Fatty Acid Regulation of Gene Expression - The Journal of Biological ...

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

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Fatty Acid Regulationof Gene Expression TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL MECHANISMS* (Received for publication, September 2, 1991)

Robert J. DistelS, GregoryS. Robinsone, and BruceM. Spiegelmann From 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 and 1984). aP2l belongs to a large family of intracellular lipid may also be involved in pathological syndromes such carrier proteins that includes liver, intestine, kidney, and as the insulin resistance of diabetes and obesity. We heart fatty acid-binding proteins, as well as myelin P2, and demonstrate here thatfatty acids can regulate specific the cellular retinol- and retinoic acid-binding proteins. The gene expression; mRNAs encoding thefatty acid bind- precise function of aP2 is not known, although it is believed to play a role in fatty acid transport or protection against the ing proteinadipocyte P2 (aP2) andtheFos-related transcription factor Fral are specifically induced at detergent-like effects of fatty acids (see Matarese and Bernleast 20-fold upon treatmentofpreadipocytes with lohr (1989) for review). The adipocyte P2 gene has served as a model for differenoleate. For aP2, the effect requires long chain fatty acids and occurs without a generalized activation of tiation-dependent gene expression in this cell type. The gene the genes linked to adipocyte differentiation. Other is transcriptionally activated during 3T3-adipocyte differenfibroblastic cells without preadipocyte characteristics tiation (Cook et al.,1985; Bernlohr et al., 1985). Several cisdo not induce aP2 mRNA in response to fatty acids. and trans-actingregulatory components of the gene have been Unlike aP2, Fral induction by fatty acids also can be identified, including an AP-1 sequence at -120,where we detected in NIH 3T3 and 3T3-C2 fibroblasts. Nuclear first identified sequence-specific interactions between Fostranscription assays in 3T3-F442A preadipocytes containing protein complexes and DNA (Distel et al., 1987; demonstrate that fatty acids elicit no transcriptional Rauscher et al., 1988). In addition, a binding site for the CAAT/enhancer-binding protein (C/EBP) located 140 base increase in the aP2 gene. Fral, ontheotherhand, shows a 3-4-fold increase in transcription. Thesere- pairs upstream of the start of transcription, has been desults demonstrate at least two distinct mechanisms by scribed (Christy et al., 1989; Herrera et al., 1989). The differentiation-dependence and tissue specificity of the aP2 gene which fatty acids may influence gene expression. and its relatively high level of expression appear to be determined by a potent fat-specific enhancer located 5.4 kilobases upstream of the startof transcription, which functions in both Adipose cells are the major depot for energy storage in cultured adipocytes and transgenic mice (Ross et al., 1990; vertebrate animals. Much or most of the lipid in these cells is Graves et al., 1991).A binding site for a member of the nuclear factor 1 family and several other nuclear factors play an derived from the uptake of free fatty acids released by the important role in this enhancer (Graves et al., 1991).’ hydrolysis of circulating triglyceride-rich lipoproteins or from Since adipocytes and preadipocytes are exposed to changing circulating fatty acid bound to serum albumin. The export of levels of fatty acids under various normal and pathological energy from fat cells during periods of nutritional deprivation metabolic states, we have specifically investigated whether also involves fatty acids; these arehydrolyzed from the cellular these substratesfor the aP2protein might regulate expression triacylglyceride droplet and released into the circulatory sys- of the aP2 gene. We have also examined the effects of fatty tem. acids on the transcription factorsthat may modulate aP2 gene Given the centralrole that fattyacids play in adipose tissue expression. These include C/EBP(Herrera et al., 1989; biology andthe known detergent-likeproperties of these Christy et al., 1989) and members of the Fos and Jun tranamphipathic molecules, it is not surprising thatfat cells scription factor family. We show here that fatty acids dracontain a specific fatty acid-binding protein. This molecule, matically activate the expression of mRNA for aP2 and the termed adipocyte P2, was cloned from adipocyte cDNA li- Fos-related gene Fral in preadipocytes, without initiating the braries and is highly abundant and relatively fat cell-specific general program of adipocyte differentiation. However, these in its expression (Spiegelman et al., 1983; Bernlohr et al., inductions appear to utilize different mechanisms, both transcriptional and post-transcriptional. * This work was supported by Grant DK31405 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by a fellowship from the Charles King Trust. I Supported by National Research Service Award Fellowship GM13033. 1 Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dana-Farber Cancer Institute, Mayer 813, 44 Binney St., Boston, MA 02115. Tel.: 617-7323567: Fax: 617-735-8971.

EXPERIMENTALPROCEDURES

Cell Culture-3T3-F442A cells were grown in Dulbecco’s modified essential medium with 10% calf serum and supplemented with glutamine 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-aminoethanesulfonic acid). R. A. Graves, P. Tontonoz, and B. M. Spiegelman, manuscript in preparation.

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Fatty Acid Regulation of Gene Expression

Cells were allowed to differentiate as described previously (Cook et al., 1985). In all experiments,sodium salts of fatty acids (Sigma) were added in a 6 1 molar ratio of fatty acid to fattyacid-free bovine serum albumin (Sigma). For the fatty acid and control treatments of cells, 10 ng/ml insulin was included to prevent spontaneous lipolysis from interfering with the assessment of fatty acid effects. Northern Blots-RNA isolation was performed by the acidified guanidinethiocyanate method as described by Chomczynski and Sacchi (1987). The RNA was size-fractionated on 2.2 M formaldehyde agarose gels and transferred to Biotrans (Pall BioSupport Group) nylon membrane filters.The RNA was photocross-linked and hybridized as described by Virca et al. (1990). Probes were labeled by the random prime technique using [a-""P]dCTP. Blots were washed in 0.5 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 X SSC at 65 "C. Nuclear Transcription-Nuclei were pooled from five 10-cm dishes of confluent preadipocytes or adipocytes as described by Greenberg and Ziff (1984). Transcription reactions were performed as described by Greenberg and Ziff (1984). Reactions were initiated by adding an equal volume of 2 X reaction buffer (10 mM Tris-HCI, pH 8.0, 5.0 mM MgCl,, 300 mM KCl, 5.0 mM dithiothreitol, 1.0 mM ATP, 1.0 mM GTP, 1.0 mM CTP, and 200 pCi [oc-"'P]UTP (800 Ci/mmol) (Du Pont-New England Nuclear) to 2 X lo7 nuclei (in about 150 pl). The reaction was carried out for 30 min at 30 "C with agitation every 5 min.RNase-freeDNase was added at 20 pg/ml, the nuclei were incubated for 10 min at 30 "C, and this reaction was terminated with 100 pg of yeast tRNA and 10 volumes (about 3 ml) of 4 M guanidine thiocyanate, 25 mM trisodium citrate, pH 7.0, 0.5% sodium sarcosyl, and 0.1 mM 0-mercaptoethanol. The RNA was then isolated as described by Chomczynski and Sacchi (1987), except that the final RNA pellet was washed three times with 70% ethanol, resuspended in 100 p1 of distilled water, and precipitated in 5% trichloroacetic acid, 10 mM sodium pyrophosphate, and the total incorporated counts determined. The RNA was resuspended in 100 pl of 10 mM Tes, pH 7.4. Ten pg of denatured plasmid DNA was slot-blotted onto nitrocellulose, according to the manufacturer (Schliecher & Schuell). The slot blots were prehybridized for 12 h at 65 "C in 10 mM Tes, pH 7.4, 300 mM NaCl, 10 mM EDTA, 50 mM sodium pyrophosphate, 2 X Denhardt's solution (50 X Denhardt's: 10 mg/ml Ficoll, 10 mg/ml polyvinylpyrrolidone, and 10 mg/ml BSA), and 0.2% sodium dodecyl sulfate. Hybridization was in 1 mlof 10 mM Tes, pH 7.4, 300 mM NaCI, 10 mM EDTA, 0.2% sodium dodecyl sulfate, and 10 X loficpm of radiolabeled RNA for 48 h at 65 "C. The blots were washed for 2 h at 55 "C in 2 X SSC and then treated with 10 pg/ml DNase-free RNase A in 2 X SSC at 37 "C and washed for 1 h in 2 X SSC a t 37 "C. Hybridization was quantitated using Phosphor-Imager (Molecular Dynamics).

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FIG. 1. Northern blot analysis of fatty acid induction of the aP2 gene. 3T3-F442A preadipocytes or adipocytes were treated for 24 h with Dulbecco's modified essential medium, 10 ng/ml insulin, and 0.16 mM fatty acid-free BSA with (+) or without (-) the addition of 1.0 mM oleate. Total RNA was isolated from these cells, and 15 p g of total RNA was hybridized on Northern blots with both 0-actin and aP2 cDNAs (Spiegelman et al., 1983). The preadipocyte lanes were exposed longer than the adipocyte ones to detect an aP2signal in the untreated cells.

AP2

aP2

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FIG. 2. Northern blot analysis of the aP2 mRNA response to long and short chain fatty acids. A, Northern blot analysis of the aP2 mRNA response to increasing doses of oleate. Preadipocytes were treated with 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mM oleate complexed to fatty acid-free BSA in an oleate to BSA ratio of 6:l. Controls cells were treated with the lowest and highest concentrations RESULTS of fatty acid-free BSA: 0.016 mM (CJ and 0.23 mM (Ch).B, Northern Long Chain Fatty Acids Can Induce aP2 Gene Expression blot analysis of aP2 mRNA levels in preadipocytes treated with in Preadipocytes-Since the adipocyte P2 protein appears to various long chain fatty acid and a long chain fatty alcohol. Preadiwere treated for 24 h with 0.05 mM fatty acid-free BSA alone be the major carrier of long chain fatty acids in adipocytes, pocytes ( A ) , 0.3 mMof the sodium salts of stearic acid (B), oleic acid (C), we asked whether the levels of exogenous fatty acid could palmitic acid (D), palmitoleic acid ( E ) ,and oleyl alcohol ( F ) commodulate the level of aP2 mRNA. 3T3-F442A adipocytes were plexed with fatty acid-free BSA in Dulbecco's modified essential treated with mediacontaining either1.0 mM oleate complexed medium. C, aP2 mRNA levels in preadipocytes treated with short t o fatty acid-free BSA (6:l) or BSA alone. After 24 h, North- and medium chain length fatty acids. Preadipocytes were treated with ern blotanalysis of the RNA from adipocytes treated with1.0 0.05 mM fatty acid-free BSA ( A ) , 0.3 mM sodium butyrate (B), 0.3 mM sodium decanoate (C), and 0.3 mM oleate ( D )in Dulbecco's mM oleate showed little or no inductionover the already high modified essential medium. All media contained 10 ng/ml insulin. level of aP2 mRNA in these cells (Fig. 1).We observed very Total RNA was isolated from these cells, and 15 p g of RNA was little change in the mRNA level over a broad range of oleate hybridized on Northern blots with the aP2 cDNA. Each experiment concentrations (0.5-3.0mM, not shown). The RNA was si- was repeated with similar results.

multaneously probed withmouse b-actin as a control for both efficiency of loading and RNA transfer. In contrast, when oleate typically reached between 25 and 50% of the message preadipocytes were treatedwith 1.0 mM oleate, theaP2 level found in untreated adipocytes. It is therefore clear that mRNA was induced 20-fold over cells treated with BSA alone a very significant level of aP2 mRNA can be induced in of the preadipocytes by oleate. (Fig. 1).Note thatin order to determine the magnitude accumulation of aP2 mRNA in the fatty acid-treated preadi- Fatty acids are typically found in fasting or fed mice at pocytes over that of the controlpreadipocytes, the RNA blots plasma levels in a range between 0.1 and 1.2 mM (Baker et were exposed long enough to obtain a signal for aP2 mRNA al., 1978). To investigate whether the response of the aP2 in the control lane (Fig. 1).Thus, the @-actinsignal appears mRNA to fatty acids occurred in a physiological range, premuch higher in preadipocytes than adipocytes. The induction adipocytes were treated with 0.1-1.4 mM oleate for 24 h (Fig. of aP2 mRNA in preadipocytes after a 24-h treatment with 2 A ) . The addition of fatty acid-free BSA alone at the lowest

Fatty Acid Regulation of Gene Expression (0.016mM) and the highest (0.23mM) concentrations used had no effect on the levels of aP2 mRNA, as demonstrated in lanes Cr and Ch. RNA isolated from the oleate-treated cells showed a %fold increase in the level of aP2 mRNA at a concentration of 0.1 mM oleate. The maximum response after 24 h occurred at doses between 0.6 and 0.8 mM. Higher doses of up to 1.4 mM oleate gave reduced levels of aP2 message, although these levels of fatty acid did not have an obvious deleterious effect on the cells. Levels greater than 2.5 mM routinely killed preadipocytes. A similar dose response curve was obtained with linoleate (C,,,,) (data not shown). Other long chain fatty acids, such as palmitate (C,,,,), palmitoleate (C,,,,), and stearate (Cls:0), also were capable of causing induction of theaP2 mRNA (Fig. 2B), whereas shortand medium chain length fatty acids, butyric (C,) and decanoic acids (Cl,J, were not as effective (Fig. 2C).Interestingly, oleyl alcohol, which cannot be directly oxidized by the P-oxidation pathway, also was effective at increasing the steady state level of the aP2mRNA (Fig. 2B). To better elucidate the nature of the mechanism of aP2 mRNA induction by fatty acids, we examined the time course of induction. Preadipocytes were treated with either BSA alone or 0.5 mM oleate for up to 48 h (Fig. 3). The first detectable change in thelevel of aP2 mRNA occurred between 5 and 10 h after the beginning of the treatment. Thelevel of aP2 mRNA was nearly maximal at 24 h and increased only slightly more at 48 h, when the experiment was terminated. Since it has been demonstrated that the transcriptional activation of aP2 occurs in theprocess of fat cell differentiation, a key question is whether the aP2 gene is specifically regulated by fatty acid treatments or whether these agents induce differentiation, leading to theexpression of the entire program of adipocyte genes. We therefore examined the effects of fatty acid on the expression of three other fat cell genes induced during differentiation: adipsin, glycerophosphate dehydrogenase, and C/EBP. Although very low levels of glycerophosphate dehydrogenase and adipsin mRNAs could be detected during this treatment, there was no difference between the fatty acid treated and the control samples (Fig. 4). In addition, C/EBP mRNA, which codes for a transcription factor that may contribute to the activation of the aP2 gene in adipocytes (Christy et al., 1989; Herrera et al., 1989), is not induced by this treatment. The small amountof glycerophosphate dehydrogenase and adipsin mRNA, detected after a long exposure of the Northern blots, presumably represents the signal from a small number of cells that differentiate spontaneously in thesecultures. Thus, no overall effect of the fatty acids on the basic differentiation program could be seen;

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FIG. 3. The time course of fatty acid induction of aP2 mRNA. Preadipocytes were treated with 0.1 mM fatty acid free-BSA (0) or 0.6 mM oleate complexed to 0.1 mM BSA (0).Total RNA was collected after 0, 2, 5, 10, 24, and 48 h from each treatment and hybridized on Northern blots with radiolabeled aP2 cDNA. Autoradiographs of the Northern blots were scanned with an Pharmacia LKB Biotechnology Inc. laser densitometer.

5939 Preadipocyte hdipocyte I 2 hr 2 4 hr

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GPD ADlPSlN

C/EBP

FIG. 4. Induction of other adipocyte specific genes by fatty acids. Northern blots of RNA from preadipocytes treated with 0.1 mM fatty acid-free BSA (-) or 0.6 mM oleate complexed to 0.1 mM BSA (+) for 10 and 24 h were hybridized with radiolabeled aP2, glycerophosphate dehydrogenase ( G P D ) ,adipsin (Spiegelman et al., 1983), or C/EBP (Xanthopoulos et al., 1989) cDNAs. A lane of adipocyte RNA was hybridized to the C/EBP cDNA as a positive control for C/EBP hybridization. C, C,, . I . 2 .4 .6 .8 1.01.2 1.4 Fra 1

m M Oleate F442a

m M Oleate Fra I

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FIG. 5. Northern blot analysis of the induction of Fral by increasing levels of oleate. Preadipocytes were treated with 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mM oleate complexed to fatty acidfree BSA in an oleate to BSA ratio of 6:l. Control cells were treated with the lowest and highest concentrations of fatty acid-free BSA 0.016 mM (CJ and 0.23 mM (Cr).All media contained 10 ng/ml insulin. Total RNA was isolated from these cells, and 15 pgof RNA was hybridized on Northern blots with the rat Fral cDNA (Cohen and Curran, 1988).

rather they appear to effect aP2 mRNA expression specifically. In data not shown, we sought to determine whether induction of aP2 mRNA by fatty acids was specific to adipocyte precursor cells.We therefore treated another preadipocyte cell line, 3T3-L1, with oleate and determined that these cells also could be induced to make aP2 mRNA in response to fatty acid treatment. Under the same conditions, two celllines that do not undergo adipose differentiation, 3T3-C2 and NIH3T3 fibroblasts, did not show any detectable induction of aP2. Interestingly, levels of fatty acid tolerated well by preadipocyte cell lines were lethal to nonpreadipocyte cells. Another determined cell line, myoblast clone B (a cell line capable of differentiating into myotubes (Harrington et aL, 1988)) demonstrated no induction of aP2 mRNA after treatment with up to0.8 mM oleate. Thus, theability of fatty acids to induce the aP2gene may belimited to determined preadipocytes and is not seen in other fibroblastic cells. Fatty Acid Induction of Fral-Considering the possibility that the induction of the aP2 mRNA occurred through a signal transduction pathway involving the AP1 cis-acting element of the aP2 gene, we examined the ability of long chain fatty acids to induce members of the nuclear transcription factor family capable of recognizing the AP1 site. We found a robust induction of the mRNA for the immediateearly gene Fos-related antigen (Fral; see Fig. 5). Fral was induced at thelowest dose (0.1 mM oleate) (compare lanes CI and . l ) and reached a maximum at 1.0 mM oleate. Although the highest level of BSA had no effect on aP2 mRNA expres-

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Fatty Acid Regulation of Gene Expression

sion (Fig. 2A), a small amount of Fral mRNA induction was observed (lane Ch).The addition of oleate, however, induced a many-fold induction in Fral mRNA over the control (1.4 mM). Identical blots were probed with c-Fos, Jun C, and Jun B without detecting a similar induction by fatty acid (data not shown). In addition, when treated with oleate, NIH 3T3 cells showed an increase in steady state levels of Fral mRNA, although these cells didnot express aP2 mRNA whentreated with fatty acids (Fig. 5). Role of Transcription in Fatty Acid-induced Gene Expression-In order to determine whether and towhat extent the induction of the Fral and aP2 genes by fatty acids was the result of increased transcription, we performed nuclear runon transcription experiments. Preadipocytes were treated with either fatty acid-free BSA or fatty acid-free BSA plus 0.5 mM oleate for 5, 10, 15, and 20 h. Nuclei from these cells and untreated adipocytes were isolated, and the amount of initiated mRNA transcription was measured for aP2, @-actin, @-tubulin,and Fral by filter hybridization. The control plasmid pGem3, into which all these cDNAs were cloned, was included. Although we have shown that fatty acid treatment of preadipocytes produced aP2 mRNA steady state levels of up to 50%of the high levelfound in adipocytes, transcription of aP2 mRNA in treated as compared with untreated preadipocytes showed no detectable increase relative to the pGem control plasmid (Fig. 6). The assay was clearly capable of detecting elevated rates of transcription, as adipocyte nuclei showed a greater than 15-fold increase in transcription over preadipocytes,as previously reported (Cook et al., 1985;Bernlohr etal., 1985). Although no detectable increase in transcription of the aP2gene wasfound after fattyacid treatment, the preadipocyte nuclei actively transcribe both @-actinand ptubulin genes. Fatty acid treatment caused no change in the transcription of either of these genes. However,fatty acids do cause increased transcription.of the Fral gene. Fral transcription was reproducibly increased at each time point with a 4.5fold induction at 5 and 20 h and2.5- and %fold inductions at 10 and 15 h. The average transcriptional induction of Fral from these experiments was 3.6-fold.

they also are an integral part of certain pathological conditions. In poorly treated diabetic states, fatty acids can reach extremely high levelsand cause significant medical problems. Liver oxidation of these excess fatty acids leads to elevated levels of circulating ketone bodies, a dangerous condition known as ketoacidosis. In addition, data has suggested that elevated levels of fatty acids themselves (which frequently occur in both obese and diabetic individuals) might play a role in the generation of an insulin-resistant state (Grunfeld et al., 1981; Svedberg et al., 1990). Because fatty acid levels fluctuate in important metabolic and pathological conditions, it should be considered that fatty acids may serve as biological effectors, as well as metabolic substrates. In the present study, we have asked whether fatty acids can regulate gene expression in several fibroblast cell lines, as well as in preadipocytes and adipocytes. It is clear from our data that such gene regulatory mechanisms exist in preadipocytes; long chain fatty acids are able to induce the steady state level of aP2 mRNA to 50% of the adipocyte level within 24 h, and asecond, at least partly independent mechanism, activates the Fral gene. In order to understand the mechanism of aP2 induction, it is important to determine whether treatment with fatty acid leads to initiation of a program of differentiation. Three genes activated during adipocyte differentiation, glycerophosphatedehydrogenase, adipsin, and C/EBP, are not affected by fatty acids. Thus, fatty acid induction of aP2 mRNA is apparently independent of the differentiation-dependent activation of theaP2 gene; rather, it appears to represent a separate regulatory mechanism. The level at which these cells are responsive to fatty acids is within the range of circulating levels of fatty acid found in the mouse and maywellbe at or below the locallevels preadipocytes would be exposed to during lipolysis. The absence of an effect from shorter chain fatty acids reflects the specificity of the response. Others have reported that sodium butyrate can activate aP2 gene expression. However, substantially higher levels are required (5.0 mM), and the effect appears to accompany the induction of differentiation (Toscani etal., 1990). Wealso asked whether oleyl alcohol, which DISCUSSION is not adirect substrate for acyl-CoA synthetase, was capable Fatty acids play a critical role in both normal metabolism of inducing aP2. It was at least as effective as oleate, suggestand certain metabolic diseases. They serve as the primary ing that acyl-CoA or other metabolic intermediates of @energy source for heart andskeletal muscle and also represent oxidation may not be the actual effector causing the induction. the major carrier of metabolic energythat is ultimately stored On the other hand, it is knownthat fibroblasts are capable of in adipose cellsin periods of nutritional abundance. In fasted oxidizing fatty alcohols (Rizzo et al., 1987). The precise fatty states, fattyacids are exported from fat at anelevated rate to acid metabolite responsible for aP2 mRNA induction remains spare glucose forthe brain. This major glucose-utilizingtissue to be determined. can also be adapted upon a prolonged fast to utilize ketone Although it has been reported that dexamethasone can bodies derived fromfatty acids by @-oxidationin the liver. In induce aP2 mRNA fibroblast cell lines (Toscani et al., 1988), addition to these obviously beneficialfunctions of fatty acids, the induction of aP2 by long chain fatty acids appears to be specific to preadipocytes. We found no induction in other 5 10 15 20 hrr. fibroblast lines or committed cell lines.The induction of aP2 + + + + A .“ -. by fatty acids is among the earliest markers for commitment &em ACI in to adipocyte differentiation so far described. aP 2 The data presented here suggest that long chain fatty acids Tubulin act to induce message accumulation by two different mechaF nI nisms in preadipocytes. aP2 mRNA induction in preadipoFIG. 6. Nuclear transcription in preadipocytes treated with cytes occurs primarily at the post-transcriptional level, fatty acid. Nuclear run-on transcripts were isolated from equal whereas Fral induction has a clear transcriptional componumbers of nuclei from preadipocytes treated with 0.083 mM fatty nent. Fatty acid treatment of preadipocytes increases the acid-free BSA (-) or fatty acid-free BSA plus 0.5 mM oleate (+) for steady state amount of aP2 mRNA to levels that approach 5, 10, 15, and 20 h and untreated adipocytes ( l a n e A ) as described 50% of the high level seen in adipocytes. This occurs without under “Experimental Procedures.” The radiolabeled transcripts were hybridized to slot-blotted cDNAs for @-actin,aP2, @-tubulin(Bond any detectable increase in aP2 transcription. Since the inet al., 1984),Fral (Cohen and Curran,1988),and topGem3 (Promega creased transcription that accompanies adipocyte differentiation is verystriking, it is clear that any increase in transcripCorp.), the vector into which all the insertswere cloned. ”

Fatty Acid ~ e g ~ ~of tGene ~ oExpress~on n tion occurring in the aP2 gene due to fatty acid treatment must be small relative to induction of the mRNA. We therefore conclude that a significant portion of the aP2 mRNA induction in preadipocytes occurs at thepost-transcriptional level. These experiments cannot rule out thepossibility that a small increase intranscription, below detectable levels, might contribute to the accumulation of aP2 mRNA. This lack of detectable increase in transcription suggests that a large increase in the steady state levels of the aP2 mRNA is likely to be the result of post-transcriptional mechanisms, most likely message stabilization. On the other hand, long chain fatty acid treatment of preadipocytes produced an increase between 2- and &fold in Fral transcription during the 20-h treatment. Unlike the aP2 gene, Fral mRNA induction by fatty acids has a clear transcriptional component. The lack of significant transcriptional induction of the aP2 gene does appear to preclude a direct role for Fral as transcriptional trans-activator of the aP2 gene via its AP-1 site. However, it does not rule out a role for Fral in activating the mechanisms by which aP2 mRNA may be stabilized. Cellular regulation of Fral message stability isalso implied by the fact that its mRNA induction is also very much greater than the concomitant increase in nuclear transcription. Other studies using protein synthesis inhibitors and growth factors have suggested that Fral mRNA stability may be highly regulated (Cohen and Curran, 1988). The possibility that the specific induction of the aP2 gene by fatty acids occurs by altering mRNA stability rather than by transcriptional regulation offers an obvious explanation for the cell type specificity of the fatty acid effect. We observe a low level of aP2 mRNA in preadipocytes but not in most other fibroblastic cells, implying that the gene is transcribed at some low level in preadipocytes. If fatty acids induce aP2 mRNA in part via mRNA stabilization, it is clear that only cells that transcribe this gene could effectively undergo the fatty acid induction. The operative regulatory mechanisms of fatty acid gene induction at thetranscriptional level for Fral and the post-transcriptional level for aP2 and Fral warrant future examination.

5941

report similar findings for the ability of long chain fatty acids to induce aP2 mRNA in the Ob1771 preadipocyte cell line.

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