Identification and characterization of GLP-1 receptor-expressing cells using a new transgenic mouse model

Abstract

GLP-1 is an intestinal hormone with widespread actions on metabolism. Therapies based on GLP-1 are highly effective because they increase glucose-dependent insulin secretion in people with type 2 diabetes, but many reports suggest that GLP-1 has additional beneficial or, in some cases, potentially dangerous actions on other tissues, including the heart, vasculature, exocrine pancreas, liver, and central nervous system. Identifying which tissues express the GLP-1 receptor (GLP1R) is critical for the development of GLP-1-based therapies. Our objective was to use a method independent of GLP1R antibodies to identify and characterize the targets of GLP-1 in mice. Using newly generated glp1r-Cre mice crossed with fluorescent reporter strains, we show that major sites of glp1r expression include pancreatic β- and δ-cells, vascular smooth muscle, cardiac atrium, gastric antrum/pylorus, enteric neurones, and vagal and dorsal root ganglia. In the central nervous system, glp1r-fluorescent cells were abundant in the area postrema, arcuate nucleus, paraventricular nucleus, and ventromedial hypothalamus. Sporadic glp1r-fluorescent cells were found in pancreatic ducts. No glp1r-fluorescence was observed in ventricular cardiomyocytes. Enteric and vagal neurons positive for glp1r were activated by GLP-1 and may contribute to intestinal and central responses to locally released GLP-1, such as regulation of intestinal secretomotor activity and appetite.

Fig. 1. Glp1r-cre transgenic mice label pancreatic β and δ-cells

A. Schematic depicting the insert of the bacterial artificial chromosome (BAC) RP23-408N20, and replacement of the glp1r coding region from exons (ex) 1-13 with iCre. Lengths (in kb) of the murine genomic sequence 3′ and 5′ of the glp1r coding region in the BAC are indicated. B. Dispersed pancreatic islet cells from glp1r-cre/ROSA26-YFP mice, analysed by FACS. Green (yellow) fluorescence was measured by excitation at 488 nm and emission at 530/20 nm and is plotted against forward scatter in arbitrary units (AU). C. Cells with high red fluorescence from glp1r-cre/ROSA26-tdRFP mice were FACS separated into populations with low and high side scatter (SSC). These were analysed by qRT-PCR for expression of hormones (D), or glp1r (E). n=3 each, *** p<0.001 by Student’s t-test. F,G. Fixed pancreatic slices were co-immunostained RFP or YFP (representing glp1r-fluorescence) together with glucagon and insulin (F) or somatostatin (G). Nuclei are visualised with Hoechst stain. In F, a cell triple positive for glucagon, insulin and glp1r-fluorescence is marked by the asterisk.

Fig. 2. Glp1r in pancreatic α-cells

A. Islet cells from glp1r-cre/ROSA26-tdRFP/GLU-Venus mice, FACS analysed to show red fluorescence (representing expression of glp1r) or green fluorescence (representing expression of gcg). B. Single or double positive cells were purified from quadrants Q1-Q3 marked in A, and analysed for expression of ins and gcg by qRT-PCR. *** p<0.001 by ANOVA with post hoc Bonferroni test, n=5-7. C. Active GLP-1 hormone concentrations in cells collected from different quadrants of A as indicated. Q1-4 all islet cells, Q1 + high SSC β-cells, Q2 + Q3 all α-cells, Q2 double fluorescent cells. (mean and SEM of n=4 FACS sorts, each of pooled islets from 1-2 mice). D-F. Pancreatic sections from glp1r-cre/ROSA26-YFP mice co-immunostained for α-smooth muscle actin (αSMA) or cytokeratin (CK) together with YFP. The marked area in E is shown at higher magnification in F.

Fig. 3. Glp1r in the cardiovascular system

Sections of aorta (A), kidney (C), intestine (D,E), and cardiac ventricle (G) or atrium (H) from glp1r-cre/ROSA26-YFP mice were co-immunostained for YFP (representing glp1r-fluorescence) and α-smooth muscle actin (αSMA), NG2 or renin, as indicated. In kidney sections, G depicts the position of glomeruli. B. qRT-PCR analysis of glp1r expression in aorta, atrium and ventricle (geometric mean + 1SEM, of n=5-6 each). F. Blood vessels in intestine from a glp1r-cre/ROSA26-tdRFP mouse: upper panel light microscopy (A arteriole, V venule), lower panel tdRFP fluorescence.

Fig. 4. Glp1r in the brain and afferent neuronal ganglia

A. Coronal brain sections from glp1r-cre/ROSA26-tdRFP mouse, immunostained for RFP. In caudal brainstem (top), a high density of fluorescent cells was observed in the area postrema (AP), whereas only few were evident in the nucleus of the solitary tract (NTS), and dorsal vagal nucleus (DMNX) and none were found in the hypoglossal nucleus (HN). In hypothalamus (middle and lower panels), large numbers of RFP fluorescent cells were observed in the arcuate nucleus (Arc), ventromedial hypothalamus (VMH) and paraventricular nucleus (PVN). Abbreviations: ME, median eminence; CC, central canal; 3V, 3rd ventricle. Scale bar = 150μm. B. Section of nodose ganglion (NG) from glp1r-cre/ROSA26-tdRFP mouse, showing RFP positive cells. C. Dorsal root ganglion (DRG) section from a glp1r-cre/ROSA26-tdRFP mouse, co-stained for RFP and calcitonin gene related peptide (CGRP). D. Whole NG or DRG were analysed for glp1r vs actb expression by qRT-PCR (geometric mean +1SEM of n=3 each). E. NG from glp1r-cre/ROSA26-tdRFP mice were studied by fura2 Ca²⁺ imaging in primary culture. Representative trace from an RFP positive NG neuron to ATP (0.5 μM) applied in the absence or presence of GLP-1 (1 nM), as indicated by the horizontal bars. F. Mean 340/380 nm fluorescence ratios from 13 RFP positive cells (RFP+, open bars) and 12 RFP negative cells (RFP−, filled bars), monitored as in E and normalised to baseline recorded in the absence of test agent. ** p<0.01 by Wilcoxon signed rank test G. Ca²⁺ responses of RFP+ (open bars) and RFP− (filled bars) cells to cholecystokinin (CCK, 10 nM), leptin (10 nM) or 5HT (10 μM), normalised to baseline in control solution. From left to right, columns represent data from n=18, 41, 9, 31, 14 and 35 cells, respectively. *p<0.05, *** p<0.001 by Mann Whitney test.

Fig. 5. Glp1r in enteric neurons

A. Fixed tissue section of liver from a glp1r-cre/ROSA26-tdRFP mouse, showing glp1r-fluorescence in structures surrounding a portal vein. B,C. Antral area of the stomach from a glp1r-cre/ROSA26-tdRFP/ROSA26-YFP mouse, immunostained in B for YFP (green) and αSMA (red) and in C for RFP (green) and nNOS (red). D. Nested PCR amplification of glp1r and β-actin, performed on 3 hand-picked glp1r-fluorescent enteric ganglia from glp1r-cre/ROSA26-tdRFP mice. E. Representative recording of a partially dissociated small intestinal glp1r-fluorescent enteric neuron in primary culture, monitored in whole cell current clamp, following addition of GLP-1 (10 nM). F. Mean action potential (AP) frequency of 7 cells under basal conditions and following GLP-1 application. * p<0.05 by Wilcoxon signed rank test. G. Enteric neurons, recorded as in E, were divided into AH-type (with characteristic hump on the action potential downstroke and after-hyperpolarisation) and S-type. The dashed line represents the 0 mV line. Small intestinal (H) and colonic (I) tissue sections from a glp1r-cre/ROSA26-tdRFP mouse co-stained for RFP together with nNOS or calretinin, as indicated. As direct RFP fluorescence is lost on fixation, a green secondary antibody was used to visualise RFP in the upper panel of H. J. Intestinal tissue section from a glp1r-cre/ROSA26-tdRFP/GLU-Venus mouse showing proximity of an L-cell (green) and a red glp1r-fluorescent fibre.