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1.  n – 3 polyunsaturated fatty acids suppress phosphatidylinositol 4,5-bisphosphate-dependent actin remodelling during CD4+ T-cell activation 
The Biochemical journal  2012;443(1):10.1042/BJ20111589.
n – 3 PUFA (polyunsaturated fatty acids), i.e. DHA (docosahexaenoic acid), found in fish oil, exhibit anti-inflammatory properties; however, the molecular mechanisms remain unclear. Since PtdIns(4,5)P2 resides in raft domains and DHA can alter the size of rafts, we hypothesized that PtdIns(4,5)P2 and downstream actin remodelling are perturbed by the incorporation of n – 3 PUFA into membranes, resulting in suppressed T-cell activation. CD4+ T-cells isolated from Fat-1 transgenic mice (membranes enriched in n – 3 PUFA) exhibited a 50% decrease in PtdIns(4,5)P2. Upon activation by plate-bound anti-CD3/anti-CD28 or PMA/ionomycin, Fat-1 CD4+ T-cells failed to metabolize PtdIns(4,5)P2. Furthermore, actin remodelling failed to initiate in Fat-1 CD4+ T-cells upon stimulation; however, the defect was reversed by incubation with exogenous PtdIns(4,5)P2. When Fat-1 CD4+ T-cells were stimulated with anti-CD3/anti-CD28-coated beads, WASP (Wiskott–Aldrich syndrome protein) failed to translocate to the immunological synapse. The suppressive phenotype, consisting of defects in PtdIns(4,5)P2 metabolism and actin remodelling, were recapitulated in CD4+ T-cells isolated from mice fed on a 4% DHA triacylglycerol-enriched diet. Collectively, these data demonstrate that n – 3 PUFA, such as DHA, alter PtdIns(4,5)P2 in CD4+ T-cells, thereby suppressing the recruitment of WASP to the immunological synapse, and impairing actin remodelling in CD4+ T-cells.
doi:10.1042/BJ20111589
PMCID: PMC3814172  PMID: 22250985
actin remodelling; immunological synapse; n – 3 polyunsaturated fatty acid; PtdIns(4,5)P2; T-cell activation; Wiskott–Aldrich syndrome protein
2.  Interactive effects of fatty acid and butyrate-induced mitochondrial Ca2+ loading and apoptosis in colonocytes 
Cancer  2011;117(23):5294-5303.
BACKGROUND
The combination of fish oil-derived docosahexaenoic acid (DHA, 22:6, n-3) and butyrate (4:0), a fiber fermentation product, synergize to enhance colonocyte apoptosis by inducing a p53-independent, oxidation sensitive, mitochondrial Ca2+-dependent (intrinsic) pathway.
METHODS
In this study, we probed the specificity of n-6 and n-3 polyunsaturated fatty acid induction of Ca2+-dependent proapoptotic events in immortalized YAMC colonocytes. We also determined whether combinations of polyunsaturated fatty acid and butyrate trigger endoplasmic stress (ER) stress conditions, thereby promoting mitochondrial Ca2+ overload. Cultures were treated with 0–50 μM of DHA (22:6, n-3), EPA (20:5, n-3), AA (20:4, n-6), LA (18:2, n-6) or OA (18:1, n-9) for a total of 72 h ± RU-360, to inhibit the mitochondrial Ca2+ uniporter, for 30 min prior to butyrate (0 or 5 mM) co-treatment.
RESULTS
DHA and butyrate combination maximally induced apoptosis and mitochondrial-to-cytosolic Ca2+ levels. In comparison, EPA, a precursor to DHA, was minimally effective. Similarly, AA and OA in combination with butyrate had no effect on mitochondrial Ca2+ or apoptosis compared to butyrate alone. DHA ± butyrate co-treatment minimally altered ER stress regulated genes, CHOP and eIF2α.
CONCLUSION
These data indicate that butyrate and DHA, but not EPA, work coordinately to trigger an ER-independent, Ca2+-dependent intrinsic mitochondrial-mediated apoptotic pathway in colonocytes.
doi:10.1002/cncr.26205
PMCID: PMC3156959  PMID: 21563175
chemoprevention; ER stress; fish oil; dietary fiber; colon cancer; combination chemotherapy; Young adult mouse colon (YAMC) cells
3.  Dietary fish oil promotes colonic apoptosis and mitochondrial proton leak in oxidatively stressed mice 
An alteration of mitochondrial function can result in disruption of redox homeostasis, and is associated with abnormal cancer cell growth. Manganese superoxide dismutase (SOD2) and glutathione peroxidase 4 (Gpx4) are two of the most important antioxidant defense enzymes that protect cells against oxidative stress. We have previously shown that n-3 polyunsaturated fatty acids (PUFA) promote colonocyte apoptosis, a marker of colon cancer risk, in part by enhancing phospholipid oxidation. To elucidate the mechanisms regulating oxidative stress-induced apoptosis in vivo, we fed heterozygous SOD2Het, Gpx4Het and transgenic Gpx4TG mice diets containing either 15% corn oil by weight (CO, enriched in n-6 PUFA) or 3.5% CO + 11.5% fish oil (FO, enriched in n-3 PUFA) for 4 wk. Our data show that (i) genetic pre-deposition to oxidative stress facilitates apoptosis in the mouse colon (Gpx4Het > SOD2Het > Wt > Gpx4Tg), (ii) dietary n-3 PUFA have an additive effect on the induction of apoptosis in Gpx4Het and SOD2Het mice; and (iii) dietary n-3 PUFA reverse the phenotype in oxidatively protected Gpx4Tg mice by elevating apoptosis to a level observed in wild type (control) animals. Complimentary experiments examining colonic mitochondrial bioenergetic profiles indicate that FO fed mice exhibit a significantly (p<0.05) increased respiration-induced proton leak relative to control CO treatment. This finding is consistent with a loss of membrane potential in response to chronic oxidative stress, and supports the contention that n-3 PUFA alter mitochondrial metabolic activity, thereby enhancing apoptosis and reducing colon cancer risk.
doi:10.1158/1940-6207.CAPR-10-0368
PMCID: PMC3137683  PMID: 21490130
apoptosis; n-3 PUFA; oxidation; colon; mitochondria
4.  Linoleic Acid and Butyrate Synergize to Increase Bcl-2 Levels in Colonocytes 
The biological properties of polyunsaturated fatty acid (PUFA) classes have been the source of much contention. For example, n-3 PUFA are chemoprotective, while n-6 PUFA may promote tumor development. Since dietary components can have combinatorial effects, we further examined the apoptotic properties of n-3 or n-6 fatty acids when combined with different fiber sources. Mice were fed diets supplemented with either fish oil (enriched in n-3 PUFA) or corn oil (enriched in n-6 PUFA) and non-fermentable (cellulose) or fermentable (pectin) fiber sources. In complementary experiments, immortalized young adult mouse colonic (YAMC) cells were treated with docosahexaenoic acid (DHA, 22:6n-3) or linoleic acid (LA, 18:2n-6) with or without butyrate. Mice fed a fish oil and pectin diet had significantly (p<0.05) increased levels of apoptosis in colonocytes compared to all other diets. Similarly, apoptosis was highly induced in DHA and butyrate co-treated YAMC cells. In contrast, in both YAMC and mouse models, LA/corn oil with butyrate/pectin treatment reduced apoptosis and enhanced expression of bcl-2. The LA and butyrate induced anti-apoptotic phenotype was reversed by knocking down bcl-2 using targeted siRNA. In comparison, overexpression of bcl-2 blocked the pro-apoptotic effect of DHA and butyrate. These data provide new mechanistic insights into the regulation of apoptosis by dietary PUFA and fiber.
doi:10.1002/ijc.25323
PMCID: PMC2962699  PMID: 20232381
Fatty acids; Fiber; Bcl-2; Apoptosis
5.  Incorporation of a Dietary Omega 3 Fatty Acid Impairs Murine Macrophage Responses to Mycobacterium tuberculosis 
PLoS ONE  2010;5(5):e10878.
Background
Beside their health benefits, dietary omega 3 polyunsaturated fatty acids (n-3 PUFA) might impair host resistance to Mycobacterium tuberculosis (Mtb) by creating an immunosuppressive environment. We hypothesized that incorporation of n-3 PUFA suppresses activation of macrophage antimycobacterial responses and favors bacterial growth, in part, by modulating the IFNγ-mediated signaling pathway.
Methodology/Principal Findings
Murine macrophage-like J774A.1 cells were incubated with bovine serum albumin (BSA)-conjugated docosahexaenoic acid (DHA; 22:6n-3) or BSA alone, activated with recombinant IFNγ, and infected with a virulent strain (H37Rv) of M. tuberculosis. The fatty acid composition of macrophage membranes was modified significantly by DHA treatment. DHA-treated macrophages were less effective in controlling intracellular mycobacteria and showed impaired oxidative metabolism and reduced phagolysosome maturation. Incorporation of DHA resulted in defective macrophage activation, as characterized by reduced production of pro-inflammatory cytokines (TNFα, IL-6 and MCP-1), and lower expression of co-stimulatory molecules (CD40 and CD86). DHA treatment impaired STAT1 phosphorylation and colocalization of the IFNγ receptor with lipid rafts, without affecting surface expression of IFNγ receptor.
Conclusions/Significance
We conclude that DHA reduces the ability of J774A.1 cells to control M. tuberculosis in response to activation by IFNγ, by modulation of IFNγ receptor signaling and function, suggesting that n-3 PUFA-enriched diets may have a detrimental effect on host immunity to tuberculosis.
doi:10.1371/journal.pone.0010878
PMCID: PMC2878322  PMID: 20526363
6.  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 formation1 
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 PKCθ, PLCγ-1 and F-actin into the IS was suppressed. In addition, both the phosphorylation status of PLCγ-1 at the IS and cell proliferation as assessed by CFSE labeling and [3H]-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.
PMCID: PMC2597670  PMID: 18941214
T cells; Signal Transduction; Cell Activation; fat-1; immunological synapse; lipid rafts; nutrition
7.  fat-1 transgene expression prevents cell culture-induced loss of membrane n-3 fatty acids in activated CD4+ T-cells☆ 
In order to evaluate the effects of fatty acids on immune cell membrane structure and function, it is often necessary to maintain cells in culture. However, cell culture conditions typically reverse alterations in polyunsaturated fatty acid (PUFA) composition achieved by dietary lipid manipulation. Therefore, we hypothesized that T-cells from transgenic mice expressing the Caenorhabditis elegans n-3 desaturase (fat-1) gene would be resistant to the culture-induced loss of n-3 PUFA and, therefore, obviate the need to incorporate fatty acids or homologous serum into the medium. CD4+ T-cells were isolated from (i) control wild type (WT) mice fed a safflower oil-n-6 PUFA enriched diet (SAF) devoid of n-3 PUFA, (ii) fat-1 transgenic mice (enriched with endogenous n-3 PUFA) fed a SAF diet, or (iii) WT mice fed a fish oil (FO) based diet enriched in n-3 PUFA. T-cell phospholipids isolated from WT mice fed FO diet (enriched in n-3 PUFA) and fat-1 transgenic mice fed a SAF diet (enriched in n-6 PUFA) were both enriched in n-3 PUFA. As expected, the mol% levels of both n-3 and n-6 PUFA were decreased in cultures of CD4+ T-cells from FO-fed WT mice after 3 d in culture. In contrast, the expression of n-3 desaturase prevented the culture-induced decrease of n-3 PUFA in CD4+ T-cells from the transgenic mice. Carboxyfluorescein succinidyl ester (CFSE) -labeled CD4+ T-cells from fat-1/SAF vs. WT/SAF mice stimulated with anti-CD3 and anti-CD28 for 3 d, exhibited a reduced (P<0.05) number of cell divisions. We conclude that fat-1-containing CD4+ T-cells express a physiologically relevant, n-3 PUFA enriched, membrane fatty acid composition which is resistant to conventional cell culture-induced depletion.
doi:10.1016/j.plefa.2008.09.017
PMCID: PMC2718534  PMID: 18977126
n-3 fatty acid desaturase; Fish oil; Phospholipids; Lymphocyte
8.  Reduced colitis-associated colon cancer in fat-1 (n-3 fatty acid desaturase) transgenic mice 
Cancer research  2008;68(10):3985-3991.
Bioactive food components containing n-3 polyunsaturated fatty acids (PUFA) modulate multiple determinants which link inflammation to cancer initiation and progression. Therefore, in this study, fat-1 transgenic mice which convert endogenous n-6 PUFA to n-3 PUFA in multiple tissues, were injected with azoxymethane followed by 3 cycles of dextran sodium sulphate (DSS) to induce colitis-associated cancer. fat-1 mice exhibited a reduced number of colonic adenocarcinomas per mouse (1.05±0.29 vs 2.12±0.51, p=0.033), elevated apoptosis (p=0.03) and a decrease in n-6 PUFA-derived eicosanoids, compared to wild type (wt) mice. To determine whether the chemoprotective effects of n-3 PUFA could be attributed to its pleiotropic anti-inflammatory properties, colonic inflammation and injury scores were evaluated 5 d after DSS exposure followed by either a 3 d or 2 wk recovery period. There was no effect of n-3 PUFA at 3 d. However, following a 2 wk recovery period, colonic inflammation and ulceration scores returned to pretreatment levels compared to 3 d recovery only in fat-1 mice. For the purpose of examining the specific reactivity of lymphoid elements in the intestine, CD3+ T cells, CD4+ T helper cells and macrophages from colonic lamina propria were quantified. Comparison of 3 d vs 2 wk recovery time points revealed that fat-1 mice exhibited decreased (p<0.05) CD3+, CD4+ T helper, and macrophage cell numbers per colon as compared to wt mice. These results suggest that the anti-tumorigenic effect of n-3 PUFA may be mediated in part via its anti-inflammatory properties.
doi:10.1158/0008-5472.CAN-07-6251
PMCID: PMC2648804  PMID: 18483285
inflammation; chemoprevention; dextran sodium sulphate; tumor promotion
9.  Bioactive dietary long chain fatty acids: Emerging mechanisms of action 
The British journal of nutrition  2008;100(6):1152-1157.
The plasma membrane of all eukaryotic cells contain heterogeneous self organizing intrinsically unstable liquid ordered domains or lipid assemblies in which key signal transduction proteins are localized. These assemblies are classified as “lipid rafts” (10–200 nm), which are composed mostly of cholesterol and sphingolipid microdomains and therefore do not integrate well into the fluid phospholipid bilayers. In addition, caveolae represent a subtype of lipid raft macrodomain that form flask-shaped membrane invaginations containing structural proteins, i.e., caveolins. With respect to the diverse biological effects of long chain polyunsaturated fatty acids (PUFA), increasing evidence suggests that n-3 PUFA and perhaps conjugated fatty acids uniquely alter the basic properties of cell membranes. Because of its polyunsaturation, docosahexaenoic acid (DHA) and possibly conjugated linoleic acid (CLA) are sterically incompatible with sphingolipid and cholesterol and, therefore, appear to alter lipid raft behavior and protein function. This review examines the evidence indicating that dietary sources of n-3 PUFA can profoundly alter the biochemical make up of lipid rafts/caveolae microdomains, thereby influencing cell signaling, protein trafficking, and cell cytokinetics.
doi:10.1017/S0007114508992576
PMCID: PMC2648819  PMID: 18492298
membrane rafts; omega-3 fatty acids; conjugated fatty acids; microdomains
10.  Docosahexaenoic Acid Alters the Size and Distribution of Cell Surface Microdomains 
Biochimica et biophysica acta  2007;1778(2):466-471.
Summary
We recently generated nutritional data suggesting that chemoprotective dietary n-3 polyunsaturated fatty acids (n-3 PUFA) are capable of displacing acylated proteins from lipid raft microdomains in vivo (Ma et al., FASEB J. 18:1040, 2004; Fan et al., J. Immunol. 173:6151, 2004). A primary source of very long chain n-3 PUFA in the diet is derived from fish enriched with docosahexaenoic acid (DHA, 22:6n-3). In this study, we sought to determine the effect of DHA on cell surface microdomain organization in situ. Using immuno-gold electron microscopy of plasma membrane sheets coupled with spatial point analysis of validated microdomain markers, morphologically featureless microdomains were visualized in HeLa cells at high resolution. Clustering of probes within cholesterol-dependent (GFP-tH) versus cholesterol-independent (GFP-tK) nanoclusters was differentially sensitive to n-3 PUFA treatment of cells. Univariate K-function analysis of GFP-tH (5 nm gold) revealed a significant increase in clustering (p<0.05) by pre-treatment with DHA and linoleic acid (LA, 18:2Δ9,12) compared to control fatty acids; whereas LA significantly (p<0.05) reduced GFP-tK clustering. These novel data suggest that the plasma membrane organization of inner leaflets is fundamentally altered by PUFA-enrichment. We speculate that our findings may help define a new paradigm to better understand the complexity of n-3 PUFA modulation of signaling networks.
doi:10.1016/j.bbamem.2007.11.003
PMCID: PMC2244794  PMID: 18068112
Dynamic domains; nanoclusters; omega-3 fatty acid; microdomains

Results 1-10 (10)