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D Ricquier

Publications and source records attributed to D Ricquier.

At least 91 records · Page 5Linked to original sources
PubMed1994

Failure to detect brown adipose tissue uncoupling protein mRNA in benign symmetric lipomatosis (Madelung's disease).

We report a case of benign symmetric lipomatosis with hypothyroidism. Functional abnormalities and distribution of lipomas in benign symmetric lipomatosis suggest that the lipomas in this disorder may represent brown adipose tissue. In conditions where mRNAs of uncoupling protein, which is believed to be unique for brown adipose tissue mitochondria, were detected in one microgram of poly (A+) rat brown fat RNA, no signal at all was found in the lipomatous tissue, suggesting that the masses of benign symmetric lipomatosis are not functional brown adipose tissue.

Adipose Tissue, Brown↗
PubMed1994

DNA polymorphism in the uncoupling protein (UCP) gene and human body fat.

The objective of this study was to identify DNA sequence variation in the UCP gene and to investigate its relationship with some obesity phenotypes. Two studies were carried out: (1) association study in unrelated subjects, and (2) sib-pair linkage analysis study in brothers and sisters. The subjects were 261 individuals from the Québec Family Study (123 parents and 138 offsprings from 64 families). The following were measured: Body mass index, percent body fat (measured by hydrostatic weighing), and subcutaneous fat (estimated by the sum of 6 skinfolds) were measured in 1978-81 and again 12 years later. Resting metabolic rate (RMR) was measured only in 1989-93. Genetic analyses were performed using Southern blotting technique and a human UCP genomic probe. (1) A BcII restriction fragment length polymorphism was identified with two alleles of 8.3 and 4.5 kb in length, and respective frequencies of 0.28 and 0.72. (2) In unrelated adults from the parental generation, a cross-sectional analysis of the 1989-93 data showed no difference in body fat and RMR between the UCP genotypes. No significant difference for the absolute changes in body fat over the 12-year period among the UCP genotypes was observed. However, a higher frequency (P < 0.05) of the 8.3-kb allele was found in high gainers compared to low gainers (i.e., above and below the median value) for percent body fat over the 12-year period. (3) No evidence of linkage between any of the obesity phenotypes and the UCP BcII marker was found. For the first time, the presence of DNA polymorphism in the human UCP gene is reported. Although, no significant association and linkage were found between the UCP BcII gene marker and body fat in the cohort of the Quebec Family Study, a higher frequency of the 8.3-kb allele was found in individuals who gained more body fat over time.

Adipose Tissue↗
PubMed1993

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.

Base Sequence↗
PubMed1993

Changes in uncoupling protein and GLUT4 glucose transporter expressions in interscapular brown adipose tissue of diabetic rats: relative roles of hyperglycaemia and hypoinsulinaemia.

We have studied the time course and relative effects of hypoinsulinaemia and hyperglycaemia on concentrations of uncoupling protein (UCP) and glucose transporter (GLUT4) and their mRNAs in brown adipose tissue (BAT) during the early phase of diabetes induced by streptozotocin. Two days after intravenous injection of streptozotocin, plasma insulin concentration was at its lowest and glycaemia was higher than 22 mmol/l. After 3 days, a 60% decrease in BAT UCP mRNA concentration and a 36% decrease in UCP was observed. Concomitantly, there was an 80% decrease in GLUT4 mRNA and a 44% decrease in GLUT4 levels. When hyperglycaemia was prevented by infusing phlorizin into diabetic rats, BAT UCP mRNA and protein levels were further decreased (respectively 90% and 60% lower than in control rats). In contrast, the marked decreases in GLUT4 mRNA and protein concentrations in BAT were similar in hyperglycaemic and normoglycaemic diabetic rats. Infusion of physiological amounts of insulin restored normoglycaemia in diabetic rats, and BAT UCP and GLUT4 mRNA and protein concentrations were maintained at the level of control rats. When insulin infusion was stopped, a 75% decrease in BAT UCP mRNA level and a 75% decrease in GLUT4 mRNA level were observed after 24 h, but UCP and GLUT4 concentrations did not decrease. This study shows that insulin plays an important role in the regulation of UCP and GLUT4 mRNA and protein concentrations in BAT. Hyperglycaemia partially prevents the rapid decrease in concentration of UCP and its mRNA observed in insulinopenic diabetes whereas it did not affect the decrease in GLUT4 mRNA and protein concentration. It is suggested that UCP is produced by a glucose-dependent gene.

Adipose Tissue, Brown↗
PubMed1993

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.

ATP-Binding Cassette Transporters↗
PubMed1993

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.

Adipose Tissue↗
PubMed1993

Immunohistochemical detection of human brown adipose tissue uncoupling protein in an autopsy series.

We used an immunohistochemical method for the inner mitochondrial membrane uncoupling protein (UCP) to examine whether human brown adipose tissue UCP could be detected by an anti-rat UCP antibody. Samples of human brown adipose tissue were obtained at medicolegal autopsies. Fat tissue was excised from around the common carotid arteries and in the subscapular region and from around the thoracic aorta. The subjects were either known alcohol consumers, in which thermogenically active brown adipose tissue (BAT) is often found, or victims of sudden infant death syndrome (SID). UCP was detected in all the cases examined, even when the post-mortem time from death to autopsy reached several days, but the intensity of the immunostaining was variable. Intense staining was observed in three cases with a post-mortem time under 24 hr, but in the SID cases a strong positive staining was seen even with a post-mortem delay of 4 days. These results show that an anti-rat UCP antibody can be used for immunohistochemical detection of UCP in human brown adipose tissue and that it provides a useful method for distinguishing between white and brown fat in paraffin-embedded samples. It can be used to detect UCP in the BAT of obese and diabetic individuals and probably also in the histopathological diagnosis of brown adipose tissue lipoma, known as hibernoma.

Adipose Tissue, Brown↗
PubMed1993

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.

Adipose Tissue, Brown↗
PubMed1993

The beta 3-adrenoceptor agonist ICI-D7114 is not as efficient on reinduction of uncoupling protein mRNA in sheep as it is in dogs and smaller species.

Adipose tissue in newborn lambs is brown, but within a few days it is transformed into white adipose tissue. In the same way, preadipocytes cultured in serum-free chemically defined medium achieve full differentiation and express uncoupling protein (UCP), a marker of brown adipose tissue, when isolated from perirenal adipose tissue of the newborn, whereas they no longer express UCP when isolated from older lambs. The effects of a chronic stimulation of adipose tissue by novel beta 3-adrenoceptor agonist (ICI D7114) on the maintenance after birth and on the reinduction in older lambs of UCP mRNA in adipose tissue were studied. Treatment of newborn lambs with this agonist for 25 d maintained a slight level of UCP mRNA in perirenal and pericardiac, but not in omental and inguinal, adipose tissue depots. Preadipocytes isolated from perirenal adipose tissue of treated animals differentiated, in vitro, into adipocytes, but no UCP mRNA could be detected either in the absence or in the presence of the beta 3-adrenoceptor agonist in the culture medium. Treatment of 1-mo-old lambs with ICI D7114 for 12 d restored UCP mRNA in perirenal and pericardiac adipose tissues of two of four treated lambs, but at a much lower level than in the same tissues at birth. In both experiments, the final BW and the ADG of lambs treated with ICI D7114 were not statistically different from controls. These results are quite different from those obtained with the same beta 3-adrenoceptor agonist in dogs and rodents.

Adipocytes↗
PubMed1993

Effect of acute magnesium deficiency on the masking and unmasking of the proton channel of the uncoupling protein in rat brown fat.

The short term regulation of heat production in brown adipose tissue mitochondria (BAT) of acutely Mg-deficient rats was demonstrated by comparing several parameters of mitochondrial energization. Mg deficiency in vivo had absolutely no effect on the BAT uncoupling protein concentration (UCP) which was only modified by thermal conditions. The same high concentration was observed 10 d cold exposed control and Mg-deficient rats. Four days of warm re-exposure at thermal neutrality led to a moderate 26 per cent decrease with both diets which was not modified by cold stress for 1 h. Proton conductance. CmH+, and proton motive force, delta p, were calculated from membrane potential and respiration rate measurements. The same high level CmH+ was observed in cold exposed rats with both diets. Compared to warm re-exposed control rats, CmH+ was threefold higher in the corresponding Mg-deficient group which indicated a much lower masking of the proton channel of UCP with the Mg-deficient diet. This difference was not dependent on the presence of magnesium in vitro. The basal CmH+, independent of UCP, was not altered by magnesium deficiency. These results emphasize that acute regulation of thermogenic BAT activity through the masking and unmasking process is altered when magnesium supply is limited in vivo.

Adipose Tissue, Brown↗
PubMed1992

Activation by ATP of a proton-conducting pathway in yeast mitochondria.

The growth of Saccharomyces cerevisiae cells under aerobic conditions, in the presence of an energy-rich source, leads to production of an excess of NAD(P)H. Since the redox balance must be maintained, it has been postulated that NAD(P)H reoxidation is accelerated by the activation of energy-dissipating reactions, which would, in turn, explain the low growth efficiencies observed. It has been demonstrated already in S. cerevisiae cultures that these putative energy-dissipating reactions are stimulated both by oxygen and high cytosolic ATP levels. In this paper, we show that ATP induces a proton-permeability pathway in mitochondria at concentrations which are within the physiological range, as revealed both from the ATP stimulation of respiration and from the induction of H(+)-dependent swelling. We also demonstrate that phosphate acts as a competitive inhibitor of the nucleotide, and since activation is observed even in the presence of atractylate, we postulate that the ATP-binding site is located in the outer face of the mitochondrial inner membrane.

Adenosine Triphosphate↗
PubMed1992

Hibernoma formation in transgenic mice and isolation of a brown adipocyte cell line expressing the uncoupling protein gene.

Transgenic mice were produced containing the adipocyte-specific regulatory region from the adipocyte P2 (aP2) gene linked to the simian virus 40 transforming genes. Most of the transgenic mice developed brown fat tumors (hibernomas) in their interscapular brown adipose tissue. Hibernoma formation was noticeable in some of the mice as early as 1 day after birth and most of the mice developed very large tumors by 1 month of age. All of the tumor tissue expressed the brown fat-specific uncoupling protein (UCP) gene as well as the aP2 gene. Several of the tumors have been used to establish cultured cell lines and at least one of these lines can be induced to differentiate into brown adipocytes. The cultured adipocytes express mRNA for UCP upon stimulation with N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, norepinephrine, isoproterenol or D7114, a beta 3 adrenergic agonist. Thus, regulation of the key thermogenic gene UCP can now be studied in an established cell line.

Adipose Tissue, Brown↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

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.

Adipose Tissue, Brown↗
PubMed1992

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.

Animals↗
PubMed1992

Regulation of UCP gene expression in brown adipocytes differentiated in primary culture. Effects of a new beta-adrenoceptor agonist.

Primary cultures of precursor cells from mouse and rat brown adipose tissue (BAT) were used to study the effect of a new beta-agonist (ICI D7114) on the uncoupling protein (UCP) gene expression. ICI 215001 (the active metabolite of D7114) increased the expression of UCP and its mRNA in brown adipocytes differentiating in vitro in a dose-dependent manner. This stimulating effect was not inhibited by propranolol, a non-specific beta-antagonist, but was partially reduced by bupranolol, a beta 3-antagonist. No expression of UCP mRNA was ever induced by ICI 215001 in white adipocytes differentiated in vitro. It was concluded that the drug could affect the brown adipose cells through a beta 3-pathway. It could clearly modulate the expression of UCP in brown adipocytes differentiated in vitro, but was not able by itself to turn on the gene.

Adipose Tissue, Brown↗