Serge Raimbault

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.

An uncoupling protein homologue putatively involved in facultative muscle thermogenesis in birds

The cDNA of an uncoupling protein (UCP) homologue was obtained by screening a chicken skeletal-muscle library. The predicted 307-amino-acid sequence of avian UCP (avUCP) is 55, 70, 70 and 46% identical with mammalian UCP1, UCP2 and UCP3 and plant UCP respectively. avUCP mRNA expression is restricted to skeletal muscle and its abundance was increased 1.3-fold in a chicken line showing diet-induced thermogenesis, and 3.6- and 2.6-fold in cold-acclimated and glucagon-treated ducklings developing muscle non-shivering thermogenesis respectively. The present data support the implication of avUCP in avian energy expenditure.

Characterization of a melatonin binding site in Siberian hamster brown adipose tissue

Melatonin has been shown, in various rodent species, to mediate photoperiodic effects on body weight and, consequently, fat mass. Pharmacological investigations indicated that the brown adipose tissue of Siberian hamsters possesses a melatonin binding site with a dissociation constant of 570+/-300 pM and a density of 3.2+/-1.8 fmol/mg protein. This binding site can also be detected on mature brown adipocyte membranes. The rank order of potency of a variety of drugs to displace 2-[125I]iodomelatonin from binding sites on Siberian hamster brown adipose tissue was as follows: 2-iodomelatonin > melatonin = prazosin > GR135531 (5-methoxycarbonylamino-N-acetyltryptamine) > N-acetylserotonin > 6-chloromelatonin > S20304 (N-(2-(1-naphthyl)ethyl)cyclobutanecarboxamide) >> methoxamine, phenylephrine, serotonin. Mel(1a) mRNA was not detected by RT-PCR (reverse transcription-polymerase chain reaction) in brown adipose tissue. Melatonin had no effect on either basal or stimulated lipolysis. Moreover, melatonin did not modify intracellular cAMP accumulation or inositol phosphate content. Together, these results suggest that the melatonin binding site characterized in brown adipose tissue is clearly different from the Mel(1) cloned subtype and has some features different from those of the Mel2 subtype.

Comment to Shinohara et al. (1991) FEBS Letters 293, 1731̄74 The uncoupling protein is not expressed in rat liver

Using Northern blot analysis, immunoblotting with purified antibodies and Polymerase Chain Reaction analysis, we were unable to detect the Uncoupling Protein‐UCP or its mRNA in liver of control, cold‐exposed or newborn rats. The unique expression of this protein in brown adipocytes was confirmed. These data refute the surprising recent report on UCP expression in rat liver (Shinohara (1991) FEBS Lett. 293, 173–174). Moreover we report that the hybridization signal obtained by these authors is probably non‐specific and due to the 3′ non‐coding domain of the UCP cDNA probe.

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.

Contribution to the Identification and Analysis of the Mitochondrial Uncoupling Proteins

This review is primarily focused on the contribution of our laboratory to study of themitochondrial uncoupling UCPs. The initial stage was the description of a 32-kDamembranous protein specifically induced in brown adipose tissue mitochondria of cold-adaptedrats. This protein was then shown by others to be responsible for brown fat thermogenesisand was referred to as the uncoupling protein-UCP (recently renamed UCP1). cDNA andgenomic clones of UCP1 were isolated and used to investigate the topology and functionalorganization of the protein in the membrane and the mechanisms of control of UCP1 genetranscription. Orientation of the transmembrane fragments was proposed and specificamino acid residues involved in the inhibition of UCP1 by purine nucleotides wereidentified in recombinant yeast. A potent enhancer mediating the response of the UCP1gene to retinoids and controlling the specific transcription in brown adipocytes wasidentified using transgenic mice. More recently, we identified UCP2, an UCP homologwidely expressed in human and rodent tissues we also collaborated to characterize theplant UCP. Although the biochemical activities and physiological roles of the novel UCPsare not well understood, these recent data stimulate research on mitochondrial carriers,mitochondrial bioenergetics, and energy expenditure.

Expression of the brown fat mitochondria uncoupling protein in Xenopus oocytes and import into mitochondrial membrane

Non shivering thermogenesis of brown adipose tissue is due to the uncoupling protein (UCP), located in the inner mitochondrial membrane, which functions as a proton translocator and can thus uncouple mitochondrial respiration. We describe here the expression of UCP in Xenopus laevis oocytes after injection of UCP mRNA, which was transcribed in vitro. UCP seems to be correctly transported into mitochondria and integrated into the membrane, but we were not able to establish definitely the functionality of this UCP. We conclude that this expression system could be suitable for the study of the mitochondrial import mechanism but not for the examination of physiological properties of UCP.

A Molecular Biology Study of the Uncoupling Protein of Brown Fat Mitochondria. A Contribution to the Analysis of Genes of Mitochondrial Carriers

The uncoupling protein (UCP) is a specialized mitochondrial carrier unique to brown adipose tissue mitochondria. It acts as a proton carrier able to dissipate the proton gradient, to bypass ATP synthesis and to dissipate energy as heat (Nicholls & Locke, 1984). The proton translocating activity of UCP is inhibited by di- and triphosphate purine nucleotides and activated by free fatty acids (Nicholls et al., 1986). Moreover, UCP can be used as a marker to identify thermogenic adipocytes.

The mitochondrial uncoupling protein UCP / genetic and structural studies

Heat production by brown adipocytes is due to uncoupling of the mitochondrial respiratory chain by the uncoupling protein UCP, a nucleotide-inhibitable and free fatty acid-activable proton carrier in the inner membrane, unique to brown adipocyte mitochondria. Our laboratory is studying the mechanisms that restrict UCP gene transcription to brown adipocytes, and the functional organization of the UCP which belongs to the family of mitochondrial transporters. Using cell transfection and transgenic mice evidence was obtained that a region encompassing 3 kb of DNA upstream the transcription start site contains positive and negative elements controlling UCP gene transcription. Transfection experiments based on DNA-CAT constructs identified a strong enhancer at −2.4 kb. This enhancer as well as the proximal region of the promoter were analyzed in detail using DNAse I footprint analysis and band-shift experiments. To study UCP topological organization, antibodies directed against certain subsequences were selected and used. The orientation of 5 out of 6 predicted α-helices was determined and allowed to propose a membranous folding. In collaboration with E. Rial (Madrid), wild and mutated UCP was expressed in yeasts. This strategy was used to demonstrate that none cysteine is essential for UCP and that lysine 268 and glycine 269 are involved in its inhibition by nucleotides.

Molecular Studies of the Mitochondrial Uncoupling Protein

The brown adipocytes play an important role in the regulation of body temperature in hibernating as well as in small and newborn mammals. These thermogenic adipocytes express an Uncoupling Protein — UCP. This mitochondrial carrier, unique to these cells, uncouples mitochondrial ATP synthesis from the respiratory chain activity and is responsible for heat production by brown adipocytes. Besides its important physiological role, UCP is a proton membranous transporter proving the validity of Mitchell’s chemiosmotic theory (Nicholls and Locke 1984, Klingenberg 1990). UCP is also able to transport Cl- ions electrophoretically (Garlid 1990). Both H+ and Cl- transports are inhibited by purine nucleotides and free fatty acids activate H+ translocation (Rial and Nicholls 1989). Another characteristic property of UCP, is that it is a member of an expanding family of mitochondrial ion transporters sharing sequence and structural homologies. In addition to UCP, the main members forming this family are the Adenine Nucleotide Translocator, the Phosphate Carrier and the Oxoglutarate Carrier (Walker 1992).