Essential fatty acids: biochemistry, physiology and pathology

Essential fatty acids (EFAs), linoleic acid (LA), and alpha-linolenic acid (ALA) are essential for humans, and are freely available in the diet. Hence, EFA deficiency is extremely rare in humans. To derive the full benefits of EFAs, they need to be metabolized to their respective long-chain metabolites, i.e., dihomo-gamma-linolenic acid (DGLA), and arachidonic acid (AA) from LA; and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from ALA. Some of these long-chain metabolites not only form precursors to respective prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LTs), but also give rise to lipoxins (LXs) and resolvins that have potent anti-inflammatory actions. Furthermore, EFAs and their metabolites may function as endogenous angiotensin-converting enzyme and 3-hdroxy-3-methylglutaryl coenzyme A reductase inhibitors, nitric oxide (NO) enhancers, anti-hypertensives, and anti-atherosclerotic molecules. Recent studies revealed that EFAs react with NO to yield respective nitroalkene derivatives that exert cell-signaling actions via ligation and activation of peroxisome proliferator-activated receptors. The metabolism of EFAs is altered in several diseases such as obesity, hypertension, diabetes mellitus, coronary heart disease, schizophrenia, Alzheimer's disease, atherosclerosis, and cancer. Thus, EFAs and their derivatives have varied biological actions and seem to be involved in several physiological and pathological processes.     

Functional characterization of desaturases involved in the formation of the terminal double bond of an unusual 16:3Delta(9,12,150) fatty acid isolated from Sorghum bicolor root hairs

Sorgoleone, produced in root hair cells of sorghum (Sorghum bicolor), is likely responsible for much of the allelopathic properties of sorghum root exudates against broadleaf and grass weeds. Previous studies suggest that the biosynthetic pathway of this compound initiates with the synthesis of an unusual 16:3 fatty acid possessing a terminal double bond. The corresponding fatty acyl-CoA serves as a starter unit for polyketide synthases, resulting in the formation of 5-pentadecatrienyl resorcinol. This resorcinolic intermediate is then methylated by an S-adenosylmethionine-dependent O-methyltransferase and subsequently dihydroxylated, yielding the reduced (hydroquinone) form of sorgoleone. To characterize the corresponding enzymes responsible for the biosynthesis of the 16:3 fatty acyl-CoA precursor, we identified and cloned three putative fatty acid desaturases, designated SbDES1, SbDES2, and SbDES3, from an expressed sequence tag (EST) data base prepared from isolated root hairs. Quantitative real-time RT-PCR analyses revealed that these three genes were preferentially expressed in sorghum root hairs where the 16:2 and 16:3 fatty acids were exclusively localized. Heterologous expression of the cDNAs in Saccharomyces cerevisiae revealed that recombinant SbDES2 converted palmitoleic acid (16:1Delta(9)) to hexadecadienoic acid (16:2Delta(9,12)), and that recombinant SbDES3 was capable of converting hexadecadienoic acid into hexadecatrienoic acid (16:3Delta(9,12,15)). Unlike other desaturases reported to date, the double bond introduced by SbDES3 occurred between carbons 15 and 16 resulting in a terminal double bond aliphatic chain. Collectively, the present results strongly suggest that these fatty acid desaturases represent key enzymes involved in the biosynthesis of the allelochemical sorgoleone.     

Long-chain polyunsaturated fatty acids, endothelial lipase and atherosclerosis

Endothelial lipase (EL), a new member of the lipase gene family, was recently cloned and has been shown to have a significant role in modulating the concentrations of plasma high-density lipoprotein levels (HDL). EL is closely related to lipoprotein and hepatic lipases both in structure and function. It is primarily synthesized by endothelial cells, functions at the cell surface, and shows phospholipase A1 activity. Overexpression of EL decreases HDL cholesterol levels whereas blocking its action increases concentrations of HDL cholesterol. Pro-inflammatory cytokines suppress plasma HDL cholesterol concentrations by enhancing the activity of EL. On the other hand, physical exercise and fish oil (a rich source of eicosapentaenoic acid and docosahexaenoic acid) suppress the activity of EL and this, in turn, enhances the plasma concentrations of HDL cholesterol. Thus, EL plays a critical role in the regulation of plasma HDL cholesterol concentrations and thus modulates the development and progression of atherosclerosis. The expression and actions of EL in specific endothelial cells determines the initiation and progression of atherosclerosis locally explaining the patchy nature of atheroma seen, especially, in coronary arteries. Both HDL cholesterol and EPA and DHA enhance endothelial nitric oxide (eNO) and prostacyclin (PGI2) synthesis, which are known to prevent atherosclerosis. On the other hand, pro-inflammatory cytokines augment free radical generation, which are known to inactivate eNO and PGI2. Thus, interactions between EL, pro- and anti-inflammatory cytokines, polyunsaturated fatty acids, and the ability of endothelial cells to generate NO and PGI2 and neutralize the actions of free radicals may play a critical role in atherosclerosis.     

Cloning and characterization of unusual fatty acid desaturases from Anemone leveillei: identification of an acyl-coenzyme A C20 Delta5-desaturase responsible for the synthesis of sciadonic acid

The seed oil of Anemone leveillei contains significant amounts of sciadonic acid (20:3Delta(5,11,14); SA), an unusual non-methylene-interrupted fatty acid with pharmaceutical potential similar to arachidonic acid. Two candidate cDNAs (AL10 and AL21) for the C(20) Delta(5cis)-desaturase from developing seeds of A. leveillei were functionally characterized in transgenic Arabidopsis (Arabidopsis thaliana) plants. The open reading frames of both Delta(5)-desaturases showed some similarity to presumptive acyl-coenzyme A (CoA) desaturases found in animals and plants. When expressed in transgenic Arabidopsis, AL21 showed a broad range of substrate specificity, utilizing both saturated (16:0 and 18:0) and unsaturated (18:2, n-6 and 18:3, n-3) substrates. In contrast, AL10 did not show any activity in wild-type Arabidopsis. Coexpression of AL10 or AL21 with a C(18) Delta(9)-elongase in transgenic Arabidopsis plants resulted in the production of SA and juniperonic fatty acid (20:4Delta(5,11,14,17)). Thus, AL10 acted only on C(20) polyunsaturated fatty acids in a manner analogous to "front-end" desaturases. However, neither AL10 nor AL21 contain the cytochrome b(5) domain normally present in this class of enzymes. Acyl-CoA profiling of transgenic Arabidopsis plants and developing A. leveillei seeds revealed significant accumulation of Delta(5)-unsaturated fatty acids as acyl-CoAs compared to the accumulation of these fatty acids in total lipids. Positional analysis of triacylglycerols of A. leveillei seeds showed that Delta(5)-desaturated fatty acids were present in both sn-2 and sn-1 + sn-3 positions, although the majority of 16:1Delta(5), 18:1Delta(5), and SA was present at the sn-2 position. Our data provide biochemical evidence for the A. leveillei Delta(5)-desaturases using acyl-CoA substrates.     

Identification of a fatty acid Delta11-desaturase from the microalga Thalassiosira pseudonana

A set of genomic DNA sequences putatively encoding front-end desaturases were identified by in silico analysis of the draft genome of the marine microalga Thalassiosira pseudonana. Among these candidate genes, an open reading frame named TpdesN was found to be full-length, intronless, and constitutively expressed during cell cultivation. The predicted amino acid sequence of the corresponding protein, TpDESN, exhibited typical features of desaturases involved in the production of polyunsaturated fatty acids (PUFAs) in algae, i.e. a cytochrome b5-like domain at the N-terminus and three conserved histidine-rich motifs in the desaturase domain. Expression of TpDESN in Saccharomyces cerevisiae revealed that this enzyme was not involved in PUFA synthesis, but specifically desaturated palmitic acid 16:0 to 16:1Delta11. To our knowledge, until this report, Delta11-desaturase activity had only been detected in insect cells.     

A bifunctional Delta12,Delta15-desaturase from Acanthamoeba castellanii directs the synthesis of highly unusual n-1 series unsaturated fatty acids

The free-living soil protozoon Acanthamoeba castellanii synthesizes a range of polyunsaturated fatty acids, the balance of which can be altered by environmental changes. We have isolated and functionally characterized in yeast a microsomal desaturase from A. castellanii, which catalyzes the sequential conversion of C(16) and C(18) Delta9-monounsaturated fatty acids to di- and tri-unsaturated forms. In the case of C(16) substrates, this bifunctional A. castellanii Delta12,Delta15-desaturase generated a highly unusual fatty acid, hexadecatrienoic acid (16:3Delta(9,12,15)(n-1)). The identification of a desaturase, which can catalyze the insertion of a double bond between the terminal two carbons of a fatty acid represents a new addition to desaturase functionality and plasticity. We have also co-expressed in yeast the A. castellanii bifunctional Delta12,Delta15-desaturase with a microsomal Delta6-desaturase, resulting in the synthesis of the highly unsaturated C(16) fatty acid hexadecatetraenoic acid (16:4Delta(6,9,12,15)(n-1)), previously only reported in marine microorganisms. Our work therefore demonstrates the feasibility of the heterologous synthesis of polyunsaturated fatty acids of the n-1 series. The presence of a bifunctional Delta12,Delta15-desaturase in A. castellanii is also considered with reference to the evolution of desaturases and the lineage of this protist.     

A defect in the activity of Delta6 and Delta5 desaturases may be a factor predisposing to the development of insulin resistance syndrome

GLUT-4 (glucose transporter) receptor, tumor necrosis factor-alpha (TNF-alpha), interleukins-6 (IL-6), daf-genes and PPARs (peroxisomal proliferation activator receptors) play a role in the development of insulin resistance syndrome and associated conditions. But, the exact interaction between these molecules/factors and the mechanism(s) by which they produce insulin resistance syndrome is not clear. I propose that a defect in the activity of the enzymes Delta6 and Delta5 desaturases that are essential for the formation of long chain metabolites of essential fatty acids, linoleic acid and alpha-linolenic acid, is a factor in the development of insulin resistance syndrome. Long chain polyunsaturated fatty acids (LCPUFAs) increase cell membrane fluidity and enhance the number of insulin receptors and the affinity of insulin to its receptors; suppress TNF-alpha, IL-6, macrophage migration inhibitory factor (MIF) and leptin synthesis; increase the number of GLUT-4 receptors, serve as endogenous ligands of PPARs, modify lipolysis, and regulate the balance between pro- and anti-oxidants, and thus, play a critical role in the pathogenesis of insulin resistance. In the nematode, Caenorhabditis elegans, the protein encoded by daf-2 is 35% identical to the human insulin receptor; daf-7 codes a transforming growth factor-beta (TGF-beta) type signal and daf-16 enhances superoxide dismutase (SOD) expression. Melatonin has anti-oxidant actions similar to daf-16, TGF-beta and SOD. Calorie restriction enhances the activity of Delta6 and Delta5 desaturases, melatonin production, decreases daf-2 signaling, free radical generation, and augments anti-oxidant defenses that may explain the beneficial effect of diet control in the management of obesity, insulin resistance, and type II diabetes mellitus. These evidences suggest that the activities of Delta6 and Delta5 enzymes play a critical role in the expression and regulation of GLUT-4, TNF-alpha, IL-6, MIF, daf-genes, melatonin, and leptin by modulating the synthesis and tissue concentrations of LCPUFAs. Caloric restriction delays ageing by activating Sir 2 deacetylase in yeast, and expression of Sir 2 (SIRT1) in human cells. Both insulin and insulin-like growth factor-1 (IGF-1) attenuated this response. SIRT1 sequesters the proapoptotic factor Bax, prevents stress-induced apoptosis of cells, and thus, prolongs survival. In addition, SIRT1 repressed PPAR-gamma, and overexpression of SIRT1 attenuated adipogenesis, and upregulation of SIRT in differentiated fat cells triggered lipolysis and loss of fat, events that are known to attenuate insulin resistance and prolong life span. It remains to be seen whether LCPUFAs have a regulatory role in SIRT1 expression and control Sir 2 deacetylase activity. Thus, calorie restriction or reduced food intake has a role not only in the pathobiology of insulin resistance, but also in other associated conditions such as obesity, type II diabetes mellitus, ageing, and longevity.     

Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt stress

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Delta(9) position. We expressed two sunflower (Helianthus annuus) oleate Delta(12) desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Delta(9,12), the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15 degrees C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp(+) or Trp(-) strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30 degrees C or 15 degrees C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.     

Epigallocatechin gallate increase the prostacyclin production of bovine aortic endothelial cells

We describe the effect of (-) epigallocatechin gallate (EGCg), one of catechins known in tea, on the prostacyclin (PGI) production by bovine aortic endothelial cells. The amounts of 6-keto-PGF(1alpha) and Delta(17)-6-keto-PGF(1alpha), stable metabolites of PGI(2) and PGI(3), released in culture medium were measured using gas chromatography/selected ion monitoring (GC/SIM). The prostacyclin production of endothelial cells was increased by EGCg in a dose- and time-dependent manner. The effect by EGCg was stronger than any other catechins (catechin, epicatechin, epigallocatechin, and epicatechin gallate). When endothelial cells incubated with EGCg and arachidonic acid (AA) or eicosapentaenoic acid (EPA), PGI(2), and PGI(3) production were increased greater than those incubated with AA or EPA alone. Furthermore, gallic acid, that also has a pyrogallol structure, increased PGI(2) production. These observations indicate that catechins increase the prostacyclin production and that the pyrogallol structure is significant to this function.     

Dimorphecolic acid is synthesized by the coordinate activities of two divergent Delta12-oleic acid desaturases

Dimorphecolic acid (9-OH-18:2Delta(10)(trans)(,12)(trans)) is the major fatty acid of seeds of Dimorphotheca species. This fatty acid contains structural features that are not typically found in plant fatty acids, including a C-9 hydroxyl group, Delta(10),Delta(12)-conjugated double bonds, and trans-Delta(12) unsaturation. Expressed sequence tag analysis was conducted to determine the biosynthetic origin of dimorphecolic acid. cDNAs for two divergent forms of Delta(12)-oleic acid desaturase, designated DsFAD2-1 and Ds-FAD2-2, were identified among expressed sequence tags generated from developing Dimorphotheca sinuata seeds. Expression of DsFAD2-1 in Saccharomyces cerevisiae and soybean somatic embryos resulted in the accumulation of the trans-Delta(12) isomer of linoleic acid (18: 2Delta(9)(cis)(,12)(trans)) rather than the more typical cis-Delta(12) isomer. When co-expressed with DsFAD2-1 in soybean embryos or yeast, DsFAD2-2 converted 18:2Delta(9)(cis)(,12)(trans) into dimorphecolic acid. When DsFAD2-2 was expressed alone in soybean embryos or together with a typical cis-Delta(12)-oleic acid desaturase in yeast, trace amounts of the cis-Delta(12) isomer of dimorphecolic acid (9-OH-18:2Delta(10)(trans,)(12)(cis)) were formed from DsFAD2-2 activity with cis-Delta(12)-linoleic acid [corrected]. These results indicate that DsFAD2-2 catalyzes the conversion of the Delta(9) double bond of linoleic acid into a C-9 hydroxyl group and Delta(10)(trans) double bond and displays a substrate preference for the trans-Delta(12), rather than the cis-Delta(12), isomer of linoleic acid. Overall these data are consistent with a biosynthetic pathway of dimorphecolic acid involving the concerted activities of DsFAD2-1 and DsFAD2-2. The evolution of two divergent Delta(12)-oleic acid desaturases for the biosynthesis of an unusual fatty acid is unprecedented in plants.     

Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.     

Essential fatty acid metabolism in patients with essential hypertension, diabetes mellitus and coronary heart disease

Mortality and morbidity from coronary heart disease (CHD), diabetes mellitus (DM) and essential hypertension (HTN) are higher in people of South Asian descent than in other groups. There is evidence to believe that essential fatty acids (EFAs) and their metabolites may have a role in the pathobiology of CHD, DM and HTN. Fatty acid analysis of the plasma phospholipid fraction revealed that in CHD the levels of gamma-linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are low, in patients with HTN linoleic acid (LA) and AA are low, and in patients with non-insulin dependent diabetes mellitus (NIDDM) and diabetic nephropathy the levels of dihomo-gamma-linolenic acid (DGLA), AA, alapha-linolenic acid (ALA) and DHA are low, all compared to normal controls. These results are interesting since DGLA, AA and EPA form precursors to prostaglandin E₁, (PGE₁), prostacyclin (PGI₂), and PGI₃, which are potent platelet anti-aggregators and vasodilators and can prevent thrombosis and atherosclerosis. Further, the levels of lipid peroxides were found to be high in patients with CHD, HTN, NIDDM and diabetic nephropathy. These results suggest that increased formation of lipid peroxides and an alteration in the metabolism of EFAs are closely associated with CHD, HTN and NIDDM in Indians. Since insulin resistance and hyperinsulinemia and features of obesity, NIDDM, HTN and CHD, diseases that are common in Indians, and as decreased insulin sensitivity is associated with decreased concentrations of polyunsaturated fatty acids (PUFAs) in skeletal muscle phospholipids and, possibly, in the plasma, the possibility is raised that changes in the metabolism of EFAs may have a fundamental role in the pathobiology of these conditions. If this is true, this suggests that supplementation of GLA, DGLA, AA, EPA and/or DHA may be indicated to prevent CHD, HTN and NIDDM in Indians.     

Influence of testosterone on polyunsaturated fatty acid biosynthesis in Sertoli cells in culture

Sertoli cells play a central role in spermatogenesis, its development and regulation. They are target cells for androgen action in the seminiferous tubules. The Sertoli cell is considered to be the most important cell type in the testis with regard to essential fatty acid metabolism. We studied the response to testosterone of cultured Sertoli cells from immature rats by determining the fatty acid composition of total cellular lipids as well as by the biosynthesis of polyunsaturated fatty acids. Fatty acid methyl esters were analysed by gas liquid chromatography and radiochromatography. Two doses of testosterone were tested (150 and 300 ng ml(-1)). Significant differences were found in fatty acids derived from total cellular lipids after 8 days in culture in the presence of testosterone (300 ng ml(-1), for 48 h). Compared to controls, the hormone produced a significant increase of 16:1 and 18:1 n-9, and of 18:2 n-6, and a decrease of 20:4 and 22:5 n-6 in total cellular lipids. The decrease in the n-6 fatty acid ratios 20:4/20:3, 20:4/18:2 and 24:5/24:4, and the increase in 18:1n-9/18:0 and 16:1n-9/16:0 ratios were taken as an indirect signal of testosterone effects on Delta5, Delta6 and Delta9 desaturase activities. The drop in Delta5 and Delta6 desaturase activities was corroborated by analysing the transformation of [1-14C]20:3 n-6 into its higher homologues. We concluded that testosterone modifies the fatty acid pattern of cultured Sertoli cells, and this hormone is involved in polyunsaturated fatty acid biosynthesis, modulating Delta5 and Delta6 desaturases activity.     

Identification of a Delta 4 fatty acid desaturase from Thraustochytrium sp. involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea

The existence of Delta 4 fatty acid desaturation in the biosynthesis of docosahexanoic acid (DHA) has been questioned over the years. In this report we describe the identification from Thraustochytrium sp. of two cDNAs, Fad4 and Fad5, coding for Delta 4 and Delta 5 fatty acid desaturases, respectively. The Delta 4 desaturase, when expressed in Saccharomyces cerevisiae, introduced a double bond at position 4 of 22:5(n-3) and 22:4(n-6) resulting in the production of DHA and docosapentanoic acid. The enzyme, when expressed in Brassica juncea under the control of a constitutive promoter, desaturated the exogenously supplied substrate 22:5(n-3), resulting in the production of DHA in vegetative tissues. These results support the notion that DHA can be synthesized via Delta 4 desaturation and suggest the possibility that DHA can be produced in oilseed crops on a large scale.