Polyunsaturated fatty acids regulate lipogenic and peroxisomal gene expression by independent mechanisms

Prostaglandins, Leukotrienes and Essential Fatty Acids (1997)57(1), 65-69

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Polyunsaturated fatty acids regulate lipogenic and peroxisomal gene expression by independent mechanisms S. D. Clarke, M. Turini, D. Jump Division of Nutritional Sciences, Department of Human Ecology, 115 GEA, The University of Texas, Austin, Texas 78712, USA

Summary Polyunsaturated fatty acids of the (n-6) and (n-3) families uniquely coordinate hepatic lipid synthesis and oxidation by suppressing the transcription of hepatic genes encoding lipogenic and glycolytic enzymes while concomitantly inducing the activity of enzymes in mitochondrial and peroxisomal fatty acid oxidation. Recently a group of fatty acid activated nuclear transcription factors termed peroxisome proliferator activated receptors (PPARs) were cloned. The discovery of PPARs led us to hypothesize that polyunsaturated fatty acids coordinately modulated the transcription of lipogenic and oxidative genes via a PPAR mediated process. Rats and mice were fed a potent PPAR activator, 5,8,11,14-eicosatetraynoic acid (ETYA), to ascertain if the expression of hepatic fatty acid synthase and peroxisomal acyI-CoA oxidase were coordinately suppressed and induced in response to PPAR activation. Expectedly, ETYA increased peroxisomal acyI-CoA oxidase mRNA abundance, but PPAR activation neither suppressed fatty acid synthase transcription nor reduced the level of fatty acid synthase mRNA. In fact, ETYA prevented the suppression of hepatic fatty acid synthase expression that characteristically results from feeding corn oil. Fatty acid composition analyses indicated that ETYA interfered with 18:2 (n-6) conversion to 20:4 (n-6). Thus, it appears that PPAR is not the sole factor responsible for the coordinate regulation of lipid synthesis and oxidation by polyunsaturated fatty acids. In addition, our data indicate that the active polyenoic fatty acid responsible for the regulation of gene transcription must undergo delta-6 desaturation. INTRODUCTION Polyunsaturated fatty acids of the (n-6) and (n-3) families have a unique ability to suppress the transcription of hepatic genes encoding lipogenic and glycolytic enzymes. 1,2On the hand, these genes are not inhibited by saturated nor monounsaturated fatty acids. This transcriptional control by the polyunsaturated fatty acids does not involve changes in cellular signaling for insulin or glucagon, 2,3 nor does it involve altered hormonal secretion patterns. 4 Moreover, several eicosanoid inhibitor studies have failed to demonstrate a requirement for eicosanoid synthesis. 5,6 However, the mechanism does require that the fatty acid be constantly present in the diet. 2,7If the (n-6) or (n-3) polyenoic fatty acid is removed from a high carbohydrate diet, the suppression of gene transcription is lost in < 3 h. 7 Similarly, replacing oleic Correspondence to: Steven D. Clarke, Office of the Chairman, Department of Human Ecology, 115 GEA, The University of Texas, Austin, Texas 78712, USA, Tel. 001 512 471 6714; Fax. 001 512 471 5630

acid with (n-6) or (n-3) polyunsaturated fatty acids initiates the inhibition of gene transcription in < 3 h. 2 Such rapid transcriptional responses suggest that the fatty acids directly modulate nuclear events rather than mediate their effects via changes in signal transduction resulting from alterations in membrane fatty acid composition. Recently a polyunsaturated fatty acid response region was identified in the proximal promoter of the pyruvate kinase and S14 genes. 8 Interestingly this DNA target region appears to be the same enhancer sequence targeted by carbohydrate and insulin? While it is tempting to propose that dietary polyunsaturated fatty acids and dietary carbohydrate may function in opposition to each other via regulation of a common c/s-acting element, and/or nuclear trans-acting protein, the precise nuclear factors involved in the fatty acid/carbohydrate modulation of gene transcription remain unclear. However, a nuclear transcription factor termed peroxisome proliferator-activated receptor (PPAR) was cloned from 65

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several tissues including liver and adipose tissue? °,H PPARs are members of the steroid receptor super-family of transcription factors; ~2 and when activated by fatty acids or a collection of non-nutritive compounds such as fibrates, the PPARs form heterodimers with other transcription factors (i.e. T3 or RxR) and modulate rates of gene transcription by bInding to specific DNA sequences in the 5"-flanking region of target genes? 2,13 Because fatty acids are among the factors which activate PPARs, we have hypothesized that the polyunsaturated fatty acid suppression of lipogenic and glycolytic genes may Involve a PPAR dependent mechanism. Specifically, the arachidonic acid analog and potent PPAR activator 5,8,11,14-eicosatetraynoic acid (ETYA) was employed to determine if ETYA would function like a polyunsaturated fatty acid, and suppress hepatic fatty acid synthase gene transcription while concomitantly inducing peroxisomal acyl-CoA oxidase.

undetectable levels of hepatic fatty acid synthase mRNA, but both diets significantly induced the level of hepatic acyl-CoA oxidase mRNA (Fig. 1). Interestingly, the corn off/fish off mixture not only increased acyl-CoA oxidase mRNA In the liver, but very effectively induced peroxisomal acyl-Co,a. oxidase expression in several additional tissues including skeletal muscle, kidney, and small intestine (Fig. 2). Given that peroxisomal fatty acid oxidation yields 30% less ATP than does mitochondrial fatty acid oxidation, 19 the 10-fold rise in skeletal muscle acyl-CoA oxidase expression may have significant meaning in terms of overall energy balance. From the data of Figures 1 and 2, it would appear that the fatty acid activation of PPAR observed in isolated cell models also occurs In vivo) °-12 In addition, it is feasible that fatty acid activation of PPAR may possibly explain

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RESULTS I n d u c t i o n of a c y l - C o A o x i d a s e by p o l y u n s a t u r a t e d fats

The ingestion of fat rich in (n-6) and (n-3) fatty acids results in a rapid (< 3 h) inhibition of hepatic lipogenic gene transcription, which in turn leads to a reduction In lipogenic enzyme content and subsequently a decrease in the rate of fatty acid biosynthesis) 4 Interestingly, dietary polyunsaturated fatty acids also increase hepatic fatty acid oxidation both at the mitochondrial and peroxisomal levels.IS We have hypothesized that polyunsaturated fatty acids coordinate fatty acid oxidation and lipid synthesis by governing the expression of rate limiting enzymes in the respective pathways (e.g. acyl-CoA oxidase and fatty acid synthase). Moreover, we proposed that such coordinate regulation employs a nuclear mechanism which is common to genes of both pathways. In this respect, a group of transcription factors termed PPARs have been identified which are activated by fatty acids, and which function to govern the transcription of a number of genes involved in several aspects of lipid metabolism. '°-~2 Included in this list of PPAR regulated genes are malic enzyme, 16mitochondrial HMG-CoA synthase, 17peroxisomal acyl-CoA oxidase, '° and hepatic and adipose fatty acid binding proteins. H,~aBecause of the emerging widespread involvement of PPARs as regulators of gene transcription, we have explored the possibility that dietary polyunsaturated fatty acids may coordinate the downregulation of lipogenic enzymes and the up regulation of fatty acid oxidative enzymes via a common PPAR mediated process. In our initial studies rats were fed for 7-14 days a diet containing 60% calories as corn off, or as a mixture of corn oil and menhaden oil (40%/20%). Both diets yielded

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40% Corn oil

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Fig. 1 Induction of hepatic peroxisomal acyI-CoA oxidase by dietary polyunsaturated fats. Rats were fed a high glucose, low fat (5% w/w) diet, or an isocaloric diet in which 60% of the energy was replaced with corn oil, or a mixture of corn oil (40%) and fish oil (20%). After 7-14 days total hepatic RNA was extracted, and the level of acyI-CoA oxidase was quantified by Northern analyses. 7

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Muscle

S. I n t H Tissue

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Fig. 2 Peroxisomalacyl-CoA oxidase induction by polyunsaturated fat in various rat tissues. Rats were fed a high glucose, low fat (5% w/w) diet or an isocaloric high fat diet (40% corn oil/20% fish oil). After 7 days the total RNA was extracted from various tissues and acyI-CoA oxidase (AOX) mRNA abundance was quantified by Northern analyses. 7

Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 57(1), 65-69

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Polyunsaturated fatty acids regulate lipogenic and peroxisomal gene expression by independent mechanisms

how polyunsaturated fatty acids coordinately regulate lipid synthesis and oxidation. However, the time course for the polyunsaturated fatty acid inhibition of hepatic fatty acid synthase expression and the induction of acyl-CoA oxidase are greatly different, i.e. 3 h vs 14 days, respectively (Fig. 1).2,7These data suggest that the mechanism for coordinate regulation of lipid metabolism by dietary fats may involve a more complex process than simply modulation of PPAR activation.

the expression of enzymes of lipid metabolism. This conclusion is supported by our earlier work showing that the polyunsaturated response region of the pyruvate kinase gene does not contain the direct repeat sequence required for PPAR binding. 8,12Moreover, PPAR would not bind to the polyunsaturated fatty acid element of the S14 gene, but was capable of interacting with the PPARresponse element of the acyl-CoA oxidase gene. 2°

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P P A R activation does not s u p p r e s s hepatic fatty acid synthase expression

To further explore the role that PPAR activation may play in the polyunsaturated fatty acid regulation of gene expression, mice were fed for 5 days a high glucose diet containing 10% triolein or corn oil + the potent PPAR activator, ETYA (200 mg/kg diet). Corn oil, which is rich in 18:2 (n-6), inhibited fatty acid synthase gene transcription and reduced the hepatic content of fatty acid synthase mRNA while fatty acid synthase expression was unaffected by the diet high in 18:1 (n-9) (Figs 3 and 4). In contrast to the fatty acid synthase gene, hepatic acyl-CoA oxidase gene expression was significantly induced by either dietary triolein or corn oil (Fig. 5). The selective inhibition of fatty acid synthase gene expression by polyunsaturated fats suggests that fatty acids may modulate the expression of acyl-CoA oxidase and fatty acid synthase by different mechanisms. In further support of this conclusion, we found that supplementing the triolein diet with the PPAR activator ETYA significantly increased the level of acyl-CoA oxidase but ETYA had no ability to suppress the hepatic level of fatty acid synthase mRNA (Fig. 3). More importantly, when ETYA was supplemented to the corn oil diet, the characteristic reduction in fatty acid syuthase mRNA was completely blocked (Fig. 3). Nuclear run-on assays revealed that the effect of ETYA was to prevent the suppression of fatty acid synthase gene transcription generally observed with corn oil or 18:2 (n-6) ingestion (Fig. 4). In a similarly designed experiment, ETYA reversed the inhibition of hepatic fatty acid synthase by 11,14,17-20:3 (n-3), i.e. the relative hepatic abundance of fatty acid syuthase mRNA in mice fed 11,14,17-20:3 (n-3) without and with ETYA in the diet was 4 +__0.5 and 30 +_4, respectively. The response of the fatty acid synthase gene to ETYA is not what would be predicted if PPAR activation was responsible for the polyunsaturated fatty acid inhibition of fatty acid synthase gene transcription. Collectively these data strongly indicate that the polyunsaturated fatty acid inhibition of fatty acid synthase transcription, and the fatty acid induction of acyl-CoA oxidase expression involve independent mechanisms; and that fatty acid activation of PPAR is not solely responsible for the apparent coordinate changes in © Pearson Professional Ltd 1997

67

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Dietary Fat Fig. 3 Response of hepatic fatty acid synthase to triolein and corn oil with/without PPAR activation by ETYA. Mice (n=4) were fed a high glucose diet containing no fat (FF), 10% triolein, or 10% corn oil with/without 200 mg/kg diet ETYA (±E). After 5 days of feeding total hepatic RNA was extracted and fatty acid synthase (FAS) mRNA abundance was quantified by Northern analyses.7 The asterisk denotes the value is significantly lower than the FF group (P<0.05). Fatty acid composition of the microsomal membranes of these mice is summarized in the Table.

ETYA Blocks 18:2 Inhibition of FAS Transcription

18:1

18:1 + ETYA

Corn Oil

Corn Oil + ETYA

mm Fig. 4 PPAR activation by ETYA blocks suppression of fatty acid synthase gene transcription by dietary corn oil. Nuclei were prepared from mice described in Figure 3, and utilized to quantify rates of fatty acid synthase (FAS) gene transcription using the nuclear run-on assay. 1

Prostaglandins, Leukotrienes and Essential FattyAcids (1997) 57(1), 65-69

Clarke et al

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mice fed corn oil or triolein with and without ETYA supplementation revealed that feeding ETYA increased the hepatic content of 18:2 (n-6) by 80-125%, and with the corn oil diet decreased the hepatic content of 20:4 (n-6) by 40% (Table). The rise in 18:2 (n-6) and the drop in 20:4 (n-6) is a classic metabolite cross-over pattern that strongly indicates that 18:2 (n-6) conversion to 20:4 (n-6) is impaired. Thus, the inhibition of lipogenic gene expression exerted by dietary polyunsaturated fatty acids would appear to require that 18:2 (n-6) and 18:3 (n-3) undergo desaturation by the delta-6 desaturase in order for the fatty acid to exert its inhibitory action. While the delta-6 desaturation requirement is consistent with a possible role for an eicosanoid mediator, numerous studies have failed to detect an eicosanoid i n v o l v e m e n t . ~,e'22

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CONCLUSION

Polyunsaturated fatty acids of the (n-6) and (n-3) families, but neither saturated nor monounsaturated fatty acids, uniquely suppress the transcription of hepatic lipogenic and glycolytic genes. 14 With the recent discovery of the fatty acid activated PPAR nuclear transcription factors has come the suggestion that the polyunsaturated fatty acid regulation of gene expression is a PPAR-mediated process. However, several lines of evidence argue strongly against such a hypothesis. First, hepatic peroxisomal acyl-CoA oxidase mRNA was significantly increased by either 18:1 (n-9) or corn oil (Fig. 2), but only corn oil suppressed hepatic fatty acid synthase transcription (Fig. 4). Second, the induction of acyl-CoA oxidase expression by corn off required several days (Fig. 1) while fatty acid synthase transcription is inhibited by polyunsaturated fatty acids within 3 h. 2 Third, the potent PPAR activator, ETYA, significantly induced acyl-CoA oxidase expression, but PPAR activation did not suppress fatty acid synthase tran-

Fig. 5 Induction of hepatic peroxisomal acyI-CoA oxidase by dietary fat and ETYA. Mice were fed as described for Figure 3. AcyI-CoA oxidase (AOX) mRNA abundance was quantified by Northern analyses. ~ The asterisk denotes values are significantly higher than the fat-free (FF) dietary group ( P < 0.05). ± E denotes +/- ETYA supplementation (200 mg/kg diet).

E T Y A i n h i b i t s t h e c o n v e r s i o n of 1 8 : 2 ( n - 6 ) to 2 0 : 4 (n-6)

The ability of ETYA to block the corn off and 20:3 (n-3) suppression of fatty acid synthase expression could be explained if ETYA either functioned as an inducer of fatty acid synthase transcription, or if it inhibited the formation of an inhibitory metabolite derived from 18:2 (n-6) or 18:3 (n-3). We have previously observed that, when ETYA is supplemented to diets containing fish oil or 20:4 (n-6), the ETYA does not prevent the suppression of fatty acid synthase gene expression? 1 Moreover, fatty acid compositional analyses of microsomes taken from the

Table The impact of dietary fat and ETYA on hepatic microsomal fatty acid composition Diet Trioleln -ETYA Microsomal fatty acid 16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4

(n-7) (n-9) (n-6) (n-6) (n-6) (n-6)

28.0 0.6 20.6 21.7 2.2 0.6 1.0 7.7

+ ± + ± ± + ± +

Corn oil +ETYA -ETYA +ETYA (mole % of total fatty acids)

1.2a 0.0 a'b 1.7a'b 0.6 b 0.3 d 0.0 a'~ 0.1 a 1.2¢

26.1 0.7 16.6 25.8 5.0 0.8 1.0 7.9

± ± ± ± ± ± ± ±

1.0a 0.0 a 1.1 ~'° 0.9 a 0.3 c 0.1 a 0.0' 0.5 a

27.5 0.3 26.3 5.4 11.4 0.3 1.0 16.7

± ± ± ± ± ± ± ±

0.3 a 0.0 b 1.8" 1.1 b 0.2 b 0.1 c 0.0~ 0.5"

26.4 0.3 24.1 6.6 20.3 0.3 0.9 10.5

± ± ± ± ± ± ± ±

0.5 a 0.0b 0.5 a 0.1 b 0.2" 0.1 c 0.1 a 0.6 b

Mice (n=-4) were fed a high glucose diet containing 10% triolein or 10% corn oil + ETYA (200 mg/kg diet) for 5 days. Hepatic microsomes were isolated and total lipid extracted for fatty acid compositional analyses using gas-liquid chromatography.

Prostaglandins, Leukott~enesand Essential Fatty Acids (1997) 57(1), 65-69

© Pearson ProfessionalLtd 1997

Polyunsaturated fatty acids regulate lipogenic and peroxisomal gene expression by independent mechanisms

scription (Figs 3 a n d 4). In fact, ETYA actually i n d u c e d fatty a c i d s y n t h a s e t r a n s c r i p t i o n in rats fed c o r n oil. This s t i m u l a t o r y effect of ETYA a p p e a r e d to result f r o m a n i n h i b i t i o n of 18:2 (n-6) c o n v e r s i o n to 20:4 (n-6). Finally, a r e c e n t l y identified p o l y u n s a t u r a t e d fatty a c i d r e s p o n s e e l e m e n t in t h e p y r u v a t e kinase g e n e does n o t c o n t a i n a s e q u e n c e to w h i c h PPAR will bind. s Clearly, t h e m e c h a n i s m b y w h i c h p o l y u n s a t u r a t e d fatty acids c o o r d i n a t e l y m o d u l a t e rates of lipid s y n t h e s i s a n d o x i d a t i o n is m o r e c o m p l e x t h a n simple i n t e r a c t i o n of a single t r a n s c r i p t i o n factor.

ACKNOWLEDGEMENTS This work was supported by grants from the USDA (NRICGP/USDA 92-3700-7465, SDC), and the National Institutes of Health (DK 43220, DBJ); Mead-Johnson Grant in Aid (SDC); and by the M.M. Love Chair for Nutritional, Cellular, and Molecular Sciences (SDC) at The University of Texas.

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interfere with the insulin glucose activation of L-type pyruvate kinase. Mol EndoPt~nol 1994; 8:1147-1153. 9. Thompson I¢. S., Towle H. C. Localization of the carbohydrate response elements of the rat L-type pyruvate kinase gene. J Biol Chem 1990; 266: 8679-8682. 10. Isseman I., Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome prolfferators. Nature 1990; 347: 645-650. 11. Tontonoz P., Hu E., Spiegelman B. M. Stimulation of adipogenesis in fibroblasts by PPARg2 a lipid-activated transcription factor. Cell 1994; 79:1147-1156. 12. Keller H., Deyer C., Medin J., Nahfoudi A., Ozato K., Wahli W. Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activatedreceptor-retinoid X receptor heterodirners. Proc NatAcad SH USA 1993; 90: 2160-2164. 13. Ren B., Thelen A., Jump D. B. Peroxisome prolfferator-activated receptor alpha inhibits $14 gene transcription. ]Biol C'hem 1996; 271: 17167-17173. 14. Clarke S. D., Jump D. B. Dietary polyunsaturated fatty acid regulation of gene transcription. Annu Rev Nutr 1994; 14: 83-98. 15. Takada 1L, Saitoh M., Mori T. Dietary gamma-linolenic acidenriched oil reduces body fat content and induces liver enzyme activities relating to fatty acid beta-oxidation in rats. J Nutr 1994; 124: 469-474. 16. Castelein H., Gulick T., Declercq P. E., Mannaerts G. P., Moore D. D., Baes M. I. The peroxisome proliferator activated receptor regulates malic enzyme gene expression. ]Biol Chem 1994; 43: 26754-26758. 17. Rodriguez J. C., Gil-Gomez G., Hegardt F. G., Haro D. Peroxisome proliferator-activatedreceptor mediates induction of the mitochondrial 3-hydroxy-3-methylglutaryl-CoAsynthase gene by fatty acids. JBiol Chem 1994; 269: 18767-18772. 18. Khan S. H., Sorof S. Liver fatty acid-binding protein: Specific mediator of thermogenesis induced by two classes of carcinogenic peroxisome prolfferators. Proc Natl Acad Sci USA 1994; 91: 848-852. 19. Reddy J. K., Mannaerts G. P. Peroxisomal lipid metabolism. Annu Rev Nutr 1994; 14: 343-370. 20. Jump D. B., Ren B., Clarke S. D., Thelen A. Effects of fatty acids on hepatic gene expression. Prostaglandins Leukot Essent Fatty AHds 1995; 52:107-111. 21. Clarke S. D., Jump D. B. Fatty acid regulation of gene expression: a unique role for polyunsaturated fats. In: Berdanier C, Hargrove J, eds. Nutrition and Gene Expression. Boca Raton: CRC Press, 1993: 227. 22. Clarke S. D., Turini M., Jump D. B., Abraham S., Reedy M. Polyunsaturated fatty acid inhibition of fatty acid synthase transcription is independent of PPAR activation (in press).

Prostaglandins, Leukotrienesand Essential Fatty Acids (1997) 57(1), 65-69