Lipid derivatives activate GPR119 and trigger GLP-1 secretion in primary murine L-cells

Highlights • GPR119, a putative fat sensor, is a potential target for metabolic disease.• KO of GPR119 in murine L-cells reduced GLP-1 response to fat in vivo.• Primary L-cells secreted GLP-1 in response to GPR119 agonists.• GPR119 agonists increased L-cell cAMP, with greatest efficacy in the colon.• Our data support the use of GPR119 agonists to raise GLP-1 levels.


Introduction
Glucagon-like peptide-1 (GLP-1) has multiple anti-diabetic effects, most notably enhancing insulin secretion, suppressing glucagon release and slowing gastric emptying [1]. Current incretin-based therapies focus on preventing the breakdown of GLP-1 by dipeptidyl peptidase-IV or administrating GLP-1 mimetics [2]. The benefits of increasing endogenous GLP-1 secretion are currently under evaluation, supported by evidence that gastric bypass surgery improves glucose tolerance, at least in part by increased GLP-1 secretion [3].
GPR119 is one of a number of candidate G-protein coupled receptors currently under investigation as a potential target for elevating GLP-1 and insulin release. GLP-1 is secreted from enteroendocrine L-cells in the intestinal epithelium, which express a variety of receptors and transporters capable of detecting ingested nutrients, including carbohydrates, lipids and proteins [4]. GPR119 is a G␣ s -coupled receptor, linked to the elevation of intracellular cAMP concentrations [5][6][7][8][9][10][11]. Physiological GPR119 ligands include oleoylethanolamide (OEA) [6], produced locally within tissues  [12][13][14], and 2-oleoyl glycerol (2-OG) [15] generated by luminal triacylglycerol digestion [16]. OEA as well as small molecule GPR119 agonists, increase GLP-1 and insulin release in rodent models [9,[17][18][19]. Indeed, GPR119 agonists were developed for human studies and taken into clinical trials in patients with type 2 diabetes, but were not found to improve metabolic control [20]. The reasons for the poor translatability remain uncertain, and the physiological roles and therapeutic potential of GPR119 are still under investigation.
The aim of this study was to investigate the physiological role of GPR119, and the signaling events triggered by GPR119 agonists in native murine L-cells. Using a fluorescent reporter providing a readout of cAMP concentrations in living native L-cells, we show that OEA, 2-OG, and a specific GPR119 agonist elevated cytoplasmic cAMP concentrations and enhanced GLP-1 secretion in primary cultured L-cells. We further present a new conditional knockout (KO) mouse model lacking GPR119 in proglucagon expressing cell populations including L-cells and alpha-cells. Oral oil tolerance tests in wild type (WT) and KO mice revealed that lipid-triggered plasma GLP-1 excursions are highly dependent on activation of GPR119 in L-cells.

Animal models
The flox Gpr119 mouse (Gpr119 fl ) was created using the embryonic stem cell method by AstraZeneca Transgenics and Comparative Genetics, Mölndal, Sweden. Genotyping for Gpr119 fl was performed using the primers: Forward, TGCAGAGAGGGAG-CAAATATCAGG; Reverse, TCTTGTTGTAACAAGCCTTCCAGG. Conditional Gpr119 knockout mice were created by crossing homozygous Gpr119 fl with heterozygous GLUCre12 mice, which express Cre recombinase under proglucagon promoter control [21]. The mice were selectively bred to produce homozygous females or hemizygous males (Gpr119 is located on the X-chromosome) for Gpr119 fl . All mice were on a C57BL/6 background. Details of generation of Glu-Epac21 mice are described elsewhere [22]. Briefly, this is a transgenic strain in which the cAMP FRET sensor, Epac2-camps, is expressed under control of the mouse glucagon receptor, using the same starting BAC and technique as used previously to generate GLU-Venus mice [23]. The L-cell-specificity of Epac2-camps expression was confirmed by immunofluorescence staining of fixed intestinal tissue slices. Mice were kept in individually ventilated cages according to UK Home Office regulations and the 'Principles of laboratory animal care.' All procedures were approved by a local ethical review committee.

Primary murine intestinal cell culture
Mice aged six weeks to six months were killed by cervical dislocation. Intestines were collected into ice-cold Leibovitz's L-15 medium (PAA, Yeovil, UK) and primary intestinal culture performed was as previously described [23]. Duodenum/jejunum was taken as a 10 cm length distal to the pylorus; 10 cm of ileum was taken proximal to the ileocecal junction, and colon included all tissue distal to the caecum. Minced tissue was digested with 0.4 mg/ml collagenase XI in Dulbecco's Modified Eagle Medium (DMEM) containing 4.5 g/l glucose. Crypts were pelleted at 100 g for 3 min before resuspension in DMEM containing 10% fetal bovine serum, 2 mmol/l L-glutamine, 100 units/ml penicillin, and 0.1 mg/mL streptomycin. 10 mol/l of the Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor Y27632 was added to small intestinal cultures. Cells were plated onto 24-well plates (secretion) or glass-bottom dishes (imaging) coated with a 1:100 dilution of Matrigel (BD Biosciences, Oxford, UK). Each 24-well culture plate contained crypt suspensions from a single mouse. Cultures were incubated at 37 • C and 5% CO 2 .

Intestinal cell secretion
Secretion studies were carried out 20-24 h post-plating, as described previously [23]. Total GLP-1 concentrations were analyzed in test solutions and cell lysates by immunoassay (MesoScale Discovery, Gaithersburg, MD, USA). Hormone levels in the test solution and cell lysate were summed to give the total well content. GLP-1 secretion was expressed as a percentage of this total.

Lipid gastric gavage
Mixed male and female adult mice were used for the gavage study, and the groups did not differ significantly in body weight. GLP-1 levels were similar in the male and female mice, so data were combined. Mice were fasted overnight (<16 h). Intragastric gavage of a 1:1 mix of olive:corn oils was administered (10 ml/kg body weight). Control wild type mice received a gavage of phosphate buffered saline. 25 min later, mice were anaesthetized with isoflurane, and terminal blood samples taken at 30 min by cardiac puncture. Plasma was separated immediately and frozen. Total GLP-1 in the plasma was measured by immunoassay (Mesoscale Discovery).

cAMP Imaging
Single-cell measurements of cAMP levels were made using the Förster resonance energy transfer (FRET)-based sensor Epac2camps, using tissues from Glu-Epac21 mice maintained in mixed primary culture for 20-78 h. The use of Epac2-camps for monitoring cAMP concentrations in GLP-1 expressing cell lines has been described previously [24]. Maximum time-averaged CFP/YFP ratios, representing [cAMP], were determined at baseline and following test reagent application.

Data analysis
Data were analyzed using Microsoft Excel and GraphPad Prism v5.0 (Graphpad Software, San Diego, USA), using Student's t-tests, ANOVA and post-hoc Bonferroni tests, as indicated in the figure legends.

GPR119 involvement in lipid-sensing
The contribution of GPR119 to GLP-1 secretion in vivo was investigated by the administration of a lipid gastric gavage to Gpr119-KO and WT mice. In WT mice, oil gavage triggered an approximate 3-fold elevation of plasma total GLP-1 concentrations at 30 min, compared with control mice gavaged with saline (Fig. 1). GLP-1 after oil gavage was significantly lower in KO animals compared to WT controls (Fig. 1), indicating that GPR119 in L-cells plays
The same ligands were then applied to cultures from Gpr119-KO mice (Cre-positive/Gpr119 fl ) and Cre-negative/Gpr119 fl mice (henceforth called WT). Secretion was measured separately from the colon, ileum, and duodenum/jejunum (Fig. 2B-D). AR231453 significantly increased GLP-1 release 4.6-fold from the colon and 2.9-fold from the ileum of WT mice; OEA significantly enhanced GLP-1 release by 3.9-fold in the colon and 2.1-fold in the duodenum/jejunum; 2-OG only increased secretion significantly in the colon (2.1-fold). Secretory responses to all three GPR119 ligands were significantly impaired in colonic cultures from Gpr119-KO mice (Fig. 2D). In ileal cultures, the response to AR231453 was reduced in Gpr119-KO tissue (Fig. 2C), whereas in duodenal/jejunal cultures, the enhanced secretion triggered by OEA was not impaired by Gpr119-KO (Fig. 2B). elevations of L-cell cAMP (Fig. 3). Particularly in the upper intestine, we observed that not all cells responded to test agents, and cells were allocated as responders if they showed a change of the FRET signal of >2% above baseline. In the duodenum 50% of L-cells (13 out of 26) exhibited cAMP responses to AR231453, compared with 45% (5/11) of L-cells in the ileum and 71% (15/21) in the colon. The mean amplitude of the cAMP response to AR231453 was not significantly different across intestinal tissues (Fig. 3D).

Discussion
Following the de-orphanization of GPR119, small molecules targeting this receptor were developed as potential new treatments for diabetes that would increase secretion from intestinal L-cells [25]. Although subsequent trials have not yet demonstrated that metabolic improvements can be brought about by the use of GPR119 agonists in humans with type 2 diabetes [20], there is still a high level of academic and commercial interest in GPR119 as a potential drug target [26,27]. Our results show that L-cell GPR119 is a critical component of the sensing mechanism responsible for GLP-1 responses to ingested lipid, and that L-cells in the distal intestine respond to GPR119 agonists with elevated cAMP and GLP-1 secretion.
We show here that GPR119 ligands increase GLP-1 release from primary cultured ileal and colonic L-cells in a GPR119-dependent manner. Of the three GPR119 agonists tested, OEA and AR231453 were more effective than 2-OG. The magnitude of the secretory response triggered by the different GPR119 ligands increased progressively from the upper small intestine to the colon. Indeed, L-cell knockout of Gpr119 largely abolished responses to OEA, 2-OG and AR231453 in the colon. In the ileum, where the secretory response was smaller, only OEA and AR231453 raised secretion in WT tissues above that found in the Gpr119-KO, and in the duodenum/jejunum, none of the ligands had a greater effect in WT than KO cultures. While our results suggest that the small response to OEA in the duodenum/jejunum of WT tissue is independent of GPR119, we cannot exclude the possibility that the proportion of L-cells undergoing Cre-dependent GPR119 excision differed between tissues and that more residual L-cells expressed GPR119 in the upper intestine. Arguing against this idea, however, AR231453 had little effect on GLP-1 secretion in the WT duodenum/jejunum, and OEA has been reported to activate other pathways such as PPAR␣ that might influence GLP-1 secretion even in the absence of Gpr119 [28].
The GLU-Epac transgenic mouse enabled us to monitor cAMP responses to GPR119 ligands in individual primary cultured L-cells. Not all L-cells were found to be responsive to AR231453, suggesting there may be a subpopulation of L-cells that do not express functional GPR119. There was a tendency for smaller and less frequent cAMP responses to AR231453 in the small intestine compared with the colon, although this did not reach statistical significance. These results do, however, mirror the gradient of GLP-1 secretory responses in cultures from the different regions. In line with these findings, we also reported previously that Gpr119 expression appeared higher in colonic than small intestinal L-cells by qRT-PCR [23].
Mice with targeted deletion of Gpr119 in L-cells exhibited a marked reduction of plasma GLP-1 levels after gastric oil gavage. This suggests that GPR119-dependent detection of luminallygenerated 2-monoacylglycerols or locally-released OEA plays a major role in the post-prandial GLP-1 secretory response to orally ingested triglycerides. While long chain free fatty acids are also released during the luminal digestion of corn and olive oils, and are sensed by GPR119-independent pathways, likely involving GPR40 and GPR120 [4], our findings suggest that these pathways play a relatively minor role compared with GPR119 in mediating the GLP-1 secretory response to oral lipids. While our data support the development of GPR119 agonists to enhance GLP-1 secretion, the role of different intestinal regions in post-prandial physiology and as drug targets deserves further attention.