Characterizing zebrafish enteroendocrine cells.

Micro-RNAs (miRNAs) are crucial for many biological processes, but their role in the enteroendocrine development and differentiation has been neglected due to the elusive nature of the enteroendocrine cells. However, transgenic mice expressing fluorescent reporter proteins under the control of promoters for Cck, Gpr41, and Lgr5, ie, two different enteroendocrine markers and a marker for the stem cells, now enables identification and FACS purification of enteroendocrine cells at different stages of their differentiation along the crypt-villus axis. Surprisingly few of the 746 analyzed miRNAs differed in their expression pattern between enteroendocrine and nonenteroendocrine cells of the gut mucosa and between enteroendocrine cells of the crypt versus the villus. Thus, only let-7g-3p, miR-7b-5p (miR-7b), and miR-375-3p (miR-375) were up-regulated in the enteroendocrine cells of both the crypt and villus compared with nonenteroendocrine cells, and in situ hybridization confirmed colocalization of miR-375 with the enteroendocrine cells. Finally, functional assays using miR-375 inhibitor and mimetic in organoid cultures revealed miR-375 as a potential regulator of the enteroendocrine lineage. Overexpression of miR-375 inhibited enteroendocrine lineage development, whereas inhibition of miR-375 stimulated the development of enteroendocrine cells in vitro. Thus, through an unbiased expression screening of all miRNA, we find very few miRNAs that are differentially expressed in the gastrointestinal mucosa. Of these, miR-375 is found to be both highly expressed and enriched in the enteroendocrine cells. Additionally, miR-375 appears to negatively regulate the development of enteroendocrine cells. Consequently, miR-375 emerges as a potential target to modulate the function of the enteroendocrine system.

The intestinal epithelium consists of specialized cell populations essential for the uptake of vital nutrients from food and the secretion of mucous, hormones, and antimicrobial factors. A small population of hormone-producing enteroendocrine cells (EECs), which comprise only ~1% of the intestinal epithelium, are crucial for maintaining endocrine homeostasis. There are different types of EECs: serotonin-producing enterochromaffin cells that regulate gastrointestinal motility, insulin release, and inflammation, and non-enterochromaffin cells, which produce various other hormones that regulate glucose metabolism, appetite, and satiety. Dysregulated EEC function contributes to several metabolic and gastrointestinal diseases, including obesity, type 2 diabetes, and inflammatory bowel disease. Moreover, while EECs are present throughout the stomach and small and large intestines, the types and functions of EECs differ depending on their location along the digestive tract. However, there is still a limited understanding of how the differentiation, regional distribution, and function of these cells are controlled. A recent study focusing on EEC differentiation identified selective expression of the transcription factor PRDM16 along the enteroendocrine cell differentiation trajectory. PRDM16 is a zinc finger-containing transcription factor highly expressed in the proximal small intestine. Preliminary data from our lab show that Prdm16 deletion, specifically within the EEC lineage, strongly decreased the expression of many enterochromaffin cell marker genes. PRDM16 is a known regulator of fatty acid oxidation and mitochondrial biogenesis in several cell types, including the intestinal epithelium. EECs are essential for sensing changes in nutrients and respond by secreting hormones that allow for adaptation to different dietary changes. For example, EEC composition and function can change in response to increased fat absorption during a high-fat diet. Given PRDM16’s regional expression in the small intestine and its known role regulator as a of fatty acid oxidation, this gene could mediate the pathways involved in fat metabolism in the EEC lineage and regulate the differentiation trajectory of EECs in a region-specific manor. Therefore, I will investigate the role of PRDM16 during the development of the EEC lineage and determine how PRDM16 mediates the response of mature EECs to changes in dietary nutrients. A T32 HD083185 Award to NW, a K01 from NIDDK to RS, a Pilot and Feasibility Grant to RS from the CMSDLD, and a Pilot and Feasibility Award to RS from the PennCHOP Microbiome Center. This abstract was presented at the American Physiology Summit 2025 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.

The intestinal enteroendocrine cells (EECs) are specialized hormone-secreting cells that respond to both circulating and luminal cues. Collectively, EECs constitute the largest endocrine organ of the body and signal to a multitude of targets including locally to neighboring intestinal cells, enteric neurons, as well as systemically to other organs, such as the pancreas and brain. To accomplish their wide range of downstream signaling effects, EECs secrete multiple hormones; however, the mechanisms that influence EEC development in the embryo and differentiation in adults are not well defined. This review highlights the recent discoveries in EEC differentiation and function while also discussing newly revealed roles of transcription factors and signaling networks involved in the allocation of EEC subtypes that were discovered using a combination of novel intestinal model systems and genetic sequencing. We also discuss the potential of these new experimental models that study the mechanisms regulating EEC function and development both to uncover novel therapeutic targets. Several EEC hormones are being used to treat various metabolic disorders, such as type 2 diabetes and obesity. Therefore, understanding the signaling pathways and gene regulatory networks that facilitate EEC formation is paramount to the development of novel therapies.

In the gastrointestinal (GI) tract, food is digested and absorbed while GI hormones are secreted from the enteroendocrine cells (EECs). These hormones regulate food intake, glucose homeostasis, digestion, GI motility, and metabolism. Although EECs may express more than a single hormone, the EECs usually secrete only one or a few hormones. The pattern of EEC secretion varies along the length of the GI tract as the different EEC types are scattered in different densities along the GI tract. Following bariatric surgery, a postprandial hypersecretion of certain GI hormones occurs which contributes to the postsurgery weight loss. Mimicking this postprandial hypersecretion of GI hormones by targeting endogenous EEC secretion, using specific modulators of receptors, ion channels, and transporters found on specific EECs, to induce weight loss is a current research aim. To achieve this, a more complete understanding of the release mechanisms, expression of receptors, transporters, and the secretion pattern of the different EEC types is needed. Using the vascularly perfused intestinal model, it is possible to obtain a detailed knowledge of these release mechanisms by evaluating the effects on secretion of blocking or stimulating specific receptors, ion channels, and transporters as well as evaluating nutrient handling and absorption in each of the different sections of the intestine. This mini-review will focus on how the isolated perfused intestine has been used in our group as a model to investigate the nutrient-induced release mechanisms of EECs with a focus on glucagon-like peptide-1 secreting cells.

Gut Microbiota Remodeling and Intestinal Adaptation to Lipid Malabsorption After Enteroendocrine Cell Loss in Adult Mice

Caudo-rostral migration of pathological forms of α-synuclein from the gut to the brain is proposed as an early feature in Parkinson’s disease pathogenesis, but the underlying mechanisms remain unknown. Intestinal epithelial enteroendocrine cells sense and respond to numerous luminal signals, including bacterial factors, and transmit this information to the brain via the enteric nervous system and vagus nerve. There is evidence that gut bacteria composition and their metabolites change in Parkinson’s disease patients, and these alterations can trigger α-synuclein pathology in animal models of the disorder. Here, we investigated the effect of toll-like receptor and free fatty acid receptor agonists on the intracellular level of α-synuclein and its release using mouse secretin tumour cell line 1 enteroendocrine cells. Secretin tumour cell line 1 enteroendocrine cells were treated for 24 or 48 h with toll-like receptor agonists (toll-like receptor 4 selective lipopolysaccharide; toll-like receptor 2 selective Pam3CysSerLys4) and the free fatty acid receptor 2/3 agonists butyrate, propionate and acetate. The effect of selective receptor antagonists on the agonists’ effects after 24 hours was also investigated. The level of α-synuclein protein was measured in cell lysates and cell culture media by western blot and enzyme-linked immunosorbent assay. The level of α-synuclein and tumour necrosis factor messenger RNA was measured by quantitative reverse transcription real-time polymerase chain reaction. Stimulation of secretin tumour cell line 1 enteroendocrine cells for 24 and 48 hours with toll-like receptor and free fatty acid receptor agonists significantly increased the amount of intracellular α-synuclein and the release of α-synuclein from the cells into the culture medium. Both effects were significantly reduced by antagonists selective for each receptor. Toll-like receptor and free fatty acid receptor agonists also significantly increased tumour necrosis factor transcription, and this was effectively inhibited by corresponding antagonists. Elevated intracellular α-synuclein increases the likelihood of aggregation and conversion to toxic forms. Factors derived from bacteria induce α-synuclein accumulation in secretin tumour cell line 1 enteroendocrine cells. Here, we provide support for a mechanism by which exposure of enteroendocrine cells to specific bacterial factors found in Parkinson’s disease gut dysbiosis might facilitate accumulation of α-synuclein pathology in the gut.

Enteroendocrine cells (EECs) are the principal nutrient sensors in the gastrointestinal (GI) tract and release hormones such as glucagon-like-peptide 1 (GLP-1) that modulate GI function and appetite. While some of the molecules involved in nutrient sensing within EECs have been described, there have been no systematic studies to map the relevant genes and pathways. Here, we developed a strategy to perform a high-throughput screen for genes that are required for nutrient-induced activation of EECs, and we applied this to probe mechanisms for sensing dietary protein. We found that all of the genes previously proposed to function as protein sensors in EECs are, collectively, dispensable for protein sensing in an EEC cell line. Instead, a screen of >20,000 sgRNAs identified numerous genes associated with mitochondrial respiration as being necessary for this process. We showed through secondary assays that impairing oxidative phosphorylation (OXPHOS) reduced EEC activation and GLP-1 release in response to nutrients but not in response to a non-nutritive stimulus. On the other hand, boosting OXPHOS increased EEC activation and GLP-1 release. These data reveal that intracellular metabolism within EECs controls the detection of dietary protein, possibly by monitoring the entry of ingested amino acids into the TCA cycle. More broadly, these findings suggest a general strategy to screen for genes and pathways that might be used to boost the nutrient-regulated release of gut peptides such as GLP-1.

Cell lineage markers in premalignant and malignant colonic mucosa

Sucrase-isomaltase (SI), a gene expressed exclusively in absorptive enterocytes, was used to examine the molecular mechanisms that regulate cell-specific gene expression in the intestinal epithelium. Transgenic mice were made with a construct containing nucleotides -8,500 to +54 of the mouse SI gene linked to a human growth hormone reporter gene. In adult transgenic animals, high-level transgene expression was limited to the small intestine, with low levels of ectopic expression in the colon. In contrast to the endogenous gene that is expressed only in enterocytes, the transgene was expressed in all four cell lineages, including enterocytes, enteroendocrine, goblet, and Paneth cells. To examine this process of lineage-specific expression further we studied Caco-2 and COLO DM cell lines, which model enterocytes and enteroendocrine cells, respectively. Reminiscent of results in transgenic animals, only Caco-2 cells transcribed the endogenous SI gene, whereas both Caco-2 and COLO DM cells supported transcription from chimeric SI reporter gene constructs. Taken together, these data suggest that each intestinal cell lineage has the cellular machinery to transcribe the SI gene. Moreover, these findings imply that transcription is normally repressed in nonenterocytic cells, possibly via a transcriptional silencer residing outside of the region of the SI gene examined in these studies.

DIARRHEA IN EARLY LIFE: PROGRESS IN DIAGNOSIS AND CONTROL OF DISEASE

The enteric nervous and enteroendocrine systems regulate different processes in the small intestine. Ablation of myenteric plexus with benzalkonium chloride (BAC) stimulates epithelial cell proliferation, whereas endocrine serotonin cells may inhibit the process. To evaluate the connection between the systems and the influence of myenteric plexus on serotoninergic cells in rats during postnatal development, the ileal plexus was partially removed with BAC. Rats were treated at 13 or 21 days and sacrificed after 15 days. The cell bodies of myenteric neurons were stained by beta NADH-diaphorase to detect the extension of denervation. The number of enteroendocrine cells in the ileum was estimated in crypts and villi in paraffin sections immunostained for serotonin. The number of neurons was reduced by 27.6 and 45% in rats treated on the 13th and 21st days, respectively. We tried to establish a correlation of denervation and the serotonin population according to the age of treatment. We observed a reduction of immunolabelled cells in the crypts of rats treated at 13 days, whereas this effect was seen in the villi of rats denervated at 21 days. These results suggest that the enteric nervous system might control the enteroendocrine cell population and this complex mechanism could be correlated to changes in cell proliferation.

Enteroendocrine Cells and Appetite Dysregulation in Crohn's Disease

BackgroundPrevious reports have suggested that the VEGF receptor neuropilin-1 (NRP-1) is expressed in a singly dispersed subpopulation of cells in the normal colonic epithelium, but that expression becomes dysregulated during colorectal carcinogenesis, with higher levels in tumour suggestive of a poor prognosis. We noted that the spatial distribution and morphology if NRP-1 expressing cells resembles that of enteroendocrine cells (EEC) which are altered in response to disease state including cancer and irritable bowel syndrome (IBS). We have shown that NRP-1 is down-regulated by butyrate in colon cancer cell lines in vitro and we hypothesized that butyrate produced in the lumen would have an analogous effect on the colon mucosa in vivo. Therefore we sought to investigate whether NRP-1 is expressed in EEC and how NRP-1 and EEC respond to butyrate and other short-chain fatty acids (SCFA - principally acetate and propionate). Additionally we sought to assess whether there is a field effect around adenomas.MethodologyBiopsies were collected at the mid-sigmoid, at the adenoma and at the contralateral wall (field) of 28 subjects during endoscopy. Samples were fixed for IHC and stained for either NRP-1 or for chromogranin A (CgA), a marker of EEC. Stool sampling was undertaken to assess individuals' butyrate, acetate and propionate levels.ResultNRP-1 expression was inversely related to SCFA concentration at the colon landmark (mid-sigmoid), but expression was lower and not related to SCFA concentration at the field. Likewise CgA+ cell number was also inversely related to SCFA at the landmark, but was lower and unresponsive at the field. Crypt cellularity was unaltered by field effect. A colocalisation analysis showed only a small subset of NRP-1 localised with CgA. Adenomas showed extensive, weaker staining for NRP-1 which contrastingly correlated positively with butyrate level. Field effects cause this relationship to be lost. Adenoma tissue shows dissociation of the co-regulation of NRP-1 and EEC.ConclusionNRP-1 is inversely associated with levels of butyrate and other SCFA in vivo and is expressed in a subset of CgA expressing cells. EEC number is related to butyrate level in the same way.

The enteroendocrine cell (EEC) lineage is important for intestinal homeostasis and systemic energy balance. Notably, progenitors of EECs exhibit stem cell potential and contribute to intestinal epithelial proliferation and renewal; however, the molecular mechanisms are not well understood. In this study, we first report that miR-7 and miR-375 (previously thought to be specific to pancreatic beta cells) are very highly expressed along the EEC lineage trajectory and are among the most enriched miRNAs in Prox1+ EEC progenitors relative to Lgr5+ intestinal stem cells. Next, by studying intestinal stem/progenitor cell responses to microbiota, high-fat diet, and bariatric surgery, we show that miR-7 and miR-375 expression levels are inversely correlated with intestinal epithelial proliferative status. We then demonstrate by both loss- and gain-of function assays in ex vivo mouse enteroids that both miRNAs exert robust control of intestinal epithelial proliferation and enteroendocrine cell maturation. We also show by single cell RNA-sequencing, intestinal phenotyping, and metabolic phenotyping in a miR-375 knockout mouse model, that miR-375 controls intestinal stem/progenitor cell dynamics, especially with increasing age. Overall, this study demonstrates for the first time that two miRNAs highly enriched in the EEC lineage are critical regulators of intestinal epithelial proliferation and likely mediators of the effects of high-fat diet on the intestinal crypt. Ongoing studies are focused on the role of these two miRNAs in the rescue of intestinal crypt behavior after bariatric surgery. Disclosure P. Sethupathy: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-16-ACE-47)

Gastric endocrine cell hormones contribute to the control of the stomach and to signalling to the brain. In other gut regions, enteroendocrine cells (EECs) exhibit extensive patterns of colocalisation of hormones. In the current study, we characterise EECs in the human gastric fundus and corpus. We utilise immunohistochemistry to investigate EECs with antibodies to ghrelin, serotonin (5-HT), somatostatin, peptide YY (PYY), glucagon-like peptide 1, calbindin, gastrin and pancreastatin, the latter as a marker of enterochromaffin-like (ECL) cells. EECs were mainly located in regions of the gastric glands populated by parietal cells. Gastrin cells were absent and PYY cells were very rare. Except for about 25% of 5-HT cells being a subpopulation of ECL cells marked by pancreastatin, colocalisation of hormones in gastric EECs was infrequent. Ghrelin cells were distributed throughout the fundus and corpus; most were basally located in the glands, often very close to parietal cells and were closed cells i.e., not in contact with the lumen. A small proportion had long processes located close to the base of the mucosal epithelium. The 5-HT cells were of at least three types: small, round, closed cells; cells with multiple, often very long, processes; and a subgroup of ECL cells. Processes were in contact with their surrounding cells, including parietal cells. Mast cells had very weak or no 5-HT immunoreactivity. Somatostatin cells were a closed type with long processes. In conclusion, four major chemically defined EEC types occurred in the human oxyntic mucosa. Within each group were cells with distinct morphologies and relationships to other mucosal cells.