Age-related increase in mitochondrial proton leak and decrease in ATP turnover reactions in mouse hepatocytes

Mary-Ellen Harper1, Shadi Monemdjou1, Jon J. Ramsey2, and Richard Weindruch2,3

1 Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5; 2 Wisconsin Regional Primate Research Center, University of Wisconsin, Madison 53715; and 3 Department of Medicine, University of Wisconsin, and Veterans Administration Geriatric Research, Education and Clinical Center, Madison, Wisconsin 53705

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Age-related changes in mitochondria, including decreased respiratory control ratios and altered mitochondrial inner membrane lipid composition, led us to study oxidative phosphorylation in hepatocytes from old (30 mo) and young (3 mo) male C57BL/J mice. Top-down metabolic control analysis and its extension, elasticity analysis, were used to identify changes in the control and regulation of the three blocks of reactions constituting the oxidative phosphorylation system: substrate oxidation, mitochondrial proton leak, and the ATP turnover reactions. Resting oxygen consumption of cells from old mice was 15% lower (P < 0.05) than in young cells. This is explained entirely by a decrease in oxygen consumption supporting ATP turnover reactions. At all values of mitochondrial membrane potential assessed, the proportion of total oxygen consumption used to balance the leak was greater in the old cells than in the young cells. Metabolic control coefficients indicate a shift in control over respiration and phosphorylation away from substrate oxidation toward increased control by leak and by ATP turnover reactions. Control of the actual number of ATP molecules synthesized by mitochondria for each oxygen atom consumed by the ATP turnover and leak reactions was greater in old than in young cells, showing that efficiency in older cells is more sensitive to changes in these two blocks of reactions than in young cells.

oxidative phosphorylation; uncoupling; oxidative stress; free radicals; aging

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

THE CONCEPT THAT FREE RADICALS are involved in key processes resulting in aging was first proposed over 40 years ago (17, 23). In recent years evidence has corroborated and extended the original free radical theory of aging, and together the evidence has been amalgamated into the now widely supported oxidative stress hypothesis of aging (1, 41, 42). The latter implicates endogenous oxidants in senescence-related loss of cellular function. The underlying tenet of the original free radical theory was that free radicals caused the progressive and irreversible damage and deterioration of cellular function. In contrast to the free radical theory, the oxidative stress hypothesis of aging does not limit the source of the damaging metabolites to oxygen-derived free radicals; moreover, the scope has been expanded from oxidant-induced molecular damage to include oxidant modulation of gene expression, signal transduction, and other normal processes (40, 41, 46, 48).

Despite over 40 years of research, there is a distinct lack of integrative data on the effects of oxidative stress on the metabolism of intact cells. Gerontological investigations of mitochondrial function have been limited almost exclusively to isolated mitochondria. Mitochondrial function, as assessed by analyses of isolated mitochondria and mitochondrial enzymes, is affected by aging (22, 29, 40). Mitochondrial components damaged through oxidative mechanisms include mitochondrial DNA (which encodes for several proteins of the electron transport chain), proteins, and lipids. Compared with nuclear DNA, the extent of oxidative damage to mitochondrial DNA (mtDNA) is much greater. Damage to mtDNA from rat liver or human brain regions is at least tenfold more than that in nuclear DNA (33, 39). There are a number of reasons explaining the vulnerability of mtDNA to oxidative damage. Beyond the fact that mitochondria are responsible for the production of the majority of free radicals in cells, the DNA is immediately juxtaposed to the electron transport chain in the mitochondrial inner membrane (MIM), where the oxidants are produced. Moreover, mtDNA lacks the protective histones and extensive DNA repair capabilities that protect nuclear DNA. The function of many mitochondrial proteins is impaired with age. For example, the activities of complexes I, II, and IV decrease with age in postmitotic tissues (3, 5, 14, 34, 43).

Effects of aging on MIM lipids include increased levels of long-chain polyunsaturated fatty acids such as 22:4 and 22:5 and decreases in 18:2, 18:1, and 16:1. As a result, with increases in age there is an increased probability of membrane lipid peroxidation (the "peroxidizability index") (30, 49). Linoleic acid (18:2) is thought to be necessary for the optimal interactions of cardiolipin with many substrate transporters and components of the MIM (28, 29). Shigenaga et al. (40) hypothesized that these decreases in inner membrane lipids containing 18:2 and altered cardiolipin-protein interactions may account for the decreased State 3-to-State 4 ratio (State 3/State 4) and contribute to the loss of efficiency in mitochondrial function. Our results support and extend this line of thought.

Although isolated mitochondria have been extensively investigated in aging studies, it is known that populations of mitochondria from tissues of old and young organisms can be differentially damaged by mitochondrial isolation procedures. Histological studies have shown that, with increasing age, mitochondria tend to be larger, there is increased matrix vacuolization, cristae are shorter, and there is a decrease in dense granules (47). Importantly, it has been estimated that only about one-half of these enlarged mitochondria are retained in mitochondrial isolations from tissues of older animals (47). Thus the quality of the mitochondrial preparation from old organisms may, in fact, be improved by the loss of unhealthy mitochondria during the isolation procedures (47), leading potentially to artifactual conclusions about changes in mitochondrial function with age. Moreover, oxidative damage occurs throughout the cell, emphasizing the importance of metabolic studies conducted using intact cells and tissues.

The experimental approach that we have used is one that has been useful in identifying the sites of action of hormones and other external effectors in metabolic pathways in intact cells as well as in mitochondrial preparations. The approach is referred to as top-down elasticity analysis (7), and it is an extension of metabolic control analysis. Whereas metabolic control analysis allows the identification of the important sites of control within metabolic pathways, top-down elasticity analysis is used in the comparison of varying metabolic conditions to identify differences in pathway regulation. The latter is extremely useful, for example, in the identification of the "sites of action" of hormones, drugs, and metabolic defects. Some of the useful measurements to emerge from an elasticity analysis include "elasticity coefficients," "flux control coefficients," and "concentration control coefficients." In very general terms, an elasticity coefficient (often referred to simply as an "elasticity") describes the responsiveness of a branch of a metabolic pathway to changes in the amount of an intermediate in that pathway. If the elasticity for a branch differs between the drug-treated and the control pathways being compared, then one site of action of the drug is located within the reactions encompassed by that branch. Other measurements include flux control and concentration control coefficients. Values of the latter describe, respectively, the relative proportion of control by branches of the pathway over the rate of the pathway and over the amounts of intermediates. Several useful reviews on this approach have been published recently (see Refs. 8, 26). In intact cells it has been successfully used to investigate the sites of action of glucagon (9), thyroid hormones (19, 20, 24, 25), and butylated hydroxyanisole (16) in mitochondria, and of fatty acids in isolated hepatocytes of rats (37). Here we use the approach to quantitatively identify in mouse hepatocytes the effects of aging on reactions that are central to oxidative phosphorylation.

On the basis of the above documented age-related changes in mitochondria, particularly the decreases in the State 3/State 4 and changes in MIM lipid composition, we hypothesized that the mitochondrial proton leak might be greater in cells from old compared with young mice. Thus we aimed, in the following set of experiments, to compare the overall kinetics of the mitochondrial proton leak in hepatocytes from old (30 mo) and young (3 mo) mice. We also conducted the first complete metabolic control analysis on oxidative phosphorylation in intact cells from old mice and report herein that the overall kinetics of the mitochondrial proton leak are altered by aging. Moreover, we show that there is a significant decrease in the amount of oxygen used to support the synthesis and use of ATP in old compared with young cells. Top-down metabolic control analysis showed that there is a shift in control away from substrate oxidation reactions toward increased control by the leak and by ATP turnover reactions in hepatocytes from old mice.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Treatment of animals. Twenty-seven male 30-mo-old C57BL/6J mice were received from the Veterans Administration Geriatric Research, Education, and Clinical Center at the University of Wisconsin (Madison, WI). These mice were part of a cohort purchased at 1 mo of age from Charles River Laboratories (Wilmington, DE), group housed (three per cage), and given free access to Purina 5001 chow diet and water. Twenty-seven young (3 mo) control C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). On receipt in Ottawa, mice were caged individually at 23°C with light from 0700 to 1900 and were given free access to a Purina 5001 chow diet and water. For the isolation of hepatocytes, nonfasted mice were anesthetized with 1 mg of pentobarbitone/100 g body weight and were killed between 0730 and 1100. Mice used in this study were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care, the Institute of Laboratory Animal Resources (National Research Council, USA), and with the Guiding Principles for Research Involving Animals and Human Beings.

Isolation and incubation of hepatocytes. Hepatocytes were isolated and incubated as earlier described (19). The viability of cells was >92% as determined by the exclusion of 0.3% (wt/vol) trypan blue. Before incubations, the cells were stored on ice in the isolation medium containing (in mM) 148 NaCl, 5 KCl, 0.81 MgSO4 · 7H2O, 0.83 Na2HPO4, 0.14 KH2PO4, 1 CaCl2, 25 NaHCO3, and 15 glucose.

For incubations, the cells were diluted approximately sevenfold in an incubation medium containing (in mM) 106 NaCl, 5 KCl, 25 NaHCO3, 0.41 MgSO4, 10 Na2HPO4, 2.5 CaCl2, 10 glucose, 10 lactate, 1 pyruvate, and 2.25% (wt/vol) defatted BSA. Stock 9% BSA was defatted by the method of Chen (12) and dialyzed against 153 mM NaCl and 11 mM KCl. Cell suspensions (3-5 ml of 6-9 mg dry wt cells/ml) were incubated in 20-ml stoppered glass vials at 37°C in a shaking water bath (100 cycles/min). To allow equilibration of the medium to a pH of 7.4, the gas phase above each suspension during incubations was 95% air-5% CO2. The cells were preincubated at 37°C in the shaking water bath for 10 min to allow the hepatocytes to reestablish ion gradients after being stored on ice. Cells were then incubated a further 20 min in the presence of the various inhibitors, uncouplers, and isotopes before aliquots were taken for the measurements of oxygen consumption and mitochondrial membrane potential (Delta Psi m). (Refer to Application of top-down elasticity analysis and top-down control analysis and to legends of Figs. 1-4 for the specific concentrations of inhibitors and uncouplers used in each experiment.)

Measurement of oxygen consumption. The respiration rate of hepatocytes was measured using a Hansatech (Norfolk, UK) Clark-type oxygen electrode, the incubation chamber of which was maintained at 37°C and magnetically stirred. Each rate was assessed in duplicate using 1.0 ml of cell suspension. Respiration rates are reported here as per 106 cells rather than per milligram dry weight of cells, as is more normal for rat hepatocyte preparations. This is simply because the yield of hepatocytes from a mouse is about one-tenth of that from a rat, and an inordinate amount of the final cell preparation would be needed to accurately assess dry cell weight. Cell counts were determined with a hemocytometer. All respiration rates were determined simultaneously and in parallel with the measurements of Delta Psi m. The resting respiration rate was defined as the oxygen consumption rate in the absence of inhibitors and uncouplers. Nonmitochondrial oxygen consumption was determined after the incubation of cells with maximal concentrations of oligomycin (1 µg/ml) and antimycin (5 µM) and with valinomycin (0.1 µM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (20 µM).

Measurement of Delta Psi m. Total aqueous volume, extracellular volume, and Delta Psi m were estimated from the distributions of 3H2O, [14C]methoxyinulin, and [3H]methyltriphenylphosphonium cation (TPMP+) as described previously (25, 36). Delta Psi m can be calculated by knowing the proportion of cytoplasmic volume that is occupied by the mitochondrial matrix [mitochondrial volume/cell volume (Vm/Vc)], the apparent activity coefficient of TPMP+ in each compartment (ae, ac, and am; where subscripts e, c, and m represent extracellular, cytoplasmic plus nuclear, and mitochondrial), and the extent of the accumulation of TPMP+ into the whole cell ([TPMP+]t/[TPMP+]e) and into the cytoplasm in relation to the external medium ([Cl-]e/[Cl-]t). The relationship between Delta Psi m and TPMP+ distribution at 37°C is shown below
&Dgr;&PSgr;<SUB>m</SUB> 
= −61.5 log <FENCE><FR><NU>V<SUB>c</SUB> · <IT>a</IT><SUB>m</SUB></NU><DE>V<SUB>m</SUB> · <IT>a</IT><SUB>c</SUB></DE></FR> <FENCE><FR><NU>[C1<SUP>−</SUP>]<SUB>t</SUB>[TPMP]<SUB>t</SUB> <IT>a</IT><SUB>c</SUB> (V<SUB>c</SUB> + V<SUB>m</SUB>)</NU><DE>[C1<SUP>−</SUP>]<SUB>e</SUB>[TPMP]<SUB>e</SUB> <IT>a</IT><SUB>e</SUB> V<SUB>c</SUB></DE></FR> − 1 </FENCE> </FENCE>
The determination and the actual values of these correction factors are outlined briefly in the following paragraphs.

Vc and Vm values were determined using quantitative morphometric cytology (45) as described previously (25). Briefly, isolated cells were fixed with glutaraldehyde, and electron micrographs of osmium-stained cells were prepared at a final magnification of 4,950-fold. Mitochondrial volume was determined from the number of intersections of a 1-cm grid overlaying the micrographs. Then the volume was calculated as the total number of intersections in mitochondria divided by the total number of intersections in cells (less the total number of intersections in lipid droplets). The cellular volume was corrected for the volume of lipid droplets, because TPMP+ is not taken up into fat (13). Mitochondrial matrix volume was calculated as 56.5% of total mitochondrial volume on the basis of the work of Loud (32), who calculated this weighted mean from the percentage matrix volume of midzonal, peripheral, and central liver cells and the percentage of these cell types in the whole liver.

[Cl-]e/[Cl-]t and plasma membrane potential (Delta Psi p) were determined from the distribution of 36Cl (35). Delta Psi p was 36.0 mV ± 6.2 (n = 3) and 39.3 mV ± 0.7 (n = 3) in cells from old and young mice, respectively.

The proportion of TPMP+ that is free (i.e., not bound) in mitochondria (am) and the cytoplasm (ac) was determined as described by Nobes et al. (36). Values for am were 0.247 (± 0.039; n = 2) and 0.171 (± 0.073; n = 3) for old and young, respectively. The ac values were 0.356 (± 0.019; n = 2) and 0.322 (± 0.015; n = 2) for old and young, respectively.

At the end of each incubation, triplicate aliquots (0.70 ml) were removed and pipetted into 1.5-ml minitubes and immediately centrifuged in a minicentrifuge for 2 min. Then 200-µl aliquots of the supernatant were removed and pipetted into scintillation vials and immediately mixed with scintillant. The residual supernatant was aspirated; the sides of each tube were wiped dry, and 40 µl of 20% (vol/vol) Triton X-100 were added. After the suspension of the pellet by vortex mixing, the bottom of the tube was cut off into a scintillation vial and the pellet was resuspended in 3.0 ml of scintillant. The radioactivities of the supernatant and pellet were determined by dual-channel scintillation counting for 3H and 14C by use of the appropriate quench and crossover corrections.

The apparent volume of pellet available to each isotope (its space in µl) was calculated as disintegrations per minute in total pellet divided by disintegrations per minute of supernatant sample. The [3H]TPMP+ accumulation ratio, ([TPMP+]t/[TPMP+]e), was calculated as ([3H]TPMP+ space - [14C]methoxyinulin space)/(3H2O space - [14C]methoxyinulin space).

Application of top-down elasticity analysis and top-down control analysis. To quantitatively determine the important sites of effects of aging on oxidative phosphorylation processes, we used the top-down elasticity analytic approach described by Brand (7). We defined the oxidative phosphorylation system as the tripartite system shown in Fig. 1 and then determined the overall elasticities to changes in Delta Psi m of the reactions that produce Delta Psi m (cellular catabolic reactions, the citric acid cycle, and the electron transport chain) and those that consume it (ATP synthesis and consumption and the proton leak). The kinetic response (or elasticity) of the Delta Psi m producers to Delta Psi m was measured by titrating the Delta Psi m consumers with oligomycin (0.01-0.05 µg/ml). The kinetic response of the leak to Delta Psi m was assessed by titrating with antimycin (0.05-0.25 µM), an inhibitor of complex III of the respiratory chain, in the presence of saturating amounts of oligomycin (1.0 µg/ml). The elasticity of the phosphorylating subsystem to Delta Psi m was measured from titrations with antimycin alone (0.10-0.20 µM). However, because the latter titrations provide the kinetics of both Delta Psi m-consuming subsystems (i.e., the phosphorylating and leak subsystems), corrections were made for the amount of oxygen required to balance the rate of the proton leak at each Delta Psi m measured. This was done using the proton leak titration curve.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1.   The oxidative phosphorylation system in hepatocytes. The intermediate within the system, the mitochondrial membrane potential (Delta Psi m), is produced by the substrate oxidation subsystem, which comprises all of the reactions including and following the oxidation of glucose, lactate, pyruvate, and endogenous substrates. Delta Psi m is consumed by the proton leak and phosphorylating subsystems. The proton leak subsystem consists of the leak of protons and any cation cycles across the mitochondrial inner membrane. The phosphorylating subsystem includes Delta Psi m-dependent ATP synthesis and all cellular ATP-consuming reactions.

To determine the distribution of control over respiration rate and over Delta Psi m in cells from old and young mice, we used top-down control analysis and the published equations for elasticities, flux control coefficients, and concentration control coefficients (11, 18).

Statistical analysis. Data were analyzed using unpaired Student's t-tests or ANOVA, which was followed by Tukey's post hoc tests. Linear regression lines were compared by analysis of covariance with use of Prism 2 for Windows. A P value of <0.05 was considered statistically significant. Unless otherwise stated, results are presented as means ± SE.

Materials. Oligomycin, antimycin, valinomycin, BSA (fraction V), collagenase (type IV), inulin, and trypan blue were from Sigma Chemical. FCCP and TPMP bromide were from Aldrich. 3H2O, Na[36Cl], [86Rb]Cl, [14C]methoxyinulin, and [3H]TPMP bromide were from Du Pont NEN. Water-insoluble compounds were dissolved in dimethyl sulfoxide.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Resting respiration rates and Delta Psi m values in hepatocytes from old and young mice. The resting respiration rate of hepatocytes from old mice was significantly less than that of hepatocytes from young controls (P < 0.04). Values were 70.9 ± 3.4 (n = 10) and 83.8 ± 5.1 (n = 7) nmol O2 · min-1 · 106 cells-1, respectively.

Resting state Delta Psi m was not significantly different between cells from old and young mice. Values were 149 ± 4.4 mV (n = 8) and 147 ± 3.4 mV (n = 7) in cells from old and young mice, respectively. State 4 Delta Psi m values were 155 ± 5.0 mV (n = 8) and 154 ± 3.0 mV (n = 7), respectively. Respiration rates and Delta Psi m values are indicated in Fig. 2. The Delta Psi m results indicate that any age-induced changes in the Delta Psi m consumers (i.e., leak and ATP turnover in the resting state, and leak alone in State 4) are matched by any changes in the Delta Psi m producers (i.e., substrate oxidation reactions).


View larger version (19K):
[in this window]
[in a new window]
 


View larger version (16K):
[in this window]
[in a new window]
 


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Kinetic responses of the mitochondrial proton leak (A), substrate oxidation (B), and phosphorylating (C) subsystems to Delta Psi m in hepatocytes from old and young rats. Oxygen consumption rates are corrected for nonmitochondrial oxygen consumption. Open symbols, old cells; filled symbols, young cells. Each symbol marked with an asterisk in B and C represents a resting point [i.e., in the absence of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and oligomycin]. A: kinetic response of mitochondrial proton leak to Delta Psi m (antimycin titration of oligomycin-inhibited respiration). B: kinetic response of substrate oxidation subsystem to Delta Psi m. Lines were fitted by linear regression to oligomycin titration points, showing no difference in kinetics (P > 0.05 by analysis of covariance) of substrate oxidation subsystem. C: kinetic response of the phosphorylating subsystem to Delta Psi m (antimycin titration of resting respiration rate from which rate of oxygen consumption used to balance the proton leak has been subtracted). Each point represents mean ± SE. Results are from cell preparations from the following nos. of old and young mice, respectively: 8 and 6 (A), 8 and 7 (B), and 5 and 5 (C). Each Delta Psi m determination was carried out in triplicate; each oxygen consumption determination was carried out simultaneously in duplicate.

Comparison of the kinetic responses of the mitochondrial proton leak, substrate oxidation, and phosphorylation subsystems to Delta Psi m in hepatocytes from old and young mice. The kinetic responses of the mitochondrial proton leak to Delta Psi m in hepatocytes are shown in Fig. 2A. The oxygen consumption rates are corrected for nonmitochondrial oxygen consumption, as described in EXPERIMENTAL PROCEDURES. These results show that, over a wide range of Delta Psi m values, the amount of oxygen used to support the mitochondrial proton leak is greater in cells from old mice. Because the kinetics of the proton leak subsystem are nonlinear, an analysis of covariance could not be used to test for statistically significant differences. However, taken together, the results show that the overall kinetics of the mitochondrial proton leak are altered in hepatocytes from old mice in relation to the results from young mice.

The kinetic responses of the substrate oxidation subsystem to Delta Psi m in old and young hepatocytes are compared in Fig. 2B. The results indicate that, at any value of Delta Psi m, there is no difference in the rate of the Delta Psi m-producing reactions between old and young hepatocytes. Thus there are no age-related differences in the overall kinetics of the substrate oxidation reactions.

A comparison of the kinetic responses of the phosphorylating subsystem to Delta Psi m in old and young hepatocytes revealed marked differences (P < 0.05; analysis of covariance) (Fig. 2C). At the resting state, the rate of the phosphorylating subsystem was ~30% lower in the old cells than in the young cells at identical values of Delta Psi m (147 mV).

Quantitative analysis of the effects of the altered kinetics of the mitochondrial proton leak and phosphorylating subsystems to Delta Psi m on respiration rate in old hepatocytes. The titrations of cellular respiration rate in old and young hepatocytes that were used to determine the kinetics of the subsystems described in Fig. 2 can be used to quantify the oxygen consumption that is used to sustain blocks of energy-dissipating reactions: nonmitochondrial oxygen consumption, proton leak reactions, and ATP turnover reactions as described by Brand (7). The proportion of resting cellular oxygen consumption that is nonmitochondrial was identified as that which was insensitive to saturating amounts of antimycin, oligomycin, valinomycin, and FCCP. There was no significant difference in the amounts of nonmitochondrial oxygen consumption; values for old and young cells were 22.1 ± 3.7 (n = 8) and 21.5 ± 1.2 (n = 7) nmol O2 · min-1 · 106 cells-1, respectively. The total mitochondrial oxygen consumption and the amounts used to balance the proton leak and ATP turnover reactions at the resting value of Delta Psi m are shown in Fig. 3. Despite the fact that the proportion of resting mitochondrial respiration used to balance the mitochondrial proton leak is doubled in the cells from old mice compared with young, there is a small but significant decrease in respiration (P < 0.04). This decrease can thus be accounted for entirely by a decrease (P < 0.03) in the rate of the only other block of reactions responsible for the dissipation of Delta Psi m, i.e., ATP synthesis and consumption reactions.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   Proportions of resting mitochondrial oxygen consumption due to proton leak and ATP turnover reactions. Total oxygen consumption is the resting rate of mitochondrial oxygen consumption measured in the absence of any inhibitors or uncouplers and represents the sum of the oxygen required to fuel the phosphorylation and leak pathways. Total oxygen consumption is significantly lower in the old (open bars) than in the young (solid bars, *P < 0.05). Oxygen used to balance mitochondrial proton leak is determined by extrapolation from titration curves shown in Fig. 2A; oxygen consumption attributable to leak at resting Delta Psi m is subtracted from total resting mitochondrial oxygen consumption. Statistical analyses show that the apparently greater leak in old cells does not reach statistical significance. Data were analyzed using ANOVA and Tukey's post hoc tests. The SE (for total mitochondrial oxygen consumption) and pooled SE (for proton leak-dependent and ATP turnover-dependent oxygen consumption) are indicated; the SE for proton leak at resting Delta Psi m was estimated as the mean of the SE for the 2 data points adjacent to it on proton leak curve in Fig. 2A.

Application of top-down control analysis. As well as being useful in the identification of the sites of action of an external effector and in the quantitative determination of the importance of changes induced within the system by an external effector, top-down elasticity analyses provide all the data needed for a top-down control analysis of the system (11, 18). A top-down control analysis was completed using the data from the present elasticity analyses around Delta Psi m in old and young hepatocytes. The results for cells in the resting state and in State 4 are shown in Tables 1-3. The results were calculated using mitochondrial respiration rates; similar elasticities and control coefficients were obtained when calculations were based on total cellular respiration rates. The elasticities to Delta Psi m of the substrate oxidation, phosphorylating, and proton leak subsystems are given in Table 1. All of the data needed for the calculation of elasticity and control coefficients can be obtained from Delta Psi m and oxygen consumption values in Fig. 2, A, B, and C. Values of each, and the inverse slopes of the respective elasticity lines, are then used in the published series of equations (18) for the calculation of control coefficients.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Overall elasticities to Delta Psi m for substrate oxidation, phosphorylating, and proton leak subsystems in hepatocytes from old and young control mice

The flux control coefficients of the three subsystems over the rate of each of the subsystems are shown in Table 2, A-C. The flux control coefficients describing the distribution of control over mitochondrial oxygen consumption (JS) are shown in Table 2A. The results from the young control hepatocytes in the resting state indicate that the substrate oxidation reactions (0.51; i.e., 51% of the control) and phosphorylation reactions (0.45) exert most of the control over JS, whereas the remainder of the control is through the proton leak (0.04). These results are similar to those obtained with hepatocytes from euthyroid rats (24, 25); however, the amount of control exerted by the proton leak is smaller here than with the latter. This is most likely due to the respiration state in these cells under our resting incubation conditions; it is possible that these hepatocytes are respiring at a state closer to State 3. As mitochondrial respiration approaches State 3, the amount of control exerted by the leak over oxygen consumption approaches zero (11). Moreover, these metabolic control data are the first from cells of mice, and these differences may indeed reflect a species effect. Results from old mice, compared with those from young controls, show an interesting shift in control over JS away from substrate oxidation reactions toward the phosphorylation and leak reactions. In old cells, 54 and 12% of the control over resting mitochondrial oxygen consumption are mediated through phosphorylation and leak reactions, respectively. The corresponding values in young control cells are 45 and 4%.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Flux control coefficients over subsystem fluxes and concentration control coefficients over Delta Psi m in hepatocytes from old and young control mice

Results shown in Table 2B similarly show that, in old cells compared with young, there is a shift in control over the phosphorylation reactions away from substrate oxidation reactions toward phosphorylation reactions and leak reactions. Control by the substrate oxidation reactions and phosphorylation reactions over the flux through the mitochondrial proton leak reactions (Table 2C) in old cells compared with young shows that there are decreases to roughly equal but opposite extents for these two blocks of reactions. Control over leak flux by the leak reactions themselves remains high in both old and young.

The concentration control coefficients of the three subsystems over the intermediate in the system, Delta Psi m, are shown in Table 2D. Control coefficients describe the distribution of control by blocks of reactions in a system over the amount of the intermediate in the system, and, unlike flux control coefficients that sum to unity, concentration control coefficients sum to zero. The values are also similar to those determined for hypothyroid cells (25) and for euthyroid cells (11, 25). The results for old and young cells are roughly similar and show that most of the control over the amount of the intermediate, Delta Psi m, is exerted by the substrate oxidation subsystem, and that the remainder of the control is through the activity of the Delta Psi m consumers: the proton leak and phosphorylating subsystems.

As described by Brand et al. (6), it is possible to estimate the actual number of ATP molecules synthesized by mitochondria for each oxygen atom consumed (effective P/O ratio) and the distribution of control (i.e., control coefficients) over the effective P/O by use of data such as those described above. Because phosphorylation flux is measured as the oxygen consumed to drive the phosphorylation reactions, the ratio of JP divided by JS provides the fraction of oxygen consumption that is used to support phosphorylation, irrespective of the true value of the maximum P/O. The remaining fraction of the oxygen consumption (JL/JS) is used to support mitochondrial proton leak reactions. Thus the effective P/O at any rate between State 3 and State 4 is this ratio multiplied by the maximum P/O, P/Omax. The effective P/O values, based on mitochondrial oxygen consumption data, are shown in Table 3. In theory, the oxidation of glucose by cells produces a maximum of 31 molecules of ATP per molecule of glucose (27); this corresponds to a P/Omax of 2.58. The values shown in Table 3 for mitochondrial respiration are higher than the previously published values (6) and again support the postulate that cells are metabolically positioned close to State 3 respiration.

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Control coefficients over effective P/O ratio in hepatocytes from old and young control mice

Brand et al. (6) reported the derivation of flux control coefficients, which quantitatively describe the control by the three blocks of reactions over the effective P/O
<IT>C</IT><SUP>P/O</SUP><SUB>S</SUB> = <IT>C</IT><SUP><IT>J</IT>p</SUP><SUB>S</SUB> − <IT>C</IT><SUP><IT>J</IT>s</SUP><SUB>S</SUB>
<IT>C</IT><SUP>P/O</SUP><SUB>P</SUB> = <IT>C</IT><SUP><IT>J</IT>p</SUP><SUB>P</SUB> − <IT>C</IT><SUP><IT>J</IT>s</SUP><SUB>P</SUB>
<IT>C</IT><SUP>P/O</SUP><SUB>L</SUB> = <IT>C</IT><SUP><IT>J</IT>p</SUP><SUB>L</SUB> − <IT>C</IT><SUP><IT>J</IT>s</SUP><SUB>L</SUB>
These equations were used with the flux control coefficients shown in Table 2 by use of values from the resting respiration states; the resulting coefficients are shown in Table 3. Because these control coefficients describe control over the fraction of the oxygen flux that is used to drive ATP synthesis (i.e., over the ratio JP/JS), their values are independent of assumed or calculated values of P/Omax.

Similar to the results for hepatocytes described earlier (6), the block of reactions with the least amount of control is that of substrate oxidation reactions, showing that increases in substrate supply on their own produce only very small changes in the effective P/O. The control coefficients for the leak reactions over the effective P/O are relatively large and negative, indicating that increases in mitochondrial proton leak reactions would cause substantial decreases in the effective P/O. The coefficients from cells of old compared with those of young mice differ in that the amounts of control by phosphorylation and leak reactions are more than double in each case. Control by substrate oxidation reactions remains low in old and young cells. These results indicate that, in hepatocytes from old mice, the efficiency of oxidative phosphorylation is more sensitive to changes in the amount of mitochondrial proton leak and in the rate of ATP synthesis and turnover reactions.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Our initial hypothesis was that increases in the mitochondrial proton leak occur with aging as an effect of oxidative damage to the lipids of the MIM. This damage then affects the integrity of the lipid bilayer through altered lipid-lipid or lipid-protein interactions, the outcome being an increased proton leak. Changes in the mitochondrial proton leak with aging were expected to be rather small, but it was anticipated that a kinetic approach, such as elasticity analysis, in which the activities of blocks of reactions are assessed over a range of metabolic "challenges," would identify and quantify any such changes. Our findings do indeed show that the overall kinetics of the mitochondrial proton leak are altered by age such that over a range of Delta Psi m values, there is an increased rate of oxygen consumed to balance the rate of the proton leak. Chronic increases in mitochondrial proton leak may have negative implications, because even minor increases are likely to initiate compensatory increases in the activity of the proton pumps of the electron transport chain to maintain protonmotive force. This may, in turn, result in the increased production of free radicals at the level of the electron transport chain. As discussed in more detail below, our results do not indicate any changes in the kinetics of the substrate oxidation reactions. The results do, however, show a marked decrease in the ATP turnover reactions.

Changes in the overall kinetics of the mitochondrial proton leak are consistent with the recent report of Hagen et al. (21), who studied mitochondrial membrane potentials, cellular production of oxidants, and levels of age-associated mtDNA deletions in rat hepatocytes. Delta Psi m was assessed using rhodamine 123 accumulation. Three distinct hepatocyte populations from old rats were collected by centrifugal elutriation and were found to have differing Delta Psi m values. One population of cells was collected from the young rats. In the largest population of old cells (~66% of cells in the preparation), membrane potential was ~40% lower than that of the young cells. A smaller subset of hepatocytes from the livers of old rats (~25% of the cells) had only slightly lower fluorescence than those from young rats. In the third subset, membrane potential was equal or greater than that of the young cells. Their rhodamine 123 fluorescence results were corroborated by quantitative determinations of potentials by assessing mitochondrial uptake of radiolabeled tetraphenylphosphonium ion. Membrane potential in young hepatocytes was 154.3 ± 20.4 mV; this value is similar to that reported herein, 147 ± 3.4 mV. In their three populations of old hepatocytes, values were 70.4 mV (66% of cells), 92.6 mV (25% of cells), and 154.4 mV (remainder of cells). The resting Delta Psi m in our old cells was 149 ± 4.4 mV, which is clearly not different from our mean value in young cells. Hagen et al. also examined resting oxygen consumption rates of hepatocytes. Although their absolute values appear high, their finding that oxygen consumption was 36% lower in the largest fraction of hepatocytes compared with young cells supports our findings. Finally, in support of the oxidative stress theory, they found that both the largest and the smallest fractions of cells produced significantly more oxidants than cells from young rats.

The very recent findings of Brookes et al. (10) also support our findings. In their study on the effects of peroxynitrite on brain mitochondria, they found that three sequential additions of 200 µM peroxynitrite (initial concentration) to rat brain mitochondria (0.2 mg protein/ml) significantly stimulated mitochondrial proton leak. Cyclosporin A did not affect the stimulation, suggesting no involvement of the mitochondrial permeability transition pore. However, the stimulation was prevented by the vitamin E analog Trolox, supporting the involvement of lipid peroxidation, a proposed mechanism of peroxynitrite cytotoxicity. The authors link lipid peroxidation-mediated increases in mitochondrial proton leak to certain neurodegenerative disorders thought to proceed via mechanisms of mitochondrial oxidative damage.

The mechanism of proton leakage warrants discussion, particularly in light of recent developments in this area. Potential mechanisms underlying the mitochondrial proton leak are depicted in Fig. 4. Within the past year, two uncoupling proteins (UCP2 and UCP3) were identified and found to be distinct from the brown adipose tissue-specific uncoupling protein (UCP1) (4, 15, 44). These proteins explain at least some of the proton leak that has been assessed in mitochondria from a variety of tissues. Thus far, none of the three currently known uncoupling proteins have been found in parenchymal cells, which constitute 95-97% of the cells isolated using the techniques employed (2). Fleury et al. (15) report low levels of UCP2 mRNA in liver; however, recent findings localize UCP2 to the Kuppfer cells of the liver (31). Nevertheless, it is possible, and likely, that there is an as-yet-unidentified uncoupling protein in the parenchymal cells. Furthermore, it is possible that there are changes in the amount and/or activity of such an uncoupling protein in these cells. However, again, our underlying hypothesis, founded on a substantial supportive literature, implicates free radical damage to lipids, thus affecting the integrity of the bilayer at the lipid-lipid or lipid-protein interfaces.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 4.   Proposed mechanisms for mitochondrial proton leak. Activity of redox proton pumps of electron transport chain creates protonmotive force. The latter drives ATP synthase activity. Mitochondrial proton leak, regardless of its mechanism, allows protons to bypass ATP synthase, and protonmotive force is dissipated. Thus additional oxygen is used by the chain to maintain protonmotive force. The mechanism of the leak in parenchymal cells of the liver is as yet unknown but may involve an as-yet-unidentified uncoupling protein and/or bilayer lipid composition of the mitochondrial inner membrane.

On the basis of previous reports of changes in the amounts and activities of a wide range of enzymes involved in substrate oxidation and specifically in components of the electron transport chain (3, 22, 43), we expected to observe changes in the kinetics of the substrate oxidation reactions. We hypothesized that we might observe results reflecting a decreased activity of this block of reactions. Alternatively, it was reasoned that, as a result of the increased proton leak, there may be a compensatory increase in the rate of the substrate oxidation reactions in an effort to restore protonmotive force to normal values; this would then accelerate the production of reactive oxygen metabolites, which in turn would induce further oxidative damage to cellular components. Our data (Fig. 2B) show clearly that any such changes as measured under our conditions are quantitatively insignificant. This does not indicate that free radical damage has not occurred but shows only that this group of reactions responds normally to imposed changes in the rate of its activity and may indicate also that the functioning of this block of reactions is at some level protected.

Our results describing decreases in the proportion of resting oxygen consumption of hepatocytes and altered kinetics of the phosphorylation reactions were not anticipated. Beyond the extensive analyses of respiratory control ratios in the literature (see for example Ref. 22), data on age-related alterations in ATP synthesis and turnover reactions are lacking. Thus it is difficult to speculate about the specific mechanisms responsible for the decreased amount of oxygen used by cells to support these reactions. As described in Fig. 1, ATP synthesis and turnover mechanisms include ATP synthetic reactions, such as the adenine nucleotide carrier and the phosphate transporter. This block of reactions also includes all cellular ATP-consuming processes, such as those involved in maintaining ion gradients across membranes (e.g., Ca2+-ATPase, Na+-K+-ATPase) and in protein, DNA, and RNA synthetic reactions. Consistent with the oxidative stress theory, these findings may be related to known age-related increases in oxidative damage to mitochondrial proteins and DNA and should be examined further.

Importantly, the findings herein provide the first metabolic control analysis of oxidative phosphorylation in relation to the metabolic effects of aging. The data provide quantitative information about the control over resting oxygen consumption, over other blocks of reactions, and over Delta Psi m. In old compared with young cells, there is a shift in control over resting oxygen consumption away from the substrate oxidation reactions toward phosphorylation and leak reactions. Thus oxygen consumption of old cells is more sensitive to changes in the rate of ATP turnover and in mitochondrial proton leak rate. Similarly, there is a shift in control over phosphorylation reactions away from substrate oxidation reactions toward phosphorylation reactions and the leak in old cells.

In addition, the control coefficients describing the control over the effective P/O denote that a greater amount of control is possessed by the ATP turnover reactions and the leak in old hepatocytes compared with young. This suggests an augmented capability of the latter blocks of reactions to affect changes in the efficiency of oxidative phosphorylation.

Overall, our findings, gathered from a relatively novel experimental perspective, extend our understanding of the effects of aging on oxidative phosphorylation in hepatocytes. The findings confer additional support for the oxidative stress theory of aging. They provide new quantitative data on the altered kinetics of the mitochondrial proton leak and of ATP turnover reactions and show shifts in metabolic control with aging.

    ACKNOWLEDGEMENTS

The authors thank Rod Nicholls for assistance with the electron microscopy of cells.

    FOOTNOTES

This study was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada (M. E. Harper) and the National Institutes of Health (RR-00167, R. Weindruch and J. J. Ramsey, and PO1 AG-11915, R. Weindruch).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: M. E. Harper, Dept. of Biochemistry, Microbiology and Immunology, Faculty of Medicine, Univ. of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada K1H 8M5.

Received 15 January 1998; accepted in final form 23 April 1998.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

1.   Ames, B., M. Shigenaga, and T. Hagen. Oxidants, antioxidants and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA 90: 7915-7922, 1993[Abstract/Free Full Text].

2.   Berry, M. N., A. M. Edwards, and G. J. Barritt. Isolated Hepatocytes---Preparation, Properties and Applications. Amsterdam: Elsevier, 1991.

3.   Boffoli, D., S. Scacco, R. Vergari, G. Solarino, G. Santacroce, and S. Papa. Decline with age of the respiratory chain activity in human skeletal muscle. Biochim. Biophys. Acta 1226: 73-82, 1994[Medline].

4.   Boss, O., S. Samec, A. Paolonigiacobino, C. Rossier, A. Dulloo, J. Seydoux, P. Muzzin, and J. P. Giacobino. Uncoupling protein-3---a new member of the mitochondrial family with tissue-specific expression. FEBS Lett. 408: 39-42, 1997[Medline].

5.   Bowling, A., E. Mutisya, L. Walker, D. Price, L. Cork, and M. Beal. Age-dependent impairment of mitochondrial function in primate brain. J. Neurochem. 60: 1964-1967, 1993[Medline].

6.   Brand, M., M.-E. Harper, and H. C. Taylor. Control of the effective P/O ratio of oxidative phosphorylation in liver mitochondria and hepatocytes. Biochem. J. 291: 739-748, 1993[Medline].

7.   Brand, M. D. The proton leak across the mitochondrial inner membrane. Biochim. Biophys. Acta 1018: 128-133, 1990[Medline].

8.   Brand, M. D. Top down metabolic control analysis. J. Theoret. Biol. 182: 351-360, 1996[Medline].

9.   Brand, M. D., D. A. L. H. Reis, and R. P. Hafner. Stimulation of the electron transport chain in mitochondria isolated from rats treated with mannoheptulose or glucagon. Arch. Biochem. Biophys. 283: 278-284, 1990[Medline].

10.   Brookes, P. S., J. M. Land, J. B. Clark, and S. J. R Heales. Peroxynitrite and brain mitochondria: evidence for increased proton leak. J. Neurochem. 70: 2195-2202, 1998[Medline].

11.   Brown, G., R. P. Hafner, and M. D. Brand. A "top-down" approach to the determination of control coefficients in metabolic control theory. Eur. J. Biochem. 188: 321-325, 1990[Abstract].

12.   Chen, R. F. Removal of fatty acids from serum albumin by charcoal treatment. J. Biol. Chem. 242: 173-181, 1967[Abstract/Free Full Text].

13.   Davis, R. J., M. D. Brand, and B. R. Martin. The effect of insulin on plasma-membrane and mitochondrial-membrane potentials in isolated fat-cells. Biochem. J. 196: 133-147, 1981[Medline].

14.   Di Monte, D., M. Sandy, L. DeLanney, S. Jewell, P. Chan, I. Irwin, and J. Langston. Age-dependent changes in mitochondrial energy production in striatum and cerebellum of the monkey brain. Neurodegeneration 2: 93-99, 1993.

15.   Fleury, C., M. Neverova, S. Collins, S. Raimbault, O. Champigny, C. Levi-Meyrueis, F. Bouillard, M. Seldin, R. Surwit, D. Ricquier, and C. Warden. Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat. Genet. 15: 269-272, 1997[Medline].

16.   Fusi, F., G. Sgaragli, and M. P. Murphy. Interaction of butylated hydroxyanisole with mitochondrial oxidative phosphorylation. Biochem. Pharmacol. 43: 1203-1208, 1992[Medline].

17.   Gerschman, R., D. Gilbert, S. Nye, P. Dwyer, and W. Fenn. Oxygen poisoning and x-irradiation: a mechanism in common. Science 119: 623-629, 1954.

18.   Hafner, R., G. C. Brown, and M. D. Brand. Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and protonmotive force in isolated mitochondria using the "top-down" approach of metabolic control theory. Eur. J. Biochem. 188: 313-319, 1990[Abstract].

19.   Hafner, R. P., G. C. Brown, and M. D. Brand. Thyroid hormone control of state 3 respiration in isolated rat liver mitochondria. Biochem. J. 265: 731-734, 1990[Medline].

20.   Hafner, R. P., C. D. Nobes, A. D. McGown, and M. D. Brand. Altered relationship between protonmotive force and respiration rate in non-phosphorylating liver mitochondria isolated from rats of different thyroid hormone status. Eur. J. Biochem. 178: 511-518, 1988[Abstract].

21.   Hagen, T. M., D. L. Yowe, J. C. Bartholomew, C. M. Wehr, K. L. Do, J.-Y. Park, and B. N. Ames. Mitochondrial decay in hepatocytes from old rats: membrane potential declines, heterogeneity and oxidants increase. Proc. Natl. Acad. Sci. USA 94: 3064-3069, 1997[Abstract/Free Full Text].

22.   Hansford, R. Bioenergetics in aging. Biochim. Biophys. Acta 726: 41-80, 1983[Medline].

23.   Harman, D. Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 11: 298-300, 1956[Medline].

24.   Harper, M.-E., and M. D. Brand. Hyperthyroidism stimulates mitochondrial proton leak and ATP turnover in rat hepatocytes but does not change the overall kinetics of substrate oxidation reactions. Can. J. Physiol. Pharmacol. 72: 899-908, 1994[Medline].

25.   Harper, M.-E., and M. D. Brand. The quantitative contributions of mitochondrial proton leak and ATP turnover reactions to the changed respiration rates of hepatocytes from rats of different thyroid hormone status. J. Biol. Chem. 268: 14850-14860, 1993[Abstract/Free Full Text].

26.   Harper, M.-E., and M. D. Brand. Use of top-down elasticity analysis to identify sites of thyroid hormone-induced thermogenesis. Proc. Soc. Exp. Biol. Med. 208: 228-237, 1995[Abstract].

27.   Hinkle, P. C., M. A. Kumar, A. Resetar, and D. L. Harris. Mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Biochemistry 30: 3576-3582, 1991[Medline].

28.   Hoch, F. Lipids and thyroid hormones. Prog. Lipid Res. 27: 199-270, 1988[Medline].

29.   Hoch, F. Cardiolipins and biomembrane function. Biochim. Biophys. Acta 1113: 71-133, 1992[Medline].

30.   Laganiere, S., and B. P. Yu. Modulation of membrane phospholipid fatty acid composition by age and food restriction. Gerontology 39: 7-18, 1993[Medline].

31.   Larrouy, D., P. Laharrague, G. Carrera, N. Vigueriebascands, C. Levi-Meyrueis, C. Fleury, C. Pecqueur, M. Nibbellink, M. Andre, L. Casteilla, and D. Ricquier. Kuppfer cells are a dominant site of uncoupling protein 2 expression in rat liver. Biochem. Biophys. Res. Commun. 235: 760-764, 1997[Medline].

32.   Loud, A. V. A quantitative stereological description of the ultrastructure of normal rat liver parenchymal cells. J. Cell Biol. 37: 27-46, 1968[Abstract/Free Full Text].

33.   Mecocci, P., U. MacGarvey, A. Kaufman, D. Koontz, J. Shoffner, D. Wallace, and M. Beal. Oxidative damage to mitochondrial DNA shows marked age-dependent increases in human brain. Ann. Neurol. 33: 609-616, 1993.

34.   Müller-Höcker, J. Cytochrome c oxidase deficient fibres in the limb muscle and diaphragm of man without muscular disease: an age-related alteration. J. Neurol. Sci. 100: 14-21, 1990[Medline].

35.   Nobes, C. D., and M. D. Brand. A quantitative assessment of the use of 36 Cl- distribution to measure plasma membrane potential in isolated hepatocytes. Biochim. Biophys. Acta 987: 115-123, 1989[Medline].

36.   Nobes, C. D., G. C. Brown, P. N. Olive, and M. D. Brand. Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes. J. Biol. Chem. 265: 12903-12909, 1990[Abstract/Free Full Text].

37.   Nobes, C. D., W. W. Hay, Jr., and M. D. Brand. The mechanism of stimulation of respiration by fatty acids in isolated hepatocytes. J. Biol. Chem. 265: 12910-12915, 1990[Abstract/Free Full Text].

38.   Nobes, C. D., P. L. Lakin-Thomas, and M. D. Brand. The contribution of ATP turnover by the Na+/K+-ATPase to the rate of respiration of hepatocytes. Effects of thyroid status and fatty acids. Biochim. Biophys. Acta 987: 115-123, 1989[Medline].

39.   Richter, C., J. Park, and B. Ames. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc. Natl. Acad. Sci. USA 85: 6465-6467, 1988[Abstract].

40.   Shigenaga, M., T. Hagen, and B. Ames. Oxidative damage and mitochondrial decay in aging. Proc. Natl. Acad. Sci. USA 91: 10771-10778, 1994[Abstract/Free Full Text].

41.   Sohal, R. S., and R. Weindruch. Oxidative stress, caloric restriction, and aging. Science 273: 59-63, 1996[Abstract].

42.   Stadtman, E. Protein oxidation and aging. Science 257: 1220-1224, 1992[Medline].

43.   Torii, K., S. Sugiyama, K. Takagi, T. Satake, and T. Ozawa. Age-related decrease in respiratory muscle mitochondrial function in rats. Am. J. Respir. Cell Mol. Biol. 6: 88-92, 1992[Medline].

44.   Vidalpuig, A., G. Solanes, D. Grujic, J. S. Flier, and B. B. Lowell. Ucp3---an uncoupling protein homologue expressed abundantly in skeletal muscle and brown adipose tissue. Biochem. Biophys. Res. Commun. 235: 79-82, 1997[Medline].

45.   Weibel, E. Stereological principles for morphology in electron microscopic cytology. Int. J. Rev. Cytol. 26: 235-302, 1969.

46.   Weindruch, R., and R. S. Sohal. Caloric intake and aging. New Eng. J. Med. 337: 986-994, 1997[Free Full Text].

47.   Wilson, P. D., and L. M. Franks. The effect of age on mitochondrial ultrastructure and enzymes. Adv. Exp. Med. Biol. 53: 171-183, 1975[Medline].

48.   Yu, B. P. Aging and oxidative stress---modulation by dietary restriction. Free Radical Biol. Med. 21: 651-668, 1996[Medline].

49.   Yu, B. P., E. Suescun, and S. Yang. Effect of age related lipid peroxidation on membrane fluidity and phospholipase A2: modulation by dietary restriction. Mech. Ageing Dev. 65: 17-33, 1992[Medline].


Am J Physiol Endocrinol Metab 275(2):E197-E206
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society