Genome sequence of a diabetes-prone rodent reveals a mutation hotspot around the ParaHox gene cluster.

The sand rat Psammomys obesus is a gerbil species native to deserts of North Africa and the Middle East, and is constrained in its ecology because high carbohydrate diets induce obesity and type II diabetes that, in extreme cases, can lead to pancreatic failure and death. We report the sequencing of the sand rat genome and discovery of an unusual, extensive, and mutationally biased GC-rich genomic domain. This highly divergent genomic region encompasses several functionally essential genes, and spans the ParaHox cluster which includes the insulin-regulating homeobox gene Pdx1. The sequence of sand rat Pdx1 has been grossly affected by GC-biased mutation, leading to the highest divergence observed for this gene across the Bilateria. In addition to genomic insights into restricted caloric intake in a desert species, the discovery of a localized chromosomal region subject to elevated mutation suggests that mutational heterogeneity within genomes could influence the course of evolution.

GLP-1 Induces Barrier Protective Expression in Brunner's Glands and Regulates Colonic Inflammation.

BACKGROUND: Beneficial roles for glucagon-like peptide 1 (GLP-1)/GLP-1R signaling have recently been described in diseases, where low-grade inflammation is a common phenomenon. We investigated the effects of GLP-1 in Brunner's glands and duodenum with abundant expression of GLP-1 receptors, as well as GLP-1 effect on colonic inflammation. METHODS: RNA from Brunner's glands of GLP-1R knockout and wild-type mice were subjected to full transcriptome profiling. Array results were validated by quantitative reverse transcription polymerase chain reaction in wild-type mice and compared with samples from inflammatory bowel disease (IBD) patients and controls. In addition, we performed a detailed investigation of the effects of exogenous liraglutide dosing in a T-cell driven adoptive transfer (AdTr) colitis mouse model. RESULTS: Analyses of the Brunner's gland transcriptomes of GLP-1R knockout and wild-type mice identified 722 differentially expressed genes. Upregulated transcripts after GLP-1 dosing included IL-33, chemokine ligand 20 (CCL20), and mucin 5b. Biopsies from IBD patients and controls, as well as data from the AdTr model, showed deregulated expression of GLP-1R, CCL20, and IL-33 in colon. Circulating levels of GLP-1 were found to be increased in mice with colitis. Finally, the colonic cytokine levels and disease scores of the AdTr model indicated reduced levels of colonic inflammation in liraglutide-dosed animals. CONCLUSIONS: We demonstrate that IL-33, GLP-1R, and CCL20 are deregulated in human IBD, and that prophylactic treatment with 0.6 mg/kg liraglutide improves disease in AdTr colitis. In addition, GLP-1 receptor agonists upregulate IL-33, mucin 5b, and CCL20 in murine Brunner's glands. Taken together, our data indicate that GLP-1 receptor agonists affect gut homeostasis in both proximal and distal parts of the gut.

Transcriptomic analysis of the lesser spotted catshark (Scyliorhinus canicula) pancreas, liver and brain reveals molecular level conservation of vertebrate pancreas function.

BACKGROUND: Understanding the evolution of the vertebrate pancreas is key to understanding its functions. The chondrichthyes (cartilaginous fish such as sharks and rays) have often been suggested to possess the most ancient example of a distinct pancreas with both hormonal (endocrine) and digestive (exocrine) roles. The lack of genetic, genomic and transcriptomic data for cartilaginous fish has hindered a more thorough understanding of the molecular-level functions of the chondrichthyan pancreas, particularly with respect to their "unusual" energy metabolism (where ketone bodies and amino acids are the main oxidative fuel source) and their paradoxical ability to both maintain stable blood glucose levels and tolerate extensive periods of hypoglycemia. In order to shed light on some of these processes, we carried out the first large-scale comparative transcriptomic survey of multiple cartilaginous fish tissues: the pancreas, brain and liver of the lesser spotted catshark, Scyliorhinus canicula. RESULTS: We generated a mutli-tissue assembly comprising 86,006 contigs, of which 44,794 were assigned to a particular tissue or combination of tissues based on mapping of sequencing reads. We have characterised transcripts encoding genes involved in insulin regulation, glucose sensing, transcriptional regulation, signaling and digestion, as well as many peptide hormone precursors and their receptors for the first time. Comparisons to mammalian pancreas transcriptomes reveals that mechanisms of glucose sensing and insulin regulation used to establish and maintain a stable internal environment are conserved across jawed vertebrates and likely pre-date the vertebrate radiation. Conservation of pancreatic hormones and genes encoding digestive proteins support the single, early evolution of a distinct pancreatic gland with endocrine and exocrine functions in jawed vertebrates. In addition, we demonstrate that chondrichthyes lack pancreatic polypeptide (PP) and that reports of PP in the literature are likely due cross-reaction with PYY and/or NPY in the pancreas. A three hormone islet organ is therefore the ancestral jawed vertebrate condition, later elaborated upon only in the tetrapod lineage. CONCLUSIONS: The cartilaginous fish are a great untapped resource for the reconstruction of patterns and processes of vertebrate evolution and new approaches such as those described in this paper will greatly facilitate their incorporation into the rank of "model organism".

GLP-1 receptor localization in monkey and human tissue: novel distribution revealed with extensively validated monoclonal antibody.

Glucagon-like peptide 1 (GLP-1) analogs are increasingly being used in the treatment of type 2 diabetes. It is clear that these drugs lower blood glucose through an increase in insulin secretion and a lowering of glucagon secretion; in addition, they lower body weight and systolic blood pressure and increase heart rate. Using a new monoclonal antibody for immunohistochemistry, we detected GLP-1 receptor (GLP-1R) in important target organs in humans and monkeys. In the pancreas, GLP-1R was predominantly localized in ß-cells with a markedly weaker expression in acinar cells. Pancreatic ductal epithelial cells did not express GLP-1R. In the kidney and lung, GLP-1R was exclusively expressed in smooth muscle cells in the walls of arteries and arterioles. In the heart, GLP-1R was localized in myocytes of the sinoatrial node. In the gastrointestinal tract, the highest GLP-1R expression was seen in the Brunner's gland in the duodenum, with lower level expression in parietal cells and smooth muscle cells in the muscularis externa in the stomach and in myenteric plexus neurons throughout the gut. No GLP-1R was seen in primate liver and thyroid. GLP-1R expression seen with immunohistochemistry was confirmed by functional expression using in situ ligand binding with (125)I-GLP-1. In conclusion, these results give important new insight into the molecular mode of action of GLP-1 analogs by identifying the exact cellular localization of GLP-1R.

The islet ghrelin cell.

The islets of Langerhans are key regulators of glucose homeostasis and have been known as a structure for almost one and a half centuries. During the twentieth century several different cell types were described in the islets of different species and at different developmental stages. Six cell types with identified hormonal product have been described so far by the use of histochemical staining methods, transmission electron microscopy, and immunohistochemistry. Thus, glucagon-producing α-cells, insulin-producing ß-cells, somatostatin-producing δ-cells, pancreatic polypeptide-producing PP-cells, serotonin-producing enterochromaffin-cells, and gastrin-producing G-cells have all been found in the mammalian pancreas at least at some developmental stage. Species differences are at hand and age-related differences are also to be considered. Eleven years ago a novel cell type, the ghrelin cell, was discovered in the human islets. Subsequent studies have shown the presence of islet ghrelin cells in several animals, including mouse, rat, gerbils, and fish. The developmental regulation of ghrelin cells in the islets of mice has gained a lot of interest and several studies have added important pieces to the puzzle of molecular mechanisms and the genetic regulation that lead to differentiation into mature ghrelin cells. A body of evidence has shown that ghrelin is an insulinostatic hormone, and the potential for blockade of ghrelin signalling as a therapeutic avenue for type 2 diabetes is intriguing. Furthermore, ghrelin-expressing pancreatic tumours have been reported and ghrelin needs to be taken into account when diagnosing pancreatic tumours. In this review article, we summarise the knowledge about islet ghrelin cells obtained so far.

Gastrin: a distinct fate of neurogenin3 positive progenitor cells in the embryonic pancreas.

Neurogenin3(+) (Ngn3(+)) progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach, where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors, but the lineage and regulators of pancreatic gastrin(+) cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin(+) cells in the developing pancreas co-express glucagon, ghrelin or pancreatic polypeptide, but many gastrin(+) cells do not express any other islet hormone. Pancreatic gastrin(+) cells express the transcription factors Nkx6.1, Nkx2.2 and low levels of Pdx1, and derive from Ngn3(+) endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3, Nkx2.2, NeuroD1 and Arx, but not Pax4 or Pax6. Finally, gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus, gastrin(+) cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells.

Beta cell workshop 2013 Kyoto.

The very modern Kyoto International Conference Center provided the site for the 8th workshop on Beta cells on April 23-26, 2013. The preceding workshops were held in Boston, USA (1991); Kyoto, Japan (1994); Helsingør, Denmark (1997); Helsinki, Finland (2003); El Perello, Spain (2006); Peebles, Scotland (2009); and Helsingør, Denmark (2011). The Kyoto meeting drew more than 200 attendees from 18 different countries. There were 47 main oral presentations, and approximately 75 posters covered virtually all aspects of the pancreas function, development and genetics of disease. Here we will review some of the newest highlights.

Divalent metal transporter 1 regulates iron-mediated ROS and pancreatic ß cell fate in response to cytokines.

Reactive oxygen species (ROS) contribute to target-cell damage in inflammatory and iron-overload diseases. Little is known about iron transport regulation during inflammatory attack. Through a combination of in vitro and in vivo studies, we show that the proinflammatory cytokine IL-1ß induces divalent metal transporter 1 (DMT1) expression correlating with increased ß cell iron content and ROS production. Iron chelation and siRNA and genetic knockdown of DMT1 expression reduce cytokine-induced ROS formation and cell death. Glucose-stimulated insulin secretion in the absence of cytokines in Dmt1 knockout islets is defective, highlighting a physiological role of iron and ROS in the regulation of insulin secretion. Dmt1 knockout mice are protected against multiple low-dose streptozotocin and high-fat diet-induced glucose intolerance, models of type 1 and type 2 diabetes, respectively. Thus, ß cells become prone to ROS-mediated inflammatory damage via aberrant cellular iron metabolism, a finding with potential general cellular implications.

Short-term effects of INGAP and Reg family peptides on the appearance of small ß-cells clusters in non-diabetic mice.

The Reg3 peptides INGAP-PP and human Reg3α/ß (HIP) have been hypothesized to stimulate ß-cell neogenesis in the pancreas. Administration of INGAP-PP has been shown to cause an increase in ß-cell mass in multiple animal models, reverse streptozotocin (STZ) induced diabetes in mice and reduces HbA1c levels in type 2 diabetic humans. In this study, we have examined the ability of the INGAP-PP and HIP peptides to induce ß-cell formation in vivo in normal mice through short-term administration of the peptides. We assessed the peptides ability to induce an increase in extra-islet insulin-positive cell clusters by looking at ß-cell number by point counting morphometry on pancreata that had been randomized using the smooth fractionator principle in non-diabetic NMRI mice after short-term injections of the peptides (5 d). Five day continuous BrdU labeling was used to determine if the new ß-cells were derived from replicating ß-cells. Real time quantitative RT-PCR and immuno-histochemistry was used to analyze changes in pancreatic transcription factor expression. A 1.5- to 2-fold increase in the volume of small extra-islet insulin-positive clusters post 5 d treatment with INGAP-PP and HIP as compared with mice treated with a non-peptide control or scrambled peptide (p<0.05) (n = 7) was found. Five day continuous BrdU infusion during the 5 d period showed little or no incorporation in islets or small insulin clusters. Five days of treatment with INGAP-PP or HIP, showed a tendency toward increased levels of pancreatic progenitor markers such as Ngn3, Nkx6.1, Sox9 and Ins. These are the first studies to compare and indicate that the human Reg3 α/ß (HIP) peptide has similar bioactivity in vivo as INGAP by causing formation of small ß-cell clusters in extra-islet pancreatic tissue after only 5 d of treatment. Upregulation of pancreatic transcription factors may be part of the mechanism of action.

Intracranial ectopic pancreatic tissue.

In 2007 a young Japanese female was reported to suffer from a congenital brain malformation with a non-functioning pancreatic endocrine tumor arising from intracranial ectopic pancreatic tissue. Ectopic pancreas is normally confined to other endodermally-derived organs and not previously reported to be found in the brain. Therefore, we sought to better understand the true pancreatic nature of the tissue and to further understand the mechanism by which ectopic pancreas could appear in the brain. A detailed immunohistochemical analysis for pancreatic hormones, transcription factors, ductal/exocrine markers and stem cell markers on sections from the resected tumor tissue was performed. All five endocrine cell types are observed but pancreatic polypeptide cells are quite rare and ghrelin and glucagon cells are more numerous than in normal human pancreas. Insulin immunoreactive cells stain for c-peptide. The ß-cell specific transcription factor, Nkx6.1, is expressed only in the insulin immunoreactive cells while neither Ptf1a or PDX-1 immunoreactive cells can be observed. Duct-like structures stain strongly for pan-cytokeratin and E-cahderin. The exocrine like tissue stains strongly for pancreatic amylase, lipase and chymotrypsin. Ngn-3 cells were very rare and not in the pancreatic area. Examining for endodermal markers we observed Sox17 had a weak staining in some areas of the pancreatic tissue but was much less widely expressed than FoxA2. The tumor tissue did not stain for the stem cell markers, Oct-4 and Sox2. It is speculated that the ectopic pancreas domain may arise from misexpression of homeodomain transcription factors related to Pdx1 within a domain of Ptf1a expression.

The comparative anatomy of islets.

In the past 20 years, numerous publications on a variety of mammalian and non-mammalian species have appeared in the literature to supplement the excellent comparative work performed in the 70s and 80s by the Falkmer, Epple, and Youson groups. What emerges is that islets are much more complex than once thought and show a lot of similarities in rodents and higher primates. The diversity of lifestyles, metabolic demands, and diets has most likely influenced the great diversity in both structure and cell-type content of islets in lower vertebrate species. In this chapter, I try to provide an overview of the evolution from endocrine cell types in invertebrates to the higher mammals and focus on what has been reported in the literature and some of our own experiences and also include a description of other hormones reported to be found in islets.

Liraglutide, but not vildagliptin, restores normoglycaemia and insulin content in the animal model of type 2 diabetes, Psammomys obesus.

In order to investigate the effect and mechanism of liraglutide and vildagliptin in diabetic Psammomys obesus, we examined proliferation and apoptosis of beta-cells, beta-cell mass (BCM), and pancreatic insulin content after zero, six and fourteen days of treatment compared to control groups. One group of animals was kept on low-energy diet and seven groups were given high-energy diet (HED) that induced diabetes over a four week period. Non-fasting morning blood glucose, body weight, HbA(1C) and pancreatic insulin content were measured and beta cell mass (BCM), proliferation and apoptosis frequencies were determined using stereological point counting. Liraglutide significantly reduced blood glucose and even normalized it in all animals treated for six days and in 11 out of 17 animals treated for fourteen days. HED increased BCM and treatment with liraglutide did not change this. However, compared to the vehicle-treated animals pancreatic insulin content was normalized in animals treated for six and fourteen days with liraglutide. In contrast, vildagliptin, in doses causing full inhibition of plasma DPP-IV activity, neither reduced blood glucose nor altered HED-induced increases in BCM or pancreatic insulin content. These results suggest that liraglutide restores normoglycaemia and improves glycaemic control in P. obesus by increasing their insulin content and improving the function of the beta-cells. In contrast, vildagliptin does not improve glycaemic control in P. obesus nor affect beta-cell insulin content.

Immunohistochemistry of pancreatic development in cattle and pig.

The aim of this study was to characterize bovine and porcine pancreatic development by immunohistochemistry. In the pig, staining for both glucagon and insulin was noted at day 19. In cattle, glucagon staining was observed at day 25 and insulin staining from day 26. In both species, glucagon-stained cells were abundant initially, but later insulin-stained cells became most abundant. A few cells displayed co-localization of glucagon and insulin staining during initial development in both species. Initially, most of the cells of the pancreatic primordia and the duodenal epithelium displayed Pdx-1-staining. All insulin-stained cells displayed Pdx-1-stained nuclei, whereas no glucagon-stained cells did so. Many Pdx-1-stained cells lacked insulin staining, but with development, the relative number of these cells diminished. Nkx6.1-staining was initially seen in a pattern similar to that for Pdx-1, but was lacking duodenal staining. Subsequently, the number of Nkx6.1-stained cells diminished, but increased again to a level where practically all insulin-stained cells also presented Nkx6.1-staining. Glucagon-stained cells, on the other hand, never had Nkx6.1 staining. In conclusion, the localization of the two transcription factors, Pdx-1 and Nkx6.1, demonstrated that pancreas development appears to be controlled by mechanisms comparable with those operating in humans.

Investigation and characterization of the duct cell-enriching process during serum-free suspension and monolayer culture using the human exocrine pancreas fraction.

OBJECTIVES: We aimed to characterize a serum-free culture system resulting in highly enriched duct cells from human exocrine pancreas. In addition, we tested the effect of vascular endothelial growth factor (VEGF) on endothelial cell proliferation and endocrine differentiation of the duct cells. METHODS: The exocrine pellet fraction was cultivated in suspension followed by monolayer culture. Time course analysis of multiple acinar and duct cell markers was performed using reverse transcription-polymerase chain reaction and immunocytochemistry. The effects of VEGF and placental growth factor on the quantities of endothelial, duct, and endocrine cells and fibroblasts were investigated using computerized imaging analysis. RESULTS: Suspension culture of the exocrine material efficiently enriched the cultures for duct cells. Frequent acinar cell death as well as cell selective adherence of acinar cells to the culture dish was the underlying cause of the enrichment. Confocal microscopy demonstrated the virtual absence of cells coexpressing duct cell- and acinar cell-specific markers. The endothelial immunoreactivity of the suspension culture system could be increased 2-fold by VEGF treatment, yet no effect was observed on endocrine cell numbers. CONCLUSIONS: We have characterized a serum-free in vitro culture system to enrich human duct cells and further show that the contribution of acinoductal transdifferentiation to the enrichment of duct cells is negligible.

Acomys, the closest relatives to Gerbils, do express Pdx-1 protein and have similar islet morphology to Gerbils.

Acomys, also called spiny mice, were once used as a diabetes model. We have recently demonstrated that the closest relatives to the Acomys, members of the family Gerbillinae, lack the transcription factor Pdx-1. Therefore, we sought to determine if members of this family also lack Pdx-1, and describe the pancreatic morphology in three different species of Acomys: Acomys cahirinus (Egyptian spiny mouse), Acomys cilicicus (Asia Minor spiny mouse) and Acomys dimidiatus (eastern spiny mouse). We successfully cloned the Acomys Pdx-1 gene and we demonstrate by immunocytochemistry that the Pdx-1 protein is expressed in the pancreatic insulin immunoreactive cells and in a subset of the somatostatin cells. The basic islet structure is very similar to other rodents - with the insulin cells in the center, and glucagon, somatostatin, PP and occasional PYY cells in the periphery. No ghrelin or CART cells were identified. Nkx6.1 was localized specifically to the insulin immunoreactive cells, while Nkx2.2 was found in all endocrine cells except the somatostatin immunoreactive cells. Both MafA and MafB were expressed in the islets; MafA being specific for the insulin cells, while MafB was primarily in the glucagon cells but also found in some insulin cells. Isl-1 was localized in all endocrine cell types. In conclusion, the closest relatives to the Gerbils express a Pdx-1 protein that is 90% similar to other rodents but also has a unique 3 amino acid insert compared to other species.  During the evolution of the spiny mice and the gerbils, it appears that the Pdx-1 gene was lost.

The morphology of islets of Langerhans is only mildly affected by the lack of Pdx-1 in the pancreas of adult Meriones jirds.

The Meriones Jirds belong to the genus of Gerbillinae (Rodentia: Muridae). We and others have previously reported the lack of the pancreatic beta-cell transcription factor, Pdx-1 in the fat sand rat, Psammomys obesus. The aim of the study was to investigate the expression and localization of Pdx-1 in phylogenetically related members of the Gerbillinae subfamily. In addition, we characterized by IHC the expression pattern of islet hormones and additional important pancreatic transcription factors in order to evaluate overall endocrine pancreas appearance. PCR showed that Pdx-1 was easily amplified from a wide range of phylogenetically distant species but not from 13 different gerbilline species. Identical to P. obesus the important beta-cell transcription factor Pdx-1 was absent from all five jirds. However, expression of other critical islet transcription factors and islet hormones was generally normal. Insulin was localized in the center of the islets with glucagon, somatostatin and pancreatic polypeptide (PP) found in the islet mantle. PYY cells were also observed and colocalized with PP cells. The NKX family of transcription factors were localized to the same cell types as seen in other rodents. MafA was nuclear localized in some of the insulin immunoreactive but not in other cell types, while MafB was found not only in the glucagon cells but also in many of the insulin cells. In conclusion, Pdx-1 appears to be lacking in all gerbils and despite the lack of Pdx-1, the Meriones Jirds have islets that are morphologically similar to other rodents and express hormones and transcription factors in the expected pattern except for MafA and MafB.

Expression of the GLP-1 receptor in mouse, rat, and human pancreas.

We studied the intra-islet localization of the glucagon-like peptide 1 receptor (GLP-1R) by colocalization studies of the GLP-1R mRNA and protein with islet cell hormones in mice, rats, and humans. In contrast to previous reports, we show that the GLP-1R is selectively located on the beta cells. The localization of GLP-1R in islets and ducts was studied using ISH and double and triple fluorescence microscopy. In normal pancreatic tissue from mice and rats, GLP-1R mRNA was only detectable in the beta cells. Double and triple immunofluorescence using two different GLP-1R antisera and combinations of insulin, glucagon, pancreatic polypeptide, and somatostatin showed that GLP-1R protein is almost exclusively colocalized with insulin. The same pattern was observed in human pancreas, but the GLP-1R expression was more heterogeneous, with populations of insulin immunoreactive cells with high and low expression. This is the first time that the GLP-1R has been localized in human islets. Furthermore, GLP-1R immunoreactivity was found in the pancreatic ducts in mouse, rat, and human pancreas. As an important confirmation of the specificity of our methods, we found no signals for GLP-1R mRNA or protein in pancreatic tissue from gene-targeted GLP-1R-deficient mice. In conclusion, our data suggest that the GLP-1 receptor is restricted to the pancreatic beta cells and the lack of receptor immunoreactivity on delta cells cannot be explained suitably to correspond with published in vivo and in vitro data. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

An immunohistochemical study of the endocrine pancreas of the African ice rat, Otomys sloggetti robertsi.

The African ice rat, Otomys sloggetti robertsi, is a member of the subfamily Otomyinae, in the superfamily of Muroidea, to which all rodents belong. Very little is known about this unique family of rodents. The study reported here examines the endocrine pancreas of this species using immunohistochemical techniques. The islets of Langerhans were scattered in the exocrine pancreas and tended to be quite small. Scattered single endocrine cells (mostly immunoreactive for insulin) were found in the exocrine pancreas and were not generally associated with ducts (as marked by pan-cytokeratin labeling). The normal islet architecture of insulin in the center and glucagon, somatostatin (SS) and pancreatic polypeptide (PP) in the rim was observed, but the islets tended to have 2-3 layers of glucagon immunoreactive cells. Examining for rarer endocrine cell types, we found that cocaine amphetamine regulated transcript (CART) immunoreactive cells were co-localized with SS; and peptide YY (PYY) immunoreactive cells could be found that were singly immunoreactive or co-localized with either PP or glucagon. Ghrelin cells were not found. MafA co-localized only with the insulin cells, while MafB, which localizes to the glucagon cells, also showed a low level of immunoreactivity in most insulin immunoreactive cells. The Nkx family of transcription factors (Nkx6.1 and 2.2) and PDX-1 were all detected in the pancreas in a similar manner to that seen in mouse and rat. In conclusion, the endocrine pancreas of the African ice rat is quite similar to that of other studied rodents, but these animals have more glucagon and SS cells than rat (Rattus) or mouse (Mus) species.

Developmental biology of the Psammomys obesus pancreas: cloning and expression of the Neurogenin-3 gene.

The desert gerbil Psammomys obesus, an established model of type 2 diabetes (T2D), has previously been shown to lack pancreatic and duodenal homeobox gene 1 (Pdx-1) expression. Pdx-1 deficiency leads to pancreas agenesis in both mice and humans. We have therefore further examined the pancreas of P. obesus during embryonic development. Using Pdx-1 antisera raised against evolutionary conserved epitopes, we failed to detect Pdx-1 immunoreactivity at any time points. However, at E14.5, Nkx6.1 immunoreactivity marks the nuclei of all epithelial cells of the ventral and dorsal pancreatic buds and the only endocrine cell types found at this time point are glucagon and PYY. At E18.5 the pancreas is well branched and both glucagon- and ghrelin-positive cells are scattered or found in clusters, whereas insulin-positive cells are not found. At E22.5, the acini of the exocrine pancreas are starting to mature, and amylase and carboxypeptidase A immunoreactivity is found scattered and not in all acini. Ghrelin-, glucagon-, PYY-, gastrin-, somatostatin (SS)-, pancreatic polypeptide (PP)-, and insulin-immunoreactive cells are found scattered or in small groups within or lining the developing ductal epithelium as marked by cytokeratin 19. Using degenerate PCR, the P. obesus Neurogenin-3 (Ngn-3) gene was cloned. Nucleotide and amino acid sequences show high homology with known Ngn-3 sequences. Using specific antiserum, we can observe that Ngn-3-immunoreactive cells are rare at E14.5 but readily detectable at E18.5 and E22.5. In conclusion, despite the lack of detection of Pdx-1, the P. obesus pancreas develops similarly to Muridae species, and the Ngn-3 sequence and expression pattern is highly conserved in P. obesus.

The effect of neurogenin3 deficiency on pancreatic gene expression in embryonic mice.

To understand the molecular mechanisms regulating pancreatic endocrine development and function, pancreatic gene expression was compared between Ngn3-deficient mice and littermate controls on embryonic days 13 and 15. Microarray analysis identified 504 genes with significant differences in expression. Fifty-two of these showed at least twofold reduction in Ngn3 knockouts compared to controls. Many of them were previously described to be involved in endocrine development and function. Among the genes not previously characterized were Rhomboid veinlet-like 4, genes involved in tetrahydrobiopterin biosynthesis and the Iroquois-type homeobox gene Irx1, the latter was selected for further investigation. In situ hybridisation demonstrated that two Iroquois genes, Irx1 and Irx2, were expressed in pancreatic endoderm of wild-type, but not Ngn3 mutant embryos. Furthermore, ectopic Ngn3 induced prominent Irx2 expression in chicken endoderm. Co-labelling established that Irx1 and Irx2 mRNA is located to glucagon-, but not insulin- or somatostatin-producing cells in mice and chicken. These data suggest that Irx1 and Irx2 serve an evolutionary conserved role in the regulation of alpha-cell-specific gene expression.