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J. Biol. Chem., Vol. 277, Issue 30, 26821-26830, July 26, 2002

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The Gene Encoding the Acyl-CoA-binding Protein Is Activated by Peroxisome Proliferator-activated Receptor  through an Intronic Response Element Functionally Conserved between Humans and Rodents^*

Torben Helledie, Lars Grøntved, Søren S. Jensen, Pia Kiilerich, Luc Rietveld, Tatjana Albrektsen^§, Maria S. Boysen, Jane Nøhr, Leif K. Larsen, Jan Fleckner^§, Hendrik G. Stunnenberg, Karsten Kristiansen, and Susanne Mandrup^¶

From the Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark, the  Department of Molecular Biology, Nijmegen Center for Molecular Life Sciences, 6525 GA Nijmegen, The Netherlands, and ^§ Novo, Nordisk A/S, 2880 Bagsvrd, Denmark

Received for publication, November 27, 2001, and in revised form, May 14, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular protein that specifically binds acyl-CoA esters with high affinity and is structurally and functionally conserved from yeast to mammals. In vitro studies indicate that ACBP may regulate the availability of acyl-CoA esters for various metabolic and regulatory purposes. The protein is particularly abundant in cells with a high level of lipogenesis and de novo fatty acid synthesis and is significantly induced during adipocyte differentiation. However, the molecular mechanisms underlying the regulation of ACBP expression in mammalian cells have remained largely unknown. Here we report that ACBP is a novel peroxisome proliferator-activated receptor (PPAR) target gene. The rat ACBP gene is directly activated by PPAR/retinoid X receptor  (RXR) and PPAR/RXR, but not by PPAR/RXR, through a PPAR-response element in intron 1, which is functionally conserved in the human ACBP gene. The intronic PPAR-response element (PPRE) mediates induction by endogenous PPAR in murine adipocytes and confers responsiveness to the PPAR-selective ligand BRL49653. Finally, we have used chromatin immunoprecipitation to demonstrate that the intronic PPRE efficiently binds PPAR/RXR in its natural chromatin context in adipocytes. Thus, the PPRE in intron 1 of the ACBP gene is a bona fide PPAR-response element.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The acyl-CoA-binding protein (ACBP)¹ is an intracellular lipid-binding protein that selectively binds medium and long chain acyl-CoA esters with high affinity. The protein has been found in all eukaryotes investigated to date from mammals to yeasts and plants and is structurally and functionally highly conserved through evolution (1). However, with the exception of the recently identified testis-specific protein known as endozepine-like peptide, ACBPs diverge significantly at a structural and functional level from other mammalian lipid-binding proteins. Whereas members of the fatty acid-binding protein family have a so-called -barrel structure and bind a broad spectrum of fatty acids and fatty acid derivatives, ACBP has a four -helix bundle structure and binds specifically acyl-CoA esters by docking the hydrocarbon chain into the lipophilic pocket and using the CoA moiety as a lid (1).

In vitro investigations have shown that ACBP is able to protect efficiently acyl-CoA esters from hydrolysis by thioesterases and that it can function both as acceptor and donor of acyl-CoA esters (2). Thus, ACBP relieves acyl-CoA inhibition of long chain acyl-CoA synthetase, acetyl-CoA carboxylase, and adenine nucleotide transferase (2) and regulates acyl-CoA:cholesterol acyltransferase activity (3). Furthermore, ACBP is able to transport acyl-CoA esters and donate these to mitochondrial -oxidation (4-6), microsomal glycerolipid synthesis (4), and phospholipid synthesis (7, 8). Overexpression of ACBP in yeast significantly increases the acyl-CoA pool size, indicating that ACBP can generate an intracellular acyl-CoA pool (9, 10).

The mammalian ACBP gene is a typical housekeeping gene (11), and ACBP appears to be ubiquitously expressed from early stages of mammalian embryogenesis (12) as well as in adult tissues (reviewed in Ref. 13). However, the level of ACBP differs markedly among different cell types. Cell types with a high level of ACBP expression include hepatocytes, steroidogenic cells, and adipocytes.

The expression of the mammalian ACBP gene is regulated with the feeding status. Fasting of rats results in a significant decrease in ACBP mRNA and protein in the liver, whereas the level in heart and kidney is unaffected (6, 14). In contrast, conditions that induce de novo fatty acid synthesis appear to induce ACBP expression (15). Similarly, ACBP expression is induced during in vitro differentiation of 3T3-L1 preadipocytes (16), a process that is accompanied by a marked triglyceride accumulation and de novo fatty acid synthesis. The proximal promoter of the human ACBP gene has been shown to contain a sterol regulatory element that was activated by sterol regulatory element-binding protein (SREBP-1)/adipocyte determination and differentiation factor 1 in transient transfections (17). This transcription factor has been shown recently to be involved in the coordinated induction by insulin of genes involved in lipogenesis (18, 19), and it is therefore possible that SREBP-1 regulates ACBP in response to fasting and feeding.

Interestingly, ACBP expression is not only increased by conditions that favor de novo fatty acid synthesis. High fat feeding of rats leads to elevated levels of ACBP in the liver (6), and various peroxisome proliferators, which are known as potent inducers of liver mitochondrial and peroxisomal -oxidation, induce ACBP mRNA and protein expression in the liver (14, 20) and ACBP protein expression in isolated hepatocytes (21). The stimulation of liver lipid catabolism by these compounds is known to be mediated by an activation of the peroxisome proliferator-activated receptor  (PPAR) (22, 23), suggesting that ACBP may be a PPAR target gene.

Although ACBP expression has been reported to increase in the liver by prolonged exposure to PPAR activators, a functional PPAR-response element (PPRE) has never been identified in the ACBP gene, and it is unknown whether the ACBP gene is a direct target of the PPARs. In this report we demonstrate unequivocally that ACBP is a PPAR target gene, which is activated by PPAR-selective ligands in adipose tissue as well as in adipocyte cell lines, and we demonstrate the existence of a functional PPRE in intron 1 of the rat ACBP gene. This PPRE confers PPAR and PPAR responsiveness to the promoter and is functionally conserved between humans and rodents. In adipocytes, the intronic PPRE mediates transcriptional activation in response to treatment with the PPAR-selective ligand BRL49653. Chromatin immunoprecipitation shows that the PPRE binds PPAR/RXR in vivo. These results show that ACBP expression is directly regulated by PPAR and possibly also PPAR through the intronic PPRE.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animal Experiments-- C57BL-Ks db/db mice at the age of 12 weeks were divided into groups of 6 animals and dosed once daily for 10 days with rosiglitazone (1.0, 3, or 10 mg/kg/dosing) or vehicle (0.2% CMC + 0.4% Tween 80 in saline) by oral gavage. At the end of the dosing period, animals were killed by decapitation, and white adipose tissue (epididymal fat pads) was removed and frozen in liquid nitrogen, and RNA was isolated by RNazol (BioSite, Täby, Sweden) according to the manufacturer's instructions. Equal amounts of total RNA from all animals in each group were pooled. All animal experiments were conducted in accordance with the Danish law.

Quantitative PCR-- Pooled total RNA isolated from the white adipose tissue was DNase-treated, and three independent reverse transcription reactions were performed using Superscript II reverse transcriptase (Invitrogen) following the manufacturer's instructions. mRNA expression levels were determined using real time fluorescent detection in a Lightcycler instrument (Roche Molecular Biochemicals) and the following primer combinations: 5'-AGCCAACTGATGAAGAGATG-f-3' and 5'-AGGCATTATGTCCTCACAGG-r-3' for ACBP, 5'-ATGCCTTTGTGGGAACCTGG-f-3'; 5'-CCCAGTTTGAAGGAAATCTCGG-r-3' for adipocyte lipid-binding protein (aP2). mRNA expression levels were determined twice in each 1st strand synthesis reaction and normalized to the expression levels of 18 S rRNA as described by the vendor (Applied Biosystems).

Cell Culture-- 3T3-L1, NIH-3T3, and 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing 4500 mg/liter glucose supplemented with 100 µg/ml streptomycin, 62.5 µg/ml penicillin, 8 µg/ml biotin, and 8 µg/ml pantothenic acid. Standard 3T3-L1 and NIH-3T3 culture media contained 10% calf serum (Sigma). Standard 293 media contained 10% fetal calf serum (FCS) (Invitrogen). Medium was exchanged every other day.

Differentiation of 3T3-L1 cells was obtained by exposing 2-day post-confluent cells (designated day 0 cells) to DMEM containing 10% FCS (Invitrogen) supplemented with 1 µM dexamethasone (Sigma), 0.5 mM 3-isobutyl-1-methylxanthine (Aldrich), and 1 µg/ml insulin (Roche Molecular Biochemicals). At day 2 cells were fed DMEM containing FCS and 1 µg/ml insulin, thereafter cells were maintained in DMEM and FCS.

Northern Blotting-- RNA was isolated (24) from 3T3-L1 cells at day 2, 0, 1-4, 6, 8, and 10 of differentiation. At day 10, cells were exposed to Me₂SO, 1 µM BRL49653 in Me₂SO, 15 µg/ml cycloheximide, or 1 µM BRL49653 + 15 µg/ml cycloheximide. RNA was isolated after 2, 6, and 12 h of incubation.

RNA was analyzed by Northern blotting with a ³²P-labeled rat ACBP cDNA fragment as probe. Signals were quantified by PhosphorImaging. The membranes were stripped and reprobed with a labeled mouse adipocyte lipid-binding protein (ALBP) cDNA probe and finally with the human 28 S rRNA probe.

Western Blotting-- 3T3-L1 cells from day 2, 0, 1-4, 6, 8, and 10 were lysed in 0.5 ml of 2.5% SDS sample buffer per 10-cm dish. Lysates were subjected to SDS-PAGE. Approximately 20 µg of cellular protein was loaded per lane. The separated proteins were transferred to a polyvinylidene difluoride membrane and stained with Ponceau S for control of equal loading. The membranes were blocked in 5% (w/v) nonfat dry milk, incubated with affinity-purified rabbit anti-mouse ACBP for 1 h, and horseradish peroxidase-conjugated secondary antibody (Dako) for another hour. Immunoreactive protein bands were detected by enhanced chemiluminescence (Amersham Biosciences).

Electrophoretic Mobility Shift Assay-- Nuclei were purified from 3T3-L1 preadipocytes or adipocytes by a modification (25) of the procedure of Dignam et al. (26). Nuclear extracts were prepared as described by Lavery and Schibler (27) using a 1× NUN solution (0.3 M NaCl, 1 M urea, 1% Nonidet P-40, 25 mM HEPES, pH 7.9, and 1 mM dithiothreitol). Protein concentrations were determined using Bradford protein assay reagent (Bio-Rad).

Rat liver nuclear extract was prepared from 3-month-old Sprague-Dawley rats weighing ~300 g according to the procedure described by Gorski et al. (28) except that the second purification of the nuclei was omitted, and protease inhibitors (1 µg/µl leupeptin, 1 µg/µl antipain, 1 µg/µl pepstatin, and 0.01 TIU/ml aprotinin) were added to all buffers just prior to use.

In vitro translations were performed using TNT kit according to the recommendations of the manufacturer (Promega). Double-stranded oligonucleotides corresponding to the rat ACBP intron 1 DR-1, rat ACBP upstream DR-1, and human ACBP intron 1 DR-1 were labeled using [-³²P]ATP and polynucleotide kinase (Roche Molecular Biochemicals).

Nuclear extracts (2-4 µg) or in vitro translated proteins were incubated 20 min on ice in binding buffer (10 mM Tris, pH 8.0, 40 mM KCl, 1 mM dithioerythritol, 4% glycerol, and 0.05% Nonidet P-40, 2.4 µg of poly(dI-dC)). Subsequently 2 × 10⁵ cpm of ³²P-labeled oligonucleotide was added, and the mixture was incubated for 20 min at room temperature. For competition assays, 10-fold excess of a homologous or heterologous competitor was mixed with the labeled probe and added to the preincubation mixture. Free DNA and DNA-protein complexes were resolved by electrophoresis in 0.5× TBE, 5% polyacrylamide gels.

Plasmids-- Plasmids used in transient transfections were pTK-3x- PPRE-luc (29), pTK-3xACBP-PPRE-luc, and pTK-luc. The plasmid pTK3xACBP-PPRE was made from pTK-3xPPRE-luc by replacement of the three copies of ACO PPRE with three copies of the rat ACBP intronic PPRE inserted in the same orientation and with the same spacing as the original ACO PPREs. Rat ACBP promoter reporter constructs rACBP(392/+1)-luc, rACBP(1512/+1)-luc, and rACBP(1535/+1)-luc were constructed by inserting the respective promoter fragments in the pGL3-basic vector (Promega). Rat ACBP promoter construct rACBP(392/+979)-luc, rACBP(1512/+979)-luc, rACBP(1535/+979)-luc, rACBP(392/+979)PPRE-luc, rACBP(1512/+979)PPRE-luc, and rACBP(1535/+979)-luc were constructed by inserting the respective promoter/exon 1 and intron 1 sequence in pGL3-basic so that the reading frame of ACBP exon 2 was fused in-frame with that of luciferase. The intronic PPRE (GGGACAGAGGTCA) was mutated to GTTTTTTTTGTCA in the PPRE constructs. Human ACBP promoter reporter constructs hACBP(516/+5)-luc, hACBP(516/+1136)-luc, and hACBP(516/+1136)PPRE-luc were constructed similarly. The intronic PPRE (GGGACAGAGGTCG) was mutated to GTTTTTTTTGTCG in the PPRE constructs. For expression of PPARs and RXR, the expression plasmids pSG5-PPAR (30), pSG5-PPAR (31), pSPORT-PPAR2 (32), and pCMX-mRXR (33) were used.

pCMV--galactosidase-control (Promega) was used for normalization, and pBluescriptKS (Stratagene), pSG5 (Stratagene), and/or pcDNA were used to adjust to equal DNA load and promoter load, respectively.

Transient Transfections-- NIH-3T3 and 293 cells were transfected at 50-70% confluency in 60-mm dishes or 6-well plates using the DC-Chol lipofection procedure (34) and a total of 5 or 2.5 µg, respectively, of DNA/plate/well. Following 6 h of incubation with DNA mixture, the medium was changed to DMEM and 10% resin-charcoal-stripped calf serum (for NIH-3T3) or FCS (for 293) supplemented with PPAR activator or vehicle (Me₂SO) alone. 3T3-L1 cells were transfected at day 4 of differentiation using the LipofectAMINE Plus procedure (Invitrogen). Transfections were performed in 12-well plates with a total of 1 µg/well. After 3 h of incubation with the DNA mixture, medium was changed to serum-free DMEM supplemented with PPAR activator or vehicle (Me₂SO) alone. Cells were harvested 20 h later in lysis buffer (Tropix), and the lysates were stored at 80 °C. All transfections were performed as triplicates. Luciferase and -galactosidase assays were performed as described previously (35).

The PPAR activators Wy14643 (Calbiochem), BRL49653 (Novo Nordisk A/S), and activator L-165041 (Merck) were used in the indicated concentrations to activate PPAR, PPAR, and PPAR, respectively. Serum was stripped with AG-1X-8 resin and activated charcoal powder as described previously (35).

Chromatin Cross-linking-- Day 4 3T3-L1 adipocytes were removed from plates by trypsinization and resuspended in DMEM + 10% FCS (18 ml per four 15-cm plates). Cells were fixed in vivo for 10 min at room temperature by addition of 2 ml of formaldehyde solution (11% formaldehyde, 0.1 M NaCl, 1 mM EDTA, 0.5 mM EGTA, and 50 mM HEPES, pH 8.0). Fixation was stopped by addition of glycine to 0.125 M final concentration, and cells were collected by centrifugation (1000 × g, 4 °C for 5 min), washed once in phosphate-buffered saline, and incubated in 10 ml of Triton lysis buffer for 10 min at 4 °C (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl, pH 8.0). Chromatin was collected by centrifugation (1000 × g, 4 °C, 5 min), washed once in 10 ml of NaCl washing buffer (0.2 M NaCl, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl, pH 8.0), resuspended in 3 ml of resuspension buffer (10 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl, pH 8.0), and transferred to 15-ml tubes. Shearing of chromatin was done by sonicating each sample 7 times for 30 s at 0 °C using a Branson 250 sonicator (output control set at 5). Samples were adjusted to 0.5% sarcosyl and swirled for 10 min. Cell debris was collected by centrifugation for 5 min at 13,000 × g. Samples were adjusted to 1.42 g/cm³ CsCl and brought to a volume of 4 ml with 1.42 g/cm³ CsCl in resuspension buffer. The cross-linked chromatin complexes were separated from free protein, lipids, DNA, and RNA by isopycnic ultracentrifugation for 72 h at 20 °C, 40,000 rpm in a Beckman SWTi60 rotor. Fractions of 0.5 ml were collected from the bottom of the gradient with syringe and needle, and the samples containing chromatin were identified on a 0.8% agarose gel. The samples containing chromatin were pooled and dialyzed overnight at 4 °C against dialysis buffer (5% glycerol, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl, pH 8.0). Dialyzed chromatin was sonicated again for 30 s as above. The cross-linked chromatin was aliquoted and stored at 80 °C.

Chromatin Immunoprecipitation-- 120 µl of protein A/G beads (Santa Cruz Biotechnology) were washed three times in immunoprecipitation (IP) dilution buffer (1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 300 mM NaCl and 1:50 Complete protease inhibitor mixture (Roche Molecular Biochemicals)). Beads were then incubated with 10 µg of sheared salmon sperm DNA and 60 µg of bovine serum albumin for 30 min at 4 °C in IP dilution buffer, washed three times in IP dilution buffer, and finally resuspended in IP dilution buffer. To 200-µl aliquots of isolated cross-linked chromatin an equal volume of 2-fold concentrated IP dilution buffer was added, and the chromatin was incubated with half of the protein A/G beads for 1 h at 4 °C on a rotating wheel. Beads were removed by centrifugation (13,000 × g, 20 s, 4 °C) and the supernatant was incubated overnight at 4 °C with 5 µl of antibody (PPAR polyclonal (Santa Cruz Biotechnology) or pan-RXR polyclonal antibody (Santa Cruz Biotechnology)) on a rotating wheel. Complexes containing PPAR and RXR were immunoprecipitated by adding the other half of the beads and incubating 11/2 h at 4 °C on a rotating wheel. Immune complexes were washed 5 min on a rotating wheel with buffer 1 (1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, and 150 mM NaCl), twice with buffer 2 (1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), and twice with TE buffer. Immune complexes were eluted from the beads with 200 µl of 1% SDS, 0.1 M NaHCO₃ for 15 min. The elution was repeated; eluates were combined; 16 µl of 5 M NaCl was added, and chromatin was decross-linked for 4 h at 65 °C. DNA was purified by phenol/chloroform extraction, precipitated with 20 µg of glycogen as carrier, and finally dissolved in 50 µl of TE buffer. For input control, 200 µl of input chromatin was decross-linked, phenol/chloroform-extracted, precipitated, and dissolved in 100 µl of TE.

Real Time PCR on Immunoprecipitated DNA-- Precipitated DNA was quantified using the GeneAmp 5700 Sequence Detection system (PE Biosystem) and real time PCR kit (Eurogenetics). Primer sets were designed to amplify the intronic PPRE of the ACBP gene, the PPRE of the adipocyte lipid-binding protein promoter (32), and the PPRE of the lipoprotein lipase promoter (36), respectively. As a negative background control, a primer set located ~8 kb downstream of the intronic PPRE of the ACBP gene was used. Primers were designed so that all PCR products were between 81 and 87 bp. Primers were as follows: ACBP intron 1 PPRE, forward 5'-TCCCACTTGCCTCTCCCTAA-3' and reverse 5'-CAGCTGGTCCCTTCCTACAGG-3'; LPL PPRE, forward 5'-CCTCCCGGTAGGCAAACTG-3' and reverse 5'-AACGGTGCCAGCGAGAAG-3'; ALBP PPRE (ARE7), forward 5'-GAGAGCAAATGGAGTTCCCAGA-3' and reverse 5'-TTGGGCTGTGACACTTCCAC-3'; and background control, forward 5'-ACACCACTGGCCGTGATGTT-3' and reverse 5'-CATCGGCGTACTCTGCTGTG-3'. The PCR amplification was carried out as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 96 °C for 15 s and combined annealing and extension at 60 °C for 60 s. The GeneAmp 5700 software was used to perform analysis of the real time fluorescence signal from SyBR Green I bound to double-stranded DNA. A threshold cycle was determined for each sample, using the exponential growth phase and base-line data of the fluorescent amplification plots. Dissociation curves were subsequently used to identify PCR products. To correct for differences in efficiency of the different PCRs, all PCR signals from immunoprecipitated DNA were normalized to PCR signals from non-precipitated input DNA. The normalized signal from the PCR obtained with the background control primer set was arbitrarily set to 1, and other PCR signals were expressed as fold above background.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ACBP Expression Is Increased in Adipose Tissue of db/db Mice Treated with BRL49653-- Peroxisome proliferators have been shown to increase the expression of ACBP in rat liver (14, 20), and we have shown previously (16) that ACBP expression is significantly increased during in vitro adipocyte differentiation, a process during which the expression of a number of PPAR target genes is up-regulated. To investigate whether ACBP expression can be induced by PPAR ligands in vivo, 12 week-old db/db mice were fed either vehicle or BRL49653 at different concentrations for 10 days. The db/db model is commonly used to study the antidiabetic effects of thiazolidinediones, and ACBP mRNA levels are similar in adipose tissue of db/db mice and wild type C57BL6 mice (results not shown). Adipose tissue was isolated, and the expression of ACBP was compared with that of the ALBP, which is a well established PPAR target gene (32). Both ACBP and ALBP are expressed at very high levels in adipocytes (37, 38); however, despite these high basal levels, the expression of ACBP as well ALBP mRNA was significantly and dose-dependently increased by BRL49653 (Fig. 1). This suggests that ACBP might be a PPAR target gene.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Expression of ACBP is induced in parallel with ALBP in adipose tissue of db/db mice treated with the PPAR ligand BRL49653. Twelve-week-old db/db mice were dosed daily with BRL49653 (1, 3, or 10 mg/kg/dosing) or vehicle for 10 days. RNA was isolated from epididymal fat pads, and after reverse transcription, mRNA expression levels were determined using real time PCR. Each bar represents the average RNA level and S.E. of the mean from six animals. Values are normalized to expression levels of 18 S rRNA. RNA level of vehicle-treated animals was set to 1.

ACBP Expression Is Induced by PPAR Ligands in the Absence of Protein Synthesis-- We have reported previously (16) that ACBP mRNA and protein are induced during adipocyte differentiation of 3T3-L1 cells. Fig. 2A confirms this observation using standard differentiation conditions instead of the limited differentiation mixture that was used in the original publication. To investigate whether the ACBP gene is directly regulated by PPAR, we added the PPAR-selective ligand BRL49653 to fully differentiated 3T3-L1 cells, which express high amounts of PPAR. The PPAR ligand significantly induced the expression of ACBP in parallel with ALBP (Fig. 2B). The observation that no significant increase is observed at the 6-h but only at the 12-h time point probably reflects the fact that the transcripts of both ACBP and ALBP are highly abundant already in the differentiated adipocytes. Addition of cycloheximide did not prevent induction of either ACBP or ALBP by the PPAR ligand, indicating that ACBP similar to ALBP is a direct PPAR target gene.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Expression of ACBP is induced in parallel with ALBP by the PPAR ligand BRL49653 in mature adipocytes in the absence of protein synthesis. 3T3-L1 cells were cultured and differentiated following the standard differentiation protocol. A, RNA and protein extracts were prepared at different times during adipocyte differentiation. RNA was analyzed by Northern blotting using 32P-labeled rat ACBP cDNA as probe. Protein extracts were analyzed by Western blotting using affinity-purified rabbit anti-rat ACBP antibody. B, at day 10, BRL49653 (1 µM) was added in the presence or absence of cycloheximide (CHX) and incubated (Inc. and Incub) for 2, 6, and 12 h. RNA was isolated, and the expression of ACBP and ALBP was determined by northern blotting. Signals were quantified by PhosphorImaging and normalized to 28 S rRNA.

Intron 1 Contains a Potential PPRE, Which Is Conserved between Rat, Mouse, and Humans-- We and others (39, 40) have reported the identification of potential regulatory elements in the promoter of the rat ACBP gene, but functional elements have not yet been described. We reported the identification of a potential PPRE in the rat ACBP gene at position 1525 and showed that it bound PPAR/RXR heterodimers in electrophoretic mobility shift assays (39). However, cloning and sequencing of the mouse and human ACBP genes showed that it was neither conserved in the human nor in the mouse ACBP sequence (Fig. 3A). Careful analysis of the entire 10.9 kb that had been sequenced from the rat ACBP gene (from ~2.2 kb upstream of exon 1 to 0.2 kb downstream of the last exon) revealed an almost perfect DR-1 (direct repeat of AGGTCA with 1 bp spacing) in intron 1. This DR-1 conformed better to the PPRE consensus, both with respect to core sequence as well as with respect to its 5'-flanking sequence, than any other sequence within the 10.9 kb. Importantly, this DR-1 is well conserved between rat, mouse, and human (Fig. 3A). Unlike most PPREs, the intronic DR-1 has a "G" as spacer between the two direct repeats. Interestingly the ARE7 PPRE, which is the most important of the two PPREs in the enhancer of ALBP (41), shows considerable sequence identity with the ACBP DR-1 and has a G as spacer as well.

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Potential PPREs in the rat ACBP gene. A, structure of the rat ACBP promoter region and intron 1 with two potential PPREs indicated. The potential PPRE in intron 1 is conserved between human and rodents, whereas the upstream potential PPRE reported previously (Elholm et al. (39)) is only present in the rat gene. The numbers, 1535, 1512, 392, +1 and +979, refer to position relative to translation start site and indicate the extension of the promoter fragments cloned into the promoter-reporter constructs. B, comparison of the potential PPREs of the rat, mouse, and human ACBP genes with well characterized functional PPREs. The potential upstream PPRE of the rat gene is shown in italics. The indicated consensus is from Ref. 53.

To investigate whether the intronic DR-1 could bind PPAR·RXR complexes in vitro, we performed electrophoretic mobility shift assays using adipocyte and hepatocyte nuclear extracts as well as in vitro translated proteins. As seen in Fig. 4A, the intronic DR-1 of the rat and human ACBP genes gives rise to retarded bands with adipocyte and hepatocyte nuclear extracts. The bands comigrate with the band formed with the previously identified upstream DR-1 in the rat promoter. The band formed with adipocyte nuclear extracts comigrates with in vitro translated PPAR/RXR and is supershifted by PPAR as well as RXR antibodies (Fig. 4B). In keeping with the very low levels of PPAR expression in preadipocytes, the retarded band is not formed with preadipocyte nuclear extract.

View larger version (56K): [in this window] [in a new window]   Fig. 4.   The potential PPRE in intron 1 binds PPAR·RXR complexes in vitro. Electrophoretic mobility shift assays using the intronic DR-1 and in vitro translated proteins or nuclear extracts. A, protein complexes from adipocyte and rat liver nuclear extracts bind to the intronic DR-1 of both the rat and the human ACBP gene as well as to the upstream DR-1 of the rat gene. B, the intronic DR-1 binds a protein complex from 3T3-L1 adipocytes but not preadipocytes. This complex comigrates with in vitro translated PPAR/RXR and is effectively supershifted by antibodies against PPAR and RXR.

The Intronic DR-1 Is a Functional PPRE in the Context of a Heterologous Promoter-- To investigate whether the DR-1 in intron 1 of the rat ACBP gene could function as a PPRE upstream of a heterologous promoter, we cloned three copies of this DR-1 in front of the thymidine kinase gene basal promoter. This was done by replacing the three copies of the rat acyl-CoA oxidase (ACO) PPRE in TK-3xPPRE-luc (29) with three copies of the rat ACBP DR-1 keeping orientation and spacing of the DR-1s identical to that of the ACO PPREs. The resulting constructs were transfected into NIH-3T3 cells, a cell line that we routinely use for investigating activation by specific PPAR subtypes because it has low endogenous levels of all PPARs. As shown in Fig. 5, the multimerized rat ACBP DR-1 functions as a PPAR-, PPAR-, and PPAR-responsive PPRE in NIH-3T3 cells.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   The DR-1 in intron 1 of the ACBP gene is a functional PPRE in the context of a heterologous promoter. NIH-3T3 cells were transfected with either TK-luc reporter, 3× rACBP-PPRE-TK-luc or 3× rACO-PPRE-TK-luc, and pSV40-mPPAR, pSV40-mPPAR2, pSV40-mPPAR, or pCMV-HNF4 expression vectors as indicated. pCMV-RXR was cotransfected with all PPAR constructs and pCMV--galactosidase expression vector as control. Following transfection cells were treated for 24 h with 1 µM BRL49653, 100 µM Wy14643, 1 µM L-165041, or Me2SO vehicle as indicated. All transfections were performed as triplicate DC-Chol lipofections in 6-cm plates with a total of 5 µg of DNA/plate. Empty expression vectors were added to compensate for promoter load. Luciferase values have been normalized to -galactosidase values. Transfection values are shown as fold induction compared with a transfection with TK-luc reporter and normalization vector alone. Transfections were done in triplicate. Standard deviations are indicated. The results are representative of three or more independent experiments.

Interestingly, the ACBP PPRE appeared to be much less efficient than the ACO PPRE in mediating PPAR transactivation, whereas the efficiency of mediating PPAR transactivation was similar for the ACBP and ACO PPREs. PPAR transactivation was mediated more efficiently by the ACBP PPRE than by the ACO PPRE. Although the ACBP PPRE is a relatively poor PPRE for PPAR transactivation, PPAR still transactivates the 3xACBP-TK-luc severalfold better than PPAR2. Whether this is due to a much higher expression level of PPAR or to a higher transactivation potential of PPAR in these cells is not clear.

Because electrophoretic mobility shift assays showed that in vitro translated HNF4 bound strongly to the ACBP DR-1 (results not shown), we tested whether this site could mediate HNF4 transactivation. However, when assayed in the context of the thymidine kinase gene basal promoter, HNF4 was unable to direct transcriptional activation through this DR-1 element and gave only a very minor activation through the ACO PPRE. Parallel transfections with an HNF4 reporter construct showed that HNF4 was indeed transcriptionally active in NIH-3T3 cells (data not shown).

The Intronic DR-1 Confers PPAR and PPAR Responsiveness to the Rat ACBP Promoter-- To investigate the functionality of the rat intronic PPRE in situ, we prepared constructs in which the rat ACBP promoter regions, exon 1 and intron 1, were fused with the luciferase gene such that the luciferase gene was inserted in-frame with the ACBP reading frame of exon 2 (Fig. 6A). The NIH-3T3 cell line was used due to their low levels of endogenous PPARs. The rACBP(392/+1)-luc as well as the rACBP(392/+979)-luc reporter constructs were highly active in NIH-3T3 cells. However, cotransfection of PPAR/RXR and addition of the PPAR-selective ligand Wy14643 significantly increased luciferase expression in NIH-3T3 cells. Similarly, cotransfection with PPAR/RXR and addition of the PPAR-selective ligand BRL49653 increased luciferase expression, whereas cotransfection with PPAR/RXR and addition of the PPAR-selective ligand L-165041 had no effect on promoter activity. HNF4 was also in the ACBP promoter context unable to activate transcription in NIH-3T3 cells. Mutation of the intronic PPRE abolished the PPAR-mediated transactivation demonstrating that this PPRE is a functional PPRE in the natural gene context as well as when linked to a heterologous promoter. Thus, the intronic PPRE confers PPAR and PPAR responsiveness to the ACBP promoter. However, despite the fact that the multimerized intronic PPRE in the thymidine kinase gene basal promoter context is responsive to PPAR in NIH-3T3 cells, PPAR is unable to activate transcription from the ACBP promoter via this PPRE.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   The DR-1 in intron 1 but not the upstream DR-1 of the rat ACBP gene is a functional PPRE in situ, which is activated by PPAR/RXR and PPAR2/RXR. A, the seven different constructs used for analysis of the rat intron 1 PPRE and upstream DR-1, in rACBP(392/+979)PPRE-luc the rACBP intron 1 PPRE, have been mutated. B, NIH-3T3 cells were transfected with either rACBP(392/+1)-luc, rACBP(392/+979)-luc, or rACBP(392/+979)PPRE-luc reporter constructs and pSV40-mPPAR pSV40-mPPAR2, pSV40-mPPAR, or pCMV-HNF4 expression vectors as indicated. pCMV-RXR was cotransfected with all PPAR constructs, and pCMV--galactosidase expression vector was used as control. Following transfection, cells were treated for 24 h with 1 µM BRL49653, 100 µM Wy14643, 1 µM L-165041, or Me2SO vehicle as indicated. Transfection and normalization performed as indicated in Fig. 5. Normalized luciferase values are shown as fold induction compared with a transfection with reporter alone. C, NIH-3T3 cells were transfected with either rACBP(1512/+1)-luc, rACBP(1512/+979)-luc, or rACBP(1535/+1)-luc and rACBP(1535/+979)-luc reporter constructs and pSV40-mPPAR expression vector as indicated. pCMV-RXR was cotransfected with all PPAR constructs, and pCMV--galactosidase expression vector was used as control. Following transfection cells were treated for 24 h with 100 µM Wy14643 or Me2SO vehicle as indicated. The 1535 clone but not the 1512 clone contain the upstream PPRE. Transfection and normalization were performed as indicated in Fig. 5. Normalized luciferase values are shown as fold induction compared with a transfection with reporter alone. Transfections were done in triplicate. Standard deviations are indicated. The results are representative of three or more independent experiments.

The functionality of the previously identified potential PPRE upstream of the rat ACBP gene has not been investigated. We therefore accessed whether this element could contribute to the PPAR responsiveness of the rat promoter. As seen in Fig. 6C, this DR-1 was unable to mediate PPAR/RXR transactivation and did not cooperate with the intronic PPRE in mediating PPAR/RXR transactivation. Thus, the upstream DR-1 is not a functional PPRE in transient transfections.

The Intronic PPRE Is Functionally Conserved in the Human ACBP Gene-- Electrophoretic mobility shift assay showed that the intronic DR-1 in the human ACBP gene was able to bind PPAR/RXR and PPAR/RXR. To investigate whether the functionality of the intron 1 PPRE was conserved in the human ACBP gene, we prepared reporter plasmids similar to the rat constructs by fusing the respective human ACBP promoter region, exon 1 and intron 1, sequences with the luciferase gene (Fig. 7A). These constructs were cotransfected with human PPAR expression plasmids into the human embryonic kidney cell line 293, which has low levels of endogenous PPARs. As for the corresponding rat constructs, the basal activity of this reporter constructs was very high. However, despite the high basal activity, hPPAR/RXR as well as hPPAR/RXR were able to activate further luciferase expression from this construct, whereas cotransfection with PPAR/RXR only marginally increased the promoter activity (Fig. 7B). PPAR-mediated transactivation was abolished by mutating the PPRE, indicating that the intronic PPRE is functionally conserved between rodents and humans. Similar to the transfections with the rat constructs the relative activation by PPAR and PPAR cannot be directly compared.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   The intronic PPRE is functionally conserved between rats and humans. A, the three different constructs used for analysis of the human intron 1 PPRE, and in hACBP-(3516/+1136)PPRE-luc the hACBP intron 1 PPRE has been mutated. B, 293 cells were transfected with either hACBP(516/+5)-luc, hACBP(516/+1136)-luc, or hACBP(516/+1136)PPRE-luc reporter constructs and pCMV-hPPAR, pCMV-hPPAR, or pCMV-hPPAR and pCMV-mRXR expression vectors where indicated. pCMV--galactosidase expression vector was cotransfected as control. Following transfection cells were treated for 24 h with media containing 1 µM BRL49653, 100 µM Wy14643, 1 µM L-165041, or Me2SO vehicle where indicated. Transfection and normalization were performed as indicated in Fig. 5 but with a total of 2.5 µg of DNA/well. Normalized luciferase values are shown as fold induction compared with a transfection with reporter alone. Transfections were done in triplicate. Standard deviations are indicated. The results are representative of three or more independent experiments.

The Intronic PPRE Mediates Thiazolidinedione Responsiveness of the ACBP Promoter in Adipocytes-- If the intronic PPRE functions as a PPAR-response element it should instigate thiazolidinedione activation of the ACBP promoter in adipocytes, which express high levels of PPAR. We therefore transiently transfected 3T3-L1 adipocytes with the rACBP(392/+979)-luc or rACBP(392/+979)PPRE-luc constructs and incubated the cells with or without the thiazolidinedione BRL49653. As seen in Fig. 8A, BRL49653 efficiently activated the rACBP(392/+979)-luc construct but not the rACBP(392/+979)PPRE-luc construct. Similarly, BRL49653 activated the longer reporter constructs prACBP(1535/+979)-luc and prACBP(1512/+979)-luc but not the corresponding constructs where the intronic PPRE had been mutated (Fig. 8B). This clearly indicates that this PPRE mediates activation by endogenous PPAR in adipocytes and that the activation of the ACBP promoter by thiazolidinedione is dependent on the intronic PPRE. In keeping with the cotransfection experiments using NIH-3T3 cells (Fig. 6C), the potential PPRE between 1535 and 1512 was unable to function as a PPRE in transient transfections of adipocytes.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   The intronic PPRE is necessary for ligand-dependent activation of ACBP promoter constructs by endogenous PPAR in adipocytes. 3T3-L1 cells were cultured and differentiated following the standard differentiation protocol. At day 4 cells were transfected with rACBP(392/+979)-luc or rACBP(392/+979)PPRE-luc, rACBP(1512/+979)-luc, rACBP(1512/+979)PPRE-luc, rACBP(1535/+979)-luc, or rACBP(1535/+979)PPRE-luc reporter constructs as indicated and pCMV--galactosidase expression vector using LipofectAMINE Plus. Cells were treated for 24 h with media containing 1 µM BRL49653 or Me2SO as control. Luciferase values have been normalized to -galactosidase values, and the normalized luciferase values are shown as fold induction compared with the Me2SO control. Transfections were done in triplicate. Standard deviations are indicated. The results are representative of three or more independent experiments.

PPAR and RXR Bind to the Intronic PPRE on Chromatin in Adipocytes-- Finally, to confirm that the intronic PPRE was also functional in vivo in the chromatin context, we isolated fragmented formaldehyde cross-linked chromatin from mature 3T3-L1 adipocytes and subjected this to chromatin immunoprecipitation using PPAR-selective and pan-RXR antibodies. PPAR and RXR cross-linking to chromatin was verified using Western blotting (data not shown). Following DNA extraction of the immunoprecipitated chromatin, real time PCR was used to determine the relative occupancy of different PPREs compared with a control fragment located ~8 kb downstream of the intronic PPRE. As shown in Fig. 9, PPAR and pan-RXR antibodies efficiently precipitated DNA fragments spanning the ACBP PPRE as well as fragments covering the ALBP and lipoprotein lipase (LPL) PPREs. The relative occupancies of the three PPREs were similar in the immunoprecipitates, indicating that these PPREs recruit PPAR/RXR with equal efficiency to their cognate sites embedded in chromatin.

View larger version (12K): [in this window] [in a new window]   Fig. 9.   PPAR and RXR associates with the intronic PPRE on chromatin in adipocytes. 3T3-L1 cells were cultured and differentiated following the standard differentiation protocol. Chromatin was isolated from day 4 adipocytes and used for chromatin immunoprecipitation with PPAR- and RXR-specific antibodies, respectively. The recovered DNA was quantified by real time PCR using primer sets amplifying the ACBP, ALBP, or LPL PPRE, respectively. A fragment located ~8 kb downstream of the intronic ACBP PPRE was used as background control. Results are expressed as relative occupancy of the respective PPREs compared with a control fragment. The figure illustrates the results from a representative experiment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ACBP is an evolutionarily highly conserved protein that is thought to play a role in the intracellular transport of medium to long chain acyl-CoA esters. The structure and biochemical properties of ACBP are well described, but the precise function of this protein in vivo remains unresolved. Because it is highly conserved from yeast to mammals and is ubiquitously expressed in mammals, it is likely that ACBP carries out basic cellular functions. In keeping with this, the mammalian ACBP gene displays all the hallmarks of a housekeeping gene (11). However, expression levels vary considerably between different cell types and in response to different metabolic conditions, indicating that during evolution ACBP may have acquired additional specialized functions. Induction of ACBP expression appears to be correlated at least to some extent with increased lipogenesis. In addition, induction of ACBP expression by PPAR activators, which induce mitochondrial and peroxisomal -oxidation in liver, has been reported. However, the molecular mechanisms underlying the regulation of ACBP expression in mammalian cells have remained largely unknown.

The only functional regulatory elements described to date in a mammalian ACBP gene is an SREBP-binding site and an NFY-binding site identified in the proximal promoter of the human gene (17). We have shown that these elements, at least in transient transfections, are functionally conserved in the rat ACBP gene,² but they remain to be characterized in a chromatin context.

Previous observations suggested that the ACBP gene might also be a PPAR target gene. First, PPAR ligands induce ACBP expression in the liver (14, 20). Second, ACBP expression is significantly induced during the course of adipocyte differentiation (16), and the induction parallels that of ALBP, a well known PPAR target gene. In this report we show that the expression of ACBP in adipose tissue is significantly induced in a dose-dependent manner when db/db mice are fed a diet containing the PPAR-specific ligand BRL49653. Furthermore, we show that the expression of ACBP is induced in 3T3-L1 adipocytes by BRL49653 independently of protein synthesis, indicating that ACBP is a direct PPAR target gene.

In keeping with this notion we identify a functional PPRE in intron 1 of the rat ACBP gene and show that it mediates PPAR/RXR transactivation in NIH-3T3 cells. The PPRE is functionally conserved in the human ACBP gene. The previously identified potential PPRE at 1525 in the rat gene is neither conserved in the human nor in the mouse gene and is not functional in the rat promoter context. The intronic PPRE mediates induction by endogenous PPAR in murine adipocytes and confers responsiveness to the PPAR-selective ligand BRL49653. Finally, we have used chromatin immunoprecipitation to demonstrate that the intronic PPRE efficiently binds PPAR/RXR in the chromatin context in adipocytes. The relative occupancy of the intronic PPRE was similar to that of the well characterized PPREs on the ALBP and LPL genes, respectively. Thus, the PPRE in intron 1 of the ACBP gene is a bone fide PPAR-response element. To our knowledge this is the first reported chromatin immunoprecipitation assay determining PPAR/RXR binding to a PPRE.

PPAR and the CCAAT/enhancer-binding protein  (C/EBP) are known to be the major and determining adipogenic transcription factors (reviewed in Ref. 42). The finding that ACBP is a direct PPAR target gene indicates that PPAR plays a central role in the up-regulation of ACBP expression during adipogenesis. SREBP-1 may be less important in the induction during differentiation, but it is likely that it modulates the expression of ACBP at the differentiated stage, like it modulates a number of other adipocyte genes (reviewed in Ref. 43). We have identified previously a potential C/EBP-responsive element at position 791 in the rat ACBP gene (39). This element bound proteins from rat liver nuclear extracts, which express high levels of C/EBP, and we therefore suggested that it might be involved in the activation of ACBP expression during adipocyte differentiation. However, this element is neither conserved in the mouse nor in the human ACBP gene (data not shown), and transfection with rat ACBP promoter constructs encompassing the region from 2310 to +979 showed that this region of the promoter was not activated by coexpression of C/EBP (results not shown). Thus, the previously identified potential C/EBP-response element in the rat gene does not seem to be functional, and no other functional C/EBP-response elements seems to be present in the region 2310 to +979 of the rat ACBP gene

The results presented in this paper show that the intronic PPRE also confers PPAR responsiveness to the ACBP promoter constructs, indicating that the ACBP gene may be a natural PPAR target gene. This is supported by the fact that ACBP expression is induced in the liver following prolonged exposure to PPAR ligands. However, the finding that ACBP expression is down-regulated in the liver by fasting when most PPAR target genes are induced (44, 45) indicates that if ACBP is a direct PPAR target gene, PPAR induction during fasting must be hampered by the absence of other transcription factors like SREBP-1, for example, or overruled by the presence of inhibitory transcription factors.

Interestingly, the sequence of the ACBP PPRE deviates from classical PPAR-responsive elements by having a G as spacer between the two repeats. When directly compared in transient transfections, the ACBP PPRE was much less efficient in mediating PPAR-dependent transactivation than the ACO PPRE, a "classical" PPAR target. This is in keeping with the finding that an "A" between the two repeats gives a stronger binding of PPAR/RXR (46). Our results indicate that an A in this position is far less critical for PPAR/RXR than for PPAR/RXR binding and function. This observation is in keeping with the PPAR-responsive ARE7 PPRE of the ALBP enhancer also having a G in this position and showing preference for PPAR binding (41). Interestingly, the sequences of the ARE7 PPRE and the ACBP PPRE including their 5'-flanking sequences are strikingly similar and different from that of PPREs of most PPAR-responsive genes. Thus, PPAR may be able to transactivate the ACBP gene, but ACBP does not appear to belong to the classical PPAR target genes.

PPAR/RXR were able to bind to the intronic ACBP PPRE in electrophoretic mobility shift assays, and the element efficiently mediated PPAR/RXR transactivation of a heterologous promoter in NIH-3T3 cells. Interestingly, however, PPAR/RXR was unable to transactivate natural ACBP promoter constructs in this cell type. HNF4 bound strongly to the intronic PPRE in electrophoretic mobility shift assays but was unable to transactivate via this element in the thymidine kinase gene promoter context as well as in the natural ACBP promoter context in NIH-3T3 cells. Thus, at least under the conditions used for our investigations the rat ACBP gene is neither a PPAR nor an HNF4 target gene. Whether HNF4 and PPAR/RXR by binding to the intronic PPRE interfere with PPAR/RXR binding and transactivation (47, 48), and thereby inhibit the transcription of the ACBP gene, remains to be established.

Regulatory elements are frequently observed in intronic regions; however, to our knowledge this is the first functional PPRE to be reported in an intronic region. Interestingly, the intronic PPRE is located in a highly conserved region of intron 1 (49) and overlaps with an alternative splice donor in the human ACBP gene (50). We have recently found that this alternative splicing is neither conserved in rats nor mice,³ indicating that the conservation of the area between humans and rodents cannot be explained by functional importance of such an alternative splicing. It is therefore likely that the PPRE and possibly other regulatory elements yet to be identified have contributed to the sequence conservation of this region in intron 1.

The finding that PPARs directly regulate the expression of ACBP is particularly interesting in the light of our recent results showing that acyl-CoA esters function as PPAR antagonists in vitro (51, 52) and that overexpression of lipid-binding proteins including ACBP decrease activation of PPARs by exogenous fatty acids (35). How ACBPs repress PPAR-mediated transactivation induced by exogenous fatty acids is unknown. Our data would be in keeping with a model in which ACBP increase the esterification of exogenously added fatty acids, thereby decreasing their availability as PPAR ligands. Whether these effects of ACBP on PPAR transactivation are physiologically relevant are unclear at this point. In another study we have shown that antisense ACBP inhibits the ability of 3T3-L1 preadipocytes to undergo differentiation and to induce key adipogenic transcription factors like PPAR (38). In the present report we show that ACBP is also a PPAR target gene, indicating that ACBP acts both upstream and downstream of PPAR in the adipogenic process. Thus, in the context of adipocyte differentiation ACBP appears to function in a positive feedback loop to increase its own expression.

In summary, we have demonstrated that the ACBP gene is a novel PPAR/RXR target gene and that PPAR/RXR activates transcription through an intronic PPRE in humans and rodents. This PPRE may also mediate transactivation by PPAR/RXR, but final proof for that awaits investigations of the interaction of endogenous PPAR/RXR with this element in a chromatin context.

	ACKNOWLEDGEMENTS

We thank Dr. U. B. Jensen for preparation of DC-Chol for transfections; Drs. P. Grimaldi, E.-Z. Zoubir, B. M. Spiegelman, and J. D. Tugwood for the PPAR, PPAR, and PPAR expression plasmids; Dr. R. M. Evans for the pTK-3xPPRE-Luc reporter and the RXR expression plasmids; and Novo Nordisk A/S and Merck for supplying ligands.

	FOOTNOTES

^* This work was supported by the Danish Natural Science Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Southern Denmark, Odense University, Campusvej 55, 5230 Odense M, Denmark, Tel.: 45-6550- 2340; Fax: 45-6550-2467; E-mail: s.mandrup@bmb.sdu.dk.

Published, JBC Papers in Press, May 15, 2002, DOI 10.1074/jbc.M111295200

² M. S. Boysen and S. Mandrup, unpublished results.

³ L. K. Larsen, J. Nøhr, K. Kristiansen, and S. Mandrup, unpublished results.

	ABBREVIATIONS

The abbreviations used are: ACBP, acyl-CoA-binding protein; ACO, acyl-CoA oxidase; ALBP, adipocyte lipid-binding protein; C/EBP, CCAAT/enhancer-binding protein; DMEM, Dulbecco's modified Eagle's medium; Me₂SO, dimethyl sulfoxide; DR-1, a direct repeat with a spacing of one nucleotide; FCS, fetal calf serum; HNF4, hepatocyte nuclear factor 4; LPL, lipoprotein lipase; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-responsive element; RXR, retinoid X receptor; SREBP, sterol regulatory element-binding protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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