Oxygen Consumption and Usage During Physical Exercise: The Balance Between Oxidative Stress and ROS-Dependent Adaptive Signaling

A. ROS and antioxidants

A great deal of epidemiological data has shown that exercise decreases the incidence of oxidative stress-associated diseases (174, 405). This beneficial effect is due to the fact that exercise-induced ROS production is necessary for oxidative stress-related adaptations. There are a number of ROS generating systems that increase blood flow to skeletal muscle during physical exercise, and more or less maintain blood flow to the brain, and significantly decrease oxygen supply to the liver and kidney (309). The mitochondrial electron transport chain is one main ROS generators found in skeletal muscle (295). As a result of exhaustive exercise, ROS production of complex I and III, with pyruvate/malate and succinate substrates, was increased by 187% and 138%, respectively, compared to the nonexercising group (342). Complex III has been suggested to yield superoxide on the cytoplasmic site of the mitochondrial membrane (47), which could be important for redox signaling. Moreover, mitochondria isolated from skeletal muscle after contraction showed significantly increased levels of H₂O₂ generation compared to noncontracting values (415). Sahlin et al. have shown that complex I is the major ROS generator in skeletal muscle of ultraendurance runners, as assessed by Amplex red reagents (343). However, it must be noted that the earlier estimation of about 1%–5% of the oxygen that enters mitochondria can be released as ROS (44) could be an overestimation, and the real value could be more than an order of magnitude lower (373). Upon this and related observations, it was suggested that mitochondria are not the main source of ROS production during exercise (298). On the other hand, a recent report suggests that mitochondria might be important sources of ROS, at complex I and III through peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) (19). In this particular study, a positive association was found between ROS production and mitochondrial respiration (19). The effects of the anabolic steroid, stanozolol, were investigated on mitochondrial ROS production, and it was found that complex I- and complex II-linked substrate generated H₂O₂ after exercise sessions, which was significantly attenuated by stanozolol (342). Therefore, despite the fact that the resting efflux of ROS is a magnitude lower than was suggested, results from the measurements of ROS/H₂O₂ efflux indicate that mitochondria are generating enhanced levels of ROS during intensive exercise. On the other hand, the release of ROS from each mitochondrial complex could differ significantly.

For example, at complex I, the iron–sulfur clusters, flavoprotein, and oxidoreductase, and at complex III, Q10 semiquinones are suggested to be the main sites of ROS generation (244, 373). Recently, a novel method has been developed, which allows detection of superoxide production in the mitochondrial matrix by the stochastic quantal events of superoxide (mitochondrial superoxide flashes [mSOFs]) (429). It has been shown that during muscle contraction, the activity of mSOF increases in mitochondria and is dependent on the activity of the electron transport chain and, furthermore, has a biphasic nature, showing an increase in brief and a decrease in longer tetanic contractions (429). This finding certainly does not rule out extracellular sources of ROS during contraction, which have been suggested as being important (304, 325, 448). Indeed, 5-lipoxygenase, cyclooxygenase, sarcolemmal NADPH oxidase, and xanthine oxidase (XO) have been implicated in superoxide generation in skeletal muscle (272, 295). NADPH oxidase is one of the major ROS generators during exercise (26, 297), since in the presence of ADP and Fe³⁺, NADPH oxidase catalyzes electron transfer from NADPH to molecular oxygen to form superoxide (21). XO is especially involved in ROS production during anaerobic exercise, and a linear relationship has been reported between XO and lactic acid levels (304). One of the reasons for this is the fact that XO activity is strongly dependent on the availability of substrate (437). During intense exercise due to the high rate of ATP degradation, AMP generation hypoxanthine and xanthine are formed and serve as substrates for XO, which uses molecular oxygen to generate ROS. Interestingly, we could detect increased XO activity in the liver 1 day after exhaustive acute exercise (305), and the protective effects of administered SOD derivatives showed that endothelium-associated XO is one source of ROS generation during intense exercise (304). Recently, myostatin has emerged as a potential ROS-inducing factor, especially during sarcopenia (372). It has been demonstrated that ablation of the myostatin gene resulted in attenuated loss of muscle mass with aging. Moreover, it turns out that myostation can induce ROS production through tumor necrosis factor-α (TNF-α) and NADPH oxidase (372). The role of exercise on myostation-mediated redox signaling is still unclear, and research is warranted on this topic. Activation of ryanodine receptor 1 (RyR1) in the sarcoplasmic reticulum of skeletal muscle is necessary for Ca²⁺ release and consequent generation of cross-bridge-related force production. With the aging of skeletal muscle, continuous Ca²⁺ leak were observed in RyR1 channels, which was associated with decreased tension, exercise capacity, and increased ROS production. Normalization of RyR1 function by pharmacological intervention that stabilized binding of calstabin1 to RyR1 significantly reduced Ca²⁺ leakage and increased endurance capacity (14).

It has been shown that a single bout of exercise and regular exercise modulate the ROS production in neutrophils differently; namely acute exercise caused apoptosis and reduction of mitochondrial membrane potential, while regular exercise increased the resistance against oxidative challenge (385). Muscle contraction generates heat, which has been shown to enhance ROS production (447). Needless to say, ROS production is an essential physiological process for muscle contraction, and it is estimated that the level of H₂O₂ can be increased by 100 nM during contractions (155). Indeed, it has been shown that low levels of exogenous H₂O₂ treatment increase, and the addition of catalase decreases, force production of the diaphragm (324). H₂O₂ was shown to modulate muscle contraction via Ca²⁺ channels (15). Administration of N-acetylcysteine (NAC) is often used to appraise the effects of ROS in muscle fatigue (70, 74, 229, 326), and it has been shown to decrease the rate of fatigue at 80% of VO2max, but not at higher intensities (74). NAC has been suggested to act through improved K⁺ regulation (229). Infusion of NAC appears to interact with some exercise-induced signaling pathways, as shown by attenuation of the exercise-induced activation of JNK. However, phosphorylation of p38 mitogen-activated protein kinase (MAPK), ERK1/2, or p65 subunit of nuclear factor-kappaB (NF-κB) was not affected (283).

Accumulating evidence suggests that ROS are generated during exercise and modulate signaling pathways and the level of muscle contraction, indicating that ROS content and the muscle function–dose relationship can be described by a hormesis curve (310). Low levels of ROS stimulate force production, while high levels do attenuate it.

To curb the toxic and deleterious effects of ROS, cells are well equipped with enzymatic and nonenzymatic antioxidant systems. The exercise-induced ROS generation results in increased activity of enzymatic antioxidants, which then results in increased resistance to oxidative challenges, including a wide variety of oxidative stress-related diseases. One of the key issues of this adaptive response is the feature of intermittent exercise. Exercise-induced ROS production, in the recovery period, is mostly overcompensated by the upregulated antioxidant system (111, 112, 160). Chronic exposure to high levels of ROS can exhaust the nonenzymatic antioxidant system and lead to impaired cellular function, macromolecule damage, apoptosis, and necrosis. The intermittent nature of exercise gives a preconditioning role to exercise. After a bout of exercise that gives a metabolic and oxidative challenge, followed by a rest period, the system is allowed to adapt to the challenges caused by this stressor (318). Indeed, during resting periods, the body does not simply store more glycogen in the skeletal muscle, which would allow better performance, but also upregulates antioxidant and oxidative damage repair systems (Fig. 4) (111, 163). Subjects involved in regular exercise, due to an adaptive response, demonstrate higher levels of mitochondrial content, and produce lower levels of ROS at the given intensity than those who are untrained. Therefore, excess physical exercise is detrimental to poorly trained individuals, but progressive training allows the cells to more easily detoxify a larger amount of ROS, partly by the induced activity of the antioxidant systems. Moreover, it has to be mentioned that severe and extreme exercise regimens or intensities may pose serious threat of oxidative damage, even in healthy subjects, and those who are poorly trained and/or not well nourished are in higher risk to suffer oxidative stress.

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FIG. 4.

The adaptive response to a single bout of exercise is limited, and oxidative damage often occurs. Moderate levels of oxidative damage could be important to the induction of the oxidative damage repair system. The regular exercise induced adaptation, due to the intermittent feature of exercise and rest periods, allows induction of the antioxidant and damage repair systems, which results in enhanced protection against oxidative stress, attenuates the aging process, and promotes health with increased functional capacities. MDA, malondialdehyde; RCD, reactive carbonyl derivatives; 8-OHdG, 8-hydroxy-2′-deoxyguanosine. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

For example, runners who often suffer from anemia, which could be associated with increased free-iron concentrations in the blood stream, are subjected to enhanced levels of oxidative damage. Moreover, exercise at high altitude also increases the risk of the accumulation of oxidative damage markers (83).

Chronic elevation of ROS is dangerous, as it could easily exhaust the system, while bouts of exposure, like preconditioning ischemia/reperfusion repeats, significantly increase the resistance to oxidative stress (128). Both acute and regular exercise could induce the activity of antioxidant enzymes, including manganese superoxide dismutase (Mn-SOD), copper–zinc superoxide dismutase (Cu,Zn-SOD), extracellular superoxide dismutase (EC-SOD), catalase, and glutathione peroxidase (GPX) (112, 138, 160, 269). The mechanism behind the induction of the antioxidant system complex most probably involves epigenetics, since it was shown that Cu,Zn-SOD knockdown results in decreased methylation of DNA (33). Therefore, it cannot be ruled out that exercise results in changes in DNA methylation and/or histone acetylation, which leads to enhanced activation of antioxidant enzyme genes. The exercise-induced ROS generation is naturally a powerful stimulus to activate the expression of antioxidant enzymes. Mn-SOD could be readily induced by ROS-mediated activation of TNF-α (428) or by NF-kB (440) among others.

B. Thiols and redox signaling in exercise

Current evidence indicates that in ROS-mediated signaling, redox-sensitive thiols play a critical role, coupling the intracellular changes in the redox state to regulation of cellular processes (34, 361). Thiols react faster than other amino acids with oxidizing species, and most of their oxidation products can be reversibly reduced. Therefore, reversible redox modification of active-site Cys residues provides a redox switch, an on-off mechanism for control of cellular signaling and function (167). On the other hand, like a rheostat, through allosteric regulation and by S-glutathione-derivatives, thiols can modulate protein function (177). Furthermore, cross-linking of proteins through disulfides yields mechanisms for protein aggregation and trafficking. The endogenous thiols, the glutathione (GSH) and thioredoxin (TRX) systems, play a central role in the antioxidant defenses that control cellular events and regulate protection against oxidative stress (361), a condition that alters normal redox control of cellular signaling, especially by disruption of thiol-redox circuits (166). In addition to their central role in supporting a large network of antioxidant defenses, GSH and TRX have various biological functions such as regulation of enzyme activity, receptors, transcription factors, and ultimately redox-sensitive signal transduction, short-term storage of cysteine, protein structure, cell growth, proliferation, and programmed cell death (358).

GSH (l-gamma-glutamyl-l-cysteinylglycine) is the predominant nonprotein thiol in the cell. GSH plays an essential role in antioxidant protection and provides an appropriate reducing milieu inside the cell. During strenuous physical exercise where oxygen consumption is increased many fold, GSH is oxidized, and as a consequence, the disulfide glutathione (GSSG) accumulates. The balance of oxidized to reduced glutathione (GSSG/GSH) acts as a redox control node to regulate sulfur switches for responses to acute exercise-induced oxidative stress and also to develop adaptations of various defense mechanisms during regular physical training (358).

Oxidation of thiols to disulfides has also been used as a sensitive marker of exercise-induced oxidative stress (203). Since 1985, a growing number of exercise studies have utilized GSSG levels and GSSG/GSH ratios as sensitive markers of oxidative stress, but the results are equivocal (109, 361). Major factors that may affect GSH oxidation response to acute exercise are the type and intensity of the exercise. For example, GSH oxidation is more prominent during exercise performed at higher intensities (362). Another factor may be the training status of the organism. In the trained individual, enhanced antioxidant defenses couple successfully with pro-oxidants to protect the organism against increased oxidative stress. As a result, GSH oxidation, which is a sensitive marker of oxidative stress, occurs less after exercise in trained individuals. Recently, we observed that in the regularly trained horse, with exercise or during recovery, there were no significant changes in the muscle GSSG- or the GSH-redox ratio, although postexercise free-radical amounts correlated positively with postexercise GSSG concentration (184). This can be explained by the well-known fact that regular exercise training of all types enhances GSH levels by inducing GSH synthesis and also by increasing GSH regeneration from GSSG (361).

Data on the role of endogenous GSH on exercise performance, especially endurance capacity, are limited. In one early study, endurance capacity for treadmill running in GSH-deficient rats was found to be reduced by half (360). Although there is a controversial report in the literature regarding the impact of GSH deficiency on prolonged swimming performance in mice (201), one may still postulate that endogenous GSH not only plays a critical role in circumventing exercise-induced oxidative stress but also determines exercise performance.

The TRX system, comprising NADPH, thioredoxin reductase (TrxR), and TRX itself, is critical for cellular redox balance and antioxidant function. TRX is a small multifunctional protein with a dithiol/disulfide active center and is the major cellular protein disulfide reductase. Located extracellularly, TRX shows a cytoprotective activity against oxidative stress-induced apoptosis. TRX has also a cytokine-like function acting as an autocrine growth factor (410). Intracellularly, TRX-1 in the cytosol and TRX-2 in the mitochondria are involved in the regulation of protein–protein or protein–nucleic acid interactions through the reduction/oxidation of protein cysteine residues. In response to a variety of cellular stresses, including oxidative stress, TRX translocates from the cytosol into the nucleus to regulate the expression of various genes. A growing number of transcription factors require TRX reduction for DNA binding (209). TRX and GSH are considered to be functionally and kinetically distinct systems. In both cells and plasma, GSH/GSSG redox is not equilibrated with other major thiol/disulfide couples (TRX and Cys/CySS), providing the basis to consider discrete redox circuits for signaling and control (167). Nevertheless, loss of cellular TRX-1 is known to result in elevated GSH levels (56). Furthermore, after an acute bout of exercise, during recovery, TRX-1 activity correlated negatively with the GSSG/TGSH ratio of oxidized GSH in skeletal muscle of horses (184). These results underline a cross-talk between two major thiol antioxidants in response to exercise-induced oxidative stress (Fig. 5).

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FIG. 5.

Thiols are important antioxidants, and the GSH/GSSG ratio modulates vital cellular processes, including antioxidant systems, apoptosis, cellular growth, and signal transduction among others. Thiols are involved in the extracellular and intracellular oxidative defense, including the mitochondrial and nuclear compartments (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.) GSH, glutathione; GSSG, disulfide glutathione; TRX, thioredoxin.

In a setting of thiol antioxidant (alpha-lipoic acid [LA]) supplementation, we observed a negative correlation between the activity of TrxR at rest and postexercise levels of free radicals after LA supplementation (184). Acute exhaustive exercise induced TRX-1 mRNA levels (192), and 8 weeks of endurance training increased TRX-1 protein levels in the rat brain (193). In line with these results, a limited number of studies from other groups also provide evidence that TRX contributes to antioxidant defense against oxidative stress induced by acute exercise of variable intensity and duration. In mouse peripheral blood mononuclear cells, 30 min of swimming exercise induced TRX protein expression 12 and 24 h after exercise (381). Similarly, plasma TRX concentrations increased continuously during an ultramarathon race (228).

Thioredoxin-interacting protein (TXNip, also termed VDUP1 for Vitamin D-upregulated protein or TBP2 for thioredoxin-binding protein) has been identified as an endogenous inhibitor of TRX in a redox-dependent fashion (261). Redox-dependent interaction of TRX with TXNip only exists between the oxidized form of TXNip and the reduced form of TRX (277) and may modulate apoptosis signal kinase (ASK-1), redox factor 1, Forkhead box class O4 (FoxO4), and nod-like receptor proteins, which offers a novel mechanism of redox regulation (442). What is, to our knowledge, the only study in the literature to investigate the role of exercise on TXNip levels found that 8 weeks of exercise training did not influence TXNip levels in the rat brain despite an increase in TRX-1 protein. Interestingly, in the same set of experiments, streptozotocin-induced diabetes increased both TXNip levels and oxidative stress, measured as increased GSSG/TGSH ratio (193), suggesting a link between disturbed redox control and disease.

C. Oxidative damage and housekeeping

The term oxidative damage is often referred to as a condition where there is a large increase in the stable product of the interaction of ROS with lipids, proteins, and DNA. The deleterious effects of the oxidative damage on cell function are well known and have been reviewed elsewhere (6, 84, 215, 357, 400, 423). The extent of oxidative damage caused by an acute bout of physical exercise, even if it is exhausting, does not cause viability risking increase in oxidative damage, and it can be argued that this might be a necessary factor of adaptation. The fact that the most often measured macromolecule damage markers of oxidative stress, such as lipid peroxidation-derived aldehydes, protein oxidation byproducts, carbonyl groups, or 8-oxoG, are always detectable even with high antioxidant levels raises the possibility that these markers could play a significant physiological role, which, to date, has not been reported. Aging increases the level of oxidative damage, but the elevation could be dangerous after the last quarter of the life span, which can be attenuated by regular exercise (310). On the other hand, the extent of damage is easily measurable at a young age (Fig. 6).

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FIG. 6.

The free radical-dependent aging theory is well accepted, and it is clearly demonstrated that the extent of oxidative damage increases in the last quarter of the life span. However, it is also clear that the oxidative modification of lipids, proteins, and DNA is well detectable even at a young age, and this could indicate that a moderate level of oxidative damage is not dangerous to cells; moreover, it even can be necessary. On the other hand, significant elevation of oxidative damage at an advanced age is associated with increased incidence of a wide range of diseases and impaired physiological function. Regular exercise has been shown to attenuate the age-associated increase in oxidative damage, and it also attenuates the deleterious effects of aging on organ function (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

It is clear that ROS are crucial players in aging, although a number of other factors are also involved. In neurons, cardiomyocytes, skeletal myocytes, sensory cells, and other postmitotic cells, the role of ROS could be more pronounced in aging, due to the ROS-mediated damage and accumulation of damage and mutations that are believed to be important in loss of function in these cells. The substitution of aged molecules for mutated ones with loss-of-function molecules leads to a progressive deterioration of such tissues. The increase in oxidative damage in the last quarter of the lifespan is probably the combined effects of the increased level of ROS production (75, 155) and the failure of the oxidative damage repair systems (117, 189).

Lipid peroxidation appears to be an unavoidable process of aerobic life, and despite the well-developed antioxidant systems, certain products of lipid peroxidation are always detectable. Moreover, it has been shown that lipid peroxidation can be generated enzymatically by lipoxygenase. The physiological role of controlled lipid peroxidation is enigmatic, but it could be important in the physiological remodeling of cellular membranes (256). Moreover, targeted, controlled lipid peroxidation by lipoxygenase has been shown to be important for the degradation of mitochondrial membranes during the maturation of erythrocytes (322). Lipoxygenation or lipid peroxidation could modify whole membrane structures, the chemical composition, and the physicochemical properties of phospholipids, resulting in altered permeability, surface charge, and passive electric properties (191).

The phospholipase A₂ (PLA₂) enzyme family, which consists of more than 20 enzymes, hydrolyzes the sn-2 ester bond of glycerophospholipids and generates free fatty acids and lysophospholipids with physiological activity (349). Ca²⁺-independent phospholipase A₂ (iPLA₂-beta) appears to be one enzyme that could repair oxidized cardiolipin, a component of the mitochondrial membrane (185). However, most of the enzymes in the family of PLA₂ are generators of oxidative stress and lipid peroxidation during prostaglandin synthesis from arachidonic acid, products of PLA₂ reaction, rather than repair of lipid peroxidation (5). The levels of lipid peroxidation in various organs, including skeletal muscle and the brain (290, 384, 398), increase with aging. However, not all the lipid peroxidation products present to the same degree or at the same time. Lipid antioxidants, especially lipid-soluble vitamins and GSH, glutathione-S-transferases, and b-alanyl-l-histidine, which can quench lipid peroxidation-derived aldehydes, including 4-hydroxy-2-nonenal (HNE), are naturally occurring phenomena. Albumin and apolipoproteins and maybe some other proteins with high intracellular concentrations can bind and buffer HNE, resulting in detoxification of the cellular milieu. The platelet-activating factor acetylhydrolase family represents a unique group of PLA₂ that exhibits high substrate specificity toward oxidized phospholipids, and has been suggested to act as a Ca²⁺-independent PLA₂ in the repair of lipid peroxidation (256). This enzyme has been shown to be activated in blood samples of well-trained triathletes, to curb the damage caused by lipoproteins (50). Diet and exercise intervention have also been shown to exhibit increased activity of platelet-activating factor acetylhydrolase (332).

A large number of studies have demonstrated increased levels of lipid peroxidation products, including malondialdehyde and F₂-isoprostane, after acute exercise bouts (159, 257, 318, 359). However, regular exercise training, in general, results in decreased levels of lipid peroxidation (159, 255, 259, 359). The regular exercise-associated attenuation of lipid peroxidation levels could be due to the increased capacity of the antioxidant systems, but the enhanced activity of lipid repair cannot be ruled out.

All amino acid residues can be modified when attacked by ROS, generating oxidation of amino acid side chains. The level of oxidative damage to proteins is a magnitude higher than that observed in lipids and DNA (319). Oxidized proteins readily generate cross-linkages and aggregate in cells. The accumulation of this oxidative junk can jeopardize the cellular function and fate. However, oxidative damage to proteins renders them susceptible to degradation by increasing the hydrophobicity of the surface of proteins (376), which could serve as a tag for the proteasome system, and lead to degradation (375). This process does not consume energy and is done without ubiquitination (366). It has been shown that proteolytic susceptibility of proteins in relation to oxidative modifications shows a biphasic response, while at moderate oxidant concentrations, proteolytic susceptibility increases, and further oxidation leads to a decline in the proteolytic susceptibility, sometimes even below the basal degradation. Such behavior seems to be a common feature of all globular, soluble proteins with defined secondary and tertiary structures, independent of the origin of the proteins (49, 118, 170, 411).

The feature that carbonylated proteins are targeted for removal by proteasomes suggests that carbonylation may act as a signal ensuring that damaged proteins enter the degradation pathway rather than the chaperone/repair pathway, since carbonylation is an irreversible/unrepairable modification (264). Increased levels of protein carbonyls are linked to a variety of age-associated diseases and often correlate with the progress of disorders and diseases (375). Carbonyl derivatives are formed by a direct metal-catalyzed oxidative attack on the amino acid side chains of arginine, lysine, proline, and threonine. Moreover, carbonyl derivatives on cysteine, histidine, and lysine can be also generated by secondary reactions with reactive carbonyl compounds on sugars, lipids, and advanced glycation/lipoxidation end products (375). Methionine and cysteine residues are even more prone to oxidation than others due to the sulfur content of these residues. The oxidative modifications of these two residues are reversible, indicating a redox-dependent signaling of methionine and cysteine residues. Cysteine residues are converted to disulfide by oxidation and reduced back to cysteine under mild conditions such as in the presence of reducing agents such as NADH without enzyme reaction. Methionine residues are transformed to methionine sulfoxide, which in turn can be repaired by methionine sulfoxidereductases, which catalyze the reduction of methionine sulfoxide back to methionine residues (181). The role of methionine reductase in exercise studies remains to be evaluated. On the other hand, carbonylation, which is an irreversible modification, is very well studied in exercise studies. An acute bout of intensive exercise increases the rate and accumulation of carbonyl derivatives (36, 258, 316), and regular exercise either maintains or decreases carbonyl levels (45, 68, 114, 318). Increased activity of the proteasome system is important for protein remodeling and, of course, to prevent the accumulation of damaged proteins that could impair cell function. The effects of exercise on the proteasome system and carbonyl accumulation could be dependent on whether the exercise is acute or chronic and also the type of exercise (resistance or endurance) (230, 246, 293, 369). Mitochondria, the main site of ROS production, need a very efficient system to prevent the accumulation of oxidized proteins. The main quality control of mitochondrial proteins is mediated by Lon protease and HSP78 (42, 43, 253, 337, 411). Indeed, it has been shown that Lon expression can be readily induced by a moderate level of oxidative stress, which prevents the accumulation of oxidized proteins and protects cell viability (254). Ischemia, for instance, significantly induced the expression of Lon protease, indicating that endoplasmic reticulum-related stress could have an impact on mitochondrial biogenesis and the degradation of mitochondrial proteins (146). Moreover, it has been shown that silencing of Lon protease resulted in hepatic insulin resistance, and the restoration of Lon activity normalized insulin handling in the liver (199). Thus, Lon can be regarded as a stress protein (253).

Lon levels decline with age, which could result in the accumulation of oxidized, or dysfunctional proteins in the mitochondria (42, 43, 196), and perhaps in the loss of mitochondrial function. We have recently observed that regular exercise can prevent the age-related decline in Lon protease (187), and therefore rejuvenate proper quality control of proteins in older cells (411). We have also shown that exercise training induced HSP78 levels in both young and old animals (187), which indicates that regular exercise has beneficial effects on mitochondrial protein quality control.

8-oxoG is the most often measured form of oxidative damage to DNA and has been reported to increase with aging, ischemia/reperfusion, radiation, and a wide range of pathological disorders (37, 248, 260, 378). Human 8-oxoguanine-DNA glycosylase (OGG1) plays a major role in the base excision repair pathway by removing 8-oxoguanine base lesions generated by ROS. OGG1 activities are primarily regulated by p300/CBP-mediated acetylation, predominantly on Lys338/Lys341 (32, 148, 388). An increased deacetylase activity of sirtuins may lead to decreases in acetylation levels of proteins, which, in turn, would result in a decline in enzymatic activity of OGG1. We have recently shown that aging increased 8-oxoG content in the hippocampus, with increased levels of OGG1. However, the acetylation of OGG1 was very low in the aged animals (189). Physical activity, on the other hand, increases the acetylation of OGG1 and decreases the age-associated accumulation of OGG1 in human skeletal muscle (319). Indeed, we and others have shown that acute and regular exercise increases the activity of OGG1 (249, 303, 306, 313, 314, 348). Therefore, the upregulated DNA repair could be an important tool by which regular exercise maintains DNA purity and could curb the incidence of certain cancers (333).

It is highly possible that evolution of the biological system has selected guanine as the potential target of ROS. As a result of low redox potential, attractiveness to ROS is apparent and makes it the most oxidizable nucleic acid base, and this finding suggests that the generation of 8-oxoG could have a significant physiological role (307, 377), as other markers of oxidative damage, such as malondialdyde or protein carbonyls 8-oxoG, are always detectable in cells. An interesting observation made on OGG1 knockout animals is that lipopolysaccharide-induced inflammation was more tolerated than in the wild types (221). The lack of OGG1, hence, resulted in increased levels of 8-oxoG, and reduced removal of this damaged base, which actually provided better protection against inflammation. Thus, the inverse correlation between the activity of OGG1 in this inflammation model suggests that certain amounts of 8-oxoG might be more tolerable to the cells than the excised form that generates inflammation (221). It was recently suggested that oxidation of guanine could be important in the transformation of heterochromatin to euchromatin (307). In addition, it has been demonstrated that exposure of cells to estrogen increases 8-oxoG levels in DNA and recruits OGG1 and topoisomerase IIβ to estrogen-responsive DNA elements in the promoter region of 17β-estradiol (E2)-responsive genes (282). Therefore, 8-oxoG, besides its damaging affect on transversion base pairs, could be important for the generation of signals that are necessary for specific gene transcription. Indeed, it has been shown that the 8-oxoG-OGG1 complex can interact with Ras proteins and work as a guanine nuclear exchange factor (38). In addition, it could be very important to redox signaling that the 8-oxoG-OGG1 complex-mediated Ras activation results in phosphorylation of the mitogen-activated kinases MEK1,2/ERK1,2 and increasing downstream gene expression. This novel information on the interaction of free 8-oxoG, but not other oxidized bases such as 8-oxodG, FapyG, or 8-oxoA, with OGG1 leads to redox signaling and could partly explain why OGG1 knockout mice are more resistant to inflammation than wild types (221).

Data from exercise studies suggest that a moderate level of oxidative stress, which could slightly increase the levels of certain oxidative damage markers, might not actually jeopardize cell function, but rather could be necessary to initiate an adaptive response (Fig. 7). Physical exercise is a natural stimulator of ROS production and a mild oxidative stressor, as demonstrated by a moderate level of oxidative damage, which can lead to an enhanced physiological function.

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FIG. 7.

Lipid peroxidation byproducts, carbonyl groups, and the 8-oxoG levels are easily detectable, suggesting that these ROS-induced modifications could be necessary for cells. Lipid peroxidation can be induced by enzymatic processes and could be important to membrane remodeling. Carbonylation of amino acid residues could be an important mediator of protein turnover, since carbonylation can serve as a tag for proteolytic degradation. 8-oxoG is necessary for transcription of specific genes and for the opening of chromatin (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.) 8-oxoG, 8-Oxo-7,8 dihydroguanine; OGG1, 8-oxoguanine-DNA glycosylase.

A. Sirtuins as redox-sensitive energy sensors of exercise

Nicotinamide adenine dinucleotide (NAD), with the ability to carry two electron equivalents, serves as an energy source, and at the same time, the cytoplasmic NAD/NADH ratio is a sensitive marker of the cellular redox state (235, 432, 441). NAD-dependent lysine deacetylases, sirtuins, control various cellular processes, including chromatin structure by the deacetylation of lysine residues of histones (271, 396), apoptosis and cell survival, by the interaction with p53 and FOXO transcription factors (233, 354, 444), mitochondrial biogenesis via deacetylation of PGC1a (252), lipid metabolism by the interaction with SREBP-1c among other proteins (291, 352, 390), insulin homeostasis (98, 205), DNA repair (171, 224, 231), inflammation by modulating the activity of NF-κB (344, 439), and oxygen sensing by deacetylating hypoxia-inducible factor-1a (HIF-1α) and HIF2α (66, 82, 210) among other mechanisms. Sirtuin-mediated deacetylation of these transcription factors would readily effect the expression of pro- and antioxidant genes, such as PUMA, NOXA, PIG3, GADD45, Nrf2, and Mn-SOD, and stress-activated protein kinases etc., which can readily regulate redox signaling. Besides the interaction between SIRT1 and poly(ADP-ribose) polymerase (PARP) to control metabolism (22), the latter is implicated in apoptosis (88), DNA repair (134), ischemia/reperfusion (403), diabetes (387), and inflammation (386).

Sirtuins (silent information regulator 2 [Sir2] proteins) belong to an ancient family of evolutionary-conserved NAD⁺-dependent enzymes with deacetylase and/or mono-ADP-ribosyltransferase activity. Seven Sir2 homologs, sirtuins (SIRT) 1 to 7, have been identified in mammals. The crystal structure of human SIRT1, which is a homolog of yeast Sir2, revealed a large groove that was intersected by a pocket lined with hydrophobic residues conserved with class-specific protein-binding sites of each Sir2 class (92). Enzymes from the sirtuin family are ubiquitously distributed. However, their level/activity has organ specificity. SIRT 3–5 are predominantly localized in the mitochondria.

The NAD/NADH ratio is changing readily during muscle contraction, due to the reduction of NAD to NADH that is catalyzed by glyceraldehyde 3-phosphate dehydrogenase and the conversion of NADH to NAD by the glycerol phosphate shuttle and lactate dehydrogenase (LDH). LDH facilitates the pyruvate uptake of mitochondria (52) that further influences the NAD/NADH ratio, and one can expect decreased SIRT1 activity right after intensive exercise bouts, due to the increased lactate/pyruvate ratio, which, in turn, decreases the NAD/NADH ratio. However, exercise bouts increase the activity of SIRT1 (188, 308, 383), indicating that within a certain range, SIRT1 activity is independent from the NAD/NADH ratio (9). On the other hand, significant oxidative stress could down-regulate SIRT1 (7).

Sirtuins, especially SIRT1, are implicated in the aging process of different organisms (119, 391), and it has been shown that changes in SIRT1 activity by agents, such as caloric restriction (CR) or resveratrol or SRT501, increase lifespan via a wide range of processes, including suppressed apoptosis, and inflammation, or enhanced DNA repair (2, 292). Although, the beneficial effects of sirtuin activators on mammals are still unclear, some data indicate that sirtuins do play both protective and proaging roles (206). Accumulating evidence suggests that exercise alters the activity, content, and expression of different sirtuins (Fig. 8). Indeed, there are reports that have demonstrated an exercise-associated oxidative challenge that results in increased expression, content, and activity of SIRT1 in humans (73, 225, 308). It seems that besides AMPK, SIRT1 is a sensitive metabolic sensor in skeletal muscle (96), and the fact that histone lysine residues are one of the targets of SIRT1 implies that the redox-sensitive energy sensor SIRT1 links energy metabolism to transcriptional regulation. In addition to this, SIRT1 and p300/CBP have been shown to control myogenic transcription factor, which is associated with dynamic changes to the NAD/NADH ratio (99), and points out the role of redox signaling via SIRT1 in the development of skeletal muscle. SIRT1 is downstream in ROS signaling, but can be importantly upstream in regulating cellular levels such as activation of FOXO3 (164), muscle ring-finger protein 1 (MuRF1) (8), and protein kinase B (Akt) (382). Therefore, these NAD-dependent metabolic sensors are regulating redox signaling in a wide array (222).

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FIG. 8.

Sirtuins are NAD⁺-dependent histone deacetylases and sensitive markers of metabolic and redox processes. SIRT-mediated deacetylation of target proteins is involved in metabolic processes, biogenesis of mitochondria, oxygen sensing, inflammation, apoptosis, and epigenetics, among others. Factors that are modifying redox balance, such as aging, caloric restriction, or physical exercise, readily alter the activity of sirtuins. The exercise-induced adaptive response, which includes metabolic and redox processes, involves the sirtuin protein family. NAD, nicotinamide adenine dinucleotide. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

Aging decreases the activity of SIRT1, which results in increased levels of lysine acetylation in the skeletal muscle and brain of rats (188, 189, 227). The effects of acetylation/deacetylation for the function of proteins have been extensively investigated in a number of laboratories. However, the mechanism is still not well understood. It has been shown that acetylation of lysine residues could affect ubiquitination of the same residue, and therefore could impact the stability of proteins. Mechanistically, larger degrees of acetylation, as we have observed with aging, could slow down ubiquitin-mediated degradation resulting in enhanced half-life and then larger accumulation of carbonylation, which could lead to an impaired physiological function. This hypothesis seems to be very attractive and concurs with some well-observed phenomena, such as age-associated increases in the half-life of proteins (116), decreased rates of protein degradation with aging (115), and impaired function as a result of aging. Exercise, depending on the intensity and duration, on the other hand, increases the activity of SIRT1, decreases the level of lysine acetylation, increases the turnover rate of proteins, decreases the accumulation of carbonyl groups, and improves cellular function (Fig. 9). It is important to note that the effects of a single bout of exercise and/or regular exercise could be completely opposite to oxidative stress, ubiquitination, and acetylation. The findings from a recent report indicate that aging does not significantly alter the expression or protein content of MuRF1, while patients with chronic heart failure showed increased expression and content of MuRF1, which was downregulated by 4wk of exercise training (107). Although this study did not examine the levels of sirtuins, it is clear that exercise training on healthy subjects and on patients with diseases would have different effects on the rate of ubiquitination and degradation. The stabilization of HIF-1α, in cardiac muscle during diseases, in the short term could be beneficial, but detrimental in the long term (144). Acetylation on protein stability varies significantly, and HIF-1α, for example, is ubiquitinated at normoxic conditions and degraded by proteasome (200), and acetylation of HIF-1α improves the interaction between ubiquitin ligase, and hence prepare protein for degradation. On the other hand, during hypoxia, SIRT-dependent deacetylation of HIF1α stabilizes the protein (200, 210).The fact that caloric restriction and physical exercise increase the activity of SIRT1 and attenuate the age-associated increase in lysine acetylation is intriguing, and needs further investigation. It is clear that the effects of lysine acetylation/deacetylation, in relation to SIRT1 and protein stability, result in a number of interesting findings.

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FIG. 9.

The suggested mechanisms between acetylation, protein stability, aging, and cellular function. Lysine acetylation could prevent the ubiquitination of the same lysine residue, and hence effect protein stability and half-life. Longer half-life results in significantly increased carbonylation of protein residues, which results in impaired cellular function.

SIRT2 is mostly localized in neurons and nerves, and preferentially deacetylates tubulin and histone H4. Moreover, a recent finding suggests that SIRT2 is activating myelin formation in Schwann cells, thus controlling an essential polarity pathway during myelin assembly (25). The effects of exercise on SIRT2 are generally unknown.

Another isoform of the sirtuin family, SIRT6, is closely associated with chromatin and increases the resistance of DNA to oxidative DNA damage and facilitates base-excision repair (239). However, recent data suggest that SIRT6, besides its crucial role in chromatin structure, is a regulator of carbohydrate metabolism. SIRT6-deficient cells show nutrient-replete conditions and shift to lactic acid glycolysis, and transfer metabolism to the survival mode from normal aerobic conditions in the presence of oxygen (445). SIRT6 deficiency activates the gene of pyruvate dehydrogenase kinase and inhibits pyruvate dehydrogenase, resulting in impairment of pyruvate conversion to acetyl-CoA to fuel the TCA cycle (445). In accordance with this, we have observed decreased levels of SIRT6, after a single bout of exercise, in the skeletal muscle of young and old individuals (308). The expression of SIRT6 was not affected by aging in human skeletal muscle, but rat skeletal muscle increased the levels of SIRT6 protein, while regular exercise training attenuated this increase, suggesting that the age-associated changes in metabolism and/or in chromatin structure are normalized by an exercise-driven adaptation (188). SIRT6 appears to be differently regulated by exercise than SIRT1 (188), which could be due to the chromatin tightness, controlling the role of SIRT6. A single bout of exercise results in large metabolic and oxidative challenges to skeletal muscle, which, to regulate an adaptive response, must open chromatin to allow gene expression to cope with the exercise stressor (308). The downregulation of SIRT6 is in accordance with this hypothesis.

SIRT7 is the only mammalian sirtuin that is mainly localized in the nucleoli. When the nucleoli disintegrate, SIRT7 associates with condensed chromosomes (232). The molecular targets of SIRT7 have not been identified, but data obtained from SIRT7 knockout mice reveal that SIRT7 deficiency leads to progressive heart hypertrophy, accompanied by inflammation and decreased stress resistance (412). At least a part of this phenotype may be explained by the fact that SIRT7 deacetylates p53, leading to hyperacetylation of p53 in vivo along with various other changes in the signaling network of cardiomyocytes (412). The effect of exercise on SIRT7 remains to be explored.

Mitochondria, the powerhouse of ATP production, also host sirtuins. One is SIRT3, which could be present in the nuclei and mitochondria (351), and it has been suggested to control metabolism and oxidative stress of mitochondria (29). Overexpression of this enzyme positively regulates the oxidative capacity of mitochondria and attenuates the generation of ROS (190), indicating that SIRT3 plays a key function in redox regulation.

Moreover, it appears that the activation of Mn-SOD is dependent on SIRT3- (302) mediated deacetylation of 122 lysine residues (393). In addition, SIRT3 has a powerful deacetylase activity on histone H4 peptides and is a major regulator of mitochondrial deacetylation (216), affecting fatty acid oxidation (137) via the deacetylation of mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase 2 (364). SIRT3 activates isocitrate dehydrogenase 2 and glutamate dehydrogenase by deacetylation, and these enzymes are involved in the production of mitochondrial NADPH. The elevated NADPH, in turn, is necessary for GSH reductase, which reduces GSSG to GSH, the cofactor necessary for mitochondrial GPX function (29).

We have found that aging increases SIRT3 levels in the rat hippocampus, and is attenuated by exercise training (188). On the other hand, exercise training and diet have been shown to increase the levels of SIRT3 (141, 274) in skeletal muscle and AMP-activated protein kinase (AMPK) as well as cAMP-response element-binding protein (CREB), indicating upstream modulators of SIRT3 (274). These data suggest that SIRT3 is a potent redox-dependent modulator of cellular metabolism and is readily modified by physical exercise.

SIRT4 is also localized in the mitochondria and has been implicated in the regulation of insulin secretion in beta-cells. Knockdown of SIRT4 increased fat oxidation in the liver and skeletal muscle (251), indicating that SIRT4, without significant deacetylase, but with high ADP-ribosyltransferase activity, also modulates cellular metabolism. The effects of exercise on SIRT4 are poorly investigated, but it appears that a single bout of exercise significantly decreases the mRNA expression of this enzymes in peripheral blood mononuclear cells (225).

SIRT5 is hosted by the mitochondria, and recent reports of this poorly studied sirtuin have revealed that SIRT5 is involved in the detoxification of ammonia by deacetylating carbamoyl phosphate synthetase 1, which is one of the key enzymes in the urea cycle, which catalyzes condensation of ammonia with bicarbonate to form carbamoyl phosphate (267). Ammonia is readily elevated during intensive exercise through deamination of AMP and branched-chain amino acids. Therefore, it is very likely that SIRT5 is modulated by exercise, and the adaptive response to regular exercise training involves SIRT5. Moreover, a recent report suggests that SIRT5, besides its deacetylase activity, also shows protein lysine desuccinylase demalonylase activity in the in vitro condition, implying that this post-translational modification might have a physiological role, controlled by redox signaling (86).

The available information strongly suggests that NAD-dependent sirtuins are very sensitive to metabolic challenges, and are modulated by redox signaling. Therefore, sirtuins are readily modified by physical exercise, and data suggest so far that sirtuins could play an important role in exercise-induced adaptations, including physical fitness, health promotion, and the attenuation of the progress of aging.

B. Redox signaling and biogenesis of mitochondria

It is clear that metabolic stress (in particular through AMPK/PGC-1α-dependent signaling processes) is one of the well-characterized ways to increase mitochondrial mass in skeletal muscle (123). However, it is also well characterized that increased metabolism is associated with changes in redox regulation (62). Therefore, oxidative stress has been suggested to be one of the causative signals of mitochondrial biogenesis (77), which is an important mechanism to reduce the exercise-induced leak of ROS from the mitochondrial network. For the same ATP production, more mitochondria can work at a lower respiratory capacity; hence, less ROS are produced, and therefore mitochondrial biogenesis can be regarded as part of the antioxidant system. Proteomic analyses yielded over 900 distinct mitochondrial proteins in human muscle and all, except the 13 encoded by mitochondrial DNA, are imprinted on nuclear DNA (273). In skeletal muscle fibers, the mass of mitochondria is dependent on the muscle subtype. The numbers and functions of mitochondria required to maintain fiber homeostasis are determined by regulatory circuits/programs that are under synchronized regulation between the mitochondrial and nuclear genes, to maintain function of respiratory electron transport complexes and proteins that are required for physiological functions of mitochondrial networks, including oxidative phosphorylation, ionic homeostasis, and mitochondrial uncoupling (thermogenesis).

In addition, skeletal muscle is equipped with two types of mitochondria: intermyofibrillar (IMF) and subsarcolemmal (SS). Although the location of mitochondria could be related to the level of ATP production, significant differences were not noted after oxidative stress or exercise training in the subpopulations (64, 65, 169). The findings of a recent study revealed that SIRT3 is present in IMF and SS, and chronic stimulation increased the content of IMF, SS, and SIRT3 in a parallel manner (122).

Mitochondrial gene expression is regulated by mitochondrial transcription factor A (TFAM), while the gene expression of nuclear DNA-encoded mitochondrial proteins is controlled by respiratory factors 1 and 2 (NRF1 and NRF2) (371). The coactivation of the transcription factors to promoter regions is carried out by PGC-1α, which is the master regulator of mitochondrial biogenesis (380). Overexpression of PGC-1α leads to a large increase in mitochondrial biogenesis, cytochrome oxidase II and cytochrome oxidase IV content, ATP synthase, myoglobin, and citrate synthase activity (212). Moreover, PGC-1α appears to have the capacity to increase mitochondrial electron transport activity while stimulating a broad range of anti-ROS mechanisms, including regulation of the expressions of Cu,Zn-SOD, GPX, catalase, uncoupling protein 2 (UCP2), and UCP3 (374). Indeed, knockout of PGC-1α leads to significant decreases in Cu,Zn-SOD, Mn-SOD, and GPX content, compared to those found in wild types of mice (202). Moreover, studies on PGC-1α knockout fibroblasts revealed significantly attenuated responses to H₂O₂ treatment by Mn-SOD, catalase, and GPX (374). Moreover, transgenic mice with overexpression of PGC-1α showed lower levels of loss in muscle mass as a result of aging, and the rate of oxidative damage was also attenuated, indicating that the exercise-associated upregulation of this transcription factor could cause similar health-promoting effects (431).

These observations confirm that PGC-1α actively is involved in antioxidant defense. Inflammation is associated with increased ROS production and oxidative damage, and the antioxidant role of PGC-1α is further supported by the anti-inflammatory effects of this coactivator. It has been shown that PGC-1α knockout mice, besides reduced endurance activity, exhibit increased levels of inflammation (129). HeLa cells were treated with ethidium bromide to deplete mitochondrial DNA, which resulted in increased oxidative stress and induction of TFAM and NRF-1, suggesting that ROS could be an important signal for mitochondrial biogenesis (236). Homocysteine-induced ROS production, which resulted in an increased expression of NRF-1 and TFAM, was attenuated by antioxidant treatments, again supporting the importance of ROS in mitochondrial biogenesis (281). Aging results in a chronic redox shift toward an oxidized milieu, which, in a significant way, can be attenuated by regular physical activity affecting a number of players in the mitochondrial biogenesis. Indeed, it seems that PGC-1α, TFAM, and NRF1 are all sensitive to redox balance and induced by ROS, leading to increased mitochondrial biogenesis and readily modified by exercise bouts (Fig. 10). Exercise resulted in increased ATP and ROS production by the mitochondria of skeletal muscle of subjects with normal glucose tolerance and impaired glucose tolerance by the induction of TFAM and NRF1 (106). Moreover, it was observed that aging and impaired glucose tolerance resulted in decreased ATP production and maintained levels of ROS (106).

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FIG. 10.

Exercise results in large metabolic challenges to skeletal muscle that cause mitochondrial biogenesis and alteration of the mitochondrial network affecting fusion and fission. ROS are important signaling molecules for muscle contractions, PGC-1α, MAPK, as well as for transcription factor NF-κB. NAD⁺/NADH levels are readily modified by ROS and could affect the activity of sirtuins (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.) AP-1, activator protein-1; iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; Mn-SOD, manganese superoxide dismutase; NF-κB, nuclear factor-kappaB; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator 1α.

Most of the protein components of the mitochondria are encoded by nuclear DNA, including RNAs, and are important to mitochondrial biogenesis. Recent data have revealed that mammalian polynucleotide phosphorylase (PNPase) is localized in the mitochondrial intermembrane space to regulate the translocation of RNA into mitochondria (425). Silencing the PNPase with RNA interference results in decreases in mitochondrial membrane potential, ATP synthesis, and increased production of lactic acid (63). PNPase-deficient cells accumulate 8-oxoG in cellular RNA, indicating that PNPase is involved in the antioxidant/damage repair process (436). Indeed, PNPase is bound in a higher degree to oxidized RNA (436). Overexpression of PNPase could also be harmful to cells, since it can cause increased production of ROS and inflammation (347). Thus, both up- and downregulation of PNPase could impair cell function (133). The available information on the effects of exercise on PNPase is very limited. Our unpublished observations suggest that aging decreases the mRNA levels of PNPase in human skeletal muscle, while exercise decreases it in young and increases the expression in old subjects (41). On the other hand, in skeletal muscle of rats, we have observed increased amounts of PNPase with aging, and this was attenuated with exercise training (187). These data suggest that the relationship between exercise and PNPase is very complex.

SIRT1 has been proposed to be one of the activators of PGC-1α during exercise-induced mitochondrial biogenesis, although a recent report showed no interaction between SIRT1 and PGC-1α (284). Moreover, besides acetylation, other post-translational modifications such as phosphorylation appear to represent an immediate mechanism to activate PGC-1α- and initiate PGC-1α-dependent gene expression (67). The results of a study with human subjects has revealed that NAC supplementation blocked exercise-associated activation of JNK, but the mRNA level of PGC-1α was not affected by the supplementation of this antioxidant (283). Moreover, it is not clear at the moment whether PGC-1α phosphorylation is significantly affected by a redox milieu.

GLUT4, the insulin-dependent carbohydrate transporter, can be modulated by ROS (136), which means a shift in the redox state toward an oxidative milieu suppresses carbohydrate metabolism and increases insulin resistance (48, 430). During exercise, the need for carbohydrate enhances carbohydrate uptake, but during rest, the upregulated antioxidant system provides excellent conditions for the insulin-dependent carbohydrate uptake (213) with increased concentrations of the GLUT4 transporter.

The regulatory role of ROS in mitochondrial biogenesis was suggested many years ago, and a significant body of reference material has accumulated supporting this idea. Moderate levels of ROS appear to stimulate mitochondrial biogenesis via PGC-1α, SIRT1, and TFAM, while the increased levels of mitochondria decrease ROS production, in addition to the antioxidant role of PGC-1α and PNPase.

A. Hypoxia in skeletal muscle

A gradient of oxygen partial pressure (pO₂) exists within the body, due to ∼160 mmHg in inspired gas, ∼115 mmHg in arterial blood, and ∼38 mmHg in capillary blood (328). Within tissues, the pO₂ gradient depends on the distance of cells from the closest O₂-supplying blood vessels and the metabolic activity and consequent O₂ consumption of the resident cells (395). For example, pO₂ in intramyocellular cells falls to as low as 3.1 mmHg under normoxia and 2.1 mmHg under hypoxia, respectively (328). Mitochondria house the final biochemical steps in the production of reducing equivalents that react at the terminal oxidases of the respiratory chain, with molecular oxygen provided by the respiratory system from the environment. In mitochondria, O₂ finally disappears, and oxygen partial pressure goes to zero (145). Mitochondria thus are an effective oxygen sink, and this allows organisms to use all of the available oxygen partial pressure of the actual environment to drive the respiratory cascade from the lungs through circulation to the mitochondria (394).

The transcription factor, HIF-1α, acts as a master regulator for the expression of genes involved in the hypoxic response of most mammalian cells (355, 365). Namely, HIF-1α is hydroxylated and degraded in normoxia, but is stable in hypoxia and translocates into the nucleus to form an active complex with HIF-1β, which is constitutively expressed (154, 355). Nevertheless, it is clearly expressed in various tissues such as the brain, kidney, liver, heart, and skeletal muscle in mice kept in normoxic conditions (21% O₂), whereas it is undetectable in the lungs (379). Further, when mice were kept in extreme hypoxia (6% O₂), the expression levels of HIF-1α in the brain, kidney, liver, heart, and skeletal muscle were significantly increased, while expression in the lungs was not affected (379, 443). These findings indicate that there is a clear pO₂ gradient between the lungs and other tissues, and that with the exception of relatively well-oxygenated lungs, low pO₂ and HIF-1α play physiologically essential roles in homeostasis of various tissues. The proposed explanation might be rather simplistic as factors as oxygen uptake and supply and extraction by individual organs could play an important role in determining the HIF response. It cannot be ruled out that pO2-dependent HIF response after induction is often bell-shaped, and results from both HIF mRNA expression induction and HIF protein stabilization in the absence of oxygen.

Mounier et al. (240) have shown, in untrained human skeletal muscle under normoxic conditions, a higher HIF-1α protein expression in predominantly oxidative muscles than in predominantly glycolytic muscles. The HIF-1α mRNA expression pattern however was not in agreement with the HIF-1α protein level. Interestingly, none of the HIF-1α target genes, such as the most studied angiogenic factor involved in muscle angiogenesis and vascular endothelial growth factor (VEGF), exhibited a muscle fiber-specific related mRNA expression at rest in normoxia. However, soleus presented a significantly higher VEGF protein content than vastus lateralis and muscles of triceps. Taken together, such findings indicate that there are muscle-specific differences in HIF-1α and VEGF expression within human skeletal muscle at rest in normoxic conditions.

Thus, the following section focuses on the significance of various conditions, such as hypoxia, oxidative stress, and physical exercise, in the HIF-1α- and VEGF-signaling pathways, with emphasis on skeletal muscle.

B. HIF-1α-independent regulation of VEGF and angiogenesis

Peroxisome proliferator-activated receptor (PPAR)-γ coactivator 1α (PGC-1α) regulates mitochondrial biogenesis in multiple cell types through coactivation of key transcription factors, including NRF-1 and NRF-2, estrogen-related receptor-α (ERRα), Gabpa/b, PPARs, and the thyroid hormone receptor (16, 265). PGC-1α is a mediator of signaling in response to deprivation of nutrients and oxygen, and it strongly regulates VEGF and other angiogenic factors to elicit neovascularization in vivo. The regulation of VEGF in response to hypoxia is thought to be mediated primarily through the well-known HIF factors (355). Surprisingly, the novel PGC-1α/ERR-α pathway (16) is apparently independent of the HIF pathway. PGC-1α^−/− mice are viable, suggesting that PGC-1α is not essential in embryonic vascularization (16). Angiogenesis in the adult occurs in both physiological and pathological contexts (57). The robust induction of vascularization by PGC-1α and its critical function in the response to limb ischemia strongly implicate PGC-1α in the angiogenic response to ischemia, providing protection against further ischemic insults (16).

On the other hand, PGC-1α is coupled to HIF signaling through the regulation of intracellular oxygen availability, allowing cells and tissues to match increased oxygen demand after mitochondrial biogenesis with increased oxygen supply, because of intracellular hypoxia (265). PGC-1α-dependent induction of HIF target genes under physiological oxygen concentrations is not through transcriptional coactivation of HIF or upregulation of HIF-1α mRNA, but through HIF-1α protein stabilization.

Other candidates are mentioned for the HIF-1α-independent regulation of VEGF and angiogenesis. Overexpression of suppressor of cytokine signaling 3 (SOCS3), a well-established negative regulator of signal transducer and activator of transcription 3 (STAT3), a mediator of cytokine signaling, which is a crucial factor for myogenesis, enhances the mRNA expression of downstream targets of STAT3, c-FOS, and VEGF, despite inhibition of STAT3 phosphorylation, and is correlated with enhanced mRNA expression of genes associated with muscle maturation and hypertrophy (55). The mechanism by which SOCS3 may mediate these responses however is unknown and will require further investigation. The decline in the regenerative capacity of skeletal muscle with age may be linked to inefficient STAT3/SOCS3 signaling. Actually, a single bout of maximal leg-extension exercise (resistance exercise) markedly increased the STAT3 signaling, including the SOCS3 gene in human skeletal muscle, accompanied by significant increases in levels of mRNAs for downstream genes of STAT3, c-FOS, JUNB, c-MYC, and VEGF, but probably not via HIF signaling (407). In addition, the levels of HIF-1 mRNA subunits did not change in response to short-term one-legged exercise training, suggesting no change in HIF-1 mRNA transcript levels in the regulation of training-induced VEGF expression (124).

Increased production of ROS, such as H₂O₂, also induces the expression of VEGF-A, the prototype VEGF ligand, by an HIF-independent and Sp1-dependent mechanism, in which ligation of VEGF-A to VEGF receptor 2 (VEGFR2) results in signal transduction leading to tissue vascularization. Such ligation generates H₂O₂ via an NADPH oxidase-dependent mechanism (1). That the function of one mitogen, VEGF, largely depends on signaling driven by another mitogen, H₂O₂, generates a novel paradigm with major therapeutic implications for a variety of angiogenesis-related disorders (338). Furthermore, oxygen–glucose deprivation generates ROS in cerebral endothelial cells, and in turn induces VEGF signaling via its receptor, Flk-1 (VEGFR2), and activates extracellular signal-regulated kinase (ERK) 1/2, thereby suggesting that VEGF acts via ERK 1/2 through oxidative-stress-dependent mechanisms, showing that ERK 1/2 can be considered a molecular target for stroke therapy (250). Interestingly, high glucose blunts VEGF response to hypoxia in immortalized rat proximal tubular cells, this effect being mediated by the oxidative stress-regulated HIF–hypoxia-responsible element pathway (173).

C. p38γ MAPK/PGC-1α signaling regulation by physical exercise and its significance for mitochondrial biogenesis and angiogenesis in skeletal muscle

p38γ MAPK, but not p38α MAPK or p38β, is required for PGC-1α upregulation in mitochondrial biogenesis and angiogenesis in response to voluntary wheel running and nerve stimulation in mice. None of the p38 MAPK isoforms was required for endurance exercise-induced IIb-to-IIa fiber-type transformation in these mice (213). Meanwhile, calcineurin (CnA) nuclear factor of activated T-cell (NFAT) regulatory axis controls fiber-type transformation in endurance exercise-induced skeletal muscle adaptation (288). Collectively, endurance exercise on one hand induces activation of the Ca²⁺-dependent CnA-NFAT pathway in control of fiber-type transformation, and on the other hand activates p38γ MAPK, which promotes PGC-1α activity and expression in control of mitochondrial biogenesis and angiogenesis (213, 288).

An acute bout of sprinting exercise that increased ROS production, mainly through a nonmitochondrial enzyme, XO, stimulated PGC-1α expression and other key proteins in the mitochondrial biogenic pathway, such as NRF-1 and TFAM. This nuclear-initiated signaling event was accompanied by activation of p38 MAPK and CREB phosphorylation. Inhibition of XO by allopurinol severely attenuated exercise activation of the PGC-1α signaling pathway, thus providing strong evidence that mitochondrial biogenesis in skeletal muscle is controlled, at least in part, by a redox-sensitive mechanism (143, 172). Moreover, stretch-stimulated glucose uptake in skeletal muscle is mediated by an ROS- and p38 MAPK-dependent mechanism that appears to be AMPKα2- and phosphatidylinositol 3-kinase (PI3-K)-independent (60). This novel signaling mechanism is distinct from canonical contraction- and insulin-stimulated signaling that increases glucose uptake, probably providing an alternative pathway for the development of novel therapeutic drugs to overcome insulin resistance. MAPK activation is also involved in the contraction-induced secretion of VEGF protein from skeletal muscle, which is partially mediated via adenosine acting on A_2B adenosine receptors (140).

D. Two-faced ROS in HIF activation

ROS are widely believed to cause or aggravate several human pathologies such as neurodegenerative diseases, cancer, stroke, and many ailments. Antioxidants are assumed to counteract the harmful effects of ROS and therefore prevent or treat oxidative stress-related diseases (93). On the other hand, there is growing evidence that ROS have deleterious or beneficial effects; this dual nature of ROS means that ROS act as intracellular signaling molecules as defense mechanisms against microorganisms (108). For instance, the mechanisms by which ROS and cytosolic H₂O₂ levels are involved in stabilization/activation and eventual degradation/silencing of HIF-1α and other redox-sensitive transcription factors, such as MAPK and PI-3K/Akt, are discussed (179).

Mitochondria and mitochondrion-derived ROS were required for the stabilization of HIF-1α by MAPK and by exposure of hepatoma cells to 6 h of hypoxia (1.5% O₂) (61). This hypoxic stabilization of HIF-1α in hepatoma cells was blocked by the overexpression of catalase, which catalyzes dismutation of H₂O₂ into H₂O and O₂, and is an inhibitor of the mitochondrion super anion channel. Also, HIF-1α and VEGF overexpression in pulmonary artery smooth muscle was induced by CoCl₂, which mimics the hypoxic response, via ROS (28). Such overexpression was inhibited by Vitamin C and by overexpression of GPX and catalase. In contrast, the addition of H₂O₂ in the absence of a hypoxic stimulus causes stabilization of HIF-1α (61). Anoxia, on the other hand, may stabilize HIF-1α essentially through depletion of the cosubstrate dioxygen of prolyl-hydroxylases, indicating that increases in cytosolic H₂O₂ in consequence of hypoxia-driven ROS production may cause HIF-1α accumulation in hypoxia through reduction of its hydroxylation (145, 12). Actually, during the acute increase phase of HIF-1α (between 0 and 2 h), 8-hydroxy-2′-deoxyguanosine (8-OHdG) was positively correlated with HIF-1α during sustained hypoxia in humans (285). Moreover, ROS-activated signaling events involving MAPK and PI-3K/Art have been implicated in the modulation of the transcriptional activity of HIF-1α (234). Further, angiotensin II in vivo and in vitro upregulates renal VEGF expression by a mechanism that involves HIF-1 activation and oxidative stress (345).

Thus, it now seems possible that ROS have important roles in the regulation of cell signaling, although these substances are potentially cell damaging. For example, there appears to be a potential feedback loop in which PGC-1α regulates antioxidant mechanisms via regulation of mitochondrial respiration and ROS. In turn, ROS might regulate antioxidant defense genes through activation of redox-sensitive transcription factors, supporting the view that ROS are important signaling molecules in adaptations to exercise (350) (Fig. 12). While increasing evidence suggests that mitochondria play a less-important role in oxidant production in contracting skeletal muscles than was previously predicted, and that the primary site of contraction-induced ROS production in muscle fibers remains unclear (294, 297), PGC-1α is transiently induced by acute exercise (20, 143). This induction has been suggested to occur in response to altered energy demands and because of PGC-1α interacting with the metabolic enzyme AMPK (130). PGC-1α has also been proposed to be a key mediator of long-term adaptations to exercise (130). Indeed, basal levels of PGC-1α gene expression increased approximately twofold after a period of endurance training in humans (339).

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FIG. 12.

Exercise results in enhanced ROS production, which activates the p38γ MAPK, AMPK, and HIF1α pathways leading to mitochondrial biogenesis and angiogenesis that are an important part of exercise-induced adaptation. AMPK, AMP-activated protein kinase; HIF-1α, hypoxia-inducible factor-1a; VEGF, vascular endothelial growth factor.

Intriguingly, and in conflict with the free radical theory of aging (131), longevity and health-promoting effects of caloric restriction and specifically reduced glucose metabolism may be due to increased formation of ROS within the mitochondria. This causes an adaptive response that culminates in subsequently increased stress resistance, assumed to ultimately cause a long-term reduction of oxidative stress, which is an adaptive response more specifically named mitochondrial hormesis or mitohormesis (330). Molecular mediators of endogenous ROS defense (Cu,Zn-SOD, Mn-SOD, and GPX) are also induced by exercise, and this effect is blocked by antioxidant supplementation (331). Consistent with the concept of mitohormesis, exercise-induced oxidative stress ameliorates insulin resistance and causes an adaptive response promoting endogenous antioxidant defense capacity. As expectedly or unexpectedly, supplementation with antioxidants may preclude these health-promoting effects of exercise in humans. Taken together, physical exercise induces numerous molecular regulators of insulin sensitivity and antioxidant defense, most of which are almost completely inhibited by antioxidant pretreatment in healthy young men (329 –331).

E. Effect of exercise on HIF and VEGF signaling

The study on acute exercise by Ameln et al. (11) was the first to show that several components of the HIF-1 pathway, involving VEGF and erythropoietin, are activated in response to acute changes in oxygen demand in healthy human skeletal muscle, due to a concurrent decrease in von Hippel-Lindau tumor suppressor protein (VHL) levels. This finding suggests that oxygen-sensitive pathways could be relevant for adaptations to physical activity by increasing capillary growth in human skeletal muscle, and supports the general importance of the HIF pathway (11). VEGF mRNA is further increased when blood flow to the exercising leg is restricted.

Several studies have revealed increased levels of the HIF-1 pathway in human skeletal muscle after acute exercise (125, 220). The positive response of mRNAs for HIF-1α and HIF-2α with acute exercise however is blunted with training (220). Moreover, a positive correlation was found between exercise-induced changes in VEGF mRNA on one hand, and those in HIF-1α mRNA, HIF-1β mRNA, and lactate on the other, while no significant differences between the restricted and nonrestricted groups were observed (125), unlike the results found in the study of Ameln et al. (11). Gustafsson et al. (125) noted that blood flow restriction, made possible by application of external pressure 50 mmHg higher than atmospheric pressure, caused about a 20% decrease in blood flow at same work load, creating a condition that allowed the study of the interaction between HIF and VEGF during exercise. This lack of a further increase in VEGF mRNA when flow restriction was added may be explained by the smaller further reduction in oxygen saturation and oxygen tension compared with the rest-to-exercise transition, even if this difference was large enough to induce a greater lactate concentration increase in the restricted condition. Another explanation could be that even if there was no significant VEGF mRNA difference before exercise between the nonrestricted and restricted conditions, a strong influence of the pre-exercise value of VEGF mRNA on the exercise-induced increase makes a difference between the two conditions more difficult to detect.

Exercise induces several pathways that are known to be important for regulating angiogenesis. VEGF is critical for basal and activity-induced regulation of skeletal muscle capillarization, whereas the importance of other growth factor pathways remains to be elucidated. Although several signaling pathways have been identified (104), significant work remains before the intracellular signaling pathways and transcription factors involved in basal and exercise-induced angiogenesis are fully understood.

For example, in addition to AMPK, exercise activates several signaling pathways that may be the links between metabolism and capillarization, including calcium (Ca²⁺)-regulated pathways (104). Large, but transient, increases in intracellular Ca²⁺ occur during exercise as a result of neural activation and muscle cell depolarization. Ca²⁺ is intimately involved in actin–myosin cross-bridge formation and is responsible in part for exercise-induced improvements in insulin sensitivity that occur, in part, through increases in glucose transporter 4. Increasing intracellular Ca²⁺ leads to the activation of several downstream signaling proteins, including calmodulin-dependent kinase and CnA, possibly resulting in increased expression of VEGF mRNA in primary human myotubes in vitro. This suggests that Ca²⁺ may be involved in muscle VEGF regulation (104), although much more work needs to be done before these studies are finalized. In addition, PPAR-β, which is also known as PPAR-δ and is involved in muscle development and metabolism, increases VEGF and promotes muscle angiogenesis through a CnA-dependent pathway (103).

Moreover, NO is involved in muscle VEGF regulation (104, 438). nNOS-mediated NO regulates, in part, muscle mitochondrial respiration, whereas endothelial NO is involved in blood flow regulation. Disrupting normal NO production (via L-NAME) eliminates the increase in collateral blood flow induced by training, but does not disturb the increase in muscle capillarity within the active muscle. Similarly, inhibiting VEGFR kinase activity eliminates the increase in collateral-dependent blood flow, and lessens, but does not eliminate, angiogenesis within the calf muscle, illustrating distinctions between the processes influencing angiogenesis and arteriogenesis (Fig. 13) (438).

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FIG. 13.

Exercise induces marked release of Ca²⁺ from the sarcoplasmic reticulum, which binds to troponin to allow the generation of cross bridges between myosin and actin filaments. Moreover, it activates calmodulin-dependent protein kinase (CaMK) that could lead to enhanced expression of GLUT4 glucose transporter. Then, as a result of insulin-mediated signaling, more GLUT4 can translocate to the membranes leading to increased glucose uptake by skeletal muscle. CaMK can also activate increased capillarization via VEGF. PPAR, peroxisome proliferator-activated receptor. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

In general, the effects of endurance training on the activity of the HIF pathway in human skeletal muscle under hypoxic conditions appear to be definitely higher than those under normoxic conditions (219, 422, 446), although there are several exceptions (97, 241), indicating that combining hypoxia with exercise training appears to improve some aspects of muscle O₂ transport and/or metabolism (Fig. 14) (219). Although there were no significant changes in the levels of mRNAs for HIF-1α or VEGF in human skeletal muscle after endurance training under normoxic conditions, the changes in HIF-1α mRNA expression correlated well with those in VEGF mRNA expression (270), suggesting that HIF-1α influences the training-induced VEGF gene expression, or alternatively that HIF-1α and VEGF expression is coregulated at the transcriptional level in human skeletal muscle. Taken together, it is envisioned that cumulative effects of transient changes in transcription during recovery from successive bouts of exercise may represent the underlying kinetic basis for the cellular adaptations associated with endurance training. Furthermore, no plasma VEGF contents in overweight men aged 50–60 years were affected by long-term endurance exercise under normoxic conditions (51). Likewise, low- or high-intensity-resistance exercise did not significantly alter serum VEGF content in humans (334).

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FIG. 14.

Oxygen sensing and angiogenesis are dependent on the HIF-1-VEGF axis, which is mediated by hydrogen peroxide-dependent signaling. The figure shows some of the key elements of the suggested signaling pathways (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.) Akt, protein kinase B; ERK, extracellular signal-regulated kinase; PI3-K, phosphatidylinositol 3-kinase.