Activation of respiration and loss of thermodynamic control in hyperthyroidism. Is it due to increased slipping in mitochondrial proton pumps?

T3 administration increases the extent of non-linearity in the flow-force relationship between pump proton conductance and protonmotive force. The effect is present also at the ATPase proton pump. These effects are not accompanied by changes in passive proton conductance. Incubation of mitochondria at 45 degrees C also causes an increased non-linearity, accompanied by a partial increase of proton conductance. It appears that the increase of respiratory activity following T3 administration is due to loss of thermodynamic control within or at the proton pumps, an effect which might be attributed to increased slipping.     

Thyroid hormone effects on the State IV proton motive force in rat liver mitochondria

In order to further investigate the mechanisms regulating the control of mitochondrial respiration by thyroid hormone, the proton motive force was measured during State IV respiration in liver mitochondria isolated from euthyroid, hyperthyroid, hypothyroid and T₃-treated hypothyroid rats. The proton motive force was significantly higher in the hyperthyroid group due to an increased pH. The proton motive force of hypothyroid mitochondria was lower than controls due to a decreased membrane potential. The proton motive force for the T3-treated hypothyroid group did not differ from the euthyroid group due to negating changes in the pH gradient and the membrane potential. The intramitochondrial volume was decreased in the hyperthyroid group and unchanged in the other groups. The results indicate that the thyroid status alters the proton motive force in State IV through individual changes in the pH and membrane potential components of the force. The component that changes in hyperthyroid mitochondria is different from that changing in hypothyroid mitochondria.     

Intrinsic uncoupling of mitochondrial proton pumps. 2. Modeling studies

The thermodynamic and kinetic properties associated with intrinsic uncoupling in a six-state model of a redox proton pump have been studied by computing the flow-force relations for different degrees of coupling. Analysis of these relations shows the regulatory influence of the thermodynamic forces on the extent and relative contributions of redox slip and proton slip. Inhibition has been introduced into the model in two different ways, corresponding to possible modes of action of experimental inhibitors. Experiments relating the rate of electron transfer to delta microH at static head upon progressive inhibition of the pumps have been simulated considering (1) the limiting case that the nonzero rate of electron transfer at static head is only due to intrinsic uncoupling (no leaks) and (2) the experimentally observed case that about 30% of the nonzero rate of electron transfer at static head is due to a constant proton leakage conductance in parallel with the pumps, the rest being due to intrinsic uncoupling. The same simulations have been performed for experiments in which the rate of electron transfer is varied by varying the substrate concentration rather than by using an inhibitor. The corresponding experimental results obtained by measuring delta microH and the rate of electron transfer at different succinate concentrations in rat liver mitochondria are presented. Comparison between simulated behavior and experimental results leads to the general conclusion that the typical relationship between rate of electron transfer and delta microH found in mitochondria at static head could certainly be a manifestation of some degree of intrinsic uncoupling in the redox proton pumps.(ABSTRACT TRUNCATED AT 250 WORDS)     

The acute regulation of mitochondrial proton conductance in cells and mitochondria from the brown fat of cold-adapted and warm-adapted guinea pigs

Cells and mitochondria were prepared from the brown adipose tissue of adult guinea-pigs adapted to either 4-7 degrees C or 22-25 degrees C. The cold-adapted cells displayed noradrenaline-stimulated, propranolol-sensitive respiration, but noradrenaline failed to increase the respiration of the warm-adapted cells. Purine-nucleotide-sensitive proton conductance was greater in cold-adapted mitochondria than in warm-adapted controls. At the same time cold-adapted mitochondria were extremely sensitive to the uncoupling effect of endogenous and infused fatty acids, and resembled the mitochondria from the brown adipose tissue of cold-adapted hamsters. Warm-adapted mitochondria were ninefold less sensitive, and resembled liver mitochondria. With cold-adapted, but not warm-adapted mitochondria, respiration increased proportionately to the rate of fatty acid infusion. It is concluded that the presence of the 32000-Mr proton conductance pathway is necessary for the expression of a high sensitivity to fatty acid uncoupling, suggesting that the fatty acids interact directly with this protein to modulate the proton conductance during the acute regulation of thermogenesis.     

Active proton leak in mitochondria: A new way to regulate substrate oxidation

The main function of mitochondria is energy transduction, from substrate oxidation to the free energy of ATP synthesis, through oxidative phosphorylation. For physiological reasons, the degree of coupling between these two processes must be modulated in order to adapt redox potential and ATP turnover to cellular needs. Such a modulation leads to energy wastage. To this day, two energy wastage mechanisms have been described: the membrane passive proton conductance (proton leak) and the decrease in the coupling efficiency between electrons transfer and proton extrusion at the proton pumps level (redox or proton slipping). In this paper, we describe a new energy wastage mechanism of interest. We show that in isolated yeast mitochondria, the membrane proton conductance is strictly dependent on the external dehydrogenases activity. An increase in their activity leads to an increase in the membrane proton conductance. This proton permeability is independent of the respiratory chain and ATP synthase proton pumps. We propose to name this new mechanism “active proton leak.” Such a mechanism could allow a wide modulation of substrate oxidation in response to cellular redox constraints.     

The relative proton stoichiometries of the mitochondrial proton pumps are independent of the proton motive force

Several different proton pumps were used to generate a proton motive force (delta p, proton motive force across the mitochondrial inner membrane) in isolated rat liver mitochondria, and the relationship between delta p and pump rate was investigated by titrating with various inhibitors of the pumps. It was found that this relationship was the same for mitochondria respiring on succinate irrespective of whether respiration was inhibited with malonate, antimycin or cyanide, indicating that the relationship was independent of the redox state of the respiratory chain. When delta p was generated by either the cytochrome bc1 complex, cytochrome oxidase, both together, or ATP hydrolysis (and transport), the reaction rates (in moles of electrons or ATP) were in the ratio of close to 3:1.5:1:1, respectively, at all accessible values of delta p. This suggests that the proton stoichiometries (H+/e and H+/ATP, where H+/e is the number of protons translocated vectorially across the inner membrane per electron transferred by the respiratory chain and H+/ATP is the number of protons translocated vectorially per ATP molecule hydrolyzed externally) were in the ratio of close to 1:2:3:3, respectively, at all values of delta p. Possible reasons for previous contradictory results are suggested.     

Uncoupling of oxidative phosphorylation. 1. Protonophoric effects account only partially for uncoupling

The mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-trifluoromethoxy)phenylhydrazone (FCCP), a typical weak acid protonophore, oleic acid, a fatty acid, and chloroform, a general anesthetic, has been investigated by measuring in mitochondria their effect on (i) the transmembrane proton electrochemical potential gradient (delta mu H) and the rates of electron transfer and adenosine 5'-triphosphate (ATP) hydrolysis in static head, (ii) delta mu H and the rates of electron transfer and ATP synthesis in state 3, and (iii) the membrane proton conductance. Both FCCP and oleic acid increase the membrane proton conductance, and accordingly, they cause a depression of delta mu H [generated by either the redox proton pumps or the adenosinetriphosphatase (ATPase) proton pumps]. Although their effects on ATP synthesis/hydrolysis, respiration, and delta mu H are qualitatively consistent with a pure protonophoric uncoupling mechanism and an additional inhibitory action of oleic acid on both the ATPases and the electron-transfer enzymes, a quantitative comparison between the dissipative proton influx and the rate of either electron transfer or ATP hydrolysis (multiplied by either the H+/e- or the H+/ATP stoichiometry, respectively) at the same delta mu H shows that the increase in membrane conductance induced by FCCP and oleic acid accounts for the stimulation of the rate of ATP hydrolysis but not for that of the rate of electron transfer. Chloroform (at concentrations that fully inhibit ATP synthesis) only very slightly increases the proton conductance of the mitochondrial membrane and causes only a little depression of delta mu H.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Hyperthermia impairs liver mitochondrial function in vitro

The effects of temperature on the relationships among the rates of pyruvate carboxylation, O(2) uptake (J(o)), oxidative phosphorylation (J(p)), and the free energy of ATP hydrolysis (G(p)) were studied in liver mitochondria isolated from 250-g female rats. Pyruvate carboxylation was evaluated at 37, 40, and 43 degrees C. In disrupted mitochondria, pyruvate carboxylase maximal reaction velocity increased from 37 to 43 degrees C with an apparent Q(10) of 2.25. A reduction in ATP/ADP ratio decreased enzyme activity at all three temperatures. In contrast, in intact mitochondria, increasing temperature failed to increase pyruvate carboxylation (malate + citrate accumulation) but did result in increased J(o) and decreased extramitochondrial G(p). J(p) was studied in respiring mitochondria at 37 and 43 degrees C at various fractions of state 3 respiration, elicited with a glucose + hexokinase ADP-regenerating system. The relationship between J(o) and G(p) was similar at both temperatures. However, hyperthermia (43 degrees C) reduced the J(p)/J(o) ratio, resulting in lower G(p) for a given J(p). Fluorescent measurements of membrane phospholipid polarization revealed a transition in membrane order between 40 and 43 degrees C, a finding consistent with increased membrane proton conductance. It is concluded that hyperthermia augments nonspecific proton leaking across the inner mitochondrial membrane, and the resultant degraded energy state offsets temperature stimulation of pyruvate carboxylase. As a consequence, at high temperatures approaching 43 degrees C, the pyruvate carboxylation rate of intact liver mitochondria may fail to exhibit a Q(10) effect.     

Proton leak in hepatocytes and liver mitochondria from archosaurs (crocodiles) and allometric relationships for ectotherms

It has previously been shown that mitochondrial proton conductance decreases with increasing body mass in mammals and is lower in a 250-g lizard than the laboratory rat. To examine whether mitochondrial proton conductance is extremely low in very large reptiles, hepatocytes and mitochondria were prepared from saltwater crocodiles ( Crocodylus porosus) and freshwater crocodiles ( Crocodylus johnstoni). Respiration rates of hepatocytes and liver mitochondria were measured at 37 degrees C and compared with values obtained for rat or previously measured for other species. Respiration rates of hepatocytes from either species of crocodile were similar to those reported for lizards and approximately one fifth of the rates measured using cells from mammals (rat and sheep). Ten-to-thirty percent of crocodile hepatocyte respiration was used to drive mitochondrial proton leak, similar to the proportion in other species. Respiration rates of crocodile liver mitochondria were similar to those of mammalian species. Proton leak rate in isolated liver mitochondria was measured as a function of membrane potential. Contrary to our prediction, the mitochondrial proton conductance of liver mitochondria from crocodiles was greater than that of liver mitochondria from lizards and was similar to that of rats. The acyl composition of liver mitochondrial phospholipids from the crocodiles was more similar to that in mitochondria from rats than in mitochondria from lizards. The relatively high mitochondrial proton conductance was associated with a relatively small liver, which seems to be characteristic of crocodilians. Comparison of data from a number of diverse ectothermic species suggested that hepatocyte respiration rate may decrease with body mass, with an allometric exponent of about -0.2, similar to the exponent in mammalian hepatocytes. However, unlike mammals, liver mitochondrial proton conductance in ectotherms showed no allometric relationship with body size.     

Altered relationship between protonmotive force and respiration rate in non-phosphorylating liver mitochondria isolated from rats of different thyroid hormone status

We have determined the relationship between rate of respiration and protonmotive force in oligomycin-inhibited liver mitochondria isolated from euthyroid, hypothyroid and hyperthyroid rats. Respiration rate was titrated with the respiratory-chain inhibitor malonate. At any given respiration rate mitochondria isolated from hypothyroid rats had a protonmotive force greater than mitochondria isolated from euthyroid controls, and mitochondria isolated from hyperthyroid rats had a protonmotive force less than mitochondria isolated from euthyroid controls. In the absence of malonate mitochondrial respiration rate increased in the order hypothyroid less than euthyroid less than hyperthyroid, while protonmotive force increased in the order hyperthyroid less than euthyroid less than hypothyroid. These findings are consistent with a thyroid-hormone-induced increase in the proton conductance of the inner mitochondrial membrane or a decrease in the H+/O ratio of the respiratory chain at any given protonmotive force. Thus the altered proton conductance or H+/O ratio of mitochondria isolated from rats of different thyroid hormone status controls the respiration rate required to balance the backflow of protons across the inner mitochondrial membrane. We discuss the possible relevance of these findings to the control of state 3 and state 4 respiration by thyroid hormone.     

Nature of proton cycling during gramicidin uncoupling of oxidative phosphorylation

Addition of gramicidin D to liver mitochondria, incubated in low- or high-salt media, results in stimulation of respiration in the absence or presence of depression of delta muH, respectively. Gramicidin D concentrations 2 orders of magnitude higher are required in the low-salt media with full uncoupling at 1 nmol of gramicidin.mg-1. The stimulation of respiration is not accompanied by increased passive proton influx in low-salt media. In high-salt media, the extent of respiratory stimulation and the extent of delta muH depression differ according to the nature and concentration of cation. The flow-force relationship is very steep when gramicidin D induced uncoupling occurs in low-salt media and much less steep in high-salt media. A multiplicity of flow-force relationship, respiratory rate vs delta muH, is obtained, the slope of which depends on the nature and concentration of cation, and which can be reproduced by computer simulation by introducing a variable extent of proton cycling either in the membrane or in the pump. The apparent proton conductance, as analyzed in the relationship of Je/delta muH vs delta muH, increases in the so-called ohmic and nonohmic regions according to whether gramicidin D is added in high-salt or low-salt media, respectively. Titration with antimycin of the respiratory control ratio (RCR) in gramicidin D treated mitochondria leads to a depression of the RCR in high-salt but not in low-salt media. The view is discussed that in low-salt media the gramicidin D induced uncoupling is due to a cycling of protons within a proton domain operationally located at or near the proton pump.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of respiration in non-phosphorylating mitochondria is shared between the proton leak and the respiratory chain.

We measured the relationship between rate of respiration and membrane potential in isolated mitochondria titrated with malonate (to inhibit the electron transport chain) or with uncoupler (to increase the proton conductance of the inner membrane). We used the flux control summation and connectivity theorems of metabolic control theory to calculate the control over non-phosphorylating respiration exerted by the respiratory chain (and associated reactions) and by the leak of protons across the inner membrane. At 37 degrees C the flux control coefficient of the leak over respiration was 0.66; the flux control coefficient of the chain over respiration was 0.34. At 25 degrees C the values were 0.75 and 0.25 respectively. We argue that the basis for previous conclusions that all the control is exerted by the proton leak under similar conditions is invalid.     

Tissue-specific depression of mitochondrial proton leak and substrate oxidation in hibernating arctic ground squirrels

A significant proportion of standard metabolic rate is devoted to driving mitochondrial proton leak, and this futile cycle may be a site of metabolic control during hibernation. To determine if the proton leak pathway is decreased during metabolic depression related to hibernation, mitochondria were isolated from liver and skeletal muscle of nonhibernating (active) and hibernating arctic ground squirrels (Spermophilus parryii). At an assay temperature of 37 degrees C, state 3 and state 4 respiration rates and state 4 membrane potential were significantly depressed in liver mitochondria isolated from hibernators. In contrast, state 3 and state 4 respiration rates and membrane potentials were unchanged during hibernation in skeletal muscle mitochondria. The decrease in oxygen consumption of liver mitochondria was achieved by reduced activity of the set of reactions generating the proton gradient but not by a lowered proton permeability. These results suggest that mitochondrial proton conductance is unchanged during hibernation and that the reduced metabolism in hibernators is a partial consequence of tissue-specific depression of substrate oxidation.     

Effects of the local anesthetic bupivacaine on oxidative phosphorylation in mitochondria. Change from decoupling to uncoupling by formation of a leakage type ion pathway specific for H+ in cooperation with hydrophobic anions

The effects of the local anesthetic bupivacaine on the oxidative phosphorylation in rat liver mitochondria were examined. Bupivacaine caused a maximum of about 7-fold stimulation of state 4 respiration at about 3 mM, released oligomycin-inhibited state 3 respiration, and activated ATPase to a similar extent to that by the weakly acidic uncoupler SF 6847. These effects were greatly enhanced by the addition of certain hydrophobic anions such as 1-anilino-8-naphthalenesulfonate, tetraphenyl borate, and picrate. In the absence of these anions, bupivacaine did not increase the proton conductance in either energized or nonenergized mitochondrial membranes or in artificial bilayer lipid membranes and did not have any effect on the proton motive force. However, it greatly enhanced the proton conductivity of these membrane systems and collapsed the proton motive force in the presence of hydrophobic anions. The results of noise analysis of artificial lipid bilayer membranes indicated that an ion pair complex of bupivacaine with hydrophobic anions formed a leakage-type ion pathway. Thus it is concluded that bupivacaine acts as a decoupler in the absence of added hydrophobic anions but in cooperation with certain anions as an uncoupler of oxidative phosphorylation due to formation of a H(+)-specific pathway in the membranes.     

Stimulation of mitochondrial functions by glucagon treatment, starvation and by treatment of isolated mitochondria with glycogen-bound enzymes

Liver mitochondria isolated from rats starved overnight, or fed rats injected with glucagon, exhibited a similar increase of the respiration rate with succinate (by 30-40%) and glutamate plus malate (by 20-30%), as compared to mitochondria from control fed animals. The content of mitochondrial adenine nucleotides was elevated by 30-45% by glucagon treatment or starvation. Mitochondrial respiration and citrulline synthesis were stimulated by 30-40% when mitochondria isolated from fed rats were briefly preincubated with the extract from liver glycogen granules, ATP and MgCl2. This effect was abolished by heating the extract at 100 degrees C.     

Bioenergetics in aging: mitochondrial proton leak in aging rat liver, kidney and heart

Aging is associated with a decline in performance in many organs and loss of physiological performance can be due to free radicals. Mitochondria are incompletely coupled: during oxidative phosphorylation some of the redox energy is dissipated as natural proton leak across the inner membrane. To verify whether proton leak occurs in mitochondria during aging, we measured the mitochondrial respiratory chain activity, membrane potential and proton leak in liver, kidneys and heart of young and old rats. Mitochondria from old rats showed normal rates of Complex I and Complex II respiration. However, they had a lower membrane potential compared to mitochondria from younger rats. In addition, they exhibited an increased rate of proton conductance which partially dissipated the mitochondrial membrane potential when the rate of electron transport was suppressed. This could compromise energy homeostasis in aging cells in conditions that require additional energy supply and could minimize oxidative damage to DNA.     

Effects of hyperthyroidism on stimulation of [1-14C]oleate oxidation to14CO2 in isolated hepatocytes from fed rats by the catecholamines,vasopressin, and angiotensin II

Possible effects of adrenaline, noradrenaline, vasopressin, and angiotensin II to increase 14CO2 production from [1-14C]oleate were examined in hepatocytes from fed L-triiodothyronine (T3)-treated or control rats. Rates of 14CO2 production were decreased and rates of ketogenesis increased in hepatocytes from T3-treated rats. These changes were accompanied by a marked shift of the 3-hydroxybutyrate:acetoacetate concentration ratio towards acetoacetate. Rates of glucose and lactate release were decreased. Whereas the Ca2+-mobilizing hormones increased 14CO2 production from [1-14C]oleate by 64-84% with hepatocytes from control rats, they increased 14CO2 production from [1-14C]oleate by on 24-32% with hepatocytes from T3-treated rats. The magnitude of the response to the Ca2+-mobilizing hormones in hepatocytes from T3-treated rats was increased by the addition of 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, to the incubation medium (increases of 52-88%). In the presence of 3-mercaptopicolinate, the 3-hydroxybutyrate:acetoacetate concentration ratio in hepatocytes from fed, T3-treated rats was similar to that in hepatocytes from control rats in the absence of 3-mercaptopicolinate. The results demonstrate that hyperthyroidism per se does not lead to a loss of sensitivity, in terms of oleate oxidation, either to the catecholamines or to vasopressin and angiotensin II. The impaired ability of hepatocytes from T3-treated rats to respond to these hormones is a consequence of decreased net glycolytic flux or a more oxidized mitochondrial redox state.     

The basal proton conductance of skeletal muscle mitochondria from transgenic mice overexpressing or lacking uncoupling protein-3.

The ability of native uncoupling protein-3 (UCP3) to uncouple mitochondrial oxidative phosphorylation is controversial. We measured the expression level of UCP3 and the proton conductance of skeletal muscle mitochondria isolated from transgenic mice overexpressing human UCP3 (UCP3-tg) and from UCP3 knockout (UCP3-KO) mice. The concentration of UCP3 in UCP3-tg mitochondria was approximately 3 microg/mg protein, approximately 20-fold higher than the wild type value. UCP3-tg mitochondria had increased nonphosphorylating respiration rates, decreased respiratory control, and approximately 4-fold increased proton conductance compared with the wild type. However, this increased uncoupling in UCP3-tg mitochondria was not caused by native function of UCP3 because it was not proportional to the increase in UCP3 concentration and was neither activated by superoxide nor inhibited by GDP. UCP3 was undetectable in mitochondria from UCP3-KO mice. Nevertheless, UCP3-KO mitochondria had unchanged respiration rates, respiratory control ratios, and proton conductance compared with the wild type under a variety of assay conditions. We conclude that uncoupling in UCP3-tg mice is an artifact of transgenic expression, and that UCP3 does not catalyze the basal proton conductance of skeletal muscle mitochondria in the absence of activators such as superoxide.     

Mitochondrial UCPs: new insights into regulation and impact

Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins sustaining an inducible proton conductance. They weaken the proton electrochemical gradient built up by the mitochondrial respiratory chain. Brown fat UCP1 sustains a free fatty acid (FA)-induced purine nucleotide (PN)-inhibited proton conductance. Inhibition of the proton conductance by PN has been considered as a diagnostic of UCP activity. However, conflicting results have been obtained in isolated mitochondria for UCP homologues (i.e., UCP2, UCP3, plant UCP, and protist UCP) where the FFA-activated proton conductance is poorly sensitive to PN under resting respiration conditions. Our recent work clearly indicates that the membranous coenzyme Q, through its redox state, represents a regulator of the inhibition by PN of FFA-activated UCP1 homologues under phosphorylating respiration conditions. Several physiological roles of UCPs have been suggested, including a control of the cellular energy balance as well as the preventive action against oxidative stress. In this paper, we discuss new information emerging from comparative proteomics about the impact of UCPs on mitochondrial physiology, when recombinant UCP1 is expressed in yeast and when UCP2 is over-expressed in hepatic mitochondria during steatosis.