FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2064488696> ?p ?o ?g. }

• W2064488696 endingPage "26393" @default.
• W2064488696 startingPage "26385" @default.
• W2064488696 abstract "The ligand-inducible nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) plays a key role in the differentiation, maintenance, and function of adipocytes and is the molecular target for the insulin-sensitizing thiazoledinediones (TZDs). Although a number of PPARγ target genes that may contribute to the reduction of circulating free fatty acids after TZD treatment have been identified, the relevant PPARγ target genes that may exert the anti-lipolytic effect of TZDs are unknown. Here we identified the anti-lipolytic human G-protein-coupled receptor 81 (GPR81), GPR109A, and the (human-specific) GPR109B genes as well as the mouse Gpr81 and Gpr109A genes as novel TZD-induced genes in mature adipocytes. GPR81/Gpr81 is a direct PPARγ target gene, because mRNA expression of GPR81/Gpr81 (and GPR109A/Gpr109A) increased in mature human and murine adipocytes as well as in vivo in epididymal fat pads of mice upon rosiglitazone stimulation, whereas small interfering RNA-mediated knockdown of PPARγ in differentiated 3T3-L1 adipocytes showed a significant decrease in Gpr81 protein expression. In addition, chromatin immunoprecipitation sequencing analysis in differentiated 3T3-L1 cells revealed a conserved PPAR:retinoid X receptor-binding site in the proximal promoter of the Gpr81 gene, which was proven to be functional by electromobility shift assay and reporter assays. Importantly, small interfering RNA-mediated knockdown of Gpr81 partly reversed the inhibitory effect of TZDs on lipolysis in 3T3-L1 adipocytes. The coordinated PPARγ-mediated regulation of the GPR81/Gpr81 and GPR109A/Gpr109A genes (and GPR109B in humans) presents a novel mechanism by which TZDs may reduce circulating free fatty acid levels and perhaps ameliorate insulin resistance in obese patients. The ligand-inducible nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) plays a key role in the differentiation, maintenance, and function of adipocytes and is the molecular target for the insulin-sensitizing thiazoledinediones (TZDs). Although a number of PPARγ target genes that may contribute to the reduction of circulating free fatty acids after TZD treatment have been identified, the relevant PPARγ target genes that may exert the anti-lipolytic effect of TZDs are unknown. Here we identified the anti-lipolytic human G-protein-coupled receptor 81 (GPR81), GPR109A, and the (human-specific) GPR109B genes as well as the mouse Gpr81 and Gpr109A genes as novel TZD-induced genes in mature adipocytes. GPR81/Gpr81 is a direct PPARγ target gene, because mRNA expression of GPR81/Gpr81 (and GPR109A/Gpr109A) increased in mature human and murine adipocytes as well as in vivo in epididymal fat pads of mice upon rosiglitazone stimulation, whereas small interfering RNA-mediated knockdown of PPARγ in differentiated 3T3-L1 adipocytes showed a significant decrease in Gpr81 protein expression. In addition, chromatin immunoprecipitation sequencing analysis in differentiated 3T3-L1 cells revealed a conserved PPAR:retinoid X receptor-binding site in the proximal promoter of the Gpr81 gene, which was proven to be functional by electromobility shift assay and reporter assays. Importantly, small interfering RNA-mediated knockdown of Gpr81 partly reversed the inhibitory effect of TZDs on lipolysis in 3T3-L1 adipocytes. The coordinated PPARγ-mediated regulation of the GPR81/Gpr81 and GPR109A/Gpr109A genes (and GPR109B in humans) presents a novel mechanism by which TZDs may reduce circulating free fatty acid levels and perhaps ameliorate insulin resistance in obese patients. Because of a high calorie diet and a sedentary lifestyle, obesity and its associated co-morbidities like hypertension, type II diabetes, and atherosclerosis rapidly increase worldwide (1James W.P. J. Intern. Med. 2008; 263: 336-352Crossref PubMed Scopus (511) Google Scholar). Adipose tissue is the major site of lipid storage in the body and plays a pivotal role in the regulation of whole body metabolic homeostasis and therefore in the pathophysiology of obesity (2Rosen E.D. Spiegelman B.M. Nature. 2006; 444: 847-853Crossref PubMed Scopus (1547) Google Scholar). After a meal, excess fuel substrates are partitioned to adipose tissue where they are processed and stored as triglycerides (TAG). 2The abbreviations used are: TAGtriglycerideChIPchromatin immunoprecipitationEMSAelectromobility shift assayFAfatty acidFFAfree fatty acidGPRG-protein-coupled receptorLPLlipoprotein lipasehMADShuman multipotent adipose-derived stem cellsPPARperoxisome proliferator-activated receptorPPREPPAR-response elementRXRretinoid X receptorSGBSSimpson-Golabi Behmel syndromeTZDthiazolidinedionePPARγperoxisome proliferator-activated receptor γChIP-seqsequencing ChIPsiRNAsmall interfering RNARTreverse transcription. 2The abbreviations used are: TAGtriglycerideChIPchromatin immunoprecipitationEMSAelectromobility shift assayFAfatty acidFFAfree fatty acidGPRG-protein-coupled receptorLPLlipoprotein lipasehMADShuman multipotent adipose-derived stem cellsPPARperoxisome proliferator-activated receptorPPREPPAR-response elementRXRretinoid X receptorSGBSSimpson-Golabi Behmel syndromeTZDthiazolidinedionePPARγperoxisome proliferator-activated receptor γChIP-seqsequencing ChIPsiRNAsmall interfering RNARTreverse transcription. Conversely, during fasting TAGs are hydrolyzed to free fatty acids (FFA) and glycerol, and the FFA released into the bloodstream can subsequently be used by other organs as energy substrates. The latter process, termed lipolysis, is tightly regulated by hormones and cytokines (3Duncan R.E. Ahmadian M. Jaworski K. Sarkadi-Nagy E. Sul H.S. Annu. Rev. Nutr. 2007; 27: 79-101Crossref PubMed Scopus (606) Google Scholar). The three main hormones that regulate lipolysis in humans are insulin, which inhibits lipolysis, and catecholamines (adrenaline and noradrenaline) and glucagon, which stimulate lipolysis. In rodents, inhibition of lipolysis by adenosine presents an additional regulatory pathway. Lipolysis is deregulated in obesity; basal lipolysis rates are increased (4Reynisdottir S. Langin D. Carlström K. Holm C. Rössner S. Arner P. Clin. Sci. 1995; 89: 421-429Crossref PubMed Scopus (75) Google Scholar), whereas the stimulation of lipolysis by catecholamines (5Large V. Peroni O. Letexier D. Ray H. Beylot M. Diabetes Metab. 2004; 30: 294-309Crossref PubMed Scopus (186) Google Scholar) as well as the anti-lipolytic action of insulin (6Jensen M.D. Haymond M.W. Rizza R.A. Cryer P.E. Miles J.M. J. Clin. Invest. 1989; 83: 1168-1173Crossref PubMed Scopus (535) Google Scholar) are inhibited. The impairment of hormonal control of lipolysis may be due to high levels of tumor necrosis factor-α, which is overproduced by adipose tissue in obese humans and rodents (7Hotamisligil G.S. Shargill N.S. Spiegelman B.M. Science. 1993; 259: 87-91Crossref PubMed Scopus (5970) Google Scholar). Deregulated lipolysis results in increased circulating FFA levels and lipid accumulation in nonadipose tissues, ultimately contributing to insulin resistance and other obesity-related metabolic disorders (8Bays H. Mandarino L. DeFronzo R.A. J. Clin. Endocrinol. Metab. 2004; 89: 463-478Crossref PubMed Scopus (503) Google Scholar). triglyceride chromatin immunoprecipitation electromobility shift assay fatty acid free fatty acid G-protein-coupled receptor lipoprotein lipase human multipotent adipose-derived stem cells peroxisome proliferator-activated receptor PPAR-response element retinoid X receptor Simpson-Golabi Behmel syndrome thiazolidinedione peroxisome proliferator-activated receptor γ sequencing ChIP small interfering RNA reverse transcription. triglyceride chromatin immunoprecipitation electromobility shift assay fatty acid free fatty acid G-protein-coupled receptor lipoprotein lipase human multipotent adipose-derived stem cells peroxisome proliferator-activated receptor PPAR-response element retinoid X receptor Simpson-Golabi Behmel syndrome thiazolidinedione peroxisome proliferator-activated receptor γ sequencing ChIP small interfering RNA reverse transcription. One of the key regulators of adipocyte differentiation, maintenance, and function is peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear hormone receptor superfamily of ligand-inducible transcription factors (9Lehrke M. Lazar M.A. Cell. 2005; 123: 993-999Abstract Full Text Full Text PDF PubMed Scopus (1123) Google Scholar). PPARγ exists in two isoforms, PPARγ1 and PPARγ2. PPARγ2 has an additional 30 amino acids at the N terminus, and its expression is restricted to adipose tissue, while PPARγ1 is more widely distributed (e.g. adipocytes, lower intestine, monocytes, and macrophages). In vitro and in vivo studies showed that PPARγ is both necessary and sufficient to induce adipogenesis (9Lehrke M. Lazar M.A. Cell. 2005; 123: 993-999Abstract Full Text Full Text PDF PubMed Scopus (1123) Google Scholar). PPARγ bind as an obligate heterodimer with the retinoic acid X receptors (RXRs) to PPAR-responsive elements (PPREs), which consist of two direct repeats of six nucleotides (AGGTCA) interspaced by one nucleotide (DR-1). Upon binding of ligand these proteins undergo a conformational change, which allows the interaction with so-called coactivators, starting a cascade of protein interactions and modifications that finally results in the induction of specific target genes (10Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). Although the endogenous ligands for PPARγ have not been firmly established, natural compounds like polyunsaturated fatty acids and eicosanoids have been shown to activate PPARγ. In addition, the antidiabetic drugs, such as thiazolidinediones (TZDs) act as high affinity PPARγ ligands (11Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3435) Google Scholar). Administration of these TZDs to obese and/or insulin-resistant patients has been shown to reduce circulating FFAs and thereby improve insulin sensitivity. Part of these effects may be explained by the stimulatory effect of TZDs on adipocyte differentiation, thereby increasing lipid storage capacity in adipose tissue. In addition, PPARγ also regulates a number of genes essential for the adipocytic phenotype, such as genes involved in lipid uptake, lipid synthesis, lipid droplet stabilization, glycerol/FA recycling, and FA oxidation (12Nielsen R. Pedersen T.A. Hagenbeek D. Moulos P. Siersbaek R. Megens E. Denissov S. Børgesen M. Francoijs K.J. Mandrup S. Stunnenberg H.G. Genes Dev. 2008; 22: 2953-2967Crossref PubMed Scopus (426) Google Scholar). Because elevated levels of serum FFAs promote insulin resistance (13Boden G. Diabetes. 1997; 46: 3-10Crossref PubMed Scopus (0) Google Scholar), an important potential mechanism for the beneficial effects of TZDs is therefore the net partitioning of lipids in adipose tissue. Consistent with this notion, genes encoding proteins involved in lipid uptake in adipocytes, such as lipoprotein lipase, CD36, and the oxidized LDL receptor have been reported to be directly regulated by PPARγ (9Lehrke M. Lazar M.A. Cell. 2005; 123: 993-999Abstract Full Text Full Text PDF PubMed Scopus (1123) Google Scholar). In addition, PPARγ directly regulates the expression of genes directly involved in lipid storage, like the lipid-droplet proteins perilipin and S3-12 (14Dalen K.T. Schoonjans K. Ulven S.M. Weedon-Fekjaer M.S. Bentzen T.G. Koutnikova H. Auwerx J. Nebb H.I. Diabetes. 2004; 53: 1243-1252Crossref PubMed Scopus (167) Google Scholar). PPARγ also regulates genes (potentially) involved in the “futile cycle” (9Lehrke M. Lazar M.A. Cell. 2005; 123: 993-999Abstract Full Text Full Text PDF PubMed Scopus (1123) Google Scholar, 15Kershaw E.E. Schupp M. Guan H.P. Gardner N.P. Lazar M.A. Flier J.S. Am. J. Physiol. Endocrinol. Metab. 2007; 293: E1736-E1745Crossref PubMed Scopus (168) Google Scholar, 16Yajima H. Kobayashi Y. Kanaya T. Horino Y. Biochem. Biophys. Res. Commun. 2007; 352: 526-531Crossref PubMed Scopus (24) Google Scholar), the re-esterification of fatty acids and glycerol to triglycerides. Several findings suggest that PPARγ and TZDs may also be implicated in the regulation of lipolysis. First, the TZD troglitazone has been shown to lower basal lipolysis rates in differentiated adipocytes (this study and see Refs. (17Lenhard J.M. Kliewer S.A. Paulik M.A. Plunket K.D. Lehmann J.M. Weiel J.E. Biochem. Pharmacol. 1997; 54: 801-808Crossref PubMed Scopus (107) Google Scholar, 18Wang P. Renes J. Bouwman F. Bunschoten A. Mariman E. Keijer J. Diabetologia. 2007; 50: 654-665Crossref PubMed Scopus (55) Google Scholar, 19McTernan P.G. Harte A.L. Anderson L.A. Green A. Smith S.A. Holder J.C. Barnett A.H. Eggo M.C. Kumar S. Diabetes. 2002; 51: 1493-1498Crossref PubMed Scopus (106) Google Scholar)) as well as tumor necrosis factor-α-activated lipolysis (20Souza S.C. Yamamoto M.T. Franciosa M.D. Lien P. Greenberg A.S. Diabetes. 1998; 47: 691-695Crossref PubMed Scopus (151) Google Scholar, 21Okazaki H. Osuga J. Tamura Y. Yahagi N. Tomita S. Shionoiri F. Iizuka Y. Ohashi K. Harada K. Kimura S. Gotoda T. Shimano H. Yamada N. Ishibashi S. Diabetes. 2002; 51: 3368-3375Crossref PubMed Scopus (97) Google Scholar). Second, introduction of a dominant-negative form of PPARγ in mature adipocytes resulted in increased lipolysis, suggesting that PPARγ normally inhibits this process (22Tamori Y. Masugi J. Nishino N. Kasuga M. Diabetes. 2002; 51: 2045-2055Crossref PubMed Scopus (236) Google Scholar). Finally, treatment of diabetic patients with TZDs has been shown to restore insulin-mediated suppression of lipolysis (23Miles J.M. Wooldridge D. Grellner W.J. Windsor S. Isley W.L. Klein S. Harris W.S. Diabetes. 2003; 52: 675-681Crossref PubMed Scopus (87) Google Scholar, 24Boden G. Cheung P. Mozzoli M. Fried S.K. Metabolism. 2003; 52: 753-759Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 25Mayerson A.B. Hundal R.S. Dufour S. Lebon V. Befroy D. Cline G.W. Enocksson S. Inzucchi S.E. Shulman G.I. Petersen K.F. Diabetes. 2002; 51: 797-802Crossref PubMed Scopus (546) Google Scholar, 26Racette S.B. Davis A.O. McGill J.B. Klein S. Metabolism. 2002; 51: 169-174Abstract Full Text PDF PubMed Scopus (23) Google Scholar). However, the relevant PPARγ target genes that may exert the anti-lipolytic effect of TZDs are unknown. To identify novel target genes that may play a role in the effects of TZDs on lipid metabolism, we performed a transcriptome analysis in human adipocytes treated with the TZD rosiglitazone. In this study we show that TZDs induce the expression of two anti-lipolytic G-protein-coupled receptors, GPR81/Gpr81 and GPR109A/Gpr109A, in human and murine adipocytes. In addition, a third anti-lipolytic GPR, the human-specific GPR109B, is also induced by rosiglitazone. This PPARγ-mediated activation may occur through a conserved PPRE located in the GPR81 promoter. The coordinated PPARγ-mediated regulation of the GPR81/Gpr81 and GPR109A/Gpr109A genes (and GPR109B in humans) presents a novel mechanism by which TZDs may reduce circulating FFA levels and perhaps ameliorate insulin resistance in obese patients. Rosiglitazone and GW9662 were purchased from Alexis Biochemicals and Cayman Chemical, respectively. Anti-PPARγ antibody (2345S) was from Cell Signaling; anti-GPR81 antibody (NLS2095) was from Novus Biologicals; anti-GPR109A (GTX12610) was from GeneTex, and anti-tubulin (T5168) was from Sigma. Anti-PPARγ (sc-7196) and anti-RXR (sc-774) were used for ChIP assays. Anti-PPARγ (sc-7273), anti-RXRα (sc-553), and anti-Gal4 (sc-510) antibodies used for EMSA were from Santa Cruz Biotechnology. All recombinant DNA work was performed according to standard procedures (27Ausubel F.M. Brent R. Kingston R. Moore D. Seidman J.J. Smith J. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1993Google Scholar). The murine Gpr81 reporter, Gpr81(−1059/+28)-luc, was generated by inserting the respective promoter into the XhoI/HindIII site of the PGL3-basic vector (Promega). All mutations were generated by QuickChange mutagenesis (Stratagene) and verified by sequencing. All other plasmids have been described earlier (28Jeninga E.H. van Beekum O. van Dijk A.D. Hamers N. Hendriks-Stegeman B.I. Bonvin A.M. Berger R. Kalkhoven E. Mol. Endocrinol. 2007; 21: 1049-1065Crossref PubMed Scopus (34) Google Scholar). Culturing of cells was performed as described (28Jeninga E.H. van Beekum O. van Dijk A.D. Hamers N. Hendriks-Stegeman B.I. Bonvin A.M. Berger R. Kalkhoven E. Mol. Endocrinol. 2007; 21: 1049-1065Crossref PubMed Scopus (34) Google Scholar, 29Wabitsch M. Brenner R.E. Melzner I. Braun M. Möller P. Heinze E. Debatin K.M. Hauner H. Int. J. Obes. Relat Metab. Disord. 2001; 25: 8-15Crossref PubMed Scopus (404) Google Scholar, 30Rodriguez A.M. Elabd C. Delteil F. Astier J. Vernochet C. Saint-Marc P. Guesnet J. Guezennec A. Amri E.Z. Dani C. Ailhaud G. Biochem. Biophys. Res. Commun. 2004; 315: 255-263Crossref PubMed Scopus (251) Google Scholar). Differentiation of 3T3-L1 (28Jeninga E.H. van Beekum O. van Dijk A.D. Hamers N. Hendriks-Stegeman B.I. Bonvin A.M. Berger R. Kalkhoven E. Mol. Endocrinol. 2007; 21: 1049-1065Crossref PubMed Scopus (34) Google Scholar), the human multipotent adipose-derived stem cells (hMADs) (30Rodriguez A.M. Elabd C. Delteil F. Astier J. Vernochet C. Saint-Marc P. Guesnet J. Guezennec A. Amri E.Z. Dani C. Ailhaud G. Biochem. Biophys. Res. Commun. 2004; 315: 255-263Crossref PubMed Scopus (251) Google Scholar), and the human Simpson-Golabi Behmel syndrome cell line (SGBS) (29Wabitsch M. Brenner R.E. Melzner I. Braun M. Möller P. Heinze E. Debatin K.M. Hauner H. Int. J. Obes. Relat Metab. Disord. 2001; 25: 8-15Crossref PubMed Scopus (404) Google Scholar) have been described earlier. Reporter assays were performed exactly as described (28Jeninga E.H. van Beekum O. van Dijk A.D. Hamers N. Hendriks-Stegeman B.I. Bonvin A.M. Berger R. Kalkhoven E. Mol. Endocrinol. 2007; 21: 1049-1065Crossref PubMed Scopus (34) Google Scholar). 3T3-L1, SGBS, and hMADs were differentiated as described above and at days 6, 8, and 17, respectively, treated with rosiglitazone or DMSO for 6 h. Micro-array experiments were performed as described before (31Stienstra R. Duval C. Keshtkar S. van der Laak J. Kersten S. Müller M. J. Biol. Chem. 2008; 283: 22620-22627Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In short, total RNA was isolated using TRIzol reagent. Concentrations and purity were determined on a NanoDrop ND-1000 spectrophotometer (Isogen). RNA integrity was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies) with 6000 NanoChips. Part of the RNA samples from four 6-cm dishes was used for quantitative RT-PCR (see under “RNA Isolation and Real Time PCR”). Remaining RNA samples from four 6-cm dishes were pooled and used for microarray analysis. Samples were hybridized on human NUGO arrays from Affymetrix. A detailed description of the analysis method is available on request. Animal study was performed as described earlier (31Stienstra R. Duval C. Keshtkar S. van der Laak J. Kersten S. Müller M. J. Biol. Chem. 2008; 283: 22620-22627Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In short, Sv129 male mice were purchased at The Jackson Laboratory (Bar Harbor, ME). At 20 weeks of age, the diet of half of the mice group was supplemented with rosiglitazone (0.01% w/w) for a week. At the end of the experiment epididymal white adipose tissue was dissected, weighed, and used for RNA isolation. The animal experiments were approved by the animal experimentation committee of Wageningen University. 3T3-L1 fibroblasts were differentiated as described above. Three independent samples of total RNA were extracted at different time points using TRIzol reagent (Invitrogen). cDNA was synthesized using the superscript first strand synthesis system (Invitrogen) according to manufacturer's protocol. Gene expression levels were determined by quantitative real time PCR with the MyIq cycler (Bio-Rad) using SYBR-green (Bio-Rad) and normalized to TFIIb expression. The primers used were as follows: murine TFIIb forward primer, 5′-TCCTCCTCAGACCGCTTTT-3′, and reverse primer, 5′-CCTGGTTCATCATCGCTAATC-3′; murine Gpr81 forward primer, 5′-GGTGGCACGATGTCATGTT-3′, and reverse primer 5′-GACCGAGCAGAACAAGATGATT-3′; murine Gpr109A forward primer, 5′-TCCAAGTCTCCAAAGGTGGT-3′, and reverse primer, 5′-TGTTTCTCTCCAGCACTGAGTT-3′; murine Fapb4 forward primer, 5′-GAAAACGAGATGGTGACAAGC-3′, and reverse primer, 5′-TTGTGGAAGTCACGCCTTT-3′; human 36B4 forward primer, 5′-CGGGAAGGCTGTGGTGCTG-3′, and reverse primer 5′- GTGAACACAAAGCCCACATTCC-3′; human GPR109A forward primer, 5′-TTCAGAGAATGCGATTTAGGG-3′, and reverse primer 5′-ACACCTTGCAACCAGTCTCC-3′; human GPR109B forward primer, 5′-TTCTGTGGGGCATCACTGT-3′, and reverse primer, 5′-GCCATTCTGGATCAGCAACT-3′; and human GPR81 forward primer, 5′-AATTTGGCCGTGGCTGATTTC-3′, and reverse primer, 5′-ACCGTAAGGAACACGATGCTC-3′. For Western blot analyses, differentiated 3T3-L1 cells were lysed in RIPA lysis buffer (200 mm Tris-HCl, pH 8,0; 0,1% SDS, 1% Triton X-100; 10 mm EDTA; 150 mm NaCl; 1% sodium deoxycholate containing protease inhibitors). Total cell lysate was diluted in 4× Laemmli sample buffer, subjected to SDS-PAGE, and transferred to Immobilon membranes (Millipore). α-PPARγ, α-GPR81, α-GPR109A, α-FABP4, and α-tubulin antibodies were used to probe for the respective proteins. ECL Plus (PerkinElmer Life Sciences) was used for detection, and signals were quantified using a densitometer. ChIP assays were performed exactly as described earlier (32Nielsen R. Grøntved L. Stunnenberg H.G. Mandrup S. Mol. Cell. Biol. 2006; 26: 5698-5714Crossref PubMed Scopus (64) Google Scholar). The primers used for ChIP assays were as follows: murine Gpr81 forward primer, 5′-AGTGCCAGAGAGGGGAGACT-3′, and reverse primer 5′-CGTTTCTCTGCAGACCTTCC-3′; murine β-globin forward primer, 5′-CCTGCCCTCTCTATCCTGTG-3′, and reverse primer 5′-GCAAATGTGTTGCCAAAAAG-3′; human GPR81 forward primer, 5′-CTGGAGAGCACACAAAGCTG-3′, and reverse primer 5′-CCACTCCAGGAAATGTTTGG-3′; and human β-globin forward primer, 5′-TGGTATGGGGCCAAGAGATA-3′, and reverse primer 5′-TAGATGCCTCTGCCCTGACT-3′. ChIP-seq was performed as described earlier (12Nielsen R. Pedersen T.A. Hagenbeek D. Moulos P. Siersbaek R. Megens E. Denissov S. Børgesen M. Francoijs K.J. Mandrup S. Stunnenberg H.G. Genes Dev. 2008; 22: 2953-2967Crossref PubMed Scopus (426) Google Scholar). EMSAs were performed as described earlier (28Jeninga E.H. van Beekum O. van Dijk A.D. Hamers N. Hendriks-Stegeman B.I. Bonvin A.M. Berger R. Kalkhoven E. Mol. Endocrinol. 2007; 21: 1049-1065Crossref PubMed Scopus (34) Google Scholar). The sequences of the double-stranded DNA oligomers used, containing the wild type or mutant PPRE from the mouse G-protein-coupled receptor 81 promoter (between −128 and −98 of the Gpr81 gene), were as follows: mGpr81 wild type, 5′-CCGGGGACGGGTAGTCAGGCAAAGGTTAGGGAGGA-3′; mGpr81 mutant A, 5′-CCGGGGACGGCAAGTCACCCAAAGGTTAGGGAGGA-3′; and mGpr81 mutant B, 5′-CCGGGACGGGTAGTCTCGCAAACCTTAGGGAGGA-3′. 3T3-L1 cells were differentiated as described above. At day 6 cells were detached using 5× trypsin/EDTA (Invitrogen), washed in medium containing 4% glycerol. For each reaction 2 million cells were resuspended in buffer L (AMAXA cell line Nucleofector kit L), and control (D-001210-01-20; Dharmacon), murine-specific PPARγ (number 2 J-040712-06 Dharmacon), or custom-made Gpr81 (5′-ACCTGGAAGTCAAGCACTATT; Dharmacon) siRNA oligonucleotides were delivered into adipocytes (500 nm of each siRNA/2 million cells) by electroporation (AMAXA Nucleofector II). Cells were reseeded, and 20 h post-electroporation cells were incubated with 1 μm rosiglitazone for an additional 28 h. Subsequently, cells were harvested for Western blot analysis, and media (n = 4) were collected for glycerol measurements. Glycerol levels were determined according to the manufacturer's instructions (Instruchemie). To identify novel TZD-regulated genes in mature human adipocytes, we performed transcriptome analysis in differentiated hMADs (30Rodriguez A.M. Elabd C. Delteil F. Astier J. Vernochet C. Saint-Marc P. Guesnet J. Guezennec A. Amri E.Z. Dani C. Ailhaud G. Biochem. Biophys. Res. Commun. 2004; 315: 255-263Crossref PubMed Scopus (251) Google Scholar). Out of 361 genes that were up-regulated after 6 h of treatment with the TZD rosiglitazone (data not shown), we selected the human-specific G-protein-coupled receptor 109B (GPR109B) to explore in more detail. Together with GPR109A and GPR81, GPR109B belongs to the class A rhodopsin-like G-protein-coupled receptors. GPR109A (also called puma-g) and the human-specific GPR109B are 96% homologous (33Lee D.K. Nguyen T. Lynch K.R. Cheng R. Vanti W.B. Arkhitko O. Lewis T. Evans J.F. George S.R. O'Dowd B.F. Gene. 2001; 275: 83-91Crossref PubMed Scopus (160) Google Scholar) and expressed in adipose tissue, spleen, and immune cells (34Wise A. Foord S.M. Fraser N.J. Barnes A.A. Elshourbagy N. Eilert M. Ignar D.M. Murdock P.R. Steplewski K. Green A. Brown A.J. Dowell S.J. Szekeres P.G. Hassall D.G. Marshall F.H. Wilson S. Pike N.B. J. Biol. Chem. 2003; 278: 9869-9874Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 35Tunaru S. Kero J. Schaub A. Wufka C. Blaukat A. Pfeffer K. Offermanns S. Nat. Med. 2003; 9: 352-355Crossref PubMed Scopus (645) Google Scholar, 36Soga T. Kamohara M. Takasaki J. Matsumoto S. Saito T. Ohishi T. Hiyama H. Matsuo A. Matsushime H. Furuichi K. Biochem. Biophys. Res. Commun. 2003; 303: 364-369Crossref PubMed Scopus (280) Google Scholar), whereas GPR81 expression is almost exclusively restricted to adipose tissue (37Schaub A. Fütterer A. Pfeffer K. Eur. J. Immunol. 2001; 31: 3714-3725Crossref PubMed Scopus (108) Google Scholar, 38Ge H. Weiszmann J. Reagan J.D. Gupte J. Baribault H. Gyuris T. Chen J.L. Tian H. Li Y. J. Lipid Res. 2008; 49: 797-803Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). GPR109A has been identified as the receptor for the anti-lipolytic drug nicotinic acid, and in GPR109A knock-out mice it has been shown that GPR109A was the receptor mediating the lipid-lowering effects of nicotinic acid (34Wise A. Foord S.M. Fraser N.J. Barnes A.A. Elshourbagy N. Eilert M. Ignar D.M. Murdock P.R. Steplewski K. Green A. Brown A.J. Dowell S.J. Szekeres P.G. Hassall D.G. Marshall F.H. Wilson S. Pike N.B. J. Biol. Chem. 2003; 278: 9869-9874Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 35Tunaru S. Kero J. Schaub A. Wufka C. Blaukat A. Pfeffer K. Offermanns S. Nat. Med. 2003; 9: 352-355Crossref PubMed Scopus (645) Google Scholar, 36Soga T. Kamohara M. Takasaki J. Matsumoto S. Saito T. Ohishi T. Hiyama H. Matsuo A. Matsushime H. Furuichi K. Biochem. Biophys. Res. Commun. 2003; 303: 364-369Crossref PubMed Scopus (280) Google Scholar). Recently, the ketone body β-hydroxybutyrate was reported as an endogenous agonist for GPR109A (39Taggart A.K. Kero J. Gan X. Cai T.Q. Cheng K. Ippolito M. Ren N. Kaplan R. Wu K. Wu T.J. Jin L. Liaw C. Chen R. Richman J. Connolly D. Offermanns S. Wright S.D. Waters M.G. J. Biol. Chem. 2005; 280: 26649-26652Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar), whereas aromatic d-amino acids can activate GPR109B (40Irukayama-Tomobe Y. Tanaka H. Yokomizo T. Hashidate-Yoshida T. Yanagisawa M. Sakurai T. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 3930-3934Crossref PubMed Scopus (51) Google Scholar). In addition, very recently two reports (41Cai T.Q. Ren N. Jin L. Cheng K. Kash S. Chen R. Wright S.D. Taggart A.K. Waters M.G. Biochem. Biophys. Res. Commun. 2008; 377: 987-991Crossref PubMed Scopus (181) Google Scholar, 42Liu C. Wu J. Zhu J. Kuei C. Yu J. Shelton J. Sutton S.W. Li X. Yun S.J. Mirzadegan T. Mazur C. Kamme F. Lovenberg T.W. J. Biol. Chem. 2009; 284: 2811-2822Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) showed that GPR81 functions as a receptor for lactate, which reduces lipolysis in vitro and in vivo (43Boyd 3rd, A.E. Giamber S.R. Mager M. Lebovitz H.E. Metabolism. 1974; 23: 531-542Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 44De Pergola G. Cignarelli M. Nardelli G. Garruti G. Corso M. Di Paolo S. Cardone F. Giorgino R. Horm. Metab. Res. 1989; 21: 210-213Crossref PubMed Scopus (21) Google Scholar). Interestingly, the GPR81, GPR109A, and GPR109B genes are colocalized on human chromosome 12 and share synteny with murine Gpr81 and Gpr109A on mouse chromosome 5 (Fig. 1A) (45Offermanns S. Trends Pharmacol. Sci. 2006; 27: 384-390Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). For this reason, expression of the GPR109A and GPR81 genes, which were not represented on the microarray, was determined together with the GPR109B gene in differentiated hMADs cells. Using quantitative RT-PCR, mRNA expression of these three genes was found to increase 4–5-fold after treatment with rosiglitazone for 6 h (Fig. 1B). The same experiment was performed in another human adipocyte cell line, the SGBS cell line (29Wabitsch M. Brenner R.E. Melzner I. Braun M. Möller P. Heinze E. Debatin K.M. Hauner H. Int. J. Obes. Relat Metab. Disord. 2001; 25: 8-15Crossref PubMed Scopus (404) Google Scholar). In these cells a similar mRNA expression profile was observed (Fig. 1C). To investigate whether conserved mechanisms of regulation exist in mouse adipocytes, we examined the effect of rosiglitazone treatment on Gpr81 and Gpr109A mRNA expression in differentiated 3T3-L1 adipocytes. As was observed for the human adipocytes, treatment of murine adipocytes with rosiglitazone stimulated the mRNA expression levels of Gpr81 and Gpr109A up to 4-fold (Fig. 1D). Finally, we examined the effect of rosiglitazone treatment" @default.
• W2064488696 created "2016-06-24" @default.
• W2064488696 creator A5012889438 @default.
• W2064488696 creator A5028342693 @default.
• W2064488696 creator A5035669874 @default.
• W2064488696 creator A5036095832 @default.
• W2064488696 creator A5040542231 @default.
• W2064488696 creator A5050694210 @default.
• W2064488696 creator A5060538191 @default.
• W2064488696 creator A5074578811 @default.
• W2064488696 creator A5084915485 @default.
• W2064488696 creator A5085068425 @default.
• W2064488696 creator A5089547723 @default.
• W2064488696 date "2009-09-01" @default.
• W2064488696 modified "2023-10-13" @default.
• W2064488696 title "Peroxisome Proliferator-activated Receptor γ Regulates Expression of the Anti-lipolytic G-protein-coupled Receptor 81 (GPR81/Gpr81)" @default.
• W2064488696 cites W1488559297 @default.
• W2064488696 cites W149212049 @default.
• W2064488696 cites W1965004625 @default.
• W2064488696 cites W1976762010 @default.
• W2064488696 cites W1980950685 @default.
• W2064488696 cites W1981478386 @default.
• W2064488696 cites W1997323800 @default.
• W2064488696 cites W2004243328 @default.
• W2064488696 cites W2004798972 @default.
• W2064488696 cites W2012650972 @default.
• W2064488696 cites W2016443141 @default.
• W2064488696 cites W2032386138 @default.
• W2064488696 cites W2033204507 @default.
• W2064488696 cites W2036468147 @default.
• W2064488696 cites W2046833545 @default.
• W2064488696 cites W2047591801 @default.
• W2064488696 cites W2050399087 @default.
• W2064488696 cites W2050442804 @default.
• W2064488696 cites W2052495959 @default.
• W2064488696 cites W2060275151 @default.
• W2064488696 cites W2064520545 @default.
• W2064488696 cites W2066721136 @default.
• W2064488696 cites W2071741407 @default.
• W2064488696 cites W2086554547 @default.
• W2064488696 cites W2087710728 @default.
• W2064488696 cites W2088986234 @default.
• W2064488696 cites W2090600090 @default.
• W2064488696 cites W2100514340 @default.
• W2064488696 cites W2101951807 @default.
• W2064488696 cites W2113637800 @default.
• W2064488696 cites W2121241298 @default.
• W2064488696 cites W2129316761 @default.
• W2064488696 cites W2129977761 @default.
• W2064488696 cites W2131820599 @default.
• W2064488696 cites W2135907315 @default.
• W2064488696 cites W2138825018 @default.
• W2064488696 cites W2144701449 @default.
• W2064488696 cites W2146845495 @default.
• W2064488696 cites W2151025367 @default.
• W2064488696 cites W2151845029 @default.
• W2064488696 cites W2152543396 @default.
• W2064488696 cites W2152825164 @default.
• W2064488696 cites W2153965051 @default.
• W2064488696 cites W2156575042 @default.
• W2064488696 cites W2158025896 @default.
• W2064488696 cites W2159926700 @default.
• W2064488696 cites W2161204617 @default.
• W2064488696 cites W2168443670 @default.
• W2064488696 cites W2170211828 @default.
• W2064488696 cites W2174415873 @default.
• W2064488696 cites W2414472597 @default.
• W2064488696 cites W2422651944 @default.
• W2064488696 doi "https://doi.org/10.1074/jbc.m109.040741" @default.
• W2064488696 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2785326" @default.
• W2064488696 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19633298" @default.
• W2064488696 hasPublicationYear "2009" @default.
• W2064488696 type Work @default.
• W2064488696 sameAs 2064488696 @default.
• W2064488696 citedByCount "77" @default.
• W2064488696 countsByYear W20644886962012 @default.
• W2064488696 countsByYear W20644886962013 @default.
• W2064488696 countsByYear W20644886962014 @default.
• W2064488696 countsByYear W20644886962015 @default.
• W2064488696 countsByYear W20644886962016 @default.
• W2064488696 countsByYear W20644886962017 @default.
• W2064488696 countsByYear W20644886962018 @default.
• W2064488696 countsByYear W20644886962019 @default.
• W2064488696 countsByYear W20644886962020 @default.
• W2064488696 countsByYear W20644886962021 @default.
• W2064488696 countsByYear W20644886962022 @default.
• W2064488696 countsByYear W20644886962023 @default.
• W2064488696 crossrefType "journal-article" @default.
• W2064488696 hasAuthorship W2064488696A5012889438 @default.
• W2064488696 hasAuthorship W2064488696A5028342693 @default.
• W2064488696 hasAuthorship W2064488696A5035669874 @default.
• W2064488696 hasAuthorship W2064488696A5036095832 @default.
• W2064488696 hasAuthorship W2064488696A5040542231 @default.
• W2064488696 hasAuthorship W2064488696A5050694210 @default.
• W2064488696 hasAuthorship W2064488696A5060538191 @default.
• W2064488696 hasAuthorship W2064488696A5074578811 @default.
• W2064488696 hasAuthorship W2064488696A5084915485 @default.
• W2064488696 hasAuthorship W2064488696A5085068425 @default.

• next