CART knock out mice have impaired insulin secretion and glucose intolerance, altered beta cell morphology and increased body weight

CART peptides are anorexigenic and are widely expressed in the central and peripheral nervous systems, as well as in endocrine cells in the pituitary, adrenal medulla and the pancreatic islets. To study the role of CART in islet function, we used CART null mutant mice (CART KO mice) and examined insulin secretion in vivo and in vitro, and expression of islet hormones and markers of beta-cell function using immunocytochemistry. We also studied CART expression in the normal pancreas. In addition, body weight development and food intake were documented. We found that in the normal mouse pancreas, CART was expressed in numerous pancreatic nerve fibers, both in the exocrine and endocrine portion of the gland. CART was also expressed in nerve cell bodies in the ganglia. Double immunostaining revealed expression in parasympathetic (vasoactive intestinal polypeptide (VIP)-containing) and in fewer sensory fibers (calcitonin gene-related peptide (CGRP)-containing). Although the expression of islet hormones appeared normal, CART KO islets displayed age dependent reduction of pancreatic duodenal homeobox 1 (PDX-1) and glucose transporter-2 (GLUT-2) immunoreactivity, indicating beta-cell dysfunction. Consistent with this, CART KO mice displayed impaired glucose-stimulated insulin secretion both in vivo after an intravenous glucose challenge and in vitro following incubation of isolated islets in the presence of glucose. The impaired insulin secretion in vivo was associated with impaired glucose elimination, and was apparent already in young mice with no difference in body weight. In addition, CART KO mice displayed increased body weight at the age of 40 weeks, without any difference in food intake. We conclude that CART is required for maintaining normal islet function in mice.     

CART is a novel islet regulatory peptide

CART peptides have emerged as important islet regulators. CART is expressed both in islet endocrine cells and in parasympathetic and sensory nerves innervating the islets. In adult rats the intra-islet expression of CART is limited to the somatostatin producing delta-cells, while in adult mice CART is mainly expressed in nerve fibers. During development islet CART is upregulated; in rats in almost all types of islet endocrine cells, including the insulin-producing beta-cells, and in mice mainly in the alpha-cells. This pattern of expression peaks around birth. CART is also expressed in human pancreatic nerves and in islet tumours where the expression level of CART may be related to the degree of differentiation of the tumour. Interestingly, in several rat models of type 2 diabetes CART expression is robustly upregulated in the beta-cells, and is prominent during the phase of beta cell proliferation and hypertrophy. While CART inhibits glucose stimulated insulin secretion from rat islets it augments insulin secretion amplified by cAMP. Mice lacking CART, on the other hand, have islet dysfunction, and humans with a missense mutation in the cart gene are prone to develop type 2 diabetes. These data favor a role of CART in normal islet function and in the pathophysiology of type 2 diabetes.     

BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system

The enteric nervous system (ENS) is derived from neural crest cells that migrate along the gastrointestinal tract to form a network of neurons and glia that are essential for regulating intestinal motility. Despite the number of genes known to play essential roles in ENS development, the molecular etiology of congenital disorders affecting this process remains largely unknown. To determine the role of bone morphogenetic protein (BMP) signaling in ENS development, we first examined the expression of bmp2, bmp4, and bmprII during hindgut development and find these strongly expressed in the ENS. Moreover, functional BMP signaling, demonstrated by the expression of phosphorylated Smad1/5/8, is present in the enteric ganglia. Inhibition of BMP activity by noggin misexpression within the developing gut, both in ovo and in vitro, inhibits normal migration of enteric neural crest cells. BMP inhibition also leads to hypoganglionosis and failure of enteric ganglion formation, with crest cells unable to cluster into aggregates. Abnormalities of migration and ganglion formation are the hallmarks of two human intestinal disorders, Hirschsprung's disease and intestinal neuronal dysplasia. Our results support an essential role for BMP signaling in these aspects of ENS development and provide a basis for further investigation of these proteins in the etiology of neuro-intestinal disorders.     

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia

Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss‐of‐function disorders such as Hirschsprung disease.     

Enteric glia inhibit intestinal epithelial cell proliferation partly through a TGF-beta1-dependent pathway

Although recent studies have shown that enteric neurons control intestinal barrier function, the role of enteric glial cells (EGCs) in this control remains unknown. Therefore, our goal was to characterize the role of EGCs in the control of intestinal epithelial cell proliferation using an in vivo transgenic and an in vitro coculture model. Assessment of intestinal epithelial cell proliferation after ablation of EGCs in transgenic mice demonstrated a significant increase in crypt cell hyperplasia. Furthermore, mucosal glial network (assessed by immunohistochemical detection of S-100beta) is altered in colon adenocarcinoma compared with control tissue. In an in vitro coculture model of subconfluent Caco-2 cells seeded onto Transwell filters with EGCs, Caco-2 cell density and [3H]thymidine incorporation were significantly lower than in control (Caco-2 cultured alone). Flow cytometry analysis showed that EGCs had no effect on Caco-2 cell viability. EGCs induced a significant increase in Caco-2 cell surface area without any sign of cellular hypertrophy. These effects by EGCs were also seen in various transformed or nontransformed intestinal epithelial cell lines. Furthermore, TGF-beta1 mRNA was expressed, and TGF-beta1 was secreted by EGCs. Exogenously added TGF-beta1 reproduced partly the EGC-mediated effects on cell density and surface area. In addition, EGC effects on Caco-2 cell density were significantly reduced by a neutralizing TGF-beta antibody. In conclusion, EGCs have profound antiproliferative effects on intestinal epithelial cells. Functional alterations in EGCs may therefore modify intestinal barrier functions and be involved in pathologies such as cancer or inflammatory bowel diseases.     

Contribution of neurotensin in the immune and neuroendocrine modulation of normal and abnormal enteric function

Among various hormones, which are synthesized by intestinal cells and influence enteric function, neurotensin (NT) has gained scientific attention the last three decades. This neuropeptide, mainly located in neuronal synaptic vesicles of hypothalamus and in neuroendocrine cells of the small bowel, participates in enteric digestive processes, gut motility and intestinal inflammatory mechanisms by cooperating with other regulators such as histamine, substance P and somatostatin. NT plays an important role mainly in intestinal lipid metabolism by cooperating with cholecystokinin and establishes a hormonal brain-gut-adipose tissue connection, which could adjust appetite, weight status and generally eating behavior with the amount and the content (particularly fat) of food intake. Moreover, NT achieves a multi-level control of intestinal motility by cooperating with the enteric- and central nervous system, and other enteric hormones (such as somatostatin). NT regulates motility patterns related to the efficiency of the digestive process, stool emptying, transition from the fasted to the postprandial state and reestablishment of the fasted status. In addition, NT possesses a long-term enteroprotective role towards the intestinal tract, despite the fact that under certain circumstances NT may participate in short-term subcellular pathways promoting an acute inflammatory response. The aim of this review is two-fold. First, is to provide an up-to-date synopsis of the available knowledge regarding the involvement of neurotensin in enteric functional status, and highlight its significance in physiological and pathological conditions. Second, is to propose new research directions concerning the role of neurotensin and other intestinal regulatory peptides in the establishment of the brain-gut axis and in the development of functional disorders of the abdominal tract. Conclusively, to clarify the areas, in which an experimental therapeutic intervention, based on NT analogs, may lead to encouraging results.     

Involvement of a Stat3 binding site in inflammation-induced enteric apelin expression

Apelin is the endogenous ligand for the APJ receptor; both are expressed in the gastrointestinal tract. Experimental colitis in rodents and inflammatory bowel disease in humans are associated with increased intestinal apelin production. Our aim was to use LPS and proinflammatory cytokine-treated (IL-6 and IFN-gamma) rodents or enteric cells to identify signaling mechanisms underlying inflammation-induced enteric apelin expression. LPS, IL-6, or IFN-gamma treatment of rodents increased enteric apelin expression. Pharmacological blockade of Jak/Stat signaling or IL-6 antibody administration inhibited elevations in enteric apelin expression. Transient transfection experiments showed that LPS, IL-6, or IFN-gamma increased apelin expression by stimulation of apelin promoter activity, and blockade of Jak/Stat signaling abolished elevations in apelin promoter activity. A chromatin immunoprecipitation assay showed that IL-6 induced binding of phospho-Stat3 to a putative Stat3 site in the apelin promoter; mutation of this site abrogated the LPS-induced elevation in apelin promoter activity. Together, our findings indicate that binding of phospho-Stat3 to the apelin promoter is the final step underlying proinflammatory cytokine-induced enteric apelin expression during intestinal inflammation.     

The Orexin System in the Enteric Nervous System of the Bottlenose Dolphin (Tursiops truncatus)

This study provides a general approach to the presence and possible role of orexins and their receptors in the gut (three gastric chambers and intestine) of confined environment bottlenose dolphin. The expression of prepro-orexin, orexin A and B and orexin 1 and 2 receptors were investigated by single immunostaining and western blot analysis. The co-localization of vasoactive intestinal peptide and orexin 1 receptor in the enteric nervous system was examined by double immunostaining. Also, orexin A concentration were measured in plasma samples to assess the possible diurnal variation of the plasma level of peptide in this species. Our results showed that the orexin system is widely distributed in bottlenose dolphin enteric nervous system of the all gastrointestinal tract examined. They are very peculiar and partially differs from that of terrestrial mammals. Orexin peptides and prepro-orexin were expressed in the main stomach, pyloric stomach and proximal intestine; while orexin receptors were expressed in the all examined tracts, with the exception of main stomach where found no evidence of orexin 2 receptor. Co-localization of vasoactive intestinal peptide and orexin 1 receptor were more evident in the pyloric stomach and proximal intestine. These data could suggest a possible role of orexin system on the contractility of bottlenose dolphin gastrointestinal districts. Finally, in agreement with several reports, bottlenose dolphin orexin A plasma level was higher in the morning during fasting. Our results emphasize some common features between bottlenose dolphin and terrestrial mammals. Certainly, further functional investigations may help to better explain the role of the orexin system in the energy balance of bottlenose dolphin and the complex interaction between feeding and digestive physiology.