Brown Fat UCP1 Is Specifically Expressed in Uterine Longitudinal Smooth Muscle Cells

Until now, uncoupling protein 1 (UCP1) was considered as unique to brown adipocytes. It supports a highly regulated uncoupling of oxidative phosphorylation that is associated with diet as well as with non-shivering thermogenesis. Here we report that UCP1 is not specific to brown adipocytes and can be expressed in longitudinal smooth muscle layers. In the uterus, this conclusion was drawn from different convergent data. A specific antibody against mouse UCP1 revealed, in mitochondrial fractions, a protein with the same molecular weight as brown fat UCP1. Sensitive and specific reverse transcriptase-polymerase chain reaction detected a mRNA whose sequence was totally homologous to that of brown fat UCP1 mRNA. Antibody against UCP1 as well as a UCP1 antisense probe specifically stained uterine longitudinal smooth muscles. UCP1 was also expressed in longitudinal smooth muscle of digestive and male reproductive tracts but was never expressed in other types of smooth muscle, including those of arterial vessels. In uterine tract, UCP1 content was increased after cold exposure or β-adrenergic agonist treatment. It was also up-regulated during the postovulatory period after sexual cycle synchronization. Its content transiently increased during gestation and decreased markedly after birth. These regulations strongly argue about a role for UCP1 in thermogenesis as well as in relaxation of longitudinal smooth muscle layers. Until now, uncoupling protein 1 (UCP1) was considered as unique to brown adipocytes. It supports a highly regulated uncoupling of oxidative phosphorylation that is associated with diet as well as with non-shivering thermogenesis. Here we report that UCP1 is not specific to brown adipocytes and can be expressed in longitudinal smooth muscle layers. In the uterus, this conclusion was drawn from different convergent data. A specific antibody against mouse UCP1 revealed, in mitochondrial fractions, a protein with the same molecular weight as brown fat UCP1. Sensitive and specific reverse transcriptase-polymerase chain reaction detected a mRNA whose sequence was totally homologous to that of brown fat UCP1 mRNA. Antibody against UCP1 as well as a UCP1 antisense probe specifically stained uterine longitudinal smooth muscles. UCP1 was also expressed in longitudinal smooth muscle of digestive and male reproductive tracts but was never expressed in other types of smooth muscle, including those of arterial vessels. In uterine tract, UCP1 content was increased after cold exposure or β-adrenergic agonist treatment. It was also up-regulated during the postovulatory period after sexual cycle synchronization. Its content transiently increased during gestation and decreased markedly after birth. These regulations strongly argue about a role for UCP1 in thermogenesis as well as in relaxation of longitudinal smooth muscle layers. uncoupling protein brown adipose tissue interscapular BAT reverse transcriptase-polymerase chain reaction 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium base pairs The primary function of mitochondria is to supply the cell with energy as ATP. They are also involved in heat production and have other functions that are not directly related to energy transduction, such as redox state control and Ca2+ homeostasis. (1Zorov D.B. Krasnikov B.F. Kuzminova A.E. Vysokikh M. Zorova L.D. Biosci. Rep. 1997; 17: 507-520Crossref PubMed Scopus (70) Google Scholar, 2Duchen M.R. J. Physiol. (Lond.). 1999; 516: 1-17Crossref Scopus (527) Google Scholar). In all the functions, membrane potential and permeability play key roles. In specialized thermogenic cells, such as the brown adipocyte, increased proton conductance across the mitochondrial inner membrane was first postulated by Nicholls and Locke (3Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1324) Google Scholar). The protein supporting this activity was later purified, named uncoupling protein (UCP),1 and cloned. This protein belongs to the mitochondrial anion carrier family. Its insertion into the mitochondrial inner membrane allows dissipation of the proton electrochemical gradient across this membrane with associated heat production and decreased ATP synthesis. Its ectopic expression in any eukaryotic cell is sufficient to reproduce similar uncoupled mitochondrial phenotypes (4Casteilla L. Blondel O. Klaus S. Raimbault S. Diolez P. Moreau F. Bouillaud F. Ricquier D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5124-5128Crossref PubMed Scopus (37) Google Scholar, 5Bathgate B. Freebairn E.M. Greenland A.J. Reid G.A. Mol. Microbiol. 1992; 6: 363-370Crossref PubMed Scopus (33) Google Scholar). Until now, its expression was only detected in mitochondria from brown adipocytes. The recent cloning and expression studies of other UCPs, such as UCP2 and UCP3, suggest that similar mechanisms may exist in numerous cells. New perspectives with regard to UCPs and particularly UCP1 are now being revealed with the discoveries that UCPs are modulators of mitochondrial reactive oxygen species generation and that coenzyme Q is an obligatory partner for their uncoupling activity (6Negre-Salvayre A. Hirtz C. Carrera G. Cazenave R. Troly M. Salvayre R. Pénicaud L. Casteilla L. FASEB J. 1997; 11: 809-815Crossref PubMed Scopus (676) Google Scholar,7Echtay K.S. Winkler E. Klingenberg M. Nature. 2000; 408: 609-613Crossref PubMed Scopus (287) Google Scholar). The function of the different UCPs in the maintenance of body temperature has been debated (8Ricquier D. Bouillaud F. Biochem. J. 2000; 345: 161-179Crossref PubMed Scopus (745) Google Scholar). Until now, only UCP1 has been clearly linked to thermogenesis and energy balance. Firstly, physiological regulation during cold exposure and starvation are consistent with this function, whereas this is not the case for other UCPs. Secondly, transgenic ablation of brown fat using a transgene overexpressing toxin under the control of UCP1 promoter (UCP1-DTA mice) led to a decrease in energy expenditure, hypergia, and obesity (9Lowell Susulic V.S. Hamann A. Lawitts V.S.J.A. Himms-Hagen J. Boyer B.B. Kozak L.P. Flier J.S. Nature. 1993; 366: 740-742Crossref PubMed Scopus (893) Google Scholar). Thirdly, UCP1 knockout mice failed to maintain body temperature after cold exposure (10Enerback S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1060) Google Scholar). None of these phenotypes were observed inucp3 −/− and ucp2 −/−mice (9Lowell Susulic V.S. Hamann A. Lawitts V.S.J.A. Himms-Hagen J. Boyer B.B. Kozak L.P. Flier J.S. Nature. 1993; 366: 740-742Crossref PubMed Scopus (893) Google Scholar, 10Enerback S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1060) Google Scholar). Nevertheless, two particular features of transgenic models could not be easily explained: (i) in UCP1-DTA mice, altered reproductive function was described and (ii) in ucp1 −/− mice, the phenotype is more prominent in female compared with male mice (9Lowell Susulic V.S. Hamann A. Lawitts V.S.J.A. Himms-Hagen J. Boyer B.B. Kozak L.P. Flier J.S. Nature. 1993; 366: 740-742Crossref PubMed Scopus (893) Google Scholar, 10Enerback S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1060) Google Scholar, 11Hamann A. Flier J.S. Lowell B.B. Z. Eraehrwiss. 1998; 37: 1-7PubMed Google Scholar). According to these phenotypes, we postulated and investigated the putative ectopic expression of UCP1 protein in the female reproductive tract. Experiments were performed on female OF1 (IOPS caw) mice (IFFA-CREDO, L'Arbresle, France) weighing 26–30 g. Animals were housed in a controlled environment (12-h light/dark cycle and 21 °C) with free access to water and standard chow diet. During cold exposure, mice were caged individually at 4 °C for 6 days. To test the effect of β-adrenergic treatment, 0.5 μg/g of mouse/day of isoproterenol were injected intraperitoneally for 5 days. To synchronize mice hormonally, one group received 5 units of intraperitoneal pregnant mare's serum gonadotropin (to mimic follicle-stimulating hormone effect), and 46 h later, this group received 5 units of human chorionic gonadotropin (luteinizing hormone effect) to produce ovulation. The control group received normal saline. 24 or 36 h after the second injection, mice were killed by cervical dislocation after CO2anesthesia. The serum and uterus were immediately removed. The efficiency of hormonal treatment was confirmed by the measurement of sexual hormones by radioimmunoassay (Sorin Biomedica). The uteri were dissected free of adipose tissue and weighed; specimens were fixed on 3.6% formaldehyde-phosphate-buffered saline, pH 7, for histological analysis. Fragments were rapidly frozen in liquid nitrogen and stored at −80 °C until RNA analysis. The remaining tissue was quickly homogenized in sucrose buffer (with anti-proteases) to prepare mitochondrial fractions. Pregnant females were killed at various stages of gestation as indicated and 48 h after spontaneous delivery. Mitochondrial fractions were purified by differential centrifugation of tissue homogenates as described previously (12Miroux B. Casteilla L. Klaus S. Raimbault S. Grandin S. Clement J.M. Ricquier D. Bouillaud F. J. Biol. Chem. 1992; 267: 13603-13609Abstract Full Text PDF PubMed Google Scholar). 20 μg of protein from total homogenate or mitochondrial fractions were electrophoresed in 10% SDS-polyacrylamide gel and transferred (at 1 h) onto nitrocellulose. These membranes were systematically stained with Ponceau red to check that equal amounts of proteins were analyzed. Overnight incubation with the same rabbit IgG against mouse UCP1 (UCP1 11A, 0.25 μg/ml) and washings were performed. Peroxidase activity of the second antibodies (diluted 1:8000) was revealed using the ECL kit and Hyperfilm ECL-TM. The blots were exposed for 2–10 min. Positive control was performed with 0.05–2 μg of BAT mitochondria purified from rat BAT. After overnight fixation, tissues were dehydrated and paraffin-embedded. Sections (5–7 μm) were incubated for 1 h at room temperature with 0.5 μg/ml UCP 11A. The second antibody coupled to alkaline phosphatase (1:200) was visualized using BCIP/NBT. Endogenous alkaline phosphatase activity was inhibited by levamisole. Slides were counterstained with nuclear red. With the double labeling method, UCP1 was revealed using Texas Red-conjugated AffiniPure F(ab′)2 fragment goat anti-rabbit IgG (H+L), diluted 1:100, as the second antibody. Monoclonal mouse anti-human smooth muscle actin, diluted 1:50, was used as a marker for actin, and fluorescein isothiocyanate-conjugated horse anti-mouse IgG (H+L), diluted 1:100, was used as the second antibody. Control experiments were performed using purified rabbit IgG and yielded no staining. These experiments were performed on paraffin sections. All prehybridization, hybridization, and stringent washing steps were similar to our previously published method (13Brauner P. Nibbelink M. Flachs P. Vitkova I. Kopecky P. Mertelikova I. Janderova L. Penicaud L. Casteilla L. Plavka R. Kopecky J. Pediatr. Res. 2001; 49: 440-447Crossref PubMed Scopus (10) Google Scholar). Briefly, prehybridization was performed (3 h/42 °C) in 80 μl of 50% formamide in 5× SSC and salmon sperm DNA (250 μg/ml). Prehybridized sections were hybridized for 15 min at 70 °C and overnight at 42 °C in 50 μl of the same solution containing only 40 μg of ssDNA and the DIG-labeled riboprobes (0.5 μg/ml for UCP2 and 0.1 μg/ml for UCP1). Stringent washes consisted of 30 min in 2× SSC (room temperature), 60 min in 2× SSC (50 °C), 60 min in 0.2× SSC (50 °C), and equilibration (5 min) in Tris-HCl buffer, pH 7.4. Subsequently, bound probe was detected with alkaline phosphatase-conjugated antidigoxigenin antibody (1:500 diluted) (Roche Molecular Biochemicals, catalog no. 1093274) and BCIP/NBT substrate. Sections were counterstained with nuclear red. The probes were prepared using the DIG RNA Labeling Kit (Roche Molecular Biochemicals, catalog no. 1-175-025) by in vitro transcription. ABamHI-EcoRI fragment (180 bp) of UCP1 cDNA was ligated into BS-Ad1. A 261-bp antisense riboprobe was generated using T3 RNA polymerase (Stratagene) after SST2 digestion of the plasmid; this probe hybridizes with the 3′-non-coding sequence. A 220-bp sense was generated with T7 polymerase afterEcoRV digestion. A 182-bp mouse UCP2 riboprobe was obtained by PCR (with the primers 5′-CAGGTCACTGTGCCCTTACC-3′ and 5′-CATGGAGAGGCTCAGAAAGG-3′) and cloned into the pGEM-T Easy vector. We obtained antisense riboprobe (272 bp) with T7 polymerase after SalI digestion and sense probe (286 bp) with SP6 polymerase and NcoI digestion. Total RNAs were prepared from pulverized tissues of non-pregnant female mice by the acidic guanidium isothiocyanate method (14Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar). After a 10-min denaturation at 70 °C, 3 μg of these RNAs were incubated for 50 min in a 20-μl reverse transcriptase reaction mix containing 50 mm Tris-HCl, pH 8.3, 75 mm KCl, 3 mm MgCl2, 10 mmdithiothreitol, 1 mm dNTP, 5.5 μmoligo(dT)15 primer, 10 units of RNase inhibitor (Promega, Madison, WI), and 200 units of RT SuperscriptTM II (Life Technologies, Inc.). Negative controls of the reverse transcription reaction were performed in the same conditions but without reverse transcriptase. The following primers were subsequently used to amplify a 198-bp fragment of UCP-1 cDNA (from position 584 to 763): sense 5′-AGACATCATCACCTTCCC-3′ and antisense 5′-CAGCTGTTCAAAGCACAC-3′ primers followed by nested sense 5′-GTGAAGGTCAGAATGCAAGC-3′ and antisense 5′-AGGGCCCCCTTCATGAGGTC-3′ primers. A 2-μl aliquot of reverse transcription reactions was used for UCP-1 cDNA amplification in a 50-μl reaction mix containing 10 mm Tris-HCl, pH 9, 50 mm KCl, 1.5 mm MgCl2, 0.1% Triton X-100, 200 μm dNTP, 1.25 units of Taq DNA polymerase (Promega, Madison, WI), and 400 nm first sense and antisense primers. Following a 5-min denaturation at 94 °C, the PCR steps consisted of 30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C for several cycles, as indicated in the figure legends, followed by 7 min at 72 °C. 1 μl of a 1:50 dilution of this PCR reaction was used in the nested amplification, using nested sense and antisense primers. The conditions were exactly the same as in the first PCR. In parallel, a 499-bp fragment of actin cDNA (from position 165 to 664) was amplified using sense 5′-CGACGAGGCCCAGAGCAAG-3′ and antisense 5′-CTAGGGCAACATAGCACAGC-3′ primers in the same conditions as the first UCP-1 amplification but with 25 cycles. Amplification products were run onto 1.5% agarose gel in 0.5× TBE (90 mm Tris, 90 mm boric acid, and 50 mm EDTA, pH 8) containing ethidium The molecular of the PCR were using the Technologies, or the molecular rabbit IgG against mouse UCP1 were from Monoclonal mouse anti-human smooth muscle actin alkaline phosphatase-conjugated rabbit anti-mouse IgG BCIP/NBT and and were all obtained from phosphatase-conjugated AffiniPure F(ab′)2 fragment anti-rabbit IgG and Texas Red-conjugated AffiniPure goat anti-rabbit IgG were obtained from antibody (from membranes ECL kit and Hyperfilm were obtained DIG RNA Labeling Kit and alkaline phosphatase-conjugated antidigoxigenin antibody were obtained Molecular fluorescein isothiocyanate-conjugated horse anti-mouse IgG was by Pregnant mare's serum gonadotropin and human chorionic gonadotropin were from were obtained from To the of UCP1 was compared with an antibody against UCP1 purified from rat BAT to UCP2 D. J.M. M. Biochem. J. PubMed Scopus Google Scholar). the detected a in brown fat and in mitochondrial fractions to highly the UCP1 antibody only revealed one protein in BAT Negative were also obtained with mitochondrial fractions purified from and in is highly expressed not UCP1 expression in the uterus was investigated using the specific UCP1 antibody and in this antibody in experiments with uterine protein detected a at the same molecular weight as that detected in brown fat This was in proteins from mitochondrial fractions in total protein fractions. This is consistent with the mitochondrial of the To the of this with the and nested was performed. no was observed in control in or cell used as controls and not In a at the bp) was obtained from uterine RNA Its cloning and confirmed the with the published mouse UCP1 mRNA sequence A. U. Kozak L.P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). in the of the was to the of PCR To the cell UCP1 experiments were performed on the reproductive tract. in the uterus was clearly stained with antibody 3 was observed the UCP1 antibody was not The of antibody against smooth muscle actin that the to the longitudinal of smooth 3 no labeling was observed in smooth 3 The was confirmed by double labeling at a revealed into smooth muscle 3 We never observed brown fat cells. The same antibody was to any in or muscle strongly in the different of to highly UCP2 not To the of the detected in experiments were performed and 3 The of UCP2 mRNA by of the probe could be obtained with the UCP2 antisense probe were different and not to muscle 3 The of other tissues containing smooth muscle that in male and digestive UCP1 antibody specifically stained the smooth muscle of the longitudinal muscle could be detected in including those of arterial 4 of UCP1 in male and and blue BCIP/NBT following alkaline phosphatase red. UCP1 UCP1 is specifically detected in smooth of all longitudinal muscle those of the In the UCP1 is also expressed in the The is indicated on the the of tissues in UCP1 expression was we our on the uterine tract. and is a of the UCP1 the UCP family. To test for similar we performed of mitochondrial fractions from mice exposed to cold for 6 or with a β-adrenergic In the detected by antibodies A and These were confirmed by histological of the uterus from control compared with mice and The in uterine UCP1 content was also observed after in mice by hormonal treatment 5 we investigated in UCP1 content during gestation UCP1 content in mitochondrial fractions increased during the of gestation and its during the of This content decreased after to to the control The of this are that UCP1 expression can no be considered as to brown adipocytes and that in the tissues is specifically expressed in longitudinal smooth muscle layers. expression of UCP1 been described in and N. A. M. I. M. J. Physiol. 1998; Google Scholar, A. M. H. PubMed Scopus Google Scholar). These have been to the different UCPs N. A. M. I. M. J. Physiol. 1998; Google Scholar, D. Raimbault S. O. B. Bouillaud F. 1992; PubMed Scopus Google Scholar). The of other UCPs, and the of UCP2 confirmed the of UCP1 expression in brown adipocytes. To any we the different and we The of the UCP1 antibody was confirmed by a of data. Firstly, this antibody is to proteins in or muscle, whereas other antibodies Secondly, these were confirmed by using RNA such as or in In the the on the uterus using UCP2 antisense probe any with the sequence of the fragment that UCP1 expression is not unique to brown adipocytes but can in smooth muscles. The of this expression of UCP1 is in all tissues only in longitudinal smooth muscle layers. To the physiological of such we our on the uterine tract. this one is to and the of the different muscle is well to physiological Pediatr. Res. 1998; PubMed Scopus Google Scholar, S. J. Physiol. 1993; PubMed Google Scholar). In this the UCP1 content in the uterus could be to be that detected in brown fat This the of UCP1 by such as H. S. M. 1999; PubMed Scopus Google Scholar). A of the UCP1 is its by and β-adrenergic agonist treatment L.P. Harper M.E. Rev. 2000; PubMed Scopus Google Scholar, D. Bouillaud F. J. Physiol. 2000; Scopus Google Scholar). strongly the protein in the uterus as These the of the detected This strongly that the uterine expression of UCP1 could be involved in non-shivering thermogenesis and is also consistent with the −/− females to cold exposure (10Enerback S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1060) Google Scholar). A role could be an in the in body temperature that during the cycle after J. PubMed Scopus Google Scholar). This is by the postovulatory of UCP1 content in uterine In the uterus, the longitudinal and smooth muscle control the of the the uterus during to the and to muscle without Pediatr. Res. 1998; PubMed Scopus Google Scholar). During this are the two uterine smooth muscle the effect the on and the effect β-adrenergic until in the and longitudinal After is by different mechanisms S. J. Physiol. 1993; PubMed Google Scholar). These are consistent with the of UCP1 expression and its after β-adrenergic and during The uterine role of UCP1 could of an effect on an and state in the smooth cells. This body of is for to that UCP1 its uncoupling could control the mitochondria functions of longitudinal smooth muscle cells. these functions, ATP is the but not the of UCP1 in reactive oxygen species and (6Negre-Salvayre A. Hirtz C. Carrera G. Cazenave R. Troly M. Salvayre R. Pénicaud L. Casteilla L. FASEB J. 1997; 11: 809-815Crossref PubMed Scopus (676) Google Scholar). in could of longitudinal smooth muscle Its expression in these could be involved in the different of uterine muscle that the maintenance of the during the gestation and its Pediatr. Res. 1998; PubMed Scopus Google Scholar). This with the reproductive of UCP1-DTA mice A. Flier J.S. Lowell B.B. Z. Eraehrwiss. 1998; 37: 1-7PubMed Google Scholar). It is in this physiological UCP1 could be by the of during gestation that the uncoupling activity of UCP1. this report that UCP1 is not specific to brown adipocytes. In our suggest that as well as its in thermogenesis after cold exposure or during the sexual may play a role in other functions such as well with uterine and be the physiological and of UCP1 in numerous by we can that UCP1 may in the of the and reproductive This the of mitochondria and their in these We D. Ricquier for the of antibodies against rat the and particularly J. M. for M. and N. for P. for and in L. for G. for mice, and for