Enteroendocrine cells (EECs) are specialized sensory cells widely distributed throughout the villi and crypts of the intestinal epithelium. EECs secrete the majority of all gut hormones in response to nutrient and microbial cues which regulate various systemic processes, including glucose regulation and satiety. With new model systems, like organoids, novel roles for EECs in regulating their local environment are beginning to emerge, particularly in the intestinal epithelium. We recently identified EECs as important regulators of intestinal epithelial barrier integrity and permeability via unknown mechanisms. Here, we used human intestinal enteroids to test the hypothesis that EECs regulate the stability of junctional proteins embedded in sphingolipid-rich lipid membrane microdomains. We compared control human intestinal enteroids with those harboring a genetic loss of NEUROG3 (EEC-deficient). Loss of EECs altered gene expression of many enzymes involved in sphingolipid metabolism alongside significant increases in several ceramide species, but only in crypt-like enteroids. Manipulation of ceramide levels by knockdown of key enzymes or exogenous addition of long-chain and very long-chain ceramides directly impacted barrier permeability. Treatment of EEC-deficient enteroids with exogenous PYY or octreotide (OCT, a synthetic analog of somatostatin) restored gene expression and ceramide levels to wild-type, visibly tightened the apical-lateral junctions between cells by TEM, restored mRNA levels of junctional proteins, and improved their subcellular localization. PYY and OCT activated the PI3K/AKT and mTOR signaling pathways, which are upstream regulators of sphingolipid metabolism and abundance. These findings in human enteroids correlate with increased barrier permeability in EEC-deficient mice. Our data support a novel mechanism by which EECs regulate intestinal barrier permeability. To our knowledge, this is the first report connecting nutrient-sensitive EECs with structural and bioactive sphingolipids. Funding sources: This project was supported by the NIH, 1K01 DK125341 (HAM) and R01 DK132079 (AJS). The Microscopy Services Laboratory, Department of Pathology and Laboratory Medicine, is supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center. The Advanced Analytics Core is supported in part by P30 DK034987 to the Center for Gastrointestinal Biology and Disease at UNC. The Analytical Chemistry Shared Resource Core in the University of Arizona Cancer Center is supported in part by P30 CA023074 and S10 OD032134 (JMS). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
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