Effects of protein supplementation frequency on physiological responses associated with reproduction in beef cows.

The objective of this experiment was to determine if frequency of protein supplementation impacts physiological responses associated with reproduction in beef cows. Fourteen nonpregnant, nonlactating beef cows were ranked by age and BW and allocated to 3 groups. Groups were assigned to a 3 × 3 Latin square design, containing 3 periods of 21 d and the following treatments: 1) soybean meal supplementation daily (D), 2) soybean meal supplementation 3 times/week (3WK), and 3) soybean meal supplementation once/week (1WK). Within each period, cows were assigned to an estrus synchronization protocol: 100 µg of GnRH + controlled internal drug release device (CIDR) containing 1.38 g of progesterone (P4) on d 1, 25 mg of PGF2α on d 8, and CIDR removal + 100 µg of GnRH on d 11. Grass-seed straw was offered for ad libitum consumption. Soybean meal was individually supplemented at a daily rate of 1 kg/cow (as-fed basis). Moreover, 3WK was supplemented on d 0, 2, 4, 7, 9, 11, 14, 16, and 18 whereas 1WK was supplemented on d 4, 11, and 18. Blood samples were collected from 0 (before) to 72 h after supplementation on d 11 and 18 and analyzed for plasma urea-N (PUN). Samples collected from 0 to 12 h were also analyzed for plasma glucose, insulin, and P4 (d 18 only). Uterine flushing fluid was collected concurrently with blood sampling at 28 h for pH evaluation. Liver biopsies were performed concurrently with blood sampling at 0, 4, and 28 h and analyzed for mRNA expression of carbamoyl phosphate synthetase I (CPS-I; h 28) and CYP2C19 and CYP3A4 (h 0 and 4 on d 18). Plasma urea-N concentrations were greater (P < 0.01) for 1WK vs. 3WK from 20 to 72 h and greater (P < 0.01) for 1WK vs. D from 16 to 48 h and at 72 h after supplementation (treatment × hour interaction, P < 0.01). Moreover, PUN concentrations peaked at 28 h after supplementation for 3WK and 1WK (P < 0.01) and were greater (P < 0.01) at this time for 1WK vs. 3WK and D and for 3WK vs. D. Expression of CPS-I was greater (P < 0.01) for 1WK vs. D and 3WK. Uterine flushing pH tended (P ≤ 0.10) to be greater for 1WK vs. 3WK and D. No treatment effects were detected (P ≥ 0.15) on expression of CYP2C19 and CYP3A4, plasma glucose, and P4 concentrations, whereas plasma insulin concentrations were greater (P ≤ 0.03) in D and 3WK vs. 1WK. Hence, decreasing frequency of protein supplementation did not reduce uterine flushing pH or plasma P4 concentrations, which are known to impact reproduction in beef cows.

Effects of calcium salts of soybean oil on factors that influence pregnancy establishment in Bos indicus beef cows.

The objective of this experiment was to compare fatty acid (FA) concentrations in plasma and reproductive tissues as well as hormones and expression of genes associated with pregnancy establishment in beef cows supplemented or not with Ca salts of soybean oil (CSSO) beginning after timed AI. Ninety nonlactating multiparous Nelore (Bos indicus) cows were timed inseminated on d 0 of the experiment and divided into 18 groups of 5 cows/group. Groups were randomly assigned to receive (as-fed basis) 100 g of a protein-mineral mix plus 100 g of ground corn per cow daily in addition to 1) 100 g/cow daily of CSSO (n = 9) or 2) 100 g/cow daily of kaolin (CON; rumen-inert indigestible substance; n = 9). All groups were maintained in a single Brachiaria brizanta pasture (24 ha) with ad libitum access to forage and water. However, groups were segregated daily and offered treatments individually at the working facility during the experimental period (d 0 to 18). Blood samples were collected and transrectal ultrasonography was performed to verify ovulation and estimate corpus luteum (CL) volume immediately before AI (d 0) and on d 7 and 18 of the experiment. On d 19, 36 cows (18 cows/treatment; 2 cows/group) diagnosed without the presence of a CL on d 0 but with a CL greater than 0.38 cm(3) in volume on d 7 and 18 were slaughtered for collection of conceptus, uterine luminal flushing, and tissue samples from the CL and endometrium. Cows receiving CSSO had greater concentrations of linoleic and other ω-6 FA in plasma (P < 0.01), endometrium (P ≤ 0.05), CL (P ≤ 0.05), and conceptus (P ≤ 0.08) compared to CON. On d 7 of the experiment, CSSO-supplemented cows had greater plasma progesterone concentrations (P < 0.01) and CL volume (P = 0.02) compared to CON, whereas no treatment effects were detected (P ≥ 0.15) for these parameters on d 18 (treatment × day interaction; P < 0.01). Cows receiving CSSO tended (P = 0.09) to have greater concentrations of interferon-tau in the uterine flushing media compared with CON. However, no treatment effects were detected for mRNA expression genes associated with pregnancy establishment in endometrial, CL, and conceptus samples (P ≥ 0.12). In summary, supplementing beef cows with 100 g of CSSO beginning after AI favored incorporation of ω-6 FA into their circulation, reproductive tissues, and conceptus, without impacting expression of genes associated with pregnancy establishment on d 19 of gestation.

Functional interaction between sterol regulatory element-binding protein-1c, nuclear factor Y, and 3,5,3'-triiodothyronine nuclear receptors.

Sterol regulatory element binding protein-1c (SREBP-1c) 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-1c.NF-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-1c.NF-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.

Evidence against the peroxisome proliferator-activated receptor alpha (PPARalpha) as the mediator for polyunsaturated fatty acid suppression of hepatic L-pyruvate kinase gene transcription.

The glycolytic enzyme, L-pyruvate kinase (L-PK), plays an important role in hepatic glucose metabolism. Insulin and glucose induce L-PK gene expression, while glucagon and polyunsaturated fatty acids (PUFA) inhibit L-PK gene expression. We have been interested in defining the PUFA regulation of L-PK. The cis-regulatory target for PUFA action includes an imperfect direct repeat (DR1) that binds HNF-4. HNF4 plays an ancillary role in the insulin/glucose-mediated transactivation of the L-PK gene. Because the fatty acid-activated nuclear receptor, peroxisome proliferator-activated receptor (PPARalpha), binds DR1-like elements and has been reported to interfere with HNF4 action, we examined the role PPARalpha plays in the regulation of L-PK gene transcription. Feeding rats either fish oil or the potent PPARalpha activator, WY14,643, suppressed rat hepatic L-PK mRNA and gene transcription. The PPARalpha-null mouse was used to evaluate the role of the PPARalpha in hepatic transcriptional control of L-PK. While WY14,643 control of L-PK gene expression required the PPARalpha, PUFA regulation of L-PK gene expression was independent of the PPARalpha. Transfection studies in cultured primary hepatocytes localized the cis-regulatory target for WY14,643/PPARalpha action to the L-PK HNF4 binding site. However, PPARalpha/RXRalpha heterodimers did not bind this region. Although both WY14,643 and PUFA suppress L-PK gene transcription through the same element, PUFA regulation of L-PK does not require the PPARalpha and PPARalpha/RXRalpha does not bind the L-PK promoter. These studies suggest that other intermediary factors are involved in both the PUFA and PPARalpha regulation of L-PK gene transcription.

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 alpha (PPARalpha)- 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 PPARalpha-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 alpha, 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.

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.

Regulation of gene expression by dietary fat.

Dietary fat is an important macronutrient for the growth and development of all organisms. In addition to its role as an energy source and its effects on membrane lipid composition, dietary fat has profound effects on gene expression, leading to changes in metabolism, growth, and cell differentiation. The effects of dietary fat on gene expression reflect an adaptive response to changes in the quantity and type of fat ingested. Specific fatty acid-regulated transcription factors have been identified in bacteria, amphibians, and mammals. In mammals, these factors include peroxisome proliferator-activated receptors (PPAR alpha, -beta, and -gamma), HNF4 alpha, NF kappa B, and SREBP1c. These factors are regulated by (a) direct binding of fatty acids, fatty acyl-coenzyme A, or oxidized fatty acids; (b) oxidized fatty acid (eicosanoid) regulation of G-protein-linked cell surface receptors and activation of signaling cascades targeting the nucleus; or (c) oxidized fatty acid regulation of intracellular calcium levels, which affect cell signaling cascades targeting the nucleus. At the cellular level, the physiological response to fatty acids will depend on (a) the quantity, chemistry, and duration of the fat ingested; (b) cell-specific fatty acid metabolism (oxidative pathways, kinetics, and competing reactions); (c) cellular abundance of specific nuclear and membrane receptors; and (d) involvement of specific transcription factors in gene expression. These mechanisms are involved in the control of carbohydrate and lipid metabolism, cell differentiation and growth, and cytokine, adhesion molecule, and eicosanoid production. The effects of fatty acids on the genome provide new insight into how dietary fat might play a role in health and disease.

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 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.

Arachidonic acid and PGE2 regulation of hepatic lipogenic gene expression.

N-6 polyunsaturated fatty acids (PUFA) suppress hepatic and adipocyte de novo lipogenesis by inhibiting the transcription of genes encoding key lipogenic proteins. In cultured 3T3-L1 adipocytes, arachidonic acid (20:4,n-6) suppression of lipogenic gene expression requires cyclooxygenase (COX) activity. In this study, we found no evidence to support a role for COX-1 or -2 in the 20:4,n-6 inhibition of hepatocyte lipogenic gene expression. In contrast to L1 preadipocytes, adipocytes and rat liver, RT-PCR and Western analyses did not detect COX-1 or COX-2 expression in cultured primary hepatocytes. Moreover, the COX inhibitor, flurbiprofen, did not affect the 20:4,n-6 regulation of lipogenic gene expression in primary hepatocytes. Despite the absence of COX-1 and -2 expression in primary hepatocytes, prostaglandins (PGE2 and PGF2alpha) suppressed fatty acid synthase, l-pyruvate kinase, and the S14 protein mRNA, while having no effect on acyl-CoA oxidase or CYP4A2 mRNA. Using PGE2 receptor agonist, the PGE2 effect on lipogenic gene expression was linked to EP3 receptors. PGE2 inhibited S14CAT activity in transfected primary hepatocytes and targeted the S14 PUFA-response region located -220 to -80 bp upstream from the transcription start site. Taken together, these studies show that COX-1 and COX-2 do not contribute to the n-6 PUFA suppression of hepatocyte lipogenic gene expression. However, cyclooxygenase products from non-parenchymal cells can act on parenchymal cells through a paracrine process and mimic the effects of n-6 PUFA on lipogenic gene expression.

Arachidonic acid inhibits lipogenic gene expression in 3T3-L1 adipocytes through a prostanoid pathway.

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 beta-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 ED50 of 800, 50, and 400 microM, 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 (PGE2 and PGF2alpha at 10 microM) inhibited CAT activity through a pertussis toxin-sensitive Gi/Go-coupled 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.

Polyunsaturated fatty acid inhibition of fatty acid synthase transcription is independent of PPAR activation.

Polyunsaturated fatty acids (PUFA) of the (n-6) and (n-3) families inhibit the rate of gene transcription for a number of hepatic lipogenic and glycolytic genes, e.g., fatty acid synthase (FAS). In contrast, saturated and monounsaturated fatty acids have no inhibitory capability. The suppression of gene transcription resulting from the addition of PUFA to a high carbohydrate diet: occurs quickly (< 3 h) after its addition to a high glucose diet; can be recreated with hepatocytes cultured in a serum-free medium containing insulin and glucocorticoids; can be demonstrated in diabetic rats fed fructose; and is independent of glucagon. While the nature of the intracellular PUFA inhibitor is unclear, it appears that delta-6 desaturation is a required step in the process. Recently, the fatty acid activated nuclear factor, peroxisome-proliferator activated receptor (PPAR) was suggested to be the PUFA-response factor. However, the potent PPAR activators ETYA and Wy-14643 did not suppress hepatic expression of FAS, but did induce the PPAR-responsive gene, acyl-CoA oxidase (AOX). Similarly, treating rat hepatocytes with 20:4 (n-6) suppressed FAS expression but had no effect on AOX. Thus, it appears that the PUFA regulation of gene transcription involves a PUFA-response factor that is independent from PPAR.

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 (TRbeta) 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 TRbeta functionally interact to confer T3 control to the S14 gene.

Fatty acid regulation of gene expression. Its role in fuel partitioning and insulin resistance.

Dietary polyenoic (n-6) and (n-3) fatty acids uniquely regulate fatty acid biosynthesis and fatty acid oxidation. They exercise this effect by modulating the expression of genes coding for key metabolic enzymes and, in doing this, PUFA govern the intracellular as well as the interorgan metabolism of glucose and fatty acids. During the past 20 years, we have gradually elucidated the cellular and molecular mechanism by which dietary PUFA regulate lipid metabolism. Central to this mechanism has been our ability to determine that dietary PUFA regulate the transcription of genes. We have only begun to elucidate the nuclear mechanisms by which PUFA govern gene expression, but one point is clear and that is that it is unlikely that one mechanism will explain the variety of genes governed by PUFA. The difficulty in providing a unifying hypothesis at this time stems from (a) the many metabolic routes taken by PUFA upon entering a cell and (b) the lack of identity of a specific PUFA-regulated trans-acting factor. Nevertheless, our studies have revealed that PUFA are not only utilized as fuel and structural components of cells, but also serve as important mediators of gene expression, and that in this way they influence the metabolic directions of fuels and they modulate the development of nutritionally related pathophysiologies such as diabetes.

Polyunsaturated fatty acid suppression of hepatic fatty acid synthase and S14 gene expression does not require peroxisome proliferator-activated receptor alpha.

Dietary polyunsaturated fatty acids (PUFA) induce hepatic peroxisomal and microsomal fatty acid oxidation and suppress lipogenic gene expression. The peroxisome proliferator-activated receptor alpha (PPARalpha) has been implicated as a mediator of fatty acid effects on gene transcription. This report uses the PPARalpha-deficient mouse to examine the role of PPARalpha in the PUFA regulation of mRNAs encoding hepatic lipogenic (fatty acid synthase (FAS) and the S14 protein (S14)), microsomal (cytochrome P450 4A2 (CYP4A2)), and peroxisomal (acyl-CoA oxidase (AOX)) enzymes. PUFA ingestion induced mRNAAOX (2.3-fold) and mRNACYP4A2 (8-fold) and suppressed mRNAFAS and mRNAS14 by >/=80% in wild type mice. In PPARalpha-deficient mice, PUFA did not induce mRNAAOX or mRNACYP4A2, indicating a requirement for PPARalpha in the PUFA-mediated induction of these enzymes. However, PUFA still suppressed mRNAFAS and mRNAS14 in the PPARalpha-deficient mice. Studies in rats provided additional support for the differential regulation of lipogenic and peroxisomal enzymes by PUFA. These studies provide evidence for two distinct pathways for PUFA control of hepatic lipid metabolism. One requires PPARalpha and is involved in regulating peroxisomal and microsomal enzymes. The other pathway does not require PPARalpha and is involved in the PUFA-mediated suppression of lipogenic gene expression.

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.

A GC-rich region containing Sp1 and Sp1-like binding sites is a crucial regulatory motif for fatty acid synthase gene promoter activity in adipocytes. Implication In the overactivity of FAS promoter in obese Zucker rats.

We have previously shown that the proximal 2-kb sequence of the fatty acid synthase (FAS) promoter transfected into rat adipocytes was highly sensitive to the cellular context, displaying an overactivity in obese (fa/fa) versus lean Zucker rat adipocytes. Using deletional analysis, we show here that FAS promoter activity mainly depends on a region from -200 to -126. This sequence exerts a strong negative effect on FAS promoter in adipocytes from lean rats but not in those from obese rats, resulting in a marked overtranscriptional activity in the latter cells. This region, fused to a heterologous promoter, the E1b TATA box, induced differential levels of gene reporter activity in lean and obese rat adipocytes, indicating it harbors fa-responsive element(s). Whatever the rat genotype, adipocyte nuclear proteins were shown to footprint the same protected sequence within the fa-responsive region, and supershift analysis demonstrated that Sp1 or Sp1-like proteins were bound to this DNA subregion. Compelling evidence that the Sp1 binding site contained in this sequence was implicated in the differential promoter activity in lean versus obese rats, was provided by the observation that mutations at this Sp1 site induced a 2.5-fold increase in FAS promoter activity in adipocytes from lean rats, whereas they had no effect in adipocytes from obese rats.

Peroxisome proliferator-activated receptor alpha inhibits hepatic S14 gene transcription. Evidence against the peroxisome proliferator-activated receptor alpha as the mediator of polyunsaturated fatty acid regulation of s14 gene transcription.

The peroxisome proliferator-activated receptor (PPARalpha) 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 PPARalpha plays in fatty acid regulation of hepatic lipogenic gene transcription. Both PPARalpha and the potent peroxisome proliferator, WY14643, inhibit S14CAT activity in transfected primary hepatocytes. WY14643 and PPARalpha 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 PPARalpha treatment. Gel shift analysis showed that PPARalpha, either alone or with RXRalpha, did not bind the S14TRR. However, PPARalpha interfered with TRbeta/RXRalpha binding to a TRE (DR+4). Functional studies showed that co-transfected RXRalpha, but not T3 receptor beta1 (TRbeta1), abrogated the inhibitory effect of PPARalpha on S14 gene transcription. These results suggest that WY14643 and PPARalpha functionally interfere with T3 regulation of S14 gene transcription by inhibiting TRbeta1/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/PPARalpha and PUFA are functionally and spatially distinct argues against PPARalpha as the mediator of PUFA suppression of S14 gene transcription.

Polyunsaturated fatty acid regulation of hepatic gene transcription.

Polyunsaturated fatty acids (PUFA) modulate the rate of gene transcription for a number of different genes including hepatic lipogenic and glycolytic genes, adipose Glut-4 and stearoyl-CoA desaturase and interleukins. Some of the transcriptional effects of PUFA appear to be mediated by eicosanoids, but the PUFA suppression of lipogenic and glycolytic genes is independent of eicosanoid synthesis and appears to involve a nuclear mechanism directly modified by PUFA. With the recent cloning of a fatty acid-activated nuclear factor termed peroxisome-proliferator- activated receptor (PPAR) has come the suggestion that PPAR may be the PUFA response factor. However, this review presents several lines of evidence that indicate that the PPAR and PUFA regulation of gene transcription involves separate and independent mechanisms, and the PPAR is not the PUFA response factor.

Polyunsaturated fatty acid regulation of hepatic gene transcription.

Polyunsaturated fatty acids (PUFA) of the n-6 and n-3 families inhibit transcription of a number of hepatic lipogenic and glycolytic genes, e.g. fatty acid synthase. In contrast, saturated and monounsaturated fatty acids exert no suppressive action on lipogenic gene expression. The unique PUFA regulation of gene expression extends beyond the liver to include genes such as adipocyte glucose transporter-4, lymphocyte stearoyl-CoA desaturase 2, and interleukins. Some of the transcriptional effects of PUFA appear to be mediated by eicosanoids, but PUFA suppression of lipogenic and glycolytic genes is independent of eicosanoid synthesis and appears to involve a nuclear mechanism directly modified by PUFA. With the recent cloning of a fatty acid-activated nuclear factor termed peroxisome-proliferator-activated receptor (PPAR) has come the suggestion that PPAR may be the PUFA response factor. This review, however, presents several lines of evidence that indicate that the PPAR and n-6 and n-3 PUFA regulation of lipogenic and glycolytic gene transcription involve separate and independent mechanisms. Thus PPAR appears not to be the PUFA response factor.