Steven D. Clarke

Cloning, Expression, and Nutritional Regulation of the Mammalian Δ-6 Desaturase

Arachidonic acid (20:4(n-6)) and docosahexaenoic acid (22:6(n-3)) have a variety of physiological functions that include being the major component of membrane phospholipid in brain and retina, substrates for eicosanoid production, and regulators of nuclear transcription factors. The rate-limiting step in the production of 20:4(n-6) and 22:6(n-3) is the desaturation of 18:2(n-6) and 18:3(n-3) by Δ-6 desaturase. In this report, we describe the cloning, characterization, and expression of a mammalian Δ-6 desaturase. The open reading frames for mouse and human Δ-6 desaturase each encode a 444-amino acid peptide, and the two peptides share an 87% amino acid homology. The amino acid sequence predicts that the peptide contains two membrane-spanning domains as well as a cytochrome b 5-like domain that is characteristic of nonmammalian Δ-6 desaturases. Expression of the open reading frame in rat hepatocytes and Chinese hamster ovary cells instilled in these cells the ability to convert 18:2(n-6) and 18:3(n-3) to their respective products, 18:3(n-6) and 18:4(n-3). When mice were fed a diet containing 10% fat, hepatic enzymatic activity and mRNA abundance for hepatic Δ-6 desaturase in mice fed corn oil were 70 and 50% lower than in mice fed triolein. Finally, Northern analysis revealed that the brain contained an amount of Δ-6 desaturase mRNA that was several times greater than that found in other tissues including the liver, lung, heart, and skeletal muscle. The RNA abundance data indicate that prior conclusions regarding the low level of Δ-6 desaturase expression in nonhepatic tissues may need to be reevaluated.

Cloning, Expression, and Fatty Acid Regulation of the Human Δ-5 Desaturase

Arachidonic (20:4(n-6)), eicosapentaenoic (20:5(n-3)), and docosahexaenoic (22:6(n-3)) acids are major components of brain and retina phospholipids, substrates for eicosanoid production, and regulators of nuclear transcription factors. One of the two rate-limiting steps in the production of these polyenoic fatty acids is the desaturation of 20:3(n-6) and 20:4(n-3) by Δ-5 desaturase. This report describes the cloning and expression of the human Δ-5 desaturase, and it compares the structural characteristics and nutritional regulation of the Δ-5 and Δ-6 desaturases. The open reading frame of the human Δ-5 desaturase encodes a 444-amino acid peptide which is identical in size to the Δ-6 desaturase and which shares 61% identity with the human Δ-6 desaturase. The Δ-5 desaturase contains two membrane-spanning domains, three histidine-rich regions, and a cytochrome b5 domain that all align perfectly with the same domains located in the Δ-6 desaturase. Expression of the open reading frame in Chinese hamster ovary cells instilled the ability to convert 20:3(n-6) to 20:4(n-6). Northern analysis revealed that many human tissues including skeletal muscle, lung, placenta, kidney, and pancreas expressed Δ-5 desaturase mRNA, but Δ-5 desaturase was most abundant in the liver, brain, and heart. However, in all tissues, the abundance of Δ-5 desaturase mRNA was much lower than that observed for the Δ-6 desaturase. When rats were fed a diet containing 10% safflower oil or menhaden fish oil, the level of hepatic mRNA for Δ-5 and Δ-6 desaturase was only 25% of that found in the liver of rats fed a fat-free diet or a diet containing triolein. Finally, a BLAST and Genemap search of the human genome revealed that the Δ-5 and Δ-6 desaturase genes reside in reverse orientation on chromosome 11 and that they are separated by <11,000 base pairs.

Sterol regulatory element binding protein-1 expression is suppressed by dietary polyunsaturated fatty acids. A mechanism for the coordinate suppression of lipogenic genes by polyunsaturated fats.

Polyunsaturated fatty acids (PUFA) coordinately suppress the transcription of a wide array of hepatic lipogenic genes including fatty acid synthase (FAS) and acetyl-CoA carboxylase. Interestingly, the over-expression of sterol regulatory element binding protein-1 (SREBP-1) induces the expression of all of the enzymes suppressed by PUFA. This observation led us to hypothesize that PUFA coordinately inhibit lipogenic gene transcription by suppressing the expression of SREBP-1. Our initial studies revealed that the SREBP-1 and FAS mRNA contents of HepG2 cells were reduced by 20:4(n-6) in a dose-dependent manner (i.e. EC50 ∼10 μm), whereas 18:1(n-9) had no effect. Similarly, supplementing a fat-free, high glucose diet with oils rich in (n-6) or (n-3) PUFA reduced the hepatic content of precursor and nuclear SREBP-1 60 and 85%, respectively; however, PUFA had no effect on the nuclear content of upstream stimulatory factor (USF)-1. The PUFA-dependent decrease in nuclear content of mature SREBP-1 was paralleled by a 70–90% suppression in FAS gene transcription. In contrast, dietary 18:1(n-9),i.e. triolein, had no inhibitory influence on the expression of SREBP-1 or FAS. The decrease in hepatic expression of SREBP-1 and FAS associated with PUFA ingestion was mimicked by supplementing the fat-free diet with the PPARα-activator, WY 14,643. Interestingly, nuclear run-on assays revealed that changes in SREBP-1 mRNA abundance were not accompanied by changes in SREBP-1 gene transcription. These results support the concept that PUFA coordinately inhibit lipogenic gene transcription by suppressing the expression of SREBP-1 and that the PUFA regulation of SREBP-1 appears to occur at the post-transcriptional level.

Polyunsaturated fatty acid regulation of gene transcription: a mechanism to improve energy balance and insulin resistance.

This review addresses the hypothesis that polyunsaturated fatty acids (PUFA), particularly those of the n-3 family, play essential roles in the maintenance of energy balance and glucose metabolism. The data discussed indicate that dietary PUFA function as fuel partitioners in that they direct glucose toward glycogen storage, and direct fatty acids away from triglyceride synthesis and assimilation and toward fatty acid oxidation. In addition, the n-3 family of PUFA appear to have the unique ability to enhance thermogenesis and thereby reduce the efficiency of body fat deposition. PUFA exert their effects on lipid metabolism and thermogenesis by upregulating the transcription of the mitochondrial uncoupling protein-3, and inducing genes encoding proteins involved in fatty acid oxidation (e.g. carnitine palmitoyltransferase and acyl-CoA oxidase) while simultaneously down-regulating the transcription of genes encoding proteins involved in lipid synthesis (e.g. fatty acid synthase). The potential transcriptional mechanism and the transcription factors affected by PUFA are discussed. Moreover, the data are interpreted in the context of the role that PUFA may play as dietary factors in the development of obesity and insulin resistance. Collectively the results of these studies suggest that the metabolic functions governed by PUFA should be considered as part of the criteria utilized in defining the dietary needs for n-6 and n-3 PUFA, and in establishing the optimum dietary ratio for n-6:n-3 fatty acids.

Omega-3 polyunsaturated fatty acid regulation of gene expression.

This review describes the mechanisms by which polyunsaturated fatty acids regulate the activity of the nuclear transcription factors, peroxisome proliferator-activated receptor and sterol regulatory element binding protein-1, and it describes the role that the peroxisome proliferator-activated receptor and sterol regulatory element binding protein-1 play in coordinating the regulation of lipid synthesis, lipid oxidation, and thermogenesis. Finally, the requirement for dietary polyunsaturated fatty acids, particularly n-3 fatty acids, is defined in terms of the effects polyunsaturated fatty acids exert on gene expression and the role that these effects play in overall energy balance.

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.

Fatty Acid Regulation of Gene Expression

Abstract: 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.

Coordinate induction of peroxisomal acyl-CoA oxidase and UCP-3 by dietary fish oil: a mechanism for decreased body fat deposition.

Rats fed dietary fats rich in 20- and 22-carbon polyenoic fatty acids deposit less fat and expend more energy at rest than rats fed other types of fats. We hypothesized that this decrease in energetic efficiency was the product of: (a) enhanced peroxisomal fatty acid oxidation and/or (b) the up-regulation of genes encoding proteins that were involved with enhanced heat production, i.e. mitochondrial uncoupling proteins (UCP-2, UCP-3) and peroxisomal fatty acid oxidation proteins. Two groups of male Fisher 344 rats 3-4 week old (n=5 per group) were pair fed for 6 weeks a diet containing 40% of its energy fat derived from either fish oil or corn oil. Epididymal fat pads from rats fed the fish oil diet weighed 25% (P < 0.05) less than those found in rats fed corn oil. The decrease in fat deposition associated with fish oil ingestion was accompanied by a significant increase in the abundance of skeletal muscle UCP-3 mRNA. The level of UCP-2 mRNA skeletal muscle was unaffected by the type of dietary oil, but the abundance of UCP-2 mRNA in the liver and heart were significantly lower (P < 0.05) in rats fed fish oil than in rats fed corn oil. In addition to inducing UCP-3 expression, dietary fish oil induced peroxisomal acyl-CoA oxidase gene expression 2-3 fold in liver, skeletal muscle and heart. These data support the hypothesis that dietary fish oil reduces fat deposition by increasing the expression of mitochondrial uncoupling proteins and increasing fatty acid oxidation by the less efficient peroxisomal pathway.

Polyunsaturated fatty acids and gene expression.

Purpose of reviewThis 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 findingsRecent 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. SummaryThe 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.

Cellular lipid binding proteins: expression, function, and nutritional regulation.

The membrane transport and cytosolic solubilization of hydrophobic ligands, including sterols, fatty acids, retinoids, and certain hydrophobic carcinogens, are facilitated by a group of similar low molecular weight proteins: plasma membrane transport protein, fatty acid binding proteins, sterol carrier protein, and retinoid binding proteins. The cellular content of these proteins, which establishes the capacity of a cell to utilize the various ligands, is determined by events regulating transcription and translation, e.g., the mRNA abundance of liver‐ and gut‐type FABPs is increased by dietary fat, and translation of hepatic FABP appears to be stimulated by insulin. Functions attributable to these lipid binding proteins remain unclear, but data are presented that indicate physiological roles in 1) fatty acid transport, esterification, and oxidation, 2) steroidogenesis, and 3) retinoid uptake, retinaldehyde reduction, and retinol esterification. An exciting and novel prospect for cellular trafficking proteins is the role they may play in regulating gene expression. In this respect, cellular lipid binding proteins, e.g., retinoid binding proteins, may deliver their ligands to nuclear trans‐acting proteins, and thereby modulate genes coding for key proteins involved in lipid metabolism or differentiation. Even though the functions of these proteins still need to be unequivocally established, it is clear that they are important in the overall homeostasis of lipid metabolism.—Clarke, S. D.; Armstrong, M. K. Cellular lipid binding proteins: expression, function, and nutritional regulation. FASEB J. 3: 2480‐2487; 1989.