Daniel Eberhard

‘Giving and taking’: endothelial and β-cells in the islets of Langerhans

The beta-cells of the islets of Langerhans are embedded in a dense capillary network. The blood vessels supply the islet cells with nutrients and oxygen, and in turn take up the secreted islet hormones to deliver them to target tissues. In addition, vessels provide a basement membrane, which optimizes islet function. In this review we focus on the dynamic interactions between blood vessels and beta-cells, which are pivotal for enhancing insulin expression and beta-cell proliferation in response to increased insulin demand during body growth, pregnancy, and virtually all conditions associated with insulin resistance. Importantly, a failure in this adaptive response might contribute to the onset of type 2 diabetes mellitus.

Characterization of pancreatic NMDA receptors as possible drug targets for diabetes treatment

In the nervous system, NMDA receptors (NMDARs) participate in neurotransmission and modulate the viability of neurons. In contrast, little is known about the role of NMDARs in pancreatic islets and the insulin-secreting beta cells whose functional impairment contributes to diabetes mellitus. Here we found that inhibition of NMDARs in mouse and human islets enhanced their glucose-stimulated insulin secretion (GSIS) and survival of islet cells. Further, NMDAR inhibition prolonged the amount of time that glucose-stimulated beta cells spent in a depolarized state with high cytosolic Ca2+ concentrations. We also noticed that, in vivo, the NMDAR antagonist dextromethorphan (DXM) enhanced glucose tolerance in mice, and that in vitro dextrorphan, the main metabolite of DXM, amplified the stimulatory effect of exendin-4 on GSIS. In a mouse model of type 2 diabetes mellitus (T2DM), long-term treatment with DXM improved islet insulin content, islet cell mass and blood glucose control. Further, in a small clinical trial we found that individuals with T2DM treated with DXM showed enhanced serum insulin concentrations and glucose tolerance. Our data highlight the possibility that antagonists of NMDARs may provide a useful adjunct treatment for diabetes.

Age- and diet-dependent requirement of DJ-1 for glucose homeostasis in mice with implications for human type 2 diabetes

Elderly patients often suffer from multiple age-related diseases. Here we show that the expression of DJ-1, an antioxidant protein with reduced expression in the central nervous system of patients with Parkinsons disease, is reduced in pancreatic islets of patients with type 2 diabetes mellitus (T2DM). In contrast, under non-diabetic conditions, DJ-1 expression increases in mouse and human islets during aging. In mouse islets, we show that DJ-1 prevents an increase in reactive oxygen species levels as the mice age. This antioxidant function preserves mitochondrial integrity and physiology, prerequisites for glucose-stimulated insulin secretion. Accordingly, DJ-1-deficient mice develop glucose intolerance and reduced β cell area as they age or gain weight. Our data suggest that DJ-1 is more generally involved in age- and lifestyle-related human diseases and show for the first time that DJ-1 plays a key role in glucose homeostasis and might serve as a novel drug target for T2DM.

Localization of GFP in Frozen Sections from Unfixed Mouse Tissues: Immobilization of a Highly Soluble Marker Protein by Formaldehyde Vapor

Green fluorescent protein (GFP) and its variants, such as enhanced GFP (EGFP), have been introduced into mammalian cells by transgenes, e.g., to distinguish donor from host cells after transplantation. Free GFP is extremely soluble and leaks out from liquid-covered cryostat sections so that fixation of whole organs before sectioning has been mandatory. This precludes the analysis of serial sections with respect to fixation-sensitive enzyme activities and antigens. We describe here a vapor fixation for sections from unfixed cryostat blocks of tissue that allows unrestricted enzyme and immunohistochemistry on adjacent sections, as demonstrated for cross-striated muscle and other tissues from EGFP transgenic “green mice” and for a transplantation experiment.

The pancreatic β-cell in the islet and organ community

The endocrine pancreas consists of highly vascularized and innervated endocrine mini-organs--the islets of Langerhans. These contain multiple types of hormone-producing cells, including the insulin-secreting beta-cell. The major task of the fully differentiated beta-cell is the tight regulation of blood glucose levels by secreting insulin into the blood stream. This requires molecular features to measure glucose and produce, process, and release insulin by exocytosis. Now multiple interactions with endocrine and nonendocrine islet cells as well as with other organs have been shown to affect the developing as well as the mature beta-cell. Therefore, failure of any of these interactions can inhibit beta-cell differentiation and glucohomeostasis. Here we review recent reports on intrapancreatic cell-cell interactions as well as signals derived from extrapancreatic organs that affect the pancreatic beta-cell.

β cells occur naturally in extrahepatic bile ducts of mice

Insulin-secreting β cells were thought to reside only in the pancreas. Here, we show that β cells are also present in the extra-hepatic bile ducts of mice. They are characterised by insulin and C-peptide content, the presence of secretory granules that are immunoreactive for insulin, and the ducts exhibit glucose-stimulated insulin secretion. Genetic lineage labelling shows that these β cells arise from the liver domain rather than the pancreas and, by histological study, they appear to be formed directly from the bile duct epithelium in late embryogenesis. Other endocrine cell types (producing somatostatin and pancreatic polypeptide) are also found in close association with the bile-duct-derived β cells, but exocrine pancreatic tissue is not present. This discovery of β cells outside the mammalian pancreas has implications for regenerative medicine, indicating that biliary epithelium might offer a new source of β cells for the treatment of diabetes. The finding also has evolutionary significance, because it is known that certain basal vertebrates usually form all of their β cells from the bile ducts. The mammalian bile-duct-derived β cells might therefore represent an extant trace of the evolutionary origin of the vertebrate β cell.

Molecular and Cellular Basis of Regeneration and Tissue Repair

Abstract.The ability to produce differentiated cell types at will offers one approach to cell therapy and therefore the treatment and cure of degenerative diseases such as diabetes and liver failure. Until recently it was thought that differentiated cells could only be produced from embryonic or adult stem cells. However, we now know that this is not the case, and there is a growing body of evidence to show that one differentiated cell type can convert into a completely different phenotype (transdifferentiation). Understanding the cellular and molecular basis of transdifferentiation will allow us to reprogram cells for transplantation. This approach will complement the use of embryonic and adult stem cells in the treatment of degenerative disorders. In this review, we will focus on some well-documented examples of transdifferentiation. (Part of a Multi-author Review)

Dexamethasone Treatment Induces the Reprogramming of Pancreatic Acinar Cells to Hepatocytes and Ductal Cells

Background The pancreatic exocrine cell line AR42J-B13 can be reprogrammed to hepatocytes following treatment with dexamethasone. The question arises whether dexamethasone also has the capacity to induce ductal cells as well as hepatocytes. Methodology/Principal Findings AR42J-B13 cells were treated with and without dexamethasone and analyzed for the expression of pancreatic exocrine, hepatocyte and ductal markers. Addition of dexamethasone inhibited pancreatic amylase expression, induced expression of the hepatocyte marker transferrin as well as markers typical of ductal cells: cytokeratin 7 and 19 and the lectin peanut agglutinin. However, the number of ductal cells was low compared to hepatocytes. The proportion of ductal cells was enhanced by culture with dexamethasone and epidermal growth factor (EGF). We established several features of the mechanism underlying the transdifferentiation of pancreatic exocrine cells to ductal cells. Using a CK19 promoter reporter, we show that a proportion of the ductal cells arise from differentiated pancreatic exocrine-like cells. We also examined whether C/EBPβ (a transcription factor important in the conversion of pancreatic cells to hepatocytes) could alter the conversion from acinar cells to a ductal phenotype. Overexpression of an activated form of C/EBPβ in dexamethasone/EGF-treated cells provoked the expression of hepatocyte markers and inhibited the expression of ductal markers. Conversely, ectopic expression of a dominant-negative form of C/EBPβ, liver inhibitory protein, inhibited hepatocyte formation in dexamethasone-treated cultures and enhanced the ductal phenotype. Conclusions/Significance These results indicate that hepatocytes and ductal cells may be induced from pancreatic exocrine AR42J-B13 cells following treatment with dexamethasone. The conversion from pancreatic to hepatocyte or ductal cells is dependent upon the expression of C/EBPβ.

Origin of pancreatic endocrine cells from biliary duct epithelium

Abstract.We describe an explant culture system to study the formation of pancreatic-type endocrine cells by the biliary tract. In this model, β-cells and other endocrine cells appear in the biliary duct epithelium and their number increases. Evidence for an origin from the duct epithelium is threefold. Firstly, differentiating cells transiently co-express insulin and bind Dolichos lectin. Secondly, β-cells in cultures isolated from Alb-Cre-R26R-LacZ mice are β-galactosidase positive. Thirdly, co-culture of biliary epithelium and ROSA26 pancreatic buds shows that endocrine cells do not migrate from the pancreas. The expression of the pancreatic transcription factors Pdx1, HNF6 and Sox9 is widespread, as is Hes1, which represses endocrine development, while that of Ngn3, which is a proendocrine transcription factor, is transient, consistent with an early stage of endocrine cell differentiation. Nicotinamide will increase the number of β-cells formed, while EGF+LIF completely inhibits their formation.

Reprogramming of Various Cell Types to a Beta-Like State by Pdx1, Ngn3 and MafA

The three transcription factors, PDX1, NGN3 and MAFA, are very important in pancreatic development. Overexpression of these three factors can reprogram both pancreatic exocrine cells and SOX9-positive cells of the liver into cells resembling pancreatic beta cells. In this study we investigate whether other cell types can be reprogrammed. Eight cell types are compared and the results are consistent with the idea that reprogramming occurs to a greater degree for developmentally related cells (pancreas, liver) than for other types, such as fibroblasts. Using a line of mouse hepatocyte-derived cells we screened 13 compounds for the ability to increase the yield of reprogrammed cells. Three are active and when used in combination they can increase the yield of insulin-immunopositive cells by a factor of six. These results should contribute to the eventual ability to develop a new cure for diabetes based on the ability to reprogram other cells in the body to a beta cell phenotype.