Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia.

A mitochondrial protein called uncoupling protein (UCP1) plays an important role in generating heat and burning calories by creating a pathway that allows dissipation of the proton electrochemical gradient across the inner mitochondrial membrane in brown adipose tissue, without coupling to any other energy-consuming process. This pathway has been implicated in the regulation of body temperature, body composition and glucose metabolism. However, UCP1-containing brown adipose tissue is unlikely to be involved in weight regulation in adult large-size animals and humans living in a thermoneutral environment (one where an animal does not have to increase oxygen consumption or energy expenditure to lose or gain heat to maintain body temperature), as there is little brown adipose tissue present. We now report the discovery of a gene that codes for a novel uncoupling protein, designated UCP2, which has 59% amino-acid identity to UCP1, and describe properties consistent with a role in diabetes and obesity. In comparison with UCP1, UCP2 has a greater effect on mitochondrial membrane potential when expressed in yeast. Compared to UCP1, the gene is widely expressed in adult human tissues, including tissues rich in macrophages, and it is upregulated in white fat in response to fat feeding. Finally, UCP2 maps to regions of human chromosome 11 and mouse chromosome 7 that have been linked to hyperinsulinaemia and obesity. Our findings suggest that UCP2 has a unique role in energy balance, body weight regulation and thermoregulation and their responses to inflammatory stimuli.

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.

Is alpha-fetoprotein decline a prognostic factor of childhood non-seminomatous germ cell tumours? Results of the French TGM95 study.

PURPOSE: In adults' non-seminomatous germ cell tumours (NS-GCT), alpha-fetoprotein (AFP) decline was identified as an important prognostic factor. We investigated its prognostic value in the French TGM95 study for childhood NS-GCT. PATIENTS AND METHODS: Three risk groups were defined: low risk (LR: localised and completely resected pS1, AFP<15000 ng/ml), with a 'wait-and-see' strategy; intermediate-risk (IR: localised incompletely resected, AFP<15000 ng/ml) with 3-5 vinblastine-bleomycine-cisplatin courses; high risk (HiR: AFP≥15000 ng/ml and/or metastatic) with 4-6 etoposide-ifosfamide-cisplatin courses. The multivariable prognostic analysis for progression-free survival (PFS) included age (±10 years), primary tumour site (1-testis, 2-ovary, 3-extragonadal), extent of disease (1-pS1, 2-loco-regional dissemination, 3-metastasis) and AFP (±10,000 ng/ml). AFP decline prognostic value was investigated in IR + HiR groups using predicted time to normalisation (TTN), AFP change, and difference between observed and expected (based on AFP half-life) area under the curve (O-E AUC). RESULTS: From January 1995 to December 2005, 239 patients (median age = 3years, 60 LR, 65 IR, 114 HiR) were included. Main sites were testis (n = 66), ovary (n = 77) and sacrococcygeal (n = 57). Five-year PFS and OS were 85% (95% confidence interval [CI] = 80-89%) and 93% (89-95%), respectively. Age ≥ 10 years (hazard ratio [HR] = 4.6, 95% CI = 2.1-10.1, p = 0.0001) and extragonadal primary (HR = 6.3, 95% CI = 2.0-19.9, p = 0.005) were significant prognostic factors. In AFP decline analysis (n = 151, 17 events), TTN (p = 0.61) and AFP change (p = 0.10) were not prognostic, whereas we showed a significant effect of O-E AUC (HR = 2.1, 95% CI = 1.0-4.2, p = 0.05). CONCLUSION: Age ≥ 10 years and extragonadal tumours remain as poor prognostic factors. Contrary to adults, TTN is not reliable in paediatric NS-GCT. The prognostic value of O-E AUC should be investigated in larger studies.

Tissue distribution of beta 3-adrenergic receptor mRNA in man.

Expression of mRNA for beta 1-, beta 2-, and beta 3-adrenergic receptors (beta 1-, beta 2-, and beta 3-AR) was investigated in human tissues. beta 1- and beta 2-AR mRNA distribution correlated with that of the cognate receptors established by pharmacological studies. beta 3-AR transcripts were abundant in infant perirenal brown adipose tissue, characterized by the presence of uncoupling protein (UCP) mRNA. In adult whole adipose tissues, beta 3-AR mRNA levels were high in deep deposits such as perirenal and omental, and lower in subcutaneous. In these deposits, UCP mRNA levels paralleled those of beta 3-AR. However, isolated omental and subcutaneous adipose cells, enriched in white adipocytes, expressed beta 3-AR but no UCP transcripts. beta 3-AR mRNA was highly expressed in gallbladder, and to a much lower extent in colon, independently of UCP mRNA. Quadriceps or abdominal muscles, heart, liver, lung, kidney, thyroid, and lymphocytes did not express intrinsic beta 3-AR mRNA. This study demonstrates that substantial amounts of brown adipocytes exist throughout life in adipose deposits, which are generally classified as white. These deposits are the main sites of beta 3-AR expression, which also occurs in gallbladder and colon. beta 3-AR may thus be involved in the control of lipid metabolism, possibly from fat assimilation in the digestive tract, to triglyceride storage and mobilization in adipose tissues.

Tissue-specific and beta-adrenergic regulation of the mitochondrial uncoupling protein gene: control by cis-acting elements in the 5'-flanking region.

Uncoupling protein (UCP) gene expression is tightly restricted to thermogenic brown adipocytes and is rapidly activated by norepinephrine released after cold exposure. To identify cis-acting regulatory elements controlling this gene, a region encompassing 4.5 kilobases of DNA upstream of the transcription start site was analyzed using hybrid UCP-chloramphenicol acetyltransferase reporter gene constructs. Evidence for the presence of both tissue-specific and beta-adrenergic response elements in this 4.5-kilobase region was obtained by comparing the expression of these reporter genes in transfected brown adipocytes (in vitro differentiated), brown preadipocytes, white adipocytes, and Chinese hamster ovary (CHO) cells and from experiments in transgenic animals. Deletion analyses in transfected cells indicated that the minimal region exhibiting promoter activity and tissue specificity is located between -157 and -57 base pairs (bp). A 211-bp activator element located between -2494 and -2283 bp was necessary for full expression in brown adipocytes. This element also activated expression of the homologous -157-bp promoter and expression of a heterologous promoter in both brown adipocytes and CHO cells. A second region, downstream of the activator and possibly located between positions -400 and -157 bp, inhibited the UCP promoter in CHO cells. In mice transgenic for a chloramphenicol acetyltransferase reporter gene containing these elements, expression was both tissue specific and regulatable by environmental temperature changes. These results indicate that both positive and negative cis-acting elements participate in the regulation of UCP gene expression.

Comment to Shinohara et al. (1991) FEBS Letters 293, 173-174. 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.

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.

The gene for rat uncoupling protein: complete sequence, structure of primary transcript and evolutionary relationship between exons.

The complete nucleotide sequence of rat Uncoupling Protein gene has been determined. 4.5 Kb of the 5'-flanking region have also been sequenced. The site of transcription start as well as 3'-end extremities were identified. Transcription unit spans 8.4 Kb and contains 6 exons and 5 introns. Uncoupling protein as well as related mitochondrial carriers such as ADP/ATP carrier and phosphate carrier has a triplicated structure and each repeat of Uncoupling Protein corresponds to 2 exons. Two gene duplications could explain the triplicated structure of UCP and the more recent event of duplication concerned exons III and V. Evidence for homology between Uncoupling Protein and Neurospora crassa ADP/ATP carrier at the gene level was also noticed.

Occurrence of brown adipocytes in rat white adipose tissue: molecular and morphological characterization.

Brown adipocytes are thermogenic cells which play an important role in energy balance. Their thermogenic activity is due to the presence of a mitochondrial uncoupling protein (UCP). Until recently, it was admitted that in rodents brown adipocytes were mainly located in classical brown adipose tissue (BAT). In the present study, we have investigated the presence of UCP protein or mRNA in white adipose tissue (WAT) of rats. Using polymerase chain reaction or Northern blot hybridization, UCP mRNA was detected in mesenteric, epidydimal, retroperitoneal, inguinal and particularly in periovarian adipose depots. The uncoupling protein was detected by Western blotting in mitochondria from periovarian adipose tissue. When rats were submitted to cold or to treatment with a beta-adrenoceptor agonist, UCP expression was increased in this tissue as in typical brown fat. Moreover, the expression was decreased in obese fa/fa rats compared to lean controls. Morphological studies showed that periovarian adipose tissue of rats kept at 24 degrees C contained cells with numerous typical BAT mitochondria with or without multilocular lipid droplets. Immunocytochemistry confirmed that multilocular cells expressed mitochondrial UCP. Furthermore, the number of brown adipocytes and the density of mitochondrial cristae increased in parallel with exposure to cold. These results demonstrate that adipocytes expressing UCP are present in adipose deposits considered as white fat. They suggest the existence of a continuum in rodents between BAT and WAT, and a great plasticity between adipose tissue phenotypes. The physiological importance of brown adipocytes in WAT and the regulation of UCP expression remain open questions.

Transcriptional regulation of the mouse uncoupling protein-2 gene. Double E-box motif is required for peroxisome proliferator-activated receptor-gamma-dependent activation.

Uncoupling protein-2 (UCP2) is present in many tissues with relevance to fuel metabolism, and its expression is increased in fat and muscle in response to elevated circulating free fatty acids resulting from fasting and high fat feeding. We proposed a role for peroxisome proliferator-activated receptor-gamma (PPARgamma) as a mediator of these physiological changes in UCP2, because thiazolidinediones also increase expression of UCP2 in these cell types (). To determine the molecular basis for this regulation, we isolated the 7.3-kilobase promoter region of the mouse UCP2 gene. The -7.3-kilobase/+12-base pair fragment activates transcription of a reporter gene by 50-100-fold. Deletion and point mutation analysis, coupled with gel shift assays, indicate the presence of a 43-base pair enhancer (-86/-44) that is responsible for the majority of both basal and PPARgamma-dependent transcriptional activity. The distal (-86/-76) part of the enhancer specifically binds Sp1, Sp2, and Sp3 and is indistinguishable from a consensus Sp1 element in competition experiments. Point mutation in this sequence reduces basal activity by 75%. A second region (-74/-66) is identical to the sterol response element consensus and specifically binds ADD1/SREBP1. However, deletion of this sequence does not affect basal transcriptional activity or the response to PPARgamma. The proximal portion of the enhancer contains a direct repeat of two E-Box motifs, which contributes most strongly to basal and PPARgamma-dependent transcription of the UCP2 promoter. Deletion of this region results in a 10-20-fold reduction of transcriptional activity and complete loss of PPARgamma responsiveness. Point mutations in either E-Box, but not in the spacer region between them, eliminate the stimulatory response to PPARgamma. However, gel shift assays show that PPARgamma does not bind to this region. Taken together, these data indicate that PPARgamma activates the UCP2 gene indirectly by altering the activity or expression of other transcription factors that bind to the UCP2 promoter.

Detection of brown adipose tissue uncoupling protein mRNA in adult patients by a human genomic probe.

1. Studies on human brown adipose tissue require specific molecular probes. A human genomic library has been screened with a complementary DNA corresponding to the uncoupling protein (UCP) of rat brown adipose tissue mitochondria. 2. Two recombinant phages were isolated, carrying genomic sequences of human UCP. From them we have subcloned a 0.5 kilobase fragment. This fragment, H-Ucp-0.5, contained two intronic regions and two exonic regions. Exonic regions encoded a sequence of 84 amino acids which exhibited a strong homology with central domain at rat UCP. The organization of H-Ucp-0.5 was confirmed by SI mapping analysis. 3. A Southern analysis suggested that the gene is single type in the human, as it is in rodents. 4. In Northern analysis experiments, H-Ucp-0.5 detected a specific 1.8 kb mRNA in human brown adipose tissue obtained from six patients with phaeochromocytoma and from one patient with a hibernoma. This molecular probe is a new, sensitive and reliable tool with which to study human brown adipocytes.

Expression of the brown fat mitochondria uncoupling protein in Xenopus oocytes and important 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.

The topology of the brown adipose tissue mitochondrial uncoupling protein determined with antibodies against its antigenic sites revealed by a library of fusion proteins.

The uncoupling protein (UCP) of brown adipose tissue mitochondria is a specialized member of the family of evolutionarily related mitochondrial membrane transporters, which also includes the ADP/ATP translocator and the phosphate carrier. We have generated a library of bacterial clones randomly expressing short subsequences of the UCP fused to the MalE periplasmic protein of Escherichia coli. Anti-UCP sera were used to select clones expressing antigenic sequences of the UCP. Ten different fusion proteins representing eight non-overlapping subsequences of the UCP were obtained. The ability of fusion proteins to select antibodies directed against a short segment of the UCP was used to study the topological organization of the UCP in the inner mitochondrial membrane. Four different fusion proteins were used to determine the orientation of the N-terminal extremities of the first, second, third and fourth predicted alpha-helices of the UCP. This topological study together with previous data on the UCP provides an experimental basis for the predicted structure of the UCP and for other homologous carrier proteins.

Stable expression of functional mitochondrial uncoupling protein in Chinese hamster ovary cells.

The mitochondrial uncoupling protein (UCP) is a membranous proton carrier exclusively synthesized in brown adipocytes. The cDNA for the rat UCP was placed in an expression vector and transfected into mammalian cells. Its expression was tested in transiently transfected CHO cells. In these cells the UCP was detected in mitochondria by using antibodies. Permanent expression of the UCP was achieved in stable transformed CHO cell lines. In these cells the UCP was characterized in mitochondrial membranes, by using antibodies and hydroxyapatite purification. The protein expressed in CHO cells displayed the functional characteristics of brown adipocyte UCP. It induced the uncoupling of respiration in isolated CHO mitochondria. The membrane potential of transformed mitochondria was also significantly lowered, as a result of the proton translocating activity of the UCP. GDP is known to inhibit the proton pathway in brown fat mitochondria. Addition of GDP to CHO mitochondria containing UCP resulted in a recoupling of respiration and an increase in membrane potential. Thus we conclude that functional UCP is expressed in CHO cells and that the insertion of the UCP alone in any mitochondria is sufficient to induce the uncoupling of respiration. This approach should allow studies on the structure-function relationship of the UCP and of several other related mitochondrial carriers.

Human uncoupling protein gene: structure, comparison with rat gene, and assignment to the long arm of chromosome 4.

The uncoupling protein (UCP) gene encodes a unique mammalian mitochondrial proton carrier that induces heat production in brown adipocytes. Human UCP gene was isolated and its organization analyzed. A comparison was made with rat UCP gene. Human UCP gene spans 13 Kb and contains a transcribed region that covers 9 Kb of the human genome. All of the exons were also sequenced except the extreme end of the 3' untranslated region. Two Kb DNA upstream the TATA box were also sequenced. This region contains several fragments that are highly homologous to the gene of rat UCP. Neither CCAAT sequence nor Sp 1 binding motif were detected. Human UCP gene is split into six exons. The complete amino acid sequence of the protein was determined. Human UCP has 305 amino acids and a molecular weight of 32,786. It has no N-terminal targeting sequence. It is 79% homologous to rat UCP both at nucleotidic and amino acid levels. The primary structure of UCP is significantly homologous to the primary structure of the human T1 ADP/ATP carrier, particularly in the C-terminal extremity, which is supposed to contain a nucleotide-binding site in both proteins. Human UCP gene is single type, as it is in rodents. Two genomic fragments were used to detect a 1.9 Kb mRNA in human perirenal brown adipose tissue. Using in situ hybridization, UCP gene was assigned in humans to chromosome 4 in q31. Interestingly, the T1 gene encoding the heart-skeletal muscle ADP/ATP carrier has recently been shown to be on the same chromosome (Li et al. Biol Chem 264:13998, 1989).

Diet-induced changes in uncoupling proteins in obesity-prone and obesity-resistant strains of mice.

Uncoupling protein 2 (UCP2) maps to a region on distal mouse chromosome 7 that has been linked to the phenotypes of obesity and type II diabetes. We recently reported that UCP2 expression is increased by high fat feeding in adipose tissue of the A/J strain of mice, which is resistant to the development of dietary obesity. More recently, a third UCP (UCP3) was identified, which is expressed largely in skeletal muscle and brown adipose tissue. The UCP2 and UCP3 genes are located adjacent to one another on mouse chromosome 7. Thus, the roles of these UCPs in both metabolic efficiency and the linkage to obesity and diabetes syndromes is unclear. For this reason, we examined the expression of UCP2 and UCP3 in white adipose tissue and interscapular brown adipose tissue and in gastrocnemius/soleus muscle preparations from the obesity-resistant A/J and C57BL/KsJ (KsJ) strains and the obesity-prone C57BL/6J (B6) mouse strain. In both KsJ and A/J mice, UCP2 expression in white fat was increased approximately 2-fold in response to 2 weeks of a high fat diet, but there was no effect of diet on UCP2 levels in B6 mice. In skeletal muscle and in brown fat, neither UCP2 nor UCP3 expression was affected by diet in A/J, B6, or KsJ mice. However, in brown fat, we observed a 2-3-fold increase in the expression of UCP1 in response to dietary fat challenge, which may be related to diet-induced elevations in plasma leptin levels. Together, these results indicate that the consumption of a high fat diet selectively regulates UCP2 expression in white fat and UCP1 expression in brown fat and that resistance to obesity is correlated with this early, selective induction of UCP1 and UCP2 and is not associated with changes in expression of UCP3.

Functional organization of the human uncoupling protein-2 gene, and juxtaposition to the uncoupling protein-3 gene.

Human and mouse UCP2 genes were cloned and sequenced. Transcriptional start sites were identified using primer extension analysis. The transcription unit of UCP2 gene is made of 2 untranslated exons followed by 6 exons encoding UCP2. In vitro translation analysis demonstrated that an open-reading-frame for a putative peptide of 36 residues present in exon 2 did not prevent UCP2 translation and confirmed that the initiation site of translation was in exon 3 as predicted from sequencing data. Short (bp -125 to +93) and long (bp -1383 and +93) CAT-constructs containing DNA upstream of the transcriptional start site of the human gene were made and transfected in adipocytes or HeLa cells allowing characterization of a potent promoter. Analysis of several genomic clones encompassing UCP2 and/or UCP3 genes demonstrated that the 2 genes are adjacent, the human UCP2 gene being located 7 kb downstream of the UCP3 gene.

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 the mitochondrial uncoupling UCPs. The initial stage was the description of a 32-kDa membranous protein specifically induced in brown adipose tissue mitochondria of cold-adapted rats. This protein was then shown by others to be responsible for brown fat thermogenesis and was referred to as the uncoupling protein-UCP (recently renamed UCP1). cDNA and genomic clones of UCP1 were isolated and used to investigate the topology and functional organization of the protein in the membrane and the mechanisms of control of UCP1 gene transcription. Orientation of the transmembrane fragments was proposed and specific amino acid residues involved in the inhibition of UCP1 by purine nucleotides were identified in recombinant yeast. A potent enhancer mediating the response of the UCP1 gene to retinoids and controlling the specific transcription in brown adipocytes was identified using transgenic mice. More recently, we identified UCP2, an UCP homolog widely expressed in human and rodent tissues we also collaborated to characterize the plant UCP. Although the biochemical activities and physiological roles of the novel UCPs are not well understood, these recent data stimulate research on mitochondrial carriers, mitochondrial bioenergetics, and energy expenditure.

Antibodies selected from whole antiserum by fusion proteins as tools for the study of the topology of mitochondrial membrane proteins. Evidence that the N-terminal extremity of the sixth alpha-helix of the uncoupling protein is facing the matrix.

The reactivity to freeze-thawed mitochondria or submitochondrial particles of a whole antiserum raised against the uncoupling protein has been investigated. Incubation with freeze-thawed brown adipose tissue mitochondria trapped antibodies reactive toward accessible parts of the uncoupling protein. One-third to one-half of antibodies against uncoupling protein which were present in the serum remained free. These antibodies were highly reactive with the vesicles obtained by sonication of mitochondria, in which the matricial side of the inner membrane was made accessible. To define epitopes recognized by the antiserum, different fusion proteins made up of MalE protein and uncoupling protein fragments were used. Immunoaffinity chromatography, using an immobilized purified fusion protein containing amino acids 253 to 290 of uncoupling protein, selected antibodies specifically directed against this part of the protein. A more precise localization of the main epitope recognized by these antibodies is proposed. These purified antibodies reacted with the protein only in submitochondrial particles, indicating a matricial orientation of this epitope. This result, associated with other data concerning uncoupling protein or related mitochondrial carriers such as the ADP/ATP translocator and the phosphate carrier, allowed us to determine the orientation of the sixth alpha-helix of the uncoupling protein.