Polyunsaturated fatty acid suppression of fatty acid synthase (FASN): evidence for dietary modulation of NF-Y binding to the Fasn promoter by SREBP-1c.

Dietary PUFAs (polyunsaturated fatty acids) co-ordinately suppress transcription of a group of hepatic genes encoding glycolytic and lipogenic enzymes. Suppression of Fasn (fatty acid synthase) transcription involves two PUFA-responsive regions, but the majority of PUFA sensitivity maps to a region within the proximal promoter containing binding sites for NF-Y (nuclear factor-Y), Sp1 (stimulatory protein 1), SREBP (sterol-regulatory-elementbinding protein), and USF (upstream stimulatory factor). Promoter activation assays indicate that altered NF-Y is the key component in regulation of Fasn promoter activity by PUFA. Using electrophoretic mobility-shift assay and chromatin immunoprecipitation analysis, we demonstrate for the first time that PUFAs decrease in vivo binding of NF-Y and SREBP-1c to the proximal promoter of the hepatic Fasn gene and the promoters of three additional genes, spot 14, stearoyl-CoA desaturase and farnesyl diphosphate synthase that are also down-regulated by PUFA. The comparable 50% decrease in NF-Y and SREBP-1c binding to the promoters of the respective PUFA-sensitive genes occurred despite no change in nuclear NF-Y content and a 4-fold decrease in SREBP-1c. Together, these findings support a mechanism whereby PUFA reciprocally regulates the binding of NF-Y and SREBP-1c to a subset of genes which share similar contiguous arrangements of sterol regulatory elements and NF-Y response elements within their promoters. PUFA-dependent regulation of SREBP-1c and NF-Y binding to this unique configuration of response elements may represent a nutrient-sensitive motif through which PUFA selectively and co-ordinately targets subsets of hepatic genes involved in lipid metabolism.

A newly discovered member of the fatty acid desaturase gene family: a non-coding, antisense RNA gene to delta5-desaturase.

The rate limiting steps in the conversion of 18-carbon unsaturated fatty acids to 20- and 22-carbon products are catalyzed by two desaturase enzymes (Delta5-desaturase and Delta6-desaturase) found within a lipid desaturase gene cluster. Careful examination of this cluster revealed the existence of a conventionally spliced (human) and an intronless (mouse and rat) non-coding RNA gene, reverse Delta5-desaturase, which is transcribed from the opposite strand of the Delta5-desaturase gene. The 654 bp human reverse Delta5-desaturase transcript contains 269 nucleotides that are complementary to exon 1 and intron 1 of the Delta5-desaturase transcript, and the 3'-end of this sequence contains a 143 nucleotide stretch that is 100% complementary to the 5'-end of the Delta5-desaturase. The rat and mouse transcripts are 1355 and 690 bp long and complementary to a portion of the first intron and the entire first exon of their respective Delta5-desaturases. All reverse Delta5-desaturase transcripts contain several stop codons in all frames suggesting that they do not encode a peptide. Reverse Delta5-desaturase RNA was detected in all rat tissues where Delta5-desaturase is expressed, and the transition between fasting and refeeding produced a significant increase in reverse Delta5-desaturase RNA relative to Delta5-desaturase mRNA. Transient expression of reverse Delta5-desaturase in CHO cells stably transformed with Delta5-desaturase produced a modest decrease in Delta5-desaturase mRNA (30%), but lowered Delta5-desaturase enzymatic activity by >70%. More importantly, a diet enriched in fish oil produced a reciprocal increase in reverse Delta5-desaturase mRNA and decrease in Delta5-desaturase mRNA that was accompanied by a 5-6-fold decrease in Delta5-desaturase enzyme activity. These findings support a significant role for reverse Delta5-desaturase as a natural antisense regulator of Delta5-desaturase.

Hepatocyte nuclear factor-4alpha contributes to carbohydrate-induced transcriptional activation of hepatic fatty acid synthase.

Refeeding a carbohydrate-rich meal after a fast produces a co-ordinated induction of key glycolytic and lipogenic genes in the liver. The transcriptional response is mediated by insulin and increased glucose oxidation, and both signals are necessary for optimal induction of FAS (fatty acid synthase). The glucose-regulated component of FAS promoter activation is mediated in part by ChREBP [ChoRE (carbohydrate response element)-binding protein], which binds to a ChoRE between -7300 and -7000 base-pairs in a carbohydrate-dependent manner. Using in vivo footprinting with nuclei from fasted and refed rats, we identify an imperfect DR-1 (direct repeat-1) element between -7110 and -7090 bp that is protected upon carbohydrate refeeding. Electrophoretic mobility-shift assays establish that this DR-1 element binds HNF-4alpha (hepatocyte nuclear factor 4alpha), and chromatin immunoprecipitation establishes that HNF-4alpha binding to this site is increased approx. 3-fold by glucose refeeding. HNF-4alpha transactivates reporter constructs containing the distal FAS promoter in a DR-1-dependent manner, and this DR-1 is required for full glucose induction of the FAS promoter in primary hepatocytes. In addition, a 3-fold knockdown of hepatocyte HNF-4alpha by small interfering RNA produces a corresponding decrease in FAS gene induction by glucose. Co-immunoprecipitation experiments demonstrate a physical interaction between HNF-4alpha and ChREBP in primary hepatocytes, further supporting an important complementary role for HNF-4alpha in glucose-induced activation of FAS transcription. Taken together, these observations establish for the first time that HNF-4alpha functions in vivo through a DR-1 element in the distal FAS promoter to enhance gene transcription following refeeding of glucose to fasted rats. The findings support the broader view that HNF-4alpha is an integral component of the hepatic nutrient sensing system that co-ordinates transcriptional responses to transitions between nutritional states.

Dietary polyunsaturated fatty acids enhance hepatic AMP-activated protein kinase activity in rats.

Polyunsaturated fatty acids (PUFA) and a number of drugs (metformin, thiazolidinediones) and hormones (leptin, adiponectin) that activate AMP-activated protein kinase (AMPK) have been reported to improve insulin sensitivity. To determine whether PUFA activate AMPK, Sprague-Dawley rats were adapted to a 3h meal-feeding regimen using a fat-free diet (FFD) supplemented with fish oil (n-3) or triolein (n-9) for 7 days. No differences in hepatic AMPK activity were observed between the groups after 21h of fasting. On the other hand, hepatic AMPK phosphorylation was decreased in rats refed the FFD, the FFD+triolein, and the FFD+PUFA by 80%, 75%, and 50%, respectively, when assessed 2h after completion of a meal. In keeping with these changes, decreases in acetyl-CoA carboxylase phosphorylation and carnitine palmitoyl transferase-1 mRNA and increases in fatty acid synthase gene expression were greatest in rats fed the FFD and least in the PUFA-fed rats. The results indicate that dietary PUFA enhance hepatic AMPK activity in vivo, and implicate AMPK as a component of the nutrient-sensing mechanism through which dietary fatty acids and especially PUFA influence the regulation of hepatic lipid metabolism and gene expression.

The multi-dimensional regulation of gene expression by fatty acids: polyunsaturated fats as nutrient sensors.

PURPOSE OF REVIEW: A diet that provides 2-5% of energy as highly unsaturated 20- and 22-carbon omega-6 or omega-3 fatty acids is associated with an inhibition of hepatic lipogenesis, a stimulation of hepatic fatty acid oxidation, and consequently a lowering of blood triglyceride levels. The purpose of this review is to demonstrate that highly unsaturated fatty acids regulate lipid metabolism by modulating protein expression at many levels including gene transcription, messenger RNA processing, mRNA decay, and post-translational protein modifications. Although the intracellular signaling mechanisms employed by highly unsaturated fatty acids are unknown, this review presents a summary of the emerging knowledge regarding highly unsaturated fatty acids as kinase cascade activators. RECENT FINDINGS: Highly unsaturated fatty acids suppress lipogenic gene transcription by reducing the DNA binding activity of several transcription factors, notably sterol regulatory-element binding protein 1 and nuclear factor Y. Highly unsaturated fatty acids inhibit the proteolytic release of sterol regulatory-element binding protein 1 from its membrane-anchored precursor through a ceramide-dependent signal, and impart a post-translational modification to nuclear factor Y. Highly unsaturated fatty acids accelerate sterol regulatory-element binding protein 1 mRNA decay and may function as antagonistic ligands for liver receptor X, thereby interfering with the liver receptor X stimulation of sterol regulatory-element binding protein 1 gene transcription. Highly unsaturated fatty acid activation of peroxisome proliferator-activated receptor alpha combined with their displacement of the oxysterol from liver receptor X may 'trap' liver receptor X as transcriptionally inactive peroxisome proliferator-activated receptor alpha/liver receptor X heterodimer. The gene expression consequences of liver receptor X 'trapping' may explain how dietary highly unsaturated fatty acids lead to a repartitioning of fatty acids away from storage and towards oxidation. SUMMARY: The liver appears to use the highly unsaturated fatty acid status as a nutrient sensor to determine whether fatty acids are to be stored or oxidized. In this way highly unsaturated fatty acids may function as nutritional factors that reduce the risk of developing hepatic lipotoxicity and insulin resistance.

Polyunsaturated fatty acids and gene expression.

PURPOSE OF REVIEW: This review focuses on the effect(s) of n-3 polyunsaturated fatty acids on gene transcription as determined by data generated using cDNA microarrays. Introduced within the past decade, this methodology allows detection of the expression of thousands of genes simultaneously and, hence, is a potentially powerful tool for studying the regulation of physiological mechanisms that are triggered or inhibited by nutrients. RECENT FINDINGS: Recent data generated with cDNA microarrays not only confirm the effects of n-3 polyunsaturated fatty acids on regulation of lipolytic and lipogenic gene expression as determined by more traditional methods but also emphasize the tissue specificity of this regulation. cDNA microarray experiments also have expanded our understanding of the role of n-3 polyunsaturated fatty acids in regulation of expression of genes involved in many other pathways. These include: oxidative stress response and antioxidant capacity; cell proliferation; cell growth and apoptosis; cell signaling and cell transduction. SUMMARY: The cDNA microarray studies published to date show clearly that n-3 polyunsaturated fatty acids, usually provided as fish oil, modulate expression of a number of genes with such broad functions as DNA binding, transcriptional regulation, transport, cell adhesion, cell proliferation, and membrane localization. These effects, in turn, may significantly modify cell function, development and/or maturation.

Regulation of human delta-6 desaturase gene transcription: identification of a functional direct repeat-1 element.

The rate-limiting step in 20:4(n-6) and 22:6(n-3) synthesis is the desaturation of 18:2(n-6) and 18:3(n-3) by Delta-6 desaturase. In this report, we demonstrate that n-6 and n-3 PUFAs suppressed the hepatic expression of rodent Delta-6 desaturase by inhibiting the rate of Delta-6 desaturase gene transcription. In contrast, consumption of the peroxisome proliferator-activated receptor (PPAR)alpha activator WY 14,643 significantly enhanced the transcription of hepatic Delta-6 desaturase by more than 500%. Transfection reporter assays with HepG2 cells revealed that the PUFA response region for the human Delta-6 desaturase gene involved the proximal promoter region of -283/+1 human Delta-6 desaturase gene, while the WY 14,643 response element (RE) was identified as an imperfect direct repeat (DR-1) located at -385/-373. The WY 14,643 induction of the human Delta-6 desaturase promoter activity was dependent upon the expression of PPARalpha. Electrophoretic mobility shift assays revealed that nuclear proteins extracted from HepG2 cells expressing PPARalpha specifically interacted with the -385/-373 DR-1 sequence of the human Delta-6 desaturase gene. The interaction was eliminated by the unlabeled PPARalpha RE of the rat acyl-CoA oxidase gene, and the protein-DNA complex was super-shifted by treatment with anti-PPARalpha. The -385/-373 sequence also interacted with a mixture of in vitro translated PPARalpha-retinoic acid receptor X (RXR)alpha, but by themselves neither PPARalpha nor RXRalpha could bind to the Delta-6 desaturase DR-1. These data indicate that the 5'-flanking region of the human Delta-6 desaturase gene contains a DR-1 that functions in the regulation of human Delta-6 desaturase gene transcription, and thereby plays a role in the synthesis of 20- and 22-carbon polyenoic fatty acids.

Dietary polyunsaturated fats regulate rat liver sterol regulatory element binding proteins-1 and -2 in three distinct stages and by different mechanisms.

Male Sprague-Dawley rats, trained to consume their daily energy needs in a single 3-h meal (0900-1200 h), were used to examine the hypothesis that polyunsaturated fatty acids (PUFA) lowered the nuclear content of sterol regulatory element binding protein (SREBP)-1 and/or -2 by suppressing the proteolytic release of mature SREBP from the membrane-anchored precursor pool. The nuclear concentrations of hepatic SREBP-1 and -2 were 50 and 42% lower (P < 0.05) in rats that consumed a single PUFA-supplemented meal (i.e., 10 g fish oil/100 g fat-free diet) than in rats fed the fat-free diet alone. This was paralleled by 63 and 52% reductions in the expression of the SREBP-1 and -2 target genes, fatty acid synthase and HMG-CoA synthase, respectively; but the marked increase in the amount of precursor SREBP-1 and -2 resulting from meal ingestion was unaffected. After the consumption of a second meal of fish oil, the nuclear level of mature SREBP-1 was only 16% of that in rats fed the fat-free diet, but the amount of nuclear SREBP-2 was not different from the level in rats fed the fat-free diet. Again, the sizes of the SREBP-1 and -2 precursor pools were not reduced. A decrease in the hepatic concentration of precursor SREBP-1 did not occur until rats had consumed 5 meals of fish oil. At this point, the nuclear content of SREBP-2 was actually twofold higher (P < 0.05) in rats fed fish oil or safflower oil, but the amount of precursor SREBP-2 was unaffected. These data indicate that PUFA suppress the in vivo proteolytic release of SREBP-1 and -2, but the effect on SREBP-2 is transitory, possibly reflecting the ability of PUFA to enhance cholesterol losses via bile acid synthesis.

The E-box like sterol regulatory element mediates the suppression of human Delta-6 desaturase gene by highly unsaturated fatty acids.

Delta-6 Desaturase (D6D) catalyzes the first step of the synthesis of highly unsaturated fatty acids (HUFA) that play pivotal roles in many biological functions. The D6D expression is under feedback regulation by dietary HUFA. We co-transfected D6D promoter-reporter constructs to HepG2 cells with an expression vector of nuclear form sterol regulatory element binding protein-1c (SREBP-1c). A 90-bp region of the D6D promoter was required for the activation by SREBP-1c as well as for the suppression of the promoter activity by HUFA. The region contained two candidates of sterol regulatory element (SRE). Mutation analysis identified E-box like SRE (SRE-2) as essential for both SREBP-1c activation and HUFA suppression. SRE-2 has a core sequence of CAGCAG, and is also conserved in stearoyl CoA desatruases. Because HUFA are primarily incorporated into phospholipids (PL), our results suggest that the primary role of SREBP-1c in liver is the regulation of fatty acid supply for PL rather than for triglycerides.

Fatty acid regulation of gene expression: a genomic explanation for the benefits of the mediterranean diet.

The development of obesity and associated insulin resistance involves a multitude of gene products, including proteins involved in lipid synthesis and oxidation, thermogenesis, and cell differentiation. The genes encoding these proteins are in essence the blueprints that we have inherited from our parents. However, what determines the way in which blueprints are interpreted is largely dictated by a collection of environmental factors. The nutrients we consume are among the most influential of these environmental factors. During the early stages of evolutionary development, nutrients functioned as primitive hormonal signals that allowed the early organisms to turn on pathways of synthesis or storage during periods of nutrient deprivation or excess. As single-cell organisms evolved into complex life forms, nutrients continued to be environmental factors that interacted with hormonal signals to govern the expression of genes encoding proteins involved in energy metabolism, cell differentiation, and cell growth. Nutrients govern the tissue content and activity of different proteins by functioning as regulators of gene transcription, nuclear RNA processing, mRNA degradation, and mRNA translation, as well as functioning as posttranslational modifiers of proteins. One dietary constituent that has a strong influence on cell differentiation, growth, and metabolism is fat. The fatty acid component of dietary lipid not only influences hormonal signaling events by modifying membrane lipid composition, but fatty acids have a very strong direct influence on the molecular events that govern gene expression. In this review, we discuss the influence that (n-9), (n-6), and (n-3) fatty acids exert on gene expression in the liver and skeletal muscle and the impact this has on intra- and interorgan partitioning of metabolic fuels.

NF-Y involvement in the polyunsaturated fat inhibition of fatty acid synthase gene transcription.

Dietary polyunsaturated fats (PUFA) reduce the hepatic content of SREBP-1 65-75%, and this is paralleled by a comparable decrease in the expression of fatty acid synthase (FAS) gene. The close association between the nuclear content of SREBP-1 and FAS transcription has led to the conclusion that PUFA inhibit lipogenic gene transcription by suppressing SREBP-1 expression, but this conclusion is based upon correlative data. When in fact the SREBP-1/USF sites of the insulin response element of FAS were mutated, only 25% of the PUFA inhibition of FAS promoter activity was lost. On the other hand, mutating the -99/-93 NF-Y site reduced overall promoter activity 85%, and eliminated 50% of the PUFA suppression of FAS promoter activity. In addition, extended cloning and transfection-reporter assays revealed that the FAS gene contains a second PUFA response region (PUFA-RR) in the distal area of -7382/-6970. Interestingly, the distal PUFA-RR(FAS) has many similarities to the PUFA-RR of l-pyruvate kinase gene while the proximal PUFA-RR(FAS) is comparable to the PUFA-RR of the S14 and stearoyl-CoA desaturase genes.