High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells.

Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.

Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping.

Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation.

Microbiota-derived acetate activates intestinal innate immunity via the Tip60 histone acetyltransferase complex.

Microbe-derived acetate activates the Drosophila immunodeficiency (IMD) pathway in a subset of enteroendocrine cells (EECs) of the anterior midgut. In these cells, the IMD pathway co-regulates expression of antimicrobial and enteroendocrine peptides including tachykinin, a repressor of intestinal lipid synthesis. To determine whether acetate acts on a cell surface pattern recognition receptor or an intracellular target, we asked whether acetate import was essential for IMD signaling. Mutagenesis and RNA interference revealed that the putative monocarboxylic acid transporter Tarag was essential for enhancement of IMD signaling by dietary acetate. Interference with histone deacetylation in EECs augmented transcription of genes regulated by the steroid hormone ecdysone including IMD targets. Reduced expression of the histone acetyltransferase Tip60 decreased IMD signaling and blocked rescue by dietary acetate and other sources of intracellular acetyl-CoA. Thus, microbe-derived acetate induces chromatin remodeling within enteroendocrine cells, co-regulating host metabolism and intestinal innate immunity via a Tip60-steroid hormone axis that is conserved in mammals.

The people behind the papers - Alfahdah Alsudayri and Lihua Ye.

Enteroendocrine cells (EECs) are specialised cells in the intestinal epithelium that sense nutrients to regulate feeding behaviour. In a new study, Lihua Ye and colleagues demonstrate that the gut microbiota are crucial in supporting EEC maturation and mitochondrial function during early postnatal development in zebrafish. To find out more about the behind the paper story, we caught up with first author Alfahdah Alsudayri and corresponding author Lihua Ye, Assistant Professor at Ohio State University.

Gut microbiota regulate maturation and mitochondrial function of the nutrient-sensing enteroendocrine cell.

Enteroendocrine cells (EECs) are crucial for sensing ingested nutrients and regulating feeding behavior. How gut microbiota regulate the nutrient-sensing EEC activity is unclear. Our transcriptomic analysis demonstrates that commensal microbiota colonization significantly increases the expression of many genes associated with mitochondrial function. Using new methods to image EEC cytoplasmic and mitochondrial Ca2+ activity in live zebrafish, our data revealed that it is dynamically regulated during the EEC development process. Mature EECs display an increased mitochondrial-to-cytoplasmic Ca2+ ratio. Mitochondria are evenly distributed in the cytoplasm of immature EECs. As EECs mature, their mitochondria are highly localized at the basal membrane where EEC vesicle secretion occurs. Conventionalized (CV) EECs, but not germ-free (GF) EECs, exhibit spontaneous low-amplitude Ca2+ fluctuation. The mitochondrial-to-cytoplasmic Ca2+ ratio is significantly higher in CV EECs. Nutrient stimulants, such as fatty acid, increase cytoplasmic Ca2+ in a subset of EECs and promote a sustained mitochondrial Ca2+ and ATP increase. However, the nutrient-induced EEC mitochondrial activation is nearly abolished in GF zebrafish. Together, our study reveals that commensal microbiota are crucial in supporting EEC mitochondrial function and maturation.

Reciprocal interactions between neuropeptide F and RYamide regulate host attraction in the mosquito Aedes aegypti.

Female mosquitoes produce eggs in gonadotrophic cycles that are divided between a previtellogenic and vitellogenic phase. Previtellogenic females consume water and sugar sources like nectar while also being attracted to hosts for blood feeding. Consumption of a blood meal activates the vitellogenic phase, which produces mature eggs and suppresses host attraction. In this study, we tested the hypothesis that neuropeptide Y-like hormones differentially modulate host attraction behavior in the mosquito Aedes aegypti. A series of experiments collectively indicated that enteroendocrine cells (EECs) in the posterior midgut produce and release neuropeptide F (NPF) into the hemolymph during the previtellogenic phase which stimulates attraction to humans and biting behavior. Consumption of a blood meal, which primarily consists of protein by dry weight, down-regulated NPF in EECs until mature eggs developed, which was associated with a decline in hemolymph titer. NPF depletion depended on protein digestion but was not associated with EEC loss. Other experiments showed that neurons in the terminal ganglion extend axons to the posterior midgut and produce RYamide, which showed evidence of increased secretion into circulation after a blood meal. Injection of RYamide-1 and -2 into previtellogenic females suppressed host attraction, while coinjection of RYamides with or without short NPF-2 also inhibited the host attraction activity of NPF. Overall, our results identify NPF and RYamide as gut-associated hormones in A. aegypti that link host attraction behavior to shifts in diet during sequential gonadotrophic cycles.

The olfactory receptor Olfr78 promotes differentiation of enterochromaffin cells in the mouse colon.

The gastrointestinal epithelium constitutes a chemosensory system for microbiota-derived metabolites such as short-chain fatty acids (SCFA). Here, we investigate the spatial distribution of Olfr78, one of the SCFA receptors, in the mouse intestine and study the transcriptome of colon enteroendocrine cells expressing Olfr78. The receptor is predominantly detected in the enterochromaffin and L subtypes in the proximal and distal colon, respectively. Using the Olfr78-GFP and VilCre/Olfr78flox transgenic mouse lines, we show that loss of epithelial Olfr78 results in impaired enterochromaffin cell differentiation, blocking cells in an undefined secretory lineage state. This is accompanied by a reduced defense response to bacteria in colon crypts and slight dysbiosis. Using organoid cultures, we further show that maintenance of enterochromaffin cells involves activation of the Olfr78 receptor via the SCFA ligand acetate. Taken together, our work provides evidence that Olfr78 contributes to colon homeostasis by promoting enterochromaffin cell differentiation.

Mapping prohormone processing by proteases in human enteroendocrine cells using genetically engineered organoid models.

Enteroendocrine cells (EECs) secrete hormones in response to ingested nutrients to control physiological processes such as appetite and insulin release. EEC hormones are synthesized as large proproteins that undergo proteolytic processing to generate bioactive peptides. Mutations in EEC-enriched proteases are associated with endocrinopathies. Due to the relative rarity of EECs and a paucity of in vitro models, intestinal prohormone processing remains challenging to assess. Here, human gut organoids in which EECs can efficiently be induced are subjected to CRISPR-Cas9-mediated modification of EEC-expressed endopeptidase and exopeptidase genes. We employ mass spectrometry-based analyses to monitor peptide processing and identify glucagon production in intestinal EECs, stimulated upon bone morphogenic protein (BMP) signaling. We map the substrates and products of major EECs endo- and exopeptidases. Our studies provide a comprehensive description of peptide hormones produced by human EECs and define the roles of specific proteases in their generation.

Alliances of the gut and bone axis.

Gut hormones secreted from enteroendocrine cells following nutrient ingestion modulate metabolic processes including glucose homeostasis and food intake, and several of these gut hormones are involved in the regulation of the energy demanding process of bone remodelling. Here, we review the gut hormones considered or known to be involved in the gut-bone crosstalk and their role in orchestrating adaptions of bone formation and resorption as demonstrated in cellular and physiological experiments and clinical trials. Understanding the physiology and pathophysiology of the gut-bone axis may identify adverse effects of investigational drugs aimed to treat metabolic diseases such as type 2 diabetes and obesity and new therapeutic candidates for the treatment of bone diseases.

Independently paced Ca2+ oscillations in progenitor and differentiated cells in an ex vivo epithelial organ.

Cytosolic Ca2+ is a highly dynamic, tightly regulated and broadly conserved cellular signal. Ca2+ dynamics have been studied widely in cellular monocultures, yet organs in vivo comprise heterogeneous populations of stem and differentiated cells. Here, we examine Ca2+ dynamics in the adult Drosophila intestine, a self-renewing epithelial organ in which stem cells continuously produce daughters that differentiate into either enteroendocrine cells or enterocytes. Live imaging of whole organs ex vivo reveals that stem-cell daughters adopt strikingly distinct patterns of Ca2+ oscillations after differentiation: enteroendocrine cells exhibit single-cell Ca2+ oscillations, whereas enterocytes exhibit rhythmic, long-range Ca2+ waves. These multicellular waves do not propagate through immature progenitors (stem cells and enteroblasts), of which the oscillation frequency is approximately half that of enteroendocrine cells. Organ-scale inhibition of gap junctions eliminates Ca2+ oscillations in all cell types - even, intriguingly, in progenitor and enteroendocrine cells that are surrounded only by enterocytes. Our findings establish that cells adopt fate-specific modes of Ca2+ dynamics as they terminally differentiate and reveal that the oscillatory dynamics of different cell types in a single, coherent epithelium are paced independently.

Mechanical regulation of stem-cell differentiation by the stretch-activated Piezo channel.

Somatic stem cells constantly adjust their self-renewal and lineage commitment by integrating various environmental cues to maintain tissue homeostasis. Although numerous chemical and biological signals have been identified that regulate stem-cell behaviour, whether stem cells can directly sense mechanical signals in vivo remains unclear. Here we show that mechanical stress regulates stem-cell differentiation in the adult Drosophila midgut through the stretch-activated ion channel Piezo. We find that Piezo is specifically expressed in previously unidentified enteroendocrine precursor cells, which have reduced proliferation ability and are destined to become enteroendocrine cells. Loss of Piezo activity reduces the generation of enteroendocrine cells in the adult midgut. In addition, ectopic expression of Piezo in all stem cells triggers both cell proliferation and enteroendocrine cell differentiation. Both the Piezo mutant and overexpression phenotypes can be rescued by manipulation of cytosolic Ca2+ levels, and increases in cytosolic Ca2+ resemble the Piezo overexpression phenotype, suggesting that Piezo functions through Ca2+ signalling. Further studies suggest that Ca2+ signalling promotes stem-cell proliferation and differentiation through separate pathways. Finally, Piezo is required for both mechanical activation of stem cells in a gut expansion assay and the increase of cytosolic Ca2+ in response to direct mechanical stimulus in a gut compression assay. Thus, our study demonstrates the existence of a specific group of stem cells in the fly midgut that can directly sense mechanical signals through Piezo.

Intestinal enteroendocrine cells rely on ryanodine and IP3 calcium store receptors for mechanotransduction.

Enteroendocrine cells (EECs) are specialized sensors of luminal forces and chemicals in the gastrointestinal (GI) epithelium that respond to stimulation with a release of signalling molecules such as serotonin (5-HT). For mechanosensitive EECs, force activates Piezo2 channels, which generate a very rapidly activating and inactivating (∼10 ms) cationic (Na+ , K+ , Ca2+ ) receptor current. Piezo2 receptor currents lead to a large and persistent increase in intracellular calcium (Ca2+ ) that lasts many seconds to sometimes minutes, suggesting signal amplification. However, intracellular calcium dynamics in EEC mechanotransduction remain poorly understood. The aim of this study was to determine the role of Ca2+ stores in EEC mechanotransduction. Mechanical stimulation of a human EEC cell model (QGP-1) resulted in a rapid increase in cytoplasmic Ca2+ and a slower decrease in ER stores Ca2+ , suggesting the involvement of intracellular Ca2+ stores. Comparing murine primary colonic EECs with colonocytes showed expression of intercellular Ca2+ store receptors, a similar expression of IP3 receptors, but a >30-fold enriched expression of Ryr3 in EECs. In mechanically stimulated primary EECs, Ca2+ responses decreased dramatically by emptying stores and pharmacologically blocking IP3 and RyR1/3 receptors. RyR3 genetic knockdown by siRNA led to a significant decrease in mechanosensitive Ca2+ responses and 5-HT release. In tissue, pressure-induced increase in the Ussing short circuit current was significantly decreased by ryanodine receptor blockade. Our data show that mechanosensitive EECs use intracellular Ca2+ stores to amplify mechanically induced Ca2+ entry, with RyR3 receptors selectively expressed in EECs and involved in Ca2+ signalling, 5-HT release and epithelial secretion. KEY POINTS: A population of enteroendocrine cells (EECs) are specialized mechanosensors of the gastrointestinal (GI) epithelium that respond to mechanical stimulation with the release of important signalling molecules such as serotonin. Mechanical activation of these EECs leads to an increase in intracellular calcium (Ca2+ ) with a longer duration than the stimulus, suggesting intracellular Ca2+ signal amplification. In this study, we profiled the expression of intracellular Ca2+ store receptors and found an enriched expression of the intracellular Ca2+ receptor Ryr3, which contributed to the mechanically evoked increases in intracellular calcium, 5-HT release and epithelial secretion. Our data suggest that mechanosensitive EECs rely on intracellular Ca2+ stores and are selective in their use of Ryr3 for amplification of intracellular Ca2+ . This work advances our understanding of EEC mechanotransduction and may provide novel diagnostic and therapeutic targets for GI motility disorders.

The bHLH transcription factor ASCL1 promotes differentiation of endocrine cells in the stomach and is regulated by Notch signaling.

Notch signaling regulates gastrointestinal stem cell proliferation and differentiation yet Notch-regulated transcriptional effectors of gastric epithelial cell differentiation are poorly understood. Here we tested the role of the bHLH transcription factor Achaete-Scute homolog 1 (ASCL1) in gastric epithelial cell differentiation, and its regulation by Notch. Newborn Ascl1 null mice showed a loss of expression of markers of neurogenin-3-dependent enteroendocrine cells, with normal expression of enterochromaffin-like cells, mucous cells, chief cells, and parietal cells. In adult mice, Ascl1 gene expression was observed in the stomach, but not the intestine, with higher expression in antral than corpus epithelium. Lineage tracing in Ascl1-CreERT2; Rosa26-LSL-tdTomato mice revealed single, scattered ASCL1+ cells in the gastric epithelium, demonstrating expression in antral gastrin- and serotonin-producing endocrine cells. ASCL1-expressing endocrine cells persisted for several weeks posttamoxifen labeling with a half-life of approximately 2 months. Lineage tracing in Gastrin-CreERT2 mice demonstrated a similar lifespan for gastrin-producing cells, confirming that gastric endocrine cells are long-lived. Finally, treatment of Ascl1-CreERT2; Rosa26-LSL-tdTomato mice with the pan-Notch inhibitor dibenzazepine increased the number of lineage-labeled cells in the gastric antrum, suggesting that Notch signaling normally inhibits Ascl1 expression. Notch regulation of Ascl1 was also demonstrated in a genetic mouse model of Notch activation, as well as Notch-manipulated antral organoid cultures, thus suggesting that ASCL1 is a key downstream Notch pathway effector promoting endocrine cell differentiation in the gastric epithelium.NEW & NOTEWORTHY Although Notch signaling is known to regulate cellular differentiation in the stomach, downstream effectors are poorly described. Here we demonstrate that the bHLH transcription factor ASCL1 is expressed in endocrine cells in the stomach and is required for formation of neurogenin-3-dependent enteroendocrine cells but not enterochromaffin-like cells. We also demonstrate that Ascl1 expression is inhibited by Notch signaling, suggesting that ASCL1 is a Notch-regulated transcriptional effector directing enteroendocrine cell fate in the mouse stomach.

Specialized Mechanosensory Epithelial Cells in Mouse Gut Intrinsic Tactile Sensitivity.

BACKGROUND AND AIMS: The gastrointestinal (GI) tract extracts nutrients from ingested meals while protecting the organism from infectious agents frequently present in meals. Consequently, most animals conduct the entire digestive process within the GI tract while keeping the luminal contents entirely outside the body, separated by the tightly sealed GI epithelium. Therefore, like the skin and oral cavity, the GI tract must sense the chemical and physical properties of the its external interface to optimize its function. Specialized sensory enteroendocrine cells (EECs) in GI epithelium interact intimately with luminal contents. A subpopulation of EECs express the mechanically gated ion channel Piezo2 and are developmentally and functionally like the skin's touch sensor- the Merkel cell. We hypothesized that Piezo2+ EECs endow the gut with intrinsic tactile sensitivity. METHODS: We generated transgenic mouse models with optogenetic activators in EECs and Piezo2 conditional knockouts. We used a range of reference standard and novel techniques from single cells to living animals, including single-cell RNA sequencing and opto-electrophysiology, opto-organ baths with luminal shear forces, and in vivo studies that assayed GI transit while manipulating the physical properties of luminal contents. RESULTS: Piezo2+ EECs have transcriptomic features of synaptically connected, mechanosensory epithelial cells. EEC activation by optogenetics and forces led to Piezo2-dependent alterations in colonic propagating contractions driven by intrinsic circuitry, with Piezo2+ EECs detecting the small luminal forces and physical properties of the luminal contents to regulate transit times in the small and large bowel. CONCLUSIONS: The GI tract has intrinsic tactile sensitivity that depends on Piezo2+ EECs and allows it to detect luminal forces and physical properties of luminal contents to modulate physiology.

Single-Cell Transcriptomics Reveals a Conserved Metaplasia Program in Pancreatic Injury.

BACKGROUND & AIMS: Acinar to ductal metaplasia (ADM) occurs in the pancreas in response to tissue injury and is a potential precursor for adenocarcinoma. The goal of these studies was to define the populations arising from ADM, the associated transcriptional changes, and markers of disease progression. METHODS: Acinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate post-injury. Transcripts of more than 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Developmental trajectories were generated. Data were compared with gastric metaplasia, KrasG12D-induced neoplasia, and human pancreatitis. Results were confirmed by immunostaining and electron microscopy. KrasG12D was expressed in injury-induced ADM using several inducible Cre drivers. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining. RESULTS: scRNA-seq of ADM revealed emergence of a mucin/ductal population resembling gastric pyloric metaplasia. Lineage trajectories suggest that some pyloric metaplasia cells can generate tuft and enteroendocrine cells (EECs). Comparison with KrasG12D-induced ADM identifies populations associated with disease progression. Activation of KrasG12D expression in HNF1B+ or POU2F3+ ADM populations leads to neoplastic transformation and formation of MUC5AC+ gastric-pit-like cells. Human pancreatitis samples also harbor pyloric metaplasia with a similar transcriptional phenotype. CONCLUSIONS: Under conditions of chronic injury, acinar cells undergo a pyloric-type metaplasia to mucinous progenitor-like populations, which seed disparate tuft cell and EEC lineages. ADM-derived EEC subtypes are diverse. KrasG12D expression is sufficient to drive neoplasia when targeted to injury-induced ADM populations and offers an alternative origin for tumorigenesis. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases.

Trp-Tyr is a dipeptide structure that potently stimulates GLP-1 secretion in a murine enteroendocrine cell model, identified by comprehensive analysis.

Dietary peptides potently stimulate glucagon-like peptide-1 (GLP-1) secretion, however, the underlying molecular mechanisms, such as structure-activity relationships and sensing mechanisms are only partly elucidated. In this study, we used a dipeptide library to identify dipeptides that potently stimulate GLP-1 release and to clarify the underlying structure-activity relationship. Murine enteroendocrine GLUTag cells were exposed to 339 dipeptides for 60 min, and the concentration of GLP-1 released into the supernatant was measured. Subsequently, selected dipeptides were examined for their reproducibility and dose responsiveness. In addition, we investigated the role of constituent amino acids in the secretion of GLP-1, and whether tripeptides containing the active dipeptide structures maintained their activity. In a concentration range of 1-5 mg/mL, twelve dipeptides had reproducible and concentration-dependent GLP-1-releasing activity. Among them, nine dipeptides (FY, KF, NI, PM, QL, QY, WF, WN, WY) were novel, with WY exhibiting the most potent activity. The reverse sequences and most free amino acids did not induce GLP-1 secretion, indicating that GLP-1-producing cells recognize the structure of each peptide to induce GLP-1 secretion. However, no apparent similarities were found between the active peptides. A comparison between the six tripeptides composed of F, W, and Y revealed the further potent tripeptides FWY and WYF, than WY. In the present study, a comprehensive analysis revealed nine novel dipeptides with high potential to stimulate GLP-1 secretion. Furthermore, the results indicate that 'WY' is a specific dipeptide sequence that potently stimulates GLP-1 secretion.

The orphan MRGPRF receptor is expressed in entero-endocrine cells of the human gut mucosa.

In the past years, it has become clear that the family of Mas-related G protein-coupled receptors plays a central role in neuro-immune communication at mucosal barrier surfaces, in particular in the skin. Remarkably, MRGPR expression at other mucosal surfaces remains poorly characterized. To fill this gap in our understanding, the present study was undertaken to screen and verify the expression of the human MRGPR family members in the mucosal biopsies of the human gastrointestinal (GI) tract. Our findings revealed that, of all human MRGPRs family members, only MRGPRF mRNA is expressed at detectable levels in human mucosal biopsies of both terminal ileum and sigmoid colon. Furthermore, immunohistochemical stainings revealed that MRGPRF is specifically expressed by mucosal entero-endocrine cells (EECs). Overall, this study showed for the first time that the human ileum and colonic mucosa represent a novel expression site for the orphan MRGPRF, more specifically in EECs.

The roles of enteroendocrine cell distribution and gustatory receptor expression in regulating peptide hormone secretion in the midgut of Bombyx mori larvae.

To regulate physiological homeostasis and behavior in Bombyx mori, more than 20 peptide hormones in the midgut of larvae are secreted upon detection of food substances at the lumen. Although it is logical to assume that the timings of peptide hormone secretions are regulated, little is known about the mechanisms. In this study, the distributions of enteroendocrine cells (EECs) producing five peptide hormones and EECs expressing gustatory receptors (Grs), as candidate receptors for luminal food substances and nutrients, were examined via immunostaining in B. mori larvae. Three patterns of peptide hormone distribution were observed. Tachykinin (Tk)- and K5-producing EECs were located throughout the midgut; myosuppressin-producing EECs were located in the middle-to-posterior midgut; and allatostatin C- and CCHamide-2-producing EECs were located in the anterior-to-middle midgut. BmGr4 was expressed in some Tk-producing EECs in the anterior midgut, where food and its digestive products arrived 5 min after feeding began. Enzyme-linked immunosorbent assay (ELISA) revealed secretion of Tk starting approximately 5 min after feeding began, suggesting that food sensing by BmGr4 may regulate Tk secretion. BmGr6 was expressed in a few Tk-producing EECs in the middle-to-posterior midgut, although its significance was unclear. BmGr6 was also expressed in many myosuppressin-producing EECs in the middle midgut, where food and its digestive products arrived 60 min after feeding began. ELISA revealed secretion of myosuppressin starting approximately 60 min after feeding began, suggesting that food sensing by BmGr6 may regulate myosuppressin secretion. Finally, BmGr9 was expressed in many BmK5-producing EECs throughout the midgut, suggesting that BmGr9 may function as a sensor for the secretion of BmK5.

Synergistic enhancement of glucagon-like peptide-1 release by γ-aminobutyric acid and L-phenylalanine in enteroendocrine cells-searching active ingredients in a water extract of corn zein protein.

This study investigated the glucagon-like peptide-1 (GLP-1)-releasing activity of an aqueous extract (ZeinS) from corn zein protein and aimed to identify the active compounds responsible for this activity. Glucagon-like peptide-1-releasing activity was evaluated using a murine enteroendocrine cell line (GLUTag). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on purified fractions of ZeinS to identify active molecules. ZeinS stimulated more GLP-1 secretion from GLUTag cells compared to zein hydrolysate. Fractions displaying biological activity were determined by solid-phase extraction and high-performance liquid chromatography (HPLC) fractionation. Subsequent LC-MS/MS analysis identified several amino acids in the active fractions of ZeinS. In particular, γ-aminobutyric acid (GABA) exhibited significant GLP-1-releasing activity both alone and synergistically with L-phenylalanine (Phe). Moreover, ZeinS-induced GLP-1 secretion was attenuated by antagonists for the GABA receptor and calcium sensing receptor. These results demonstrate that GABA and Phe identified in ZeinS synergistically stimulate GLP-1 secretion in enteroendocrine cells.

A cell atlas of the adult Drosophila midgut.

Studies of the adult Drosophila midgut have led to many insights in our understanding of cell-type diversity, stem cell regeneration, tissue homeostasis, and cell fate decision. Advances in single-cell RNA sequencing provide opportunities to identify new cell types and molecular features. We used single-cell RNA sequencing to characterize the transcriptome of midgut epithelial cells and identified 22 distinct clusters representing intestinal stem cells, enteroblasts, enteroendocrine cells (EEs), and enterocytes. This unbiased approach recovered most of the known intestinal stem cells/enteroblast and EE markers, highlighting the high quality of the dataset, and led to insights on intestinal stem cell biology, cell type-specific organelle features, the roles of new transcription factors in progenitors and regional variation along the gut, 5 additional EE gut hormones, EE hormonal expression diversity, and paracrine function of EEs. To facilitate mining of this rich dataset, we provide a web-based resource for visualization of gene expression in single cells. Altogether, our study provides a comprehensive resource for addressing functions of genes in the midgut epithelium.