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1.  Analysis of Epidermal Growth Factor Receptor Dimerization by BS3 Cross-Linking 
Dimerization of receptor tyrosine kinases is a well-characterized process. It is imperative for the activation of many receptors, including the epidermal growth factor receptor (EGFR). EGFR has been shown to be regulated by a number of factors, including lipid raft localization. For example, alteration of the lipid raft localization of EGFR has been demonstrated to modify receptor dimerization. This protocol describes an assay to quantify EGFR dimers using BS3 cross-linking. BS3 cross-linking is well suited for this purpose because of its length, water solubility, and membrane impermeability. Although this protocol is written specifically for EGFR, the assay can be extrapolated in order to characterize dimerization of other receptor tyrosine kinases.
PMCID: PMC4327833  PMID: 25319886
Epidermal growth factor receptor; Dimerization; Lipid rafts; BS3; Cross-linking; DHA
2.  Dietary fish oil and DHA down-regulate antigen-activated CD4+ T-cells while promoting the formation of liquid-ordered mesodomains 
The British journal of nutrition  2013;111(2):254-260.
We have demonstrated previously that n-3 PUFA endogenously produced by fat-1 transgenic mice regulate CD4+ T-cell function by affecting the formation of lipid rafts, liquid-ordered mesodomains in the plasma membrane. In the present study, we tested the effects of dietary sources of n-3 PUFA, i.e. fish oil (FO) or purified DHA, when compared with an n-6 PUFA-enriched maize oil control diet in DO11.10 T-cell receptor transgenic mice. Dietary n-3 PUFA were enriched in CD4+ T-cells, resulting in the increase of the n-3:n-6 ratio. Following antigen-specific CD4+ T-cell activation by B-lymphoma cells pulsed with the ovalbumin 323–339 peptide, the formation of liquid-ordered mesodomains at the immunological synapse relative to the whole CD4+ T-cell, as assessed by Laurdan labelling, was increased (P<0·05) in the FO-fed group. The FO diet also suppressed (P<0·05) the co-localisation of PKCθ with ganglioside GM1 (monosialotetrahexosylganglioside), a marker for lipid rafts, which is consistent with previous observations. In contrast, the DHA diet down-regulated (P<0·05) PKCθ signalling by moderately affecting the membrane liquid order at the immunological synapse, suggesting the potential contribution of the other major n-3 PUFA components of FO, including EPA.
PMCID: PMC4327854  PMID: 23962659
n-3 PUFA; DHA; Fish oil; Lipid rafts; CD4+ T-cells
3.  Antagonizing Arachidonic Acid-Derived Eicosanoids Reduces Inflammatory Th17 and Th1 Cell-Mediated Inflammation and Colitis Severity 
Mediators of Inflammation  2014;2014:917149.
During colitis, activation of two inflammatory T cell subsets, Th17 and Th1 cells, promotes ongoing intestinal inflammatory responses. n-6 polyunsaturated fatty acid- (PUFA-) derived eicosanoids, such as prostaglandin E2 (PGE2), promote Th17 cell-mediated inflammation, while n-3 PUFA antagonize both Th17 and Th1 cells and suppress PGE2 levels. We utilized two genetic mouse models, which differentially antagonize PGE2 levels, to examine the effect on Th17 cells and disease outcomes in trinitrobenzene sulfonic acid- (TNBS-) induced colitis. Fat-1 mice contain the ω3 desaturase gene from C. elegans and synthesize n-3 PUFA de novo, thereby reducing the biosynthesis of n-6 PUFA-derived eicosanoids. In contrast, Fads1 Null mice contain a disrupted Δ5 desaturase gene and produce lower levels of n-6 PUFA-derived eicosanoids. Compared to Wt littermates, Fat-1 and Fads1 Null mice exhibited a similar colitic phenotype characterized by reduced colonic mucosal inflammatory eicosanoid levels and mRNA expression of Th17 cell markers (IL-17A, RORγτ, and IL-23), decreased percentages of Th17 cells and, improved colon injury scores (P ≤ 0.05). Thus, during colitis, similar outcomes were obtained in two genetically distinct models, both of which antagonize PGE2 levels via different mechanisms. Our data highlight the critical impact of n-6 PUFA-derived eicosanoids in the promotion of Th17 cell-mediated colonic inflammation.
PMCID: PMC4127240  PMID: 25136149
4.  Non-invasive analysis of intestinal development in preterm and term infants using RNA-Sequencing 
Scientific Reports  2014;4:5453.
The state and development of the intestinal epithelium is vital for infant health, and increased understanding in this area has been limited by an inability to directly assess epithelial cell biology in the healthy newborn intestine. To that end, we have developed a novel, noninvasive, molecular approach that utilizes next generation RNA sequencing on stool samples containing intact epithelial cells for the purpose of quantifying intestinal gene expression. We then applied this technique to compare host gene expression in healthy term and extremely preterm infants. Bioinformatic analyses demonstrate repeatable detection of human mRNA expression, and network analysis shows immune cell function and inflammation pathways to be up-regulated in preterm infants. This study provides incontrovertible evidence that whole-genome sequencing of stool-derived RNA can be used to examine the neonatal host epithelial transcriptome in infants, which opens up opportunities for sequential monitoring of gut gene expression in response to dietary or therapeutic interventions.
PMCID: PMC4071321  PMID: 24965658
5.  A chemoprotective fish oil/pectin diet enhances apoptosis via Bcl-2 promoter methylation in rat azoxymethane-induced carcinomas 
We have demonstrated that diets containing fish oil and pectin (FO/P) reduce colon tumor incidence relative to control (corn oil and cellulose [CO/C]) in part by inducing apoptosis of DNA-damaged colon cells. Relative to FO/P, CO/C promotes colonocyte expression of the antiapoptotic modulator, Bcl-2, and Bcl-2 promoter methylation is altered in colon cancer. To determine if FO/P, compared with CO/C, limits Bcl-2 expression by enhancing promoter methylation in colon tumors, we examined Bcl-2 promoter methylation, mRNA levels, colonocyte apoptosis and colon tumor incidence in azoxymethane (AOM)-injected rats. Rats were provided diets containing FO/P or CO/C, and were terminated 16 and 34 weeks after AOM injection. DNA isolated from paraformaldehyde-fixed colon tumors and uninvolved tissue was bisulfite modified and amplified by quantitative reverese transcriptase-polymerase chain reaction to assess DNA methylation in Bcl-2 cytosine–guanosine islands. FO/P increased Bcl-2 promoter methylation (P = 0.009) in tumor tissues and colonocyte apoptosis (P = 0.020) relative to CO/C. An inverse correlation between Bcl-2 DNA methylation and Bcl-2 mRNA levels was observed in the tumors. We conclude that dietary FO/P promotes apoptosis in part by enhancing Bcl-2 promoter methylation. These Bcl-2 promoter methylation responses, measured in vivo, contribute to our understanding of the mechanisms involved in chemoprevention of colon cancer by diets containing FO/P.
PMCID: PMC3999967  PMID: 23354397
Bcl-2; DNA methylation; epigenetics; fish oil; pectin
6.  Colon cancer cell apoptosis is induced by combined exposure to the n-3 fatty acid docosahexaenoic acid and butyrate through promoter methylation 
DNA methylation and histone acetylation contribute to the transcriptional regulation of genes involved in apoptosis. We have demonstrated that docosahexaenoic acid (DHA, 22:6 n-3) and butyrate enhance colonocyte apoptosis. To determine if DHA and/or butyrate elevate apoptosis through epigenetic mechanisms thereby restoring the transcription of apoptosis-related genes, we examined global methylation; gene-specific promoter methylation of 24 apoptosis-related genes; transcription levels of Cideb, Dapk1, and Tnfrsf25; and global histone acetylation in the HCT-116 colon cancer cell line. Cells were treated with combinations of (50 μM) DHA or linoleic acid (18:2 n-6), (5 mM) butyrate or an inhibitor of DNA methyltransferases, and 5-aza-2′-deoxycytidine (5-Aza-dC, 2 μM). Among highly methylated genes, the combination of DHA and butyrate significantly reduced methylation of the proapoptotic Bcl2l11, Cideb, Dapk1, Ltbr, and Tnfrsf25 genes compared to untreated control cells. DHA treatment reduced the methylation of Cideb, Dapk1, and Tnfrsf25. These data suggest that the induction of apoptosis by DHA and butyrate is mediated, in part, through changes in the methylation state of apoptosis-related genes.
PMCID: PMC3999970  PMID: 24495951
docosahexaenoic acid; butyrate; apoptosis; DNA methylation; epigenetics
7.  Effects of Fatty Acids on Benzo[a]pyrene Uptake and Metabolism in Human Lung Adenocarcinoma A549 Cells 
PLoS ONE  2014;9(3):e90908.
Dietary supplementation with natural chemoprotective agents is receiving considerable attention because of health benefits and lack of toxicity. In recent in vivo and in vitro experimental studies, diets rich in n-3 polyunsaturated fatty acids have been shown to provide significant anti-tumor action. In this investigation, the effects of control fatty acids (oleic acid (OA), linoleic acid (LA)) and n-3 PUFA, e.g., docosahexaenoic acid (DHA) on the uptake and metabolism of the carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP) was investigated in A549 cells, a human adenocarcinoma alveolar basal epithelial cell line. A549 cells activate BaP through the cytochrome P450 enzyme system to form reactive metabolites, a few of which covalently bind to DNA and proteins. Therefore, multiphoton microscopy spectral analysis combined with linear unmixing was used to identify the parent compound and BaP metabolites formed in cells, in the presence and absence of fatty acids. The relative abundance of select metabolites was associated with altered P450 activity as determined using ethoxyresorufin-O-deethylase activity in cells cultured in the presence of BSA-conjugated fatty acids. In addition, the parent compound within cellular membranes increases significantly in the presence of each of the fatty acids, with the greatest accumulation observed following DHA treatment. DHA treated cells exhibit significantly lower pyrene-like metabolites indicative of lower adducts including DNA adducts compared to control BSA, OA or LA treated cells. Further, DHA reduced the abundance of the proximate carcinogen BaP 7,8-dihydrodiol and the 3-hydroxybenzo[a]pyene metabolites compared to other treatments. The significant changes in BaP metabolites in DHA treated cells may be mediated by the effects on the physicochemical properties of the membrane known to affect enzyme activity related to phase I and phase II metabolism. In summary, DHA is a highly bioactive chemo-protective agent capable of modulating BaP-induced DNA adducts.
PMCID: PMC3961214  PMID: 24651647
8.  Folate Transport Gene Inactivation in Mice Increases Sensitivity to Colon Carcinogenesis 
Cancer research  2005;65(3):887-897.
Low dietary folate intake is associated with an increased risk for colon cancer; however, relevant genetic animal models are lacking. We therefore investigated the effect of targeted ablation of two folate transport genes, folate binding protein 1 (Folbp1) and reduced folate carrier 1 (RFC1), on folate homeostasis to elucidate the molecular mechanisms of folate action on colonocyte cell proliferation, gene expression, and colon carcinogenesis. Targeted deletion of Folbp1 (Folbp1+/− and Folbp1−/−) significantly reduced (P < 0.05) colonic Folbp1 mRNA, colonic mucosa, and plasma folate concentration. In contrast, subtle changes in folate homeostasis resulted from targeted deletion of RFC1 (RFC1+/−). These animals had reduced (P < 0.05) colonic RFC1 mRNA and exhibited a 2-fold reduction in the plasma S-adenosylmethionine/S-adenosylhomocysteine. Folbp1+/− and Folbp1−/− mice had larger crypts expressed as greater (P < 0.05) numbers of cells per crypt column relative to Folbp1+/+ mice. Colonic cell proliferation was increased in RFC1+/− mice relative to RFC1+/+ mice. Microarray analysis of colonic mucosa showed distinct changes in gene expression specific to Folbp1 or RFC1 ablation. The effect of folate transporter gene ablation on colon carcinogenesis was evaluated 8 and 38 weeks post-azoxymethane injection in wild-type and heterozygous mice. Relative to RFC1+/+ mice, RFC1+/− mice developed increased (P < 0.05) numbers of aberrant crypt foci at 8 weeks. At 38 weeks, RFC1+/− mice developed local inflammatory lesions with or without epithelial dysplasia as well as adenocarcinomas, which were larger relative to RFC1+/+ mice. In contrast, Folbp1+/− mice developed 4-fold (P < 0.05) more lesions relative to Folbp1+/+ mice. In conclusion, Folbp1 and RFC1 genetically modified mice exhibit distinct changes in colonocyte phenotype and therefore have utility as models to examine the role of folate homeostasis in colon cancer development.
PMCID: PMC3938162  PMID: 15705887
9.  Membrane lipid raft organization is uniquely modified by n-3 polyunsaturated fatty acids 
Fish oil, enriched in bioactive n-3 polyunsaturated fatty acids (PUFA), has been shown to play a role in prevention of colon cancer. The effects of n-3 PUFA are pleiotropic and multifaceted, resulting in an incomplete understanding of their molecular mechanisms of action. Here, we focus on a highly conserved mechanism of n-3 PUFA, which is the alteration of the organization of the plasma membrane. We highlight recent work demonstrating that enrichment of n-3 PUFA in the plasma membrane alters the lateral organization of membrane signaling assemblies (i.e. lipid rafts). This mechanism is central for n-3 PUFA regulation of downstream signaling, T-cell activation, transcriptional activation, and cytokine secretion. We conclude that these studies provide strong evidence for a predominant mechanism by which n-3 PUFA function in colon cancer prevention.
PMCID: PMC3404206  PMID: 22515942
n-3 polyunsaturated fatty acids; DHA; EPA; lipid rafts; colon cancer; chemoprevention; T-lymphocytes
10.  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.
PMCID: PMC3814172  PMID: 22250985
actin remodelling; immunological synapse; n – 3 polyunsaturated fatty acid; PtdIns(4,5)P2; T-cell activation; Wiskott–Aldrich syndrome protein
11.  Alteration of colonic stem cell gene signatures during the regenerative response to injury 
Biochimica et biophysica acta  2012;1822(10):1600-1607.
Since aberrant wound healing and chronic inflammation can promote malignant transformation, we determined whether dietary bioactive fish oil (FO)-derived n-3 polyunsaturated fatty acids (n-3 PUFA) modulate stem cell kinetics in a colitis-wounding model. Lgr5-LacZ and Lgr5-EGFP-IRES-creERT2 mice were fed diets enriched with n-3 PUFA vs n-6 PUFA (control) and exposed to dextran sodium sulfate (DSS) for 5 days in order to induce crypt damage and colitis throughout the colon. Stem cell number, cell proliferation, apoptosis, expression of stem cell (Lgr5, Sox9, Bmi1, Hopx, mTert, Ascl2, and DCAMKL-1) and inflammation (STAT3) markers were quantified. DSS treatment resulted in the ablation of Lgr5+ stem cells in the distal colon, concurrent with the loss of distal crypt structure and proliferating cells. Lgr5, Ascl2 and Hopx mRNA expression levels were decreased in damaged colonic mucosa. Lgr5+ stem cells reappeared at day 5 of DSS recovery, with normal levels attained by day 6 of recovery. There was no effect of diet on the recovery of stem cells. FO fed animals exhibited higher levels of phospho-STAT3 at all time points, consistent with a higher wounding by DSS in FO feeding. n-3 PUFA-fed mice exhibited a reduction in stem cell associated factors, Ascl2, Axin2 and EphB3. These results indicate that rapidly cycling Lgr5+ stem cells residing at position 1 in the colon epithelium are highly susceptible to DSS-induced damage and that dietary cues can impact stem cell regulatory networks.
PMCID: PMC3418394  PMID: 22750333
Lgr5; dextran sodium sulfate; fish oil; Wnt signaling; colon
12.  Host-Microbe Interactions in the Neonatal Intestine: Role of Human Milk Oligosaccharides123 
Advances in Nutrition  2012;3(3):450S-455S.
The infant intestinal microbiota is shaped by genetics and environment, including the route of delivery and early dietary intake. Data from germ-free rodents and piglets support a critical role for the microbiota in regulating gastrointestinal and immune development. Human milk oligosaccharides (HMO) both directly and indirectly influence intestinal development by regulating cell proliferation, acting as prebiotics for beneficial bacteria and modulating immune development. We have shown that the gut microbiota, the microbial metatranscriptome, and metabolome differ between porcine milk–fed and formula-fed (FF) piglets. Our goal is to define how early nutrition, specifically HMO, shapes host-microbe interactions in breast-fed (BF) and FF human infants. We an established noninvasive method that uses stool samples containing intact sloughed epithelial cells to quantify intestinal gene expression profiles in human infants. We hypothesized that a systems biology approach, combining i) HMO composition of the mother’s milk with the infant’s gut gene expression and fecal bacterial composition, ii) gene expression, and iii short-chain fatty acid profiles would identify important mechanistic pathways affecting intestinal development of BF and FF infants in the first few months of life. HMO composition was analyzed by HLPC Chip/time-of-flight MS and 3 HMO clusters were identified using principle component analysis. Initial findings indicated that both host epithelial cell mRNA expression and the microbial phylogenetic profiles provided strong feature sets that distinctly classified the BF and FF infants. Ongoing analyses are designed to integrate the host transcriptome, bacterial phylogenetic profiles, and functional metagenomic data using multivariate statistical analyses.
PMCID: PMC3649482  PMID: 22585924
13.  Insight into Hypoxia Tolerance in Cowpea Bruchid: Metabolic Repression and Heat Shock Protein Regulation via Hypoxia-Inducible Factor 1 
PLoS ONE  2013;8(4):e57267.
Oxygen is of fundamental importance for most living organisms including insects. Hermetic storage uses airtight containment facilities to withhold oxygen required for development, thus preventing damage by insect pests in stored grain. Cowpea bruchid (Callosobruchus maculatus) ceases feeding and growth when exposed to 2% oxygen. However, although population expansion is temporarily arrested, the bruchids (especially late stage larvae) can survive extended periods of hypoxia and recover development if normoxic conditions resume, an ability rarely found in mammals. To begin to understand fundamental mechanisms that enable insects to cope with oxygen deprivation, we constructed a 3′-anchored cDNA library from 4th instar larvae subjected to normoxic and hypoxic treatments (respectively), and performed 454-pyrosequencing. Quality filtering and contig assembly resulted in 20,846 unique sequences. Of these, 5,335 sequences had hits in BlastX searches (E  = 10−6), constituting a 2,979 unigene set. Further analysis based on gene ontology terms indicated that 1,036 genes were involved in a diverse range of cellular functions. Genes encoding putative glycolytic and TCA cycle enzymes as well as components of respiratory chain complexes were selected and assessed for transcript responses to low oxygen. The majority of these genes were down-regulated, suggesting that hypoxia repressed metabolic activity. However, a group of genes encoding heat shock proteins (HSPs) was induced. Promoter analyses of representative HSP genes suggested the involvement of hypoxia-inducible transcription factor 1 (HIF1) in regulating these hypoxia-induced genes. Its activator function has been confirmed by transient co-transfection into S2 cells of constructs of HIF1 subunits and the HSP promoter-driven reporter.
PMCID: PMC3625201  PMID: 23593115
14.  n-3 Polyunsaturated fatty acids exert immunomodulatory effects on lymphocytes by targeting plasma membrane molecular organization 
Molecular aspects of medicine  2011;33(1):46-54.
Fish oil, enriched in bioactive n-3 polyunsaturated fatty acids (PUFA), has therapeutic value for the treatment of inflammation-associated disorders. The effects of n-3 PUFAs are pleiotropic and complex; hence, an understanding of their cellular targets and molecular mechanisms of action remains incomplete. Here we focus on recent data indicating n-3 PUFAs exert immunosuppressive effects on the function of effector and regulatory CD4+ T cells. In addition, we also present emerging evidence that n-3 PUFAs have immunomodulatory effects on B cells. We then focus on one multifaceted mechanism of n-3 PUFAs, which is the alteration of the biophysical and biochemical organization of the plasma membrane. This mechanism is central for downstream signaling, eicosanoid production, transcriptional regulation and cytokine secretion. We highlight recent work demonstrating n-3 PUFA acyl chains in the plasma membrane target the lateral organization of membrane signaling assemblies (i.e. lipid rafts or signaling networks) and de novo phospholipid biosynthesis. We conclude by proposing new functional and mechanistic questions in this area of research that will aid in the development of fish oil as adjuvant therapy for treating unresolved chronic inflammation.
PMCID: PMC3246093  PMID: 22020145
Fish oil; T cells; B cells; plasma membrane domains
15.  Mechanistic insights into the role of microRNAs in cancer: influence of nutrient crosstalk 
Frontiers in Genetics  2012;3:305.
A plethora of studies have described the disruption of key cellular regulatory mechanisms involving non-coding RNAs, specifically microRNAs (miRNA) from the let-7 family, the miR-17 family, miR-21, miR-143, and the miR-200 family, which contribute to aberrant signaling and tumor formation. Certain environmental factors, such as bioactive dietary agents, e.g., folate, curcumin, polyunsaturated fatty acids, are also thought to impact the progression and severity of cancer. In terms of the chemoprotective mechanisms of action, these bioactive dietary agents appear to act, in part, by modulating tissue levels of miR-16, miR-17 family, miR-26b, miR-106b, and miR-200 family miRNAs and their target genes. However, the mechanisms of nutrient action are not yet fully understood. Therefore, additional characterization of the putative underlying mechanisms is needed to further our understanding of the biology, early diagnosis, prevention, and the treatment of cancer. For the purpose of elucidating the epigenetic landscape of cancer, this review will summarize the key findings from recent studies detailing the effect of bioactive dietary agents on miRNA regulation in cancer.
PMCID: PMC3531809  PMID: 23293655
microRNAs; cancer; chemoprevention; diet; epigenetics
16.  Interactive effects of fatty acid and butyrate-induced mitochondrial Ca2+ loading and apoptosis in colonocytes 
Cancer  2011;117(23):5294-5303.
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.
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.
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.
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.
PMCID: PMC3156959  PMID: 21563175
chemoprevention; ER stress; fish oil; dietary fiber; colon cancer; combination chemotherapy; Young adult mouse colon (YAMC) cells
17.  Dietary n-3 Polyunsaturated Fatty Acids (PUFA) Decrease Obesity-Associated Th17 Cell-Mediated Inflammation during Colitis 
PLoS ONE  2012;7(11):e49739.
Clinical and experimental evidence suggests that obesity-associated inflammation increases disease activity during colitis, attributed in part to the effects of Th17 cells. Using a model of concurrent obesity and colitis, we monitored changes in critical immune cell subsets and inflammatory biomarker expression in three key tissues: visceral adipose tissue, colon (local inflammatory site) and spleen (systemic inflammatory site), and we hypothesized that n-3 PUFA would reduce the percentage of inflammatory immune cell subsets and suppress inflammatory gene expression, thereby improving the disease phenotype. Obesity was induced in C57BL/6 mice by feeding a high fat (HF) diet (59.2% kcal) alone or an isocaloric HF diet supplemented with fish oil (HF-FO) for 12 weeks. Colitis was induced via a 2.5% trinitrobenzene sulfonic acid (TNBS) enema. The HF-FO diet improved the obese phenotype by reducing i) serum hormone concentrations (leptin and resistin), ii) adipose tissue mRNA expression of inflammatory cytokines (MCP-1, IFNγ, IL-6, IL17F and IL-21) and iii) total (F4/80+ CD11b+) and inflammatory adipose tissue M1 (F4/80+ CD11c+) macrophage content compared to HF (P<0.05). In addition, the HF-FO diet reduced both colitis-associated disease severity and colonic mRNA expression of the Th17 cell master transcription factor (RORγτ) and critical cytokines (IL-6, IL-17A, IL-17F, IL-21, IL-23 and IFNγ) versus HF (P<0.05). Compared to HF, the percentage of both splenic Th17 and Th1 cells were reduced by the HF-FO group (P<0.05). Under ex vivo polarizing conditions, the percentage of HF-FO derived CD4+ T cells that reached Th17 cell effector status was suppressed (P = 0.05). Collectively, these results indicate that n-3 PUFA suppress Th1/Th17 cells and inflammatory macrophage subsets and reconfigure the inflammatory gene expression profile in diverse tissue sites in obese mice following the induction of colitis.
PMCID: PMC3500317  PMID: 23166761
18.  n-3 Fatty acids uniquely affect anti-microbial resistance and immune cell plasma membrane organization 
Chemistry and physics of lipids  2011;164(7):626-635.
It is now well established that dietary lipids are incorporated into macrophage and T-cell membrane microdomains, altering their structure and function. Within cell membranes, there are specific detergent-resistant domains in which key signal transduction proteins are localized. These regions are classified as “lipid rafts”. Rafts are composed mostly of cholesterol and sphingolipids and therefore do not integrate well into the fluid phospholipid bilayers causing them to form microdomains. Upon cell activation, rafts compartmentalize signal-transducing molecules, thus providing an environment conducive to signal transduction. In this review, we discuss recent novel data describing the effects of n-3 PUFA on alterations in the activation and functions of macrophages and T-cells. We believe that the modifications in these two disparate immune cell types are linked by fundamentally similar changes in membrane lipid composition and transmembrane signaling functions. We conclude that the outcomes of n-3 PUFA-mediated immune cell alterations may be beneficial (e.g., anti-inflammatory) or detrimental (e.g., loss of microbial immunity) depending upon the cell type interrogated.
PMCID: PMC3183364  PMID: 21798252
Macrophage; tuberculosis; phagosome maturation; lipid rafts; polyunsaturated fatty acids; fish oil; fat-1 mice
19.  A High Legume Low Glycemic Index Diet Improves Serum Lipid Profiles in Men 
Lipids  2010;45(9):765-775.
Clinical studies have shown that fiber consumption facilitates weight loss and improves lipid profiles; however, the beneficial effects of high fermentable fiber low glycemic index (GI) diets under conditions of weight maintenance are unclear. In the Legume Inflammation Feeding Experiment, a randomized controlled cross-over feeding study, 64 middle-aged men who had undergone colonoscopies within the previous 2 years received both a healthy American (HA) diet (no legume consumption, fiber consumption = 9 g/1,000 kcal, and GI = 69) and a legume enriched (1.5 servings/1,000 kcal), high fiber (21 g/1,000 kcal), low GI (GI = 38) diet (LG) in random order. Diets were isocaloric and controlled for macronutrients including saturated fat; they were consumed each for 4 weeks with a 2–4 week break separating dietary treatments. Compared to the HA diet, the LG diet led to greater declines in both fasting serum total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) (P <0.001 and P <0.01, respectively). Insulin-resistant (IR) subjects had greater reductions in high density lipoprotein cholesterol (HDL-C; P <0.01), and triglycerides (TAG)/HDL-C (P = 0.02) after the LG diet, compared to the HA diet. Insulin-sensitive (IS) subjects had greater reductions in TC (P <0.001), LDL-C (P <0.01), TC/HDL-C (P <0.01), and LDL-C/HDL-C (P = 0.02) after the LG diet, compared to the HA diet. In conclusion, a high legume, high fiber, low GI diet improves serum lipid profiles in men, compared to a healthy American diet. However, IR individuals do not achieve the full benefits of the same diet on cardiovascular disease (CVD) lipid risk factors.
PMCID: PMC3461593  PMID: 20734238
Legume intake; Lipids; Insulin resistance
20.  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.
PMCID: PMC3137683  PMID: 21490130
apoptosis; n-3 PUFA; oxidation; colon; mitochondria
21.  Alteration of EGFR Spatiotemporal Dynamics Suppresses Signal Transduction 
PLoS ONE  2012;7(6):e39682.
The epidermal growth factor receptor (EGFR), which regulates cell growth and survival, is integral to colon tumorigenesis. Lipid rafts play a role in regulating EGFR signaling, and docosahexaenoic acid (DHA) is known to perturb membrane domain organization through changes in lipid rafts. Therefore, we investigated the mechanistic link between EGFR function and DHA. Membrane incorporation of DHA into immortalized colonocytes altered the lateral organization of EGFR. DHA additionally increased EGFR phosphorylation but paradoxically suppressed downstream signaling. Assessment of the EGFR-Ras-ERK1/2 signaling cascade identified Ras GTP binding as the locus of the DHA-induced disruption of signal transduction. DHA also antagonized EGFR signaling capacity by increasing receptor internalization and degradation. DHA suppressed cell proliferation in an EGFR-dependent manner, but cell proliferation could be partially rescued by expression of constitutively active Ras. Feeding chronically-inflamed, carcinogen-injected C57BL/6 mice a fish oil containing diet enriched in DHA recapitulated the effects on the EGFR signaling axis observed in cell culture and additionally suppressed tumor formation. We conclude that DHA-induced alteration in both the lateral and subcellular localization of EGFR culminates in the suppression of EGFR downstream signal transduction, which has implications for the molecular basis of colon cancer prevention by DHA.
PMCID: PMC3384615  PMID: 22761867
22.  A metagenomic study of diet-dependent interaction between gut microbiota and host in infants reveals differences in immune response 
Genome Biology  2012;13(4):r32.
Gut microbiota and the host exist in a mutualistic relationship, with the functional composition of the microbiota strongly affecting the health and well-being of the host. Thus, it is important to develop a synthetic approach to study the host transcriptome and the microbiome simultaneously. Early microbial colonization in infants is critically important for directing neonatal intestinal and immune development, and is especially attractive for studying the development of human-commensal interactions. Here we report the results from a simultaneous study of the gut microbiome and host epithelial transcriptome of three-month-old exclusively breast- and formula-fed infants.
Variation in both host mRNA expression and the microbiome phylogenetic and functional profiles was observed between breast- and formula-fed infants. To examine the interdependent relationship between host epithelial cell gene expression and bacterial metagenomic-based profiles, the host transcriptome and functionally profiled microbiome data were subjected to novel multivariate statistical analyses. Gut microbiota metagenome virulence characteristics concurrently varied with immunity-related gene expression in epithelial cells between the formula-fed and the breast-fed infants.
Our data provide insight into the integrated responses of the host transcriptome and microbiome to dietary substrates in the early neonatal period. We demonstrate that differences in diet can affect, via gut colonization, host expression of genes associated with the innate immune system. Furthermore, the methodology presented in this study can be adapted to assess other host-commensal and host-pathogen interactions using genomic and transcriptomic data, providing a synthetic genomics-based picture of host-commensal relationships.
PMCID: PMC3446306  PMID: 22546241
23.  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.
PMCID: PMC2962699  PMID: 20232381
Fatty acids; Fiber; Bcl-2; Apoptosis
24.  Classification of Diet-Modulated Gene Signatures at the Colon Cancer Initiation and Progression Stages 
Digestive diseases and sciences  2011;56(9):2595-2604.
The effects of dietary polyunsaturated (PUFAs) and monounsaturated fatty acids (MUFAs) on intestinal cytokinetics within the context of colon cancer initiation and progression have been extensively studied. n-3 PUFAs have received the most attention due to their potential protective role. However, further investigation of the epigenetic perturbations caused by fatty acids in the context of colon cancer development is needed.
We used DNA microarrays to identify discriminative gene signatures (gene combinations) for the purpose of classifying n-3 PUFA-fed, carcinogen-injected, Sprague–Dawley rats at the initiation and progression stages. Animals were assigned to three dietary treatments differing only in the type of fat (corn oil/n-6 PUFA, fish oil/n-3 PUFA, or olive oil/n-9 monounsaturated fatty acid).
The effects of diet on colonic mucosal gene expression signatures during tumor initiation and progression were subsequently compared (12 h and 10 weeks after azoxymethane injection). Microarray analysis revealed that the number of differentially expressed (DE) genes in each of the three diet comparisons increased with the progression of colon cancer. Each dietary lipid source exhibited its own unique transcriptional profile, as assessed by linear discriminant analysis. Applying this novel approach, we identified the single genes and the two- to three-gene combinations that best distinguished the dietary treatment groups. For the chemoprotective (fish oil) diet, mediators of stem cell homeostasis, e.g., ephrin B1 and bone morphogenic protein 4, were the top-performing gene classifiers.
These results suggest that dietary chemoprotective n-3 PUFA impact genes that regulate the colon stem cell niche and tumor evolution.
PMCID: PMC3139012  PMID: 21409376
Chemoprevention; Cancer initiation and progression; Fish oil; Linear discriminant analysis
25.  Regulatory Activity of Polyunsaturated Fatty Acids in T-Cell Signaling 
Progress in lipid research  2010;49(3):250-261.
n-3 polyunsaturated fatty acids (PUFA) are considered to be authentic immunosuppressors and appear to exert beneficial effects with respect to certain immune-mediated diseases. In addition to promoting T-helper 1 (Th1) cell to T-helper 2 (Th2) cell effector T-cell differentiation, n-3 PUFA may also exert anti-inflammatory actions by inducing apoptosis in Th1 cells. With respect to mechanisms of action, effects range from the modulation of membrane receptors to gene transcription via perturbation of a number of second messenger cascades. In this review, the putative targets of anti-inflammatory n-3 PUFA, activated during early and late events of T-cell activation will be discussed. Studies have demonstrated that these fatty acids alter plasma membrane micro-organization (lipid rafts) at the immunological synapse, the site where T-cells and antigen presenting cells (APC) form a physical contact for antigen initiated T-cell signaling. In addition, the production of diacylglycerol and the activation of different isoforms of protein kinase C (PKC), mitogen activated protein kinase (MAPK), calcium signaling, and nuclear translocation/activation of transcriptional factors, can be modulated by n-3 PUFA. Advantages and limitations of diverse methodologies to study the membrane lipid raft hypothesis, as well as apparent contradictions regarding the effect of n-3 PUFA on lipid rafts will be critically presented.
PMCID: PMC2872685  PMID: 20176053

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