Identification of a novel type of polyunsaturated fatty acid synthase involved in arachidonic acid biosynthesis

Arachidonic acid (ARA) is a polyunsaturated fatty acid (PUFA) and an essential component of membrane lipids. However, the PUFA synthase required for ARA biosynthesis has not been identified in any organism. To identify the PUFA synthase producing ARA, we determined the draft genome sequence of the marine bacterium Aureispira marina, which produces a high level of ARA, and found a gene cluster encoding a putative PUFA synthase for ARA production. Expression of the gene cluster in Escherichia coli induced production of ARA, demonstrating that the gene cluster encodes a PUFA synthase required for ARA biosynthesis.     

The influence of dietary docosahexaenoic acid and arachidonic acid on central nervous system polyunsaturated fatty acid composition

Numerous studies on perinatal long-chain polyunsaturated fatty acid nutrition have clarified the influence of dietary docosahexaenoic acid (DHA) and arachidonic acid (ARA) on central nervous system PUFA concentrations. In humans, omnivorous primates, and piglets, DHA and ARA plasma and red blood cells concentrations rise with dietary preformed DHA and ARA. Brain and retina DHA are responsive to diet while ARA is not. DHA is at highest concentration in cells and tissues associated with high energy consumption, consistent with high DHA levels in mitochondria and synaptosomes. DHA is a substrate for docosanoids, signaling compounds of intense current interest. The high concentration in tissues with high rates of oxidative metabolism may be explained by a critical role related to oxidative metabolism.     

Production of the polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid by the fungus Pythium ultimum

Several strains of species of the fungal genus Pythium, and of Phytophthora cinnamomi, were screened for content of the polyunsaturated fatty acids (PUFAs) arachidonic acid (AA) and eicosapentaenoic acid (EPA). The aim of the investigation was to establish alternative sources of these PUFAs, which are of importance in human nutrition. As a relatively prolific producer of EPA and AA, P. ultimum strain #144 was selected for a study of conditions that enhance their production over baseline levels that are present in the fungus when cultured for 6 d at 25 degrees C with rotary shaking (120 r.p.m.) in Vogel's medium containing sucrose as the carbon substrate. The levels of AA and EPA under these conditions were 133 +/- 27 and 138 +/- 25 mg l-1 (n = 5), respectively. Maximal production of these fatty acids was accomplished by the following sequence of steps. (1) Incubate the cultures for 6 d after inoculation under the conditions described above. Then (2) add glucose to the cultures (2%, w/v, final concentration) and incubate for a further 6 d at 13 degrees C. Under these conditions, the AA content of the mycelium was 205% higher than baseline levels and the EPA content was 198% higher. (3) Allow the cultures to remain stationary for 10 d which increases the AA content to 253% above baseline levels and the EPA content by 236%. Using such a procedure, 322 mg AA l-1 and 383 mg EPA 1-1 were produced.     

Dietary polyunsaturated fatty acids as inducers of apoptosis: implications for cancer

It has recently become clear the role played by alterations in apoptosis during the development of several chronic diseases (i.e. inflammatory, neurodegenerative and neoplastic pathologies). For this reason, the research for possible therapeutic strategies involving the modulation of the apoptotic pathways has attracted considerable interest in the past few years. In particular, it has been shown that apoptosis may be induced or inhibited by a variety of nutritional compounds providing health benefits. The aim of this review is to examine the ability of different dietary polyunsaturated fatty acids (PUFAs) to induce apoptosis, especially in the cancer field. The molecular effects of different PUFAs found in dairy products, meat, fish, vegetable seeds and oils, and known to affect the incidence and progression of cancer and other chronic diseases, will be analyzed. To this aim, our effort will concentrate in critically reviewing the published works concerning the effects of: (a) the n-6 PUFAs γ-linolenic acid, arachidonic acid, and conjugated linoleic acid; (b) the n-3 PUFAs eicosapentaenoic acid and docosahexaenoic acid on the apoptotic process. We will also pay attention to the recent findings regarding the possible role of PUFAs as regulators of the endoplasmic reticulum stress-pathway of apoptosis.     

Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria

When docosahexaenoic acid (DHA)-producing Moritella marina strain MP-1 was cultured in the medium containing 0.5 μ g cerulenin ml⁻¹, an inhibitor for fatty acid biosynthesis, the cells grew normally, but the␣content of DHA in the total fatty acids increased from 5.9–19.4%. The DHA yield of M. marina strain MP-1 cells also increased from 4 to 13.7 mg l⁻¹ by cerulenin treatment. The same effect of cerulenin was observed in eicosapentaenoic acid (EPA)-producing Shewanella marinintestina strain IK-1 grown in the medium containing 7.5 μg cerulenin ml⁻¹, and the cerulenin treatment increased the EPA yield from 1.6 to 8 mg l⁻¹. The use of cerulenin is, therefore, advantageous to increase the content of intracellular polyunsaturated fatty acids (PUFA) in particular PUFA-containing phospholipids in bacterial cells.An erratum to this article can be found at .     

Essential fatty acid for the mosquito Culex pipiens: arachidonic acid.

A nutrient associated with animal-derived phospholipids has previously been found essential for newly-emerged adults of the mosquito Culex pipiens to fly and survive more than a few days. Pure arachidonic acid was completely effective in supporting the emergence of viable flying adults; in combination with synthetic dipalmitoyl lecithin, which slightly improves larval growth rate without inducing adult flight, it wholly adequately replaces animal phospholipids. Linoleic and linolenic acids, which have satisfied the needs of all insects hitherto shown to require an essential fatty acid, were ineffective for C. pipiens, with or without synthetic lecithin. An optimal effect on adult flight was obtained with 0.05 mg of arachidonic acid per 100 ml of dietary medium, a concentration much lower than the linoleic/linolenic concentrations needed by other insects with an essential fatty acid requirement. The relationship of this unique mosquito fatty acid requirement to the essential fatty acid needs of both vertebrates and insects in general is discussed.     

Conversion of alpha-linolenic acid to longer-chain polyunsaturated fatty acids in human adults

The principal biological role of alpha-linolenic acid (alphaLNA; 18:3n-3) appears to be as a precursor for the synthesis of longer chain n-3 polyunsaturated fatty acids (PUFA). Increasing alphaLNA intake for a period of weeks to months results in an increase in the proportion of eicosapentaenoic acid (EPA; 20:5n-3) in plasma lipids, in erythrocytes, leukocytes, platelets and in breast milk but there is no increase in docosahexaenoic acid (DHA; 22:6n-3), which may even decline in some pools at high alphaLNA intakes. Stable isotope tracer studies indicate that conversion of alphaLNA to EPA occurs but is limited in men and that further transformation to DHA is very low. The fractional conversion of alphaLNA to the longer chain n-3 PUFA is greater in women which may be due to a regulatory effect of oestrogen. A lower proportion of alphaLNA is used for beta-oxidation in women compared with men. Overall, alphaLNA appears to be a limited source of longer chain n-3 PUFA in humans. Thus, adequate intakes of preformed long chain n-3 PUFA, in particular DHA, may be important for maintaining optimal tissue function. Capacity to up-regulate alphaLNA conversion in women may be important for meeting the demands of the fetus and neonate for DHA.     

15-lipoxygenase metabolites of gamma-linolenic acid/eicosapentaenoic acid suppress growth and arachidonic acid metabolism in human prostatic adenocarcinoma cells: possible implications of dietary fatty acids

Although gammalinolenic acid (GLA) and eicosapentaenoic acid (EPA) have independently been reported to suppress growth of cancer cells, their relative potencies are unknown. To determine the possible attenuating efficacies of dietary GLA or EPA on prostate carcinogenesis, we hereby report the in vitro effects of GLA, EPA and their 15-lipoxygenase (15-LOX) metabolites: 15(S)-HETrE and 15(S)-HEPE, respectively, on growth and arachidonic acid (AA) metabolism in human androgen-dependent (LNCaP) and androgen-independent (PC-3) prostatic cancer cells in culture. Specifically, both cells were preincubated respectively with the above PUFAs. Growth was determined by [3H]thymidine uptake and AA metabolism by HPLC analysis of the extracted metabolites. Our data revealed increased biosynthesis of prostaglandin E2 (PGE2) and 5-hydroxyeicosatetraenoic acid (5(S)-HETE) by both cells. Preincubation of the cells with 15(S)-HETrE or 15(S)-HEPE more markedly inhibited cellular growth and AA metabolism when compared to precursor PUFAs. Notably, 15(S)-HETrE exerted the greatest inhibitory effects. These findings therefore imply that dietary GLA rather than EPA should better attenuate prostate carcinogenesis via its in vivo generation of 15(S)-HETrE, thus warranting exploration.     

Interactions of saturated, n-6 and n-3 polyunsaturated fatty acids to modulate arachidonic acid metabolism

Anti-thrombotic effects of omega-3 (n-3) fatty acids are believed to be due to their ability to reduce arachidonic acid levels. Therefore, weanling rats were fed n-3 acids in the form of linseed oil (18:3n-3) or fish oil (containing 20:5n-3 and 22:6n-3) in diets containing high levels of either saturated fatty acids (hydrogenated beef tallow) or high levels of linoleic acid (safflower oil) for 4 weeks. The effect of diet on the rate-limiting enzyme of arachidonic acid biosynthesis (delta 6-desaturase) and on the lipid composition of hepatic microsomal membrane was determined. Both linseed oil- or fish oil-containing diets inhibited conversion of linoleic acid to gamma-linolenic acid. Inhibition was greater with fish oil than with linseed oil, only when fed with saturated fat. delta 6-Desaturase activity was not affected when n-3 fatty acids were fed with high levels of n-6 fatty acids. Arachidonic acid content of serum lipids and hepatic microsomal phospholipids was lower when n-3 fatty acids were fed in combination with beef tallow but not when fed with safflower oil. Similarly, n-3 fatty acids (18:3n-3, 20:5n-3, 22:5n-3, and 22:6n-3) accumulated to a greater extent when n-3 fatty acids were fed with beef tallow than with safflower oil. These observations indicate that the efficacy of n-3 fatty acids in reducing arachidonic acid level is dependent on the linoleic acid to saturated fatty acid ratio of the diet consumed.     

Metabolic engineering of plants for polyunsaturated fatty acid production

Very-long-chain polyunsaturated fatty acids (VLCPUFAs) have demonstrated health benefits. Currently, the main sources for these fatty acids are oils from fish and microbes. However, shrinking fish populations and the high cost of microbial oil extraction are making the economic sustainability of these sources questionable. Metabolic engineering of oilseed crops could provide a novel and sustainable source of VLCPUFAs. Recently, genes encoding desaturases and elongases from microbes have been identified and successfully expressed in oilseed plants. However, the levels of VLCPUFAs produced in transgenic plants expressing these genes are still much lower than those found in native microbes. This review assesses the recent progress and future perspectives in the metabolic engineering of PUFAs in plants.     

Inhibition of polyunsaturated fatty acid accumulation in plants expressing a fatty acid epoxygenase

Earlier, we described the isolation of a Crepis palaestina cDNA (Cpal2) which encoded a Delta12-epoxygenase that could catalyse the synthesis of 12,13-epoxy-cis-9-octadecenoic acid (18:1E) from linoleic acid (18:2). When the Cpal2 gene was expressed under the control of a seed-specific promoter in Arabidopsis, plants were able to accumulate small amounts 18:1E and 12,13-epoxy-cis-9,15-octadec-2-enoic acid in their seed lipids. In this report we give results obtained from a detailed analysis of transgenic Arabidopsis plants containing the Cpal2 gene. The seeds from these plants accumulate varying levels of 18:1E, but show a marked increase in 18:1 and equivalent decrease in 18:2 and 18:3. We further observed that the co-expression of a C. palaestina Delta12-desaturase in Arabidopsis appears to return the relative proportions of the C(18) seed fatty acids to normal levels and results in a 2-fold increase in total epoxy fatty acids.     

A neuronal two P domain K+ channel stimulated by arachidonic acid and polyunsaturated fatty acids.

TWIK-1, TREK-1 and TASK K+ channels comprise a class of pore-forming subunits with four membrane-spanning segments and two P domains. Here we report the cloning of TRAAK, a 398 amino acid protein which is a new member of this mammalian class of K+ channels. Unlike TWIK-1, TREK-1 and TASK which are widely distributed in many different mouse tissues, TRAAK is present exclusively in brain, spinal cord and retina. Expression of TRAAK in Xenopus oocytes and COS cells induces instantaneous and non-inactivating currents that are not gated by voltage. These currents are only partially inhibited by Ba2+ at high concentrations and are insensitive to the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine and Cs+. A particularly salient feature of TRAAK is that they can be stimulated by arachidonic acid (AA) and other unsaturated fatty acids but not by saturated fatty acids. These channels probably correspond to the functional class of fatty acid-stimulated K+ currents that recently were identified in native neuronal cells but have not yet been cloned. These TRAAK channels might be essential in normal physiological processes in which AA is known to play an important role, such as synaptic transmission, and also in pathophysiological processes such as brain ischemia. TRAAK channels are stimulated by the neuroprotective drug riluzole.     

Brain phospholipid arachidonic acid half-lives are not altered following 15 weeks of N-3 polyunsaturated fatty acid adequate or deprived diet

Previous studies have infused radiolabeled arachidonic acid (AA) into rat brains and followed AA esterification into phospholipids for up to 24 h; however, the half-life of AA in rat brain phospholipids is unknown. Eighteen day old rats were fed either an n-3 PUFA adequate or deprived diet for 15 weeks. Following the 15 weeks, 40 µCi of [³H] AA was injected intracerebroventricularly into the right lateral ventricle using stereotaxic surgery and returned to their dietary treatment. From 4–120 days after [³H] AA administration, brains were collected for chemical analyses. The half-life of AA in rat brain phospholipids was 44 ± 4 days for the n-3 PUFA adequate group and 46 ± 4 days for the n-3 PUFA deprived group, which closely approximates the predicted half-life previously reported, based on the rate of entry from the plasma unesterified pool, suggesting the plasma unesterified pool is a major contributor to brain uptake of AA. Furthermore, unlike a previous report in which the half-life of brain phospholipid docosahexaenoic acid (DHA) was increased in n-3 PUFA deprived rats, n-3 PUFA deprivation did not significantly alter the AA half-life, suggesting different mechanisms exist to maintain brain concentrations of AA and DHA.     

Regulation of intracellular calcium levels by polyunsaturated fatty acids, arachidonic acid and docosahexaenoic acid, in astrocytes: possible involvement of phospholipase A2

Pathological conditions in the brain, such as ischemia, trauma and seizure are accompanied by increased levels of free n-6 and n-3 polyunsaturated fatty acids (PUFA), mainly arachidonic acid (AA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3). A neuroprotective role has been suggested for PUFA. For investigation of the potential molecular mechanisms involved in neuroprotection by PUFA, we studied the regulation of the concentration of intracellular Ca2+ ([Ca2+]i) in rat brain astrocytes. We evaluated the presence of extracellular PUFA and the release of intracellular PUFA. Interestingly, only the constitutive brain PUFA AA and DHA, but not eicosapentaenoic acid (EPA) had prominent effects on intracellular Ca2+. AA and DHA suppressed [Ca2+]i oscillation, inhibited store-operated Ca2+ entry, and reduced the amplitudes of Ca2+ responses evoked by agonists of G protein-coupled receptors. Moreover, prolonged exposure of astrocytes to AA and DHA brought the cells to a new steady state of a moderately elevated [Ca2+]i level, where the cells became virtually insensitive to external stimuli. This new steady state can be considered as a mechanism of self-protection. It isolates disturbed parts of the brain, because AA and DHA reduce pathological overstimulation in the tissue surrounding the damaged area. In inflammation-related events, frequently AA and DHA exhibit opposite effects. However, in astrocytes AA and DHA exerted comparable effects on [Ca2+]i. Extracellularly added AA and DHA, but not EPA, were also able to induce the release of [3H]AA from prelabeled astrocytes. Therefore, we also suggest the involvement of phospholipase A2 activation and lysophospholipid generation in the regulation of intracellular Ca2+ in astrocytes.     

Effect of fatty acid anilides on the generation of arachidonic acid by human polymorphonuclear leukocytes

The addition of oleoylanilide or linoleylanilide to human polymorphonuclear leukocytes induces a time- and dose-dependent generation of arachidonic acid. Half-maximal effect is caused by a dose of 0.2 mg linoleylanilide/ml. Fatty acid anilides also produce a time- and dose-dependent inhibition of the synthesis of triacylglycerol. Half-maximal effect is caused by 1 microgram linoleylanilide/ml. These results indicate that fatty acid anilides, which have been found in the illegal cooking oil which intoxicated thousands of Spaniards, alter lipid metabolism in human polymorphonuclear leukocytes.     

Effects of fatty acids on mitochondria: implications for cell death

Fatty acids have prominent effects on mitochondrial energy coupling through at least three mechanisms: (i) increase of the proton conductance of the inner mitochondrial membrane; (ii) respiratory inhibition; (iii) opening of the permeability transition pore (PTP). Furthermore, fatty acids physically interact with membranes and possess the potential to alter their permeability; and they are also excellent respiratory substrates that feed electrons into the respiratory chain. Due to the complexity of their actions, the effects of fatty acids on mitochondrial function in situ are difficult to predict. We have investigated the mitochondrial and cellular effects of fatty acids of increasing chain length and degree of unsaturation in relation to their potential to affect mitochondrial function in situ and to cause cell death. We show that saturated fatty acids have little effect on the mitochondrial membrane potential in situ, and display negligible short-term cytotoxicity for Morris Hepatoma 1C1 cells. The presence of double bonds increases both the depolarizing effects and the cytotoxicity, but these effects are offset by the hydrocarbon chain length, so that more unsaturations are required to observe an effect as the hydrocarbon chain length is increased. With few exceptions, depolarization and cell death are due to opening of the PTP rather than to the direct effects of fatty acids on energy coupling.