低温暴露和恢复对棘胸蛙雌性亚成体生存力及能量物质消耗的影响

棘胸蛙(Paa spinosa)亚成体在人工驯养过程中容易出现越冬困难,非正常的冬眠可能会影响次年的繁育。以雌性棘胸蛙亚成体(1—2龄)为对象,研究该蛙在人工低温暴露(4℃保持90 d)条件下的生存力、机体能量物质消耗、肥满度、脏器系数的变化特征,以及这些参数在温度恢复至正常(由4℃缓慢升至22℃后保持7 d)后的变化情况。结果表明,该蛙在低温暴露过程中存活率逐渐降低,恢复期无死亡。肥满度(K)和体重/体长(Kwl)在低温暴露期间有逐渐升高的趋势,但两者在经历恢复期(22℃,7 d)后均恢复至初始水平(P>0.05)。胃系数和脾系数在低温暴露期呈明显的上升趋势(P<0.05),且两者在第90天均显著大于初始水平(P<0.05)。恢复期肝系数显著减小(P<0.05)。在低温暴露期各阶段肝脏和肌肉脂肪含量与初始无统计差异(P>0.05);肝脏水分在低温暴露期间呈明显下降趋势(P<0.05),而肌肉水分则与之相反;肝脏非脂肪干物质含量呈显著上升趋势(P<0.05),而肌肉非脂肪干物质则呈相反趋势。肝糖原含量随暴露时间的延长呈现显著上升趋势(P<0.05),低温暴露第60天和第90天肝糖原含量与初始相比分别增加59.4%和60.1%,而恢复期肝糖原含量则降至初始水平(P>0.05)。根据结果可以看出,在低温暴露过程中肝脏和肌肉脂肪含量变化不显著,同时肥满度、肝系数、肝脏非脂肪干物质和肝糖原含量均有不同程度的升高,而肌肉非脂肪干物质则显著减少(P<0.05),说明该蛙雌性亚成体在低温期主要消耗的能量物质不是脂肪而是肌肉非脂肪干物质,或者肌肉非脂肪干物质在组织间发生了大量转运。     

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.     

Effect of treatment with alpha-p-chlorophenoxyisobutyrate and of cold exposure on the distribution of lipids in hepatic mitochondria.

Administration of alpha-p-chlorophenoxyisobutyrate (0.25% in the diet) to rats increased the liver weight, hepatic contents of ubiquinone and mitochondrial protein with no effect on the sterols. The increase was progressive with the period of drug treatment and was potentiated by simultaneous cold exposure. Withdrawal of the drug treatment as well as the cold stress resulted in a return of the liver weight and mitochondrial content to normal levels but this was not so for the ubiquinone content. Treatment with alpha-p-chlorophenoxyisobutyrate with or without cold exposure also resulted in a small but significant increase in the mitochondrial lipids which could be accounted for completely by an increase in the phospholipids with no change in the neutral lipid content. Analysis of the individual phospholipids showed that the drug treatment per se resulted in a specific increase in phosphatidylethanolamine content whereas simultaneous cold exposure or cold per se showed an increase in phosphatidylcholine. Cardiolipin content was unaffected. Mitochondria isolated from drug-treated animals maintained at an ambient or low environmental temperature showed a small but significant decrease in the respiratory control index for the oxidation of glutamate and malate whereas the coupled oxidation rates and ADP/O ratios were normal. Such a feature was also observed in the animals exposed to short periods of cold stress without the drug treatment. In all the cases the oxidation of succinate was unaffected. The role of accumulated phospholipids in the mitochondrial membranes in drug treatment and cold exposure is discussed in relation to the possible involvement in increased thermogenesis.     

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.     

Effect of cold-induced hyperthyroidism on H2O2 production and susceptibility to stress conditions of rat liver mitochondria

Previous studies have shown that T3 treatment and cold exposure induce similar biochemical changes predisposing rat liver to oxidative stress. This suggests that the liver oxidative damage observed in experimental and functional hyperthyroidism is mediated by thyroid hormone. To support this hypothesis we investigated whether middle-term cold exposure (2 and 10 days), like T3 treatment, also increases H2O2 release by liver mitochondria. We found that the rate of H2O2 release increased only during State 4 respiration, but faster flow of reactive oxygen species (ROS) from mitochondria to the cytosolic compartment was ensured by the concomitant increase in tissue mitochondrial proteins. Cold exposure also increased the capacity of mitochondria to remove H2O2. This indicates that cold causes accelerated H2O2 production, which might depend on enhanced autoxidizable carrier content and should lead to increased mitochondrial damage. Accordingly, mitochondrial levels of hydroperoxides and protein-bound carbonyls were higher after cold exposure. Levels of low-molecular weight antioxidants were not related to the extent of oxidative damage, but susceptibility to both in vitro oxidative challenge and Ca2+-induced swelling increased in mitochondria from cold exposed rats. The cold-induced changes in several parameters, including susceptibility to swelling, were time dependent, because they were apparent or greater after 10 days cold exposure. The cold-induced increase in swelling may be a feedback mechanism to limit tissue oxidative stress, purifying the mitochondrial population from ROS-overproducing mitochondria, and the time course for such change is consistent with the gradual development of cold adaptation.     

Mitochondrial adaptations to steatohepatitis induced by a methionine- and choline-deficient diet

Nonalcoholic fatty liver disease (NAFLD) has become common liver disease in Western countries. There is accumulating evidence that mitochondria play a key role in NAFLD. Nevertheless, the mitochondrial consequences of steatohepatitis are still unknown. The bioenergetic changes induced in a methionine- and choline-deficient diet (MCDD) model of steatohepatitis were studied in rats. Liver mitochondria from MCDD rats exhibited a higher rate of oxidative phosphorylation with various substrates, a rise in cytochrome oxidase (COX) activity, and an increased content in cytochrome aa3. This higher oxidative activity was associated with a low efficiency of the oxidative phosphorylation (ATP/O, i.e., number of ATP synthesized/natom O consumed). Addition of a low concentration of cyanide, a specific COX inhibitor, restored the efficiency of mitochondria from MCDD rats back to the control level. Furthermore, the relation between respiratory rate and protonmotive force (in the nonphosphorylating state) was shifted to the left in mitochondria from MCDD rats, with or without cyanide. These results indicated that, in MCDD rats, mitochondrial ATP synthesis efficiency was decreased in relation to both proton pump slipping at the COX level and increased proton leak although the relative contribution of each phenomenon could not be discriminated. MCDD mitochondria also showed a low reactive oxygen species production and a high lipid oxidation potential. We conclude that, in MCDD-fed rats, liver mitochondria exhibit an energy wastage that may contribute to limit steatosis and oxidative stress in this model of steatohepatitis.     

Decreased activities of ubiquinol:ferricytochrome c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobalamin[c-lactam]-induced methylmalonic aciduria.

Rats treated with hydroxycobalamin[c-lactam] (HCCL), a cobalamin analogue that induces methylmalonic aciduria, have increased hepatic mitochondrial content and increased oxidative metabolism of pyruvate and palmitate per hepatocyte. The present studies were undertaken to characterize oxidative metabolism in isolated liver mitochondria from rats treated with HCCL. After 5-6 weeks, state 3 oxidation rates for diverse substrates are reduced in mitochondria from HCCL-treated rats. Similar reductions of mitochondrial oxidation rates are obtained with dinitrophenol-uncoupled mitochondria excluding defective phosphorylation as a cause for the observed decrease in mitochondrial oxidation. The activities of mitochondrial oxidases are reduced in HCCL-treated rats and demonstrate a defect in complex IV. Investigation of the complexes of the respiratory chain reveals a 32% decrease of ubiquinol:ferricytochrome c oxidoreductase (complex III) activity and a 72% decrease of ferrocytochrome c:oxygen oxidoreductase (complex IV) activity in mitochondria from 5-6-week HCCL-treated rats as compared with controls. Liver mitochondria from HCCL-treated rats also demonstrate decreased cytochrome content per mg of mitochondrial protein (25% decrease of cytochrome b and 52% decrease of cytochrome a + a3 as compared with control rats). The HCCL-treated rat represents an animal model for the study of the consequences of respiratory chain defects in liver mitochondria.     

Cold exposure differently influences mitochondrial energy efficiency in rat liver and skeletal muscle

This study deals with mitochondrial energy efficiency in liver and skeletal muscle mitochondria in 15 days cold exposed rats. Cold exposure strongly increases the sensitivity to uncoupling by added palmitate of skeletal muscle but not liver mitochondria, while mitochondrial energy coupling in the absence of fatty acids is only slightly affected by cold in liver and skeletal muscle. In addition, uncoupling protein 3 content does not follow changes in skeletal muscle mitochondrial coupling. It is therefore concluded that skeletal muscle could play a direct thermogenic role based on fatty acid-induced mild uncoupling of mitochondrial oxidative phosphorylation.     

Oxidative activity in mitochondria isolated from rat liver at different stages of development

The purpose of this study was to evaluate the oxidative capacities in hepatic mitochondria isolated from prepubertal, young adult and adult rats (40, 90 and 180 days of age, respectively). In these rats, mitochondrial respiratory rates using FAD- and NAD-linked substrates as well as mitochondrial protein mass were measured. The results show that only the oxidative capacity of FAD-linked pathways significantly declined in mitochondria from 180-day-old rats compared with those from younger animals. When we consider FAD-linked respiration expressed per g liver, no significant difference was found among rats of different ages because of an increased mitochondrial protein mass found in 180-day-old rats. However, when FAD-linked and lipid-dependent respiratory rates were expressed per 100 g body weight, significant decreases occurred in 180-day-old rats. Therefore, the decrease in liver weight expressed per 100 g body weight rather than an impaired hepatic cellular activity may be the cause of body energy deficit in 180-day-old rats. Copyright © 1998 John Wiley & Sons, Ltd.     

Organophosphorous plant growth regulator Melaphen: Resistance of plant and animal cells to stress factors

The addition of the organophosphorous plant growth regulator Melaphen (4 × 10^?12 M) to the incubation medium increases the maximum rate of oxidation of NAD-dependent substrates in rat liver and sugar beet root mitochondria. In addition, Melaphen stimulates electron transport during oxidation of succinate by rat liver mitochondria, but has no effect on the rate of this substrate oxidation in sugar beet root mitochondria. In storage organs of plants, the rate of oxidation of NAD-dependent substrates by mitochondria is relatively low. By stimulating the activity of NAD-dependent dehydrogenases, Melaphen stimulates energy metabolism in the cells and manifests adaptogenic activity by accelerating the germination of seeds. Melaphen does not influence the fluorescence of lipid peroxidation (LPO) products in mitochondria non-exposed to stress, but decreases 1.5–2 fold the LPO fluorescence in rat liver mitochondria exposed to cold stress and artificially “aged” sugar beet root mitochondria. Besides, Melaphen increases the rate of electron transport in a terminal site of respiratory chains of plant and animal mitochondria and decreases LPO. The data obtained testify to antistress activity of Melaphen.     

The effect of cold exposure on rat liver mitochondrial respiratory capacity.

1. The effect of cold exposure on the respiratory capacity of rat liver mitochondria has been studied using succinate as the substrate. 2. The mitochondria obtained in this study were well coupled, as shown by the RCR and ADP/O ratios. 3. In addition, durohydroquinone was used to eliminate the regulation of substrate supply. Likewise, we measured uncoupled respiration to evaluate the maximal electron flow through the respiratory chain. 4. We found that oxygen consumption using succinate or durohydroquinone + FCCP as substrates, as well as ATP production were not affected by cold exposure. 5. Our results also show that, when succinate is used, the maximal capacity of the respiratory chain is measured. 6. The data obtained do not support a role of the electron transport chain as a target of cold action.     

The effect of the prostaglandin analogue-misoprostol on rat liver mitochondria after chronic alcohol feeding

Rats fed ethanol (36% of total calories in a nutritionally adequate liquid diet) for 5 weeks develop functional alterations of hepatic mitochondria and steatosis of the liver. At the fatty liver stage, ADP-stimulated respiration of mitochondria was depressed in ethanol fed rats by 30% (p less than 0.001) with glutamate + malate and by 23% (p less than 0.001) with succinate as substrates. A similar decrease was noted in the respiratory control ratio (RCR) (34% and 29%, respectively). The total lipid content of the liver increased 2.6 fold (p less than 0.001). Mitochondrial dysfunction could be prevented, in part, by the treatment with a synthetic derivative of prostaglandin E1, misoprostol, at a mean daily dose of 80 micrograms/kg of body weight. The RCR with glutamate + malate as substrates was improved by 36% (p less than 0.05). We conclude that misoprostol attenuates several functional alterations in liver mitochondria during alcohol feeding.     

Adaptive thermogenesis in Brandt's vole (Lasiopodomys brandti) during cold and warm acclimation

Brandt's voles (Lasiopodomys brandti) exposed to cold (5±1 °C) or warm (23±1 °C) showed some physiological and biochemical variations which might be important in adaptation to their environments. Cold acclimation induced increases in resting metabolic rate (RMR) and the serum triiodothyronine (T₃) level, the state-4 respiration of liver and muscle mitochondria were activated after 7 days when animals exposed to cold, and the activity of cytochrome c oxidase (COX) of liver and muscle mitochondria tended to rise with cold exposure. RMR and T₃ level decreased during warm acclimation. The state-4 respiration of liver mitochondria declined after 3 days and muscle after 7 days when animals exposed to warm, and the activities of COX of liver and muscle mitochondria tended to decrease with warm acclimation. The cold activation of liver and muscle mitochondrial respiration (regulated by T₃) was one of the cytological mechanisms of elevating RMR. Both state-4 respiration and COX activity of brown adipose tissue (BAT) mitochondria increased significantly during cold acclimation and decreased markedly after acclimated to warm. The uncoupling protein 1 (UCP1) contents in BAT increased after exposure to cold and decreased after warm acclimation. Nonshivering thermogenesis (NST) plays an important role in the process of thermoregulation under cold acclimation for Brandt's voles. Changes in thermogenesis is a important way to cold adaptation for Brandt's voles in natural environments.     

Cardiolipin content is involved in liver mitochondrial energy wasting associated with cancer-induced cachexia without the involvement of adenine nucleotide translocase

Cancer-induced cachexia describes the progressive skeletal muscle wasting associated with many cancers leading to shortened survival time in cancer patients. We previously reported that cardiolipin content and energy-wasting processes were both increased in liver mitochondria in a rat model of peritoneal carcinosis (PC)-induced cachexia. To increase the understanding of the cellular biology of cancer cachexia, we investigated the involvement of adenine nucleotide translocator (ANT) in mitochondrial energy-wasting processes in liver mitochondria of PC and pair-fed control rats and its interactions with cardiolipin in isolated liver mitochondria from healthy rats exposed to cardiolipin-enriched liposomes. We showed in this study that functional ANT content was decreased in liver mitochondria from PC rats but without any effects on the efficiency of ATP synthesis. Moreover, non-phosphorylating energy wasting was not affected by saturating concentrations of carboxyatractylate (CAT), a potent inhibitor of ANT, in liver mitochondria from PC rats. Decreased efficiency of ATP synthesis was found in normal liver mitochondria exposed to cardiolipin-enriched liposomes, with increased non-phosphorylating energy wasting, thus mimicking mitochondria from PC rats. However, the functional ANT content in these cardiolipin-enriched mitochondria was unchanged, although non-phosphorylating energy wasting was reduced by CAT-induced inhibition of ANT. Finally, non-phosphorylating energy wasting was increased in cardiolipin-enriched mitochondria with substrates for complexes 1 and 2, but not for complex 4. In conclusion, increased energy wasting measured in liver mitochondria from rats with cancer cachexia is dependent on cardiolipin but independent of ANT. Interactions between ANT and cardiolipin are modified when cancer cachexia occurs.     

Role of the Ca2+ cycle in uncoupling of oxidative phosphorylation in liver mitochondria of cold-acclimated rats

Cold acclimation of Wistar rats for 2-4 weeks at about 3 degrees C resulted in an increased respiration rate and a reduced ADP/O ratio in liver mitochondria. With increasing duration of acclimation up to 10-12 weeks, these parameters returned to a normal level. The increase in the respiration rate and the decline of the mitochondrial ADP/O ratio were associated with a significant activation of the electroneutral release of Ca2+. When the animals were acclimated for 10-12 weeks the rate of Ca2+ release reduced to control values. The addition of 1 microM ruthenium red resulted in a decrease in the rates of mitochondrial respiration in control and cold-acclimated rats to approximately equal values and in a partial restoration of the ADP/O ratio in liver mitochondria of rats kept in the cold for 2-4 weeks. The respiratory activity of mitochondria isolated in the presence of 1 mM EGTA unaffected by ruthenium red.     

The oxidation of different substrates by liver mitochondria from rat pups in early postnatal development

We have studied the respiratory ratio of liver mitochondria during oxidation of substrates of the carbohydrate and lipid metabolism in newborn rats and during early postnatal development. High rate of respiration coupled with the synthesis of ATP from ADP and phosphate has been found during oxidation of carbohydrate substrates (pyruvate + malate); however, caprilate, a substrate of lipid metabolism, does not support such respiration. However, in the young rats aging from 2 to 30 days utilization of carbohydrate and lipid substrates via the phosphorylating pathway proceeds with similar efficacy.     

Effects of short-term ozone exposure on lung mitochondrial oxidative and energy metabolism

Ozone effects on lung mitochondrial oxidative metabolism were examined after short-term exposure of rats and monkeys to O₃. Exposure of animals to 2 ppm O₃ for 8 hr or to 4 ppm O₃ for 4 hr caused a 15–27% (P < 0.05) depression of lung mitochondrial O₂ consumption, using 2-oxoglutarate, succinate, and glycerol-1-phosphate. but not ascorbate plus Wurster's blue as substrates. Under these exposure conditions (4 ppm 4 hr) the ADP:O ratios dropped 25–36% (P < 0.05) and the respiratory control indices decreased 27–33% (P < 0.02) for oxidation of all substrates examined. Lung mitochondria from control animals were relatively impermeable to added NADH, but those from O₃-exposed animals showed an increased permeability as judged from NADH oxidation at a rate 3-fold higher than the control. Likewise, added cytochrome c caused a 22% (P < 0.01) stimulation of succinate oxidation in exposed lung mitochondria as against 5% (nonsignificant) in controls. Ozone exposure also caused a 20% (P < 0.01) oxidation of thiol groups in lung mitochondria, but no lipid peroxidation products were detectable in O₃-exposed lung tissue. The depression of substrate utilization, coupled phosphorylation and respiratory control observed in lung mitochondria of O₃-exposed animals might be related to alteration of membrane permeability, and inhibition of respiratory enzymes (dehydrogenases) due to oxidation of functional thiol groups.     

Nutritional effects on mitochondrial bioenergetics. Alterations in calcium uptake by rat liver mitochondria

The uptake of Ca2+ by energized liver mitochondria was compared in normal fed as well as in protein-energy malnourished rats. In the presence of phosphate, mitochondria obtained from both groups were able to accumulate Ca2+ from the suspending medium and eject H+ during oxidation of common substrates which activate different segments of the respiratory chain. The rate of Ca2+ uptake was significantly lower in mitochondria from protein-energy malnourished rats. The rates of oxygen consumption and H+ ejection were decreased by 20-30% during oxidation of substrates at the three coupling sites. Similarly, mitochondria from protein-energy malnourished rats exhibit a 34% decrease in the maximal rate of Ca2+ uptake and a 25% lower capacity for Ca2+ load. The stoichiometric relationship of Ca2+/2e- remained unaffected. In steady state, with succinate as a substrate in the presence of rotenone and N-ethylmaleimide, mitochondria from normal fed and protein-energy malnourished rats showed a similar rate of Ca2+ uptake. Furthermore in both groups the stoichiometry of the H+/O ratio was close to 8.0 (H+/site ratio close to 4.0), and of Ca2+/site was close to 2.0. The diminished rate of Ca2+ uptake observed in mitochondria from protein-energy malnourished rats could be explained on the basis of a depressed rate of electron transport in the respiratory chain rather than by an effect at the level of the Ca2+ or H+ transport mechanism per se.     

Hepatic selective adjustments in short-term cold exposed rats.

The metabolic adjustments which occur in rat liver cells during the first days of cold exposure have been investigated. We have measured both mitochondrial mass and oxidative capacities as well as ATP production after five and 10 days of cold exposure. In addition, we have measured plasma membrane Na+/K(+)-ATPase activity. Cold exposure elicited a significant increase in mitochondrial mass as well as in state 3 oxidative rates and ATP production in isolated mitochondria using various substrates. Moreover, our results show that Na(+)-pumping activity significantly increased after both five and 10 days of cold exposure. Our results suggest that during the first days of cold exposure liver cells undergo alterations which are useful for survival in the cold.     

Differential response of rat brown and white adipose tissue to environmental or nutritional stress.

1. In vivo fatty acid synthesis by brown adipose tissue was enhanced in rats exposed to cold (5 degrees C) or altitude (4300 m) for 7 days but was unaltered in rats exposed to heat (35 degrees C) for an equivalent period. In vivo fatty acid synthesis by white adipose tissue was depressed by cold exposure while altitude and heat exposure had no effect. 2. In vitro, CO2 production and lipid synthesis were elevated in brown adipose tissue from rats fasted for 4 days. Refeeding (4 days) such rats reversed these effects, leading to depressed values relative to those of control rats. In contrast, these metabolic events in white adipose tissue were decreased by fasting and increased compared to controls during subsequent refeeding.