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Signalling pathways involved in the detection of peptones by murine small intestinal enteroendocrine L-cells - PubMed

Signalling pathways involved in the detection of peptones by murine small intestinal enteroendocrine L-cells

Ramona Pais et al. Peptides. 2016 Mar.

Abstract

Glucagon like peptide-1 is an insulinotropic hormone released from intestinal L-cells in response to food ingestion. Here, we investigated mechanisms underlying the sensing of peptones by primary small intestinal L-cells. Meat, casein and vegetable-derived peptones (5 mg/ml), the L-amino acids Phe, Trp, Gln and Ala (20 mM each), and the dipeptide glycine-sarcosine (20 mM) stimulated GLP-1 secretion from primary cultures prepared from the small intestine. Further mechanistic studies were performed with meat peptone, and revealed the elevation of intracellular calcium in L-cells. Inhibition of the calcium sensing receptor (CaSR), transient receptor potential (TRP) channels and Q-type voltage gated calcium channels (VGCC) significantly attenuated peptone-stimulated GLP-1 release and reduced intracellular Ca(2+) responses. CaSR inhibition also attenuated the GLP-1 secretory response to Gln. Targeting these pathways in L-cells could be used to increase endogenous production of GLP-1 and offer exploitable avenues for the development of therapeutics to treat diabetes and obesity.

Keywords: Enteroendocrine; GLP-1; L-cell; Peptones.

Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

Fig. 1
Fig. 1

Peptones and amino acids stimulate GLP-1 secretion from small intestinal cultures. (a) Peptones (5 mg/ml) from three different sources stimulated GLP-1 secretion from mixed primary cultures from murine small intestine. Cultures were incubated for 2 h under control conditions or in the presence of meat, milk or vegetable peptones. GLP-1 secretion in each well is expressed relative to the basal secretion (control) measured in parallel on the same day. Data represent the mean ± SEM. ***p  < 0.001 compared with controls by one way ANOVA followed by Dunnet's multiple comparison test. n represents the number of wells, 42 for controls and 31, 12 and 12 for peptones from meat, milk and vegetable source, respectively. (b) Peptones from meat show a dose dependent stimulation of GLP-1 secretion. Cultures were incubated for 2 h under control conditions or in the presence of 0.5, 5 or 50 mg/ml meat peptones. GLP-1 secretion in each well is expressed relative to the basal secretion (control) measured in parallel on the same day. Data represent the mean ± SEM. ***p < 0.001 compared with controls by one way ANOVA followed by Dunnet's multiple comparison test. n = 12 wells each for controls and peptones. (c) GLP-1 secretion stimulated by a range of amino acids and the dipeptide Gly-Sar, all tested at 20 mM. Data represent the mean ± SEM. ***p < 0.001 compared with controls by one way ANOVA followed by Dunnet's multiple comparison test. n represents the number of wells for each condition, 36 for controls, 24 for Gln, 15 for Ala, 9 for Leu, 18 for Phe and 12 for Gly-Sar.

Fig. 2
Fig. 2

Peptones trigger intracellular calcium concentration elevation in L-cells. (a) A representative trace showing the change in GCaMP3 fluorescence before, during and after the application of meat peptones (pep, 5 mg/ml) to a primary duodenal L-cell cultured from GLU-Cre/ROSA26-GCaMPe mice. Peptones caused reversible increase in cytosolic calcium. KCl (30 mM) was used as a positive control to show that the cell was still viable after peptone application. (b) Mean calcium changes in L-cells following the addition of meat peptone (pep, 5 mg/ml, n = 29 cells) and KCl (30 mM, n = 24)

Fig. 3
Fig. 3

Role of voltage gated calcium channels in peptone-stimulated GLP-1 secretion. (a) A representative trace showing intracellular calcium changes in a primary duodenal L-cell before, during and after the application of meat peptone (pep, 5 mg/ml) and co-application of cobalt chloride (CoCl2, 5 mM). (b) Mean calcium changes in L-cells following the addition of peptone in the presence (n = 10) and absence (n = 10) of cobalt chloride and KCl (n = 8). Cobalt chloride significantly inhibited the increase in intracellular calcium by peptones. Data represent the mean ± SEM. *p < 0.05, ***p < 0.001 compared with baseline and between conditions by one- and two-sample Student’s t test. (c) Expression of different cacn α-subunit mRNAs in small intestine L-cells (black bars) and control small intestine cells (open bars) assessed by Affymetrix microarray. Expression was evaluated by RMA analysis, and is depicted on an arbitrary scale on which values >100 represent expression that can be reliably detected by quantitative RT-PCR (n = 2–3 per cell type). (d) Relative GLP-1 secretion from murine small intestine cultures incubated for 2 h in the presence of meat peptones (n = 9) and calcium channel inhibitors, nifedipine (Nif, 10 μM, n = 9), ω-conotoxin MVIIC (ω-cono Tx, 1 μM n = 9) and ω-agatoxin IVA (Aga Tx, 0.2 μM, n = 9). Data represent the mean ± SEM. *p < 0.05, ***p < 0.001 compared with their respective controls by one-way ANOVA with post hoc Bonferroni test.

Fig. 4
Fig. 4

Role of transient receptor potential channels in peptone-stimulated GLP-1 secretion. (a) A representative trace showing intracellular calcium changes in a primary duodenal L-cell before, during and after the application of meat peptones (pep, 5 mg/ml) and co-application of lanthanum chloride (LaCl3, 50 μM). (b) Mean calcium changes in L-cells following the addition of peptone in the presence (n = 9) and absence (n = 9) of lanthanum chloride and KCl (n = 7). Co-application of lanthanum chloride significantly inhibited peptone- stimulated rise in intracellular calcium. Data represent the mean ± SEM. **p < 0.01, ***p < 0.001 compared with baseline and between conditions by one- and two-sample Student’s t test. (c) Representative trace showing intracellular calcium changes in primary duodenal L-cells before, during and after the application of KCl (30 mM) and co-application of lanthanum chloride (LaCl3, 50 μM). (d) Representative trace showing intracellular calcium changes in primary duodenal L-cells before, during and after the application of KCl (30 mM) and co-application of cobalt chloride (CoCl2, 5 mM). (e) Mean calcium changes in L-cells following the addition of KCl in the absence (n = 17) and presence of lanthanum chloride (n = 16) and cobalt chloride (n = 6). Co-application of cobalt chloride significantly inhibited KCl- stimulated rise in intracellular calcium. Data represent the mean ± SEM. **p < 0.01, ***p < 0.001 compared with baseline and ##p < 0.01 compared with KCl alone by one- and two-sample Student’s t test. (f) GLP-1 secretion from murine small intestine cultures incubated for 2 h in the presence of peptones (n = 19) and TRP channel inhibitors, lanthanum chloride (LaCl3, 50 μM, n = 19). GLP-1 secretion in each well is expressed relative to the basal secretion (control) measured in parallel on the same. Data represent the mean ± SEM. **p < 0.01, ***p < 0.01 compared with their respective controls by one-way ANOVA with post hoc Bonferroni analysis.

Fig. 5
Fig. 5

Role of CaSR in peptone-stimulated GLP-1 secretion. (a) CaSR inhibition reduced peptone-stimulated GLP-1 secretion from mixed primary cultures incubated for 2 h under control conditions (standard bath solution, n = 19), meat peptone alone (Pep, 5 mg/ml, n = 19) or in the additional presence of the CaSR inhibitor, Calhex 231HCl (Calhex, 10 μM, n = 16). GLP-1 secretion in each well is expressed relative to the basal secretion (control) measured in parallel on the same. Data represent the mean ± SEM. **p < 0.01, compared with their respective controls by one-way ANOVA with post hoc Bonferroni analysis. (b) Cultures were incubated with Gln (20 mM, n = 12) and Phe (20 mM, n = 12) in the presence or absence of Calhex 231HCl (10 μM, n = 12). GLP-1 secretion in each well is expressed relative to the basal secretion (control) measured in parallel on the same day. Data represent the mean ± SEM. ***p < 0.01, compared with control or ##p < 0.01 compared to their respective controls by one-way ANOVA with post hoc Bonferroni analysis.

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