Fatty Acid Regulation of Gene Expressionl

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TH E OURNAL F BIOLOGICALHEMISTRY0 1992by The American Societyfor Biochemistry andMolecular Biology, Inc.

Val. 267, No . 9, Issue of March 25, pp. 5937-5941, 1992Printed in U .S.A.

Fatty Acid Regulationof Gene ExpressionTRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL MECHANISMS*

(Received for publication, September 2, 1991)

Robert J. DistelS, GregoryS. Robinsone, and BruceM. SpiegelmannFrom the Dana-Farber Cancer Institute and the Department of Biological Chemistry and Molecular Pharmacology,Harvard Medical School, Boston,Massachusetts 02115

Fatty acids are important metabolic substrates andmay also be involved in pathological syndromes suchas the insulin resistance of diabetes and obesity. Wedemonstrate here thatatty acids can regulate specificgene expression; mRNAs encoding the atty acid bind-ing proteinadipocyte P2 (aP2) andtheFos-relatedtranscription factor Fral are specifically induced atleast 20-fold upon treatment of preadipocytes witholeate. For aP2, the effect requires long chain fattyacids and occurs without a generalized activation ofthe genes inked to adipocyte differentiation. Otherfibroblastic cells without preadipocyte characteristicsdo not induce aP2 mRNA in response to fatty acids.Unlike aP2, Fral induction by fatty acids also can bedetected in NIH 3T3 and 3T3-C2 fibroblasts. Nucleartranscription assays in 3T3-F442A preadipocytesdemonstrate that fatty acids elicit no transcriptionalincrease n the aP2 gene. Fral , on the other hand,shows a 3-4-fold increase in transcription. Thesere-sults demonstrate at least two distinct mechanisms bywhich fatty acids may influence gene expression.

Adipose cells are the major depot for energy storage invertebrate animals. Much or most of the lipid in these cells isderived from the uptake of free fatty acids released by thehydrolysis of circulating triglyceride-rich lipoproteins or fromcirculating fatty acid bound to serum albumin. The export ofenergy from fat cells during periods of nutritional deprivationalso involves fatty acids; these are ydrolyzed from the cellulartriacylglyceride droplet and released into the circulatory sys-tem.

Given the central role that fattyacids play in adipose tissuebiology and he known detergent-likeproperties of theseamphipathic molecules, it is not surprising that at cellscontain a specific fatty acid-binding protein. This molecule,termed adipocyte P2, was cloned from adipocyte cDNA li-braries and is highly abundant andrelatively fat cell-specificin ts expression (Spiegelman et al., 1983; Bernlohr et al.,

* This work was supported by Grant DK31405 from the NationalInst itutes of Health. T he costs of publication of thi s article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked advertisement in accordance with 18U.S.C. Section 1734 solely to indicate thi s fact.$ Supported by a fellowship from the Charles King Trust .GM13033.I Supported by National Research Service Award Fellowship1Established Investigator of the American Heart Association. Towhom correspondence should be addressed: Dana-Farber Cancer In-sti tute, Mayer 813, 44 Binney St., Boston, MA 02115. Tel.: 617-732-3567: Fax: 617-735-8971.

1984). aP2l belongs to a large family of intracellular lipidcarrier proteins that includes liver, intestine, kidney, andheart fatty acid-binding proteins, as well as myelin P2, andthe cellular retinol- and retinoic acid-binding proteins. Theprecise function of aP2 is not known, although it is believedto play a role in fatty acid transport orprotection against thedetergent-like effects of fatty acids (see Matarese and Bern-lohr (1989) for review).The adipocyte P2 gene has served as a model for differen-tiation-dependent gene expression in this cell type. The geneis transcriptionally activated during 3T3-adipocyte differen-tiation (Cook et al.,1985; Bernlohr e t al., 1985). Several cis-and trans-acting egulatory components of the gene have beenidentified, including an AP-1 sequence at -120,where wefirst identified sequence-specific interactions between Fos-containing protein complexes and DNA (Distel et al., 1987;Rauscher et al., 1988). In addition, a binding site for theCAAT/enhancer-binding protein (C/EBP) located 140 basepairs upstream of the start of transcription, has been de-scribed (Christy et al., 1989; Herrera e t al., 1989). The differ-entiation-dependence and tissue specificity of the aP2 geneand its relatively high level of expression appear to be deter-mined by a potent fat-specific enhancer located 5.4 kilobasesupstream of the sta rt f transcription, which functions in bothcultured adipocytes and transgenic mice (Ross et al., 1990;Graves et al., 1991).A binding site for a member of the nuclearfactor 1 family and several other nuclear factors play animportant role in this enhancer (Graves et al., 1991).Since adipocytes and preadipocytes are exposed to changinglevels of fatty acids under various normal and pathologicalmetabolic states, we have specifically investigated whetherthese substrates or the aP2 rotein might regulate expressionof the aP2 gene. We have also examined the effects of fattyacids on the transcription factors hat may modulate aP2 geneexpression. These include C/EBP Herrera et al., 1989;Christy et al., 1989) and members of the Fos and Jun t ran-scription factor family. We show here that fat ty acids dra-matically activate the expression of mRNA for aP2 and theFos-related gene Fr al in preadipocytes, without initiating thegeneral program of adipocyte differentiation. However, theseinductions appear to utilize different mechanisms, both tran-scriptional and post-transcriptional.

EXPERIMENTALPROCEDURESCell Culture-3T3-F442A cells were grown in Dulbeccos modifiedessential medium with 10% calf serum and supplemented with glu-tamine and penicillin/streptomycin except where otherwise noted.

The abbreviations used are: aP2, adipocyte P2; C/EBP, CAAT/enhancer-binding protein; BSA, bovine serum albumin; Tes, ( N -tris(hydroxymethyl)methyl-2-aminoethanesulfoniccid).R. A. Graves, P. Tontonoz, and B. M. Spiegelman, manuscript inpreparation.5937


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5938 Fatty Acid Regulation of Gene ExpressionCells were allowed to differentiate as described previously (Cook etal., 1985). In all experiments, sodium salts of fatty acids (Sigma) wereadded in a 6 1 molar ratio of fatty acid to fatty cid-free bovine serumalbumin (Sigma). For the fatty acid and control treatments of cells,10 ng/ml insulin was included to prevent spontaneous lipolysis frominterfering with the assessment of fatty acid effects.Northern Blots-RNA isolation was performed by the acidifiedguanidine hiocyanate method as described by Chomczynski andSacchi (1987). The RNA was size-fractionated on 2.2 M formaldehydeagarose gels and transferred to Biotrans (Pall BioSupport Group)nylon membrane filters.The RNA was photocross-linked and hybrid-ized as described by Virca et al. (1990). Probes were labeled by therandom prime technique using [a-""P]dCTP. Blots were washed in0.5 X ssc (1x ssc:0.15 M NaCl, 0.015 M trisodium citrate , pH 7.0),5% sodium dodecyl sulfate at 65 "C, followed by two washes in 0.5 XSSC at 65 "C.

Nuclear Transcription-Nuclei were pooled from five 10-cm dishesof confluent preadipocytes or adipocytes as described by Greenbergand Ziff (1984). Transcription reactions were performed as describedby Greenberg and Ziff (1984). Reactions were initiated by adding anequal volume of 2 X reaction buffer (10 mM Tris-HCI, pH 8.0, 5.0mM MgCl,, 300 mM KCl, 5.0 mM dithiothreitol, 1.0 mM ATP, 1.0 mMGTP, 1.0 mM CTP, and 200 pCi [oc-"'P]UTP (800 Ci/mmol) (DuPont-New England Nuclear) to 2 X lo7 nuclei (in about 150 pl). Thereaction was carried out for 30 min at 30 "C with agitation every 5min.RNase-freeDNase was added at 20 pg/ml, the nuclei wereincubated for 10 min a t 30 "C, and this reaction was terminated with100 pg of yeast tRNA and 10 volumes (about 3 ml) of 4 M guanidinethiocyanate, 25 mM trisodium citra te, pH 7.0, 0.5% sodium sarcosyl,and 0.1 mM 0-mercaptoethanol. The RNA was then isolated asdescribed by Chomczynski and Sacchi (1987), except that the finalRNA pellet was washed three times with 70% ethanol, resuspendedin 100 p1 of distilled water, and precipitated in 5% trichloroaceticacid, 10 mM sodium pyrophosphate, and the tota lncorporated countsdetermined. The RNA was resuspended in 100 pl of 10 mM Tes , pH7.4. Ten pg of denatured plasmid DNA was slot-blotted onto nitro-cellulose, according to the manufacturer (Schliecher & Schuell). Theslot blots were prehybridized for 12 h a t 65 "C in 10 mM Tes, pH 7.4,300 mM NaCl, 10 mM EDTA, 50 mM sodium pyrophosphate, 2 XDenhardt's solution (50 X Denhardt's: 10 mg/ml Ficoll, 10 mg/mlpolyvinylpyrrolidone, and 10 mg/ml BSA), and 0.2% sodium dodecylsulfate. Hybridization was in 1 ml of 10 mM Tes , pH 7.4, 300 mMNaCI, 10 mM EDTA, 0.2% sodium dodecyl sulfate, and 10 X lofi pmof radiolabeled RNA for 48 h a t 65 "C. The blots were washed for 2h at 55 "C in 2 X SSC and then treated with 10 pg/ml DNase-freeRNase A in 2 X SSC at 37 "C and washed for 1h in 2 X SSC at 37 "C.Hybridization was quantitated using Phosphor-Imager (MolecularDynamics).

RESULTSLong Chain Fatty Acids Can Induce aP2 Gene Expressionin Preadipocytes-Since th e adipocyte P 2 prote in app ears tobe the major carrier of long chain fatty acids in adipocytes,we asked wh eth er the levels of exogenous fatty acid couldmod ulate the level of aP 2 mRNA . 3T3-F442A adipocytes weretreated with mediacon tainin g eithe r .0 mM oleate complexed

t o fatty acid-free BSA (6:l) or BSA alone. After 24 h, North-ern blo t nalysis of the RNA from adipocytes treated with1.0mM oleate showed little or no ind uctio n ver th e already h ighlevel of aP2 mRNA in these cells (Fig. 1).We observed verylittle change in th e mRNA level over a broad range of oleateconcentrations (0.5-3.0mM, not show n). The RNA was si-multaneously probed withmouse b-actin as a control for bothefficiency of loading and RNA transfer. In contrast , whenpreadipocytes were treatedwi th 1.0 mM oleate, th eaP 2mR NA was induced 20-fold over cells treated with BSA alone(Fig. 1).Note that n order to determine the m agnitudef theaccumulation of aP2 mRNA in the fat ty acid- treated preadi-pocytes over th at of the con trol readipocytes, the RNA blotswere exposed long enough to o btain a signal for aP 2 mR NAin the control lan e (Fig. 1).Thus, the @ -actin ignal appearsmuch higher in preadipocytes tha n adipocytes. The indu ctionof aP2 mRNA in preadipocytes after a 24-h treatment with

Actin

aP2

- + - +

AdipocylcrcadipocytcFIG.1. Northern blot analysis of fatty acid induction of theaP2 gene. 3T3-F442A preadipocytes or adipocytes were treated for24 h with Dulbecco's modified essential medium, 10 ng/ml insulin,and 0.16 mM fatty acid-free BSA with (+) or without (-) the additionof 1.0 mM oleate. Total RNA was isolated from these cells, and 15p g

of total RNA was hybridized on Northern blots with both 0-actin andaP2 cDNAs (Spiegelman et al., 1983). The preadipocyte lanes wereexposed longer than the adipocyte ones to detect an aP2 ignal in theuntreated cells.

AP2

aP2

aP2

C A 13 C I)

FIG. 2. Northern blot analysis of the aP2 mRNA responseto long and short chain fatty acids. A , Northern blot analysis ofthe aP2 mRNA response to increasing doses of oleate. Preadipocyteswere treated with 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mM oleatecomplexed to fatty acid-free BSA in an oleate to BSA ratio of 6:l.Controls cells were treated with the lowest and highest concentrationsof fat ty acid-free BSA: 0.016 mM (CJ and 0.23 mM (Ch). , Northernblot analysis of aP2 mRNA levels in preadipocytes treated withvarious long chain fatty acid and a long chain fatty alcohol. Preadi-pocytes were treated for 24 h with 0.05 mM fatty acid-free BSA alone( A ) ,0.3 mMof the sodium sal ts of stearic acid (B ), oleic acid (C),palmitic acid (D),almitoleic acid ( E ) , nd oleyl alcohol ( F ) com-plexed with fatty acid-free BSA in Dulbecco's modified essentialmedium. C, aP2 mRNA levels in preadipocytes treated with shortand medium chain length fatty acids. Preadipocytes were treated with0.05 mM fatty acid-free BSA ( A ) ,0.3 mM sodium butyrate (B ), 0.3mM sodium decanoate (C), and 0.3 mM oleate ( D)n Dulbecco'smodified essential medium. All media contained 10 ng/ml insulin.Total RNA was isolated from these cells, and 15 p g of RNA washybridized on Northern blots with the aP2 cDNA. Each experimentwas repeated with similar results.

oleate typically reached between 25 and 50% of the messagelevel found in untre ated adipocytes. It is therefore clear tha ta very significant level of aP2 mRNA can be induced inpreadipocytes by oleate.Fat ty acids are typically found in fasting or fed mice atplasma levels in a range between 0.1 and 1.2 mM (Bak er etal., 1978). To investigate w heth er the response of the aP2mRNA to fatty acids occurred in a physiological range, pre-adipo cytes were tre ate d with 0.1-1.4 mM oleate for 24 h (Fig.2 A ) . The additio n of fatty acid-free BSA alone at th e owest


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Fatty Acid Regulation of Gene Expression 5939(0.016 mM) and the highest (0.23 mM) concentrations usedhad no effect on the levels of aP2 mRNA, as demonstrated inlanes Cr and Ch . RNA isolated from the oleate-treated cellsshowed a %fold increase in the level of aP2 mRNA at aconcentration of 0.1 mM oleate. The maximum response after24 h occurred at doses between 0.6 and 0.8 mM. Higher dosesof up to 1.4 mM oleate gave reduced levels of aP2 message,although these levels of fatty acid did not have an obviousdeleterious effect on the cells. Levels greater than 2.5 mMroutinely killed preadipocytes. A similar dose response curvewas obtained with linoleate (C,,,,) (data not shown). Otherlong chain fatty acids, such as palmitate (C,,,,), palmitoleate(C,,,,), and stearate (Cls:0), also were capable of causing in-duction of theaP2 mRNA (Fig. 2B), whereas shortandmedium chain length fatty acids, butyric (C,) and decanoicacids (Cl,J, were not as effective (Fig. 2C). Interestingly, oleylalcohol, which cannot be directly oxidized by the P-oxidationpathway, also was effective at increasing the steady state levelof the aP2mRNA (Fig. 2B).To better elucidate the nature of the mechanism of aP2mRNA induction by fatty acids, we examined the time courseof induction. Preadipocytes were treated with either BSAalone or 0.5 mM oleate for up to 48 h (Fig. 3). The firstdetectable change in theevel of aP2 mRNA occurred between5 and 10 h after the beginning of the treatment. Theevel ofaP2 mRNA was nearly maximal at 24 h and increased onlyslightly more at 48 h, when the experiment was terminated.Since it has been demonstrated that the transcriptionalactivation of aP2 occurs in theprocess of fat cell differentia-tion, a key question is whether the aP2 gene is specificallyregulated by fatty acid treatments or whether these agentsinduce differentiation, leading to theexpression of the entireprogram of adipocyte genes. We therefore examined the ef-fects of fatty acid on the expression of three other fat cellgenes induced during differentiation: adipsin, glycerophos-phate dehydrogenase, and C/EBP. Although very low levelsof glycerophosphate dehydrogenase and adipsin mRNAs couldbe detected during this treatment, there was no differencebetween the fatty acid treated and the control samples (Fig.4). In addition, C/EBP mRNA, which codes for a transcrip-tion factor tha t may contribute to the activation of the aP2gene in adipocytes (Christy et al., 1989; Herrera et al., 1989),is not induced by this treatment. Themall amount f glycero-phosphate dehydrogenase and adipsin mRNA, detected aftera long exposure of the Northern blots, presumably representsthe signal from a small number of cells that differentiatespontaneously in thesecultures. Thus, no overall effect of thefatty acids on the basic differentiation program could be seen;

a, 3 t / I

O T I0 10 2 0 30 40 5 0Hours

FIG.3. The time course of fatty acid induction of aP2mRNA. Preadip ocytes were treate d with 0.1 mM f atty acid free-BSA(0)r 0.6 mM o leat e comp lexed to 0.1 mM B SA (0) . otal RNA wascollected after 0, 2, 5, 10, 24, and 48 h from each trea tme nt andhybridized on Northern blots with radiolabeled aP2 cDNA. Autora-diographs of the North ern blots were scanned with an PharmaciaLK B Biotechnology Inc. laser densitometer.

Preadipocytedipocyte-2 hr 24 hr- + -aP 2

GPD

ADlPSlN

C/EBPFIG.4. Induction of other adipocyte specific genes by fattyacids. Northern blots of RNA from preadipocytes treated with 0.1mM fa tty acid -free BSA (-) or 0.6 mM oleate com plexe d to 0.1 mMBS A (+) for 10 and 24 h were hybridized with radiolabeled aP 2,glycerophosphate dehydrogenase ( G P D ) , dipsin (Spiegelman et al.,1983), or C/EBP (Xanthopoulos et al., 1989) cDNAs. A lane ofadipocyte RNA was hybridized to the C/EB P cDNA a s a positivecontrol for C/EB P hybridization.

C , C , , . I . 2 .4 . 6 .8 1.01.2 1.4 m M OleateFra1 F442a

m M OleateFra I I IH 3T3

FIG.5. Northern blot analysis of the induction of Fral byincreasing levels of oleate. Preadipocytes were treated with 0.1,0.2, 0.4, 0.6, 0.8, 1.0, 1.2, a nd 1.4 mM oleate com plexed to fatt y acid-free BSA in an oleate to BSA ra tio of 6:l. Con trol cells were treatedwith th e lowest and highest concentrations of fatty acid-free BS A0.016 mM (CJ an d 0.23 mM (Cr).All media containe d 10ng/ml insulin.Tot al RN A was isolated from these cells, and 15 pgof RNA washybridized on No rthern blots with the ra t Fral cDNA (Cohen andCurran, 1988).rather they appear to effect aP2 mRNA expression specifi-cally.In data not shown, we sought to determine whether induc-tion of aP2 mRNA by fatty acids was specific to adipocyteprecursor cells. We therefore treated another preadipocytecell line, 3T3-L1, with oleate and determined that these cellsalso could be induced to make aP2 mRNA in response to fattyacid treatment. Under the same conditions, two cell ines thatdo not undergo adipose differentiation, 3T3-C2 and NIH3T3fibroblasts, did not show any detectable induction of aP2.Interestingly, levels of fatty acid tolerated well by preadipo-cyte cell lines were lethal to nonpreadipocyte cells. Anotherdetermined cell line, myoblast clone B (a cell line capable ofdifferentiating into myotubes (Harrington et aL, 1988)) dem-onstrated no induction of aP2 mRNA after treatment withup to0.8 mM oleate. Thus, the bility of fatty acids to inducethe aP2gene may beimited to determined preadipocytes andis not seen in other fibroblastic cells.Fatty Acid Induction of Fral-Considering the possibilitythat the induction of the aP2 mRNA occurred through asignal transduction pathway involving the AP1 cis-actingelement of the aP2 gene, we examined the ability of longchain fatty acids to induce members of the nuclear transcrip-tion factor family capable of recognizing the AP1 site. Wefound a robust induction of the mRNA for the immediate-early gene Fos-related antigen (Fra l; see Fig. 5). Fral wasinduced at the owest dose (0.1 mM oleate) (compare lanes CIand . l ) and reached a maximum at 1.0 mM oleate. Althoughthe highest level of BSA had no effect on aP2 mRNA expres-


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5940 Fat ty Acid Regulation of Gene Expressionsion (Fig. 2A ) , a small amount of Fral mRNA induction wasobserved (lane Ch).The addition of oleate, however, induceda many-fold induction in Fra l mRNA over the control (1.4mM). Identical blots were probed with c-Fos, Jun C, and JunB without detecting a similar induction by fatty acid (datanot shown). In addition, when treated with oleate, NIH 3T3cells showed an increase in steady state levels of Fra l mRNA,although these cells didnot express aP2 mRNA when reatedwith fatty acids (Fig. 5).Role of Transcription in Fatty Acid-induced Gene Expres-sion-In order to determine whether and towhat extent theinduction of the Fral and aP2 genes by fatty acids was theresult of increased transcription, we performed nuclear run-on transcription experiments. Preadipocytes were treatedwith either fatty acid-free BSA or fatty acid-free BSA plus0.5 mM oleate for 5, 10, 15, and 20 h. Nuclei from these cellsand untreated adipocytes were isolated, and the amount ofinitiated mRNA transcription was measured for aP2, @-actin,@-tubulin, nd Fr al by filter hybridization. The control plas-mid pGem3, into which all these cDNAs were cloned, wasincluded. Although we have shown th at fatty acid treatmentof preadipocytes produced aP2 mRNA steady state levels ofup to 50%of the high level found in adipocytes, transcriptionof aP2 mRNA in treated as compared with untreated preadi-pocytes showed no detectable increase relative to the pGemcontrol plasmid (Fig. 6). The assay was clearly capable ofdetecting elevated rates of transcription, as adipocyte nucleishowed a greater than 15-fold increase in transcription overpreadipocytes, as previously reported (Cook etal.,1985; Bern-lohr et l. , 1985). Although noetectable increase in transcrip-tion of the aP2gene was ound after fattyacid treatment, thepreadipocyte nuclei actively transcribe both @-actin and p-tubulin genes. Fatty acid treatment caused no change in thetranscription of either of these genes. However, atty acids docause increased transcription.of the Fr al gene. Fr al transcrip-tion was reproducibly increased at each time point with a 4.5-fold induction at 5 and 20 h and2.5- and %fold inductions a t10 and 15 h. The average transcriptional induction of Fra lfrom these experiments was 3.6-fold.

DISCUSSIONFatty acids play a critical role in both normal metabolismand certain metabolic diseases. They serve as the primaryenergy source foreart and keletal muscle and also representthe major carrier of metabolic energy ha t is ultimately storedin adipose cells n periods of nutritional abundance. In fastedstates, fattyacids are exported from fat at an levated rate tospare glucose for he brain. This major glucose-utilizing issuecan also be adapted upon a prolonged fast to utilize ketonebodies derived from atty acids by @-oxidation n the iver. In

addition to these obviously beneficial unctions of fatty acids,5 10 15 20 hrr .+ - + - + - + A&em . -.AC I inaP 2TubulinF nFIG. 6. Nuclear transcription in preadipocytes treated withfatty acid. Nuclear run-on transcripts were isolated from equalnumbers of nuclei from preadipocytes treated with 0.083 mM fattyacid-free BSA (-) or fatty acid-free BSA plus 0.5 mM oleate (+) for5,10,15, and 20 h and untreated adipocytes ( l a n e A ) as describedunder Experimental Procedures. The radiolabeled transcripts were

hybridized to slot-blotted cDNAs for @-actin,aP2, @-tubulin(Bondet al., 1984),Fra l (Cohen and Curran,1988),and to Gem3 (PromegaCorp.), the vector into which all the insertswere cloned.

they also are an integral part of certain pathological condi-tions. In poorly treated diabetic states, fatty acids can reachextremely high levelsand cause significant medical problems.Liver oxidation of these excess fatty acids leads to elevatedlevels of circulating ketone bodies, a dangerous conditionknown as ketoacidosis. In addition, data has suggested thatelevated levels of fatty acids themselves (which frequentlyoccur in both obese and diabetic individuals) might play arole in the generation of a n insulin-resistant state (Grunfeldet al., 1981; Svedberg et al., 1990).Because fatty acid levels fluctuate in important metabolicand pathological conditions, it should be considered that fattyacids may serve as biological effectors, as well as metabolicsubstrates. In the present study, we have asked whether fattyacids can regulate gene expression in several fibroblast celllines, as well as in preadipocytes and adipocytes. It is clearfrom our data that such gene regulatory mechanisms exist inpreadipocytes; long chain fatty acids are able to induce thesteady state level of aP2 mRNA to 50% of the adipocyte levelwithin 24 h, and a econd, at least partly independent mech-anism, activates the Fral gene. In order to understand themechanism of aP2 induction, it is important to determinewhether treatment with fatty acid leads to initiation of aprogram of differentiation. Three genes activated during adi-pocyte differentiation, glycerophosphatedehydrogenase, adip-sin, and C/EBP, are not affected by fatty acids. Thus, fattyacid induction of aP2 mRNA is apparently independent ofthe differentiation-dependent activation of theaP2 gene;rather, it appears to represent a separate regulatory mecha-nism.The level a t which these cells are responsive to fattyacidsis within the range of circulating levels of fatty acid found inthe mouse and maywellbe at or below the local evelspreadipocytes would be exposed to during lipolysis. The ab-sence of an effect from shorter chain fatty acids reflects thespecificity of the response. Others have reported that sodiumbutyrate can activate aP2 gene expression. However, substan-tially higher levels are required (5.0 mM), and he effectappears to accompany the induction of differentiation (Tos-cani etal., 1990). Wealso asked whether oleyl alcohol, whichis not adirect substrate for acyl-CoA synthetase, was capableof inducing aP2. It was a t least as effective as oleate, suggest-ing that acyl-CoA or other metabolic intermediates of @-oxidation may not be the actual effector causing the induction.On the other hand, i t is known hat fibroblasts are capable ofoxidizing fatty alcohols (Rizzo et al., 1987). The precise fattyacid metabolite responsible for aP2 mRNA induction remainsto be determined.Although it has been reported that dexamethasone caninduce aP2 mRNA fibroblast cell lines (Toscani et al., 1988),the induction of aP2 by long chain fatty acids appears to bespecific to preadipocytes. We found no induction in otherfibroblast lines or committed cell lines. The induction of aP2by fatty acids is among the earliest markers for commitmentto adipocyte differentiation so far described.The data presented here suggest that long chain fatty acidsact to induce message accumulation by two different mecha-nisms in preadipocytes. aP2 mRNA induction in preadipo-cytes occurs primarily at the post-transcriptional level,whereas Fr al induction has a clear transcriptional compo-nent. Fatty acid treatment of preadipocytes increases thesteady state amount of aP2 mRNA to levels that approach50%of the high level seen in adipocytes. This occurs withoutany detectable increase in aP2 transcription. Since the in-creased transcription that accompanies adipocyte differentia-tion is very striking, i t is clear tha t any increase in transcrip-


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Fatty Acid ~ e g ~ ~ t ~ o nf GeneExpress~on 5941tion occurring in the aP2 gene due to fatty acid treatmentmust be small relative to induction of the mRNA. We there-fore conclude that a significant portion of the aP2 mRNAinduction in preadipocytes occurs at thepost-transcriptionallevel. These experiments cannot rule out thepossibility thata small increase in ranscription, below detectable levels,might contribute to the accumulation of aP2 mRNA. Thislack of detectable increase in transcription suggests that alarge increase in the steady state levels of the aP2mRNA islikely to be the result of post-transcriptional mechanisms,most likely message stabilization. On the other hand, longchain fatty acid treatment of preadipocytes produced an in-crease between 2- and &fold in Fral transcription during the20-h treatment. Unlike the aP2 gene, Fral mRNA inductionby fatty acids has a clear transcriptional component.The lack of significant transcriptional induction of the aP2gene does appear to preclude a direct role for Fra l as tran-scriptional trans-activator of the aP2 gene via its AP-1 site.However, it does not rule out a role for Fral in activating hemechanisms by which aP2 mRNA may be stabilized. Cellularregulation of Fral message stability is lso implied by the factthat its mRNA induction is also very much greater than theconcomitant increase in nuclear transcription. Other studiesusing protein synthesis inhibitors and growth factors havesuggested that Fral mRNA stability may be highly regulated(Cohen and Curran, 1988).The possibility that the specific induction of the aP2 geneby fatty acids occurs by altering mRNA stability rather thanby transcriptional regulation offers an obvious explanationfor the cell type specificity of the fatty acid effect. We observea low level of aP2 mRNA in preadipocytes but not in mostother fibroblastic cells, implying tha t the gene is transcribedat some low level in preadipocytes. If fatty acids induce aP2mRNA in part via mRNA stabilization, it is clear that onlycells that transcribe this gene could effectively undergo thefatty acid induction. The operative regulatory mechanisms offatty acid gene induction at the ranscriptional level for Fraland the post-transcriptional level fo r aP2 and Fral warrantfuture examination.

A c k n o ~ l e ~ m e n ~ s - W ehank R. Graves, P. Tontonoz, and G.Hotamisligil for their careful reading of the manuscript.NoteAdded n Proof-Amri et al. (Amri, E.-Z., Bertrand, B.,Ailhaud, G., and Grimaldi, P. (1991) . Lipid Res. 32, 1457-1463)

report similar findings for the ability of long chain fatty acids toinduce aP2 mRNA in the Ob1771 preadipocyte cell line.REF~RENCES

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Fatty Acid Regulation of Gene Expressionl