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OBJECTIVE—Islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP) is selectively expressed in islet β-cells and is a major autoantigen in both mouse and human type 1 diabetes. This study describes the use of a combination of transgenic and transfection approaches to characterize the gene regions that confer the islet-specific expression of IGRP. RESEARCH DESIGN AND METHODS—Transgenic mice were generated containing the IGRP promoter sequence from −306, −911, or −3911 to +3 ligated to a LacZ reporter gene. Transgene expression was monitored by 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside staining of pancreatic tissue. RESULTS—In all the transgenic mice, robust LacZ expression was detected in newborn mouse islets, but expression became mosaic as animals aged, suggesting that additional elements are required for the maintenance of IGRP gene expression. VISTA analyses identified two conserved regions in the distal IGRP promoter and one in the third intron. Transfection experiments demonstrated that all three regions confer enhanced luciferase reporter gene expression in βTC-3 cells when ligated to a minimal IGRP promoter. A transgene containing all three conserved regions was generated by using a bacterial recombination strategy to insert a LacZ cassette into exon 5 of the IGRP gene. Transgenic mice containing a 15-kbp fragment of the IGRP gene were then generated. This transgene conferred LacZ expression in newborn mouse islets; however, expression was still suppressed as animals aged. CONCLUSIONS—The data suggest that long-range enhancers 5′ or 3′ of the IGRP gene are required for the maintenance of IGRP gene expression in adult mice.

OBJECTIVE—Zinc transporter eight (SLC30A8) is a major target of autoimmunity in human type 1A diabetes and is implicated in type 2 diabetes in genome-wide association studies. The type 2 diabetes nonsynonymous single nucleotide polymorphism (SNP) affecting aa325 lies within the region of highest ZnT8 autoantibody (ZnT8A) binding, prompting an investigation of its relationship to type 1 diabetes. RESEARCH DESIGN AND METHODS—ZnT8A radioimmunoprecipitation assays were performed in 421 new-onset type 1 diabetic Caucasians using COOH-terminal constructs incorporating the known human aa325 variants (Trp, Arg, and Gln). Genotypes were determined by PCR-based SNP analysis. RESULTS—Sera from 224 subjects (53%) were reactive to Arg325 probes, from 185 (44%) to Trp325probes, and from 142 (34%) to Gln325probes. Sixty subjects reacted only with Arg325 constructs, 31 with Trp325 only, and 1 with Gln325 only. The restriction to either Arg325 or Trp325 corresponded with inheritance of the respective C- or T-alleles. A strong gene dosage effect was also evident because both Arg- and Trp-restricted ZnT8As were less prevalent in heterozygous than homozygous individuals. The SLC30A8 SNP allele frequency (75% C and 25% T) varied little with age of type 1 diabetes onset or the presence of other autoantibodies. CONCLUSIONS—The finding that diabetes autoimmunity can be defined by a single polymorphic residue has not previously been documented. It argues against ZnT8 autoimmunity arising from molecular mimicry and suggests a mechanistic link between the two major forms of diabetes. It has implications for antigen-based therapeutic interventions because the response to ZnT8 administration could be protective or immunogenic depending on an individual's genotype.

The molecular mechanism of pancreatic morphogenesis and differentiation is a fundamental enigma during pancreas development. Early pancreatic organogenesis shares a common pool of pancreatic progenitor cells that originate from the foregut endoderm1, 2. The dynamic signaling interactions between surrounding mesoderm, in an autocrine and paracrine manner leads to the branching morphogenesis of the pancreatic epithelium3. The key transcription factors like Pdx1, Hlxb9, Ptf1a, Nkx6-1, and Nkx2-2, Nkx6-2, Sox 9 and many more unidentified such factors mark the pancreatic progenitor cells in the undifferentiated epithelium. These known and unknown entities allow an orchestrated series of complex differentiation and proliferation steps leading to the parallel differentiation of endocrine and exocrine (acinar and ductal) compartments4. The critical events governing the differentiation of endocrine cells occur when Neurog3 is transiently expressed in a subset of cells derived from the pancreatic epithelium from E9.5 to E18.55, 6. These Neurog3 expressing cells are identifiable by lineage tracing as endocrine progenitor cells7, 8. We have previously shown that endocrine differentiation in the human fetal pancreas also takes place in the islands of epithelial tissue but within a much larger volume of mesenchymal tissue9. In humans, endocrine progenitor marker, NGN3-positive cells also persist from 9–23 weeks of gestation9, 10. In mice, unlike humans where secondary transition has not been documented9, the differentiation of the acinar compartment is synchronized with that of endocrine differentiation during the secondary transition11 and is governed by the shift and segregation in expression of Ptf1a from the central to the expanding tips of the pancreatic epithelium3. Unfortunately, the data regarding ductal development is relatively sparse12–14. Tremendous progress has been made in understanding the molecular dynamics of some of these known factors mentioned above and the identification and validation of novel entities that drive the differentiation process may further enhance our ability to generate therapeutic beta cells from ES and or iPS cells15–17. To document the transcriptome of the developing pancreas in the mouse and highlight the qualitative and quantitative features of global gene expression that contributes to the specification, growth and differentiation of the major endocrine and exocrine (acinar and ductal) cell types, we performed microarray analysis at E12.5, E13.5, E15.5 and E18.5 on murine embryonic pancreas. In order to expedite the discovery of novel factors that govern pancreatic differentiation, we have synergized the differential analysis of significant genes with an advanced computational approach called the Hanalyzer18, 19. The Hanalyzer extracts information from external resources, either by parsing structured data or by using biomedical language processing to glean information from unstructured data, and tracks knowledge provenance about genes. The use of the tool was critical in the identification of four novel genes whose roles have not been extensively characterized in the murine pancreatic developmental context; Cystathionine beta synthase (Cbs), Growth Factor Independent 1 (Gfi1), Meis homeobox 1 (Meis 1) and Aldehyde dehydrogenase 18 family member a1 (Aldh18a1). In the current study, we validated the temporal expression of Cbs, Gfi1, Meis1 and Aldh18a1 and further documented the localization of Cbs in embryonic and adult pancreas. We identified Cbs as one of the previously unknown cellular markers expressed in early pancreatic epithelium that may contribute to the acinar differentiation in the murine pancreas.

More than one-half of the ~50 human chemokines have been associated with or implicated in the pathogenesis of type 1 diabetes, yet their actual expression patterns in the islet environment of type 1 diabetic patients remain, at present, poorly defined. Here, we have integrated a human islet culture system, murine models of virus-induced and spontaneous type 1 diabetes, and the histopathological examination of pancreata from diabetic organ donors with the goal of providing a foundation for the informed selection of potential therapeutic targets within the chemokine/receptor family. Chemokine (C-C motif) ligand (CCL) 5 (CCL5), CCL8, CCL22, chemokine (C-X-C motif) ligand (CXCL) 9 (CXCL9), CXCL10, and chemokine (C-X3-C motif) ligand (CX3CL) 1 (CX3CL1) were the major chemokines transcribed (in an inducible nitric oxide synthase–dependent but not nuclear factor-κB–dependent fashion) and translated by human islet cells in response to in vitro inflammatory stimuli. CXCL10 was identified as the dominant chemokine expressed in vivo in the islet environment of prediabetic animals and type 1 diabetic patients, whereas CCL5, CCL8, CXCL9, and CX3CL1 proteins were present at lower levels in the islets of both species. Of importance, additional expression of the same chemokines in human acinar tissues emphasizes an underappreciated involvement of the exocrine pancreas in the natural course of type 1 diabetes that will require consideration for additional type 1 diabetes pathogenesis and immune intervention studies.

Competing Interests: MKS and DRP work for Lexicon Pharmaceuticals and generated the mice described in this study. There are no pending patents, products in development or marketed products associated with these mice or this study. This relationship between Lexicon and Vanderbilt does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. Funding: The Vanderbilt Hormone Assay & Analytical Services Core and the Vanderbilt Islet Procurement and Analysis Core are both supported by National Institutes of Health (NIH) grants DK20593, to the Vanderbilt Diabetes Research Training Center (VDRTC), and DK59637, to the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC). The authors also thank personnel in the MMPC for advice on performing glucose tolerance tests and Mary Dawes for performing electron microscopy. The electron microscopy core is part of the VUMC Cell Imaging Shared Resource, which is supported by NIH grants CA68485, DK20593, DK58404, HD15052, DK59637 and EY08126. Research in the laboratory of RO was supported by NIH grant DK92589. Research in the laboratory of JCH was supported by the American Diabetes Association (9901-116), NIH grant DK052068, Juvenile Diabetes Research Association grant 4-2007-1056 and the Barbara Davis Center Diabetes and Endocrinology Research Center (P30 DK57516). Research in the laboratory of DWP was supported by NIH grant DK085064. Research in the laboratory of DAJ was supported by K01 DK081666. Research in the laboratory of OPM was supported by NIH grants DK043748 and DK078188. SAS was supported by NIH grant DK080193 and JDRF grant 1-2008-1021. The Vanderbilt Hormone Assay & Analytical Services Core, the Vanderbilt Islet Procurement and Analysis Core and the Vanderbilt Cell Imaging Shared Resource are all supported by NIH grant P60 DK20593, to the Vanderbilt Diabetes Research Training Center (VDRTC). LDP was supported by the Vanderbilt Molecular Endocrinology Training Program grant 5T32 DK07563. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The effects of increasing intracellular cAMP levels on IL-1 alpha and IL-1 beta mRNA expression and IL-1 production in human monocytes and nonlymphoid hematopoietic cell lines were examined. Peripheral monocytes and myelomonocytic cell lines could be stimulated by LPS or phorbol myristate acetate (PMA) to express IL-1 mRNA. Dibutyryl cAMP, 8-bromo-cAMP, forskolin, cholera toxin, PGE1, and PGE2 synergized with PMA or LPS to increase the accumulation in cell lines of IL-1 alpha mRNA by up to 50-fold and that of IL-1 beta mRNA by 10- to 20-fold compared to LPS or PMA alone. This increase in IL-1 alpha and IL-1 beta mRNA accumulation was more modest in monocytes. The synergistic stimulation was due to enhanced IL-1 gene transcription rate rather than increased IL-1 mRNA stability. Despite this marked increase in IL-1 mRNA accumulation, IL-1 protein synthesis in these cells was increased by only twofold. Thus, IL-1 synthesis in monocytes and myelomonocytic cell lines is under stringent translational control.

The ability to induce the oncogenic activation of the human prolactin receptor (PRLR) was examined by deleting 178 amino acids of the extracellular ligand-binding domain. Expression of this deletion mutant in the interleukin-3 (IL-3)-dependent murine myeloid cell line 32Dcl3 resulted in the induction of growth factor-independent proliferation. Parental 32Dcl3 cells proliferated only in the presence of exogenous murine IL-3 (mIL-3), while 32Dcl3 cells transfected with the long form of the human PRLR were able to proliferate in response to mIL-3, ovine prolactin, or human PRL. Cells expressing the Delta178 deletion mutant contained numerous phosphotyrosine-containing proteins in the absence of stimulation with either mIL-3 or ovine prolactin. Growth factor stimulation increased the number of proteins phosphorylated and the intensity of phosphorylation. These proteins included constitutively phosphorylated Janus kinase 2, signal transducer and activator of transcription 5, and SHC. Activated extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) were observed in unstimulated 32Dcl3 cells expressing the Delta178 mutant. Likewise, transfection of Nb2 cells with the Delta178 deletion mutant induced growth factor-independent proliferation and constitutive activation of Janus kinase 2, ERK1, and ERK2. In addition to the induction of a growth factor-independent state, the expression of the Delta178 deletion mutant also suppressed the apoptosis that occurs when 32Dcl3 cells are cultured in the absence of growth factors such as IL-3. These data suggest that the constitutive activation of the PRLR can be achieved by deletion of the ligand binding domain and that this mutation leads to the oncogenic activation of the receptor as determined by the ability of the receptor to induce growth factor-independent proliferation of factor-dependent hematopoietic cells.