Proton leak and hydrogen peroxide production in liver mitochondria from energy-restricted rats

Jon J. Ramsey,1 Kevork Hagopian,1 Teresa M. Kenny,1 Edward K. Koomson,1 Lisa Bevilacqua,2 Richard Weindruch,3 and Mary-Ellen Harper2

1Department of Molecular Biosciences, University of California, Davis, California 95616; 2Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5; and 3Department of Medicine, University of Wisconsin, Madison, Wisconsin 53606

Submitted 23 June 2003 ; accepted in final form 10 September 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Energy restriction (ER), without malnutrition, is the only environmental intervention that consistently increases maximum life span in laboratory rodents. One theory proposes that a reduction in energy expenditure and reactive oxygen species production is the mechanism responsible for this action of ER. To further test this theory, proton leak, H2O2 production, lipid peroxidation, and protein carbonyls were measured in mitochondria from FBNF1 rats fed either a control or 40% ER diet (onset at 6 mo of age). Liver mitochondria were isolated at 7 and 12 mo of age. Liver weight decreased 25 and 36% at 1 and 6 mo of ER, respectively (P < 0.05). ER resulted in an increase (P < 0.05) in percent total polyunsaturates, n-6 polyunsaturates, and total unsaturates (6 mo only) in mitochondrial lipids. These changes, however, were not associated with significant alterations in mitochondrial function. State 4 respiration and membrane potential were not different (P > 0.05) between groups at either assessment period. Similarly, proton leak kinetics were not different between control and ER animals. Top-down metabolic control analysis and its extension, elasticity analysis, were used at the 6-mo assessment and revealed no difference in control of the oxidative phosphorylation system between control and ER rats. H2O2 production with either succinate or pyruvate/malate substrates was also not different (P > 0.05) between groups at either time point. In conclusion, ER did not alter proton leak or H2O2 production at this age or stage of restriction in liver.

calorie restriction; energy expenditure; mitochondrial lipids; aging; oxidative stress


ENERGY RESTRICTION (ER), without malnutrition, is the only intervention that has consistently been shown to increase maximum life span in laboratory rodents (55). The mechanism responsible for the retardation of aging by ER, however, is unknown. Two theories of aging, the rate of living theory (40) and the free radical theory (21), support the idea that a hypometabolic state of decreased oxygen consumption and/or decreased mitochondrial reactive oxygen species (ROS) production may be causal. Both theories have been proposed to explain the mechanism for ER, suggesting that a decrease in oxygen consumption and ROS production may be central to the action of ER. Despite the important role that alterations in energy metabolism and oxygen consumption may play in the retardation of aging, little is known about the effects of long-term ER on the processes regulating mitochondrial energy metabolism.

Whole animal studies have reported either a decrease or no change in weight-adjusted oxygen consumption with long-term ER (44). These studies, however, do not provide information about possible changes in energy metabolism within cells of specific tissues. Because of the many assumptions required with data normalization procedures, whole animal studies can at best provide a rough indication of possible changes in cellular/mitochondrial energy metabolism. ER does not produce a miniature anatomic version of the control but induces a nonuniform change in the weight of tissues/organs (16, 55) and thus makes accurate normalization between groups very difficult. Measurements of tissue-specific cellular energy metabolism pathways are necessary to determine precisely the effects of ER on energy metabolism.

The present study focuses on energy metabolism in liver. We chose to study liver because it is a major contributor to whole animal energy expenditure (44) and it rapidly changes weight in response to ER (44).

Proton leak, protein turnover, and Na+-K+-ATPase are the major processes contributing to resting energy expenditure (44, 45). In this study, we have focused on mitochondrial proton leak because it is responsible for ~25% of resting oxygen consumption (45) and because changes in mitochondrial lipid composition and circulating thyroid hormone levels that occur with ER suggest a possible reduction in proton leak. A few studies have reported a positive correlation between docosahexaenoic acid (22:6n-3) and proton leak and a negative correlation between linoleic acid (18:2n-6) and proton leak (5, 7, 43). ER has been reported to increase mitochondrial linoleic acid and decrease docosahexaenoic acid content (31). Also, ER has been shown to decrease circulating triiodothyronine concentrations in rats (25), and hypothyroidism has been shown to decrease proton leak (22, 36). In addition to correlative data, we have shown that mitochondrial proton leak is increased with aging in intact hepatocytes (24) and decreased by ER in mitochondria isolated from skeletal muscle (33).

Correlative studies may also be used to indicate a direct relationship between mitochondrial proton leak rate and rate of H2O2 production. When compared across species, life span is inversely related to the rate of mitochondrial H2O2 production (41, 49). With the use of the same species, proton leak data (42) can be used to demonstrate a positive correlation between proton leak and the rate of mitochondrial H2O2 production. Similarly, hypothyroidism has been shown to decrease proton leak (22) and H2O2 production (52). Also, our previous findings that proton leaks are increased with aging (24) and decreased with ER (33) correspond with reports that H2O2 production is increased with aging and decreased with ER (50).

This correlative evidence is against the popular theory that proton leak is related to a reduction in ROS production. Lately, work with the recently identified "uncoupling" proteins (uncoupling proteins-2, -3, etc.) have further fueled speculation that proton leaks (and uncoupling proteins) function primarily to inhibit ROS production. This role of proton leak as a protector against ROS formation stems from work showing that addition of uncoupling agents to isolated mitochondria decreases ROS formation (2). It has now been clearly demonstrated that many uncoupling agents decrease ROS production when added to isolated mitochondria. Although these findings are important, caution should be taken in extrapolating these results to long-term proton leak changes in vivo. Isolated mitochondria studies do not allow for any possible gene transcription changes that may be associated with proton leak alterations, and too often the assumption is made that sustained proton leak changes will occur without other mitochondrial adaptations. It is possible therefore that short- and long-term uncoupling have very different affects on ROS formation. We believe that proton leak is at minimum a marker of mitochondrial membrane changes that produce a mitochondrial environment of decreased ROS production and may actually be the driving force for such changes.

Accordingly, we have hypothesized that a reduction in proton leak may decrease ROS production and could play a role in the retardation of aging by ER (44). To better characterize the effects of ER on proton leak, we determined proton leak and H2O2 production in liver of adult rats fed a 40% ER diet. Mitochondrial lipid composition and markers of oxidative stress were also measured. Top-down metabolic control analysis and its extension, elasticity analysis, were performed to determine potential differences in the regulation of oxidative phosphorylation.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and diets. Male FBNF1 rats were purchased from the National Institute on Aging (NIA) Aging Rodent Colony (Bethesda, MD) and were used for all phases of this study. Before the start of the study, 30 4-mo-old rats were purchased and given ad libitum access to an AIN-93M purified diet (Research Diets, New Brunswick, NJ). Food intake was measured daily for 2 mo. The average daily food intake of the group was taken as the ad libitum food intake amount that would be used for all dietary restriction adjustments. At the completion of this 2-mo period, all rats were randomly divided into control and restricted groups. In addition, other male FBNF1 rats were purchased from the NIA Aging Rodent Colony at 5 mo of age (fed ad libitum for 1 mo). These additional rats were used to supplement the numbers from the initial group and give a total number of animals sufficient for a long-term study. All rats were 6 mo of age at the time of initiation of ER. The control group was fed the AIN-93M diet at 95% of the calculated ad libitum food intake amount. The restricted group was fed a modified AIN-93M diet (Research Diets) at 60% of the calculated ad libitum food intake amount. The corn starch (carbohydrate) component of the restricted diet was decreased, resulting in an increase in the percentage of protein, fat, minerals, and vitamins in the restricted diet and thus achieving equal absolute daily intakes of essential nutrients between groups. The fat source used in both diets was soybean oil. The energy intakes were 61.6 kcal/day for the control and 37.6 kcal/day for the restricted rats. All animals were fed between 8:00 and 9:00 AM each morning. All rats were caged individually at 23°C with light from 7:00 AM to 7:00 PM. The rats were allowed free access to water during all stages of the study. At the 1-mo time point, seven control and five ER animals were used, and at the 6-mo time point seven control and ER animals were used. Not all animals were studied in every assay; animal numbers are accordingly indicated in Tables 1, 2, 3, 4, 5, Figs. 1, 2, 3, 4, and METHODS. All animal protocols were approved by the University of California Davis internal animal care and use committee and are in accordance with the guidelines of the Institute of Laboratory Animal Resources (National Research Council).


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Table 1. Organ weights in control and energy-restricted rats

 

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Table 2. Overall elasticities to {Delta}{Psi}m for substrate oxidation, phosphorylation, and proton leak subsystems in liver mitochondria from control and restricted rats after 6 mo of ER

 

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Table 3. Flux control coefficients over subsystem fluxes and concentration control coefficients over {Delta}{Psi}m in liver mitochondria from control and restricted animals after 6 mo of calorie restriction

 

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Table 4. Fatty acid composition of mitochondria from control and energy-restricted rats

 


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Fig. 1. Liver mitochondrial proton leak kinetics in control (n = 4) and energy-restricted (ER; n = 4) FBNF1 rats at 1 mo of ER. Proton leak kinetics were completed with 10 mM succinate and 8 µg/mg protein oligomycin and were titrated with 0.3-10 mM malonate. Conditions used for these measurements are described in METHODS.

 


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Fig. 2. Liver mitochondrial substrate oxidation (A), phosphorylation (B), and proton leak (C) kinetics in control (C; n = 6) and ER (n = 7) FBNF1 rats at 6 mo of ER. A: substrate oxidation kinetics were completed with 100 µM ATP and 10 mM succinate and titrated with hexokinase. B: phosphorylation kinetics were completed with 100 µM ADP, 1.50 U/ml hexokinase, and 10 mM succinate and titrated with malonate (0.3-10 mM). C: proton leak kinetics were completed with 10 mM succinate and oligomycin and were titrated with malonate. Conditions used for these measurements are described in METHODS.

 


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Fig. 3. Liver mitochondrial H2O2 generation in control and ER FBNF1 rats at 1 mo (A) and 6 mo (B) of ER. Assays were performed as described in text. Seven control and four ER rats were used at the 1-mo (A) measurements and six control and seven ER rats were used for the 6-mo (B) measurements. Sub, substrate; AA, antimycin A; Rot, rotenone; Succ, succinate; Pyr, pyruvate; Mal, malate. *P < 0.02 for comparison of 1-mo control with succinate vs. 1-mo ER with succinate.

 


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Fig. 4. Liver mitochondrial markers of oxidative stress at 1 and 6 mo of ER in FBNF1 rats. Lipid peroxidation (A) and protein carbonyls (B) were determined as described in text. Solid bars, controls; open bars; ER. A: for lipid peroxidation, 3 control and 4 ER rats were used at the 1-mo measurements, and 6 control and 6 ER rats were used for the 6-mo measurements. B: for carbonyls, 5 control and 5 ER rats were used at the 1-mo measurements, and 6 control and 7 ER rats were used for the 6-mo measurements. TBARS, thiobarbituric acid reactive substances.

 

Chemicals. All chemicals were purchased from Sigma (St. Louis, MO) except for hexokinase, which was purchased from Roche (Indianapolis, IN). Bio-Rad protein assay kits were from Bio-Rad Laboratories (Hercules, CA), and the bicinchoninic acid (BCA) protein assay kits and BSA standards were from Pierce (Rockford, IL). Percoll was from Amersham Biosciences (Piscataway, NJ).

Mitochondrial isolation and organ weight measurements. Mitochondria were isolated from liver using previously described methods (12). All animals were food deprived overnight before being killed. The rats were killed by decapitation, and the liver was removed rapidly, weighed, and placed in ice-cold isolation medium (250 mM sucrose, 10 mM Tris·HCl, 1 mM EGTA, and 0.1% BSA, pH 7.4). The liver was then cut into small cubes with a razor blade and rinsed with cold isolation medium to remove blood. Tissue (10% wt/vol in isolation medium) was transferred to an ice-cold glass-Teflon homogenizer and homogenized with six strokes of the pestle. The homogenate was transferred to chilled centrifuge tubes and centrifuged at 1,000 g for 5 min at 4°C. The supernatant was decanted into clean centrifuge tubes and centrifuged at 12,000 g for 10 min (4°C). The supernatant was discarded, and the pellet was gently suspended in isolation medium using a cooled glass rod and centrifuged at 12,000 g. This procedure was repeated two times. The pellet was then resuspended in isolation medium without BSA, centrifuged at 12,000 g, suspended once again in isolation medium without BSA, and stored on ice.

Internal organs (brain, heart, kidneys, lungs, stomach, small intestine, and large intestine) were removed from the animals after they were killed, cleaned of fat or connective tissue, and weighed. Stomach and intestines were cleaned of ingesta or feces before being weighed.

Fractionation of mitochondria using Percoll gradients. Mitochondria isolated from the previous step were further fractionated on a 30% (vol/vol) Percoll gradient in 0.225 M mannitol, 1 mM EGTA, and 25 mM HEPES buffer, pH 7.4, and centrifuged at 95,000 g for 30 min at 4°C in a Beckman L8-80M ultracentrifuge using a 60Ti Beckman rotor (47). Mitochondria were collected from the lower band near the bottom of the tube, washed two times with a 2 mM HEPES buffer containing 0.22 M mannitol, 70 mM sucrose, and 0.5 mM EGTA, pH 7.4, and centrifuged after each wash at 6,400 g for 10 min at 4°C. The washed mitochondria were then washed two times with 0.15 M KCl and centrifuged as above, and the final pellet was resuspended in the above wash buffer. The use of KCl washes with liver mitochondria was critical in the removal of catalase, which comes not only from tissue homogenization but also from the lysis of red blood cells (Dr. Cecilia Giulivi, personal communication).

Measurement of mitochondrial oxygen consumption. Mitochondrial oxygen consumption was measured using previously described methods (33). Briefly, oxygen consumption was measured using a Clark-type oxygen electrode (Hansatech, Norfolk, UK), with an incubation chamber maintained at 37°C. All measurements were completed in duplicate using mitochondria (1.0 mg mitochondrial protein/ml) in incubation medium (100 mM KCl, 20 mM sucrose, 20 mM glucose, 10 mM KH2PO4, 5 mM HEPES, 2 mM MgCl2, 1 mM EGTA, 5 µM rotenone, and 0.4 µg nigericin/mg mitochondrial protein, pH 7.2). Rotenone is used to block complex I and ensure that energy flux through the electron transport chain is controlled through FADH2-linked substrates such as succinate. Nigericin, in the presence of KCl, is used to convert the {Delta}pH component of the protonmotive force to units of mitochondrial membrane potential (mV). State 3 respiration was defined as the oxygen consumption rate in the presence of 10 mM succinate, 1.50 U/ml hexokinase, and 100 µM ADP. Later experiments showed that additional ADP could further stimulate respiration by ~20%, so the reported measurements will be referred to as 100 µM ADP in Tables 1, 2, 3, 4 and in RESULTS. Although these measurements are short of the intended state 3 respiration rates, they are still of physiological relevance. Because mitochondria normally exist between states 3 and 4, the 100 µM ADP measurements represent a physiologically relevant condition of respiration stimulated by a relatively high ADP concentration. State 4 oxygen consumption was determined in the presence of the ATP synthase inhibitor oligomycin (8 µg/mg mitochondrial protein).

Measurement of mitochondrial membrane potential. Membrane potential was measured with a methyltriphenylphosphonium (TPMP+)-sensitive electrode using previously published methods (3, 33). Mitochondrial membrane potential ({Delta}{psi}m) was calculated using the modified Nernst equation

where [TPMPmat+] is the concentration of TPMP+ in the mitochondrial matrix and [TPMPext+] is the concentration of TPMP+ outside the mitochondria. The factor am is the mitochondrial TPMP binding correction, and the value used for liver was 0.4 (46). Membrane potentials were assessed simultaneously with all of the oxygen consumption measurements, as described above.

Top-down metabolic control analysis and proton leak kinetics. Top-down elasticity analysis, an extension of top-down metabolic control analysis, was used to determine regulation of oxidative phosphorylation (4, 23). For this study, the oxidative phosphorylation system was divided into substrate oxidation, proton leak, and phosphorylation components. The kinetics (or elasticity) to changes in {Delta}{psi}m were measured for both reactions that produce (substrate oxidation) and consume (proton leak and phosphorylation) {Delta}{psi}m, their common intermediate. The kinetic response of the proton leak to {Delta}{psi}m was determined by titrating the electron transport chain with malonate (0.3-10 mM), an inhibitor of complex II of the chain, in the presence of oligomycin (8 µg/mg protein). The kinetic response of the substrate oxidation pathway to {Delta}{psi}m was determined by titrating with hexokinase (0.25-1.5 U/ml) in the presence of 100 µM ATP. The kinetic response of the phosphorylation system to {Delta}{psi}m was determined by titrating with malonate (0.3-10 mM) in the presence of 100 µM ADP and 1.5 U/ml hexokinase. Because this measurement contains components of both phosphorylation and proton leak, a correction was subsequently made for the proton leak-related oxygen consumption at each membrane potential. Top-down metabolic control analysis and previously published equations for elasticities, flux control coefficients, and concentration control coefficients were used to determine the distribution of control over the respiration rate and {Delta}{psi}m in the mitochondria from animals at the 6-mo assessment (18).

Measurement of H2O2 generation by mitochondria. The rate of H2O2 generation by the mitochondria was determined fluorometrically, according to Hyslop and Sklar (26). Briefly, mitochondria (0.5 mg mitochondrial protein) were added to 3 ml of 10 mM potassium phosphate buffer, pH 7.4, containing 154 mM KCl, 0.1 mM EGTA, 3 mM MgCl2, 500 µg/assay p-hydroxyphenylacetate, and 4 units of horseradish peroxidase, using 10 mM succinate or 10 mM pyruvate/5 mM malate as substrates. No superoxide dismutase was included in the reaction mixture. The increase in fluorescence at 37°C was monitored by a Perkin-Elmer LS 55 luminescence spectrometer equipped with a water Peltier heating system and a magnetic stirring sample compartment. The excitation and emission wavelengths were 320 and 400 nm, respectively, and the final assay volume was 3 ml. Levels of H2O2 were expressed as picomoles of H2O2 per minute per milligram protein. Rates were determined by converting fluorescence readings using a standard curve generated over a range of H2O2 concentrations.

Determination of protein carbonyls. Mitochondrial protein carbonyls were determined according to the method of Levine et al. (34) using 2,4-dinitrophenylhydrazine. The protein content of each assay was kept ~1 mg, and samples were first treated with streptomycin sulfate (1% final concentration) to remove contaminating nucleic acids. Carbonyl content was measured spectrophotometrically, using a Perkin-Elmer Lambda 25 spectrophotometer set at 366 nm, and calculations were made by using a molar extinction coefficient of 22,000 M-1/cm. Results were expressed as nanomole carbonyl per milligram protein.

Determination of lipid peroxidation. Lipid peroxidation levels or thiobarbituric acid reactive substances (TBARS) were determined as malondialdehyde-thiobarbituric acid aducts according to the method of Beuge and Aust (8). Because the mitochondria were isolated in sucrose-containing medium, they were centrifuged for 3 min at 12,000 g at 4°C, and the supernatant was discarded. The mitochondrial pellet was then resuspended in a volume of 0.1 M potassium phosphate buffer, pH 7.4, equal to the discarded supernatant and centrifuged as above. This step was repeated two times to help eliminate sucrose from the sample that interferes with the assay (8). Samples were then treated with butylated hydroxytoluene to avoid generation and overestimation of peroxidation developed during the heating step (17). Peroxidation levels were then measured spectrophotometrically in a Perkin-Elmer Lambda 25 spectrophotometer set at 535 nm, using a molar extinction coefficient of 1.56 x 105 M-1/cm and expressed as nanomole TBARS per milligram protein.

Protein assays. Protein concentrations were determined in all samples using a Bio-Rad protein assay kit (Bio-Rad Laboratories), except in the case of protein carbonyl assays, where a BCA protein assay kit from Pierce was used, since the presence of high guanidine hydrochloride in samples interfered with the Bio-Rad assay system. In both cases, BSA was used to generate standard curves. No differences in mitochondrial protein yield (mg protein/g wet wt of liver) were observed in control vs. ER rats.

Mitochondrial fatty acid composition. Fatty acid composition of liver mitochondria was determined by a previously described method (53) in Percoll isolated mitochondria that were stored under nitrogen. Lipids were extracted from the mitochondria with chloroform-methanol (2:1 vol/vol) and methylated with 3 N methanolic hydrochloride. Fatty acid methyl esters were separated and quantified by gas chromatography.

Statistical analysis. Comparisons between groups were completed using the Student's t-tests, and comparisons within a treatment group were completed using paired Student's t-tests. Respiration and membrane potential comparisons were also made between groups using ANOVA in which the factors were individual animal, group (control or ER), and inhibitor concentration. All statistical analyses were completed using JMP software (SAS Institute, Cary, NC). Results are presented as means ± SE.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Body and organ weights. These data are summarized in Table 1. At 1 mo of ER, body weight was 12.0% lower (P < 0.05) in ER compared with control rats. Liver, heart, and large intestine weights were also reduced (P < 0.05) in ER animals. At 6 mo of ER, body weight was 29.6% lower (P < 0.05) in ER compared with control rats. All organ weights were reduced (P < 0.05) in the ER group at the 6-mo assessment point. Liver, with a reduction of 25.2 or 36.3%, showed the greatest change with ER at 1 or 6 mo of treatment, respectively. Liver is the only organ that showed a reduction in weight similar to the level of ER at the 6-mo assessment.

Mitochondrial respiration and kinetics of proton leak, substrate oxidation, and phosphorylation systems. Proton leak kinetics at the 1-mo assessment are summarized in Fig. 1. Maximal leak-dependent respiration and membrane potential (the furthest points to the right in the graph) were not different between groups. The membrane potentials in the restricted group tended to be higher than the controls at similar respiration rates at some parts in Fig. 1, suggesting a trend toward a very slight decrease in proton leak with ER. The kinetics of substrate oxidation, phosphorylation, and proton leak systems are summarized for the 6-mo assessment period in Fig. 2. No changes were observed between groups for each of the kinetics plots. Membrane potentials and respiration rates were not different (P > 0.05) between ER and control animals under any of the conditions used for the points in the plots in Fig. 2. These results indicate no difference in substrate oxidation (Fig. 2A), phosphorylation (Fig. 2B), and proton leak (Fig. 2C) kinetics between ER and control rats at this assessment period.

State 4 and state 3 respiration rates and membrane potentials were not different (P > 0.05) between control and ER animals at either assessment period. Respiratory control ratios (RCR) were 2.48 ± 0.08 and 2.66 ± 0.15 for the control and ER groups, respectively, at the 6-mo assessment. Also, RCR were 2.38 ± 0.21 and 2.34 ± 0.11 for the control and ER groups, respectively, at the 1-mo assessment. Although these RCR values are similar to previously reported values with succinate as a substrate (28, 54), it is important to note that these values were calculated under conditions that did not reach a true state 3. Thus they are underestimates of the correct RCR. The high membrane potentials we observed in both groups provide further support that the mitochondria used in this study were well coupled.

Metabolic control analysis. Elasticities of the substrate oxidation, phosphorylation, and proton leak systems to {Delta}{psi}m are summarized in Table 2. The data used to calculate the elasticity values were obtained from Fig. 2. The elasticity values of each of the oxidative phosphorylation subsystems were similar in ER and control rats for both resting (100 µM ADP) and state 4 states of respiration at 6 mo of ER.

Flux control coefficients of the subsystems over the rate of each subsystem are summarized in Table 3. The flux control coefficients describing control over a particular subsystem (i.e., substrate oxidation) add up to one, with a value of one indicating complete control, zero indicating no control, and a value of 0.50 indicating 50% control. From the data in Table 3, it is clear that the processes controlling oxidative phosphorylation in the liver were not altered in adult animals after 6 mo of ER. This is true in both 100 µM ADP and state 4 conditions of respiration. Substrate oxidation was primarily controlled by the substrate oxidation and phosphorylation reactions in both groups of animals. The control exerted by proton leak was higher than would be expected for state 3 conditions. Our values were similar to those reported previously for hepatocytes in the resting state from euthyroid rats (22). This reflects the fact that the conditions we intended to produce state 3 did not actually stimulate a maximal respiration (as mentioned in METHODS).

The phosphorylation reactions exerted most of the control (0.71 in both control and ER) over phosphorylation flux in the resting (100 µM ADP) state, with significant control also from substrate oxidation reactions and a smaller negative control from proton leak in both groups of rats. The proton leak exerted the bulk of the control (0.93 in both control and ER) over flux through the proton leak pathway. Under state 4 conditions, the proton leak subsystem was primarily responsible for controlling flux through both substrate oxidation and proton leak.

Concentration control coefficients of the three subsystems over {Delta}{psi}m (the intermediate of the system) are also summarized in Table 3. Concentration control coefficients add up to zero, and the systems exerting the greatest control will have the largest absolute values. Concentration control coefficients were similar between control and ER animals for each of three subsystems.

Partial internal response coefficients may be calculated by multiplying the elasticities in Table 2 by the concentration control coefficients in Table 3. Because elasticities and flux control coefficients were very similar between treatment groups, partial internal response coefficients between control and ER groups were also similar (data not shown).

Mitochondrial H2O2 production and markers of oxidative damage. At the 1-mo assessment (Fig. 3A), H2O2 production was not different (P > 0.05) between control and ER groups for any substrate or substrate plus inhibitor, except for measurements using succinate alone where H2O2 was higher (P < 0.02) in the ER compared with controls. H2O2 production was not different (P > 0.05) between control and ER groups for any substrate or substrate plus inhibitor at the 6-mo assessment (Fig. 3B).

H2O2 production in the presence of substrate plus antimycin A was higher (P < 0.05) than either substrate alone or substrate plus rotenone for each treatment group and assessment period. From a substrate standpoint, succinate generated higher rates of H2O2 production than pyruvate/malate at both 1 and 6 mo. Rotenone significantly (P < 0.05) decreased H2O2 production with succinate as a substrate and increased (P < 0.05) H2O2 production with pyruvate/malate for both assessment periods.

Markers of oxidative damage in mitochondrial lipids and proteins were measured. Lipid peroxidation (Fig. 4A) and protein carbonyls (Fig. 4B) did not show significant differences (P > 0.05) between control and ER groups at either 1- or 6-mo assessments.

Mitochondrial fatty acid composition. Fatty acid compositions of mitochondria from control and ER rats are summarized in Table 4. At the 1-mo assessment, 18:3n-6, unsaturation index, percent polyunsaturates, and percent n-6 polyunsaturates were increased (P < 0.05) in ER compared with control animals. In contrast, 22:5n-3 was decreased (P < 0.05) in the ER group. At the 6-mo assessment, 18:2n-6, 18:3n-3, 18:3n-6, 22:4n-6, percent unsaturates, percent polyunsaturates, and percent n-6 polyunsaturates were all higher (P < 0.05), whereas 16:0 and 20:3n-6 were lower (P < 0.05) in ER compared with controls.


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We sought to determine the effect of ER on mitochondrial proton leak and H2O2 production in liver. In addition to these measures, we also used elasticity analysis, an extension of metabolic control analysis, to investigate the effects of ER on oxidative phosphorylation. Changes in mitochondrial lipid composition, organ weight, and markers of oxidative stress were also studied.

Strong conclusions have been made about ER-induced changes in energy metabolism in whole animal studies. However, organ-to-organ variations in the response to ER are likely, so we decided to determine changes in organ weight to better understand possible alterations in body composition and energy metabolism with this treatment. Previous studies have shown that ER does not result in a uniform change in the weight of internal organs (16, 20, 55). A 40% ER was started at 1 mo of age in the study summarized by Weindruch and Sohal (55), whereas it was initiated at 12-14 wk of age for the rats in the other studies (16, 20). In all of these studies, the magnitudes of body weight and energy intake reductions were similar with long-term ER. However, organ weight changes did not always mirror the reductions in body weight and energy intake. Liver showed a decrease in weight comparable to, or slightly greater than, the level of restriction. Brain weight was not greatly altered, whereas the weights of other organs were reduced by roughly 30% with ER. Similar to the other studies, the magnitude of the 6-mo ER change in liver weight in our study was comparable to the reduction in energy intake (Table 1). The magnitude of the other organ weight changes in our study tended to be lower than those reported previously. This could be because either the duration of ER (6 mo) was less than the previous studies or the age at initiation of ER was greater in our study. These changes further indicate the importance of investigating energy metabolism in specific organs, since different organs show great variations in their ability to alter weight when subjected to ER.

The role that changes in energy metabolism may play in the mechanism of ER has been discounted by some, based on whole animal energy expenditure measurements (37). Our results and those of others clearly indicate that the ER animal is not a miniaturized version of the control animal. A question that remains unanswered is what is the most appropriate method of normalizing or even expressing results from whole animal studies of ER? To properly determine the role energy metabolism might play in the actions of ER, it is thus necessary to investigate specific energy metabolism processes within individual organs/tissues.

This study focused on changes in mitochondrial metabolism within liver, a tissue that exhibits very large and rapid reductions in weight in response to ER. Initial decreases in liver weight may be attributable to reductions in liver glycogen stores; however, sustained ER appears to induce changes in liver metabolism beyond simple reductions in glycogen content. Several studies using arterio-venous difference techniques in growing (11, 13) and adult (39) sheep have shown that ER causes a rapid and dramatic decrease in liver oxygen consumption. Although these studies were not completed in rats, a study comparing liver weight and whole animal energy expenditure in rats having different levels of energy intake concluded that liver weight was strongly correlated with fasting energy expenditure (30). This result supports the idea that ER induces a substantial reduction in liver oxygen consumption. A goal of our study was to determine if alterations in mitochondrial proton leak could contribute to reductions in liver oxygen consumption with ER.

Toward this goal, mitochondrial proton leak, a major cellular energy-consuming process, was determined. No differences between control and ER animals, however, were found (Figs. 1 and 2). Although state 4 membrane potentials did show a trend toward higher values in ER animals at 1 mo of ER, any hint of differences between groups had disappeared by the 6-mo assessment. This was not the finding that we predicted based on previous studies indicating a decrease in proton leak with hypothyroidism (22) and a correlation between proton leak and linoleic (negative correlation) or docosahexenoic (positive correlation) acid content in mitochondria (5, 7, 43). ER has been shown to decrease circulating triiodothyronine levels (25) and alter mitochondrial fatty acids in a manner resulting in a decreased concentration of docosahexaenoic acid and an increased concentration of linoleic acid (31). Also, we have shown that long-term ER results in a decrease in maximal leak-dependent respiration in mitochondria of skeletal muscle. Although these results led us to predict a reduction in proton leak with ER, there are a number of important differences between the studies, which may explain the lack of a change in proton leak with 6 mo of ER.

Although ER has been shown to decrease circulating triiodothyronine in rats, the magnitude of this reduction is small, and circulating thyroxine levels are not changed (25) with ER. This is in contrast to the conditions used in proton leak studies (22), where hypothyroidism is induced by 6-n-propyl-2-thiouracil and both serum triiodothyronine and thyroxine levels are decreased (19). It is likely that the ER used in our study did not produce a change in the magnitude of thyroid hormones sufficient to elicit an alteration in liver mitochondrial proton leak.

The mitochondrial fatty acid changes that we observed in our study (Table 4) were different from those previously reported for rat liver mitochondria with long-term ER (31, 32). The latter two studies reported an increase in linoleic acid with ER; this is consistent with our observation at the 6-mo ER time point. However, we also observed an increase in the percentage of unsaturated and polyunsaturated fatty acids (but no significant change in docosahexaenoic acid) in mitochondria. These results could reflect the fact that we used a different dietary fat source (soybean oil) than the corn oil (31) or semipurified diet of unreported fat source (32) used in the previous studies. The difference in results likely reflects the fact that the mitochondria used for fatty acid measurements in our study were isolated on Percoll gradients, whereas the previous studies used unpurified mitochondria. Contamination of mitochondria with broken plasma membranes or endoplasmic reticulum could well account for the fatty acid differences observed between these previous studies (31, 32) and our study. Differences in rat strain and age of initiation of ER could also contribute to the differences between the studies. Regardless, the fatty acid changes that we observed were not associated with a significant alteration in proton leak.

We have previously shown that long-term ER is associated with a reduction in maximum leak-dependent respiration in skeletal muscle mitochondria (33), and current studies indicate that changes in maximal leak-dependent respiration and ROS production occur as early as 2 wk after initiation of ER (1a). These results indicate that liver and skeletal muscle mitochondria respond very differently to ER. Muscle is a postmitotic tissue that shows considerable oxidative damage and atrophy with aging (50), whereas the liver is affected less by aging (27) and tends to show oxidative damage only at an advanced age (14, 51). Also, liver shows a rapid reduction in weight after initiation of ER, whereas changes in skeletal muscle weight occur at a slower rate (44). Considering these differences between tissues, it is not surprising that liver and skeletal muscle may show different metabolic responses to ER. It is possible that liver adapts to ER primarily by decreasing weight without significant changes in mitochondrial energy metabolism, whereas skeletal muscle may respond to ER by decreasing proton leak or the activity of other energy-consuming pathways. It is also possible that alterations in skeletal muscle proton leak with ER are driven by alterations in uncoupling proteins. However, because liver parenchymal cells do not normally express either uncoupling protein-2 or -3 (38) an uncoupling protein-induced change in proton leak is unlikely. However, it is possible that ER could induce changes in liver proton leak if given a sufficient amount of time. We have previously shown that proton leak is greater in hepatocytes from old (30 mo) compared with young (3 mo) mice (24), consistent with the idea that oxidative damage with aging may underlie the increased leak. At 6 mo of ER, the rats used in our study were only 12 mo of age and still in early middle age. It will be important in future studies of older animals to determine if ER can counteract any aging-induced increases in proton leak.

In addition to proton leak, top-down metabolic control analysis and its extension, elasticity analysis, showed no differences in the regulation of oxidative phosphorylation between treatments (Table 3). Our results were similar to those obtained for adult euthyroid rats (22), and this further strengthens our observation that 6 mo of ER did not alter liver mitochondrial function.

Markers of oxidative damage (TBARS for lipid peroxidation and carbonyls for protein oxidative damage) did not show significant differences between control and ER groups (Fig. 4). Several studies have measured markers of oxidative stress in liver mitochondria from control and ER rats. One study measuring the time course for lipid peroxidation changes in liver reported that significant differences between control and ER rats did not occur until 30 mo of age (14). Another study measuring lipid peroxidation in liver found no difference between control and ER groups at 12 wk of ER but reported a reduction in lipid peroxidation with ER at 24 wk (56). A report by Kaneko et al. (29), in which 8-hydroxydeoxyguanosine (8-OHDG) was measured as a marker of oxidative damage to nuclear DNA, found no difference between control and ER until 30 mo of age. These studies suggest that changes in markers of oxidative damage in liver only occur after long-term maintenance of ER or after animals have reached advanced age. One study, however, has reported differences in 8-OHDG between ER and control rats at only 6 wk of ER (15). Our results support the idea that ER does not lower oxidative stress at 6 mo of ER. Although we are not aware of any other studies measuring carbonyls in liver from ER rats, a previous report does indicate an increase in liver carbonyls with age, although this increase did not become significant until 24 mo of age (51). The animals in our study were only 12 mo of age at the 6-mo assessment and thus may not have reached an age where significant oxidative damage could be observed.

Measures of mitochondrial H2O2 production did not show significant differences between control and ER groups except for H2O2 production with succinate, which was higher with ER at the 1-mo assessment (Fig. 3A). This increase occurred at a time when liver was still undergoing a change in weight and may reflect possible cellular remodeling with decreases in hepatocyte size and mitochondrial number. Previous studies have reported a decrease in hepatocyte size with ER in sheep (10) and fasting (72-h duration) in rats (9). Although liver changes appear to occur rapidly with ER, a study of 30% ER in sheep found that ER-induced changes in liver oxygen consumption required 21-29 days to reach equilibrium (13). If rats behave in a similar manner, then liver remodeling could still be underway at the 1-mo assessment. The low liver weights at the 6-mo assessment hint that liver adjustments in size could still be happening through 1 mo of ER. However, it is still purely speculation that changes in liver size and possibly mitochondrial number could have any effect on H2O2 production. It has recently been shown that hepatic apoptosis is increased in ER rats compared with controls at 2 mo of ER (48). It is possible that the increased ROS production with succinate in the ER group reflects this increased apoptosis. A relatively small number of cells (and mitochondria) may be responsible for this elevated ROS production, and this may explain why proton leak measures are not altered. Also, it is important to note that only H2O2 production with succinate alone is significantly altered by ER, and since all other measures of H2O2 production (including measures of succinate with inhibitors) are not different between groups strong conclusions about the relationship between proton leak and ROS production should probably not be drawn from this single finding.

At the 6-mo assessment, H2O2 production was similar between control and ER animals for both substrates (Fig. 3B). Only a few studies have measured H2O2 production in liver after initiation of ER (15, 35). Long-term ER (12 mo) resulted in a reduction in H2O2 production in liver mitochondria respiring on either succinate or pyruvate/malate (35). Short-term ER (6 wk) also resulted in a reduction in H2O2 with mitochondria respiring on pyruvate/malate, but no change was observed with succinate as a substrate (15). Our results differ from these findings in that we did not observe reductions in H2O2 production at 6 mo of ER with either pyruvate/malate or succinate substrates. In these previous studies, ER was initiated at either a younger (35) or older (15) age than the 6-mo age that we used for the start of ER. Also, the previous studies both used Wistar rats and did not purify their mitochondrial preparations using Percoll. It is possible that these differences in experimental design could have influenced the results that we have observed. With each of these H2O2 studies, it is also possible that differences in manganese superoxide dismutase (Mn-SOD) activity between control and ER animals could influence the results. However, it is unlikely that the lack of difference in H2O2 production that we observed between ER and control rats is the result of increases in Mn-SOD activity with ER. Activities of antioxidant enzymes have not been shown to follow a consistent pattern with ER (50), and we are not aware of strong evidence to support a large alteration in Mn-SOD activity with ER.

Our study and the two previous studies with H2O2 measurements (15, 35), however, did show similar changes in the pattern of H2O2 production with complex I vs. complex II substrates or with the addition of inhibitors to mitochondria respiring on either substrate. H2O2 production was greater with succinate than with pyruvate/malate as substrate. Addition of rotenone to succinate greatly decreased H2O2 production, indicating that much of the ROS production with succinate occurs at complex I. Addition of rotenone to pyruvate/malate increased H2O2 production, which also points to complex I as a major site for ROS production. In the case of succinate, this complex I-linked H2O2 production is the result of a backflow of electrons from complex II to complex I, whereas inhibition of complex I by rotenone would likely increase ROS production with pyruvate/malate by maintaining complex I in a reduced state. As expected, addition of antimycin A to either substrate produced maximal rates of H2O2 production. This is because the electron transport chain on the substrate side of antimycin A is maintained in a reduced state (2) and thus would stimulate ROS production from both complexes I and III.

Although ER did not produce a change in liver mitochondrial proton leak or H2O2 production, this should not be taken as proof that alterations in either process play no role in the actions of ER. Compared with other organ systems, the liver preserves its function fairly well with aging, and old age is not associated with any particular liver disease (1, 27). It is possible that the actions of ER occur primarily through tissue other than liver and changes in liver may be small compared with those in postmitotic tissues. Also, liver may be a tissue that requires long periods of time to show changes in mitochondrial function with ER.

The results of our study indicate that 6 mo of ER, initiated in adult rats, does not result in changes in liver mitochondrial proton leak and H2O2 production. At this stage, it appears that liver may adapt to ER primarily by decreasing size and not greatly altering mitochondrial energy metabolism. Therefore, future studies of long-term ER (>6 mo) are needed to determine the role that changes in mitochondrial energy metabolism may play in the mechanism of action of ER.


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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The work was supported by National Institue on Aging Grant RO1 AG-17902.


    ACKNOWLEDGMENTS
 
We thank Dr. Bruce German (UC-Davis) for performing the fatty acid analysis as part of National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-35747-17/04, Dr. Cecilia Giulivi (Univ. of Minnesota-Duluth) for advice on Percoll gradients, and Barbara Sohal (Univ. of Southern California) for advice on H2O2 assays.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. J. Ramsey, Dept. of Molecular Biosciences, School of Veterinary Medicine, Univ. of California, Davis, CA 95616 (E-mail: jjramsey{at}ucdavis.edu).

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. Section 1734 solely to indicate this fact.


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