Mitochondrial redox metabolism in aging: Effect of exercise ... - Core

Available online at www.sciencedirect.com

Journal of Sport and Health Science 2 (2013) 67e74 www.jshs.org.cn

Review

Mitochondrial redox metabolism in aging: Effect of exercise interventions Hai Bo a,b, Ning Jiang a, Li Li Ji a,c, Yong Zhang a,* a

Tianjin Key Laboratory of Exercise Physiology and Sports Medicine, Department of Health & Exercise Science, Tianjin University of Sport, Tianjin 300381, China b Department of Military Training Medicines, Logistics University of Chinese People’s Armed Police Force, Tianjin 300162, China c Laboratory of Physiological Hygiene and Exercise Science, School of Kinesiology, University of Minnesota, Minneapolis, MN 55455, USA Received 12 December 2012; revised 20 January 2013; accepted 22 February 2013

Abstract Mitochondrial redox metabolism has long been recognized as being central to the effects of aging and the development of age-related pathologies in the major oxidative organs. Consistent evidence has shown that exercise is able to retard the onset and impede the progression of aging by modifying mitochondrial oxidanteantioxidant homeostasis. Here we provide a broad overview of the research evidence showing the relationship between mitochondrial redox metabolism, aging and exercise. We address part aspects of mitochondrial reactive oxygen species (ROS) metabolism, from superoxide production to ROS detoxification, especially antioxidant enzymes and uncoupling protein. Furthermore, we describe mitochondrial remodeling response to aging and exercise, which is accompanied by bioenergetics and redox regulation. In addition, potential mechanisms for redox signaling involved in mitochondrial remodeling and redox metabolism regulation are also reviewed. Copyright Ó 2013, Shanghai University of Sport. Production and hosting by Elsevier B.V. Open access under CC BY-NC-ND license. Keywords: Aging; Exercise; Mitochondrial remodeling; PGC-1a; Reactive oxygen species

1. Introduction The aging process results in a gradual and progressive structural and functional deterioration of biomolecules that is associated with many pathological conditions, including cancer, neurodegenerative diseases, sarcopenia, and liver dysfunction. In the 1950s, Harman1 first proposed the “free radical theory of aging”. This theory contended that free radicals, produced from normal metabolism, lead to

* Corresponding author. E-mail address: [email protected] (Y. Zhang) Peer review under responsibility of Shanghai University of Sport

Production and hosting by Elsevier

irreversible cell damage and an overall functional decline during aging. In the 1970s, he extended the theory to implicate mitochondrial production of reactive oxygen species (ROS, such as O2 and H2O2) as the main cause for age-related macromolecular damage.2 Epidemiological studies clearly demonstrate that endurance exercise reduces the risk of chronic diseases and extends life expectancy.3 Consistent evidence has shown that exercise is able to retard the onset and impede the progression of aging by modifying mitochondrial oxidanteantioxidant homeostasis.4 However, there are still unanswered basic scientific questions in the implication of exercise in modulating aging. Especially, how increased ROS production during physical exercise could be involved in exerciseinduced health-promoting benefits. In this review, we consider the evidence that specific aspects of mitochondrial redox metabolism affect aging. We then discuss redoxsensitive signal transduction pathways associated with exercise in modulating aging.

2095-2546 Copyright Ó 2013, Shanghai University of Sport. Production and hosting by Elsevier B.V. Open access under CC BY-NC-ND license. http://dx.doi.org/10.1016/j.jshs.2013.03.006

68

H. Bo et al.

2. Mitochondrial ROS production and aging In the mitochondrial respiratory chain, under conditions in which complexes I and III are highly reduced, a significant proportion of electrons are diverted from them directly to molecular oxygen, thus forming the radical superoxide (O2 ). In addition, it recently has been shown that p66shc, a protein present in the intermembrane space, can also catalyze O2 production via electron transfer from reduced cytochrome c to molecular oxygen.5 Although O2 is not membrane permeable, it can be spontaneously or catalytically dismutated to hydrogen peroxide (H2O2), which can efficiently pass through biomembranes and therefore acts not only at its site of formation but also elsewhere. At increased abundance, ROS are able to damage cellular components such as proteins, nucleic acids, and lipids by the abstraction of electrons. The damaging potential of mitochondrial ROS is the kernel of the “mitochondrial free radical theory of aging”, which suggests that mitochondrial ROS lead to an accumulation of molecular damage over time and, after passing certain thresholds, to cellular degeneration and death. In general, ROS production is found to be increased in cells and is associated with aging. Furthermore, genetic studies in worm, fly, and mouse have linked enhanced stress resistance or reduced free radical production with increased lifespan. Because mitochondria are the major producer of ROS in mammalian cells, the close proximity to ROS places mitochondrial prone to oxidative damage. During aging, the increase in ROS production is associated with increased levels of 8-oxoguanine (8-oxoG) from mitochondrial DNA (mtDNA) oxidation, increased nitration of complex II, and altered carbonylation of complex I, complex V, and isocitrate dehydrogenase. Oxidative damage induced impaired function of respiratory chain will in turn trigger further accumulation of ROS, which results in a vicious cycle leading to energy depletion and ultimately cell death.

3. Mitochondrial antioxidant enzymes in aging To avoid the potentially damaging effects of ROS, cells use a variety of systems to keep ROS within tolerable limits. This is especially important in mitochondria because the enzymes that drive ATP production are rich in thiol residues that can be irreversibly oxidized. Superoxide generated by mitochondria is quickly dismutated to H2O2 by either manganese (Mn) or copper/zinc (Cu/Zn)-dependent superoxide dismutase (SOD), which are located in the matrix and intermembrane space, respectively. Although H2O2 is the main ROS used in signaling, it is degraded by a variety of enzymes and low molecular weight molecules to prevent oxidative stress. The chief H2O2-quenching systems in mitochondria include the GSH/glutathione peroxidase (GPx) system, peroxiredoxins, thioredoxins, and glutaredoxins. Catalase (CAT) also degrades H2O2 but only when H2O2 reaches high concentrations. MnSOD is an intramitochondrial free radical scavenging enzyme that is the first line of defense against O2 produced. Over-expression of MnSOD in Drosophila melanogaster was

shown to have beneficial effects, extending lifespan.6 Furthermore, the tissue-specific over-expression of MnSOD in motor neurons was sufficient to mediate the same effect. Conversely, complete ablation of this enzyme in mice causes severe oxidative damage to mitochondria and early postnatal death associated with dilated cardiomyopathy and neurodegeneration.7 MnSOD activity, both in rats and men, are reported to increase significantly with aging and to respond to age-related oxidative stress in the mitochondria by upregulation. However, MnSOD protein and mRNA levels was unchanged or decreased with age in most tissues. In addition, binding of transcription factor nuclear factor-kB (NF-kB) and transcription factor activator protein-1 (AP-1) to corresponding oligonucleotide sequences present in the MnSOD gene were decreased in aged cells.8 Both NF-kB and AP-1 binding sites are present in the promoter of the mammalian MnSOD gene, and oxidative stress has been shown to activate their binding. A decrease in the binding of these nuclear factors despite increased ROS generation observed in aged cells suggests that molecular signaling of MnSOD gene expression is retarded due to aging. The observed increase in MnSOD activity in some tissues appears to be due to a posttranslational modification (activation) of the enzyme molecules during aging. CAT is widely distributed in the cell, with the majority of activity occurring in the mitochondria and peroxisomes, where it functions to catalyze the decomposition of H2O2 to water and oxygen. Transgenic mice that overexpress mitochondrial CAT have increased median and maximum lifespans, attenuated age-related changes including decline of diastolic function, myocardial performance as well as ventricular fibrosis.9 CAT largely determines the functional antioxidant capacity of mitochondria and is the enzyme that is most affected in aging.10 In rats and mice cellular CAT levels drop with age, which is accompanied by an increase in oxidative stress. Chronically reducing CAT activity causes cells to display a cascade of accelerated aging reactions.11 Sirtuin1 (Sirt1) has been shown to affect CAT expression and be a determinant of cell apoptosis by regulating cellular ROS levels. NADdependent deacetylase Sirt1 maintains cell survival by regulating CAT expression and by preventing the depletion of ROS required for cell survival. In contrast, excess ROS upregulates Sirt1, which activates CAT leading to rescuing apoptosis.12 4. Exercise interventions affecting mitochondrial antioxidant enzymes There is an abundance of literature reporting muscle antioxidant adaptation to chronic exercise training and several reviews have also addressed this topic thoroughly. Among antioxidant enzymes in skeletal muscle, MnSOD activity has consistently been shown to increase with exercise training in an intensity-dependent manner. GPx activity has also shown to increase after endurance training by most authors. GSH plays a critical role in muscle antioxidant defense during exercise. Several studies have demonstrated that GSH content and the rate-limiting enzyme for GSH synthesis, a-glutamylcysteine

Mitochondrial redox metabolism in aging and exercise

synthetase (GCS), can be induced by endurance training in rat skeletal muscle. However, training effect on CAT activity has been inconsistent and controversial.13 As mentioned previously several oxidative stress-sensitive signaling pathways are operational in mammalian systems and play an important role in maintaining cellular oxidanteantioxidant balance. One of the most important involves the NF-kB. Several antioxidant enzymes contain NF-kB binding sites in their gene promoter region, such as MnSOD, inducible nitric oxide synthetase, and GCS. Therefore, they can be potential targets for exercise-activated upregulation via the NF-kB signaling pathway. Ji et al.14 reported that an acute bout of treadmill running activated NF-kB binding in rat skeletal muscle 2 h after exercise. Furthermore, the NF-kB signaling pathway was activated in a redox-sensitive manner during muscular contraction.14 Consistently, Gomez-Cabrera et al.15 have demonstrated that marathon running induces activation of the p50 subunit of the NF-kB complex in lymphocytes. This effect was prevented by treatment with allopurinol. It is well documented that a single episode of muscular contraction can activate the MAPK pathway in human skeletal muscle. As members of MAPK family, extracellular signalregulated kinase (ERK) and p38 MAPK activity can lead to the sequential phosphorylation of a series of proteins, resulting in increased expression AP-1, which is an important DNAbinding site on many genes responsive to oxidative stress.16 It was also found that ROS generated during exercise activate MAPKs, which in turn activate NF-kB, resulting in an increased expression of important enzymes associated with cell antioxidant defense (MnSOD and GPx). Prevention of ROS formation by inhibition of XO abolishes these effects.17 This result showed the role of cross-talk in the MAPK and NF-kB pathway in exercise-activated antioxidant signaling. 5. Uncoupling proteins, mitochondrial ROS production, and aging It is well established that there is a strong positive correlation between mitochondrial membrane potential (Dj) and ROS production. It has been reported that, at high Dj, even a small increase in membrane potential gives rise to a large stimulation of H2O2 production. Similarly, only a small decrease in Dj (10 mV), which is termed “mild uncoupling”, is able to inhibit H2O2 production by 70%. Hence, the mild uncoupling of mitochondrial oxidative phosphorylation may represent the first line of defense against oxidative stress. Brand18 first proposed the “uncoupling to survive” hypothesis, which states that increased uncoupling leads to greater oxygen consumption and reduces the proton motive force by reducing Dj which subsequently reduces ROS generation. Further evidence supports this hypothesis as a number of studies show higher mitochondrial respiration and greater mitochondrial uncoupling increases lifespan in a diverse range of organisms. Uncoupling proteins (UCPs), a heterogeneous family of proteins located in the mitochondrial inner membrane, appear to be a logical candidate to achieve mild uncoupling. UCP2

69

and UCP3 are w73% homologous to each other and w58% homologous to the thermogenic protein, UCP1. UCP3 is highly selectively expressed in skeletal muscle and brown fat, whereas UCP2 is ubiquitously expressed. It has been critically examined that UCP2/UCP3 become true uncoupling proteins when stimulated by ROS or 4-hydroxynonenal, and thus become capable of diminishing oxidative damage by a feedback mechanism activating mild uncoupling.19 Evidence for this includes observations that mice deficient in UCP3 have higher levels of membrane lipid peroxidative damage.20 Numerous studies have demonstrated that UCP2 overexpression diminishes ROS production in a large number of tissues examined, including the heart, pancreas, adipose tissue, muscle, immune system, spleen, and steatotic liver.21 Andrews and Horvath22 reported that UCP2 gene deletion dramatically reduces maximum lifespan in mice, and transgenic UCP2 overexpression can partially rescue the early neonatal lethality of MnSOD deficiency. Similar observations have been made with UCP3. Mitochondria from UCP2 knockout mice produce more ROS and contain more oxidative damage in comparison to wild-type.23 In addition, overexpression of UCP3 has been found to lower the increased ROS formation associated with aging.24 However, simultaneous overexpression of UCP2 and UCP3 has minimal effect on longevity.19 This could be related to the requirement of these proteins for effector molecules. Maximal activity of UCPs requires stimulation with fatty acids, including end products of fatty acid peroxidation. UCPs activities are also inhibited by adenine nucleotides. Thus, increasing UCP expression without modifying levels of stimulators and inhibitors of their activities may not affect membrane potential under normal conditions. It has been repeatedly demonstrated that aging is associated with an increase in oxidative stress, and therefore an increase in uncoupling may be expected with aging. However, there was a dramatic reduction of UCP3 content associated with decreased state 4 respiration of skeletal muscle mitochondria from old rats.25 In addition, it was found that muscle with the most active fiber type (type I) and higher resting O2 uptake had mild uncoupling in adults but stable mitochondrial function into old age. This protection of mitochondrial function suggests that mild uncoupling is a mechanism that reduces the ROS production, ameliorates mitochondrial damage, and assists fiber survival with age. In contrast, muscle with greater content of the least active fiber type (type II) and lower resting O2 uptake was well coupled in adults yet showed defect in mitochondrial coupling and depletion of ATP with age consistent with ROS damage. Thus, mild uncoupling may explain the paradox of greater longevity in the most active fibers by providing a protective mechanism that impacts the pace of cellular aging.26 6. Mitochondrial uncoupling in response to exercise Several studies have shown that UCP3 expression was increased in response to an acute bout of exercise or contractile activity in mammalian skeletal muscle.27 We

70

H. Bo et al.

investigated UCP2 and UCP3 expression along with mitochondrial respiratory function and ROS generation in rat heart and skeletal muscle during and after an acute bout of prolonged exercise up to 2.5 h. The data demonstrate that UCP2/ UCP3 expression can be rapidly upregulated during prolonged exercise, and a coordinated regulation to reduce ROS generation and to preserve mitochondrial respiratory function.28,29 Noticeably, MnSOD expression and enzyme activity was not up-regulated until after exercise under this experimental condition. Thus, our data suggest that during an acute bout of demanding exercise, the primary strategy of mitochondria to reduce oxidative stress is decreasing the production of O2 by overexpressing UCP2/UCP3, instead of enhancing the removal of O2 by overexpressing MnSOD. Our study also demonstrated that endurance training attenuated acute stress-induced UCP2/UCP3 expression and activity in rat. Meanwhile, endurance training significantly elevated MnSOD activity and protein level.28,30 Similarly, Noland et al.31 reported that acute exercise increases skeletal muscle UCP3 expression in untrained but not trained humans. These findings raised some interesting questions regarding the relationship among mitochondrial respiratory uncoupling, ROS generation, antioxidant defense, and ATP synthesis. An ameliorated exercise response of UCP2/UCP3 after training could be viewed as a compensatory mechanism to avoid excessive sacrifice of oxidative phosphorylation efficiency and heat production. McLeod et al.32 reported that in mitochondria from SOD2 knockout mice, ATP production was markedly reduced due to a higher O2 concentration which activated UCP2 excessively. Taken together, exercise training adaptation of MnSOD may enhance mitochondrial tolerance to ROS production and hence a smaller UCP2/UCP3 activation during intensive exercise, thus protecting the efficiency of oxidative phosphorylation.

7. Mitochondrial remodeling is impaired in aging Mitochondria are highly mobile and remarkably plastic organelles that continuously undergo organellar turnover and morphology remodeling. Evidence is accumulating that mitochondrial dynamic remodeling are sensitive to various physiological and pathological stimuli, and mitochondrial remodeling are no epiphenomena, but central to cell function and survival. Mitochondria are able to change their architecture from individual structures to complex tubular networks through fusion and fission. It has been established that this dynamic structure is regulated by proteins controlling fission, such as mitochondrial fission 1 protein (Fis1) and dynamin-related protein 1 (Drp1), and fusion, such as mitofusin homologs Mfn1/2 and Dynamin-like 120 kDa protein OPA1. Genetic defects in the proteins involved in mitochondrial fusion and fission lead to severely altered mitochondrial shape, loss of mtDNA integrity, increased oxidative stress and apoptotic cell death.33 In healthy cells, mitochondrial fusion provides a synchronized internal cable for translocating metabolites and intramitochondrial mixing during biogenesis, whereas

mitochondrial division facilitates the equal distribution of mitochondria into daughter cells during mitosis and allows the selective degradation of damaged mitochondria through autophagy. However, it has become increasingly clear that these protective mechanisms are markedly impaired in aging and that faulty mitochondrial dynamics might be involved in the aging process. Crane et al.34 reported that the expression of Mfn2 and Drp1 genes is reduced in the skeletal muscle of aging individuals. Furthermore, Using RNA interference to reduce Fis1 in mammalian cells, it was found that mitochondria become enlarged and flattened and these morphological changes are correlated with increased a-galactosidase activity, a marker of cell senescence, and decreased mitochondrial membrane potential, increased ROS generation, and DNA damage.35 The constant renewal of mitochondria is crucial for maintaining healthy mitochondria with age. Accurate organellar turnover requires the coordination of two key cellular processes: mitochondrial biogenesis and selective degradation (autophagy). The capacity for mitochondrial biogenesis diminishes with age and this is an important parameter in the mitochondrial dysfunction associated with aging.36 If biogenesis is affected, it is reasonable to expect that mitochondrial turnover must be slower and the accumulation of modified lipids, proteins and DNA must also increase, further aggravating the situation resulting from the deficient activity of aged mitochondria. The precise reason for the decrease in the rate of mitochondrial biogenesis during aging is currently unknown. However, it seems that both extra- and intracellular regulatory factors of mitochondrial biogenesis are implicated. In fact, the pleiotropic signaling of mitochondria, H2O2 and NO diffusion to the cytosol is modified in aged animals and contributes to the decreased mitochondrial biogenesis in old animals.37 Autophagy appears to decline with age, and several key players in the autophagic pathway (e.g., ATG5 and ATG7) show decreased expression in the brains of aging individuals. Stimulation of autophagy can increase the healthy lifespan in multiple model organisms, including mice and primates.38 Autophagy can mitigate inflammatory reactions through several mechanisms. Recently, it was found that autophagy can inhibit NLRP3 activation by removing permeabilized or ROS producing mitochondria.39 Because pathological aging is accompanied by chronic inflammation, these anti-inflammatory effects of autophagy may mediate additional health benefits. Mitochondrial dynamics are also important for proper organellar turnover in that they affect mitochondrial degradation pathways. Fis1 expression was found to be reduced in abnormal mitochondria, whereas overexpression of this protein blocked the senescence-related phenotype and maintained cells in a proliferating state. The link between fission proteins and mitochondrial turnover has also been illustrated in neuronal cells, in which Fis1 was shown to activate autophagy and the selective degradation of depolarized mitochondria.40 In summary, aging compromises the remodeling of mitochondria by disrupting the homeostatic regulation of


71

mitochondrial fusion and fission pathways. The presence of fragmented mitochondria owing to a decline in fusion and/or an increase in fission events can compromise mtDNA integrity, mitochondrial structural and functional complementation, and mitochondrial biogenesis, all of which can lead to mitochondrial dysfunction. Conversely, the formation of enlarged mitochondria as a result of decreased fission and/or increased fusion events can diminish mitochondrial turnover by impairing autophagy and biogenesis, leading to the accumulation of damaged mitochondria in aged cells. In both cases, the abnormal mitochondria are unable to fulfill their lifesustaining roles. Therefore, age associated alterations in mitochondrial fusion and fission dynamics might play a causative role in mitochondrial remodeling impairment and increase susceptibility to cell death in response to various types of stress during progressive aging.

old rats. It was shown that an age-associated lack of expression of PGC-1a in response to not only training but also to cold exposure or triiodothyronine. Recently, Farhoud et al.48 reported that chronic oxidative stress suppresses the gene expression of PGC-1a through activation of the ubiquitinproteasome system, thereby inhibiting its downstream mitochondrial biogenesis and metabolic gene expression programs. In addition, chronic inhibition of NF-kB via the pharmacological agent pyrolidine dithiocarbamate (PDTC) was found to increase PGC-1a level suggesting inactivity might downregulate PGC-1a due to elevated NF-kB activity.49 It could be presumed that aging related inflammation and subsequent activation of NF-kB could negatively influence the expression of PGC-1a and its effect on mitochondrial biogenesis. These results showed that loss of mitochondrial biogenesis during aging may be due to a lack of induction of PGC-1a.

8. PGC-1a, mitochondrial remodeling, and aging

9. Mitochondrial remodeling, ROS, and exercise

Despite the complexity of the various signaling pathways that regulate mitochondrial remodeling, they all seem to share proliferator-activated receptor-a coactivator 1a (PGC-1a) family of transcription factors. PGC-1a appears to act as a master regulator of energy metabolism and mitochondrial remodeling by coordinating the activity of multiple transcription factors. PGC-1a strongly co-activates NRF-1, which is an intermediate transcription factor to stimulate the synthesis of mitochondrial transcription factor A (Tfam). Tfam can be considered the most important mammalian transcription factor for mtDNA because it stimulates mitochondrial DNA transcription and replication. Recently, Aquilano et al.41 showed that PGC-1a and Sirt1 not only colocalize with the nuclear, but also interact with Tfam in the mitochondria to form multiprotein complexes. In addition, PGC-1a has been shown to be involved in mitochondrial network remodeling through controlled fusion and fission. H2O2-stimulated Mfn1/2 expression was dramatically attenuated in PGC-1a knockout C2C12 muscle cells,42 whereas PGC-1a over-expression markedly enhances Mfn2 mRNA and protein levels in cultured muscle cells.43 PGC-1a also stimulates the transcriptional activity of the human Mitofusin 2 (Mfn2) gene promoter, which requires the integrity of the nuclear hormone estrogen-related receptor ERRa-binding element.44 Finally, PGC-1a controls most of the enzymes and proteins affecting oxidanteantioxidant balance such as UCP2/UCP3, MnSOD, and GPx expression.45 Kang et al.46 reported that aged (24 months) rats had significantly lower gene expression in the PGC-1a signaling pathways, shown by decreased mRNA and protein contents for PGC-1a, Tfam and cytochrome c compared to young (4 months) rats. Furthermore, phosphorylation of the upstream enzymes AMP kinase and p38 MAPK, as well as cAMP response element nuclear binding, was down-regulated. Derbre et al.47 studied mitochondrial biogenesis and PGC-1a expression in old rats and compared them with PGC-1a knockout mice. They found a striking similarity between the response to exercise training in PGC-1a knockout mice and in

The term Hormesis has been adopted to explain how a mild oxidative stress associated with exercise can result in favorable adaptations that protect the body against more severe stresses and disorders derived from physical stress or other etiological origin. The typical reaction to ROS can be described by a bellshaped curve: low concentrations have a stimulating effect (signaling, receptor stimulation, and enzymatic stimulation), while a massive level of ROS inhibits enzyme activity and causes apoptosis or necrosis. Recent evidence suggests that suppression of ROS production fails to extend lifespan in worms and may even decrease lifespan in humans, presumably due to the reduction of the ROS signaling, which seems to be important for different cellular processes.50,51 It was suggested that exercise could increase the formation of ROS to a level that may induce significant, but tolerable, damage that can in turn induce beneficial adaptations. Short-term ROS production is apparently important in prevention of aging by induction of a process named mitohormesis and redox signaling.52 This process seems to be particularly important for the health effect of exercise. Recently, we studied the effect of aging and exercise on mitochondrial remodeling in skeletal muscle because it contains mainly post-mitotic cells and because of the importance of this organ in aging (Fig. 1). Mfn1 and Drp1 protein were unchanged at 15 months. Mfn1 was decreased 39% at 20 months vs. 5 months ( p < 0.05), whereas Drp1 showed w70% ( p < 0.01) increase at 20 months vs. 5 months. Compared with control group, 12-week treadmill training resulted in an increase in Mfn1 in 15 months ( p < 0.01), whereas in a small decrease in 20 months ( p < 0.05) (Fig. 1A). In contrast to Mfn1, compared with control group, training resulted significantly increased in Drp1 protein both at 15 and 20 months vs. 5 months ( p < 0.01 and p < 0.05, respectively) (Fig. 1B). Furthermore, cytochrome c oxidase subunit IV (COXIV), the marker of mitochondrial biogenesis, showed a 30% increase at 20 months vs. 5 months ( p < 0.05). Compared with control group, training increased COXIV content by w30% at 5 and 15 months old rats ( p < 0.05), and by w20% at 20 months old rats ( p < 0.05)

72

H. Bo et al.

Fig. 1. Western blot analysis of Mfn1 (A), Drp1 (B), COXIV (C), and mitochondrial ATP synthase activity (D) in rat skeletal muscle of 12-week treadmill trained 5-, 15-, and 20-month-old rats and controls. yp < 0.05, yyp < 0.01: trained group compared with control group at various ages; *p < 0.05, **p < 0.01: in control group, compared with 5 months; #p < 0.05, ##p < 0.01: in trained group, compared with 5 months.

(Fig. 1C). We also measured ATP synthase activity as indication of mitochondrial energy production. ATP synthase activity was unchanged at 15 and 20 months vs. 5 months, whereas training increased its activity at various ages, 5-, 15-, and 20-month-old, compared with control group ( p < 0.01, p < 0.05, and p < 0.05, respectively) (Fig. 1D). Taken together, our data suggest that regular exercise training stimulates mitochondrial biogenesis, a rejuvenation of the mitochondrial network via fission and fusion, and an improved efficient of mitochondrial energy transfer. All of these properties, working in conjunction with one another, would improve the overall functionality of mitochondria in aged cells (Zhang et al., unpublished data). Gomez-Cabrera et al.53 demonstrated a reduction in mitochondrial biogenesis and protein quality control factors compared to placebo control, following supplementation of vitamin C during a 6-week endurance protocol in rats. It is clearly shown that exercise induced mitochondrial mass remodeling is a redox-sensitive process. Recently, we found that exercise induced mitochondrial ROS may contribute to the rapidly alteration in mitochondrial fusion/fission protein expression.54 Koopman et al.55 reported that vitamin E supplementation reduced ROS production and restored aberrant mitochondrial morphology in fibroblasts with mitochondrial complex I deficiency, suggesting that ROS is involved in controlling mitochondrial shape in these cells. Using a nonapoptotic concentration of H2O2 in long-term treatment,

originally long and interconnected mitochondrial tubules were transiently shortened and weakly fragmented.56 In addition, low doses H2O2 in a transient exposure induce mitochondria hyperfuse and form a highly interconnected network in cells.57 Based on the above evidence it can be concluded that ROS induce remodeling of the mitochondrial network depending on the concentration of ROS and duration of the treatment. ROS is also an important signal for induction of autophagy. Starvation-induced autophagy can be suppressed by antioxidants suppressing the well-known prosurvival function of starvationinduced autophagy.58 Recently, it is demonstrated that exercise is a potent inducer of autophagy, and that acute and chronic exercise enhances glucose metabolism in mice capable of inducing autophagy but not in autophagy-deficient mice.59 Several studies have shown that an acute bout of endurance exercise and stimulated muscle contraction can upregulate PGC1a and activate mitochondrial protein synthesis and proliferation.60 Furthermore, repeated exercise bouts (exercise training) could result in accumulation of PGC-1a, NRF-1, and Tfam protein levels.61 These observations were thought that PGC-1a plays an important role in mediating mitochondrial adaptation to exercise, such as elevated respiratory activity, increased expression of Krebs cycle enzymes, promoted endogenous antioxidant defense capacity, enhanced fatty acid oxidation, and mitochondrial morphological changes. Garnier et al.62 demonstrated in healthy humans that VO2peak was dependent on


coordinated expression of PGC-1a, which was lineally correlated to Mfn2 and Drp1 levels in human leg muscle. In C2C12 skeletal muscle cells, increasing the production of ROS by incubation with 300 mM H2O2, results in increased PGC-1a promoter activity and PGC-1a mRNA expression requiring the activation of AMPK.63 Ristow et al.64 demonstrated exercise-induced oxidative stress elevated PGC-1a and PGC-1b expression and ameliorated insulin resistance, which were precluded by antioxidants supplementation. 10. Conclusion A vast body of data has accumulated linking mitochondrial redox metabolism to the aging process. Therefore, the accumulation of oxidative damage can be decreased either by lowering the generation of ROS (caloric restriction) or by regular exposure to a small amount of ROS (such as mild exercise) that could result in slight oxidative damage, which then leads to up-regulation of antioxidant systems and amelioration of mitochondrial remodeling. Signal transduction by coordinated action of PGC-1, NF-kB, and MAPK (p38) may be potential regulators in these events. These products include (1) antioxidant enzymes (e.g., MnSOD, GPx, GCS); (2) molecules controlling metabolic status and thus ROS production, (e.g., UCPs, enzymes in fatty acid and glucose metabolism); (3) transcription factors for mitochondrial biogenesis (e.g., PGC-1a, NRF-1, Tfam), and (4) a wide range of proteins that could influence mitochondrial dynamic remodeling, such as mitochondrial fusion/fission and autophagy proteins. Exercise training is a useful and inexpensive intervention for preventing aging and degenerative disease, in which mitochondrial redox metabolism plays a key role. Acknowledgments This work was supported by research grants from the National Natural Science Foundation of China (No. 31110103919, 31200894, 31000523, 30771048, 30470837, 31071040, and 30270638), Tianjin Municipal Sci-tech-innovation Base Project (No. 10SYSYJC28400), Tianjin Science and Technology Planning Project (No. 12JCQNJC07900), and General Administration of Sport of China Basic Project (No. 10B058). References 1. Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol 1956;11:298e300. 2. Harman D. The biologic clock: the mitochondria? J Am Geriatr Soc 1972;20:145e7. 3. Charansonney OL. Physical activity and aging: a life-long story. Discov Med 2011;12:177e85. 4. Bo H, Zhang Y, Ji LL. Redefining the role of mitochondria in exercise: a dynamic remodeling. Ann N Y Acad Sci 2010;1201:121e8. 5. Pani G, Koch OR, Galeotti T. The p53-p66shc-manganese superoxide dismutase (MnSOD) network: a mitochondrial intrigue to generate reactive oxygen species. Int J Biochem Cell Biol 2009;41:1002e5. 6. Sun J, Folk D, Bradley TJ, Tower J. Induced overexpression of mitochondrial Mn-superoxide dismutase extends the life span of adult Drosophila melanogaster. Genetics 2002;161:661e72.

73 7. Jang YC, Remmen VH. The mitochondrial theory of aging: insight from transgenic and knockout mouse models. Exp Gerontol 2009;44:256e60. 8. Lustgarten MS, Jang YC, Liu Y, Qi W, Qin Y, Dahia PL, et al. MnSOD deficiency results in elevated oxidative stress and decreased mitochondrial function but does not lead to muscle atrophy during aging. Aging Cell 2011;10:493e505. 9. Treuting PM, Linford NJ, Knoblaugh SE, Emond MJ, Morton JF, Martin GM, et al. Reduction of age-associated pathology in old mice by overexpression of catalase in mitochondria. J Gerontol A Biol Sci Med Sci 2008;63:813e22. 10. Terlecky SR, Koepke JI, Walton PA. Peroxisomes and aging. Biochim Biophys Acta 2006;1763:1749e54. 11. Koepke JI, Wood CS, Terlecky LJ, Walton PA, Terlecky SR. Progeric effects of catalase inactivation in human cells. Toxicol Appl Pharmacol 2008;232:99e108. 12. Hasegawa K, Wakino S, Yoshioka K, Tatematsu S, Hara Y, Minakuchi H. Sirt1 protects against oxidative stress-induced renal tubular cell apoptosis by the bidirectional regulation of catalase expression. Biochem Biophys Res Commun 2008;372:51e6. 13. Gomez-cabrera MC, Vinã J, Ji LL. Interplay of oxidants and antioxidants during exercise: implications for muscle health. Phys Sportsmed 2009;37:116e23. 14. Ji LL, Gomez-cabrera MC, Steinhafel N, Vinã J. Acute exercise activates nuclear factor (NF)-kappaB signaling pathway in rat skeletal muscle. FASEB J 2004;18:1499e506. 15. Gomez-Cabrera MC, Martinez A, Santangelo G, Pallardo´ FV, Sastre J, Vinã J. Oxidative stress in marathon runners: interest of antioxidant supplementation. Br J Nutr 2006;96(Suppl. 1):S31e3. 16. Chongthammakun V, Sanvarinda Y, Chongthammakun S. Reactive oxygen species production and MAPK activation are implicated in tetrahydrobiopterin-induced SH-SY5Y cell death. Neurosci Lett 2009;449:178e82. 17. Gomez-cabrera MC, Borras C, Pallardo FV, Sastre J, Ji LL, Vinã J. Decreasing xanthine oxidase-mediated oxidative stress prevents useful cellular adaptations to exercise in rats. J Physiol 2005;567(Pt 1):113e20. 18. Brand MD. Uncoupling to survive? The role of mitochondrial inefficiency in ageing. Exp Gerontol 2000;35:811e20. 19. Echtay KS, Esteves TC, Pakay JL, Jekabsons MB, Lambert AJ, PorteroOtıń M, et al. A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling. EMBO J 2003;22:4103e10. 20. Brand MD, Pamplona R, Portero-otin M, Requena JR, Roebuck SJ, Buckingham JA, et al. Oxidative damage and phospholipid fatty acyl composition in skeletal muscle mitochondria from mice underexpressing or overexpressing uncoupling protein 3. Biochem J 2002;368(Pt 2):597e603. 21. Yonezawa T, Kurata R, Hosomichi K, Kono A, Kimura M, Inoko H. Nutritional and hormonal regulation of uncoupling protein 2. IUBMB Life 2009;61:1123e31. 22. Andrews ZB, Horvath TL. Uncoupling protein-2 regulates lifespan in mice. Am J Physiol Endocrinol Metab 2009;296:E621e7. 23. Seifert EL, Bezaire V, Estey C, Harper ME. Essential role for uncoupling protein-3 in mitochondrial adaptation to fasting but not in fatty acid oxidation or fatty acid anion export. J Biol Chem 2008;283:25124e31. 24. Nabben M, Hoeks J, Briede JJ, Glatz JF, Moonen-Kornips E, Hesselink MK, et al. The effect of UCP3 overexpression on mitochondrial ROS production in skeletal muscle of young versus aged mice. FEBS Lett 2008;582:4147e52. 25. Kerner J, Turkaly PJ, Minkler PE, Hoppel CL. Aging skeletal muscle mitochondria in the rat: decreased uncoupling protein-3 content. Am J Physiol Endocrinol Metab 2001;281:E1054e62. 26. Amara CE, Shankland EG, Jubrias SA, Marcinek DJ, Kushmerick MJ, Conley KE. Mild mitochondrial uncoupling impacts cellular aging in human muscles in vivo. Proc Natl Acad Sci USA 2007;104:1057e62. 27. Hesselink MK, Schrauwen P, Holloszy JO, Jones TE. Divergent effects of acute exercise and endurance training on UCP3 expression. Am J Physiol Endocrinol Metab 2003;284:E449e50. 28. Bo H, Jiang N, Ma G, Qu J, Zhang G, Cao D, et al. Regulation of mitochondrial uncoupling respiration during exercise in rat heart: role of

74

29.

30.

31.

32.

33. 34.

35.

36. 37.

38. 39.

40. 41.

42.

43.

44.

45. 46.

H. Bo et al. reactive oxygen species (ROS) and uncoupling protein 2. Free Radic Biol Med 2008;44:1373e81. Jiang N, Zhang G, Bo H, Qu J, Ma G, Cao D, et al. Upregulation of uncoupling protein-3 in skeletal muscle during exercise: a potential antioxidant function. Free Radic Biol Med 2009;46:138e45. Bo H, Wang YH, Li HY, Zhao J, Zhang HY, Tong CQ. Endurance training attenuates the bioenergetics alterations of rat skeletal muscle mitochondria submitted to acute hypoxia: role of ROS and UCP3. Sheng Li Xue Bao 2008;60:767e76. Noland RC, Hickner RC, Jimenez-linan M, Vidal-Puig A, Zheng D, Dohm GL, et al. Acute endurance exercise increases skeletal muscle uncoupling protein-3 gene expression in untrained but not trained humans. Metabolism 2003;52:152e8. McLeod CJ, Aziz A, Hoyt Jr RF, McCoy Jr JP, Sack MN. Uncoupling proteins 2 and 3 function in concert to augment tolerance to cardiac ischemia. J Biol Chem 2005;280:33470e6. Otera H, Mihara K. Molecular mechanisms and physiologic functions of mitochondrial dynamics. J Biochem 2011;149:241e51. Crane JD, Devries MC, Safdar A, Hamadeh MJ, Tarnopolsky MA. The effect of aging on human skeletal muscle mitochondrial and intramyocellular lipid ultrastructure. J Gerontol A Biol Sci Med Sci 2010;65:119e28. Lee S, Jeong SY, Lim WC, Kim S, Park YY, Sun X, et al. Mitochondrial fission and fusion mediators, hFis1 and OPA1, modulate cellular senescence. J Biol Chem 2007;282:22977e83. Lopez-lluch G, Irusta PM, Navas P, de Cabo R. Mitochondrial biogenesis and healthy aging. Exp Gerontol 2008;43:813e9. Seo AY, Joseph AM, Dutta D, Hwang JC, Aris JP, Leeuwenburgh C. New insights into the role of mitochondria in aging: mitochondrial dynamics and more. J Cell Sci 2010;123(Pt 15):2533e42. Green DR, Galluzzi L, Kroemer G. Mitochondria and the autophagy-inflammation-cell death axis in organismal aging. Science 2011;333:1109e12. Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC, et al. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol 2011;12:222e30. Gomes LC, Scorrano L. High levels of Fis1, a pro-fission mitochondrial protein, trigger autophagy. Biochim Biophys Acta 2008;1777:860e6. Aquilano K, Vigilanza P, Baldelli S, Pagliei B, Rotilio G, Ciriolo MR. Peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) and sirtuin 1 (SIRT1) reside in mitochondria: possible direct function in mitochondrial biogenesis. J Biol Chem 2010;285:21590e9. St-pierre J, Drori S, Uldry M, Silvaggi JM, Rhee J, Ja¨ger S, et al. Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators. Cell 2006;127:397e408. Liesa M, Borda-d’Agua B, Medina-Go´mez G, Lelliott CJ, Paz JC, Rojo M, et al. Mitochondrial fusion is increased by the nuclear coactivator PGC-1beta. PLoS One 2008;3:e3613. Soriano FX, Liesa M, Bach D, Chan DC, Palacıń M, Zorzano A. Evidence for a mitochondrial regulatory pathway defined by peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, estrogen-related receptor-alpha, and mitofusin 2. Diabetes 2006;55:1783e91. Wenz T. Mitochondria and PGC-1alpha in aging and age-associated diseases. J Aging Res 2011;2011:810619. Kang C, Chung E, Diffee G, Ji LL. Exercise training attenuates agingassociated reduction in mitochondrial biogenesis in rat skeletal muscle. Med Sci Sports Exerc 2009;41:S44.

47. Derbre F, Gomez-cabrera MC, Nascimento AL, Sanchis-Gomar F, Martinez-Bello VE, Tresguerres JA, et al. Age associated low mitochondrial biogenesis may be explained by lack of response of PGC-1alpha to exercise training. Age (Dordr) 2012;34:669e79. 48. Farhoud MH, Nijtmans LG, Wanders RJ, Wessels HJ, Lasonder E, Janssen AJ, et al. Impaired ubiquitin-proteasome-mediated PGC-1alpha protein turnover and induced mitochondrial biogenesis secondary to complex-I deficiency. Proteomics 2012;12:1349e62. 49. Feng H, Kang C, Dickman J, Koenig R, Awoyinka I, Zhang Y, et al. Training-induced mitochondrial adaptation: role of PGC-1a, NF-kB and b-blockade. Exp Physiol 2012;98:784e95. 50. Yang W, Hekimi S. A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans. PLoS Biol 2010;8:e1000556. 51. Christen WG, Glynn RJ, Sesso HD, Kurth T, MacFadyen J, Bubes V, et al. Age-related cataract in a randomized trial of vitamins E and C in men. Arch Ophthalmol 2010;128:1397e405. 52. Ristow M, Zarse K. How increased oxidative stress promotes longevity and metabolic health: the concept of mitochondrial hormesis (mitohormesis). Exp Gerontol 2010;45:410e8. 53. Gomez-Cabrera MC, Domenech E, Romagnoli M, Arduini A, Borras C, Pallardo FV, et al. Oral administration of vitamin C decreases muscle mitochondrial biogenesis and hampers training-induced adaptations in endurance performance. Am J Clin Nutr 2008;87:142e9. 54. Ding H, Jiang N, Liu H, Liu X, Liu D, Zhao F, et al. Response of mitochondrial fusion and fission protein gene expression to exercise in rat skeletal muscle. Biochim Biophys Acta 2010;1800:250e6. 55. Koopman WJ, Verkaart S, van Emst-de Vries SE, Grefte S, Smeitink JA, Nijtmans LG, et al. Mitigation of NADH: ubiquinone oxidoreductase deficiency by chronic Trolox treatment. Biochim Biophys Acta 2008;1777:853e9. 56. Jendrach M, Mai S, Pohl S, Vo¨th M, Bereiter-Hahn J. Short- and long-term alterations of mitochondrial morphology, dynamics and mtDNA after transient oxidative stress. Mitochondrion 2008;8:293e304. 57. Tondera D, Grandemange S, Jourdain A, Karbowski M, Mattenberger Y, Herzig S, et al. SLP-2 is required for stress-induced mitochondrial hyperfusion. EMBO J 2009;28:1589e600. 58. Scherz-shouval R, Elazar Z. Regulation of autophagy by ROS: physiology and pathology. Trends Biochem Sci 2011;36:30e8. 59. He C, Bassik MC, Moresi V, Sun K, Wei Y, Zou Z, et al. Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis. Nature 2012;481:511e5. 60. Baar K, Wende AR, Jones TE, Marison M, Nolte LA, Chen M, et al. Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC-1. FASEB J 2002;16:1879e86. 61. Cartoni R, Le´ger B, Hock MB, Praz M, Crettenand A, Pich S, et al. Mitofusins 1/2 and ERRalpha expression are increased in human skeletal muscle after physical exercise. J Physiol 2005;567(Pt 1):349e58. 62. Garnier A, Fortin D, Zoll J, N’Guessan B, Mettauer B, Lampert E, et al. Coordinated changes in mitochondrial function and biogenesis in healthy and diseased human skeletal muscle. FASEB J 2005;19:43e52. 63. Irrcher I, Ljubicic V, Hood DA. Interactions between ROS and AMP kinase activity in the regulation of PGC-1alpha transcription in skeletal muscle cells. Am J Physiol Cell Physiol 2009;296:116e23. 64. Ristow M, Zarse K, Oberbach A, Klo¨ting N, Birringer M, Kiehntopf M, et al. Antioxidants prevent health-promoting effects of physical exercise in humans. Proc Natl Acad Sci USA 2009;106:8665e70.