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The Effects of Dietary Restriction on Mitochondrial Dysfunction in Aginga

RITCHIE J. FEUERSb

Department of Genetic Toxicology, National Center for Toxicological Research,3900 NCTR Road, Jefferson, Arkansas 72079

ABSTRACT: Age-associated alterations in the mitochondrial electron transport sys-tem (ETS) may lead to free radical generation and contribute to aging. The com-plexes of the ETS were screened spectrophotometrically in gastrocnemius of young(10 month) as well as older (20 and 26 month) B6C3F1 female mice fed an ad libitum(AL) diet or a restricted (DR) in total calories diet (40% less food than AL mice). Theactivities of complexes I, III, and IV decreased significantly by 62%, 54%, and 74%,respectively, in old AL mice (AL20) compared to young AL mice (AL10). Complexes I,III, and IV from DR10 mice had activities that were significantly lower than thoseseen in AL10 mice (suggesting a lower total respiratory rate or improved efficiency).By contrast, complex II activity did not decrease with age (actually increased, but notsignificantly) in AL20 mice. Complex II was decreased across age in DR mice. Km forubiquinol-2 of complex III was significantly increased in AL10 animals (0.33 mM vs.0.26 mM in DR10 mice) and was further increased with aging (0.44 mM in AL20 vs.0.17 mM in DR20 mice). This suggests obstruction of binding, inhibition of electronflow in aging, which could yield premature product release as a free radical. Totalcomplex IV by Vmax was highest in AL10 mice, but the proportion of complex as high-affinity sites was lower (69%) than in either DR10 (80%) or DR20 (80%). The per-centage of high-affinity sites decreased to only 45% in AL20 mice, and Vmax wasreduced by 75 percent. In AL26 mice high-affinity sites decreased to 33 percent. Atphysiologic concentration of reduced cytochrome c, significant dysfunction of com-plex IV in AL20 or AL26 mice would be expected with obstruction of overall electrontransport. The age-associated loss of activity and function of complexes I, III, and IVmay contribute to increased free radical production. Lack of sufficient DNA repairin mitochondria and juxtaposition to the ETS adds to susceptibility and accumula-tion of mtDNA and other mitochondrial macromolecular damage. DR seems toretard this deterioration of mitochondrial respiratory function by preserving enzy-matic activities and function.

It was initially suggested by Harman1 that free radicals may induce molecular damage andcontribute to aging, and a growing body of evidence suggests that oxidative metabolismplays a central role in any mechanism of aging. The generation of ATP takes place in mito-chondria where four electrons are added to O2 with subsequent production of H2O. How-ever, it has been estimated that there may be as much as a 1–2% error for 1 electronadditions.2 Thus, electron transport (ETS) and oxidative phosphorylation have evolved asefficient systems, but a significant number of reactive oxygen species (ROS) may be pro-duced over time within mitochondria.3–7 In an escalating process, mitochondria may act asan initiating source of ROS, promoting their own disarray and destruction.8

aThis work was supported in part by the National Center for Toxicological Research and theNational Institute on Aging.

bTel: 870/543-7437 or -7330; fax: 870/543-7136; e-mail: [email protected]

FEUERS: DIETARY RESTRICTION AND ELECTRON TRANSPORT IN AGING 193

It can be argued that the rate of aging is dependent upon the balance between ROS andquantitative and qualitative aspects of cellular antioxidant systems. These opposing forcesproduce an acceptable steady state level of ROS due to antioxidant advantages in younganimals.9 However, this steady state level of ROS may increase with age as antioxidantsystems are compromised.10 For example, it has been shown that quantitative and qualita-tive activities of glucose 6-phosphate dehydrogenase, glutathione peroxidase, and catalaseare compromised with age,11,12 and in such oxidatively active tissues as skeletal muscle,antioxidant enzyme systems are induced within the mitochondrial fraction.13

Ames14 has estimated that the number of oxidative lesions per cell per day to the DNAmay be as high as 100,000 in the rat. This damage has been shown to be rather ubiquitousbecause it involves not only nuclear DNA damage, but damage to mtDNA, proteins,15

membranes, and other macromolecules. However, much of the damage is repaired.14

Unfortunately, as with the antioxidant systems, these repair processes are not perfectlyefficient and limited, as is the case with mtDNA repair. It has been shown that as many as2 million DNA lesions/cell may accumulate in aging (2 years) rat tissue,16 and proteinlesions also accumulate with age.15 Inasmuch as oxidative damage increases and mito-chondrial function seem to decay with age,14 a case may be made that mitochondrial dys-function plays a central role in any mechanism of aging. A growing literature suggests thatalterations in mitochondrial ETS may be involved. Decreases with age in the activities ofthe four ETS complexes have been demonstrated for human skeletal muscle,17–19 whereasdeclines in the activities of the ETS complexes with age in rat muscle have also beennoted.8,20,21

Dietary restriction (DR) extends maximum life span and retards both the rate of biolog-ical aging and the development of age-associated degenerative diseases.22–24 The mecha-nism of DR’s action is not clear, but it has been speculated that, in part, DR may act byreducing mitochondrial free radical generation.7,8,23–25

MATERIAL AND METHODS

Animals and Housing

Female B6C3F1 mice were bred and raised in a specific pathogen-free animal facilityat the National Center for Toxicological Research (NCTR), using procedures describedpreviously.26 This is a long-lived genotype. Females at the NCTR colony fed the NIH-31diet ad libitum have an average life span of 30 months and a maximum life span (i.e., themean for the longest-lived decile) of 36 months. Animals were maintained individually inplastic cages with wire metal tops and hardwood chip bedding at 23°C. Cages werechanged weekly, and fresh water was always available.

Experimental Design and Dietary Regimen

All mice were weaned at 3 weeks of age and fed the standard NIH-31 diet (containing0.315% vitamin mixture and 0.185% mineral mixture) until they reached 14 weeks of age.The mice were then randomly assigned to either a control group, which was fed the stan-dard NIH-31 diet ad libitum (AL) or a group subjected to DR, which received 60% of the

194 ANNALS NEW YORK ACADEMY OF SCIENCES

AL intake of the NIH-31 diet supplemented with vitamins and a mineral mixture at 1.67times that in the standard diet. In our colony, this DR regimen increases the average andmaximum life spans to 41 and 47 months.

Tissue Collection and Preparation

A total of 40 mice were studied: 18 (n = 9 AL, n = 9 DR) were 10-month-old mice, 18 (n= 9 AL, n = 9 DR) were 20-month-old mice, and 4 were 26-month-old mice fed the ALdiet. For two weeks prior to experimentation, DR animals were fed at the beginning of thedark span (1600–0400 h). All mice were killed by cervical dislocation between 1800 and1900 hours. The gastrocnemius muscle was removed, frozen in liquid nitrogen, and thenstored at –70°C. Later, the tissues were thawed on ice, weighed, minced, and homogenizedusing a Tekmar homogenizer. All tissues were homogenized at 4°C in 50 mM phosphatebuffer (pH 7.4) in volumes 10 times the weight of tissue. Crude homogenates were centri-fuged at 1000 g for 10 min at 4°C in a Beckman Model LS-75 centrifuge using a 50 Tirotor. The supernatants were collected and centrifuged again at 12,000 g for 10 min at 4°C.The resultant pellets were suspended in 1 mL of buffer (250 mM mannitol, 70 mM sucrose,1 mM EDTA, pH 7.4) and recentrifuged at 12,000 g for 10 min at 4°C. Finally, the 12,000 gpellets were resuspended in 0.5 mL of second buffer at –70°C until time of assay.

Enzyme Analyses

All four complexes of the mitochondrial ETS were measured spectrophotometricallyon a COBAS FARA II autoanalyzer (Roche, New York) using modifications of themethod of Ragan et al.27 All the chemicals except ubiquinol were from Sigma (St. Louis,MO). Ubiquinol was graciously supplied by Dr. T. Ichien (Eisai Co., Tokyo, Japan). Com-plex I (NADH-ubiquinone oxidoreductase; EC 1.6.99.3) activity was measured by moni-toring the oxidation of NADH to NAD by ubiquinone-1 at 30°C. The reaction mixturecontained potassium phosphate buffer (KPB, 10 mM, pH 8.0), NADH (5 mM), lecithin(15 mg/mL), and the mitochondrial fraction (9 µL). The reaction was started by additionof ubiquinone-1 (10 mM) to the reaction mixture, and the decrease in absorbance wasmeasured at 340 nm. Complex II (succinate-ubiquinone oxidoreductase; EC 1.3.5.1) activ-ity was measured as a rate of reduction of ubiquinone-2 by succinate at 30°C followed bythe secondary reduction of 2,6-dichlorophenolindophenol (DPIP) by the ubiquinolformed. The reaction mixture contained KPB (1 mM, pH 7.4) and distilled water. Thedecrease in absorbance was measured at 600 nm after the addition of a mixture of DPIP (1mM), ubiquinone (2.5 mM), and the mitochondrial fraction (4 µL) to the above reactionmixture. Complex III (ubiquinol-cytochrome c oxidoreductase; EC 1.10.2.2) activity wasassayed by following the rate of reduction of cytochrome c by ubiquinol-2 at 30°C. Thereaction was started by addition of ubiquinol-2 to KPB (50 mM), EDTA (100 mM), cyto-chrome c (1 mM), and the mitochondrial fraction (5 µL) diluted 1:10 with sucrose (250mM): Tris-HCl (10 mM) buffer (pH 7.8) containing potassium cyanide (50 mM). Thereduced cytochrome c was measured as an increase in absorbance at 550 nm. Complex IV(cytochrome c oxidase; EC 1.9.3.1) activity was measured as a rate of oxidation ofreduced cytochrome c (10 µM) by the enzyme at 30°C. The decrease in reduced cyto-


chrome c was monitored at 550 nm. Protein concentrations were determined spectrophoto-metrically by the biuret method using a kit provided by COBAS FARA and bovine serumalbumin as a standard.

Michaelis-Menten Kinetic Analysis

Complexes III and IV were measured at various ubiquinol-2 concentrations (0.03–3.0 mM) and reduced cytochrome c concentrations (0.5–50 µM), respectively, under theassay conditions described above. Mathematical analysis was performed using the meth-ods described by Segel,28 using a nonlinear curve-fitting program in Sigma Plot 5.0 (Jan-del Scientific, Corte Madera, CA). Km and Vmax were calculated using the Hill equationfor complex III and modifications of the Hill equation for a situation where two enzymesact on a single substrate for complex IV.

Statistical Analysis

All results are expressed as means ± standard error of the mean (SEM). Data were ana-lyzed statistically by using the Student’s independent or paired t-test, and p values of 0.05or less were considered statistically significant. Km and Vmax values were calculated foreach sample and then summed for each group prior to statistical analysis. Additionally,data from each sample were pooled prior to calculation of Km and Vmax. Statistical signifi-cance was similar regardless of approach.

RESULTS

The specific activity of complex I from gastrocnemius was determined in 10- and 20-month-old AL and DR mice.8 The 20-month-old animals fed ad libitum (AL20) displayeda significant decline (65%, p < 0.05) in enzyme activity compared to 10-month-old micefed ad libitum (AL10). Ten-month-old DR mice (DR10) showed much lower enzyme activ-ities compared to AL10 (71% decrease, p < 0.05). There was no age-associated change inactivity for complex I for the 20-month-old DR mice (DR20). For the AL-fed mice therewas no significant age-associated change in specific activity of complex II. At 10 monthsof age there was no influence of diet on complex II activity. However, at 20 months of agemice on DR showed a significantly (69%, p < 0.05) lower enzyme activity compared toAL-fed mice. Similar to complex I, AL20 mice showed an overt age-associated decrease(55%, p < 0.05) in complex III activity compared to AL10 mice. At 10 months of age theactivity of complex III was lower (40%, p < 0.05) in mice subjected to DR than in con-trols. Among the four complexes studied, complex IV showed the most severe reduction inactivity between 10 and 20 months of age in the AL-fed mice (75%, p < 0.001). The activ-ity observed in 26-month-old AL mice was also very low compared to AL10 mice. Theinfluence of DR was age dependent. At 10 months of age, the activity of preparations fromDR mice was less (36%, p < 0.05) than that from AL10 mice. However, at 20 months ofage, the average activity of samples from DR mice exceeded (121%, p < 0.05) that of AL20

mice. The activities of DR10 and DR20 mice did not differ.8


Kinetic analysis of complex III indicated an age-associated reduction in the bindingaffinity of the enzyme to its substrate in AL mice (TABLE 1). A higher Km value (0.44 ±0.05 mM) was found for AL20 than for AL10 (0.34 ± 0.03 mM). In mice subjected to DR,the Km values were significantly lower than in AL mice in both 10 months (0.26 ± 0.05mM) and 20 months (0.18 ± 0.02 mM) of age. Total complex III present as indicated byVmax value suggests that old mice had higher amounts of complex III than all other groups(p < 0.05). The number of binding sites was calculated to be one for all age and dietgroups.

Biphasic kinetic behavior, which is characteristic for complex IV, was observed. Thisresults from the existence of both high- and low-affinity binding sites for complex IV.The Km of the high-affinity binding site ranged from 5.8 to 8.9 µM, whereas the Km ofthe low-affinity binding site was 21 µM in all 10- and 20-month groups (TABLE 2). At 26months, the Km for the high-affinity site increased to 14.0 µM. The Km for the low-affin-

TABLE 1. Km and Vmax Determinations for Complex III8,a

DietAge Vmax Km

AL10 214.1 ± 7.7a 0.34 ± .03b

DR10 160.6 ± 12.2b 0.26 ± .05c

AL20 149.9 ± 7.6b 0.44 ± .05a

DR20 243.8 ± 8.3a 0.18 ± .02c

aKm and Vmax were calculated directly from velocity versus ubiquinol-2 concentration data usingthe Michaelis-Menten equation (v = Vmax [S]/Km + [S]).Vmax units = U/mg protein.Km units = nMubiquinol-2. Different superscript letters indicate statistically significant differences (p < 0.05).

TABLE 2. Km and Vmax Determinations for Complex IV8,a

High affinity Low affinity

DietAge Vmax Km Vmax Km

Percent High-

affinity Sites

AL10 41.2 ± 4.0a 8.9 ± 1.2a 19.8 ± 2.4a 21.3 ± 0.2a 68

DR10 31.7 ± 5.1b 7.6 ± 2.4a 8.1 ± 2.8b 21.5 ± 0.3a 80

AL20 7.2 ± 0.6c 5.8 ± 1.8a 8.5 ± 0.4b 21.4 ± 0.2a 46

DR20 33.4 ± 3.2b 7.9 ± 1.6a 8.4 ± 1.3b 21.4 ± 0.3a 80

AL26 3.9 ± 0.7d 14.0 ± 0.4b 8.0 ± 3.8b 28.4 ± 32.2a 33aKm and Vmax values were calculated from velocity data versus reduced cytochrome c concentra-

tions using the Hill equation modeled for two enzymes acting on a single substrate (v = Vmax1 [S]n/Km1 + [S]n +Vmax2 [S]n/Km2 +[S]n). The number of binding sites per complex (n in the Hill equation)is not reported but ranged between 1.4 and 1.8 in all cases except for low-affinity sites of AL micewhere n = 12. Vmax units = nmol/min/mg protein. Km units = µM reduced cytochrome c. Differentsuperscript letters indicate statistically significant differences (p < 0.05).


ity site for samples from AL26 mice could not be confidently determined using the Hillequation. For AL mice, the Vmax attributed to high-affinity sites declined from 83%from 10 to 20 months of age and was 91% less than the 10-month value for AL26 mice.No age-associated changes in Vmax for high-affinity sites was observed in the DR mice;however, these values were 19–23% less than values for AL10 mice. The Vmax attribut-able to low-affinity sites was highest in the AL10 mice, with values for AL20, AL26,DR10, and DR20 being ~50% lower. The fraction of complex IV binding sites of highaffinity was significantly reduced to 46% in AL20 compared to values of 69 and 80% inAL10 and DR10 mice, respectively. The DR20 mice maintained this same level (80%) ofhigh-affinity binding sites. The fraction of high-affinity sites for AL26 mice was 33 per-cent.

DISCUSSION

Major declines ranging from 54 to 74% in the specific activities of complexes I, III,and IV in mitochondria prepared from the gastrocnemius muscle of 20-month-old mice(late middle age in this long-lived strain) compared to 10-month-old animals wereobserved. By contrast, complex II did not show statistically significant changes with age.Decreased complex I and IV activities agrees with the data of Torri et al.20 who studiedmitochondria from a limb muscle (psoas major) of young Wistar rats of three ages (~2, 8,and 13 months). The activities found for complexes I and IV in the 13-month-old rats wereonly 49 and 76%, respectively, that of the 2-month-old rats. Significant age-relatedchanges in complexes II and III were not observed. Therefore, it is clear that largedecreases in mitochondrial ETS activities occur many months before these animals areold. This group extended their findings to include older rats (21–23 months) and did notobserve further changes in activities from those recorded at 13 months of age.21 Humanskeletal muscle has also been studied 29 for the influences of aging on ETS activities, withthe most consistent declines reported for complexes I and IV.17–19

The present data8 are the first to describe the influence of life span-prolonging DR onthe ETS activities and kinetic properties in muscle mitochondria. Several strong changeswere induced by DR. At 10 months of age, the activities of complexes I, III, and IV were33–64% lower in DR mice than in AL mice. By contrast, at 20 months of age, the activi-ties for DR mice for complexes I and III were not different from those of controls, andcomplex IV activity was 53% higher than the control value. This outcome at 20 monthswas largely a consequence of 54–75% declines in complex I, III, and IV activities between10 and 20 months in AL mice, whereas no significant changes were occurring in the activ-ities of DR mice. The kinetic analysis of complex III revealed a 29% increase in the Km forubiquinol-2 from 10 to 20 months in AL mice with no change in Km in DR mice. A 90%decline was observed in the Vmax calculated for the high-affinity site of complex IV from10 to 26 months of age in AL mice, and through 20 months of age, this change was atten-uated by DR. The Km for high-affinity sites was uninfluenced by age or diet through 20months but was approximately twofold higher for the 26-month AL mice. Therefore, thisincrease in Km for complex IV represents an age-associated change for AL mice not occur-ring until after 20 months of age in this model. Another kinetic parameter, the percentageof total binding sites, which were of high affinity, fell progressively in AL mice from 68%


at 10 months, to 46% at 20 months and 33% at 26 months. This value was 80% for DRmice at both ages (10 and 20 months) studied.

To date, gerontologic studies of the ETS have emphasized activities of the complexes.Although valuable, activity data are less able than are kinetic data to elucidate mechanisticproperties of these enzyme complexes. Complex I consists of 41 subunits, 7 of which areencoded by mtDNA, whereas complex II is unique in its being the smallest complex (4subunits) all of nuclear origin.30 With limited remaining sample, the kinetic properties ofcomplexes III and IV were studied. Complex III consists of 11 subunits (10 of nuclear ori-gin), with the one of mitochondrial origin (cytochrome b apoprotein) being a redox centerand constituting 18% of the mass of complex III.31,32 Complex IV consists of 13 subunits,3 of which (subunits 1, 2, and 3) are encoded by mtDNA and constitute 55% of the massof complex IV. Subunits 1 and 2 contain redox centers whereas subunit 3 acts in protontransport.18,33 The binding site is formed by subunit 2 from one monomer and subunit 3from the adjacent monomer.31

The age-associated reduction in the Vmax for complex III from AL muscle suggests adecrease in total enzyme content. By contrast, Vmax actually increased over 10 to 20months in DR mice. This is especially impressive when coupled to the maintenance of ahigh affinity for substrate with age in the DR mice, suggesting not only more of the com-plex, but higher catalytic efficiency. Conversely, in the AL situation, Km increased whileVmax fell, indicating less of the complex with lower catalytic efficiency. A reduced poten-tial for substrate binding, or the poorly controlled binding just described, may contributeto age-associated increases in superoxide and hydrogen peroxide production (which wereameliorated by DR), as Sohal’s group recently reported.10 A large part of mitochondrialfree radical production is thought to arise between NADH dehydrogenase and ubiquinone/cytochrome b2. If ubiquinol-2 binding is poor, two predictions might follow: (1) anincrease in ubiquinol-2 levels would occur in order to achieve “youthful” activities and,(2) free radical production would increase due to premature release of a free radical prod-uct. Additionally, it is known that the cytochrome b apoprotein is associated with the bind-ing site for ubiquinol-2. Changes in the ability of this molecule to bind substrate mayimply structural changes within this mtDNA-coded subunit.

The active site of complex IV is composed by subunits of mtDNA origin. Therefore,it may be argued that the lack of age or dietary changes in Km for either high- or low-affinity sites through 20 months of age indicates a lack of structural change in these sub-units. This could suggest that the mtDNA regions encoding these proteins had accumu-lated insufficient damage to yield reduced substrate affinity. However, at 26 months, Kmdid increase substantially in AL mice, which supports the possibility of mtDNA damageof consequence. The Vmax data indicate that the amount of total enzyme complex fellwith age in AL mice while the proportion of high-affinity sites decreased. In as much asthe number of high-affinity sites decrease at a faster rate than the low-affinity sites, it canbe argued that some catastrophic alteration (or array of alterations, including modifica-tions of membrane structures affecting these membrane-bound complexes) results ineventual loss of activity at the high-affinity site. Interestingly, the proportion of total Vmax

derived from high-affinity sites was marginally higher at 10 months of age in DR mice(80% vs. 68%), and this same level was maintained in DR20 mice. In short, all of thekinetic changes observed with age in AL mice were opposed by DR. These differencesthat occur with DR would improve the ability of the complex to catalyze conversions ofsubstrate to product at lower concentrations of reduced cytochrome c.


Age-associated ETS dysfunction, especially decreases in total complex coupled withdecreased binding affinity, and the accumulation of a higher proportion of low-affinitysites provide a potential for a biochemical mechanism for age-associated increases in freeradical generation. Additionally, alterations in catalysis would likely obstruct normal elec-tron flow (a kind of electron “traffic jam”), further increasing the potential for free radicalgeneration. Support for this idea comes from Sohal,34 who found that partial inhibition ofcomplex IV in Drosophila stimulated mitochondrial hydrogen peroxide production. Anyof the age-associated alterations in ETS activities or kinetic parameters could causereduced ATP synthesis and compromise cellular function. All of the age-associated alter-ations were opposed by DR.

The data presented herein illustrates a situation where free radicals are generated at lowrates in the younger animal with abundant and efficient ATP generation. The data suggestthat there is an obstruction of electron flow though complex I as the amount of the com-plex diminishes, and through complex III due to loss of total complex and increases in Km

with age. This builds a kind of a dam against electron flow (most notably at complex III).Thus, fewer electrons can pass through the ETS and ultimately to complex IV. Duringaging, high-affinity binding sites of complex IV are lost, further impeding electron flow. Itis suggested that due to these problems more free radicals would be generated at thesesites along the ETS. One can envision that as oxidative lesions accumulate, a mechanismexists where a single electron is passed, and due to poor binding, a premature free radicalproduct would be released prior to the second electron passage. Caloric restrictionresolves these qualitative and quantitative aging problems associated with complexes I,III, and IV, and this may be one mechanism through which caloric restriction limits freeradical generation, leading to extension of maximum achievable life span.

It is suggested that future efforts should center on identification of new nutritional andendocrine interventions that act to improve enzyme efficiency. These types of interven-tions would obviously be compatible and perhaps act synergistically with emerging anti-oxidant interventions.

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