AbstractEnteroendocrine (EEC) cells have a pivotal role in intestinal nutrient sensing and release hormones that orchestrate food digestion and regulate appetite. EEC cells are found scattered throughout the intestine and have typically been classified based on the primary hormone they contain. I cells represent a subset of EEC cells that secrete cholecystokinin (CCK) and are mainly localized to the duodenum. Recent studies have shown that I cells express mRNAs encoding several gut hormones. In this study, we investigated the hormonal profile of murine fluorescence-activated cell sorting-sorted duodenal I cells using semiquantitative RT-PCR, liquid chromatography tandem mass spectrometry, and immunostaining methods. We report that I cells are enriched in mRNA transcripts encoding CCK and also other key gut hormones, including neurotensin, glucose-dependent insulinotropic peptide (GIP), secretin, peptide YY, proglucagon, and ghrelin (Ghrl). Furthermore, liquid chromatography tandem mass spectrometry analysis of fluorescence-activated cell sorting-purified I cells and immunostaining confirmed the presence of these gut hormones in duodenal I cells. Immunostaining highlighted that subsets of I cells in both crypts and villi coexpress differential amounts of CCK, Ghrl, GIP, or peptide YY, indicating that a proportion of I cells contain several hormones during maturation and when fully differentiated. Our results reveal that although I cells express several key gut hormones, including GIP or proglucagon, and thus have a considerable overlap with classically defined K and L cells, approximately half express Ghrl, suggesting a potentially important subset of duodenal EEC cells that require further consideration.