Calorie restriction, oxidative stress and longevity

Studies on the relationship between oxidative stress and ageing in different vertebrate species and in calorie-restricted animals are reviewed. Endogenous antioxidants inversely correlate with maximum longevity in animal species and experiments modifying levels of these antioxidants can increase survival and mean life span but not maximum life span (MLSP). The available evidence shows that long-living vertebrates consistently have low rates of mitochondrial free radical generation, as well as a low grade of fatty acid unsaturation on cellular membranes, which are two crucial factors determining their ageing rate. Oxidative damage to mitochondrial DNA is also lower in long-living vertebrates than in short-living vertebrates. Calorie restriction, the best described experimental strategy that consistently increases mean and maximum life span, also decreases mitochondrial reactive oxygen species (ROS) generation and oxidative damage to mitochondrial DNA. Recent data indicate that the decrease in mitochondrial ROS generation is due to protein restriction rather than to calorie restriction, and more specifically to dietary methionine restriction. Greater longevity would be partly achieved by a low rate of endogenous oxidative damage generation, but also by a macromolecular composition highly resistant to oxidative modification, as is the case for lipids and proteins.     

Aging in vertebrates,and the effect of caloric restriction: a mitochondrial free radical production–DNA damage mechanism?

Oxygen is toxic to aerobic animals because it is univalently reduced inside cells to oxygen free radicals. Studies dealing with the relationship between oxidative stress and aging in different vertebrate species and in caloric-restricted rodents are discussed in this review. Healthy tissues mainly produce reactive oxygen species (ROS) at mitochondria. These ROS can damage cellular lipids, proteins and, most importantly, DNA. Although antioxidants help to control this oxidative stress in cells in general, they do not decrease the rate of aging, because their concentrations are lower in long- than in short-lived animals and because increasing antioxidant levels does not increase vertebrate maximum longevity. However, long-lived homeothermic vertebrates consistently have lower rates of mitochondrial ROS production and lower levels of steady-state oxidative damage in their mitochondrial DNA than short-lived ones. Caloric-restricted rodents also show lower levels of these two key parameters than controls fed ad libitum. The decrease in mitochondrial ROS generation of the restricted animals has been recently localized at complex I and the mechanism involved is related to the degree of electronic reduction of the complex I ROS generator. Strikingly, the same site and mechanism have been found when comparing a long- with a short-lived animal species. It is suggested that a low rate of mitochondrial ROS generation extends lifespan both in long-lived and in caloric-restricted animals by determining the rate of oxidative attack and accumulation of somatic mutations in mitochondrial DNA.     

The rate of free radical production as a determinant of the rate of aging: evidence from the comparative approach

The relationship of oxidative stress with maximum life span (MLSP) in different vertebrate species is reviewed. In all animal groups the endogenous levels of enzymatic and non-enzymatic antioxidants in tissues negatively correlate with MLSP and the most longevous animals studied in each group, pigeon or man, show the minimum levels of antioxidants. A possible evolutionary reason for this is that longevous animals produce oxygen radicals at a low rate. This has been analysed at the place where more than 90% of oxygen is consumed in the cell, the mitochondria. All available work agrees that, across species, the longer the life span, the lower the rate of mitochondrial oxygen radical production. This is true even in animal groups that do not conform to the rate of living theory of aging, such as birds. Birds have low rates of mitochondrial oxygen radical production, frequently due to a low free radical leak in their respiratory chain. Possibly the low rate of mitochondrial oxygen radical production of longevous species can decrease oxidative damage at targets important for aging (like mitochondrial DNA) that are situated near the places of free radical generation. A low rate of free radical production can contribute to a low aging rate both in animals that conform to the rate of living (metabolic) theory of aging and in animals with exceptional longevities, like birds and primates. Available research indicates there are at least two main characteristics of longevous species: a high rate of DNA repair together with a low rate of free radical production near DNA. Simultaneous consideration of these two characteristics can explain part of the quantitative differences in longevity between animal species. Accepted: 12 December 1997     

Mitochondrial Oxygen Radical Generation and Leak: Sites of Production in States 4 and 3, Organ Specificity, and Relation to Aging and Longevity

Studies in heart and nonsynaptic brain mitochondria from two mammals and three birds showthat complex I generates oxygen radicals in heart and nonsynaptic brain mitochondria in States4 and 3, whereas complex III does it only in heart mitochondria and only in State 4. Theincrease in oxygen consumption during the State 4 to 3 transition is not accompanied by aproportional increase in oxygen radical generation. This will protect mitochondria and tissuesduring bursts of activity. Comparisons between young and old rodents do not show a consistentpattern of variation in mitochondrial oxygen radical production during aging. However, allthe interspecies comparisons performed to date between different mammals, and betweenmammals and birds, agree that animals with high maximum longevities have low rates ofmitochondrial oxygen radical production, irrespective of the value of their basal specificmetabolic rate. The sites and mechanisms allowing this, the recently described low degree ofmembrane fatty acid unsaturation of longevous animals, and their relation to longevity andaging are discussed.     

Lifelong treatment with atenolol decreases membrane fatty acid unsaturation and oxidative stress in heart and skeletal muscle mitochondria and improves immunity and behavior,without changing mice longevity

The membrane fatty acid unsaturation hypothesis of aging and longevity is experimentally tested for the first time in mammals. Lifelong treatment of mice with the β1‐blocker atenolol increased the amount of the extracellular‐signal‐regulated kinase signaling protein and successfully decreased one of the two traits appropriately correlating with animal longevity, the membrane fatty acid unsaturation degree of cardiac and skeletal muscle mitochondria, changing their lipid profile toward that present in much more longer‐lived mammals. This was mainly due to decreases in 22:6n‐3 and increases in 18:1n‐9 fatty acids. The atenolol treatment also lowered visceral adiposity (by 24%), decreased mitochondrial protein oxidative, glycoxidative, and lipoxidative damage in both organs, and lowered oxidative damage in heart mitochondrial DNA. Atenolol also improved various immune (chemotaxis and natural killer activities) and behavioral functions (equilibrium, motor coordination, and muscular vigor). It also totally or partially prevented the aging‐related detrimental changes observed in mitochondrial membrane unsaturation, protein oxidative modifications, and immune and behavioral functions, without changing longevity. The controls reached 3.93 years of age, a substantially higher maximum longevity than the best previously described for this strain (3.0 years). Side effects of the drug could have masked a likely lowering of the endogenous aging rate induced by the decrease in membrane fatty acid unsaturation. We conclude that it is atenolol that failed to increase longevity, and likely not the decrease in membrane unsaturation induced by the drug.     

Protein Restriction Without Strong Caloric Restriction Decreases Mitochondrial Oxygen Radical Production and Oxidative DNA Damage in Rat Liver

Previous studies have shown that caloric restriction decreases mitochondrial oxygen radical production and oxidative DNA damage in rat organs, which can be linked to the slowing of aging rate induced by this regime. These two characteristics are also typical of long-lived animals. However, it has never been investigated if those decreases are linked to the decrease in the intake of calories themselves or to decreases in specific dietary components. In this study the possible role of the dietary protein was investigated. Using semipurified diets, the ingestion of proteins of Wistar rats was decreased by 40% below that of controls while the other dietary components were ingested at the same level as in animals fed ad libitum. After seven weeks in this regime the liver of the protein restricted animals showed 30–40% decreases in mitochondrial production of reactive oxygen species (ROS) and in oxidative damage to nuclear and mitochondrial DNA. The decreases in ROS generation occurred specifically at complex~I. They also occurred without changes in mitochondrial oxygen consumption. Instead, there was a decrease in the percent free radical leak (the percentage of total electron flow leading to ROS generation in the respiratory chain). These results are strikingly similar to those previously obtained after 40% caloric restriction in the liver of Wistar rats. Thus, the results suggest that part of the decrease in aging rate induced by caloric restriction can be due to the decreased intake of proteins acting through decreases in mitochondrial ROS production and oxidative DNA damage. Interestingly, these tissue oxidative stress-linked parameters can be lowered by restricting only the intake of dietary protein, probably a more feasible option than caloric restriction for adult humans.     

Lowered methionine ingestion as responsible for the decrease in rodent mitochondrial oxidative stress in protein and dietary restriction possible implications for humans

Available information indicates that long-lived mammals have low rates of reactive oxygen species (ROS) generation and oxidative damage at their mitochondria. On the other hand, many studies have consistently shown that dietary restriction (DR) in rodents also decreases mitochondrial ROS (mtROS) production and oxidative damage to mitochondrial DNA and proteins. It has been observed that protein restriction also decreases mtROS generation and oxidative stress in rat liver, whereas neither carbohydrate nor lipid restriction change these parameters. This is interesting because protein restriction also increases maximum longevity in rodents (although to a lower extent than DR) and is a much more practicable intervention for humans than DR, whereas neither carbohydrate nor lipid restriction seem to change rodent longevity. Moreover, it has been found that isocaloric methionine restriction also decreases mtROS generation and oxidative stress in rodent tissues, and this manipulation also increases maximum longevity in rats and mice. In addition, excessive dietary methionine also increases mtROS generation in rat liver. These studies suggest that the reduced intake of dietary methionine can be responsible for the decrease in mitochondrial ROS generation and the ensuing oxidative damage that occurs during DR, as well as for part of the increase in maximum longevity induced by this dietary manipulation. In addition, the mean intake of proteins (and thus methionine) of Western human populations is much higher than needed. Therefore, decreasing such levels to the recommended ones has a great potential to lower tissue oxidative stress and to increase healthy life span in humans while avoiding the possible undesirable effects of DR diets.     

Lowered methionine ingestion as responsible for the decrease in rodent mitochondrial oxidative stress in protein and dietary restriction: Possible implications for humans

Available information indicates that long-lived mammals have low rates of reactive oxygen species (ROS) generation and oxidative damage at their mitochondria. On the other hand, many studies have consistently shown that dietary restriction (DR) in rodents also decreases mitochondrial ROS (mtROS) production and oxidative damage to mitochondrial DNA and proteins. It has been observed that protein restriction also decreases mtROS generation and oxidative stress in rat liver, whereas neither carbohydrate nor lipid restriction change these parameters. This is interesting because protein restriction also increases maximum longevity in rodents (although to a lower extent than DR) and is a much more practicable intervention for humans than DR, whereas neither carbohydrate nor lipid restriction seem to change rodent longevity. Moreover, it has been found that isocaloric methionine restriction also decreases mtROS generation and oxidative stress in rodent tissues, and this manipulation also increases maximum longevity in rats and mice. In addition, excessive dietary methionine also increases mtROS generation in rat liver. These studies suggest that the reduced intake of dietary methionine can be responsible for the decrease in mitochondrial ROS generation and the ensuing oxidative damage that occurs during DR, as well as for part of the increase in maximum longevity induced by this dietary manipulation. In addition, the mean intake of proteins (and thus methionine) of Western human populations is much higher than needed. Therefore, decreasing such levels to the recommended ones has a great potential to lower tissue oxidative stress and to increase healthy life span in humans while avoiding the possible undesirable effects of DR diets.     

Independent and additive effects of atenolol and methionine restriction on lowering rat heart mitochondria oxidative stress

A low rate of mitochondrial ROS production (mitROSp) and a low degree of fatty acid unsaturation are characteristic traits of long-lived animals and can be obtained in a single species by methionine restriction (MetR) or atenolol (AT) treatments. However, simultaneous application of both treatments has never been performed. In the present investigation it is shown that MetR lowers mitROSp and complex I content. Both the MetR and the AT treatments lower protein oxidative modification and oxidative damage to mtDNA and the fatty acid unsaturation degree in rat heart mitochondria. The decrease in fatty acid unsaturation seems to be due, at least in part, to decreases in desaturase and elongase activities or peroxisomal β-oxidation. Furthermore, the phosphorylation of extracellular signal-regulated kinase (ERK) was stimulated by MetR and AT. The decrease in membrane fatty acid unsaturation and protein oxidation, and the changes in fatty acids and p-ERK showed additive effects of both treatments. In addition, the increase in mitROSp induced by AT observed in the present investigation was totally avoided with the combined MetR + AT treatment. It is concluded that the simultaneous treatment with MetR plus atenolol is more beneficial than either single treatment alone to lower oxidative stress in rat heart mitochondria, analogously to what has been reported in long-lived animal species.     

Low Mitochondrial Free Radical Production Per Unit O2 Consumption Can Explain the Simultaneous Presence of High Longevity and High Aerobic Metabolic Rate in Birds

Birds are unique since they can combine a high rate of oxygen consumption at rest with a high maximum life span (MLSP). The reasons for this capacity are unknown. A similar situation is present in primates including humans which show MLSPs higher than predicted from their rates of O₂ consumption. In this work rates of oxygen radical production and O₂ consumption by mitochondria were compared between adult male rats (MLSP = 4 years) and adult pigeons (MLSP = 35 years), animals of similar body size. Both the O₂ consumption of the whole animal at rest and the O₂ consumption of brain, lung and liver mitochondria were higher in the pigeon than in the rat. Nevertheless, mitochondrial free radical production was 2-4 times lower in pigeon than in rat tissues. This is possible because pigeon mitochondria show a rate of free radical production per unit O₂ consumed one order of magnitude lower than rat mitochondria: bird mitochondria show a lower free radical leak at the respiratory chain. This result, described here for the first time, can possibly explain the capacity of birds to simultaneously increase maximum longevity and basal metabolic rate. It also suggests that the main factor relating oxidative stress to aging and longevity is not the rate of oxygen consumption but the rate of oxygen radical production. Previous inconsistencies of the rate of living theory of aging can be explained by a free radical theory of aging which focuses on the rate of oxygen radical production and on local damage to targets relevant for aging situated near the places where free radicals are continuously generated.     

Effect of methionine dietary supplementation on mitochondrial oxygen radical generation and oxidative DNA damage in rat liver and heart

Methionine restriction without energy restriction increases, like caloric restriction, maximum longevity in rodents. Previous studies have shown that methionine restriction strongly decreases mitochondrial reactive oxygen species (ROS) production and oxidative damage to mitochondrial DNA, lowers membrane unsaturation, and decreases five different markers of protein oxidation in rat heart and liver mitochondria. It is unknown whether methionine supplementation in the diet can induce opposite changes, which is also interesting because excessive dietary methionine is hepatotoxic and induces cardiovascular alterations. Because the detailed mechanisms of methionine-related hepatotoxicity and cardiovascular toxicity are poorly understood and today many Western human populations consume levels of dietary protein (and thus, methionine) 2–3.3 fold higher than the average adult requirement, in the present experiment we analyze the effect of a methionine supplemented diet on mitochondrial ROS production and oxidative damage in the rat liver and heart mitochondria. In this investigation male Wistar rats were fed either a L-methionine-supplemented (2.5 g/100 g) diet without changing any other dietary components or a control (0.86 g/100 g) diet for 7 weeks. It was found that methionine supplementation increased mitochondrial ROS generation and percent free radical leak in rat liver mitochondria but not in rat heart. In agreement with these data oxidative damage to mitochondrial DNA increased only in rat liver, but no changes were observed in five different markers of protein oxidation in both organs. The content of mitochondrial respiratory chain complexes and AIF (apoptosis inducing factor) did not change after the dietary supplementation while fatty acid unsaturation decreased. Methionine, S-AdenosylMethionine and S-AdenosylHomocysteine concentration increased in both organs in the supplemented group. These results show that methionine supplementation in the diet specifically increases mitochondrial ROS production and mitochondrial DNA oxidative damage in rat liver mitochondria offering a plausible mechanism for its hepatotoxicity.     

Comparative studies of oxidative stress and mitochondrial function in aging

The oxidative stress theory and its correlate the mitochondrial theory of aging are among the most studied and widely accepted of all hypotheses of the mechanism of aging. To date, most of the supporting evidence for these theories has come from investigations using common model organisms such as Caenorhabditis elegans, Drosophila melanogaster, and laboratory rodents. However, comparative data from a wide range of endotherms provide equivocal support as to whether oxidative stress is merely a correlate, rather than a determinant, of species' maximum lifespan. The great majority of studies in this area have been devoted to the relationship between reactive oxygen species and maximal longevity in young adult organisms, with little emphasis on mitochondrial respiratory efficiency, age-related alterations in mitochondrial physiology or oxidative damage. The advantage of studying a broader spectrum of species is the broad range of virtually every biological phenotype/trait, such as lifespan, body weight and metabolic rate. Here we summarize the results from a number of comparative studies in an effort to correlate oxidant production and oxidative damage among many species with their maximal lifespan and briefly discuss the pitfalls and limitations. Based on current information, it is not possible to accept or dispute the oxidative stress theory of aging, nor can we exclude the possibility that private mechanisms might offer an explanation for the longevity of exceptionally long-lived animal models. Thus, there is need for more thorough and controlled investigations with more unconventional animal models for a deeper understanding of the role of oxidative stress in longevity.     

Prevention of flight activity prolongs the life span of the housefly, Musca domestica, and attenuates the age-associated oxidative damamge to specific mitochondrial proteins

The purpose of this study was to explore the mechanisms by which oxidative stress affects the aging process. The hypothesis that the rate of accumulation of oxidative damage to specific mitochondrial proteins is linked to the life expectancy of animals was tested in the housefly. The rate of oxygen consumption and life expectancy of the flies were experimentally altered by confining the flies in small jars, where they were unable to fly. Prevention of flight activity decreased the rate of oxygen utilization of flies and almost tripled their life span as compared to those permitted to fly. Rate of mitochondrial H(2)O(2) generation at various ages was lower in the low activity flies than in the high activity flies. Oxidative damage to mitochondrial proteins, adenine nucelotide translocase, and aconitase, detected as carbonyl modifications, was attenuated; and the loss in their functional activity occurring with age was retarded in the long-lived low activity flies as compared to the short-lived high activity flies. The two proteins were previously identified to be the only mitochondrial proteins exhibiting age-related increases in carbonylation. Results support the hypothesis that accrual of oxidative damage to specific protein targets and the consequent loss of their function may constitute a mechanism by which oxidative stress controls the aging process.     

Forty percent methionine restriction lowers DNA methylation, complex I ROS generation, and oxidative damage to mtDNA and mitochondrial proteins in rat heart

Methionine dietary restriction (MetR), like dietary restriction (DR), increases rodent maximum longevity. However, the mechanism responsible for the retardation of aging with MetR is still not entirely known. As DR decreases oxidative damage and mitochondrial free radical production, it is plausible to hypothesize that a decrease in oxidative stress is the mechanism for longevity extension with MetR. In the present investigation male Wistar rats were subjected to isocaloric 40% MetR during 7 weeks. It was found that 40% MetR decreases heart mitochondrial ROS production at complex I during forward electron flow, lowers oxidative damage to mitochondrial DNA and proteins, and decreases the degree of methylation of genomic DNA. No significant changes occurred for mitochondrial oxygen consumption, the amounts of the four respiratory complexes (I to IV), and the mitochondrial protein apoptosis-inducing factor (AIF). These results indicate that methionine can be the dietary factor responsible for the decrease in mitochondrial ROS generation and oxidative stress, and likely for part of the increase in longevity, that takes place during DR. They also highlight some of the mechanisms involved in the generation of these beneficial effects.     

Effects of fasting on oxidative stress in rat liver mitochondria

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O₂ consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N^epsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.     

Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals.

DNA damage is considered of paramount importance in aging. Among causes of this damage, free radical attack, particularly from mitochondrial origin, is receiving special attention. If oxidative damage to DNA is involved in aging, long-lived animals (which age slowly) should show lower levels of markers of this kind of damage than short-lived ones. However, this possibility has not heretofore been investigated. In this study, steady-state levels of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) referred to deoxyguanosine (dG) were measured by high performance liquid chromatography (HPLC) in the mitochondrial (mtDNA) and nuclear (nDNA) DNA from the heart of eight and the brain of six mammalian species ranging in maximum life span (MLSP) from 3.5 to 46 years. Exactly the same digestion of DNA to deoxynucleosides and HPLC protocols was used for mtDNA and nDNA. Significantly higher (three- to ninefold) 8-oxodG/dG values were found in mtDNA than in nDNA in all the species studied in both tissues. 8-oxodG/dG in nDNA did not correlate with MLSP across species either in the heart (r=-0.68; P<0.06) or brain (r = 0.53; P<0.27). However, 8-oxodG/dG in mtDNA was inversely correlated with MLSP both in heart (r=-0.92; P<0.001) and brain (r=-0.88; P<0.016) tissues following the power function y = a(.)x(b), where y is 8-oxodG/dG and x is the MLSP. This agrees with the consistent observation that mitochondrial free radical generation is also lower in long-lived than in short-lived species. The results obtained agree with the notion that oxygen radicals of mitochondrial origin oxidatively damage mtDNA in a way related to the aging rate of each species.-Barja, G., Herrero, A. Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals.     

Mitochondrial oxidative stress, aging and caloric restriction: the protein and methionine connection

Caloric restriction (CR) decreases aging rate and mitochondrial ROS (MitROS) production and oxidative stress in rat postmitotic tissues. Low levels of these parameters are also typical traits of long-lived mammals and birds. However, it is not known what dietary components are responsible for these changes during CR. It was recently observed that 40% protein restriction without strong CR also decreases MitROS generation and oxidative stress. This is interesting because protein restriction also increases maximum longevity (although to a lower extent than CR) and is a much more practicable intervention for humans than CR. Moreover, it was recently found that 80% methionine restriction substituting it for l-glutamate in the diet also decreases MitROS generation in rat liver. Thus, methionine restriction seems to be responsible for the decrease in ROS production observed in caloric restriction. This is interesting because it is known that exactly that procedure of methionine restriction also increases maximum longevity. Moreover, recent data show that methionine levels in tissue proteins negatively correlate with maximum longevity in mammals and birds. All these suggest that lowering of methionine levels is involved in the control of mitochondrial oxidative stress and vertebrate longevity by at least two different mechanisms: decreasing the sensitivity of proteins to oxidative damage, and lowering of the rate of ROS generation at mitochondria.     

Effects of fasting on oxidative stress in rat liver mitochondria

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O₂ consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N^epsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.     

Lower levels of F2-isoprostanes in serum and livers of long-lived Ames dwarf mice

F₂-isoprostanes (IsoPs), lipid peroxidation products, are markers that quantitatively measure levels of oxidative stress. IsoP levels increase in tissues and serum of aging animals suggesting an increase in oxidative stress. This supports the Free Radical Theory of Aging, which proposes that elevated levels of reactive oxygen species (ROS) cause macromolecular damage, and is a factor in the age-associated decline in tissue function. Numerous studies have shown that the longevity of long-lived mutant mice correlates with their resistance to oxidative stress. However, although the Ames dwarf (DW) mice show resistance to oxidative stress, it has not been shown that these mice have inherently lower levels of ROS. Our results show that the serum and liver IsoP levels in DW mice are lower at all ages suggesting that the lower levels of endogenous ROS production in DW mice may be a factor in their resistance to oxidative stress and longevity.     

Carbohydrate restriction does not change mitochondrial free radical generation and oxidative DNA damage

Many previous investigations have consistently reported that caloric restriction (40%), which increases maximum longevity, decreases mitochondrial reactive species (ROS) generation and oxidative damage to mitochondrial DNA (mtDNA) in laboratory rodents. These decreases take place in rat liver after only seven weeks of caloric restriction. Moreover, it has been found that seven weeks of 40% protein restriction, independently of caloric restriction, also decrease these two parameters, whereas they are not changed after seven weeks of 40% lipid restriction. This is interesting since it is known that protein restriction can extend longevity in rodents, whereas lipid restriction does not have such effect. However, before concluding that the ameliorating effects of caloric restriction on mitochondrial oxidative stress are due to restriction in protein intake, studies on the third energetic component of the diet, carbohydrates, are needed. In the present study, using semipurified diets, the carbohydrate ingestion of male Wistar rats was decreased by 40% below controls without changing the level of intake of the other dietary components. After seven weeks of treatment the liver mitochondria of the carbohydrate restricted animals did not show changes in the rate of mitochondrial ROS production, mitochondrial oxygen consumption or percent free radical leak with any substrate (complex I- or complex II-linked) studied. In agreement with this, the levels of oxidative damage in hepatic mtDNA and nuclear DNA were not modified in carbohydrate restricted animals. Oxidative damage in mtDNA was one order of magnitude higher than that in nuclear DNA in both dietary groups. These results, together with previous ones, discard lipids and carbohydrates, and indicate that the lowered ingestion of dietary proteins is responsible for the decrease in mitochondrial ROS production and oxidative damage in mtDNA that occurs during caloric restriction.