Selective Activation of Estrogen Receptor α Activation Function-1 Is Sufficient to Prevent Obesity, Steatosis, and Insulin Resistance in Mouse
2017; Elsevier BV; Volume: 187; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2017.02.013
ISSN1525-2191
AutoresMaéva Guillaume, Sandra Handgraaf, Aurélie Fabre, Isabelle Raymond‐Letron, Élodie Riant, Alexandra Montagner, Alexia Vinel, Mélissa Buscato, Natalia Smirnova, Coralie Fontaine, Hervé Guillou, Jean‐François Arnal, Pierre Gourdy,
Tópico(s)Adipose Tissue and Metabolism
ResumoEstrogen receptor α (ERα) regulates gene transcription through two activation functions (ERα-AF1 and ERα-AF2). We recently found that the protection conferred by 17β-estradiol against obesity and insulin resistance requires ERα-AF2 but not ERα-AF1. However, the interplay between the two ERα-AFs is poorly understood in vivo and the metabolic influence of a specific ERα-AF1 action remains to be explored. To this end, wild-type, ERα-deficient, or ERα-AF1–deficient ovariectomized female mice were fed a high-fat diet and concomitantly administered with vehicle or tamoxifen, a selective ER modulator that acts as a ERα-AF1 agonist/ERα-AF2 antagonist. In ovariectomized wild-type mice, tamoxifen significantly reduced food intake and totally prevented adiposity, insulin resistance, and steatosis. These effects were abolished in ERα-deficient and ERα-AF1–deficient mice, revealing the specific role of ERα-AF1 activation. Finally, hepatic gene expression changes elicited by tamoxifen in wild-type mice were abrogated in ERα-AF1–deficient mice. The combination of pharmacologic and transgenic approaches thus indicates that selective ERα-AF1 activation by tamoxifen is sufficient to elicit metabolic protection, contrasting with the specific requirement of ERα-AF2 in the metabolic actions of 17β-estradiol. This redundancy in the ability of the two ERα-AFs to separately mediate metabolic prevention strikingly contrasts with the contribution of both ERα-AFs in breast cancer proliferation, shedding new light on the therapeutic potential of selective ER modulation. Estrogen receptor α (ERα) regulates gene transcription through two activation functions (ERα-AF1 and ERα-AF2). We recently found that the protection conferred by 17β-estradiol against obesity and insulin resistance requires ERα-AF2 but not ERα-AF1. However, the interplay between the two ERα-AFs is poorly understood in vivo and the metabolic influence of a specific ERα-AF1 action remains to be explored. To this end, wild-type, ERα-deficient, or ERα-AF1–deficient ovariectomized female mice were fed a high-fat diet and concomitantly administered with vehicle or tamoxifen, a selective ER modulator that acts as a ERα-AF1 agonist/ERα-AF2 antagonist. In ovariectomized wild-type mice, tamoxifen significantly reduced food intake and totally prevented adiposity, insulin resistance, and steatosis. These effects were abolished in ERα-deficient and ERα-AF1–deficient mice, revealing the specific role of ERα-AF1 activation. Finally, hepatic gene expression changes elicited by tamoxifen in wild-type mice were abrogated in ERα-AF1–deficient mice. The combination of pharmacologic and transgenic approaches thus indicates that selective ERα-AF1 activation by tamoxifen is sufficient to elicit metabolic protection, contrasting with the specific requirement of ERα-AF2 in the metabolic actions of 17β-estradiol. This redundancy in the ability of the two ERα-AFs to separately mediate metabolic prevention strikingly contrasts with the contribution of both ERα-AFs in breast cancer proliferation, shedding new light on the therapeutic potential of selective ER modulation. In the past two decades, estrogen receptor α (ERα) has been identified as a key regulator of energy and glucose homeostasis and consequently proposed as a promising target to develop new therapeutic strategies to fight against obesity-related metabolic disorders, such as type 2 diabetes and nonalcoholic fatty liver disease.1Hevener A.L. Clegg D.J. Mauvais-Jarvis F. Impaired estrogen receptor action in the pathogenesis of the metabolic syndrome.Mol Cell Endocrinol. 2015; 418 Pt 3: 306-321Crossref PubMed Scopus (63) Google Scholar However, understanding the mechanisms of the metabolic protection conferred by ERα activation remains a crucial challenge for optimizing pharmacologic approaches for selective ERα modulation, especially to avoid or at least to minimize the classic estrogen-induced proliferative effects on reproductive tissues. As a member of the nuclear receptor superfamily, ERα regulates transcription through two activation functions (AFs) located in the N-terminal (ERα-AF1) and C-terminal (ERα-AF2) domains. Both AFs are fully activated by estrogens such as 17β-estradiol (E2), although ERα-AF1 can also mediate ligand-independent transcriptional activation.2Metzger D. Ali S. Bornert J.M. Chambon P. Characterization of the amino-terminal transcriptional activation function of the human estrogen receptor in animal and yeast cells.J Biol Chem. 1995; 270: 9535-9542Crossref PubMed Scopus (208) Google Scholar ERα-AF2 resides in the well-ordered ligand-binding domain, and crystallographic structures revealed how the conformational changes induced by various ligands modulate its interactions with conserved motifs of coregulatory proteins.3Smith C.L. O'Malley B.W. Coregulator function: a key to understanding tissue specificity of selective receptor modulators.Endocr Rev. 2004; 25: 45-71Crossref PubMed Scopus (799) Google Scholar, 4Lonard D.M. O'Malley B.W. Nuclear receptor coregulators: judges, juries, and executioners of cellular regulation.Mol Cell. 2007; 27: 691-700Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar In contrast, ERα-AF1, located in the intrinsically disordered AB domain, has hitherto eluded crystallization and high-resolution structure determinations.5Simons Jr., S.S. Edwards D.P. Kumar R. Minireview: dynamic structures of nuclear hormone receptors: new promises and challenges.Mol Endocrinol. 2014; 28: 173-182Crossref PubMed Scopus (60) Google Scholar Most steroid hormone receptors are characterized by a relatively large N-terminal domain (400 to 600 amino acids), and AF1 is generally the transcriptional AF with the highest activity.5Simons Jr., S.S. Edwards D.P. Kumar R. Minireview: dynamic structures of nuclear hormone receptors: new promises and challenges.Mol Endocrinol. 2014; 28: 173-182Crossref PubMed Scopus (60) Google Scholar However, the ERα N-terminal domain only comprises 184 amino acids, suggesting that ERα-AF1 could play a less prominent role compared with ERα-AF2. Nevertheless, in vitro experiments and the recent generation of mouse models with a selective deletion in either ERα-AF1 or ERα-AF2 revealed that the respective roles of ERα-AF1 and ERα-AF2 are cell type dependent and even related on differentiation stages.6Merot Y. Metivier R. Penot G. Manu D. Saligaut C. Gannon F. Pakdel F. Kah O. Flouriot G. The relative contribution exerted by AF-1 and AF-2 transactivation functions in estrogen receptor alpha transcriptional activity depends upon the differentiation stage of the cell.J Biol Chem. 2004; 279: 26184-26191Crossref PubMed Scopus (68) Google Scholar Indeed, although the activation of both AFs is required for uterus7Abot A. Fontaine C. Raymond-Letron I. Flouriot G. Adlanmerini M. Buscato M. Otto C. Berges H. Laurell H. Gourdy P. Lenfant F. Arnal J.F. The AF-1 activation function of estrogen receptor alpha is necessary and sufficient for uterine epithelial cell proliferation in vivo.Endocrinology. 2013; 154: 2222-2233Crossref PubMed Scopus (56) Google Scholar and probably also for breast cancer cell proliferation,8Carreau C. Flouriot G. Bennetau-Pelissero C. Potier M. Respective contribution exerted by AF-1 and AF-2 transactivation functions in estrogen receptor alpha induced transcriptional activity by isoflavones and equol: consequence on breast cancer cell proliferation.Mol Nutr Food Res. 2009; 53: 652-658Crossref PubMed Scopus (24) Google Scholar, 9Penot G. Le Peron C. Merot Y. Grimaud-Fanouillere E. Ferriere F. Boujrad N. Kah O. Saligaut C. Ducouret B. Metivier R. Flouriot G. The human estrogen receptor-alpha isoform hERalpha46 antagonizes the proliferative influence of hERalpha66 in MCF7 breast cancer cells.Endocrinology. 2005; 146: 5474-5484Crossref PubMed Scopus (84) Google Scholar, 10Klinge C.M. Riggs K.A. Wickramasinghe N.S. Emberts C.G. McConda D.B. Barry P.N. Magnusen J.E. Estrogen receptor alpha 46 is reduced in tamoxifen resistant breast cancer cells and re-expression inhibits cell proliferation and estrogen receptor alpha 66-regulated target gene transcription.Mol Cell Endocrinol. 2010; 323: 268-276Crossref PubMed Scopus (61) Google Scholar ERα-AF2 activation is required and is sufficient for E2's protection against osteoporosis11Börjesson A.E. Farman H.H. Engdahl C. Koskela A. Sjögren K. Kindblom J.M. Stubelius A. Islander U. Carlsten H. Antal M.C. Krust A. Chambon P. Tuukkanen J. Lagerquist M.K. Windahl S.H. Ohlsson C. The role of activation functions 1 and 2 of estrogen receptor-α for the effects of estradiol and selective estrogen receptor modulators in male mice.J Bone Miner Res. 2013; 28: 1117-1126Crossref PubMed Scopus (21) Google Scholar and atheroma12Billon-Gales A. Fontaine C. Filipe C. Douin-Echinard V. Fouque M.J. Flouriot G. Gourdy P. Lenfant F. Laurell H. Krust A. Chambon P. Arnal J.F. The transactivating function 1 of estrogen receptor {alpha} is dispensable for the vasculoprotective actions of 17{beta}-estradiol.Proc Natl Acad Sci U S A. 2009; 106: 2053-2058Crossref PubMed Scopus (103) Google Scholar, 13Billon-Gales A. Krust A. Fontaine C. Abot A. Flouriot G. Toutain C. Berges H. Gadeau A.P. Lenfant F. Gourdy P. Chambon P. Arnal J.F. Activation function 2 (AF2) of estrogen receptor-{alpha} is required for the atheroprotective action of estradiol but not to accelerate endothelial healing.Proc Natl Acad Sci U S A. 2011; 108: 13311-13316Crossref PubMed Scopus (98) Google Scholar, 14Smirnova N.F. Fontaine C. Buscato M. Lupieri A. Vinel A. Valera M.C. Guillaume M. Malet N. Foidart J.M. Raymond-Letron I. Lenfant F. Gourdy P. Katzenellenbogen B.S. Katzenellenbogen J. Laffargue M. Arnal J.F. The activation function-1 of estrogen receptor alpha prevents arterial neointima development through a direct effect on smooth muscle cells.Circ Res. 2015; 117: 770-778Crossref PubMed Scopus (38) Google Scholar and also to preserve energy and glucose homeostasis.15Handgraaf S. Riant E. Fabre A. Waget A. Burcelin R. Liere P. Krust A. Chambon P. Arnal J.F. Gourdy P. Prevention of obesity and insulin resistance by estrogens requires ERalpha activation function-2 (ERalphaAF-2), whereas ERalphaAF-1 is dispensable.Diabetes. 2013; 62: 4098-4108Crossref PubMed Scopus (73) Google Scholar Indeed, we recently found that the prevention of high-fat diet (HFD)–induced obesity, insulin resistance, and hyperglycemia by either endogenous estrogens or E2 administration absolutely requires ERα-AF2, whereas ERα-AF1 appears to be dispensable.15Handgraaf S. Riant E. Fabre A. Waget A. Burcelin R. Liere P. Krust A. Chambon P. Arnal J.F. Gourdy P. Prevention of obesity and insulin resistance by estrogens requires ERalpha activation function-2 (ERalphaAF-2), whereas ERalphaAF-1 is dispensable.Diabetes. 2013; 62: 4098-4108Crossref PubMed Scopus (73) Google Scholar However, the question of whether a pharmacologically selective activation of ERα-AF1 retains the ability to confer such a metabolic protection remains to be addressed. Several selective estrogen receptor modulators (SERMs) have been developed for clinical use. Their SERM activity relies on the induction of ligand-specific alterations in the conformation of ERα ligand-binding domain that, thereby, influences the ability of the ER-ligand complex to interact with coactivators and corepressors.3Smith C.L. O'Malley B.W. Coregulator function: a key to understanding tissue specificity of selective receptor modulators.Endocr Rev. 2004; 25: 45-71Crossref PubMed Scopus (799) Google Scholar Furthermore, it has been postulated that the relative balance of coactivator and corepressor expression determines the relative agonist versus antagonist activity displayed by SERMs in a specific cellular target. Tamoxifen, the prototypic SERM recognized for >30 years as the gold standard for endocrine treatment of ER-positive breast cancer,16Early Breast Cancer Trialists' Collaborative GroupTamoxifen for early breast cancer: an overview of the randomised trials.Lancet. 1998; 351: 1451-1467Abstract Full Text Full Text PDF PubMed Scopus (3911) Google Scholar has been characterized to exert ERα-AF1 agonist and ERα-AF2 antagonist effects.7Abot A. Fontaine C. Raymond-Letron I. Flouriot G. Adlanmerini M. Buscato M. Otto C. Berges H. Laurell H. Gourdy P. Lenfant F. Arnal J.F. The AF-1 activation function of estrogen receptor alpha is necessary and sufficient for uterine epithelial cell proliferation in vivo.Endocrinology. 2013; 154: 2222-2233Crossref PubMed Scopus (56) Google Scholar, 17Berry M. Metzger D. Chambon P. Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen.EMBO J. 1990; 9: 2811-2818Crossref PubMed Scopus (663) Google Scholar Interestingly, although clinical data are scarce regarding the influence of tamoxifen on energy homeostasis and body composition, experimental observations suggested that it could exert a significant protection against body weight gain and adipose tissue accumulation.18Wade G.N. Heller H.W. Tamoxifen mimics the effects of estradiol on food intake, body weight, and body composition in rats.Am J Physiol. 1993; 264: R1219-R1223PubMed Google Scholar, 19Wallen W.J. Belanger M.P. Wittnich C. Sex hormones and the selective estrogen receptor modulator tamoxifen modulate weekly body weights and food intakes in adolescent and adult rats.J Nutr. 2001; 131: 2351-2357Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Thus, to explore the ability of in vivo ERα-AF1 selective activation to confer protection against obesity and associated metabolic disorders, we combined the use of tamoxifen, as a pharmacologic tool, with genetically modified mouse models deleted for either the whole ERα (ERα−/−) or specifically ERα-AF1 (ERα-AF1−/−). Although experimental data have suggested that the mechanisms of tamoxifen's action and its metabolites could not be restricted to ERα activation,20de Medina P. Paillasse M.R. Segala G. Khallouki F. Brillouet S. Dalenc F. Courbon F. Record M. Poirot M. Silvente-Poirot S. Importance of cholesterol and oxysterols metabolism in the pharmacology of tamoxifen and other AEBS ligands.Chem Phys Lipids. 2011; 164: 432-437Crossref PubMed Scopus (44) Google Scholar, 21de Medina P. Payre B.L. Bernad J. Bosser I. Pipy B. Silvente-Poirot S. Favre G. Faye J.C. Poirot M. Tamoxifen is a potent inhibitor of cholesterol esterification and prevents the formation of foam cells.J Pharmacol Exp Ther. 2004; 308: 1165-1173Crossref PubMed Scopus (62) Google Scholar, 22Segala G. de Medina P. Iuliano L. Zerbinati C. Paillasse M.R. Noguer E. Dalenc F. Payre B. Jordan V.C. Record M. Silvente-Poirot S. Poirot M. 5,6-Epoxy-cholesterols contribute to the anticancer pharmacology of tamoxifen in breast cancer cells.Biochem Pharmacol. 2013; 86: 175-189Crossref PubMed Scopus (44) Google Scholar we found that ERα mediates the tamoxifen-induced prevention of obesity, steatosis, and insulin resistance in HFD-fed ovariectomized female mice. We then found that tamoxifen-induced ERα-AF1 activation is sufficient to mediate a full protection against HFD-induced dysmetabolic features. Wild-type (WT) C57BL6/J female mice were purchased from Charles River (Saint-Germain-sur-l'Arbresle, France). ERα-deficient (ERα−/−), ERα-AF1–deficient (ERα-AF1−/−) mice, and their respective littermates (ERα+/+, ERα-AF1+/+), all on a C57BL6/J background, were generated as previously described12Billon-Gales A. Fontaine C. Filipe C. Douin-Echinard V. Fouque M.J. Flouriot G. Gourdy P. Lenfant F. Laurell H. Krust A. Chambon P. Arnal J.F. The transactivating function 1 of estrogen receptor {alpha} is dispensable for the vasculoprotective actions of 17{beta}-estradiol.Proc Natl Acad Sci U S A. 2009; 106: 2053-2058Crossref PubMed Scopus (103) Google Scholar and kindly provided by Prof. Pierre Chambon (IGBMC, Strasbourg, France). WT littermates were systematically used as controls. Mice were housed in cages in groups of six and kept in a specific pathogen-free and temperature-controlled facility on a 12-hour light to dark cycle. Each experimental group included at least six animals. All procedures that involved experimental animals were performed in accordance with the principles and guidelines established by the Institute National de la Santé et de la Recherche Médicale and were approved by the local ethical committee of animal care. All female mice underwent bilateral ovariectomy at 4 weeks of age, and 2 weeks later, they were subcutaneously implanted with pellets releasing either tamoxifen (1.5 mg for 60 days, ie, 1.2 mg/kg/d; Innovative Research of America, Sarasota, FL) or vehicle (placebo pellet). The dosage of tamoxifen is in the range of those currently prescribed in clinics and previously reported to exert protective effects on bone, endothelium, and body weight in rodents.18Wade G.N. Heller H.W. Tamoxifen mimics the effects of estradiol on food intake, body weight, and body composition in rats.Am J Physiol. 1993; 264: R1219-R1223PubMed Google Scholar, 23Ke H.Z. Chen H.K. Simmons H.A. Qi H. Crawford D.T. Pirie C.M. Chidsey-Frink K.L. Ma Y.F. Jee W.S. Thompson D.D. Comparative effects of droloxifene, tamoxifen, and estrogen on bone, serum cholesterol, and uterine histology in the ovariectomized rat model.Bone. 1997; 20: 31-39Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 24Liu L. Zou P. Zheng L. Linarelli L.E. Amarell S. Passaro A. Liu D. Cheng Z. Tamoxifen reduces fat mass by boosting reactive oxygen species.Cell Death Dis. 2015; 6: e1586Crossref PubMed Scopus (52) Google Scholar, 25Lamas A.Z. Caliman I.F. Dalpiaz P.L. de Melo Jr., A.F. Abreu G.R. Lemos E.M. Gouvea S.A. Bissoli N.S. Comparative effects of estrogen, raloxifene and tamoxifen on endothelial dysfunction, inflammatory markers and oxidative stress in ovariectomized rats.Life Sci. 2015; 124: 101-109Crossref PubMed Scopus (48) Google Scholar, 26Grainger D.J. Witchell C.M. Metcalfe J.C. Tamoxifen elevates transforming growth factor-beta and suppresses diet-induced formation of lipid lesions in mouse aorta.Nat Med. 1995; 1: 1067-1073Crossref PubMed Scopus (120) Google Scholar Mice were concomitantly fed a HFD (energy content: 45% fat, 20% protein, and 35% carbohydrate; 3.7 kcal/g; Research Diets, New Brunswick, NJ) for 6 to 12 weeks. Mice were anesthetized with 10 mg/kg i.p. injection of ketamine (Merial, Lyon, France) and 1 mg/kg xylazine (Sigma-Aldrich, Isle d'Abeau Chesnes, France) before ovariectomy or with 2% isoflurane before pellet implantation. Food intake (measurement of food consumption within each cage, expressed as mean weight of food per mice and per day) and body weight were recorded weekly. One week before sacrifice, body composition was analyzed by magnetic resonance imaging (EchoMRI) in live animals. Then i.p. glucose tolerance tests were performed at 9:00 AM after 6 and 12 weeks of treatment in overnight fasted mice. Blood glucose concentrations were monitored with a glucose meter (Roche Diagnostic, Grenoble, France) at −30, 0, 30, 60, and 90 minutes after glucose injection (1 g/kg). Mice were sacrificed at 11:00 AM, after 3 hours of fasting with free access to water. Blood samples were collected from the retro-orbital venous plexus and stored at −20°C. Mice were euthanized by cervical dislocation, and organs were carefully removed, weighed, frozen in liquid nitrogen, and stored at −80°C. One week before sacrifice, basal metabolism was measured in additional tamoxifen- or vehicle-treated ovariectomized female WT mice fed a HFD for 12 weeks by indirect calorimetry after 24 hours of acclimatization in individual cages. Oxygen consumption, carbon dioxide production, and food and water intakes were measured (Phenomaster; TSE Systems, Bad Homburg vor der Höhe, Germany) in individual mice at 10-minute intervals during a 24-hour period at constant temperature (20°C). We calculated the respiratory exchange ratio as the ratio of carbon dioxide production to oxygen consumption and energy expenditure (kilocalories of heat produced). Ambulatory physical activity was monitored by an infrared photocell beam interruption method. A pair-feeding procedure was performed in ovariectomized WT female mice fed a chow diet and receiving daily s.c. injections of tamoxifen (0.5 mg/kg, dissolved at a concentration of 0.125 mg/mL in sesame oil that contained 1% ethanol; Sigma-Aldrich) or vehicle (4 μL/g) for 5 days. Each day, the vehicle-treated group received a quantity of diet corresponding to the mean amount eaten the day before by tamoxifen-treated animals. Mice were sacrificed 4 hours after the final injection. Liver tissues were quickly excised, fixed in 10% buffered formalin, and embedded in paraffin, and sections (3 μm) were stained with hematoxylin and eosin (H&E). Additional fresh liver samples were immersed in Tissue-Tek O.C.T. Compound (Sakura Finetek Japan Co., Ltd., Tokyo, Japan) then frozen in isopentane cooled by liquid nitrogen, and cryosections (7 μm) were stained with Oil Red O (ORO) to assess neutral lipid accumulation as previously described with minor modifications.27Matic M. Bryzgalova G. Gao H. Antonson P. Humire P. Omoto Y. Portwood N. Pramfalk C. Efendic S. Berggren P.O. Gustafsson J.A. Dahlman-Wright K. Estrogen signalling and the metabolic syndrome: targeting the hepatic estrogen receptor alpha action.PLoS One. 2013; 8: e57458Crossref PubMed Scopus (41) Google Scholar Images of each sample were obtained with Eclipse Ci Nikon microscope and using DS-FI camera driven by NIS-AR element software version 4.13 (Nikon, Tokyo, Japan) (H&E sections) or scanned (ORO sections) with nanozoomer scanner (Hamamatsu Photonics, Hamamatsu, Japan) and analyzed with NDP view software version 1.2.47 (Hamamatsu Photonics). Hepatic levels of triglycerides, free cholesterol, and cholesterol esters were determined using the Bligh and Dyer method,28Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can J Biochem Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42694) Google Scholar and lipid extracts were analyzed by gas-liquid chromatography as previously described.29Barrans A. Collet X. Barbaras R. Jaspard B. Manent J. Vieu C. Chap H. Perret B. Hepatic lipase induces the formation of pre-beta 1 high density lipoprotein (HDL) from triacylglycerol-rich HDL2: a study comparing liver perfusion to in vitro incubation with lipases.J Biol Chem. 1994; 269: 11572-11577PubMed Google Scholar Serum samples were used to measure alanine aminotransferase and lipid profile (free fatty acids, triglycerides, total and high-density lipoprotein cholesterol). Plasma insulin and adipokines (resistin, leptin, adiponectin) levels were determined using the Multiplex Immunoassay Technology Xmap (MADKMAG-71K-05 and MADPNMAG-70K-01; Millipex, Millipore, Saint-Quentin-en-Yveline, France). A commercial enzyme-linked immunosorbent assay kit was used to measure serum levels of fibroblast growth factor 21 (EZRMFGF21-26K; Millipore) according to the manufacturer's instructions. Total mRNA were extracted from homogenized liver and subcutaneous white adipose tissue samples using a Precellys tissue homogenizer (Bertin Technology, Saint-Quentin-en-Yveline, France) and the GenElute Mammalian Total RNA Miniprep kit (Sigma-Aldrich), then reverse transcribed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). For hepatic gene expression profiling in WT mice, we performed a quantitative PCR (Fluidigm Dynamic Array, Fluidigm platform, GenoToul, Toulouse, France) from a set of 42 genes based on published microarrays databases available from https://www.ncbi.nlm.nih.gov/gds (accession numbers GSE70329 and GSE70350) and bibliographic analysis highlighting genes reported to be involved in hepatic lipid and glucose metabolic pathways or sensitive to estrogens. Real-time quantitative PCR was performed using an ABI ViiA 7 apparatus. We then performed real-time quantitative PCR on ABI StepOnePlus (Life Technologie, GeT facility, GenoToul) for a set of hepatic genes regulated by tamoxifen in WT mice to assess hepatic expression changes in ERα-AF1 mice. Similarly, real-time quantitative PCR was performed to evaluate adipose tissue gene expression from a set of eight genes involved in lipid and glucose metabolic pathways. Primers were validated by testing PCR efficiency using standard curves (efficiency, 95% to 105%). Primers used in this study are listed in Table 1. Gene expression was quantified using the comparative CT method. Hypoxanthine guanine phosphoribosyl transferase 1 was used as reference.Table 1Oligonucleotide Sequences for Real-Time PCRGeneNCBI refseqForward primerReverse primerAcaa2NM_1774705′-AGTCGTGGGCTACTTCGTGTC-3′5′-GGCAAAAGCTTCGTTCACGTCT-3′AcacaNM_1333605′-TTACAGGATGGTTTGGCCTTTC-3′5′-CAAATTCTGCTGGAGAAGCCAC-3′AcacbNM_1339045′-CCTGAATCTCACGCGCCTA-3′5′-CAGATGGAGTCCAGACATGCTG-3′Acox1NM_0157295′-CAAAGAAACCCCTCCAGCC-3′5′-GCTGTGTGCCGTCTGGAGT-3′AcadlNM_0073815′-AGAAGTTCATCCCCCAGATGAC-3′5′-GGCGTTCGTTCTTACTCCTTGT-3′Apoa1NM_0096925′-TGGGCCAACAGCTGAACC-3′5′-TCCCAGAAGTCCCGAGTCAA-3′Apoa4NM_0074685′-ACCCAGCTAAGCAACAATGCC-3′5′-GTCCTGGAAGAGGGTACTGAGC-3′Apoa5NM_0804345′-AAGCACTCAGGCACGGAAGAG-3′5′-GCTGCCTTTCAGGTTCTCCTGT-3′ApobNM_0096935′-AATCTGTGGTTTCATCATGAGGAC-3′5′-GGCCAGCTTGAGTTCGTACCT-3′CebpaNM_0076785′-AGGGACTTAGGTGTTGGGGATT-3′5′-GTGCAAAAAGCAAGGGATTAGG-3′Cpt1aNM_0134955′-GAAGAAGAAGTTCATCCGATTCAAG-3′5′-GATATCACACCCACCACCACG-3′Cyp17aNM_0078095′-CATCCCACACAAGGCTAACA-3′5′-CAGTGCCCAGAGATTGATGA-3′Cyp7a1NM_0078245′-AGAGCAACTAAACAACCTGCCAGTA-3′5′-GCACTGGAGAGCCGCAGA-3′Dgat2NM_0263845′-ACAGCTGCAGGTCATCTCAGTACTA-3′5′-AGCACAGCTATCAGCCAGCA-3′Fabp1NM_0173995′-AAAGTCAAGGCAGTCGTCAAGC-3′5′-CAATGTCGCCCAATGTCATGGT-3′Fabp4NM_0244065′-AAATCACCGCAGACGACAGGAA-3′5′-TGTGGTCGACTTTCCATCCCA-3′FasnNM_0079885′-AGTCAGCTATGAAGCAATTGTGGA-3′5′-CACCCAGACGCCAGTGTTC-3′Fgf21NM_0200135′-AAAGCCTCTAGGTTTCTTTGCCA-3′5′-CCTCAGGATCAAAGTGAGGCG-3′G6pcNM_0080615′-CTCACTTTCCCCACCAGGTC-3′5′-GCTGAAAGTTTCAGCCACAGC-3′Gdf15NM_0118195′-TGGGACCCCAATCTCACCTCT-3′5′-AGCCGAGAGGACTCGAACTCA-3′GckNM_0102925′-TCGCAGGTGGAGAGCGA-3′5′-TCGCAGTCGGCGACAGA-3′HprtNM_0135565′-ACAGGCCAGACTTTGTTGGATT-3′5′-TTGCGCTCATCTTAGGCTTTGT-3′HmgcrNM_0082555′-CTTGTGGAATGCCTTGTGATTG-3′5′-GAAGAATGTCATGAACACAAAGTAGTTG-3′InsrNM_0105685′-CACTGTCATCAATGGGCAGTTT-3′5′-CATCAGGTTCCGAACAGTTGC-3′Irs2NM_0010812125′-GGAGCCGGACCCGTAGCCTT-3′5′-GGCTGGTAGCGCTTCACTCTTTC-3′LepNM_0084935′-CAGTGCCTATCCAGAAAGTCCAG-3′5′-AATGAAGTCCAAGCCAGTGACC-3′LeprNM_1461465′-ATTCCCTCGGCGCTTCCCTT-3′5′-ACAGCTGCTGCTCAGGGGAT-3′LplNM_0085095′-ATGGCAAGCAACACAACCAG-3′5′-TGTGGAAACCTCGGGCAG-3′MlxiplNM_0214555′-ACTCAGGGAATACACGCCTACAG-3′5′-GAAGAAGGAATTCAGAGCTCAGAAA-3′Nr1h3NM_0138395′-GGAGTGTCGACTTCGCAAATG-3′5′-TCAAGCGGATCTGTTCTTCTGAC-3′Nr1h4NM_0091085′-CCACCGGCTGTCAGGATT-3′5′-CGCGTGTTCTGTTAGCATACCTT-3′Pck1NM_0110445′-GGCCACAGCTGCTGCAG-3′5′-GGTCGCATGGCAAAGGG-3′PemtNM_0012900125′-ACTCATGCATGCTAGTCCCA-3′5′-AGCAGTGAAGGGCTCTTCAT-3′PltpNM_0111255′-GGATTAAAGTGTCCAATGTCTCCTG-3′5′-GTGGAGAAAAAGTTATACATCCTCCTG-3′Plin2NM_0074085′-CCATTTCTCAGCTCCACTCCAC-3′5′-GTGTCGTCGTAGCCGATGC-3′Pnpla2NM_0011636895′-AGTGTCCTTCACCATCCGCTT-3′5′-GGATATCTTCAGGGACATCAGGC-3′PparaNM_0111445′-CCCTGTTTGTGGCTGCTATAATTT-3′5′-GGGAAGAGGAAGGTGTCATCTG-3′PpardNM_0111455′-AAGTGGCCATGGGTGACG-3′5′-TGGTCCAGCAGGGAGGAAG-3′PpargNM_0111465′-ATGGGTGAAACTCTGGGAGATTCT-3′5′-CTTGGAGCTTCAGGTCATATTTGTA-3′Sirt1NM_0198125′-GCTGTGAAGTTACTGCAGGAGTGT-3′5′-CCGCAAGGCGAGCATAGATA-3′Scd1NM_0091275′-CAGTGCCGCGCATCTCTAT-3′5′-CTGACTGGCAAATATAGCTGTATCCT-3′Srebf1NM_0114805′-CAGACACTGGCCGAGATGTG-3′5′-CTTGGTTGTTGATGAGCTGGAG-3′Slc2a4NM_0092045′-AAAAGTGCCTGAAACCAGAG-3′5′-TCACCTCCTGCTCTAAAAGG-3′Ucp2NM_0116715′-CATGGTAGCCACCGGCA-3′5′-CTTCAATCGGCAAGACGAGAC-3′NCBI refseq, National Center for Biotechnology Information reference sequence. Open table in a new tab NCBI refseq, National Center for Biotechnology Information reference sequence. Results are expressed as means ± SEM. Statistical analyses were performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA; www.graphpad.com). t-tests were used to analyze the effects of treatment and genotype on all parameters, except for body weight evolution and i.p. glucose tolerance tests for which two-way analysis of variance tests with repeated measures were used to test the interaction between treatment and genotypes. Bonferroni posttests were subsequently performed in case of interaction. P < 0.05 was considered statistically significant. To first determine whether ERα-AF1 pharmacologic activation confers metabolic protection in our experimental settings, ovariectomized WT female mice were fed with a HFD and concomitantly treated with 1.2 mg/kg/d of tamoxifen or vehicle for 12 weeks. As expected, tamoxifen induced a proliferative effect on the uterus with a wet uterine weight of 33.5 ± 2.8 mg in tamoxifen-treated mice versus 13.0 ± 2.2 mg in control mice (P = 0.0002). Tamoxifen administration prevented HFD-induced body weight gain (Figure 1A), an effect associated with a significant reduction in food intake compared with control mice (Figure 1B). In terms of body composition, tamoxifen prevented HFD-induced fat mass accumulation but did not influence lean mass (Figure 1C). The prevention of fat mass expansion not only concerned subcutaneous but also visceral (ie, perigonadic and mesenteric) white adipose tissues (Figure 1D). As respectively attested by glucose tolerance tests (Figure 1E) and homeostatic model assessment of insulin resistance values (Figure 1F), tamoxifen-treated WT mice were also protected from HFD-induced glucose intolerance and insulin resistance, which rapidly occurred in control mice as expected. Moreover, tamoxifentreatment protected WT mice from fatty liver development, as suggested by histologic staining with H&E and ORO and conf
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