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Am J Physiol Cell Physiol 293: C1302-C1308, 2007. First published July 25, 2007; doi:10.1152/ajpcell.00203.2007
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CELLULAR METABOLISM

Sexual dimorphism in liver mitochondrial oxidative capacity is conserved under caloric restriction conditions

A. Valle, R. Guevara, F. J. García-Palmer, P. Roca, and J. Oliver

Grup de Metabolisme Energètic i Nutrició, Departament de Biologia Fonamental i Ciències de la Salut; Institut Universitari d'Investigació en Ciències de la Salut, Universitat de les Illes Balears, and Ciber Fisiopatología Obesidad y Nutrición (CB06/03) Instituto Salud Carlos III, Palma de Mallorca; Spain

Submitted 17 May 2007 ; accepted in final form 19 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Caloric restriction (CR) without malnutrition has been shown to increase maximal life span and delay the rate of aging in a wide range of species. It has been proposed that reduction in energy expenditure and oxidative damage may explain the life-extending effect of CR. Sex-related differences also have been shown to influence longevity and energy expenditure in many mammalian species. The aim of the present study was to determine the sex-related differences in rat liver mitochondrial machinery, bioenergetics, and oxidative balance in response to short-term CR. Mitochondria were isolated from 6-mo-old male and female Wistar rats fed ad libitum or subjected to 40% CR for 3 mo. Mitochondrial O2 consumption, activities of the oxidative phosphorylation system (complexes I, III, IV, and V), antioxidative activities [MnSOD, glutathione peroxidase (GPx)], mitochondrial DNA and protein content, mitochondrial H2O2 production, and markers of oxidative damage, as well as cytochrome C oxidase and mitochondrial transcription factor A levels, were measured. Female rats showed a higher oxidative capacity and GPx activity than males. This sexual dimorphism was not modified by CR. Restricted rats showed slightly increased oxygen consumption, complex III activity, and GPx antioxidant activity together with lower levels of oxidative damage. In conclusion, the sexual dimorphism in liver mitochondrial oxidative capacity was unaffected by CR, with females showing higher mitochondrial functionality and ROS protection than males.

oxidative phosphorylation; free radicals; antioxidant enzymes; mitochondrial transcription factor A

CALORIC RESTRICTION (CR) without malnutrition is the only known experimental intervention that has been shown to extend life span and to delay the onset of age-related diseases in mammals (34, 49). Although the benefits of CR are well established, the mechanisms underlying its effects remain unclear. During the last decades increasing evidence has suggested that oxidative damage to proteins, lipids, and DNA may be responsible for normal aging, and CR may operate by decreasing the accumulation of this damage to macromolecules (10, 45). Since electron transport chain is the main source of free radical production in the cell, mitochondria are likely to play a central role in the life-extending mechanism underlying CR.

Naturally occurring episodes of CR in animal populations are common due to adverse climatic or biological changes such as drought, cold, or plagues. The need to postpone reproduction until more energy-favorable periods seems to be the selective pressure responsible for the development of these anti-aging mechanisms (21). However, some authors have argued that because of their higher relative importance for reproduction and the survival of the species, females have been subjected to more severe selection pressures to be more resistant to CR than males (19, 51). In fact, several studies on rodents have shown that CR has a greater and more permanent effect on physical growth in male than in female rats (8, 18). Recently, in our laboratory (47, 48), CR was described to produce a higher deactivation of brown adipose tissue by means of a loss of mitochondrial recruitment in female rats, which contributes to a large extent to overall energy saving.

During nutritional interventions, the liver is one of the most affected tissues, since it orchestrates the supply of energy substrates to different tissues. Although CR has been shown to decrease oxidative stress in liver mitochondria (15, 16), the influence of sex in this response has not been explored to date. Previous works have demonstrated that liver from female rats shows highly differentiated mitochondria with greater machinery per mitochondrion (20). On the other hand, mitochondria from female rats exhibit higher antioxidant gene expression (4), which seems to be induced by estrogens (3).

Taking this into account, the aim of this study was to determine the sex-related differences in rat liver mitochondrial machinery, bioenergetics, and oxidative balance in response to short-term CR. To tackle this aim, we measured mitochondrial O2 consumption, oxidative phosphorylation (OXPHOS) activities, protein content, and the levels of mitochondrial transcription factor A (TFAM) in male and female rats maintained on a 40% CR diet for 3 mo. H2O2 production, antioxidative activities, and markers of protein and lipid oxidative damage were also measured.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Routine chemicals were supplied by Sigma-Aldrich (St. Louis, MO), Panreac (Barcelona, Spain), and Amersham Pharmacia Biotech (Little Chalfont, UK). Real-time PCR reagents and oligonucleotide primer sequences were supplied by Roche Diagnostics (Basel, Switzerland).

Animals and diets. All animals were treated in accordance with the university bioethical committee guidelines for animal care and European Union regulations (86/609/EEC) and were approved by the ethics committee. Male and female Wistar rats ages 3 mo were purchased from Charles River (Barcelona, Spain) and housed individually in wire-bottom cages at 22°C with 12 h light-dark cycle. To study the effects of CR in both sexes, 12 males and 12 female rats were randomly divided into four groups (n = 6): control male and control female groups fed ad libitum (AL) with standard chow pellets (A04; supplied by Panlab, Barcelona, Spain) and restricted male and restricted female groups subjected to 40% food restriction (CR) for 3 mo. In restricted animals, food was supplied on a daily basis at the beginning of the dark cycle and was weekly updated compared with ad libitum rats to correct for growth requirements.

Death and mitochondria isolation. Animals were killed by decapitation, and livers were removed rapidly, weighed, and placed in ice-cold isolation buffer (250 mM sucrose, 5 mM Tris·HCl, and 2 mM EGTA, pH 7.4). Liver was finely chopped and rinsed in the isolation buffer to remove excess blood. Liver samples (5 g) were homogenized in 35 ml of ice-cold isolation buffer with a Teflon/glass homogenizer. Aliquots were stored at –80°C for determination of total DNA (46), triglycerides (43), and protein content (5). The rest of the homogenate was used for isolation of mitochondria by differential centrifugation. Briefly, nuclei and cell debris were removed by centrifugation at 500 g for 10 min, and supernatants were centrifuged at 8,000 g to yield the mitochondrial pellet. Pellets were washed once by resuspension and centrifuged (8,000 g), and the final pellets were resuspended in the same buffer. Mitochondrial protein was measured using the Bradford method (5).

Measurement of mitochondrial O2 consumption. Liver mitochondrial O2 consumption was measured polarographically as described previously (31) with minor modifications. Mitochondria were incubated in a water-thermostatically regulated chamber with a computer-controlled Clark-type O2 electrode (Oxygraph; Hansatech, Norfolk, UK) at a concentration of 1 mg/ml mitochondrial protein in respiration buffer (145 mM KCl, 30 mM HEPES, 5 mM KH2PO4, 3 mM MgCl2, 0.1 mM EGTA, and 0.1% BSA, pH 7.4 at 37 °C). Glutamate/malate (2.5 mM/2.5 mM) or succinate (5 mM) was used as substrate in the absence (state 4) and in the presence (state 3) of 500 µM ADP. Mitochondrial viability was checked by the respiratory control ratio (state 3/state 4).

Mitochondrial activities. The measurement of the specific activities of the OXPHOS complex I (NADH:ubiquinone oxidoreductase; EC 1.6.99.5 [EC] ) (39), complex III (ubiquinol:cytochrome c reductase; EC 1.10.2.2 [EC] ) (24), and complex IV or COX (cytochrome c oxidase; EC 1.9.3.1) were performed as described and adapted to microtiter plate assay with some modifications. Mitochondrial antioxidant superoxide dismutase (MnSOD) (38) and glutathione peroxidase (GPx) (44) activities were also assayed. The assays were performed in a 200-µl final volume with 1–5 µg of mitochondrial proteins. COX activity was also measured in homogenates to calculate mitochondrial recovery.

Detection of H2O2 production in mitochondria. The rate of H2O2 production in mitochondria was determined by using the oxidation of the fluorogenic indicator Amplex red (Molecular Probes, Paisley, UK) in the presence of horseradish peroxidase. Mitochondria (0.25 mg protein/ml) were incubated at 37°C in respiration buffer containing 0.1 U/ml horseradish peroxidase and 50 µM Amplex red. H2O2 production was initiated in mitochondria by adding succinate (5 mM) as substrate. Background fluorescence was measured in parallel in wells containing all reactants except substrate. Fluorescence was recorded in a microplate reader (FLx800; Bio-Tek Instruments, Winooski, VT) with 530-nm excitation and 590-nm emission wavelengths. Levels of H2O2 were expressed as fluorescence minus background (pmol·mg protein–1·min–1). Rates were determined by converting fluorescence readings, using standard curves generated over a range of H2O2 concentrations.

Extraction and quantification of mitochondrial DNA. Mitochondrial DNA (mtDNA) was extracted by digestion with proteinase K (100 µg/µl) in a buffer containing 50 mM KCl, 10 mM Tris·HCl, 2.5 mM MgCl2, and 0.5% Tween 20. Mitochondria samples were incubated overnight at 37°C and then boiled for 5 min to inactivate the enzyme. Mitochondrial DNA was linearized by digestion with BclI restriction enzyme for 3 h at 50°C and boiled for 5 min. Samples were centrifuged at 7,000 g for 5 min, and the resulting supernatant was used for amplification. A quantitative PCR assay was adapted to the LightCycler technology from Koekemoer et al. (22). PCR was performed to amplify a 162-nt fragment of the mitochondrial NADH dehydrogenase subunit 4 gene. The primer sequences were 5'-TACACGATGAGGCAACCAAA-3' and 5'-GGTAGGGGGTGTGTTGTGAG-3'. The concentration of the purified template was determined spectrophotometrically. Increasing amounts of template were amplified in parallel reactions to obtain a standard curve. Amplification was carried out in a LightCycler rapid thermal cycler system (Roche) using a total volume of 10 µl containing 0.375 µM of each primer, 3 mM MgCl2, 1 µl of LightCycler FastStart DNA Master SYBR green I (Roche), and 2.5 µl of sample prepared as described above. The PCR reactions were cycled 35 times after initial denaturation (95°C, 10 min), with the following parameters: denaturation at 95°C for 10 s, annealing at 60°C for 12 s, and extension at 72°C for 12 s.

TFAM and COX II Western blotting. For TFAM and COX II, 40 µg of mitochondrial and 30 µg of homogenate protein, respectively, were fractioned by SDS-PAGE (12% polyacrylamide gel) and electrotransferred onto nitrocellulose filters. Ponceau S staining was used to provide visual evidence of correct loading and electrophoretic transfer of proteins to nitrocellulose filter. Membranes were incubated overnight at 4–6°C in a blocking solution of 5% nonfat powdered milk in Tris-buffered saline (20 mM Tris·HCl, 0.13 mM NaCl, and 0.1% Tween 20). Developments of the immunoblots were performed using an enhanced chemiluminescence Western blotting analysis system (Amersham). Bands in films were analyzed using scanner photodensitometry and quantified using Kodak 1D Image Analysis software.

Measurement of carbonyl content. Carbonyl groups were quantified using the Oxyblot protein oxidation detection kit (Chemicon, Chandlers Ford, UK). 2,4-Dinitrophenylhydrazine (DNPH) derivatization was carried out for 15 min on 15 µg of homogenate protein following the manufacturer's instructions. Proteins were transferred to nitrocellulose filters by means of a slot-blot system (Bio-Rad, Hercules, CA). After incubation with anti-DNP antibody, blots were developed using a chemiluminescence detection system (Amersham). Bands in films were analyzed using scanner photodensitometry and quantified using Kodak 1D Image Analysis software. To determine specificity, the oxidized proteins provided by the kit were included as a positive control. Treatment of sample with a control solution served as a negative control to the DNPH derivatization.

Measurement of thiobarbituric acid-reactive substances. Lipid peroxidation levels or thiobarbituric acid-reactive substances (TBARS) were determined as malondialdehyde-thiobarbituric acid adducts according to Buege et al. (6). Peroxidation levels were measured spectrophotometrically at 532 nm, using a molar extinction coefficient of 1.56 x 105 M–1/cm, and expressed as nanomoles of TBARS per milligram of protein.

Statistics. Results are means ± SE. Statistical analysis was carried out using the Statistical Program for the Social Sciences software (SPSS 14.0). Statistical significance of the data was assessed using two-way ANOVA. The statistical factors analyzed were restriction diet (R) and sex (S). Student's t-test was used to determine the differences between the groups involved. Statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Liver mass and composition. The effects of CR and sex on liver mass and composition are summarized in Table 1. As expected, body and liver mass were significantly lower in CR compared with AL-fed rats of both sexes. Liver mass loss in restricted male rats seemed to be slightly greater compared that in female rats (28 vs. 21%). No significant differences were found in total protein, DNA, or triglyceride content.


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  		Table 1. Effect of sex and CR on biometric parameters and liver composition

 
Liver mitochondrial content. The levels of several mitochondrial markers such as mitochondrial protein, mtDNA, or COX II content are compiled in Table 2. No differences were observed in mitochondrial protein content per gram of tissue in any of the studied groups. Nevertheless, mtDNA levels per gram of tissue were significantly higher in males compared with females in both control and restricted rats. Thus the protein/mtDNA ratio was higher in female rats, indicating a higher protein content per mitochondria in that sex.


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  		Table 2. Effect of sex and CR on mtDNA, mitochondrial protein, TFAM, and COX II protein levels in liver

 
This greater amount in mitochondrial protein adjusted to mtDNA is in agreement with the higher levels of TFAM found in females (representative Western blots are shown in Fig. 1). COX subunit II also showed higher levels in female compared with male rats fed under AL conditions. No CR effects were found on these parameters.


Figure 1
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  		Fig. 1. Western blot of mitochondrial transcription factor A (TFAM) and cytochrome c oxidase (COX II) in liver of male and female rats. Total protein (40 µg) for TFAM and COX II (30 µg) were fractioned in 12% SDS-PAGE. Ponceau S staining was used to provide visual evidence of correct loading and electrophoretic transfer of proteins to nitrocellulose filter. Representative bands from 2 animals of each group are shown. AL, ad libitum; CR, 40% caloric restriction.

 
O2 consumption. The rate of mitochondrial O2 consumption was measured with glutamate/malate or succinate as substrates in states 4 and 3. As shown in Fig. 2, female rats showed higher oxidative capacity than male rats independently of group, state, or substrate used.


Figure 2
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  		Fig. 2. Effect of sex and CR on liver mitochondrial O2 consumption. The substrate/ADP titration protocol is described in detail in MATERIALS AND METHODS. Bars represent means ± SE of 6 animals per group. ANOVA: S, effect of sex (P < 0.05). Student's t-test: *P < 0.05, male vs. female. {dagger}P < 0.05, control (AL) vs. restricted (CR).

 
Under CR conditions, O2 consumption showed a trend for increase in both sexes that only marginally failed to reach significance (P = 0.063). Nevertheless, this increase was statistically significant in male liver mitochondria when respiring in state 3 with glutamate/malate as substrate.

Mitochondrial activities. Figure 3 shows the activities of several electron transport chain complexes key in oxidative and phosphorylative capacities of mitochondria. Complex I and III activities were significantly higher in female rats in both dietary conditions. CR had no effects on complex I activity, whereas complex III activity was significantly increased by CR in both sexes. Complex IV or COX activity was unaffected by sex or CR. With regard to ATPase, phosphorylative capacity of liver mitochondria was unchanged in either sex or dietary intervention.


Figure 3
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  		Fig. 3. Effect of sex and CR on activity of mitochondrial oxidative phosphorylation (OXPHOS) complexes (CI, complex I; CIII, complex III) in liver mitochondria. Bars represent means ± SE of 6 animals per group (au, arbitrary units). ANOVA: S (P < 0.05); R, effect of diet (P < 0.05); NS, not significant. Student's t-test: *P < 0.05, male vs. female. {dagger}P < 0.05 control vs. restricted.

 
Mitochondrial H2O2 production. In view of the greater activity of some mitochondrial complexes in female rats and the increasing tendency induced by CR in these parameters, we measured the production of H2O2 in mitochondria using succinate as substrate (Fig. 4). The rate of H2O2 production of liver mitochondria from female AL-fed rats was significantly higher than that of males. This sex-related difference in H2O2 production disappeared under CR by decreasing female H2O2 production rate.


Figure 4
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  		Fig. 4. Effect of sex and CR on H2O2 production in liver mitochondria. Mitochondria were incubated with succinate as substrate in the presence of Amplex red as probe for H2O2 production as described in MATERIALS AND METHODS. Bars represent means ± SE of 6 animals per group. ANOVA: NS. Student's t-test: *P < 0.05, male vs. female.

 
Markers of oxidative damage. To estimate the profile of oxidative damage, we measured the protein carbonyl and TBARS content in the rat liver of the studied groups (Fig. 5). For protein carbonyls, no sex-related differences were observed between AL groups, whereas CR induced a decrease in carbonyl content that was statistically significant in female rats. Lipid peroxidation measured as TBARS content was also found to be reduced in both sexes by CR.


Figure 5
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  		Fig. 5. Effect of sex and CR on markers of oxidative stress. Protein carbonyls and thiobarbituric acid-reactive substances (TBARS) were determined as described in MATERIALS AND METHODS. Bars represent means ± SE of 6 animals per group. ANOVA: R (P < 0.05). Student's t-test: {dagger}P < 0.05, control vs. restricted.

 
Mitochondrial antioxidative activities. Figure 6 shows the specific activity of the main antioxidant enzymes MnSOD and GPx in mitochondrial H2O2 scavenging. MnSOD activity was similar between sexes but was slightly increased by CR in female rats (P < 0.1). GPx activity in female rats was twice that in male rats, and CR showed a trend to increase this activity in both sexes (P < 0.1).


Figure 6
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  		Fig. 6. Effect of sex and CR on liver mitochondrial antioxidative activities. Mitochondrial antioxidant superoxide dismutase (MnSOD) and glutathione peroxidase (GPx) activity was determined as described in MATERIALS AND METHODS. Bars represent means ± SE of 6 animals per group. ANOVA: S (P < 0.05); NS, not significant. Student's t-test: *P < 0.05, male vs. female.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Sexual dimorphism in liver mitochondria. Several studies have demonstrated that there is a sexual dimorphism in mitochondrial metabolism in rodents, especially affecting tissues with an important involvement in the energy metabolism such as liver, muscle, or brown adipose tissue (7, 20, 43). Our laboratory (20) previously reported that female rats exhibit functional and morphological differences in liver mitochondria showing greater machinery and differentiation degree than males. In the present study, comparisons between mitochondria from control male and female rats confirmed our previous findings but also highlighted differences in liver mitochondrial number between sexes. Considering that mtDNA content is indicative of number of mitochondria (9, 23), female rats showed a lower mitochondrial content per cell compared with males. However, the content of protein per mitochondria, and its specific O2 consumption, indicates a greater machinery or differentiation state of these mitochondria in agreement with the higher membrane potential previously described in female liver mitochondria (20). Although female rats presented a lower number of mitochondria, their total levels of COX II exceeded that of males (see Table 2). This suggests that the higher maturation degree of mitochondria from female rats counteracts their lower number of mitochondria, even exceeding the total oxidative capacity of males as interpreted by O2 consumption data. Nevertheless, although COX II protein levels and mitochondrial respiration rate were higher in female compared with male rats, mitochondrial COX activity was found not to be significantly different between sexes, indicating that additional factors may be modulating the activity of this flux-generating complex in mitochondria (28).

The aforementioned differences in mitochondrial characteristics imply variations in the proliferation and differentiation patterns. In this sense, TFAM plays a key role in mitochondrial biogenesis, because it is basic to the initiation of both replication and expression of mtDNA, which codes for part of the mitochondrial proteins such as COX II (11, 37). It has been postulated that small amounts of TFAM are necessary to initiate mtDNA replication, whereas expression of mtDNA is activated only at high concentrations of this factor (13, 33). Our data agree with this idea, since mitochondria from female rats showed higher TFAM levels in concert with their higher differentiation state, whereas the lower levels of males agree with their greater mitochondria number as interpreted by their mtDNA content.

It has been shown that increased mitochondrial membrane potential and/or elevated O2 consumption may result in elevated reactive oxygen species (ROS) (36). In our study, the higher hydrogen peroxide production observed in mitochondria from female rats is consistent with this notion. However, these results differ from other studies, which found a lower hydrogen peroxide production in mitochondria from female rats (3, 4). These studies consider that the higher antioxidative activities such as GPx in female rat mitochondria are responsible for the lower ROS production. Our study and those previously mentioned agree in the finding of higher antioxidant activity in female rats but differ in the involvement of these enzymes in the net ROS production in the in vitro assay conditions. In this way, end products of oxidative damage such as carbonyl content or TBARS could be better markers of oxidative stress, since they are the result of the balance among total peroxide production, antioxidant defenses, and repairing systems. No sex-related differences were found in these markers of oxidative damage, indicating that the higher GPx activity observed in the current study in mitochondria from female rats, or any other repairing system, may counteract the higher peroxide production.

Effect of sex on liver mitochondrial response to short-term CR. Taking into account the sexual dimorphism observed in liver mitochondria bioenergetics, our purpose in this study was to determine whether the CR effects on mitochondria are dependent on sex. To our knowledge, this is the first work that looks for sex-related differences in the liver mitochondrial phenotype in response to CR. Previous works in male rats have found that CR does not modify O2 consumption or metabolic rate in mitochondria and hepatocytes isolated from liver of rats fed under CR conditions (26, 50). In contrast, a recent study reported that hepatocytes isolated from CR male rats reduce their total O2 consumption but increase their mitochondria pool (30). Our results are in agreement with the former set of reports, with CR not exerting significant changes in mitochondrial O2 consumption or activities. However, in the current study we noticed that CR induced a significant increase in complex III activity, with a similar trend in both COX activity (P = 0.067) and mitochondrial respiration rate (P = 0.063). This increase in mitochondrial respiration rate was even statistically significant in mitochondria of restricted male rats respiring in state 3 with glutamate/malate. These findings are in agreement with a previous work showing a time-dependent increment of mitochondrial oxidative capacity in liver of male rats subjected to moderate CR (26%) (2). This tendency to increase mitochondrial oxidative capacity could be related to a higher energy demand due to the role the liver plays in the adaptation of the organism to nutritional changes. Thus, in the case of CR, animals are subjected to intermittent and more prolonged fasting periods, increasing insulin sensitivity, liver beta-oxidation, and ketone bodies synthesis (17, 32), functions that could be affecting liver energy demands. Interestingly, intermittent fasting (diets with reduced meal frequencies such as every-other-day fasting) also have been shown to increase life span similarly to CR, even when there is little or no overall decrease in calorie intake (1, 14). Further studies are necessary to clarify whether the benefits of CR come from the reduction in calories or from prolonging fasting periods.

With regard to oxidative stress, previous works in male rats have demonstrated that long-term CR (>12 mo) attenuates oxidative damage to macromolecules by lowering H2O2 production (15, 16, 31). However, when animals are subjected to short-term CR (<6 mo), a reduction or no change has been reported depending on the study (15, 25, 41). In our 3-mo CR assessment, we did not find a statistically significant reduction in H2O2 production, although females showed a trend toward a reduction (P = 0.08), achieving levels similar to that in males. The levels of markers of protein and lipid oxidation were also decreased by CR in agreement with previous reports (12, 27). No sex-related effects were found in these parameters in response to CR, although carbonyls showed a profile in accordance with higher CR effects in females. It is worth noting that females started from a higher oxidative capacity in liver mitochondria that was increased by CR. However, as shown in Fig. 5, the levels of oxidative damage were reduced, showing levels similar to that in males. This fact may be explained on the basis of the higher GPx activity found in the liver mitochondria from female rats. Nevertheless, the likelihood of other mechanisms operating in this attenuation of oxidative damage, such as changes in mitochondrial efficiency, protein turnover, or other antioxidant systems, cannot be ruled out.

In relation to this, CR induced an upward trend in GPx activity in agreement with previous reports (40, 52), whereas MnSOD activity remained almost unchanged (29). However, the different studies in the literature do not support a clear-cut pattern of CR-related changes in antioxidant defenses, with reports showing controversial effects on these activities (35, 42). Differences in rodent model, strain, age of initiation, and duration of CR may be responsible for the differences between these studies.

The intrinsic sex-related differences in GPx activity were not modified by restricted feeding, with female rats showing higher activity than males. This may explain, at least in part, the aforementioned similar-sex attenuation of oxidative damage despite the higher oxidative capacity of female rats. It is worth noting that the activity of the glutathione system depends on the availability of reducing equivalents. Whether greater GPx activity in females also involves sex-related differences in the NADPH mitochondrial production system awaits further experimental exploration.

In summary, liver mitochondria from female rats show signs of a higher oxidative capacity with a greater differentiation degree than that of male rats. These mitochondrial features are consistent with the higher hydrogen peroxide production but not with elevated oxidative damage, probably in relation with the greater antioxidant protection in females. Furthermore, the results also demonstrate that CR does not alter the sexual dimorphism in liver mitochondrial oxidative capacity and antioxidant defenses.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Spanish Government (FIS PI042294, PI042377, PI060266, and PI060293) and by the Conselleria d'Innovació i Energia of the Comunitat Autònoma de les Illes Balears (PROGECIB-40A). A. Valle was funded by a grant from the Ministerio de Educación y Ciencia of the Spanish Government. R. Guevara was funded by a grant from the Comunitat Autònoma de les Illes Balears.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Hidetoshi Inagaki for providing the antiserum against TFAM.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Oliver, Dept. de Biologia Fonamental i Ciències de la Salut, Universitat de les Illes Balears, Cra. Valldemossa km 7.5, E-07122 Palma de Mallorca, Spain (e-mail: jordi.oliver{at}uib.es)

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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 DISCUSSION
 GRANTS
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