Enzymes and receptors of prostaglandin pathways with arachidonic acid-derived versus eicosapentaenoic acid-derived substrates and products

Dietary fish oil containing omega 3 highly unsaturated fatty acids has cardioprotective and anti-inflammatory effects. Prostaglandins (PGs) and thromboxanes are produced in vivo both from the omega 6 fatty acid arachidonic acid (AA) and the omega 3 fatty acid eicosapentaenoic acid (EPA). Certain beneficial effects of fish oil may result from altered PG metabolism resulting from increases in the EPA/AA ratios of precursor phospholipids. Here we report in vitro specificities of prostanoid enzymes and receptors toward EPA-derived, 3-series versus AA-derived, 2-series prostanoid substrates and products. The largest difference was seen with PG endoperoxide H synthase (PGHS)-1. Under optimal conditions purified PGHS-1 oxygenates EPA with only 10% of the efficiency of AA, and EPA significantly inhibits AA oxygenation by PGHS-1. Two- to 3-fold higher activities or potencies with 2-series versus 3-series compounds were observed with PGHS-2, PGD synthases, microsomal PGE synthase-1 and EP1, EP2, EP3, and FP receptors. Our most surprising observation was that AA oxygenation by PGHS-2 is only modestly inhibited by EPA (i.e. PGHS-2 exhibits a marked preference for AA when EPA and AA are tested together). Also unexpectedly, TxA(3) is about equipotent to TxA(2) at the TP alpha receptor. Our biochemical data predict that increasing phospholipid EPA/AA ratios in cells would dampen prostanoid signaling with the largest effects being on PGHS-1 pathways involving PGD, PGE, and PGF. Production of 2-series prostanoids from AA by PGHS-2 would be expected to decrease in proportion to the compensatory decrease in the AA content of phospholipids that would result from increased incorporation of omega 3 fatty acids such as EPA.     

Polyunsaturated fatty acids differentially alter PGF(2alpha) and PGE(2) release from bovine trophoblast and endometrial tissues during short-term culture

Two studies tested the hypothesis that eicosapentaenoic (20:5omega3; EPA), docosahexaenoic acids (22:6omega3; DHA) or linoleic acid (C18:2omega6; LIN) reduced bovine endometrial and trophoblast prostaglandin F(2alpha) (PGF(2alpha)) and prostaglandin E(2) (PGE(2)) release during short-term culture. In Study 1, endometrial tissues were collected from non-lactating, non-pregnant cows and endometrial plus trophoblast tissues from pregnant cows 16 days post-insemination. In Study 2, endometrial and trophoblast tissues were collected on day 17 of pregnancy, from cows synchronised using a double prostaglandin (PG) or Ovagentrade mark synchronisation. Tissues were incubated in medium only (M) or media supplemented with fatty acids: eicosapentaenoic (20:5omega3; EPA), docosahexaenoic acids (22:6omega3; DHA) or linoleic acid (C18:2omega6; LIN). In Study 1, PGE(2) release from 'pregnant' endometria was higher (P=0.094) than from 'non-pregnant' endometria, while PGF(2alpha) concentrations were similar. Fatty acids treatment had no effect on PGF(2alpha) or PGE(2) release from either pregnant or non-pregnant endometria. Individual fatty acid treatments had no effect on the ratio of PGF(2alpha) to PGE(2) from trophoblast tissues, but when the data from the 3 fatty acid treatments were combined (EPA, DHA and LIN treatment groups) the ratio of PGF(2alpha) to PGE(2) was reduced (P=0.026) when compared to medium only. In Study 2, PGE(2) concentrations were higher (P=0.013) from the trophoblast collected from Ovagentrade mark cows as compared to that of the PG synchrony group. When the data from the 3-omega fatty acids were combined (DHA and EPA treatment groups), the 3-omega treatments decreased (P<0.05) PGE(2) biosynthesis from both endometrial and trophoblast tissues from animals synchronised following PG synchrony but not Ovagentrade mark synchrony. Short-term culture with low concentrations of 3-omega fatty acids tended to reduce prostaglandin release from trophoblast collected 16 days after insemination, with the type of synchrony modifying PGE(2) production from the trophoblast tissues collected 17 days after insemination. The ability of exogenous fatty acids to modify embryonic prostaglandin release needs to be examined in the context of supplementing dairy cows with different sources of fats. Synchronisation method altered trophoblast PGE(2) release, highlighting the importance of the hormonal environment in modifying embryonic prostaglandin synthesis and release.     

In chronic fatigue syndrome, the decreased levels of omega-3 poly-unsaturated fatty acids are related to lowered serum zinc and defects in T cell activation

There is now evidence that major depression is accompanied by decreased levels of omega3 poly-unsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). There is a strong comorbidity between major depression and chronic fatigue syndrome (CFS). The present study has been carried out in order to examine PUFA levels in CFS. In twenty-two CFS patients and 12 normal controls we measured serum PUFA levels using gas chromatography and mass spectrometry. We found that CFS was accompanied by increased levels of omega6 PUFAs, i.e. linoleic acid and arachidonic acid (AA), and mono-unsaturated fatty acids (MUFAs), i.e. oleic acid. The EPA/AA and total omega3/omega6 ratios were significantly lower in CFS patients than in normal controls. The omega3/omega6 ratio was significantly and negatively correlated to the severity of illness and some items of the FibroFatigue scale, i.e. aches and pain, fatigue and failing memory. The severity of illness was significantly and positively correlated to linoleic and arachidonic acid, oleic acid, omega9 fatty acids and one of the saturated fatty acids, i.e. palmitic acid. In CFS subjects, we found significant positive correlations between the omega3/omega6 ratio and lowered serum zinc levels and the lowered mitogen-stimulated CD69 expression on CD3+, CD3+ CD4+, and CD3+ CD8+ T cells, which indicate defects in early T cell activation. The results of this study show that a decreased availability of omega3 PUFAs plays a role in the pathophysiology of CFS and is related to the immune pathophysiology of CFS. The results suggest that patients with CFS should respond favourably to treatment with--amongst other things--omega3 PUFAs, such as EPA and DHA.     

Prostaglandin E precursor fatty acids inhibit human IL-2 production by a prostaglandin E-independent mechanism

Essential fatty acids, from which PG derive, can participate in development and regulation of immune responses and have been shown to suppress inflammation and tissue injury in animal models. In this report, we investigate the effects of the immediate (DGLA, precursor to PGE1), arachidonic acid (AA, PGE precursors, dihomogamma linolenic acid (DGLA, precursor to PGE1), arachidonic acid (AA, precursor to PGE2), and eicosapentaenoic acid (EPA, precursor to PGE3) on IL-2 production by PHA-stimulated human PBMC. DGLA and AA inhibited IL-2 production in a dose-dependent manner: half-maximal inhibition was obtained by using the fatty acids at the dose of 10 micrograms/ml without significant effects on cell viability. EPA inhibited IL-2 production by PBMC of only some donors. Incubation of cells in the presence of oleic, stearic, and palmitic acids, which are not PG precursors, did not affect mitogen-induced IL-2 production. A progressive increase in incorporation of DGLA into cellular lipids was observed over a 48-h incubation period. IL-2 production was reduced also when PBMC were pretreated overnight with DGLA or AA and washed before exposure to PHA. Whereas addition of the cyclo-oxygenase inhibitor, indomethacin, at the time of mitogenic stimulation led to increased IL-2 production and prevented mitogen- and fatty acid-induced increases in PGE release, it had no significant effect on the capacity of the fatty acids to suppress IL-2 production. Time course experiments showed that DGLA and AA inhibited IL-2 production even at times of minimal or no PGE release by the treated cultures. Moreover, DGLA and AA inhibited IL-2 production by the human leukemia T cell line Jurkat which, when appropriately induced, is able to release high levels of IL-2 in the absence of accessory cells and measurable PGE production. Taken together, these data indicate that essential fatty acids inhibit IL-2 production directly without conversion into their cyclo-oxygenase pathway products, and suggest that human lymphocyte function may be altered profoundly by small changes in their fatty acid profile.     

Polyenoic fatty acid ratios alter fibroblast collagen production via PGE2 and PGE receptor subtype response

Previous experiments have shown that dietary n-6 and n-3 polyenoic fatty acids (PFA) have different effects on collagen production, a process that may be related to the formation of prostaglandins (PG). This study tested the hypothesis that fibroblast collagen production could be regulated by different n- 6:n-3 PFA ratios and that the effects were mediated by PGE(2) and altered signaling via the different PGE receptor subtypes. Compared to a bovine serum albumin control, eicosapentaenoic acid (EPA; 20:5 n-3) treated cells significantly (P < 0.05) increased both collagen production and collagen as a percentage of total cellular protein (C-PTP), but arachidonic acid (AA; 20:4 n-6) reduced collagen production and C-PTP. As the amount of AA decreased and that of EPA increased, collagen production and C-PTP increased, especially when ratio of n-6:n-3 PFA was less than 1:1. C-PTP was significantly correlated with the amount of PGE(2) in the medium. AA- or EPA-treated cells produced similar C-PTP when incubated with 10(-6) M indomethacin, a cyclooxygenase inhibitor. Addition of exogenous PGE(2) to cell cultures treated with 10(-6) M indomethacin for 48 hrs decreased C-PTP in both AA and EPA groups. Decreased C-PTP was observed in AA-treated cells exposed to EP1, EP2, and EP4 PGE receptor agonists and in EPA-treated cells exposed to EP2 and EP4 agonists. AA-treated cell responded to activators of cyclic adenosine monophosphate and protein kinase C by decreasing C-PTP, but EPA-treated cells were unresponsive. In conclusion, collagen production in 3T3-Swiss fibroblasts induced by different n-6:n-3 PFA ratios was correlated with PGE(2) production and altered responsiveness and signaling via the different PGE receptor subtypes.     

Cellular interactions between n-6 and n-3 fatty acids: a mass analysis of fatty acid elongation/desaturation, distribution among complex lipids, and conversion to eicosanoids.

The biologic effect of eicosanoids depends in large measure upon the relative masses in tissues of eicosanoids derived from the n-6 fatty acids, dihomogammalinolenic acid and arachidonic acid, and the n-3 fatty acid, eicosapentaenoic acid. Generation of this tissue balance is related to the relative cellular masses of these precursor fatty acids, the competition between them for entry into and release from cellular phospholipids, and their competition for the enzymes that catalyze their conversion to eicosanoids. In order to better understand these processes, we studied the cellular interactions of n-6 and n-3 fatty acids using an essential fatty acid-deficient, PGE-producing, mouse fibrosarcoma cell line, EFD-1. Unlike studies using cells with endogenous pools of n-6 and n-3 fatty acids, the use of EFD-1 cells enabled us to examine the metabolic fate of each family of fatty acids both in the presence and in the absence of the second family of fatty acids. Thus, the specific effects of one fatty acid family on the other could be directly assessed. In addition, we were able to replete the cells with dihomogammalinolenic acid (DHLA), arachidonic acid (AA), and eicosapentaenoic acid (EPA) of known specific activities; thus the masses of cellular DHLA, AA, and EPA, and their metabolites, PGE1, PGE2, and PGE3, respectively, could be accurately quantitated. The major findings of this study were: 1) n-6 fatty acids markedly stimulated the elongation of EPA to 22:5 whereas n-3 fatty acids inhibited the delta 5 desaturation of DHLA to AA and the elongation of AA to 22:4; 2) n-6 fatty acids caused a specific redistribution of cellular EPA from phospholipid to triacylglycerol; 3) n-3 fatty acids reduced the mass of DHLA and AA only in phosphatidylinositol whereas n-6 fatty acids reduced the mass of EPA to a similar extent in all cellular phospholipids; and 4) n-3 fatty acids caused an identical (33%) reduction in the bradykinin-induced release of PGE1 and PGE2, whereas n-6 fatty acids stimulated PGE3 release 2.3-fold. Together, these highly quantitative metabolic data increase our understanding of the regulation of both the cellular levels of DHLA, AA, and EPA, and their availability for eicosanoid synthesis. In addition, these findings provide a context for the effective use of these fatty acids in dietary therapies directed at modulation of eicosanoid production.     

Effect of supplementation of arachidonic acid (AA) or a combination of AA plus docosahexaenoic acid on breastmilk fatty acid composition

We investigated whether supplementation with arachidonic acid (20:4 omega 6; AA), or a combination of AA and docosahexaenoic acid (22:6 omega 3; DHA) would affect human milk polyunsaturated fatty acid (PUFA) composition. Ten women were daily supplemented with 300 mg AA, eight with 300 mg AA, 110 mg eicosapentaenoic acid (20:5 omega 3; EPA) and 400 mg DHA, for one week and eight women served as unsupplemented controls. Milk samples were collected on days 0, 1 and 7. The fatty acid composition of the milk was analyzed by capillary gas chromatography with flame ionisation detection. Supplementation with AA alone had no effect on breastmilk AA, but tended to reduce EPA and DHA levels. Administration of a combination of AA, EPA and DHA tended to increase both milk AA and long chain PUFA (LCPUFA)omega 3 content. A larger simultaneous increase of milk AA, DHA and EPA than observed in the present study can probably be accomplished by the use of a combination of a lower LCPUFA omega 6/LCPUFA omega 3 ratio and higher AA, EPA and DHA dosages.     

Effect of randomized supplementation with high dose olive, flax or fish oil on serum phospholipid fatty acid levels in adults with attention deficit hyperactivity disorder

Dietary intake of omega-3 fatty acids has been positively correlated with cardiovascular and neuropsychiatric health in several studies. The high seafood intake by the Japanese and Greenland Inuit has resulted in low ratios of the omega-6 fatty acid arachidonic acid (AA, 20:4n-6) to eicosapentaenoic acid (EPA, 20:5n-3), with the Japanese showing AA:EPA ratios of approximately 1.7 and the Greenland Eskimos showing ratios of approximately 0.14. It was the objective of this study to determine the effect of supplementation with high doses (60 g) of flax and fish oils on the blood phospholipid (PL) fatty acid status, and AA/EPA ratio of individuals with Attention Deficit Hyperactivity Disorder (ADHD), commonly associated with decreased blood omega-3 fatty acid levels. Thirty adults with ADHD were randomized to 12 weeks of supplementation with olive oil (< 1% omega-3 fatty acids), flax oil (source of alpha-linolenic acid; 18:3n-3; alpha-LNA) or fish oil (source of EPA and docosahexaenoic acid; 22:6n-3; DHA). Serum PL fatty acid levels were determined at baseline and at 12 weeks. Flax oil supplementation resulted in an increase in alpha-LNA and a slight decrease in the ratio of AA/EPA, while fish oil supplementation resulted in increases in EPA, DHA and total omega-3 fatty acids and a decrease in the AA/EPA ratio to values seen in the Japanese population. These data suggest that in order to increase levels of EPA and DHA in adults with ADHD, and decrease the AA/EPA ratio to levels seen in high fish consuming populations, high dose fish oil may be preferable to high dose flax oil. Future study is warranted to determine whether correction of low levels of long-chain omega-3 fatty acids is of therapeutic benefit in this population.     

β-oxidation modulates metabolic competition between eicosapentaenoic acid and arachidonic acid regulating prostaglandin E2 synthesis in rat hepatocytes–Kupffer cells

The ability of n-3 PUFA to competitively inhibit the use of arachidonic acid (AA) for membrane phospholipid synthesis and prostaglandin E₂ (PGE₂) production has been well demonstrated in single cell models. In the present study, we investigated the metabolic competition between AA and eicosapentaenoic acid (EPA) for PGE₂ synthesis in a rat hepatocyte–Kupffer cell (HPC/KC) co-culture system when the cellular oxidation capacity was enhanced by exogenous l-carnitine. We demonstrate that in the absence of l-carnitine, 1) β-oxidation rates of EPA and AA were comparable in HPCs and in KCs; 2) AA and not EPA was preferentially incorporated into glycerolipids; and 3) addition of EPA significantly decreased AA-dependent PGE₂ synthesis in HPCs and cyclooxygenase-2 (COX-2) expression in co-cultured HPCs/KCs. However, enhancing the cellular oxidation capacity by the addition of l-carnitine 1) significantly increased β-oxidation of EPA in HPCs, but only marginally elevated the oxidation of AA in HPCs and the oxidation of both fatty acids in KCs; 2) decreased the esterification, but did not alter the preferential incorporation of AA into glycerolipids; and 3) alleviated the significant competitive inhibition of AA-dependent PGE₂ synthesis and COX-2 expression by EPA. Taken together, the results strongly suggest that l-carnitine affects competition between AA and EPA in PG synthesis in liver cells by enhancing oxidation of EPA in HPCs. This implies that the beneficial effects of n-3 PUFA, especially EPA, are affected by the cellular oxidation capacity.     

Variations of lipid and fatty acid contents during the reproductive cycle of the anthozoan <Emphasis Type="Italic">Renilla koellikeri</Emphasis>

Lipid and fatty acid contents were monitored in the sea pansy Renilla koellikeri during its reproductive cycle. The fluctuations of lipids and fatty acids were closely related to sexual development and gonads represented the principal site of lipid storage in the colony. Between April and May, fatty acids and lipids greatly accumulated just prior to spawning. The subsequent decrease of lipid and fatty acid contents in June probably resulted from loss due to spawning. Fatty acid composition was assessed for the whole animal and was compared between male and female gonads, and between gonads and somatic tissues. Arachidonic acid (AA) was the principal fatty acid in the whole colony followed by palmitic acid and eicosapentaenoic acid (EPA). The examination of fatty acid variations in gonads showed that EPA and AA were highly incorporated in eggs during the month preceding spawning, whereas in the same period, EPA was the only fatty acid to accumulate in spermatophores. The differences of fatty acid levels observed in gonads reflected sex differences observed in colonies. The seasonal variations of the omega6/omega3 ratio of the colonies were largely influenced by EPA and AA, and appeared as a reliable indicator of the sexual maturity of the sea pansy.     

Dietary omega-3 fatty acids modulate the eicosanoid profile in man primarily via the CYP-epoxygenase pathway

Cytochrome P450 (CYP)-dependent metabolites of arachidonic acid (AA) contribute to the regulation of cardiovascular function. CYP enzymes also accept EPA and DHA to yield more potent vasodilatory and potentially anti-arrhythmic metabolites, suggesting that the endogenous CYP-eicosanoid profile can be favorably shifted by dietary omega-3 fatty acids. To test this hypothesis, 20 healthy volunteers were treated with an EPA/DHA supplement and analyzed for concomitant changes in the circulatory and urinary levels of AA-, EPA-, and DHA-derived metabolites produced by the cyclooxygenase-, lipoxygenase (LOX)-, and CYP-dependent pathways. Raising the Omega-3 Index from about four to eight primarily resulted in a large increase of EPA-derived CYP-dependent epoxy-metabolites followed by increases of EPA- and DHA-derived LOX-dependent monohydroxy-metabolites including the precursors of the resolvin E and D families; resolvins themselves were not detected. The metabolite/precursor fatty acid ratios indicated that CYP epoxygenases metabolized EPA with an 8.6-fold higher efficiency and DHA with a 2.2-fold higher efficiency than AA. Effects on leukotriene, prostaglandin E, prostacyclin, and thromboxane formation remained rather weak. We propose that CYP-dependent epoxy-metabolites of EPA and DHA may function as mediators of the vasodilatory and cardioprotective effects of omega-3 fatty acids and could serve as biomarkers in clinical studies investigating the cardiovascular effects of EPA/DHA supplementation.     

In vivo assessment of precursor induced prostaglandin release within the rat gastric lumen

Gastroprotection associated with the intragastric administration of prostaglandin (PG) precursor fatty acids such as linoleic (LA), gamma-linolenic (GLA), and arachidionic acid (AA) has been reported to be mediated via their conversion to PGs. This report examines the relationship between gastroprotection and the extent/rate of PG-release in rats intragastrically administered PG biosynthetic precursors: LA, AA, dihomo-gamma-linolenic acid (DHGL) or oleic acid (OA, a nonprecursor fatty acid). At various times following intragastic administration of a fatty acid, gastric fluid was collected, extracted, chromatographed, and assayed for PGE₁ or PGE₂ by specific radioimmunoassay. AA and DHGL dose dependently elevated gastric PGE₂ and PGE₁ levels, respectively. Maximal PGE elevation, 200–400 ng/stomach, was over 400-fold above basal values, and observed within 5–10 minutes of administration. Conversely, OA and LA elicited only a minor (2–10 fold) stimulation of PGE release. In contrast to effects on PG release, all four fatty acids protected the gastric mucosa against macroscopic damage induced by ethanol. The apparent rank order of potency was AA > DHGL = LA > OA (the difference in potency between DHGL or LA and OA was not significant). Since LA and OA (a nonprecursor) only marginally elevated lumenal PGs relative to DHGL or AA, yet were equally efficacious in the gastroprotection assay, it is likely the other fatty acid-related mechanisms play an important role in protecting the stomach against ethanol-induced injury.     

Regulation of fatty acid biosynthesis as a possible mechanism for the mitoinhibitory effect of fumonisin B1 in primary rat hepatocytes

The mitoinhibitory effect of fumonisin B1 (FB1) on the mitogenic response of epidermal growth factor (EGF) was investigated in primary hepatocyte cultures with respect to the alterations in the omega6 fatty acid metabolic pathway. Fatty acid analyses of hepatocytes showed that EGF treatment resulted in a significant decrease in the relative levels of 20:4omega6 (arachidonic acid) and an increase in 18:2omega6 (linoleic acid). Supplementation of the hepatocyte cultures with 20:4omega6 in the absence of EGF resulted in an increase in the total omega6 and omega6/omega3 fatty acid ratio. Addition of 20:5omega3 (eicosapentaenoic acid) resulted in an increase of the relative levels of the long chain omega3 fatty acids at the expense of the omega6 fatty acids. When 20:4omega6 and 20:5omega3 was added in the presence of EGF, the mitogenic response of EGF was increased and decreased respectively. When compared to the fatty acid profiles in the absence of EGF, the decreased mitogenic response coincided with a decrease of total omega6 fatty acids and total polyunsaturated fatty acids (PUFA). In addition, the saturated and mono-unsaturated fatty acids increased and the polyunsaturated/saturated (P/S) fatty acid ratio decreased which implied a more rigid membrane structure. Addition of prostaglandin E2 (PGE2) and prostaglandin E1 (PGE1) stimulated and inhibited the mitogenic response respectively. Ibuprofen, a known cyclooxygenase inhibitor, and FB1 inhibited the EGF-induced mitogenic response in a dose-dependent manner. The mitoinhibitory effect of FB1 on the EGF response was counteracted by the addition of PGE2. FB1 also disrupts the omega6 fatty acid metabolic pathway in primary hepatocytes, resulting in the accumulation of C18:2omega6 in phospatidylcholine and triacylglicerol. The disruption of the omega6 fatty acid metabolic pathway and/or prostaglandin synthesis is likely to be an important event in the mitoinhibitory effect of FB1 on growth factor responses.     

Role of omega-3 polyunsaturated fatty acids on cyclooxygenase-2 metabolism in brain-metastatic melanoma

Cyclooxygenase-2 (COX-2) is important in the progression of epithelial tumors. Evidence indicates that omega-6 PUFAs such as arachidonic acid (AA) promote the growth of tumor cells; however, omega-3 fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] inhibit tumor cell proliferation. We investigated the effects of omega-3 PUFA on the expression and function of COX-2 in 70W, a human melanoma cell line that metastasizes to the brain in nude mice. We show that 1) tumor necrosis factor-alpha upregulates the expression of both COX-2 mRNA and prostaglandin E2 (PGE2) production, and 2) omega-3 and omega-6 PUFA regulate COX-2 mRNA expression and PGE2 production. AA increased COX-2 mRNA expression and prostaglandin production in omega-6-stimulated 70W cells. Conversely, COX-2 mRNA expression decreased in cells incubated with EPA or DHA. AA increased Matrigel invasion 2.4-fold, whereas EPA or DHA did not. Additionally, PGE2 increased in vitro invasion 2.5-fold, whereas exposure to PGE3 significantly decreased invasion. Our results demonstrate that incubation of 70W cells with either AA or PGE2 increased invasiveness, whereas incubation with EPA or DHA downregulated both COX-2 mRNA and protein expression, with a subsequent decrease in Matrigel invasion. Taken together, these results indicate that omega-3 PUFA regulate COX-2-mediated invasion in brain-metastatic melanoma.     

The role of triacylglycerol as a reservoir of polyunsaturated fatty acids for the rapid production of chloroplastic lipids in certain microalgae

Many microalgae are known to accumulate triacylglycerols (TAGs), especially under nitrate starvation. Generally, these TAGs are predominantly constructed of saturated and monounsaturated fatty acids. In contrast, the TAGs of the red microalga Porphyridium cruentum are rich in arachidonic acid (AA, 20:4 omega6) and eicosapentaenoic acid (EPA, 20:5 omega3). A mutant of this alga, impaired of growth at suboptimal temperatures, was shown to have reduced levels of EPA and of the eukaryotic molecular species of monogalactosyldiacylglycerol (MGDG) and an elevated level of TAG. Labelling experiments have shown that labelling of wild-type TAGs decreased, whereas that of the mutant remained high. Contemporarily, eukaryotic MGDG of the mutant was less labelled. Similarly, TAGs of the green alga Tl2, which can grow at a low temperature, are extremely rich in AA. We have labelled exponentially growing cultures of T12 kept at 25 degrees C with radioactive AA and cultivated the cultures for a further 12 h at 25 degrees C, 12 degrees C or 4 degrees C. At low temperatures, labelled AA was transferred from TAGs to polar lipids. These findings may indicate that polyunsaturated fatty acids that can be incorporated into the membranes, enabling the organism to quickly respond to low-temperature-induced stress.     

Long chain polyunsaturated fatty acids alter membrane-bound RANK-L expression and osteoprotegerin secretion by MC3T3-E1 osteoblast-like cells

Inflammation triggers an increase in osteoclast (bone resorbing cell) number and activity. Osteoclastogenesis is largely controlled by a triad of proteins consisting of a receptor (RANK), a ligand (RANK-L) and a decoy receptor (osteoprotegerin, OPG). Whilst RANK is expressed by osteoclasts, RANK-L and OPG are expressed by osteoblasts. The long chain polyunsaturated fatty acid (LCPUFA) arachidonic acid (AA, 20:4n-6) and its metabolite prostaglandin E2 (PGE2), are pro-inflammatory and PGE2 is a potent stimulator of RANKL expression. Various LCPUFAs such as eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and gamma-linolenic acid (GLA, 18:3n-6) have anti-inflammatory activity. We aimed to determine if AA itself can stimulate RANKL expression and whether EPA, DHA and GLA inhibit RANKL expression in osteoblasts. MC3T3-E1/4 osteoblast-like cells were cultured under standard conditions with each of the LCPUFAs (5microg/ml) for 48h. Membrane-bound RANKL expression was measured by flow cytometry and OPG secretion measured by ELISA. In a second experiment, RANKL expression in MC3T3-E1/4 cells was stimulated by PGE2 treatment and the effect of EPA, DHA and GLA on membrane-bound RANKL expression and OPG secretion determined. The percentage of RANKL-positive cells was higher (p<0.05) than controls following treatment with AA or GLA but not after co-treatment with the cyclooxygenase inhibitor, indomethacin. DHA and EPA had no effect on membrane-bound RANKL expression under standard cell culture conditions. Secretion of OPG was lower (p<0.05) in AA-treated cells but not significantly different from controls in GLA, EPA or DHA treated cells. Treatment with prostaglandin E2 (PGE2) resulted in an increase (p<0.05) in the percentage of RANK-L positive cells and a decrease (p<0.05) in mean OPG secretion. The percentage of RANKL positive cells was significantly lower following co-treatment with PGE2 and either DHA or EPA compared to treatment with PGE2 alone. Mean OPG secretion remained lower than controls in cells treated with PGE2 regardless of co-treatment with EPA or DHA. Results from this study suggest COX products of GLA and AA induce membrane-bound RANKL expression in MC3T3-E1/4 cells. EPA and DHA have no effect on membrane-bound RANKL expression in cells cultured under standard conditions however both EPA and DHA inhibit the PGE2-induced increase in RANKL expression in MC3T3-E1/4 cells.     

Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants

We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in the omega3/6 Delta8 -desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids. Arabidopsis thaliana was transformed sequentially with genes encoding a Delta9 -specific elongating activity from Isochrysis galbana, a Delta8 -desaturase from Euglena gracilis and a Delta5 -desaturase from Mortierella alpina. Instrumental in the successful reconstitution of these C20 polyunsaturated fatty acid biosynthetic pathways was the I. galbana C18-Delta9 -elongating activity, which may bypass rate-limiting steps present in the conventional Delta6 -desaturase/elongase pathways. The accumulation of EPA and AA in transgenic plants is a breakthrough in the search for alternative sustainable sources of fish oils.     

Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are valuable commodities that provide important human health benefits. We report the transgenic production of significant amounts of AA and EPA in Brassica juncea seeds via a stepwise metabolic engineering strategy. Using a series of transformations with increasing numbers of transgenes, we demonstrate the incremental production of VLCPUFAs, achieving AA levels of up to 25% and EPA levels of up to 15% of total seed fatty acids. Both fatty acids were almost exclusively found in triacylglycerols, with AA located preferentially at sn-2 and sn-3 positions and EPA distributed almost equally at all three positions. Moreover, we reconstituted the DHA biosynthetic pathway in plant seeds, demonstrating the practical feasibility of large-scale production of this important omega-3 fatty acid in oilseed crops.