Marc Goubern

Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production

The gene Ucp2 is a member of a family of genes found in animals and plants, encoding a protein homologous to the brown fat uncoupling protein Ucp1 (refs 1–3). As Ucp2 is widely expressed in mammalian tissues, uncouples respiration and resides within a region of genetic linkage to obesity, a role in energy dissipation has been proposed. We demonstrate here, however, that mice lacking Ucp2 following targeted gene disruption are not obese and have a normal response to cold exposure or high-fat diet. Expression of Ucp2 is robust in spleen, lung and isolated macrophages, suggesting a role for Ucp2 in immunity or inflammatory responsiveness. We investigated the response to infection with Toxoplasma gondii in Ucp2−/− mice, and found that they are completely resistant to infection, in contrast with the lethality observed in wild-type littermates. Parasitic cysts and inflammation sites in brain were significantly reduced in Ucp2−/− mice (63% decrease, P<0.04). Macrophages from Ucp2 −/− mice generated more reactive oxygen species than wild-type mice (80% increase, P<0.001) in response to T. gondii, and had a fivefold greater toxoplasmacidal activity in vitro compared with wild-type mice (P<0.001 ), which was absent in the presence of a quencher of reactive oxygen species (ROS). Our results indicate a role for Ucp2 in the limitation of ROS and macrophage-mediated immunity.

Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition

Upon induction of permeability transition with different agents (Ca2+, tert‐butyl hydroperoxide, atractyloside), mouse hepatocyte mitochondria manifest a disruption of outer membrane integrity leading to the release of cytochrome c and apoptosis‐inducing factor (AIF), two proteins which are involved in programmed cell death (apoptosis). Chelation of Ca2+ shortly (within 2 min) after its addition to isolated mitochondria reestablished the mitochondrial transmembrane potential (ΔΨm), prevented induction of large amplitude swelling and release of both cytochrome c and AIF. In contrast, late Ca2+ chelation (10 min after addition of Ca2+) failed to affect these parameters. Cytochrome c appears to be released through a mechanically damaged outer mitochondrial membrane rather than via a specific release mechanism. These findings clarify the mechanisms through which irreversible permeability transition occurs with subsequent large amplitude swelling culminating in the release of intermembrane proteins from mitochondria. Moreover, they confirm the hypothesis formulated by Skulachev [FEBS Lett. 397 (1996) 7–10 and Q. Rev. Biophys. 29 (1996) 169–202] linking permeability transition to activation of the apoptogenic catabolic enzymes.

Retinoids activate proton transport by the uncoupling proteins UCP1 and UCP2

In mammalian brown adipose tissue, thermogenesis is explained by uncoupling mitochondrial respiration from ATP synthesis. Uncoupling protein‐1 (UCP1) is responsible for this uncoupled state, because it allows proton re‐entry into the matrix and thus dissipates the proton gradient generated by the respiratory chain. Proton transport by UCP1 is regulated negatively by nucleotides and positively by fatty acids. Adrenergic stimulation of brown adipocytes stimulates lipolysis and therefore enhances uncoupling and thermogenesis. Adrenergic stimulation also boosts ucp1 gene transcription. Since retinoic acid also promotes ucp1 gene transcription and its structure makes it a possible activator of UCP1, we hypothesized that retinoic acid, like noradrenaline, could have a dual action and trigger the activity of the protein UCP1 itself. Here we show that retinoic acid strongly increases proton transport by UCP1 in brown adipose tissue mitochondria and that it is much more potent than fatty acids. These data are corroborated with yeast mitochondria where UCP1 was introduced by genetic manipulation. The yeast expression system allows the comparison of the UCP1 with the newly described homologues UCP2 and UCP3. The search for regulators of UCP2 has demonstrated that it is positively regulated by retinoids in a pH‐dependent manner.

Long term treatment with olanzapine mixed with the food in male rats induces body fat deposition with no increase in body weight and no thermogenic alteration.

Body weight gain is a worrying side effect of many new antipsychotic drugs. The mechanisms by which antipsychotic drugs increase weight in humans are not known. Attempts to model the metabolic effects of antipsychotic drugs in the animal have not been successful. Female rats appear to be sensitive to the effects of antipsychotics, but male rats less, and this does not match the clinical situation in humans. In previous rodent studies, antipsychotics were always given by daily gavage or injections. Antipsychotics have different pharmacokinetics in rodents and humans, and in the present study, we tested the hypothesis that the insensitivity of male rats to the effects of antipsychotics could be related to their mode of administration. Thus, we administered antipsychotic drugs mixed with the food. Animals were treated during 6 weeks with haloperidol (1mg/kg), olanzapine (1mg/kg), ziprasidone (10mg/kg), or a control solution. Animals were allowed to self-select food among three macronutrients (carbohydrates, lipids and proteins). Food selection was measured throughout the study. At the end of the study, body composition was measured by dissection and weighing of the rats main organs and tissues. Mitochondrial thermogenesis was measured in brown adipose tissue in olanzapine-treated animals. Circulating leptin, insulin, glucose, triglycerides, cholesterol, high-density lipoprotein (HDL) were also assayed at the end of the study. The results show that none of the antipsychotic treatments modified caloric intake, food selection or body weight. Olanzapine did not alter mitochondrial thermogenesis. However, haloperidol and olanzapine induced a significant increase in adiposity and circulating leptin. Ziprasidone produced a moderate fat accumulation. It is concluded that mixing antipsychotic treatments with the food provides a reliable animal model of antipsychotic-induced fat accumulation.

Variations in energization parameters and proton conductance induced by cold adaptation and essential fatty acid deficiency in mitochondria of brown adipose tissue in the rat.

Male weanling Long-Evans rats were fed on a low-fat semipurified diet (control diet, 2% sunflower oil; essential fatty acid (EFA) deficient diet, 2% hydrogenated coconut oil) for 9 weeks. In order to modulate need for non-shivering thermogenesis, groups of rats on each diet were exposed at 28 degrees C (thermoneutrality) and at 5 degrees C (cold acclimation) for the last 5 weeks. In brown adipose tissue (BAT) mitochondria, several parameters of mitochondrial energization, protonmotive force (delta p) and its components delta pH and membrane potential, delta psi, were investigated. Simultaneous measurement of oxygen consumption and delta psi (the main component of delta p) was performed by varying alpha-glycerophosphate concentration and the force/flux relationship of the mitochondria was established by comparison of proton conductance, CmH+, over the whole range of protonmotive force. delta p. In the absence of GDP, at 28 degrees C, EFA deficiency induced a marked increase in CmH+. Cold acclimation led to comparable enhanced CmH+ in control and EFA-deficient mitochondria. In the presence of GDP which binds and inhibits the BAT 32 kDa uncoupling protein, CmH+ was the same in 28 degrees C and 5 degrees C control mitochondria, but EFA deficiency led to an enhanced GDP independent CmH+ at 28 degrees C and to a lesser extent at 5 degrees C. These results are discussed with reference to substantial changes in mitochondrial lipid composition induced by the deficiency.

Intraperitoneal leptin modifies macronutrient choice in self-selecting rats

This study aimed to evaluate the consequences on food intake and body weight (BW) of leptin administration in rats receiving a choice between the three macronutrients. Two studies were performed: during the first, rats received an acute intraperitoneal (IP) leptin administration (1 mg/kg) twice (at 8 and 14 weeks of age), at the beginning of the nocturnal cycle, while during the second, they received a chronic leptin infusion (osmotic minipump, 7 days). The total 24-h food intake after acute leptin injections was reduced by 14% and 17%, respectively. Body weight gain (BWG) after leptin injections was about half that seen on control days. Chronic leptin infusion reduced total intake, affecting mainly protein (P). Fat intake increased slightly since day 2 and became significant on the fourth day. After the leptin infusion, carbohydrate (CHO) eaters (>35% carbohydrate/total energy) significantly reduced the carbohydrate proportion in their total energy intake. There was no difference concerning macronutrient selection by fat eaters (Hfat). Leptin infusion reduced the number of mixed meals on the first day. In addition, the thermogenesis of brown adipose tissue (BAT) was higher in leptin than in control (C) rats. Consequently, leptin injections reduced food intake and BWG and increased thermogenesis, thus acting on the two terms of the energy balance. Moreover, leptin has different effects on macronutrient preferences, dependent upon age (tests 1 and 2) and the type (acute or chronic) of injection. High leptinemia level related to age or to minipump infusion lead to leptin resistance as found in old or obese subjects. It could explain our results.

Exercise-training reduces BAT thermogenesis in rats

In the energy balance equation, physical activity represents one component of energy expenditure. From various studies it appears that exercise-training does not affect clearly thermogenesis which depends on brown adipose tissue (BAT) activity. In the present work we examine how exercise-training can influence food intake and body weight regulation in relation to BAT thermogenesis. The proton conductance of the uncoupling protein of BAT was examined in male adult Wistar trained 2 h/day for 20 days and compared to that of sedentary (2 h of fasting instead of exercise) or control animals. All animals were provided with separate sources of the 3 macronutrients (protein, fat and carbohydrate) containing an identical percentage of vitamins, salt mixture and cellulose powder. At the end of training, rats were placed at 5 degrees C during 10 days, then during 4 days at 28 degrees C. This condition has been demonstrated to favour and amplify BAT responsiveness to moderate modifications of stimulation. The body weight of trained rats became significantly lower than that of the control and sedentary rats and this difference persisted all throughout the experiment. When placed at 5 degrees C, all rats increased their total ingestion: control rats enhanced fat intake, while sedentary and trained rats enhanced carbohydrate ingestion. When placed at 28 degrees C, all rats had identical total energy and that of the 3 items intakes. BAT proton conductance was about 40% lower in the trained compared with the sedentary plus the control rats. This indicated a lower BAT thermogenic activity in the trained animals. It could be concluded that exercise-training in rats induces negative energy balance; the reduced BAT activity could restrain weight loss and overeating.

Energy balance in an inbred strain of rats: comparison with the Wistar strain.

Food intake and body weight gain were examined in two groups of male rats (7 weeks): an inbred strain, Dark Agouti (DA, n = 12) and a noninbred strain, Wistar (n = 13). The animals were allowed to select their diet from separate sources of the three macronutrients protein, fat, and carbohydrate. After 10 days of adaptation to the diets, body weights and food intakes were measured for 3 weeks. During this period, meal patterns were recorded for at least 5 days in each rat. Then, rats were switched to a chow diet (UAR, A.O4) for 10 days. The total caloric intake of DA rats was 60% that of Wistar rats, while their body weight gain was 25% that of Wistar rats (1.3 g/day in DA vs. 5.3 g in Wistar). However, when energy intake was related to total body weight, there was no difference in energy ingestion. It was observed that DA rats ingested mainly proteins (45%) and fats (41%), while Wistar rats ingested an identical proportion of proteins and carbohydrates (40%). The percent of total white adipose tissue to total body weight was identical in both strains (6% on average). Brown adipose tissue thermogenic activity of DA rats was threefold higher than in Wistar rats. This could be one of the elements responsible for the lower body weight gain of this group of rats. Self-selected food intake of the inbred DA strain of rats, in contrast to what was expected, was greatly variable.

Effect of sympathetic de-activation on thermogenic function and membrane lipid composition in mitochondria of brown adipose tissue

Male Long-Evans rats (9 weeks of age) were exposed to cold (5 degrees C) for 10 days. Then, sympathetic de-activation of brown adipose tissue (BAT) was performed either by BAT surgical denervation (Sy) or by warm re-exposure at 28 degrees C (WE) for 4 days. The incidence of the two treatments on thermogenic activity of BAT mitochondrial membranes and their lipid composition was investigated. Sy and WE induced a large decrease in GDP binding on the uncoupling protein (UCP) (43% and 82%, respectively). Several parameters of mitochondrial energization were investigated. Sy and WE substantially decreased UCP-dependent proton conductance (CmH+) over the whole range of protonmotive force. CmH+ showed greater variation than GDP binding. The low basal UCP-independent CmH+ was the same in all groups. Comparison of GDP binding and CmH+ with UCP content which is not modified revealed a masking of both the nucleotide binding site and the proton channel. Sy and WE induced the same increase of phosphatidylcholine to phosphatidylethanolamine ratio (16%) but had opposite effects on fatty acid unsaturation. The results were discussed with reference to functional significance of these variations in BAT mitochondrial thermogenic activity and lipid composition.

Concomitant food intake and adipose tissue responses under chronic insulin infusion in rats

Body weight (BW), food intake (FI), and activity of white adipose tissue (WAT) and brown adipose tissue (BAT) were studied in adult male rats under chronic insulin infusion. Insulin was infused for 4, 7 or 10 days via implanted minipumps. Insulin-treated rats gained more BW than control rats until 7th day of infusion. At 10 days, the difference in BW decreased. The average cumulative FI was significantly higher after 4, 7 and 10 days of insulin infusion. Feed efficiency (FE) was increased in insulin-treated rats after 4 and 7 days. An increase in WAT weight was observed in insulin-treated rats together with an increased activity of lipogenic enzymes. BAT weight was augmented after 4 days of insulin infusion. This was due mainly to lipid accumulation. Specific mitochondrial guanosine diphosphate (GDP) binding was significantly decreased by 58% in insulin-treated rats after 4 days of infusion. This reduced thermogenic activity, along with the increased FI and FE were responsible for the rapid BW gain observed during the first 7 days of insulin infusion.