Differentiation of pancreatic endocrine progenitors reversibly blocked by premature induction of MafA.

Specification and maturation of insulin(+) cells accompanies a transition in expression of Maf family of transcription factors. In development, MafA is expressed after specification of insulin(+) cells that are expressing another Maf factor, MafB; after birth, these insulin(+) MafA(+) cells stop MafB expression and gain glucose responsiveness. Current differentiation protocols for deriving insulin-producing ß-cells from stem cells result in ß-cells lacking both MafA expression and glucose-stimulated insulin secretion. So driving expression of MafA, a ß-cell maturation factor in endocrine precursors could potentially generate glucose-responsive MafA(+) ß cells. Using inducible transgenic mice, we characterized the final stages of ß-cell differentiation and maturation with MafA pause/release experiments. We found that forcing MafA transgene expression, out of its normal developmental context, in Ngn3(+) endocrine progenitors blocked endocrine differentiation and prevented the formation of hormone(+) cells. However, this arrest was reversible such that with stopping the transgene expression, the cells resumed their differentiation to hormone(+) cells, including α-cells, indicating that the block likely occurred after progenitors had committed to a specific hormonal fate. Interestingly, this delayed resumption of endocrine differentiation resulted in a greater proportion of immature insulin(+)MafB(+) cells at P5, demonstrating that during maturation the inhibition of MafB in ß-cell transitioning from insulin(+)MafB(+) to insulin(+)MafB(-) stage is regulated by cell-autonomous mechanisms. These results demonstrate the importance of proper context of initiating MafA expression on the endocrine differentiation and suggest that generating mature Insulin(+)MafA(+) ß-cells will require the induction of MafA in a narrow temporal window to achieve normal endocrine differentiation.

Synergizing genomic analysis with biological knowledge to identify and validate novel genes in pancreatic development.

OBJECTIVE: This study investigated the utility of advanced computational techniques to large-scale genome-based data to identify novel genes that govern murine pancreatic development. METHODS: An expression data set for mouse pancreatic development was complemented with high-throughput data analyzer to identify and prioritize novel genes. Quantitative real-time polymerase chain reaction, in situ hybridization, and immunohistochemistry were used to validate selected genes. RESULTS: Four new genes whose roles in the development of murine pancreas have not previously been established were identified: cystathionine ß-synthase (Cbs), Meis homeobox 1, growth factor independent 1, and aldehyde dehydrogenase 18 family, member A1. Their temporal expression during development was documented. Cbs was localized in the cytoplasm of the tip cells of the epithelial chords of the undifferentiated progenitor cells at E12.5 and was coexpressed with the pancreatic and duodenal homeobox 1 and pancreas-specific transcription factor, 1a-positive cells. In the adult pancreas, Cbs was localized primarily within the acinar compartment. CONCLUSIONS: In silico analysis of high-throughput microarray data in combination with background knowledge about genes provides an additional reliable method of identifying novel genes. To our knowledge, the expression and localization of Cbs have not been previously documented during mouse pancreatic development.

Bombesin receptor subtype-3 (BRS-3) regulates glucose-stimulated insulin secretion in pancreatic islets across multiple species.

Bombesin receptor subtype-3 (BRS-3) regulates energy homeostasis, and BRS-3 agonism is being explored as a possible therapy for obesity. Here we study the role of BRS-3 in the regulation of glucose-stimulated insulin secretion (GSIS) and glucose homeostasis. We quantified BRS-3 mRNA in pancreatic islets from multiple species and examined the acute effects of Bag-1, a selective BRS-3 agonist, on GSIS in mouse, rat, and human islets, and on oral glucose tolerance in mice. BRS-3 is highly expressed in human, mouse, rhesus, and dog (but not rat) pancreatic islets and in rodent insulinoma cell lines (INS-1 832/3 and MIN6). Silencing BRS-3 with small interfering RNA or pharmacological blockade with a BRS-3 antagonist, Bantag-1, reduced GSIS in 832/3 cells. In contrast, the BRS-3 agonist (Bag-1) increased GSIS in 832/3 and MIN6 cells. The augmentation of GSIS by Bag-1 was completely blocked by U73122, a phospholipase C inhibitor. Bag-1 also enhanced GSIS in islets isolated from wild-type, but not Brs3 knockout mice. In vivo, Bag-1 reduced glucose levels during oral glucose tolerance test in a BRS-3-dependent manner. BRS-3 agonists also increased GSIS in human islets. These results identify a potential role for BRS-3 in islet physiology, with agonism directly promoting GSIS. Thus, in addition to its potential role in the treatment of obesity, BRS-3 may also regulate blood glucose levels and have a role in the treatment of diabetes mellitus.

Regenerating pancreatic beta-cells: plasticity of adult pancreatic cells and the feasibility of in-vivo neogenesis.

PURPOSE OF REVIEW: Diabetes results from inadequate functional mass of pancreatic beta-cells and therefore replenishing with new glucose-responsive beta-cells is an important therapeutic option. In addition to replication of pre-existing beta-cells, new beta-cells can be produced from differentiated adult cells using in-vitro or in-vivo approaches. This review will summarize recent advances in in-vivo generation of beta-cells from cells that are not beta-cells (neogenesis) and discuss ways to overcome the limitations of this process. RECENT FINDINGS: Multiple groups have shown that adult pancreatic ducts, acinar and even endocrine cells exhibit cellular plasticity and can differentiate into beta-cells in vivo. Several different approaches, including misexpression of transcription factors and tissue injury, have induced neogenesis of insulin-expressing cells in vivo and ameliorated diabetes. SUMMARY: Recent breakthroughs demonstrating cellular plasticity of adult pancreatic cells to form new beta-cells are a positive first step towards developing in-vivo regeneration-based therapy for diabetes. Currently, neogenesis processes are inefficient and do not generate sufficient amounts of beta-cells required to normalize hyperglycemia. However, an improved understanding of mechanisms regulating neogenesis of beta-cells from adult pancreatic cells and of their maturation into functional glucose-responsive beta-cells can make therapies based on in-vivo regeneration a reality.

GeneSpeed Beta Cell: an online genomics data repository and analysis resource tailored for the islet cell biologist.

OBJECTIVE: We here describe the development of a freely available online database resource, GeneSpeed Beta Cell, which has been created for the pancreatic islet and pancreatic developmental biology investigator community. RESEARCH DESIGN AND METHODS: We have developed GeneSpeed Beta Cell as a separate component of the GeneSpeed database, providing a genomics-type data repository of pancreas and islet-relevant datasets interlinked with the domain-oriented GeneSpeed database. RESULTS: GeneSpeed Beta Cell allows the query of multiple published and unpublished select genomics datasets in a simultaneous fashion (multiexperiment viewing) and is capable of defining intersection results from precomputed analysis of such datasets (multidimensional querying). Combined with the protein-domain categorization/assembly toolbox provided by the GeneSpeed database, the user is able to define spatial expression constraints of select gene lists in a relatively rigid fashion within the pancreatic expression space. We provide several demonstration case studies of relevance to islet cell biology and development of the pancreas that provide novel insight into islet biology. CONCLUSIONS: The combination of an exhaustive domain-based compilation of the transcriptome with gene array data of interest to the islet biologist affords novel methods for multidimensional querying between individual datasets in a rapid fashion, presently not available elsewhere.

Mouse pancreatic endocrine cell transcriptome defined in the embryonic Ngn3-null mouse.

OBJECTIVE: To document the transcriptome of the pancreatic islet during the early and late development of the mouse pancreas and highlight the qualitative and quantitative features of gene expression that contribute to the specification, growth, and differentiation of the major endocrine cell types. A further objective was to identify endocrine cell biomarkers, targets of diabetic autoimmunity, and regulatory pathways underlying islet responses to physiological and pathological stimuli. RESEARCH DESIGN AND METHODS: mRNA expression profiling was performed by microarray analysis of e12.5-18.5 embryonic pancreas from neurogenin 3 (Ngn3)-null mice, a background that abrogates endocrine pancreatic differentiation. The intersection of this data with mRNA expression in isolated adult pancreatic islets and pancreatic endocrine tumor cell lines was determined to compile lists of genes that are specifically expressed in endocrine cells. RESULTS: The data provided insight into the transcriptional and morphogenetic factors that may play major roles in patterning and differentiation of the endocrine lineage before and during the secondary transition of endocrine development, as well as genes that control the glucose responsiveness of the beta-cells and candidate diabetes autoantigens, such as insulin, IA-2 and Slc30a8 (ZnT8). The results are presented as downloadable gene lists, available at https://www.cbil.upenn.edu/RADQuerier/php/displayStudy.php?study_id=1330, stratified by predictive scores of relative cell-type specificity. CONCLUSIONS: The deposited data provide a rich resource that can be used to address diverse questions related to islet developmental and cell biology and the pathogenesis of type 1 and 2 diabetes.

Requirement of inositol pyrophosphates for full exocytotic capacity in pancreatic beta cells.

Inositol pyrophosphates are recognized components of cellular processes that regulate vesicle trafficking, telomere length, and apoptosis. We observed that pancreatic beta cells maintain high basal concentrations of the pyrophosphate diphosphoinositol pentakisphosphate (InsP7 or IP7). Inositol hexakisphosphate kinases (IP6Ks) that can generate IP7 were overexpressed. This overexpression stimulated exocytosis of insulin-containing granules from the readily releasable pool. Exogenously applied IP7 dose-dependently enhanced exocytosis at physiological concentrations. We determined that IP6K1 and IP6K2 were present in beta cells. RNA silencing of IP6K1, but not IP6K2, inhibited exocytosis, which suggests that IP6K1 is the critical endogenous kinase. Maintenance of high concentrations of IP7 in the pancreatic beta cell may enhance the immediate exocytotic capacity and consequently allow rapid adjustment of insulin secretion in response to increased demand.

The cation efflux transporter ZnT8 (Slc30A8) is a major autoantigen in human type 1 diabetes.

Type 1 diabetes (T1D) results from progressive loss of pancreatic islet mass through autoimmunity targeted at a diverse, yet limited, series of molecules that are expressed in the pancreatic beta cell. Identification of these molecular targets provides insight into the pathogenic process, diagnostic assays, and potential therapeutic agents. Autoantigen candidates were identified from microarray expression profiling of human and rodent pancreas and islet cells and screened with radioimmunoprecipitation assays using new-onset T1D and prediabetic sera. A high-ranking candidate, the zinc transporter ZnT8 (Slc30A8), was targeted by autoantibodies in 60-80% of new-onset T1D compared with <2% of controls and <3% type 2 diabetic and in up to 30% of patients with other autoimmune disorders with a T1D association. ZnT8 antibodies (ZnTA) were found in 26% of T1D subjects classified as autoantibody-negative on the basis of existing markers [glutamate decarboxylase (GADA), protein tyrosine phosphatase IA2 (IA2A), antibodies to insulin (IAA), and islet cytoplasmic autoantibodies (ICA)]. Individuals followed from birth to T1D showed ZnT8A as early as 2 years of age and increasing levels and prevalence persisting to disease onset. ZnT8A generally emerged later than GADA and IAA in prediabetes, although not in a strict order. The combined measurement of ZnT8A, GADA, IA2A, and IAA raised autoimmunity detection rates to 98% at disease onset, a level that approaches that needed to detect prediabetes in a general pediatric population. The combination of bioinformatics and molecular engineering used here will potentially generate other diabetes autoimmunity markers and is also broadly applicable to other autoimmune disorders.

ETS-family genes in pancreatic development.

ETS-family factors play major roles in development and cancer, notably as critical targets for extra-cellular signaling pathways, including MAPK-signaling. Given the presently limited knowledge on the role of ETS-factors in pancreatic development, we here sought to characterize all 26 individual members of the ETS-family in relation to pancreatic development using a combination of genomics, RT-PCR, and histological techniques. This analysis uncovers 22 ETS family genes displaying select spatial and temporal expression patterns in the developing pancreas. Highly specific expression of ETS-family components is observed in pancreatic progenitor cells or the associated embryonic mesenchyme. Other members are linked to the differentiation of more mature pancreatic cells, including exocrine and endocrine cell types. We find that two members of the Etv subfamily, Etv4 and Etv5, are expressed in cells proximal to pancreatic mesenchyme, and, furthermore, induced in FGF10-arrested pancreatic progenitors suggesting that these factors mediate mesenchymal-to-epithelial signaling.

C16:0 sulfatide inhibits insulin secretion in rat beta-cells by reducing the sensitivity of KATP channels to ATP inhibition.

Sulfatide (3'-sulfo-beta-galactosyl ceramide) is a glycosphingolipid present in mammalians in various fatty acid isoforms of which the saturated 16 carbon-atom length (C16:0) is more abundant in pancreatic islets than in neural tissue, where long-chain sulfatide isoforms dominate. We previously reported that sulfatide isolated from pig brain inhibits glucose-induced insulin secretion by activation of ATP-sensitive K+ channels (K(ATP) channels). Here, we show that C16:0 sulfatide is the active isoform. It inhibits glucose-stimulated insulin secretion by reducing the sensitivity of the K(ATP) channels to ATP. (The half-maximal inhibitory concentration is 10.3 and 36.7 micromol/l in the absence and presence of C16:0 sulfatide, respectively.) C16:0 sulfatide increased whole-cell K(ATP) currents at intermediate glucose levels and reduced the ability of glucose to induce membrane depolarization, reduced electrical activity, and increased the cytoplasmic free Ca2+ concentration. Recordings of cell capacitance revealed that C16:0 sulfatide increased Ca2+-induced exocytosis by 215%. This correlated with a stimulation of insulin secretion by C16:0 sulfatide in intact rat islets exposed to diazoxide and high K+. C24:0 sulfatide or the sulfatide precursor, beta-galactosyl ceramide, did not affect any of the measured parameters. C16:0 sulfatide did not modulate glucagon secretion from intact rat islets. In betaTC3 cells, sulfatide was expressed (mean [+/-SD] 0.30 +/- 0.04 pmol/microg protein), and C16:0 sulfatide was found to be the dominant isoform. No expression of sulfatide was detected in alphaTC1-9 cells. We conclude that a major mechanism by which the predominant sulfatide isoform in beta-cells, C16:0 sulfatide, inhibits glucose-induced insulin secretion is by reducing the K(ATP) channel sensitivity to the ATP block.

Secretory phospholipase A2 is released from pancreatic beta-cells and stimulates insulin secretion via inhibition of ATP-dependent K+ channels.

The release of sPLA(2) from single mouse pancreatic beta-cells was monitored using a fluorescent substrate of the enzyme incorporated in the outer leaflet of the plasma membrane. Stimulation of beta-cells with agents that increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) induced a rapid release of sPLA(2) to the extracellular medium. Exogenous sPLA(2) strongly stimulated insulin secretion in mouse pancreatic islets at both basal and elevated glucose concentrations. The stimulation of insulin secretion by sPLA(2) was mediated via inhibition of ATP-dependent K(+) channels and an increase in [Ca(2+)](i). Measurements of cell capacitance in single beta-cells revealed that sPLA(2) did not modify depolarisation-induced exocytosis. Our data suggest that a positive feedback regulation of insulin secretion by co-released sPLA(2) is operational in pancreatic beta-cells and point to this enzyme as an autocrine regulator of insulin secretion.

cPLA2alpha-evoked formation of arachidonic acid and lysophospholipids is required for exocytosis in mouse pancreatic beta-cells.

Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A2 (cPLA2alpha) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca2+-dependent exocytosis in single mouse pancreatic beta-cells. cPLA2alpha dose dependently (EC50 = 86 nM) stimulated depolarization-evoked exocytosis by 450% without affecting the whole cell Ca2+ current or cytoplasmic Ca2+ levels. The stimulatory effect involved priming of secretory granules as reflected by an increase in the size of the readily releasable pool of granules from 70-80 to 280-300. cPLA2alpha-stimulated exocytosis was antagonized by the specific cPLA2 inhibitor AACOCF3. Ca2+-evoked exocytosis was reduced by 40% in cells treated with AACOCF3 or an antisense oligonucleotide against cPLA2alpha. The action of cPLA2alpha was mimicked by a combination of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl- uptake through ClC-3 Cl- channels. Accordingly, the stimulatory action of cPLA2alpha was inhibited by the Cl- channel inhibitor DIDS and in cells pretreated with ClC-3 Cl- channel antisense oligonucleotides. We propose that cPLA2alpha has an important role in controlling the rate of exocytosis in beta-cells. This effect of cPLA2alpha reflects an enhanced transgranular Cl- flux, leading to an increase in the number of granules available for release, and requires the combined actions of arachidonic acid and lysophosphatidylcholine.