Free Radical Research, March 2014; 48(3): 322–332 © 2014 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2013.867959

ORIGINAL ARTICLE

Effect of training and vitamin E administration on rat liver oxidative metabolism P. Venditti, G. Napolitano, D. Barone & S. Di Meo

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Department of Biology, University of Naples “Federico II”, Naples, Italy Abstract We studied vitamin E effects on metabolic changes and oxidative damage elicited by swim training in rat liver. Training reduced mitochondrial aerobic capacity but increased liver content of mitochondrial proteins, so that tissue aerobic capacity was not different in trained and sedentary animals. Vitamin E supplementation prevented the training-induced mitochondrial changes. Training and vitamin E effects were consistent with the changes in tissue content of factors involved in mitochondrial biogenesis (peroxisomal proliferator-activated receptor-γ coactivator and nuclear respiratory factors 1 and 2). Tissue and mitochondrial oxidative damage was reduced by training decreasing the rate of mitochondrial reactive oxygen species (ROS) production and enhancing glutathione levels and glutathione peroxidase and glutathione reductase activities. The effects of vitamin E were different when it was administered to sedentary or trained rats. In the former, vitamin E reduced liver preparations oxidative damage decreasing ROS production rate and increasing GSH content without any effect on antioxidant enzyme activities. In the latter, vitamin E did not modify ROS production and oxidative damage but decreased antioxidant levels. This decrease was likely responsible for the enhanced susceptibility to in vitro oxidative attack of the hepatic tissue from trained rats following vitamin E supplementation. These results indicate that vitamin E integration, which can be healthy for animals subjected to acute exercise, is not advisable during training because it prevents or reduces the favourable effects of the physical activity. They also support the idea that the stimulus for training-induced adaptive responses can derive from the increased ROS production that accompanies the single sessions of the training program. Keywords: vitamin E, training, oxidative damage, rat liver, mitochondria

Introduction It is long known that acute physical exercise can produce significant damages, including alterations in membranes of mitochondria and sarcoplasmic and endoplasmic reticulum [1–4] of skeletal muscles and other tissues. Since reactive oxygen species (ROS) involvement in the exercise-induced damage was firstly demonstrated by Davies et al. [3], research in the area is greatly grown and it is now clear that intense muscular contractile activity results in oxidative stress, as indicated by decreased levels of reduced glutathione (GSH) and increased oxidation of proteins, lipids and DNA [5]. Moreover, protein and lipid oxidative damage is associated with both decrease in muscle force production and fatigue appearance [6]. Differently from acute exercise, regular physical activity (training) has several healthy effects, including the maintenance of insulin sensitivity and cardiorespiratory fitness, so that it is able to prevent type 2 diabetes and coronary heart diseases [7,8]. Furthermore, training enhances the tissue metabolism, inducing mitochondrial proliferation, and the effectiveness of the antioxidant defense system, increasing the activity of antioxidant enzymes [4,9]. The observation that the expression of antioxidant enzymes is induced by exposure of living

organisms to low concentrations of ROS [10] led to think that ROS, generated during the single exercise sessions, act as signals regulating molecular events crucial for adaptive responses to exercise, including up-regulation of antioxidant genes [11]. In agreement with the different actions of the ROS in acute and chronic exercises, the effects of antioxidant supplementation seem to depend on the type of performed exercise. The oxidative damage due to non-eccentric exercise is prevented by antioxidant supplementation [12,13], which is also able to prolong endurance to physical exercise [14], thus supporting the idea of a relationship between fatigue and free radicals during exercise. Conversely, when the ROS, produced during exercise, act as signals regulating molecular events underlying muscular cell adaptive responses, antioxidant supplementation appears to prevent such responses. Actually, vitamin C supplementation during training prevents the increase in antioxidant enzyme expression in skeletal muscle and health promoting effects on insulin sensitivity [15]. Furthermore, it decreases training-induced mitochondrial biogenesis and endurance performance [16]. Little is known about the effects of antioxidant supplementation on swim training-induced adaptive responses of hepatic tissue, which plays a central role as an energy supplier for the working muscles [17].

Correspondence: Paola Venditti, Department of Biology, University of Naples “Federico II”, Via Mezzocannone 8, I-80134 Naples, Italy. Tel: ⫹ 39 081 2535076. Fax: ⫹ 39 081 2535090. E-mail: [email protected] (Received date: 5 September 2013; Accepted date: 18 November 2013; Published online: 13 December 2013)

Training and vitamin E

Thus, to throw light on the possible ROS involvement in hepatic adaptation to swim training, we investigated the effects of the vitamin E supplementation of the trained rat diet on parameters affecting oxidative damage of hepatic preparations. We also investigated the effects of the supplementation on metabolic responses to exercise and possible underlying mechanisms, including changes in expression of activators and co-activators of the mitochondrial biogenesis. Methods

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Ethrane anaesthesia (Abbot, Aprilia, Italy) and hearts and livers were rapidly excised. The hearts were weighed after great vessels and valves were trimmed away and ventricles and atria rinsed free of blood. The livers were freed of connective tissue, weighed, finely minced, and washed with ice-cold homogenisation medium (HM) (220 mM mannitol, 70 mM sucrose, 1 mM EDTA, 0.1% fatty acid-free albumin, 10 mM Tris, pH 7.4). Finally, tissue fragments were gently homogenised (20% w/v) in the same solution using a glass Potter-Elvehjem homogeniser set at a standard velocity (500 rpm) for 1 min. Aliquots of homogenates were used for analytical procedures and preparation of mitochondrial fractions.

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Animals Preparation of mitochondria Male Wistar rats of 50-days old, supplied by Nossan (Correzzana, Italy) at Day 45 of age, were randomly divided into sedentary and trained animals. Swimming, used as the exercise for the trained rats, was administered 5 days per week for 10 weeks. Our program provided initially brief swimming periods and their gradual lengthening to both facilitate the learning process and to reduce stress reaction. Therefore, in the first week, the rats were made to swim for 15 min daily to familiarize with the water immersion. The training session lasted 50 min in the second week, 60 min in the third, 70 min in the fourth, and 90 min from the fifth to the tenth week. Swimming was performed in a plastic container that was 100 cm high filled to a depth of 45 cm with water maintained at 35°C. Untrained animals were kept in a small chamber holding about 3 cm water maintained at 35°C. Up to Day 50 of age, animals were provided with the same control diet, a commercial rat chow purchased from Mucedola (Settimo Milanese Mi Italy) containing 70 mg of vitamin E (α-tocopherol)/Kg (~105 UI/Kg). From Day 50, at the onset of the training program, half of sedentary and trained rats received a vitamin E-supplemented diet consisting of commercial rat chow to which α-tocopherol was added to a final concentration of 700 mg/Kg (~1050 UI/Kg), whereas the other rats received the control diet. Supplementation dose was found to be able to protect against changes in heart electrical activity induced by oxidant treatment [18]. Thus, there were four groups of eight rats each: sedentary untreated (S), sedentary vitamin E-treated (S⫹VE), trained untreated (T), and trained vitamin E-treated (T⫹VE). All rats were subjected to the same conditions (one per cage, constant artificial circadian cycle of 12 h of light and 12 h of darkness, and 50 ⫾ 10% relative humidity), and received water and food on an ad libitum basis. The treatment of animals in these experiments was in accordance with the guidelines set forth by the University’s Animal Care Review Committee. Liver homogenate preparation Twenty-four hours following the end of the training program, at about 120 days of age, all animals were weighed and sacrificed by decapitation while under

The homogenates, diluted 1:1 with HM, were freed of debris and nuclei by centrifugation at 500 g for 10 min at 4°C. The resulting supernatants were centrifuged at 10,000 g for 10 min. The mitochondrial pellets were resuspended in washing buffer (WB) (220 mM mannitol, 70 mM sucrose, 1 mM EGTA, 20 mM Tris, pH 7.4) and centrifuged at same sedimentation velocity. Mitochondrial preparations were washed in this manner twice before final suspension in WB. Mitochondrial protein was measured by the biuret method [19]. Vitamin E content Determination of liver vitamin E content in rat homogenates was performed using a high-performance liquid chromatography procedure [20]. Cytochrome oxidase activity Cytochrome oxidase (COX) activity was determined by the procedure of Barré et al. [21]. Oxygen consumption Oxygen consumption of homogenates and mitochondria was monitored at 30°C by an Hansatech respirometer in 1.0 mL of incubation medium (145 mM KCl, 30 mM Hepes, 5 mM KH2PO4, 3 mM MgCl2, 0.1 mM EGTA, pH 7.4) with 50 μL of homogenate or 0.25 mg of mitochondria per mL and succinate (10 mM), plus 5 μM rotenone (Rot), or pyruvate/malate (10/2.5 mM) as substrates, in the absence (State 4) and in the presence (State 3) of 500 μM ADP. Furthermore, the ratio between States 3 and 4 respiration rates (respiratory control ratio, RCR) was calculated. Oxidative damage The extent of the peroxidative processes in liver homogenates and mitochondrial preparations was determined by measuring the levels of lipid hydroperoxides (HPs) according to Heath and Tappel [22]. Determination of protein oxidative damage was performed measuring protein-bound

324 P. Venditti et al. carbonyl levels by the procedure of Reznick and Packer [23] for homogenates and by the modified procedure of Schild et al. [24] for mitochondria. Susceptibility to oxidative stress

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The susceptibility of liver homogenates to in vitro oxidative stress was evaluated by the change in hydroperoxide levels induced by treatment of 10% tissue homogenate with Fe and ascorbate (Fe/As), at a concentration of 100/1000 μM, for 10 min at room temperature. The reaction was terminated by the addition of 0.2% 2,6-di-t-butylp-cresol (BHT) and the hydroperoxide levels were evaluated as previously described. Antioxidants Glutathione peroxidase (GPX) activity was assayed according to Flohé and Günzler [25]. Glutathione reductase (GR) activity was measured according to Carlberg and Mannervik [26]. Reduced (GSH) and oxidized (GSSG) glutathione concentration was measured as described by Griffith [27]. Mitochondrial H2O2 release The rate of mitochondrial H2O2 release was measured at 30°C following the increase in fluorescence (excitation at 320 nm, emission at 400 nm) due to oxidation of p-hydroxyphenylacetate (PHPA) by H2O2 in the presence of horseradish peroxidase (HRP) [28] in a computercontrolled Jasko fluorometer equipped with a thermostatically controlled cell-holder. The reaction mixture consisted of 0.1 mg mL⫺ 1 mitochondrial proteins, 6 U mL⫺ 1 HRP, 200 μg mL⫺ 1 PHPA, and either 10 mM succinate, plus 5 μM rotenone, or 10 mM pyruvate/2.5 mM malate added at the end to start the reaction in a medium containing 145 mM KCl, 30 mM Hepes, 5 mM KH2PO4, 3 mM MgCl2, 0.1 mM EGTA, pH 7.4. Measurements with the different substrates were also performed in the presence of 500 μM ADP. Furthermore, the effects of two respiratory inhibitors were investigated: rotenone (Rot), which blocks the transfer of electrons from Complex I to ubiquinone [29], and antimycin A (AA), which interrupts electron transfer within the ubiquinone-cytochrome b site of Complex III [30]. Inhibitor concentrations (5 μM Rot and 10 μM AA) which do not interfere with the detection PHPA-HRP system were used [31]. Western blot analysis The levels of expression of the peroxisomal proliferatoractivated receptor-γ coactivator (PGC-1), nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) and cytochrome c were determined by Western Blot analysis. Liver fragments were gently homogenized (1:10, w/v) in 500 mM NaCl, 0.5% nonidet P-40, 6 mM EDTA, 6 mM EGTA, 1 mM dithiotreitol, 40 mM Tris-HCl, pH 8.0, in the presence of antiprotease mixture including 40 μg/mL PMSF,

5 μg/mL leupeptin, 5 g/mL aprotinin, 7 g/mL pepstatin. Homogenates were centrifuged at 1000 g for 10 min at 4°C and the resulting supernatants were used for sample preparation. Samples were prepared by diluting 10 μL of supernatant containing 1.5 mg/mL of proteins with 5 μL of 3% SDS, 30% glycerol, 15% β-mercaptoethanol, 0.1% bromophenol blue, 0.187 M Tris base, pH 6.8, and were boiled for 5 min before loading on the gel. Then, samples were electrophoresed through 6% stacking and 12% running SDS-PAGE gel according to Laemmli [32]. Gel was run in the mini protean equipment (Bio-Rad) for about 1 h at constant voltage (25 V). Separated hepatic proteins were transferred to nitrocellulose membranes by electroblotting. Membranes were incubated with a 1:1000 dilution of antibodies to PGC-1 (SC-13067), NRF-1 (SC-33771), NRF-2 (SC-22810), and cytochrome c (SC-7159) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in 154 mM NaCl, 10 mM Tris-HCl, pH 8.0, 2.5% non-fat dry milk, 10% Tween 20. Rabbit polyclonal antibodies raised against amino acids 1–300 mapping near the N-terminus of PGC-1, 204–503 mapping at the C-terminus of NRF-1, 1–180 mapping near the N-terminus of NRF-2α, and 1–104 of cytochrome c were used. Antibody binding was detected by carrying out secondary antibody incubations using peroxidase-conjugated anti first IgG antibodies (Santa Cruz Biotechnology) diluted 1:4000. Secondary antibody was detected using the ECL system according to the manufacturer’s recommendation (Santa Cruz Biotechnology). The blots were stripped by treating them for 10 min with 0.2 M NaOH followed by 5-min wash with H2O and two 5-min washes with 154 mM NaCl, 10 mM Tris-HCl, pH 8.0, 0.1% Tween 20. The blots were again blocked for 30 min with 154 mM NaCl, 10 mM Tris-HCl, pH 8.0, 2.5% non-fat dry milk, 10% Tween 20, washed as above, and incubated for 2 h with a 1:2000 dilution of anti-actin antibody (Santa Cruz Biotechnology) in blocking solution. Remaining procedures, as described for other antibodies, were followed. The actin was used for loading standardization. To compare protein expression levels among groups, a standard sedentary sample was run on each gel and all group values were then compared with such a sample that was assigned a value of 1. Statistical analysis The data obtained in eight different experiments are expressed as means⫹standard error. Data were analysed with a two-way analysis of variance method. When a significant F ratio was found, the Student–Newman–Keuls multiple range test was used to determine the statistical significance between means. Probability values (P) less than 0.05 were considered significant. Results The body masses were 399 ⫾ 5, 387 ⫾ 15, 353 ⫾ 9.6, and 334 ⫾ 7.3 g, heart masses were 0.89 ⫾ 0.02, 0.84 ⫾ 0.03,

0.96 ⫾ 0.02, and 0.93 ⫾ 0.03 g, heart/body mass ratios were 2.18 ⫾ 0.05, 2.13 ⫾ 0.03, 2.68 ⫾ 0.07, and 2.76 ⫾ 0.09 for S, S⫹VE, T, and T⫹VE rats, respectively. Body mass was not significantly (p ⬎ 0.05) affected by vitamin E treatment in both sedentary and trained rats. Training decreased body mass in both vitamin E-treated and -untreated rats. Heart mass was increased (p ⬍ 0.05) by training in vitamin E-treated and -untreated rats. Heart/ body mass ratio was increased (p ⬍ 0.05) in vitamin E-treated and -untreated rats by training which is well recognized to be effective in inducing myocardial hypertrophy [33]. Tissue vitamin E content was 40.6 ⫾ 2.8, 115.0 ⫾ 1.1, 39.9 ⫾ 1.6, and 99.5 ⫾ 2.9 nmol/g of tissue, for S, S⫹VE, T, and T⫹VE rats, respectively. Such a content was increased (p ⬍ 0.05) by diet antioxidant supplementation, and was decreased (p ⬍ 0.05) by training in vitamin E-fed animals. Cytochrome oxidase activity Homogenate COX activity was not modified by training and vitamin E treatment. Training reduced mitochondrial COX activity in untreated, but not in vitamin-treated rats (Figure 1, upper panel).

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Cytochrome c expression Information on liver content of mitochondrial proteins was also obtained determining expression of cytochrome c, a typical marker protein of mitochondria. Western blot experiments were conducted using identical amounts of total protein extract from livers of control or vitamin E-fed animals, sedentary or trained. Proteins were loaded onto an SDS-PAGE, and blotted according to standard protocols. The high specificity of the antibody and the molecular weight markers allowed us to easily identify the cytochrome c protein. Cytochrome c level was increased by training in untreated rats and decreased by in vitamin E treatment in trained animals (Figure 2), confirming the effects of training and vitamin E treatment on mitochondrial protein content reported in Figure 1.

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The ratio between COX activities in homogenates and mitochondria provided a rough estimate of tissue content of mitochondrial proteins. Such a content was not modified by vitamin E treatment and was increased by training in untreated but not in vitamin E-treated animals (Figure 1, lower panel).

COX activity

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Training and vitamin E

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Figure 1. Cytochrome oxidase activities (upper panel) and mitochondrial protein content (lower panel) in rat liver preparations. Values are means ⫾ S.E.M of eight different experiments. Cytochrome oxidase activity is expressed as μmol O min⫺ 1 per mg of mitochondrial protein or g tissue. Mitochondrial protein is expressed as mg of protein per g of tissue. S, sedentary untreated rats; S ⫹ VE, sedentary vitamin E-treated rats; T, trained untreated rats; T ⫹ VE, trained vitamin E-treated rats. aSignificant difference for trained rats versus respective sedentary controls; bSignificant difference for vitamin E-treated animals versus respective untreated controls. The level of significance was chosen as p ⬍ 0.05.

Figure 2. Effect of training and vitamin E treatment on hepatic levels of cytochrome c in rat liver. Liver total proteins from sedentary untreated (S), sedentary rats vitamin E-treated (S ⫹ VE), trained (T) and trained vitamin E-treated (T ⫹ VE) rats, were isolated and analyzed using Western blot analysis. Representative blot cytochrome c and actin protein expressions are shown (above). Analysis was performed as described in Materials and Methods. Bar graphs (below) correspond to the respective densitometric quantification (means ⫾ S.E.M. of three independent experiments). Actin was used for loading standardization. Ratios of band intensities to the β-actin band intensities were compared with those of a standard sedentary untreated sample that was assigned a value of 1. aSignificant difference for trained rats versus respective sedentary controls; bSignificant difference for vitamin E-treated animals versus respective untreated controls. The level of significance was chosen as p ⬍ 0.05.

326 P. Venditti et al. S

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Figure 3. Rates of O2 consumption by rat liver homogenates and mitochondria. Values are means ⫾ S.E.M. For each value eight rats were used. Oxygen consumption is expressed as μmol O min⫺ 1 per g of tissue and nmol O min⫺ 1 per mg of mitochondrial protein. S, sedentary untreated rats; S ⫹ VE, sedentary vitamin E-treated rats; T, trained untreated rats; T ⫹ VE, trained vitamin E-treated rats. Rates of O2 consumption were measured in the absence (State 4) and in the presence (State 3) of ADP with Complex II (succinate) and Complex I (pyruvate/malate) linked substrates. aSignificant difference for trained rats versus respective sedentary controls; bSignificant difference for vitamin E-treated animals versus respective untreated controls. The level of significance was chosen as p ⬍ 0.05.

O2 consumption The rates of O2 consumption by liver homogenates are reported in Figure 3, upper panel. During State 3, the rates of succinate-supported O2 consumption were increased by training and such an increase was prevented by vitamin E. During the State 4, the rates of oxygen consumption were not modified by training and were reduced by vitamin E treatment in trained animals. RCR values, which were 4.65 ⫾ 0.21, 4.57 ⫾ 0.23, 4.34 ⫾ 0.21, and 4.62 ⫾ 0.08, for S, S⫹VE, T, and T⫹VE rats, respectively, were not modified by training and antioxidant treatment. The rates of pyruvate/malate-supported O2 consumption, during State 3, were not modified by training and antioxidant treatment, while, during State 4, were increased by training in vitamin E-treated and -untreated rats. RCR values, which were 3.09 ⫾ 0.11, 3.22 ⫾ 0.18, 2.61 ⫾ 0.10, and 2.36 ⫾ 0.04 for S, S ⫹ VE,T, and T⫹VE rats, respectively, were significantly (p ⬍ 0.05) decreased by training in vitamin E-treated and -untreated rats.

The rates of O2 consumption by liver mitochondria are reported in Figure 3, lower panel. During State 3, the rates of succinate-supported O2 consumption, were decreased by training in vitamin E-treated and -untreated rats and decreased by vitamin E in sedentary and trained animals. State 4 respiration rates were decreased by training in vitamin E-treated and -untreated rats and by vitamin E in trained animals. RCR values, which were 4.70 ⫾ 0.09, 4.26 ⫾ 0.14, 5.12 ⫾ 0.09, and 4.79 ⫾ 0.16 for S, S⫹VE, T, and T⫹VE rats, respectively, were unaffected (p ⬎ 0.05) by training and significantly decreased (p ⬍ 0.05) by vitamin E in sedentary rats. During State 3, the rates of pyruvate/malate-supported O2 consumption were decreased by training in vitamin E-treated and -untreated rats, and increased by vitamin E in trained animals. During State 4, the rate of oxygen consumption was decreased by training in vitamin E-treated and -untreated rats, and by vitamin E in sedentary animals. RCR values, which were 1.93 ⫾ 0.11, 2.35 ⫾ 0.17, 2.38 ⫾ 0.06, and 2.30 ⫾ 0.06 for S, S⫹VE, T, and T⫹VE rats, respectively, were significantly (p ⬍ 0.05) increased by training in untreated and by vitamin E in sedentary animals. Protein expression In order to verify whether treatments modified concentrations of protein abundance of activators (NRF-1, NRF-2) and co-activator (PGC-1) of mitochondrial biogenesis, an analysis by western blot was performed. Western blot experiments were conducted using identical amounts of total protein extract from livers of control or vitamin E-fed animals, sedentary or trained. Proteins were loaded onto an SDS-PAGE, and blotted according to standard protocols. The high specificity of the antibodies and the molecular weight markers allowed us to easily identify NRF-1, and NRF-2 proteins. The specificity of the PGC-1 antibody was relatively lower, but the protein was clearly identified by its molecular weight. The results reported in Figure 4 show that PGC-1, NRF-1, and NRF-2 levels were increased by training in both vitamin-treated and -untreated rats and decreased by vitamin E treatment in trained animals. Oxidative damage The levels of lipid hydroperoxides and protein-bound carbonyls are reported in Figure 5. In both liver homogenates and mitochondria, the levels of hydroperoxides were significantly reduced by training in vitamin E-treated and -untreated rats. Vitamin E significantly reduced the lipid hydroperoxide levels in sedentary, but not in trained animals (Figure 5, upper panel). The carbonyl content of liver homogenates (Figure 5, lower panel) was reduced by training in vitamin E-treated and -untreated rats, and by vitamin E in sedentary rats. Conversely, the mitochondrial carbonyl content was decreased by training in untreated rats and by vitamin E in sedentary rats.

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Figure 5. Effect of training and vitamin E treatment on rat tissue and mitochondrial oxidative damage. Lipid hydroperoxides are expressed as pmol NADPH min⫺ 1 per g of tissue or mg of mitochondrial protein. Protein-bound carbonyls are expressed as nmol per mg of tissue or mitochondrial protein. S, sedentary untreated rats; S ⫹ VE, sedentary vitamin E-treated rats; T, trained untreated rats; T ⫹ VE, trained vitamin E-treated rats. Values are means ⫾ S.E.M. For each value eight rats were used. aSignificant difference for trained rats versus respective sedentary controls; bSignificant difference for vitamin E-treated animals versus respective untreated controls. The level of significance was chosen as p ⬍ 0.05.

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Figure 4. Effect of training and vitamin E treatment on hepatic levels of NRF-1, NRF-2, and PGC-1 in rat liver. Liver total proteins from sedentary untreated (S), sedentary rats vitamin E-treated (S ⫹ VE), trained (T) and trained vitamin E-treated (T ⫹ VE) rats, were isolated and analyzed using Western blot analysis. Representative blots of PGC-1, NRF-1, NRF-2, and actin protein expressions are shown (above). Analysis is performed as described in Materials and Methods. Bar graphs (below) correspond to the respective densitometric quantification (means ⫾ S.E.M. of three independent experiments). Actin was used for loading standardization. Ratios of band intensities to the β-actin band intensities were compared with those of a standard sedentary untreated sample that was assigned a value of 1. aSignificant difference for trained rats versus respective sedentary controls; bSignificant difference for vitamin E-treated animals versus respective untreated controls. The level of significance was chosen as p ⬍ 0.05.

Tissue and mitochondrial GSH levels and GSH/GSSG ratios In Figure 6, the GSH (upper panel) levels and GSH/GSSG ratios (lower panel) for liver preparations are reported. In homogenates, GSH content was increased by training in

untreated rats and decreased in vitamin E-treated rats. Moreover, it was increased by vitamin E treatment in sedentary and decreased in trained rats. GSH/GSSG ratio was increased by training in untreated and decreased in vitamin E-treated rats. In mitochondria, the GSH content was increased by training in untreated rats and by vitamin E in sedentary animals. The GSH/GSSG ratio was increased in vitamin E-untreated rats by training, whereas was increased in sedentary rats and decreased in trained rats by vitamin E. Mitochondrial H2O2 release The rates of H2O2 release are reported in Figure 7. During both States 4 and 3 respiration, the rates of succinate supported H2O2 mitochondrial release were decreased by training in vitamin E-treated and -untreated rats and by vitamin E in sedentary animals. In the presence of pyruvate malate, in both respiration states, the H2O2 release rate was reduced by training in untreated and by vitamin E in sedentary rats. Effect of inhibitors on H2O2 release As shown in the Table I, the rate of mitochondrial succinate-supported H2O2 release was decreased by training in

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Figure 7. Effects of treatments on rates of H2O2 release by rat liver mitochondria in States 4 and 3 of respiration. Values are means ⫾ S.E.M. For each value eight rats were used. Mitochondrial H2O2 release rate is expressed as pmol min⫺ 1 per mg of mitochondrial protein. S, sedentary untreated rats; S ⫹ VE, sedentary vitamin E-treated rats; T, trained untreated rats; T ⫹ VE, trained vitamin E-treated rats. aSignificant difference for trained rats versus respective sedentary controls; bSignificant difference for vitamin E-treated animals versus respective untreated controls. The level of significance was chosen as p ⬍ 0.05.

Mitochondria

Figure 6. Effect of training and vitamin E treatment on rat tissue and mitochondrial reduced glutathione content (GSH) (upper panel) and GSH/GGSG ratio (lower panel). GSH is expressed as μmol NADPH min⫺ 1 per g of tissue and nmol NADPH min⫺ 1 per mg of mitochondrial protein. S, sedentary untreated rats; S ⫹ VE, sedentary vitamin E-treated rats; T, trained untreated rats; T ⫹ VE, trained vitamin E-treated rats. Values are means ⫾ S.E.M. For each value eight rats were used. aSignificant difference for trained rats versus respective sedentary controls; bSignificant difference for vitamin E-treated animals versus respective untreated controls. The level of significance was chosen as p ⬍ 0.05.

vitamin E-treated and -untreated animals and by vitamin E in sedentary rats. Rotenone addition decreased the rates of H2O2 release in all groups, stopping what was occurring at Complex I, due to the to reverse electron flow from

coenzyme Q [34]. However, the effects of training and antioxidant treatment were not different from those observed in the absence of rotenone. After further addition of antimycin A, H2O2 release rates increased in all groups, but the H2O2 release was decreased by training in vitamin E-treated and -untreated rats while vitamin E attenuated the training-induced reduction. Addition of antimycin to pyruvate/malate supported mitochondria increased the H2O2 release rates in all groups, but training decreased the release in vitamin E-treated and -untreated rats. The addiction of rotenone to pyruvate malate respiring mitochondria increased the H2O2 release rates in all groups. Such rates were reduced by training in vitamin E-treated and -untreated rats, while were decreased by vitamin E in sedentary and increased in trained rats.

Table I. Effects of inhibitors, specific for different segments of the respiratory chain, on rates of H2O2 release by rat liver mitochondria. Group Substrate Succinate (Succ) Succ ⫹ rotenone Succ ⫹ rot ⫹ antimycin A Pyruvate/Malate (Pyr/Mal) Pyr/Mal ⫹ antimycin A Pyruvate/Malate (Pyr/Mal) Pyr/Mal ⫹ rotenone

S

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T

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121.4 ⫾ 1.2 105.2 ⫾ 1.3# 787.1 ⫾ 6.8# 202.7 ⫾ 1.5 966.9 ⫾ 9.2# 201.7 ⫾ 2.1 229.1 ⫾ 2.1#

112.5 ⫾ 0.6b 98.0 ⫾ 0.6#,b 786.8 ⫾ 1.8# 186.5 ⫾ 1.2b 960.0 ⫾ 1.0# 189.5 ⫾ 1.3b 222.9 ⫾ 2.0b,#

101.5 ⫾ 0.6a 88.9 ⫾ 1.9a,# 719.4 ⫾ 1.6a,# 178.5 ⫾ 2.0a 920.3 ⫾ 2.1a,# 178.4 ⫾ 2.0a 210.8 ⫾ 1.1a,#

101.3 ⫾ 1.1a 87.1 ⫾ 1.7a,# 738.0 ⫾ 4.2a,b,# 182.1 ⫾ 2.2 935.6 ⫾ 3.4a,b,# 182.0 ⫾ 2.2 217.4 ⫾ 0.8a,b,#

Values are means ⫾ S.E.M. For each value eight rats were used. Mitochondrial H2O2 release rate is expressed in pmol min⫺ 1 mg⫺ 1protein. S, sedentary untreated rats; S ⫹ VE, sedentary vitamin E-treated rats; T, trained untreated rats; T ⫹ VE, trained vitamin E-treated rats. aSignificant difference for trained rats versus respective sedentary controls. bSignificant difference for vitamin E-treated animals versus respective untreated controls. #Significant effect of the last inhibitor added versus mitochondria under same conditions without that inhibitor. The level of significance was chosen as P ⬍ 0.05.

Training and vitamin E

Activities of antioxidants enzymes

S

Tissue susceptibility to oxidative stress In Figure 9 are reported the changes induced by Fe/As treatment in the lipid hydroperoxide levels as a measure of the susceptibility of homogenate to oxidative attack. The changes indicate that such a susceptibility is decreased

S+VE

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HOMOGENATES 90

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0 GR

MITOCHONDRIA 1200

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ΔHp levels

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Figure. 9 Effect of training and vitamin E treatment on liver susceptibility to oxidative stress, evaluated by change in Hp levels (pmol NADPH min⫺ 1 per g of tissue) after Fe/As treatment. S, sedentary untreated rats; S ⫹ VE, sedentary vitamin E-treated rats; T, trained untreated rats; T ⫹ VE, trained vitamin E-treated rats. Values are means ⫾ S.E.M. For each value eight rats were used. aSignificant difference for trained rats versus respective sedentary controls; bSignificant difference for vitamin E-treated animals versus respective untreated controls. The level of significance was chosen as p ⬍ 0.05.

Discussion

Enzyme activity 0 GPX

T+VE

10

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b

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by training in untreated and increased in vitamin E-treated rats. Moreover, the susceptibility is decreased by vitamin E in sedentary and increased in trained rats.

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The activities of antioxidant enzymes in liver homogenates and mitochondria are reported in Figure 8. In tissue homogenate (upper panel), training increased GPX and GR activities in untreated rats, and such an increase was prevented by vitamin E integration. In the mitochondria (lower panel), GPX activity was increased by training in both vitamin E-treated and -untreated rats and decreased by vitamin E in trained rats. Mitochondrial GR activity was increased by training in vitamin E-treated and -untreated rats, but was not affected by vitamin E treatment.

S+VE

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GR

Figure 8. Effect of training and vitamin E treatment on the enzyme activities of Glutathione peroxidase (GPX) and reductase (GR) in liver homogenate and mitochondria. GPX and GR activities are expressed as μmol NADPH min⫺ 1 per g of tissue or mg of mitochondrial protein. S, sedentary untreated rats; S ⫹ VE, sedentary vitamin E-treated rats; T, trained untreated rats; T ⫹ VE, trained vitamin E-treated rats. Values are means ⫾ S.E.M. For each value eight rats were used. aSignificant difference for trained rats versus respective sedentary controls; bSignificant difference for vitamin E-treated animals versus respective untreated controls. The level of significance was chosen as p ⬍ 0.05.

Our results show that liver aerobic capacity, evaluated by COX activity, is not affected by training, being the reduction in mitochondrial aerobic capacity compensated by a mitochondrial proliferation, as suggested by the increased liver content of mitochondrial proteins. Liver response to chronic swimming is different from that of the skeletal muscle, which exhibits an increase in the number of functionally unmodified mitochondria and, hence, in the tissue metabolic capacity [4]. These different responses are consistent with the different functions of liver and skeletal muscle as energy supplier and consumer, respectively [17], during aerobic long-lasting exercise. Vitamin E treatment prevents the training-induced hepatic metabolic changes, reinstating the control values for both mitochondrial aerobic capacity and liver content of mitochondrial proteins. The effects of the treatments on the oxidative capacity of the liver preparations mirror only partially those on ADP-stimulated respiration, which is decreased by training in the mitochondria irrespective of substrate, but is increased in the homogenates in the presence of succinate. Moreover, vitamin E administration to trained rats prevents the increase in the succinate-sustained State 3 respiration in the homogenates, whereas, in mitochondria, it induces further reduction in succinate-sustained State 3 respiration and partially prevents the reduction in the pyruvate/malate sustained respiration. Because vitamin E does not affect the State 3 respiration of preparations from sedentary animals, it appears to be able to modify the metabolic characteristics of the hepatic tissue only when administered to animals subjected to a training program,

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330 P. Venditti et al. interfering with mitochondrial biogenesis and altering the ratios among the respiratory chain components. Thus, our results indicate that in the liver, like in the muscle [16], exercise induces adaptive responses, including an increase in mitochondrial protein content, which is prevented by antioxidant supplementation. Mitochondrial biogenesis involves the orchestrated expression of the mitochondrial genome and the nuclear genes that encode mitochondrial proteins. Mitochondrial biogenesis is controlled by PGC-1, a co-activator of nuclear regulatory proteins which play a role in the transcriptional control of many genes, including those involved in mitochondrial biogenesis, adaptive thermogenesis, glucose and fatty acid metabolism, fiber-type switching in skeletal muscle, and heart development [35]. Studies on skeletal muscle showed rapid increases in PGC-1 expression after a single bout of exercise [36] or stimulation of muscle contractions [37] and increases in PGC-1 transcription after endurance training [38]. In this paper, for the first time, evidence is reported that, in rat liver, swim training increases PGC-1, NRF-1, and NRF-2 expression, which is reduced by vitamin E supplementation. This result supports the idea of a relationship between liver PGC-1, NRF-1, and NRF-2 levels and mitochondrial protein content, and shows that the antioxidant supplementation, through its effects on such factors, counteracts training-induced metabolic adaptations. On the other hand, the absence of training-induced increase in liver aerobic capacity indicates a differential induction of the mitochondrial protein expression for which the available data do not allow to provide a straightforward explanation. It has been reported that PGC-1 expression is up-regulated by ROS [39] and that most of the transcriptional factors are under ROS regulation [40,41]. Increases in PGC-1expression and oxidative damage extent were found in liver from cold exposed rats [42], while a similar relationship is not found in the liver from rats subjected to moderate training and antioxidant treatment. Indeed, in liver preparations, lipid and protein oxidation is significantly reduced by training, in agreement with previous reports [4,9]. Moreover, vitamin E treatment reduces lipid and protein oxidation in sedentary rats without modifying PGC-1, NRF-1, and NRF-2 expression, and does not induce further reduction in oxidative damage in trained animals, although it reduces PGC-1, NRF-1, and NRF-2 expression. The lack of relationship between PGC-1, NRF-1, and NRF-2 levels and oxidative damage extent is confirmed by the changes, elicited by training and vitamin E treatment, in GSH levels and GSH/GSSG ratios, whose decreases are considered as indices of oxidative stress. These results can be readily explained if adaptive responses to training and their prevention by vitamin E also involve changes in the ROS production rate and antioxidant system effectiveness, which determine tissue oxidative damage. To date, there is a contention about the cellular sources for superoxide generation. Within mitochondria,

superoxide radical, generated by univalent oxygen reduction at Complex I [43] and Complex III [44], undergoes rapid dismutation to H2O2. The hydrogen hydroperoxide escaping antioxidant removal systems is released in the cytosol and converted into hydroxyl radical, which plays a major role in determining the extent of tissue oxidative damage. Although some authors [45] have reappraised the early report that 2–5% of the total oxygen consumed by mitochondria is reduced to superoxide [46], mitochondria are yet cited as the main cellular source of ROS in tissues [47]. On the other hand, ROS production from the various cellular sources can modify during exercise in tissue-dependent manner. It was proposed that, in the liver, the exercise and recovery period after exercise mimic the ischemia/reperfusion phenomenon, which causes increased free radical production in which xanthine dehydrogenase–xanthine oxidase (XD-XO) conversion and auto-oxidation of respiratory chain components are involved [48]. The involvement of the XD–XO conversion was supported by the observation that exercise-induced delayed increase in liver lipid peroxidation and uric acid content were reduced by rat treatment with allopurinol [49]. However, such changes were elicited by exhaustive exercise and it is unlikely that the tissue damage, resulting in conversion of XD in XO, happens during the single swimming sessions of short duration provided by our training program. If so, mitochondrial respiratory should supply strong contribution to production of ROS, which prime adaptive responses to exercise. Significant reduction in ROS production was found in liver homogenates from rats trained to run, but the sources of ROS production were not investigated [50]. Subsequently, we found that training reduces the rate of H2O2 release by liver mitochondria from swimming rats [9]. The present results confirm this training effect and suggest that it can contribute to prevent the massive ROS efflux and tissue damage which are usually associated to increases in mitochondrial protein content. Our results also show that training effect is not modified by vitamin E, which, on the other hand, is able to reduce H2O2 release rate in sedentary animals. A possible explanation for this observation is provided by the examination of H2O2 release rates measured in the presence of inhibitors of the respiratory chain. The block in the electron flux, operated by such inhibitors, renders the electron carriers, including the autoxidizable ones, on the substrate site, completely reduced so that their concentration becomes the only factor affecting ROS production rate. On this basis, our results indicate that the training reduces the concentration of autoxidizable carriers located at Complexes I and III, and the vitamin increases such a concentration, particularly that at Complex III, in trained rats, without any effect in sedentary rats. These results suggest that, in trained rats, the increased production of the H2O2 precursor, due to higher autoxidizable carriers concentration, is compensated by its increased removal, likely due to ability of

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Training and vitamin E

both α-tocopherol [51] and α-tocopheroxyl radical [52] to scavenge superoxide radical. It has been hypothesized that ROS generated during muscle contraction play a role in muscle adaptation to exercise by activating redox-sensitive transduction of antioxidant enzymes [53]. The increases in GPX and GR activities found in homogenates from trained rats and the prevention of such effects by vitamin E suggest that a similar role could also be played by ROS in hepatic tissue. Interestingly, in mitochondrial preparations, vitamin E attenuates the training-linked increases in GPX activity and does not modify those in GR activity. Mitochondrial proteins, encoded by nuclear genes, are translocated into the mitochondria via the protein import machinery primarily consisting of two multisubunit complexes, referred to as translocases of the outer (TOM complex) and inner (TIM complex) membrane [54]. Thus, our observation that the antioxidant enzyme activities remain high in mitochondria from vitamin E-trained rats could be explained by the finding that the protein import into the mitochondria is accelerated by chronic contractile activity [11]. Because the GPX and GR combined action is the major determinant of the tissue GSH content, it is not surprising that the training and vitamin E-linked changes in liver GSH levels reflect those in enzyme activities. Thus, the persistence of high mitochondrial GSH levels, following vitamin E administration to trained rats, can be explained by high GR activity in mitochondrial compartment. The changes found in GPX and GR activities and GSH content suggest that liver from trained rats is able to more effectively oppose an oxidative attack and such an ability is reduced by vitamin E treatment. This idea is supported by our observation that the training-induced reduction in liver susceptibility to in vitro oxidative stress, is prevented by vitamin E treatment, confirming that the vitamin interferes with the biochemical adaptation to training that makes the tissues more resistant to oxidative insults. In conclusion, the results reported in this paper confirm the idea that, at low doses, radicals and other ROS are able to regulate various cellular functions. In particular, our results show that ROS, produced during a training program, can act as signals regulating molecular events crucial for adaptive responses of hepatic tissue, including mitochondrial biogenesis and increase in antioxidant enzyme activity. Vitamin E, the major chain-breaking antioxidant, at present is among the most commonly consumed dietary supplements due to the belief that it provides health benefits against diseases associated with oxidative damage, even though results of the present and other studies [55] do not seem to encourage the use of indiscriminate vitamin E supplementation. Indeed, available data indicate that vitamin E integration, which can be useful to decrease tissue oxidative damage elicited by acute exercise, can be harmful during a training program because it reduces the benefits resulting from regular physical activity.

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Effect of training and vitamin E administration on rat liver oxidative metabolism.

We studied vitamin E effects on metabolic changes and oxidative damage elicited by swim training in rat liver. Training reduced mitochondrial aerobic ...
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