The beneficial effect of caloric restriction (CR) in extending maximal lifespan was first reported by Osborne and colleagues (25) nearly a century ago. In the years since then, CR has become one of the most widely studied interventions that slow aging. Although many different regimes of CR have been identified as effective in slowing aging (e.g., varying degrees of energy restriction on a daily basis versus every other day feeding), the key component is a reduction in the energy intake without adversely affecting the intake of essential nutrients (the latter sometimes requiring supplementation of the diet to ensure normal nutrient levels). One of the most widely studied CR regimes involves restricting energy intake to 60% of ad libitum (AL). This degree of energy restriction evokes an extension of maximal lifespan by approximately 25%-40% in rodents and better preserves organ function throughout the organism at least in proportion to the extension of lifespan. Although many different molecular pathways are affected by CR, many of these converge on the mitochondria (2), lending important insight into the mechanisms involved in the protective effects of CR. Similarly, recent data (8) suggest that many of the same molecular pathways are activated with CR in humans as in animal models, and although it is speculated that the maximal life extension effect in humans may not be as pronounced, the health benefits of CR in humans are considerable (14,23). This article reviews evidence in relation to the hypothesis that CR reduces mitochondrial reactive oxygen species (ROS) production and promotes mitochondrial renewal via enhanced drive on mitochondrial biogenesis and autophagy.
MOLECULAR PATHWAYS INDUCED BY CR: CONVERGENCE ON THE MITOCHONDRIA
Many different ideas have been advanced to explain the beneficial effects of CR in slowing aging and attenuating the associated deterioration of cellular function. Given the organism-wide benefits of CR, the most strongly supported explanations have been those that consider the effects of CR on specific elements of cellular function (e.g., molecular signaling pathways like the sirtuins). As previously noted, a critical role for a CR-induced alteration of mitochondrial function has come to the fore, and this continues to be an exciting area of interrogation. The appeal of a central role for mitochondria is that it provides a means by which the molecular signaling pathways within individual cells and the systemic alterations seen with CR may be integrated to account for CR's organism-wide benefits. Specifically, a CR-induced reduction in cellular energy charge, based upon an increased ratio of nicatinamide adenine dinucleotide (NAD+) and its protonated form, NADH (NAD+/NADH ratio), activates sirtuin 1 (SIRT1) in mammals. Sirtuin 1 not only modulates global gene expression via its effects on histone acetylation state (some of which support and/or amplify the more direct effects of CR on mitochondria) but also stimulates mitochondrial biogenesis in particular (8,20) via the activation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) secondary to both increased PGC-1α expression and its deacetylation by SIRT1 (13). Among the effects of SIRT1 on global gene expression that may amplify the effects of CR on mitochondria, CR increases skeletal muscle insulin sensitivity by enhancing phosphatidylinositol signaling (22). This increase in insulin sensitivity is likely central to mediating the decline in circulating insulin seen with CR, which, in turn, is necessary to permit a reduction in mitochondrial ROS generation (17). The importance of some circulating factor(s) (such as insulin) in the CR-induced alteration of mitochondrial function is consistent with observations that cells cultured with serum from rats subjected to CR exhibit a reduced mitochondrial ROS generation (20). Thus, CR can impact mitochondria both by stimulating biogenesis of new organelles and by fundamentally altering their function (e.g., reduced ROS generation). On the other hand, controversy remains concerning whether CR induces more efficient (less proton leak) or less efficient (greater proton leak) mitochondrial coupling. Specifically, whereas one research group advocates that an increase in mitochondrial proton leak by CR facilitates a reduction in mitochondrial ROS generation (17), another group finds exactly the opposite, with CR reducing both mitochondrial proton leak and ROS production (3). Regardless of the controversy herein, a reduction in mitochondrial ROS generation and increase in mitochondrial protein turnover seem to be well-established aspects of the effects of CR on mitochondria. The putative significance of these CR-induced effects on the mitochondria in the context of preserving skeletal muscle mitochondrial function with aging will be discussed in the sections that follow.
AGING AND SKELETAL MUSCLE MITOCHONDRIAL FUNCTION
Although the term mitochondrial dysfunction is used fairly liberally in the literature, the criteria used in making this distinction are often far from rigorous nor without alternate interpretation. One criterion that will be considered here in making the judgment of a decline in mitochondrial function with aging is whether the capacity for energy provision per unit of mitochondrial volume declines. This is an important distinction from simply a reduction in mitochondrial volume per unit of muscle that results in a reduced oxidative capacity of the muscle. Specifically, although a reduced skeletal muscle mitochondrial volume could occur in response to reduced physical activity with aging and reduce muscle oxidative capacity without impacting the capacity of individual mitochondria to generate energy, a reduction in the capacity for energy provision per unit of mitochondrial volume (with or without an accompanying reduction in muscle oxidative capacity) implies mitochondrial defect.
Whereas many studies have examined changes in muscle oxidative capacity with aging, only a few have directly addressed the capacity of the mitochondria with aging per se. In this latter respect, Conley and colleagues (9) showed previously that there was a greater decline in the oxidative capacity of human vastus lateralis muscle (inferred from phosphocreatine recovery after knee extensor exercise) of aged subjects than could be accounted for by the reduction in mitochondrial volume density (measured by electron microscopy in muscle cross-sections taken from biopsy samples), revealing a reduced oxidative capacity per volume of mitochondria in aged human skeletal muscle (Fig. 1). Others have examined function of mitochondria isolated from muscles of aged individuals or organisms and, although there is some support for impaired mitochondrial function in aged muscles, there is also some indication that isolating mitochondria may underestimate the potential for mitochondrial dysfunction with aging by selectively harvesting the healthiest mitochondria (29). Although this has never been tested experimentally, it has been hypothesized that because of increasing fragility of some mitochondria with aging (28), this would result in selective harvest of the healthiest mitochondria in the aged muscles, thereby leading to an underestimate of mitochondrial dysfunction in isolated mitochondrial fractions (29). Furthermore, as mitochondria in skeletal muscle exist in varying degrees of a reticulum, experimental isolation of mitochondria would disrupt this structural arrangement, which could also obscure important changes in mitochondrial function in vivo. These important issues remain to be adequately addressed in the literature.
Another indication of impaired mitochondrial function with aging is a disproportionate change in one enzyme versus another, as the activities of different mitochondrial enzymes normally scale proportionally across a wide range of muscle oxidative capacity (10). Interestingly, complex IV of the electron transport chain often exhibits a disproportionate decine in activity with aging relative to other mitochondrial enzymes (24). This has also been seen in aged skeletal muscles (15). The reasons for a greater decline in complex IV activity remain to be fully accounted for, but strong possibilities include the accumulation of oxidative damage and/or incorrect assembly of the subunit proteins. Collectively, therefore, it seems that aging is associated with a reduction in skeletal muscle oxidative capacity, which exceeds that explainable by simply a reduction in muscle mitochondrial content. This suggests that whereas physical inactivity may be a contributor to declining muscle oxidative capacity with aging, aging-specific effects on the mitochondria themselves are also an important consideration.
FACTORS ACCOUNTING FOR MITOCHONDRIAL DYSFUNCTION IN AGED MUSCLES
Mitochondrial protein exhibits a continual turnover, with the enzymes having a half-life of approximately 7 d (5). One reason for this turnover is that mitochondria normally produce some ROS, which in turn can oxidatively damage the mitochondrial proteins and impair enzyme function. This impaired enzyme function, particularly if it occurs in the electron transport chain, could elevate ROS production and lead to a downward spiral in mitochondrial function. Consistent with the idea that accumulation of oxidative damage can impair mitochondrial enzyme activity, elevating oxidative stress in aging muscle can reduce aconitase enzyme activity without reducing its protein content (6). The significance of this observation is that aconitase has an iron-sulfur center, which renders it particularly susceptible to oxidative damage, and thus, it provides a useful biomarker of oxidative damage in mitochondria.
In accounting for impaired mitochondrial function in aged skeletal muscles, it is relevant that a major enzyme involved in the degradation of oxidatively damaged mitochondrial proteins (Lon protease) declines with aging (6), and mitochondrial protein synthesis rate declines in aged muscle (27). Furthermore, mitochondrial autophagy, whereby whole organelles are engulfed and enzymatically degraded in lysosomes, is thought to be impaired in aging muscles (28). Collectively, these changes show that mitochondrial protein turnover declines with aging and that this is likely due to combined effects of reduced mitochondrial protein synthesis, impaired removal of oxidatively damaged mitochondrial proteins, and reduced mitochondrial autophagy. As previously implied, the expected impact of this reduced mitochondrial turnover would be manifest as not only a reduced oxidative capacity per unit of mitochondrial volume because the longer mitochondrial protein dwell-time would facilitate accumulation of more oxidative damage but also an increase in mitochondrial ROS generation secondary to, for example, a relatively greater reduction in complex IV activity (by allowing oxygen to accumulate to higher levels, this favors production of ROS). This is consistent with reports showing elevated mitochondrial ROS generation in both aged rodent (21) and human (7) skeletal muscles.
In summary, a decline in mitochondrial function in aged skeletal muscles occurs at least in part secondary to a reduction in mitochondrial turnover rate that leads to accumulation of defective mitochondria. This accumulation of defective mitochondria would further exacerbate the problem by increasing ROS production, leading to more oxidative damage, creating a vicious cycle (Fig. 2). On this basis, it seems logical to propose that maintaining mitochondrial turnover across the lifespan would be beneficial in maintaining mitochondrial function.
CR, MITOCHONDRIA, AND AGING
One of the first studies to investigate the impact of CR on skeletal muscle mitochondrial function with aging was that of Desai and colleagues (11). They observed that the age-related decline in activities of electron transport chain complexes seen in mice with AL food access was completely prevented by CR. Interestingly, however, they also observed that the CR animals began in young adulthood with lower activities of the electron transport chain complexes compared with age-matched AL mice (Fig. 3). In addition, the affinity of complex IV for reduced cytochrome c was higher in the mitochondria from CR mice, which may be more functionally relevant in vivo than the reduced maximal in vitro activity of the complex because metabolic substrates in working muscle are not in saturating amounts in vivo.
More recently, we observed that CR completely prevented the age-related decline in skeletal muscle aerobic function during high-intensity muscle contractions in situ (15) (Fig. 4). Similar to the results of Desai and colleagues (11), we also observed that the activities of several mitochondrial enzymes were lower in the muscles of young adult CR rats, despite the fact that the skeletal muscle maximal aerobic function of CR rats in young adulthood was identical to that of the young adult AL rats. In addition, we also observed that CR completely prevented the age-related decline in complex IV activity seen in the AL animals and that this benefit remained even after accounting for the life extension by CR, meaning that the functional health span of the muscles was also extended by CR (1). It should also be pointed out that we saw no evidence of reduced mitochondrial coupling efficiency by CR because the quotient of oxygen uptake and force production tended to be lower (not higher) in the CR rats (15). These results point to subtle differences in the mitochondria of CR rats that may be important to help them remain healthy with aging, as will be further explored below.
A number of factors have been identified that play an important role in the preservation of mitochondrial function with aging by CR. Among the most important is a reduction in mitochondrial ROS generation. Several laboratories have now shown that CR induces a reduction in mitochondrial ROS generation, and this effect can be seen in as little as 2 wk of initiating CR (4). It has also been shown that this adaptation can be prevented by normalizing the reduction in circulating insulin levels seen with CR (17) or by increasing fat intake in animals undergoing CR (12). The impact of this kind of adaptation is that the mitochondrial enzymes (and, indeed, the other cellular components) would be subject to a lower level of oxidative stress on a moment-to-moment basis, which, all other things being equal, would assist in preserving mitochondrial function by reducing accumulation of mitochondrial oxidative damage. This point is consistent with observations of lower oxidatively damaged mitochondrial proteins in aged CR animals (18).
Another important clue related to the protection of mitochondrial function with aging by CR involves the rate at which the mitochondrial proteins, and the mitochondria themselves, are turned over. It was previously mentioned that aging is associated with not only a reduced rate of mitochondrial protein synthesis but also a reduced rate of mitochondrial protein degradation (e.g., via the Lon protease) and reduced mitochondrial autophagy. A major driver of mitochondrial biogenesis involves the action of PGC-1α, a transcription factor that coordinates the expression of genes involved in generating new mitochondria. Activation of PGC-1α is thought to occur via a number of pathways, one of which is in response to a decrease in cellular high energy state, via induction of 5′adenosine monophosphate-activated protein kinase. Interestingly, aging is associated with a decreased skeletal muscle 5′adenosine monophosphate-activated protein kinase activity (26) and PGC-1α gene expression (1). This decline in the drive on mitochondrial biogenesis with aging likely forms the basis of the reduced rate of mitochondrial protein synthesis observed with aging (27). On the other hand, CR augments the PGC-1α signaling pathway (13), resulting in increased mitochondrial biogenesis in vitro (20) and an attenuation of the age-related decline in skeletal muscle PGC-1α messenger RNA levels (1). On the other hand, whether CR results in a net increase in mitochondrial content remains unclear. Although in vitro results suggest that CR increases mitochondrial content (20), there was no difference in citrate synthase protein (a marker for mitochondrial content) in plantaris or gastrocnemius muscle of young adult rats that had been on a CR diet for 4 months versus their age-matched counterparts (1).
As previously noted, it has been suggested that impaired mitochondrial autophagy, whereby whole mitochondria are engulfed and degraded within lysosomes, becomes impaired with aging, and this contributes to the accumulation of abnormal mitochondria (28). On this basis, Wohlgemuth and colleagues (30) recently showed that CR increased the appearance of autophagic vacuoles in the heart of aged rats. This was associated with an increase in the expression of proteins involved in the regulation of autophagy. It has also recently been found that the SIRT1 pathway, which is up-regulated with CR, can increase the activity of important components of the autophagy pathway via deacetylating autophagy proteins (19). This suggests that CR may enhance mitochondrial protein turnover not only by stimulating mitochondrial biogenesis but also by enhancing mitochondrial degradation via autophagy. The net result of these effects is that CR keeps the mitochondria pristine with aging by ensuring their regular maintenance and replacement (Fig. 5).
CONCLUSIONS AND PERSPECTIVES
The profound benefits of CR on skeletal muscle mitochondria teach us an important lesson: the impact of aging on skeletal muscle mitochondrial function can be delayed. Although there is evidence that CR in humans induces many of the same molecular responses as seen in other species, including an induction of the SIRT1 pathway in skeletal muscle (8), CR does not represent a very practical solution because of the discipline necessary to remain on the CR diet. As such, CR is being used by researchers principally to help us understand what physiological responses permit maintenance of healthy mitochondria with aging. This in turn will be pivotal to identifying novel strategies that may involve dietary and/or physical activity manipulations, to yield similar benefits of CR without having to endure years of hunger pangs.
Considerable effort is now focusing on clarifying the mechanisms by which sirtuins, like SIRT1, mediate the protective effects of CR and identifying other interventions that mimic CR's effects without requiring energy restriction. One particularly promising example of such an agent is resveratrol, a compound found in the skin of grapes, which has been shown to activate SIRT1, deacetylate PGC-1α, and induce mitochondrial biogenesis (16). Although the cynical may rail against such discoveries, suggesting that this will herald in a new era of sedentary existence where people need only take a pill to get benefits, a more optimistic outlook is that these kinds of interventions will facilitate better health and vitality for all of us, including those who choose to remain physically active well into the "golden years," by protracting the period of frailty and associated disability.
The author thanks the Canadian Institutes of Health Research and Alberta Heritage Foundation for Medical Research for support of this work.
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