Opposing roles of the entero-pancreatic hormone urocortin-3 in glucose metabolism in rats.

AIM/HYPOTHESIS: Urocortin-3 (UCN3) is a glucoregulatory peptide produced in the gut and pancreatic islets. The aim of this study was to clarify the acute effects of UCN3 on glucose regulation following an oral glucose challenge and to investigate the mechanisms involved. METHODS: We studied the effect of UCN3 on blood glucose, gastric emptying, glucose absorption and secretion of gut and pancreatic hormones in male rats. To supplement these physiological studies, we mapped the expression of UCN3 and the UCN3-sensitive receptor, type 2 corticotropin-releasing factor receptor (CRHR2), by means of fluorescence in situ hybridisation and by gene expression analysis. RESULTS: In rats, s.c. administration of UCN3 strongly inhibited gastric emptying and glucose absorption after oral administration of glucose. Direct inhibition of gastrointestinal motility may be responsible because UCN3's cognate receptor, CRHR2, was detected in gastric submucosal plexus and in interstitial cells of Cajal. Despite inhibited glucose absorption, post-challenge blood glucose levels matched those of rats given vehicle in the low-dose UCN3 group, because UCN3 concomitantly inhibited insulin secretion. Higher UCN3 doses did not further inhibit gastric emptying, but the insulin inhibition progressed resulting in elevated post-challenge glucose and lipolysis. Incretin hormones and somatostatin (SST) secretion from isolated perfused rat small intestine was unaffected by UCN3 infusion; however, UCN3 infusion stimulated secretion of somatostatin from delta cells in the isolated perfused rat pancreas which, unlike alpha cells and beta cells, expressed Crhr2. Conversely, acute antagonism of CRHR2 signalling increased insulin secretion by reducing SST signalling. Consistent with these observations, acute drug-induced inhibition of CRHR2 signalling improved glucose tolerance in rats to a similar degree as administration of glucagon-like peptide-1. UCN3 also powerfully inhibited glucagon secretion from isolated perfused rat pancreas (perfused with 3.5 mmol/l glucose) in a SST-dependent manner, suggesting that UCN3 may be involved in glucose-induced inhibition of glucagon secretion. CONCLUSIONS/INTERPRETATION: Our combined data indicate that UCN3 is an important glucoregulatory hormone that acts through regulation of gastrointestinal and pancreatic functions.

Expression Profile of the GLP-1 Receptor in the Gastrointestinal Tract and Pancreas in Adult Female Mice.

Therapies based on glucagon-like peptide-1 receptor (GLP-1R) agonism are highly effective in treating type 2 diabetes and obesity, but the localization of GLP-1Rs mediating the antidiabetic and other possible actions of GLP-1 is still debated. The purpose with this study was to identify sites of GLP-1R mRNA and protein expression in the mouse gastrointestinal system by means of GLP-1R antibody immunohistochemistry, Glp1r mRNA fluorescence in situ hybridization, and 125I-exendin (9-39) autoradiography. As expected, GLP-1R staining was observed in almost all ß-cells in the pancreatic islets, but more rarely in α- and δ-cells. In the stomach, GLP-1R staining was found exclusively in the gastric corpus mucous neck cells, known to protect the stomach mucosa. The Brunner glands were strongly stained for GLP-1R, and pretreatment with GLP-1 agonist exendin-4 caused internalization of the receptor and mucin secretion, while pretreatment with phosphate-buffered saline or antagonist exendin (9-39) did not. In the intestinal mucosa, GLP-1R staining was observed in intraepithelial lymphocytes, lamina propria lymphocytes, and enteroendocrine cells containing secretin, peptide YY, and somatostatin, but not cholecystokinin. GLP-1R staining was seen in nerve fibers within the choline acetyl transferase- and nitric oxide-positive myenteric plexuses from the gastric corpus to the distal large intestine being strongest in the mid- and hindgut area. Finally, intraperitoneal administration of radiolabeled exendin (9-39) strongly labeled myenteric fibers. In conclusion, this study expands our knowledge of GLP-1R localization and suggests that GLP-1 may serve an important role in modulating gastrointestinal health and mucosal protection.

Dietary Neu5Ac Intervention Protects Against Atherosclerosis Associated With Human-Like Neu5Gc Loss-Brief Report.

Objective: Species-specific pseudogenization of the CMAH gene during human evolution eliminated common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) biosynthesis from its precursor N-acetylneuraminic acid (Neu5Ac). With metabolic nonhuman Neu5Gc incorporation into endothelia from red meat, the major dietary source, anti-Neu5Gc antibodies appeared. Human-like Ldlr-/-Cmah-/- mice on a high-fat diet supplemented with a Neu5Gc-enriched mucin, to mimic human red meat consumption, suffered increased atherosclerosis if human-like anti-Neu5Gc antibodies were elicited. Approach and Results: We now ask whether interventional Neu5Ac feeding attenuates metabolically incorporated Neu5Gc-mediated inflammatory acceleration of atherogenesis in this Cmah-/-Ldlr-/- model system. Switching to a Neu5Gc-free high-fat diet or adding a 5-fold excess of Collocalia mucoid-derived Neu5Ac in high-fat diet protects against accelerated atherosclerosis. Switching completely from a Neu5Gc-rich to a Neu5Ac-rich diet further reduces severity. Remarkably, feeding Neu5Ac-enriched high-fat diet alone has a substantial intrinsic protective effect against atherosclerosis in Ldlr-/- mice even in the absence of dietary Neu5Gc but only in the human-like Cmah-null background. Conclusions: Interventional Neu5Ac feeding can mitigate or prevent the red meat/Neu5Gc-mediated increased risk for atherosclerosis, and has an intrinsic protective effect, even in the absence of Neu5Gc feeding. These findings suggest that similar interventions should be tried in humans and that Neu5Ac-enriched diets alone should also be investigated further.

The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection.

We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone 1 , a compound in clinical trials for anti-fibrotic and anti-inflammatory applications 2 , as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry 3 . We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir 4 . Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone's translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

Development of a bispecific immune engager using a recombinant malaria protein.

As an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.

Adhesion receptor ADGRG2/GPR64 is in the GI-tract selectively expressed in mature intestinal tuft cells.

OBJECTIVE: GPR64/ADGRG2 is an orphan Adhesion G protein-coupled receptor (ADGR) known to be mainly expressed in the parathyroid gland and epididymis. This investigation aimed to delineate the cellular expression of GPR64 throughout the body with focus on the gastrointestinal (GI) tract. METHODS: Transgenic Gpr64mCherry reporter mice were histologically examined throughout the body and reporter protein expression in intestinal tuft cells was confirmed by specific cell ablation. The GPCR repertoire of intestinal Gpr64mCherry-positive tuft cells was analyzed by quantitative RT-PCR analysis and in situ hybridization. The Gpr64mCherry was crossed into the general tuft cell reporter Trpm5GFP to generate small intestinal organoids for time-lapse imaging. Intestinal tuft cells were isolated from small intestine, FACS-purified and transcriptionally compared using RNA-seq analysis. RESULTS: Expression of the Gpr64mCherry reporter was identified in multiple organs and specifically in olfactory microvillous cells, enteric nerves, and importantly in respiratory and GI tuft cells. In the small intestine, cell ablation targeting Gpr64-expressing epithelial cells eliminated tuft cells. Transcriptional analysis of small intestinal Gpr64mCherry -positive tuft cells confirmed expression of Gpr64 and the chemo-sensors Sucnr1, Gprc5c, Drd3, and Gpr41/Ffar3. Time-lapse studies of organoids from Trpm5GFP:Gpr64mCherry mice revealed sequential expression of initially Trpm5GFP and subsequently also Gpr64mCherry in maturing intestinal tuft cells. RNA-seq analysis of small intestinal tuft cells based on these two markers demonstrated a dynamic change in expression of transcription factors and GPCRs from young to mature tuft cells. CONCLUSIONS: GPR64 is expressed in chemosensory epithelial cells across a broad range of tissues; however, in the GI tract, GPR64 is remarkably selectively expressed in mature versus young immunoregulatory tuft cells.

Selective release of gastrointestinal hormones induced by an orally active GPR39 agonist.

OBJECTIVES: Obesity is a complex disease associated with a high risk of comorbidities. Gastric bypass surgery, an invasive procedure with low patient eligibility, is currently the most effective intervention that achieves sustained weight loss. This beneficial effect is attributed to alterations in gut hormone signaling. An attractive alternative is to pharmacologically mimic the effects of bariatric surgery by targeting several gut hormonal axes. The G protein-coupled receptor 39 (GPR39) expressed in the gastrointestinal tract has been shown to mediate ghrelin signaling and control appetite, food intake, and energy homeostasis, but the broader effect on gut hormones is largely unknown. A potent and efficacious GPR39 agonist (Cpd1324) was recently discovered, but the in vivo function was not addressed. Herein we studied the efficacy of the GPR39 agonist, Cpd1324, on metabolism and gut hormone secretion. METHODS: Body weight, food intake, and energy expenditure in GPR39 agonist-treated mice and GPR39 KO mice were studied in calorimetric cages. Plasma ghrelin, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and peptide YY (PYY) levels were measured. Organoids generated from murine and human small intestine and mouse colon were used to study GLP-1 and PYY release. Upon GPR39 agonist administration, dynamic changes in intracellular GLP-1 content were studied via immunostaining and changes in ion transport across colonic mucosa were monitored in Ussing chambers. The G protein activation underlying GPR39-mediated selective release of gut hormones was studied using bioluminescence resonance energy transfer biosensors. RESULTS: The GPR39 KO mice displayed a significantly increased food intake without corresponding increases in respiratory exchange ratios or energy expenditure. Oral administration of a GPR39 agonist induced an acute decrease in food intake and subsequent weight loss in high-fat diet (HFD)-fed mice without affecting their energy expenditure. The tool compound, Cpd1324, increased GLP-1 secretion in the mice as well as in mouse and human intestinal organoids, but not in GPR39 KO mouse organoids. In contrast, the GPR39 agonist had no effect on PYY or GIP secretion. Transepithelial ion transport was acutely affected by GPR39 agonism in a GLP-1- and calcitonin gene-related peptide (CGRP)-dependent manner. Analysis of Cpd1324 signaling properties showed activation of Gαq and Gαi/o signaling pathways in L cells, but not Gαs signaling. CONCLUSIONS: The GPR39 agonist described in this study can potentially be used by oral administration as a weight-lowering agent due to its stimulatory effect on GLP-1 secretion, which is most likely mediated through a unique activation of Gα subunits. Thus, GPR39 agonism may represent a novel approach to effectively treat obesity through selective modulation of gastrointestinal hormonal axes.

Using a Reporter Mouse to Map Known and Novel Sites of GLP-1 Receptor Expression in Peripheral Tissues of Male Mice.

Glucagon-like peptide-1 receptor (GLP-1R) activation is used in the treatment of diabetes and obesity; however, GLP-1 induces many other physiological effects with unclear mechanisms of action. To identify the cellular targets of GLP-1 and GLP-1 analogues, we generated a Glp1r.tdTomato reporter mouse expressing the reporter protein, tdTomato, in Glp1r-expressing cells. The reporter signal is expressed in all cells where GLP-1R promoter was ever active. To complement this, we histologically mapped tdTomato-fluorescence, and performed Glp-1r mRNA in situ hybridization and GLP-1R immunohistochemistry on the same tissues. In male mice, we found tdTomato signal in mucus neck, chief, and parietal cells of the stomach; Brunner's glands; small intestinal enteroendocrine cells and intraepithelial lymphocytes; and myenteric plexus nerve fibers throughout the gastrointestinal tract. Pancreatic acinar-, ß-, and δ cells, but rarely α cells, were tdTomato-positive, as were renal arteriolar smooth muscle cells; endothelial cells of the liver, portal vein, and endocardium; aortal tunica media; and lung type 1 and type 2 pneumocytes. Some thyroid follicular and parafollicular cells displayed tdTomato expression, as did tracheal cartilage chondrocytes, skin fibroblasts, and sublingual gland mucus cells. In conclusion, our reporter mouse is a powerful tool for mapping known and novel sites of GLP-1R expression in the mouse, thus enhancing our understanding of the many target cells and effects of GLP-1 and GLP-1R agonists.

Decreased number of colonic tuft cells in quiescent ulcerative colitis patients.

BACKGROUND: Colonic tuft cells are epithelial chemosensory cells involved in barrier integrity, modulation of inflammatory responses and gut homeostasis. Recent evidence indicates an involvement of tuft cells in ulcerative colitis pathogenesis, though mechanisms remain largely unknown.Here, we quantified the colonic tuft cell population in patients with quiescent ulcerative colitis as compared to patients without identified colonic disease (controls). METHODS: In this retrospective study, we obtained endoscopic colonic sigmoid biopsies from 14 patients with quiescent ulcerative colitis and from 17 controls. In a blinded central-reading design, we identified tuft cells by immunohistochemistry using a cyclooxygenase-1 antibody as a marker and performed a simple counting by visual inspection. Poisson regression was employed for statistics and results were adjusted for gender, age and smoking status. RESULTS: Ulcerative colitis patients demonstrated a 55% reduced tuft cell count in colonic mucosa compared with the control group (95% confidence limit: range 31-71%, P = 0.0002). Ulcerative colitis patients had a mean tuft cells count of 46 tuft cells/mm2 (95% CI, 36-59), while controls demonstrated a mean of 104 tuft cells/mm2 (95% CI, 79-136). No interactions of other covariates, such as age, smoking status, total duration of ulcerative colitis disease and duration of clinical remission prior to study inclusion were detected between ulcerative colitis patients and controls. CONCLUSION: Quiescent ulcerative colitis patients have a relatively low number of colonic tuft cells. Further studies are warranted to explore the potential involvement of tuft cells in ulcerative colitis pathogenesis.

Secretin release after Roux-en-Y gastric bypass reveals a population of glucose-sensitive S cells in distal small intestine.

OBJECTIVES: Gastrointestinal hormones contribute to the beneficial effects of Roux-en-Y gastric bypass surgery (RYGB) on glycemic control. Secretin is secreted from duodenal S cells in response to low luminal pH, but it is unknown whether its secretion is altered after RYGB and if secretin contributes to the postoperative improvement in glycemic control. We hypothesized that secretin secretion increases after RYGB as a result of the diversion of nutrients to more distal parts of the small intestine, and thereby affects islet hormone release. METHODS: A specific secretin radioimmunoassay was developed, evaluated biochemically, and used to quantify plasma concentrations of secretin in 13 obese individuals before, 1 week after, and 3 months after RYGB. Distribution of secretin and its receptor was assessed by RNA sequencing, mass-spectrometry and in situ hybridization in human and rat tissues. Isolated, perfused rat intestine and pancreas were used to explore the molecular mechanism underlying glucose-induced secretin secretion and to study direct effects of secretin on glucagon, insulin, and somatostatin secretion. Secretin was administered alone or in combination with GLP-1 to non-sedated rats to evaluate effects on glucose regulation. RESULTS: Plasma postprandial secretin was more than doubled in humans after RYGB (P < 0.001). The distal small intestine harbored secretin expressing cells in both rats and humans. Glucose increased the secretion of secretin in a sodium-glucose cotransporter dependent manner when administered to the distal part but not into the proximal part of the rat small intestine. Secretin stimulated somatostatin secretion (fold change: 1.59, P < 0.05) from the perfused rat pancreas but affected neither insulin (P = 0.2) nor glucagon (P = 0.97) secretion. When administered to rats in vivo, insulin secretion was attenuated and glucagon secretion increased (P = 0.04), while blood glucose peak time was delayed (from 15 to 45 min) and gastric emptying time prolonged (P = 0.004). CONCLUSIONS: Glucose-sensing secretin cells located in the distal part of the small intestine may contribute to increased plasma concentrations observed after RYGB. The metabolic role of the distal S cells warrants further studies.

Paracrine crosstalk between intestinal L- and D-cells controls secretion of glucagon-like peptide-1 in mice.

DPP-4 inhibitors, used for treatment of type 2 diabetes, act by increasing the concentrations of intact glucagon-like peptide-1 (GLP-1), but at the same time, they inhibit secretion of GLP-1, perhaps by a negative feedback mechanism. We hypothesized that GLP-1 secretion is feedback regulated by somatostatin (SS) from neighboring D-cells, and blocking this feedback circuit results in increased GLP-1 secretion. We used a wide range of experimental techniques, including gene expression analysis, immunohistochemical approaches, and the perfused mouse intestine to characterize the paracrine circuit controlling GLP-1 and SS. We show that 1) antagonizing the SS receptor (SSTr) 2 and SSTr5 led to increased GLP-1 and SS secretion in the mouse, 2) SS exhibits strong tonic inhibition of GLP-1 secretion preferentially through SSTr5, and 3) the secretion of S was GLP-1 receptor dependent. We conclude that SS is a tonic inhibitor of GLP-1 secretion, and interventions in the somatostain-GLP-1 paracrine loop lead to increased GLP-1 secretion.

Long-Acting Neurotensin Synergizes With Liraglutide to Reverse Obesity Through a Melanocortin-Dependent Pathway.

Neurotensin (NT), a gut hormone and neuropeptide, increases in circulation after bariatric surgery in rodents and humans and inhibits food intake in mice. However, its potential to treat obesity and the subsequent metabolic dysfunctions have been difficult to assess owing to its short half-life in vivo. Here, we demonstrate that a long-acting, pegylated analog of the NT peptide (P-NT) reduces food intake, body weight, and adiposity in diet-induced obese mice when administered once daily for 6 days. Strikingly, when P-NT was combined with the glucagon-like peptide 1 mimetic liraglutide, the two peptides synergized to reduce food intake and body weight relative to each monotherapy, without inducing a taste aversion. Further, P-NT and liraglutide coadministration improved glycemia and reduced steatohepatitis. Finally, we show that the melanocortin pathway is central for P-NT-induced anorexia and necessary for the full synergistic effect of P-NT and liraglutide combination therapy. Overall, our data suggest that P-NT and liraglutide combination therapy could be an enhanced treatment for obesity with improved tolerability compared with liraglutide monotherapy.

Neurotensin Is Coexpressed, Coreleased, and Acts Together With GLP-1 and PYY in Enteroendocrine Control of Metabolism.

The 2 gut hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are well known to be coexpressed, costored, and released together to coact in the control of key metabolic target organs. However, recently, it became clear that several other gut hormones can be coexpressed in the intestinal-specific lineage of enteroendocrine cells. Here, we focus on the anatomical and functional consequences of the coexpression of neurotensin with GLP-1 and PYY in the distal small intestine. Fluorescence-activated cell sorting analysis, laser capture, and triple staining demonstrated that GLP-1 cells in the crypts become increasingly multihormonal, ie, coexpressing PYY and neurotensin as they move up the villus. Proglucagon promoter and pertussis toxin receptor-driven cell ablation and reappearance studies indicated that although all the cells die, the GLP-1 cells reappear more quickly than PYY- and neurotensin-positive cells. High-resolution confocal fluorescence microscopy demonstrated that neurotensin is stored in secretory granules distinct from GLP-1 and PYY storing granules. Nevertheless, the 3 peptides were cosecreted from both perfused small intestines and colonic crypt cultures in response to a series of metabolite, neuropeptide, and hormonal stimuli. Importantly, neurotensin acts synergistically, ie, more than additively together with GLP-1 and PYY to decrease palatable food intake and inhibit gastric emptying, but affects glucose homeostasis in a more complex manner. Thus, neurotensin is a major gut hormone deeply integrated with GLP-1 and PYY, which should be taken into account when exploiting the enteroendocrine regulation of metabolism pharmacologically.

Research Resource: A Chromogranin A Reporter for Serotonin and Histamine Secreting Enteroendocrine Cells.

Chromogranin A (ChgA) is an acidic protein found in large dense-core secretory vesicles and generally considered to be expressed in all enteroendocrine cells of the gastrointestinal (GI) tract. Here, we characterize a novel reporter mouse for ChgA, ChgA-humanized Renilla reniformis (hr)GFP. The hrGFP reporter was found in the monoamine-storing chromaffin cells of the adrenal medulla, where ChgA was originally discovered. hrGFP also was expressed in enteroendocrine cells throughout the GI tract, faithfully after the expression of ChgA, as characterized by immunohistochemistry and quantitative PCR analysis of fluorescence-activated cell sorting-purified cells, although the expression in the small intestine was weak compared with that of the stomach and colon. In the stomach, hrGFP was highly expressed in almost all histamine-storing enterochromaffin (EC)-like cells, at a lower level in the majority of serotonin-storing EC cells and ghrelin cells, in a small fraction of somatostatin cells, but was absent from gastrin cells. In the small intestine, the hrGFP reporter was selectively, but weakly expressed in EC cells, although not in any peptide-storing enteroendocrine cells. In the colon, hrGFP was exclusively expressed in EC cells but absent from the peptide-storing enteroendocrine cells. In contrast, in the pancreas, hrGFP was expressed in ß-cells, α-cells, and a fraction of pancreatic polypeptide cells. It is concluded that ChgA-hrGFP in the GI tract functions as an effective reporter, particularly for the large populations of still poorly characterized monoamine-storing enteroendocrine cells. Furthermore, our findings substantiate the potential function of ChgA as a monoamine-binding protein that facilitates the regulated endocrine secretion of large amounts of monoamines from enteroendocrine cells.

Transcriptional and Functional Characterization of the G Protein-Coupled Receptor Repertoire of Gastric Somatostatin Cells.

In the stomach, somatostatin (SST) acts as a general paracrine negative regulator of exocrine secretion of gastric acid and pepsinogen and endocrine secretion of gastrin, ghrelin, and histamine. Using reporter mice expressing red fluorescent protein (RFP) under control of the SST promotor, we have characterized the G protein-coupled receptors expressed in gastric Sst-RFP-positive cells and probed their effects on SST secretion in primary cell cultures. Surprisingly, besides SST, amylin and PYY were also highly enriched in the SST cells. Several receptors found to regulate SST secretion were highly expressed and/or enriched. 1) The metabolite receptors calcium-sensing receptor and free fatty acid receptor 4 (GPR120) functioned as positive and negative regulators, respectively. 2) Among the neurotransmitter receptors, adrenergic receptors α1a, α2a, α2b, and ß1 were all highly expressed, with norepinephrine and isoproterenol acting as positive regulators. The muscarinic receptor M3 acted as a positive regulator, whereas M4 was conceivably a negative regulator. 3) Of the hormone receptors, the GLP-1 and GIP receptors, CCKb (stimulated by both CCK and gastrin) and surprisingly the melanocortin MC1 receptor were all positive regulators. 4) The neuropeptide receptors for calcitonin gene-related peptide, adrenomedullin, and vasoactive intestinal peptide acted as positive regulators, no effect was observed using galanin and nociceptin although transcripts for the corresponding receptors appeared highly expressed. 5) The SST receptors 1 and 2 functioned in an autocrine negative feedback loop. Thus, the article provides a comprehensive map of receptors through which SST secretion is regulated by hormones, neurotransmitters, neuropeptides and metabolites that act directly on the SST cells in the gastric mucosa.

GPR41/FFAR3 and GPR43/FFAR2 as cosensors for short-chain fatty acids in enteroendocrine cells vs FFAR3 in enteric neurons and FFAR2 in enteric leukocytes.

The expression of short-chain fatty acid receptors GPR41/FFAR3 and GPR43/ free fatty acid receptor 2 (FFAR2) was studied in the gastrointestinal tract of transgenic monomeric red fluorescent protein (mRFP) reporter mice. In the stomach free fatty acid receptor 3 (FFAR3)-mRFP was expressed in a subpopulation of ghrelin and gastrin cells. In contrast, strong expression of FFAR3-mRFP was observed in all cholecystokinin, glucose-dependent insulinotropic peptide (GIP), and secretin cells of the proximal small intestine and in all glucagon-like peptide-1 (GLP-1), peptide YY, and neurotensin cells of the distal small intestine. Throughout the colon and rectum, FFAR3-mRFP was strongly expressed in the large population of peptide YY and GLP-1 cells and in the neurotensin cells of the proximal colon. A gradient of expression of FFAR3-mRFP was observed in the somatostatin cells from less than 5% in the stomach to more than 95% in the rectum. Substance P-containing enterochromaffin cells displayed a similar gradient of FFAR3-mRFP expression throughout the small intestine. Surprisingly, FFAR3-mRFP was also expressed in the neuronal cells of the submucosal and myenteric ganglia. Quantitative PCR analysis of fluorescence-activated cell sorting (FACS) purified FFAR3-mRFP positive cells confirmed the coexpression with the various peptide hormones as well as key neuronal marker proteins. The FFAR2-mRFP reporter was strongly expressed in a large population of leukocytes in the lamina propria of in particular the small intestine but surprisingly only weakly in a subpopulation of enteroendocrine cells. Nevertheless, synthetic ligands specific for either FFAR3 or FFAR2 each released GLP-1 from colonic crypt cultures and the FFAR2 agonist mobilized intracellular Ca²âº in FFAR2 positive enteroendocrine cells. It is concluded that FFAR3-mRFP serves as a useful marker for the majority of enteroendocrine cells of the small and large intestine and that FFAR3 and FFAR2 both act as sensors for short-chain fatty acids in enteroendocrine cells, whereas FFAR3 apparently has this role alone in enteric neurons and FFAR2 in enteric leukocytes.

A major lineage of enteroendocrine cells coexpress CCK, secretin, GIP, GLP-1, PYY, and neurotensin but not somatostatin.

Enteroendocrine cells such as duodenal cholecystokinin (CCK cells) are generally thought to be confined to certain segments of the gastrointestinal (GI) tract and to store and release peptides derived from only a single peptide precursor. In the current study, however, transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the CCK promoter demonstrated a distribution pattern of CCK-eGFP positive cells that extended throughout the intestine. Quantitative PCR and liquid chromatography-mass spectrometry proteomic analyses of isolated, FACS-purified CCK-eGFP-positive cells demonstrated expression of not only CCK but also glucagon-like peptide 1 (GLP-1), gastric inhibitory peptide (GIP), peptide YY (PYY), neurotensin, and secretin, but not somatostatin. Immunohistochemistry confirmed this expression pattern. The broad coexpression phenomenon was observed both in crypts and villi as demonstrated by immunohistochemistry and FACS analysis of separated cell populations. Single-cell quantitative PCR indicated that approximately half of the duodenal CCK-eGFP cells express one peptide precursor in addition to CCK, whereas an additional smaller fraction expresses two peptide precursors in addition to CCK. The coexpression pattern was further confirmed through a cell ablation study based on expression of the human diphtheria toxin receptor under the control of the proglucagon promoter, in which activation of the receptor resulted in a marked reduction not only in GLP-1 cells, but also PYY, neurotensin, GIP, CCK, and secretin cells, whereas somatostatin cells were spared. Key elements of the coexpression pattern were confirmed by immunohistochemical double staining in human small intestine. It is concluded that a lineage of mature enteroendocrine cells have the ability to coexpress members of a group of functionally related peptides: CCK, secretin, GIP, GLP-1, PYY, and neurotensin, suggesting a potential therapeutic target for the treatment and prevention of diabetes and obesity.