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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 mnol/l, 60 min) dose dependently increased the secretion of mucins (210 [+ or -] 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/1, 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. Muc2; Muc3; Muc4; MUC5AC; mucus: intestinal barrier: phosphatidylinositol 3-kinase doi:101152/agpgi.00091.2007

We have recently shown that [beta]-casomorphin-7, a milk opioid peptide, strongly stimulates mucin secretion in the rat jejunum through a nervous pathway and opioid receptor activation. In this study, the hypothesis that [beta]-casomorphin-7 may also act directly on intestinal goblet cells was investigated in vitro in rat and human intestinal mucin-producing cells (DHE and HT29-MTX) using quantitative and semiquantitative RT-PCR and ELISA. The presence of [mu]-opioid receptors was demonstrated in rat goblet cells in the upper half of the colonic crypt and in the two cell lines by immunohistochemistry and RT-PCR. In rat DHE cells, [beta]-casomorphin-7 increased the expression of rat mucin (rMuc)2 and rMuc3 but not rMuc1, rMuc4, and rMuc5AC. This effect was time and dose dependent, with the maximum of increase in transcripts being noticed for a concentration of [10.sup.-4] M after 2 h of stimulation for rMuc2 (225% of controls) and 4 h of stimulation for rMuc3 (208% of controls). Mucin secretion was maximally increased after 8 h of stimulation. Interestingly, these effects were prevented by pretreatment of the cells with the [mu]-opioid antagonist cyprodime. In human HT29-MTX cells, [beta]-casomorphin-7 ([10.sup.-4] M) also increased MUC5AC mRNA levels (219% after 24 h of stimulation) and the secretion of this mucin (169% of controls). In conclusion, [beta]-casomorphin-7 may contribute significantly to mucin production via a direct effect on intestinal goblet cells and the activation of [micro]-opioid receptors. Because intestinal mucins have a crucial mucosal protective function, dairy products containing [beta]-casomorphin-7 may improve intestinal protection and could have dietary and health applications. rat mucin 2; rat mucin 3; mucin 5AC; mucus; milk bioactive peptides

Intestinal mucus is critically involved in the protection of the mucosa. An enzymatic casein hydrolysate and [beta]-casomorphin-7, a [mu]-opioid peptide generated in the intestine during bovine casein digestion, markedly induce mucus discharge. Because shorter [mu]-opioid peptides have been described, the effects of the opioid peptides in casein, [beta]-casomorphin-7, -6, -4, -4N[H.sub.2] and -3, and of opioid neuropeptides met-enkephalin, dynorphin A and (D-Ala2,N-Me-Phe4,glycinol5)enkephalin (DAMGO) on intestinal mucus secretion were investigated. The experiments were conducted with isolated perfused rat jejunum. Mucus secretion under the influence of [beta]-casomorphins and opioid neuropeptides administered intraluminally or intra-arterially was evaluated using an ELISA for rat intestinal mucus. Luminal administration of [beta]-casomorphin-7 (1.2 x [10.sup.-4] mol/L) provoked a mucus discharge (500% of controls) that was inhibited by naloxone, a specific opiate receptor antagonist. Luminal [beta]-casomorphin-6, -4 and -4N[H.sub.2] did not modify basal mucus secretion, whereas intra-arterial administration of [beta]-casomorphin-4 (1.2 x [10.sup.-6] mol/L) induced a mucus discharge. In contrast, intra-arterial administration of the nonopioid peptide [beta]-casomorphin-3 did not release mucus. Among the opioid neuropeptides, intra-arterial infusion of Met-enkephalin or dynorphin-A did not provoke mucus secretion. In contrast, [beta]-endorphin (1.2 x [10.sup.-8] to 1.2 x [10.sup.-6] mol/L) induced a dose-dependent release of mucus (maximal response at 500% of controls). DAMGO (1.2 x [10.sup.-6] mol/L), a [mu]-receptor agonist, also evoked a potent mucus discharge. Our findings suggest that [mu]-opioid neuropeptides, as well as [beta]-casomorphins after absorption, modulate intestinal mucus discharge. Milk opioidderived peptides may thus be involved in defense against noxious agents and could have dietary and health applications. J. Nutr. 133: 3499-3503, 2003. KEY WORDS: * mucus * intestine * milk * casomorphin * opioid

The hypothesis that dietary proteins or their hydrolysates may regulate intestinal mucin discharge was investigated in the isolated vascularly perfused rat jejunum using an enzyme-linked immunosorbent assay for rat intestinal mucins. On luminal administration, casein hydrolysate [0.05-5% (wt/vol)] stimulated mucin secretion in rat jejunum (maximal response at 417% of controis). Lactalbumin hydrolysate (5%) also evoked mucin discharge. In contrast, casein, and a mixture of amino acids was without effect. Chicken egg albumin and its hydrolysate or meat hydrolysate also did not modify mucin release. Interestingly, casein hydrolysate-induced mucin secretion was abolished by intra-arterial TTX or naloxone (an opioid antagonist). [beta]-Casomorphin-7, an opioid peptide released from [beta]-casein on milk ingestion, induced a strong mucin secretion (response at 563% of controls) that was inhibited by naloxone. Intra-arterial [beta]-casomorphin-7 also markedly increased mucin secretion (410% of controls). In conclusion, two enzymatic milk protein hydrolysates (casein and lactalbumin hydrolysates) and [beta]-casomorphin-7, specifically, induced mucin release in rat jejunum. The casein hydrolysate-induced mucin secretion is triggered by a neural pathway and mediated by opioid receptor activation. goblet cells; casein; lactalbumin; [beta]-casomorphin; isolated perfused intestine

Dietary protein might modulate mucin flow and intestinal mucin gene expression. Since unheated phaseolin from Phaseolus vulgaris bean is resistant to digestion and increases gut endogenous protein losses, we hypothesised that unheated phaseolin influences mucin flow and gene expression, and that phaseolin heat treatment reverses these effects. The hypothesis was tested using a control diet containing casein as the sole protein source and three other diets with casein being replaced by 33 and 67 % of unheated and 67 % of heated phaseolin. The rats were fed for 6 d and euthanised. Digesta and faeces were collected for determining digestibility and mucin flow. Gut tissues were collected for mucin (Muc1, Muc2, Muc3 and Muc4) and Trefoil factor 3 (Tff3) gene expressions. Colonic mucin flow decreased linearly with increasing the dietary level of unheated phaseolin (P < 0·05). Unheated phaseolin increased N flow in ileum, colon and faeces (P < 0·05), and reduced apparent N digestibility linearly (P < 0·01). Heat treatment reversed all these changes (P < 0·05 to < 0·001), except mucin flow. The expressions of Muc mRNA in gut tissues were influenced by dietary phaseolin level (ileum and colon: Muc3 and Muc4) and thermal treatment (ileum: Muc2; colon: Muc2, Muc3, Muc4 and Tff3) (P < 0·05 to 0·001). In conclusion, phaseolin modulates mucin flow and Muc gene expression along the intestines differentially.

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.