Epithelial-mesenchymal interaction protects normal colonocytes from 4-HNE-induced phenotypic transformation.

INTRODUCTION: Recent studies have shown that epithelial-stromal interactions could play a role in the development of colorectal cancer. Here, we investigated the role of fibroblasts in the transformation of normal colonocytes induced by 4-HNE. METHODS: Normal Co colonocytes and nF fibroblasts from the same mouse colon were exposed, in monoculture (m) or coculture (c), to 4-HNE (5 µM) twice weekly for 3 weeks. Gene expression was then analysed and the ability of Co colonocytes to grow in anchorage-independent conditions was tested in soft agar. Fibroblasts previously treated or not with 4-HNE were also seeded in culture inserts positioned above the agar layers to allow paracrine exchanges with colonocytes. RESULTS: First, 60% of the genes studied were modulated by coculture in Co colonocytes, with notably increased expression of BMP receptors. Furthermore, while 4-HNE increased the ability of monoculture-treated Co colonocytes to form colonies, this effect was not observed in coculture-treated Co colonocytes. Adding a selective BMPR1 inhibitor during the treatment phase abolished the protective effect of coculture. Conversely, addition of a BMP4 agonist to the medium of monoculture-treated Co colonocytes prevented phenotypic transformation by 4-HNE. Second, the presence of nF(m)-HNE fibroblasts during the soft agar assay increased the number and size of Co(m) colonocyte colonies, regardless of whether these cells had been previously treated with 4-HNE in monoculture. For soft agar assays performed with nF(c) and Co(c) cells initially treated in coculture, only the reassociation between Co(c)-HNE and nF(c)-HNE resulted in a small increase in the number of colonies. CONCLUSIONS: During the exposure phase, the epithelial-mesenchymal interaction protected colonocytes from 4-HNE-induced phenotypic transformation via activation of the BMP pathway. This intercellular dialogue also limited the ability of fibroblasts to subsequently promote colonocyte-anchorage-independent growth. In contrast, fibroblasts pre-exposed to 4-HNE in monoculture strongly increased the ability of Co(m) colonocytes to form colonies.

Effects of sodium nitrite reduction, removal or replacement on cured and cooked meat for microbiological growth, food safety, colon ecosystem, and colorectal carcinogenesis in Fischer 344 rats.

Epidemiological and experimental evidence indicated that processed meat consumption is associated with colorectal cancer risks. Several studies suggest the involvement of nitrite or nitrate additives via N-nitroso-compound formation (NOCs). Compared to the reference level (120 mg/kg of ham), sodium nitrite removal and reduction (90 mg/kg) similarly decreased preneoplastic lesions in F344 rats, but only reduction had an inhibitory effect on Listeria monocytogenes growth comparable to that obtained using the reference nitrite level and an effective lipid peroxidation control. Among the three nitrite salt alternatives tested, none of them led to a significant gain when compared to the reference level: vegetable stock, due to nitrate presence, was very similar to this reference nitrite level, yeast extract induced a strong luminal peroxidation and no decrease in preneoplastic lesions in rats despite the absence of NOCs, and polyphenol rich extract induced the clearest downward trend on preneoplastic lesions in rats but the concomitant presence of nitrosyl iron in feces. Except the vegetable stock, other alternatives were less efficient than sodium nitrite in reducing L. monocytogenes growth.

Study of the colonic epithelial-mesenchymal dialogue through establishment of two activated or not mesenchymal cell lines: Activated and resting ones differentially modulate colonocytes in co-culture.

Continuous and rapid renewal of the colonic epithelium is crucial to resist the plethora of luminal deleterious agents. Subepithelial fibroblasts contribute to this turnover by regulating epithelial proliferation and differentiation. However, when intestinal homeostasis is disturbed, fibroblasts can acquire an activated phenotype and play a major role in the progression of intestinal pathologies. To evaluate the involvement of fibroblasts in the regulation of colonocytes under homeostatic or pathological conditions, we established resting and activated conditionally immortalized fibroblast cell lines (nF and mF) from mouse colonic mucosa. We then studied the epithelial-mesenchymal interactions between activated or resting fibroblasts and the normal mouse colonocytes (Co) using a co-culture model. Both fibroblastic cell lines were characterized by RT-qPCR, western blot and immunofluorescence assay. Our results showed that nF and mF cells were positive for fibroblastic markers such as vimentin and collagen 1, and negative for cytokeratin 18 and E-cadherin, attesting to their fibroblastic type. They also expressed proteins characteristic of the epithelial stem cell niche such as Grem1, CD90 or Wnt5a. Only rare nF fibroblasts were positive for α-SMA, whereas all mF fibroblasts strongly expressed this marker, supporting that mF cells were activated fibroblasts/myofibroblasts. In coculture, nF fibroblasts and Co cells strongly interacted via paracrine exchanges resulting in BMP4 production in nF fibroblasts, activation of BMP signaling in Co colonocytes, and decreased growth of colonocytes. Activated-type mF fibroblasts did not exert the same effects on Co cells, allowing colonocytes free to proliferate. In conclusion, these two colonic fibroblast lines, associated with Co cells in coculture, should allow to better understand the role of mesenchymal cells in the preservation of homeostasis and the development of intestinal pathologies.

Milk Polar Lipids in a High-Fat Diet Can Prevent Body Weight Gain: Modulated Abundance of Gut Bacteria in Relation with Fecal Loss of Specific Fatty Acids.

SCOPE: Enhanced adiposity and metabolic inflammation are major features of obesity associated with altered gut microbiota and intestinal barrier. How these metabolic outcomes can be impacted by milk polar lipids (MPL), naturally containing 25% of sphingomyelin, is investigated in mice fed a mixed high-fat (HF) diet . METHODS AND RESULTS: Male C57Bl/6 mice receive a HF-diet devoid of MPL (21% fat, mainly palm oil, in chow), or supplemented with 1.1% or 1.6% of MPL (HF-MPL1; HF-MPL2) via a total-lipid extract from butterserum concentrate for 8 weeks. HF-MPL2 mice gain less weight versus HF (p < 0.01). Diets do not impact plasma markers of inflammation but in the liver, HF-MPL2 tends to decrease hepatic gene expression of macrophage marker F4/80 versus HF-MPL1 (p = 0.06). Colonic crypt depth is the maximum in HF-MPL2 (p < 0.05). In cecal microbiota, HF-MPL1 increases Bifidobacterium animalis versus HF (p < 0.05). HF-MPL2 decreases Lactobacillus reuteri (p < 0.05), which correlates negatively with the fecal loss of milk sphingomyelin-specific fatty acids (p < 0.05). CONCLUSION: In mice fed a mixed HF diet, MPL can limit HF-induced body weight gain and modulate gut physiology and the abundance in microbiota of bacteria of metabolic interest. This supports further exploration of how residual unabsorbed lipids reaching the colon can impact HF-induced metabolic disorders.

Oleic Acid Uptake Reveals the Rescued Enterocyte Phenotype of Colon Cancer Caco-2 by HT29-MTX Cells in Co-Culture Mode.

Gastrointestinal epithelium is the unique route for nutrients and for many pharmaceuticals to enter the body. The present study aimed to analyze precisely whether co-culture of two colon cancer cell lines, mucus-producing cells HT29-MTX and enterocyte-like Caco-2 cells, ameliorate differentiation into an in vitro intestinal barrier model and the signaling pathways involved. Differentiated Caco-2 cells gene datasets were compared first to intestinal or cancer phenotypes and second to signaling pathway gene datasets. Experimental validations were performed in real-time experiments, immunochemistry, and gene expression analyses on Caco-2 versus co-cultures of Caco-2 and HT29-MTX (10%) cells. Partial maintenance of cancer-cell phenotype in differentiated Caco-2 cells was confirmed and fatty acids merged as potential regulators of cancer signaling pathways. HT29-MTX cells induced morphological changes in Caco-2 cells, slightly increased their proliferation rate and profoundly modified gene transcription of phenotype markers, fatty acid receptors, intracellular transporters, and lipid droplet components as well as functional responses to oleic acid. In vitro, enterocyte phenotype was rescued partially by co-culture of cancer cells with goblet cells and completed through oleic acid interaction with signaling pathways dysregulated in cancer cells.

Soybean polar lipids differently impact adipose tissue inflammation and the endotoxin transporters LBP and sCD14 in flaxseed vs. palm oil-rich diets.

Obesity and type 2 diabetes are nutritional pathologies, characterized by a subclinical inflammatory state. Endotoxins are now well recognized as an important factor implicated in the onset and maintain of this inflammatory state during fat digestion in high-fat diet. As a preventive strategy, lipid formulation could be optimized to limit these phenomena, notably regarding fatty acid profile and PL emulsifier content. Little is known about soybean polar lipid (SPL) consumption associated to oils rich in saturated FA vs. anti-inflammatory omega-3 FA such as α-linolenic acid on inflammation and metabolic endotoxemia. We then investigated in mice the effect of different synthetic diets enriched with two different oils, palm oil or flaxseed oil and containing or devoid of SPL on adipose tissue inflammation and endotoxin receptors. In both groups containing SPL, adipose tissue (WAT) increased compared with groups devoid of SPL and an induction of MCP-1 and LBP was observed in WAT. However, only the high-fat diet in which flaxseed oil was associated with SPL resulted in both higher WAT inflammation and higher circulating sCD14 in plasma. In conclusion, we have demonstrated that LPS transporters LBP and sCD14 and adipose tissue inflammation can be modulated by SPL in high fat diets differing in oil composition. Notably high-flaxseed oil diet exerts a beneficial metabolic impact, however blunted by PL addition. Our study suggests that nutritional strategies can be envisaged by optimizing dietary lipid sources in manufactured products, including fats/oils and polar lipid emulsifiers, in order to limit the inflammatory impact of palatable foods.

Variants of ß-casofensin, a bioactive milk peptide, differently modulate the intestinal barrier: In vivo and ex vivo studies in rats.

ß-Casofensin is a bioactive milk peptide that modulates the intestinal barrier, particularly through its action on goblet cells. ß-Casofensin corresponds to fragment (f) 94-123 of the bovine ß-casein (ß-CN) A2 variant. Fifteen genetic variants of bovine ß-CN (A1-3, B-G, H1-2, I-L) are known, of which the A2, A1, and B forms are the most common. These variants differ from each other by the substitution of one or more amino acids, some of which are localized in f94 to 123. The aim of our study was to compare the intestinal effects of ß-casofensin A2 and its 3 main variants: A1, A3, and B. For this purpose, a solution (0.1 µM; 10 µL/g of body weight, postnatal d 10-20) containing ß-casofensin A2, one of its variants (A1, A3, or B), or drinking water (control; CT) was administered to rat pups orally. After euthanasia (postnatal d 20), intestinal segments were collected for biochemical and histochemical analysis and also used to determine paracellular permeability to fluorescein isothiocyanate-labeled 4-kDa dextran in an Ussing chamber. We also studied the direct effects of ß-casofensin A2 and its A1 variant on the paracellular permeability of jejunum segments of adult rats. ß-Casofensin A2 and its B variant significantly increased the population of goblet cells compared with the CT, A1, and A3 groups. The mucin 2 mRNA level was significantly higher in the ß-casofensin A2 group than in the CT, A3, and B groups. Our results also revealed that the protein expression of zonula occludens-1 and occludin was reduced in the jejunum of rats in the A1, A3, and B groups compared with the CT group. However, the A1 variant was the only peptide to decrease jejunal permeability compared with the CT group. This variant, tested directly in the apical compartment of an Ussing chamber at a concentration of 0.1 nM, also reduced jejunal permeability. In conclusion, the substitution of a single amino acid alters the effect of ß-CN sequence f94 to 123 on goblet cells and on intestinal permeability. A genetic polymorphism of ß-CN can affect the biological activity of peptides derived from this protein. These data should be taken into account in the production of bioactive foods.

Protective effects of ß-casofensin, a bioactive peptide from bovine ß-casein, against indomethacin-induced intestinal lesions in rats.

SCOPE: ß-casofensin, also known as peptide ß-CN(94-123), is a milk bioactive peptide that modulates the intestinal barrier through its action on goblet cells. Here, we evaluated whether oral administration of ß-casofensin can prevent indomethacin-induced injury of the jejunum in rats. METHODS AND RESULTS: Rats received ß-casofensin (0.01-100 µM) or tap water by daily gavage (4 µL/g) for eight days, then two subcutaneous injections of indomethacin (10 mg/kg, days 9 and 10) and were euthanized on day 12. In vitro, we investigated the effects of ß-casofensin on the restitution of a wounded monolayer. Preventive administration of ß-casofensin (100 µM) reduced intestinal macroscopic and microscopic damage induced by indomethacin. ß-casofensin also prevented the depletion of goblet cells and increased myeloperoxidase activity, as well as tumor necrosis factor-ɑ (TNF-ɑ) expression and immunostaining of active caspase-3 in the jejunum of rats treated with indomethacin. In wound healing experiments, ß-casofensin promoted epithelial restitution with no effect on cell proliferation. This effect was inhibited by pre-incubation with an anti-CC chemokine receptor 6 (CCR6) neutralizing antibody. CONCLUSIONS: ß-casofensin exerts protective effects in indomethacin-induced enteritis through preservation of goblet cells and improvement in wound healing. ß-casofensin could therefore become vital in nutritional programs for the prevention of intestinal diseases.

Dietary emulsifiers from milk and soybean differently impact adiposity and inflammation in association with modulation of colonic goblet cells in high-fat fed mice.

SCOPE: Enhanced adiposity and metabolic inflammation are major features of obesity that could be impacted by dietary emulsifiers. We investigated in high-fat fed mice the effects of using a new polar lipid (PL) emulsifier from milk (MPL) instead of soybean lecithin (soybean PL [SPL]) on adipose tissue and intestinal mucosa function. METHODS AND RESULTS: Four groups of C57BL6 mice received for 8 wks a low-fat (LF) diet or a high-fat diet devoid of PLs or an high-fat diet including MPL (high-fat-MPL) or SPL (high-fat-SPL). Compared with high-fat diet, high-fat-SPL diet increased white adipose tissue (WAT) mass (p < 0.05), with larger adipocytes (p < 0.05) and increased expression of tumor necrosis factor alpha, monochemoattractant protein-1, LPS-binding protein, and leptin (p < 0.05). This was not observed with high-fat-MPL diet despite similar dietary intakes and increased expression of fatty acid transport protein 4 and microsomal TG transfer protein, involved in lipid absorption, in upper intestine (p < 0.05). High-fat-MPL mice had a lower expression in WAT of cluster of differentiation 68, marker of macrophage infiltration, versus high-fat and high-fat-SPL mice (p < 0.05), and more goblet cells in the colon (p < 0.05). CONCLUSIONS: Unlike SPL, MPL in the high-fat diet did not induce WAT hypertrophy and inflammation but increased colonic goblet cells. This supports further clinical exploration of different sources of dietary emulsifiers in the frame of obesity outbreak.

Saturated and Unsaturated Fatty Acids Differently Modulate Colonic Goblet Cells In Vitro and in Rat Pups.

BACKGROUND: High-fat diets induce intestinal barrier alterations and promote intestinal diseases. Little is known about the effects of long-chain fatty acids (LCFAs) on mucin 2 (MUC2) production by goblet cells, which are crucial for intestinal protection. OBJECTIVE: We investigated the effects of LCFAs on the differentiation of colonic goblet cells, MUC2 expression, and colonic barrier function. METHODS: Upon reaching confluence, human colonic mucus-secreting HT29-MTX cells were stimulated (21 d) with a saturated LCFA (palmitic or stearic acid), a monounsaturated LCFA (oleic acid), or a polyunsaturated LCFA (linoleic, γ-linolenic, α-linolenic, or eicosapentaenoic acid). In addition, rat pups underwent oral administration of oil (palm, rapeseed, or sunflower oil) or water (10 µL/g body weight, postnatal days 10-15). Subsequently, colon goblet cells were studied by Western blotting, reverse transcriptase-quantitative polymerase chain reaction, and immunohistochemistry and colonic transmucosal electrical resistance was measured by using Ussing chambers. RESULTS: In vitro, palmitic acid enhanced MUC2 production (140% of control) and hepatocyte nuclear factor 4α expression, whereas oleic, linoleic, γ-linolenic, α-linolenic, and eicosapentaenoic acids reduced MUC2 expression (at least -50% of control). All unsaturated LCFAs decreased the expression of human atonal homolog 1, a transcription factor controlling goblet cell differentiation (at least -31% vs. control). In vivo, rats fed palm oil had higher palmitic acid concentrations (3-fold) in their colonic contents and increased mucus granule surfaces in their goblet cells (>2-fold) than did all other groups. Palm oil also increased colonic transmucosal electrical resistance (245% of control), yet had no effect on occludin and zonula occludens-1 expression. In contrast, sunflower and rapeseed oils decreased goblet cell number when compared with control (at least -10%) and palm oil (at least -14%) groups. CONCLUSIONS: Palm oil in rat pups and palmitic acid in HT29-MTX cells increase the production of MUC2 and strengthen the intestinal barrier. In contrast, unsaturated LCFAs decrease MUC2 expression. These data should be taken into account in the context of preventive or therapeutic nutritional programs.

Increasing fat content from 20 to 45 wt% in a complex diet induces lower endotoxemia in parallel with an increased number of intestinal goblet cells in mice.

The impacts of high-fat diets (HFDs) on the onset of metabolic endotoxemia and low-grade inflammation are well established in rodent models. However, the dose-effect of dietary lipid intakes on these parameters is not known. We hypothesized that increasing dietary lipid amounts could be linked to parallel increases of endotoxemia, low-grade inflammation, and metabolic and intestinal alterations. Six-week-old male C57BL/6J mice were fed a low-fat diet (LFD, 2.6 wt% of lipids), a moderate HFD (mHFD, 22 wt% of lipids), or a very HFD (vHFD, 45 wt% of lipids) formulated mainly using chow ingredients and milk fat. After 12 weeks, white adipose tissues, liver, intestine, distal colon contents, and plasma were collected. Only vHFD mice significantly increased body weight and fat mass vs LFD mice. This was associated with increases of plasma concentrations of triglycerides, leptin and adiponectin, and liver lipids. No such differences were observed between LFD and mHFD mice. However, mHFD developed metabolic endotoxemia and inflammation, unlike vHFD mice. In turn, vHFD mice showed more goblet cells in all intestine segments vs both other groups and a decrease of Bacteroides-Prevotella in their microbiota vs LFD mice. Finally, mHFD mice colon exhibited a decrease in lactobacilli and in the levels of occludin phosphorylation. Altogether, using complex HFD, no associations were observed between dietary lipid amounts and the magnitude of endotoxemia, inflammation, and physiological alterations developed. These results reveal the impact of the diet composition on intestinal goblet cells and mucus coat, bringing new insights about further consequences on HFD-induced metabolic disorders.

ß-Casein(94-123)-derived peptides differently modulate production of mucins in intestinal goblet cells.

We recently reported the identification of a peptide from yoghurts with promising potential for intestinal health: the sequence (94-123) of bovine ß-casein. This peptide, composed of 30 amino acid residues, maintains intestinal homoeostasis through production of the secreted mucin MUC2 and of the transmembrane-associated mucin MUC4. Our study aimed to search for the minimal sequence responsible for the biological activity of ß-CN(94-123) by using several strategies based on (i) known bioactive peptides encrypted in ß-CN(94-123), (ii) in silico prediction of peptides reactivity and (iii) digestion of ß-CN(94-123) by enzymes of intestinal brush border membranes. The revealed sequences were tested in vitro on human intestinal mucus-producing HT29-MTX cells. We demonstrated that ß-CN(108-113) (an ACE-inhibitory peptide) and ß-CN(114-119) (an opioid peptide named neocasomorphin-6) up-regulated MUC4 expression whereas levels of the secreted mucins MUC2 and MUC5AC remained unchanged. The digestion of ß-CN(94-123) by intestinal enzymes showed that the peptides ß-CN(94-108) and ß-CN(117-123) were present throughout 1·5 to 3 h of digestion, respectively. These two peptides raised MUC5AC expression while ß-CN(117-123) also induced a decrease in the level of MUC2 mRNA and protein. In addition, this inhibitory effect was reproduced in airway epithelial cells. In conclusion, ß-CN(94-123) is a multifunctional molecule but only the sequence of 30 amino acids has a stimulating effect on the production of MUC2, a crucial factor of intestinal protection.

Maternally acquired genotoxic Escherichia coli alters offspring's intestinal homeostasis.

The neonatal gut is rapidly colonized by a newly dominant group of commensal Escherichia coli strains among which a large proportion produces a genotoxin called colibactin. In order to analyze the short- and long-term effects resulting from such evolution, we developed a rat model mimicking the natural transmission of E. coli from mothers to neonates. Genotoxic and non-genotoxic E. coli strains were equally transmitted to the offspring and stably colonized the gut across generations. DNA damage was only detected in neonates colonized with genotoxic E. coli strains. Signs of genotoxic stress such as anaphase bridges, higher occurrence of crypt fission and accelerated renewal of the mature epithelium were detected at adulthood. In addition, we observed alterations of secretory cell populations and gut epithelial barrier. Our findings illustrate how critical is the genotype of E. coli strains acquired at birth for gut homeostasis at adulthood.

Pasture v. standard dairy cream in high-fat diet-fed mice: improved metabolic outcomes and stronger intestinal barrier.

Dairy products derived from the milk of cows fed in pastures are characterised by higher amounts of conjugated linoleic acid and α-linolenic acid (ALA), and several studies have shown their ability to reduce cardiovascular risk. However, their specific metabolic effects compared with standard dairy in a high-fat diet (HFD) context remain largely unknown; this is what we determined in the present study with a focus on the metabolic and intestinal parameters. The experimental animals were fed for 12 weeks a HFD containing 20 % fat in the form of a pasture dairy cream (PDC) or a standard dairy cream (SDC). Samples of plasma, liver, white adipose tissue, duodenum, jejunum and colon were analysed. The PDC mice, despite a higher food intake, exhibited lower fat mass, plasma and hepatic TAG concentrations, and inflammation in the adipose tissue than the SDC mice. Furthermore, they exhibited a higher expression of hepatic PPARα mRNA and adipose tissue uncoupling protein 2 mRNA, suggesting an enhanced oxidative activity of the tissues. These results might be explained, in part, by the higher amounts of ALA in the PDC diet and in the liver and adipose tissue of the PDC mice. Moreover, the PDC diet was found to increase the proportions of two strategic cell populations involved in the protective function of the intestinal epithelium, namely Paneth and goblet cells in the small intestine and colon, compared with the SDC diet. In conclusion, a PDC HFD leads to improved metabolic outcomes and to a stronger gut barrier compared with a SDC HFD. This may be due, at least in part, to the protective mechanisms induced by specific lipids.

High-fat diet action on adiposity, inflammation, and insulin sensitivity depends on the control low-fat diet.

Animal studies using a high-fat diet (HFD) have studied the effects of lipid overconsumption by comparing a defined HFD either with a natural-ingredient chow diet or with a defined low-fat diet (LFD), despite the dramatic differences between these control diets. We hypothesized that these differences in the control diet could modify the conclusions regarding the effects that an increase of fat in the diet has on several metabolic parameters. For 11 weeks, C57bl6/J mice were fed a low-fat chow diet (8% energy from fat), a typical semisynthetic LFD (12%), or a semisynthetic HFD (sy-HF) (40%). Conclusions about the effect of sy-HF on body weight gain, subcutaneous adipose tissue, insulin sensitivity, and adipose tissue inflammation were modified according to the control LFD. Conversely, conclusions about epididymal and retroperitoneal adipose tissue; fat intake effects on liver and muscular lipids, cholesterol, free fatty acids, and markers of low-grade inflammation; and of adipose tissue macrophage infiltration were the same regardless of the use of low-fat chow diet or semisynthetic LFD. For some physiological outcomes, conflicting conclusions were even reached about the effects of increased fat intake according to the chosen low-fat control. Some deleterious effects of sy-HF may not be explained by lipid overconsumption but rather by the overall quality of ingredients in a semisynthetic diet. According to the control LFD chosen, conclusions on the lipid-related effects of HFDs must be formulated with great care because some end points are profoundly affected by the ingredient composition of the diet rather than by fat content.

A novel bioactive peptide from yoghurts modulates expression of the gel-forming MUC2 mucin as well as population of goblet cells and Paneth cells along the small intestine.

Several studies demonstrated that fermented milks may provide a large number of bioactive peptides into the gastrointestinal tract. We previously showed that beta-casomorphin-7, an opioid-like peptide produced from bovine ß-casein, strongly stimulates intestinal mucin production in ex vivo and in vitro models, suggesting the potential benefit of milk bioactive peptides on intestinal protection. In the present study, we tested the hypothesis that the total peptide pool (TPP) from a fermented milk (yoghurt) may act on human intestinal mucus-producing cells (HT29-MTX) to induce mucin expression. Our aim was then to identify the peptide(s) carrying the biological activity and to study its impact in vivo on factors involved in gut protection after oral administration to rat pups (once a day, 9 consecutive days). TPP stimulated MUC2 and MUC4 gene expression as well as mucin secretion in HT29-MTX cells. Among the four peptide fractions that were separated by preparative reversed-phase high-performance liquid chromatography, only the C2 fraction was able to mimic the in vitro effect of TPP. Interestingly, the sequence [94-123] of ß-casein, present only in C2 fraction, also regulated mucin production in HT29-MTX cells. Oral administration of this peptide to rat pups enhanced the number of goblet cells and Paneth cells along the small intestine. These effects were associated with a higher expression of intestinal mucins (Muc2 and Muc4) and of antibacterial factors (lysozyme, rdefa5). We conclude that the peptide ß-CN(94-123) present in yoghurts may maintain or restore intestinal homeostasis and could play an important role in protection against damaging agents of the intestinal lumen.

Dietary oxidized n-3 PUFA induce oxidative stress and inflammation: role of intestinal absorption of 4-HHE and reactivity in intestinal cells.

Dietary intake of long-chain n-3 PUFA is now widely advised for public health and in medical practice. However, PUFA are highly prone to oxidation, producing potentially deleterious 4-hydroxy-2-alkenals. Even so, the impact of consuming oxidized n-3 PUFA on metabolic oxidative stress and inflammation is poorly described. We therefore studied such effects and hypothesized the involvement of the intestinal absorption of 4-hydroxy-2-hexenal (4-HHE), an oxidized n-3 PUFA end-product. In vivo, four groups of mice were fed for 8 weeks high-fat diets containing moderately oxidized or unoxidized n-3 PUFA. Other mice were orally administered 4-HHE and euthanized postprandially versus baseline mice. In vitro, human intestinal Caco-2/TC7 cells were incubated with 4-hydroxy-2-alkenals. Oxidized diets increased 4-HHE plasma levels in mice (up to 5-fold, P < 0.01) compared with unoxidized diets. Oxidized diets enhanced plasma inflammatory markers and activation of nuclear factor kappaB (NF-κB) in the small intestine along with decreasing Paneth cell number (up to -19% in the duodenum). Both in vivo and in vitro, intestinal absorption of 4-HHE was associated with formation of 4-HHE-protein adducts and increased expression of glutathione peroxidase 2 (GPx2) and glucose-regulated protein 78 (GRP78). Consumption of oxidized n-3 PUFA results in 4-HHE accumulation in blood after its intestinal absorption and triggers oxidative stress and inflammation in the upper intestine.

Resistin-like molecule beta regulates intestinal mucous secretion and curtails TNBS-induced colitis in mice.

BACKGROUND: Resistin and resistin-like molecule (RELM)beta comprise a novel class of cysteine-rich proteins secreted into the circulation implicated in hepatic insulin resistance and inflammation. RELMbeta is specifically produced by intestinal goblet cells but regulation of its expression and much of its local function are not elucidated. RELMbeta has been suggested to regulate colonic inflammation susceptibility, which is dependent on the mucosal barrier integrity. METHODS: In this work we explored the physiopathological role of RELMbeta in the colon. Among agents tested, carbachol and gastrin were strong inhibitors of RELMbeta mRNA accumulation. We examined the effect of recombinant RELMbeta on mucin secretion by human mucus-secreting HT29-Cl.16E cells in culture and by mouse colonic goblet cells in vivo. RESULTS: RELMbeta upregulated MUC2 and M1/MUC5AC gene expression in HT29-Cl.16E cells. RELMbeta enhanced M1/MUC5AC secretion by human colonic HT29-Cl.16E cells and MUC2 secretion by murine intestinal goblet cells. RELMbeta exerted its action exclusively on the apical side of HT29-Cl.16E cells, in agreement with its luminal mucosecretagogue effect in mice. Its action required calcium, protein kinase C, tyrosine kinases, and extracellular-regulated protein kinase activities and was synergized by carbachol. An intracolonic RELMbeta challenge was performed in the trinitrobenzene sulfonic acid (TNBS)-murine model of colitis and macroscopic and histological scores were monitored. The macroscopic and histopathological severity of TNBS-induced colitis was significantly attenuated by RELMbeta pretreatment. CONCLUSIONS: A direct participation in maintaining the mucosal defense barrier can be ascribed to RELMbeta in line with a regulatory role in intestinal inflammation.

Leptin modulates the expression of secreted and membrane-associated mucins in colonic epithelial cells by targeting PKC, PI3K, and MAPK pathways.

Mucins play an essential role in the protection and repair of gastrointestinal mucosa. We recently showed that luminal leptin strongly stimulated mucin secretion in vivo in rat colon. In the present study, we challenged the hypothesis that leptin may act directly on goblet cells to induce mucin expression in rat and human intestinal mucin-producing cells (DHE and HT29-MTX). The endoluminal effect of leptin was also studied in vivo in rat perfused colon model. The presence of leptin receptors was demonstrated in the two cell lines by Western blot and RT-PCR. In rat DHE cells, leptin (0.01-10 nmol/l, 60 min) dose dependently increased the secretion of mucins (210 +/- 3% of controls) and the expression of Muc2, Muc3, and Muc4 (twofold basal level) but not of Muc1 and Muc5AC. Luminal perfusion of leptin (60 min, 0.1-100 nmol/l) in rat colon also increased the mRNA level of Muc2, Muc3, and Muc4 but not of Muc1. In human HT29-MTX cells, leptin (0.01-10 nmol/l, 60 min) dose dependently enhanced MUC2, MUC5AC, and MUC4 mRNA levels. These effects were prevented by pretreatment of cells with the leptin mutein L39A/D40A/F41A, which acts as a receptor antagonist. Finally, pathway inhibition experiments suggest that leptin increased mucin expression by activating PKC-, phosphatidyl inositol 3-kinase-, and MAPK-dependent pathways but not the JAK/STAT pathway. In conclusion, leptin may contribute significantly to membrane-associated and secreted mucin production via a direct stimulation of colonic epithelial cells and the activation of leptin receptors. These data are consistent with a role for leptin in regulation of the intestinal barrier function.

Intestinal MUC2 and gastric M1/MUC5AC in preneoplastic lesions induced by 1,2-dimethylhydrazine in rat: a sequential analysis.

Our study was performed to sequentially analyze the expression of the intestinal mucin MUC2 and of the gastric mucin MUC5AC as indicators during progression of preneoplastic biomarkers in rat colon. F344 rats were sacrificed 2, 4, 8, 12, 24 and 36 weeks after injection of 1,2-dimethylhydrazine (DMH, 200 mg/kg, i.p.). The expression of MUC2 and of MUC5AC was studied by immunohistochemistry in preneoplastic lesions classified in two categories: histologically altered foci (HAF) and beta-catenin accumulated crypts (BCAC). HAF appeared 4 weeks after DMH injection. Their crypt multiplicity stagnated with time (3-4 crypts/foci) but gastric MUC5AC mucin was always observed in some goblet cells of the lesions of this category. In contrast, MUC2-immunostaining was not modified compared to the adjacent crypts. Double-immunofluorescence revealed that goblet cells which produced MUC5AC continued to express MUC2. In BCAC, crypt multiplicity and mucin expression strongly evolved with time. These lesions were observed only 8 weeks after DMH-injection. At this stage, 20% of BCAC showed a decreased MUC2 expression and 33% were MUC5AC immunopositive. At the 36-week point, 43% of BCAC had a reduced MUC2 staining and 90% were positive for MUC5AC. This immunopositivity was often observed in all the cells of these lesions. Seldom, some BCAC were depleted at the same time in MUC2 and in MUC5AC. Similar alterations in mucin expression were observed in human colonic pre-neoplastic lesions. These findings suggest that a decrease in MUC2 expression and staining of MUC5AC in non-goblet-like cells predicts histological progression of preneoplastic lesions.