L-Cell Differentiation Is Induced by Bile Acids Through GPBAR1 and Paracrine GLP-1 and Serotonin Signaling
Article Figures & Tables
Figures
- Figure 1
Activation of GPBAR1 by the bile acid LCA and a synthetic agonist increases L-cell number in organoids. A: Representative images of control and organoids treated with 10 μmol/L LCA. L cells are labeled by Gcg-YFP expression in this and following images unless otherwise indicated. Nuclei labeled by DAPI. Scale bars, 50 μm. B: L-cell numbers in control (Ctrl) organoids (n = 70) and organoids treated with LCA (n = 78). In this and following figures, the data are mean ± SEM (unless otherwise indicated). C: Representative images of control and L3740-treated organoids. Scale bars, 50 μm. D: L-cell numbers in control (n = 38) and L3740-treated organoids (n = 30). E: GLP-1 secretion from L3740-treated organoids stimulated by 18 mmol/L glucose and 2 mmol/L l-glutamine. Data from three experiments were performed with five replicas. F: Gene expression of intestinal hormones in L3740-treated organoids (n = 4–10 in each series). G: Gene expression of intestinal cell type markers and transcription factors directing L-cell development. Lgr5 and CD133, proliferating cells; ITF, goblet cells; Lyz1, Paneth cells; I-Fabp, enterocytes; Ngn3, NeuroD1, Arx, and Foxa1/2, L-cell transcription factors (n = 4–10 experiments in each series). H: L-cell numbers, identified by immunostaining, in control and GPBAR1 KO and wild-type (WT) mouse organoids after 48-h treatment with L3740 (n = 21–25). I: Expression of L-cell differentiation markers in control and L3740-treated GPBAR1 KO organoids (n = 3 for each series). J: GLP-1 release in response to LCA and L3740 in GPBAR1 KO organoids (n = 5 for each series). *P < 0.05, **P < 0.01, ***P < 0.001.
- Figure 2
In-feed L3740 treatment increases oral glucose tolerance, GLP-1 secretion, and L-cell number in mice. A: Blood glucose excursion during an oral glucose tolerance test after L3740 treatment (n = 10 for each series). B: Area under the curve (AUC) from data in panel A. Ctrl, control. C: Plasma GLP-1 concentrations in control and L3740-treated mice before the test and 10 min after glucose gavage (n = 8 for each series). D: L-cell percentage in mouse ileum sections (n = 4 for each series). E: L cells (Gcg-YFP, green) in the ileum of control and L3740-treated mice. Nuclei labeled by DAPI (blue). Scale bars, 50 μm. F: Expression of Gcg and Ngn3 in mouse ileal epithelium (n = 4 for each series). *P < 0.05, **P < 0.01, ***P < 0.001.
- Figure 3
GPBAR1 stimulation increases L-cell abundance in human organoids. A: L-cell numbers in control (Ctrl; n = 66) and L3740-treated human small intestine organoids (n = 44). B: Representative images of control and L3740-treated human organoids. L cells are labeled by GLP-1 immunostaining (white arrows), and nuclei are labeled by DAPI. Scale bars, 50 µm. C: A shift in the organoid distribution by the cell number after treatment with L3740. Percentages of organoids with various L-cell numbers are presented from data in panel A. P < 0.05 by χ2 contingency test. D: Expression of NGN3, NEUROD1 (ND1), and GCG (n = 4 for each series, generated from two human organoid lines). *P < 0.05, **P < 0.01.
- Figure 4
GPBAR1 induction of L-cell differentiation requires cross talk with serotonin signaling. A: Effect of L3740 in GLP1R KO mouse organoids (n = 47 for control [Ctrl] and n = 58 for L3740-treated series). B: Effect of 5-HTR3 and 5-HTR4 inhibitor tropisetron (TRP) and 5-HTR4 inhibitor RS-39604 in organoids alone and in combination with L3740. C: Htr4 and Htr3 in mouse ileum identified by in situ hybridization. Nucleus labeling by DAPI (blue). White arrows indicate positive Htr3 staining outside of the epithelial layer. Scale bars, 50 µm. D: L3740 increases Htr4 expression in organoids (n = 7 for control and n = 6 for L3740-treated series). E: Coexpression of Htr4 and cell markers Lgr5, CD133, Math1, and Ngn3 in mouse ileal crypts identified by in situ hybridization. White arrows indicate positive Htr4 staining. Nuclei are labeled with DAPI (blue). Scale bars, 20 µm. F: Effect of 5-HT4R agonist BIMU 8 and L3740 on the L-cell number (n = 17–35 in panels A and B). G: BIMU 8 increases L-cell number in GLP1R KO mouse organoids, while L3740 has no effect on the L cells (n = 47 for control and n = 20 for L3740-treated series). H: BIMU 8 increases L-cell numbers in GPBAR1 KO mouse organoids (n = 21 for control and n = 23 for L-3740-treated series). I: FFAR1 agonist AM-1638 and GPR119 agonist AR53 have a similar effect on the number of L cells, while addition of RS-39604 counteracts this effect (n = 33–42). **P < 0.01, ***P < 0.001.
In this Issue
