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Methionine and its hydroxy analogue (MHA) have been shown to benefit mouse intestinal regeneration. The intestinal organoid is a good model that directly reflects the impact of certain nutrients or chemicals on intestinal development. Here, we aimed to establish a chicken intestinal organoid culture method first and then use the model to explore the influence of methionine deficiency and MHA on intestinal organoid development. The results showed that 125-μm cell strainer exhibited the highest efficiency for chicken embryo crypt harvesting. We found that transforming growth factor-β (TGF-β) inhibitor (A8301) supplementation promoted enterocyte differentiation at the expense of the proliferation of intestinal stem cells (ISC). The mitogen-activated protein kinase p38 inhibitor (SB202190) promoted intestinal organoid formation and enterocyte differentiation but suppressed the differentiation of enteroendocrine cells, goblet cells and Paneth cells. However, the suppression of enteroendocrine cell and Paneth cell differentiation by SB202190 was alleviated at the presence of A8301. The glycogen synthase kinase 3 inhibitor (CHIR99021), valproic acid (VPA) alone and their combination promoted chicken intestinal organoid formation and enterocyte differentiation at the expense of the expression of Paneth cells and goblet cells. Chicken serum significantly improved organoid formation, especially in the presence of A8301, SB202190, CHIR99021, and VPA, but inhibited the differentiation of Paneth cells and enteroendocrine cells. Chicken serum at a concentration of 0.25% meets the requirement of chicken intestinal organoid development, and the beneficial effect of chicken serum on chicken intestinal organoid culture could not be replaced by fetal bovine serum and insulin-like growth factor-1 (IGF-1). Moreover, commercial mouse organoid culture medium supplemented with A8301, SB202190, CHIR99021, VPA, and chicken serum promotes chicken organoid budding. Based on the chicken intestinal organoid model, we found that methionine deficiency mimicked by cycloleucine suppressed organoid formation and organoid size, and this effect was reinforced with increased cycloleucine concentrations. Methionine hydroxy analogue promoted regeneration of ISC but decreased cell differentiation compared with the results obtained with L-methionine. In conclusion, our results provide a potentially excellent guideline for chicken intestinal organoid culture and insights into methionine function in crypt development.

Background & Aims: Intestinal stem cells (ISCs) are sensitive to dietary alterations and nutrient availability. Neurotensin (NT), a gut peptide localized predominantly to the small bowel and released by fat ingestion, stimulates the growth of intestinal mucosa under basal conditions and during periods of nutrient deprivation, suggesting a possible role for NT on ISC function. Methods: Leucine-rich repeat-containing G-protein coupled receptor 5-Enhanced Green Fluorescent Protein (Lgr5-EGFP) NT wild type (Nt+/+) and Lgr5-EGFP NT knockout (Nt-/-) mice were fed ad libitum or fasted for 48 hours. Small intestine tissue and crypts were examined by gene expression analyses, fluorescence-activated cell sorting, Western blot, immunohistochemistry, and crypt-derived organoid culture. Drosophila expressing NT in midgut enteroendocrine cells were fed a standard diet or low-energy diet and esg-green fluorescent protein+ ISCs were quantified via immunofluorescence. Results: Loss of NT impaired crypt cell proliferation and ISC function in a manner dependent on nutrient status. Under nutrient-rich conditions, NT stimulated extracellular signal-regulated kinases 1 and 2 signaling and the expression of genes that promote cell-cycle progression, leading to crypt cell proliferation. Under conditions of nutrient depletion, NT stimulated WNT/β-catenin signaling and promoted an ISC gene signature, leading to enhanced ISC function. NT was required for the induction of WNT/β-catenin signaling and ISC-specific gene expression during nutrient depletion, and loss of NT reduced crypt cell proliferation and impaired ISC function and Lgr5 expression in the intestine during fasting. Conversely, the expression of NT in midgut enteroendocrine cells of Drosophila prevented loss of ISCs during nutrient depletion. Conclusions: Collectively, our findings establish an evolutionarily conserved role for NT in ISC maintenance during nutritional stress. GSE182828

Enteroendocrine (EE) cells are the most abundant hormone-producing cells in humans and are critical regulators of energy homeostasis and gastrointestinal function. Challenges in converting human intestinal stem cells (ISCs) into functional EE cells, ex vivo, have limited progress in elucidating their role in disease pathogenesis and in harnessing their therapeutic potential. To address this, we employed small molecule targeting of the endocannabinoid receptor signaling pathway, JNK, and FOXO1, known to mediate endodermal development and/or hormone production, together with directed differentiation of human ISCs from the duodenum and rectum. We observed marked induction of EE cell differentiation and gut-derived expression and secretion of SST, 5HT, GIP, CCK, GLP-1 and PYY upon treatment with various combinations of three small molecules: rimonabant, SP600125 and AS1842856. Robust differentiation strategies capable of driving human EE cell differentiation is a critical step towards understanding these essential cells and the development of cell-based therapeutics., Hormone-producing enteroendocrine cells (EEC) regulate of energy homeostasis and gastrointestinal function. Here the authors report protocols to induce human intestinal stem cells into EECs producing multiple gut hormones, including SST, 5-HT, CCK and GIP, using directed differentiation with small molecules targeting FOXO1, JNK and CB1 signalling.

Publisher Summary This chapter discusses various functions of the gastrointestinal tract that is essential for the orderly digestion, absorption, and transportation of food and residue. The motor activity of the gut is one of the integrated functions that are essential for the normal assimilation of food. Gut motility facilitates the transportation of nutrients, brings together digestive enzymes and their substrates, temporarily stores content, particularly in the distal small bowel and right colon, for optimal absorption, and finally, excretes nondigestible residue by defecation in a well-coordinated function under voluntary control. The extrinsic autonomic nervous system (ANS) is critically important for almost all secretory and motor functions in the digestive tract. The function of the gastrointestinal smooth muscle is intimately controlled by the release of peptides and transmitters by the intrinsic (or enteric) nervous system; the modulation of the latter input arises in the extrinsic autonomic nerves, the craniospinal parasympathetic excitatory input, and the thoracolumbar sympathetic outflow, which is predominantly inhibitory to the gut but excitatory to the sphincter. Gastrointestinal smooth muscle forms an electrical syncytium whereby the impulse that induces the contraction of the first muscle cell results in efficient transmission to a sheet of sequentially linked cells in the transverse and longitudinal axes of the intestine. The pacemaker of the intestinal muscle syncytium is the network of interstitial cells of Cajal that serve to coordinate contraction circumferentially and longitudinally along the gut.

The anterior pituitary gland regulates growth, metabolism, and reproduction by secreting hormones. Folliculo-stellate (FS) cells are non-endocrine cells located among hormone-producing cells in the anterior pituitary glands. They form follicular lumens, which are sealed by tight junctions (TJs). Although FS cells are hypothesized to contribute to fine-tuning of endocrine cells, little is known about the exact roles of FS cells. Here, we investigated the molecular composition of TJs in FS cells. We demonstrated that occludin is a good marker for TJs in the pituitary gland and examined the structure of the lumens surrounded by FS cells. We also found that claudin-9 is a major component of TJs in the FS cells. In immunoelectron microscopy, claudin-9 was specifically localized at TJs of the FS cells. The expression of claudin-9 was gradually increased in the pituitary gland after birth, suggesting that claudin-9 is developmentally regulated and performs some specific functions on the paracellular barrier of follicles in the pituitary gland. Furthermore, we found that angulin-1, angulin-2, and tricellulin are localized at the tricellular contacts of the FS cells. Our findings provide a first comprehensive molecular profile of TJs in the FS cells, and may lead us towards unveiling the FS cell functions.

Cyprinid fishes have one of the simplest types of gastrointestinal tract among vertebrates. Those fish species do not possess a true stomach that is replaced by a simple dilatation at the anterior part of the intestine called the intestinal bulb. Twenty adult specimens of grass carp were used in the present study to identify the cellular components as well as the immunohistochemical and surface architectural characteristics of the intestinal bulb. The mucosa of the intestinal bulb shows numerous, deep longitudinal folds arranged in zigzagging-like patterns. The epithelium is composed mainly of absorptive columnar cells covered by microvilli and mucous goblet cells. Spindle-shaped enteroendocrine cells and some migratory immune cells such as intraepithelial lymphocytes and rodlet cells could be identified between the absorptive cells. The epithelium also contains many secretory granules and large numbers of vacuoles containing digestive enzymes mostly in the basal part. The immunohistochemistry revealed that CD20-positive B-lymphocytes are immunolocalized mainly in the connective tissue core lamina propria of the mucosal folds. However, CD3-immunopositive T-lymphocytes are highly concentrated in the lamina propria. In addition, intraepithelial T-lymphocytes expressed immunopositivity to CD3. The current study presented many types of immune cells and suggests their essential immunological role for the intestinal blub.

Aims/hypothesis Pancreatic polypeptide (PP) cells, which secrete PP (encoded by the Ppy gene), are a minor population of pancreatic endocrine cells. Although it has been reported that the loss of beta cell identity might be associated with beta-to-PP cell-fate conversion, at present, little is known regarding the characteristics of Ppy-lineage cells. Methods We used Ppy-Cre driver mice and a PP-specific monoclonal antibody to investigate the association between Ppy-lineage cells and beta cells. The molecular profiles of endocrine cells were investigated by single-cell transcriptome analysis and the glucose responsiveness of beta cells was assessed by Ca2+ imaging. Diabetic conditions were experimentally induced in mice by either streptozotocin or diphtheria toxin. Results Ppy-lineage cells were found to contribute to the four major types of endocrine cells, including beta cells. Ppy-lineage beta cells are a minor subpopulation, accounting for 12–15% of total beta cells, and are mostly (81.2%) localised at the islet periphery. Unbiased single-cell analysis with a Ppy-lineage tracer demonstrated that beta cells are composed of seven clusters, which are categorised into two groups (i.e. Ppy-lineage and non-Ppy-lineage beta cells). These subpopulations of beta cells demonstrated distinct characteristics regarding their functionality and gene expression profiles. Ppy-lineage beta cells had a reduced glucose-stimulated Ca2+ signalling response and were increased in number in experimental diabetes models. Conclusions/interpretation Our results indicate that an unexpected degree of beta cell heterogeneity is defined by Ppy gene activation, providing valuable insight into the homeostatic regulation of pancreatic islets and future therapeutic strategies against diabetes. Data availability The single-cell RNA sequence (scRNA-seq) analysis datasets generated in this study have been deposited in the Gene Expression Omnibus (GEO) under the accession number GSE166164 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE166164). Graphical abstract

Morphofunctional characteristics of the large intestine are rarely explored to understand the physiology, behavior and ecology of neotropical primates. In this study, we analyzed the histometric parameters of the large intestine of hybrid marmosets (Callithrix sp.) captured in forest fragments of Viçosa-Brazil, under seasonal interference. These animals were predominantly insectivorous in the rainy season and gummivores in the dry season. Large intestine fragments were collected and processed according to histological methods and stained with toluidine blue for general analysis, periodic acid of Schiff (PAS) and Alcian blue (AB) for goblet cells, Grimelius and Masson Fontana for argyrophil and argentaffin endocrine cells. Several histometric parameters were more expressive in the large intestine of the rainy season marmosets: greater thickness of the parietal layers, greater number of argyrophil and argentaffin endocrine cells, and AB-positive goblet cells, characteristics favor secretomotor functions and reduce the passage time of the fecal bolus, which is consistent with an insectivorous diet. In contrast, parameters such as crypt width, height of the absorptive cells and striated border, and the number of PAS-positive cells were more expressive in the dry season marmosets, reflecting the need for longer passage time for digestion and absorption of food items from tree gum, which are more complex and demand the action of microorganisms present in the large intestine, as well as greater protection against the abrasive action of dietary fibers and against microorganisms. Thus, it can be said that the marmoset's large intestine has morphological adaptations to maximize energy intake from the diet, which alternates under the influence of seasonality.

This study investigated the histomorphological, ultrastructural and morphometrical postnatal developmental changes in the rabbit fundic region, especially during changing of the feeding intake. Seventy-two New Zealand rabbits (V-Line breed) at the ages of 1, 7, 15, 23, 30 and 60 days were obtained for light and electron microscopy and morphometric studies of the fundic region. The newborn rabbit's fundic wall was thin and organized into mucosa, submucosa, musclosa and serosa, with a significant increase in thickness with ageing. The fundic glands were few at the first week of life, then increased in length and diameter compared to the preceding age with prominent zonation at 23 days. The gastric pits appeared wide and deep at the first week of life then became typically narrow and shallow at the third week. The mucous cells were the main cell types lining the fundic glands in the first week of life. These cells showed remoulding with a marked increase in Periodic Acid-Schiff reactivity with age. Parietal cells were differentiated earlier (on the first day of life) than the chief cells and distributed at the neck and basal zones. Chief cells differentiated at 15 days old at the base of the glands, followed by an increase in the number and activity. Few active enteroendocrine cells were first seen at 15 days old and then widely distributed throughout the glands. Conclusion: Pronounced histomorphological changes in the fundic mucosal layer, especially the surface and glandular epithelium, correlate with the postnatal rabbit-feeding intake changes.

We detail a 6-stage planar differentiation methodology for generating human pluripotent stem cell (hPSC)-derived pancreatic β cells (SC-β cells) that secrete high amounts of insulin in response to glucose stimulation. This protocol first induces definitive endoderm by treatment with Activin A and CHIR99021, then generates PDX1+/NKX6–1+ pancreatic progenitors through the timed application of keratinocyte growth factor (KGF), SANT1, TPPB, LDN193189, and retinoic acid (RA). Endocrine induction and subsequent SC-β cell specification is achieved with a cocktail consisting of the cytoskeletal depolymerizing compound latrunculin A combined with XXI, T3, ALK5 inhibitor II, SANT1, and RA. The resulting SC-β cells and other endocrine cell types can then be aggregated into islet-like clusters for analysis and transplantation. This differentiation methodology takes approximately 35 days to generate functional SC-β cells, plus an additional 1–2 weeks for initial stem cell expansion and final cell assessment. This protocol builds upon a large body of previous work for generating β-like cells. In this iteration, we have eliminated the need for three-dimensional culture during endocrine induction, allowing for the generation of highly functional SC-β cells to be done entirely on tissue culture polystyrene (TCP). This change simplifies the differentiation methodology, requiring only basic stem cell culture experience as well as familiarity with assessment techniques common in biology labs. In addition to expanding protocol accessibility and simplifying SC-β cell generation, we demonstrate that this planar methodology is amenable for differentiating SC-β cells from a wide variety of cell lines from various sources, broadening its applicability.

There are individual differences in the tolerance to hypoxia and stress. Stress can contribute to the development of various diseases, including inflammatory bowel disease. It was found that inflammatory bowel diseases in animals susceptible to hypoxia runs more severe course than in tolerant animals. We studied morphofunctional changes in the colon under conditions of modeled cold stress in male C57BL/6 mice susceptible and tolerant to hypoxia. The animals were daily subjected to cold stress (20 min at -20°C) for 2 weeks. Cold stress was followed by an increase in the volume fraction of goblet cells in the colon and production of mucins by these cells in mice tolerant to hypoxia and an increase in cell content in the lamina propria of the colon mucous membrane in animals susceptible to hypoxia. The number of serotoninproducing endocrine cells increased in both groups, but these changes were more pronounced in mice susceptible to hypoxia.

The current COVID-19 pandemic, which continues to spread across the globe, is caused by severe acute respiratory syndrome coronavirus (SARS-Cov-2). Soon after the pandemic emerged in China, it became clear that the receptor-binding domain (RBD) of angiotensin-converting enzyme 2 (ACE2) serves as the primary cell surface receptor for SARS-Cov-2. Subsequent work has shown that diabetes and hyperglycemia are major risk factors for morbidity and mortality in COVID-19 patients. However, data on the pattern of expression of ACE2 on human pancreatic β cells remain contradictory. Additionally, there is no consensus on whether the virus can directly infect and damage pancreatic islets and hence exacerbate diabetes. In this mini-review, we highlight the role of ACE2 receptor and summarize the current state of knowledge regarding its expression/co-localization in human pancreatic endocrine cells. We also discuss recent data on the permissiveness of human pancreatic β cells to SARS-Cov-2 infection.

The objective of this study was to investigate the plasma concentrations of insulin-like growth factor-2 (IGF-2) as well as its expression in the uterus and ovary of healthy dogs and those with cystic endometrial hyperplasia (CEH)–pyometra complex. Group 1 (n = 10) included bitches with open cervix pyometra, while Group 2 (n = 7) consisted of clinically healthy bitches in dioestrus. The number of IGF-2 immunopositive interstitial cells was significantly higher in Group 1, whereas in Group 2 there were only two cases in which a few cells were IGF-2 immunopositive. IGF-2 immunopositivity was observed in the endometrial glandular epithelium in both groups. Additionally, interstitial fibroblasts and macrophages in the endometrium were also positive in Group 1. The concentration of plasma IGF-2 was higher in Group 1 than in Group 2 (P < 0.05). The concentration was positively correlated with IGF-2 expression in the endometrial glands (r = 0.926; P < 0.001) in Group 1. However, a negative correlation was present between plasma IGF-2 concentration and IGF-2 expression in the interstitial endocrine cells of the ovary in Group 1 (r = −0.652; P < 0.05). The results suggest that IGF-2 plays an important role during the inflammatory process occurring in bitches with CEH–pyometra complex as well as in the endometrium of healthy bitches in dioestrus.

Conventional methods for obtaining pancreatic β cells are based on simulating the embryonic development phase of endocrine cells via hierarchical differentiation of pluripotent stem cells (PSCs). Accordingly, we attempted to modify the protocols for obtaining insulin-secreting cells (ISCs) by sequential differentiation of a human embryonic stem cell (hESC), using the HS181 cell line. Furthermore, we hypothesize that actual pancreatic endocrine cells may arise from trans-differentiation of mature ductal cells after the embryonic developmental stage and throughout the rest of life. According to the hypothesis, ductal cells are trans-differentiated into endocrine and exocrine cells, undergoing a partial epithelial to mesenchymal transition (EMT). To address this issue, we developed two new protocols based on hESC differentiation to obtain ductal cells and then induce EMT in cells to obtain hormone-secreting islet-like cells (HSCs). The ductal (pre-EMT exocrine) cells were then induced to undergo partial EMT by treating with Wnt3a and activin A, in hypoxia. The cell derived from the latter method significantly expressed the main endocrine cell-specific markers and also β cells, in particular. These experiments not only support our hypothetical model but also offer a promising approach to develop new methods to compensate β cell depletion in patients with type 1 diabetes mellitus (T1DM). Although this protocol of generating islet-like cells from ductal cells has a potential to treat T1DM, this strategy may be exploited to optimize the function of these cells in an animal model and future clinical applications.

Dispersed single cells from pancreatic islets can configure the 3D islet-like architecture (pseudo-islets) with insulin secretion potential and controllable size through their aggregation property. The present study was designed to investigate whether cryopreservation of islets or islet cells can contribute to the efficient pseudo-islet fabrication in the rat model. In control group (CT), islet single cells were prepared by trypsin digestion of 50-400-µm o fresh control islets, and then cultured for 3 days in the U-bottom micro-well to fabricate pseudo-islets. In vitrification-warming group (VW), islet single cells were prepared from post-warm islets cryopreserved by vitrification on nylon mesh device, and then cultured for 3 days. In freezing group (FR), islet single cells originated from fresh islets were subjected to a conventional Bicell® freezing, and post-thaw cells were cultured for 3 days. To generate 1 IEQ pseudo-islets (150 µm o) by the sphere culture, 1250 CT cells, 1250 VW cells, and 1500 FR cells were seeded to each micro-well. The viability of the pseudo-islets was comparable among the three groups (93.9-96.9%). Furthermore, the insulin secretion assay showed that those pseudo-islets responded sufficiently to the high glucose stimulation. Immunostaining for insulin and glucagon showed that the endocrine cell arrangement of those pseudo-islets is similar to that of native and isolated islets. These islets/pseudo-islets had the β-cells in core and the α-cells in mantle, which was typical characteristic of the rodent islets. However, some clusters of α-cells were observed inside the FR pseudo-islets. Interestingly, the VW pseudo-islets had significantly fewer α-cells than the CT or FR pseudo-islets. These results suggest that the sphere culture of islet cells is useful tool to generate the pseudo-islets with the customized size and normal functionality, even after islet cryopreservation. This article is protected by copyright. All rights reserved.

Single-cell RNA sequencing combined with spatial information on landmark genes enables reconstruction of spatially-resolved tissue cell atlases. However, such approaches are challenging for rare cell types, since their mRNA contents are diluted in the spatial transcriptomics bulk measurements used for landmark gene detection. In the small intestine, enterocytes, the most common cell type, exhibit zonated expression programs along the crypt-villus axis, but zonation patterns of rare cell types such as goblet and tuft cells remain uncharacterized. Here, we present ClumpSeq, an approach for sequencing small clumps of attached cells. By inferring the crypt-villus location of each clump from enterocyte landmark genes, we establish spatial atlases for all epithelial cell types in the small intestine. We identify elevated expression of immune-modulatory genes in villus tip goblet and tuft cells and heterogeneous migration patterns of enteroendocrine cells. ClumpSeq can be applied for reconstructing spatial atlases of rare cell types in other tissues and tumors., Combining scRNA-seq with spatial information to enable the reconstruction of spatially-resolved cell atlases is challenging for rare cell types. Here the authors present ClumpSeq, an approach for sequencing small clumps of tissue attached cells, and apply it to establish spatial atlases for all secretory cell types in the small intestine.

The literature review highlights the questions of the interaction of Helicobacter pylori and the human body. Modern data on the structure of the pathogenicity island in the Helicobacter pylori genome are presented. There is given a detailed description of both well-known virulence and pathogenicity factors of the infection (genes encoding the formation of urease subunits, in particular urel, cytotoxin associated gene A, vacuolating cytotoxin gen A, blood group associated binding adhesion, induced by contact with epithelium) and less studied ones (sialic acid-binding adhesion, adhesion-associated lipoprotein A and B, adhesin gene of Helicobacter pylori, Hp outer membrane protein). The significance of individual genes and proteins encoded by them in the development of chronic inflammatory process in diseases of the upper digestive tract, as well as in ulcer and carcinogenesis is analyzed. Mechanisms of interaction of bacteria with epithelial cells of the gastric mucosa, adhesive and cytotoxic effects of Helicobacter pylori, factors of biofilm formation are described. The influence of the genetic structure of Infect on cytological composition of the gastric glands in the form of reduction of specialized glandular cells chief and parietal cells of pyloric glands and the increase of endocrine cells in the pool is assessed. It is shown that colonization of the gastric mucosa by highly pathogenic strains of Helicobacter pylori contributes to the development of widespread pronounced and active inflammation in it, the appearance of morphological signs of atrophy. The role of the genetic characteristics of the infection in the failure of anti-helicobacter therapy is emphasized. Separately, the question of the effect of combined infection of the gastric mucosa with highly pathogenic strains of Helicobacter pylori and Epstein-Barr virus is highlighted.

Precise regulation of circulating glucose is crucial for human health and ensures a sufficient supply to the brain, which relies almost exclusively on glucose for metabolic energy. Glucose homeostasis is coordinated by hormone-secreting endocrine cells in the pancreas, as well as glucose utilization and production in peripheral metabolic tissues including the liver, muscle, and adipose tissue. Glucose-regulatory tissues receive dense innervation from sympathetic, parasympathetic, and sensory fibers. In this review, we summarize the functions of peripheral nerves in glucose regulation and metabolism. Dynamic changes in peripheral innervation have also been observed in animal models of obesity and diabetes. Together, these studies highlight the importance of peripheral nerves as a new therapeutic target for metabolic disorders.

Wasting marmoset syndrome (WMS) is a serious disease in captive common marmoset (Callithrix jacchus) colonies. Because of the high mortality rates, elucidation of the underlying mechanisms is essential. In this study, we compared the histopathology, the number of each epithelial cell in the jejunum and colon, and the expression patterns of some molecular markers between healthy and WMS-affected marmosets. Atrophy of villi in the jejunum and mononuclear cell infiltration in the lamina propria were observed in the intestinal tract of WMS-affected marmosets. Although the numbers of transient amplifying cells and tuft cells were increased, the number of goblet cells was obviously decreased in the jejunum and colon of WMS-affected marmosets compared to healthy marmosets. In addition, the number of enterocytes in the jejunum was decreased in WMS animals. There was no apparent difference in the numbers of stem cells, enteroendocrine cells, or Paneth cells. The expression of β-catenin and Tcf7l2 was increased in WMS, and the co-existence of β-catenin and Tcf7l2/Cyclin D1 was observed around the crypts in WMS-affected marmosets. These findings suggest that cell proliferation continues, but cell differentiation is halted in the intestinal tract due to the enhanced β-catenin/Tcf7l2/Cyclin D1signaling pathway in WMS, which results in malfunction of the villus and mucosa.

Secretogranin III (SgIII) is a granin protein involved in secretory granule formation in peptide-hormone-producing endocrine cells. In this study, we analyzed the expression of the LacZ reporter in the SgIII knockout mice produced by gene trapping ( SgIII-gtKO) for the purpose of comprehensively clarifying the expression patterns of SgIII at the cell and tissue levels. In the endocrine tissues of SgIII-gtKO mice, LacZ expression was observed in the pituitary gland, adrenal medulla, and pancreatic islets, where SgIII expression has been canonically revealed. LacZ expression was extensively observed in brain regions, especially in the cerebral cortex, hippocampus, hypothalamic nuclei, cerebellum, and spinal cord. In peripheral nervous tissues, LacZ expression was observed in the retina, optic nerve, and trigeminal ganglion. LacZ expression was particularly prominent in astrocytes, in addition to neurons and ependymal cells. In the cerebellum, at least four cell types expressed SgIII under basal conditions. The expression of SgIII in the glioma cell lines C6 and RGC-6 was enhanced by excitatory glutamate treatment. It also became clear that the expression level of SgIII varied among neuron and astrocyte subtypes. These results suggest that SgIII is involved in glial cell function, in addition to neuroendocrine functions, in the nervous system

Purpose Many studies about bariatric surgery have analyzed the effect of sleeve gastrectomy (SG) on glucose improvement, beta-cell mass, and islet size modification. The effects of SG on the other endocrine cells of the pancreas, such as the alpha-cell population, and their regulatory mechanisms remain less studied. Materials and Methods We focused our work on the changes in the alpha-cell population after SG in a healthy model of Wistar rats. We measured alpha-cell mass, glucose tolerance, and insulin release after oral glucose tolerance tests and plasma glucagon secretion patterns after insulin infusion. Three Wistar rat groups were employed: SG-operated, surgical control (Sham), and fasting control. Results The results obtained showed significant increases in the alpha-cell population after SG. The result was an increase in beta-cell transdifferentiation; it was shown by some expressed molecules (the loss of expression of Pdx-1 and the increase in Arx and Pax6 cells/mm2 of islet). The serum results were enhanced plasma glucagon secretion pattern after insulin infusion assays and normal glucose tolerance and insulin release after OGTT. Conclusion We concluded that SG leads to an expansion of the alpha-cell population, at expense of beta-cell; this expansion of alpha-cells is related to transdifferentiation. Plasma glucose level was not affected due to an increased glucagon response.

Recent studies have demonstrated that genes related to bitter taste receptors (TAS2Rs) on various chromosomes are expressed in extra-oral organs of various animals. The bitter taste receptor TAS2R14 is conserved among primate species and shows broad ligand sensitivity. Mice have a number of orthologues to primate TAS2R14 located in tandem on chromosome 16; however, their expression patterns are not unique. We characterized the expression of TAS2R14 in various cell types in the intestines of the rhesus macaque and evaluated its role in hormone production in the gut. TAS2R14 expression was examined in the intestines of rhesus macaques, a common non-human primate model, by RT-qPCR and immunohistochemical staining. Mean expression levels of TAS2R14 in the duodenum, ileum, and colon were similar to each other and were lower than those in circumvallate papillae. An immunohistochemical analysis revealed TAS2R14 immunoreactivity in enteroendocrine cells positive for cholecystokinin, serotonin, and the G protein GNAT3. These results suggest that primate TAS2R14 is broadly expressed in the intestine, mainly in enteroendocrine cells, and promotes gut hormone secretion in response to bitter stimuli.

5-HT containing enteroendocrine cells (EEC), the most abundant type of EEC in the gut, regulate many functions including motility, secretion and inflammatory responses. We examined the morphologies of 5-HT cells from stomach to rectum, patterns of hormone co-expression in the stomach and colon, and the relationship of 5-HT cells with nerve fibres. We also reviewed some of the relevant literature. The morphologies of 5-HT cells were distinct, depending on their location in the gut. A noticeable feature of some 5-HT cells in the antrum and colon was their long basal processes, which resembled processes of neurons, whereas 5-HT cells in the small intestinal mucosa lacked basal processes. In the stomach, numerous 5-HT cells, including cells with basal processes, were identified as enterochromaffin-like cells by their expression of histidine decarboxylase. In the colon, we observed a small number of 5-HT cells that were in close contact with, but distinct from, oxyntomodulin (OXM) and PYY immunoreactive EEC. We did not find specific relationships between nerve fibres and the processes of colonic 5-HT cells. We conclude that five major features, i.e., gut region, morphology, hormone content, receptor repertoire and cell lineage, can be used to define 5-HT cells.

Serotonin (5-HT)-containing gastrointestinal endocrine cells contribute to regulation of numerous bodily functions, but whether these functions are related to differences in cell shape is not known. The current study identified morphologies and localization of subtypes of 5-HT-containing enteroendocrine cells in the mouse large intestine. 5-HT cells were most frequent in the proximal colon compared with cecum and distal colon. The large intestine harbored both open (O) cells, with apical processes that reached the lumen, and closed (C) cells, not contacting the lumen, classified into O1, O2, and O3 and C1, C2, and C3 cells, by the lengths of their basal processes. O1 and C1 cells, with basal processes sometimes longer that 100 µm, were most common in the distal colon. Their long basal processes ran against the inner surfaces of the mucosal epithelial cells and were strongly immunoreactive for 5-HT; these processes are ideally placed to communicate with the epithelium and to react to mechanical forces. O2 and C2 cells that had similar but shorter basal processes were also most common in the distal colon. O3 and C3 cells had no or very short basal processes. The O3 open type 5-HT cells were abundant in the proximal colon, particularly at the luminal surface, where they could release 5-HT into the lumen to act on luminal 5-HT receptors. Numerous O3 type 5-HT cells occurred in the lower (submucosal) region of the crypts in all segments and might release 5-HT to influence cell renewal in the crypt proliferative zones.

Studies on localization and distribution of enteroendocrine cells (EECs) are important for better understanding of their role in the ontogenetic development of intestines. Information about the distribution of the most important endocrine cells in the digestive tract of the ostrich is very limited; therefore, the aim of the present study was to identify gastrin and somatostatin EECs in the small intestine of the ostrich (Struthio camelus var. domesticus) chicks at different ages. Six embryos along with 42 ostriches of both sexes from hatching up to 60 days post-hatching, including six embryos, were obtained from an ostrich farm in Latvia. Duodenum, jejunum and ileum were investigated using routine histology and immunohistochemistry methods. Gastrin and somatostatin EECs were examined in 10 microscopic fields of the intestinal mucosa in each tissue sample. The cells were detected in all age groups as well as the embryos. The number of both types of EEC in the mucosa of the ileum was significantly lower (p

This paper overviews the development and differentiation of the hypothalamus and pituitary gland from embryonic stem (ES) and induced pluripotent stem (iPS) cells. It is important to replicate the developmental process in vivo to create specific cells/organoids from ES/iPS cells. We also introduce the latest findings and discuss future issues for clinical application. Neuroectodermal progenitors are induced from pluripotent stem cells by strictly removing exogenous patterning factors during the early differentiation period. The induced progenitors differentiate into rostral hypothalamic neurons, in particular magnocellular vasopressin+ neurons. In three-dimensional cultures, the ES/iPS cells differentiate into hypothalamic neuroectoderm as well as non-neural head ectoderm back to back. Rathke's pouch-like structures self-organize at the interface between the two layers and generated various endocrine cells, including corticotrophs and somatotrophs from the Rathke's pouch-like structures. Our next objective is to sophisticate our stepwise methodology to establish the novel transplantation treatment for hypopituitarism and to apply it to developmental disease models.

Adenomatous polyposis coli (APC), a well-known anti-oncogene, is considered to have multiple functions through its several binding domains. We have continuingly studied APC1638T/1638T mice (APC1638T mice) to elucidate the functions of APC other than tumor suppression. A distinctive feature of the APC1638T mice is they are tumor free and live as long as APC+/+ mice (WT mice). Previously, we found the length of crypt-villus axis in the jejunum was significantly elongated in APC1638T mice compared with that of WT mice. The populations of goblet cells, Paneth cells, and enteroendocrine cells were also disordered in APC1638T mice. Here, we further analyzed the intestinal dyshomeostasis in APC1638T mice, focusing on the proliferation and differentiation of intestinal stem cell (ISC) lineages, and apoptotic cell shedding at the villus tips. We found that the proliferation of ISC lineages was normally controlled; however, the shedding process of apoptosis cells was significantly delayed in the APC1638T mouse jejunum. Furthermore, the number of microfold cells (M cells) was significantly increased in the APC1638T mouse jejunum. Our data suggested both differentiation process of ISCs and turnover process of intestinal epithelia were disturbed in APC1638T mice, and that contributed to the villus elongation in the APC1638T mouse jejunum.

ContextMaturity-onset diabetes of the young (MODY) 8 is a rare form of monogenic diabetes characterized by a mutation in CEL (carboxyl ester lipase) gene, which leads to exocrine pancreas dysfunction, followed by β cell failure. Induced pluripotent stem cells can differentiate into functional β cells. Thus, β cells from MODY8 patients can be generated in vitro and used for disease modelling and cell replacement therapy.MethodsA genetic study was performed in a patient suspected of monogenic diabetes.ResultsA novel heterozygous pathogenic variant in CEL (c.1818delC) was identified in the proband, allowing diagnosis of MODY8. Three MODY8-iPSC (induced pluripotent stem cell) clones were reprogrammed from skin fibroblasts of the patient, and their pluripotency and genomic stability confirmed. All 3 MODY8-iPSC differentiated into β cells following developmental stages. MODY8-iPSC–derived β cells were able to secrete insulin upon glucose dynamic perifusion. The CEL gene was not expressed in iPSCs nor during any steps of endocrine differentiation.ConclusioniPSC lines from a MODY8 patient with a novel pathogenic variant in the CEL gene were generated; they are capable of differentiation into endocrine cells, and β cell function is preserved in mutated cells. These results set the basis for in vitro modelling of the disease and potentially for autologous β cell replacement.

Aims/hypothesis We hypothesised that human beta cells are structurally and functional polarised with respect to the islet capillaries. We set out to test this using confocal microscopy to map the 3D spatial arrangement of key proteins and live-cell imaging to determine the distribution of insulin granule fusion around the cells. Methods Human pancreas samples were rapidly fixed and processed using the pancreatic slice technique, which maintains islet structure and architecture. Slices were stained using immunofluorescence for polarity markers (scribble, discs large [Dlg] and partitioning defective 3 homologue [Par3]) and presynaptic markers (liprin, Rab3-interacting protein [RIM2] and piccolo) and imaged using 3D confocal microscopy. Isolated human islets were dispersed and cultured on laminin-511-coated coverslips. Live 3D two-photon microscopy was used on cultured cells to image exocytic granule fusion events upon glucose stimulation. Results Assessment of the distribution of endocrine cells across human islets found that, despite distinct islet-to-islet complexity and variability, including multi-lobular islets, and intermixing of alpha and beta cells, there is still a striking enrichment of alpha cells at the islet mantle. Measures of cell position demonstrate that most beta cells contact islet capillaries. Subcellularly, beta cells consistently position polar determinants, such as Par3, Dlg and scribble, with a basal domain towards the capillaries and apical domain at the opposite face. The capillary interface/vascular face is enriched in presynaptic scaffold proteins, such as liprin, RIM2 and piccolo. Interestingly, enrichment of presynaptic scaffold proteins also occurs where the beta cells contact peri-islet capillaries, suggesting functional interactions. We also observed the same polarisation of synaptic scaffold proteins in islets from type 2 diabetic patients. Consistent with polarised function, isolated beta cells cultured onto laminin-coated coverslips target insulin granule fusion to the coverslip. Conclusions/interpretation Structural and functional polarisation is a defining feature of human pancreatic beta cells and plays an important role in the control of insulin secretion. Graphical abstract

Psychobiotics are a special class of probiotics, which deliver mental health benefits to individuals. They differ from conventional probiotics in their ability to produce or stimulate the production of neurotransmitters, short-chain fatty acids, enteroendocrine hormones and anti-inflammatory cytokines. Owing to this potential, psychobiotics have a broad spectrum of applications ranging from mood and stress alleviation to being an adjuvant in therapeutic treatment for various neurodevelopment and neurodegenerative disorders. The common psychobiotic bacteria belong to the family Lactobacilli, Streptococci, Bifidobacteria, Escherichia and Enterococci. The two-way crosstalk between the brain and the gastrointestinal system is influenced by these bacteria. The neurons present in the enteric nervous system interact directly with the neurochemicals produced by microbiota of the gut, thereby influencing the signaling to central nervous system. The present review highlights the scope and advancements made in the field, enlisting numerous commercial psychobiotic products that have flooded the market. In the latter part we discuss the potential concerns with respect to psychobiotics, such as the effects due to withdrawal, compatibility with immunocompromised patients, and the relatively unregulated probiotic market.

A detailed understanding of intestinal stem cell (ISC) self-renewal and differentiation is required to treat chronic intestinal diseases. However, the different models of ISC lineage hierarchy1–6 and segregation7–12 are subject to debate. Here, we have discovered non-canonical Wnt/planar cell polarity (PCP)-activated ISCs that are primed towards the enteroendocrine or Paneth cell lineage. Strikingly, integration of time-resolved lineage labelling with single-cell gene expression analysis revealed that both lineages are directly recruited from ISCs via unipotent transition states, challenging the existence of formerly predicted bi- or multipotent secretory progenitors7–12. Transitory cells that mature into Paneth cells are quiescent and express both stem cell and secretory lineage genes, indicating that these cells are the previously described Lgr5+ label-retaining cells7. Finally, Wnt/PCP-activated Lgr5+ ISCs are molecularly indistinguishable from Wnt/β-catenin-activated Lgr5+ ISCs, suggesting that lineage priming and cell-cycle exit is triggered at the post-transcriptional level by polarity cues and a switch from canonical to non-canonical Wnt/PCP signalling. Taken together, we redefine the mechanisms underlying ISC lineage hierarchy and identify the Wnt/PCP pathway as a new niche signal preceding lateral inhibition in ISC lineage priming and segregation. Polarity cues regulate intestinal stem cell fate. Bottcher et al. demonstrate that mouse intestinal stem cells, which express the Wnt/planar cell polarity reporter Flattop, are primed either towards the enteroendocrine or Paneth cell lineage.

The intestines have been recognized as important tissues for metabolic regulation, including glycemic control, but their vital role in promoting the anti-diabetic effects of bitter melon, the fruit of Momordica charantia L, has seldom been characterized, nor acknowledged. Evidence suggests that bitter melon constituents can have substantial interactions with the intestinal epithelial cells before circulating to other tissues. We therefore characterized the effects of bitter melon extract (BME) on intestinal epithelial cells. BME was found to contain substantial amounts of carbohydrates, proteins, and triterpenoids. TNF-α induced insulin resistance in an enterocyte cell line of IEC-18 cells, and BME promoted glucose utilization of the insulin-resistant cells. Further analysis suggested that the increased glucose consumption was a result of the combined effects of insulin sensitizing and insulin substitution functions of BME. The functions of insulin substitution were likely generated due to the activation of AMP-activated protein kinase. Meanwhile, BME acted as a glucagon-like peptide 1 (GLP-1) secretagogue on enteroendocrine cells, which may be mediated by the activation of bitter-taste receptors. Therefore, BME possesses insulin sensitizing, insulin substitution, and GLP-1 secretagogue functions upon intestinal cells. These effects of BME on intestinal cells likely play a significant part in the anti-diabetic action of bitter melon.

Human intestinal organoids (HIOs) are a promising in vitro model consisting of different intestinal cell types with a 3D microarchitecture resembling native tissue. In the current study, we aimed to assess the expression of the most common intestinal CYP enzymes in a human induced pluripotent stem cell (hiPSC)-derived HIO model, and the suitability of that model to study chemical-induced changes in CYP expression and activity. We compared this model with the commonly used human colonic adenocarcinoma cell line Caco-2 and with a human primary intestinal epithelial cell (IEC)-based model, closely resembling in vivo tissue. We optimized an existing protocol to differentiate hiPSCs into HIOs and demonstrated that obtained HIOs contain a polarized epithelium with tight junctions consisting of enterocytes, goblet cells, enteroendocrine cells and Paneth cells. We extensively characterized the gene expression of CYPs and activity of CYP3A4/5, indicating relatively high gene expression levels of the most important intestinal CYP enzymes in HIOs compared to the other models. Furthermore, we showed that CYP1A1 and CYP1B1 were induced by β-naphtoflavone in all three models, whereas CYP3A4 was induced by phenobarbital and rifampicin in HIOs, in the IEC-based model (although not statistically significant), but not in Caco-2 cells. Interestingly, CYP2B6 expression was not induced in any of the models by the well-known liver CYP2B6 inducer phenobarbital. In conclusion, our study indicates that hiPSC-based HIOs are a useful in vitro intestinal model to study biotransformation of chemicals in the intestine. Electronic supplementary material The online version of this article (10.1007/s00204-020-02953-6) contains supplementary material, which is available to authorized users.

The Drosophila adult midgut is a model epithelial tissue composed of a few major cell types with distinct regional identities. One of the limitations to its analysis is the lack of tools to manipulate gene expression based on these regional identities. To overcome this obstacle, we applied the intersectional split-GAL4 system to the adult midgut and report 653 driver combinations that label cells by region and cell type. We first identified 424 split-GAL4 drivers with midgut expression from ∼7300 drivers screened, and then evaluated the expression patterns of each of these 424 when paired with three reference drivers that report activity specifically in progenitor cells, enteroendocrine cells, or enterocytes. We also evaluated a subset of the drivers expressed in progenitor cells for expression in enteroblasts using another reference driver. We show that driver combinations can define novel cell populations by identifying a driver that marks a distinct subset of enteroendocrine cells expressing genes usually associated with progenitor cells. The regional cell type patterns associated with the entire set of driver combinations are documented in a freely available website, providing information for the design of thousands of additional driver combinations to experimentally manipulate small subsets of intestinal cells. In addition, we show that intestinal enhancers identified with the split-GAL4 system can confer equivalent expression patterns on other transgenic reporters. Altogether, the resource reported here will enable more precisely targeted gene expression for studying intestinal processes, epithelial cell functions, and diseases affecting self-renewing tissues.

Background/objectives Distribution and activity of ghrelin cells in the stomach of obese subjects are controversial. Subjects/methods We examined samples from stomachs removed by sleeve gastrectomy in 49 obese subjects (normoglycemic, hyperglycemic and diabetic) and quantified the density of ghrelin/chromogranin endocrine cells by immunohistochemistry. Data were compared with those from 13 lean subjects evaluated by gastroscopy. In 44 cases (11 controls and 33 obese patients) a gene expression analysis of ghrelin and its activating enzyme ghrelin O-acyl transferase (GOAT) was performed. In 21 cases (4 controls and 17 obese patients) the protein levels of unacylated and acylated-ghrelin were measured by ELISA tests. In 18 cases (4 controls and 14 obese patients) the morphology of ghrelin-producing cells was evaluated by electron microscopy. Results The obese group, either considered as total population or divided into subgroups, did not show any significant difference in ghrelin cell density when compared with control subjects. Inter-glandular smooth muscle fibres were increased in obese patients. In line with a positive trend of the desacylated form found by ELISA, Ghrelin and GOAT mRNA expression in obese patients was significantly increased. The unique ghrelin cell ultrastructure was maintained in all obese groups. In the hyperglycemic obese patients, the higher ghrelin expression matched with ultrastructural signs of endocrine hyperactivity, including expanded rough endoplasmic reticulum and reduced density, size and electron-density of endocrine granules. A positive correlation between ghrelin gene expression and glycemic values, body mass index and GOAT was also found. All obese patients with type 2 diabetes recovered from diabetes at follow-up after 5 months with a 16.5% of weight loss. Conclusions Given the known inhibitory role on insulin secretion of ghrelin, these results suggest a possible role for gastric ghrelin overproduction in the complex architecture that takes part in the pathogenesis of type 2 diabetes.

The endocrine and exocrine pancreas have been studied separately by endocrinologists and gastroenterologists as two organ systems. The pancreatic islet, consisting of 1–2% mass of the whole pancreas, has long been believed to be regulated independently from the surrounding exocrine tissues. Particularly, islet blood flow has been consistently illustrated as one-way flow from arteriole(s) to venule(s) with no integration of the capillary network between the endocrine and exocrine pancreas. It is likely linked to the long-standing dogma that the rodent islet has a mantle of non–β-cells and that the islet is completely separated from the exocrine compartment. A new model of islet microcirculation is built on the basis of analyses of in vivo blood flow measurements in mice and an in situ three-dimensional structure of the capillary network in mice and humans. The deduced integrated blood flow throughout the entire pancreas suggests direct interactions between islet endocrine cells and surrounding cells as well as the bidirectional blood flow between the endocrine and exocrine pancreas, not necessarily a unidirectional blood flow as in a so-called insuloacinar portal system. In this perspective, we discuss how this conceptual transformation could potentially affect our current understanding of the biology, physiology, and pathogenesis of the islet and pancreas.

Context The mechanisms underlying Roux-en-Y gastric bypass (RYGB) surgery-induced weight loss and the immediate postoperative beneficial metabolic effects associated with the operation remain uncertain. Enteroendocrine cell (EEC) secretory function has been proposed as a key factor in the marked metabolic benefits from RYGB surgery. Objective To identify novel gut-derived peptides with therapeutic potential in obesity and/or diabetes by profiling EEC-specific molecular changes in obese patients following RYGB-induced weight loss. Subjects and Methods Genome-wide expression analysis was performed in isolated human small intestinal EECs obtained from 20 gut-biopsied obese subjects before and after RYGB. Targets of interest were profiled for preclinical and clinical metabolic effects. Results Roux-en-Y gastric bypass consistently increased expression levels of the inverse ghrelin receptor agonist, liver-expressed antimicrobial peptide 2 (LEAP2). A secreted endogenous LEAP2 fragment (LEAP238-47) demonstrated robust insulinotropic properties, stimulating insulin release in human pancreatic islets comparable to the gut hormone glucagon-like peptide-1. LEAP238-47 showed reciprocal effects on growth hormone secretagogue receptor (GHSR) activity, suggesting that the insulinotropic action of the peptide may be directly linked to attenuation of tonic GHSR activity. The fragment was infused in healthy human individuals (n = 10), but no glucoregulatory effect was observed in the chosen dose as compared to placebo. Conclusions Small intestinal LEAP2 expression was upregulated after RYGB. The corresponding circulating LEAP238-47 fragment demonstrated strong insulinotropic action in vitro but failed to elicit glucoregulatory effects in healthy human subjects.

Small intestinal neuroendocrine tumors (SI-NET) are serotonin-secreting well-differentiated neuroendocrine tumors of putative enterochromaffin (EC) cell origin. Recent studies recognize a subset of EC cells that is label-retaining at the +4 position in the crypt and functions as a reserve intestinal stem cell. Importantly, this +4 reserve EC cell subset not only contributes to regeneration of the intestinal epithelium during injury and inflammation but also to basal crypt homeostasis at a constant rate. The latter function suggests that the +4 EC cell subset serves as an active reserve stem cell via a constant rate of dedifferentiation. Characterization of early tumor formation of SI-NET, observed as crypt-based EC cell clusters in many cases of familial SI-NETs, suggests that the +4 active reserve EC cell subset is the cell of origin. This newly discovered active reserve stem cell property of EC cells can account for unique biological mechanisms and processes associated with the genesis and development of SI-NETs. The recognition of this property of the +4 active reserve EC cell subset may provide novel opportunities to explore NETs in the gastrointestinal tract and other organs.

Introduction. Parasympathetic fibers innervating the pancreas are involved in the regulation of both exo-crine and endocrine function, in the regulation of endocrine cell proliferation, and are also implicated in the pathogenesis of type 1 diabetes. Nonetheless, data concerning the distribution of parasympathetic fibers within the human pancreas in prenatal development are absent in the literature. Our aim was to evaluate the possibility of using the markers of cholinergic neurons and nerve fibers, namely vesicular acetylcholine transporter (VAChT) and acetylcholinesterase (AChE) in studies of prenatal develop-ment of parasympathetic innervation of the human pancreas. Materials and methods. The study was performed on 10 autopsies of the fetal pancreas (gestational age 10-34 weeks) using immunoperoxidase labeling with antibodies to VAChT and AChE. Results. Immunopositive reaction to AChE was detected in bundles of nerve fibers of various diameters, networks of thin nerve fibers as well as in individual neurons of the intramural ganglia. The structures of the nervous system were immunonegative to VAChT. In the exocrine pancreas, that is, in the interlobular connective tissue, near the ducts and inside the forming lobules, thin cholinergic fibers prevailed on the studied developmental periods. In pancreatic islets, cholinergic fibers were detected less frequently and were located at the periphery.Immunopositive reaction with antibodies to AChE and mouse monoclonal antibodies to VAChT was also detected in some endocrine cells in the pancreatic islets. Conclusion. We have shown that antibodies to AChE detect cholinergic neurons and nerve fibers in the developing human pancreas. We have also demonstrated that in the fetal pancreas thin cholinergic fibers prevail in the exocrine part and rarely are detected in the pancreatic islets, which is typical in adults. The results showing the VAChT and AChE immunoreactivity in the endocrine cells of fetal pancreatic islets are in agreement with data obtained in the adult human pancreas and suggest that the endocrine cells can be a source of acetylcholine. Keywords: pancreas, human development, parasympathetic innervation, VAChT, AChE

Recent studies have revealed the presence of zinc and the expression of zinc transporter (ZnT) family members in most endocrine cell types. It was demonstrated that ZnT family plays an important role in the synthesis and secretion of many hormones. Moreover, recently ZnT8 was described as a newly islet autoantigen in type 1 diabetes.We studied the expression of ZnT8 transporter in thyroid tissues from patients with immune and non-immune thyroid diseases. The study was performed in thyroid tissues after thyroidectomy from patients with thyroid non-toxic nodular goitre (NTNG

Serotonin (5-hydroxytryptamine [5-HT]) synthesized and released in enterochromaffin (EC) cells participates in various functions in the gastrointestinal tract by acting on a diverse range of 5-HT receptors (HTRs) expressed on smooth muscle, enteric neurons, and epithelial cells. We previously observed that genes encoding HTR2A, HTR2B, and HTR4 are expressed in murine intestinal organoids, suggesting the expression of these HTRs in intestinal epithelial cells. The present study investigated the localization of these HTRs in the murine intestine by immunofluorescence staining. HTR2A, HTR2B, and HTR4 localized in individual solitary cells in the epithelium, while HTR2C was observed in the lamina propria. In the epithelium, HTR2A, HTR2B, and HTR4 colocalized with 5-HT, and HTR4 colocalized with glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). Murine intestinal organoids show a colocalization pattern that is similar to in vivo HTR2A and HTR4 with 5-HT, GLP-1, and PYY. Intraperitoneal and intragastric administration of tegaserod, an HTR4 agonist, failed to alter plasma GLP-1 levels in fasted mice. However, intragastric but not intraperitoneal administration of tegaserod reduced dietary lipid-induced increases of plasma GLP-1 levels. This action of tegaserod was inhibited by co-administration of RS39604, an HTR4 antagonist. These results suggest that murine ileal GLP-1/PYY-producing enteroendocrine (EE) cells express HTR4, while 5-HT-producing EC cells express HTR2A, HTR2B, and HTR4. In addition, the observations regarding in vivo GLP-1 secretion suggest that HTR4 signaling in ileal EE cells suppresses dietary lipid-induced GLP-1 secretion. We thus propose that EC and EE cells may interact with each other through paracrine signaling mechanisms.

Farm animals play an important role in translational research as large animal models of the gastrointestinal (GI) tract. The mechanistic investigation of zoonotic diseases of the GI tract, in which animals can act as asymptomatic carriers, could provide important information for therapeutic approaches. In veterinary medicine, farm animals are no less relevant, as they can serve as models for the development of diagnostic and therapeutic approaches of GI diseases in the target species. However, farm animal-derived cell lines of the intestinal epithelium are rarely available from standardised cell banks and, in addition, are not usually specific for certain sections of the intestine. Immortalised porcine or bovine enterocytic cell lines are more widely available, compared to goat or sheep-derived cell lines; no continuous cell lines are available from the chicken. Other epithelial cell types with intestinal section-specific distribution and function, such as goblet cells, enteroendocrine cells, Paneth cells and intestinal stem cells, are not represented in those cell line-based models. Therefore, intestinal organoid models of farm animal species, which are already widely used for mice and humans, are gaining importance. Crypt-derived or pluripotent stem cell-derived intestinal organoid models offer the possibility to investigate the mechanisms of inter-cell or host–pathogen interactions and to answer species-specific questions. This review is intended to give an overview of cell culture models of the intestinal epithelium of farm animals, discussing species-specific differences, culture techniques and some possible applications for intestinal organoid models. It also highlights the need for species-specific pluripotent stem cell-derived or crypt-derived intestinal organoid models for promotion of the Three Rs principles ( replacement, reduction and refinement).

The ability to absorb ingested nutrients is an essential function of all metazoans and utilizes a wide array of nutrient transporters found on the absorptive enterocytes of the small intestine. A unique population of patients has previously been identified with severe congenital malabsorptive diarrhea upon ingestion of any enteral nutrition. The intestines of these patients are macroscopically normal, but lack enteroendocrine cells (EECs), suggesting an essential role for this rare population of nutrient-sensing cells in regulating macronutrient absorption. Here, we use human and mouse models of EEC deficiency to identify an unappreciated role for the EEC hormone peptide YY in regulating ion-coupled absorption of glucose and dipeptides. We find that peptide YY is required in the small intestine to maintain normal electrophysiology in the presence of vasoactive intestinal polypeptide, a potent stimulator of ion secretion classically produced by enteric neurons. Administration of peptide YY to EEC-deficient mice restores normal electrophysiology, improves glucose and peptide absorption, diminishes diarrhea and rescues postnatal survival. These data suggest that peptide YY is a key regulator of macronutrient absorption in the small intestine and may be a viable therapeutic option to treat patients with electrolyte imbalance and nutrient malabsorption., Enteroendocrine cells (EECs) are specialized gastrointestinal cells that have a role in nutrient sensing and hormone secretion. Here the authors show that peptide YY from EECs regulates nutrient absorption in intestinal organoids.

Aim: The aim of the study was to identify the distribution pattern and frequency of endocrine cell types in the digestive tract of Varanus salvator. Materials and Methods: The presence of endocrine cells (glucagon, somatostatin, and serotonin) in the digestive tract (esophagus, stomach, and intestine) was detected using the avidin-biotin complex (ABC) method. Results: Three types of endocrine cells immunoreactive to antisera glucagon, serotonin, and somatostatin were found in the caudal portion of the small and large intestines but were not observed in the esophagus, stomach, and caput and medial sections of the small intestine. Endocrine cells distributed in the digestive tract of V. salvator vary in color intensity, from weak to sharp, in response to the primer antibody. Conclusion: Endocrine cells in the digestive tract that is immunoreactive to glucagon, somatostatin, and serotonin are those found in the caudal portion of the small and large intestines. They are varied in distribution pattern, frequency, and color intensity.

Background The incretin hormone glucagon-like peptide-l (GLP-1) is an important regulator of post-prandial insulin secretion, acting through a G protein-coupled cell surface receptor (GLP-1R). In addition to its expression in pancreatic β-cells, several studies suggested that GLP-1R is located in extra-pancreatic tissues. Objectives In this study, we examined for the first time the testicular distribution of the GLP-1R, both in normal human and neoplastic testicular tissues as well as in rodent testis and rodent testicular cell lines. Methods and methods The GLP-1R distribution in testicular section has been evaluated by immunohistochemistry, the specificity of IHC was validated by demonstrating a positive staining for GLP-1RmRNA by RISH technology. While GLP-1R expression in terms of protein was detected by western blot analysis, Moreover, mRNA levels were determined in human testis, in rodent Leydig, and Sertoli cell lines. Results Using immunohistochemistrya specific staining for GLP-1R was detected in Leydig cells. The specificity of IHC was validated by demonstrating a positive staining for GLP-1RmRNA only in these cell types. Species differences in the GLP-1R expression between humans and rodents were observed. Interestingly, a decreased expression of the receptor in rodent tumor Leydig cell line and an absence in human Leydig tumor samples was detected. Discussion It may be hypothesized that GLP-1R acts like an oncosuppressor in Leydig tumors. A role in regulation of hormone secretion by GLP-1 has been shown in other endocrine cells, therefore we hypothesized that GLP-1R is able to modulate somehow the Leydig cell function. Conclusion In our findings, a careful evaluation of human testicular tissues and rodent testis revealed Leydig cells as a potential target for GLP-1. Collectively, an effect of GLP-1R in Leydig cell function may be presumed although future studies are needed to ascertain the GLP-1R's role both in normal and tumor Leydig cells.

Pulmonary neuroendocrine cells (PNECs) – a unique cell population identified at all levels in the epithelium of the respiratory tract, histophysiology of which is still poorly understood. Given its important role as one of the main regulators of the respiration processes and homeostasis, its studying is one of the urgent tasks of medicine. According to the International Terms for Human Cytology and Histology published by the Federative International Committee on Anatomical Terminology (FICAT) under the writing of Wolters Kluwer and Lippincott Williams & Wilkins (2008), these cells are called respiratory neuroendocrine cells (in the trachea) or respiratory endocrine cells (in the bronchial tree). However, these cells have documented in modern international scientific literature as pulmonary neuroendocrine cells. The aim of this work is to analyze the modern scientific literature data on the effect of PNECs on the lung homeostasis in normal and pathological conditions. PNECs and their clusters – neuroepithelial bodies act as factors that regulate lung growth and maturation in embryogenesis via secretion of serotonin and gastrin-releasing hormone. In postnatal ontogenesis, PNEC secretion products – amines and neuropeptides, are characterized by participation in various physiological and pathological processes in the lung. PNECs normally maintain neurohumoral control over vascular and airway smooth muscle tone, act as peripheral chemoreceptors, and also are responsible for regulation of cell proliferation, differentiation, and mucus production from the respiratory epithelium. In case of respiratory tract damage, PNECs are capable of transdifferentiation by activating the Notch signaling pathway and renewal of other cellular types of respiratory epithelium. PNECs have a neuroimmunomodulating effect by means of neuropeptides and neurotransmitters secretion, which maintain and enhance the airways inflammatory response to an allergen. After the allergen exposition, PNECs activate type 2 innate lymphoid cells (ILC2) which being modulated by the neuropeptide CGRP produce type 2 cytokines IL-5 and IL-13, thereby contributing to an allergic response in the airways. At the same time, secreted by the PNECs neurotransmitter γ-aminobutyric acid (GABA) interacts with IL-13 to activate goblet cell mucus secretion. ILC2 induce eosinophilic inflammation and airways hypersensitivity. Recent studies have shown that ILC2 cells also stimulate Th2-associated immune response. Thus, CGRP and GABA are the key products of PNEC, which stimulate the Th2-associated immune response in the lung. Conclusions. Pulmonary neuroendocrine cells together with immune cells form a neuroimmunological module for the reception and response to environmental chemoattractants. The data on the role of pulmonary neuroendocrine cells in the airways allergic inflammation are still controversial in the literature, which necessitates further study of this issue.

The islet of Langerhans contains at least five types of endocrine cells producing distinct hormones. In response to nutrient or neuronal stimulation, islet endocrine cells release biochemicals including peptide hormones to regulate metabolism and to control glucose homeostasis. It is now recognized that malfunction of islet cells, notably insufficient insulin release of β-cells and hypersecretion of glucagon from α-cells, represents a causal event leading to hyperglycemia and frank diabetes, a disease that is increasing at an alarming rate to reach an epidemic level worldwide. Understanding the mechanisms regulating stimulus-secretion coupling and investigating how islet β-cells maintain a robust secretory activity are important topics in islet biology and diabetes research. To facilitate such studies, a number of biological systems and assay platforms have been developed for the functional analysis of islet cells. These technologies have enabled detailed analyses of individual islets at the cellular level, either in vitro, in situ, or in vivo.

The secretory pathway of neurons and endocrine cells contains a variety of mechanisms designed to combat cellular stress. These include not only the unfolded protein response pathways but also diverse chaperone proteins that collectively work to ensure proteostatic control of secreted and membrane-bound molecules. One of the least studied of these chaperones is the neural- and endocrine-specific molecule known as proSAAS. This small chaperone protein acts as a potent anti-aggregant both in vitro and in cellulo and also represents a cerebrospinal fluid biomarker in Alzheimer’s disease. In the present study, we have examined the idea that proSAAS, like other secretory chaperones, might represent a stress-responsive protein. We find that exposure of neural and endocrine cells to the cell stressors tunicamycin and thapsigargin increases cellular proSAAS mRNA and protein in Neuro2A cells. Paradoxically, proSAAS secretion is inhibited by these same drugs. Exposure of Neuro2A cells to low concentrations of the hypoxic stress inducer cobalt chloride, or to sodium arsenite, an oxidative stressor, also increases cellular proSAAS content and reduces its secretion. We conclude that the cellular levels of the small secretory chaperone proSAAS are positively modulated by cell stress.

INTRODUCTION: The intestinal epithelium contains specialized cells including enterocytes, goblet, Paneth, enteroendocrine, and stem cells. Impaired barrier integrity in Inflammatory Bowel Disease is characterized by elevated levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α). Prior studies in immortalized lines such as Caco-2, without native epithelial heterogeneity, demonstrate the amelioration of TNF-α compromised barrier integrity via nicotinic (nAChR) or muscarinic (mAChR) acetylcholine receptor activation. METHODS: A tissue-engineered model of primary human small intestinal epithelium was derived from dissociated organoids cultured on collagen-coated Transwells. Differentiation was accomplished with serum-containing media and compared to Caco-2 and HT-29 regarding alkaline phosphatase expression, transepithelial electrical resistance (TEER), and IL-8 secretion. Inflammation was modeled via basal stimulation with TNF-α (25 ng/mL) with or without nicotine (nAChR agonist) or bethanechol (mAChR agonist). Apoptosis, density (cells/cm(2)), TEER, lucifer yellow permeability, 70 kDa dextran transport, cell morphology, and IL-8 secretion were characterized. RESULTS: Primary intestinal epithelium demonstrates significant functional differences compared to immortalized cells, including increased barrier integrity, IL-8 expression, mucus production, and the presence of absorptive and secretory cells. Exposure to TNF-α impaired barrier integrity, increased apoptosis, altered morphology, and increased secretion of IL-8. Stimulation of nAChR with nicotine did not ameliorate TNF-α induced permeability nor alter 70 kDa dextran transport. However, stimulation of mAChR with bethanechol decreased transport of 70 kDa dextran but did not ameliorate TNF-α induced paracellular permeability. CONCLUSIONS: A primary model of intestinal inflammation was evaluated, demonstrating nAChR or mAChR activation does not have the same protective effects compared to immortalized epithelium. Inclusion of other native stromal support cells are underway. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12195-020-00633-0) contains supplementary material, which is available to authorized users.

Objective When total pancreatectomy with islet autotransplantation (TPIAT) is performed for chronic pancreatitis, the pancreas and most of the duodenum are removed, with Roux-en-Y reconstruction of the gastrointestinal tract. Enteroendocrine cells in the intestines and pancreas secrete hormones coordinating digestion and motility, but anatomic reconstruction alters transit of nutrients to these cells. We hypothesized that TPIAT leads to changes in enteroendocrine hormones. Methods Glucagon-like peptide 1 (GLP-1), peptide YY (PYY), and pancreatic polypeptide (PP) were measured from mixed-meal tolerance tests of 34 clinical trial participants before and 18 months after TPIAT. Area under the curve of GLP-1 and PYY-stimulated responses were calculated by trapezoidal method, and the PP response was measured as the stimulated max minus baseline (ΔPP). Results Area under the curve of GLP-1 and PYY increased significantly after TPIAT (GLP-1 average +553.1 pg/mL per minute, P = 0.004; PYY average +4647.9 pg/mL per minute, P = 0.02). ΔPP trended toward lower after TPIAT (average, -52.2 pg/mL, P = 0.06). Conclusions In this novel study of enteroendocrine hormones in TPIAT patients, stimulated levels of GLP-1 and PYY were significantly higher after versus before TPIAT. ΔPP was lower after TPIAT, but not significantly. These hormone changes have potential clinical implications that warrant further research.