Just Accepted by Free Radical Research
Vitamin E supplementation modifies adaptive responses to training in rat skeletal muscle Paola Venditti, Gaetana Napolitano, Daniela Barone and Sergio Di Meo 10.3109/10715762.2014.937341
Free Radic Res Downloaded from informahealthcare.com by University of Maastricht on 06/25/14 For personal use only.
Abstract Aim of the present study was to test, by vitamin E treatment, the hypothesis that muscle adaptive responses to training are mediated by free radicals produced during the single exercise sessions. Therefore, we determined aerobic capacity of tissue homogenates and mitochondrial fractions, tissue content of mitochondrial proteins and expression of factors (PGC-1, NRF-1, and NRF-2) involved in mitochondrial biogenesis. Moreover, we determined the oxidative damage extent, antioxidant enzyme activities, and glutathione content in both tissue preparations, mitochondrial ROS production rate,. Finally we tested mitochondrial ROS production rate and muscle susceptibility to oxidative stress. The metabolic adaptations to training, consisting in increased muscle oxidative capacity coupled with the proliferation of a mitochondrial population with decreased oxidative capacity, were generally prevented by antioxidant supplementation. Accordingly, the expression of the factors involved in mitochondrial biogenesis, which were increased by training, was restored to the control level by the antioxidant treatment. Even the training -induced increase in antioxidant enzyme activities, glutathione level and tissue capacity to oppose to an oxidative attach were prevented by vitamin E treatment. Our results support the idea that the stimulus for training-induced adaptive responses derives from the increased production, during the training sessions, of reactive oxygen species that stimulates the expression of PGC-1, which is involved in mitochondrial biogenesis and antioxidant enzymes expression. On the other hand, the observation that changes induced by training in some parameters are only attenuated by vitamin E treatment, suggests that other signaling pathways, which are activated during exercise and impinge on PGC-1, can modify the response to the antioxidant integration.
© 2014 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
Vitamin E supplementation modifies adaptive responses to training in rat skeletal muscle
PT ED
Paola Venditti, Gaetana Napolitano, Daniela Barone and Sergio Di Meo
Dipartimento delle Scienze Biologiche - Sezione di Fisiologia, Università di Napoli, I-
Correspondence: Paola Venditti - Dipartimento di Biologia, Università di Napoli “Federico II”, Via Mezzocannone 8, I-80134, Napoli, Italy. Tel.: +39 081 2535076. Fax: +39 081 2535090. E-mail address: [email protected]
Short title: Training and Vitamin E
CE
Abstract
ST
AC
Aim of the present study was to test, by vitamin E treatment, the hypothesis that muscle adaptive responses to training are mediated by free radicals produced during the single exercise sessions. Therefore, we determined aerobic capacity of tissue homogenates and mitochondrial fractions, tissue content of mitochondrial proteins and expression of factors (PGC-1, NRF-1, and NRF-2) involved in mitochondrial biogenesis. Moreover, we determined the oxidative damage extent, antioxidant enzyme activities, and glutathione content in both tissue preparations, mitochondrial ROS production rate,. Finally we tested mitochondrial ROS production rate and muscle susceptibility to oxidative stress. The metabolic adaptations to training, consisting in increased muscle oxidative capacity coupled with the proliferation of a mitochondrial population with decreased oxidative capacity, were generally prevented by antioxidant supplementation. Accordingly, the expression of the factors involved in mitochondrial biogenesis, which were increased by training, was restored to the control level by the antioxidant treatment. Even the training -induced increase in antioxidant enzyme activities, glutathione level and tissue capacity to oppose to an oxidative attach were prevented by vitamin E treatment. Our results support the idea that the stimulus for training-induced adaptive responses derives from the increased production, during the training sessions, of reactive oxygen species that stimulates the expression of PGC-1, which is involved in mitochondrial biogenesis and antioxidant enzymes expression. On the other hand, the observation that changes induced by training in some parameters are only attenuated by vitamin E treatment, suggests that other signaling pathways, which are activated during exercise and impinge on PGC-1, can modify the response to the antioxidant integration.
JU
Free Radic Res Downloaded from informahealthcare.com by University of Maastricht on 06/25/14 For personal use only.
80134 Napoli, Italy
Key words: Vitamin E, Training, Oxidative damage, Rat muscle, Mitochondria Introduction
As it is known, unaccustomed (acute) exercise can produce significant damage in skeletal muscle [1]. The contraction form most damaging to skeletal muscle is that in which the muscle is contracting while being lengthened (eccentric contraction).
PT ED
During such a contraction, disruption of cytoskeletal structures, loss of muscle force generation and influx of phagocytic cells and neutrophils into the damaged fibers
induces muscular adaptations, which also lead to an increased ability to perform prolonged strenuous exercise. Adaptive responses to training include increases in mitochondrial mass and respiratory capacity of skeletal muscle [3, 4], and reduction in membrane sensitivity to exhaustive exercise-induced damage [5].
CE
Since Davies et al. [6] suggested that an increased free radical production contributes to the muscle damage induced by acute exercise, several studies showed elevated levels of oxidative damage markers in various tissues after acute long-term
AC
exercise [7]. ROS involvement in muscle damage induced by acute exercise was also supported by the observation that such damage can be prevented by antioxidant supplementation [8, 9].
Differently from acute exercise, training exerts protective effects against tissue
ST
injury under conditions leading to increased free radical production, including ischemia-reperfusion [10] and experimental hyperthyroidism [11]. Training also slows down peroxidative processes [5, 12] and the appearance of other signs of free radical generation [5] induced by acute exercise in rat skeletal muscle. This effect is associated with increased antioxidant defenses [5, 13] and decreased free radical
JU
Free Radic Res Downloaded from informahealthcare.com by University of Maastricht on 06/25/14 For personal use only.
occur [2]. Conversely, aerobic physical activity regularly performed (training),
activity [14, 15]. It has been suggested that the ROS generated during the single exercise sessions act as signals regulating molecular events crucial for the adaptive responses to training. It has also been proposed that antioxidant supplementation, decreasing ROS formation, prevents useful adaptations for muscular cells [16].
Such an idea has been tested determining the effects of the antioxidant supplementation on exercise-induced adaptive responses. However, investigations concerning the effects of higher intakes of vitamin C and/or E on exercise performance and redox homeostasis, have supplied contrasting results [17]. This has prompted us to examine the effects of the vitamin E supplementation on muscle
PT ED
adaptive responses to swim training involved in muscle respiratory capacity and
Methods Animals
Male Wistar rats 50 days old, supplied by Nossan (Correzzana, Italy) at day 45 of age, were randomly divided into sedentary and trained animals. Swimming, used as
CE
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 reduce stress reaction.
AC
Therefore, in the first week, the rats were made to swim for 15 min daily to familiarize with the water immersion. In the second week, the training session lasted 50 min and from the third to the tenth week the rats swam for 60 min a day. Swimming was performed in a plastic container that was 100 cm high filled to a
ST
depth of the 45 cm with water maintained at 35°C. Such a protocol was found to increase endurance in rats subjected to acute exercise [5]. Untrained animals were kept in 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
JU
Free Radic Res Downloaded from informahealthcare.com by University of Maastricht on 06/25/14 For personal use only.
redox homeostasis.
commercial rat chow purchased from Nossan, containing 70 mg/Kg of vitamin E (αtocopherol). 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 the α-tocopherol (Sigma-Aldrich), was added to a final concentration of 700 mg/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 preventing oxidative damage[18].Thus, there were four groups of rats: sedentary untreated (S), sedentary vitamin E-treated (S+VE), trained untreated (T), trained vitamin E-treated (T+VE). All rats were subjected to the same conditions (one per cage, constant artificial
PT ED
circadian cycle of 12 h of light and 12 h of darkness, and 50±10 % relative humidity), and received water on and food an ad libitum basis.
set forth by the University’s Animal Care Review Committee. Muscle 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 ether
CE
anaesthesia. Heart and gastrocnemius muscles were rapidly excised and placed in ice-cold isolation medium (IM) (220 mM mannitol, 70 mM sucrose, 1 mM EDTA, 20 mM Tris, pH 7.4) containing 0.1% fatty acid-free albumin. The hearts were
AC
weighed after great vessels and valves were trimmed away and ventricles and atria rinsed free of blood.
The gastrocnemius muscles were freed of extra-cellular fat and connective tissue, weighed, finely minced, and washed with IM. Tissue fragments were incubated for 5
ST
min with IM containing 0.1 mg/ml nagarse, washed and gently homogenized in IM (1:10 w/v) using a Potter-Elvejem homogenizer set at a standard velocity (500 rpm) for 2 min. Aliquots of the homogenates were used for analytical procedures and preparation of mitochondrial fractions.
JU
Free Radic Res Downloaded from informahealthcare.com by University of Maastricht on 06/25/14 For personal use only.
The treatment of animals in these experiments was in accordance with the guidelines
Preparation of mitochondria Homogenates were freed of debris and nuclei by centrifugation at 500 g for 10 min at 4°C and the resulting supernatants were centrifuged at 3000 g for 10 min. The mitochondrial pellets were re-suspended 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]. Analytical procedures Determination of muscle vitamin E content was performed using a high-performance
PT ED
liquid chromatography procedure [20]. Cytochrome oxidase (COX) activity was determined by the procedure of Barré et al. [21] using muscle homogenates and
preparations contained per ml either 100 mg of tissue or 0.2 mg of mitochondrial proteins. The ratio between COX activities in homogenates and mitochondria provided a rough estimate of tissue content of mitochondrial proteins.
Oxygen consumption of homogenates and mitochondria was monitored at 30° C by
CE
a Hansatech respirometer in 1.6 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
AC
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. The extent of the peroxidative processes in muscle homogenates and mitochondrial preparations was determined by measuring the level of lipid hydroperoxides (HPs)
ST
according to Heath & Tappel [22]. Determination of protein oxidative damage was performed measuring protein-bound carbonyl levels by the procedure of Reznick & Packer [23] for homogenates and by the modified procedure of Schild et al. [24] for mitochondria.
JU
Free Radic Res Downloaded from informahealthcare.com by University of Maastricht on 06/25/14 For personal use only.
mitochondrial suspensions diluted with modified Chappel-Perry medium so that the
The tissue susceptibility to oxidative stress was evaluated by the change in hydroperoxide levels induced by treatment of 10 % tissue homogenate with iron 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-butyl-pcresol (BHT) and the hydroperoxide levels were evaluated as previously described.
Glutathione peroxidase (GPX) activity was assayed at 37°C according to Flohé & Günzler [25] with H2O2 as substrate. Glutathione reductase (GR) activity was measured at 30°C according to Carlberg & Mannervik [26]. Reduced glutathione (GSH) concentration was measured as described by Griffith [27]. The rate of mitochondrial H2O2 release was measured at 30° C following the
PT ED
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
thermostatically controlled cell-holder. The reaction mixture consisted of 0.1 mg ml1
mitochondrial proteins, 6 U ml-1 HRP, 200 μg ml-1 PHPA, and 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
CE
mM MgCl2, 0.1 mM EGTA, pH 7.4. Measurements with the different substrates in the presence of 500 μM ADP were also performed. Furthermore, the effects of two respiratory inhibitors were investigated: rotenone (Rot), which blocks the transfer of
AC
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, 10 μM AA) which do not interfere with the detection PHPA-HRP system were used [31].
ST
The levels of expression of the peroxisomal proliferator-activated 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. Muscle fragments were gently homogenized (1:10, w/v) in 500 mM NaCl, 0.5% nonidet P-40, 6 mM EDTA,
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peroxidase (HRP) [28] in a computer-controlled Jasko fluorometer equipped with a
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 SDSPAGE 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
PT ED
transferred to nitrocellulose membranes by electroblotting. Membranes were incubated with a 1:1000 dilution of antibodies to PGC-1 (SC-13067), NRF-1 (SC-
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
CE
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
AC
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
ST
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 antiactin antibody (Santa Cruz Biotechnology) in blocking solution. Remaining procedures, as described for other antibodies, were followed. The actin was used for
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33771), NRF-2 (SC-22810), and cytochrome c (SC-7159) (Santa Cruz
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) < 0.05 were considered significant. Results
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Rat 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
respectively. Body masses, heart masses and heart mass/body mass ratios were not significantly (p>0.05) affected by vitamin E treatment. Body masses were decreased (p
Vitamin E supplementation modifies adaptive responses to training in rat skeletal muscle Paola Venditti, Gaetana Napolitano, Daniela Barone and Sergio Di Meo 10.3109/10715762.2014.937341
Free Radic Res Downloaded from informahealthcare.com by University of Maastricht on 06/25/14 For personal use only.
Abstract Aim of the present study was to test, by vitamin E treatment, the hypothesis that muscle adaptive responses to training are mediated by free radicals produced during the single exercise sessions. Therefore, we determined aerobic capacity of tissue homogenates and mitochondrial fractions, tissue content of mitochondrial proteins and expression of factors (PGC-1, NRF-1, and NRF-2) involved in mitochondrial biogenesis. Moreover, we determined the oxidative damage extent, antioxidant enzyme activities, and glutathione content in both tissue preparations, mitochondrial ROS production rate,. Finally we tested mitochondrial ROS production rate and muscle susceptibility to oxidative stress. The metabolic adaptations to training, consisting in increased muscle oxidative capacity coupled with the proliferation of a mitochondrial population with decreased oxidative capacity, were generally prevented by antioxidant supplementation. Accordingly, the expression of the factors involved in mitochondrial biogenesis, which were increased by training, was restored to the control level by the antioxidant treatment. Even the training -induced increase in antioxidant enzyme activities, glutathione level and tissue capacity to oppose to an oxidative attach were prevented by vitamin E treatment. Our results support the idea that the stimulus for training-induced adaptive responses derives from the increased production, during the training sessions, of reactive oxygen species that stimulates the expression of PGC-1, which is involved in mitochondrial biogenesis and antioxidant enzymes expression. On the other hand, the observation that changes induced by training in some parameters are only attenuated by vitamin E treatment, suggests that other signaling pathways, which are activated during exercise and impinge on PGC-1, can modify the response to the antioxidant integration.
© 2014 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
Vitamin E supplementation modifies adaptive responses to training in rat skeletal muscle
PT ED
Paola Venditti, Gaetana Napolitano, Daniela Barone and Sergio Di Meo
Dipartimento delle Scienze Biologiche - Sezione di Fisiologia, Università di Napoli, I-
Correspondence: Paola Venditti - Dipartimento di Biologia, Università di Napoli “Federico II”, Via Mezzocannone 8, I-80134, Napoli, Italy. Tel.: +39 081 2535076. Fax: +39 081 2535090. E-mail address: [email protected]
Short title: Training and Vitamin E
CE
Abstract
ST
AC
Aim of the present study was to test, by vitamin E treatment, the hypothesis that muscle adaptive responses to training are mediated by free radicals produced during the single exercise sessions. Therefore, we determined aerobic capacity of tissue homogenates and mitochondrial fractions, tissue content of mitochondrial proteins and expression of factors (PGC-1, NRF-1, and NRF-2) involved in mitochondrial biogenesis. Moreover, we determined the oxidative damage extent, antioxidant enzyme activities, and glutathione content in both tissue preparations, mitochondrial ROS production rate,. Finally we tested mitochondrial ROS production rate and muscle susceptibility to oxidative stress. The metabolic adaptations to training, consisting in increased muscle oxidative capacity coupled with the proliferation of a mitochondrial population with decreased oxidative capacity, were generally prevented by antioxidant supplementation. Accordingly, the expression of the factors involved in mitochondrial biogenesis, which were increased by training, was restored to the control level by the antioxidant treatment. Even the training -induced increase in antioxidant enzyme activities, glutathione level and tissue capacity to oppose to an oxidative attach were prevented by vitamin E treatment. Our results support the idea that the stimulus for training-induced adaptive responses derives from the increased production, during the training sessions, of reactive oxygen species that stimulates the expression of PGC-1, which is involved in mitochondrial biogenesis and antioxidant enzymes expression. On the other hand, the observation that changes induced by training in some parameters are only attenuated by vitamin E treatment, suggests that other signaling pathways, which are activated during exercise and impinge on PGC-1, can modify the response to the antioxidant integration.
JU
Free Radic Res Downloaded from informahealthcare.com by University of Maastricht on 06/25/14 For personal use only.
80134 Napoli, Italy
Key words: Vitamin E, Training, Oxidative damage, Rat muscle, Mitochondria Introduction
As it is known, unaccustomed (acute) exercise can produce significant damage in skeletal muscle [1]. The contraction form most damaging to skeletal muscle is that in which the muscle is contracting while being lengthened (eccentric contraction).
PT ED
During such a contraction, disruption of cytoskeletal structures, loss of muscle force generation and influx of phagocytic cells and neutrophils into the damaged fibers
induces muscular adaptations, which also lead to an increased ability to perform prolonged strenuous exercise. Adaptive responses to training include increases in mitochondrial mass and respiratory capacity of skeletal muscle [3, 4], and reduction in membrane sensitivity to exhaustive exercise-induced damage [5].
CE
Since Davies et al. [6] suggested that an increased free radical production contributes to the muscle damage induced by acute exercise, several studies showed elevated levels of oxidative damage markers in various tissues after acute long-term
AC
exercise [7]. ROS involvement in muscle damage induced by acute exercise was also supported by the observation that such damage can be prevented by antioxidant supplementation [8, 9].
Differently from acute exercise, training exerts protective effects against tissue
ST
injury under conditions leading to increased free radical production, including ischemia-reperfusion [10] and experimental hyperthyroidism [11]. Training also slows down peroxidative processes [5, 12] and the appearance of other signs of free radical generation [5] induced by acute exercise in rat skeletal muscle. This effect is associated with increased antioxidant defenses [5, 13] and decreased free radical
JU
Free Radic Res Downloaded from informahealthcare.com by University of Maastricht on 06/25/14 For personal use only.
occur [2]. Conversely, aerobic physical activity regularly performed (training),
activity [14, 15]. It has been suggested that the ROS generated during the single exercise sessions act as signals regulating molecular events crucial for the adaptive responses to training. It has also been proposed that antioxidant supplementation, decreasing ROS formation, prevents useful adaptations for muscular cells [16].
Such an idea has been tested determining the effects of the antioxidant supplementation on exercise-induced adaptive responses. However, investigations concerning the effects of higher intakes of vitamin C and/or E on exercise performance and redox homeostasis, have supplied contrasting results [17]. This has prompted us to examine the effects of the vitamin E supplementation on muscle
PT ED
adaptive responses to swim training involved in muscle respiratory capacity and
Methods Animals
Male Wistar rats 50 days old, supplied by Nossan (Correzzana, Italy) at day 45 of age, were randomly divided into sedentary and trained animals. Swimming, used as
CE
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 reduce stress reaction.
AC
Therefore, in the first week, the rats were made to swim for 15 min daily to familiarize with the water immersion. In the second week, the training session lasted 50 min and from the third to the tenth week the rats swam for 60 min a day. Swimming was performed in a plastic container that was 100 cm high filled to a
ST
depth of the 45 cm with water maintained at 35°C. Such a protocol was found to increase endurance in rats subjected to acute exercise [5]. Untrained animals were kept in 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
JU
Free Radic Res Downloaded from informahealthcare.com by University of Maastricht on 06/25/14 For personal use only.
redox homeostasis.
commercial rat chow purchased from Nossan, containing 70 mg/Kg of vitamin E (αtocopherol). 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 the α-tocopherol (Sigma-Aldrich), was added to a final concentration of 700 mg/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 preventing oxidative damage[18].Thus, there were four groups of rats: sedentary untreated (S), sedentary vitamin E-treated (S+VE), trained untreated (T), trained vitamin E-treated (T+VE). All rats were subjected to the same conditions (one per cage, constant artificial
PT ED
circadian cycle of 12 h of light and 12 h of darkness, and 50±10 % relative humidity), and received water on and food an ad libitum basis.
set forth by the University’s Animal Care Review Committee. Muscle 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 ether
CE
anaesthesia. Heart and gastrocnemius muscles were rapidly excised and placed in ice-cold isolation medium (IM) (220 mM mannitol, 70 mM sucrose, 1 mM EDTA, 20 mM Tris, pH 7.4) containing 0.1% fatty acid-free albumin. The hearts were
AC
weighed after great vessels and valves were trimmed away and ventricles and atria rinsed free of blood.
The gastrocnemius muscles were freed of extra-cellular fat and connective tissue, weighed, finely minced, and washed with IM. Tissue fragments were incubated for 5
ST
min with IM containing 0.1 mg/ml nagarse, washed and gently homogenized in IM (1:10 w/v) using a Potter-Elvejem homogenizer set at a standard velocity (500 rpm) for 2 min. Aliquots of the homogenates were used for analytical procedures and preparation of mitochondrial fractions.
JU
Free Radic Res Downloaded from informahealthcare.com by University of Maastricht on 06/25/14 For personal use only.
The treatment of animals in these experiments was in accordance with the guidelines
Preparation of mitochondria Homogenates were freed of debris and nuclei by centrifugation at 500 g for 10 min at 4°C and the resulting supernatants were centrifuged at 3000 g for 10 min. The mitochondrial pellets were re-suspended 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]. Analytical procedures Determination of muscle vitamin E content was performed using a high-performance
PT ED
liquid chromatography procedure [20]. Cytochrome oxidase (COX) activity was determined by the procedure of Barré et al. [21] using muscle homogenates and
preparations contained per ml either 100 mg of tissue or 0.2 mg of mitochondrial proteins. The ratio between COX activities in homogenates and mitochondria provided a rough estimate of tissue content of mitochondrial proteins.
Oxygen consumption of homogenates and mitochondria was monitored at 30° C by
CE
a Hansatech respirometer in 1.6 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
AC
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. The extent of the peroxidative processes in muscle homogenates and mitochondrial preparations was determined by measuring the level of lipid hydroperoxides (HPs)
ST
according to Heath & Tappel [22]. Determination of protein oxidative damage was performed measuring protein-bound carbonyl levels by the procedure of Reznick & Packer [23] for homogenates and by the modified procedure of Schild et al. [24] for mitochondria.
JU
Free Radic Res Downloaded from informahealthcare.com by University of Maastricht on 06/25/14 For personal use only.
mitochondrial suspensions diluted with modified Chappel-Perry medium so that the
The tissue susceptibility to oxidative stress was evaluated by the change in hydroperoxide levels induced by treatment of 10 % tissue homogenate with iron 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-butyl-pcresol (BHT) and the hydroperoxide levels were evaluated as previously described.
Glutathione peroxidase (GPX) activity was assayed at 37°C according to Flohé & Günzler [25] with H2O2 as substrate. Glutathione reductase (GR) activity was measured at 30°C according to Carlberg & Mannervik [26]. Reduced glutathione (GSH) concentration was measured as described by Griffith [27]. The rate of mitochondrial H2O2 release was measured at 30° C following the
PT ED
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
thermostatically controlled cell-holder. The reaction mixture consisted of 0.1 mg ml1
mitochondrial proteins, 6 U ml-1 HRP, 200 μg ml-1 PHPA, and 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
CE
mM MgCl2, 0.1 mM EGTA, pH 7.4. Measurements with the different substrates in the presence of 500 μM ADP were also performed. Furthermore, the effects of two respiratory inhibitors were investigated: rotenone (Rot), which blocks the transfer of
AC
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, 10 μM AA) which do not interfere with the detection PHPA-HRP system were used [31].
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The levels of expression of the peroxisomal proliferator-activated 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. Muscle fragments were gently homogenized (1:10, w/v) in 500 mM NaCl, 0.5% nonidet P-40, 6 mM EDTA,
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peroxidase (HRP) [28] in a computer-controlled Jasko fluorometer equipped with a
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 SDSPAGE 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
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transferred to nitrocellulose membranes by electroblotting. Membranes were incubated with a 1:1000 dilution of antibodies to PGC-1 (SC-13067), NRF-1 (SC-
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
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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
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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
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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 antiactin antibody (Santa Cruz Biotechnology) in blocking solution. Remaining procedures, as described for other antibodies, were followed. The actin was used for
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33771), NRF-2 (SC-22810), and cytochrome c (SC-7159) (Santa Cruz
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) < 0.05 were considered significant. Results
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Rat 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
respectively. Body masses, heart masses and heart mass/body mass ratios were not significantly (p>0.05) affected by vitamin E treatment. Body masses were decreased (p
