Brigitte Sibille

Uncoupling protein and ATP/ADP carrier increase mitochondrial proton conductance after cold adaptation of king penguins

Juvenile king penguins develop adaptive thermogenesis after repeated immersion in cold water. However, the mechanisms of such metabolic adaptation in birds are unknown, as they lack brown adipose tissue and uncoupling protein‐1 (UCP1), which mediate adaptive non‐shivering thermogenesis in mammals. We used three different groups of juvenile king penguins to investigate the mitochondrial basis of avian adaptive thermogenesis in vitro. Skeletal muscle mitochondria isolated from penguins that had never been immersed in cold water showed no superoxide‐stimulated proton conductance, indicating no functional avian UCP. Skeletal muscle mitochondria from penguins that had been either experimentally immersed or naturally adapted to cold water did possess functional avian UCP, demonstrated by a superoxide‐stimulated, GDP‐inhibitable proton conductance across their inner membrane. This was associated with a markedly greater abundance of avian UCP mRNA. In the presence (but not the absence) of fatty acids, these mitochondria also showed a greater adenine nucleotide translocase‐catalysed proton conductance than those from never‐immersed penguins. This was due to an increase in the amount of adenine nucleotide translocase. Therefore, adaptive thermogenesis in juvenile king penguins is linked to two separate mechanisms of uncoupling of oxidative phosphorylation in skeletal muscle mitochondria: increased proton transport activity of avian UCP (dependent on superoxide and inhibited by GDP) and increased proton transport activity of the adenine nucleotide translocase (dependent on fatty acids and inhibited by carboxyatractylate).

Overexpression of UCP3 in cultured human muscle lowers mitochondrial membrane potential, raises ATP/ADP ratio, and favors fatty acid vs. glucose oxidation

The skeletal muscle mitochondrial uncoupling protein‐3 (UCP3) promotes substrate oxidation, but direct evidence for its metabolic role is lacking. Here, we show that UCP3 overexpression in cultured human muscle cells decreased mitochondrial membrane potential (ΔΨm). Despite this, the ATP content was not significantly decreased compared with control cells, whereas ADP content was reduced and thus the ATP/ADP ratio raised. This finding was in contrast with the effect caused by the chemical protonophoric uncoupler, CCCP, which lowered ΔΨm, ATP, and the ATP/ADP ratio. UCP3‐overexpression enhanced oxidation of oleate, regardless of the presence of glucose, whereas etomoxir, which blocks fatty acid entry to mitochondria, suppressed the UCP3 effect. Glucose oxidation was stimulated in UCP3‐overexpressing cells, but this effect was inhibited by oleate. UCP3 caused weak increase of both 2‐Deoxyglucose uptake and glycolytic rate, which differed from the marked stimulation by CCCP. We concluded that UCP3 promoted nutrient oxidation by lowering ΔΨm and enhanced fatty acid‐dependent inhibition of glucose oxidation. Unlike the uncoupler CCCP, however, UCP3 raised the ATP/ADP ratio and modestly increased glucose uptake and glycolysis. We propose that this differential effect provides a biological significance to UCP3, which is up‐regulated in metabolic stress situations where it could be involved in nutrient partitioning.

Up-regulation of avian uncoupling protein in cold-acclimated and hyperthyroid ducklings prevents reactive oxygen species production by skeletal muscle mitochondria

BackgroundAlthough identified in several bird species, the biological role of the avian homolog of mammalian uncoupling proteins (avUCP) remains extensively debated. In the present study, the functional properties of isolated mitochondria were examined in physiological or pharmacological situations that induce large changes in avUCP expression in duckling skeletal muscle.ResultsThe abundance of avUCP mRNA, as detected by RT-PCR in gastrocnemius muscle but not in the liver, was markedly increased by cold acclimation (CA) or pharmacological hyperthyroidism but was down-regulated by hypothyroidism. Activators of UCPs, such as superoxide with low doses of fatty acids, stimulated a GDP-sensitive proton conductance across the inner membrane of muscle mitochondria from CA or hyperthyroid ducklings. The stimulation was much weaker in controls and not observed in hypothyroid ducklings or in any liver mitochondrial preparations. The production of endogenous mitochondrial reactive oxygen species (ROS) was much lower in muscle mitochondria from CA and hyperthyroid ducklings than in the control or hypothyroid groups. The addition of GDP markedly increased the mitochondrial ROS production of CA or hyperthyroid birds up to, or above, the level of control or hypothyroid ducklings. Differences in ROS production among groups could not be attributed to changes in antioxidant enzyme activities (superoxide dismutase or glutathione peroxidase).ConclusionThis work provides the first functional in vitro evidence that avian UCP regulates mitochondrial ROS production in situations of enhanced metabolic activity.

Overexpression of mitochondrial uncoupling protein-3 does not decrease production of the reactive oxygen species, elevated by palmitate in skeletal muscle cells

Fatty acids induced an increase in reactive oxygen species (ROS) and enhanced NF‐κB activation in L6 myotubes differentiated in culture. Palmitate proved more effective than oleate in eliciting these effects. The induction of uncoupling protein‐3 (UCP3) at levels similar to those occurring in vivo, attained through the use of an adenoviral vector, led to a reduction of mitochondrial membrane potential in L6 myotubes. However, the capacity of palmitate to increase ROS was not reduced but, quite the opposite, it was moderately enhanced due to the presence of UCP3. The presence of UCP3 in mitochondria did not modify the expression of genes encoding ROS‐related enzymes, either in basal conditions or in the presence of palmitate. However, in the presence of UCP3, UCP2 mRNA expression was down‐regulated in response to palmitate. We conclude that UCP3 does not act as a protective agent against palmitate‐dependent induction of ROS production in differentiated skeletal muscle cells.

Uncoupling protein-3 sensitizes cells to mitochondrial-dependent stimulus of apoptosis†

The mitochondrial uncoupling protein‐3 is a member of the mitochondrial carrier protein family. As a homologue of the thermogenic brown fat uncoupling protein‐1, it possesses a mitochondrial uncoupling activity and thus can influence cell energy metabolism but its exact biological function remains unclear. In the present study, uncoupling protein‐3 was expressed in 293 cells using the tetracycline‐inducible system and its impact on cell bioenergetics and responsiveness to the apoptotic stimulus was determined. The induction of uncoupling protein‐3 expression in mitochondria did not lead to uncontrolled respiratory uncoupling in intact cells. However, it caused a GDP‐inhibition of state 4 respiration and a GDP‐induced re‐polarization of the inner mitochondrial membrane in the presence of fatty acids, in agreement with its expected physiological behavior as an uncoupling protein (UCP). Uncoupling protein‐3 expression did not cause apoptosis per se but increased the responsiveness of the cells to a mitochondrial apoptotic stimulus (i.e., addition of staurosporine in the culture medium). It enhanced caspase 3 and caspase 9 activation and favored cytochrome c release. Moreover, cells in which uncoupling protein‐3 expression had been induced showed a higher mitochondrial Bax/Bcl‐2 ratio essentially due to enhanced translocation of Bax from cytosol to mitochondria. Finally, the induction of uncoupling protein‐3 also increased the sensitivity of mitochondria to open the permeability transition pore in response to calcium. It is concluded that the presence of uncoupling protein‐3 in mitochondria sensitizes cells to apoptotic stimuli involving mitochondrial pathways.

Chronic ethanol ingestion increases efficiency of oxidative phosphorylation in rat liver mitochondria

The efficiency of oxidative phosphorylation was compared between rats chronically fed with ethanol and controls. (i) Results showed that the liver mitochondria state 4 respiratory rate was strongly inhibited, while the corresponding proton‐motive force was not affected; (ii) the cytochrome oxidase content and activity were decreased and (iii) the oxidative‐phosphorylation yield was increased in the ethanol exposed group. Furthermore, oxidative phosphorylation at coupling site II was not affected by ethanol. Cytochrome oxidase inhibition by sodium‐azide mimicked the effects of ethanol intoxication in control mitochondria. This indicates that the decrease in cytochrome oxidase activity induced by ethanol intoxication directly increases the efficiency of oxidative phosphorylation.

Reptilian uncoupling protein: functionality and expression in sub-zero temperatures

SUMMARY Here we report the partial nucleotide sequence of a reptilian uncoupling protein (repUCP) gene from the European common lizard (Lacerta vivipara). Overlapping sequence analysis reveals that the protein shows 55%, 72% and 77% sequence homology with rat UCP1, UCP2 and UCP3, respectively, and 73% with bird and fish UCPs. RepUCP gene expression was ubiquitously detected in 4°C cold-acclimated lizard tissues and upregulated in muscle tissues by a 20 h exposure to sub-zero temperatures in a supercooling state or after thawing. In parallel, we show an increase in the co-activators, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and peroxisome proliferator-activated receptors (PPAR), mRNA expression, suggesting that the mechanisms regulating UCP expression may be conserved between mammals (endotherms) and reptiles (ectotherms). Furthermore, mitochondria extracted from lizard skeletal muscle showed a guanosine diphosphate (GDP)-sensitive non phosphorylating respiration. This last result indicates an inhibition of extra proton leakage mediated by an uncoupling protein, providing arguments that repUCP is functional in lizard tissues. This result is associated with a remarkable GDP-dependent increase in mitochondrial endogenous H2O2 production. All together, these data support a physiological role of the repUCP in superoxide limitation by lizard mitochondria in situations of stressful oxidative reperfusion following a re-warming period in winter.

Oxidative phosphorylation in intact hepatocytes: Quantitative characterization of the mechanisms of change in efficiency and cellular consequences

Two mechanisms may affect the yield of the oxidative phosphorylation pathway in isolated mitochondria: (i) a decrease in the intrinsic coupling of the proton pumps (H+/2ea or H+/ATP), and (ii) an increase in the inner membrane conductance (proton or cation leak). Hence three kinds of modifications can occur and each of them have been characterized in isolated rat liver mitochondria (see preceding chapter by Rigoulet et al.). In intact isolated hepatocytes, these modifications are linked to specific patterns of bioenergetic parameters, i.e. respiratory flux, mitochondrial redox potential, DY, and phosphate potential.

2,4 DINITROPHENOL-UNCOUPLING EFFECT ON DELTA PSI IN LIVING HEPATOCYTES DEPENDS ON REDUCING-EQUIVALENT SUPPLY

Mitochondrial uncouplers, such as 2,4 dinitrophenol (DNP), increase the cellular respiration by decreasing mitochondrial membrane potential (delta psi). We show that this respiratory effect can be transient or even prevented in isolated liver cells depending on the exogenous substrate used (dihydroxyacetone vs. octanoate or proline). Moreover the decrease in ATP/ADP ratio induced by DNP is partially restored by addition of octanoate or proline. By using rhodamine 123 (Rh123) monitored by flow cytometry in living hepatocytes, we were able to follow in time delta psi in such DNP-uncoupled cells incubated with various substrates. The ability of this method to evaluate delta psi changes was assessed by using myxothiazol (3.6 microM), an inhibitor of the b-c1 complex of the respiratory chain which decreased delta psi (65%), or oligomycin (6 microg/ml), an inhibitor of the F0F1-ATPase which increased it (50%). Although DNP induced a dose-dependent decrease of delta psi, we found that octanoate or proline addition prevented such effect. We propose that octanoate or proline may counteract the uncoupling effect of DNP by providing a high supply of reducing equivalents to the respiratory chain.

Liver mitochondrial properties from the obesity-resistant Lou/C rat

Objective:The first objective was to evaluate the influence of caloric intake on liver mitochondrial properties. The second objective was aimed at determining the impact of increasing fat intake on these properties.Design:Lou/C rats, displaying an inborn low caloric intake and resistant to diet-induced obesity, were compared to Wistar rats fed either ad libitum or pair-fed. An additional group of Lou/C rats were allowed to increase their fat intake by adjusting their diet from a standard high carbohydrate low-fat diet to a high-fat carbohydrate-free diet.Measurements:Hydrogen peroxide (H2O2) generation, oxygen consumption rate (J O2), membrane potential (ΔΨ), activity of respiratory chain complexes, cytochrome contents, oxidative phosphorylation efficiency (OPE) and uncoupling protein 2 (UCP2) expression were determined in liver mitochondria.Results:H2O2 production was higher in Lou/C than Wistar rats with glutamate/malate and/or succinate, octanoyl-carnitine, as substrates. These mitochondrial features cannot be mimicked by pair-feeding Wistar rats and remained unaltered by increasing fat intake. Enhanced H2O2 production by mitochondria from Lou/C rats is due to an increased reverse electron flow through the respiratory-chain complex I and a higher medium-chain acyl-CoA dehydrogenase activity. While J O2 was similar over a large range of ΔΨ in both strains, Lou/C rats were able to sustain higher membrane potential and respiratory rate. In addition, mitochondria from Lou/C rats displayed a decrease in OPE that cannot be explained by increased expression of UCP2 but rather to a slip in proton pumping by cytochrome oxidase.Conclusions:Liver mitochondria from Lou/C rats display higher reactive oxygen species (ROS) generation but to deplete upstream electron-rich intermediates responsible for ROS generation, these animals increased intrinsic uncoupling of cytochrome oxidase. It is likely that liver mitochondrial properties allowed this strain of rat to display higher insulin sensitivity and resist diet-induced obesity.