Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes

The mitochondrial membrane potential in isolated hepatocytes was measured using the distribution of the lipophilic cation triphenylmethylphosphonium (TPMP+) with appropriate corrections for plasma membrane potential, cytoplasmic and mitochondrial binding of TPMP+, and other factors. The relationship between mitochondrial membrane potential and respiration rate in hepatocytes was examined as the respiratory chain was titrated with myxothiazol in the presence of oligomycin. This relationship was nonproportional and similar to results with isolated mitochondria respiring on succinate. This shows that there is an increased proton conductance of the mitochondrial inner membrane in situ at high values of membrane potential. From the respiration rate and mitochondrial membrane potential of hepatocytes in the absence of oligomycin, we estimate that the passive proton permeability of the mitochondrial inner membrane accounts for 20-40% of the basal respiration rate of hepatocytes. The relationship between log[TPMP+]tot/[TPMP+]e and respiration rate in thymocytes was also nonproportional suggesting that the phenomenon is not peculiar to hepatocytes. There is less mitochondrial proton leak in hepatocytes from hypothyroid rats. A large proportion of the difference in basal respiration rate between hepatocytes from normal and hypothyroid rats can be accounted for by differences in the proton permeability characteristics of the mitochondrial inner membrane.     

Hypothyroidism in rats decreases mitochondrial inner membrane cation permeability

We investigated the cation permeability of liver mitochondria isolated from hypothyroid or euthyroid rats by measuring the rate of swelling of respiring mitochondria in acetate salts as a function of membrane potential. Mitochondria from hypothyroid rats have a decreased permeability of roughly 3-fold in the presence of monovalent cations K and tetramethylammonium at any (measured) membrane potential. Since the monovalent cation leak and the proton leak are known to respond similarly to membrane potential our results support the theory that the difference in non-phosphorylating respiration rate between mitochondria from hypothyroid and euthyroid rats is due to a difference in proton leak.     

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.     

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.     

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.     

Thyroid hormone status and membrane n-3 fatty acid content influence mitochondrial proton leak

Proton leak, as determined by the relationship between respiration rate and membrane potential, was lower in mitochondria from hypothyroid rats compared to euthyroid controls. Moreover, proton leak rates diminished even more when hypothyroid rats were fed a diet containing 5% of the lipid content as n-3 fatty acids. Similarly, proton leak was lower in euthyroid rats fed the 5% n-3 diet compared to one containing only 1% n-3 fatty acids. Lower proton leaks rates were associated with increased inner mitochondrial membrane levels of n-3 fatty acids and a decrease in the ratio of n-6/n-3 fatty acids. This trend was evident in the phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin phospholipid fractions. These results suggest that a significant portion of the effect of thyroid hormone status on proton leak is due to alterations in membrane fatty acid composition, primarily changes in n-3 content. Both the hypothyroid state and dietary effects appear to be mediated in part by inhibition of the Delta6- and Delta5-desaturase pathways.     

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.     

The contribution of the leak of protons across the mitochondrial inner membrane to standard metabolic rate

This paper presents and assesses the hypothesis that the proton leak across the mitochondrial inner membrane is an important contributor to standard metabolic rate, and that increases in the amount of mitochondrial inner membrane may be important in causing changes in proton leak and in the standard metabolic rate. The standard metabolic rate of an animal is known to be a function of body mass, phylogeny and thyroid status, and is largely attributed to the metabolically active internal organs. The total area of mitochondrial inner membrane in these organs correlates well with standard metabolic rate over a wide range of body masses in both ectotherms and endotherms. In hepatocytes isolated from rats, proton leak across the mitochondrial inner membrane accounts for about 30% of the resting oxygen consumption, and the distribution of control over respiration suggests that changes in mitochondrial inner membrane surface area will be accompanied by significant changes in the proton leak. This change in the leak will result in significant changes in resting oxygen consumption, but changes in ATP demand may also have a role to play in determining resting respiration rate. Extrapolation of these results to other tissues and other animals suggests that the hypothesis has the potential to explain a substantial proportion of the variation in standard metabolic rate with body mass, phylogeny and thyroid status. However, in most cases the quantitative contribution of proton leak compared to cellular ATP turnover has yet to be experimentally determined.     

Uncoupling protein-2 contributes significantly to high mitochondrial proton leak in INS-1E insulinoma cells and attenuates glucose-stimulated insulin secretion

Proton leak exerts stronger control over ATP/ADP in mitochondria from clonal pancreatic beta-cells (INS-1E) than in those from rat skeletal muscle, due to the higher proton conductance of INS-1E mitochondria [Affourtit and Brand (2006) Biochem. J. 393, 151-159]. In the present study, we demonstrate that high proton leak manifests itself at the cellular level too: the leak rate (measured as myxothiazol-sensitive, oligomycin-resistant respiration) was nearly four times higher in INS-1E cells than in myoblasts. This relatively high leak activity was decreased more than 30% upon knock-down of UCP2 (uncoupling protein-2) by RNAi (RNA interference). The high contribution of UCP2 to leak suggests that proton conductance through UCP2 accounts for approx. 20% of INS-1E respiration. UCP2 knock-down enhanced GSIS (glucose-stimulated insulin secretion), consistent with a role for UCP2 in beta-cell physiology. We propose that the high mitochondrial proton leak in beta-cells is a mechanism which amplifies the effect of physiological UCP2 regulators on cytoplasmic ATP/ADP and hence on insulin secretion.     

疲劳性运动中线粒体电子漏引起质子漏增加

以大鼠递增强度力竭性竭性跑台运动为疲劳运动模型,观察了运动后大鼠骨骼肌线粒体电子漏和质子漏的变化。结果表明,运动性疲劳状态下大鼠骨骼肌线粒体超氧阴离子生成增加,脂质过氧线粒体质子漏增多是氧化磷酸化偶联程度下降的重要因素。实验结果支持电子漏引起质子漏的假说。     

The mechanism of stimulation of respiration by fatty acids in isolated hepatocytes

Addition of fatty acids to isolated hepatocytes raised respiration rate by 92% and raised mitochondrial membrane potential (delta psi m) in situ from 155 to 162 mV suggesting that the increased fuel supply had a greater effect on respiration rate than any increases in processes that consumed mitochondrial protonmotive force (delta p). The relationship between delta psi m and respiration rate was changed by addition of fatty acids or lactate, showing that there was also stimulation of delta p-consuming reactions. In the presence of oligomycin the relationship between delta psi m and respiration rate was unaffected by substrate addition, showing that the kinetics of delta p consumption by the H+ leak across the mitochondrial inner membrane were unchanged. The stimulation of delta p consumers by fatty acids therefore must be in the pathways of ATP synthesis and turnover. Inhibition of several candidate ATP-consuming reactions had little effect on basal or fatty acid-stimulated respiration, and the nature of the ATP turnover reactions in hepatocytes remains speculative. We conclude that fatty acids (and other substrates) stimulate respiration in hepatocytes in two distinct ways. They provide substrate for the electron transport chain, raising delta p and increasing the non-ohmic proton leak across the mitochondrial inner membrane and the rate of oxygen consumption. They also directly stimulate an unidentified delta p-consuming reaction in the cytoplasm. They do not work by uncoupling or by stimulation of intramitochondrial ATP-turnover reactions.     

Superoxide stimulates a proton leak in potato mitochondria that is related to the activity of uncoupling protein

The ability of plant mitochondrial uncoupling proteins to catalyze a significant proton conductance in situ is controversial. We have re-examined conditions that lead to uncoupling of mitochondria isolated from the tubers of potato (Solanum tuberosum). Specifically, we have investigated the effect of superoxide. In the absence of superoxide, linoleic acid stimulated a proton leak in mitochondria respiring NADH that was insensitive to GTP. However, when exogenous superoxide was generated by the addition of xanthine and xanthine oxidase, there was an additional linoleic acid-stimulated proton leak that was specifically inhibited by GTP. Under these conditions of assay (NADH as a respiratory substrate, in the presence of linoleic acid and xanthine/xanthine oxidase) there was a higher rate of proton conductance in mitochondria from transgenic potato tubers overexpressing the StUCP gene than those from wild type. The increased proton leak in the transgenic mitochondria was completely abolished by the addition of GTP. This suggests that superoxide and linoleic acid stimulate a proton leak in potato mitochondria that is related to the activity of uncoupling protein. Furthermore, it demonstrates that changes in the amount of StUCP can alter the rate of proton conductance of potato mitochondria.     

Characterisation of the control of respiration in potato tuber mitochondria using the top-down approach of metabolic control analysis.

Control over oxidative phosphorylation by purified potato mitochondria was determined using the top-down approach of metabolic control analysis. The control over the respiration rate, phosphorylation rate, proton-leak rate and proton motive force exerted by the respiratory chain, phosphorylation reactions and the proton leak were measured over a range of phosphorylation rates from resting (state 4) to maximal (state 3). These rates were obtained by adding different amounts of hexokinase in the presence of glucose, or different amounts of oligomycin in the presence of ADP. The respiratory substrate was NADH or succinate, both of which feed electrons directly to ubiquinone. The rate of oxygen consumption by the alternative oxidase pathway was negligible with NADH as substrate but was measurable with succinate and was subtracted. Control over the respiration rate in potato mitochondria was predominantly exerted by the respiratory chain at all rates except close to state 4, where control by the proton leak was equally or more important. For oxidation of NADH, the flux control coefficient over the respiration rate exerted by the respiratory chain in state 3 was between 0.8 and 1.0, while in state 4, control over the respiration rate was shared about equally between the chain and the proton leak. The control over the phosphorylation rate was predominantly exerted by the respiratory chain, although at low rates control by the phosphorylation system was also important. For oxidation of NADH, the flux control coefficient over the phosphorylation rate exerted by the respiratory chain in state 3 was 0.8-1.0, while near state 4 the flux control coefficients over the phosphorylation rate were about 0.8 for the phosphorylation system and 0.25 for the chain. Control over the proton leak rate was shared between the respiratory chain and the proton leak; the phosphorylation system had negative control. For oxidation of NADH, the flux control coefficients over the leak rate in state 3 were 1.0 for the leak, 0.4 for the chain and -0.4 for the phosphorylation system, while in state 4 the flux control coefficients over leak rate were about 0.5 for the leak and 0.5 for the chain. Control over the magnitude of the protonmotive force was small, between -0.2 and +0.2, reflecting the way the system operates to keep the protonmotive force fairly constant; the respiratory chain and the phosphorylation system had equal and opposite control and there was very little control by the proton leak except near state 4.     

Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and protonmotive force in isolated mitochondria using the 'top-down' approach of metabolic control theory

The rate of respiration of isolated mitochondria was set at different values by addition of either oligomycin or an ADP-regenerating system (glucose and different amounts of hexokinase). We measured the relationship between respiration rate and membrane potential as respiration was titrated by the addition of malonate under each condition. We used the flux control summation and connectivity theorems and the branching theorem of metabolic control theory to calculate the control over respiration rate exerted by the respiratory chain (and associated reactions), phosphorylating system (and associated reactions) and proton leak at each respiration rate. The analysis also yielded the flux control coefficients of these three reactions over phosphorylation rate and proton leak rate and their concentration control coefficients over protonmotive force. We found that respiration rate was controlled largely by the proton leak under non-phosphorylating conditions, by the phosphorylating system at intermediate rates and by both the phosphorylating system and the respiratory chain in state 3. The rate of phosphorylation was controlled largely by the phosphorylating system itself in state 4 and at intermediate rates, while state 3 control was shared between the phosphorylating system and the respiratory chain; the proton leak had insignificant control. In all states the phosphorylating system had large negative control over the proton leak; the chain and the proton leak both had large positive control coefficients. The protonmotive force was controlled by the chain and by the phosphorylating system; the proton leak had little control.     

UCP1 muscle gene transfer and mitochondrial proton leak mediated thermogenesis

Mitochondrial uncoupling protein 1 (UCP1) mediates the thermogenic transport of protons through the inner mitochondrial membrane. This proton leak uncouples respiration from ATP synthesis. The current study assessed the possible contribution of UCP1 muscle gene transfer to impair mitochondrial respiration in a tissue lacking UCP1 gene expression. Rats received an intramuscular injection of plasmid pXC1 containing UCP1 cDNA in the right tibialis muscles, while left tibialis muscles were injected with empty plasmid as control. Ten days after DNA injection, mitochondria from tibialis anterior muscles were isolated and analyzed. UCP1 gene transfer resulted in protein expression as analyzed by inmunoblotting. Mitochondria isolated from UCP1-injected muscles showed a significant increase in state 2 and state 4 oxygen consumption rates and a decreased respiration control ratio in comparison to mitochondria from control muscles. Furthermore, UCP1-containing mitochondria had a lower membrane potential in those states (2 and 4) when compared with control mitochondria. Our results revealed that UCP1 muscle gene transfer is associated with an induced mitochondrial proton leak, which could contribute to increase energy expenditure.     

Mitochondrial physiology of diapausing and developing embryos of the annual killifish Austrofundulus limnaeus: implications for extreme anoxia tolerance

Diapausing embryos of the annual killifish Austrofundulus limnaeus have the highest reported anoxia tolerance of any vertebrate and previous studies indicate modified mitochondrial physiology likely supports anoxic metabolism. Functional mitochondria isolated from diapausing and developing embryos of the annual killifish exhibited VO₂, respiratory control ratios (RCR), and P:O ratios consistent with those obtained from other ectothermic vertebrate species. Reduced oxygen consumption associated with dormancy in whole animal respiration rates are correlated with maximal respiration rates of mitochondria isolated from diapausing versus developing embryos. P:O ratios for developing embryos were similar to those obtained from adult liver, but were diminished in mitochondria from diapausing embryos suggesting decreased oxidative efficiency. Proton leak in adult liver corresponded with that of developing embryos but was elevated in mitochondria isolated from diapausing embryos. In metabolically suppressed diapause II embryos, over 95% of the mitochondrial oxygen consumption is accounted for by proton leak across the inner mitochondrial membrane. Decreased activity of mitochondrial respiratory chain complexes correlates with diminished oxidative capacity of isolated mitochondria, especially during diapause. Respiratory complexes exhibited suppressed activity in mitochondria with the ATP synthase exhibiting the greatest inhibition during diapause II. Mitochondria isolated from diapause II embryos are not poised to produce ATP, but rather to shuttle carbon and electrons through the Kreb’s cycle while minimizing the generation of a proton motive force. This particular mitochondrial physiology is likely a mechanism to avoid production of reactive oxygen species during large-scale changes in flux through oxidative phosphorylation pathways associated with metabolic transitions into and out of dormancy and anoxia.     

Contribution of mitochondrial proton leak to respiration rate in working skeletal muscle and liver and to SMR

Proton pumpingacross the mitochondrial inner membrane and proton leak back throughthe natural proton conductance pathway make up a futile cycle thatdissipates redox energy. We measured respiration and averagemitochondrial membrane potential in perfused rat hindquarter withmaximal tetanic contraction of the left gastrocnemius-soleus-plantaris muscle group, and we estimate that the mitochondrial proton cycle accounted for 34% of the respiration rate of the preparation. Similarmeasurements in rat hepatocytes given substrates to cause a high rateof gluconeogenesis and ureagenesis showed that the proton cycleaccounted for 22% of the respiration rate of these cells. Combiningthese in vitro values with literature values for the contribution ofskeletal muscle and liver to standard metabolic rate (SMR), wecalculate that the proton cycle in working muscle and liver may accountfor 15% of SMR in vivo. Although this value is less than the 20% ofSMR we calculated previously using data from resting skeletal muscleand hepatocytes, it is still large, and we conclude that the futileproton cycle is a major contributor to SMR.     

In Phosphorylating <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> Mitochondria the Sensitivity of Uncoupling Protein Activity to GTP Depends on the Redox State of Quinone

In isolated Acanthamoeba castellanii mitochondria respiring in state 3 with external NADH or succinate, the linoleic acid-induced purine nucleotide-sensitive uncoupling protein activity is able to uncouple oxidative phosphorylation. The linoleic acid-induced uncoupling can be inhibited by a purine nucleotide (GTP) when quinone (Q) is sufficiently oxidized, indicating that in A. castellanii mitochondria respiring in state 3, the sensitivity of uncoupling protein activity to GTP depends on the redox state of the membranous Q. Namely, the inhibition of the linoleic acid-induced uncoupling by GTP is not observed in uninhibited state 3 respiration as well as in state 3 respiration progressively inhibited by complex III inhibitors, i.e., when the rate of quinol (QH₂)-oxidizing pathway is decreased. On the contrary, the progressive decrease of state 3 respiration by declining respiratory substrate availability (by succinate uptake limitation or by decreasing external NADH concentration), i.e., when the rate of Q-reducing pathways is decreased, progressively leads to a full inhibitory effect of GTP. Moreover, in A. castellanii mitochondria isolated from cold-treated cells, where a higher uncoupling protein activity is observed, the inhibition of the linoleic acid-induced proton leak by GTP is revealed for the same low values of the Q reduction level.     

Evidence for Involvement of Uncoupling Proteins in Cerebral Mitochondrial Oxidative Phosphorylation Deficiency of Rats Exposed to 5,000 m High Altitude

The present study aimed to investigate the change of proton leak and discuss the role of cerebral uncoupling proteins (UCPs) and its regulatory molecules non-esterified fatty acid (NEFA) in high altitude mitochondrial oxidative phosphorylation deficiency. The model group animals were exposed to acute high altitude hypoxia, and the mitochondrial respiration, protein leak, UCPs abundance/activity and cerebral NEFA concentration were measured. We found that in the model group, cerebral mitochondrial oxidative phosphorylation was severely impaired with decreased ST3 respiration rate and ATP pool. Proton leak kinetics curves demonstrated an increase in proton leak; GTP binding assay pointed out that total cerebral UCPs activity significantly increased; Q-PCR and western blot showed upregulated expression of UCP4 and UCP5. Moreover, cerebral NEFA concentration increased. In conclusion, UCPs mediated proton leak is closely related to cerebral mitochondria oxidative phosphorylation deficiency during acute high altitude hypoxia and NEFA is involved in this signaling pathway.     

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.