Asha Devi, S.; Prathima, S.; Subramanyam, M. V. Dietary vitamin E and physical exercise: II. Antioxidant status and lipofuscin-like substances in aging rat heart. Exp Gerontol 38:291-297; 2003. [74] Asha Devi, S.; Prathima, S.; Subramanyam, M. V. Dietary vitamin E and physical exercise: I. Altered endurance capacity and plasma lipid profile in ageing rats. Exp Gerontol 38:285-290; 2003. [75] Gey, G. O.; Cooper, K. H.; Bottenberg, R. A. Effect of ascorbic acid on endurance performance and athletic injury. JAMA 211:105; 1970. [76] Gerster, H. The role of vitamin C in athletic performance. J Am Coll Nutr 8:636-643; 1989. [77] Kelly, D. P.; Scarpulla, R. C. Transcriptional regulatory circuits controlling mitochondrial biogenesis and function. Genes Dev 18:357-368; 2004. [78] Keith, R. E.; Merrill, E. The effects of vitamin C on maximum grip strength and muscular endurance. J Sports Med Phys Fitness 23:253-256; 1983. [79] Jackson, M. J.; Edwards, R. H. Free radicals and trials of antioxidant therapy in muscle diseases. Adv Exp Med Biol 264:485-491; 1990. [80] Witt, E. H.; Reznick, A. Z.; Viguie, C. A.; Starke-Reed, P.; Packer, L. Exercise, oxidative damage and effects of antioxidant manipulation. J Nutr 122:766-773; 1992. [81] Goldfarb, A. H. Antioxidants: role of supplementation to prevent exercise-induced oxidative stress. Med Sci Sports Exerc 25:232-236.; 1993. [82] Weight, L. M.; Myburgh, K. H.; Noakes, T. D. Vitamin and mineral supplementation: effect on the running performance of trained athletes. Am J Clin Nutr 47:192-195; 1988. [83] Maughan, R. J. Nutritional ergogenic aids and exercise performance. Nutr Res Rev 12:255-280; 1999. [84] Tiidus, P. M.; Houston, M. E. Vitamin E status and response to exercise training. Sports Med 20:12-23.; 1995. [85] Rokitzki, L.; Logemann, E.; Huber, G.; Keck, E.; Keul, J. alpha-Tocopherol supplementation in racing cyclists during extreme endurance training. Int J Sport Nutr 4:253264; 1994. [86] Higashida, K.; Kim, S. H.; Higuchi, M.; Holloszy, J. O.; Han, D. H. Normal Adaptations to Exercise Despite Protection Against Oxidative Stress. Am J Physiol Endocrinol Metab; 2011. [87] Sharman, I. M.; Down, M. G.; Norgan, N. G. The effects of vitamin E on physiological function and athletic performance of trained swimmers. J Sports Med Phys Fitness 16:215-225; 1976. [88] Lawrence, J. D.; Smith, J. L.; Bower, R. C.; Riehl, W. P. The effect of alpha tocopherol (vitamin E) and pyridoxine HCL (vitamin B6) on the swimming endurance of trained swimmers. J Am Coll Health Assoc 23:219-222; 1975. [89] Lawrence, J. D.; Bowe, R. C.; Riehl, W. P. Effects of alpha-tocopherol acetate on the swimming endurance of trained swimmers. Am. J. Clin. Nutr. 28:205; 1975. 23

[90] Shephard, R. J.; Campbell, R.; Pimm, P.; Stuart, D.; Wright, G. R. Vitamin E, exercise, and the recovery from physical activity. Eur J Appl Physiol Occup Physiol 33:119-126; 1974. [91] Watt, T.; Romet, T. T.; McFarlane, I.; McGuey, D.; Allen, C.; Goode, R. C. Letter: Vitamin E and oxygen consumption. Lancet 2:354-355; 1974. [92] Shephard, R. J. Vitamin E and athletic performance. J Sports Med Phys Fitness 23:461470; 1983. [93] Sharman, I. M.; Down, M. G.; Sen, R. N. The effects of vitamin E and training on physiological function and athletic performance in adolescent swimmers. Br J Nutr 26:265-276; 1971. [94] Packer, L.; Gohil, K.; deLumen, B.; Terblanche, S. E. A comparative study on the effects of ascorbic acid deficiency and supplementation on endurance and mitochondrial oxidative capacities in various tissues of the guinea pig. Comp Biochem Physiol B 83:235-240; 1986. [95] Marshall, R. J.; Scott, K. C.; Hill, R. C.; Lewis, D. D.; Sundstrom, D.; Jones, G. L.; Harper, J. Supplemental vitamin C appears to slow racing greyhounds. J Nutr 132:1616S-1621S; 2002. [96] Khassaf, M.; McArdle, A.; Esanu, C.; Vasilaki, A.; McArdle, F.; Griffiths, R. D.; Brodie, D. A.; Jackson, M. J. Effect of vitamin C supplements on antioxidant defence and stress proteins in human lymphocytes and skeletal muscle. J Physiol 549:645-652; 2003. [97] Close, G. L.; Ashton, T.; Cable, T.; Doran, D.; Holloway, C.; McArdle, F.; MacLaren, D. P. Ascorbic acid supplementation does not attenuate post-exercise muscle soreness following muscle-damaging exercise but may delay the recovery process. Br J Nutr 95:976-981; 2006. [98] Teixeira, V. H.; Valente, H. F.; Casal, S. I.; Marques, A. F.; Moreira, P. A. Antioxidants do not prevent postexercise peroxidation and may delay muscle recovery. Med Sci Sports Exerc 41:1752-1760; 2009. [99] Malm, C.; Svensson, M.; Ekblom, B.; Sjodin, B. Effects of ubiquinone-10 supplementation and high intensity training on physical performance in humans. Acta Physiol Scand 161:379-384; 1997. [100] Malm, C.; Svensson, M.; Sjoberg, B.; Ekblom, B.; Sjodin, B. Supplementation with ubiquinone-10 causes cellular damage during intense exercise. Acta Physiol Scand 157:511512; 1996. [101] Richardson, R. S.; Donato, A. J.; Uberoi, A.; Wray, D. W.; Lawrenson, L.; Nishiyama, S.; Bailey, D. M. Exercise-induced brachial artery vasodilation: role of free radicals. Am J Physiol Heart Circ Physiol 292:H1516-1522; 2007. [102] Wray, D. W.; Uberoi, A.; Lawrenson, L.; Bailey, D. M.; Richardson, R. S. Oral antioxidants and cardiovascular health in the exercise-trained and untrained elderly: a radically different outcome. Clin Sci (Lond) 116:433-441; 2009. [103] Maxwell, A. J.; Schauble, E.; Bernstein, D.; Cooke, J. P. Limb blood flow during exercise is dependent on nitric oxide. Circulation 98:369-374; 1998. [104] Braakhuis, A. J. Effect of vitamin C supplements on physical performance. Curr Sports Med Rep 11:180-184; 2012 [105] Copp, S. W.; Ferreira, L. F.; Herspring, K. F.; Hirai, D. M.; Snyder, B. S.; Poole, D. C.; Musch, T. I. The effects of antioxidants on microvascular oxygenation and blood flow in skeletal muscle of young rats. Exp Physiol 94:961-971; 2009. [106] Olesen, J.; Gliemann, L.; Bienso, R.; Schmidt, J.; Hellsten, Y.; Pilegaard, H. Exercise training, but not resveratrol, improves metabolic and inflammatory status in skeletal muscle of aged men. J Physiol 592:1873-1886; 2013. [107] Braakhuis, A. J.; Hopkins, W. G.; Lowe, T. E. Effects of dietary antioxidants on training and performance in female runners. Eur J Sport Sci 14:160-168; 2014. [108] Hood, D. A. Invited Review: contractile activity-induced mitochondrial biogenesis in skeletal muscle. J Appl Physiol 90:1137-1157; 2001. 24

[109] Papanicolaou, K. N.; Phillippo, M. M.; Walsh, K. Mitofusins and the mitochondrial permeability transition: the potential downside of mitochondrial fusion. Am J Physiol Heart Circ Physiol 303:H243-255; 2012. [110] Wright, D. C.; Han, D. H.; Garcia-Roves, P. M.; Geiger, P. C.; Jones, T. E.; Holloszy, J. O. Exercise-induced mitochondrial biogenesis begins before the increase in muscle PGC-1alpha expression. J Biol Chem 282:194-199; 2007. [111] Holloszy, J. O. Biochemical adaptations in muscle. Effects of exercise on mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle. J Biol Chem 242:2278-2282; 1967. [112] Puigserver, P.; Wu, Z.; Park, C. W.; Graves, R.; Wright, M.; Spiegelman, B. M. A coldinducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92:829-839; 1998. [113] Scarpulla, R. C. Nuclear activators and coactivators in mammalian mitochondrial biogenesis. Biochim Biophys Acta 1576:1-14; 2002. [114] Scarpulla, R. C. Transcriptional paradigms in mammalian mitochondrial biogenesis and function. Physiol Rev 88:611-638; 2008. [115] Rockl, K. S.; Witczak, C. A.; Goodyear, L. J. Signaling mechanisms in skeletal muscle: acute responses and chronic adaptations to exercise. IUBMB Life 60:145-153; 2008. [116] Davies, K. J. A.; Quintanilha, A. T.; Brooks, G. A.; Packer, L. Free radicals and tissue damage produced by exercise. Biochem. Biophys. Res. Commun. 107:1198-1205; 1982. [117] St-Pierre, J.; Drori, S.; Uldry, M.; Silvaggi, J. M.; Rhee, J.; Jager, S.; Handschin, C.; Zheng, K.; Lin, J.; Yang, W.; Simon, D. K.; Bachoo, R.; Spiegelman, B. M. Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators. Cell 127:397-408; 2006. [118] Silveira, L. R.; Pilegaard, H.; Kusuhara, K.; Curi, R.; Hellsten, Y. The contraction induced increase in gene expression of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1alpha (PGC-1alpha), mitochondrial uncoupling protein 3 (UCP3) and hexokinase II (HKII) in primary rat skeletal muscle cells is dependent on reactive oxygen species. Biochim Biophys Acta 1763:969-976; 2006. [119] Kang, C.; O'Moore, K. M.; Dickman, J. R.; Ji, L. L. Exercise activation of muscle peroxisome proliferator-activated receptor-gamma coactivator-1alpha signaling is redox sensitive. Free Radic Biol Med 47:1394-1400; 2009. [120] Gomez-Cabrera, M. C.; Domenech, E.; Romagnoli, M.; Arduini, A.; Borras, C.; Pallardo, F. V.; Sastre, J.; Vina, J. Oral administration of vitamin C decreases muscle mitochondrial biogenesis and hampers training-induced adaptations in endurance performance. Am J Clin Nutr 87:142-149; 2008. [121] Ristow, M.; Zarse, K.; Oberbach, A.; Kloting, N.; Birringer, M.; Kiehntopf, M.; Stumvoll, M.; Kahn, C. R.; Bluher, M. Antioxidants prevent health-promoting effects of physical exercise in humans. Proc Natl Acad Sci U S A 106:8665-8670; 2009. [122] Strobel, N. A.; Peake, J. M.; Matsumoto, A.; Marsh, S. A.; Coombes, J. S.; Wadley, G. D. Antioxidant supplementation reduces skeletal muscle mitochondrial biogenesis. Med Sci Sports Exerc 43:1017-1024; 2011. [123] Venditti, P.; Napolitano, G.; Barone, D.; Di Meo, S. Vitamin E supplementation modifies adaptive responses to training in rat skeletal muscle. Free Radic Res 48:1179-1189; 2014. [124] Paulsen, G.; Cumming, K. T.; Holden, G.; Hallen, J.; Ronnestad, B. R.; Sveen, O.; Skaug, A.; Paur, I.; Bastani, N. E.; Ostgaard, H. N.; Buer, C.; Midttun, M.; Freuchen, F.; Wiig, H.; Ulseth, E. T.; Garthe, I.; Blomhoff, R.; Benestad, H. B.; Raastad, T. Vitamin C and E supplementation hampers cellular adaptation to endurance training in humans: a double-blind, randomised, controlled trial. J Physiol 592:1887-1901; 2014.

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[125] Bouitbir, J.; Charles, A. L.; Echaniz-Laguna, A.; Kindo, M.; Daussin, F.; Auwerx, J.; Piquard, F.; Geny, B.; Zoll, J. Opposite effects of statins on mitochondria of cardiac and skeletal muscles: a 'mitohormesis' mechanism involving reactive oxygen species and PGC-1. Eur Heart J 33:1397-1407; 2012. [126] Calabrese, E. J.; Baldwin, L. A. Tales of two similar hypotheses: the rise and fall of chemical and radiation hormesis. Hum Exp Toxicol 19:85-97; 2000. [127] Yfanti, C.; Akerstrom, T.; Nielsen, S.; Nielsen, A. R.; Mounier, R.; Mortensen, O. H.; Lykkesfeldt, J.; Rose, A. J.; Fischer, C. P.; Pedersen, B. K. Antioxidant supplementation does not alter endurance training adaptation. Med Sci Sports Exerc 42:1388-1395; 2010. [128] Gomez-Cabrera, M. C.; Ristow, M.; Vina, J. Antioxidant supplements in exercise: worse than useless? Am J Physiol Endocrinol Metab 302:E476-477; author reply E478-479; 2012.

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FIGURE LEGENDS Figure 1. Historical overview of exercise redox biology We summarize the main finding in the last60 years on the role of ROS generated during muscle contraction on function and damage and the effect of antioxidant supplements: 50’s: First evidence showing that SM contains ROS [2]; 70’s: Increased lipid peroxidation during exercise [3-4]; 80’s: ROS elevated in muscles electrically stimulated to contract [5]; 1982: Increase in ROS after exhaustive exercise (first in vivo evidence)[6]; 80’s: Vitamin E retards tissue damage that occurs during exercise [7, 8]; 90's: The role of exercise in glutathione redox status was stablished [13, 14]; 90's: ROS are involved in normal muscle contraction [17, 18]; 1994: ROS contribute to muscle fatigue, NAC reduces fatigue [25]; Late 90’s: Exercise-induced increase in the muscle antioxidant capacity [40-42]; 2000: ROS play a role in cell signaling and adaptations to exercise [48,49,51]; 2008: Evidence against supplementation with antioxidants in exercise training [116]. Figure 2. Janus faced of ROS in exercise Beneficial and negative effect of supplementation with antioxidants after one bout of exhaustive exercise and during training. Exercise-induced ROS causes muscle damage, oxidative stress and fatigue. However, ROS can act as signaling molecules and mediate the adaptive muscle responses to exercisetraining. The responses result from the cumulative effects of repeated exercise bouts. Figure 3. Mitochondrial biogenesis pathway involved in metabolism and in cell signaling A range of stimulus (conditions, diseases and injures) regulate mitochondrial biogenesis. Mitochondria are considered as the most important cellular sources and targets of reactive oxygen and nitrogen species (RONS). Mitochondria signals via RONS and Ca+2 are critical regulators of cell cycle, proliferation, and apoptosis.Mitochondria are the organelles that generate, via oxidative phosphorylation, the cellular energy carrier ATP. They are involved in several biosynthetic processes, including lipid, cholesterol, heme, and nucleotide synthesis. Figure 4. Activation of mitochondrial biogenesis by exercise training-induced ROS Mitochondrial biogenesis is driving by the coordinate regulation of nuclear and mitochondrial genomes. Exercise training-induced ROS stimulates PGC-1α production in skeletal muscle cells. This signal promotes PGC-1α gene expression in the nucleus, where PGC-1α acts to specific genes such as NRF-1 which activates the expression of mtTFA. This nuclear protein is a NRF-1 target gene which is considered the most important mammalian transcription factor for mtDNA because it stimulates mitochondrial DNA transcription and replication. Antioxidant supplements during training have a detrimental effect on mitochondrial biogenesis due to the inhibition of ROS associated with exercise and also on mitochondrial antioxidant defense system which is down-regulated.

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Redox modulation of mitochondriogenesis in exercise. Does antioxidant supplementation blunt the benefits of exercise training?

Physical exercise increases the cellular production of reactive oxygen species (ROS) in muscle, liver, and other organs. This is unlikely due to incre...
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