Duodenal enteroendocrine cells and GIP as treatment targets for obesity and type 2 diabetes.

The duodenum is an important source of endocrine and paracrine signals controlling digestion and nutrient disposition, notably including the main incretin hormone glucose-dependent insulinotropic polypeptide (GIP). Bariatric procedures that prevent nutrients from contact with the duodenal mucosa are particularly effective interventions to reduce body weight and improve glycaemic control in obesity and type 2 diabetes. These procedures take advantage of increased nutrient delivery to more distal regions of the intestine which enhances secretion of the other incretin hormone glucagon-like peptide-1 (GLP-1). Preclinical experiments have shown that either an increase or a decrease in the secretion or action of GIP can decrease body weight and blood glucose in obesity and non-insulin dependent hyperglycaemia, but clinical studies involving administration of GIP have been inconclusive. However, a synthetic dual agonist peptide (tirzepatide) that exerts agonism at receptors for GIP and GLP-1 has produced marked weight-lowering and glucose-lowering effects in people with obesity and type 2 diabetes. This appears to result from chronic biased agonism in which the novel conformation of the peptide triggers enhanced signalling by the GLP-1 receptor through reduced internalisation while reducing signalling by the GIP receptor directly or via functional antagonism through increased internalisation and degradation.

Genetic drivers of heterogeneity in type 2 diabetes pathophysiology.

Type 2 diabetes (T2D) is a heterogeneous disease that develops through diverse pathophysiological processes1,2 and molecular mechanisms that are often specific to cell type3,4. Here, to characterize the genetic contribution to these processes across ancestry groups, we aggregate genome-wide association study data from 2,535,601 individuals (39.7% not of European ancestry), including 428,452 cases of T2D. We identify 1,289 independent association signals at genome-wide significance (P < 5 × 10-8) that map to 611 loci, of which 145 loci are, to our knowledge, previously unreported. We define eight non-overlapping clusters of T2D signals that are characterized by distinct profiles of cardiometabolic trait associations. These clusters are differentially enriched for cell-type-specific regions of open chromatin, including pancreatic islets, adipocytes, endothelial cells and enteroendocrine cells. We build cluster-specific partitioned polygenic scores5 in a further 279,552 individuals of diverse ancestry, including 30,288 cases of T2D, and test their association with T2D-related vascular outcomes. Cluster-specific partitioned polygenic scores are associated with coronary artery disease, peripheral artery disease and end-stage diabetic nephropathy across ancestry groups, highlighting the importance of obesity-related processes in the development of vascular outcomes. Our findings show the value of integrating multi-ancestry genome-wide association study data with single-cell epigenomics to disentangle the aetiological heterogeneity that drives the development and progression of T2D. This might offer a route to optimize global access to genetically informed diabetes care.

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.

A hydrolyzed casein diet promotes Ngn3 controlling enteroendocrine cell differentiation to increase gastrointestinal motility in mice.

Gut hormones are produced by enteroendocrine cells (EECs) found along the intestinal epithelium, and these cells play a crucial role in regulating intestinal function, nutrient absorption and food intake. A hydrolyzed casein diet has been reported to promote the secretion of gut hormones through the regulation of EEC development, but the underlying mechanism remains unclear. Therefore, this study was conducted to investigate whether the hydrolyzed casein diet can regulate EEC differentiation by employing mouse and organoid models. Mice were fed diets containing either casein (casein group) or hydrolyzed casein (hydrolyzed casein group) as the sole protein source. The hydrolyzed casein diet upregulated the expression of transcription factors, induced EEC differentiation, increased fasting serum ghrelin concentrations and promoted gastrointestinal (GI) motility in the duodenum compared to the casein diet. Interestingly, these differences could be abolished when there is addition of antibiotics to the drinking water, suggesting a significant role of gut microbiota in the hydrolyzed casein-mediated EEC function. Further investigation showed that the hydrolyzed casein diet led to reduced microbial diversity, especially the abundance of Akkermansia muciniphila (A. muciniphila) on the duodenal mucosa. In contrast, gavage with A. muciniphila impaired EEC differentiation through attenuated neurog3 transcription factor (Ngn3) expression, mediated through the promotion of Notch signaling. Moreover, pasteurized A. muciniphila showed similar effects to enter organoids in vitro. Overall, we found that a hydrolyzed casein diet reduced the abundance of A. muciniphila and promoted Ngn3 controlling EEC differentiation and this pathway is associated with increased GI motility in mice. The findings provide new insights into the role of hydrolyzed casein in gut transit and guidelines for using hydrolyzed casein in safe formula milk.

An Approach to Intersectionally Target Mature Enteroendocrine Cells in the Small Intestine of Mice.

Enteroendocrine cells (EECs) constitute only a small proportion of Villin-1 (Vil1)-expressing intestinal epithelial cells (IECs) of the gastrointestinal tract; yet, in sum, they build the largest endocrine organ of the body, with each of them storing and releasing a distinct set of peptides for the control of feeding behavior, glucose metabolism, and gastrointestinal motility. Like all IEC types, EECs are continuously renewed from intestinal stem cells in the crypt base and terminally differentiate into mature subtypes while moving up the crypt-villus axis. Interestingly, EECs adjust their hormonal secretion according to their migration state as EECs receive altering differentiation signals along the crypt-villus axis and thus undergo functional readaptation. Cell-specific targeting of mature EEC subtypes by specific promoters is challenging because the expression of EEC-derived peptides and their precursors is not limited to EECs but are also found in other organs, such as the brain (e.g., Cck and Sst) as well as in the pancreas (e.g., Sst and Gcg). Here, we describe an intersectional genetic approach that enables cell type-specific targeting of functionally distinct EEC subtypes by combining a newly generated Dre-recombinase expressing mouse line (Vil1-2A-DD-Dre) with multiple existing Cre-recombinase mice and mouse strains with rox and loxP sites flanked stop cassettes for transgene expression. We found that transgene expression in triple-transgenic mice is highly specific in I but not D and L cells in the terminal villi of the small intestine. The targeting of EECs only in terminal villi is due to the integration of a defective 2A separating peptide that, combined with low EEC intrinsic Vil1 expression, restricts our Vil1-2A-DD-Dre mouse line and the intersectional genetic approach described here only applicable for the investigation of mature EEC subpopulations.

RET Signaling Persists in the Adult Intestine and Stimulates Motility by Limiting PYY Release From Enteroendocrine Cells.

BACKGROUND & AIMS: RET tyrosine kinase is necessary for enteric nervous system development. Loss-of-function RET mutations cause Hirschsprung disease (HSCR), in which infants are born with aganglionic bowel. Despite surgical correction, patients with HSCR often experience chronic defecatory dysfunction and enterocolitis, suggesting that RET is important after development. To test this hypothesis, we determined the location of postnatal RET and its significance in gastrointestinal (GI) motility. METHODS: RetCFP/+ mice and human transcriptional profiling data were studied to identify the enteric neuronal and epithelial cells that express RET. To determine whether RET regulates gut motility in vivo, genetic, and pharmacologic approaches were used to disrupt RET in all RET-expressing cells, a subset of enteric neurons, or intestinal epithelial cells. RESULTS: Distinct subsets of enteric neurons and enteroendocrine cells expressed RET in the adult intestine. RET disruption in the epithelium, rather than in enteric neurons, slowed GI motility selectively in male mice. RET kinase inhibition phenocopied this effect. Most RET+ epithelial cells were either enterochromaffin cells that release serotonin or L-cells that release peptide YY (PYY) and glucagon-like peptide 1 (GLP-1), both of which can alter motility. RET kinase inhibition exaggerated PYY and GLP-1 release in a nutrient-dependent manner without altering serotonin secretion in mice and human organoids. PYY receptor blockade rescued dysmotility in mice lacking epithelial RET. CONCLUSIONS: RET signaling normally limits nutrient-dependent peptide release from L-cells and this activity is necessary for normal intestinal motility in male mice. These effects could contribute to dysmotility in HSCR, which predominantly affects males, and uncovers a mechanism that could be targeted to treat post-prandial GI dysfunction.

Fundamental Neurochemistry Review: The role of enteroendocrine cells in visceral pain.

While visceral pain is commonly associated with disorders of the gut-brain axis, underlying mechanisms are not fully understood. Dorsal root ganglion (DRG) neurons innervate visceral structures and undergo hypersensitization in inflammatory models. The characterization of peripheral DRG neuron terminals is an active area of research, but recent work suggests that they communicate with enteroendocrine cells (EECs) in the gut. EECs sense stimuli in the intestinal lumen and communicate information to the brain through hormonal and electrical signaling. In that context, EECs are a target for developing therapeutics to treat visceral pain. Linaclotide is an FDA-approved treatment for chronic constipation that activates the intestinal membrane receptor guanylyl cyclase C (GUCY2C). Clinical trials revealed that linaclotide relieves both constipation and visceral pain. We recently demonstrated that the analgesic effect of linaclotide reflects the overexpression of GUCY2C on neuropod cells, a specialized subtype of EECs. While this brings some clarity to the relationship between linaclotide and visceral analgesia, questions remain about the intracellular signaling mechanisms and neurotransmitters mediating this communication. In this Fundamental Neurochemistry Review, we discuss what is currently known about visceral nociceptors, enteroendocrine cells, and the gut-brain axis, and ongoing areas of research regarding that axis and visceral pain.

Colonic L-cell impairment in aged subjects with type 2 diabetes leads to diminished GLP-1 production.

AIMS: Glucagon-like peptide 1 (GLP-1) is produced by the L subtype of enteroendocrine cells (EECs). Patients with type 2 diabetes (T2D) exhibit reduced incretin effect, but the pathophysiology and functional change of the L-cells remain unclear. Deciphering the mechanisms of the biological changes in L-cells under T2D conditions may assist in the research of gut-based strategies for T2D therapy. METHODS: We investigated the fasting serum GLP-1 levels and the distribution of colonic L-cells in young and aged participants with and without T2D. Additionally, we established an aged male T2D Wistar rat model subjected to a long-term high-fat and high-fructose (HFHF) diet. Histological investigations and single-cell RNA sequencing (scRNA-seq) analyses were performed to explore the mechanisms underlying functional changes in the colonic EECs. RESULTS: We observed a decline in circulating GLP-1 levels and a reduced number of colonic L-cells in elderly patients with T2D. The mechanisms underlying impaired L-cell formation and disturbed GLP-1 production were revealed using aged T2D rats induced by a long-term HFHF diet. The scRNA-seq results showed that the transcription factors that regulate L-cell commitment, such as Foxa1, were downregulated, and the expression of genes that participate in encoding GLP-1, GLP-1 posttranslational processing, hormone secretion, and nutrient sensing was disturbed. CONCLUSIONS: Taken together, the reduced L-cell lineage commitment and disturbed L-cell functions might be the major cause of the reduced GLP-1 production in aged populations with T2D. Our study provides new insights for identifying novel targets in colonic L-cells for improving endogenous GLP-1 production.

Disruptions in cell fate decisions and transformed enteroendocrine cells drive intestinal tumorigenesis in Drosophila.

Most epithelial tissues are maintained by stem cells that produce the different cell lineages required for proper tissue function. Constant communication between different cell types ensures precise regulation of stem cell behavior and cell fate decisions. These cell-cell interactions are often disrupted during tumorigenesis, but mechanisms by which they are co-opted to support tumor growth in different genetic contexts are poorly understood. Here, we introduce PromoterSwitch, a genetic platform we established to generate large, transformed clones derived from individual adult Drosophila intestinal stem/progenitor cells. We show that cancer-driving genetic alterations representing common colon tumor genome landscapes disrupt cell fate decisions within transformed tissue and result in the emergence of abnormal cell fates. We also show that transformed enteroendocrine cells, a differentiated, hormone-secreting cell lineage, support tumor growth by regulating intestinal stem cell proliferation through multiple genotype-dependent mechanisms, which represent potential vulnerabilities that could be exploited for therapy.

Unbiased transcription factor CRISPR screen identifies ZNF800 as master repressor of enteroendocrine differentiation.

Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach, small intestine (SI), and colon. EECs regulate aspects of metabolic activity, including insulin levels, satiety, gastrointestinal secretion, and motility. The generation of different EEC lineages is not completely understood. In this work, we report a CRISPR knockout screen of the entire repertoire of transcription factors (TFs) in adult human SI organoids to identify dominant TFs controlling EEC differentiation. We discovered ZNF800 as a master repressor for endocrine lineage commitment, which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine TF network centered on PAX4. Thus, organoid models allow unbiased functional CRISPR screens for genes that program cell fate.

Region-specific remodeling of the enteric nervous system and enteroendocrine cells in the colon of spinal cord injury patients.

Patients with spinal cord injury (SCI) suffer from major bowel dysfunction, whose exact pathophysiology, particularly the involvement of the enteric nervous system or epithelial dysfunction is poorly understood. Herein, we aimed to characterize the mucosal biopsies of the right and left colon in SCI patients vs controls (CT): (1) remodeling of key enteric neurotransmitters, (2) remodeling of enteroendocrine cells, and (3) mucosal inflammation compared to those in controls. In SCI, mucosal ACh concentration was lower in the right colon as compared to CT, but no change was observed in the left colon, and AChE expression was lower in both the right and left colons than in CT. While the VIP concentration was similar in the right and left colons, VIP mRNA expression was increased in the right colon and decreased in the left colon, in SCI patients as compared to CT. Interestingly, 5-HT concentration was reduced in the left colon but not in the right colon in SCI patients. Moreover, in SCI patients, as compared to CT, SERT mRNA expression was selectively increased in the left colon while TPH1 mRNA expression was increased in the right and left colons. Although mucosal TNFα and IL-1ß mRNA expression did not significantly differ between SCI and CT groups, we identified a significant positive correlation between TNFα and IL-1ß mRNA expression and left colon transit time in the SCI group. In conclusion, region-specific changes occur in the enteric neurotransmitter, serotonergic, and inflammatory pathways in the colon of SCI patients. The significant correlations between these pathways and clinical parameters in the left colon further set a scientific basis for designing therapeutic targets to improve colonic motor dysfunction in patients.Biobank information: Spinal cord injury patients: PHRC ConstiCAPE-clinical trial NCT02566746. Controls: Anosain-clinical trial NCT03054415 and biobank of the "Institut des Maladies de l'Appareil Digestif (IMAD)" registered under number DC-2008-402.

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.

Nutrient-driven dedifferentiation of enteroendocrine cells promotes adaptive intestinal growth in Drosophila.

Post-developmental organ resizing improves organismal fitness under constantly changing nutrient environments. Although stem cell abundance is a fundamental determinant of adaptive resizing, our understanding of its underlying mechanisms remains primarily limited to the regulation of stem cell division. Here, we demonstrate that nutrient fluctuation induces dedifferentiation in the Drosophila adult midgut to drive adaptive intestinal growth. From lineage tracing and single-cell RNA sequencing, we identify a subpopulation of enteroendocrine (EE) cells that convert into functional intestinal stem cells (ISCs) in response to dietary glucose and amino acids by activating the JAK-STAT pathway. Genetic ablation of EE-derived ISCs severely impairs ISC expansion and midgut growth despite the retention of resident ISCs, and in silico modeling further indicates that EE dedifferentiation enables an efficient increase in the midgut cell number while maintaining epithelial cell composition. Our findings identify a physiologically induced dedifferentiation that ensures ISC expansion during adaptive organ growth in concert with nutrient conditions.

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.

Dietary Vitamin A Affects the Function of Incretin-Producing Enteroendocrine Cells in Male Mice Fed a High-Fat Diet.

BACKGROUND: Retinol-binding protein 2 (RBP2) is an intracellular carrier for vitamin A in the absorptive enterocytes. Mice lacking RBP2 (Rbp2-/-) display an unexpected phenotype of obesity, glucose intolerance, and elevated glucose-dependent insulinotropic polypeptide (GIP) levels. GIP and glucagon-like peptide 1 (GLP-1) are incretin hormones secreted by enteroendocrine cells (EECs). We recently demonstrated the presence of RBP2 and other retinoid-related proteins in EECs. OBJECTIVES: Given RBP2's role in intracellular retinoid trafficking, we aimed to evaluate whether dietary vitamin A affects incretin-secreting cell function and gene expression. METHODS: Male Rbp2-/- mice and sex- and age-matched controls (n = 6-9) were fed a high-fat diet (HFD) for 18 wk containing normal (VAN, 4000 IU/kg of diet) or low (VAL, 25% of normal) vitamin A concentrations. Body weight was recorded biweekly. Plasma GIP and GLP-1 levels were obtained fasting and 30 min after an oral fat gavage at week 16. Glucose tolerance tests were also performed. Mice were killed at week 18, and blood and tissue samples were obtained. RESULTS: Rbp2-/- mice displayed greater weight gain on the VAN compared with the VAL diet from week 7 of the intervention (P ≤ 0.01). Stimulated GIP levels were elevated in Rbp2-/- mice compared with their controls fed the VAN diet (P = 0.02), whereas their GIP response was lower when fed the VAL diet (P = 0.03). Although no differences in GLP-1 levels were observed in the VAN diet group, a lower GLP-1 response was seen in Rbp2-/- mice fed the VAL diet (P = 0.02). Changes in incretin gene expression and that of other genes associated with EEC lineage and function were consistent with these observations. Circulating and hepatic retinoid levels revealed no systemic vitamin A deficiency across dietary groups. CONCLUSIONS: Our data support a role for RBP2 and dietary vitamin A in incretin secretion and gene expression in mice fed a HFD.

GPR120/FFAR4: A Potential New Therapeutic Target for Inflammatory Bowel Disease.

Inflammatory bowel disease, whose major forms are Crohn's disease and ulcerative colitis, is characterized by chronic inflammation of the gut due to the loss of tolerance toward antigens normally contained in the gut lumen. G protein-coupled receptor (GPR) 120 has gained considerable attention as a potential therapeutic target for metabolic disorders due to its implication in the production of the incretin hormone glucagon-like peptide 1 and the secretion of cholecystokinin. Recent studies have also highlighted the role of GPR120 in regulating immune system activity and inflammation. GPR120, expressed by intestinal epithelial cells, proinflammatory macrophages, enteroendocrine L cells, and CD4+ T cells, suppresses proinflammatory and enhances anti-inflammatory cytokine production, suggesting that GPR120 might have a pivotal role in intestinal inflammation and represent a possible therapeutic target in inflammatory bowel disease. This narrative review aims at summarizing the role of GPR120 in the maintenance of intestinal homeostasis through the analysis of the most recent studies.

Short-chain fatty acids, secondary bile acids and indoles: gut microbial metabolites with effects on enteroendocrine cell function and their potential as therapies for metabolic disease.

The gastrointestinal tract hosts the largest ecosystem of microorganisms in the body. The metabolism of ingested nutrients by gut bacteria produces novel chemical mediators that can influence chemosensory cells lining the gastrointestinal tract. Specifically, hormone-releasing enteroendocrine cells which express a host of receptors activated by these bacterial metabolites. This review will focus on the activation mechanisms of glucagon-like peptide-1 releasing enteroendocrine cells by the three main bacterial metabolites produced in the gut: short-chain fatty acids, secondary bile acids and indoles. Given the importance of enteroendocrine cells in regulating glucose homeostasis and food intake, we will also discuss therapies based on these bacterial metabolites used in the treatment of metabolic diseases such as diabetes and obesity. Elucidating the mechanisms gut bacteria can influence cellular function in the host will advance our understanding of this fundamental symbiotic relationship and unlock the potential of harnessing these pathways to improve human health.

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.

Mammalian Intestinal Development and Differentiation-The State of the Art.

The development of the mammalian intestine, from its earliest origins as a morphologically uniform sheet of endoderm cells during gastrulation into the complex organ system that is essential for the life of the organism, is a truly fascinating process. During midgestation development, reciprocal interactions between endoderm-derived epithelium and mesoderm-derived mesenchyme enable villification, or the conversion of a radially symmetric pseudostratified epithelium into the functional subdivision of crypts and villi. Once a mature crypt-villus axis is established, proliferation and differentiation of new epithelial cells continue throughout life. Spatially localized signals including the wingless and Int-1, fibroblast growth factor, and Hippo systems, among others, ensure that new cells are being born continuously in the crypt. As cells exit the crypt compartment, a gradient of bone morphogenetic protein signaling limits proliferation to allow for the specification of multiple mature cell types. The first major differentiation decision is dependent on Notch signaling, which specifies epithelial cells into absorptive and secretory lineages. The secretory lineage is subdivided further into Paneth, goblet, tuft, and enteroendocrine cells via a complex network of transcription factors. Although some of the signaling molecules are produced by epithelial cells, critical components are derived from specialized crypt-adjacent mesenchymal cells termed telocytes, which are marked by Forkhead box l1, GLI Family Zinc Finger 1, and platelet-derived growth factor receptor α. The crucial nature of these processes is evidenced by the multitude of intestinal disorders such as colorectal cancer, short-bowel syndrome, and inflammatory bowel disease, which all reflect perturbations of the development and/or differentiation of the intestine.

Rebaudioside A from Stevia rebaudiana stimulates GLP-1 release by enteroendocrine cells via bitter taste signalling pathways.

Glucagon-like peptide 1 (GLP-1) is a multifaceted intestinal hormone with diverse physiological functions throughout the body. Previously, we demonstrated that the steviol glycoside rebaudioside A (rebA) from Stevia rebaudiana stimulates the release of GLP-1 from mouse intestinal organoids and pig intestinal segments. To further unravel the underlying mechanisms, we examined the involvement of sweet- and bitter taste receptors and their associated signal transduction pathways. Experiments with mouse and human intestinal enteroendocrine cell lines (STC-1 and HuTu-80, respectively) confirmed that rebA stimulates GLP-1 release in a concentration-dependent manner. Experiments with selective inhibitors of sweet signalling in both the murine as well as the human enteroendocrine cells showed that the GLP-1-induced release by rebA occurs independently of the sweet taste receptor. Functional screening of 34 murine bitter taste receptors (Tas2rs) revealed an activation response with Tas2r108, Tas2r123 and Tas2r134. Moreover, we found evidence in human HuTu-80 cells, that TAS2R4 and TRPM5 are involved in rebA-induced GLP-1 secretion, suggesting a role for bitter taste signaling in gut hormone release. Interestingly, the rebA-dependent GLP-1 release may be modulated by GABA and 6-methoxyflavanone present in the diet. Together, our findings warrant further characterization of the specific metabolic effects of rebA among the non-caloric sweeteners.