Polyunsaturated fatty acids (PUFA) suppress hepatic lipogenic gene transcription through a peroxisome proliferator activated receptor ␣ (PPAR␣)-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 mRNA SREBP1 through a PPAR␣-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 ( ؊139 TCGCCTGAT ؊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 ␣, 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.

Sterol regulatory element binding protein-1c (SREBP1c) is a key hepatic transcription factor involved in lipogenic gene expression. In an effort to understand the role SREBP-1c plays in lipogenic gene transcription, we have examined the functional interaction between SREBP-1c, nuclear factor Y, 3,5,3-triiodothyronine (T 3 ) receptors, and co-activators using the S14 gene promoter as a model. T 3 , glucose, and insulin rapidly induce S14 gene transcription in rat liver and in primary hepatocytes. Linker scanning analyses of the S14 promoter showed that an SRE at ؊139/؊131 base pairs (bp) binding SREBP-1c and a Y-box at ؊104/؊99 bp binding NF-Y are indispensable for both T 3 -and SREBP-1c-mediated induction of S14 promoter activity in rat primary hepatocytes. T 3 and glucose/insulin induce S14 gene transcription through separate enhancers. Enhancer substitution studies reveal a preferential interaction between SREBP-1c⅐NF-Y and the T 3 regulatory region (؊2.8/؊2.5 kb) binding thyroid hormone receptor/RXR heterodimers. Elevating hepatocellular levels of specific co-activators (CBP, p/CAF, or GCN5) induced S14 promoter activity 2-3-fold, while SREBP-1c induced promoter activity 10-fold. The combination of these treatments induced S14 promoter activity (20 -35-fold). However, this additive effect was lost when the T 3 regulatory region was deleted. Based on these results, we suggest that the SREBP-1c⅐NF-Y complex facilitates the interaction between co-activators that are recruited to distal hormone-regulated enhancers and the general transcription machinery that binds the S14 proximal promoter. Sterol regulatory element-binding proteins (SREBPs)1 play a major role in cholesterol and lipid homeostasis in the liver and other tissues. Emerging evidence suggests that SREBP-1c is involved in the control of lipogenesis, while SREBP-2 functions in the regulation of cholesterol homeostasis (1, 2). While SREBP-1a and SREBP-1c are derived from a common gene, differential promoter usage and splicing accounts for the variations in the N termini sequence (2). Rnase protection studies suggest that SREBP-1c is the major SREBP-1 subtype expressed in human and rodent liver (3).SREBP precursors (ϳ125 kDa) are tethered to the endoplasmic reticulum and are converted to a mature (nuclear, ϳ65 kDa) form by two proteolytic cleavage steps in the endoplasmic reticulum and Golgi (2). The nuclear form of SREBP (nSREBP) is a helix-loop-helix transcription factor that binds sterol regulatory elements (SRE; PyGCPy) in sterol-responsive genes.

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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 alpha (PPAR alpha)-null mouse, we showed that dietary PUFA induction of acyl CoA oxidase (AOX) and CYP4A2 require PPAR alpha. However, PPAR alpha 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 PPAR alpha 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.

This report examines the effect of polyunsaturated fatty acids (PUFA) on lipogenic gene expression in cultured 3T3-L1 adipocytes. Arachidonic acid (20:4, n-6) and eicosapentaenoic acid (20:5, n-3) suppressed mRNAs encoding fatty acid synthase (FAS) and S14, but had no effect on ␤ -actin. Using a clonal adipocyte cell line containing a stably integrated S14CAT fusion gene, oleic acid (18:1, n-9), arachidonic acid (20:4, n-6) and eicosapentaenoic acid (20:5, n-3) inhibited chloramphenicol acetyltransferase (CAT) activity with an ED 50 of 800, 50, and 400 m , respectively. Given the high potency of 20:4, n-6, its effect on adipocyte gene expression was characterized. Arachidonic acid suppressed basal CAT activity, but did not affect glucocorticoid-mediated induction of S14CAT expression. The effect of 20:4, n-6 on S14CAT expression was blocked by an inhibitor of cyclooxygenase implicating involvement of prostanoids. Prostaglandins (PGE 2 and PGF 2 ␣ at 10 m ) inhibited CAT activity through a pertussis toxin-sensitive G i /G ocoupled signalling cascade. Our results suggest that 20:4, n-6 inhibits lipogenic gene expression in 3T3-L1 adipocytes through a prostanoid pathway. This mechanism of control differs from the polyunsaturated fatty acid-mediated suppression of hepatic lipogenic gene expression.-Mater, M. K., D. Pan, W. G. Bergen, and D. B. Jump. Arachidonic acid inhibits lipogenic gene expression in 3T3-L1 adipocytes through a prostanoid pathway.