Zebrafish MCH receptor mRNA expression and regulation by intestinal inflammation.

<div><p>A) Relative expression of zebrafish MCHR1b and MCHR2 in brain and the intestine.</p> <p>B) Expression of intestinal MCHR1b and MCHR2 mRNA following treatment with TNBS or vehicle (n=9-10 fish per group). Values in the vehicle treatment group are set to 100.</p> <p>AU: arbitrary units.</p></div

Relative mRNA expression of various cytokines in the intestine of zebrafish with TNBS-induced enterocolitis, at 6 hours post-exposure (n=9-10 fish per group).

<p>For each gene, expression in the vehicle treated group was set to 100. AU=arbitrary units.</p

MCH expression in the adult zebrafish intestine.

<div><p>A) Amino acid sequence alignments of the MCH peptides from human, mouse and zebrafish. Identical residues are highlighted.</p> <p>B) Comparative mRNA expression of the zebrafish MCH peptides in brain and the gastrointestinal tube, presented in logarithmic scale. (n=8-9 fish per group).</p></div

Adult zebrafish are susceptible to TNBS-induced enterocolitis.

<p>Kaplan-Meier survival analysis in response to different concentrations of TNBS (40mM-320mM). TNBS was administered by intrarectal infusion and doses were adjusted for body weight to a volume of 1 uL/0.1 g of body weight. Fish were monitored for a total of 96 hours in 6-12 hour intervals. For each TNBS dose, 13-14 fish per group were assigned to TNBS or vehicle (30% ethanol) treatments. </p

Vancomycin administration reduces mortality associated to TNBS-enterocolitis.

<div><p>A) Graphical representation of the study design. Vancomycin (100mg/L) was added to the fish tank water 18 hours prior to the induction of colitis.</p> <p>B) Kaplan-Meier analysis comparing the effect of treatment with vancomycin or vehicle on the survival of zebrafish with TNBS enterocolitis (n=21-25 fish per group).</p></div

Sequence alignment of MCH receptors in various species.

<div><p>A) Sequence alignment of zebrafish MCHR1a, MCHR1b and human and mouse MCHR1.</p> <p>B) Sequence alignment of MCHR2 receptors in zebrafish, human and mouse .</p> <p>Identical residues are highlighted.</p> <p>C) Graphical representation of sequence identity for MCH receptors among species.</p></div

MCH increases cell viability <i>in vitro</i> by suppressing apoptosis.

<p>Caco2 human colonic adenocarcinoma cells were serum starved overnight (2% FBS) and treated for 48 hrs with MCH (2.4 µg/ml). (A) Cell viability was measured using the MTT assay. Treatment with 20% FBS served as a positive control. (B) Cell apoptosis was measured by the Apo-One Homogenous Caspase3−/7 assay. (C) Cells were treated with MCH (2.4 µg/ml), IGF-1 (10 nM) or their combination and cell proliferation was assessed by measuring BrdU incorporation using a colorimetric assay. Results are expressed relatively to vehicle treated cells (100). Graphs depict mean±sem of 6 replicates and are representative of 2 independent experiments. ***p<0.005; ****p<0.001 (D) NCM460 non-transformed human colonic epithelial cells stably expressing human MCHR1 (NCM460/MCHR1) were treated with MCH and erk1/2 phosphorylation at different time points was analyzed by western blot. Expression of GAPDH serves as a control for equal loading.</p

Attenuated activation of the wnt/beta-catenin and ERK signaling pathways in APCmin mice lacking MCH.

<p>Paraffin sections of intestinal adenomas from WT and MCH KO mice, both of the APCmin background, were stained for (A) beta-catenin, a downstream effector of the wnt signaling pathway; (B) c-myc, a target of beta-catenin; and (C) p-erk. Quantification of differences between the groups (percentage of positive cells per adenoma) is shown in graphs on the right of each picture. Results are expressed as mean±sem. ***p<0.005; ****p<0.001.</p

MCHR1 expression in normal colonic epithelial cells and colon adenocarcinoma.

<p>(A) In human colonic biopsies, normal epithelium as well as adenocarcinoma cells were positive for MCHR1 staining. Moreover, Caco2 cells, a human colon adenocarcinoma cell line, showed MCHR1 immunoreactivity. (B) Likewise, MCHR1 was found to be expressed by mouse intestinal epithelial cells, intestinal tumors developed in APCmin mice and the MCA-38 murine colon cancer cell line. Incubation with pre-immune serum was used as a negative control.</p

Increased apoptosis in intestinal adenomas developed in MCH-deficient APCmin mice.

<p>Paraffin sections of intestinal adenomas from WT and MCH-KO mice, both of the APCmin background, were stained for (A) TUNEL, a marker of apoptosis; and (B) Ki67, a marker of proliferation. Quantification of differences between the groups (percentage of positive cells per adenoma) is shown in graphs on the right of each picture. *p<0.05.</p