Physical and chemical modulation of lipid rafts by a dietary n-3 polyunsaturated fatty acid increases ethanol-induced oxidative stress

Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported to modulate lipid raft-dependent signaling, but not yet lipid raft-dependent oxidative stress. Previously, we have shown that ethanol-induced membrane remodeling, i.e., an increase in membrane fluidity and alterations in physical and biochemical properties of lipid rafts, participated in the development of oxidative stress. Thus, we decided to study n-3 PUFA effects in this context, by pretreating hepatocytes with eicosapentaenoic acid (EPA), a long-chain n-3 PUFA, before addition of ethanol. EPA was found to increase ethanol-induced oxidative stress through membrane remodeling. Addition of EPA resulted in a marked increase in lipid raft aggregation compared to ethanol alone. In addition, membrane fluidity of lipid rafts was markedly enhanced. Interestingly, EPA was found to preferentially incorporate into nonraft membrane regions, leading to raft cholesterol increase. Lipid raft aggregation by EPA enhanced phospholipase Cγ translocation into these microdomains. Finally, phospholipase Cγ was shown to participate in the potentiation of oxidative stress by promoting lysosome accumulation, a major source of low-molecular-weight iron. To conclude, the ability of EPA to modify lipid raft physical and chemical properties plays a key role in the enhancement, by this dietary n-3 PUFA, of ethanol-induced oxidative stress.     

Dietary docosahexaenoic acid suppresses T cell protein kinase C theta lipid raft recruitment and IL-2 production

To date, the proximal molecular targets through which dietary n-3 polyunsaturated fatty acids (PUFA) suppress the inflammatory process have not been elucidated. Because cholesterol and sphingolipid-enriched rafts have been proposed as platforms for compartmentalizing dynamically regulated signaling assemblies at the plasma membrane, we determined the in vivo effects of fish oil and highly purified docosahexaenoic acid (DHA; 22:6n-3) on T cell microdomain lipid composition and the membrane subdomain distribution of signal-transducing molecules (protein kinase C (PKC)theta;, linker for activation of T cells, and Fas/CD95), before and after stimulation. Mice were fed diets containing 5 g/100 g corn oil (control), 4 g/100 g fish oil (contains a mixture of n-3 PUFA) plus 1 g/100 g corn oil, or 4 g/100 g corn oil plus 1 g/100 g DHA ethyl ester for 14 days. Dietary n-3 PUFA were incorporated into splenic T cell lipid raft and soluble membrane phospholipids, resulting in a 30% reduction in raft sphingomyelin content. In addition, polyclonal activation-induced colocalization of PKCtheta; with lipid rafts was reduced by n-3 PUFA feeding. With respect to PKCtheta; effector pathway signaling, both AP-1 and NF-kappaB activation, IL-2 secretion, and lymphoproliferation were inhibited by fish oil feeding. Similar results were obtained when purified DHA was fed. These data demonstrate for the first time that dietary DHA alters T cell membrane microdomain composition and suppresses the PKCtheta; signaling axis.     

Low dietary fish-oil threshold for myocardial membrane n-3 PUFA enrichment independent of n-6 PUFA intake in rats

Long chain n-3 PUFA docosahexaenoic acid (DHA) is important for heart and brain function. Investigations of biologically plausible mechanisms using animal models associate cardioprotection with DHA incorporation into myocardial membranes that are largely derived from supra-physiological fish oil (FO) intake. We measured the incorporation of DHA into myocardial membranes of rats from low dietary FO intake within human dietary range and quantitatively assessed the influence of dietary n-6 PUFA. With rats fed diets containing 0.16%–5% FO, equal to 0.12%–8.7% energy (%en) as eicosapentaenoic acid (EPA) and DHA (EPA+DHA), and either 1.5%en or 7.5%en n-6 PUFA (linoleic acid) for four weeks, dietary n-6:n-3 PUFA ratios ranged from 74 to 0.3. Myocardial DHA concentration increased in a log-linear fashion with a dietary threshold of 0.019%en as EPA+DHA and half maximal dietary [EPA+DHA] equal to 0.29%en (95% CI, 0.23–0.35). Dietary linoleic acid intake did not influence myocardial DHA. Myocardial membranes are sensitive to absolute dietary intake of long chain n-3 PUFA at low %en in the rat, equivalent to a human intake of one meal of fatty fish per week or less. The dietary ratio of n-6:n-3 PUFA has no influence on long chain n-3 PUFA cellular incorporation from dietary fish oil.     

Docosahexaenoic and Eicosapentaenoic Acids Segregate Differently between Raft and Nonraft Domains

Omega-3 polyunsaturated fatty acids (n-3 PUFA), enriched in fish oils, are increasingly recognized to have potential benefits for treating many human afflictions. Despite the importance of PUFA, their molecular mechanism of action remains unclear. One emerging hypothesis is that phospholipids containing n-3 PUFA acyl chains modify the structure and composition of membrane rafts, thus affecting cell signaling. In this study the two major n-3 PUFA found in fish oils, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, are compared. Using solid-state (2)H NMR spectroscopy we explored the molecular organization of 1-[(2)H(31)]palmitoyl-2-eicosapentaenoylphosphatidylcholine (PEPC-d(31)) and 1-[(2)H(31)]palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC-d(31)) in mixtures with sphingomyelin (SM) and cholesterol (chol). Our results indicate that whereas both PEPC-d(31) and PDPC-d(31) can accumulate into SM-rich/chol-rich raftlike domains, the tendency for DHA to incorporate into rafts is more than twice as great as for EPA. We propose that DHA may be the more bioactive component of fish oil that serves to disrupt lipid raft domain organization. This mechanism represents an evolution in the view of how PUFA remodel membrane architecture.     

N-3 Poly-Unsaturated Fatty Acids Shift Estrogen Signaling to Inhibit Human Breast Cancer Cell Growth

Although evidence has shown the regulating effect of n-3 poly-unsaturated fatty acid (n-3 PUFA) on cell signaling transduction, it remains unknown whether n-3 PUFA treatment modulates estrogen signaling. The current study showed that docosahexaenoic acid (DHA, C22:6), eicosapentaenoic acid (EPA, C20:5) shifted the pro-survival and proliferative effect of estrogen to a pro-apoptotic effect in human breast cancer (BCa) MCF-7 and T47D cells. 17 β-estradiol (E2) enhanced the inhibitory effect of n-3 PUFAs on BCa cell growth. The IC50 of DHA or EPA in MCF-7 cells decreased when combined with E2 (10 nM) treatment (from 173 µM for DHA only to 113 µM for DHA+E2, and from 187 µm for EPA only to 130 µm for EPA+E2). E2 also augmented apoptosis in n-3 PUFA-treated BCa cells. In contrast, in cells treated with stearic acid (SA, C18:0) as well as cells not treated with fatty acid, E2 promoted breast cancer cell growth. Classical (nuclear) estrogen receptors may not be involved in the pro-apoptotic effects of E2 on the n-3 PUFA-treated BCa cells because ERα agonist failed to elicit, and ERα knockdown failed to block E2 pro-apoptotic effects. Subsequent studies reveal that G protein coupled estrogen receptor 1 (GPER1) may mediate the pro-apoptotic effect of estrogen. N-3 PUFA treatment initiated the pro-apoptotic signaling of estrogen by increasing GPER1-cAMP-PKA signaling response, and blunting EGFR, Erk 1/2, and AKT activity. These findings may not only provide the evidence to link n-3 PUFAs biologic effects and the pro-apoptotic signaling of estrogen in breast cancer cells, but also shed new insight into the potential application of n-3 PUFAs in BCa treatment.     

n-3 PUFA and membrane microdomains: a new frontier in bioactive lipid research

In recent years, our understanding of the plasma membrane has changed considerably as our knowledge of lipid microdomains has expanded. Lipid microdomains include structures known as lipid rafts and caveolae, which are readily identified by their unique lipid constituents. Cholesterol, sphingolipids and phospholipids with saturated fatty acyl chain moieties are highly enriched in these lipid microdomains. Lipid rafts and caveolae have been shown to play an important role in the compartmentalization, modulation and integration of cell signaling. Therefore, these microdomains may have an influential role in human disease. Dietary n-3 polyunsaturated fatty acids (PUFA) ameliorate a number of human diseases including coronary heart disease, autoimmune and inflammatory disorders, diabetes, obesity and cancer, which has been generally linked to its membrane remodeling properties. Recent in vitro evidence suggests that perturbations in membrane composition alter the function of resident proteins and, consequently, cellular responses. This review examines the role of n-3 PUFA in modulating the lipid composition and functionality of lipid microdomains and its potential significance to human health.     

Diets enriched in menhaden fish oil, seal oil, or shark liver oil have distinct effects on the lipid and fatty-acid composition of guinea pig heart

The purpose of this investigation was to determine whether diets supplemented with oils from three different marine sources, all of which contain high proportions of long-chain n-3 polyunsaturated fatty acids (PUFA), result in qualitatively distinct lipid and fatty acid profiles in guinea pig heart. Albino guinea pigs (14 days old) were fed standard, nonpurified guinea pig diets (NP) or NP supplemented with menhaden fish oil (MO), harp seal oil (SLO) or porbeagle shark liver oil (PLO) (10%, w/w) for 4-5 weeks. An n-6 PUFA control group was fed NP supplemented with corn oil (CO). All animals appeared healthy, with weight gains marginally lower in animals fed the marine oils. Comparison of relative organ weights indicated that only the livers responded to the diets, and that they were heavier only in the marine-oil fed guinea pigs. Heart total cholesterol levels were unaffected by supplementing NP with any of the oils, whereas all increased the triacylglycerol (TAG) content. The fatty-acid profiles of totalphospholipid (TPL), TAG and free fatty acid (FFA) fractions of heart lipids showed that feeding n-3 PUFA significantly altered the proportions of specific fatty-acid classes. For example, all marine-oil-rich diets were associated with increases in total monounsaturated fatty acids in TPL (p < 0.05), and with decreases in total saturates in TAG (p < 0.05). Predictably, the n-3 PUFA enriched regimens significantly increased the cardiac content of n-3 PUFA and decreased that of n-6 PUFA, although the extent varied among the diets. As a result, n-6/n-3 ratios were significantly lower in all myocardial lipid classes of marine-oil-fed guinea pigs. Analyses of the profiles of individual PUFA indicated that quantitatively, the fatty acids of the three marine oils were metabolized and/or incorporated into TPL, TAG and FFA in a diet-specific manner. In animals fed MO-enriched diets in which eicosapentaenoic acid (EPA) > docosahexacnoic acid (DHA), ratios of DHA /EPA in the hearts were 1.2, 2.2 and 1.5 in TPL, TAG and FFA, respectively. In SLO-fed guinea pigs in which dietary EPA DHA, ratios of DHA/EPA were 0.9, 3.4 and 2.1 in TPL, TAG and FFA, respectively. Feeding NP + PLO (DHA/EPA = 4.8), resulted in values for DHA/EPA in cardiac tissue of 2.1, 10.6 and 2.9 in TPL, TAG and FFA, respectively. In the TAG and FFA, proportions of n-3 docosapentaenoic acid (n-3 DPA) were equal to or higher than EPA in the SLO- and PLO-fed animals. The latter group exhibited the greatest difference between the DHA/n-3 DPA ratio in the diet and in cardiac TAG and FFA fractions (7, 3.4 and 3.1, respectively). Quantitative analysis indicated that 85% of the n-3 PUFA were in TPL, 7-11% were in TAG, and 2-6% were FFA. Specific patterns of distribution of EPA, DPA and DHA depended on the dietary oil. Both the qualitative and quantitative results of this study demonstrated that in guinea pigs, n-3 PUFA in different marine oils are metabolized and/or incorporated into cardiac lipids in distinct manners. In support of the concept that the diet-induced alterations reflect changes specifically in cardiomyocytes, we observed that direct supplementation of cultured guinea pig myocytes for 2-3 weeks with EPA or DHA produced changes in the PUFA profiles of their TPL that were qualitatively similar to those observed in tissue from the dietary study. The factors that regulate specific deposition of n-3 PUFA from either dietary oils or individual PUFA are not yet known, however the differences that we observed could in some manner be related to cardiac function and thus their relative potentials as health-promoting dietary fats.     

Biophysical and biochemical mechanisms by which dietary N-3 polyunsaturated fatty acids from fish oil disrupt membrane lipid rafts

N-3 polyunsaturated fatty acids (PUFAs) from fish oil exert their functional effects by targeting multiple mechanisms. One mechanism to emerge in the past decade is the ability of n-3 PUFA acyl chains to perturb the molecular organization of plasma membrane sphingolipid/cholesterol-enriched lipid raft domains. These domains are nanometer-scale assemblies that coalesce to compartmentalize select proteins for optimal function. Here we review recent evidence on how n-3 PUFAs modify lipid rafts from biophysical and biochemical experiments from several different model systems. A central theme emerges from these studies. N-3 PUFA acyl chains display tremendous conformational flexibility and a low affinity for cholesterol and saturated acyl chains. This unique flexibility of n-3 PUFA acyl chains impacts the organization of inner and outer leaflet lipid rafts by disrupting acyl chain packing and molecular order within rafts. Ultimately, the disruption in raft organization has consequences for protein clustering and thereby signaling. Overall, elucidating the complex mechanisms by which n-3 PUFA acyl chains reorganize membrane architecture will enhance the translation of these fatty acids into the clinic for treating several diseases.     

The opposing effects of n-3 and n-6 fatty acids

Polyunsaturated fatty acids (PUFAs) can be classified in n-3 fatty acids and n-6 fatty acids, and in westernized diet the predominant dietary PUFAs are n-6 fatty acids. Both types of fatty acids are precursors of signaling molecules with opposing effects, that modulate membrane microdomain composition, receptor signaling and gene expression. The predominant n-6 fatty acid is arachidonic acid, which is converted to prostaglandins, leukotrienes and other lipoxygenase or cyclooxygenase products. These products are important regulators of cellular functions with inflammatory, atherogenic and prothrombotic effects. Typical n-3 fatty acids are docosahexaenoic acid and eicosapentaenoic acid, which are competitive substrates for the enzymes and products of arachidonic acid metabolism. Docosahexaenoic acid- and eicosapentaenoic acid-derived eicosanoids antagonize the pro-inflammatory effects of n-6 fatty acids. n-3 and n-6 fatty acids are ligands/modulators for the nuclear receptors NFkappaB, PPAR and SREBP-1c, which control various genes of inflammatory signaling and lipid metabolism. n-3 Fatty acids down-regulate inflammatory genes and lipid synthesis, and stimulate fatty acid degradation. In addition, the n-3/n-6 PUFA content of cell and organelle membranes, as well as membrane microdomains strongly influences membrane function and numerous cellular processes such as cell death and survival.     

Effect of docosahexaenoic acid on interleukin-2 receptor signaling pathway in lipid rafts

Recent studies have shown that polyunsaturated fatty acids (PUFA) regulated the functions of membrane receptors in T cells and suppressed T cell-mediated immune responses. But the molecular mechanisms of immune regulation are not yet elucidated. Lipid rafts are plasma membrane microdomains, in which many receptors localized. The purpose of this study was to investigate the effect of DHA on IL-2R signaling pathway in lipid rafts. We isolated lipid rafts by discontinuous sucrose density gradient ultracentrifugation, and found that DHA could change the composition of lipid rafts and alter the distribution of key molecules of IL-2R signaling pathway, which transferred from lipid rafts to detergent-soluble membrane fractions. These results revealed that DHA treatment increased the proportion of polyunsaturated fatty acids especially n−3 polyunsaturated fatty acids in lipid rafts and changed the lipid environment of membrane microdomains in T cells. Compared with controls, DHA changed the localization of IL-2R, STAT5a and STAT5b in lipid rafts and suppressed the expression of JAK1, JAK3 and tyrosine phosphotyrosine in soluble membrane fractions. Summarily, this study concluded the effects of DHA on IL-2R signaling pathway in lipid rafts and explained the regulation of PUFAs in T cell-mediated immune responses.     

Effect of docosahexaenoic acid on interleukin-2 receptor signaling pathway in lipid rafts

PUFAs have been shown to mediate immune re-sponse especially the functions of T cells[1]. Recent researches have demonstrated that PUFAs can in-crease membrane fluidity and modify the functions of membrane receptors and enzymes in T cell membra-ne[2,3]. M…     

Fatty acid and peptide profiles in plasma membrane and membrane rafts of PUFA supplemented RAW264.7 macrophages

The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.     

Dietary omega-6 fatty acid lowering increases bioavailability of omega-3 polyunsaturated fatty acids in human plasma lipid pools

BackgroundDietary linoleic acid (LA, 18:2n-6) lowering in rats reduces n-6 polyunsaturated fatty acid (PUFA) plasma concentrations and increases n-3 PUFA (eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)) concentrations.ObjectiveTo evaluate the extent to which 12 weeks of dietary n-6 PUFA lowering, with or without increased dietary n-3 PUFAs, alters unesterified and esterified plasma n-6 and n-3 PUFA concentrations in subjects with chronic headache.DesignSecondary analysis of a randomized trial. Subjects with chronic headache were randomized for 12 weeks to (1) average n-3, low n-6 (L6) diet; or (2) high n-3, low n-6 LA (H3–L6) diet. Esterified and unesterified plasma fatty acids were quantified at baseline (0 weeks) and after 12 weeks on a diet.ResultsCompared to baseline, the L6 diet reduced esterified plasma LA and increased esterified n-3 PUFA concentrations (nmol/ml), but did not significantly change plasma arachidonic acid (AA, 20:4n-6) concentration. In addition, unesterified EPA concentration was increased significantly among unesterified fatty acids. The H3–L6 diet decreased esterified LA and AA concentrations, and produced more marked increases in esterified and unesterified n-3 PUFA concentrations.ConclusionDietary n-6 PUFA lowering for 12 weeks significantly reduces LA and increases n-3 PUFA concentrations in plasma, without altering plasma AA concentration. A concurrent increase in dietary n-3 PUFAs for 12 weeks further increases n-3 PUFA plasma concentrations and reduces AA.     

n-3 polyunsaturated fatty acids suppress the localization and activation of signaling proteins at the immunological synapse in murine CD4+ T cells by affecting lipid raft formation

The molecular properties of immunosuppressive n-3 polyunsaturated fatty acids (PUFA) have not been fully elucidated. Using CD4(+) T cells from wild-type control and fat-1 transgenic mice (enriched in n-3 PUFA), we show that membrane raft accumulation assessed by Laurdan (6-dodecanoyl-2-dimethyl aminonaphthalene) labeling was enhanced in fat-1 cells following immunological synapse (IS) formation by CD3-specific Ab expressing hybridoma cells. However, the localization of protein kinase Ctheta, phospholipase Cgamma-1, and F-actin into the IS was suppressed. In addition, both the phosphorylation status of phospholipase Cgamma-1 at the IS and cell proliferation as assessed by CFSE labeling and [(3)H]thymidine incorporation were suppressed in fat-1 cells. These data imply that lipid rafts may be targets for the development of dietary agents for the treatment of autoimmune and chronic inflammatory diseases.     

LAT displacement from lipid rafts as a molecular mechanism for the inhibition of T cell signaling by polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) suppress immune responses and inhibit T cell activation through largely unknown mechanisms. The displacement of signaling proteins from membrane lipid rafts has recently been suggested as underlying PUFA-mediated T cell inhibition. We show here that PUFA treatment specifically interferes with T cell signal transduction by blocking tyrosine phosphorylation of LAT (linker for activation of T cells) and phospholipase Cgamma1. A significant fraction of LAT was displaced from rafts by PUFA treatment along with other signaling proteins. However, retaining LAT alone in lipid rafts effectively restored phospholipase Cgamma1/calcium signaling in PUFA-treated T cells. These data reveal LAT displacement from lipid rafts as a molecular mechanism by which PUFAs inhibit T cell signaling and underline the predominant importance of LAT localization in rafts for efficient T cell activation.     

α-Linolenic acid-enriched butter attenuated high fat diet-induced insulin resistance and inflammation by promoting bioconversion of n-3 PUFA and subsequent oxylipin formation

α-Linolenic acid (ALA) is an essential fatty acid and the precursor for long-chain n-3 PUFA. However, biosynthesis of n-3 PUFA is limited in a Western diet likely due to an overabundance of n-6 PUFA. We hypothesized that dietary reduction of n-6/n-3 PUFA ratio is sufficient to promote the biosynthesis of long-chain n-3 PUFA, leading to an attenuation of high fat (HF) diet-induced obesity and inflammation. C57BL/6 J mice were fed a HF diet from ALA-enriched butter (n3Bu, n-6/n-3=1) in comparison with isocaloric HF diets from either conventional butter lacking both ALA and LA (Bu, n-6/n-3=6), or margarine containing a similar amount of ALA and abundant LA (Ma, n-6/n-3=6). Targeted lipidomic analyses revealed that n3Bu feeding promoted the bioconversion of long-chain n-3 PUFA and their oxygenated metabolites (oxylipins) derived from ALA and EPA. The n3Bu supplementation attenuated hepatic TG accumulation and adipose tissue inflammation, resulting in improved insulin sensitivity. Decreased inflammation by n3Bu feeding was attributed to the suppression of NF-κB activation and M1 macrophage polarization. Collectively, our work suggests that dietary reduction of the n-6/n-3 PUFA ratio, as well as total n-3 PUFA consumed, is a crucial determinant that facilitates n-3 PUFA biosynthesis and subsequent lipidomic modifications, thereby conferring metabolic benefits against obesity-induced inflammation and insulin resistance.