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.

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.

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.

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.

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

The coupling of O2 consumption to ADP phosphorylation in mitochondria is partial. This is particularly obvious in brown adipocyte mitochondria which use a regulated uncoupling mechanism generating heat production from substrate oxidation, and catalysing thermogenesis in rodents or infants in response to cold, and arousing hibernators. In the case of brown adipose tissue, the uncoupling mechanism is related to a specific protein in the inner mitochondrial membrane referred to as UCP1. Although the biological importance of UCP1 in human adults is not demonstrated, genetic analysis of various human cohorts suggested a participation of UCP1 to control of fat content and body weight. Very recently, the cloning of UCP2 and UCP3, two homologues of UCP1, has renewed the field of research on the importance of respiration control in metabolic processes and metabolic diseases. UCP2 is widely expressed in organs, whereas UCP3 is mainly present in muscles. These proteins may explain why the coupling of respiration to ADP phosphorylation is less than perfect. Their biological importance should be studied. They also represent new putative targets for drugs against metabolic diseases such as obesity.

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.

BMCP1, a novel mitochondrial carrier with high expression in the central nervous system of humans and rodents, and respiration uncoupling activity in recombinant yeast.

We report here the cloning and functional analysis of a novel homologue of the mitochondrial carriers predominantly expressed in the central nervous system and referred to as BMCP1 (brain mitochondrial carrier protein-1). The predicted amino acid sequence of this novel mitochondrial carrier indicates a level of identity of 39, 31, or 30%, toward the mitochondrial oxoglutarate carrier, phosphate carrier, or adenine nucleotide translocator, respectively, and a level of identity of 34, 38, or 39% with the mitochondrial uncoupling proteins UCP1, UCP2, or UCP3, respectively. Northern analysis of mouse, rat, or human tissues demonstrated that mRNA of this novel gene is mainly expressed in brain, although it is 10-30-fold less expressed in other tissues. In situ hybridization analysis of brain showed it is particularly abundant in cortex, hippocampus, thalamus, amygdala, and hypothalamus. Chromosomal mapping indicates that BMCP1 is located on chromosome X of mice and at Xq24 in man. Expression of the protein in yeast strongly impaired growth rate. Analysis of respiration of total recombinant yeast or yeast spheroplasts and in particular of the relationship between respiratory rate and membrane potential of yeast spheroplasts revealed a marked uncoupling activity of respiration, suggesting that although BMCP1 sequence is more distant from the uncoupling proteins (UCPs), this protein could be a fourth member of the UCP family.

Association between uncoupling protein polymorphisms (UCP2-UCP3) and energy metabolism/obesity in Pima indians.

The UCP2-UCP3 gene cluster maps to chromosome 11q13 in humans, and polymorphisms in these genes may contribute to obesity through effects on energy metabolism. DNA sequencing of UCP2 and UCP3 revealed three polymorphisms informative for association studies: an Ala-->Val substitution in exon 4 of UCP2, a 45 bp insertion/deletion in the 3'-untranslated region of exon 8 of UCP2 and a C-->T silent polymorphism in exon 3 of UCP3. Initially, 82 young (mean age = 30 +/- 7 years), unrelated, full-blooded, non-diabetic Pima Indians were typed for these polymorphisms by direct sequencing. The three sites were in linkage disequilibrium ( P < 0.00001). The UCP2 variants were associated with metabolic rate during sleep (exon 4, P = 0.007; exon 8, P = 0.016) and over 24 h (exon 8, P = 0.038). Heterozygotes for UCP2 variants had higher metabolic rates than homozygotes. The UCP3 variant was not significantly associated with metabolic rate or obesity. In a further 790 full-blooded Pima Indians, there was no significant association between the insertion/deletion polymorphism and body mass index (BMI). However, when only individuals >45 years of age were considered, heterozygotes (subjects with the highest sleeping metabolic rate) had the lowest BMI (P = 0.04). The location of the insertion/deletion polymorphism suggested a role in mRNA stability; however, it appeared to have no effect on skeletal muscle UCP2 mRNA levels in a subset of 23 randomly chosen Pima Indians. In conclusion, these results suggest a contribution from UCP2 (or UCP3) to variation in metabolic rate in young Pima Indians which may contribute to overall body fat content later in life.

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.

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.

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.

Activation of the uncoupling protein by fatty acids is modulated by mutations in the C-terminal region of the protein.

The transport properties of the uncoupling protein (UCP) from brown adipose tissue have been studied in mutants where Cys304 has been replaced by either Gly, Ala, Ser, Thr, Ile or Trp. This position is only two residues away from the C-terminus of the protein, a region that faces the cytosolic side of the mitochondrial inner membrane. Mutant proteins have been expressed in Saccharomyces cerevisiae and their activity determined in situ by comparing yeast growth rates in the presence and absence of 2-bromopalmitate. Their bioenergetic properties have been studied in isolated mitochondria by determining the effects of fatty acids and nucleotides on the proton permeability and NADH oxidation rate. It is revealed that substitution of Cys304 by non-charged residues alters the response of UCP to fatty acids. The most effective substitution is Cys for Gly since it greatly enhances the sensitivity to palmitate, decreasing threefold the concentration required for half-maximal stimulation of respiration. The opposite extreme is the substitution by Ala which increases twofold the half-maximal concentration. We conclude that the C-terminal region participates in the fatty acid regulation of UCP activity. The observed correlation between yeast growth rates in the presence of bromopalmitate and the calculated activation constants for respiration in isolated mitochondria validates growth analysis as a method to screen the in situ activity of UCP mutants.

A sequence related to a DNA recognition element is essential for the inhibition by nucleotides of proton transport through the mitochondrial uncoupling protein.

The uncoupling protein (UCP) is uniquely expressed in brown adipose tissue, which is a thermogenic organ of mammals. The UCP uncouples mitochondrial respiration from ATP production by introducing a proton conducting pathway through the mitochondrial inner membrane. The activity of the UCP is regulated: nucleotide binding to the UCP inhibits proton conductance whereas free fatty acids increase it. The similarities between the UCP, the ADP/ATP carrier and the DNA recognition element found in the DNA binding domain of the estrogen receptor suggested that these proteins could share common features in their respective interactions with free nucleotides or DNA, and thus defined a putative 'nucleotide recognition element' in the UCP. This article provides demonstration of the validity of this hypothesis. The putative nucleotide recognition element corresponding to the amino acids 261-269 of the UCP was gradually destroyed, and these mutant proteins were expressed in yeast. Flow cytometry, measuring the mitochondrial membrane potential in vivo, showed increased uncoupling activities of these mutant proteins, and was corroborated with studies with isolated mitochondria. The deletion of the three amino acids Phe267, Lys268 and Gly269, resulted in a mutant where proton leak could be activated by fatty acids but not inhibited by nucleotides.

Cysteine residues are not essential for uncoupling protein function.

The uncoupling protein (UCP) of brown adipose tissue is a regulated proton carrier which allows uncoupling of mitochondrial respiration from ATP synthesis and, therefore, dissipation of metabolic energy as heat. In this article we demonstrate that, when UCP is expressed in Saccharomyces cerevisiae, it retains all its functional properties: proton and chloride transport, high-affinity binding of nucleotides and regulation of proton conductance by nucleotides and fatty acids. Site-directed mutagenesis demonstrates that sequential replacement by serine of cysteine residues in the UCP does not affect either its uncoupling activity or its regulation by nucleotides and fatty acids, and therefore establishes that none of the seven cysteine residues present in the wild-type UCP is critical for its activity. These data indicate that transport models involving essential thiol groups can be discounted and that chemical modification data require critical re-evaluation.

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.

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.

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.

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.