Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C28
Murine I-cells express G-Protein Coupled Lipid Receptors GPR40, GPR119 and GPR120 mRNA transcripts.
A. G. Sykaras1,2, C. Demenis1, X. Huang1, O. Kiss1, M. Case1, J. T. McLaughlin3, C. P. Smith1
1. Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom. 2. School of Medicine, University of Crete, Heraklion, Crete, Greece. 3. School of Translational Medicine, University of Manchester, Manchester, United Kingdom.
Cholecystokinin (CCK) is the archetypal anti-orexigenic and satiety hormone that co-ordinates digestion by inhibition of gastric emptying and release of pancreatic enzymes and bile. Following ingestion of fat, CCK is secreted by a subset of entero-endocrine cells (I-cells) that are localized in small intestine. Isolation of native I-cells will help us to understand how signals from nutrients are transmitted in the cells to stimulate CCK release. The aim of our study was to isolate native I-cells and investigate the expression of G protein coupled receptors (GPCRs) that may be crucial for nutrient sensing. We used the(Cck-EGFP)BJ203Gsat transgenic mouse model in which enhanced Green Fluorescence Protein(eGFP) is expressed under the control of the CCK gene promoter. Cryosections of duodenum were immunostained with an anti-mouse proCCK antibody. Duodenal epithelial cells were dissociated (from 4 adult mice per experiment), using a mechanical/chemical method and analyzed by fluorescence activated cell sorting (FACS). Living cells were sorted into two populations, one eGFP-positive (eGFP+) and one eGFP-negative (eGFP-). RNA was isolated from sorted cells and its integrity was analyzed on a PicoChip Bioanalyzer. After DNAse treatment, RNA was subjected to semi-quantitative RT-PCR. Duodenal eGFP cells are flask-shaped with a narrow apical and a broad basolateral membrane, typical of entero-endocrine cells.90% of eGFP-tagged cells were co-stained with an anti-CCK antibody, confirming that eGFP cells express CCK. CCK staining was more intense at basolateral region, where secretory vesicles are localized. Dissociated duodenal cells were subjected to FACS. 15000-25000 eGFP+ cells (0.3-0.7 % of total population) were isolated in each experiment. Fluorescence microscopy confirmed that the purity of eGFP+ sorted cells was higher than 90%.Semi-quantitative RT-PCR revealed that CCK mRNA was exclusively expressed in eGFP+ cells. Moreover, RT-PCR of several transcripts that are specifically expressed in goblet and epithelial cells confirmed that we obtained a pure I-cells population with minimum contamination. Then, we examined if I-cells express long chain fatty acid(LCFA) receptors GPR40 and GPR120, finding that GPR40 and GPR120 mRNA transcripts are enriched in I-cells. We also investigated if the oleoylethanolamide receptor GPR119 is present in I cells and found that GPR119 is enriched in I cells. These experiments were repeated 3 times with similar results. We report that I-cells contain mRNA transcripts of GPR40,GPR119 and GPR120.Presence of GPR40 and GPR120 in I cells indicate that they may act as fat sensors to promote CCK release.GPR119 presence in I cells is surprising and may indicate that I cells are targets of endocannabinoid system.
Where applicable, experiments conform with Society ethical requirements