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.

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.

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.

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.

Eotaxin-3/CCL26 gene expression in intestinal epithelial cells is up-regulated by interleukin-4 and interleukin-13 via the signal transducer and activator of transcription 6.

Several inflammatory processes of the bowel are characterized by an accumulation of eosinophils at sites of inflammation. The mechanisms that govern mucosal infiltration with eosinophils are not fully understood. Eotaxin-3/CCL-26 belongs to a family of CC chemokines, which are potent chemoattractants for eosinophils. In this study, we hypothesized that intestinal epithelial cells could release eotaxin-3. We demonstrate that the T helper 2 type cytokines interleukin-4 or interleukin-13 increase eotaxin-3 mRNA levels and eotaxin-3 protein expression in the human intestinal epithelial cell lines HT-29 CL.19A and T84 in a dose-dependent manner. Addition of actinomycin-D prior to interleukin-4/-13 stimulation led to decreases in eotaxin-3 mRNA levels similar to those observed in controls without interleukin-4/-13. Interleukin-4 and interleukin-13 activated signal transducer and activator of transcription 6 which was found to bind the two canonical signal transducer and activator of transcription 6 binding sites located in the eotaxin-3 promoter. Experiments with the eotaxin-3 promoter luciferase constructs revealed that the most proximal signal transducer and activator of transcription 6 binding site located between positions -62 and -71 relative to the transcriptional start was necessary for full eotaxin-3 promoter activity. Importantly, we present evidence that the signal transducer and activator of transcription 6 is necessary and sufficient for interleukin-4 or interleukin-13 mediated eotaxin-3 gene up-regulation using HT-29 CL.19A cells expressing a dominant-negative signal transducer and activator of transcription 6. Overall, these results demonstrate that epithelial eotaxin-3 is up-regulated in the context of a T helper 2 mediated inflammatory bowel disease via the signal transducer and activator of transcription 6, thus suggesting that the intestinal epithelium actively participates in the recruitment of eosinophils at the site of inflammation.

Expression of human TFF3 in relation to growth of HT-29 cell subpopulations: involvement of PI3-K but not STAT6.

The trefoil factor family (TFF) peptides 1 and 2 (TFF1 and 2) are expressed in mucus cells of the stomach, whereas TFF3 is localized in goblet cells of the intestine. In the present study, we aimed to determine whether phosphatidylinositol 3-kinase (PI3-K) or signal transducer and activator of transcription protein 6 (STAT6) is involved in the expression of goblet cell specific markers. TFF3 expression was analyzed by RT-PCR, Northern blot, and radioimmunoassay (RIA) in relation to cell growth in subclones of HT-29 cells including the CL.16E and methotrexate (MTX) cell lines, which both exhibit a phenotype of mucus-secreting intestinal cells. A 30-fold increase in TFF3 mRNA levels and a 10-fold increase in TFF3-cell content were observed between the early proliferative and the late confluency states. The levels of MUC2 and MUC3 mRNA were also increased in the course of the differentiation process. A three to fourfold increase in PI3-K and Akt activities was observed in early post-confluent cells as compared with pre-confluent cells. Exposure of pre- and post-confluent cells to LY294002, a specific PI3-K inhibitor, for 1-4 days profoundly reduced TFF3 and MUC2 expression. A marked reduction in mucin granules content was also observed in LY-treated cells. Inhibition of the mitogen-activated protein (MAP) kinase kinase (MEK) with PD98059 did not modify the course of differentiation of the goblet cell lines. Moreover, stable transfection of HT-29 CL.16E cells with a dominant negative form of STAT6 had no effect on TFF3 induction. Together, these data indicate that PI3-K promotes the expression of TFF3 and MUC2 and that the PI3-K/Akt pathway may play a pivotal role in intestinal goblet cell differentiation.

IL-4 and IL-13 up-regulate intestinal trefoil factor expression: requirement for STAT6 and de novo protein synthesis.

The development of intestinal goblet cell hyperplasia/hypertrophy during nematode infection involves the Th2 cytokines IL-4 and IL-13 via STAT6 activation. This is thought to play an important role in host protective immunity against the infection. In this study we demonstrate that IL-4 and IL-13 up-regulate the specific goblet cell product trefoil factor-3 (TFF3) from the mucus-producing HT-29 CL.16E and HT-29 cells selected by adaptation to methotrexate. Up-regulation of TFF3 mRNA and protein levels occurred in a time- and dose-dependent fashion and was accompanied by up-regulation of the goblet cell product mucin 2 (MUC2). Addition of actinomycin D before IL-4/IL-13 stimulation led to decreases in TFF3 mRNA levels similar to those observed in controls without IL-4/IL-13. Furthermore, IL-4-mediated increased TFF3 transcription required de novo protein synthesis. Stable transfection of HT-29 CL.16E cells with a truncated dominant-negative form of STAT6 produced a cell line that was unresponsive to IL-4/IL-13. Although only one consensus STAT6 binding site is contained in the TFF3 gene, located in the intron 1, it did not operate as an enhancer in the context of an SV40 promoter/luciferase construct. Thus, STAT6 activation mediates a transcriptional enhancement of TFF3 by induction of de novo synthesized protein in goblet cells.