©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning of a Protein That Mediates Transcriptional Effects of Fatty Acids in Preadipocytes
HOMOLOGY TO PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (*)

(Received for publication, September 5, 1994; and in revised form, October 31, 1994)

Ez-Zoubir Amri (1) Frédéric Bonino (1) Gérard Ailhaud (1) Nada A. Abumrad (2) Paul A. Grimaldi (1)(§)

From the  (1)Centre de Biochimie, UMR 134 CNRS, Faculté des Sciences, Parc Valrose, 06108 Nice cédex 2, France and the (2)Department of Physiology and Biophysics, Health Sciences Center, State University of New York, Stony Brook, New York 11794-8661

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Exposure of preadipocytes to long chain fatty acids induces expression of several gene markers of adipocyte differentiation. This report describes the cloning, from a preadipocyte library, of a cDNA encoding a fatty acid-activated receptor, FAAR. The cDNA had the characteristics and ligand-binding domains of nuclear hormone receptors and encoded a 440 amino acid protein related to peroxisome proliferator-activated receptors, PPAR. The deduced protein sequence was 88% homologous to that of hNUC I, isolated from human osteosarcoma cells. FAAR mRNA was abundant in adipose tissue, intestine, brain, heart, and skeletal muscles and less abundant in kidney, liver, testis, and spleen. The mRNA was undetectable in growing Ob1771 and 3T3-F442A preadipocytes, was strongly induced early during differentiation, and was increased by fatty acid. Transcription assays using hybrid receptor showed strong stimulation by fatty acid and weaker induction by fibrates. Transfection of 3T3-C2 fibroblasts, with a FAAR expression vector, conferred fatty acid inducibility of the adipocyte lipid-binding protein and the fatty acid transporter. Transcriptional induction of these genes exhibited inducer specificity identical to that described in preadipocytes. In summary, the data indicate that FAAR is likely a mediator of fatty acid transcriptional effects in preadipocytes.


INTRODUCTION

Fatty acid (FA) (^1)treatment of preadipocytes induces expression of several genes encoding proteins implicated in FA metabolism. These include the adipocyte lipid-binding protein, ALBP(1, 2, 3) , the acyl-CoA synthase (1, 2) and a recently cloned (4) membrane protein implicated in FA binding and transport, FAT. (^2)Some features of the transcriptional effects of FA in preadipocytes have been described. Both saturated and unsaturated long chain FA were effective in preadipocytes(1, 2) , unlike findings with mammary cells(5) . Induction did not require FA metabolism since it was observed with 2-bromopalmitate, which is not metabolized by preadipocytes(6) , but it required protein synthesis and was fully reversed upon FA removal(1, 2) . These effects of FA have potentially important nutritional and clinical significance. It is likely that FA, in vivo, as in vitro(7) promote adipose conversion of preadipocytes. In addition, transcriptional regulation by FA applies to cell types other than adipocytes and to multiple lipid-related genes(8, 9, 10) .

The molecular mechanism mediating the effect of FA on gene expression in preadipocytes remains unknown. Recent evidence has suggested that FA may act through nuclear receptors of the steroid-thyroid superfamily, the peroxisome proliferator-activated receptors (PPARs). Activation of these receptors by FA has been reported(8, 9, 10) , and an arachidonic acid analogue (ETYA) was shown to be even more potent than fibrates in activating one of these receptors, xPPARalpha(11) . In this report we have investigated whether a member of the PPAR family mediates the transcriptional effects of FA in preadipocytes. Using a DNA fragment containing conserved PPAR sequence, we have isolated a mouse PPAR-like protein that is activated by FA and related molecules but weakly by fibrates. Furthermore, expression of this protein in fibroblasts is shown to confer FA-responsive gene expression.


EXPERIMENTAL PROCEDURES

Materials

Culture media were obtained from Life Technologies, Inc. 2-Bromopalmitate was from Aldrich, and bovine serum and other chemical products were from Sigma. Vectors were from Stratagene (ZAPII and pSG5) and Clontech (pMAMneo). The TNT reticulocyte lysate was from Promega. Enzymes for DNA and RNA manipulation were from Boehringer Mannheim. All radioactive materials and hybridization membranes (Hybond) were from Amersham.

Cell Culture

Ob1771(12) , 3T3-F442A(13) , and 3T3-C2 (14) cells, plated at a density of 2 times 10^3/cm^2, were grown in Dulbecco's modified Eagle's medium supplemented with 200 units/ml of penicillin, 50 µg/ml of streptomycin, 33 µM biotin, 17 µM pantothenate, and containing 8% bovine serum (standard medium). To promote differentiation of Ob1771 and 3T3-F442A cells, bovine serum was replaced at confluence by fetal calf serum, and insulin (17 nM) and triiodothyronine (2 nM) were added (differentiation medium). All FA were added from concentrated (50 mM) stock solutions in ethanol to prewarmed culture media with stirring as described previously(6) . Clofibric acid was from a 0.5 M stock solution in Me(2)SO, and Wy 14,643 and ETYA were from a 50 mM stock solution in Me(2)SO. Control cell incubations included the same concentrations of ethanol or Me(2)SO.

Cloning and Sequencing of cDNA

A random-primed cDNA library was constructed in ZAPII using RNA from 1-day post-confluent Ob1771 cells maintained for 24 h in standard medium containing 250 µM linolenic acid. Positive clones were identified under low stringency conditions by probing with a P-labeled BglII-KpnI fragment(502-698) of mPPAR(9) . Sequence of both strands of the largest clone, termed FAAR, was determined by dideoxy sequencing.

RNA Analysis

RNA, prepared as described by Chomczynski and Sacchi(15) , was analyzed and quantitated by densitometry as described previously(16) . Measurements were made within the linear response of the integrated peaks as a function of immobilized RNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was the internal standard. Nuclear transcription assays were performed as described previously(12) .

DNA Binding Assays

Oligonucleotides containing the sequence (5`-CCCGAACGTGACCTTTGTCCTGGTCC-3`) of the acyl-CoA oxidase gene (-578 to -553) were annealed and labeled using Klenow DNA polymerase and [P]dCTP. A typical DNA binding assay contained, in a final volume of 20 µl, 75 pM of each protein in 25 mM HEPES buffer pH 7.8, 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 2 µg of poly(dI-dC), and 10% glycerol. The mixture was kept 10 min on ice and radiolabeled oligonucleotide (25,000 counts/min, 0.5 ng) was added without or with a 50-fold molar excess of unlabeled competitor, and the incubation was continued for another 20 min. Reaction mixtures were loaded onto a pre-run (30 min) 5% polyacrylamide gel equilibrated in 0.5 times Tris-borate/EDTA. Following electrophoresis, gels were soaked (15 min) in 5% (v/v) glycerol, dried, and autoradiographed.

Hybrid Receptor Activation Assay

The hybrid receptor hGR/FAAR was prepared by polymerase chain reaction mutagenesis using standard techniques. The hybrid receptor contained amino acid residues 1-486 (domains A/B and C) of the human glucocorticoid receptor (hGR) and amino acid residues 137-440 of the FAAR. The polymerase chain reaction-amplified DNA was cloned into the BamHI site of the pSG5 expression vector, and the sequence was confirmed by dideoxy sequencing. COS-1 cells were transfected with the plasmid MMTV-CAT, in which the expression of chloramphenicol acetyltransferase is under the control of the MMTV promoter, with or without pSG-hGR/FAAR expression vector. Transient transfection was performed in triplicate with cells maintained in 60-mm dishes with phenol red-free medium supplemented with 8% activated charcoal-treated fetal calf serum, using 2.5 µg of both plasmids and the DOTAP ( N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-triethylammonium methylsulfate) transfection reagent (Boehringer Mannheim). 8 h later, cells were washed and incubated in the same medium in the presence of various ligands as indicated. 48 h after transfection, cell extracts were prepared and assayed for CAT enzyme activity, using the CAT enzyme assay system (Promega).

Cell Transfection

An expression (pSG5-FAAR) vector was obtained by insertion of the coding sequence (nucleotides 1-1440) of FAAR into the BamHI site of pSG5 vector. 3T3-C2 cells (2 times 10^5/100-mm plate) maintained in standard medium were co-transfected with 1 µg of pMAMneo and 20 µg of pSG5 without (controls) or with FAAR cDNA (FAAR cells) using the DOTAP transfection reagent. Geneticin was added to the culture medium 48 h later, and geneticin-resistant clones were selected and tested for the response to FA as described previously for Ob1771 cells(2, 6) .


RESULTS

Cloning of FAAR cDNA

Screening of the cDNA library from FA-treated Ob1771 cells with a mPPAR fragment (BglII-KpnI), corresponding to the conserved sequence between members of the PPAR family, yielded five positive clones. One of the clones was identified as the c-erb A/thyroid receptor. The other four represented different lengths of the same cDNA, referred to as FAAR, consisting of 1543 base pairs with an open reading frame beginning at nucleotide 58 and encoding a protein with 440 amino acids and a calculated mass of 50 kDa. Consistent with this, in vitro translation of RNA from FAAR cDNA yielded a 50-kDa protein (data not shown).

The deduced amino acid sequence for FAAR was different from those previously reported (9, 17, 18) for mouse PPARs (PPARalpha and ). However, sequences with 86 and 84% identities to putative DNA-binding domains of mPPAR and mPPAR, respectively, were identified in FAAR. The putative ligand-binding domain and the D domains of mouse PPARs exhibited 70 and 50% identities, respectively, with corresponding domains of FAAR. A search of data base nucleotide sequences revealed near complete homology (99%) of FAAR sequence to that of a 503-base pair cDNA recently isolated from a mouse brain cDNA library (17) and postulated to represent the A/B and DNA-binding domains of hNUC I, a member of the PPAR family isolated from a human osteosarcoma cDNA library(19) . FAAR and hNUC I exhibited strong similarities of their DNA- binding (97%), ligand-binding (95%), and A/B (80%) and D domains (93%). The deduced protein sequences for FAAR and hNUC I were 88% similar. The above showed that FAAR was the mouse homologue of human NUC I.

Tissue Distribution of FAAR mRNA

Expression of FAAR mRNA in various mouse tissues is shown in Fig. 1. A single 3.1-kb transcript was detected and was found to be highly expressed in adipose tissue, small intestine, skeletal muscle, lung, heart, and brain. Moderate (kidney) and low expression (liver, spleen, and testis) were measured in other tissues.


Figure 1: Tissue distribution of FAAR mRNA in adult mouse. RNA (30 µg/lane) from tissues of 8-week-old mice were analyzed and quantitated as described under ``Materials and Methods.'' Actin mRNA (alpha and/or beta) is shown as internal standard. Li, liver; W, epididymal adipose tissue; I, intestine; M, skeletal muscle; K, kidney; S, spleen; Lu, lung; H, heart; T, testis; B, brain.



Regulation of FAAR mRNA by Cell Differentiation and FA Treatment

FAAR mRNA was undetectable in growing Ob1771 (Fig. 2A, lane 1) and 3T3-F442A (lane 3) cells. However, strong induction was observed with cell differentiation (lanes 2 and 4, respectively). In contrast, only a very faint signal was obtained in 3T3-C2 fibroblasts, even when maintained for 11 days after confluence in differentiation medium (lane 5). Similar faint FAAR mRNA signals were obtained with other fibroblastic cell lines such as NIH-3T3 and 10T (not shown). The time course for FAAR mRNA induction during differentiation of Ob1771 cells is shown in Fig. 2B and compared to those of PPAR (17, 26) and ALBP mRNAs. FAAR mRNA, undetectable before cell confluence, increased rapidly thereafter reaching 60% of its final level 2 days after confluence. In the same cell series, PPAR and ALBP mRNAs became detectable beginning 4 days after confluence.


Figure 2: Induction of FAAR mRNA with adipose differentiation and by fatty acids. A, RNA (20 µg/lane) was analyzed by Northern blotting as described under ``Materials and Methods.'' 1, RNA from Ob1771 subconfluent cells; 2, RNA from 11-day post-confluent differentiated Ob1771; 3, RNA from subconfluent 3T3-442A cells; 4, RNA from 11-day post-confluent differentiated 3T3-442A cells; 5, RNA from 11-day post-confluent 3T3-C2 cells. B, Ob1771 cells were maintained in differentiation medium, and RNA was prepared at the indicated times and analyzed by Northern blotting. FAAR mRNA (), PPAR (box), and ALBP mRNA (bullet) signals were quantitated by densitometry and standardized to GAPDH mRNA signals. C, 1-day post-confluent Ob1771 cells were maintained for 24 h in standard medium (lane 1) in the presence of 250 µM palmitate (lane 2) or 100 µM 2-bromopalmitate (lane 3). RNA was analyzed as in A. The results shown in A-C are representative of three separate experiments.



Fig. 2C shows that exposure of Ob1771 preadipocytes (day 1 post-confluence) to palmitate or 2-bromopalmitate increased level of FAAR mRNA and induced ALBP expression. Interestingly, PPAR mRNA, which is undetectable at day 1, remained not expressed in cells exposed for 24 h to FA.

FAAR Binds to PPRE

Relation to the PPAR family was confirmed by demonstrating FAAR binding to the peroxisome proliferator-responsive element (PPRE) (11, 21, 22, 23) and enhancement of the binding by heterodimerization with retinoid X receptors (RXR)(22, 23, 24) . Mobility shift experiments (Fig. 3) were performed using a PPRE oligonucleotide in the presence of FAAR, RXRs, and RARs translated in vitro. Incubation of PPRE with FAAR resulted in a faint retarded signal whereas incubation with RXRalpha did not. Incubation of both proteins led to appearance of a strong retarted signal indicating the formation of a DNAbulletprotein complex which was wiped out in the presence of a 50-fold molar excess of unlabeled PPRE. RXRbeta and to a lesser extent RXR were able to form heterodimers with FAAR and bind PPRE. This was not observed when RARs were used in combination with FAAR (data not shown).


Figure 3: FAAR and RXRalpha or RXRbeta bind cooperatively to the acyl-CoA oxidase PPRE. In vitro translated proteins were incubated according to the indicated combinations in the presence of labeled oligonucleotide. The proteinbulletDNA complexes were resolved by polyacrylamide gel electrophoresis as described under ``Materials and Methods.''



FAAR Is Activated by Fatty Acids

To search for a putative activator for the FAAR, an expression vector for a chimeric receptor (hGR/FAAR) consisting of the A/B and the DNA-binding domain of hGR, and the D and the ligand-binding domains of FAAR was cotransfected into COS-1 cells along with a reporter plasmid containing the CAT gene controlled by the glucocorticoid-responsive MMTV promoter. Cells transfected with MMTV-CAT and hGR plasmids demonstrated strong induction of CAT activity by dexamethasone. Little response was observed with 2-bromopalmitate (Fig. 4, inset). The opposite was observed with cells cotransfected with hGR/FAAR and MMTV-CAT plasmids. Dexamethasone was a poor inducer (column a) while 2-bromopalmitate was very effective producing a dose-dependent increase in CAT activity up to 13.5-fold at 10 µM (columns b-e). Palmitate (100 µM, column f) and ETYA (10 µM, column g) were also potent inducers. Clofibric acid (column i) and the potent peroxisome proliferator, Wy 14,643 (column j) were less effective resulting, respectively, in 3- and 5-fold increases in CAT activity at the maximal concentrations of 500 and 100 µM.


Figure 4: Activation of hGR/FAAR hybrid receptor by FA. COS-1 cells were transfected with MMTV-CAT vector and either the hGR/FAAR or hGR (insert) expression vectors. The transfected cells were treated for 40 h in the following conditions: a, 0.1 µM dexamethasone; b-e, 1, 3, 10, and 30 µM 2-bromopalmitate; f, 100 µM palmitate; g, 10 µM ETYA; h, 100 µM bromooctanoate; i, 500 µM clofibric acid; and j, 100 µM Wy 14,643. CAT enzyme activity was determined as described under ``Materials and Methods.'' Results are presented by taking as 1 the value obtained in cells maintained in control medium and are presented as the mean ± S.D. of triplicate transfections.



Expression of FAAR mRNA in 3T3-C2 Fibroblasts Confers Fatty Acid-responsive Gene Expression

The relationship between FAAR expression and regulation of gene transcription by FA was investigated in 3T3-C2 fibroblasts. These cells express very low levels of FAAR mRNA (Fig. 2, lane 5) and do not respond to FA by increasing expression of ALBP (2) or FAT genes.^2 Stably transfected cells expressing FAAR and cells containing the empty expression vector (control cells) were analyzed for FAAR expression. As shown in Fig. 5, expression of the endogenous form of FAAR (3.1 kb, upper band) was very low (lanes 1-8) in all transfected cells. FAAR-transfected cells (three clones are shown) expressed other FAAR mRNAs in addition to the faint endogenous form. Clones FAAR-45 and 47 (lanes 3-6) expressed strongly the expected transfected form (1.8 kb, lowest band). FAAR-27 cells showed a more complex pattern of FAAR mRNA expression which included additional and aberrant 2.2- and 2.8-kb species (lanes 7 and 8).


Figure 5: Stable transfectants 3T3-C2 expressing FAAR are responsive to fatty acids. A, control transfected (lanes 1 and 2), FAAR-45 (lanes 3 and 4), -47 (lanes 5 and 6), and -27 (lanes 7 and 8) cells were maintained from confluence to day 2 post-confluence in standard medium (lanes 1, 3, 5, and 7) or exposed for the last day to 100 µM 2-bromopalmitate (lanes 2, 4, 6, and 8). RNA was analyzed by Northern blot as described under ``Materials and Methods.'' Autoradiographic times were: 8 h for GAPDH mRNA, 15 h for ALBP and FAT mRNAs, and 6 h for FAAR mRNA. The data are representative of three independent experiments. B, 1-day post-confluent FAAR-27 (bullet), -45 (box), -47 (), and control cells (circle) maintained in standard medium were exposed for 24 h to increasing concentrations of 2-bromopalmitate. FAT mRNA signals were quantified as in Fig. 2B.



The effects of FA treatment of FAAR and control cells (100 µM 2-bromopalmitate for 24 h) on ALBP and FAT gene expression are shown in Fig. 5A. As expected, ALBP and FAT mRNAs remained undetectable in all (six were tested) control-transfected cells (lane 2versuslane 1). In contrast, exposure of FAAR-expressing cells to 2-bromopalmitate induced expression of ALBP and FAT mRNAs (lanes 4, 6, and 8).

The response to FA was characterized with respect to inducer concentration on FAT gene expression in confluent transfected cells (Fig. 5B). For the three FAAR expressing clones, response to 2-bromopalmitate was detectable beginning at 3 µM with a half-maximal effect observed at about 20 µM. In contrast, FAT mRNA remained undetectable in control 3T3-C2 cells subjected to the same treatments. Similar results were obtained for the ALBP gene expression (data not shown).

The specificity of FAAR-mediated FAT gene induction was next investigated in FAAR-45 cells exposed for 24 h to increasing concentrations of various inducers. Fig. 6reports the maximal response obtained for each compound. FA analogues, i.e. 2-bromopalmitate and ETYA (columns b and e) as well as natural FA, i.e. palmitate and linolenate (columns c and d), were strong inducers of FAT mRNA expression. In contrast, middle chain FA derivative, 2-bromooctanoate (column f), clofibric acid (column g), and Wy 14,643 (column h) were found to be ineffective or weak inducers of FAT mRNA. Nuclear run-on experiments (Fig. 6, inset) were carried out using nuclei from FAAR-45 cells maintained for 24 h in standard medium in the absence (lane 1) or presence (lane 2) of 100 µM 2-bromopalmitate. FAT and ALBP transcription rates, which were very low in cells maintained in standard medium, were strongly increased by FA treatment indicating that induction of FAT and ALBP mRNA expression in response to FA was primarily due to transcriptional activation of the corresponding genes.


Figure 6: Activation of FAT mRNA expression by various inducers in FAAR-45 cells. 1-day post-confluent FAAR-45 cells were maintained for 24 h in the following conditions: a, standard medium; b, 100 µM 2-bromopalmitate; c, 200 µM palmitate; d, 200 µM linolenate; e, 100 µM ETYA; f, 100 µM 2-bromooctanoate; g, 500 µM clofibric acid; and h, 100 µM Wy 14,643. RNA was analyzed as in Fig. 2. The data are representative of three independent experiments. Inset, run-on assays from nuclei of 1-day post-confluent FAAR-45 cells exposed (2) or not (1) for 24 h to 100 µM 2-bromopalmitate.




DISCUSSION

The present work, which dealt with transcriptional effects of FA in preadipocytes, reported the following: 1) a cDNA, termed FAAR, encoding a member of the PPAR superfamily, was isolated from a library constructed from FA-treated preadipocytes; 2) FAAR was activated by FA; 3) FAAR was shown to be one of the earliest markers of preadipocyte differentiation; 4) the ability of FAAR, when expressed in 3T3-C2 fibroblasts unresponsive to FA, to confer FA-specific induction of two gene markers of adipose differentiation, ALBP and FAT, was documented; 5) FA induction in preadipocytes and in FAAR-expressing 3T3-C2 cells was shown to exhibit similar features consistent with FAAR mediation of the FA effects. The findings constitute the first direct demonstration of a receptor's role in mediating FA regulation of differentiation-linked genes in preadipocytes.

The preadipocyte FAAR is homologous to hNUC I, previously isolated from a human osteosarcoma cDNA library(19) . The deduced protein sequences for FAAR and hNUC I were 88% similar. Like hNUC I, FAAR is related to the mouse (9, 17, 18) and rat (10) PPARs as evidenced by the high identity of the respective DNA- and ligand-binding domains. In addition, FAAR binds to PPREs and the binding is enhanced by heterodimerization with RXRs (Fig. 3). However, weak similarity of other domains of FAAR with those of mouse and rat PPARs suggests that FAAR, and possibly hNUC I, form a distinct subtype of the PPAR family. This is further supported by two other lines of evidence. First, the observed tissue distribution of FAAR is different from that previously reported for mPPAR. FAAR is highly expressed in adipose tissue, muscle, and intestine and weakly expressed in liver and kidney (Fig. 1) while mPPAR is preferentially expressed in liver, kidney, and heart(9) . Second, the inducer responsiveness of FAAR and PPAR are different. FAAR appears equally sensitive to saturated and unsaturated FA and is less sensitive to fibrates ( Fig. 4and Fig. 6). In contrast, PPAR are activated by fibrates and unsaturated FA better than by saturated FA(10, 11) . The data would suggest that FAAR might be typical of a subgroup of PPAR preferentially responsive to long chain FA.

Multiple lines of evidence support the interpretation that FAAR mediates the transcriptional effects of FA in preadipose cells. FAAR is not expressed in 3T3-C2 and preconfluent preadipose cells which are unresponsive to FA. Its appearance at confluence in Ob1771 preadipocytes (Fig. 2) coincides with acquisition of the response to FA (2, 6) . Expression of FAAR in FA-unresponsive 3T3-C2 fibroblasts conferred FA-responsive expression of ALBP and FAT genes. Furthermore, the response observed exhibited the same specificity previously demonstrated in Ob1771 preadipocytes. In both cases, 2-bromopalmitate was more effective than native FA ( Fig. 6and (6) ). Both saturated and unsaturated FA were effective. Fibrates were weaker inducers, and middle chain FA were ineffective ( Fig. 6and (2) and (6) ).

The mechanism of FAAR action to mediate induction of ALBP and FAT remains to be determined. FAAR, in combination with RXRs, may act by binding to the same DNA motif as PPARs(20, 21) . PPAR is thought to bind to a direct repeat-like element in the ALBP promoter (26) which has been also involved in the tissue-specific expression of ALBP(27) . A direct repeat-like element was found in the promoter of human CD36, a homologue of FAT(28) . Thus, it is tempting to speculate that such direct repeat-like elements might constitute DNA-binding motifs for FAAR. Clearly, FAAR mRNA emerged earlier than PPAR mRNA during adipose conversion process of Ob1771 cells (Fig. 2B) as well as in 3T3-L1 cells(29, 30) . It is clear also that early after confluence, the time at which the FA responsiveness appears, preadipose cells express FAAR but not PPAR mRNA (Fig. 2B). PPAR was not expressed in FAAR-transfected 3T3-C2 cells treated with FA where induction of FAT and ALBP mRNA expression was observed (data not shown). Taken together, these observations indicate that FAAR is likely involved in FA activation of lipid metabolism-related genes in preadipose cells (2, 6) and in the adipogenic action of FA which occurs during the first days of the confluent phase(7) . PPAR, which is expressed at a later stage, might mediate, possibly in combination with FAAR, regulatory effects of FA occurring in partially to fully differentiated cells(1) .

Since FA promote terminal differentiation of preadipocytes in culture (7) , it will be important to determine whether 3T3-C2 fibroblasts, expressing high levels of FAAR, can be induced to differentiate in the presence of FA. Activation of FAAR could, in vivo, constitute an important part of the molecular mechanism behind the adipogenic effects of overfeeding. The data documenting FA induction of FAT expression adds one more differentiation-linked gene to those known to be sensitive to FA in preadipocytes. Furthermore, FA induction of FAT expression would amplify the effects of FA on preadipocyte differentiation since the FAT protein is implicated in the membrane binding and transport of FA into the cell(4) . While ALBP is adipose tissue specific, FAAR and FAT are more ubiquitous in expression and might respond to FA regulation in other cells, for example in developing muscle and intestinal cells. Determination of whether FAAR mediates transcriptional effects of FA in cells other than adipocytes will be crucial to understanding differential regulation of lipid-related genes in various tissues.


FOOTNOTES

*
This work was supported by Institut National de la Santé et de la Recherche Médicale Grant CRE920708 (to P. A. G.), Centre National de la Recherche Scientifique Grant UMR 134 CNRS, and by a grant from the Taher Biomedical Fund (to N. A. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L28116[GenBank].

§
To whom correspondence should be addressed. Tel.: 33-93529923; Fax: 33-93529917; grimaldi{at}naxos.unice.fr.

(^1)
The abbreviations used are: FA, fatty acid; ALBP, adipocyte lipid-binding protein; FAT, putative adipocyte FA transporter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-responsive element; RAR, retinoic acid receptor; RXR, retinoid X receptor; ETYA, 5,8,11,14-eicosatetraynoic acid; CAT, chloramphenicol acetyltransferase; hGR, human glucocorticoid receptor; kb, kilobase(s); MMTV, mouse mammary tumor virus.

(^2)
E.-Z. Amri, P. A. Grimaldi, and N. A. Abumrad, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. P. Chambon (Strasbourg, France), M. D. Lane (Baltimore, MD), and W. Wahli (Lausanne, Switzerland) for the kind gifts of RARs/RXRs ALBP cDNAs and Wy 14,643, L. Staccini for expert technical assistance, and G. Oillaux for secretarial assistance.


REFERENCES

  1. Amri, E., Bertrand, B., Ailhaud, G., and Grimaldi, P. (1991) J. Lipid Res. 32, 1449-1456 [Abstract]
  2. Amri, E., Ailhaud, G., and Grimaldi, P. (1991) J. Lipid Res. 32, 1457-1463 [Abstract]
  3. Distel, R. J., Robinson, G. S., and Spiegelman, B. M. (1992) J. Biol. Chem. 267, 5937-5941 [Abstract/Free Full Text]
  4. Abumrad, N. A., El-Maghrabi, M., Amri, E. Z., Lopez, E., and Grimaldi, P. A. (1993) J. Biol. Chem. 268, 17665-17668 [Abstract/Free Full Text]
  5. Levay-Young, B. K., and Nandi, S. (1989) Endocrinology 125, 1513-1518 [Abstract]
  6. Grimaldi, P. A., Knobel, S. M., Whitesell, R., and N. Abumrad. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10930-10934 [Abstract]
  7. Amri, E., Ailhaud, G., and Grimaldi. P. A. (1994) J. Lipid Res. 35, 930-937 [Abstract]
  8. Green, S. (1992) Biochem. Pharmacol. 43, 393-401 [Medline] [Order article via Infotrieve]
  9. Issemann, I., and Green, S. (1990) Nature 347, 645-650 [CrossRef][Medline] [Order article via Infotrieve]
  10. Gottlicher, M., Widmark, E., Li, Q., and Gustafsson, J. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4653-4657 [Abstract]
  11. Keller, H., Dreyer, C., Medin, J., Mahfoudi, A., Ozato, K., and Wahli, W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2160-2164 [Abstract]
  12. Doglio, A., Dani, C., Grimaldi, P., and Ailhaud, G. (1986) Biochem. J. 238, 123-129 [Medline] [Order article via Infotrieve]
  13. Green, H., and Kehinde, O. (1976) Cell 7, 105-113 [Medline] [Order article via Infotrieve]
  14. Green, H., and Kehinde, O. (1974) Cell 1, 113-116
  15. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  16. Amri, E. Z., Dani, C., Doglio, A., Etienne, J., Grimaldi, P., and Ailhaud, G. (1986) Biochem. J. 238, 115-122 [Medline] [Order article via Infotrieve]
  17. Chen, F., Law, S. W., and O'Malley, B. W. (1993) Biochem. Biophys. Res. Commun. 196, 671-677 [CrossRef][Medline] [Order article via Infotrieve]
  18. Zhu, Y., Alvares, K., Huang, Q., Rao, M. S., and Reddy, J. K. (1993) J. Biol. Chem. 268, 26817-26820 [Abstract/Free Full Text]
  19. Schmidt, A., Endo, N., Rutledge, S. J., Vogel, R., Shinar, D., and Rodan, G. A. (1992) Mol. Endocrinol. 6, 1634-1641 [Abstract]
  20. Osumi, T., Wen, J. K., and Hashimoto, T. (1991) Biochem. Biophys. Res. Commun. 175, 866-871 [Medline] [Order article via Infotrieve]
  21. Tugwood, J. D., Issemann, I., Anderson, R. G., Bundell, K. R., McPheat, W. L., and Green, S. (1992) EMBO J. 11, 433-439 [Abstract]
  22. Kliewer, S. A., Umesono, K., Noonan, D. J., Heyman, R. A., and Evans, R. M. (1992) Nature 358, 771-774 [CrossRef][Medline] [Order article via Infotrieve]
  23. Bardot, O., Aldridge, T. C., Latruffe, N., and Green, S. (1993) Biochem. Biophys. Res. Commun. 192, 37-45 [CrossRef][Medline] [Order article via Infotrieve]
  24. Issemann, I., Prince, R. A., Tugwood, J. D., and Green, S. (1993) J. Mol. Endocrinol. 11, 37-47 [Abstract]
  25. Cook, K. S., Hunt, C. R., and Spiegelman, B. M. (1985) J. Cell Biol. 100, 514-520 [Abstract]
  26. Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. I., and Spiegelman, B. M. (1994) Genes & Dev. 8, 1224-1234
  27. Graves, R. A., Tontonoz, P., and Spiegelman, B. M. (1992) Mol. Cell. Biol. 12, 1202-1208 [Abstract]
  28. Armesilla, A. L., and Vegas, M. A. (1994) J. Biol. Chem. 269, 18985-18991 [Abstract/Free Full Text]
  29. Chawla, A., and Lazar, M. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1786-1790 [Abstract]
  30. Chawla, A., Schwartz, E. J., Dimaculangan, D. D., and Lazar, M. A (1994) Endocrinology 135, 798-800 1 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.