Regulation of gene expression by fatty acids: Special reference to fatty acid-binding protein (FABP)

Bi~chimie ¢t1997) 79. 12t.L133 0 SociOl6 fran~'aisc de biochimie d biologic mol,Sculaire / Eb,evier. Paris

Regu|atbn of gene e×pressbn by fatty acids: Special reference to fatty ac a-Dmam , protein FABP) I Niot, H Pokier, Ph B e s n a r d * Laboratoire de Physioh~gie de h, Nmrition, l='cole Nationale Supdrieur; de Biologic At)pliqu~;e h la Nutrition et ~) I 'Alimcntaliun {ENSBANA ), I. e.whmade Erasme, Universit6 de Bourgogne, 21000 Dijon. France

d,leceived 7 October 1996: accepted 3t) November 1996) S~t|l'nulla|'y ..... During lhc lasl years, the direct hlvolven)el|l of lipidic nutrients in the regulation of genes has been established. Fany acid,, may induce or repress the transcription rate of several genes iuw)lved h) both lipid and carbohydrate metabolisms. Gene np-regulatitm has been fuund in various tissues including liver, adipose tissue and small intestine, it is only triggered by saturated and unsaturated Ion~,-chain fatty acids or their CoA-derivatives. In contrast, gene dowmregulatiou appears It) be restricted to the liver. This ne~.ati~e effect is excrlcd only by polyunsaturated laity acids. Long-chain fatty acids are able to regulate the expression of two different genes oppositely in the same cell type. The molecular mechanism of these fairy acid-ulediated effects rermtins unclear. The involvement of members of the pCl'OXi%Olllt" proliferator-activated receptor is discussed.

fatty acids / gene regulation / lipid and e'.lrbobydrate metabolisms different organs including small intestine, liver and adipose tissue where they are essentially involved in lipid and carbohydratc metabolisms.

|ntroduction Fatty acids (FAs) play an important cellular role as energy substrates, membrane components and precursors of lipid mediators. They are also inw)lved in the permeability of ionic clmnnels, in synaptic transmission alld signal trailsduction pathway Ii, 21. During the recent years, it has become increasingly clear that FAs can likewise exert critical regulatory functions in the cell through direct acliwllion or inhibition of genes 13, 41. These I:Aqegul;.ilt~ry effects have a transcriptiomd origin anti arc fully reversible after effector removal, FA-target genes have been d~'scribed in

*Correspondence

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reprints

Abbreviations: FA, fatty acid; LCFA, long-chain fatty acid; PUFA.

polyunsaturated fatty acid; LPL, lipoprotein lipase (EC 3. I. 1.34); FAT, fatty acid transporter; ALBP, adipocyte lipid-binding protein; L-FABP, liver fatty acid-binding protein; I-FABE intestinal fatty acid-binding protein; ACS, acyI-CoA synthetase (EC 6.2.1.3); A c e , acyI-CoA oxi6ase (EC 1.3.3.6); CYP 450, cytochromc P4501VAI (9035-51-2); CPT-I, carnitine palmitoyl transferase I (EC 2.3.1.21 ); PEPCK, I~hosphoen()l!~yruwtle carboxykina.~c (EC 4.1.1.32); HMG-CoA synthetase, 3-hydroxy-3-methylglutarylCoA synthetase (EC 4.1,3.5); MCAD. mediun)-ch,'tin acyI-CoA dehydrogenase (EC 1.31; Ape A-I. apoprotein A-I: FAS, fany acid synthase (EC 6); L-PK. L-pyruvate kinase (EC 2.1.7.40); S 14, spot 14; SCD- I, slearoyI-CoA desalurase (EC I. 14.99.5); GK, glucokinase (EC 2.7.1.2); PPAR, peroxisome proliferator-actiwlted receptor; FAAR, fatty acid-activated receptor; RXR, retinoid X receptor: PPRE. peroxisonre proiiferator responsive element.

Genes up-regulated by LCFAs Long-clmin t'atty acids tI.CFA~) can dirediy inL're;tse. In1 association with hOl'illOll~2S,geliC expression in response to change in dietary lipid compositiorl. A recent example el a FA-induced gene is provided by a pn't~tcin whidl facilitates cellular uptake and cytopht.smi¢ l~,ansp~lt of LCFA~ in both liver and gut: the liver fitlty acid-binding protein (LoFABP), In the small intestine, L-FABP mRNA level is specifically increased in mice chronically fed a diet slightly enriched in sunflower oil (fig I A). In this species, the t.,xpress[ol~ of L-FABP gene, which is apl)arently silent in the dislal i[eunl, can be switched on when an adequate inducer is directly infused in tile ileal lumen [5]. Using this highly sensitive model, we demonstrated by nuclear run-on assay that sun° flower oil infusion increases the transcriptional rate of I.FABP gene in contrast to intestinal FABP (I-FABP), which is also expressed in the gttt (fig I B). The up-regulation triggered by FAs is also fimnd when lhtoleic acid (C18:2. n-BL the main LCFA found in this oil, is either directly added in the diet (fig 2). L-FABP induction occurs with saturated and unsaturated LCFAs, while mediurn-chain FAs are ineffeclive, Interestingly. a non-metabolizable FA, ~-bromol~almi o tale. appears to be a strong inducer. Since it may bc activated by CoA, the induction of the L-FABP gene may occur either with LCFAs or tlaeir CoA-derivatives (Poirier

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FIR I, Et't'¢cll of sunflower oil adminisllrallion on L-FABP mRNA levels (A| and relative transcription rate of L-FABP gene (B) m the small intestine. A, Mi~¢ were receiving by gastric garage it daily dose of 0,2 mL of sunllower oil for 7 days (grey bar). Conllro!s truly received ()~2 mL of 0,9% NaCI (black bar) *% P < 0,0!, n = 3, B, Nuclei were isolated I~om i!e:d mu¢osa !6 h lifter inlusion with 0.2 mL mineral oil (black !x~r,cot~llrol) or sunflower oil llgrey bar} and were subsequently tlsed in tnmscripdtm run-on assays. After normalization to ~-actin, the lran~l'ipllion r~.tl¢ of L~FABP g¢11¢ was expressed relative to !~FABP della i'~ond in the conlrt~l experimenll. The graph is derived l'rol|l three hldol~0adenll rUlbOli assays, ud. undcllt:¢lahle (from IS] with tile tlerllU~,niOll ~.~lIhe/='l!l~] Bi,~cheml,

¢,t eL submitted), The FA stimutatory effect on L~FABP cxo

pres~ioa is gen¢-s~cific si~¢ bFABP and ~-actin mRNA levels are u~hanged, An hormonailyomediatedeffect is uno likely, Indeed, LCFAs are able to induce L-FABP expression in the enterocyte-like Caco-2 cells cultured in return-free medium (Pokier et al, submitted), Similar regulation is f o u ~ both in rat hepatoma Fao cells and in cultured rat hcpatocytes suggesting that the L-FABP gene is also upregulatcd by LCFA in liver 161, This induction is tranmripiional and requires a de ~llm,o protein synthesis 161. To date, at least seven other genes [3, 7-131 are known to be transcriptionally up-regulated by LCFAs through nc~trhy regulatory m~hanisms than tho~ found for the LFABP gea¢ (t~ble IL The main differences arc found for the phosphoenolpyruvate carboxykinase (PEI~K) gone which is only stimulated by unsaturated FAs through a process intkpemJent of pa~tcin neosynthcsis [12, 131, At leaM three other genes, the cytochrome P450IVAI {CYP 450) 1141, the medium-chain acybCoA deshydroge-

aase (MCAD) 1151 and the 3-hydmxy-3~mcthylglutarylCoA synthetase'(HMG CoA synthetase) [ !6l, encoding fi)r enzymes involved resl~ctively in microsomal ~oxidation, mittx~hondrial l~-oxidation and in ketogenesis, are also putative FA target genes. HoweveL the transcriptional activation of native genes is still uncertain.

Down-regulation of genes by PUFAs FAs can also potentiate gene inhibition. Indeed, using a combination of in ll,ivo and in vitro studies, it has been demonstrated by nuclear run=on assay that at least four genes [4, IO, 17. 18J are tnmscriptionally down-regulated by FAs (table II), This repressive effect does not require peripheral metabolism of FAs nor a FA-mediated release of hormones since it was also tbund in cells cultured in serum-free medium, In contrast to FA-mediated up-regulation, the FA-repression is strictly restricted to PUFAs. For instance, fatty

131

acid synthase (FAS) and spo~ 14 (St4) expression in fiver are dramatically decreased only in the presence of PL]FAs containing 18 carbons and over, two double bonds in the 9 and 12 positions 1191. Moreover, this down-regulati~)n is dependent on the desaturaiion of 1Socarhon FA by the delta6 desaturase but is not mediated by a prostaglandin or thromboxane metabolites [19]. In general, C18:3, n-3 is more potent than C18:2, n-6 and the products of de|ta-6 desaturation are 2--4 times more potent suppressors than their respective FA precursors, Liver seems to be the target tissue of this down-regulation. Indeed, in rodents, PUFAs inhibit transcriptionally the lipogenic enzymes FAS and S14 in liver but have no effect in adipose tissue, lung, brain and small intestine [20, 21]. It is noteworthy that several other genes e.~coding proreins involved in both carbohydrate metabolism, as glucokinase and L-pyruvate kinase 122 I, and lipid metabolism, as acetyI-CoA carboxylase and malic enzyme 1171, are also down-legulated by PUFAs both itz vivo and i , vitro. However, to our knowledge, the transcriptional origin of this control is not yet determined by nuclear run-on analysis.

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Molecular mechanisms involved in the FA regulation of gene expression LCFAs can regulate oppositely the expression o1' two genes in a same cell. Far instance, L-FABP's mRNA level is strongly increased by linoleic acid (C 18:2, n-6) in FAO cells while FAS mRNA levels are decreased 161. Similarly, eicosapentaenoic acid (C20:5, n-3) decreases apoprotein A-I (ApoA-l) mRNA to approximately 50% of control levels, but induces signifio cantly acyI-CoA oxidase (ACO) mRNA level in primary heo patocytes l¥om rat I I(l]. Therefore, various FA-mediated regulations may exist in :l same celhilar type.

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intestinal segments Fig 2. Effects of l'atly acid administration on L-FABP rnRN A levels in the small intestine. Mice were daily force-,fcd ,,,,'ill)0.2 mL sodium salt linoleic acid (32 ~.lmollday)for3 days (:)). Controls were receiving only the vehicle (NaCI 0.gf/( ) (O). The small intestine was divided iiHo I0 st~'gll|CiII~. of 5 till iroln the pylortts to the ileo-cecal valvttle. Duod. duodenum. ]he atl)ounts of tolal RNA sampled were cllecked by ,m 18S rRNA probe. Means _~ SEM. n = 3.

"Iab Ie !,Genes Iranscriptionally up-reguhlted by FAs. Proteins

Fllnctiotts

('ell lines~tissues

Rcfercnce.~

LPL

Hydrolysis of TG-rieh lipoproteins

Pre-adipocytes

7

FAT ALBP L-FABP

Putative plasma membrane FA transporter Cellular lipid binding protein Cellular lipid binding protein

Pre-adipocytes Pre-adipocytes Hepatocytes, hepatoma cells,

8 3 6

ACS

FA activation

Pre-adipocytcs

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ACO CPT-I

FA oxidation FA oxidation

Hepatocytes Hepatocytes

It} II

PEPCK

Gluconeogenesis, glyceroneogenesis

ltepatoma cell.',, adipocyles

12. 13

LPL, lipoprolein lipase; FAT, fatty acid transporter: ALI:IP,adipocyte lipid-binding protein: L-FABP, liver fany acid-binding prolcin; ACS. aeyI-CoA synthetase" ACO, acyI-CoA oxidase; CPT-I, carnitine pahnitoyl transferase I: PEPCK, phosphoenol-pyruvate carboxykinase.

132 Tabl~ II. ~ n e ~ tran~riptionally down-reguhacdby FAs. Prowi~ls

l+'tm,+ti~ms

Cell lines~tissues

References

Ape A-I

Extracellular lipid transport

Hepatocytes/liver

I0

FAS S14

Lipogenesis Putative lipogenicenzyme

Hepatocyte~liver HepatocytesJliver

17 4

SCD+I

FA de~tumtion

Hepatocytes/liver

i8

Ape A-I, ~ p r o t e i n A+I; FAS, fatty acid synthase; S 14, spot 14; SCD-i, stearoyI-CoA desaturase-1.

The molecular mechanisms involved in the gene regulatkmby LCFAs are still unclear. Recent findings suggest that n~mbers of the nuclear receptor supergene family, the peroxisome proliferator-activated receptors (PPARs) [231 ctmld be involved in FA-mediated up-regulation of genes. Activated PPARs. heterodimerized with retinoic X ~'eceptor IRXR), n~tlify the transcription of target genes after binding to a specific nucleotidic sequence termed peroxisome proliferator-responsive element (PPRE). Three different PPARs subty~s, at, 1~ (termed also ~ or FAAR, ie fatty ~'~id-acti','at~ receptor) and y PPAR, have been identified. These nuclear receptors are activated by unsaturated and saturated LCFA 124, 251, Interestingly, most genes presently known to be regulated by LCFAs contain a PPRE+Iike sequence in their promoter 1261, Moreover, Amri et al have ~'¢ntly dem~mslrated, using stable tran~fection of 3T3+C2 fibroblast~ with a FAAR exp~ssiort vector, that expression of thi~ PPAR confers FA+spe¢ific induction of FKI" ALBP and LPL 17, 271, The mechanism by which LCFAs or !heir derivatives ac~ tivate PPARs is unknown, A ligand-de~ndent activation ha~ only ~ n demonstrated liw the PPARy subtype which hinds with high affinity araehidonic acid metabulites of the p m ~ t ~ h ~ n J2 g ~ u p (reviewed in !261), The fact ttmt various LCFAs a~able to trigger PPAR activation suggests the existence of a ligand~inde~ndent mechanism, One I~Xssibilitymight be a LCFA-mediated induction of protein kina~s involved in activation of essential tmn~ription protein(s), as PPARs and/or accessory factor(s), through modifications of permeation of ionic channels, Interestingly, the Cat+influx into rat small intestinal epithelial cell line IEC+6 is mad~edly it~reas,,~ in p~sence of LCFAs (oleic and lim)leic acids) while no effect is found with a short chain FA {capryli¢ acid, C8:0) 1281, The origin of PUFA-mediated suppression of gene tranmription is met yet established, in the liver, deletion analysis of ~+ S 14 promoter has allowed to the kmalization of a negative PUPA+response element (nPUFA-RE)+ This nucleotidic ,~6~qtl~n~,'¢is th~ target for the arachidonic acid (C20:4, n-6) and: ¢icosapentaenoic acid (C20:5, n-3) action, probably t ~ u g h its interaction with one or ~veral nuclear reccptoe(s), Recent data demonstrate that PPARct, the main heIx~tic PPAR subtype, is not involved in PUFA inhibition of

S 14 gene 1291. Therefore, the PUFA-regulated w~ms-acting factor(s) still remain to be identified. The growing knowledge in the reguialion of genes by FAs demonstrates the reality of tills control bul raises several questiorts. For instance, what is the relative effect of nutrients compared to endocrine control? How do LCFAs interact with hormones in affecting gene expression in viva? Is there a coordinated regulation of lipid and carbohydrate metabulisms by LCFAs in the cell? It is likely that additional complexities will be discovered in the interplay of nutritional, endocrine and genetic factors in the near future.

Acknowledgments H Puirier is ~upported by a fellowship from the Ministate de ia Recherche e! de la Tedmologie. This work was supported by tile MinislC:re de 1' Enseignement Sup6rieur et de ia Recherche I MESR, grant 94-G-OI77), the Ass~:iation pour la Recherche st, r le Cancer (ARC, grant lot)5) and the Conseil R6gional de Botlrgogne Igrant no q5-51 i 2-13(111 ),

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