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J Biol Chem, Vol. 275, Issue 17, 12612-12618, April 28, 2000

Induction of the Fatty Acid Transport Protein 1 and Acyl-CoA Synthase Genes by Dimer-selective Rexinoids Suggests That the Peroxisome Proliferator-activated Receptor-Retinoid X Receptor Heterodimer Is Their Molecular Target^*

Geneviève Martin^§, Hélène Poirier^¶, Nathalie Hennuyer, Diane Crombie, Jean Charles Fruchart, Richard A. Heyman^**, Philippe Besnard^¶, and Johan Auwerx^§§

From the  Département d'Athérosclérose, INSERM U325, Institut Pasteur de Lille, 1 rue du Prof. Calmette, 59019 Lille, France, ^¶ Laboratoire de physiologie de la nutrition, EP1777/CNRS/CESG, Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation, Université de Bourgogne, Campus Universitaire, 1 Esplanade Erasme, 21000 Dijon, France,  Ligand Pharmaceuticals, 10255 Science Center Drive, San Diego, California 92121, and  Institut de Génétique et Biologie Moléculaire et Cellulaire, CNRS, INSERM, Université Louis Pasteur 1 rue Laurent Fries, 67400 Illkirch, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The intracellular fatty acid content of insulin-sensitive target tissues determines in part their insulin sensitivity. Uptake of fatty acids into cells is a controlled process determined in part by a regulated import/export system that is controlled at least by two key groups of proteins, i.e. the fatty acid transport protein (FATP) and acyl-CoA synthetase (ACS), which facilitate, respectively, the transport of fatty acids across the cell membrane and catalyze their esterification to prevent their efflux. Previously it was shown that the expression of the FATP-1 and ACS genes was controlled by insulin and by peroxisome proliferator-activated receptor (PPAR) agonists in liver or in adipose tissue. The aim of this investigation was to determine the effects of retinoic acid derivatives on the expression of FATP-1 and ACS. In several cultured cell lines, it was shown that the expression of both the FATP-1 and ACS mRNAs was specifically induced at the transcriptional level by selective retinoid X receptor (RXR) but not by retinoic acid receptor (RAR) ligands. This effect was most pronounced in hepatoma cell lines. A similar induction of FATP-1 and ACS mRNA levels was also observed in vivo in Zucker diabetic fatty rats treated with the RXR agonist, LGD1069 (4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic acid). Through the use of heterodimer-selective compounds, it was demonstrated that the modulatory effect of these rexinoids on FATP-1 and ACS gene expression was mediated through activation of RXR in the context of the PPAR-RXR heterodimer. The observation that both RXR and PPAR agonists can stimulate the transcription of genes implicated in lipid metabolism, suggest that rexinoids may also act as lipid-modifying agents and support a role of the permissive PPAR-RXR heterodimer in the control of insulin sensitivity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Free fatty acids can be released from adipocytes by hormone-sensitive lipase or they can be generated from triglycerides-rich lipoproteins by the action of lipoprotein lipase. Circulating fatty acids can then cross the plasma membrane either by virtue of their lipid solubility (1) or by being actively taken up by cells in a process mediated by proteins of the fatty acid transport protein (FATP)¹ family. The best characterized of these FATPs is FATP-1, a plasma membrane protein of 63 kDa, which has been shown to actively transport long chain fatty acids (2). Another protein found to be indirectly involved in the process of long chain fatty acid uptake is the acyl-CoA synthetase (ACS). Whereas FATP acts as a transporter of fatty acids, the role of ACS is confined to prevent the efflux of fatty acids through an esterification process. Two other specialized membrane transport system for fatty acids have been described. The first one is the fatty acid translocase (FAT) or glycoprotein CD36 (3, 4), which acts in a coordinate manner with low molecular weight intracellular fatty acid-binding protein (FABPc) (5, 6). The second is a membrane bound fatty acid-binding protein, FABP_PM closely related to the mitochondrial aspartate aminotransferase (7-10).

Nutritionally derived vitamin A derivatives are affecting metabolism by changing gene expression secondary to activation of receptors of the nuclear receptor superfamily (11, 12). Three retinoic acid receptors (RARs), termed RAR, -, and -, and three retinoid X receptor (RXR), designated RXR, -, and -, are classically thought to transduce most of the effects of retinoic acid (RA) on gene expression. Both 9-cis RA (9c-RA) and all-trans-RA (at-RA) can directly bind and activate RARs, whereas RXR only binds 9c-RA. Recently, new specific ligands for RXR and/or a particular dimers have been developed (13). These selective RXR agonists, called rexinoids, function as insulin sensitizers and have beneficial effects on the hyperglycemia and hyperinsulinemia in mouse models of noninsulin-dependent diabetes mellitus and obesity (14). Some of these compounds, such as LGD1069 and LG100268 (15), selectively induce the activity of the RXR homodimer and the RXR-LXR and RXR-peroxisome proliferator-activated receptor (PPAR) heterodimers, but not of RXR-RAR or RXR-TR heterodimers. By contrast, LG100754 has been identified as an antagonist of the RXR homodimer but it activates RXR when it is paired as an heterodimer with PPAR or RAR (13). This ligand, hence, is different from the other activators of RAR-RXR heterodimers, because it was previously thought that retinoids can only activate RAR in the RAR-RXR heterodimer, whereas RXR was believed to be a silent partner in this setting. Because retinoid effects on gene expression can be determined by multiple dimer combinations, including but not limited to RXR-RAR, RXR-RXR, and RXR-PPAR, these rexinoids make the determination possible of which heterodimer is specifically involved in gene regulation and, hence, they provide tools for a better understanding of RXR action and the development of new drugs.

We suggested previously that thiazolidinedione PPAR activators might improve glucose secondary to tissue-specific co-regulation of the several proteins involved in fatty acid uptake such as lipoprotein lipase (16), FATP-1, and ACS target genes (17), which ultimately alter fatty acid partitioning. RXR-PPAR heterodimers, however, have been shown to respond to both RXR and PPAR ligands (18). Therefore, we speculated that RXR ligands could have similar effects and be potential modulators of the expression of the FATP-1 and ACS genes through activation of RXR in the RXR-PPAR heterodimer. In order to confirm the implication of the PPAR-RXR heterodimer in the improvement of glucose homeostasis, we studied the regulation of the FATP-1 and ACS genes by several synthetic retinoic acid analogues. This allowed us to determine the composition of the dimer that was involved in this process and could provide us with a better understanding of the regulation of lipid uptake by nuclear receptors. Our results implicate the PPAR-RXR heterodimer as the molecular target mediating the effects of rexinoids on fatty acid partitioning leading to an improvement of insulin sensitivity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- at-RA was purchased from Sigma. 9c-RA, TTNPB (ethyl-p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtyl)-1-propenyl]benzoic acid) (19), LGD1069 (4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic acid), LG100754 ((2E,4E,6Z)-7- (-3-n-propoxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl)-3 methylocta-2,4,6-trienoic acid), and LG100268 (6-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-cyclopropyl]nicotinic acid) (20, 15) were kind gifts from Dr. M. Boehm at Ligand Pharmaceuticals.

Cell Culture and Treatments-- FAO cells are a well-differentiated subclone, derived from the rat hepatoma H4 IIEC3 cell line. They were maintained at 37 °C in a humidified atmosphere of 5% CO₂, 95% air in Ham's F-12 medium (Life Technologies, Inc.) containing 10% fetal calf serum, penicillin (200 IU/mL), and streptomycine (50 mg/mL) according to previously published procedures (21). Culture medium was changed every 48 h. The human hepatoma cell line HepG2 was obtained from ECACC (Porton Down, Salisbury, United Kingdom), and the mouse preadipocyte cell line 3T3-L1 was from ATCC (Manassas, VA). These cells were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% lipid-depleted and dextran charcoal-treated fetal calf serum, L-glutamine, and antibiotics, unless stated otherwise. 3T3-L1 cells were differentiated initially by a 2-day treatment with dexamethasone (0.1 µM), isobutyl methyl xanthine (0.25 mM), and insulin (0.4 µM). Subsequently, the cells were maintained for an additional 8 days with insulin until complete differentiation. Caco2 cells were maintained in DMEM supplemented with 10% fetal calf serum and 1% of essential amino acids. Differentiation occurred spontaneously, and cells were treated after 7 days of differentiation process. Experiments were performed on subconfluent hepatoma cells maintained in culture under serum-free conditions. at-RA and 9c-RA were dissolved in dimethyl sulfoxide, whereas oleic acid was complexed with bovine serum albumin (BSA) before addition to the cells. Actinomycin D treatment was performed 45 min before the treatment of the cells with retinoic acid treatment.

In Vivo Study-- Male Zucker diabetic fatty (ZDF) rats were purchased from Harlan at 7 weeks of age. Animals were housed four per cage and were maintained in a 12-h light/12-h dark cycle (lights were turned on at 7 a.m.). All animals had free access to laboratory chow and tap water. LGD1069, 0.3, 1.0, 3.0, 10, and 30 mg/kg/day, was added to the diet of ZDF rats during 50 days. There were four animals per treatment group. Lean rats fed with either normal chow or chow supplemented with 30 mg/kg/day of LGD1069 served as nondiabetic controls. Animals were fasted overnight before sacrifice. Animals were anesthetized with ether and sacrificed by cervical dislocation. Tissues (liver, epididymal fat pad, skeletal muscle, and heart) were collected and immediately frozen in liquid nitrogen until analysis.

RNA Analysis-- RNA preparation, Northern blot hybridizations, and quantification of total cellular RNA were performed as described previously (22). A mouse FATP-1 cDNA probe was obtained after cloning an RT-PCR fragment from mouse adipose tissue RNA (primers, 5'-ATG CGG GCT CCT GGA GCA GGA CAG CC-3' and 5'-CTG CGT GTC AGG CAG GAT GCT CTC AGG CCC-3') into pBluescript-KS. The insert was sequenced and found to be identical to the reported mouse FATP-1 sequence (2). The rat ACS probe corresponds to the EcoRV restriction fragment of the rat ACS cDNA (23), and -actin was used as a control probe (24).

Isolation of Nuclei and Transcription Rate Assay-- Nuclei were prepared from FAO and differentiated 3T3-L1 cells that were treated either with 9c-RA or vehicle. Transcription run-on assays were performed as described by Nevins (25). Equivalent counts of nuclear RNA labeled with [-³²P]UTP (3000 Ci/mmol) were hybridized for 36 h at 65 °C to 5 µg of FATP-1, ACS, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and vector DNA (pBluescript) immobilized on Hybond-C Extra filters (Amersham Life Science, Buckinghamshire, UK). After hybridization, filters were washed once at 21 °C for 10 min in 0.5 × SSC and 0.1% SDS and twice at 65 °C for 30 min and subsequently exposed to x-ray film (BIOMAX-MS, Eastman Kodak company, Rochester, NY). Quantitative analysis was performed using scanning densitometry (Bio-Rad GS670 densitometer, Hercules, CA).

Preparation of Albumin-bound Fatty Acids and Fatty Acid Uptake Assay-- Radiolabeled [¹⁴C]oleate was added to water at 40 °C. Albumin (BSA; fraction V, fatty acid free, Sigma) was then added by gentle mixing from a concentrated stock (20 g/100 mL) to give a final molar ratio of 1/1, thereafter 2× Hanks' solution was added to obtain a 1× final solution. The radioactive fatty acid was allowed to equilibrate with the albumin for 45 min at 37 °C. The measurement of uptake of [¹⁴C]oleate (about 50 mCi/mmol, NEN Life Science Products) was carried out in 24- or 6-well plates with 10⁶ cells/mL of medium. Before treatment, the cells were washed with 1× Hanks' solution. Retinoids (doses ranging from 10⁹ to 10⁶ M) were added in fresh DMEM medium containing 10% fetal calf serum. After 48 h of treatment, cells were washed with 1× Hank's solution and incubated for one additional hour in serum-free, glucose-free medium. Cells were then washed once at 37 °C and twice at 23 °C with 1× Hanks' solution containing BSA. Hanks' solution without BSA was then added before the assay. A volume corresponding to 1 µCi of [¹⁴C]oleate albumin-bound solution was added in each well, and cells were incubated for 1 min at 21 °C. Incubation was stopped after 1 min with three washes of ice-cold 1× Hanks' solution without BSA. A complementary experiment has been performed to verify whether a specific cell surface binding of [¹⁴C]oleate could interfere with the assay. For this second assay, the cells were washed under more stringent conditions in 1× Hanks' solution containing 0.5% BSA. Cells were then lysed in 400 µL of 0.1% SDS solution. The lysate was counted for 5 min with 4 mL of scintillation solution. All assays were performed on triplicate points.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

9c-RA Induces FATP-1 and ACS mRNA Levels in Vitro-- We examined the regulation of FATP-1 and ACS gene expression upon retinoic acid treatment over a dose range from 10⁸ to 10⁵ M in FAO cells cultured during 6 h. 9c-RA produced a dose-dependent increase of both FATP-1 and ACS mRNA levels in this hepatic cell line (Fig. 1A). A maximal increase for FATP-1 (>9-fold) and ACS (14-fold) was observed with a dose of 10⁶ M 9c-RA. FATP-1 and ACS mRNA were also up-regulated by at-RA but to a much lesser extent than with 9c-RA (data not shown). We next determined whether FATP-1 levels were modified by retinoids in the human hepatoma cell line HepG2. 9c-RA also induced FATP-1 and ACS (data not shown) mRNA after 24 h in HepG2 cells. This effect was already evident at a dose of 10⁸ M of 9c-RA (Fig. 1B). However, no effect of at-RA on the expression of FATP-1 and ACS mRNA could be detected in HepG2 cells (data not shown).

View larger version (48K): [in this window] [in a new window]   Fig. 1.   A, the effects of 9c-RA on FATP-1 and ACS gene expression on FAO cells. Cells were kept 18 h in serum-free medium and treated for 6 h with increasing concentrations of 9c-RA (108 to 105 M). Control cells were incubated with vehicle alone. 30 µg of total RNA was loaded per lane. Northern blot analysis was carried out, and blots were hybridized with FATP-1, ACS, and -actin probes. Quantification was performed with an imaging densitometer (Bio-Rad GS-670). Values were normalized by comparison with a -actin control probe, and results are expressed as the percentage of the control signal in untreated cells. B, the effects of 9c-RA and at-RA on FATP-1 gene expression in Hep-G2 cells. Cells were kept 18 h in serum-free medium and treated for 6 h with increasing concentrations (108 to 105 M) of either 9c-RA and at-RA. Controls were incubated with vehicle. Northern blot analysis was carried out and blots were hybridized with an FATP-1, ACS, and -actin probe. C, the effects of 9c-RA on FATP-1 mRNA levels in differentiated 3T3-L1 cells. Differentiated 3T3-L1 cells were treated for 24 h with different concentrations 9c-RA (0-106 M). Northern blots were hybridized with an FATP-1 and ACS probe. -actin was used as control probe.  indicates that the 3T3-L1 cells are differentiated. D, induction of FATP-1 and ACS mRNA levels by 9c-RA in Caco2 cells. Caco2 cells were serum-deprived for 18 h and then treated during 6 h with 106 M of 9c-RA. Northern blot analysis was carried out as described under "Experimental Procedures." Triplicate samples are shown.

The strong induction of FATP-1 in hepatoma cells led us to investigate the regulation in other rat and human cell lines representative of tissues with a high fatty acid metabolism (i.e. adipose tissue and small intestine). 3T3-L1 preadipocyte cells were therefore induced to differentiate, with a mixture of dexamethasone, isobutyl methyl xanthine, and insulin, and then treated with 9c-RA (10⁹ to 10⁶ M during 24 h). 9c-RA induced both FATP-1 and ACS (data not shown) mRNA expression in a dose-dependent manner in these differentiated 3T3-L1 cells. A maximal increase was observed at 10⁶ M 9c-RA (Fig. 1C).

The expression of FATP-1 and ACS mRNA was evaluated after retinoid treatment in the human colon adenocarcinoma cells Caco2. Both FATP-1 and ACS gene expression increased after 6 h of treatment with 9c-RA at 10⁶ M in this cell line. Under similar conditions, however, higher levels of induction of both mRNA species are nevertheless observed in FAO cells (Fig. 1A) relative to Caco2 cells (Fig. 1D).

The Induction of FATP-1 and ACS mRNA Levels by 9c-RA Is At the Transcriptional Level-- We next determined the relative contribution of transcriptional and post-transcriptional processes to the increase in mRNA levels of FATP-1, by using the transcriptional inhibitor actinomycin D. Prior addition of actinomycin D completely abolished the increase of FATP-1 (Fig. 2A) and ACS (data not shown) mRNA in 3T3-L1 and FAO cells. To assess the role of mRNA stability in the observed changes of the FATP-1 mRNAs, RNA synthesis was blocked with actinomycin D 6 h after 9c-RA induction in FAO cells, and the relative rates of disappearance of FATP-1 mRNA were determined (Fig. 2, B and C). Disappearance rates of FATP mRNA were very similar before and after 9c-RA treatment, suggesting that 9c-RA did not change the stability of FATP-1 mRNA.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Actinomycin D treatment and stability of FATP-1 mRNA levels. A, differentiated 3T3-L1 cells were either left untreated (Control), treated with actinomycin D (ActD; at 5 µg/mL), 9c-RA (at 106 M), or both actinomycin D and 9c-RA (ActD + 9c-RA). Actinomycin D was added for 60 min before the addition of 9c-RA, which was left on the cells for for 24 h. B, FAO cells were either treated with vehicle (Control) or vehicle with 9c-RA for 6 h. Then actinomycin D (5 µg/mL) was added and FATP-1 and GAPDH mRNA levels were determined at different times after the addition of actinomycin D. The relative concentration of RNA before treatment with actinomycin D was set at 100%. C, graphical presentation of a densitometric scan of the results shown in B.

Nuclear run-on analysis was then carried out to determine whether the induction of FATP-1 and ACS expression by 9c-RA was a direct consequence of enhanced gene transcription. FAO cells were treated for 4 h with 9c-RA (10⁶ M) (Fig. 3), whereas differentiated 3T3-L1 adipocytes were treated for 12 h with 9c-RA (10⁶ M) (Fig. 3). Control cells received vehicle only. Transcription rates for the FATP-1 and ACS genes were induced 2-fold and 2.8-fold, respectively, in FAO cells and 4- and 3-fold, respectively, in differentiated 3T3-L1 cells. In the retinoic acid-treated cells, transcription of the GAPDH gene was not affected in neither of the two cell lines. Because the stability of FATP mRNA is not altered by 9c-RA treatment, the induction of FATP-1 mRNA is primarily the result of an increase in the transcription rate rather then being caused by alterations in mRNA stability.

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Nuclear run-on analysis of the transcription rates of the FATP-1, ACS, and GAPDH genes after 9c-RA treatment in FAO (A) and differentiated 3T3-L1 cells (B). FAO cells were treated for 4 h with 106 M 9c-RA after 18 h of growth in serum-deprived conditions. Differentiated 3T3-L1 cells were treated during 12 h with 106 M of 9c-RA. Transcription rates were then determined for the FATP-1, ACS, and GAPDH genes in nuclei from control cells () or 9c-RA treated cells (+). A Bluescript (BS) template was used as a control. Densitometric scanning of the results is depicted on the right.

Rexinoids, but Not RAR Agonists, Induce FATP-1 and ACS Gene Expression in Differentiated 3T3-L1 Cells-- To determine whether the effect of retinoids on the regulation of FATP-1 and ACS gene expression was mediated by RAR or RXR, both 3T3-L1 preadipocytes and differentiated 3T3-L1 adipocytes were treated either with at-RA or TTNPB (specific RAR agonists) or 9c-RA (an RAR/RXR panagonist). Interestingly, no effect of these retinoids on FATP-1 or ACS mRNA levels was observed in undifferentiated 3T3-L1 preadipocyte cells (Figs. 4 and 5). In differentiated 3T3-L1, no activation of FATP-1 and ACS gene expression was detectable after 24 h of treatment with at-RA or TTNPB. In contrast, a dose-dependent induction of both FATP-1 (Fig. 4) and ACS (Fig. 5) mRNA expression was observed 24 h after treatment with 9c-RA in the differentiated 3T3-L1 cells. Maximum induction of mRNA expression occurred at 10⁶ M 9c-RA (3.5- and 3-fold for FATP-1 and ACS, respectively). Furthermore, the fold induction of steady-state mRNA levels (Fig. 5) corresponds to the increase in transcription rates (Fig. 5) observed in 3T3-L1 cells.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Expression of FATP-1 mRNA in undifferentiated and differentiated 3T3-L1 cells after treatment with the indicated concentrations of different retinoids. Differentiated (3T3-L1 ; filled squares) or nondifferentiated (3T3-L1; open squares) cells were treated for 24 h with different concentrations of each of the retinoids indicated. Northern blot analysis was carried out and blots were hybridized with an FATP-1 probe as specified under "Experimental Procedures." FATP-1 mRNA was normalized against -actin mRNA expression, and the results are expressed as percentages of values in control cells.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Expression of ACS mRNA in undifferentiated and differentiated 3T3-L1 cells after treatment with the indicated concentrations of different retinoids. Differentiated (3T3-L1 ; filled triangles) or nondifferentiated (3T3-L1; open triangles) cells were treated for 24 h with different concentrations of each of the retinoids indicated. Northern blot analysis was carried out and blots were hybridized with an ACS probe as specified under "Experimental Procedures." ACS mRNA was normalized against -actin mRNA expression, and the results are expressed as percentages of values in control.

To specify which RXR heterodimer combination induced FATP-1 and ACS gene expression, a number of specific RXR ligands were used: 1) LGD1069 and LG100268, specific activators of RXR homodimers and PPAR-RXR or LXR-RXR, but without activity on RAR-RXR heterodimers; and 2) LG100754, an activator of PPAR-RXR and RAR-RXR heterodimers, which is unable to activate RXR homodimers. All these compounds enhance FATP-1 in a significant and dose-dependent manner in differentiated 3T3-L1 adipocytes expression (Fig. 4; 4-fold activation at 10⁶ M with LGD1069, 3.6-fold induction at 10⁶ M with LG100268, and a 4-fold induction at 10⁷ M with LG100754). In contrast, ACS expression was not affected by LG100268 and LGD1069, whereas LG100754 induced ACS mRNA levels significantly (Fig. 5). Once again, none of these specific rexinoids have an effect on undifferentiated 3T3-L1 preadipocytes (Figs. 5 and 6).

RXR Agonists, but Not RAR Agonists, Induce Oleate Uptake in Differentiated 3T3-L1 Cells-- To establish whether the induction of the FATP-1 and ACS was accompanied by a concomitant increase in fatty acid uptake into the cells, we measured [¹⁴C]oleate uptake in differentiated 3T3-L1 cells exposed to increasing doses of two prototypic retinoids, i.e. LG100754, an RXR agonist, and TTNPB, an RAR agonist (dose range 10⁹ to 10⁶ M). LG100754 induces [¹⁴C]oleate uptake dose-dependently. A maximal induction of fatty acid uptake by 2-fold was observed at a concentration of 10⁷ M. No effect on [¹⁴C]oleate uptake was observed after TTNPB, confirming that this effect was specific for the rexinoids (Fig. 6).

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Oleate uptake assay in 3T3-L1 differentiated cells. Differentiated cells 3T3-L1 (3T3-L1 ) were treated for 24 h with different concentrations of either LG100754 or TTNPB (109 to 106 M), and [14C]oleate uptake studies were carried out. Results were normalized after protein quantification and expressed as percentages of [14C]oleate uptake relative to untreated control cells. Significant differences were indicated by an asterisk (p < 0.05).

LGD1069 Induces FATP-1 mRNA Levels in the Liver and Adipose Tissue of Zucker Diabetic Fatty Rats-- We wanted to know whether these strong effects on FATP-1 and ACS gene expression in vitro could be translated into changes in mRNA expression in vivo. ZDF rats were therefore treated during 50 days with either no or increasing amounts of LGD1069 (0.3, 1.0, 3.0, 10 to 30 mg/kg/day; Fig. 7). In the liver, FATP-1 mRNA levels were significantly induced in a dose-dependent fashion by LGD1069 with a maximal effect at 30 mg/kg/day (Fig. 7). Interestingly, in adipose tissue, FATP-1 was only induced at the dose of 1 mg/kg/day (an effect lost at higher doses of LGD1069) in the ZDF rats. In the heart or skeletal muscle the expression of the FATP-1 gene was not affected by LGD1069 therapy. Similarly to FATP-1 mRNA, ACS mRNA levels were dose-dependently induced in the liver. No effect of LGD1069 was detected in adipose tissue, muscle, and heart. Also in lean animals, FATP and ACS mRNA levels were only induced in the liver, whereas the changes observed in the other tissues, such as adipose tissue, did not reach significance.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Dose-dependent regulation of FATP-1 and ACS mRNA levels by LGD1069 in Zucker diabetic fatty and normal rats. RNA was prepared from tissues of ZDF rats after 50 days of treatment with the indicated amounts of LGD1069. Northern blots were prepared and hybridized with either FATP-1 and ACS probe and the signals were normalized toward -actin. ZDF rats are represented by dark bars and lean rats by light gray bars.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results of this study demonstrate that RXR ligands control both FATP-1 and ACS gene expression in several cell types. In liver cell lines, FATP-1 and ACS mRNA levels have been shown previously to be strongly induced by PPAR activators, such as the fibrates (25-27). The transcriptional induction of the ACS gene is mediated by the interaction of the activated PPAR-RXR heterodimer with a cognate response element in the promotor region of the ACS (25). Hence, it was not too surprising to observe a strong induction of FATP-1 and ACS expression in hepatoma cell lines by specific RXR activators, which activate the other partner of the PPAR-RXR heterodimer. Long chain fatty acid transport in hepatoma cells seems to be tightly controlled by retinoic acid, because L-FABP, a cytoplasmic protein implicated in long chain fatty acid uptake, is also regulated by 9c-RA (28). The co-induction of FATP-1 and ACS expression by rexinoids, which is similar to the co-induction of both these genes by fibrates furthermore indicates that rexinoids and fibrates may have similar metabolic effects on rat and human liver. This has already been suggested by the observation that both retinoids and peroxisome proliferators stimulate transcription of genes encoding the peroxisomal -oxidation enzymes (29, 30), most likely through activation of the PPAR-RXR heterodimer (18). In adipose cell lines, which are highly insulin-sensitive, FATP-1 and ACS expression are also co-regulated by rexinoids in a dose-dependent manner, although the maximum level of induction appears lower. In contrast to the liver cells, which mainly express PPAR, PPAR is the predominant PPAR isotype expressed in adipocytes and PPAR activators have been shown to induce both FATP-1 and ACS expression (27). Hence, the induction of FATP-1 (and to a lesser extent ACS) expression in adipose cell lines by RXR agonists, suggest that RXR is also here an active partner in the PPAR-RXR heterodimer. In Caco2, FATP-1 and ACS genes are also co-regulated by rexinoids. This is the first time that FATP-1 is shown to be expressed in a human intestinal cell line. In view of the co-regulation by retinoic acids of several fatty acid transporters, such as L-FABP (28), ACS and FATP-1, in this tissue, a potential important role for retinoic acid in absorptive processes in the gut is suggested. It furthermore underscores the potential implication of FATP-1, in addition to FATP-4 in fatty acid uptake in the gut (31).

Interestingly, the strong regulatory effects of retinoids on FATP-1 and ACS expression in the liver-derived cell lines (FAO and HepG2; Fig. 1) seems to be translated in vivo (Fig. 7), underscoring the physiological relevance of these findings. In contrast, the apparent difference between the regulation of FATP-1 and ACS gene expression between our in vitro studies in adipocyte cell lines and our in vivo studies in adipose tissue seems puzzling. Whereas we observe a ±4-fold induction of the expression of both genes in adipocyte cell lines, a much smaller effect was observed in vivo in ZDF adipose tissue, suggesting that the in vivo response in ZDF adipose tissue is somehow impeded. This could be linked to differences either in the expression or activity of one of the heterodimer components (RXR/PPAR) or of one of the cofactors required for transcriptional activity. Further studies in our laboratory are addressing this issue at present.

Our studies with more selective ligands confirm the importance of the RXR-PPAR heterodimer in mediating the effects of PPAR and/or RXR agonists on gene expression. Because LG100754 induces only the activity of the RXR-RAR and RXR-PPAR heterodimers, the up-regulation of FATP-1 and ACS by LG100754 implicates one or the other heterodimer in mediating the effects. LG100268, which is specific for the RXR homodimer or RXR-LXR and RXR-PPAR heterodimers, but not RAR-RXR heterodimers, activates FATP-1 expression. Finally, TTNPB, which is an activator of RAR in the context of RXR-RAR heterodimer, has no effect on FATP-1 and ACS. The combined results of studies using these synthetic retinoids implicates the PPAR-RXR heterodimer as the molecular target of the regulation of FATP-1 and ACS by retinoic acids. Because PPAR and RXR are both active components of this heterodimer and because we have previously demonstrated that the FATP-1 and ACS genes are both regulated by PPAR activators (27), it appears that rexinoids cooperate synergistically with PPAR ligands to control the expression of these two genes involved in fatty acid metabolism. Furthermore, the fact that the co-regulation of FATP-1 and ACS genes by rexinoids depends on the differentiated state of 3T3-L1 cells points to the importance of PPAR in this process. In preadipocyte cells, PPAR is expressed at low levels and its expression increases upon adipocyte differentiation. The absence of a rexinoid response in undifferentiated 3T3-L1 cells, expressing almost no PPAR, hence also suggests that PPAR is an obligatory partner of RXR in the retinoid-dependent regulation of FATP-1 and ACS genes in adipose tissue.

It is interesting to note that RXR agonists such as LG100268 and LGD1069 function as insulin sensitizers in a similar way as PPAR agonists (14). Glucose homeostasis is improved when fatty acid concentration decreases in the circulation and more particularly in muscle tissue. The beneficial effect of PPAR activators on glucose homeostasis has been previously shown to be in part due to a redistribution of fatty acids toward adipose tissue with a relative depletion of fatty acids in the muscle (reviewed in Ref. 17). As known since Randle's work (32-34), depletion of muscle fatty acid content will result in an improvement of glucose homeostasis. Hence, it is tempting to speculate that the improvements of glucose homeostasis observed with rexinoids are, similar to the effects of PPAR agonists, linked to an altered partitioning of fatty acids, with a relative depletion of muscle fatty acid uptake and metabolism. As observed in vivo in this study, the attenuated regulation of FATP-1 and ACS expression in both heart and skeletal muscle is in fact suggestive of a role for these two genes in reducing fatty acid uptake in muscle tissue, which could contribute to the insulin-sensitizing effects of these rexinoids. In view of the distinct tissue distribution of PPAR and the various RXRs, RXR ligands might have a different tissue-specific activity, affecting mainly the liver, whereas PPAR activators will affect mainly adipose tissue. RXR agonists could, hence, provide an alternative or complimentary way to regulate the expression of genes implicated in fatty acid distribution and that ultimately affect glucose homeostasis.

This study further underscores distinct metabolic effects of retinoids versus rexinoids and demonstrates that RXR is an important signaling molecule in the regulation of two genes implicated in fatty acid metabolism. Combined with our previous studies on the effects of PPAR agonists on FATP-1 and ACS expression, the data obtained with dimer-specific rexinoids in this manuscript suggest that the PPAR-RXR complex is the molecular target by which rexinoids regulate FATP-1 and ACS gene expression. The observation, that both rexinoids and PPAR agonists control the transcription of the same genes implicated in lipid metabolism, suggests that rexinoids also act as hypolipidemic and hypoglycemic agents through activation of PPAR-RXR complex and are consistent with the hypothesis that the PPAR-RXR heterodimer is a molecular target for insulin sensitivity.

	FOOTNOTES

^* This work was supported by grants from CNRS INSERM CHUR, Association de Recherche contre le Cancer (Grant 6403), Janssen Research Foundation, Ligand Pharmaceuticals, and Fondation pour la Recherche Médicale (to J.A.) and Conseil regional de Bourgogne (to P.B.).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.

^** Current address: X-ceptor Pharmaceuticals, 4757 Nexus Centre Drive, Suite 200, San Diego, CA 92121.

^§ Supported by the Janssen Research Foundation.

^§§ A research director with CNRS. To whom correspondence should be addressed: Fax: 33-388-65-32-01; E-mail: auwerx@igbmc.u-strasbg.fr.

	ABBREVIATIONS

The abbreviations used are: FATP, fatty acid transport protein; RA, retinoic acid; at-RA, all-trans-RA; 9c-RA, 9-cis-RA; RAR, retinoic acid receptor; RXR, retinoid X receptor; TTNPB, ethyl-p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtyl)-1-propenyl]benzoic acid; LGD1069, 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic acid; LG100268, 6-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl]nicotinic acid; LG100754, (2E,4E,6Z)-7-(-3-n-propoxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl)-3 methylocta-2,4,6-trienoic acid; PPAR, peroxisome proliferator-activated receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ACS, acyl-CoA synthase; FABP, fatty acid-binding protein; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; ZDF rats, Zucker diabetic fatty rats.

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