Fatty acid desaturases introduce a double bond in a specific position of long-chain fatty acids, and are conserved across kingdoms. Degree of unsaturation of fatty acids affects physical properties of membrane phospholipids and stored triglycerides. In addition, metabolites of polyunsaturated fatty acids are used as signaling molecules in many organisms. Three desaturases, Delta9, Delta6, and Delta5, are present in humans. Delta-9 catalyzes synthesis of monounsaturated fatty acids. Oleic acid, a main product of Delta9 desaturase, is the major fatty acid in mammalian adipose triglycerides, and is also used for phospholipid and cholesteryl ester synthesis. Delta-6 and Delta5 desaturases are required for the synthesis of highly unsaturated fatty acids (HUFAs), which are mainly esterified into phospholipids and contribute to maintaining membrane fluidity. While HUFAs may be required for cold tolerance in plants and fish, the primary role of HUFAs in mammals is cell signaling. Arachidonic acid is required as substrates for eicosanoid synthesis, while docosahexaenoic acid is required in visual and neuronal functions. Desaturases in mammals are regulated at the transcriptional level. Reflecting overlapping functions, three desaturases share a common mechanism of a feedback regulation to maintain products in membrane phospholipids. At the same time, regulation of Delta9 desaturase differs from Delta6 and Delta5 desaturases because its products are incorporated into more diverse lipid groups. Combinations of multiple transcription factors achieve this sophisticated differential regulation.

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 Delta-6 desaturase. In this report, we describe the cloning, characterization, and expression of a mammalian Delta-6 desaturase. The open reading frames for mouse and human Delta-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 b5-like domain that is characteristic of nonmammalian Delta-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 Delta-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 Delta-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 Delta-6 desaturase expression in nonhepatic tissues may need to be reevaluated.

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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 ⌬-5 desaturase. This report describes the cloning and expression of the human ⌬-5 desaturase, and it compares the structural characteristics and nutritional regulation of the ⌬-5 and ⌬-6 desaturases. The open reading frame of the human ⌬-5 desaturase encodes a 444-amino acid peptide which is identical in size to the ⌬-6 desaturase and which shares 61% identity with the human ⌬-6 desaturase. The ⌬-5 desaturase contains two membrane-spanning domains, three histidine-rich regions, and a cytochrome b 5 domain that all align perfectly with the same domains located in the ⌬-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 ⌬-5 desaturase mRNA, but ⌬-5 desaturase was most abundant in the liver, brain, and heart. However, in all tissues, the abundance of ⌬-5 desaturase mRNA was much lower than that observed for the ⌬-6 desaturase. When rats were fed a diet containing 10% safflower oil or menhaden fish oil, the level of hepatic mRNA for ⌬-5 and ⌬-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 ⌬-5 and ⌬-6 desaturase genes reside in reverse orientation on chromosome 11 and that they are separated by <11,000 base pairs.

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. EC 50 ϳ10 M), 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 PPAR␣-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.Dietary polyunsaturated fatty acids (PUFA) 1 are effective hypolipidemic agents (1), and they exert this effect by coordinately suppressing hepatic lipid synthesis and secretion while inducing hepatic and skeletal muscle fatty acid oxidation (2-10). Dietary PUFA coordinately decrease the transcription of hepatic genes encoding glycolytic and lipogenic enzymes (fatty acid synthase, acetyl-CoA carboxylase, stearoyl-CoA desaturase, malic enzyme, L-pyruvate kinase, and glucokinase) (3, 11-15), whereas they concomitantly increase the transcription of genes encoding enzymes involved in fatty acid oxidation (carnitine palmitoyltransferase (16) and acyl-CoA oxidase (9)). The outcome is a decrease in hepatic lipogenesis and an increase in hepatic fatty acid oxidation and ketogenesis. Genes encoding the oxidative enzymes appear to be regulated by a common transcription factor, peroxisomal proliferator-activated receptor (PPAR) (9, 16 -21). Because PPARs are lipidactivated transcription factors, they have often been proposed as the "master switches" that regulate the expression of enzymes involved in lipid synthesis and degradation (19 -21). However, several lines of evidence, including studies with PPAR␣ knock-out mice, indicate that the PUFA suppression of lipogenic gene transcription does not directly involve PPAR␣ (22,23).De...