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.     

Longevity and antioxidant enzymes,non-enzymatic antioxidants and oxidative stress in the vertebrate lung: a comparative study

It has been proposed that antioxidants can be longevity determinants in animals. However, no comprehensive study has been conducted to try to relate free radicals with maximum life span. This study compares the lung tissue of various vertebrate species — amphibia, mammals and birds — showing very different and well known maximum life spans and life energy potentials. The lung antioxidant enzymes superoxide dismutase, catalase, Se-dependent and non-Se-dependent glutathione peroxidases, and glutathione reductase showed significantly negative correlations with maximum life span. The same was observed for the lung antioxidants, reduced glutathione and ascorbate. It is concluded that a generalized decrease in tissue antioxidant capacity is a characteristic of longevous species. It is suggested that a low rate of free radical recycling (free-radical generation and scavenging) can be an important factor involved in the evolution of high maximum animal longevities. A low free-radical production could be responsible for a low rate of damage at critical sites such as mitochondrial DNA.Abbreviations CAT catalase - COX cytochrome oxidase - GPx glutathione peroxidase - GR glutathione reductase - GSH reduced glutathione - GSSG oxidized glutathione - LEP life energy potential - MDA malondialdehyde - MLSP maximum life span - MR metabolic rate - MW molecular weight - PO₂ partial pressure of oxygen - SOD superoxide dismutase - VO₂ basal oxygen consumption     

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.     

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.     

Rate of generation of oxidative stress-related damage and animal longevity

Comparative studies about the relationship between endogenous antioxidant and pro-oxidant factors and maximum longevity of different animal species are reviewed. The majority of studies on antioxidant supplementation indicate that it can increase mean survival without changing maximum longevity. On the other hand, endogenous antioxidants are negatively correlated with maximum longevity. The same is true for the rates of mitochondrial oxygen radical generation, oxidative damage to mitochondrial DNA, and the degree of fatty acid unsaturation of cellular membranes in postmitotic tissues. The lower rate of mitochondrial oxygen radical generation of long-lived animals in relation to that of short-lived ones can be a primary cause of their slow aging rate. This is secondarily complemented in long-lived animals with low rates of lipid peroxidation due to their low degrees of fatty acid unsaturation. These two traits suggest that the rate of generation of endogenous oxidative damage determines, at least in part, the rate of aging in animals.     

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.     

Mitochondrial oxidative stress plays a key role in aging and apoptosis

Harman first suggested in 1972 that mitochondria might be the biological clock in aging, noting that the rate of oxygen consumption should determine the rate of accumulation of mitochondrial damage produced by free radical reactions. Later in 1980 Miquel and coworkers proposed the mitochondrial theory of cell aging. Mitochondria from postmitotic cells use O2 at a high rate, hence releasing oxygen radicals that exceed the cellular antioxidant defences. The key role of mitochondria in cell aging has been outlined by the degeneration induced in cells microinjected with mitochondria isolated from fibroblasts of old rats, especially by the inverse relationship reported between the rate of mitochondrial production of hydroperoxide and the maximum life span of species. An important change in mitochondrial lipid composition is the age-related decrease found in cardiolipin content. The concurrent enhancement of lipid peroxidation and oxidative modification of proteins in mitochondria further increases mutations and oxidative damage to mitochondrial DNA (mtDNA) in the aging process. The respiratory enzymes containing the defective mtDNA-encoded protein subunits may increase the production of reactive oxygen species, which in turn would aggravate the oxidative damage to mitochondria. Moreover, superoxide radicals produced during mitochondrial respiration react with nitric oxide inside mitochondria to yield damaging peroxynitrite. Treatment with certain antioxidants, such as sulphur-containing antioxidants, vitamins C and E, or the Ginkgo biloba extract EGb 761, protects against the age-associated oxidative damage to mtDNA and the oxidation of mitochondrial glutathione. Moreover, the EGb 761 extract also prevents changes in mitochondrial morphology and function associated with aging of the brain and liver.     

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.     

The long life of birds: the rat-pigeon comparison revisited

The most studied comparison of aging and maximum lifespan potential (MLSP) among endotherms involves the 7-fold longevity difference between rats (MLSP 5y) and pigeons (MLSP 35y). A widely accepted theory explaining MLSP differences between species is the oxidative stress theory, which purports that reactive oxygen species (ROS) produced during mitochondrial respiration damage bio-molecules and eventually lead to the breakdown of regulatory systems and consequent death. Previous rat-pigeon studies compared only aspects of the oxidative stress theory and most concluded that the lower mitochondrial superoxide production of pigeons compared to rats was responsible for their much greater longevity. This conclusion is based mainly on data from one tissue (the heart) using one mitochondrial substrate (succinate). Studies on heart mitochondria using pyruvate as a mitochondrial substrate gave contradictory results. We believe the conclusion that birds produce less mitochondrial superoxide than mammals is unwarranted. We have revisited the rat-pigeon comparison in the most comprehensive manner to date. We have measured superoxide production (by heart, skeletal muscle and liver mitochondria), five different antioxidants in plasma, three tissues and mitochondria, membrane fatty acid composition (in seven tissues and three mitochondria), and biomarkers of oxidative damage. The only substantial and consistent difference that we have observed between rats and pigeons is their membrane fatty acid composition, with rats having membranes that are more susceptible to damage. This suggests that, although there was no difference in superoxide production, there is likely a much greater production of lipid-based ROS in the rat. We conclude that the differences in superoxide production reported previously were due to the arbitrary selection of heart muscle to source mitochondria and the provision of succinate. Had mitochondria been harvested from other tissues or other relevant mitochondrial metabolic substrates been used, then very different conclusions regarding differences in oxidative stress would have been reached.     

Ginkgo biloba extract EGb 761 protects against mitochondrial aging in the brain and in the liver.

According to the free radical theory of aging, oxygen-derived free radicals causes the age-associated impairment at the cellular and tissue levels. The mitochondrial theory of aging points to mitochondria, and specially mitochondrial DNA, as the major targets of free radical attack upon aging. Thus, oxidative damage to mtDNA accumulate with age in human and rodent tissues and also is inversely related to maximum life span of mammals. Mitochondrial deficits, such as a decrease in mitochondrial membrane potential, occur upon aging due to oxidative damage. The age-related mitochondrial oxidative stress may be prevented by late onset administration of certain antioxidants, such as Ginkgo biloba extract EGb 761. These antioxidants may also delay the physiological impairment associated with aging.     

ADP-Regulation of Mitochondrial Free Radical Production Is Different with Complex I- or Complex II-Linked Substrates: Implications for the Exercise Paradox and Brain Hypermetabolism

In agreement with classic studies, succinate-supplemented rat and pigeon heart and nonsynaptic brain mitochondrial free radical production is stopped by ADP additions causing the stimulation of respiration from State 4 to State 3. Nevertheless, with Complex I-linked substrates, mitochondria produce free radicals in State 3 at rates similar or somewhat higher than during resting respiration. The absence of sharp increases in free radical production during intense respiration is possible due to strong decreases of free radical leak in State 3. The results indicate that Complex I is the main mitochondrial free radical generator in State 3, adding to its already known important generation of active oxygen species in State 4. The observed rate of mitochondrial free radical production with Complex I-linked substrates in the active State 3 can help to explain two paradoxes: (a) the lack of massive muscle oxidative damage and shortening of life span due to exercise, in spite of up to 23-fold increases of oxygen consumption together with the very low levels of antioxidants present in heart, skeletal muscle, and brain; (b) the presence of some degree of oxidative stress during exercise and hyperactivity in spite of the stop of mitochondrial free radical production by ADP with succinate as substrate.     

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.     

The emerging role of cardiovascular risk factor-induced mitochondrial dysfunction in atherogenesis

An important role in atherogenesis is played by oxidative stress, which may be induced by common risk factors. Mitochondria are both sources and targets of reactive oxygen species, and there is growing evidence that mitochondrial dysfunction may be a relevant intermediate mechanism by which cardiovascular risk factors lead to the formation of vascular lesions. Mitochondrial DNA is probably the most sensitive cellular target of reactive oxygen species. Damage to mitochondrial DNA correlates with the extent of atherosclerosis. Several cardiovascular risk factors are demonstrated causes of mitochondrial damage. Oxidized low density lipoprotein and hyperglycemia may induce the production of reactive oxygen species in mitochondria of macrophages and endothelial cells. Conversely, reactive oxygen species may favor the development of type 2 diabetes mellitus, mainly through the induction of insulin resistance. Similarly - in addition to being a cause of endothelial dysfunction, reactive oxygen species and subsequent mitochondrial dysfunction - hypertension may develop in the presence of mitochondrial DNA mutations. Finally, other risk factors, such as aging, hyperhomocysteinemia and cigarette smoking, are also associated with mitochondrial damage and an increased production of free radicals. So far clinical studies have been unable to demonstrate that antioxidants have any effect on human atherogenesis. Mitochondrial targeted antioxidants might provide more significant results.     

Testing the 'free radical theory of aging' hypothesis: physiological differences in long-lived and short-lived colubrid snakes

We test the 'free radical theory of aging' using six species of colubrid snakes (numerous, widely distributed, non-venomous snakes of the family Colubridae) that exhibit long (> 15 years) or short (< 10 years) lifespans. Because the 'rate of living theory' predicts metabolic rates to be correlated with rates of aging and oxidative damage results from normal metabolic processes we sought to answer whether physiological parameters and locomotor performance (which is a good predictor of survival in juvenile snakes) mirrored the evolution of lifespans in these colubrid snakes. We measured whole animal metabolic rate (oxygen consumption Vo2), locomotor performance, cellular metabolic rate (mitochondrial oxygen consumption), and oxidative stress potential (hydrogen peroxide production by mitochondria). Longer-lived colubrid snakes have greater locomotor performance and reduced hydrogen peroxide production than short-lived species, while whole animal metabolic rates and mitochondrial efficiency did not differ with lifespan. We present the first measures testing the 'free radical theory of aging' using reptilian species as model organisms. Using reptiles with different lifespans as model organisms should provide greater insight into mechanisms of aging.     

Targeted expression of the human uncoupling protein 2 (hUCP2) to adult neurons extends life span in the fly

The oxidative stress hypothesis of aging predicts that a reduction in the generation of mitochondrial reactive oxygen species (ROS) will decrease oxidative damage and extend life span. Increasing mitochondrial proton leak-dependent state 4 respiration by increasing mitochondrial uncoupling is an intervention postulated to decrease mitochondrial ROS production. When human UCP2 (hUCP2) is targeted to the mitochondria of adult fly neurons, we find an increase in state 4 respiration, a decrease in ROS production, a decrease in oxidative damage, heightened resistance to the free radical generator paraquat, and an extension in life span without compromising fertility or physical activity. Our results demonstrate that neuronal-specific expression of hUCP2 in adult flies decreases cellular oxidative damage and is sufficient to extend life span.     

Mitochondrial bioenergetics in aging

Mitochondria are strongly involved in the production of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. The mitochondrial theory of aging considers somatic mutations of mitochondrial DNA induced by oxygen radicals as the primary cause of energy decline; experimentally, complex I appears to be mostly affected and to become strongly rate limiting for electron transfer. Mitochondrial bioenergetics is also deranged in human platelets upon aging, as shown by the decreased Pasteur effect (enhancement of lactate production by respiratory chain inhibition). Cells counteract oxidative stress by antioxidants; among lipophilic antioxidants, coenzyme Q is the only one of endogenous biosynthesis. Exogenous coenzyme Q, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases.     

Dietary Restriction at Old Age Lowers Mitochondrial Oxygen Radical Production and Leak at Complex I and Oxidative DNA Damage in Rat Brain

Previous studies in mammalian models indicate that the rate of mitochondrial reactive oxygen species ROS production and the ensuing modification of mitochondrial DNA (mtDNA) link oxidative stress to aging rate. However, there is scarce information concerning this in relation to caloric restriction (CR) in the brain, an organ of maximum relevance for ageing. Furthermore, it has never been studied if CR started late in life can improve those oxidative stress-related parameters. In this investigation, rats were subjected during 1 year to 40% CR starting at 24 months of age. This protocol of CR significantly decreased the rate of mitochondrial H₂O₂ production (by 24%) and oxidative damage to mtDNA (by 23%) in the brain below the level of both old and young ad libitum-fed animals. In agreement with the progressive character of aging, the rate of H₂O₂ production of brain mitochondria stayed constant with age. Oxidative damage to nuclear DNA increased with age and this increase was fully reversed by CR to the level of the young controls. The decrease in ROS production induced by CR was localized at Complex I and occurred without changes in oxygen consumption. Instead, the efficiency of brain mitochondria to avoid electron leak to oxygen at Complex I was increased by CR. The mechanism involved in that increase in efficiency was related to the degree of electronic reduction of the Complex I generator. The results agree with the idea that CR decreases aging rate in part by lowering the rate of free radical generation of mitochondria in the brain.     

Mitochondrial ROS--radical detoxification, mediated by protein kinase D

The mitochondrial electron transport chain is the major source for the production of oxygen radicals. Mitochondria-generated reactive oxygen species (mROS) have been implicated in decreasing the life span and contributing to age-related diseases (known as the free radical theory of aging). Recently, the serine/threonine kinase protein kinase D1 (PKD1) was identified as a mitochondrial sensor for oxidative stress. mROS-activated PKD regulates a radical-sensing signaling pathway, which relays mROS production to the induction of nuclear genes that mediate cellular detoxification and survival. This PKD regulated signaling pathway is the first known mitochondria located and mitochondrially regulated antioxidant system that protects these organelles and cells from oxidative stress-mediated damage or cell death. The identification of this and further intracellular protective signaling pathways provides an opportunity to manipulate the effects of mROS, and might provide the key to targeting aging effects and age-related diseases that have been linked to mitochondrial dysfunctions.     

Differential effects of thyroid status on regional H2O2 production in slow- and fast-twitch muscle of ducklings

Birds seem to employ powerful physiological strategies to curb the harmful effects of reactive oxygen species (ROS) because they generally live longer than predicted by the free radical theory of aging. However, little is known about the physiological mechanisms that confer protection to birds against excessive ROS generation. Hence, we investigated the ability of birds to control mitochondrial ROS generation during physiologically stressful periods. In our study, we analyzed the relationship between the thyroid status and the function of intermyofibrillar and subsarcolemmal mitochondria located in glycolytic and oxidative muscles of ducklings. We found that the intermyofibrillar mitochondria of both glycolytic and oxidative muscles down regulate ROS production when plasma T₃ levels rise. The intermyofibrillar mitochondria of the gastrocnemius muscle (an oxidative muscle) produced less ROS and were more sensitive than the pectoralis muscle (a glycolytic muscle) to changes in plasma T₃. Such differences in the ROS production by glycolytic and oxidative muscles were associated with differences in the membrane proton permeability and in the rate of free radical leakage within the respiratory chain. This is the first evidence which shows that in birds, the amount of ROS that the mitochondria release is dependent on: (1) their location within the muscle; (2) the type of muscle (glycolytic or oxidative) and (3) on the thyroid status. Reducing muscle mitochondrial ROS generation might be an important mechanism in birds to limit oxidative damage during periods of physiological stress.     

Mitochondria, oxidative stress and aging

In the eighties, Miquel and Fleming suggested that mitochondria play a key role in cellular aging. Mitochondria, and specially mitochondrial DNA (mtDNA), are major targets of free radical attack. At present, it is well established that mitochondrial deficits accumulate upon aging due to oxidative damage. Thus, oxidative lesions to mtDNA accumulate with age in human and rodent tissues. Furthermore, levels of oxidative damage to mtDNA are several times higher than those of nuclear DNA. Mitochondrial size increases whereas mitochondrial membrane potential decreases with age in brain and liver.

Recently, we have shown that treatment with certain antioxidants, such as sulphur-containing antioxidants, vitamins C and E or the Ginkgo biloba extract EGb 761, protects against the age-associated oxidative damage to mtDNA and oxidation of mitochondrial glutathione. Moreover, the extract EGb 761 also prevents changes in mitochondrial morphology and function associated with aging of the brain and liver. Thus, mitochondrial aging may be prevented by antioxidants. Furthermore, late onset administration of certain antioxidants is also able to prevent the impairment in physiological performance, particularly motor co-ordination, that occurs upon aging.