Thyroid-hormone control of state-3 respiration in isolated rat liver mitochondria.

Oxidative phosphorylation can be treated as two groups of reactions; those that generate protonmotive force (dicarboxylate carrier, succinate dehydrogenase and the respiratory chain) and those that consume protonmotive force (adenine nucleotide and phosphate carriers. ATP synthase and proton leak). Mitochondria from hypothyroid rats have lower rates of respiration in the presence of ADP (state 3) than euthyroid controls. We show that the kinetics of the protonmotive-force generators are unchanged in mitochondria from hypothyroid animals, but the kinetics of the protonmotive-force consumers are altered, supporting proposals that the important effects of thyroid hormone on state 3 are on the ATP synthase or the adenine nucleotide translocator.     

Participation of the adenine nucleotide translocator in the regulation of pyruvate oxidation in heart mitochondria

A regulatory role of adenine nucleotide translocator (ANT) was determined by titration of mitochondrial respiration (state 3) with carboxyatractyloside. It was shown that ANT regulates pyruvate oxidation: the control strength is more pronounced after depletion of endogenous substrates or after the increase in extramitochondrial ATP/ADP. The rate of succinate oxidation is controlled mainly by succinate dehydrogenase, while ANT does not participate in its regulation.     

On the thyroid hormone-induced increase in respiratory capacity of isolated rat hepatocytes.

The respiratory capacities of hepatocytes, derived from hypothyroid, euthyroid and hyperthyroid rats, have been compared by measuring rates of oxygen uptake and by titrating components of the respiratory chain with specific inhibitors. Thyroid hormone increased the maximal rate of substrate-stimulated respiration and also increased the degree of ionophore-stimulated oxygen uptake. In titration experiments, similar concentrations of oligomycin or antimycin were required for maximal inhibition of respiration regardless of thyroid state, suggesting that the changes in respiratory capacity were not the result of variation in the amounts of ATP synthase or cytochrome b. However, less rotenone was required for maximal inhibition of respiration in the hypothyroid state than in cells from euthyroid or hyperthyroid rats, implying that hepatocytes from hypothyroid animals contain less NADH dehydrogenase. The concentration of carboxyatractyloside necessary for maximal inhibition of respiration was 100 microM in hepatocytes from hypothyroid rats, but 200 microM and 300 microM in hepatocytes from euthyroid and hyperthyroid rats, respectively, indicating a possible correlation between levels of thyroid hormone and the amount or activity of adenine nucleotide translocase. The increased capacity for coupled respiration in response to thyroid hormone is not associated with an increase in the components of the electron transport chain or ATP synthase, but correlates with an increased activity of adenine nucleotide translocase.     

Rapid thyroid-hormone effect on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell.

The effect of thyroid-hormone application on cytosolic and mitochondrial ATP/ADP ratio was investigated in rat liver in vivo and in the isolated perfused organ. In vivo the ATP/ADP ratio in livers from hypothyroid rats was 0.84 +/- 0.08 in the mitochondrial matrix and 5.6 +/- 0.9 in the cytosol, as was observed in euthyroid controls. In contrast, hyperthyroidism was followed by a significant decrease in the mitochondrial and by an increase in the cytosolic ATP/ADP ratio (to 0.34 +/- 0.06 and 11.3 +/- 2.8 respectively). In the perfused liver from hypothyroid animals, addition of L-3,3',5-tri-iodothyronine in the perfusate also provoked, within 2 h, a significant decrease in the mitochondrial ATP/ADP ratio, whereas the cytosolic ratio was unaffected. From these and previous data in the isolated perfused liver and in isolated mitochondria from hypothyroid and tri-iodothyronine-treated rats it is concluded that thyroid hormones increase mitochondrial respiration and ATP regeneration, which is associated with an acceleration of mitochondrial adenine nucleotide transport and significant alterations in the mitochondrial and cytosolic ATP/ADP ratios.     

Direct action of T3 on phosphorylation potential in the sheep heart in vivo

Thyroid acting through ligand binding to nuclear receptors modifies myocardial respiratory kinetics and oxidative phosphorylation in the heart. Direct nongenomic action of thyroid hormone on high-energy phosphate concentrations and respiratory kinetics has never been proven in vivo but might be responsible for observed changes in oxygen utilization efficiency immediately after triiodothyronine (T3) administration. We tested the hypothesis that T3 directly and rapidly modifies myocardial high-energy phosphate concentrations and phosphorylation potential in vivo. Anesthetized sheep (age 28-40 days) thyroidectomized shortly after birth (Thy) and euthyroid age-matched controls (Con) underwent median sternotomy and received T3 infusion (0.8 microg/kg), followed by epinephrine infusion to increase myocardial oxygen consumption (MVo2). 31P magnetic resonance spectra were monitored via a surface coil over the left ventricle. T3 increased phosphocreatine (PCr)/ATP and decreased ADP in Thy animals without causing a change in MVo2. T3 produced no changes in high-energy phosphates in Con animals. T3 did not modify the PCr/ATP or ADP response to epinephrine and elevation in MVo2 in either group. Cardiac mitochondria isolated from Thy and Con animals showed no change in respiratory rate or ADP/ATP exchange efficiency after T3 incubation. T3 infusion in a hypothyroid state decreases ADP concentration, thereby altering the equilibrium between phosphorylation potential and myocardial respiratory rate. These T3-induced effects are not due to changes in ADP/ATP exchange efficiency through action at the adenine nucleotide translocator but may be due to T3 mediation of substrate utilization, confirmed in other models.     

The role of the adenine nucleotide translocator in oxidative phosphorylation. A theoretical investigation on the basis of a comprehensive rate law of the translocator

A minimum model of adenine nucleotide exchange through the inner membrane of mitochondria is presented. The model is based on a sequential mechanism, which presumes ternary complexes formed by binding of metabolites from both sides of the membrane. The model explains the asymmetric kinetics of ADP-ATP exchange as a consequence of its electrogenic character. In energized mitochondria, a part of the membrane potential suppresses the binding of extramitochondrial ATP in competition with ADP. The remaining part of the potential difference inhibits the back exchange of internal ADP for external ATP. The assumption of particular energy-dependent conformational states of the translocator is not necessary. The model is not only compatible with the kinetic properties reported in the literature about the adenine nucleotide exchange, but it also correctly describes the response of mitochondrial respiration to the extramitochondrial ATP/ADP ratio under different conditions. The model computations reveal that the translocation step requires some loss of free energy as driving force. The size of the driving force depends on the flux rate as well as on the extra- and intramitochondrial ATP/ADP quotients. By both quotients the translocator controls the export of ATP formed by oxidative phosphorylation in mitochondria.     

Control of mitochondrial oxidative phosphorylation

The objective of this investigation is to analyze the two following problems of the regulation of mitochondrial oxidative phosphorylation: what is the extramitochondrial parameter that controls ATP production according to the cytoplasmic demands and how the control is distributed between various mitochondrial enzymes. On the basis of the data of Groen et al. (1982) it is shown that as the respiration rates ranged over 30-50% of the maximum (i.e. within the physiological region) the contribution of the adenine nucleotide translocator to the control of the ATP flux is no less than 90%, referring to the total contribution of all mitochondrial enzymes as 100%. Founding on the key role of the adenine nucleotide translocator it has been concluded that besides the extramitochondrial [ATP]/[ADP] ratio the absolute ADP concentration is another extramitochondrial signal controlling significantly the rate of oxidative phosphorylation.     

Control of adenine nucleotide metabolism in hepatic mitochondria from rats with ethanol-induced fatty liver

Male rats developed fatty liver after being fed on an ethanol-containing diet for 31 days. Liver mitochondria from these animals catalysed ATP synthesis at a slower rate when compared with mitochondria from pair-fed control rats (control mitochondria), and demonstrated lowered respiratory control with succinate as substrate, owing to a decrease in the State-3 respiratory rate. Respiration in the presence of uncoupler was comparable in mitochondria from both groups of rats. Translocation of both ATP and ADP was decreased in mitochondria from ethanol-fed rats, with ADP uptake being lowered more dramatically by ethanol feeding. Parameters influencing adenine nucleotide translocation were investigated in mitochondria from ethanol-fed rats. Experiments performed suggested that lowered adenine nucleotide translocation in these mitochondria is not the result of inhibition of the translocase by either long-chain acyl-CoA derivatives or unesterified fatty acids. Analysis of endogenous adenine nucleotides in these mitochondria revealed lowered ATP concentrations, but no decrease in total adenine nucleotides. In experiments where the endogenous ATP in these mitochondria was shifted to higher concentrations by incubation with oxidizable substrates or defatted bovine serum albumin, the rate of ADP translocation was increased, with a linear correlation being observed between endogenous ATP concentrations and the rate of ADP translocation. The depressed ATP concentration in mitochondria from ethanol-fed rats suggests that the ATP synthetase complex is replenishing endogenous ATP at a slower rate. The lowered ATPase activity of the ATP synthetase observed in submitochondrial particles from ethanol-fed animals suggests a decrease in the function of the synthetase complex. A decrease in the rate of ATP synthesis in mitochondria from ethanol-fed rats is sufficient to explain the decreased ADP translocation and State-3 respiration.     

Relation between glutamate and adenine nucleotide levels of heart mitochondria during hypoxia

The effect of acute respiratory hypoxia in rats on mitochondrial respiration, adenine nucleotides and some amino acids of the heart was studied. The decrease in the total (ATP + ADP + AMP) and exchangeable (ATP + ADP) adenine nucleotide pool of the mitochondria was accompanied by a pronounced loss of state 3 respiration with glutamate plus malate and a slight decrease with succinate plus rothenone. The uncoupled respiration of mitochondria with glutamate and malate was decreased in the same degree as in the absence of 2,4-dinitrophenol. State 4 respiration with substrates of both types was unaffected by hypoxia. These data point to a hypoxia-induced impairment of complex I of the respiratory chain. The decrease of tissue and mitochondrial glutamate was accompanied by the elevation of alanine content in the heart and an increase in intramitochondrial aspartate. The ADP-stimulated respiration of mitochondria was correlated with mitochondrial glutamate and ATP as well as with exchangeable adenine nucleotide pools during hypoxia. The experimental results suggest that mitochondrial dysfunction induced by hypoxia may also be attributed to the low level of mitochondrial glutamate.     

Control of mitochondrial respiration. The contribution of the adenine nucleotide translocator depends on the ATP- and ADP-consuming enzymes

The consequence of the complexity of the metabolic network on the amount of control strength of adenine nucleotide translocator was investigated with isolated rat liver mitochondria. Two experimental systems were compared: (i) mitochondria in the presence of yeast hexokinase (hexokinase system) and (ii) the same system plus additional pyruvate kinase (pyruvate kinase system). In both systems the control strength was analysed for the adenine nucleotide translocator by inhibitor titration studies with carboxyatractyloside and for the hexokinase or pyruvate kinase by changing their relative activities. Experimental results were compared with computer simulation of these systems and that of a third one, where the extramitochondrial ATP / ADP ratio was held constant by perifusion (perifusion system). The results demonstrate quite different flux-dependent control strength of the translocator in the three systems. In the hexokinase system the control strength of the translocator on mitochondrial respiration was zero up to respiration rates of about 60 nmol O₂/mg protein per min. For higher rates, the control strength increased until the maximum value (0.45) was reached in the fully active state. Here, the same value was also found in the pyruvate kinase system. In all other states of respiration the translocator exerts a higher control strength in the pyruvate kinase system than in the hexokinase system. This different behaviour was attributed to the various changes in the adenine nucleotide pattern caused by partial inhibition of the translocator in the hexokinase and pyruvate kinase system. The data clearly show that the sharing of control strength depends not only on the respiration rate but also on the complexity of the metabolic system.     

Regulation of oxidative phosphorylation in mitochondria of epididymal bull spermatozoa

The regulation of oxidative phosphorylation was studied with digitonin-treated epididymal bull spermatozoa in which mitochondria are directly accessible to low molecular compounds in the extracellular medium. Due to the high extramitochondrial ATPase activity in this cell preparation, it was possible to stimulate respiration to a small extent only by added hexokinase in the presence of glucose and adenine nucleotides. Added pyruvate kinase plus phosphoenol pyruvate, however, strongly suppressed the respiration. Under these conditions, the respiration was found to depend on the extramitochondrial [ATP]/[ADP] ratio in the range of 1-100. The contribution of the adenine nucleotide translocator to this dependence was determined by titration with the irreversible inhibitor carboxyatractyloside in the presence of ADP. Using lactate plus malate as substrate, the active state respiration was controlled to about 30% by the translocator, whereas 12 and 4% were determined in the presence of L-glycerol-3-phosphate and malate alone, respectively. In order to compare the results with those for intact cells, the adenine nucleotide patterns were determined in intact and digitonin-treated spermatozoa under conditions of controlled respiration in the presence of vanadate and carboxyatractyloside, respectively. About 21% of total cellular adenine nucleotides were found in digitonin-treated cells representing the mitochondrial compartment. While allowing for the intramitochondrial amount of adenine nucleotides, the cytosolic [ATP]/[ADP] ratio was estimated to be 6-times higher than the mitochondrial ratio in intact cells. It is concluded from the data presented that the principal mechanism by which oxidative phosphorylation in sperm mitochondria is regulated via the extramitochondrial [ATP]/[ADP] ratio is the same as that demonstrated for other isolated mitochondria.     

Thyroid hormone effect on gene expression of the adenine nucleotide translocase in different rat tissues

The ADP/ATP transport across the mitochondrial membrane is achieved by the adenine nucleotide translocase (ANT), an integral inner mitochondrial membrane protein. As deduced from experiments in rat liver in vivo and in isolated rat liver mitochondria this ADP/ATP transport is accelerated by thyroid hormone application, thus explaining, at least to a considerable extent, the thyroid hormone mediated increase in mitochondrial metabolic activity. The present study investigates the effect of T3 on rat liver, heart, and kidney ANT gene expression. As shown by Northern blot analysis, a cDNA for beef heart ANT-mRNA showed cross-hybridization with the ANT-mRNA from rat heart, liver, and kidney. Hypo- and hyperthyroid rats showed no differences in size nor in amounts of heart, liver, and kidney ANT-mRNA. Measurement of heart ANT-protein level revealed no major differences among the various thyroid states. Thus, the long-term action of thyroid hormones on increasing the carrier-mediated ADP/ATP translocation cannot be ascribed to an effect of T3 on ANT gene expression. The mechanism by which T3 activates this transporter system remains to be identified but some possibilities are suggested.     

Properties of succinate oxidation in tomato fruit mitochondria

Mitochondria from tomato fruit (Lycopersicon esculentum Mill.) exhibited a respiratory control ratio of 2.5 and an ADP:O ratio of 1.3 for succinate oxidation for 24 hours after isolation. They also showed a delay in response to the first addition of ADP. The addition of ATP and ADP before succinate eliminated the delayed response as did chelation of endogenous cations with ethylenediaminetetraacetic acid. The addition of ATP after succinate resulted in a longer delay in response than that obtained with ADP. Exogenous oxaloacetate in low concentration inhibited respiration in states 3 and 4 with succinate and resulted in delayed response to ADP. The function of adenine nucleotide during the delay in response may be to promote the metabolism of oxaloacetate or to decrease the affinity of oxaloacetate to its site of inhibition.     

Short-term control of mitochondrial adenine nucleotide translocator by thyroid hormone

An improved simple technique for measuring adenine nucleotide translocator activity at low medium substrate concentrations is described. Confirming previous reports, thyroidectomy was shown to lead to lowered translocator activity in rat liver mitochondria. The rapidly exchangeable portion of the matrix nucleotide also decreased in hypothyroid preparations even though the total nucleotides increased substantially. The apparent Km of translocator for ADP increased from 2.8 to 6.2 microM in hypothyroid preparations: Mg2+ ions raised this to about 20 microM. All of these changes in adenine nucleotide translocation were entirely reversed by 15 min after a single intravenous near-physiological dose of triiodothyronine.     

Sigmoidal relation between mitochondrial respiration and log ([ATP]/[ADP])out under conditions of extramitochondrial ATP utilization. Implications for the control and thermodynamics of oxidative phosphorylation

Except for close to state 3, mitochondrial respiration has been observed to vary almost linearly with the extramitochondrial phosphorylation potential. For the understanding of the control, thermodynamics, and stoichiometries of oxidative phosphorylation, it is important if this linearity corresponds to an extension of a near-equilibrium flow-force relationship. Using three methods to determine the extramitochondrial ATP/ADP ratio, we observed that at high ATP/ADP ratios the relationship between respiratory rate and log (ATP/ADP) deviated in a sigmoidal fashion from linearity, if the amount of hexokinase present was modulated. In a titration with uncoupler, the sigmoidicity at high ATP/ADP ratios was absent. This difference between the flow-force relationships of these two experiments suggests that the sigmoidicity in the former case reflects a nonproportional flow-force relationship of the adenine nucleotide translocator. In the latter case, one measures the flow-force relationship of the redox-driven proton pumps alone, which turns out to be virtually linear. We determined the flow-force relation of the adenine nucleotide translocator for two ways of varying the force and confirmed the sigmoidicity in both cases. The implication is that the near-linearity of the flow-force relationships at intermediary respiratory rates does not correspond to an Onsager-type (near equilibrium) linearity. We discuss that this phenomenon requires the application of nonclassical forms of nonequilibrium thermodynamics and may be responsible for some of the control over oxidative phosphorylation that is exerted by the cytosolic ATP consuming processes.     

The hydrophobic cationic cyanine dye inhibits oxidative phosphorylation by inhibiting ADP transport, not by electrophoretic transfer, into mitochondria

The effect of the divalent cationic cyanine dye tri-S-C4(5) on oxidative phosphorylation in rat liver mitochondria was examined. The dye at about 100 n mols per mg mitochondrial protein inhibited state 3 respiration and ATP synthesis almost completely. However, it had no effect on submitochondrial particles, like other hydrophobic cations. The dye inhibited the transport of ADP into mitochondria mediated by the adenine nucleotide translocator. Thus, the inhibition of oxidative phosphorylation by the cationic dye was concluded to be due to its action on the adenine nucleotide translocator, not to its electrophoretic transfer into the inner space of mitochondria according to the inside-negative electrochemical potential.     

Effects of thyroid hormones on the bypasses of the antimycin A block in the bc1 complex of rat liver mitochondria.

The effect of thyroid hormones on the electron flow through the bc1 complex of rat liver mitochondria was studied using two dye bypasses of the Antimycin A block of the bc1 complex by the method of Alexandre and Lehninger (Biochim. Biophys. Acta 767:120; 1984). Bypass respiration rates with both DCIP (2,6-dichlorophenolindophenol) and TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride) were elevated in the hyperthyroid rats and depressed in the hypothyroid groups compared to the euthyroid controls. T3 treatment of hypothyroid rats returned the bypass rates to control levels in 24 hours with the TMPD dye but not for the DCIP. This further demonstrates that different portions of the bc1 complex respond individually to the thyroid state.     

Control of energy transformation of mitochondria. Analysis by a quantitative model

A mathematical model of control of energy transformation in mitochondria is presented. The considered processes are: the proton translocation by the respiratory chain, the production of ATP by ATPase, the translocation of adenine nucleotides and of phosphate by their translocators, and a passive backflow of protons through the mitochondrial membrane. The mathematical equations expressing the steady-state kinetics of these processes and the relations between them were derived on the basis of current experimental data. The model predicts fairly well the values of the proton electrochemical gradient, of the ATP/ADP ratios within and outside mitochondria and of the distribution of phosphate between both compartments in different metabolic states of mitochondria. From the general agreement of model computations with experimental data, it is suggested that the electron flux through the respiratory chain is immediately controlled by the energy back-pressure of the proton electrochemical gradient, that the ATPase reaction is near equilibrium in phosphorylating mitochondria but that the adenine nucleotide exchange across the mitochondrial membrane requires some loss of energy. The latter is caused by an inhibition of the translocator by ATP from the outer side or by ADP from the inner side depending on the actual ATP/ADP in both compartments. It explains that no fixed relation exists between the rate of respiration and the phosphorylation state of extramitochondrial adenine nucleotides. The relation is modified by the concentration of phosphate and by intramitochondrial energy utilization.     

The role of adenine nucleotide translocator in the regulation of oxidative phosphorylation in heart mitochondria

The regulatory role of adenine nucleotide translocase in oxidative phosphorylation was determined by titration of respiration of isolated rabbit heart mitochondria with carboxyatractyloside in the creatine phosphokinase ADP-regenerating system, which is not rate-limiting. It was found that the respiration rate is not controlled by adenine nucleotide translocase in states 3 and 4. Within the physiological region of respiration (30-70% of the maximal rate), the control coefficient for ADP/ATP translocase is 0.62-0.75. Thus, translocase plays a key role in the regulation of oxidative phosphorylation.     

Mitochondrial alterations in livers of Sod1-/- mice fed alcohol

Chronic alcohol consumption induced liver injury in Cu,Zn-superoxide dismutase-deficient mice (Sod1-/-), with extensive centrilobular necrosis and inflammation and a reduction in hepatic ATP content. Mechanisms by which ethanol decreased ATP in these mice remain unclear. We investigated alterations in mitochondria of Sod1-/- mice produced by chronic ethanol treatment. These mitochondria had an increase in State 4 oxygen consumption with succinate and especially with glutamate plus malate compared to mitochondria from pair-fed Sod1-/- mice or mitochondria from wild-type mice fed dextrose or ethanol. This uncoupling was associated with a decrease in ADP/O and respiratory control ratios, a decline in mitochondrial membrane potential, enhanced mitochondrial permeability transition, and decreased aconitase activity. Total thiols and uncoupling protein 2 levels were elevated in the pair-fed Sod1-/- mitochondria, perhaps an adaptive response to oxidant stress. However, no such increases were found with the ethanol-fed Sod1-/- mitochondria, suggesting a failure to develop these adaptations. The mitochondria from the ethanol-fed Sod1-/- mice had elevated levels of cleaved Bax, Bak, Bcl-xl, and adenine nucleotide translocator. Immunoprecipitation studies revealed increased association of Bax and Bak with the adenine nucleotide translocator. ADP-ATP exchange was very low in the ethanol-fed Sod1-/- mitochondria. These results suggest that ethanol treatment of Sod1-/- mice produces uncoupling and a decline in Deltapsi, swelling, increased association of proapoptotic proteins involved in the permeability transition, and decreased adenine nucleotide translocator activity, which may be responsible for the decline in ATP levels and development of necrosis in this model of alcohol-induced liver injury.