Annette Thelen

Sterol response element-binding protein 1c (SREBP1c) is involved in the polyunsaturated fatty acid suppression of hepatic S14 gene transcription.

Polyunsaturated fatty acids (PUFA) suppress hepatic lipogenic gene transcription through a peroxisome proliferator activated receptor α (PPARα)- and cyclooxygenase-independent mechanism. Recently, the sterol response element-binding protein 1 (SREBP1) was implicated in the nutrient control of lipogenic gene expression. In this report, we have assessed the role SREBP1 plays in the PUFA control of three hepatic genes, fatty acid synthase, L-pyruvate kinase (LPK), and the S14 protein (S14). PUFA suppressed both the hepatic mRNASREBP1 through a PPARα-independent mechanism as well as SREBP1c nuclear content (nSREBP1c, 65 kDa). Co-transfection of primary hepatocytes revealed a differential sensitivity of the fatty acid synthase, S14, and LPK promoters to nSREBP1c overexpression. Of the three promoters examined, LPK was the least sensitive to overexpressed nSREBP1c. Promoter deletion and gel shift analyses of the S14 promoter localized a functional SREBP1c cis-regulatory element to an E-box-like sequence (−139TCGCCTGAT−131) within the S14 PUFA response region. Although overexpression of nSREBP1c significantly reduced PUFA inhibition of S14CAT, overexpression of other factors that induced S14CAT activity, such as steroid receptor co-activator 1 or retinoid X receptor α, had no effect on S14CAT PUFA sensitivity. These results suggest that PUFA regulates hepatic nSREBP1c, a factor that functionally interacts with the S14 PUFA response region. PUFA regulation of nSREBP1c may account for the PUFA-mediated suppression of hepatic S14 gene transcription.

Peroxisome Proliferator-activated Receptor α Inhibits Hepatic S14 Gene Transcription EVIDENCE AGAINST THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR α AS THE MEDIATOR OF POLYUNSATURATED FATTY ACID REGULATION OF S14 GENE TRANSCRIPTION

The peroxisome proliferator-activated receptor (PPARα) has been implicated in fatty acid regulation of gene transcription. Lipogenic gene transcription is inhibited by polyunsaturated fatty acids (PUFA). We have used the PUFA-sensitive rat liver S14 gene as a model to examine the role PPARα plays in fatty acid regulation of hepatic lipogenic gene transcription. Both PPARα and the potent peroxisome proliferator, WY14643, inhibit S14CAT activity in transfected primary hepatocytes. WY14643 and PPARα target the S14 T3 regulatory region (TRR, −2.8 to −2.5 kilobases), a region containing 3 T3 response elements (TRE). Transfer of the TRR to the thymidine kinase (TK) promoter conferred negative control to the TKCAT gene following WY14643 and PPARα treatment. Gel shift analysis showed that PPARα, either alone or with RXRα, did not bind the S14TRR. However, PPARα interfered with TRβ/RXRα binding to a TRE (DR+4). Functional studies showed that co-transfected RXRα, but not T3 receptor β1 (TRβ1), abrogated the inhibitory effect of PPARα on S14 gene transcription. These results suggest that WY14643 and PPARα functionally interfere with T3 regulation of S14 gene transcription by inhibiting TRβ1/RXR binding to S14 TREs. Previous studies had established that the cis-regulatory targets of PUFA control were located within the proximal promoter region of the S14 gene, i.e. between −220 and −80 bp. Finding that the cis-regulatory elements for WY14643/PPARα and PUFA are functionally and spatially distinct argues against PPARα as the mediator of PUFA suppression of S14 gene transcription.

Dietary polyunsaturated fatty acids and hepatic gene expression.

Dietary polyunsaturated fatty acids (PUFA) have profound effects on hepatic gene transcription leading to significant changes in lipid metabolism. PUFA rapidly suppress transcription of genes encoding specific lipogenic and glycolytic enzymes and induce genes encoding specific peroxisomal and cytochrome P450 (CYP) enzymes. Using the peroxisome proliferator-activated receptor α (PPARα)-null mouse, we showed that dietary PUFA induction of acyl PPARα is not required for the PUFA-mediated suppression of fatty acid synthase (FAS), S14, or L-pyruvate kinase (L-PK). Studies in primary rat hepatocytes and cultured 3T3-L1 adipocytes showed that metabolites of 20:4n-6, like prostaglandin E2 (PGE2), suppress mRNA encoding FAS, S14, and L-PK through a Gi/Go-coupled signal transduction cascade. In contrast to adipocytes, 20:4n-6-mediated suppression of lipogenic gene expression in hepatic parenchymal cells does not require cyclooxygenase. Transfection analysis of S14CAT fusion genes in primary hepatocytes shows that peroxisome proliferator-activated PPARα acts on the thyroid hormone response elements (−2.8/−2.5 kb). In contrast, both PGE2 and 20:4n-6 regulate factors that act on the proximal promoter (−150/−80 bp) region, respectively. In conclusion, PUFA affects hepatic gene transcription through at least three distinct mechanisms: (i) a PPAR-dependent pathway, (ii) a prostanoid pathway, and (iii) a PPAR and prostanoid-independent pathway. PUFA regulation of hepatic lipid metabolism involves an integration of these multiple pathways.

Dietary Fat, Genes, and Human Health

These studies show that a macronutrient like dietary fat plays an important role in gene expression. In the cases presented here, dietary fat regulates gene expression leading to changes in carbohydrate and lipid metabolism. The interesting outcome of these studies is the finding that the molecular targets for dietary fat action did not converge with the principal targets for hormonal regulation of gene transcription, like hormone receptors. Instead, PUFA-RF targets elements that play key ancillary roles in gene transcription. This is important because it shows how PUFA can interfere with hormone regulation of a specific gene without having generalized effect on overall hormonal control, i.e. PUFA effects are promoter-specific. How PUFA-RF interferes with gene transcription will require the isolation and characterization of PUFA-RF along with the tissue-specific factors targeted by PUFA-RF. A different story emerges when fatty acids activate PPAR. Based on the studies presented here and elsewhere, long chain-highly unsaturated fatty acids (like 20:5,n-3 and 22:6, n-3) or high levels of fat activate PPAR. PPAR directly activates genes like AOX, but also inhibits transcription of genes like S14, FAS, apolipoprotein CIII, transferrin. For S14, the mechanism of inhibition involves sequestration of RXR, a critical factor for T3 receptor binding to DNA. Thus, PPAR can have generalized effects on T3 action or on other nuclear receptors, like vit. D (VDR) and retinoic acid (RAR) receptors, that require RXR for action. For apolipoprotein CIII and transferrin, PPAR/RXR heterodimers compete for HNF-4 binding sites (DR + 1). In addition to HNF-4, COUP-TF, ARP-1 and RXR all bind the DR + 1 type motif. These factors are important for tissue-specific regulation of gene transcription. PPAR can potentially interfere with the transcription of multiple genes through disruption of nuclear receptor signaling leading to changes in phenotype. Clearly, more studies are required to assess the role PPAR plays in the fatty acid regulation of gene transcription and its contribution to chronic disease. Finally, it is clear that dietary fat has the potential to affect gene expression through multiple pathways. Depending on the gene examined, PUFA might augment or abrogate gene transcription which leads to specific phenotypic changes altering metabolism, differentiation or cell growth. These effects can be beneficial to the organism, such as the n-3 PUFA-mediated suppression of serum triglycerides or detrimental, like the saturated and n-6 PUFA-mediated promotion of insulin resistance. How such effects contribute to the onset or progression of specific neoplasia is unclear. However, studies in metabolism might provide important clues for this connection.

Multiple mechanisms for polyunsaturated fatty acid regulation of hepatic gene transcription

Dietary polyunsaturated fatty acids (PUFA) have profound effects on hepatic gene transcription leading to significant changes in lipid metabolism. Highly unsaturated n-3 PUFA suppress the transcription of genes encoding specific lipogenic enzymes and induce the expression of genes encoding specific enzymes involved in peroxisomal and microsomal fatty acid oxidation. Our studies have shown that fatty acid effects on hepatic gene expression may involve at least three distinct pathways. One pathway involves peroxisome proliferator-activated receptor (PPARalpha), a fatty acid activated nuclear receptor. PPARalpha is required for the PUFA induction of mRNAs encoding enzymes involved in fatty acid oxidation. However, PPARalpha is not required for PUFA suppression of mRNAs encoding proteins involved in lipogenesis. A second pathway involves prostanoids. In cultured 3T3-L1 adipocytes, cyclooxygenase derived 20:4 n-6 metabolites, like PGE2, suppress mRNAs encoding proteins involved in lipogenesis. However, in hepatic parenchymal cells, 20:4 n-6 suppression of lipogenic gene expression does not require a cyclooxygenase. Nevertheless, PGE2 and PGF2alpha suppress hepatic lipogenic gene expression. 20:4 n-6 cyclooxygenase products can arise from non-parenchymal cells and through a paracrine control process act on a G-protein linked receptor signaling cascade to suppress lipogenic gene expression. The fact that n-3 and n-6 PUFA suppression of lipogenic gene expression does not require PPARalpha or cyclooxygenase activity indicates the presence of a third pathway for the control of hepatic gene transcription. These studies indicate that the pleiotropic effects of PUFA on hepatic lipid metabolism cannot be attributed to a single regulatory mechanism.

The CCAAT Box Binding Factor, NF-Y, Is Required for Thyroid Hormone Regulation of Rat Liver S14 Gene Transcription

Triiodothyronine (T3) activates rat liver S14 gene transcription through T3 receptors (TRβ) binding distal thyroid hormone response elements located between −2.8 and −2.5 kilobase pairs upstream from the transcription start site. Previous studies suggested that proximal promoter elements located between −220 to −80 base pairs upstream from the 5′ end of the S14 gene were involved in hormone activation of the S14 gene. This report identifies an inverted CCAAT box (or Y box) at−104ATTGG−100 as a core cis-regulatory element. Gel shift studies using rat liver nuclear proteins show that at least three CCAAT-binding factors interact with this region as follows: NF-Y and c/EBP-related proteins formed major complexes, whereas NF-1/CTF forms a minor complex in gel shift assay. Mutation of the Y box indicated that loss of NF-Y binding, but not c/EBP or NF-1, correlated closely with a decline in basal activity and a loss of T3-mediated transactivation. Substitution of the S14 Y box in reporter genes with elements binding only NF-Y elevated basal activity and T3-mediated transactivation, whereas substitution with elements binding c/EBP-related proteins or SP1 displayed low basal activity and T3-mediated transactivation. These studies indicate that NF-Y and TRβ functionally interact to confer T3 control to the S14 gene.