Enteroendocrine cell differentiation and function in the intestine.

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

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.

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.

Intestinal organoids are used to analyze the differentiation of enteroendocrine cells (EECs) and to manipulate their density for treating type 2 diabetes. EEC differentiation is a continuous process tightly regulated in the gut by a complex regulatory network. However, the effect of chronic hyperglycemia, in the modulation of regulatory networks controlling identity and differentiation of EECs, has not been analyzed. This study aimed to investigate the effect of glucotoxicity on EEC differentiation in small intestinal organoid platforms. Mouse intestinal organoids were cultured in the presence/absence of high glucose concentrations (35 mM) for 48 h to mimic glucotoxicity. Chronic hyperglycemia impaired the expression of markers related to the differentiation of EEC progenitors (Ngn3) and L-cells (NeuroD1), and it also reduced the expression of Gcg and GLP-1 positive cell number. In addition, the expression of intestinal stem cell markers was reduced in organoids exposed to high glucose concentrations. Our data indicate that glucotoxicity impairs L-cell differentiation, which could be associated with decreased intestinal stem cell proliferative capacity. This study provides the identification of new targets involved in new molecular signaling mechanisms impaired by glucotoxicity that could be a useful tool for the treatment of type 2 diabetes.

Disorders of intestinal enteroendocrine cells (EEC) are a rare cause of congenital diarrhea and diabetes. The gene NEUROG3 is essential in EEC differentiation, and mutations in this gene lead to a paucity of EEC in the intestine and pancreas, often presenting clinically as congenital diarrhea and diabetes mellitus. We present the earliest known diagnosis of NEUROG3-associated enteric endocrinopathy, which was identified on a neonatal diabetes genetic panel sent at 4 weeks of age. Our patient presented with severe diarrhea, malnutrition, electrolyte derangements, and neonatal diabetes. He was started on parenteral nutrition at 3 months of age for nutritional and hydration support and required long-acting insulin for his diabetes. We demonstrate significant reduction in EEC, including cells expressing glucagon-like peptide-1, in intestinal biopsies from our patient, raising the possibility that loss of glucagon-like peptide-1 contributes to NEUROG3-associated diarrhea and diabetes mellitus. This case advances our understanding of the presentation, diagnosis, and management of this rare 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.

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)

Parkinson's disease (PD) is characterized by the abnormal aggregation of α-synuclein, which can originate in the gut and propagate to the brain. Recent evidence suggests a correlation between metabolic disorders, particularly diabetes, and PD pathogenesis through the gut-brain axis. Methylglyoxal (MGO), a glucose-derived metabolite produced by gut bacteria such as Proteus mirabilis, is implicated in protein misfolding and glycation. This study investigated whether MGO induced α-synuclein aggregation in intestinal enteroendocrine cells and explored the underlying mechanisms. Mouse enteroendocrine STC-1 cells were treated with MGO (0.01-1mM) for 36h, and changes in α-synuclein aggregation, neuronal markers, and relevant signaling pathways were assessed. MGO at 1mM significantly reduced cell viability and neuronal marker expression, and concentrations of 0.1 and 1mM increased α-synuclein aggregation. MGO also inhibited SIRT1 expression, leading to increased Hif-1α transcription and reduced expression of autophagy-related proteins Beclin1 and LC3B. These changes were accompanied by mitochondrial dysfunction, as evidenced by decreased Bcl2, increased cytochrome C expression, and reduced levels of the antioxidant factor HO-1. Our findings provide the first evidence that MGO directly induces α-synuclein aggregation in enteroendocrine cells via the SIRT1-Hif-1α-autophagy pathway dysregulation, establishing a potential mechanistic link between gut microbiome-derived metabolites and PD pathogenesis. These results suggest that intestinal glycation may be a critical target for preventing α-synuclein pathology originating in the gut.

Short Chain Fatty Acid Transporter/Receptor Expression and Signaling in Enteric Glial Cells

Fundamentals of Neurogastroenterology: Basic Science

Neurological and psychiatric patients have increased dramatically in number in the past few decades. However, effective treatments for these diseases and disorders are limited due to heterogeneous and unclear pathogenic mechanisms. Therefore, further exploration of the biological aspects of the disease, and the identification of novel targets to develop alternative treatment strategies, is urgently required. Systems-level investigations have indicated the potential involvement of the brain–gut axis and intestinal microbiota in the pathogenesis and regulation of neurological and psychiatric disorders. While intestinal microbiota is crucial for maintaining host physiology, some important sensory and regulatory cells in the host should not be overlooked. Intestinal epithelial enteroendocrine cells (EECs) residing in the epithelium throughout intestine are the key regulators orchestrating the communication along the brain-gut-microbiota axis. On one hand, EECs sense changes in luminal microorganisms via microbial metabolites; on the other hand, they communicate with host body systems via neuroendocrine molecules. Therefore, EECs are believed to play important roles in neurological and psychiatric disorders. This review highlights the involvement of EECs and subtype cells, via secretion of endocrine molecules, in the development and regulation of neurological and psychiatric disorders, including Parkinson’s disease (PD), schizophrenia, visceral pain, neuropathic pain, and depression. Moreover, the current paper summarizes the potential mechanism of EECs in contributing to disease pathogenesis. Examination of these mechanisms may inspire and lead to the development of new aspects of treatment strategies for neurological and psychiatric disorders in the future.

Methionine deficiency and its hydroxy analogue influence chicken intestinal 3-dimensional organoid development.

Enteroendocrine Cells and Appetite Dysregulation in Crohn's Disease

Diabetes, as a metabolic disorder, is accompanied with several gastrointestinal (GI) symptoms, like abdominal pain, gastroparesis, diarrhoea or constipation. Serious and complex enteric nervous system damage is confirmed in the background of these diabetic motility complaints. The anatomical length of the GI tract, as well as genetic, developmental, structural and functional differences between its segments contribute to the distinct, intestinal region-specific effects of hyperglycemia. These observations support and highlight the importance of a regional approach in diabetes-related enteric neuropathy. Intestinal large and microvessels are essential for the blood supply of enteric ganglia. Bidirectional morpho-functional linkage exists between enteric neurons and enteroglia, however, there is also a reciprocal communication between enteric neurons and immune cells on which intestinal microbial composition has crucial influence. From this point of view, it is more appropriate to say that enteric neurons partake in multidirectional communication and interact with these key players of the intestinal wall. These interplays may differ from segment to segment, thus, the microenvironment of enteric neurons could be considered strictly regional. The goal of this review is to summarize the main tissue components and molecular factors, such as enteric glia cells, interstitial cells of Cajal, gut vasculature, intestinal epithelium, gut microbiota, immune cells, enteroendocrine cells, pro-oxidants, antioxidant molecules and extracellular matrix, which create and determine a gut region-dependent neuronal environment in diabetes.

Enteroendocrine cells (EECs) lining the gastrointestinal (GI) wall sense chemical and mechanical changes in the gut, secreting an array of hormones and neurotransmitters in response. Enterochromaffin (EC) cells synthesize and secrete serotonin and represent approximately half of all EECs. EEC signals are transmitted to the brain via activation of afferent nerve fibers terminating in the GI tract. EECs grow neurite-like processes termed by some as "neuropods." These are proposed to contact intrinsic and extrinsic afferent nerves innervating the gut wall as the site of formation of a neuroepithelial circuit that signals from gut to both enteric and central nervous systems. This study focused on investigating, along the length of the mouse GI tract, the spatial relationship between EC cells and sensory nerve fibers, all of which contain the neuropeptide CGRP. This was analyzed using confocal laser scanning microscopy and Imaris 3-dimensional image analysis. Our aim was to determine the potential relevance of cell processes as the sites of neurotransmission on EC cells. We found the number of contacts between EC cells and CGRP fibers increases along the GI tract, with maximal contacts occurring in the distal colon. These contacts localized primarily on the cell body, and not on processes. Further, a high degree of variability in EC cell architecture, including the frequency and size of processes was seen. These findings do not support the notion that EEC processes are a primary cell location at which a neuroepithelial circuit is formed but do indicate that the likelihood of such contact points occurring increases in more distal areas of the GI tract.