Endocrine Abstracts

The I-cell is one of the critical human enteroendocrine cells essential in modulating digestion and appetite by releasing cholecystokinin (CCK). The transcriptome analysis of mouse and human I cells reveals the expression of several G-protein coupled receptors that can be activated in the presence of different free fatty acids and based on mouse enteroendocrine cell lines and preliminary data in our group from human duodenum-derived organoids, different free fatty acid receptors stimulate CCK secretion through intracellular calcium rise. However, little is known about whether the direct effect of fatty acids in human I-cells can stimulate CCK release in short-term effects. Therefore, this study aims to evaluate the acute CCK release dynamics in response to different fatty acids in human organoid-derived I-cells through the use of a biosensor assay employing CCK1R transfected HEK293 cells loaded with Fura2 to enable Ca2+-elevation monitoring as a proxy in response to CCK released from nearby cells. For this study, the plasmid expressing CCK1R and a red fluorescent protein ‘mCherry’ in a bicistronic message under a eukaryotic promoter was transfected transiently into HEK-293 cells using transit-293. Live-cell calcium imaging experiments were performed using the ratiometric calcium indicator Fura-2. We confirmed the responsiveness of the biosensor cells by perfusing them with CCK (100 nM), whereas they were unresponsive to KCl (70 mM), which will be used as a control stimulant for CCK release from I-cells, as we already know that this triggers Ca2+-elevation in I-cells and stimulates secretion assessed over longer time frames (2h) by an LC-MS/MS assay. Future work will now use the new biosensor to monitor CCK release from Venus-labeled I cells in co-culture, and different stimuli will be tested in co-cultured and isolated biosensor cells. This study deepens our understanding of the physiological processes of I-cells and holds promise for drug development.