SEARCH

SEARCH BY CITATION

Keywords:

  • aging;
  • calorie restriction;
  • mitochondria;
  • oxidative stress;
  • reactive oxygen species;
  • redox status

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. CR feeding retards the rate of accrual of tissue oxidative stress
  5. Functional effects of tissue oxidative damage
  6. Is the effect of CR feeding on oxidative tissue damage reversible?
  7. CR and mitochondrial ROS generation in vitro
  8. CR and mitochondrial ROS generation in vivo
  9. Is the effect of CR feeding on mitochondrial ROS generation a rate phenomenon?
  10. Metabolic control analysis
  11. Acknowledgments
  12. References

Accumulated oxidative stress resulting from a gradual shift in the redox status of tissues is now considered to be a key mechanism underlying the aging process. Calorie-restricted (CR) feeding, an experimental protocol to extend survival and delay aging in rodents, is recognized to slow the rate of accrual of age-related oxidative stress. This conclusion is based on the increase in tissues with age of the oxidation products of proteins, lipids and DNA. The functional consequence, however, of the accumulation of these non-specific oxidative markers is more difficult to determine. A shift in the redox status of tissues with age and calorie restriction feeding may have a greater impact on cell function through activation of redox sensitive transcription factors than through the accumulation of these non-specific oxidative markers. Activation of such transcription factors will stimulate signalling pathways that will lead to a change in the gene expression profile and cell functioning. Little research has been conducted in this area. It has been proposed that CR feeding slows the rate of accrual of oxidative damage because mitochondria in these animals have a lower rate of superoxide generation when compared with mitochondria from control animals. This proposal is based on in vitro observations using isolated mitochondria and clearly requires further confirmation in isolated cells or using an in vivo approach. The application of metabolic control analysis to identify in isolated mitochondria the mechanism underlying this response has suggested one possible explanation for the lower superoxide production rates observed.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. CR feeding retards the rate of accrual of tissue oxidative stress
  5. Functional effects of tissue oxidative damage
  6. Is the effect of CR feeding on oxidative tissue damage reversible?
  7. CR and mitochondrial ROS generation in vitro
  8. CR and mitochondrial ROS generation in vivo
  9. Is the effect of CR feeding on mitochondrial ROS generation a rate phenomenon?
  10. Metabolic control analysis
  11. Acknowledgments
  12. References

Developed from the original ideas of Harman (1956), the role of oxidative stress as a consequence of normal metabolism has emerged as one feasible molecular mechanism to explain the aging process. Attention has been focused on quantifying the effect calorie-restricted (CR) feeding has on the rate of accrual of tissue oxidative damage and in evaluating the contribution mitochondrial-generated reactive oxygen species (ROS) make to the lowered concentration of oxidative damage observed.

CR feeding retards the rate of accrual of tissue oxidative stress

  1. Top of page
  2. Summary
  3. Introduction
  4. CR feeding retards the rate of accrual of tissue oxidative stress
  5. Functional effects of tissue oxidative damage
  6. Is the effect of CR feeding on oxidative tissue damage reversible?
  7. CR and mitochondrial ROS generation in vitro
  8. CR and mitochondrial ROS generation in vivo
  9. Is the effect of CR feeding on mitochondrial ROS generation a rate phenomenon?
  10. Metabolic control analysis
  11. Acknowledgments
  12. References

Tissue oxidative damage resulting from the pro-oxidant-generating processes of normal metabolism does accumulate with age and it is evident that CR feeding regimes can attenuate the rate of accrual of oxidative damage (Yu, 1993). The tissue chemical markers on which this conclusion was originally based were the membrane lipid peroxides and the secondary decomposition products that include hydrocarbons, epoxides, aldehydes, ketones and carboxylic acids (Chipalkatti et al., 1983; De et al., 1983; Koizumi et al., 1987; Laganiere & Yu, 1987). This has subsequently included oxidative damage to bases in genomic and mitochondrial DNA (Fraga et al., 1990; Richter, 1995), tissue protein carbonyl formation (Sohal et al., 1994; Takahashi & Goto, 2002) and recently redox-sensitive transcription factors such as AP1 and NFκB (Kim & Chung, 2002; Kim et al., 2002).

Although the overall prediction has been verified, i.e. that the rate of accumulation of tissue oxidative damage is slower in animals exhibiting retarded aging induced by CR feeding, studies have revealed a number of inconsistencies and unanswered questions. For example, in the study of Chung et al. (1992), even by 3 months of age, 1.5 months after the initiation of CR feeding, a significant reduction in the concentration of 8-hydroxy-2′-deoxyguanosine (8-OHdg) was observed in rat liver nuclear DNA in comparison with control animals. However, a later study using the same strain of rat, Fischer 344, could not confirm these early effects of CR feeding in attenuating oxidative damage in genomic DNA (Kaneko et al., 1997). In this study levels of oxidative damage were similar in control and CR animals until about 24 months and only very late in life was an increase in this oxidative marker observed in control animals. CR feeding initiated in late middle age in rats supports the interpretation that CR may be most effective at attenuating a late-life acceleration in tissue oxidative damage (Aspnes et al., 1997). Conversely, Gredilla et al. (2001a) report a significant decrease in oxidative damage in rat liver for nuclear and mitochondrial DNA after only 6 weeks of 60% restricted feeding. However, utilizing the same strain of rat, feeding conditions, analytical methodology and oxidative marker they could only confirm a decrease in the concentration of 8-oxo-7,8,dihyro-2′deoxyguanosine (8-oxodG) in mitochondrial DNA at 24 months of age, there being no significant difference in the concentration of 8-oxodG in nuclear DNA between dietary groups at this age (Lopez-Torres et al., 2002).

This lack of consistency between reports of DNA oxidative damage as a function of age and CR feeding regimes may reflect recognized methodological problems in some of the earlier studies and also tissue-specific responses to CR feeding. Detailed confirmation therefore of the effect of aging on the accumulation of DNA oxidative damage and its attenuation by CR feeding requires further clarification.

When two levels of CR feeding, 35% and 50% restriction, were initiated at 17 months of age, muscle from rats on 50% CR showed better preserved fibre number and fibre type composition in the vastus lateralis muscle when examined at 30–32 months of age. Muscle from rats on 50% CR feeding had significantly fewer COX and SDH++ fibres (fibres negative for cytochrome c oxidase activity and/or fibres over-expressing succinate dehydrogenase activity), and also fewer mtDNA deletion products, as compared with muscle from rats limited to a 35% restriction of calorie intake (Aspnes et al., 1997). Therefore, even relatively late in life, certain tissue types retain their plasticity in response to CR feeding and demonstrate a reduction in oxidative damage.

A dose–response relationship is known to exist between survival and the degree of energy restriction employed in rodents (Merry, 2002) and this relationship is in agreement with the observations reported for mtDNA deletion products on CR feeding of varying intensity (Aspnes et al., 1997). Similarly, a positive correlation can be demonstrated between survival and the duration of the CR regime used (Merry, 2002). Given these relationships, little data have been published that demonstrate a similar correlative attenuation in the rate of accrual of tissue oxidative damage with the intensity or duration of CR feeding regime, raising a question mark over a causal link between survival and generalized oxidative damage of tissue.

Marked heterogeneity is seen both within and between tissue types, with regard to the severity of oxidative damage observed with age, the type of protein oxidative damage sustained and the response to CR feeding (Dubey et al., 1996). The generic conclusion that CR feeding regimes retard the accumulation of oxidative damage represents an oversimplification resulting from gross tissue averaging. Detailed observations on the rate at which different tissues and cell types accumulate oxidative stress with age and their responsiveness to the protective effect of CR feeding are lacking. From studies on brain tissue there is an indication that those regions such as the striatum that show the largest increase in carbonyl formation with age also exhibit the greatest sensitivity to CR feeding regimes. For example, in comparison with the striatum, the hippocampus carbonyl concentration shows little increase with age in the mouse and is relatively resilient to the protective effects of CR feeding (Dubey et al., 1996).

Functional effects of tissue oxidative damage

  1. Top of page
  2. Summary
  3. Introduction
  4. CR feeding retards the rate of accrual of tissue oxidative stress
  5. Functional effects of tissue oxidative damage
  6. Is the effect of CR feeding on oxidative tissue damage reversible?
  7. CR and mitochondrial ROS generation in vitro
  8. CR and mitochondrial ROS generation in vivo
  9. Is the effect of CR feeding on mitochondrial ROS generation a rate phenomenon?
  10. Metabolic control analysis
  11. Acknowledgments
  12. References

The functional consequence of an age-related increase in such biochemical markers of oxidative damage has been little studied and remains therefore largely unknown. It is possible that the concentrations of oxidative damage reported with age may fall below the threshold that a cell or tissue may tolerate with little or no direct impact on functional efficiency. Conversely, oxidative damage to key genes and proteins may result in a significant decrement in the efficiency of cell functioning. It is assumed that the second interpretation is correct but data are very limited in support of this suggestion.

Although there is a lack of detail on the quantitative relationship between oxidative damage and cell functioning, it is now apparent that a change in the redox status of tissues does occur with aging and that the transition to a more oxidized cell environment is retarded by maintaining animals on a CR diet (Kim & Chung, 2002; Kim et al., 2002; Cho et al., 2003). Such a shift in the redox status of cells acting through a range of redox-sensitive transcription factors is likely to modify the gene expression profile in cells and contribute to the aged phenotype. Details regarding the effect of CR feeding on age-related change in tissue redox status and redox-sensitive transcription factor activation are still very fragmentary (Kim et al., 2002; Kim & Chung, 2002).

The role of redox-sensitive transcription factors in aging and the mechanism of CR feeding is an area that has received little attention. It may be crucial in integrating metabolic control to mitochondrial nuclear signalling pathways and global gene expression profiles. Jazwinski (2000) draws attention to four broad physiological processes that are important in determining longevity in budding yeast and the response to CR, i.e. metabolic control, resistance to stress, gene dysregulation and genetic stability. It is suggested that there are at least three mechanisms whereby the rate of aging may be retarded in yeast, namely mitochondrial dysfunction signalled by the retrograde response to the nucleus, calorie restriction and protein synthesis that involves transcriptional silencing requiring Sir2 histone deacetylase, and the induction of ribosomal protein genes. Although it may be possible to make a tentative link between the metabolic state and Sir2 activity because of its nicotinamide adenine dinucleotide (NAD) dependence (Anderson et al., 2003), it is unclear if there is an interaction between the retrograde response and the mechanism of CR in yeast. Based on current knowledge, CR and the retrograde response appear to act through different pathways and it is unclear if a homologous retrograde response operates in higher organisms (Jazwinski, 2000).

Is the effect of CR feeding on oxidative tissue damage reversible?

  1. Top of page
  2. Summary
  3. Introduction
  4. CR feeding retards the rate of accrual of tissue oxidative stress
  5. Functional effects of tissue oxidative damage
  6. Is the effect of CR feeding on oxidative tissue damage reversible?
  7. CR and mitochondrial ROS generation in vitro
  8. CR and mitochondrial ROS generation in vivo
  9. Is the effect of CR feeding on mitochondrial ROS generation a rate phenomenon?
  10. Metabolic control analysis
  11. Acknowledgments
  12. References

An unresolved issue is the extent of tissue malleability in response to crossover-feeding studies designed to induce or reverse oxidative damage. Dubey et al. (1996) have reported that in 15-month-old mice, steady-state concentration of oxidised protein in the brain can be reversed over a 6-week period when animals are switched between control and CR feeding. However, the same malleability could not be demonstrated for protein carbonyl content in isolated mitochondria from hind limb skeletal muscle (Lass et al., 1998). Interestingly in this study, although muscle mitochondrial carbonyl concentration increased with age in fully fed animals, no age-related increase was evident in CR animals. A similar lack of an age-associated rise in mitochondrial oxidized protein in CR animals was also observed for the concentration of thiobarbituric acid reactive substances in these organelles (Lass et al., 1998). Although the rate of superoxide generation from submitochondrial particles was 41% higher at 20 months old from ad libitum fed mice than with 4-month-old control or 20-month-old CR animals, the complete ablation of an age-related increase in oxidative damage in muscle mitochondria from CR animals appears surprising.

CR and mitochondrial ROS generation in vitro

  1. Top of page
  2. Summary
  3. Introduction
  4. CR feeding retards the rate of accrual of tissue oxidative stress
  5. Functional effects of tissue oxidative damage
  6. Is the effect of CR feeding on oxidative tissue damage reversible?
  7. CR and mitochondrial ROS generation in vitro
  8. CR and mitochondrial ROS generation in vivo
  9. Is the effect of CR feeding on mitochondrial ROS generation a rate phenomenon?
  10. Metabolic control analysis
  11. Acknowledgments
  12. References

Although much of the detail is lacking, there is prima facia evidence discussed above that accrual of tissue oxidative stress may be an important mechanism underlying tissue dysfunction with age, and that CR feeding regimes retard this process. Tissue concentration of oxidative damage is a measure of the equilibrium between rates of oxidant generation and of oxidant scavenging, repair and turnover-processes (Beckman & Ames, 1998). CR feeding modifies all these separate processes in a tissue-specific and time-dependent manner (Merry, 2000) but the primary mechanism by which calorie restriction is considered to reduce oxidative tissue damage is through a reduction in the generation of mitochondrial ROS (Sohal et al., 1994; Gredilla et al., 2001a,b). This conclusion has been derived mainly from data obtained from isolated mitochondria respiring under non-physiological conditions.

Whether mitochondrial oxidant production increases with age is still unclear because there is a lack of agreement between published studies (Guarnieri et al., 1992; Nohl, 1993; Rollo et al., 1996; Hansford et al., 1997; Kwong & Sohal, 1998). This situation reflects the sensitivity of in vitro mitochondrial ROS generation to the incubation conditions employed, such as the use of complex 1 or 2 substrates, the concentration of the substrate supporting respiration, the partial pressure of oxygen in the medium and the presence or absence of specific inhibitors such as rotenone. Variability between mitochondrial preparations increases the variance of the data obtained and the standard approach of differential centrifugation to isolate mitochondria may be selective in removing abnormal or enlarged mitochondria from older tissues. This could result in a conservative estimate of the effect of age on mitochondrial ROS generation.

The application of electron paramagnetic resonance spectroscopy with a site-specific spin label to quantify ROS generation in a mixed population of brain synaptosomes and mitochondria isolated from Brown Norway rats did not confirm that CR attenuated ROS production following metabolic stimulation of mitochondria with a high concentration of succinate (20 mm), a complex II substrate (Gabbita et al., 1997). This study did confirm an age effect in the ability of mitochondria to leak electrons to oxygen following stimulation of mitchondrial respiration with 20 mm succinate. This study, however, suffers from similar limitations to those using biochemical assays for ROS determination in that the isolation procedure is selective and the respiration conditions under which the estimates of ROS generation were made were non-physiological.

CR and mitochondrial ROS generation in vivo

  1. Top of page
  2. Summary
  3. Introduction
  4. CR feeding retards the rate of accrual of tissue oxidative stress
  5. Functional effects of tissue oxidative damage
  6. Is the effect of CR feeding on oxidative tissue damage reversible?
  7. CR and mitochondrial ROS generation in vitro
  8. CR and mitochondrial ROS generation in vivo
  9. Is the effect of CR feeding on mitochondrial ROS generation a rate phenomenon?
  10. Metabolic control analysis
  11. Acknowledgments
  12. References

The direct confirmation in vivo of the in vitro observation of reduced ROS production from mitochondria isolated from CR fed animals has been limited by technical difficulties although it has been established that cells isolated from CR animals retain their altered metabolic profile under culture conditions (Lambert & Merry, 2000; de Cabo et al., 2003). If the in vitro analysis is indicative of the situation in vivo, then the question arises as to the nature of the underlying mechanism responsible for a lower rate of mitochondrial ROS generation under CR feeding conditions.

Is the effect of CR feeding on mitochondrial ROS generation a rate phenomenon?

  1. Top of page
  2. Summary
  3. Introduction
  4. CR feeding retards the rate of accrual of tissue oxidative stress
  5. Functional effects of tissue oxidative damage
  6. Is the effect of CR feeding on oxidative tissue damage reversible?
  7. CR and mitochondrial ROS generation in vitro
  8. CR and mitochondrial ROS generation in vivo
  9. Is the effect of CR feeding on mitochondrial ROS generation a rate phenomenon?
  10. Metabolic control analysis
  11. Acknowledgments
  12. References

The first study of the effect of CR feeding on the state 4 mitochondrial respiration rate used liver and brain mitochondria isolated from 3- to 7-month-old mice and reported no significant effect of CR feeding. This conclusion was independent of whether a complex 1 substrate (glutamate, malate and pyruvate, or β-hydroxybutyrate) or a complex 2 substrate (succinate with rotenone) was used to support respiration (Weindruch et al., 1980). The lack of an effect of CR feeding on mitochondrial state 4 respiration was evident irrespective of whether the rates were normalized per milligram of mitochondrial protein or to cytochrome c oxidase activity. The situation has subsequently become less clear as later reports are contradictory (Sohal et al., 1994; Gredilla et al., 2001a,b; Lal et al., 2001; Lopez-Torres et al., 2002; Lambert & Merry, 2003).

The early work on in vitro ROS production in mouse mitochondria isolated from brain, heart and kidney reported a general correlation of depressed state 4 respiration with lowered ROS generation when respiration was supported by succinate, a complex 2 substrate (Sohal et al., 1994). A significant (50%) depression of ROS generation, however, was not always associated with a lowered state 4 mitochondrial respiration rate in this study. Brain mitochondria isolated from 9- and 17-month-old control and CR fed animals showed no significant difference in state 4 respiration rate (Sohal et al., 1994), in spite of significantly lowered superoxide and hydrogen peroxide generation rates in CR mitochondria.

A significant decrease in state 4 respiration in mitochondria isolated from CR animals would be indicative of either a reduction in the proton leak of the inner mitochondrial membrane, if the inner mitochondrial membrane potential remained unaltered, or a fall in the magnitude of the membrane potential, or a combination of both these responses. The generation rate of superoxide is reported to be exquisitely sensitive to the protonmotive force (Δp), at least at complex 3 (Korshunov et al., 1997; Liu, 1997; Votyakova & Reynolds, 2001). It is clear, however, from the studies so far published that a depression in the state 4 respiration rate, using either a complex 1 or a complex 2 substrate, is not obligatory in order to observe a significant reduction in mitochondrial ROS generation under CR feeding conditions (Gredilla et al., 2001b). There is a continuing debate as to the exact location in the electron transport chain where CR has the greatest effect on reducing the electron leak to molecular oxygen. The interpretation of published data is confused further because of the lack of consistency in the use of rotenone in the incubation medium to prevent reverse electron flow at complex 1.

Barja's group have argued that the decrease in mitochondrial ROS production takes place mainly or exclusively at complex 1 under CR feeding conditions, with this complex being maintained in a less reduced state than with liver mitochondria isolated from control rats (Lopez-Torres et al., 2002). To explain a lowered ROS production in which state 4 respiration is unaltered, as observed by Lopez-Torres et al. (2002), would require that the complexes of the electron transport chain become more oxidized. This would be indicative of a fall in Δp; however, the membrane potential was not measured in this study to confirm this prediction.

Short-term CR feeding (6 weeks of 60% feeding) also significantly depressed ROS production in isolated liver mitochondria when respiration was supported with a state 1, but not a state 2 substrate (Gredilla et al., 2001a). Although no significant effect on ROS production was observed with a state 2 substrate, mean rates of H2O2 procution were depressed as when respiration was supported with a complex 1 substrate. The interanimal variability in H2O2 generation, however, was significantly greater when respiration was supported by succinate rather than with pyruvate/malate. A significant difference between control and CR mitochondrial ROS generation rates could not therefore be demonstrated when respiration was supported by a complex 2 substrate.

Metabolic control analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. CR feeding retards the rate of accrual of tissue oxidative stress
  5. Functional effects of tissue oxidative damage
  6. Is the effect of CR feeding on oxidative tissue damage reversible?
  7. CR and mitochondrial ROS generation in vitro
  8. CR and mitochondrial ROS generation in vivo
  9. Is the effect of CR feeding on mitochondrial ROS generation a rate phenomenon?
  10. Metabolic control analysis
  11. Acknowledgments
  12. References

To observe a reduction of 50% in ROS production would require a less than 10% fall in Δp, maintaining Δp within the thermodynamic range for ATP synthesis at the Fo-F1 synthase (Korshunov et al., 1997). Such an adaptation in mitochondria that are homeostatic around Δp could be achieved if the proton conductance of the inner mitochondrial membrane was increased with an unaltered state 4 respiration rate. To resolve whether CR feeding increased or decreased the proton conductance of the inner mitochondria membrane, metabolic control analysis, specifically top-down regulation analysis (Brand & Brown, 1994), has been applied to the study of mitochondrial bioenergetics with CR (Lal et al., 2001). Metabolic control analysis, specifically elasticity and control analysis, allows the important sites of control within a complex homeostatic system (the mitochondrion) to be identified. Regulation analysis allows the system to be characterized when it responds to an effector such as CR feeding (see Lambert & Merry, 2003, for details of the application of metabolic control analysis to mitochondrial bioenergetics during aging and CR feeding).

Only two studies have been published so far using this approach and they report conflicting conclusions. The initial publication reported the proton leak kinetics for mitochondria isolated from control rat hind-limb skeletal muscle of 4- and from 33-month-old control and CR animals (Lal et al., 2001). The degree of energy restriction used was 67% started at 10 months of age. This study did not therefore address the question as to the effect of CR feeding on the proton leak and magnitude of Δp in young animals. The only effect induced by CR feeding identified at 33 months was a lowered membrane potential associated with a higher proton leak over the whole range of membrane potentials measured when compared with young control animals. A large degree of variation was observed in the data describing the proton leak kinetics in mitochondria isolated from 33-month-old control animals. This high degree of variability in the old control data precluded the identification of any significant effect of CR feeding on the kinetics of the proton leak at 33 months of age (Lal et al., 2001). There is a suggestion from the data presented that the proton leak in 33-month-old CR animals is greater during state 4 respiration than that observed for the age-matched control animals, with a consequent lowering of the membrane potential. The authors report a 23% decrease in the state 4 respiration rate in 33-month-old CR mitochondria in comparison with age-matched controls. It is difficult to reconcile this decrease in electron flow with an unchanged proton leak and unaltered protonmotive force. If the proton leak is identical at 33 months of age in mitochondria from control and CR hind-limb muscle, then Δp must fall in the CR mitochondria to maintain the homeostatic system in balance.

The second study applying the metabolic control analysis approach is from my own laboratory where the effect of age, CR feeding and CR feeding with insulin supplementation is reported (Lambert & Merry, 2003). Using the same experimental and theoretical approach as before (Lal et al., 2001), CR feeding was shown to induce an increase in the proton leak of liver mitochondria from 6 to 28 months of age that could be reversed by elevating the plasma insulin concentration in CR animals by means of an implanted mini-osmotic pump. Plasma insulin concentrations in CR animals are significantly lowered in comparison with age-matched control animals.

No significant effect of CR feeding could be observed on either state 3 and 4 mitochondrial respiration rates in an age-matched comparison performed at 6, 18 and 28 months of age. The consequence of the unchanged respiration rate but increased membrane proton conductance in CR mitochondria was to lower, by approximately 10%, the inner mitochondrial membrane potential. This increase in the oxidized state of the electron transport chain complexes was interpreted as a plausible mechanism to explain the lower superoxide production rates in these mitochondria. The proposal clearly requires further verification and confirmation in other tissue types to ascertain if it is a generalized response of mitochondria to CR feeding. The compositional and structural change in the mitochondrial membrane of CR liver mitochondria that results in the increased proton conductance is not currently understood.

In conclusion, confirmation that CR feeding reduces mitochondrial ROS generation in vivo is urgently required. The mechanism proposed to explain the lowered ROS generation in mitochondria isolated from CR animals needs further verification and clarification, especially in different tissue types. Data are required also to document the effect of CR feeding on non-mitochondrial sources of ROS. An understanding of the role of redox-sensitive transcription factor activation to control age and CR feeding-induced modification of gene expression is required in order to develop an understanding of the signalling pathways mediating the CR effect on longevity.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. CR feeding retards the rate of accrual of tissue oxidative stress
  5. Functional effects of tissue oxidative damage
  6. Is the effect of CR feeding on oxidative tissue damage reversible?
  7. CR and mitochondrial ROS generation in vitro
  8. CR and mitochondrial ROS generation in vivo
  9. Is the effect of CR feeding on mitochondrial ROS generation a rate phenomenon?
  10. Metabolic control analysis
  11. Acknowledgments
  12. References
  • Anderson RM, Bitterman KJ, Wood JG, Medvedik O, Sinclair DA (2003) Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae. Nature 423, 181185.
  • Aspnes LE, Lee CM, Weindruch R, Chung SS, Roecker EB, Aiken JM (1997) Caloric restriction reduces fiber loss and mitochondrial abnormalities in aged rat muscle. FASEB J. 11, 573581.
  • Beckman KB, Ames BN (1998) The free radical theory of aging matures. Physiol. Rev. 78, 547581.
  • Brand MD, Brown GC (1994) The experimental application of control analysis to metabolic systems. In Biothermokinetics (WesterhoffHV, ed.). Andover: Intercept, pp. 2235.
  • De Cabo R, Furer-Galban S, Anson RM, Gilman C, Gorospe M, Lane MA (2003) An in vitro model of caloric restriction. Exp. Gerontol. 38, 631639.
  • Chipalkatti S, De AK, Aiyar AS (1983) Effect of diet restiction on some biochemical parameters related to aging in mice. J. Nutrition 113, 944950.
  • Cho CG, Kim HJ, Chung SW, Jung KJ, Shim KH, Yu BP, Yodoi J, Chung HY (2003) Modulation of glutathione and thioredoxin systems by calorie restriction during the aging process. Exp. Gerontol. 38, 539548.
  • Chung MH, Kasai H, Nishimura S, Yu BP (1992) Protection of DNA damage by dietary restriction. Free Rad. Biol. Med. 12, 523525.
  • De AK, Chipalkatti S, Aiyar AS (1983) Some biochemical parameters of aging in relation to dietary protein. Mech. Aging Dev. 21, 3748.
  • Dubey A, Forster MJ, Lal H, Sohal RS (1996) Effect of age and caloric-intake on protein oxidation in different brain-regions and on behavioral functions of the mouse. Arch. Biochem. Biophys. 333, 189197.
  • Fraga CG, Shigenaga MK, Park JW, Degan P, Ames BN (1990) Oxidative damage to DNA during aging: 8-hydroxy-2′-deoxyguanosine in rat organ DNA and urine. Proc. Natl Acad. Sci. USA 87, 45334537.
  • Gabbita SP, Butterfield DA, Hensley K, Shaw W, Carney JM (1997) Aging and caloric restriction affect mitochondrial respiration and lipid membrane status: an electron paramagnetic resonance investigation. Free Rad. Biol. Med. 23, 191201.
  • Gredilla R, Barja G, Lopez-Torres M (2001a) Effect of short-term caloric restriction on H2O2 production and oxidative DNA damage in rat liver mitochondria and location of the free radical source. J. Bioenerg. Biomembr. 33, 279287.
  • Gredilla R, Sanz A, Lopez-Torres M, Barja G (2001b) Calorie restriction decreases mitochondrial free radical generation at complex I and lowers oxidative damage to mitochondrial DNA in the rat heart. FASEB J. 15, U481U496.
  • Guarnieri C, Muscari C, Caldarera CM (1992) Mitochondrial Production of Oxygen Free Radicals in the Heart Muscle During the Life Span of the Rat – Peak at Middle Age. Klosterberg, Basel, Switzerland: Birkhauser-Verlag.
  • Hansford RG, Hogue BA, Mildaziene V (1997) Dependence of H2O2 formation by rat heart mitochondria on substrate availability and donor age. J. Bioenerg. Biomembr. 29, 8995.
  • Harman D (1956) Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 11, 298300.
  • Jazwinski M (2000) Metabolic control and aging. Trends Genet. 16, 506511.
  • Kaneko T, Tahara S, Matsuo M (1997) Retarding effect of dietary restriction on the accumulation of 8-hydroxy-2′-deoxyguanosine in organs of Fischer 344 rats during aging. Free Rad. Biol. Med. 23, 7681.
  • Kim H-J, Chung H-Y (2002) Molecular exploration of age-related NFκB/IKK downregulation by calorie restriction in rat kidney. Free Rad. Biol. Med. 32, 9911005.
  • Kim HJ, Jung KJ, Seo AY, Choi JS, Yu BP, Chung HY (2002) Calorie restriction modulates redox-sensitive AP-1 during the aging process. J. Am. Aging Assoc. 25, 123130.
  • Koizumi A, Weindruch R, Walford RL (1987) Influences of dietary restriction and age on liver enzyme activities and lipid peroxidation in mice. J. Nutr. 117, 361367.
  • Korshunov SS, Skulachev VP, Starkov AA (1997) High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416, 1518.
  • Kwong LK, Sohal RS (1998) Substrate and site specificty of hydrogen peroxide generation in mouse mitochondria. Arch. Biochem. Biophys. 350, 118126.
  • Laganiere S, Yu BP (1987) Anti-lipoperoxidation action of food restriction. Biochem. Biophys. Res. Commun. 145, 11851191.
  • Lal SB, Ramsey JJ, Monemdjou S, Weindruch R, Harper M-E (2001) Effects of calorie restriction on skeletal muscle mitochondrial proton leak in aging rats. J. Gerontol. 56A, B116B122.
  • Lambert AJ, Merry BJ (2000) Use of primary cultures of rat hepatocytes for the study of aging and caloric restriction. Exp. Gerontol. 35, 583594.
  • Lambert AJ, Merry BJ (2004) Effect of caloric restriction on mitochondrial reactive oxygen species production and bioenergetics – reversal by insulin. Am. J. Physiol. 286, R71 ( in press print version published at http://ajpregu.physiology.org/papbyrecent.shtml).
  • Lass A, Sohal BH, Weindruch R, Forster MJ, Sohal RS (1998) Caloric restriction prevents age-associated accrual of oxidative damage to mouse skeletal muscle mitochondria. Free Rad. Biol. Med. 25, 10891097.
  • Liu S-S (1997) Generating, partitioning, targeting and functioning of superoxide in mitochondria. Bioscience Report 17, 259272.
  • Lopez-Torres M, Gredilla R, Sanz A, Barja G (2002) Influences of aging and long-term calorie restriction on oxygen radical generation and oxidative DNA damage in rat liver mitochondria. Free Rad. Biol. Med. 32, 882889.
  • Merry BJ (2000) Calorie restriction and age-related oxidative stress. In Molecular and Cellular Gerontology (ToussaintO, OsiewaczHD, LithgowGJ, BrackC, eds). New York: New York Academy of Sciences, 908, pp. 180198.
  • Merry BJ (2002) Molecular mechanisms linking calorie restriction and longevity. IJBCB 34, 13401354.
  • Nohl H (1993) Involvement of free radicals in aging – a consequence or cause of senescence. Br. Med. Bull. 49, 653667.
  • Richter C (1995) Oxidative damage to mitochondrial-DNA and its relationship to aging. Int. J. Biochem. Cell B 27, 647653.
  • Rollo CD, Carlson J, Sawada M (1996) Accelerated aging of giant transgenic mice is associated with elevated free-radical processes. Can. J. Zool. 74, 606620.
  • Sohal RS, Ku HH, Agarwal S, Forster MJ, Lal H (1994) Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse. Mech. Aging Dev. 74, 121133.
  • Takahashi R, Goto S (2002) Effect of dietary restriction beyond middle age: accumulation of altered proteins and protein degradation. Microsc. Res. Techn. 59, 278281.
  • Votyakova TV, Reynolds IJ (2001) DeltaPsi (m)-Dependent and -independent production of reactive oxygen species by rat brain mitochondria. J. Neurochem. 79, 266277.
  • Weindruch RL, Cheung MK, Verity MA, Walford RL (1980) Modification of mitochondrial respiration by aging and dietary restriction. Mech. Aging Dev. 12, 372392.
  • Yu BP (1993) Antioxidant action of dietary restriction in the aging. J. Nutri. Sci. Vitaminol. 00, S75S83.