Minireview: The Role of Oxidative Stress in Relation to Caloric Restriction and Longevity
http://www.100md.com
《内分泌学杂志》
Department of Animal Physiology-II, Faculty of Biology, Complutense University, Madrid 28040, Spain
Abstract
Reduction of caloric intake without malnutrition is one of the most consistent experimental interventions that increases mean and maximum life spans in different species. For over 70 yr, caloric restriction has been studied, and during the last years the number of investigations on such nutritional intervention and aging has dramatically increased. Because caloric restriction decreases the aging rate, it constitutes an excellent approach to better understand the mechanisms underlying the aging process. Various investigations have reported reductions in steady-state oxidative damage to proteins, lipids, and DNA in animals subjected to restricted caloric intake. Most interestingly, several investigations have reported that these decreases in oxidative damage are related to a lowering of mitochondrial free radical generation rate in various tissues of the restricted animals. Thus, similar to what has been described for long-lived animals in comparative studies, a decrease in mitochondrial free radical generation has been suggested to be one of the main determinants of the extended life span observed in restricted animals. In this study we review recent reports of caloric restriction and longevity, focusing on mitochondrial oxidative stress and the proposed mechanisms leading to an extended longevity in calorie-restricted animals.
Introduction
RESEARCH ON CALORIC restriction (CR) and longevity has noticeably increased in the last 5 yr. During that period, the number of published reports was 30-fold higher than in the 1990s, denoting that caloric restriction currently is a widespread approach to aging research. Because it has been studied for more than 70 yr since the first report of its effect on life span (1), CR is a well-known nutritional intervention, and its beneficial effects on longevity have been described in detail. Although getting older leads to a decline in maximum functional capacities, caloric restriction decelerates such declines. Animals under a CR regimen (without malnutrition) maintain most physiological functions in a youthful state at more advanced ages. CR also retards age-related diseases, such as cardiomyopathy, nephropathy, diabetes, hypertension-related diseases, and neoplastic processes (2, 3). The beneficial effects of CR can be observed not only when initiated at a young age, but also in adulthood. Although it has been reported that CR could be ineffective or even detrimental if started late in life (4), various investigations show that positive effects can also be observed when caloric restriction is started in middle age or later (5, 6). Interest in caloric restriction has increased since investigations in nonhuman primates started, and positive effects on long-lived mammals became clearer. Although full longevity data are not yet available, ongoing studies suggest that the CR-related reduction in the aging rate reported in rodents and other short-lived species could also take place in primates (7) and thus possibly in humans.
It is currently believed that the positive effects of CR depend on the reduced intake of calories themselves (8), although it has been reported that changes in the proportion of the main dietary components can also modulate longevity (9). For instance, restriction of protein intake significantly increased mean and maximum longevities in different rat strains (10, 11, 12). Furthermore, when animals were fed a diet restricted in the essential amino acid methionine, mean and maximum life spans were enhanced as well (13). More recently, we have observed that protein restriction without strong caloric restriction has similar effects on the mitochondrial free radical generation rate and mitochondrial DNA (mtDNA) oxidative damage in rat liver as the reduction of the total intake of calories (14). All these data are consistent with the possibility that part of the effects on the aging rate induced by CR could be due to the decreased intake of particular components of the diet, such as proteins.
Caloric Restriction and Mitochondrial Oxidative Stress
Although the fundamental mechanisms of aging are still unclear, a growing body of evidence involves mitochondria and the continuous generation of free radicals at the mitochondrial inner membrane. The mitochondrial free radical theory of aging (15) currently receives extensive support from experimental investigations (16, 17, 18, 19, 20). The connection between caloric restriction and the mitochondrial free radical theory of aging has been brought about by numerous investigations describing a lower mitochondrial free radical generation rate in restricted animals compared with ad libitum-fed animals (19, 20).
Since it was proposed that CR worked, at least in part, by decreasing oxidative stress (19), several studies have shown that moderate caloric restriction leads to lower oxidative damage to cellular macromolecules. The effect of caloric restriction on protein and DNA oxidative damage has been extensively investigated in the last decades, because both macromolecules have been proposed to play key roles in the aging process. Lipids and the degree of fatty acid unsaturation have been also proposed to play an important role in longevity (21). However, studies of the degree of fatty acid unsaturation of mitochondria have shown no changes in heart from Wistar rats (22, 23) when caloric restriction was extended up to 12 months. Only after life-long CR were decreases in the unsaturation index observed in liver mitochondria of Brown-Norway rats (24) and heart mitochondria of Fischer 344 rats (25).
Regarding protein damage, several studies have reported that the levels of protein carbonyls (26, 27, 28) and dityrosine cross-links (29) are lower in CR animals than in ad libitum-fed ones. Moreover, restricted animals exhibit slower accumulation of those markers with age than fully fed animals. However, protein carbonyls are not a single defined chemical entity, and the effect of CR has been shown to diverge depending on the selected marker. Thus, when more specific protein markers, such as advanced Maillard products or specific carbonyl modifications (glutamic and aminoadipic semialdehydes), were recently investigated in heart (22, 23) and liver (24) rat mitochondria, it was found that the reduction in particular levels of protein oxidative damage after CR depends on implementation time and is tissue specific. Hence, although in heart mitochondria of restricted Wistar rats, a general reduction in protein oxidative damage was observed after 4 and 12 months, but not after 6 wk (22, 23), in liver mitochondria from Brown-Norway rats, a minor reduction in the levels of protein oxidative damage was detected only after life-long CR (24).
Different types of oxidative damage could differently influence the lower aging rate of restricted animals. Whereas damage to lipids and proteins is continuously repaired by cellular turnover using the information coded in the DNA, once a postmitotic cell loses all copies of a given gene, it cannot recuperate its information content, because such information flows from DNA to proteins, not in the reverse direction. Mitochondrial DNA has been suggested to play a determinant role in the aging process (16, 18, 30). Similarly to long-lived animals, CR rodents show a slower aging rate and extended longevity due in part to a reduced mitochondrial free radical production and a slower long-term accumulation of mutations in mtDNA (20).
In accordance with that, many investigations have consistently reported reduced levels of mutagenic oxidative modifications in mtDNA after long-term CR (31, 32, 33, 34, 35, 36, 37). To better characterize the relation among CR, mtDNA oxidative damage, and mitochondrial free radical generation, it is desirable to investigate them simultaneously. A series of studies was performed in which both parameters were measured in parallel in restricted and ad libitum-fed Wistar rats. These investigations showed that long-term CR decreased the rate of mitochondrial H2O2 production and mtDNA oxidative damage in rat liver (35), heart (34), skeletal muscle (36), and brain (37). Furthermore, the quantitative reduction of mtDNA oxidative damage was strikingly similar to that found for mitochondrial free radical generation in all cases (Table 1).
Eating Less, Living Longer: Underlying Mechanisms
As described above, one of the most consistent effects of CR is the reduction in oxidative damage in animals subjected to moderate CR, but what mechanisms cause such reductions Although various potential mechanisms could explain the protective effect of CR on oxidative damage, including an increased capacity to degrade/repair modified macromolecules or an increase in antioxidant defenses, a reduction in the rate of mitochondrial free radical generation seems the most likely candidate.
Concerning scavenger activities, results of different studies do not support an induction of antioxidant activity in CR animals. Increases in such activities were described in some CR models (38), but later studies in rat liver (39) and heart (40) and mouse skeletal muscle (26), brain, heart, and kidney (32) failed to describe any clear-cut overall pattern of CR-related changes in antioxidant defenses, ruling out antioxidants as determinants of the lower oxidative damage observed in CR animals. The attenuation of oxidative damage modifications in CR may also result from an enhancement of the rate of degradation/repair of damaged macromolecules. Supporting that idea, increases in protein synthesis and turnover have been described in restricted rodents (41) as well as increases in the rate of protein catabolism in mouse liver (42). More recently, it has been reported that peptidylglutamyl-peptide hydrolase activity of the 20S proteasome is increased in the heart of rats subjected to short-term CR (40), whereas the chymotrypsin-like activity is increased in soleus muscle after life-long CR (43). Thus, investigations suggest that an increased capacity of removal of damaged proteins occurs in restricted animals. However, if different types of oxidative damage have different impacts on the life span extension effect of CR, it is also plausible that the different mechanisms degrading/repairing oxidative damage vary in their impact on longevity. If oxidative damage to mtDNA plays a main role in determination of the aging rate, the repair systems of mtDNA could also have a key role in the life span extension effect of CR and should be studied in detail. Recent investigations have found that the main pathway repairing oxidative damage in mtDNA (base excision repair) is down-regulated in brain and kidney mitochondria, whereas no changes are detected in liver mitochondria during caloric restriction (44). Those results agree with the idea that rates of repair of endogenous damage to mtDNA would be lower in restricted and long-lived animals, because their rates of free radical attack on mtDNA are also lower (30). In fact, it has been shown that long-lived animals show lower rates of mitochondrial H2O2 generation than short-lived ones (45). Decreases in mitochondrial free radical production have been also widely observed in different tissues of restricted rodents. This effect of CR on mitochondrial free radical generation is better detected after long-term implementation (Table 2). When animals were subjected to short- or medium-term CR, a reduction or no change has been reported depending on the tissue and the study (Table 3).
Hormonal Pathways and CR Effects
Physiological changes in primates and rodents undergoing CR have been extensively described, including alterations in hormonal pathways. Reduced body size, lower body temperature, and decreases in plasma GH and IGF-I levels have been reported. Restricted animals also show lower levels of glucose and insulin in plasma along with an increased sensitivity to insulin in relation to ad libitum-fed animals. Glucocorticoid levels are enhanced, and thyroid hormones are somewhat reduced. There are also some effects on reproductive development, i.e. delay in sexual maturation and decrease in fertility.
Particularly, the modification of the insulin/IGF-I signaling pathway in CR animals has been considered of special interest, because this highly conserved system has been proposed to regulate longevity in many animals from nematodes to mammals (46). Loss of function mutation of single genes in that pathway extends the maximum life span in mice (47), and these mutant mice show some physiological characteristics similar to those of CR rodents. However, it has been reported that CR extends the maximum life span of one of those mutant mice, Ames dwarfs (48), suggesting that the pathways responsible for increasing longevity can be different in the mutants and the restricted animals. Recently, various investigations have been performed to clarify the effect of insulin-like signaling in oxidative stress during caloric restriction. Some of those studies indicate that insulin supplementation can reverse the effects of CR on mitochondrial free radical production in liver (49) as well as on lipid peroxidation in liver and heart (50), suggesting that changes in restricted animals could be due at least in part to lower insulin levels and altered insulin signaling. However, another investigation studying the effects of insulin and GH treatment on various oxidative stress-related parameters in both CR and control rats (51) shows that both prooxidant and protective effects occur depending on the tissue and the particular parameter measured. Consequently, the idea that insulin-like signaling controls mitochondrial oxidative stress in caloric restriction cannot be generalized at present. On the other hand, in Caenorhabditis elegans, the life span extension effect of CR seems to be independent of the insulin/IGF-I signaling pathway (52).
The increment in glucocorticoid levels during CR has been proposed to have an important role in the beneficial effects of CR (53). It has been hypothesized that the hypothalamic-pituitary-adrenal glucocorticoid system plays a protective role against the moderate stress of food restriction (54), and neuroprotective properties have been described for increased glucocorticoids in restricted animals (55). However, additional studies are needed to clarify whether glucocorticoids are involved in the control of mitochondrial oxidative stress during CR.
Conclusions
In summary, investigations performed during the last years in rodents clearly show that long-term reduction in caloric intake decreases the levels of oxidative damage to cellular macromolecules. Among them, mtDNA has been proposed to play an important role in the aging process, and various studies have shown that CR decreases both mitochondrial free radical generation and oxidative damage to mtDNA. The available studies suggest that the lessening of oxidative damage to mtDNA during CR is mainly due to decreases in the rate of generation of endogenous damage (mitochondrial free radical generation), rather than to greater scavenging or repair of the damage already inflicted. Whether this reduction in mitochondrial free radical generation is hormonally controlled has not been clarified, and additional studies are needed.
Footnotes
The experiments from Dr. Barja’s laboratory described in this article were supported by grants from the National Research Foundation of the Spanish Ministry of Health (no. 99/1049) and the Spanish Ministry of Science and Technology (SAF 2002-01635).
Abbreviations: CR, Caloric restriction; mtDNA, mitochondrial DNA.
References
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Abstract
Reduction of caloric intake without malnutrition is one of the most consistent experimental interventions that increases mean and maximum life spans in different species. For over 70 yr, caloric restriction has been studied, and during the last years the number of investigations on such nutritional intervention and aging has dramatically increased. Because caloric restriction decreases the aging rate, it constitutes an excellent approach to better understand the mechanisms underlying the aging process. Various investigations have reported reductions in steady-state oxidative damage to proteins, lipids, and DNA in animals subjected to restricted caloric intake. Most interestingly, several investigations have reported that these decreases in oxidative damage are related to a lowering of mitochondrial free radical generation rate in various tissues of the restricted animals. Thus, similar to what has been described for long-lived animals in comparative studies, a decrease in mitochondrial free radical generation has been suggested to be one of the main determinants of the extended life span observed in restricted animals. In this study we review recent reports of caloric restriction and longevity, focusing on mitochondrial oxidative stress and the proposed mechanisms leading to an extended longevity in calorie-restricted animals.
Introduction
RESEARCH ON CALORIC restriction (CR) and longevity has noticeably increased in the last 5 yr. During that period, the number of published reports was 30-fold higher than in the 1990s, denoting that caloric restriction currently is a widespread approach to aging research. Because it has been studied for more than 70 yr since the first report of its effect on life span (1), CR is a well-known nutritional intervention, and its beneficial effects on longevity have been described in detail. Although getting older leads to a decline in maximum functional capacities, caloric restriction decelerates such declines. Animals under a CR regimen (without malnutrition) maintain most physiological functions in a youthful state at more advanced ages. CR also retards age-related diseases, such as cardiomyopathy, nephropathy, diabetes, hypertension-related diseases, and neoplastic processes (2, 3). The beneficial effects of CR can be observed not only when initiated at a young age, but also in adulthood. Although it has been reported that CR could be ineffective or even detrimental if started late in life (4), various investigations show that positive effects can also be observed when caloric restriction is started in middle age or later (5, 6). Interest in caloric restriction has increased since investigations in nonhuman primates started, and positive effects on long-lived mammals became clearer. Although full longevity data are not yet available, ongoing studies suggest that the CR-related reduction in the aging rate reported in rodents and other short-lived species could also take place in primates (7) and thus possibly in humans.
It is currently believed that the positive effects of CR depend on the reduced intake of calories themselves (8), although it has been reported that changes in the proportion of the main dietary components can also modulate longevity (9). For instance, restriction of protein intake significantly increased mean and maximum longevities in different rat strains (10, 11, 12). Furthermore, when animals were fed a diet restricted in the essential amino acid methionine, mean and maximum life spans were enhanced as well (13). More recently, we have observed that protein restriction without strong caloric restriction has similar effects on the mitochondrial free radical generation rate and mitochondrial DNA (mtDNA) oxidative damage in rat liver as the reduction of the total intake of calories (14). All these data are consistent with the possibility that part of the effects on the aging rate induced by CR could be due to the decreased intake of particular components of the diet, such as proteins.
Caloric Restriction and Mitochondrial Oxidative Stress
Although the fundamental mechanisms of aging are still unclear, a growing body of evidence involves mitochondria and the continuous generation of free radicals at the mitochondrial inner membrane. The mitochondrial free radical theory of aging (15) currently receives extensive support from experimental investigations (16, 17, 18, 19, 20). The connection between caloric restriction and the mitochondrial free radical theory of aging has been brought about by numerous investigations describing a lower mitochondrial free radical generation rate in restricted animals compared with ad libitum-fed animals (19, 20).
Since it was proposed that CR worked, at least in part, by decreasing oxidative stress (19), several studies have shown that moderate caloric restriction leads to lower oxidative damage to cellular macromolecules. The effect of caloric restriction on protein and DNA oxidative damage has been extensively investigated in the last decades, because both macromolecules have been proposed to play key roles in the aging process. Lipids and the degree of fatty acid unsaturation have been also proposed to play an important role in longevity (21). However, studies of the degree of fatty acid unsaturation of mitochondria have shown no changes in heart from Wistar rats (22, 23) when caloric restriction was extended up to 12 months. Only after life-long CR were decreases in the unsaturation index observed in liver mitochondria of Brown-Norway rats (24) and heart mitochondria of Fischer 344 rats (25).
Regarding protein damage, several studies have reported that the levels of protein carbonyls (26, 27, 28) and dityrosine cross-links (29) are lower in CR animals than in ad libitum-fed ones. Moreover, restricted animals exhibit slower accumulation of those markers with age than fully fed animals. However, protein carbonyls are not a single defined chemical entity, and the effect of CR has been shown to diverge depending on the selected marker. Thus, when more specific protein markers, such as advanced Maillard products or specific carbonyl modifications (glutamic and aminoadipic semialdehydes), were recently investigated in heart (22, 23) and liver (24) rat mitochondria, it was found that the reduction in particular levels of protein oxidative damage after CR depends on implementation time and is tissue specific. Hence, although in heart mitochondria of restricted Wistar rats, a general reduction in protein oxidative damage was observed after 4 and 12 months, but not after 6 wk (22, 23), in liver mitochondria from Brown-Norway rats, a minor reduction in the levels of protein oxidative damage was detected only after life-long CR (24).
Different types of oxidative damage could differently influence the lower aging rate of restricted animals. Whereas damage to lipids and proteins is continuously repaired by cellular turnover using the information coded in the DNA, once a postmitotic cell loses all copies of a given gene, it cannot recuperate its information content, because such information flows from DNA to proteins, not in the reverse direction. Mitochondrial DNA has been suggested to play a determinant role in the aging process (16, 18, 30). Similarly to long-lived animals, CR rodents show a slower aging rate and extended longevity due in part to a reduced mitochondrial free radical production and a slower long-term accumulation of mutations in mtDNA (20).
In accordance with that, many investigations have consistently reported reduced levels of mutagenic oxidative modifications in mtDNA after long-term CR (31, 32, 33, 34, 35, 36, 37). To better characterize the relation among CR, mtDNA oxidative damage, and mitochondrial free radical generation, it is desirable to investigate them simultaneously. A series of studies was performed in which both parameters were measured in parallel in restricted and ad libitum-fed Wistar rats. These investigations showed that long-term CR decreased the rate of mitochondrial H2O2 production and mtDNA oxidative damage in rat liver (35), heart (34), skeletal muscle (36), and brain (37). Furthermore, the quantitative reduction of mtDNA oxidative damage was strikingly similar to that found for mitochondrial free radical generation in all cases (Table 1).
Eating Less, Living Longer: Underlying Mechanisms
As described above, one of the most consistent effects of CR is the reduction in oxidative damage in animals subjected to moderate CR, but what mechanisms cause such reductions Although various potential mechanisms could explain the protective effect of CR on oxidative damage, including an increased capacity to degrade/repair modified macromolecules or an increase in antioxidant defenses, a reduction in the rate of mitochondrial free radical generation seems the most likely candidate.
Concerning scavenger activities, results of different studies do not support an induction of antioxidant activity in CR animals. Increases in such activities were described in some CR models (38), but later studies in rat liver (39) and heart (40) and mouse skeletal muscle (26), brain, heart, and kidney (32) failed to describe any clear-cut overall pattern of CR-related changes in antioxidant defenses, ruling out antioxidants as determinants of the lower oxidative damage observed in CR animals. The attenuation of oxidative damage modifications in CR may also result from an enhancement of the rate of degradation/repair of damaged macromolecules. Supporting that idea, increases in protein synthesis and turnover have been described in restricted rodents (41) as well as increases in the rate of protein catabolism in mouse liver (42). More recently, it has been reported that peptidylglutamyl-peptide hydrolase activity of the 20S proteasome is increased in the heart of rats subjected to short-term CR (40), whereas the chymotrypsin-like activity is increased in soleus muscle after life-long CR (43). Thus, investigations suggest that an increased capacity of removal of damaged proteins occurs in restricted animals. However, if different types of oxidative damage have different impacts on the life span extension effect of CR, it is also plausible that the different mechanisms degrading/repairing oxidative damage vary in their impact on longevity. If oxidative damage to mtDNA plays a main role in determination of the aging rate, the repair systems of mtDNA could also have a key role in the life span extension effect of CR and should be studied in detail. Recent investigations have found that the main pathway repairing oxidative damage in mtDNA (base excision repair) is down-regulated in brain and kidney mitochondria, whereas no changes are detected in liver mitochondria during caloric restriction (44). Those results agree with the idea that rates of repair of endogenous damage to mtDNA would be lower in restricted and long-lived animals, because their rates of free radical attack on mtDNA are also lower (30). In fact, it has been shown that long-lived animals show lower rates of mitochondrial H2O2 generation than short-lived ones (45). Decreases in mitochondrial free radical production have been also widely observed in different tissues of restricted rodents. This effect of CR on mitochondrial free radical generation is better detected after long-term implementation (Table 2). When animals were subjected to short- or medium-term CR, a reduction or no change has been reported depending on the tissue and the study (Table 3).
Hormonal Pathways and CR Effects
Physiological changes in primates and rodents undergoing CR have been extensively described, including alterations in hormonal pathways. Reduced body size, lower body temperature, and decreases in plasma GH and IGF-I levels have been reported. Restricted animals also show lower levels of glucose and insulin in plasma along with an increased sensitivity to insulin in relation to ad libitum-fed animals. Glucocorticoid levels are enhanced, and thyroid hormones are somewhat reduced. There are also some effects on reproductive development, i.e. delay in sexual maturation and decrease in fertility.
Particularly, the modification of the insulin/IGF-I signaling pathway in CR animals has been considered of special interest, because this highly conserved system has been proposed to regulate longevity in many animals from nematodes to mammals (46). Loss of function mutation of single genes in that pathway extends the maximum life span in mice (47), and these mutant mice show some physiological characteristics similar to those of CR rodents. However, it has been reported that CR extends the maximum life span of one of those mutant mice, Ames dwarfs (48), suggesting that the pathways responsible for increasing longevity can be different in the mutants and the restricted animals. Recently, various investigations have been performed to clarify the effect of insulin-like signaling in oxidative stress during caloric restriction. Some of those studies indicate that insulin supplementation can reverse the effects of CR on mitochondrial free radical production in liver (49) as well as on lipid peroxidation in liver and heart (50), suggesting that changes in restricted animals could be due at least in part to lower insulin levels and altered insulin signaling. However, another investigation studying the effects of insulin and GH treatment on various oxidative stress-related parameters in both CR and control rats (51) shows that both prooxidant and protective effects occur depending on the tissue and the particular parameter measured. Consequently, the idea that insulin-like signaling controls mitochondrial oxidative stress in caloric restriction cannot be generalized at present. On the other hand, in Caenorhabditis elegans, the life span extension effect of CR seems to be independent of the insulin/IGF-I signaling pathway (52).
The increment in glucocorticoid levels during CR has been proposed to have an important role in the beneficial effects of CR (53). It has been hypothesized that the hypothalamic-pituitary-adrenal glucocorticoid system plays a protective role against the moderate stress of food restriction (54), and neuroprotective properties have been described for increased glucocorticoids in restricted animals (55). However, additional studies are needed to clarify whether glucocorticoids are involved in the control of mitochondrial oxidative stress during CR.
Conclusions
In summary, investigations performed during the last years in rodents clearly show that long-term reduction in caloric intake decreases the levels of oxidative damage to cellular macromolecules. Among them, mtDNA has been proposed to play an important role in the aging process, and various studies have shown that CR decreases both mitochondrial free radical generation and oxidative damage to mtDNA. The available studies suggest that the lessening of oxidative damage to mtDNA during CR is mainly due to decreases in the rate of generation of endogenous damage (mitochondrial free radical generation), rather than to greater scavenging or repair of the damage already inflicted. Whether this reduction in mitochondrial free radical generation is hormonally controlled has not been clarified, and additional studies are needed.
Footnotes
The experiments from Dr. Barja’s laboratory described in this article were supported by grants from the National Research Foundation of the Spanish Ministry of Health (no. 99/1049) and the Spanish Ministry of Science and Technology (SAF 2002-01635).
Abbreviations: CR, Caloric restriction; mtDNA, mitochondrial DNA.
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