Search

There is mounting evidence that noncoding microRNAs (miRNA) are modulated by select chemoprotective dietary agents. For example, recently we demonstrated that the unique combination of dietary fish oil (containing n-3 fatty acids) plus pectin (fermented to butyrate in the colon) (FPA) up-regulates a subset of putative tumor suppressor miRNAs in intestinal mucosa, and down-regulates their predicted target genes following carcinogen exposure as compared to control (corn oil plus cellulose (CCA)) diet. To further elucidate the biological effects of diet and carcinogen modulated miR's in the colon, we verified that miR-26b and miR-203 directly target PDE4B and TCF4, respectively. Since perturbations in adult stem cell dynamics are generally believed to represent an early step in colon tumorigenesis and to better understand how the colonic stem cell population responds to environmental factors such as diet and carcinogen, we additionally determined the effects of the chemoprotective FPA diet on miRNAs and mRNAs in colonic stem cells obtained from Lgr5-EGFP-IRES-creER(T2) knock-in mice. Following global miRNA profiling, 26 miRNAs (P

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 5days 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.

During colon inflammation, Th17 cells and immunosuppressive regulatory T cells (Treg) are thought to play promotive and preventative roles, respectively. Dietary (n-3) PUFA favorably modulate intestinal inflammation in part by downregulating T-cell activation and functionality. We used the Fat-1 mouse, a genetic model that synthesizes long-chain (n-3) PUFA de novo, to test the hypothesis that (n-3) PUFA protect against colonic inflammation by modulating the polarization of Treg and Th17 cells during colitis. Male and female wild-type (WT) and Fat-1 mice were administered dextran sodium sulfate (DSS) in the drinking water (2.5%) to induce acute (5 d DSS) or chronic (3 cycles DSS) colitis and the percentage of Treg and Th17 cells residing locally [colonic lamina propria (cLP)] and systemically (spleen) was determined by flow cytometry. The percentage of Treg in either tissue site was unaffected by genotype (P > 0.05); however, during chronic colitis, the percentage of Th17 cells residing in both the spleen and cLP was lower in Fat-1 mice compared to WT mice (P < 0.05). Colonic mucosal mRNA expression of critical Th17 cell cytokines and chemokine receptors (IL-17F, IL-21, and CCR6) were lower, whereas expression of the Th17 cell suppressive cytokine, IL-27, was greater in Fat-1 mice compared to WT mice during chronic colitis (P < 0.05). Moreover, colon histological scores were improved in Fat-1 mice (P < 0.05). Collectively, these results demonstrate for the first time, to our knowledge, that (n-3) PUFA can modulate the colonic mucosal microenvironment to suppress Th17 cell accumulation and inflammatory damage following the induction of chronic colitis.

Inflammatory bowel disease (IBD) patients are at high risk for developing folate deficiency and colon cancer. Since it is difficult to study the subtle global and gene-specific epigenetic mechanisms involved in folate-mediated tumor initiation and promotion, we have generated genetically modified mouse models by targeting the reduced folate carrier (RFC1) and folate-binding protein (Folbp1) genes. The transgenic mice were fed semi-purified diets for 8 weeks containing either normal (2 mg) or deficient (0.1 mg folate/kg diet) levels of folate. Compound heterozygous mice (Folbp1(+/-); RFC1(+/-)) fed an adequate folate diet exhibited a reduction in plasma folate concentrations compared to heterozygous (Folbp1(+/-)) and littermate wild-type mice (P.05). In contrast, no differences were observed in colonic mucosa. Consumption of a low folate diet significantly reduced (three- to fourfold) plasma and tissue folate levels in all animal models, although plasma homocysteine levels were not altered. In order to elucidate the relationship between folate status and inflammation-associated colon cancer, animals were injected with azoxymethane followed by dextran sodium sulphate treatment in the drinking water. Mice were fed a normal folate diet and were terminated 5 weeks after carcinogen injection. The number of high multiplicity aberrant crypt foci per centimeter of colon was significantly elevated (P.05) in compound Folbp1(+/-); RFC1(+/-) (3.5+/-0.4) mice as compared to Folbp1(+/-) (1.9+/-0.3) and wild-type control mice (1.1+/-0.1). These data demonstrate that the ablation of two receptor/carrier-mediated pathways for folate transport increases the risk for developing inflammation-associated colon cancer.

We recently demonstrated that (n-3) PUFA trigger the induction of apoptosis in the colon by enhancing phospholipid oxidation and mitochondrial Ca2+ accumulation. To further elucidate the mechanisms regulating oxidative stress-induced apoptosis in vivo, a 2 x 2 experiment was designed using both wild type (control) and manganese-dependent superoxide dismutase (SOD2) heterozygous knockout mice (SOD2(+/-)), which exhibit increased mitochondrial oxidative stress. Mice were fed diets differing only in the type of fat [corn oil or fish oil containing (n-3) PUFA] at 15% by weight for 4 wk. Dietary (n-3) PUFA treatment enhanced (22%) apoptosis in colonic crypts. In addition, SOD2 haploinsufficiency enhanced (20%) apoptosis, which was further increased (36%) by (n-3) PUFA feeding. Dietary lipid source and genotype interactively modulated nitrotyrosine levels (P = 0.027) and inflammation (P = 0.032). These findings demonstrate that the proapoptotic effects of (n-3) PUFA are enhanced in oxidatively stressed SOD2(+/-) mice. Thus, (n-3) PUFA appear to promote an oxidation-reduction imbalance in the intestine, which may directly or indirectly trigger apoptosis and thereby reduce colon cancer risk.

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

S 5627 is a synthetic analogue of chlorogenic acid. S 5627 is a potent linear competitive inhibitor of glucose 6-phosphate (Glc-6-P) hydrolysis by intact microsomes (K i = 41 nm) but is without effect on the enzyme in detergent- or NH4OH-disrupted microsomes.3H-S 5627 was synthesized and used as a ligand in binding studies directed at characterizing T1, the Glc-6-P transporter. Binding was evaluated using Ca2+-aggregated microsomes, which can be sedimented at low g forces. Aside from a modest reduction in K values for both substrate and S 5627, Ca2+ aggregation had no effect on glucose-6-phosphatase (Glc-6-Pase). Scatchard plots of binding data are readily fit to a simple “two-site” model, with K d = 21 nm for the high affinity site and K d = 2 μm for the low affinity site. Binding to the high affinity site was competitively blocked by Glc-6-P (K i = 9 mm), whereas binding was unaffected by mannose-6-phosphate, Pi, and PPiand only modestly depressed by 2-deoxy-d-glucose 6-phosphate, a poor substrate for Glc-6-Pase in intact microsomes. Thus the high affinity 3H-S 5627 binding site fits the criteria for T1. Permeabilization of the membrane with 0.3% (3-[(chloramidopropyl)-dimethylammonio]-1-propanesulfonate) activated Glc-6-Pase and broadened its substrate specificity, but it did not significantly alter the binding of 3H-S 5627 to the high affinity sites or the ability of Glc-6-P to block binding. These data demonstrate unequivocally that two independent Glc-6-P binding sites are involved in the hydrolysis of Glc-6-P by intact microsomes. The present findings are the strongest and most direct evidence to date against the notion that the substrate specificity and the intrinsic activity of Glc-6-Pase in native membranes are determined by specific conformational constraints imposed on the enzyme protein. These data constitute compelling evidence for the role of T1 in Glc-6-Pase activity.