Very long chain n-3 and n-6 polyunsaturated fatty acids bind strongly to liver fatty acid-binding protein

Synthesis of n-3 and n-6 very long chain-PUFAs (VLC-PUFAs) from 18-carbon essential fatty acids is differentially regulated. The predominant product arising from n-3 fatty acids is docosahexaenoic acid (22:6n-3), with the liver serving as the main site of production. The synthetic pathway requires movement of a 24-carbon intermediate from the endoplasmic reticulum to peroxisomes for retroconversion to 22:6n-3. The mechanism of this intra-organelle flux is unknown, but could be binding-protein facilitated. We thus investigated binding of a series of previously untested VLC-PUFAs to liver fatty acid-binding protein (L-FABP). Three fluorometric assays were employed, all of which showed strong binding (K(d)' approximately 10(-8) to 10(-7) M) of 20-, 22-, and 24-carbon n-3 PUFAs to L-FABP. In contrast, synthesis of the predominant n-6 PUFA product, arachidonic acid, does not require intra-organelle transport. However, we found that n-6 VLC-PUFAs bound to L-FABP with affinities (K(d)' approximately 10(-8) to 10(-7) M) comparable to their n-3 counterparts.Although these results raise the possibility that L-FABP may participate in the cytoplasmic processing of n-3 and n-6 VLC-PUFAs, there is no evidence on the basis of binding affinities that L-FABP accounts for differences in the predominant products formed by the n-3 and n-6 PUFA metabolic pathways.     

n-3 polyunsaturated fatty acids and the cardiovascular system

n-3 Polyunsaturated fatty acids, mainly those contained in fish oils, are candidates for inclusion in secondary prevention programmes for coronary heart disease, based on the results of recent randomized trials in humans. Marine n-3 polyunsaturated fatty acids retard coronary atherosclerosis and appear to prevent fatal arrhythmias; and they decrease mortality subsequent to myocardial infarction.     

Marine fish oil is more potent than plant-based n-3 polyunsaturated fatty acids in the prevention of mammary tumors

Marine-derived n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been shown to inhibit mammary carcinogenesis. However, evidence regarding plant-based α-linolenic acid (ALA), the major n-3 PUFA in the Western diet, remains equivocal. The objective of this study was to examine the effect of lifelong exposure to plant- or marine-derived n-3 PUFAs on pubertal mammary gland and tumor development in MMTV-neu(ndl)-YD5 mice. It is hypothesized that lifelong exposure to n-3 PUFA reduces terminal end buds during puberty leading to delayed tumor onset, volume and multiplicity. It is further hypothesized that plant-derived n-3 PUFAs will exert dose-dependent effects. Harems of MMTV-FVB males were bred with wild-type females and fed either a (1) 10% safflower (10% SF, n-6 PUFA, control), (2) 10% flaxseed (10% FS), (3) 7% safflower plus 3% flaxseed (3% FS) or (4) 7% safflower plus 3% menhaden (3% FO) diet. Female offspring were maintained on parental diets. Compared to SF, 10% FS and 3% FO reduced (P<.05) terminal end buds at 6 weeks and tumor volume and multiplicity at 20 weeks. A dose-dependent reduction of tumor volume and multiplicity was observed in mice fed 3% and 10% FS. Antitumorigenic effects were associated with altered HER2, pHER-2, pAkt and Ki-67 protein expression. Compared to 10% SF, 3% FO significantly down-regulated expression of genes involved in eicosanoid synthesis and inflammation. From this, it can be estimated that ALA was 1/8 as potent as EPA+DHA. Thus, marine-derived n-3 PUFAs have greater potency versus plant-based n-3 PUFAs.     

Serotoninergic neurotransmission is affected by n-3 polyunsaturated fatty acids in the rat

We explored the effects of chronic alpha-linolenic acid dietary deficiency on serotoninergic neurotransmission. In vivo synaptic serotonin (5-HT) levels were studied in basal and pharmacologically stimulated conditions using intracerebral microdialysis in the hippocampus of awake 2-month-old rats. We also studied the effects of reversion of the deficient diet on fatty acid composition and serotoninergic neurotransmission. A balanced (control) diet was supplied to deficient rats at different stages of development, i.e. from birth, 7, 14 or 21 days of age. We demonstrated that chronic n-3 polyunsaturated fatty acid dietary deficiency induced changes in the synaptic levels of 5-HT both in basal conditions and after pharmacological stimulation with fenfluramine. Higher levels of basal 5-HT release and lower levels of 5-HT-stimulated release were found in deficient than in control rats. These neurochemical modifications were reversed by supply of the balanced diet provided at birth or during the first 2 weeks of life through the maternal milk, whereas they persisted if the balanced diet was given from weaning (at 3 weeks of age). This suggests that provision of essential fatty acids is durably able to affect brain function and that this is related to the developmental stage during which the deficiency occurs.     

Intravenous infusion of n-3 polyunsaturated fatty acids.

Dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) is regarded as beneficial for the prevention and treatment of atherosclerosis and thrombosis and chronic inflammatory diseases like rheumatoid arthritis and psoriasis. It may be possible to treat some acute diseases like acute myocardial infarction or acute rejection of grafted organs if it is possible to make n-3 PUFA take effect quickly (in hours instead of days). Three sets of experiments were done. In Experiment 1, emulsion of trieicosapentaenoyl-glycerol (EPA-TG) and tridocosahexaenoyl-glycerol was infused through rabbit ear veins, and the leukotriene B4/B5 production from polymorphonuclear leukocytes was measured at different time points by high-performance liquid chromatography. In Experiment 2, delayed type hypersensitivity (DTH) of mice was measured with sheep red blood cells as an antigen. Pure n-3 PUFA emulsions or a control solution were infused through tail veins just before the second challenge of the antigen. DTH was measured 24 hr after the second challenge. In Experiment 3, human natural killer cell activity was measured using K562 target cells before and after the infusion of pure EPA-TG emulsion to an antecubital vein. Leukotriene B4 production by rabbit polymorphonuclear leukocytes was depressed by 40% by EPA-TG infusion. DTH was suppressed almost completely by n-3 PUFA infusion. Natural killer cell activity was suppressed almost completely by EPA-TG infusion in 8 hr. DTH, natural killer cell activity, and leukotriene B4 production are probably related to acute rejection of grafted organs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of n-3 polyunsaturated fatty acids on dermatitis in NC/Nga mice

The administration of n-3 polyunsaturated fatty acids (PUFAs) is known to be effective against allergic diseases by suppressing the production of eicosanoids derived from arachidonic acid. To investigate the mechanisms and efficacy of n-3 PUFA treatment in patients with atopic dermatitis (AD), we administered four different formulas of alpha-linolenic acid for 6 weeks in an AD model using NC/Nga mice. According to the doses of alpha-linolenic acid given, the levels of alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid in the red blood cell membranes increased while the levels of linoleic acid and arachidonic acid decreased. However, there was no significant difference among the four dose groups in clinical skin severity score, histopathological findings of skin lesions, or levels of total plasma IgE. Moreover, there was no significant difference in the production of leukotriene B(4) and Leukotriene C(4) from skin lesions after stimulation with A23187 among the groups, although the production of prostaglandin E(2) (PGE(2)) was significantly reduced and skin blood flow in the ear was significantly higher in the group given the highest dose of alpha-linolenic acid. Our results suggest that the administration of alpha-linolenic acid can change the fatty acid composition, PGE(2) production, and skin blood flow but may not prevent the development of dermatitis in NC/Nga mice.     

Docosahexaenoic acid synthesis from n-3 polyunsaturated fatty acids in differentiated rat brain astrocytes

DHA, the main n-3 PUFA in the brain, is synthesized from n-3 PUFA precursors by astrocytes. To assess the potential of this process to supply DHA for the brain, we investigated whether the synthesis in astrocytes is dependent on DHA availability. Rat brain astrocytes differentiated with dibutyryl cAMP and incubated in media containing 10% fetal bovine serum synthesized DHA from alpha-linolenic acid ([1-(14)C]18:3n-3), docosapentaenoic acid ([3-(14)C]22:5n-3), tetracosapentaenoic acid ([3-(14)C]24:5n-3), and tetracosahexaenoic acid ([3-(14)C]24:6n-3). When DHA was added to media containing a 5 microM concentration of these (14)C-labeled n-3 PUFA, radiolabeled DHA synthesis was reduced but not completely suppressed even when the DHA concentration was increased to 15 microM. Radiolabeled DHA synthesis also was reduced but not completely suppressed when the astrocytes were treated with 30 microM DHA for 24 h before incubation with 5 microM [1-(14)C]18:3n-3.These findings indicate that although the DHA synthesis in astrocytes is dependent on DHA availability, some synthesis continues even when the cells have access to substantial amounts of DHA. This suggests that DHA synthesis from n-3 PUFA precursors is a constitutive process in the brain and, therefore, is likely to have an essential function.     

Effect of n-6 and n-3 polyunsaturated fatty acids ingestion on rat liver membrane-associated enzymes and fluidity

The influences of diets having different fatty acid compositions on the fatty-acid content, desaturase activities, and membrane fluidity of rat liver microsomes have been analyzed. Weanling male rats (35–45 g) were fed a fat-free semisynthetic diet supplemented with 10% (by weight) marine fish oil (FO, 12.7% docosahexaenoic acid and 13.8% eicosapentaenoic acid), evening primrose oil (EPO, 7.8% γ-linolenic acid and 70.8% linoleic acid) or a mixture of 5% FO-5% EPO. After 12 weeks on the respective diets, animals fed higher proportions of (n-3) polyunsaturated fatty acids (FO group) consistently contained higher levels of 20:3(n-6), 20:5(n-3), 22:5(n-3), and 22:6(n-3), and lower levels of 18:2(n-6) and 20:4(n-6), than those of the EPO (a rich source of (n-6) polyunsaturated fatty acids) or the FO + EPO groups. Membrane fluidity, as estimated by the reciprocal of the order parameter S_DPH, was higher in the FO than in the EPO or the FO + EPO groups, and the n-6 fatty-acid desaturation system was markedly affected.     

Mammary tumor development is directly inhibited by lifelong n-3 polyunsaturated fatty acids

IntroductionDespite the advocacy that diet may be a significant contributor to cancer prevention, there is a lack of direct evidence from epidemiological and experimental studies to substantiate such claims. Experimental studies suggest that n-3 polyunsaturated fatty acids (n-3 PUFA) from marine oils may reduce breast cancer risk, however, findings are equivocal. Thus, in this study, novel transgenic mouse models were employed to provide, for the first time, direct evidence for an anti-cancer role of n-3 PUFA in mammary tumorigenesis.Methodsfat-1 Mice, which are capable of endogenous n-3 PUFA synthesis, were bred with mouse mammary tumor virus (MMTV)-neu(ndl)-YD5 mice, an aggressive breast cancer model. The resultant offspring, including novel hybrid progeny, were assessed for tumor onset, size and multiplicity as well as n-3 PUFA composition in mammary gland and tumor tissue. A complementary group of MMTV-neu(ndl)-YD5 mice were fed n-3 PUFA in the diet.ResultsMice expressing MMTV-neu(ndl)-YD5 and fat-1 displayed significant (P<.05) reductions in tumor volume (~30%) and multiplicity (~33%), as well as reduced n-6 PUFA and enriched n-3 PUFA in tumor phospholipids relative to MMTV-neu(ndl)-YD5 control mice. The effect observed in hybrid progeny was similarly observed in n-3 PUFA diet fed mice.ConclusionUsing complementary genetic and conventional dietary approaches we provide, for the first time, unequivocal experimental evidence that n-3 PUFA is causally linked to tumor prevention.     

Insulin secretion after dietary supplementation with conjugated linoleic acids and n-3 polyunsaturated fatty acids in normal and insulin-resistant mice

Conjugated linoleic acids (CLAs) and n-3 polyunsaturated fatty acids (PUFAs) improve insulin sensitivity in insulin-resistant rodents. However, the effects of these fatty acids on insulin secretion are not known but are of importance to completely understand their influence on glucose homeostasis. We therefore examined islet function after dietary supplementation consisting of 1% CLAs in combination with 1% n-3 enriched PUFAs for 12 wk to mice on a normal diet and to insulin-resistant mice fed a high-fat diet (58% fat). In the mice fed a normal diet, CLA/PUFA supplementation resulted in insulin resistance associated with low plasma adiponectin levels and low body fat content. Intravenous and oral glucose tolerance tests revealed a marked increase in insulin secretion, which nevertheless was insufficient to counteract the insulin resistance, resulting in glucose intolerance. In freshly isolated islets from mice fed the normal diet, both basal and glucose-stimulated insulin secretion were adaptively augmented by CLA/PUFA, and at a high glucose concentration this was accompanied by elevated glucose oxidation. In contrast, in high-fat-fed mice, CLA/PUFA did not significantly affect insulin secretion, insulin resistance, or glucose tolerance. It is concluded that dietary supplementation of CLA/PUFA in mice fed the normal diet augments insulin secretion, partly because of increased islet glucose oxidation, but that this augmentation is insufficient to counterbalance the induction of insulin resistance, resulting in glucose intolerance. Furthermore, the high-fat diet partly prevents the deleterious effects of CLA/PUFA, but this dietary supplementation was not able to counteract high-fat-diet-induced insulin resistance.     

Effect of n-3 and n-6 polyunsaturated fatty acids on hare reproductive performances

The study was carried out on 42 breeder couples (42 males and 42 females) of European brown hare (Lepus europaeus), divided into three groups fed three different experimental diets (14 couples/treatment). Two diets were supplemented with n-3 and n-6 polyunsaturated fatty acids (PUFAs; 2% of linseed oil and soybean oil, respectively) and were compared with a control diet supplemented with a monounsaturated fatty acids (2% of olive oil). During the experimental period (from 15 April to 30 September), the following parameters were recorded: days from the beginning of trial to the first parturition, parturition interval, number of parturitions, number of leverets born (alive and dead), dead during suckling, the total number of leverets weaned and feed intake per cage (of males, females and leverets until weaning). Feed intake was not influenced by treatments. In hares fed n-3 and n-6 diets, the days from the beginning of the trial to the first parturition and the parturition interval were similar and were lower compared with control group (63.1 v. 70.6 days, and 37.8 v. 40.9 days, respectively; P < 0.05). Hares from n-6 group had a higher (P < 0.05) number of parturitions per cage during the experimental period than the n-3 and control group that showed a similar value (3.00 v. 2.36, respectively). The total number of leverets born per cage and parturition in n-6 and n-3 groups increased with respect to those fed control diet (P < 0.05). The leverets' mortality rate at birth was higher in n-6 than in n-3 and control group (3.50 v. 2.17, respectively; P < 0.05). In control group, leverets' mortality rate during suckling was lower with respect to n-3 (P < 0.05) and n-6 (P < 0.05), showing the highest value for the latter (P < 0.05). In spite of this higher mortality, the number of leverets weaned per cage and parturition was higher (P < 0.05) in n-6 compared with n-3 group, being the latter higher than the control group (3.12, 2.79 and 2.43, respectively). Our results show that the dietary PUFAs, particularly n-6 supplementation, have a positive influence on the reproductive performances of the European brown hare.     

Nutritional n-3 polyunsaturated fatty acids deficiency alters cannabinoid receptor signaling pathway in the brain and associated anxiety-like behavior in mice

N-3 polyunsaturated fatty acids (PUFAs) cannot be synthesized de novo in mammals and need to be provided by dietary means. In the brain, the main n-3 PUFA is docosahexaenoic acid (DHA), which is a key component of neuronal membranes. A low dietary level of DHA has been associated with increased risk of developing neuropsychiatric diseases; however, the mechanisms involved remain to be determined. In this study, we found that long-term exposure to an n-3 deficient diet decreases the level of DHA in the brain and impairs the cannabinoid receptor signaling pathway in mood-controlling structures. In n-3 deficient mice, the effect of the cannabinoid agonist WIN55,212-2 in an anxiety-like behavior test was abolished. In addition, the cannabinoid receptor signaling pathways were altered in the prefrontal cortex and the hypothalamus. Consequently, our data suggest that behavioral changes linked to an n-3 dietary deficiency are due to an alteration in the endocannabinoid system in specific brain areas.     

n-6 and n-3 polyunsaturated fatty acids differentially modulate oncogenic Ras activation in colonocytes

Ras proteins are critical regulators of cell function, including growth, differentiation, and apoptosis, with membrane localization of the protein being a prerequisite for malignant transformation. We have recently demonstrated that feeding fish oil, compared with corn oil, decreases colonic Ras membrane localization and reduces tumor formation in rats injected with a colon carcinogen. Because the biological activity of Ras is regulated by posttranslational lipid attachment and its interaction with stimulatory lipids, we investigated whether docosahexaenoic acid (DHA), found in fish oil, compared with linoleic acid (LA), found in corn oil, alters Ras posttranslational processing, activation, and effector protein function in young adult mouse colon cells overexpressing H-ras (YAMC-ras). We show here that the major n-3 polyunsaturated fatty acid (PUFA) constituent of fish oil, DHA, compared with LA (an n-6 PUFA), reduces Ras localization to the plasma membrane without affecting posttranslational lipidation and lowers GTP binding and downstream p42/44(ERK)-dependent signaling. In view of the central role of oncogenic Ras in the development of colon cancer, the finding that n-3 and n-6 PUFA differentially modulate Ras activation may partly explain why dietary fish oil protects against colon cancer development.     

Differential induction of electrophile-responsive element-regulated genes by n-3 and n-6 polyunsaturated fatty acids

In this study the n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid and docosahexaenoic acid appear to be effective inducers of electrophile-responsive element (EpRE) regulated genes, whereas the n-6 PUFA arachidonic acid is not. These n-3 PUFAs need to be oxidized to induce EpRE-regulated gene expression, as the antioxidant vitamin E can partially inhibit the PUFA induced dose-dependent effect. Results were obtained using a reporter gene assay, real-time RT-PCR and enzyme activity assays. The induction of EpRE-regulated phase II genes by n-3 PUFAs may be a major pathway by which n-3 PUFAs, in contrast to n-6 PUFAs, are chemopreventive and anticarcinogenic.     

Studies on the metabolic fate of n-3 polyunsaturated fatty acids

Several different processes involved in the metabolic fate of docosahexaenoic acid (DHA, C22:6n-3) and its precursor in the biosynthesis route, C24:6n-3, were studied. In cultured skin fibroblasts, the oxidation rate of [1-14C] 24:6n-3 was 2.7 times higher than for [1-14C]22:6n-3, whereas [1-14C]22:6n-3 was incorporated 7 times faster into different lipid classes than was [1-14C]24:6n-3. When determining the peroxisomal acyl-CoA oxidase activity, similar specific activities for C22:6(n-3)-CoA and C24:6(n-3)-CoA were found in mouse kidney peroxisomes. Thioesterase activity was measured for both substrates in mouse kidney peroxisomes as well as mitochondria, and C22:6(n-3)-CoA was hydrolyzed 1.7 times faster than C24:6(n-3)-CoA. These results imply that the preferred metabolic fate of C24:6(n-3)-CoA, after its synthesis in the endoplasmic reticulum (ER), is to move to the peroxisome, where it is beta-oxidized, producing C22:6(n-3)-CoA. This DHA-CoA then preferentially moves back, probably as free fatty acid, to the ER, where it is incorporated into membrane lipids.     

The effects of n-3 and n-6 polyunsaturated fatty acids on plasma lipids and fatty acids of treated phenylketonuric children

Dietary-treated phenylketonuric patients (PKUs) display low levels of long-chain polyunsaturated fatty acids (PUFA) in plasma lipids. In a 6-month clinical trial we observed a decrease of triglycerides and an increase of n-3 long-chain PUFA in plasma of PKUs supplemented with fish oil, while no major differences in respect to the baseline values were found in a group supplemented with blackcurrant oil. A more complete source of long-chain PUFA of both the n-6 and n-3 series should be investigated for dietary supplementation of PKU patients.     

NGF blocks polyunsaturated fatty acids biosynthesis in n-3 fatty acid-supplemented PC12 cells

Regulation of polyunsaturated fatty acid (PUFA) biosynthesis in proliferating and NGF-differentiated PC12 pheochromocytoma cells deficient in n-3 docosahexaenoic acid (DHA 22:6n-3) was studied. A dose- and time-dependent increase in eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and DHA in phosphatidylethanolamine (PtdEtn) and phosphatidylserine (PtdSer) glycerophospholipids (GPL) via the elongation/desaturation pathway following alpha-linolenic acid (ALA, 18:3n-3) supplements was observed. That was accompanied by a marked reduction of eicosatrienoic acid (Mead acid 20:3n-9), an index of PUFA deficiency. EPA supplements were equally effective converted to 22:5n-3 and 22:6n-3. On the other hand, supplements of linoleic acid (LNA, 18:2n-6) were not effectively converted into higher n-6 PUFA intermediates nor did they impair elongation/desaturation of ALA. Co-supplements of DHA along with ALA did not interfere with 20:5n-3 biosynthesis but reduced further elongation to 22-hydrocarbon PUFA intermediates. A marked decrease in the newly synthesized 22:5n-3 and 22:6n-3 following ALA or EPA supplements was observed after nerve growth factor (NGF)-induced differentiation. NGF also inhibited the last step in 22:5n-6 formation from LNA. These results emphasize the importance of overcoming n-3 PUFA deficiency and raise the possibility that growth factor regulation of the last step in PUFA biosynthesis may constitute an important feature of neuronal phenotype acquisition.