Alpha-lipoic acid supplementation increases the efficacy of exercise- and diet-induced obesity treatment and induces immunometabolic changes in female mice and women.

The decrease in the regulatory T cells (Tregs) population is highly involved in adipose tissue inflammation and insulin resistance in obesity. Tregs depend on fatty acids via ß-oxidation for immunosuppressive function adapting their antioxidant systems to allow survival to oxidative stress. In this study, we have hypothesized that a dietary supplementation with alpha-lipoic acid (ALA), a powerful antioxidant, would improve immunometabolism when added to the classical strategy of obesity treatment. First, we showed by in vitro experiments that ALA favors the polarization of mice CD4 + T cells toward Tregs. Next, we have carried out a translational study where female obese mice and women were supplemented with ALA or vehicle/placebo (mice: 2.5 gALA /kgfood ; 6 weeks; women: 600 mgALA /day, 8 weeks) while following a protocol including regular exercise and a change in diet. Fatty acid oxidation potential and activity of nuclear erythroid-related factor 2 (NRF2) of mouse secondary lymphoid tissues were improved by ALA supplementation. ALA reduced visceral adipose tissue (VAT) mass and preserved Tregs in VAT in mice. In women, ALA supplementation induced significant metabolic changes of circulating CD4 + T cells including increased oxidative capacity and fatty acid oxidation, ameliorated their redox status, and improved the reduction of visceral fat mass. While appropriate biological markers are still required to be used in clinics to judge the effectiveness of long-term obesity treatment, further studies in female mice and women are needed to determine whether these immunometabolic changes would reduce VAT mass-associated risk for secondary health issues arising from obesity.

Gene Doping with Peroxisome-Proliferator-Activated Receptor Beta/Delta Agonists Alters Immunity but Exercise Training Mitigates the Detection of Effects in Blood Samples.

Synthetic ligands of peroxisome-proliferator-activated receptor beta/delta (PPARß/δ) are being used as performance-enhancing drugs by athletes. Since we previously showed that PPARß/δ activation affects T cell biology, we wanted to investigate whether a specific blood T cell signature could be employed as a method to detect the use of PPARß/δ agonists. We analyzed in primary human T cells the in vitro effect of PPARß/δ activation on fatty acid oxidation (FAO) and on their differentiation into regulatory T cells (Tregs). Furthermore, we conducted studies in mice assigned to groups according to an 8-week exercise training program and/or a 6-week treatment with 3 mg/kg/day of GW0742, a PPARß/δ agonist, in order to (1) determine the immune impact of the treatment on secondary lymphoid organs and to (2) validate a blood signature. Our results show that PPARß/δ activation increases FAO potential in human and mouse T cells and mouse secondary lymphoid organs. This was accompanied by increased Treg polarization of human primary T cells. Moreover, Treg prevalence in mouse lymph nodes was increased when PPARß/δ activation was combined with exercise training. Lastly, PPARß/δ activation increased FAO potential in mouse blood T cells. Unfortunately, this signature was masked by training in mice. In conclusion, beyond the fact that it is unlikely that this signature could be used as a doping-control strategy, our results suggest that the use of PPARß/δ agonists could have potential detrimental immune effects that may not be detectable in blood samples.

Decrease in αß/γδ T-cell ratio is accompanied by a reduction in high-fat diet-induced weight gain, insulin resistance, and inflammation.

The implication of αß and γδ T cells in obesity-associated inflammation and insulin resistance (IR) remains uncertain. Mice lacking γδ T cells show either no difference or a decrease in high-fat diet (HFD)-induced IR, whereas partial depletion in γδ T cells does not protect from HFD-induced IR. αß T-cell deficiency leads to a decrease in white adipose tissue (WAT) inflammation and IR without weight change, but partial depletion of these cells has not been studied. We previously described a mouse model overexpressing peroxisome proliferator-activated receptor ß (PPAR-ß) specifically in T cells [transgenic (Tg) T-PPAR-ß] that exhibits a partial depletion in αß T cells and no change in γδ T-cell number. This results in a decreased αß/γδ T-cell ratio in lymphoid organs. We now show that Tg T-PPAR-ß mice are partially protected against HFD-induced weight gain and exhibit decreased IR and liver steatosis independently of animal weight. These mice display an alteration of WAT-depots distribution with an increased epididymal-WAT mass and a decreased subcutaneous WAT mass. Immune cell number is decreased in both WAT-depots, except for γδ T cells, which are increased in epididymal-WAT. Overall, we show that decreasing αß/γδ T-cell ratio in WAT-depots alters their inflammatory state and mass repartition, which might be involved in improvement of insulin sensitivity.-Le Menn, G., Sibille, B., Murdaca, J., Rousseau, A.-S., Squillace, R., Vergoni, B., Cormont, M., Niot, I., Grimaldi, P. A., Mothe-Satney, I., Neels, J. G. Decrease in αß/γδ T-cell ratio is accompanied by a reduction in high-fat diet-induced weight gain, insulin resistance, and inflammation.

Complementary Immunometabolic Effects of Exercise and PPARß/δ Agonist in the Context of Diet-Induced Weight Loss in Obese Female Mice.

Regular aerobic exercise, independently of weight loss, improves metabolic and anti-inflammatory states, and can be regarded as beneficial in counteracting obesity-induced low-grade inflammation. However, it is still unknown how exercise alters immunometabolism in a context of dietary changes. Agonists of the Peroxisome Proliferator Activated-Receptor beta/delta (PPARß/δ) have been studied this last decade as "exercise-mimetics", which are potential therapies for metabolic diseases. In this study, we address the question of whether PPARß/δ agonist treatment would improve the immunometabolic changes induced by exercise in diet-induced obese female mice, having switched from a high fat diet to a normal diet. 24 mice were assigned to groups according to an 8-week exercise training program and/or an 8-week treatment with 3 mg/kg/day of GW0742, a PPARß/δ agonist. Our results show metabolic changes of peripheral lymphoid tissues with PPARß/δ agonist (increase in fatty acid oxidation gene expression) or exercise (increase in AMPK activity) and a potentiating effect of the combination of both on the percentage of anti-inflammatory Foxp3+ T cells. Those effects are associated with a decreased visceral adipose tissue mass and skeletal muscle inflammation (TNF-α, Il-6, Il-1ß mRNA level), an increase in skeletal muscle oxidative capacities (citrate synthase activity, endurance capacity), and insulin sensitivity. We conclude that a therapeutic approach targeting the PPARß/δ pathway would improve obesity treatment.

α-Lipoic acid up-regulates expression of peroxisome proliferator-activated receptor ß in skeletal muscle: involvement of the JNK signaling pathway.

We hypothesized that α-lipoic acid (α-LA) might interact with the transcriptional control of peroxisome proliferator-activated receptor (PPAR)ß in skeletal muscle. Molecular mechanisms were investigated using differentiated C2C12 myotubes treated with α-LA and/or PPARß agonist GW0742. In vivo studies with 3-mo-old C57Bl6 mice were realized: voluntary wheel running (VWR) training (7 wk), and a 6 wk diet containing (or not) α-LA (0.25% wt/wt). This last condition was combined with (or not) 1 bout of treadmill exercise (18 m/min for 1 h). Using a reporter assay, we demonstrate that α-LA is not an agonist of PPARß but regulates PPARß target gene expression through an active PPARß pathway. GW0742-induced pyruvate dehydrogenase kinase 4 mRNA is potentiated by α-LA. In C2C12, α-LA lowers the activation of the JNK signaling pathway and increases PPARß mRNA and protein levels (2-fold) to the same extent as with the JNK inhibitor SP600125. Similarly to VWR training effect, PPARß expression increases (2-fold) in vastus lateralis of animals fed an α-LA-enriched diet. However, α-LA treatment does not further stimulate the adaptive up-regulation of PPARß observed in response to 1 bout of exercise. We have identified a novel mechanism of regulation of PPARß expression/action in skeletal muscle with potential physiologic application through the action of α-LA, involving the JNK pathway.

Diabetes-Induced Changes in Macrophage Biology Might Lead to Reduced Risk for Abdominal Aortic Aneurysm Development.

Type 2 diabetes patients are less likely to develop an abdominal aortic aneurysm (AAA). Since macrophages play a crucial role in AAA development, we hypothesized that this decrease in AAA risk in diabetic patients might be due to diabetes-induced changes in macrophage biology. To test this hypothesis, we treated primary macrophages obtained from healthy human volunteers with serum from non-diabetic vs. diabetic AAA patients and observed differences in extracellular acidification and the expression of genes involved in glycolysis and lipid oxidation. These results suggest an increase in metabolism in macrophages treated with serum from diabetic AAA patients. Since serum samples used did not differ in glucose content, these changes are not likely to be caused by differences in glycemia. Macrophage functions have been shown to be linked to their metabolism. In line with this, our data suggest that this increase in macrophage metabolism is accompanied by a shift towards an anti-inflammatory state. Together, these results support a model where diabetes-induced changes in metabolism in macrophages might lead to a reduced risk for AAA development.

Invalidation of the Transcriptional Modulator of Lipid Metabolism PPARß/δ in T Cells Prevents Age-Related Alteration of Body Composition and Loss of Endurance Capacity.

Anti-inflammatory regulatory T cells (Tregs) are the most metabolically flexible CD4+ T cells by using both glycolysis and fatty acid oxidation (FAO) which allow them to migrate in tissues. With aging, Tregs accumulate in secondary lymphoid organs and are involved in impairment of skeletal muscle (SKM) regeneration and mass maintenance. In this study, we showed that a deletion of a FAO modulator, peroxisome proliferator-activated receptor beta/delta (PPARß/δ), specifically in T cells (KO-T PPARß/δ), increased the number of CD4+ T cells at day 2 following a cardiotoxin-induced SKM regeneration. Older KO-T PPARß/δ mice maintained a Tregs prevalence in lymph nodes similar to young mice. Surprisingly, KO-T PPARß/δ mice were protected from the effects of age on lean and fat mass and endurance capacity. Our results lead us to propose an original potential role of T cell metabolism in the effects of aging on the maintenance of body composition and endurance capacity.

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

BACKGROUND: Although 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. RESULTS: The 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). CONCLUSION: This work provides the first functional in vitro evidence that avian UCP regulates mitochondrial ROS production in situations of enhanced metabolic activity.

Activation of mitochondrial-driven apoptosis in skeletal muscle cells is not mediated by reactive oxygen species production.

While the acquisition of apoptosis resistance is part of the differentiation program of skeletal muscle cells, differentiated muscle cells can undergo apoptosis in response to physiological or pathological stimuli. The generation of reactive oxygen species by mitochondria plays a major role in the control of apoptosis in many cell types. Indeed their involvement in controlling apoptosis in differentiated muscle cells, or in generating resistance to apoptosis remains unknown. Moreover, differentiated muscle cells specifically express the uncoupling protein-3, a mitochondrial protein potentially involved in controlling reactive oxygen species production. To study the role of mitochondrial reactive oxygen species in the control of apoptosis in skeletal muscle cells, L6E9 myoblasts and myotubes were exposed to staurosporine, an inducer of apoptosis via mitochondrial pathways. Staurosporine activated apoptotic pathways (i.e. caspase-3 and caspase-9) increasing reactive oxygen species in myoblasts and, to a minor extent, in myotubes. However, the increase in reactive oxygen species was not needed to induce apoptosis nor was it involved in the differential sensitization of myoblasts and myotubes to apoptosis. Moreover, expression of uncoupling protein-3 in myotubes did not affect reactive oxygen species production, although it produced a slight sensitization for staurosporine-induced apoptosis. Results indicate that apoptotic activation in skeletal muscle cells mainly involves reactive oxygen species-independent mechanisms and that mitochondrial uncoupling protein-3 is not protective either for reactive oxygen species production or for apoptotic activation in muscle cells.

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-kappaB 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.

Peroxisome Proliferator Activated Receptor Beta (PPARß) activity increases the immune response and shortens the early phases of skeletal muscle regeneration.

Peroxisome Proliferator-Activated Receptor Beta (PPARß) is a transcription factor playing an important role in both muscle myogenesis and remodeling, and in inflammation. However, its role in the coordination of the transient muscle inflammation and reparation process following muscle injury has not yet been fully determined. We postulated that activation of the PPARß pathway alters the early phase of the muscle regeneration process, i.e. when immune cells infiltrate in injured muscle. Tibialis anteriors of C57BL6/J mice treated or not with the PPARß agonist GW0742 were injected with cardiotoxin (or with physiological serum for the contralateral muscle). Muscle regeneration was monitored on days 4, 7, and 14 post-injury. We found that treatment of mice with GW0742 increased, at day 4 post-damage, the recruitment of immune cells (M1 and M2 macrophages) and upregulated the expression of the anti-inflammatory cytokine IL-10 and TGF-ß mRNA. Those effects were accompanied by a significant increase at day 4 of myogenic regulatory factors (Pax7, MyoD, Myf5, Myogenin) mRNA in GW0742-treated mice. However, we showed an earlier return (7 days vs 14 days) of Myf5 and Myogenin to basal levels in GW0742- compared to DMSO-treated mice. Differential effects of GW0742 observed during the regeneration were associated with variations of PPARß pathway activity. Collectively, our findings indicate that PPARß pathway activity shortens the early phases of skeletal muscle regeneration by increasing the immune response.

A role for Peroxisome Proliferator-Activated Receptor Beta in T cell development.

Metabolism plays an important role in T cell biology and changes in metabolism drive T cell differentiation and fate. Most research on the role of metabolism in T lymphocytes focuses on mature T cells while only few studies have investigated the role of metabolism in T cell development. In this study, we report that activation or overexpression of the transcription factor Peroxisome Proliferator-Activated Receptor ß (PPARß) increases fatty acid oxidation in T cells. Furthermore, using both in vivo and in vitro models, we demonstrate that PPARß activation/overexpression inhibits thymic T cell development by decreasing proliferation of CD4-CD8- double-negative stage 4 (DN4) thymocytes. These results support a model where PPARß activation/overexpression favours fatty acid- instead of glucose-oxidation in developing T cells, thereby hampering the proliferative burst normally occurring at the DN4 stage of T cell development. As a consequence, the αß T cells that are derived from DN4 thymocytes are dramatically decreased in peripheral lymphoid tissues, while the γδ T cell population remains untouched. This is the first report of a direct role for a member of the PPAR family of nuclear receptors in the development of T cells.

Fat intake reverses the beneficial effects of low caloric intake on skeletal muscle mitochondrial H(2)O(2) production.

Food restriction is the most effective modulator of oxidative stress and it is believed that a reduction in caloric intake per se is responsible for the reduced generation of reactive oxygen species (ROS) by mitochondria. Hydrogen peroxide (H(2)O(2)) generation and oxygen consumption (O(2)) by skeletal muscle mitochondria were determined in a peculiar strain of rats (Lou/C) characterized by a self-low-caloric intake and a dietary preference for fat. These rats were fed either with a standard high-carbohydrate (HC) or a high-fat (HF) diet and the results were compared to those measured in Wistar rats fed a HC diet. H(2)O(2) production was significantly reduced in Lou/C rats fed a HC diet; this effect was not due to a lower O(2) consumption but rather to a decrease in rotenone-sensitive NADH-ubiquinone oxidoreductase activity and increased expression of uncoupling proteins 2 and 3. The reduced H(2)O(2) generation displayed by Lou/C rats was accompanied by a significant inhibition of permeability transition pore (PTP) opening. H(2)O(2) production was restored and PTP inhibition was relieved when Lou/C rats were allowed to eat a HF diet, suggesting that the reduced oxidative stress provided by low caloric intake is lost when fat proportion in the diet is increased.

Leanness of Lou/C rats does not require higher thermogenic capacity of brown adipose tissue.

Lou/C rats, an inbred strain of Wistar origin, remain lean throughout life and therefore represent a remarkable model of obesity resistance. To date, the exact mechanisms responsible for the leanness of Lou/C rats remain unknown. The aim of the present study was to investigate whether the leanness of Lou/C rats relies on increased thermogenic capacities in brown adipose tissue (BAT). Results showed that although daily energy expenditure was higher in Lou/C than in Wistar rats, BAT thermogenic capacity was not enhanced in Lou/C rats kept at thermoneutrality as demonstrated by reduced thermogenic response to norepinephrine in vivo, similar oxidative activity of BAT isolated mitochondria in vitro, similar levels of UCP1 mRNA and lower abundance of UCP1 protein in interscapular BAT depots. Relative abundance of ß3-adrenergic receptor mRNA was lower in Lou/C BAT while that of GLUT4, FABP or CPT1 was not altered. Activity-related energy expenditure was however considerably increased at thermoneutrality as Lou/C rats demonstrated an impressively high spontaneous running activity in voluntary running wheels. Prolonged cold-exposure (4 °C) depressed the spontaneous running activity of Lou/C rats while BAT thermogenic capacity was increased as reflected by rises in BAT mass, oxidative activity and UCP1 expression. It is concluded that the leanness of Lou/C rats cannot be ascribed to higher thermogenic capacity of brown fat but rather to, at least in part, increased locomotor activity. BAT is not deficient in this rat strain as it can be stimulated by cold exposure when locomotor activity is reduced suggesting some substitution between these thermogenic processes.

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

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 degrees 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 gamma coactivator-1alpha (PGC-1alpha) 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 H(2)O(2) 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.

Mitochondrial adaptations to steatohepatitis induced by a methionine- and choline-deficient diet.

Nonalcoholic fatty liver disease (NAFLD) has become common liver disease in Western countries. There is accumulating evidence that mitochondria play a key role in NAFLD. Nevertheless, the mitochondrial consequences of steatohepatitis are still unknown. The bioenergetic changes induced in a methionine- and choline-deficient diet (MCDD) model of steatohepatitis were studied in rats. Liver mitochondria from MCDD rats exhibited a higher rate of oxidative phosphorylation with various substrates, a rise in cytochrome oxidase (COX) activity, and an increased content in cytochrome aa3. This higher oxidative activity was associated with a low efficiency of the oxidative phosphorylation (ATP/O, i.e., number of ATP synthesized/natom O consumed). Addition of a low concentration of cyanide, a specific COX inhibitor, restored the efficiency of mitochondria from MCDD rats back to the control level. Furthermore, the relation between respiratory rate and protonmotive force (in the nonphosphorylating state) was shifted to the left in mitochondria from MCDD rats, with or without cyanide. These results indicated that, in MCDD rats, mitochondrial ATP synthesis efficiency was decreased in relation to both proton pump slipping at the COX level and increased proton leak although the relative contribution of each phenomenon could not be discriminated. MCDD mitochondria also showed a low reactive oxygen species production and a high lipid oxidation potential. We conclude that, in MCDD-fed rats, liver mitochondria exhibit an energy wastage that may contribute to limit steatosis and oxidative stress in this model of steatohepatitis.

Long term highly saturated fat diet does not induce NASH in Wistar rats.

BACKGROUND: Understanding of nonalcoholic steatohepatitis (NASH) is hampered by the lack of a suitable model. Our aim was to investigate whether long term high saturated-fat feeding would induce NASH in rats. METHODS: 21 day-old rats fed high fat diets for 14 weeks, with either coconut oil or butter, and were compared with rats feeding a standard diet or a methionine choline-deficient (MCD) diet, a non physiological model of NASH. RESULTS: MCDD fed rats rapidly lost weight and showed NASH features. Rats fed coconut (86% of saturated fatty acid) or butter (51% of saturated fatty acid) had an increased caloric intake (+143% and +30%). At the end of the study period, total lipid ingestion in term of percentage of energy intake was higher in both coconut (45%) and butter (42%) groups than in the standard (7%) diet group. No change in body mass was observed as compared with standard rats at the end of the experiment. However, high fat fed rats were fattier with enlarged white and brown adipose tissue (BAT) depots, but they showed no liver steatosis and no difference in triglyceride content in hepatocytes, as compared with standard rats. Absence of hepatic lipid accumulation with high fat diets was not related to a higher lipid oxidation by isolated hepatocytes (unchanged ketogenesis and oxygen consumption) or hepatic mitochondrial respiration but was rather associated with a rise in BAT uncoupling protein UCP1 (+25-28% vs standard). CONCLUSION: Long term high saturated fat feeding led to increased "peripheral" fat storage and BAT thermogenesis but did not induce hepatic steatosis and NASH.

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.

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).

Polyunsaturated fatty acid deficiency reverses effects of alcohol on mitochondrial energy metabolism.

BACKGROUND/AIMS: Polyunsaturated fatty acids (PUFA) deficiency is common in patients with alcoholic liver disease. The suitability of reversing such deficiency remains controversial. The aim was to investigate the role played by PUFA deficiency in the occurrence of alcohol-related mitochondrial dysfunction. METHODS: Wistar rats were fed either a control diet with or without alcohol (control and ethanol groups) or a PUFA deficient diet with or without alcohol (PUFA deficient and PUFA deficient+ethanol groups). After 6 weeks, liver mitochondria were isolated for energetic studies and fatty acid analysis. RESULTS: Mitochondria from ethanol fed rats showed a dramatic decrease in oxygen consumption rates and in cytochrome oxidase activity. PUFA deficiency showed an opposite picture. PUFA deficient+ethanol group roughly reach control values, regarding cytochrome oxidase activity and respiratory rates. The relationship between ATP synthesis and respiratory rate was shifted to the left in ethanol group and to the right in PUFA-deficient group. The plots of control and PUFA deficient+ethanol groups were overlapping. Phospholipid arachidonic over linoleic ratio closely correlated to cytochrome oxidase and oxygen uptake. CONCLUSIONS: PUFA deficiency reverses alcohol-related mitochondrial dysfunction via an increase in phospholipid arachidonic over linoleic ratio, which raises cytochrome oxidase activity. Such deficiency may be an adaptive mechanism.