Xiangwei Xiao

M2 macrophages promote beta-cell proliferation by up-regulation of SMAD7

Significance Here, we show how, mechanistically, inflammation-recruited macrophages may stimulate beta-cell proliferation in the pancreas, and specifically identify that TGFβ1 and EGF, which are secreted by M2 macrophages, induce SMAD7 expression in beta cells. SMAD7 not only activates cell cycle activators but also induces the nuclear exclusion of cell cycle inhibitors to promote beta-cell replication. Our study thus reveals a molecular pathway to induce beta-cell proliferation through enhanced SMAD7 activity specifically in beta cells. Determination of signaling pathways that regulate beta-cell replication is critical for beta-cell therapy. Here, we show that blocking pancreatic macrophage infiltration after pancreatic duct ligation (PDL) completely inhibits beta-cell proliferation. The TGFβ superfamily signaling inhibitor SMAD7 was significantly up-regulated in beta cells after PDL. Beta cells failed to proliferate in response to PDL in beta-cell–specific SMAD7 mutant mice. Forced expression of SMAD7 in beta cells by itself was sufficient to promote beta-cell proliferation in vivo. M2, rather than M1 macrophages, seem to be the inducers of SMAD7-mediated beta-cell proliferation. M2 macrophages not only release TGFβ1 to directly induce up-regulation of SMAD7 in beta cells but also release EGF to activate EGF receptor signaling that inhibits TGFβ1-activated SMAD2 nuclear translocation, resulting in TGFβ signaling inhibition. SMAD7 promotes beta-cell proliferation by increasing CyclinD1 and CyclinD2, and by inducing nuclear exclusion of p27. Our study thus reveals a molecular pathway to potentially increase beta-cell mass through enhanced SMAD7 activity induced by extracellular stimuli.

No evidence for β cell neogenesis in murine adult pancreas.

Whether facultative β cell progenitors exist in the adult pancreas is a major unsolved question. To date, lineage-tracing studies have provided conflicting results. To track β cell neogenesis in vivo, we generated transgenic mice that transiently coexpress mTomato and GFP in a time-sensitive, nonconditional Cre-mediated manner, so that insulin-producing cells express GFP under control of the insulin promoter, while all other cells express mTomato (INSCremTmG mice). Newly differentiated β cells were detected by flow cytometry and fluorescence microscopy, taking advantage of their transient coexpression of GFP and mTomato fluorescent proteins. We found that β cell neogenesis predominantly occurs during embryogenesis, decreases dramatically shortly after birth, and is completely absent in adults across various models of β cell loss, β cell growth and regeneration, and inflammation. Moreover, we demonstrated upregulation of neurogenin 3 (NGN3) in both proliferating ducts and preexisting β cells in the ligated pancreatic tail after pancreatic ductal ligation. These results are consistent with some recent reports, but argue against the widely held belief that NGN3 marks cells undergoing endocrine neogenesis in the pancreas. Our data suggest that β cell neogenesis in the adult pancreas occurs rarely, if ever, under either normal or pathological conditions.

TGFβ Receptor Signaling Is Essential for Inflammation-Induced but Not β-Cell Workload–Induced β-Cell Proliferation

Protection and restoration of a functional β-cell mass are fundamental strategies for prevention and treatment of diabetes. Consequently, knowledge of signals that determine the functional β-cell mass is of immense clinical relevance. Transforming growth factor β (TGFβ) superfamily signaling pathways play a critical role in development and tissue specification. Nevertheless, the role of these pathways in adult β-cell homeostasis is not well defined. Here, we ablated TGFβ receptor I and II genes in mice undergoing two surgical β-cell replication models (partial pancreatectomy or partial duct ligation), representing two triggers for β-cell proliferation, increased β-cell workload and local inflammation, respectively. Our data suggest that TGFβ receptor signaling is necessary for baseline β-cell proliferation. By either provision of excess glucose or treatment with exogenous insulin, we further demonstrated that inflammation and increased β-cell workload are both stimulants for β-cell proliferation but are TGFβ receptor signaling dependent and independent, respectively. Collectively, by using a pancreas-specific TGFβ receptor–deleted mouse model, we have identified two distinct pathways that regulate adult β-cell proliferation. Our study thus provides important information for understanding β-cell proliferation during normal growth and in pancreatic diseases.

Hypoglycemia Reduces Vascular Endothelial Growth Factor A Production by Pancreatic Beta Cells as a Regulator of Beta Cell Mass

Background: Beta cell VEGF-A is critical for islet vascularization and Insulin secretion. Results: VEGF-A release and synthesis in beta cells are regulated separately. Sustained hypoglycemia reduces beta cell mass through a decrease in Vegf-A signaling. Conclusion: Beta cell mass can be regulated via modulated Vegf-A signaling. Significance: Our data reveal a novel pathway for regulating beta cell mass physiologically. VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature, and Insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that VEGF-A secretion, but not gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with Insulin pellets, resulting in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need.

Pancreatic duct cells as a source of VEGF in mice

Aims/hypothesisVascular endothelial growth factor (VEGF) is essential for proper pancreatic development, islet vascularisation and insulin secretion. In the adult pancreas, VEGF is thought to be predominantly secreted by beta cells. Although human duct cells have previously been shown to secrete VEGF at angiogenic levels in culture, an analysis of the kinetics of VEGF synthesis and secretion, as well as elucidation of an in vivo role for this ductal VEGF in affecting islet function and physiology, has been lacking.MethodsWe analysed purified duct cells independently prepared by flow cytometry, surgical isolation or laser-capture microdissection. We infected duct cells in vivo with Vegf (also known as Vegfa) short hairpin RNA (shRNA) in an intrapancreatic ductal infusion system and examined the effect of VEGF knockdown in duct cells in vitro and in vivo.ResultsPancreatic duct cells express high levels of Vegf mRNA. Compared with beta cells, duct cells had a much higher ratio of secreted to intracellular VEGF. As a bioassay, formation of tubular structures by human umbilical vein endothelial cells was essentially undetectable when cultured alone and was substantially increased when co-cultured with pancreatic duct cells but significantly reduced when co-cultured with duct cells pretreated with Vegf shRNA. Compared with islets transplanted alone, improved vascularisation and function was detected in the islets co-transplanted with duct cells but not in islets co-transplanted with duct cells pretreated with Vegf shRNA.Conclusions/interpretationHuman islet preparations for transplantation typically contain some contaminating duct cells and our findings suggest that the presence of duct cells in the islet preparation may improve transplantation outcomes.

Rapid and simplified purification of recombinant adeno-associated virus.

Preclinical gene therapy studies both in vitro and in vivo require high purity preparations of adeno-associated virus (AAV). Current methods for purification of AAV entail the use of centrifugation over either a CsCl or iodixanol gradient, or the use of chromatography. These methods can be cumbersome and expensive, necessitating ultrahigh speed gradient centrifugation or, for chromatography the use of other expensive equipment. In addition, these methods are time consuming, and the viral yield is not high. Currently no commercial purification kits are available for other than AAV serotype 2. A simplified method was used for the purification of AAV, with a viral yield that is able to be used effectively in adult and embryo mice. The method does not require ultrahigh speed gradient centrifugation nor chromatography. Instead, polyethylene glycol (PEG)/aqueous two-phase partitioning is used to remove soluble proteins from the PEG8000 precipitated virus-protein mixture. The procedure obtained rapidly up to 95% recovery of high quality purified AAV. The entire purification process, including HEK293 cell transfection, can be completed readily within one week, with purity seemingly higher than that obtained after one round of CsCl gradient purification.

Neurogenin3 activation is not sufficient to direct duct-to-beta cell transdifferentiation in the adult pancreas

Background: Whether neurogenin3 activation represents beta cell neogenesis in adults is controversial. Results: Neurogenin3-activated pancreatic duct cells do not become beta cells. Conclusion: Neurogenin3 activation is not a signature for adult beta cell neogenesis. Significance: Our data strongly argue against the widely held belief that neurogenin3 is a marker of beta cell neogenesis in the adult pancreas, especially from duct cells. It remains controversial whether adult pancreatic ducts harbor facultative beta cell progenitors. Because neurogenin3 (Ngn3) is a key determinant of pancreatic endocrine cell neogenesis during embryogenesis, many studies have also relied upon Ngn3 expression as evidence of beta cell neogenesis in adults. Recently, however, Ngn3 as a marker of adult beta cell neogenesis has been called into question by reports of Ngn3 expression in fully-developed beta cells. Nevertheless, direct evidence as to whether Ngn3 activation in adult pancreatic duct cells may lead to duct-to-beta cell transdifferentiation is lacking. Here we studied two models of Ngn3 activation in adult pancreatic duct cells (low-dose alloxan treatment and pancreatic duct ligation) and lineage-traced Ngn3-activated duct cells by labeling them through intraductal infusion with a cell-tagging dye, CFDA-SE No dye-labeled beta cells were found during the follow-up in either model, suggesting that activation of Ngn3 in duct cells is not sufficient to direct their transdifferentiation into beta cells. Therefore, Ngn3 activation in duct cells is not a signature for adult beta cell neogenesis.

Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes

Genetic manipulations, with or without lineage tracing for specific pancreatic cell types, are very powerful tools for studying diabetes, pancreatitis and pancreatic cancer. Nevertheless, the use of Cre/loxP systems to conditionally activate or inactivate the expression of genes in a cell type– and/or temporal-specific manner is not applicable to cell tracing and/or gene manipulations in more than one lineage at a time. Here we report a technique that allows efficient delivery of dyes for cell tagging into the mouse pancreas through the duct system, and that also delivers viruses carrying transgenes or siRNA under a specific promoter. When this technique is applied in genetically modified mice, it enables the investigator to perform either double lineage tracing or cell lineage tracing combined with gene manipulation in a second lineage. The technique requires <40 min.

Intraislet Pancreatic Ducts Can Give Rise to Insulin-Positive Cells.

A key question in diabetes research is whether new β-cells can be derived from endogenous, nonendocrine cells. The potential for pancreatic ductal cells to convert into β-cells is a highly debated issue. To date, it remains unclear what anatomical process would result in duct-derived cells coming to exist within preexisting islets. We used a whole-mount technique to directly visualize the pancreatic ductal network in young wild-type mice, young humans, and wild-type and transgenic mice after partial pancreatectomy. Pancreatic ductal networks, originating from the main ductal tree, were found to reside deep within islets in young mice and humans but not in mature mice or humans. These networks were also not present in normal adult mice after partial pancreatectomy, but TGF-β receptor mutant mice demonstrated formation of these intraislet duct structures after partial pancreatectomy. Genetic and viral lineage tracings were used to determine whether endocrine cells were derived from pancreatic ducts. Lineage tracing confirmed that pancreatic ductal cells can typically convert into new β-cells in normal young developing mice as well as in adult TGF-β signaling mutant mice after partial pancreatectomy. Here the direct visual evidence of ducts growing into islets, along with lineage tracing, not only represents strong evidence for duct cells giving rise to β-cells in the postnatal pancreas but also importantly implicates TGF-β signaling in this process.

Concise Review: New Insights Into the Role of Macrophages in β-Cell Proliferation

Diabetes mellitus can potentially be treated with islet transplantation, but additional sources of β cells are necessary to overcome the short supply of donor pancreases. Although controversy still exists, it is generally believed that the postnatal expansion of the β‐cell mass is mainly through pre‐existing β‐cell replication. Thus, understanding the molecular mechanisms underlying the regulation of β‐cell proliferation might lead to clinical strategies for increasing β‐cell numbers, both in vitro and in vivo. Macrophages have a well‐recognized role in the development of insulitis as part of the pathogenesis of type 1 diabetes. However, a potential role for macrophage polarization, triggered by specific environmental stimuli, in promoting β‐cell proliferation has only recently been appreciated. In the present review, we discuss several independent studies, using different regeneration models, that demonstrate a substantial inductive role for macrophages in β‐cell proliferation. Additional dissection of the involved cell‐cell crosstalk through specific signal transduction pathways is expected to improve our understanding of β‐cell proliferation and might facilitate the current β‐cell replacement therapy.