Age-specific mortality rates from heart disease, stroke, complications of diabetes, Alzheimer disease, and cancer increase exponentially with age, which imposes a huge financial burden on the health care systems in the Western world. There is an urgent need for effective therapeutic strategies that have the potential to promote health in the elderly, simultaneously preventing or delaying the development of various diseases of aging. During the past decade, dietary supplementation with resveratrol (3,5,4′-trihydroxystilbene) has emerged as a promising approach to counteract age-related diseases. Resveratrol is a naturally occurring polyphenol found in more than 70 species of plants, including grapes (Vitis vinifera), cranberries (Vaccinium macrocarpon), and peanuts (Arachis hypogaea), which was shown to confer diverse physiological effects in laboratory animals including cancer protection, microvascular protection, neuroprotection, cardioprotection, and antidiabetic effects (4). In this review, we consider the evidence in support of the hypothesis that mitochondrial protective effects of resveratrol underlie its antiaging action that can prevent/delay the development of age-related diseases in the cardiovascular system and other organs (3,7,30). The use of resveratrol as a dietary supplement to promote mitochondrial health in the elderly and diabetic patients is discussed.
Resveratrol as a Dietary Restriction Mimetic
Dietary restriction (a dietary regimen that restricts calorie intake), when not associated with malnutrition, has been shown to improve health and slow the aging process in a wide range of animals (for a review, see (20)). Sirtuin enzymes are nicotinamide adenine dinucleotide (NAD)-dependent deacetylases that are conserved from yeast to humans, influence lifespan in lower organisms, and seem to mediate at least certain effects of dietary restriction. Resveratrol has been proposed to mimic protective effects of dietary restriction, based on the observation that it can activate the mammalian sirtuin SIRT1 (silent mating type information regulation 2, Saccharomyces cerevisiae, homolog 1) in an in vitro assay (14). However, the precise importance of sirtuins in the effects of dietary restriction and the ability of resveratrol to directly activate these enzymes in vivo remain controversial.
Even while the rationale for testing resveratrol as a dietary restriction mimetic has been challenged, evidence for dietary restriction-like effects has continued to emerge. Resveratrol has been shown to extend life in yeast, worms, and flies in a sirtuin-dependent manner (14,34), although not all groups have reproduced these results (2). Furthermore, resveratrol prevents insulin resistance, enhances mitochondrial biogenesis, improves vascular function, delays functional decline, and restores normal longevity in obese mice (3,15). Perhaps most impressively, resveratrol induces transcriptional profiles in multiple tissues that resemble those of animals consuming fewer calories (1,22). At the same time, it is clear that resveratrol fails to mimic other aspects of dietary restriction, such as slowing heart rate and decreasing core body temperature (18) or, thus far, extending lifespan in nonobese animals (19,22). Therefore, resveratrol seems to mimic some, but not all, effects of dietary restriction, and much remains to be learned about the mechanisms responsible. A common theme that emerges from studies on mammals is that both dietary restriction and resveratrol treatment can elicit changes in cellular mitochondrial content and alter mitochondrial production of reactive oxygen species (ROS). These effects are likely to have significant, possibly synergistic, impact on cellular aging processes.
Resveratrol Promotes Mitochondrial Biogenesis
Mitochondria are highly dynamic organelles, and their biogenesis likely is to be involved in the regulation of cellular metabolism, redox regulation, and signal transduction. Impairment of mitochondrial biogenesis has been described during aging, in diabetes, and in the metabolic syndrome and is thus likely to contribute to cellular energetic imbalance, oxidative stress, and organ dysfunction in these pathological conditions. The physiological improvements induced by resveratrol in rodent models of aging and the metabolic syndrome are accompanied by an increase in the mitochondrial content of liver, skeletal muscle (3,15) and blood vessels (7), which is intriguing because an increase in mitochondrial biogenesis has been observed in the liver, adipose tissue, central nervous system, and cardiovascular system of mice, as well as skeletal muscle from humans, during dietary restriction (17). Importantly, resveratrol was shown to increase cellular mitochondrial content even in lean mice, which do not show any benefit in terms of longevity (19,22).
Mitochondrial biogenesis induced by resveratrol treatment likely has important effects in liver (3) and brain (24) and seems to have significant consequences in skeletal muscle because resveratrol-treated mice exhibit an approximately two-fold increase in endurance (15). Important for the present review, mitochondria also have a crucial role in vascular pathophysiology (29). Previous studies show that dysregulation of mitochondrial biogenesis represents an early manifestation of endothelial dysfunction, shifting cell metabolism toward metabolic hypoxia in animals with impaired nitric oxide (NO) bioavailability (29). In that regard, it is significant that resveratrol treatment induces mitochondrial biogenesis in the vasculature (7).
Multiple mechanisms may explain resveratrol-induced mitochondrial biogenesis and its contribution to cytoprotection and organismal health (Figure). Activation of SIRT1 by resveratrol can promote deacetylation and activation of peroxisome proliferator activator gamma coactivator 1 alpha (PGC-1α), the "master regulator" of mitochondrial biogenesis. Alternatively, other pathways, such as activation of the energy sensor adenosine monophosphate (AMP)-activated protein kinase may allow PGC-1α to be activated independently of SIRT1, or many mechanisms may act in concert. Impaired mitochondria (e.g., in diabetes and in aging) may alter adenosine triphosphate production, the synthetic and secretory function of cells, cellular redox homeostasis, and nuclear gene expression (by changing retrograde signaling pathways). Resveratrol-induced mitochondrial biogenesis would correct this impairment. Furthermore, because mitochondrial proliferation reduces the flow of electrons per unit mitochondria, resveratrol-induced mitochondrial biogenesis may reduce mitochondrial ROS production in the cells. Indeed, resveratrol, at physiologically relevant concentrations, attenuates mitochondrial oxidative stress in endothelial cells (8).
Resveratrol Attenuates Mitochondrial Oxidative Stress
The mitochondrial theory of aging postulates that mitochondrial oxidative stress and the consequential free radical reactions underlie aging (33). According to this theory, increased production of ROS results in a variety of macromolecular oxidative modifications with age, and the accumulation of such oxidative damage of proteins, lipids, and DNA is the primary causal factor in the aging process. Many previous studies lend support to the mitochondrial theory of aging. First, there is clear evidence that aging in mammals is associated with mitochondrial oxidative stress in virtually every tissue studied (10,11,31-33). Mitochondrial oxidative stress also is observed frequently in diabetes and the metabolic syndrome, and the theory predicts that mitochondria-derived ROS would accelerate aging in these pathological conditions. Second, although the mitochondrial theory is a subject of ongoing debate because of recent findings in genetically manipulated laboratory mice that are inconsistent with it, comparative studies demonstrate that in evolutionarily distant species, an inverse correlation exists between mitochondrial ROS production and species longevity (33). Third, lifespan extension in dietary restriction has been attributed to a marked reduction of mitochondria-derived ROS production. Fourth, although many genetic manipulations designed to perturb oxidative stress have failed to produce the intended effect, targeting of the peroxide-scavenging enzyme catalase to mitochondria significantly extended life in mice (26,33). Thus, there is a continuing interest in identifying novel therapeutic approaches to attenuate mitochondrial oxidative stress in elderly patients.
Because of the original observation that resveratrol mimics some of the antiaging effects of dietary restriction (34), a large body of evidence has accumulated that resveratrol confers multifaceted antioxidant effects in virtually every organism studied. Important for the current overview, recent studies demonstrate that resveratrol attenuates both steady-state and high glucose-induced mitochondrial superoxide (O2−) production in various cell types, including primary human coronary arterial endothelial cells (30). This mitochondrial protective effect of resveratrol is, at least in part, attributed to the induction of mitochondrial antioxidant systems by resveratrol (30). The in vitro effects of resveratrol are consistent with the cytoprotective effects of resveratrol treatment in rodent models of aging and diabetes (22,36). It should be noted that nonmitochondrial sources of ROS also contribute to age-related cellular oxidative stress. In particular, tumor necrosis factor (TNF) α-mediated up-regulation of cell membrane-associated NAD(P)H oxidases seems to contribute significantly to aging-induced increases in cellular O2− levels in the cardiovascular system (29). Importantly, both TNFα and NAD(P)H oxidase activation can induce mitochondrial oxidative stress in endothelial cells. Because resveratrol treatment of aged mice results in significant down-regulation of TNFα and/or NAD(P)H oxidases in the vasculature (22) and the skeletal muscle (25), it is possible that attenuation of nonmitochondria-derived ROS production also contribute to resveratrol-mediated mitochondrial protection. This view also is supported by the findings that activation of NAD(P)H oxidases decreases the bioavailability of NO in aging animals (reviewed recently in (29)), which has been linked to the age-related impairment of mitochondrial biogenesis.
There are multiple mechanisms by which resveratrol-induced reduction of mitochondrial oxidative stress may promote organismal health (Figure). Increased ROS levels in the mitochondria are known to inactivate critical enzymes involved in mitochondrial metabolism (e.g., α-ketoglutarate dehydrogenase and aconitase), which may diminish adenosine triphosphate production. It also is likely that mitochondrial oxidative stress results in direct damage to mitochondrial DNA, which may play a further role in the development of age-related diseases. Resveratrol-induced attenuation of mitochondrial oxidative stress would correct these impairments. Resveratrol was shown to protect cultured cells against senescence-associated changes (13); however, the role of reduced mitochondrial production of ROS in these effects is presently unclear. Mitochondria-derived O2− is membrane impermeable (except in the protonated perhydroxyl radical form, which represents only a small fraction of total O2− produced), whereas H2O2 easily penetrates the mitochondrial membranes. Mitochondrion-derived H2O2 has important signaling functions, such as activation of nuclear factor (NF) κB-dependent inflammatory pathways in aging (31) and metabolic diseases. Thus, resveratrol-induced attenuation of mitochondrial oxidative stress is likely to confer antiinflammatory effects. Indeed, resveratrol treatment significantly decreases NF-κB-dependent gene expression in aortas of aged mice and mice with type 2 diabetes (22).
Taken together, these observations suggest that protection of mitochondria by resveratrol is likely to contribute to its antiaging action (3,27). Similarly, mitochondrial protection can be achieved through feeding a calorie restricted diet, which also induces pathways activated by resveratrol (6) and attenuates mitochondrial ROS production (16).
Role of SIRT1 Activation in Mitochondrial Protection
Previous studies demonstrated that SIRT1-dependent pathways and NO-dependent mechanisms contribute to the beneficial mitochondrial and cellular effects of dietary restriction (6,21). Many of the effects that have been observed in resveratrol-treated animals are consistent with the modulation of SIRT1 targets, particularly the transcriptional coactivator PGC-1α (Figure) (3,15). SIRT1 likely regulates multiple pathways involved in mitochondrial biogenesis, among which pathways regulated by endothelium-derived NO seem to play a key role (7). Resveratrol is thought to increase NO production by up-regulating endothelial NO synthase (eNOS) at the level of transcription (22). In addition, previous studies suggest that SIRT1 may directly deacetylate and activate eNOS in endothelial cells.
These findings may be seen as support for the originally proposed mechanism that resveratrol functions as a direct activator of SIRT1. However, resveratrol is known to produce a wide array of effects in mammalian cells, including activation of AMP-activated protein kinase (AMPK), an energy sensor that is involved in some of the same pathways as SIRT1 and phosphorylates PGC-1α directly (3,35). Although SIRT1 can activate the kinase upstream of AMPK, this pathway does not seem to be necessary for AMPK stimulation by resveratrol (9), and in fact, there is some evidence to suggest that SIRT1 might be downstream of AMPK under some circumstances (5,12). In a similar situation, although SIRT1 can activate eNOS, there is evidence that SIRT1 induction also is a downstream effect of NO production (21). Therefore, it will be important to carefully test the role of SIRT1 in the physiological changes induced by resveratrol.
Role of Nrf2 Activation in Mitochondrial Protection
There is increasing evidence that activation of NF-E2-related factor 2 (Nrf2) is an important mechanism by which resveratrol confers cytoprotective effects (28). Nrf2 is a basic leucine zipper transcription factor that regulates the coordinated expression of key antioxidant mechanisms in the cell by binding to the antioxidant response elements in the promoter regions of target genes (e.g., NAD(P)H:quinone oxidoreductase 1, heme oxygenase-1, enzymes involved in the regulation of glutathione synthesis), which regulate mitochondrial and cellular levels of ROS (Figure). Activation of Nrf2-dependent pathways has been shown to promote cardiovascular health and neurocognitive function in rodent models of aging and diabetes. Furthermore, Nrf2 was shown to mediate cancer protection but not increased longevity induced by caloric restriction in mice (23). Because Nrf2-driven pathways can be activated by concentrations of resveratrol readily achievable in vivo, future studies should elucidate whether Nrf2 activation contributes to the mitochondrial protective effects of resveratrol in aging and in pathophysiological conditions associated with mitochondrial dysfunction. Recent evidence obtained using Nrf2-/- mice lend support to the hypothesis that Nrf2 activation plays an important role in the vasoprotective action of resveratrol under diabetic conditions (28), but the role of mitochondria in the observed effects remain to be elucidated. Furthermore, induction of the Nrf2 target heme oxygenase-1 also has been implicated in the cardioprotective effects of resveratrol under conditions of experimentally induced ischemia, which is known to be associated with mitochondrial oxidative stress and cellular energetic imbalance. Future studies should test the possibility that SIRT1 may modulate Nrf2-driven cellular responses, and studies also are warranted to determine whether Nrf2-dependent pathways regulate SIRT1 at the level of transcription.
Although significant progress has been achieved in elucidating the cellular mechanisms activated by resveratrol, the specific roles for pathways regulating mitochondrial function, mitochondrial antioxidant defenses, and mechanisms involved in mitochondrial biogenesis require further study. There is a reasonable consensus that, in rodents, mitochondrial oxidative stress and activation of redox-sensitive proinflammatory pathways (e.g., NF-κB) play a central role in the development of many age-related diseases, which may be prevented or reversed by resveratrol treatment. Research efforts should be focused on fully elucidating the links between mitochondrial effects of resveratrol and its cardiac, cerebrovascular, and neuroprotective action in aging. Furthermore, it is unknown whether administration of resveratrol would affect mitochondrial function in primates. Thus, future studies should determine whether mitochondrial protective effects of resveratrol are manifested in primate models of aging and metabolic diseases. Moreover, clinical studies with resveratrol in the elderly should be encouraged. Because resveratrol is relatively enriched in red wine, it often is speculated that resveratrol explains some of the protective effects of wine consumption in humans; however, the doses used to study resveratrol in rodents are typically orders of magnitude higher than what could be obtained from reasonable consumption of wine. Thus, clear guidelines for the effective dose and formulation of resveratrol for human studies should be established. Given the dramatic protection against age-related diseases that is afforded by dietary restriction, every effort should be made to identify and understand compounds, such as resveratrol, that seem to be able to mimic some of its effects on mitochondria and antioxidant defense mechanisms.
The authors acknowledge the work of their colleagues, whose ideas contributed to the understanding of the role of resveratrol in prevention of age-related pathologies but whose publications could not be cited because of the limits imposed by the journal. This work was supported by grants from the American Diabetes Association (to Z.U.), American Federation for Aging Research (to A.C.), the University of Oklahoma College of Medicine Alumni Association (to A.C.), the National Institutes of Health (AG031085 to A.C.; AT006526 and HL077256 to Z.U.; AG031182 to J.A.B.; P01 AG11370 to W.E.S.), and the Intramural Research Program of the National Institute on Aging (R.d.C.).
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