Effect of experimental and cold exposure induced hyperthyroidism on H2O2 production and susceptibility to oxidative stress of rat liver mitochondria

To investigate the iodothyronine role in liver responses to cold, we examined metabolic and oxidative mitochondrial changes in cold-exposed, T3-treated, and T4-treated rats, which exhibit different T4 serum levels. All treatments increased mitochondrial respiration which reached the highest and lowest values after T3 and cold treatment, respectively. The T3- and T4-induced changes agreed with the respective increases in Complex IV activities, while those elicited by cold were inconsistent with increased activities of respiratory complexes. Mitochondrial capacity to produce H2O2 was the highest in T3-treated rats, whereas it was similar in T4-treated and cold-exposed rats. The effects of respiratory inhibitors suggested that T3 and T4 mainly increase the mitochondrial content of autoxidizable electron carrier of Complex I and Complex III, respectively. The indices of oxidative modifications of proteins exhibited increases consistent with the treatment effects on H2O2 production. The increases in indices of lipid peroxidation were also dependent on changes in lipid composition. The increased protein damage in treatment groups was confirmed using immunoblotting analysis, which also showed oxidative damage in a 133 kDa fraction, which was not expressed in T3-treated rats. Antioxidant levels were not related to the extent of oxidative damage as only mitochondrial GSH levels decreased in T3-treated rats. Mitochondrial susceptibility to in vitro oxidative challenge and Ca2+-induced swelling was increased by all treatments, but was the highest in T3-treated rats. In the whole, our results indicate T3 as main responsible for the changes in the mitochondrial population associated with cold exposure. However, a significant role is also played by T4, which appears to acts mainly modulating T3 effects, but also inducing some effects different from the T3 ones.     

Vitamin E attenuates cold-induced rat liver oxidative damage reducing H2O2 mitochondrial release

Vitamin E is a major chain-breaking antioxidant which is able to reduce liver oxidative damage without modifying aerobic capacity in T(3)-treated rats. We investigated whether vitamin E has similar effects in hyperthyroid state induced by cold exposure. Cold exposure increased aerobic capacity and O(2) consumption in homogenates and mitochondria and tissue mitochondrial protein content. Vitamin E did not modify aerobic capacity and mitochondrial protein content of cold liver, but increased ADP-stimulated respiration of liver preparations. Succinate-supported H(2)O(2) release rates were increased by cold during basal and stimulated respiration, whereas the pyruvate/malate-supported ones increased only during basal respiration. Vitamin administration to cold-exposed rats decreased H(2)O(2) release rates with both substrates during basal respiration. This effect reduced ROS flow from mitochondria to cytosol, limiting liver oxidative damage. Cold exposure also increased mitochondrial capacity to remove H(2)O(2), which was reduced by vitamin treatment, showing that the antioxidant also lowers H(2)O(2) production rate. The different effects of cold exposure and vitamin treatment on H(2)O(2) generation were also found in the presence of respiration inhibitors. Although this can suggest that the cold and vitamin induce opposite changes in mitochondrial content of autoxidizable electron carriers, it is likely that vitamin effect is due to its capacity to scavenge superoxide radical. Finally, vitamin E reduced mitochondrial oxidative damage and susceptibility to oxidants, and prevented Ca(2+)-induced swelling elicited by cold. In the whole, our results suggest that vitamin E is able to maintain aerobic capacity and attenuate oxidative stress of hepatic tissue in cold-exposed rats modifying mitochondrial population characteristics.     

Thyroid Hormone Influences Antioxidant Defense System in Adult Rat Brain

The objective of the current study was to find out whether thyroid hormone influences antioxidant defense parameters of rat brain. Several oxidative stress and antioxidant defense parameters of mitochondrial (MF) and post-mitochondrial (PMF) fractions of cerebral cortex (CC) of adult rats were compared among euthyroid (control), hypothyroid [6-n-propylthiouracil (PTU)-challenged], and hyperthyroid (T3-treatment to PTU-challenged rats) states. Oxidative stress parameters, such as thiobarbituric acid-reactive substances (TBA-RS) and protein carbonyl content (PC), in MF declined following PTU challenge in comparison to euthyroid rats. On the other hand, when PTU-challenged rats were treated with T3, a significant increase in the level of oxidative stress parameters in MF was recorded. Hydrogen peroxide content of MF as well as PMF of CC was elevated by PTU-challenge and brought to normal level by subsequent treatment of T3. Although mitochondrial glutathione (reduced or oxidized) status did not change following PTU challenge, a significant reduction in oxidized glutathione (GSSG) level was noticed in PMF following the treatment. T3 administration to PTU-challenged rats had no effect on mitochondrial glutathione status. Total and CN-resistant superoxide dismutase (SOD) activities in MF of CC augmented following PTU challenge. CN-resistant SOD activity did not change when PTU-challenged rats were treated with T3. Although CN-sensitive SOD activity of PMF remained unaltered in response to PTU challenge, its activity increased when PTU-challenged rats were treated with T3. Catalase activity in PMF of CC of PTU-challenged rats increased, whereas the activity was decreased when hypothyroid rats were treated with T3. Similarly, total and Se-dependent glutathione peroxidase (GPx) activities of MF increased following PTU challenge and reduced following administration of T3. Se-independent GPx activity of MF and PMF and glutathione reductase activity of PMF decreased following PTU challenge and did not change further when rats were treated with T3. On the other hand, glutathione S-transferase activity of MF and PMF of CC did not change following PTU challenge but decreased below detectable level following T3 treatment. Results of the current investigation suggest that antioxidant defense parameters of adult rat brain are considerably influenced by thyroid states of the body.     

Comparison of heat and cold stress to assess thermoregulatory dysfunction in hypothyroid rats

How borderline impairment of thyroid function can affect thermoregulation is an important issue because of the antithyroidal properties of a many environmental toxicants. This study compared the efficacy of heat and cold stress to identify thermoregulatory deficits in rats subjected to borderline and overt hypothyroidism via subchronic exposure to propylthiouracil (PTU). After 3 wk of exposure to PTU in the drinking water (0, 2.5, 5, 10, and 25 mg/l), rats were subjected to a heat stress challenge (34 degrees C for 2.5 h). After one more week of PTU treatment, the same rats were subjected to a cold stress challenge (7 degrees C for 2.5 h). Core temperature (T(c)) was monitored by radiotelemetry. Baseline T(c) during the light phase was reduced by treatment with 25 mg/l PTU. The rate of rise and overall increase in T(c) during heat stress was attenuated by PTU doses of 10 and 25 mg/l. Cold stress resulted in a 1.0 degrees C increase in T(c) regardless of PTU treatment. The rate of rise in T(c) during the cold stress challenge was similar in all PTU treatment groups. There was a dose-related decrease in serum thyroxine (T(4)) at PTU doses >/=5 mg/l. Serum triiodothyronine (T(3)) was reduced at PTU doses of 5 and 25 mg/l. Serum thyroid-stimulating hormone (TSH) was marginally elevated by PTU treatment. Overall, heat stress was more effective than cold stress for detecting a thermoregulatory deficit in borderline (i.e., 10 mg/l PTU) and overtly hypothyroid rats (i.e., 25 mg/l PTU). A significant thermoregulatory deficit is manifested with a 78% decrease in serum T(4). A thermoregulatory deficit is more correlated with a reduction in serum T(4) compared with T(3). Serum levels of TSH are unrelated to thermoregulatory response to heat and cold stress.     

Rat liver response elicited by long-term cold exposure.

We measured mitochondrial protein mass as well as State 4 and 3 respiratory rates using different substrates in isolated liver mitochondria from 30-day cold-exposed rats. In addition, we measured the respiration under different conditions of stimulation in isolated hepatocytes from long-term cold-exposed rats. The results show that long-term cold exposure elicits a significant increase in hepatic mass and mitochondrial protein mass. No variation was found in oxygen consumption of isolated mitochondria and hepatocytes. On the whole, the results indicate that long-term exposure elicits an increase in hepatic mitochondrial protein mass but not in hepatic oxygen consumption.     

Cold exposure as a potentiating factor of pineal actions in nonhibernating mammals

Twenty-eight-day-old male rats were used in three experiments to study whether cold exposure potentiates pineal actions in nonhibernating mammals. The following questions were considered: (a) Can cold exposure increase the antigonadal effects of light deprivation? (b) Are the effects induced by blindness plus cold exposure pineal dependent? (c) Can cold exposure modify the response of the endocrine-reproductive axis to exogenously administered melatonin? Blind cold-exposed rats showed a significant loss in body weight as well as in weights of pituitary and reproductive tract organs compared with either intact or blind animals kept at 22 degrees C, or intact rats exposed to cold; serum testosterone levels were also lowest in blind cold-exposed rats. These effects were not present in blind cold-exposed animals that were pinealectomized at the beginning of the experiment. When intact animals placed at 22 or 10 degrees C were treated with daily injections of melatonin (50 micrograms) there was a reduction of body weight and weights of the hypophyso-gonadal axis organs. Those effects of melatonin were, however, significantly greater in cold-exposed rats than in rats placed at 22 degrees C. These results suggest that cold exposure should be considered as another state which potentiates the pineal-dependent actions of light deprivation. Cold exposure probably acts by increasing the sensitivity of sites at which pineal melatonin exerts its actions.     

Muscle type difference in the regulation of UCP3 under cold conditions

We found opposite regulation of uncoupling protein 3 (UCP3) in slow-twitch soleus and fast-twitch gastrocnemius muscles of rats during cold exposure. Namely, the UCP3 mRNA level was downregulated in the soleus muscles, but upregulated in the gastrocnemius muscles after a 24-h cold exposure. In the analysis of UCP3 protein, we first succeeded in detecting UCP3 short-form as well as the long-form in vivo, which levels were decreased markedly in the cold-exposed soleus muscles. However, the levels of UCP3 and cytochrome oxidase subunit IV were well maintained in the cold-exposed gastrocnemius muscles with a rise in the total mitochondrial protein level, suggesting an increase of total oxidative ability. The fast-twitch muscle rather than the slow-twitch one may play an important role in adaptive responses, including thermogenesis under acute cold exposure.     

Role of Amino Acid-catabolizing Enzymes in Urea Synthesis for Rats Treated with the Thyroid Hormone

The purpose of the present study was to determine whether the regulation of urea synthesis was mediated through the supply of nitrogen by amino acid-catabolizing enzymes and whether the concentration of acetylCoA would control the N-acetylglutamate concentration when the thyroid status was manipulated. Experiments were conducted on three groups of rats, each being given 6-propyl-2-thiouracil (PTU, thyroid inhibitor) without a triiodothyronine (T₃) treatment, or PTU + T₃, or neither PTU nor T₃ (control), respectively. The urinary excretion of urea, the liver concentration of N-acetylglutamate, and the hepatic activities of serine dehydratase, threonine dehydratase, alanine transaminase (GPT) and aspartate transaminase (GOT) in rats given PTU + T₃ were significantly lower than those in rats given PTU alone. The activity of glutamate dehydrogenase and the concentrations of free amino acids and acetylCoA in the liver of the PTU + T3-treated group were significantly higher than those in the group treated with PTU alone. These results suggest that the higher activity of amino acid-catabolizing enzymes in the hypothyroid (with PTU alone) rats is likely to stimulate urea synthesis.     

Modulation of rat liver mitochondrial antioxidant defence system by thyroid hormone

In the present study the effect of thyroid hormone (T(3)) on oxidative stress parameters of mitochondria of rat liver is reported. Hypothyroidism is induced in male adult rats by giving 0.05% propylthiouracil (PTU) in drinking water for 30 days and in order to know the effect of thyroid hormone, PTU-treated rats were injected with 20 microg T(3)/100 g body weight/day for 3 days. The results of the present study indicate that administration of T(3) to hypothyroid (PTU-treated) rats resulted in significant augmentation of oxidative stress parameters such as thiobarbituric acid reactive substances and protein carbonyl content of mitochondria in comparison to its control and euthyroid rats. The hydrogen peroxide content of the mitochondria of liver increased in hypothyroid rats and was brought to a normal level by T(3) treatment. Induction of hypothyroidism by PTU treatment to rats also resulted in the augmentation of total and CN-sensitive superoxide dismutase (SOD) activities of the mitochondria, which was reduced when hypothyroid rats were challenged with T(3). Although CN-resistant SOD activity of the mitochondria remained unaltered in response to hypothyroidism induced by PTU treatment, its activity decreased when hypothyroid rats were injected with T(3). The catalase activity of the mitochondria decreased significantly by PTU treatment and was restored to normal when PTU-treated rats were given T(3). Total, Se-independent and Se-dependent glutathione peroxidase activities of the mitochondria were increased following PTU treatment and reduced when T(3) was administered to PTU-treated rats. The reduced and oxidised glutathione contents of the mitochondria of liver increased significantly in hypothyroid rats and their level was restored to normal when hypothyroid rats were injected with T(3). The results of the present study suggest that the mitochondrial antioxidant defence system is considerably influenced by the thyroid states of the body.     

Brown fat thermogenesis in cold-acclimated rats is not abolished by the suppression of thyroid function

The effects of long-term cold exposure on brown adipose tissue (BAT) thermogenesis in hypothyroid rats have been examined. Thyroid ablation was performed in normal rats after 2 mo of exposure to 4 degrees C, when BAT hypertrophy and thermogenic activity were maximal. After ablation, hypothyroid and normal controls remained in the cold for 2 additional months. At the end of the 4-mo cold exposure, all untreated hypothyroid rats were alive, had normal body temperature, and had gained an average 12.8% more weight than normal controls. Long-term cold exposure of hypothyroid rats markedly increased BAT weight, mitochondrial proteins, uncoupling protein (UCP)-1, mRNA for UCP-1, and oxygen consumption to levels similar to those seen in cold-exposed normal rats. The results indicate that thyroid hormones are required for increased thermogenic capacity to occur as an adaptation to long-term cold exposure. However, cold adaptation can be maintained in the absence of thyroid hormone.     

Renal responses to chronic cold exposure

The aim of this study was to assess our hypothesis that the release of antidiuretic hormone (ADH), the renal concentrating response to ADH, or both is decreased by prolonged cold exposure. Six groups (n = 6/group) of rats were used. Three groups were exposed to cold (5 degrees C), whilethe remaining three groups were kept at room temperature (25 degrees C). It was found that urine osmolality decreased significantly and serum osmolality increased significantly during cold exposure. The ratio of water/food intake was not affected by prolonged cold exposure. However, prolonged cold exposure increased the ratio of urine output/food intake in the cold-exposed rats, indicating that more urine flow is required by the cold-exposed rats to excrete the osmotic substance at a given food intake. The difference between water intake and urine output decreased significantly in the cold-exposed rats. Thus, prolonged cold exposure increases water loss from excretion. Renal concentrating responses to 24-h dehydration and Pitressin were decreased significantly in the cold-exposed rats. Plasma ADH levels remained unchanged, but renal ADH receptor (V2 receptor) mRNA was decreased significantly in the cold-exposed rats. The results strongly support the conclusion that cold exposure increases excretive water loss, and this may be due to suppression of renal V2 receptors rather than inhibition of ADH release.     

Oxidation of γ-Hydroxybutyrate to Succinic Semialdehyde by a Mitochondrial Pyridine Nucleotide-Independent Enzyme

An antibody that inhibits over 95% of the cytosolic NADP+-dependent gamma-hydroxybutyrate (GHB) dehydrogenase activity of either rat brain or kidney was found to inhibit only approximately 50% of the conversion of [1-14C]GHB to 14CO2 by rat kidney homogenate. A similar result was obtained with sodium valproate, a potent inhibitor of GHB dehydrogenase. The mitochondrial fraction from rat brain and kidney was found to catalyze the conversion of [1-14C]GHB to 14CO2. The dialyzed mitochondrial fraction also catalyzed the oxidation of GHB to succinic semialdehyde (SSA) in a reaction that did not require added NAD+ or NADP+ and which was not inhibited by sodium valproate. The enzyme from the mitochondrial fraction which converts GHB to SSA appears to be distinct from the NADP+-dependent cytosolic oxidoreductase which catalyzes this reaction.     

The effect of low ambient temperature on the febrile responses of rats to semi-purified human endogenous pyrogen

The febrile responses of Sprague-Dawley rats to semi-purified human endogenous pyrogen were studied at a thermoneutral ambient temperature (26 degrees C) and in the cold (3 degrees C). It was found that while rats developed typical monophasic febrile responses at thermoneutrality, febrile responses were absent in the cold-exposed rats. Experiments were conducted to determine whether this lack of febrile responses in cold-exposed rats was due to an inability of these animals to generate or retain heat in the cold. Thermogenesis and vasoconstriction were stimulated in cold-exposed rats by selectively cooling the hypothalamus, using chronically implanted thermodes. It was shown that, using this stimulus, metabolic rate could be increased by more than 50 percent and body temperature could be driven up at a rate of 5 degrees C/hour in rats exposed to the cold. Therefore, it was concluded that the lack of febrile responses of cold-exposed rats to pyrogen is in no way due to a physical or physiological inability to retain heat. Instead, it appears that in some manner cold exposure suppresses the sensitivity or responsiveness of the rat to pyrogenic stimuli.     

Acute and chronic hypoxia in rats. I. Effect on organismic respiration, mitochondrial protein mass in liver and succinic dehydrogenase activity in liver, kidney and heart

The objective of this investigation was to characterize organismic, organ and mitochondrial alterations in rats over the course of 27 days at 0.4 atm. In the adjustment phase (day 1 through 5) a significant decrease in systemic oxygen uptake and body weight (23% of pre-altitude values) occurred. In the acclimating state (day 7 to 27) body weight was regained but oxygen consumption remained depressed. Hematocrit increased hyperbolically from 45% in 0-day rats to 79% in 27-day rats. Liver, kidney and heart weights and total organ protein paralleled the changes observed in body weight. Total organ succinic dehydrogenase activity showed a wave-like oscillation for liver and kidney; activity was decreased in both organs by day 5, showed a transient but significant increase on days 16 through 18 and a return to diminished activity on day 27. Succinic dehydrogenase activity for heart became depressed in the adjustment phase but showed a stable level comparable to pre-altitude values in the accliminating phase, days 7 through 27. Liver mitochondrial protein mass was unchanged from pre-altitude values on days 5 and 27 even though succinic dehydrogenase activity was significantly depressed. Therefore, the changes in succinic dehydrogenase activity are not representative of altered mitochondrial mass but suggest that mitochondrial function was altered.     

Regulation of rat liver NADP+-isocitrate dehydrogenase during aging

In an attempt to understand the mechanism of aging in relation to the differences in enzyme regulation, the induction and kinetic properties of NADP⁺ -isocitrate dehydrogenase of the liver of immature (6 weeks), mature (13 weeks), adult (33 weeks) and old (85 weeks) female rats were studied. The specific activity of the cytoplasmic and mitochondrial NADP+ -isocitrate dehydrogenase increased up to the adult age (33 weeks) and decreased in the old rats (85 weeks). Overiectomy decreased and estradiol administration induced activity of both the mitochondrial and eytoplasmic enzyme in the liver ol immature, mature and adult rats but had no significant effect in old rats. However, the activity of mitochondrial NADP⁺ -isocitrate dehydrogenase decreased and eytoplasmic NADP+ -isocitrate dehydrogenase increased following ovariectomy in old rats (85 weeks). Hormone-mediated induction of enzyme activity was actinomycin D sensitive. The Km for isocitrate and NADP, Ki value for oxalomalate, heat stability and electrophoretic mobility of the purified enzyme from the cytosol fraction of the liver of immature and old rats were similar. It can he concluded that the enzyme does not change structurally with age. Part of this work was presented at the 48th Annual General Meeting of the Society of Biological Chemist, India, 1979.     

Adenosine triphosphatase activity in the osmoregulatory organs of vertebrates

Studies have been made on the activity of cation- and anion-stimulated ATPases, as well as succinic dehydrogenase in homogenates and subcellular fractions from osmoregulatory organs of marine (elasmobranch and teleost) and freshwater (teleost) fishes, amphibians, reptiles, birds and mammals. The activity of Na+, K+-ATPase was found to be rather similar in almost all osmoregulatory organs of the species investigated. The highest level of Cl-stimulated ATPase was found in microsomal fraction of the kidneys from birds and mammals. Succinic dehydrogenase activity is significantly higher in the renal tissue of mammals, both in total homogenates and in mitochondrial fraction.     

Effects of chronic cold exposure on the endothelin system.

Cold temperatures have adverse effects on the human cardiovascular system. Endothelin (ET)-1 is a potent vasoconstrictor. We hypothesized that cold exposure increases ET-1 production and upregulates ET type A (ETA) receptors. The aim of this study was to determine the effect of cold exposure on regulation of the ET system. Four groups of rats (6-7 rats/group) were used: three groups were exposed to moderate cold (6.7 +/- 2 degrees C) for 1, 3, and 5 wk, respectively, and the remaining group was maintained at room temperature (25 degrees C) and served as control. Cold exposure significantly increased ET-1 levels in the heart, mesenteric arteries, renal cortex, and renal medulla. Cold exposure increased ETA receptor protein expression in the heart and renal cortex. ET type B (ETB) receptor expression, however, was decreased significantly in the heart and renal medulla of cold-exposed rats. Cold exposure significantly increased the ratio of ETA to ETB receptors in the heart. An additional four groups of rats (3 rats/group) were used to localize changes in ETA and ETB receptors at 1, 3, and 5 wk of cold exposure. Immunohistochemical analysis showed an increase in ETA, but a decrease in ETB, receptor immunoreactivity in cardiomyocytes of cold-exposed rats. Increased ETA receptor immunoreactivity was also found in vascular smooth muscle cells of cold-exposed rats. Cold exposure increased ETA receptor immunoreactivity in tubule epithelial cells in the renal cortex but decreased ETB receptor immunoreactivity in tubule epithelial cells in the renal medulla. Therefore, cold exposure increased ET-1 production, upregulated ETA receptors, and downregulated ETB receptors.     

Subcellular localization of renin and kallikrein in rat kidney.

The subcellular localization of renin and kallikrein in rat kidney cortex homogenate was investigated using both differential and density gradient centrifugation techniques. Highest specific activity of renin was found in the heavy mitochondrial fraction. Mitochondrial localization of renin was further supported by the behaviour of succinic dehydrogenase. By differential centrifugation, highest specific activity of kallikrein was found in the light mitochondrial fraction, while by density gradient centrifugation kallikrein was almost completely recovered in the lysosomal fraction. Lysosomal localization of kallikrein is further supported by the behaviour of acid phosphatase. The different subcellular localizations of renin and kallikrein are confirmed and the suggestion that kallikrein is located in the lysosomes is advanced.