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Diabetes is characterized by insulin insufficiency due to a relative paucity of functional β-cell mass. Thus, strategies for increasing β-cell mass in situ are sought-after for therapeutic purposes. Pregnancy is a physiological state capable of inducing robust β-cell mass expansion, however, the mechanisms driving this expansion are not fully understood. Thus, the aim of this study was to characterize pregnancy-induced changes in the islet proteome at the peak of β-cell proliferation in mice. Islets from pregnant and nonpregnant littermates were compared via 2 proteomic strategies. In vivo pulsed stable isotope labeling of amino acids in cell culture was used to monitor de novo protein synthesis during the first 14.5 days of pregnancy. In parallel, protein abundance was determined using ex vivodimethyl labelling at gestational day 14.5. Comparison of the 2 datasets revealed 170 islet proteins to be up regulated as a response to pregnancy. These included several proteins, not previously associated with pregnancy-induced islet expansion, such as CLIC1, STMN1, MCM6, PPIB, NEDD4, and HLTF. Confirming the validity of our approach, we also identified proteins encoded by genes known to be associated with pregnancy-induced islet expansion, such as CHGB, IGFBP5, MATN2, EHHADH, IVD, and BMP1. Bioinformatic analyses demonstrated enrichment and activation of the biological functions: “protein synthesis” and “proliferation,” and predicted the transcription factors HNF4α, MYC, MYCN, E2F1, NFE2L2, and HNF1α as upstream regulators of the observed expressional changes. As the first characterization of the islet-proteome during pregnancy, this study provides novel insight into the mechanisms involved in promoting pregnancy-induced β-cell mass expansion and function.

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.

A young Japanese female was in 2007 reported to suffer from a congenital brain malformation with a non-functioning pancreatic endocrine tumor arising from intracranial ectopic pancreatic tissue.1 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 β-cell specific transcription factor, Nkx6.1, is expressed only in the insulin immunoreactive cells while Neither Ptf1a or PDX-1 immunoreactive cells can not be observed. Duct like structures stain strongly for pan-cytokeratin, 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.

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.

Pancreas morphogenesis and cell differentiation are highly conserved among vertebrates during fetal development. The pancreas develops through simple budlike structures on the primitive gut tube to a highly branched organ containing many specialized cell types. This review presents an overview of key molecular components and important signaling sources illustrated by an extensive three-dimensional (3D) imaging of the developing mouse pancreas at single cell resolution. The 3D documentation covers the time window between embryonic days 8.5 and 14.5 in which all the pancreatic cell types become specified and therefore includes gene expression patterns of pancreatic endocrine hormones, exocrine gene products, and essential transcription factors. The 3D perspective provides valuable insight into how a complex organ like the pancreas is formed and a perception of ventral and dorsal pancreatic growth that is otherwise difficult to uncover. We further discuss how this global analysis of the developing pancreas confirms and extends previous studies, and we envisage that this type of analysis can be instrumental for evaluating mutant phenotypes in the future.

Insufficient functional beta cell mass is a common denominator in most forms of diabetes. It will be important to be able to image the functional beta cell mass during onset and / or progression of type 1 and type 2 diabetes. It would be equally useful to possibly image beta-cell neo-formation. With the establishment of the Beta Cell Biology Consortium (BCBC) and its core facilities, one goal is to generate antibody tools against stage specific surface markers characterizing the sequential maturation from pancreatic precursors to mature beta-cells. We believe that such antibodies will also be useful for imaging the corresponding stages. Several BCBC research groups, as well as the Antibody and the Functional Genomics Cores, are involved in this project. Here we outline the overall approaches and strategies employed by the BCBC. An ultimate goal for the Consortium is that such antibody tools will allow for the optimization of in vitro culture conditions that will replicate normal pancreatic beta-cell ontogeny - a prerequisite for generating clinically relevant betacell mass for diabetes therapy.

It is well established that gut and pancreas development depend on epithelial-mesenchymal interactions. We show here that several Wnt, Frizzled, and secreted frizzled-related protein (sFRP) encoding mRNAs are present during mouse pancreatic morphogenesis. Wnt5a and 7b mRNA is broadly expressed in foregut mesenchyme starting around embryonic day 10 in mice. Other members expressed are Wnt2b, Wnt5b, and Wnt11. In addition, genes for the Wnt receptors, Frizzled2, 3, 4, 5, 6, 7, 8, and 9 are expressed. To understand potential Wnt functions in pancreas and foregut development in vivo, we analyzed transgenic F0 mouse fetuses expressing Wnt1 and 5a cDNAs under control of the PDX-1 gene promoter. In PDX-Wnt1 fetuses, the foregut region normally comprising the proximal duodenum instead resembles a posterior extension of the stomach, often associated with complete pancreatic and splenic agenesis. Furthermore, the boundary between expression domains of gastric and duodenal markers is shifted in a posterior direction. In PDX-Wnt5a fetuses, several structures derived from the proximal foregut are reduced in size, including the pancreas, spleen, and stomach, without any apparent shift in the stomach to duodenum transition. In these fetuses, overall pancreatic morphology is changed and the pancreatic epithelium is dense and compact, consistent with Wnt5A effects on cell movements and/or attachment. Taken together, these results suggest that Wnt genes participate in epithelial-mesenchymal signaling and may specify region identity in the anterior foregut. © 2002 Wiley-Liss, Inc.

ABSTRACTHepatocyte nuclear factor 6 (HNF-6) is the prototype of a new class of cut homeodomain transcription factors. During mouse development, HNF-6 is expressed in the epithelial cells that are precursors of the exocrine and endocrine pancreatic cells. We have investigated the role of HNF-6 in pancreas differentiation by inactivating its gene in the mouse. In hnf6-/-embryos, the exocrine pancreas appeared to be normal but endocrine cell differentiation was impaired. The expression of neurogenin 3 (Ngn-3), a transcription factor that is essential for determination of endocrine cell precursors, was almost abolished. Consistent with this, we demonstrated that HNF-6 binds to and stimulates the ngn3gene promoter. At birth, only a few endocrine cells were found and the islets of Langerhans were missing. Later, the number of endocrine cells increased and islets appeared. However, the architecture of the islets was perturbed, and their ß cells were deficient in glucose transporter 2 expression. Adult hnf6-/-mice were diabetic. Taken together, our data demonstrate that HNF-6 controls pancreatic endocrine differentiation at the precursor stage and identify HNF-6 as the first positive regulator of the proendocrine gene ngn3in the pancreas. They also suggest that HNF-6is a candidate gene for diabetes mellitus in humans.

Hepatocyte nuclear factor 6 (HNF-6) is the prototype of a new class of cut homeodomain transcription factors. During mouse development, HNF-6 is expressed in the epithelial cells that are precursors of the exocrine and endocrine pancreatic cells. We have investigated the role of HNF-6 in pancreas differentiation by inactivating its gene in the mouse. In hnf6−/−embryos, the exocrine pancreas appeared to be normal but endocrine cell differentiation was impaired. The expression of neurogenin 3 (Ngn-3), a transcription factor that is essential for determination of endocrine cell precursors, was almost abolished. Consistent with this, we demonstrated that HNF-6 binds to and stimulates the ngn3gene promoter. At birth, only a few endocrine cells were found and the islets of Langerhans were missing. Later, the number of endocrine cells increased and islets appeared. However, the architecture of the islets was perturbed, and their β cells were deficient in glucose transporter 2 expression. Adult hnf6−/−mice were diabetic. Taken together, our data demonstrate that HNF-6 controls pancreatic endocrine differentiation at the precursor stage and identify HNF-6 as the first positive regulator of the proendocrine gene ngn3in the pancreas. They also suggest that HNF-6is a candidate gene for diabetes mellitus in humans.

The paired box and homeodomain containing transcription factors Pax4 and Pax6 are known to be essential for development of the pancreatic endocrine cells. In this report we demonstrate that stable expression of Pax4 in a rat glucagon-producing cell line inhibits the endogenously expressed glucagon gene completely. Furthermore, Pax4 represses Pax6 independent transcription of the insulin promoter, suggesting that Pax4 can actively repress transcription in addition to acting by competition with the transcriptional activator Pax6.

Abstract:  Sixty-seven insulinomas were investigated by immunohistochemistry using site-directed antibodies against insulin, proinsulin, chromogranin A, HISL-19, and four proteins directly or indirectly involved in the proteolytic processing of proinsulin: the prohormone convertases PC2 and PC3, carboxypeptidase H (CPH) and 7B2. Results were expressed in a six-grade score according to the frequency of immunoreactive tumour cells. Insulin was expressed by all tumours, appearing in either a diffuse or a polarized pattern and being detected in more than 30% of tumour cells in all cases but three. Proinsulin was also expressed in all tumours, with more than 50% of tumour cells immunoreactive in all cases but 5. It was consistently localized in the Golgi apparatus. In about half the cases, moreover, it also showed diffuse cytoplasmic staining, usually with a very sparse distribution. Trabecular and solid insulinomas did not present specific, homogeneous patterns of insulin immunostaining. However, insulin immunoreactivity was much more abundant in trabecular than in solid neoplasms, being present in virtually all tumour cells (score 6) in 50% and 8% of cases, respectively. Virtually all insulinomas expressed PC2, PC3, CPH and 7B2, usually in 30–100% of tumour cells, with a frequency significantly related to that of insulin. However, detection of PC2 and 7B2 was slightly less frequent than that of PC3 and CPH. In consecutive sections these proteins were found to be mostly co-localized with insulin and chromogranin A but not with proinsulin. They were heavily expressed in all 10 tumours with more than 10% of cells showing cytoplasmic proinsulin immunoreactivity, indicating that the leakage of proinsulin from the Golgi compartment is not associated with faulty expression of converting enzymes and possibly reflects a saturated processing capacity. HISL-19 immunoreactivity was found in both Golgi apparatus and insulin stores, indicating that the relevant antigen is different from all other proteins investigated. These results do not support a defect in expression or localization of proinsulin-processing enzymes in most insulinomas.

The hypothalamic satiety peptide CART (cocaine and amphetamine regulated transcript) is expressed at high levels in anorectic rat glucagonomas but not in hypoglycemic insulinomas. However, a non‐anorectic metastasis derived from the glucagonoma retained high CART expression levels and produced circulating CART levels comparable to that of the anorectic tumors. Moreover, distinct glucagonoma lines derived by stable HES‐1 transfection of the insulinoma caused severe anorexia but retained low circulating levels of CART comparable to that of insulinoma bearing or control rats. Islet tumor associated anorexia and circulating CART levels are thus not correlated, and in line with this peripheral administration of CART (5–50 mg/kg) produced no effect on feeding behavior. In the rat two alternatively spliced forms of CART mRNA exist and quantitative PCR revealed expression of both forms in the hypothalamus, in the different islet tumors, and in the islets of Langerhans. Immunocytochemistry as well as in situ hybridization localized CART expression to the somatostatin producing islet D cell. A potential endocrine/paracrine role of islet CART remains to be clarified.

Novel islet cell, duct cell, and acinar cell markers have been identified by monoclonal autoantibodies (Maab) derived from prediabetic BB rats. Spleen cells from two rats that both developed diabetes after splenectomy were fused with mouse myeloma cells. A cellular immunoradiometric assay for differential reactivity toward the surface of two closely related, insulin- and noninsulin-producing rat islet tumor cell lines was used to select and clone several IgM-producing hybridomas. The supernatants were finally characterized by two-color immunofluorescence with islet hormone antisera on frozen sections of human, monkey, and rat pancreas. Maab EB52 stained PP cells, but also few A cells on rat pancreas. Maab CA812 identified a subpopulation of islet D cellson rat, human, and monkey pancreas. Although the CA81 Zreactive antigen and somatostatin were coexpressed in most D cells in adult rat pancreas, only a few islet D cells were stained in the newborn pancreas. The CA812-reactiveantigen was not detected in somatostatin-producing cells in the duct epithelium. Maab H37 and IF5 selectively stained acinar cells in rat, human, and monkey pancreas, whereas Maab DA39 identified the rat ductal epithelium including the scattered endocrine cells of the ducts. In summary, B lymphocytes producing autoantibodies to pancreatic endocrine, exocrine, and ductal markers are present in prediabetic BB rats and can be detected by use of transformed pluripotent islet cells as target. Such B lymphocytes can be immortalizedto produce monoclonal antibodies to study their role in insulin dependent diabetes mellitus pathogenesis and to clarify the development of the pancreas.

Glucose‐stimulated expression of the insulin gene in β cells is mediated by the PDX‐1 transcription factor. In this report, we show that stimulation results from effects on activation and DNA‐binding potential. Thus, glucose specifically stimulated expression in MIN6 β cells from chimeras of PDX‐1 and the GAL4 DNA‐binding domain which spanned the N‐terminal PDX‐1 activation domain located between amino acids 1 to 79. GAL4:PDX activity was induced over physiological glucose concentrations and was also regulated by effectors of this response. The level of endogenous PDX‐1 binding and phosphorylation were also induced under these conditions. We discuss how changes in PDX‐1 phosphorylation may influence activity in glucose‐treated β cells.

Genetically obese Zucker rats (fa/fa) on a diabetogenic diet rich in lard and sucrose develop chronic hyperglycemia accompanied by severe hyperinsulinemia. Non-enzymatically glycated protein content was increased in tendon (p<0.0001) and aorta (p<0.04), but not nerve, from hyperglycemic rats as compared to normoglycemic lean litter-mates on a conventional chow diet (mmol furosine/mol tyrosine in tissue hydrolysate from 61-week-old rats: tendon, 29.8±1.8 vs 25.9±1.3; aorta, 12.0±1.0 vs 11.0±1.1). In pancreatic islets, non-enzymatically glycated protein was neither found in lean rats of any age nor in the obese up to an age of 36 weeks. At an age of 61 weeks, non-enzymatically glycated protein accumulated in islets of obese animals, resulting in levels of 17.02–44.65 mmol furosine/mol tyrosine. This rise in islet glycated protein content was not accompanied by a comparable increase in plasma glycemia, but simultaneous histological examination of pancreatic tissue revealed fibrosis of islets. Fibers were probably of collagenic quality without islet amyloid polypeptide immunoreactivity. Because collagen is known to be highly susceptible to non-enzymatic glycation, we suspect that collagenic fibers but not endocrine cells are the main source of glycated protein accumulation in these islets. Hence, our data do not give evidence that non-enzymatic protein glycation plays a role in the islet degeneration occurring in hyperglycemia. Furthermore, immunohistochemical staining for various endocrine peptides did not suggest loss of any hormone-producing cell type or defective pancreatic hormone production in hyperglycemic old, obese Zucker rats. The more soluble nature of rat vs human islet amyloid polypeptide may explain the fact that hyperglycemia-induced fibrosis in rat islets does not include amyloidosis, which may contribute to late beta cell failure in human type II diabetes.

Development of a high capacity multiplex reverse transcriptase-polymerase chain reaction protocol has allowed us to screen lineage related rat islet tumors classified as α-, β-, and δ-like as judged by their hormone profile for differential expression of more than 50 selected genes. We find that in addition to insulin the insulinoma express the normal β-cell markers Pdx-1, IAPP, and Glut-2, and that these markers are absent from the glucagonoma: a reflection of the normal α-cell. Furthermore, this study suggests that the GLP-1, glucagon, GIP, IGF-1, and insulin receptors as well as E-cadherin, R-cadherin, Id-1, and Id-2 are differentially expressed within the islet of Langerhans. Importantly, insulinoma-specific expression of the recently cloned homeodomain protein Nkx 6.1 predicted β-cell-specific expression in the normal islet. Immunohistochemistry using antibodies raised against recombinant Nkx 6.1 did indeed localize Nkx 6.1 expression exclusively to the nuclei of normal islet β-cells. Apart from pancreatic islets only the antral part of the stomach contained Nkx 6.1 mRNA. We conclude that multiplex reverse transcriptase-polymerase chain reaction-based mRNA profiling is a powerful tool to identify differentially expressed genes within phenotypically related cells and propose that Nkx 6.1 is involved in specifying the unique characteristics of the β-cell.

We have carried out an immunohistochemical investigation of 15 human insulinomas applying monoclonal antibodies specifically recognizing proinsulin and insulin. Our results demonstrate that the epitopes unique to proinsulin and insulin can be detected with the respective monoclonal antibodies using the protein A-gold technique after routine formaldehyde fixation and paraffin embedding of the tissues. The immunostaining pattern for proinsulin and insulin in the insulinomas was different from the observed in B cells of pancreatic islets present in the adjacent normal pancreas. Furthermore, the pattern of immunostaining was found to vary from tumor to tumor. These findings strongly suggest the possibility of a disturbed proinsulin to insulin conversion in human insulinomas

The gastric epithelium is renewed from stem cells in the isthmus of the gastric glands. We describe that the two neuroendocrine peptides gastrin and somatostatin are coexpressed by isthmic stem cells. Bromodeoxyuridine labeling indicates that the coexpressing cells divide and differentiate into gastrin and somatostatin cells, which remain in paracrine contact during most of their migration down into the gland. The coexpressing cells display nuclear immunoreactivity for the transcription factors Isl-1 and CREB, which have been implicated in somatostatin gene expression. Differentiated gastrin cells lack Isl-1 reactivity and show variable staining for CREB while differentiated somatostatin cells display Isl-1 and CREB reactivity.

The homeodomain protein Isl-1 and the proto-oncogene Rhombotin (a LIM-only protein), share a double zinc-binding LIM domain and have both been implicated in neural and possibly endocrine development. Isl-1 is expressed in all endocrine cell-types of the islet of Langerhans while Rhombotin mRNA expression was reported in rat insulinoma cells. We have cloned and sequenced Rhombotin cDNA from rat insulinoma (99.4% identical to human and mouse sequences) and demonstrate that it is expressed in normal islets, intestinal tissue, and testis, in addition to the brain; but absent in all other organs tested. Rhombotin mRNA is expressed in phenotypically distinct islet tumours (α-, β, and δ-tumours) at levels comparable to that of normal islets. Antisera raised against two distinct epitopes contained within a short synthetic peptide representing part of the N-terminal LIM domain of Rhombotin surprisingly stain α- and δ-cells, respectively, on sections of rat pancreas. Rhombotin is undetectable by immunocytochemistry using LIM-domain antisera on intact monolayer islet tumor cells or transfected fibroblasts while readily detectable when equipped with a FLAG epitope, as detected with FLAG antiserum. In contrast, recombinant FLAG-Rhombotin is efficiently recognised by Western blotting or immunoprecipitation with all LIM-specific antisera. Almost identical results were obtained with LIM-specific versus homeodomain/C-terminal Isl-1 antisera staining α-cell cytoplasm or all islet nuclei, respectively. We conclude that Rhombotin in addition to Isl-1 is expressed in the islet of Langerhans and propose that the differential staining patterns obtained with antisera towards the LIM domains versus flanking epitopes of both proteins reflect (1) cell-specific protein-protein interactions of these domains or, alternatively, (2) islet cell type specific expression of novel homologous LIM domain proteins.

Regenerative therapy in diabetes with the capacity to reconstitute a functional β-cell mass sufficient for glycemic control holds the promise to effectively prevent the development of devastating late complications due to the unique ability of the β-cell to sense and regulate blood-glucose levels. An ability that cannot be mimicked by insulin replacement therapy or any other means of current treatment regiments for very large patient populations. Recently, Douglas A. Melton’s group from Harvard University reported the identification of a circulating protein secreted from the liver under insulin resistant states which is sufficient to dramatically and specifically increase the replication rate of β-cells in the mouse resulting in an increased functional β-cell mass over time. They re-named the factor betatrophin and described a number of exciting features of this molecule which suggested that it could be a potential candidate for development as a regenerative medicine in diabetes.1The official name of the gene encoding mouse betatrophin is Gm6484, but it has been annotated a number of times under different names: EG6242192,3, RIFL4, Lipasin5and ANGPTL8.6The official human gene name is C19orf80, but it has also been annotated as TD267, LOC559088, as well as RIFL, Lipasin, ANGPTL8 and betatrophin.

The distribution of proinsulin and insulin immunoreactivity was studied in 76 human insulinomas and in normal pancreas. One trabecular and two solid insulinomas showed the staining pattern of normal β cells. A “near normal” staining pattern (perinuclear proinsulin and diffuse or polarized insulin staining) existed in 10 of 27 trabecular and 11 of 44 solid insulinomas. An “intermediate” staining pattern (intense perinuclear as well as weaker diffuse proinsulin staining with diffuse or polarized insulin staining) was observed in 10 of 27 trabecular and 20 of 44 solid insulinomas. Different “abnormal” staining patterns were found in 6 of 27 trabecular and 6 of 44 solid insulinomas. Of the 5 glandular insulinomas, 4 exhibited a “near normal” and one an “abnormal” staining pattern. No correlation was found between any particular staining pattern and the multihormonality or malignancy of the insulinomas. The diffuse labeling for proinsulin in about 50% of the insulinomas is suggestive of abnormal prohormone processing.