Abstract
We investigated whether swim training modifies the effects of tri-iodothyronine (T3) treatment on the metabolic response and oxidative damage of rat liver. Respiratory capacities, oxidative damage, levels of antioxidants, and susceptibility to oxidative challenge of liver homogenates were determined. Mitochondrial respiratory capacities, rates of H2O2 release, and oxidative damage were also evaluated. Training modified most of the measured parameters in both thyroid states, although the extent of changes was higher in hyperthyroid preparations. T3 treatment enhanced homogenate respiratory capacity, which was further enhanced by training despite the decrease in mitochondrial respiratory capacity. Hormonal treatment also induced liver oxidative damage and glutathione depletion, and increased tissue susceptibility to oxidative challenge. These effects were lower in trained animals. The extensive oxidative damage found in liver homogenates from hyperthyroid sedentary rats was due to low tissue antioxidant protection and high mitochondrial H2O2 production rate, which were increased and decreased respectively by animal training. The training effect on H2O2 production was associated with lower oxidative damage and susceptibility to Ca2+-induced swelling of mitochondria. Measurements with respiratory inhibitors indicated that the differences in H2O2 release in hyperthyroid groups were due to differences in mitochondrial content of autoxidizable electron carrier located at Complex III. We conclude that moderate training is able to reduce hyperthyroid state-linked cellular and subcellular oxidative damage in liver increasing its antioxidant defenses and decreasing the mitochondrial generation of reactive oxygen species.
Introduction
It is long known that in vertebrates that experimentally induced hyperthyroidism leads to accelerated basal metabolic rate and oxygen consumption in several tissues (Schwartz & Oppenheimer 1978). It is also well established that the increased respiratory capacity displayed from rat tissues following prolonged thyroid hormone treatment is associated with oxidative stress, as documented by the high levels of oxidative damage indicators (Videla 2000, Venditti & Di Meo 2006). Accumulating evidence has demonstrated that in the liver such effects are primarily due to increased tissue content of mitochondrial respiratory chain components (Brand & Murphy 1987), including autoxidizable electron carriers (Venditti et al. 2003). This increase enhances tetravalent and univalent oxygen reduction, leading to higher oxygen consumption and reactive oxygen species (ROS) production respectively (Fernández & Videla 1993, Venditti et al. 2003).
Studies on the effects of tri-iodothyronine (T3) treatment on the antioxidant defense system of rat liver have shown that the changes in both the activities of antioxidant enzymes and the levels of low molecular weight scavengers are unbalanced and often opposite, although the whole antioxidant capacity significantly decreases in hyperthyroid state (Videla 2000, Venditti & Di Meo 2006). This decrease is associated with a higher susceptibility of hepatic tissue to oxidative challenge both in vitro (Venditti et al. 1997, 1999a) and in vivo (Videla et al. 1995, Troncoso et al. 1997). Interestingly, T3 effects appear to be strongly dependent on treatment duration. Indeed, ischemia-reperfusion injury, which is exacerbated by a 2-day T3 treatment (Troncoso et al. 1997), is reduced by a single T3 dose (Fernández et al. 2007).
Hyperthyroid state-linked liver oxidative damage can be attenuated by strengthening antioxidant status, as demonstrated by the observation that the administration of vitamin E (Venditti et al. 1999a) and caffeic acid phenylethyl ester (Mohamadin et al. 2007) protects against lipid peroxidation in hyperthyroid liver.
Some studies indicate that antioxidant administration and moderate physical training can have similar protective effects in conditions leading to increased free radical production. For example, the appearance of signs of increased free radical generation induced by acute swimming was prevented in rat liver by both vitamin E administration (Brady et al. 1979) and training (Venditti & Di Meo 1996). The training effect on liver was associated with an increase in tissue whole antioxidant defenses (Venditti & Di Meo 1996). However, it is not possible to exclude that, like in rat skeletal muscle (Venditti et al. 1999b, Servais et al. 2003), a reduced free radical activity contributes to delayed peroxidative reactions in the hepatic tissue.
To shed some light on this matter, we evaluated the effects of physical training on respiratory capacity and oxidative damage of liver homogenates from euthyroid and hyperthyroid rats. Moreover, because mitochondria are a possible target of ROS, respiratory capacity and oxidative damage of mitochondrial preparations were also evaluated. Markers of lipid and protein damage were related with scavenger levels and susceptibility to oxidants of liver homogenates and with mitochondrial H2O2 production rates.
Materials and Methods
Materials
All chemicals used (Sigma Chimica) were of the highest grades available. The response to oxidative stress was determined using reagents and instrumentation of the commercially available Amerlite System (Ortho-Clinical Diagnostics, Milano, Italy).
Serum levels of free T3 (FT3) and thyroxine (FT4) were determined using commercial RIA kits (DiaSorin, Salluggia, Italy).
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 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 the stress reaction. Therefore, in the first week, the rats were made to swim for 15 min daily to familiarize them 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 with water maintained at 35 °C to a depth of 55 cm. Untrained animals were kept in a small chamber holding about 3 cm water maintained at 35 °C.
Ten days before the end of the training program half of sedentary and trained rats were made hyperthyroid by the treatment with daily i.p. injections of T3 (10 μg/100 g body weight). Thus, there were four groups of rats: euthyroid sedentary (ES), euthyroid trained (ET), hyperthyroid sedentary (HS), and hyperthyroid trained (HT).
All the rats were subjected to the same conditions (one per cage, constant artificial circadian cycle of 12-h light:12-h darkness, and 50±10% relative humidity), and fed the same diet, a commercial rat chow purchased from Nossan, and water 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 killed by decapitation while under ether anesthesia. Arterial blood samples were collected and later analyzed to determine the plasma levels of FT3 and FT4. The livers were rapidly excised and placed into ice-cold homogenization medium (HM; 220 mM mannitol, 70 mM sucrose, 1 mM EDTA, 0.1% fatty acid-free albumin, 10 mM Tris, pH 7.4). Tissues were freed from connective tissue and were weighed, finely minced, and washed with HM. Finally, tissue fragments were gently homogenized (20% w/v) in the same solution using a glass Potter–Elvehjem homogenizer set at a standard velocity (500 r.p.m.) for 1 min. Aliquots of homogenates were used for analytical procedures and preparation of mitochondrial fractions.
Preparation of mitochondria
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 the same sedimentation velocity. Mitochondrial preparations were washed in this manner twice before final suspension in WB. Mitochondrial protein was measured by the biuret method (Gornall et al. 1949).
Analytical procedures
Cytochrome oxidase (COX) activity was determined by the procedure of Barré et al. (1987) using liver homogenates and mitochondrial suspensions diluted with modified Chappel–Perry medium so that the preparations contained per ml either 100 mg tissue or 0.2 mg mitochondrial proteins.
Oxygen consumption of homogenates and mitochondria was monitored at 30 °C by a Hansatech respirometer in 1.6 ml incubation medium (145 mM KCl, 30 mM HEPES, 5 mM KH2PO4, 3 mM MgCl2, 0.1 mM EGTA, pH 7.4) with 50 μl homogenate or 0.25 mg 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.
The extent of the peroxidative processes in liver homogenates and mitochondrial preparations was determined by measuring the level of lipid hydroperoxides (HPs) according to Heath & Tappel (1976). Determination of protein oxidative damage was performed measuring protein-bound carbonyl levels by the procedure of Reznick & Packer (1994) for homogenates and by the modified procedure of Schild et al. (1997) for mitochondria.
Glutathione peroxidase (GPX) activity was assayed at 37 °C according to Flohé & Günzler (1984) with H2O2 as substrate. Glutathione reductase (GR) activity was measured at 30 °C according to Carlberg & Mannervik (1985).
Ubiquinols (CoQH2) from 0.5 ml of 10% homogenate were oxidized to ubiquinones (CoQs) with 0.5 ml of 2% FeCl3 and 2.0 ml ethanol. The content of CoQs was then determined according to Lang et al. (1986). Vitamin E content was determined using the HPLC procedure of Lang et al. (1986). Reduced glutathione (GSH) concentration was measured as described by Griffith (1980).
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; Hyslop & Sklar 1984) in a computer-controlled Jasko fluorometer equipped with a thermostatically controlled cell-holder. The reaction mixture consisted of 0.1 mg/ml mitochondrial proteins, 6 U/ml HRP, 200 μg/ml 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 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, which blocks the transfer of electrons from Complex I to ubiquinone (Palmer et al. 1968), and antimycin A (AA), which interrupts electron transfer within the ubiquinone–cytochrome b site of Complex III (Turrens et al. 1985). Inhibitor concentrations (5 μM Rot, 10 μM AA) that do not interfere with the detection PHPA-HRP system were used (Venditti et al. 2003).
Capacity to remove H2O2 was determined by comparing the ability of mitochondrial samples to reduce H2O2-linked fluorescent emission with that of desferrioxamine solutions (Venditti et al. 2001). Thus, the capacity of mitochondrial samples to remove H2O2 was expressed as equivalent desferrioxamine concentration.
Response to oxidative challenge was determined as previously described (Venditti et al. 1999c). Briefly, the samples of 10% (w/v) homogenates were obtained by diluting the 20% homogenates with equal volumes of 0.2% Lubrol in 15 mM Tris, pH 8.5. Several dilutions of samples up to a tissue concentration of 0.002% were prepared in 15 mM Tris (pH 8.5). The assays were performed in microtiter plates. Enhanced chemiluminescence reactions were initiated by addition of 250 μl of the reaction mixture to 25 μl of the samples. The reaction mixture was obtained by mixing the solutions containing substrate in excess and signal generating reagents respectively in buffer at pH 8.6 (Vitros Signal Reagent). The plates were incubated at 37 °C for 30 s under continuous shaking and then transferred to a luminescence analyzer (Amerlite Analyzer). The emission values were fitted to dose–response curves using the statistical facilities of the Fig.P graphic program (Biosoft, Cambridge, UK).
Mitochondrial swelling was spectrophotometrically measured by determining the apparent absorbance at 540 nm in a medium containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES, pH 7.2, 2 mM succinate, 4 μM rotenone, 0.3 mg mitochondrial protein per ml of reaction mixture, 100 μM Ca2+, and 50 mM EGTA or 100 μM cyclosporin A (CSA) where indicated.
Mitochondrial membrane potential (ΔΨ) was estimated through the fluorescence changes of safranine (8 μM) recorded on the Jasko fluorometer (excitation wavelength 495 nm, emission wavelength 586 nm) in a medium containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES, pH 7.2, 2 mM succinate, 6 μM rotenone, 0.3 mg mitochondrial protein per ml of reaction mixture, 100 μM Ca2+. ΔΨ was calculated according to Åkerman & Wikström (1976) using a calibration curve obtained incubating mitochondria in a medium containing 200 mM sucrose, 10 mM HEPES, pH 7.2, 6 μM rotenone, 0.38 EDTA, 8 μM safranine, 38.5 ng/ml valinomycin, and KCl at concentrations from 0 to 0.96 mM.
Statistical analysis
The data obtained in eight different experiments are expressed as means±s.e.m. Data were analyzed with a two-way ANOVA method. When a significant F ratio was found, the Student–Newman–Keuls multiple range test was used to determine the statistical significance between means. The effects of respiratory inhibitors in the same group were statistically analyzed with the Student's t-test. Probability values (P) <0.05 were considered significant.
Results
The animal thyroid state was documented by their heart mass/body mass ratio and plasma levels of FT3 and FT4. Heart mass/body mass ratios were 2.25±0.04, 2.95±0.05, 3.15±0.07, and 3.30±0.12 in ES, ET, HS, and HT rats respectively. They were significantly (P<0.05) increased by training in the euthyroid rats and by T3 treatment in the sedentary and trained rats. As it is shown in Fig. 1, plasma levels of FT3 increased, whereas FT4 levels decreased in hyperthyroid rats in comparison with respective euthyroid controls. Conversely, there were not differences in FT3 and FT4 levels between sedentary and trained animals.
Serum thyroid hormone levels in euthyroid sedentary (ES), euthyroid trained (ET), hyperthyroid sedentary (HS), and hyperthyroid trained (HT) rats. Values are means±s.e.m. of eight different experiments. FT3 and FT4 are serum level of free tri-iodothyronine and thyroxine respectively. aSignificant versus euthyroid controls. The level of significance was chosen as P<0.05.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0625
COX activity
In liver homogenates, COX activity was increased by T3 administration in both sedentary and trained rats and was not modified by training in euthyroid and hyperthyroid animals. In liver mitochondria, COX activity was increased by T3 treatment in sedentary and trained group and was significantly decreased by training in both thyroid states (Table 1).
Cytochrome oxidase activities and mitochondrial protein content in rat liver
| Cytochrome oxidase | |||
|---|---|---|---|
| Homogenate | Mitochondria | Mitochondrial protein | |
| Group | |||
| ES | 69.3±1.3 | 0.94±0.01 | 72.7±3.3 |
| ET | 67.5±0.5 | 0.84±0.01† | 80.4±1.9† |
| HS | 113.0±1.6* | 1.19±0.02* | 95.0±3.0* |
| HT | 108.5±2.3* | 0.99±0.02*,† | 109.5±1.7*,† |
Values are means±s.e.m. For each value eight rats were used. Cytochrome oxidase activity (COX) is expressed in μmol O/min per milligram mitochondrial protein or g tissue. Mitochondrial protein is expressed as mg protein per g tissue. ES, euthyroid sedentary rats; ET, euthyroid trained rats; HS, hyperthyroid sedentary rats; HT, hyperthyroid trained rats. *Significant versus euthyroid controls. †Significant versus sedentary animals. Treatment×training interaction significant for mitochondrial COX. The level of significance was chosen as P<0.05.
The ratio between COX activities in homogenates and mitochondria provided a rough estimate of tissue content of mitochondrial proteins. Such a content was higher in hyperthyroid rats than in respective euthyroid controls and was increased by training in both thyroid states.
O2 consumption
The rates of O2 consumption by liver homogenates are reported in Fig. 2 (upper panel). The rates of succinate-supported O2 consumption were higher in hyperthyroid rats than in euthyroid controls during both State 4 and State 3. Respiration rates were increased by training only in hyperthyroid rats during State 4. Respiratory control ratio (RCR) values, which were 4.3±0.2, 4.0±0.3, 4.8±0.2, and 4.6±0.2 for ES, ET, HS, and HT rats respectively, were not significantly (P>0.05) affected by T3 and training. The rates of pyruvate/malate-supported O2 consumption were higher in hyperthyroid than in euthyroid controls during State 4 and State 3. Furthermore, they were increased by training in euthyroid rats during State 4. RCR values, which were 2.8±0.1, 2.1±0.1, 2.2±0.1, and 2.6±0.1 for ES, ET, HS, and HT rats respectively, were significantly (P<0.05) increased by T3 in trained rats and decreased in sedentary ones, whereas they were decreased by training in the euthyroid rats and increased in the hyperthyroid ones.
Rates of O2 consumption by rat liver homogenates (upper panel) and mitochondria (bottom panel) from euthyroid sedentary rats (ES); euthyroid trained rats (ET); hyperthyroid sedentary rats (HS); and hyperthyroid trained (HT) rats. Values are means±s.e.m. of eight different experiments. 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 versus euthyroid controls. bSignificant versus sedentary animals. Treatment×training interaction significant for State 4 homogenate respiration supported by pyruvate/malate, State 4 mitochondrial respiration supported by succinate, and State 3 mitochondrial respiration supported by succinate or pyruvate/malate. The level of significance was chosen as P<0.05.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0625
The rates of O2 consumption by liver mitochondria are reported in Fig. 2 (bottom panel). The rates of succinate-supported O2 consumption were higher in hyperthyroid rats than in euthyroid controls during both State 4 and State 3. Respiration rates were reduced by training in euthyroid rats during State 4 and in both thyroid states during State 3. RCR values, which were 4.7±0.1, 5.1±0.3, 3.8±0.1, and 3.3±0.1 for ES, ET, HS, and HT rats respectively, were significantly (P<0.05) reduced by T3 in sedentary and trained rats.
The rates of pyruvate/malate-supported O2 consumption were increased by T3 in sedentary and trained rats during State 4 and in sedentary rats during State 3. Furthermore, the rates were decreased by training in both thyroid states during State 4 and State 3. RCR values, which were 1.7±0.1, 2.3±0.1, 1.7±0.1, and 1.8±0.1 for ES, ET, HS, and HT rats respectively, were significantly (P<0.05) decreased by T3 in trained rats and increased by training in euthyroid ones.
Oxidative damage
In liver homogenates and mitochondria, the levels of hydroperoxides and protein-bound carbonyls were significantly higher in hyperthyroid rats than in respective euthyroid controls. Furthermore, they were reduced by training in both thyroid states (Fig. 3).
Oxidative damage in liver preparations from euthyroid sedentary (ES), euthyroid trained (ET), hyperthyroid sedentary (HS), and hyperthyroid trained (HT) rats. Values are means±s.e.m. of eight different experiments. Hydroperoxides (HPs) are expressed in nmol NADP min/g tissue and pmol NADP min/mg mitochondrial protein. Protein-bound carbonyls (CO) are expressed in nmol/mg protein. aSignificant versus euthyroid controls. bSignificant versus sedentary animals. Treatment×training interaction significant for mitochondrial HP and carbonyl levels. The level of significance was chosen as P<0.05.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0625
Antioxidants
The activities of antioxidant enzymes and the levels of low molecular weight antioxidants in the liver homogenates are reported in Table 2. T3 treatment increased GPX activities in sedentary rats and decreased GR activities in both sedentary and trained animals. Training increased GPX activity in euthyroid but not in hyperthyroid rats and did not affect GR activities in both thyroid states. Changes in vitamin E were found only in HT liver, which exhibited levels lower than their euthyroid and untrained controls. CoQ9 and CoQ10 levels were not modified by T3 administration or training. GSH levels were decreased by T3 treatment in trained and untrained rats, and were increased by training in both thyroid states.
Antioxidants levels in rat liver homogenates. Values are means±s.e.m. For each value eight rats were used. Glutathione peroxidase activity (GPX) is expressed in μmol NADPH min/g tissue. Glutathione reductase (GR) is expressed in μmol NADPH min/g tissue. Vitamin E (Vit E), coenzyme Q9 (CoQ9), and coenzyme Q10 (CoQ10) content is expressed in nmol/g tissue. Reduced glutathione (GSH) is expressed in μmol/g tissue
| Group | ||||
|---|---|---|---|---|
| ES | ET | HS | HT | |
| Parameter | ||||
| GPX | 75.7±1.4 | 85.0±0.8† | 85.5±2.8* | 87.6±2.1 |
| GR | 9.0±0.5 | 9.8±0.3 | 7.7±0.3* | 8.3±0.1* |
| Vit E | 49.94±1.63 | 45.44±0.69 | 47.31±0.85 | 35.70±1.50*,† |
| CoQ9 | 76.98±1.63 | 82.27±3.50 | 82.77±1.26 | 76.72±1.02 |
| CoQ10 | 7.44±0.22 | 8.59±0.31 | 7.93±0.37 | 7.76±0.62 |
| GSH | 4.79±0.09 | 5.49±0.39† | 2.61±0.07* | 3.48±0.09*,† |
ES, euthyroid sedentary rats; ET, euthyroid trained rats; HS, hyperthyroid sedentary rats; HT, hyperthyroid trained rats. *Significant versus euthyroid controls. †Significant versus sedentary animals. Treatment×training interaction significant for vitamin E and CoQ9 content. The level of significance was chosen as P<0.05.
Response to oxidative challenge
The luminescence response to changes of concentration of the homogenates (Fig. 4) was described by the equation (E=a.C/exp(b.C)). The parameters a and b, which determine the light emission maximum (Emax=a/e.b), are dependent on the concentration of substances able to induce (iron or cuprum ligands) and inhibit (antioxidants) respectively the H2O2-induced luminescent reaction. Examination of the curves in Fig. 4 and data reported in Table 3 show that the emission maximum was increased by T3 treatment in sedentary and trained rats, and was decreased by training in both thyroid states. The values of the parameters a and b indicate that the increase in emission peak induced by T3 treatment is due to lower b values and higher a values, while the decrease induced by training is mainly imputed to higher b values.
Response to in vitro oxidative challenge of liver homogenates from euthyroid sedentary (ES), euthyroid trained (ET), hyperthyroid sedentary (HS), and hyperthyroid trained (HT) rats. Tissue susceptibility to stress was evaluated by determining the variations, with concentration of homogenates, of light emission from a luminescent reaction. Emission values are given percentage of an arbitrary standard (44 ng/ml peroxidase). The curves were computed from experimental data by equation E=a.C/exp(b.C).
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0625
Parameters characterizing the response to oxidative stress of rat liver homogenates. Values are means±s.e.m. For each value eight rats were used. For explanation of symbols see text. The relation between light emission and homogenate concentration is described by the equation: E=a.C/exp(b.C). Emax=a/e.b
| Group | ||||
|---|---|---|---|---|
| ES | ET | HS | HT | |
| Parameter | ||||
| a | 72.8±5.4 | 70.3±4.2 | 98.7±3.7* | 90.0±6.5* |
| b | 1.34±0.03 | 1.81±0.12† | 0.75±0.08* | 1.03±0.10*,† |
| Emax | 20.0±1.1 | 14.3±1.5† | 48.4±2.4* | 32.12±2.1*,† |
ES, euthyroid sedentary rats; ET, euthyroid trained rats; HS, hyperthyroid sedentary rats; HT, hyperthyroid trained rats. *Significant versus euthyroid controls. †Significant versus sedentary animals. Treatment×training interaction significant for Emax. The level of significance was chosen as P<0.05.
Mitochondrial H2O2 release and capacity to remove H2O2
During both State 4 and State 3 respiration, the rates of succinate and pyruvate/malate supported H2O2 mitochondrial release were increased by T3 treatment in sedentary and trained rats, and were decreased by training in both thyroid states (Fig. 5).
Rates of H2O2 release by liver mitochondria from euthyroid sedentary (ES), euthyroid trained (ET), hyperthyroid sedentary (HS), and hyperthyroid trained (HT) rats. Values are means±s.e.m. of eight different experiments. Rates of H2O2 release 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 versus euthyroid controls. bSignificant versus sedentary animals. Treatment × training interaction significant for H2O2 release sustained by succinate during State 4 and State 3, and H2O2 release sustained by pyruvate/malate during State 3. The level of significance was chosen as P<0.05.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0625
The capacities to remove H2O2 were increased by T3 treatment (P<0.05) in trained and untrained rats and were decreased by training in both thyroid states (P<0.05). The values, expressed as equivalent concentration of desferroxamine (nmol/mg protein), were 3.18±0.09, 2.77±0.05, 4.13±0.10, and 3.45±0.07 for ES, ET, HS, and HT rats respectively.
Effect of inhibitors on H2O2 release
As shown in Table 4, T3 treatment increased the rate of mitochondrial succinate-supported H2O2 release, in the absence of rotenone, in both sedentary and trained rats, whereas training reduced it in both thyroid states. As expected, rotenone addition decreased the rates of H2O2 release in all groups, by stopping the release occurring at Complex I, due to the reverse electron flow from coenzyme Q (Ernster & Lee 1967). However, the rates were again increased by T3 and decreased by training. Analogous results were obtained after further addition of antimycin, which increased H2O2 release rates in all groups. Addition of antimycin or rotenone to pyruvate/malate supported mitochondria increased H2O2 release rates in all groups. However, whereas in the presence of antimycin, the significance of differences between groups was not modified, in the presence of rotenone the rates of H2O2 release were not different in sedentary and trained hyperthyroid rats.
Effects of inhibitors specific for different segments of the respiratory chain on rates of H2O2 release by rat liver mitochondria. 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
| Group | ||||
|---|---|---|---|---|
| ES | ET | HS | HT | |
| Substrate | ||||
| Succinate (Succ) | 115.3±1.0 | 101.2±0.8† | 179.0±2.3* | 132.6±0.6*,† |
| Succ+Rot | 101.4±1.1‡ | 92.0±2.9†,‡ | 146.5±2.4*,‡ | 111.4±0.8*,†,‡ |
| Succ+Rot+AA | 803.8±9.8‡ | 718.3±1.8†,‡ | 919±14.8*,‡ | 830.7±3.4*,†,‡ |
| Pyruvate/Malate (Pyr/Mal) | 194.7±1.5 | 181.1±1.1† | 310.4±2.7* | 300.2±4.3*,† |
| Pyr/Mal+AA | 992.9±4.2‡ | 924.4±2.2†,‡ | 1036.8±11.4*,‡ | 1020.6±17.3*,†,‡ |
| Pyr/Mal | 194.6±2.5 | 182.0±1.2† | 310.9±2.6* | 300.5±3.4*,† |
| Pyr/Mal+Rot | 227.6±2.6‡ | 214.8±1.2†,‡ | 332.8±2.4*,‡ | 329.5±2.5*,‡ |
ES, euthyroid sedentary rats; ET, euthyroid trained rats; HS, hyperthyroid sedentary rats; HT, hyperthyroid trained rats. *Significant versus euthyroid controls. †Significant versus sedentary animals; ‡significant effect of the last inhibitor added versus mitochondria under same conditions without that inhibitor. Treatment×training interaction significant for H2O2 release in the presence of succinate, succinate plus rotenone, pyruvate/malate plus antimycin or rotenone. The level of significance was chosen as P<0.05.
Mitochondrial swelling
As shown by the absorbance changes in Fig. 6, the extent of swelling was increased by T3 treatment in Ca2+-loaded mitochondria from sedentary rats, but not in mitochondria from trained rats. Mitochondrial swelling was drastically reduced by CSA or EGTA (unreported results), pointing to the role played by the permeability transition pore. Ca2+-induced swelling was preceded by a rapid decrease in membrane potential, which was greater in preparations from sedentary hyperthyroid rats than in those from trained rats. No difference in Ca2+-induced mitochondrial swelling and ΔΨ decrease were found in euthyroid preparations (Fig. 6).
Ca2+-induced swelling (upper panel) and membrane potential dissipation (lower panel) of liver mitochondria from euthyroid sedentary (ES), euthyroid trained (ET), hyperthyroid sedentary (HS), and hyperthyroid trained (HT) rats. Swelling, monitored as decrease in the absorbance at 540 nm, is expressed as percentage of the initial value before Ca2+ addition. Membrane potential (ΔΨ), estimated through fluorescence changes of safranine, was calculated using a suitable calibration curve. Its decrease is expressed as percentage of the initial value before Ca2+ addition. Values are means±s.e.m. of eight different experiments. The initial absorbance values were 0.85±0.04, 0.90±0.07, 0.86±0.05, and 0.83±0.05 for ES, ET, HS, and HT preparations respectively. Initial values of ΔΨ were 149.1±11.5, 152.7±10.4, 140.6±9.7, and 147.0±8.9 for ES, ET, HS, and HT preparations respectively. aSignificant versus euthyroid controls. bSignificant versus sedentary animals. Treatment×training interaction significant for swelling and membrane potential. The level of significance was chosen as P<0.05.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0625
Discussion
A first consideration, elicited from the results reported in this paper, concerns the effects on the liver oxidative metabolism capacity of T3 prolonged administration to sedentary and trained rats. The aerobic metabolic capacity of tissues can be evaluated measuring the in vitro COX activity, which is positively correlated to the maximal oxygen consumption (Simon & Robin 1971). It has been reported that training does not modify COX activity and, therefore, the oxidative capacity of liver (Terblanche et al. 2001), which, conversely, increases in experimental (Venditti et al. 1999a, 2006) and functional (Venditti et al. 2004, 2006) hyperthyroidism. The present results confirm the above reports and indicate that T3 induces similar increases in liver oxidative capacity in sedentary and trained rats.
These increases were due, in both animal groups, to enhanced mitochondrial aerobic capacity and tissue content of mitochondrial proteins. However, the changes in aerobic capacity were greater in HS group, whereas those in protein content were greater in HT group. Our results also suggest that training does not increase liver aerobic capacity, because the proliferation of mitochondrial population it seems to induce is accompanied by a decrease in the aerobic capacity of the organelles.
The mitochondrial proliferation found in hyperthyroid liver can be attributed to the capacity of T3 to increase the expression of peroxisomal proliferator-activated receptor gamma co-activator (PGC)-1, a transcriptional coactivator that plays a pivotal role in mitochondrial biogenesis (Weitzel et al. 2001). On the other hand, catecholamines may be responsible for mitochondrial proliferation in the liver from trained rats. In fact, recent studies have shown i) an increased PGC-1 expression in muscle from swimming trained rats (Goto et al. 2000), ii) an increased plasma catecholamine level during single swimming sessions (Park et al. 2005) and an increased PGC-1 expression in murine muscle in response to exercise linked to β-adrenergic receptor activation (Miura et al. 2007).
Interestingly, the prolonged T3 administration and training elicited changes in the oxidative capacity of homogenates and mitochondria were reflected in the changes in succinate and pyruvate/malate sustained State 3 respiration of these preparations. This suggests that both treatments modify liver mitochondrial population without alter the ratios among the components of respiratory chain.
Some changes in State 4 respiration rate induced by training in mitochondria and homogenates remain to be understood. The increase in basal respiration rates represents a compensatory response to increased leak of protons back in the mitochondrial matrix. Thus, the increases induced by T3 are consistent with an enhanced basal proton conductance dependent on oxidative modifications of inner mitochondrial membrane lipids (Brand 2000). In this light, the respiration rates should have to decrease in mitochondrial preparations from trained rats, but this does not happen in succinate-supplemented mitochondria from HT rats. Moreover, although the changes found in homogenates can be explained by the increased tissue content in mitochondrial proteins, the pyruvate/malate-supported respiration rate measured in homogenates from ET rats appears to be out of proportion to the mitochondrial protein content.
There are a few studies concerning the chronic swimming effects on liver oxidative damage in euthyroid rats, which reported discrepant results (Venditti & Di Meo 1996, 1997, Aydin et al. 2007), likely due to different training protocols. Conversely, there is full agreement about thyroid hormone promotion of liver oxidative damage (Videla 2000, Venditti & Di Meo 2006). The present results show that moderate training reduces lipid and protein oxidation in euhyroid animals and attenuates its increase associated with T3 administration, suggesting that training exerts a strong protective effect against T3-induced oxidative stress.
Oxidative stress results from a disturbance of the normal cell balance between production of ROS and the capacity to neutralize their action. Thus, in order to understand the mechanisms underlying the differential effects of T3 on liver oxidative damage in sedentary and trained rats, we examined changes in liver capacity to generate and neutralize free radicals.
In previous reports, we showed that the total antioxidant capacity of rat liver is increased by training (Venditti & Di Meo 1996, 1997) and decreased by T3 treatment (Venditti et al. 1997, 1999a). The determination of total antioxidant capacity on tissue preparations presents several limits. On the other hand, due to the complexity of the antioxidant defense systems, investigations on tissue antioxidant status based on the determination of single antioxidant compounds or enzymes often does not offer a coherent pattern. This also happens for the data here reported, concerning some components of the antioxidant defense system, which do not allow to establish whether the effectiveness of such system is differently modified by T3 treatment in sedentary and trained rats. Indeed, the levels of some antioxidants are not modified by training in hyperthyroid rats, whereas those of vitamin E and GSH undergo opposite changes, likely because the different degree of GSH depletion is not the cause, but the consequence of the different levels of oxidative stress in the two rat groups. However, some information was obtained by examining the liver sensitivity to oxidative processes by measuring the levels of light emission resulting from in vitro exposure to H2O2 of liver homogenates. As the emission maximum indicates the susceptibility of the preparations to oxidative challenge (Venditti et al. 1999c), our results reveal that such a susceptibility is reduced by training, but in greater measure in T3-treated animals. Also, the results indicate that the lower susceptibility to oxidants found in trained groups is mainly due to a higher antioxidant capacity. Thus, there is indirect evidence that a greater effectiveness of the antioxidant defense system contributes to the lower degree of oxidative damage induced by T3 in the liver from trained rats.
More clear information has been obtained on ROS generation contribution to the different extent of oxidative damage found in sedentary and trained rats. ROS are produced in several cellular sites, but mitochondria have long been recognized as the major source of free radicals (Liu et al. 2002). The enhancement in mitochondrial protein content of hyperthyroid livers should lead to increased ROS flow from mitochondria to cytoplasm. However, it is apparent that training protects the hepatic tissue against oxidative damage by inducing adaptive modifications that prevent the elevated mitochondrial H2O2 efflux. Indeed, training limits the T3-induced increase in H2O2 mitochondrial release rates. The effect concerns mainly the H2O2 release during State 4 respiration sustained by Complex II-linked substrate, so that in the trained group the rate of H2O2 release per g of tissue is 87% of that found in sedentary group. A training-induced reduction of succinate-supported ROS release occurs also in euthyroid rats. However, such reduction is small and is able only to compensate the increase in mitochondrial protein content.
The observation that the mitochondrial capacity to remove H2O2 is higher in hyperthyroid than in euthyroid groups and in sedentary than in trained groups indicates that the differences in H2O2 release reflect differences in the production of the H2O2 precursor, the superoxide radical 
Conversely, it is apparent that the prolonged T3 administration to trained rats induces a smaller increase in the mitochondrial content of the ROS generator located at Complex III. Indeed, in the presence of rotenone, succinate-supported mitochondrial H2O2 release is lower in trained group than in the sedentary one, whereas the release supported by pyruvate/malate is not significantly different in the two groups. Further support comes from the results obtained in the presence of antimycin A, which makes ROS production dependent only on the concentration of autoxidizable carriers. Such results show that H2O2 release is lower in the trained group when it depends on the Complex III generator, i.e. in the presence of succinate, whereas there are not differences between the groups when it depends on the Complex I and III generators, i.e., in the presence of pyruvate/malate. Finally, when pyruvate/malate-supplemented mitochondria are treated with rotenone, which makes H2O2 release dependent only on the concentration of the Complex I generator, such release is not significantly different in the two hyperthyroid groups. We also observed that, in the presence of rotenone, pyruvate/malate-sustained H2O2 release was lower in trained than in sedentary euthyroid rats. However, to date we are not able to offer a convincing explanation for this different effect of training on autoxidizable carrier of Complex I in euthyroid and hyperthyroid rats.
The T3 and training-dependent changes in ROS generation have important implications for the function and even the viability of mitochondria, because these organelles are the main target of the ROS they produce. It is known that hyperthyroidism leads to an increase in total liver Ca2+ concentration (Hummerich & Soboll 1989). In the presence of this ion, oxidative alterations of protein thiols of the mitochondrial inner membrane promote a membrane permeabilization referred to as mitochondrial permeability transition (Zoratti & Szabo 1995), which leads to mitochondrial swelling. Our results show that mitochondrial susceptibility to Ca2+-induced swelling is significantly lower in trained than in sedentary hyperthyroid rats. This observation is consistent with the failure of T3 to induce similar levels of oxidative stress and ROS production in mitochondria from sedentary and trained animals.
In conclusion, this work supplies strong indication that training limits the oxidative stress brought out in the hepatic tissue by prolonged thyroid hormone treatment, reducing mitochondrial ROS production and preserving the antioxidant system effectiveness. Because it has been recently shown that functional recovery from ischemia reperfusion of hyperthyroid hearts is increased by training (Venditti et al. 2008), it is apparent that, together with antioxidant integration, properly graded physical activity can protect hyperthyroid tissues against the oxidative damage-linked dysfunctions.
Acknowledgements
This work was supported by grants from Italian Ministry of University and Scientific and Technological Research. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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(All authors contributed equally to this work)
