Murine prenatal expression of cholecystokinin in neural crest, enteric neurons, and enteroendocrine cells

Cholecystokinin (CCK) is a regulatory peptide that is primarily expressed in two adult cell types: endocrine cells of the intestine and neurons of the central nervous system. To determine the ontogeny of CCK expression during intestinal organogenesis, we created a mouse strain in which the CCK gene was replaced by a lacZ reporter cassette using homologous recombination in embryonic stem cells. Initially, CCK expression in the developing intestine was limited to the myenteric plexus of the enteric nervous system. This expression pattern was widespread, extending from the proximal stomach into the colon, yet transient, being detected soon after gut tube closure [embryonic day 10.5 (E10.5)] through E15.5. Since enteric neurons are derived from the neural crest, we examined earlier (E8.5–9.5) embryos and concluded that lacZ was expressed in subpopulations of neural tube and neural crest cells. Endocrine cell expression in the intestinal epithelium occurred later, beginning at E15.5 as enteric neuronal expression was dwindling. This expression persisted to yield the adult pattern of scattered single endocrine cells in the upper small intestine. The data show that CCK is a very early marker of both neuronal and endocrine cell lineages in the developing gastrointestinal tract. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that CCK receptor transcripts were detected in embryos as early as E10.5, suggesting that CCK signaling is established early in mouse development. Dev Dyn 1999;216:190–200 . © 1999 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35165/1/9_ftp.pd

Functional characterization of enterochromaffin cells in mice and humans

Serotonin (5-HT) is a key regulator of gastrointestinal (GI) motility. Abnormal concentrations of 5-HT are found in GI disorders. About 90-95% of bodily 5-HT is produced by enterochromaffin (EC) cells in the GI. The synthesis of 5-HT is catalyzed by the enzyme tryptophan hydroxylase (Tph1). Given the dubious data collected from the previously generated animal models, to elucidate the role of EC cells in the GI tract, we have generated an EC cell-specific tamoxifen inducible Cre line (Tph1CreERT2/+), a reporter mouse (Tph1CreERT2/+;Rosa26tdTom/+), and an EC cell depleted mouse (Tph1CreERT2/+;Rosa26DTA/+). We examined changes of the EC cells and 5-HT in the GI tract of the transgenic mice using various in vivo and in vitro experimental techniques. We also measured levels of 5-HT, SERT, and TPH1 in stomach biopsy and/or blood samples from idiopathic gastroparesis patients by ELISA and western blot. We confirmed EC cell-restricted Cre activity in the reporter line (Tph1CreERT2/+;Rosa26tdTom/+). The reporter mice were then used to map EC cell distribution in the stomach (fundus, corpus, and antrum), small intestine (duodenum, jejunum, and ileum), and colon (proximal, mid, and distal colon). Proximal colon and antrum have the most EC cells while the fundus contains no observable EC cells. The distribution pattern is consistent with levels of 5-HT in the tissue of the GI tract. Furthermore, we examined EC cell turnover time using the tdTom reporter mice. We found two different types of EC cells, fast-turnover EC cells, and slow-turnover EC cells. Most fast-turnover EC cells are located in the epithelial layer and lost within ten days. Slow-turnover EC cells are located in the crypt and express stem cell markers (LGR5 and REG4) and survive over one year. Next, to access the functional role of EC cells in GI motility, we generated Tph1CreERT2/+;Rosa26DTA/+ mice. These EC cells were conditionally removed upon tamoxifen injection. 5-HT in blood and GI tissue were significantly reduced in tamoxifen injected mice. The reduction of 5-HT resulted in slower gastric emptying, slow colonic transit time, slow colonic migrating motor complex, slow total GI transit time, and decreased pellet output. We next examined human stomach biopsy samples (fundus, gastric body, and antrum) with idiopathic gastroparesis along with healthy controls. Consistent with the mouse stomach, we found the highest 5-HT concentration detected in human antrum and a lower concentration was detected in the gastric body, and levels were negligible in healthy fundus. However, in idiopathic gastroparesis samples, we found 5-HT was significantly reduced in the antrum and body from all patients. 5-HT was also reduced in the patient’s plasma and platelets. Finally, we assessed the gain-of-function of 5-HT in regulating GI motility in the mouse model via oral administration of 5-HT. The oral gavage of 5-HT to the EC cell-depleted mice rescue the gastroparesis and constipation. In summary, our new Tph1CreERT2/+ mice provide a novel and powerful tool to study the functional roles of EC cells in the GI tract which lead to the discovery of serotonin deficiency in idiopathic gastroparesis

Nkx2.2 is expressed in a subset of enteroendocrine cells with expanded lineage potential

There are two major stem cell populations in the intestinal crypt region that express either Bmi1 or Lgr5; however, it has been shown that other populations in the crypt can regain stemness. In this study, we demonstrate that the transcription factor NK2 homeobox 2 (Nkx2.2) is expressed in enteroendocrine cells located in the villus and crypt of the intestinal epithelium and is coexpressed with the stem cell markers Bmi1 and Lgr5 in a subset of crypt cells. To determine whether Nkx2.2-expressing enteroendocrine cells display cellular plasticity and stem cell potential, we performed genetic lineage tracing of the Nkx2.2-expressing population using Nkx2.2Cre/;R26RTomato mice. These studies demonstrated that Nkx2.2+ cells are able to give rise to all intestinal epithelial cell types in basal conditions. The proliferative capacity of Nkx2.2-expressing cells was also demonstrated in vitro using crypt organoid cultures. Injuring the intestine with irradiation, systemic inflammation, and colitis did not enhance the lineage potential of Nkx2.2-expressing cells. These findings demonstrate that a rare mature enteroendocrine cell subpopulation that is demarcated by Nkx2.2 expression display stem cell properties during normal intestinal epithelial homeostasis, but is not easily activated upon injury

Cell Stem Cell

The gastrointestinal (GI) epithelium is a highly regenerative tissue with the potential to provide a renewable source of insulin(+) cells after undergoing cellular reprogramming. Here, we show that cells of the antral stomach have a previously unappreciated propensity for conversion into functional insulin-secreting cells. Native antral endocrine cells share a surprising degree of transcriptional similarity with pancreatic \uce\ub2 cells, and expression of \uce\ub2 cell reprogramming factors in vivo converts antral cells efficiently into insulin(+) cells with close molecular and functional similarity to \uce\ub2 cells. Induced GI insulin(+) cells can suppress hyperglycemia in a diabetic mouse model for at least 6 months and regenerate rapidly after ablation. Reprogramming of antral stomach cells assembled into bioengineered mini-organs in vitro yielded transplantable units that also suppressed hyperglycemia in diabetic mice, highlighting the potential for development of engineered stomach tissues as a renewable source of functional \uce\ub2 cells for glycemic control.R01DK082889/DK/NIDDK NIH HHS/United StatesR01 DK084056/DK/NIDDK NIH HHS/United StatesU01 DK089536/DK/NIDDK NIH HHS/United StatesR01 DK106253/DK/NIDDK NIH HHS/United StatesP30 HD018655/HD/NICHD NIH HHS/United StatesP30DK034854/DK/NIDDK NIH HHS/United StatesP30 DK034854/DK/NIDDK NIH HHS/United StatesF32 DK107108/DK/NIDDK NIH HHS/United StatesR01 DK082889/DK/NIDDK NIH HHS/United StatesDP-0144-14-00/DP/NCCDPHP CDC HHS/United StatesP30HD18655/HD/NICHD NIH HHS/United StatesR01DK084056/DK/NIDDK NIH HHS/United StatesR00 DK077445/DK/NIDDK NIH HHS/United States2017-03-03T00:00:00Z26908146PMC477939

PDX-1 is Required for Posterior Poregut Patterning and Differentiation of the Pancreas and Duodenum

The Xenopus homeobox gene, XlHbox8, has been proposed to be involved in endodermal differentiation, specifically in pancreatic and duodenal development (Wright et al., 1988. Development 105, 787-794). To test this hypothesis directly, the mouse homolog, pdx-1, was cloned and gene targeting was used to produce two separate null alleles. In one, the second pdx-1 exon, including homeobox sequences, was replaced by a neomycin resistance cassette. In the second, a lacZ reporter was fused in-frame with the N-terminus of PDX-1, replacing most of the homeodomain. Neonatal $pdx{-}1\ {-}/{-}$ mice for both mutations are apancreatic, in confirmation of the report by Jonsson et al. (Jonsson. J., Carlsson, L., Edlund, T. and Edlund, H. 1994. Nature 371, 606-609.). However, the data presented in this dissertation show that the pancreatic buds form in homozygous mutants, with the dorsal bud undergoing limited proliferation and outgrowth to form a small, irregularly branched, ductular tree. No insulin or amylase-positive cells are found in these outgrowths, but glucagon-expressing GLUT2-positive cells are found. The rostral duodenum suffers a local absence of the normal columnar epithelial lining, villi, and Brunner\u27s glands, which are replaced by a GLUT2-positive cuboidal epithelium resembling the bile duct lining. The abundance of enteroendocrine cells in the rostral duodenal villi is greatly reduced in pdx-$1\ {-}/{-}$ embryos. The PDX-1/$\beta$-galactosidase fusion allele is expressed in the pancreatic and duodenal cells in the absence of functional PDX-1, and the majority of these cells express PDX-1/$\beta$-galactosidase fusion protein into perinatal stages without changes in the boundaries or levels of expression. These results are discussed in terms of a role for pdx-1 in posterior foregut patterning, specifically in the differentiation of the pancreas and rostral duodenum

Regulation of Pancreatic α and β Cell Function by the Bile Acid Receptor TGR5

The discovery that bile acids act as endogenous ligands of the membrane receptor TGR5 and the nuclear receptor FXR increased their significance as regulators of cholesterol, glucose and energy metabolism. Activation of TGR5, expressed on enteroendocrine L cells, by bile acids caused secretion of GLP-1, which stimulates insulin secretion from pancreatic β cells. Expression of TGR5 on pancreatic islet cells and the direct effect of bile acids on the endocrine functions of pancreas, however, are not fully understood. The aim of this study was to identify expression of TGR5 in pancreatic islet cells and determine the effect of bile acids on insulin secretion. Expression of TGR5 was identified by quantitative PCR and western blot in islets from human and mouse, and in α (αTC1-6) and β (MIN6) cells. Release of insulin, glucagon and GLP-1 were measured by ELISA. The signaling pathways coupled to TGR5 activation were identified by direct measurements such as stimulation of G proteins, adenylyl cyclase activity, PI hydrolysis and intracellular Ca2+ in response to bile acids; and confirmed by the use of selective inhibitors that block specific steps in the signaling pathway. Our studies identified expression of TGR5 receptors in β cells and demonstrated that activation of these receptors by both pharmacological ligands (oleanolic acid (OA) and INT-777) and physiological ligand (lithocholic acid, LCA) induced insulin secretion. TGR5 receptors are also expressed in α cells and, activation of TGR5 by OA, INT-777 and LCA at 5 mM glucose induced release of glucagon, which is processed from proglucagon by the selective expression of prohormone convertase 2 (PC2). However, under hyperglycemia, activation of TGR5 in α cells augmented the glucose-induced increase in GLP-1 secretion, which in turn, stimulated insulin secretion. Secretion of GLP-1 from α cells reflected TGR5-mediated increase in PC1 promoter activity and PC1 expression, which selectively converts proglucagon to GLP-1. The signaling pathway activated by TGR5 to mediate insulin and GLP-1 secretion involved Gs/cAMP/Epac/PLC-ε/Ca2+. These results provide insights into the mechanisms involved in the regulation of pancreatic α and β cell function by bile acids and may lead to new therapeutic avenues for the treatment of diabetes

Generation and use of new tools for the characterisation of gut hormone receptors

Enteroendocrine hormones released from the intestine following food intake have several roles in the control of metabolism, some of which are exploited therapeutically for the treatment of type 2 diabetes. Within this thesis, focus has been on the receptors of the gut hormones glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2). In recent years there has been a surge of interest in the enteroendocrine hormones particularly due to the success of GLP-1 mimetics in the treatment of type 2 diabetes. GLP-1 is an incretin hormone, which enhances glucose induced insulin secretion by binding GLP-1 receptors (GLP1R) on pancreatic β-cells. Despite the therapeutic success, several extra-pancreatic clinical effects of GLP-1 remain unexplained. Here, a GLP1R monoclonal antagonistic antibody that can block GLP1R signalling in vivo has been developed and characterised, providing a new tool for the study of GLP1R physiology. GIP is the second incretin hormone, initially referred to as the ‘ugly duckling’ incretin hormone due to it’s ineffectiveness in inducing insulin secretion in type 2 diabetic patients. Aside from the incretin actions, GIP is thought to be involved in the regulation of high-fat diet (HFD) induced obesity. A new transgenic mouse model expressing a fluorescent reporter under the control of the Gipr promoter has been used here to identify GIPR expressing cells. This model showed GIPR expression in the pancreas, adipose tissue, duodenum and nodose ganglia. Surprisingly GIPR expressing cells were found centrally, in areas of the hypothalamus involved in the regulation of food intake and energy expenditure. We consequently sought to investigate the function of GIPR expressing hypothalamic cells. GLP-2, unlike GLP-1 and GIP, is not an incretin hormone. Rather, GLP-2 has been implicated in the regulation of epithelial cell proliferation and apoptosis within the intestine. Therapeutically, an analogue of GLP-2 is used for the treatment of short bowel syndrome. A common missense mutation in the GLP-2 receptor (GLP2R), D470N, has been found to be associated with type 2 diabetes, and here we sought to understand the mechanism underlying this association. The D470N mutant has decreased β-arrestin recruitment, though the significance of this finding will need further research. Overall; the new monoclonal antagonistic GLP1R antibody will help to further understand GLP1R physiology, the new transgenic GIPR mouse model has contributed to the understanding of GIPR localisation, and cell based assays have identified functional implications of a polymorphism in the GLP2R associated with an increased risk of diabetes. It is hoped that further understanding of the physiology of these gut hormone receptors will be critical in the development of new therapeutics for diabetes and obesity.MedImmune funded Ph