Claire Pecqueur

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.

Uncoupling Protein 2, in Vivo Distribution, Induction upon Oxidative Stress, and Evidence for Translational Regulation

Uncoupling protein 2 (UCP2) belongs to the mitochondrial anion carrier family and partially uncouples respiration from ATP synthesis when expressed in recombinant yeast mitochondria. We generated a highly sensitive polyclonal antibody against human UCP2. Its reactivity toward mitochondrial proteins was compared between wild type and ucp2(−/−) mice, leading to non-ambiguous identification of UCP2. We detected UCP2 in spleen, lung, stomach, and white adipose tissue. No UCP2 was detected in heart, skeletal muscle, liver, and brown adipose tissue. The level of UCP2 in spleen mitochondria is less than 1% of the level of UCP1 in brown adipose tissue mitochondria. Starvation and LPS treatments increase UCP2 level up to 12 times in lung and stomach, which supports the hypothesis that UCP2 responds to oxidative stress situations. Stimulation of the UCP2 expression occurs without any change in UCP2 mRNA levels. This is explained by translational regulation of the UCP2 mRNA. We have shown that an upstream open reading frame located in exon two of theucp2 gene strongly inhibits the expression of the protein. This further level of regulation of the ucp2 gene provides a mechanism by which expression can be strongly and rapidly induced under stress conditions.

Uncoupling protein-2 controls proliferation by promoting fatty acid oxidation and limiting glycolysis-derived pyruvate utilization

Uncoupling protein‐2 (UCP2) belongs to the mitochondrial carrier family and has been thought to be involved in suppressing mitochondrial ROS production through uncoupling mitochondrial respiration from ATP synthesis. However, we show here that loss of function of UCP2 does not result in a significant increase in ROS production or an increased propensity for cells to undergo senescence in culture. Instead, Ucp2—/— cells display enhanced proliferation associated with a metabolic switch from fatty acid oxidation to glucose metabolism. This metabolic switch requires the unrestricted availability of glucose, and Ucp2— /— cells more readily activate autophagy than wild‐type cells when deprived of glucose. Altogether, these results suggest that UCP2 promotes mitochondrial fatty acid oxidation while limiting mitochondrial catabolism of pyruvate. The persistence of fatty acid catabolism in Ucp2+/ + cells during a proliferative response correlates with reduced cell proliferation and enhances resistance to glucose starvation‐induced autophagy.— Pecqueur C., Bui, T., Gelly, C., Hauchard, J., Barbot, C., Bouillaud, F., Ricquier, D., Miroux, B., Thompson C. B. Uncoupling protein‐2 controls proliferation by promoting fatty acid oxidation and limiting glycolysis‐derived pyruvate utilization. FASEB J. 22, 9–18 (2008)

In vivo resistance of lipolysis to epinephrine. A new feature of childhood onset obesity.

A decreased mobilization of triglycerides may contribute to fat accumulation in adipocytes, leading to obesity. However, this hypothesis remains to be proven. In this study, epinephrine-induced lipid mobilization was investigated in vivo in nine markedly obese children (160+/-5% ideal body weight) aged 12.1+/-0.1 yr during the dynamic phase of fat deposition, compared with six age-matched nonobese children. As an in vivo index of lipolysis, we measured glycerol flux using a nonradioactive tracer dilution approach, and plasma free fatty acid concentrations. In the basal state, the obese children had a 30% lower rate of glycerol release per unit fat mass than the lean children. To study the regulation of lipolysis, epinephrine was infused stepwise at fixed doses of 0.75 and then 1. 50 microg/min in both groups. In lean children, glycerol flux and plasma free fatty acid increased to an average of 249-246% of basal values, respectively, in response to a mean plasma epinephrine of 396+/-41 pg/ml. The corresponding increase was only 55-72% in the obese children, although their mean plasma epinephrine reached 606+/-68 pg/ml. All obese and nonobese children, except an Arg64Trp heterozygote, were homozygotes for Trp at position 64 of their beta3-adrenoreceptor. The resistance of lipolysis to epinephrine showed no relationship with the Arg64 polymorphism of the beta3-adrenoreceptor gene. In summary, in vivo lipolysis, which mostly reflects the mobilization of lipid stores from subcutaneous adipose tissue, shows a decreased sensitivity to epinephrine in childhood onset obesity. Since our study was carried out in obese children during the dynamic phase of fat accumulation, the observed resistance to catecholamines might possibly be causative rather than the result of obesity.

Genetic and physiological analysis of the role of uncoupling proteins in human energy homeostasis

Abstract. The metabolic utilization of substrates results in ATP synthesis and energy loss as heat. In tissues and cells the mitochondria reoxidize reduced coenzymes and phosphorylate ADP. A significant proportion of the energy is released through thermogenesis by mitochondria. This is due to a less than perfect coupling of cellular respiration to ATP synthesis. Previous studies of brown adipocytes, which are cells specialized in regulatory thermogenesis, have shown that heat production is due to the regulated activity and synthesis of a particular proton transporter in the inner membrane of brown adipocyte mitochondria – uncoupling protein (UCP) 1. UCP homologues have recently been identified. UCP2 is widely expressed in human tissues, whereas UCP3 is expressed predominantly in human skeletal muscles. These novel UCPs represent genes which are potentially important for regulation of metabolic pathways and energy expenditure in humans. Biochemical and genetic studies support a role for these novel UCPs in metabolic regulations in humans. However, several physiological studies question such a role. Importantly, UCP2 and UCP3 seem to be able to control the activity of mitochondria in response to oxidants.

Transcriptional Activation of the Human ucp1 Gene in a Rodent Cell Line SYNERGISM OF RETINOIDS, ISOPROTERENOL, AND THIAZOLIDINEDIONE IS MEDIATED BY A MULTIPARTITE RESPONSE ELEMENT

Uncoupling protein 1 (UCP1) is uniquely expressed in brown adipocytes and generates heat production by uncoupling respiration from ATP synthesis. The activatory effects of norepinephrine and retinoic acid (RA) on rodent ucp1gene transcription have been well characterized. These effects are mediated by a 211-base pair (bp) enhancer which is also sufficient to restrict expression to brown adipose tissue. The molecular mechanisms controlling the transcription of the human ucp1 gene are unknown. In order to study the transcriptional regulation of the human gene, we set up chloramphenicol acetyltransferase constructs containing the entire or deleted 5′ regions upstream of the transcriptional start site of the gene. These constructs were transiently transfected in a mouse cell line. A 350-bp hormone response region showing a significant homology with the rat ucp1 enhancer and located between theBclI polymorphic site and an AatII site (bp −3820/−3470) was detected. This region was sufficient to mediate the stimulation by RA and by combined treatments (RA + isoproterenol (ISO), RA + thiazolidinedione (TZD), or RA + ISO + TZD). The highest stimulation, a 26-fold increase in basal activity, was obtained by RA + ISO + TZD treatment. In contrast to the rodent gene, under our conditions, the effect of ISO and/or TZD is dependent on RA stimulation. Analysis of 105 bp inside the 350-bp element by site-directed mutagenesis and gel retardation experiments demonstrated that a multipartite response element mediates the drug stimulation. This region binds RARs and RXRs nuclear factors, CREB/ATF factors, and also PPARγ despite the absence of a consensus peroxisome-proliferator response element. The activation of the human ucp1 gene transcription by certain hormones or drugs, and the identification of the cis-elements involved, will help to identify new compounds activating fat oxidation and energy expenditure in humans.

UCP2, a metabolic sensor coupling glucose oxidation to mitochondrial metabolism?

Mitochondrial uncoupling of oxidative phosphorylation may serve a variety of purposes such as the regulation of substrate oxidation, free radical production (a major by‐product of mitochondrial respiration) and ATP production and turnover. As regulators of energy expenditure and antioxidant defenses, uncoupling proteins would seem to offer an attractive mechanism by which to explain the control of body weight, resting metabolic rate and aging. As a result, the discovery of UCP1 homologues has led to an impressive number of publications. However, 10 years after their identification, no consensus has been found concerning the function of UCP homologues, and there are controversies as to whether or not they even have physiologically significant uncoupling activity. Here, we discuss a potential new function for UCP2, as a carrier involved in the coupling between glucose oxidation and mitochondrial metabolism.

Expression of UCP3 in CHO cells does not cause uncoupling, but controls mitochondrial activity in the presence of glucose.

The proton-transport activity of UCP1 (uncoupling protein 1) triggers mitochondrial uncoupling and thermogenesis. The exact role of its close homologues, UCP2 and UCP3, is unclear. Mounting evidence associates them with the control of mitochondrial superoxide production. Using CHO (Chinese-hamster ovary) cells stably expressing UCP3 or UCP1, we found no evidence for respiration uncoupling. The explanation lies in the absence of an appropriate activator of UCP protonophoric function. Accordingly, the addition of retinoic acid uncouples the respiration of the UCP1-expressing clone, but not that of the UCP3-expressing ones. In a glucose-containing medium, the extent of the hyperpolarization of mitochondria by oligomycin was close to 22 mV in the five UCP3-expressing clones, contrasting with the variable values observed with the 15 controls. Our observations suggest that, when glycolysis and mitochondria generate ATP, and in the absence of appropriate activators of proton transport, UCPs do not transport protons (uncoupling), but rather other ions of physiological relevance that control mitochondrial activity. A model is proposed using the known passive transport of pyruvate by UCP1.

Contributions of studies on uncoupling proteins to research on metabolic diseases.

Abstract. Ricquier D, Fleury C, Larose M, Sanchis D, Pecqueur C, Raimbault S, Gelly C, Vacher D, Cassard‐Doulcier A‐M, Lévi‐Meyrueis C, Champigny O, Miroux B, Bouillaud F (Centre National de la Recherche Scientifique, Centre de Recherche sur l’Endocrinologie Moléculaire et le Développement, Meudon, France). Contributions of studies on uncoupling proteins to research on metabolic diseases. (Minisymposium: Genes & Obesity). J Intern Med 1999; 245: 637–642.

A new polymorphic site located in the human UCP1 gene controls the in vitro binding of CREB-like factor

Uncoupling protein 1 (UCP1) is uniquely expressed in brown adipose tissue (BAT) and generates heat by uncoupling respiration from ATP synthesis. A defect in BAT thermogenesis has been described in different models of rodent obesity. In humans, the implication of BAT in energy expenditure is still under discussion. A BclI polymorphism associated with fat gain over time has been described in the upstream region of the human UCP1 (hUCP1) gene. In this study, a new polymorphic site linked to the BclI site is described which results in a C to A point mutation, 89 bp downstream of the BclI site, ie at position −3737 bp. This site is located in the recently analysed regulatory region of the hUCP1 gene. The mutation disrupts a consensus site for the binding of ATF/CREB transcription factor family and inhibits the factor binding in vitro. However, transient transfection of a rodent brown adipocyte cell line shows that the isoproterenol (ISO) stimulation of the hUCP1 gene transcription is not significantly affected by this mutation. However, we postulate that the C/A substitution, in human, may contribute to a minor defect in the regulation of hUCP1 transcription and that would explain fat gain over time.