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Age-Related Mitochondrial DNA Deletion in Rat Liver Depends on Dietary Fat Unsaturation

Institute of Nutrition and Food Technology, Departments of ¹ Physiology ² Biochemistry and Molecular Biology, University of Granada, Spain.

Address correspondence to José L. Quiles, PhD, Instituto de Nutrición y Tecnología de Alimentos, C/Ramón y Cajal 4 18071 Granada, Spain. E-mail: jlquiles{at}ugr.es

	Abstract

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We fed male Wistar rats lifelong on virgin olive (rich in the monounsaturated oleic acid) or sunflower (rich in the polyunsaturated linoleic acid) oil-based diets. At 6 and 24 months, liver mitochondria were analyzed for a mitochondrial DNA (mtDNA) deletion, reactive oxygen species, antioxidants, and ultrastructural alterations. An aging-related increase in the relative amount of the deletion was observed for both dietary groups, being higher in animals fed sunflower oil. Oxidative stress was lower in virgin olive oil-fed animals. Aging led to higher superoxide dismutase, catalase, and glutathione peroxidase activities and increased -tocopherol and coenzyme Q. Mitochondria from aged animals fed sunflower oil exhibited a lower number of cristae and a higher circularity. Results suggest that the age-related increase of the relative amount of deleted mtDNA depends on fat unsaturation. Moreover, the studied mtDNA deletion was correlated with mitochondrial oxidative stress and ultrastructural alterations.

Nutrition has been related to aging, mainly at the level of caloric restriction. Caloric restriction prevents the age-related increase in frequency of mitochondrial deletions in the liver (9), and enhances mean life span in a wide range of species by a reduction in the oxidative stress level (11). However, caloric restriction results in practical and ethical difficulties that make its actual development almost impossible (12). Dietary fat type determines several biochemical parameters at the mitochondrial membrane level (13,14). The importance of dietary fatty acids resides in the fact that mitochondrial membrane adapts its lipid composition to dietary fat (15–17). In contrast, adaptations of the electron transport system in relation to dietary fat type have been widely reported (18–20). Moreover, oxidative stress is related to biological membrane composition. In that sense, a polyunsaturated fat source will lead to membranes being more prone to oxidation than would a saturated or a monounsaturated fat source. That has been widely demonstrated under a wide range of physiological and pathological situations in animal models and in humans (21–25).

The liver is the central metabolic organ of the body; therefore, dietary changes can have a major impact on aging liver and on general health (26). Moreover, the liver is critical in the protection from oxidative damage and plays a major role in the breakdown of potentially toxic lipophilic toxins (27). Although the aging liver appears to preserve its function relatively well (26), several changes have been associated with this organ during the process of aging. Elucidating the nature and mechanisms of these changes in the liver with aging may lead to a better understanding and treatment of hepatic dysfunction. According to that, the present study was designed to investigate the possible effect on a particular deletion at the liver mtDNA level and other aspects (oxidative stress and mitochondrial abnormalities) in liver mitochondria during aging by feeding rats lifelong with two different dietary fat sources. These fats were different in their lipid profile: virgin olive oil, mainly rich in the monounsaturated oleic acid and sunflower oil, mainly rich in the polyunsaturated linoleic acid.

	MATERIALS AND METHODS

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Experimental Design
Thirty-two male Wistar rats (Rattus norvegicus) initially weighing 80–90 g were allocated housed 8 per cage and maintained on a 12-hour light/dark cycle, with free access to food and drinking water. The rats were randomly assigned to two experimental groups and fed from weaning until 24 months of age on a semisynthetic and isoenergetic diet [according to the AIN93 criteria (28)] composed of (in g/100 g of diet): 26.7 casein, 13.53 starch, 45.29 sucrose, 1.0 vitamin mixture, 3.68 mineral mixture, 1.84 cellulose, 0.09 choline, 0.30 methionine, and 8.0 fat. Experimental diets differed only in the dietary fat source: virgin olive oil or sunflower oil (Table 1). Eight rats per group were killed at 6 and 24 months, respectively, from the start of the experiment. Animals were handled according to the guidelines of the Spanish Society for Laboratory Animals, and the experiment was approved by the Ethical Committee of the University of Granada. The rats were killed at the same time of the day to avoid any circadian fluctuation by cervical dislocation followed by decapitation. Blood was collected and plasma isolated. Livers were immediately removed to obtain mitochondrial fraction according to Fleischer and colleagues (29). Liver mitochondrial protein was determined according to Lowry and colleagues (30) using bovine serum albumin as a standard.

Real-Time PCR
Real-time PCR analysis was performed at the Genomic Facility from the Parque Científico de Madrid (Madrid, Spain). Two different regions of the mitochondrial genome were studied, one that is rarely affected by deletions both in humans and in rats (31,32) and another that is absent in the majority of individuals with large-scale deletions (31) and is also included in the so-called common deletion both in humans and in rats (32). The region rarely deleted is within the ND1 gene and the frequently deleted region is in the ND4 gene. Having identified these regions, we designed primers and probes using commercially available software (Primer Express; Applied Biosystems, Foster City, CA). PCR primers and fluorogenic probes for regions of ND1 (forward primer, CGCCCCAACCCTCTCC; reverse primer, GTATGCCTAGGTTGAGGTTGATAAGG; probe, ACTAGCTCTAAGCCTATGAATC) and ND4 (forward primer, CATTTTCCTGATCGAACCCCTCTAT; reverse primer, AGTTTTCCTCGTTGGGTTGTGATAA; probe, TTCCTGTGATGACAATGTT) were synthesized. Concentrations and PCR program for 10-µl assays were as follows: 0.09 µl of 0.25 ng/µl of DNA (separately amplified with the ND1 and ND4 primer/probe combinations). To each sample 4.41 µl of nanopure water, 5 µl of 2 x TaqMan Universal PCR MasterMix, and 0.5 µl of the combination primer/probe (900 nM for the primer and 250 nM for the fluorogenic probes) were added. PCR and fluorescence analysis were performed using the ABI 7900 HT (Weiterstadt, Germany). Amplification conditions were: 10 minutes at 95°C (polymerase activation) followed by 40 cycles with 15 seconds at 95°C (for denaturalization) and 1 minute at 60°C (annealing and extension). Each sample was assayed in triplicate and analyzed with SDS software (ABI) and Microsoft Excel (Redmond, WA). The method used for the real-time PCR quantification was 2^–Ct, which has been previously described (33). In this method, the relative amount of the deleted gene R = 2^–Ct, where Ct is calculated as: Cts – Ctcal; Cts is the Ct for each sample (Cts_ND1 – Cts_ND4), and Ctcal is the Ct for the calibrator (Ctcal_ND1 – Ctcal_ND4). Young animals were used as calibrators for changes in relative quantity in old groups. We performed a test (data not shown) to validate the 2^–Ct method, which is the calculation of the reaction efficiency (E). Both reactions (that from ND1 and that from ND4) propagated with a very high efficiency (close to 1). When the efficiency between two reactions is similar, Ct does not depend on the dilution series. In that situation, Ct values can be used to measure the input DNA and to quantify the relative amount of ND1 and ND4.

Plasma Biochemical Parameters
Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using Spinreact enzymatic kits (Girona, Spain).

Lipid Peroxidation Status
The ferrous oxide–xylenol orange (FOX2) method was used for determining hydroperoxides in mitochondrial membrane. Hydroperoxide levels were assayed according to the principle of the rapid peroxide-mediated oxidation of Fe²⁺ to Fe³⁺ under acidic conditions (34) using triphenylphosphine, an agent that avoids artifactual color generation in samples that might contain substantial quantities of loosely available iron. Thiobarbituric acid-reactive substances (TBARS) in mitochondrial membrane were determined according to Orrenius and colleagues (35).

Analysis of Antioxidant Enzyme Activity
Catalase activity was determined following the method described by Aebi (36), by monitoring at 240 nm the H₂O₂ decomposition as a consequence of the catalytic activity of catalase. Superoxide dismutase (SOD) was determined by the method of Crapo and colleagues (37), on the basis of the inhibition by SOD of the reduction of cytochrome c, as measured by spectrophotometry at 550 nm. Glutathione peroxidase was determined according to Flohé and colleagues (38). That method is based on the instantaneous formation of oxidized glutathione during the reaction catalyzed by glutathione peroxidase. That oxidized glutathione is continually reduced by an excess of glutathione reductase and NADPH present in the cuvette. The subsequent oxidation of NADPH to NADP⁺ was monitored spectrophotometrically at 340 nm. Tert-butyl hydroperoxide was used as substrate.

Tocopherol and Coenzyme Q Analysis
After extraction with methanol and light petroleum according to the method of Lang and Packer (39), levels of mitochondrial coenzyme Q and -tocopherol were determined by reversed-phase high-performance liquid chromatography (HPLC) using a Spherisorb S5 ODS1 (Merck, Darmstadt, Germany) column (maintained with an oven at a constant temperature of 22°C) and ethanol/purified water 97:3 (vol/vol) as mobile phase. The HPLC system was a Beckman In-line Diode Array Detector (model 168; Fullerton, CA) connected to a Waters (Milford, MA) 717 Plus Autosampler. Coenzyme Q and -tocopherol were identified by predetermining the retention times of individual standard. To assay total coenzyme Q as oxidized coenzyme Q, samples were treated with 1,4 benzoquinone (2 mg/ml), and then coenzyme Q was assayed.

Quantitative Determination of Fatty Acids From Mitochondrial Phospholipids
Initial total lipid isolation was performed according to Kolarovic and Fournier (40). Briefly, 200 µg of mitochondrial protein plus 0.5 ml of water were vortexed for 30 seconds with 100 µl of internal standard (0.857 mg/ml of heptadecanoic phospholipids [PL-17:0] dissolved in chloroform). Four milliliters of hexane/2-propanol (3:2) with 25 mg/L of butylated hydroxytoluene (BHT) were added and centrifuged for 10 minutes (4°C) at 1500 g. The organic layer was then removed to another glass tube, and the process was repeated 3 more times with the hydrophilic layer. The organic layer was evaporated to dryness under vacuum. The isolated lipids were dissolved with 200 µl of hexane/methyl-tert-butyl-ether/acetic acid (100:3:0.3, vol/vol/vol). Fatty acid composition of mitochondrial phospholipids was measured by previous separation of lipid classes using aminopropyl columns (Sep-Pak Cartridges; Waters) as described by Agren and colleagues (41). Fifty microliters of PL-17:0 were added to each sample as internal standard. Phospholipid fractions obtained from the columns were evaporated to dryness under vacuum, and 100 µl of chloroform was added to each tube. Fatty acid methyl esters were formed according to the method of Lepage and Roy (42). A gas-liquid chromatograph Model HP-5890 Series II (Hewlett Packard, Palo Alto, CA) equipped with a flame ionization detector was used to analyze fatty acids. Chromatography was performed using a 60-m-long capillary column (32-mm internal diameter and 20-mm thickness) impregnated with Sp 2330 FS (Supelco, Inc., Bellefonte, PA). The injector and the detector were maintained at 250°C and 275°C, respectively; nitrogen was used as carrier gas, and the split ratio was 29:1. Temperature programming (for a total time of 40 minutes) was as follows: initial temperature, 160°C for 5 minutes, 6°C/min to 195°C, 4°C/min to 220°C, 2°C/min to 230°C, hold 12 minutes, 14°C/min to 160°C.

Electron Microscopy Analysis of Isolated Mitochondria
After mitochondrial extraction, 400 µl of extracts was prefixed in 1.5% formaldehyde prepared in 1% cacodylate buffer, pH 7.4, for 2 hours at 4°C. After three washes in cacodylate buffer, extracts were fixed in 1% osmium tetroxide for 60 minutes at 0–4°C. The samples were dehydrated in graded ethanol and embedded in Epon resin. After overnight incubation at 65°C, ultrathin sections (70 nm) were cut with a diamond knife using an Ultracut Ultramicrotome (Reicher-Jung, Nussloch, Germany) and placed on 200-mesh copper grids. All sections were stained with uranyl acetate, counterstained with lead citrate, and viewed using a Carl Zeiss (Oberkochen, Germany) EM10C electron microscope at 40,000x magnification in the Scientific Instrument Service, University of Granada. Negatives were digitally transformed into positive images. Mitochondrial cristae were counted and expressed as number of cristae per micrometer of mitochondrial contour. The National Institutes of Health (NIH) Image J program was used for the quantification of mitochondrial size, contour, and circularity parameters in isolated mitochondria. All the chemical products and solvents, of highest grade available, were acquired from Sigma (St. Louis, MO) and Merck.

Statistical Analysis
The results represent the mean and the standard error of 8 animals. Previous to any statistical analysis, all variables were checked for normality and homogeneous variance using the Kolmogorov–Smirnoff and the Levene tests, respectively. When a variable was found not to follow normality, it was log-transformed and reanalyzed. Statistically significant differences (p <.05) were assessed by analysis of variance (ANOVA). To evaluate differences in means across groups, a multiple comparison test adjusted by Bonferroni correction was performed. Data were analyzed using the SPSS/PC statistical software package (SPSS for Windows, 11.0.1, 2001; SPSS, Inc., Chicago, IL).

	RESULTS

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Relative Increase of the Deleted mtDNA for the ND4 Gene in Old Animals
Young animals were used as calibrator, thus they become the sample with the 1x relative amount, and all other quantities are expressed as an n-fold increase to the calibrator (Figure 1). Old animals fed virgin olive oil showed a 6.5 ± 0.5-fold increase of mtDNA deleted for the ND4 gene; meanwhile, the relative increase for old animals fed sunflower was 10.4 ± 1.5, which represents a 60% higher increase in the group of old animals fed sunflower oil when compared with those fed virgin olive oil.

View larger version (7K): [in this window] [in a new window]   Figure 1. Relative increase of deleted mitochondrial DNA (mtDNA) in liver (mean ± standard error of the mean; n = 8) of rats fed virgin olive or sunflower oil. *Statistically significant difference between virgin olive oil group and sunflower oil group (p <.05)

Electron Microscopy Analysis of Isolated Mitochondria
Electron microscopy of isolated mitochondria showed that mitochondria preparations were free of contamination and that all groups exhibited similar purity (Table 4, Figure 2). The size of isolated mitochondria as well as the mitochondrial contour were similar irrespective of diet but were lower in the old groups. The circularity parameter was similar for both dietary groups at 6 months of age. In old animals, circularity was higher in animals fed sunflower oil. The number of cristae per micrometer of mitochondrial contour was lower in animals fed sunflower oil compared to those fed virgin olive oil, both at 6 and 24 months of age. Aging diminished the number of cristae in the sunflower oil-fed group.

View larger version (146K): [in this window] [in a new window]   Figure 2. Transmission electron micrograph of isolated liver mitochondria of rats fed virgin olive oil or sunflower oil. Magnification: 40,000x

	DISCUSSION

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It has been well-established that mitochondrial phospholipid fatty acids from body tissues are modulated by the diet (13,16). In this study, the different lipid profiles of diets were properly reflected in liver mitochondrial phospholipids of young and old animals (Tables 1 and 2), and suggest proper adaptation of the rats to dietary fats. These results are noteworthy because any benefit or damage derived from the intake of the two lipid sources is maintained throughout life, providing the opportunity to modulate aging by diet. Also, it is interesting to note the net increase found in all the fatty acids studied with aging, irrespective of dietary fat. As far as we know, this is the first report of this type in mitochondrial phospholipids. However, in relation to blood lipids, evidence suggests that a net increase in the different classes of lipids usually happens (46–48). The reasons for lipid accumulation during aging are several, including increased sedentarism, metabolic changes such as those related to a severe age-related impairment of 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase regulation (49), and lower activity of prenyl transferases (50,51). In relation to mitochondrial phospholipids, we have no clue at the moment about the reason of this accumulation with age at the mitochondrial membrane level.

In relation to oxidative stress, it has been described that mitochondrial lipid profiles of animals fed diets rich in PUFAn6 are associated with higher levels of oxidative stress than are those of animals fed virgin olive oil. Examples of such situations are the stress produced by xenobiotics such as doxorubicin (19), the performance of physical exercise (13), or the intake of thermally oxidized (fried) fats (22,24). We have demonstrated, even in aging, that lipid peroxidation is lower in animals fed virgin olive oil (52). In the present study it is confirmed that hydroperoxides and TBARS found in old animals fed virgin olive oil were lower than those in old animals fed sunflower oil (Table 3). Concerning oxidative stress and aging, opposing results have been reported in past years (1). However, after contradictory artifacts are avoided and appropriate biomarkers are used, overall, it seems that free radical damage increases during aging (1,53). Our study agrees with the above-mentioned assumption, which is additionally in accord with the free radical theory of aging of Harman (4). Thus, we found increased levels of damage with aging in both dietary groups, although this increase was higher in animals fed sunflower oil. This higher level of oxidative stress with age may be directly related to the higher lipid content of liver mitochondrial membranes from old animals. However, despite both dietary groups reaching similar levels of total fatty acids, the animals fed sunflower oil had higher oxidative stress figures. That result may be explained by PUFA n6 values being higher in animals fed sunflower oil. Also, as has been stated above, these fatty acids are more prone to be oxidized.

Antioxidant defenses do not decrease with aging; increases with age have been usually described (1). In the present study, higher activities of antioxidant enzymes (Table 3) were found in groups of old animals, irrespective of diet. It has been suggested that low or normal activity of antioxidant enzymes might indicate the existence of a balance between free radical production and antioxidant levels and that an increase in some of them only show an increase in the degree of oxidative stress (54). That explanation would be true in the present study, because the higher antioxidant activity correlates with the increased oxidative stress found in old animals. It is interesting that animals fed virgin olive oil, which had a lower oxidative increase with aging, reached a higher increase in -tocopherol than those fed sunflower oil. In that sense, the liver appears to accumulate these potent antioxidants at the sites at which they are needed to try to counterbalance oxidative damage (55,56) and apparently, as can be ascertained from the higher increase of -tocopherol in aged animals fed virgin olive oil and the absence of an increase of glutathione peroxidase in these animals, dietary fat may modulate the final oxidative extent associated with aging. In contrast, the increase in tocopherol and coenzyme Q with age might account only for the net increase in lipids with aging, as these antioxidants are of lipidic nature.

We investigated if changes found at the mtDNA deletion level and oxidative stress status could affect mitochondrial ultrastructure under our experimental conditions. In fact, the lifelong intake of sunflower oil led to worse preserved mitochondrial structure, as suggested by the lower number of cristae per micrometer of mitochondrial contour found in old animals fed sunflower oil compared to young animals fed the same oil; additionally, animals fed virgin olive oil had a higher number of mitochondrial cristae at both age periods. In the same way, mitochondrial circularity (an increase of which represents control loss) was higher in old animals fed sunflower oil compared to those fed virgin olive oil. It deserves to be mentioned that ultrastructural abnormalities at the mitochondrial level are related to several pathologies (57), and here we found a correlation between the mtDNA deleted, the oxidative stress status, and some of these ultrastructural abnormalities.

Summary
Present results confirm the increase in the relative amount of deleted mtDNA in liver with aging and demonstrate that this increase is differentially modulated by the lifelong intake of different dietary fats. In that sense, the use of virgin olive oil led to a lower increase in the deletion than did the intake of the more polyunsaturated sunflower oil. In contrast, under our experimental conditions, the increase in the relative amount of studied mtDNA deletion could be correlated with mitochondrial oxidative stress status and ultrastructural alterations.

	Acknowledgments

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We are grateful for financial support to the Spanish Ministry of Science and Technology (grant 1FD97-0457-c02-01). Dr. José L. Quiles, Dr. Julio J. Ochoa, and Dr. Carmen Ramirez-Tortosa are supported by a "Ramón y Cajal" contract from the Spanish Ministry of Science and Technology and the University of Granada.

We are also grateful to Dr. Ricardo Ramos (Genomic Facility of the Parque Científico de Madrid) for his assistance with the real-time PCR analyses and to Dr. Concepción Hernandez and Dr. David Porcel (Scientific Instrument Service of the University of Granada) for their assistance with the electron microscopy analyses.

	Footnotes

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Decision Editor: James R. Smith, PhD

Received January 6, 2005

Accepted July 15, 2005

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