Oxidation Of NAD-dependent Substrates Research Articles

Blockade of L-type calcium channels alters hepatic mitochondrial function in insulin-resistant rats

Mitochondria are central organelles in maintaining energy and oxidative homeostasis. Despite intensive research, the function of mitochondria and the mechanisms of its regulation under physiological conditions and in insulin resistance require detailed investigation. The aim of this study was to investigate the effect of blockade L-type calcium channels in insulin-resistant rats on hepatic mitochondrial oxidative function and changes in its oxidative status. Insulin resistance was modeled in 6-month-old male Wistar rats by 14 days of high-fat feeding. Standard fed animals served as controls. Verapamil was administered intraperitoneally at a dose of 1 mg/kg to block L-type calcium channels. Indicators of pro- and antioxidant systems (active products of tiobarbituric acid, reduced glutathione, catalase, Cu,Zn-superoxide dismutase) were assayed in the liver homogenate extracted from anesthetized animals after 3 h. Mitochondrial function was studied by the Chance polarographic method using different metabolic substrates. It was shown that in intact animals blockade of L-type calcium channels reduced the efficiency of mitochondrial respiration (V3/V4) in liver mitochondria during oxidation of all substrates through an inhibitory effect on the phosphorylation respiration (V3) and a stimulatory effect on the controlled respiration (V4), without affecting the oxidative status of the liver. In rats with insulin resistance the rate of V3 during oxidation of both NAD- and FADdependent substrates was decreased, violations of oxidative status and increased antioxidant protection were detected. However, in insulin-resistant rats blockade of L-type calcium channels significantly enhanced basic respiration (V2) during NAD-dependent substrate oxidation, V3 and V4 during palmitoyl lipid substrate oxidation, reduced the V3/V4 ratio compared to control, and partially or fully restored the violation of the oxidative status. This may indicate the involvement of calcium mechanisms in the disturbance of the oxidative status of the liver and the regulation of energy metabolism in mitochondria during insulin resistance.

Тетранитрозильный комплекс железа с тиосульфатными лигандами предотвращает дисфункцию митохондрий в условиях стресса

The effect of stress (water deficiency, high-temperature stress) and nitric oxide donor sodium μ2-dithiosulphate-tetranitosyldiferrate tetrahydrate Na2 [Fe2 (S2O3)2 (NO)4]2 × 4H2O (TNIC-thio) on the fatty acid composition and bioenergetic characteristics of 5-day etiolated pea seedling mitochondria was studied. Stressful effects caused the activation of LPO in the mitochondrial membranes. At the same time, significant changes occurred in the content of C18 and C20 fatty acids (FA). A decrease in the content of linoleic and linolenic acids, one of the main FA components of cardiolipin in higher plants, apparently caused a decrease in the maximum rates of oxidation of NAD-dependent substrates. The. treatment of pea seeds with 10-6M TNIC-thio prevented the activation of LPO, changes in the fatty acid composition of mitochondrial membranes, and contributed to the preservation of the bioenergetic characteristics of these organelles. By preventing the decline in energy metabolism, TNIC-thio probably has adaptogenic properties, that were also reflected in physiological parameters, namely, the growth of seedlings. Treatment of pea seeds and seedlings with the studied preparation prevented inhibition of root and shoot growth in conditions of water deficiency. Based on the data obtained, it can be concluded that the protective properties of TNIC-thio are due to its antioxidant activity.

Effect of High Temperature on Oxidation of NAD-Dependent Substrates and Alternative Oxidase Activity in Mitochondria of Lupine Cotyledons

Effect of High Temperature on Oxidation of NAD-Dependent Substrates and Alternative Oxidase Activity in Mitochondria of Lupine Cotyledons

Tissue-Specific Peculiarities of Vibration-Induced Hypoxia in Rabbit Liver and Kidney.

Activity of the energy production systems in rabbit liver and kidney under conditions of unfavorable vibration exposure was studied by the polarography method using a galvanic-type closed oxygen sensor. The rate of oxidation of endogenous substrates by mitochondria was determined by the tissue and was 5.2±0.6 and 8.13±1.4 (ng-atom O)×min-1×mg-1 protein for liver and kidney of intact animals, respectively. After 21 vibration sessions against the background of inhibition of NAD-dependent substrate oxidation in liver mitochondria, the rate metabolism of exogenous succinic acid increased by 44% and then decreased with prolongation of the effect, which indicated impaired function of the respiratory chain. Similar fluctuations of the parameters were revealed in kidney mitochondria, though their amplitude was lower. The study of bioenergetic mechanisms of hypoxia in various tissues makes it possible to determine the targets for the pharmacological action of antihypoxic drugs.

Substrate-Specific Reduction of Tetrazolium Salts by Isolated Mitochondria, Tissues, and Leukocytes.

Tetrazolium salts are commonly used in cytochemical and biochemical studies as indicators of metabolic activity of cells. Formazans, formed by reduction of tetrazolium salts, behave as pseudo-solutions during initial incubation, which allows monitoring their optical density throughout incubation. The criteria and conditions for measuring oxidative activity of mitochondria and dehydrogenase activity in reduction of nitroblue tetrazolium (NBT) and methyl thiazolyl tetrazolium (MTT) in suspensions of isolated mitochondria, tissue homogenates, and leukocytes were investigated in this work. We found that the reduction of these two acceptors depended on the oxidized substrate - NBT was reduced more readily during succinate oxidation, while MTT - during oxidation of NAD-dependent substrates. Reduction of both acceptors was more sensitive to dehydrogenase inhibitors that to respiratory chain inhibitors. The reduction of NBT in isolated mitochondria, in leukocytes in the presence of digitonin, and in liver and kidney homogenates was completely blocked by succinate dehydrogenase inhibitors - malonate and TTFA. Based on these criteria, activation of succinate oxidation was revealed from the increase in malonate-sensitive fraction of the reduced NBT under physiological stress. The effect of progesterone and its synthetic analogs on oxidation of NAD-dependent substrates by mitochondria was investigated using MTT. Both acceptors are also reduced by superoxide anion; the impact of this reaction is negligible or completely absent under physiological conditions, but can become detectable on generation of superoxide induced by inhibitors of individual enzyme complexes or in the case of mitochondrial dysfunction. The results indicate that the recording of optical density of reduced NBT and MTT is a highly sensitive method for evaluation of metabolic activity of mitochondria applicable for different incubation conditions, it offers certain advantages in comparison with other methods (simultaneous incubation of a large set of probes in spectral cuvettes or plates); moreover, it allows determination of activity of separate redox-dependent enzymes when selective inhibitors are available.

The effects of phenolic microbial metabolites on the activities of mitochondrial enzymes

The effects of microbial phenolic metabolites on the activities of enzymes of the tricarboxylic acid cycle were investigated in isolated mitochondria. The detection of metabolites of the tricarboxylic acid cycle in the blood of patients with sepsis as potential biomarkers of mitochondrial dysfunction was investigated. We found that microbial phenolic metabolites possess an inhibitory effect on the activity of dehydrogenases, as determined by the reduction of dichlorophenolindophenol and nitroblue tetrazolium in liver mitochondria and liver homogenates. The effect was more pronounced in the case of the oxidation of NAD-dependent substrates than succinate oxidation, as well as at lower concentrations of microbial metabolites than in the case of inhibition of respiration. Using gas chromatography coupled with mass spectrometry it was shown that the content of the tricarboxylic acid cycle metabolites is lower in the blood of patients with sepsis as compared to healthy donors. Our data demonstrate that microbial phenolic acids can significantly contribute to mitochondrial dysfunction and to metabolic suppression, both of which are characteristic of these pathologies.

Фазові зміни енергетичного обміну при адаптації до іммобілізаційного стресу

In stress, it was showed the organ and tissue changes associated with damage by lipid peroxides, and the disrupted barrier function. As a consequence, it was to lead to a syndrome of "stress-induced lung" and violation of oxygen delivery to the tissues and hypoxia. Purpose of the study was to investigate the dynamics of changes in gas exchange, blood glucose, body temperature, oxidant and antioxidant system activity, as well as mitochondrial respiration by Chance under the influence of chronic stress (6-hour immobilization daily for 3 weeks). It was identified 4 phase changes of energy metabolism in the dynamics of chronic stress. In the first phase, hypomethabolic, instability oxidative metabolism, decreased oxidation of NAD-dependent substrates, significant elevation of FAD-dependent substrates oxidation and low MRU were found. The activity of superoxide dismutase (MnSOD) was increased; it was occurred on a background low activity of glutathione peroxidase, and of misbalanced antioxidant system. After seven immobilizations, second phase-shift in energy metabolism, was observed, and then the third phase (hypermetabolic) started. It was characterized by gradual increase in oxidative metabolism, the restoration of oxidation of NAD-dependent substrates, MRU, as well as optimizing balance of oxidant and antioxidant systems. The fourth phase was started after 15 immobilizations, and characterized by the development of adaptive reactions expressed in increased tolerance of energy metabolism to the impact of immobilization. The results are correlated with changes in the dynamics of blood corticosterone. Thus, it was found the phase character of the energy metabolism rebuilding during the chronic stress.

Стан і способи фармакологічної корекції киснезалежних процесів у тканинах пародонта при тривалому іммобілізаційному стресі

Influence of prolonged immobilization (6 h strict horizontal position of rats in the tight containers daily for 2 weeks) on oxygen tension, oxygen consumption, pro-/antioxidant balance, and energetic metabolism of soft and hard periodontal tissues has been investigated. It was established that prolonged immobilization stress resulted in marked decrease in the gum tissue PO2 (36%) and in the bone tissue oxygen consumption rate (46%) compared to control. It was also determined that prolonged stress led to a reduction in the gum mitochondrial respiration rate. The latter was more expressed in case of the NAD-dependent substrate oxidation than of the FAD- dependent one. It was determined that the prolonged stress results in intensification of peroxide processes and depletion of antioxidant protection of soft tissues of periodontum. It was found that Thiotriazolin and Actovegin have modified and diminished stress-induced disorders in the soft and hard periodontal tissues oxygen homeostasis under prolonged immobilization stress.

Fatty acid composition of membrane lipids and energy metabolism in mitochondria of pea seedlings under water deficit

The impacts of water deficit and melamine salt of bis(oximethyl)phosphonic acid (melaphen) on the fatty acid (FA) composition of membrane lipids and energy metabolism in mitochondria of 5-day-old pea (Pisum sativum L., cv. Flora-2) seedlings were studied. Insufficient watering resulted in the accumulation of saturated and a decrease in the content of unsaturated FAs with 18 and 20 carbon atoms. Seed treatment with 3 × 10−10 M melaphen prevented these changes in the FA composition in the mitochondrial membrane lipids. Changes in the FA compositions of membrane lipids were correlated with changes in energy metabolism in mitochondria: the efficiency of oxidative phosphorylation and the rate of NAD-dependent substrate oxidation in the presence of ADP and FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) were reduced. A close correlation was observed between changes in the highest rates of NAD-dependent substrate oxidation and the relative content of FAs with 18 (r = 0.76489) and 20 (r = 0.9637) carbon atoms. The regulatory role of C18 and C20 unsaturated FAs in the mitochondrial energy metabolism of pea seedlings is discussed.

P 9: Taurine protects against impairment of mitochondrial function and oxidative stress in the heart of rats with fructose-induced insulin resistance

Background and aims Taurine (2-aminoethanesulfonic acid) is a free amino acid found in substantial amounts in all mammalian tissues. Recently, data is accumulating that show the effectiveness of T against diabetes mellitus, insulin resistance and its complications, including retinopathy, nephropathy, neuropathy, atherosclerosis and cardiomyopathy, independent of hypoglycemic effect. Numerous experimental and several clinical studies demonstrated that T helps the cardiovascular system through a variety of mechanisms including modulation of intracellular calcium concentration, antagonism of angiotensin II action, membrane-stabilizing, antioxidant, and lipid-lowering effects. The aim of the study was to assess the effects of T on mitochondrial respiratory chain activity and lipid peroxidation in the heart of prediabetic rats fed a high-fructose diet. Materials and Methods Male Wistar rats were divided into three groups: the control group fed on a regular diet (C, n=8), the high fructose-fed group (F, n=8), which had free access to 250 g/L solutions of fructose for 8 weeks and the fructose-fed group treated with taurine (F+T, n=8) for 8 weeks (100 mg/kg/day per os). Mitochondria were isolated by differential centrifugation from the hearts of fed rats. Oxygen consumption rate was measured polarographically at 37°C using a Clark-type oxygen electrode with either glutamate/malate or succinate as energy substrates of complex I or II, respectively. Levels of lipid hydroperoxides, reduced glutathione (GSH) and glutathione reductase (GR) activity were determined in mitochondrial preparations. Result We found reduction of NAD-dependent substrate oxidation rate in metabolic state 3 in the heart mitochondria of rats with fructose-induced insulin resistance by 30% in comparison with intact control (p Conclusion These results demonstrate that taurine protects against impairment of respiratory chain activity complex I and attenuates oxidative stress in the heart mitochondria of rats with fructose-induced insulin resistance. Thus, this action of taurine could be an important mechanism for providing benefits to the cardiovascular system in patients with insulin resistance and type 2 diabetes.

Тривала адаптація щурів до гіпобаричної гіпоксії попереджує стресорну гіперглікемію та оптимізує функціональний стан мітохондрій при гострій гіпоксії

Oxygen consumption, glucose blood level and liver mitochondrial respiration were investigated in male Wistar rats permanently living in middle altitude (2100 m, Elbrus region). The animals were characterized by reduced body oxygen consumption and blood glucose level, as well as by intensified utilization of NAD-dependent substrates in mitochondrial respiratory chain with increasing indices of ADP-stimulated respiration in comparison with plains rats. As a result of adaptive rebuilding of oxidative metabolism in rats--inhabitants of midlands, the nature and severity of metabolic responses to acute hypoxia were also changed. After lifting in barochamber to a "height" of 5600 m during 3 hours, plains rats transiently demonstrated hypometabolic and hyperglycemic reactions. A rapid adaptation of mitochondrial function occurred due to increase in the rate of FAD-dependent substrate oxidation accompanied by a decrease in the effectiveness of phosphorylation. In midland rats, by contrast, hypoglycemic reaction was developed, and further reduction of aerobic metabolism was limited. Rapid adaptation of mitochondrial function to acute hypoxia in those rats was more intense than in the plains animals. This was achieved by a significant increase in the rate of NAD-dependent substrate oxidation, especially lipids, and an improved efficiency of mitochondrial respiration and an increased economy of oxygen utilization.

Role of mitochondrial thiols of different localization in the generation of reactive oxygen species

The role of thiols of the outer and the inner membranes of mitochondria in the regulation of generation of reactive oxygen species (ROS) has been studied. It was found that N-ethylmaleimide (NEM), which penetrates through the mitochondrial membrane and binds thiols to form thioesters, at concentrations from 20 to 250 μM activates the production of superoxide anion and hydrogen peroxide during the oxidation of the substrates of complexes I and II of the respiratory chain. 5′,5′-Dithiobis-(2-nitrobenzoate) (DTNB), which does not penetrate into mitochondria and binds thiols to form disulfides, weakly activates hydrogen peroxide production during the oxidation of NAD-dependent substrates and inhibits the ROS production upon succinate oxidation. DTNB is particularly effective in inhibiting the menadione-induced formation of ROS. The differences in the ROS formation by these reagents are explained by the fact that they influence different thiol-containing proteins and enzymes. As distinct from NEM, which inhibits complex I of the respiratory chain, DTNB has no effect on the respiratory chain of mitochondria but can bind the SH-groups of NADH-quinone oxidoreductase, which is localized in the outer mitochondrial membrane and participates in the redox cycle of menadione. It was also shown that the ability to inhibit the ADP-stimulated respiration, a feature inherent in both reagents, does not significantly contribute to ROS production.

Fatty acid composition of mitochondrial membranes of pea germs exposed to insufficient watering and treated with organophosphorous plant growth regulator

The influence of insufficient watering and a melamine salt of bis(oxymethyl)-phosphonic acid (melaphen) on fatty acid composition of mitochondrial membrane of pea seedlings has been studied. It is shown that insufficient watering results in a 1.57-fold decrease in the ratio of unsaturated to saturated C18 fatty acids and in a 3.04-fold decrease in the ratio of unsaturated to saturated C20 fatty acids. The modification of fatty acid composition of mitochondrial membranes was associated with the alterations in their functional state: maximal rates of oxidation of NAD-dependent substrates and the rates of electron transport at the end of respiratory chain decreased. A preliminary treatment of the seeds with 3 × 10−9 M melaphen restored the maximal rates of oxidation of NAD-dependent substrates to the control values. We suggest that the alterations of the electron transport rates in respiratory chain in mitochondria are related to the physicochemical state of the membranes of these organelles, as the treatment with melaphen prevented the changes in fatty acid composition of the membranes of seedlings growing under the conditions of insufficient watering.

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.

Toxic effects of microbial phenolic acids on the functions of mitochondria

Low-molecular-weight phenolic acids (PhAs) phenylacetate, phenyllactate, phenylpropionаte, р-hydroxyphenyllactate, and p-hydroxyphenylacetate are essentially the products of the degradation of aromatic amino acids and polyphenols by the intestinal microflora. In sepsis, the concentrations of some of these acids in the blood increase tens of times. Assuming that these compounds can cause the mitochondrial dysfunction in sepsis, we examined their effects on respiration, the induction of pore opening, and the production of reactive oxygen species (ROS) in mitochondria. It was found that phenylpropionаte and phenylacetate produce a more toxic effect on mitochondria than the other phenolic acids. At concentrations 0.01–0.1 mM they decreased the rate of oxidation of NAD-dependent substrates and activated the Ca 2+- and menadione-induced opening of the cyclosporin A-sensitive pore and the production of ROS. The disturbances caused by these PhAs are similar to those observed in mitochondria in sepsis, and hence the rise in their level may be one of the causes of mitochondrial dysfunctions. Phenyllactate, р-hydroxyphenyllactate, and p-hydroxyphenylacetate inhibited the production of ROS and pore opening, acting as antioxidants. Thus, the ability of PhAs to affect the mitochondrial functions, as well as an increase in their concentrations in sepsis (the total concentration of these PhAs in the blood is close to 0.1 mM), suggests that PhAs can be directly involved in the development of mitochondrial failure.

Peculiarities of functioning of liver mitochondria of the river lamprey Lampetra fluviatilis and the common frog Rana temporaria at periods of suppression and activation of energy metabolism

The work dealt with study of mitochondria in reversible metabolic suppression of hepatocytes of the river lamprey Lampetra fluviatilis in the course of prespawning starvation and of liver mitochondria of the common frog Rana temporaria during hibernation and activity. In winter the metabolic depression of lamprey hepatocytes, unlike that of frog hepatocytes, has been found to be due to deactivation of complex I of the electron transport mitochondrial chain, a low rate of NAD-dependent substrate oxidation, a low content of adenine nucleotide content, and a high degree of mitochondrial membrane permeability to H+ and other monovalent ions (KCl-, K+). The mitochondrial membrane permeability decreases in the presence of ethyleneglycoldiamineethyltetraacetic acid (EGTA), cyclosporine A (CsA), adenosine-5'-diphosphate (ADP), and Mg+. These facts indicate the presence in these mitochondria of the Ca2+ -dependent unspecific pore in the low-conductance state. Histological studies showed the lamprey and the frog to have principal differences in use of energy substrates at the period of metabolic depression. Lampreys utilize predominantly lipids, whereas frogs--glycogen. The clearly pronounces activation of lipid consumption is observed at the spring period before spawning and death of lamprey. Possible causes of metabolic depression are discussed as well as similarity and difference in behavior of mitochondria of cyclostomes and amphibians throughout metabolic depression and activity.

Effect of Melafen on functional efficiency of mitochondrial membranes from sugar beet taproots

Mitochondria were isolated from sugar beet (Beta vulgaris L) taproots and incubated in the presence of low concentrations of Melafen (2 × 10−9 and 4 × 10−12 M). This treatment of mitochondrial membranes induced an appreciable decrease in microviscosity of superficial lipids in the lipid bilayer and a parallel increase in microviscosity of the deeply immersed lipid regions adjacent to membrane proteins. Melafen had no effect on fluorescence of lipid peroxidation products in membranes of freshly prepared mitochondria but declined this fluorescence to control values in artificially aged mitochondria. Melafen raised the maximum rates for oxidation of NAD-dependent substrates, elevated the efficiency of oxidative phosphorylation, and activated electron transport in the terminal (cytochrome oxidase) step of mitochondrial respiratory chain, which implies the activation of energy metabolism within the cell. The acceleration of electron transport through the terminal step of mitochondrial respiratory chain was apparently accompanied by retardation of lipid peroxidation, which prevented impairment of mitochondrial membranes under stress conditions. A proposal is put forward that some properties of Melafen are favorable for adaptogenesis because its effects on mitochondrial energy metabolism depended on the functional state of mitochondria.

Isolation and characterization of tightly coupled mitochondria from wild type and nap mutant Neurospora crassa.

A fast and reproducible procedure was elaborated for isolation of tightly coupled mitochondria from wild type and nap-mutant Neurospora crassa cells harvested at different growth stages. The isolated mitochondrial preparations had controlled metabolic states and were tightly coupled, i.e., displayed good respiratory control and had close to the theoretically expected maximal ADP/O ratios upon oxidation of Krebs cycle intermediates and exogenous NADH. They contained the fully competent respiratory chain with all three points of energy conservation. Oxidation of all examined substrates by mitochondria from both wild type and mutant cells was mediated by two alternative terminal oxidative systems, albeit to varying extent, with the more pronounced engagement of the alternative oxidase in the stationary growth phase and with a minor contribution of this non-phosphorylating pathway in the substrate oxidation by mutant mitochondria. Oxidation of NAD-dependent substrates by mitochondria from the two cell types was accommodated via both rotenone-sensitive and rotenone-insensitive pathways, while the level of rotenone-insensitive pathway in mutant cells was lower than in wild type cells. It is suggested that a more limited contribution of alternative non-phosphorylating oxidative pathways to the total respiration in mutant cells, as compared with wild type cells, could, at least partially, explain an elevated ATP level in these cells. However, the absence of principal differences in the arrangement of the respiratory chain in mitochondria of wild type and mutant cells implies that the elevated ATP level in the nap mutant is largely related to reduced ATP expenses for transport processes in these cells.

Inactivation of complex I of the respiratory chain of maize mitochondria incubated in vitro by elevated temperature

The functional stability of the ‘external’ NADH dehydrogenase and complexes I–IV of the respiratory chain of maize mitochondria was studied during mitochondria incubation in vitro at elevated temperatures. The increase in the incubation temperature from 0°C to 37°C significantly changed the stability of the respiratory chain. At 27°C and higher, the rate of oxidation of NAD-depended substrates decreased drastically, which is related to inactivation of complex I. Complexes II, III and IV of the respiratory chain and the ‘external’ NADH dehydrogenase were functionally stable at elevated temperatures. Moreover, the possibility of electron transport during oxidation of NAD-dependent substrates, in particular malate, bypasses complex I using rotenon insensitive NADH dehydrogenase.

Qualitative difference in mitochondria of endothermic and ectothermic animals

A significantly higher rate of respiration in the absence of added ADP has been revealed in mitochondria of endotherms as compared with that of the ectotherms with similar rates of respiration during phosphorylation. A high rate of ADP-independent (non-coupled with ATP synthesis) respiration is observed during oxidation of succinate, NADH and ascorbate + cytochrome c, but not with NAD-dependent substrates. It increases in the presence of cytochrome c during oxidation of succinate and NADH and is also revealed during oxidation of NAD-dependent substrates in the presence ofNAD + and cytochrome c. ADP-independent respiration is not affected by oligomycin, however, it is essentially inhibited in the presence of GDP. It is suggested that the significant difference in the value of ADP-independent respiration of endo- and ectotherms is due to the existence in endotherms of a non-coupled population of mitochondria which generates heat without preliminary synthesis of ATP.