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Type 1 diabetes (T1D) is an autoimmune disease in which pathogenic lymphocytes target autoantigens expressed in pancreatic islets, leading to the destruction of insulin-producing β-cells. Zinc transporter 8 (ZnT8) is a major autoantigen abundantly present on the β-cell surface. This unique molecular target offers the potential to shield β-cells against autoimmune attacks in T1D. Our previous work showed that a monoclonal antibody (mAb43) against cell-surface ZnT8 could home in on pancreatic islets and prevent autoantibodies from recognizing β-cells. This study demonstrates that mAb43 binds to exocytotic sites on the β-cell surface, masking the antigenic exposure of ZnT8 and insulin after glucose-stimulated insulin secretion. In vivo administration of mAb43 to NOD mice selectively increased the proportion of regulatory T cells in the islet, resulting in complete and sustained protection against T1D onset as well as reversal of new-onset diabetes. The mAb43-induced self-tolerance was reversible after treatment cessation, and no adverse effects were exhibited during long-term monitoring. Our findings suggest that mAb43 masking of the antigenic exposure of β-cells suppresses the immunological cascade from B-cell antigen presentation to T cell–mediated β-cell destruction, providing a novel islet-targeted and antigen-specific immunotherapy to prevent and reverse clinical T1D. Article Highlights: mAb43, a zinc transporter 8 (ZnT8)–specific monoclonal antibody, provides lasting protection against autoimmune diabetes in NOD mice. The in vivo islet specificity of mAb43 enables targeted therapy to enhance safety. High-affinity binding of mAb43 to the extracellular surface of ZnT8 shields β-cells from antigenic exposure. β-Cell masking results in a localized increase in regulatory T cells in the pancreatic islet. Prolonged mAb43 treatment clears destructive insulitis, preserves β-cell mass, and reverses seroconversion of insulin autoantibodies in NOD mice. The immunological processes bridging the masking of specific cell-surface autoantigens and the broad suppression of polyspecific T-cell autoimmunity are still unclear. [ABSTRACT FROM AUTHOR]

Introduction: Leptin inhibits insulin secretion from isolated islets from multiple species, but the cell type that mediates this process remains elusive. Several mouse models have been used to explore this question. Ablation of the leptin receptor (Lepr) throughout the pancreatic epithelium results in altered glucose homeostasis and ex vivo insulin secretion and Ca2+ dynamics. However, Lepr removal from neither alpha nor beta cells mimics this result. Moreover, scRNAseq data has revealed an enrichment of LEPR in human islet delta cells. Methods: We confirmed LEPR upregulation in human delta cells by performing RNAseq on fixed, sorted beta and delta cells. We then used a mouse model to test whether delta cells mediate the diminished glucose-stimulated insulin secretion in response to leptin. Results: Ablation of Lepr within mouse delta cells did not change glucose homeostasis or insulin secretion, whether mice were fed a chow or high-fat diet. We further show, using a publicly available scRNAseq dataset, that islet cells expressing Lepr lie within endothelial cell clusters. Conclusions: In mice, leptin does not influence beta-cell function through delta cells. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is a disease in which autoimmune attack is directed to the insulin-producing β-cell in the pancreatic islet. Autoantigens on the β-cell surface membrane are specific markers for molecular recognition and targets for engagement by autoreactive B lymphocytes, which produce islet cell surface autoantibody (ICSA) upon activation. Here, we report the cloning of an ICSA (mAb43) that recognizes a major T1D autoantigen, ZnT8, with a subnanomolar binding affinity and conformation specificity. We demonstrate that cell-surface binding of mAb43 protects the extracellular epitope of ZnT8 against immunolabeling by serum ICSA from a patient with T1D. Further, mAb43 exhibits in vitro and ex vivo specificity for islet cells, mirroring the exquisite specificity of islet autoimmunity in T1D. Systemic administration of mAb43 yields a pancreas-specific biodistribution in mice and islet homing of a mAb43-linked imaging payload through the pancreatic vasculature, thereby validating the in vivo specificity of mAb43. Identifying ZnT8 as a major antigenic target of ICSA allows for research into the molecular recognition and engagement of autoreactive B cells in the chronic phase of T1D progression. The in vivo islet specificity of mAb43 could be further exploited to develop in vivo imaging and islet-specific immunotherapies. [ABSTRACT FROM AUTHOR]

Type 2 diabetes mellitus (T2DM) is characterized by the inability of the insulin-producing β-cells to overcome insulin resistance. We previously identified an imprinted region on chromosome 14, the DLK1-MEG3 locus, as being downregulated in islets from humans with T2DM. In this study, using targeted epigenetic modifiers, we prove that increased methylation at the promoter of Meg3 in mouse βTC6 β-cells results in decreased transcription of the maternal transcripts associated with this locus. As a result, the sensitivity of β-cells to cytokine-mediated oxidative stress was increased. Additionally, we demonstrate that an evolutionarily conserved intronic region at the MEG3 locus can function as an enhancer in βTC6 β-cells. Using circular chromosome conformation capture followed by high-throughput sequencing, we demonstrate that the promoter of MEG3 physically interacts with this novel enhancer and other putative regulatory elements in this imprinted region in human islets. Remarkably, this enhancer is bound in an allele-specific manner by the transcription factors FOXA2, PDX1, and NKX2.2. Overall, these data suggest that the intronic MEG3 enhancer plays an important role in the regulation of allele-specific expression at the imprinted DLK1-MEG3 locus in human β-cells, which in turn impacts the sensitivity of β-cells to cytokine-mediated oxidative stress. [ABSTRACT FROM AUTHOR]

Forkhead box (Fox) transcription factors are evolutionarily conserved in organisms ranging from yeast to humans. They regulate diverse biological processes both during development and throughout adult life. Mutations in many Fox genes are associated with human disease and, as such, various animal models have been generated to study the function of these transcription factors in mechanistic detail. In many cases, the absence of even a single Fox transcription factor is lethal. In this Primer, we provide an overview of the Fox family, highlighting several key Fox transcription factor families that are important for mammalian development. [ABSTRACT FROM AUTHOR]

Human pancreatic islets consist of multiple endocrine cell types. To facilitate the detection of rare cellular states and uncover population heterogeneity, we performed single-cell RNA sequencing (RNA-seq) on islets from multiple deceased organ donors, including children, healthy adults, and individuals with type 1 or type 2 diabetes. We developed a robust computational biology framework for cell type annotation. Using this framework, we show that α- and β-cells from children exhibit less well-defined gene signatures than those in adults. Remarkably, α- and β-cells from donors with type 2 diabetes have expression profiles with features seen in children, indicating a partial dedifferentiation process. We also examined a naturally proliferating α-cell from a healthy adult, for which pathway analysis indicated activation of the cell cycle and repression of checkpoint control pathways. Importantly, this replicating α-cell exhibited activated Sonic hedgehog signaling, a pathway not previously known to contribute to human α-cell proliferation. Our study highlights the power of single-cell RNA-seq and provides a stepping stone for future explorations of cellular heterogeneity in pancreatic endocrine cells. [ABSTRACT FROM AUTHOR]

Type 2 diabetes incidence increases with age, while β-cell replication declines. The transcription factor FoxM1 is required for β-cell replication in various situations, and its expression declines with age. We hypothesized that increased FoxM1 activity in aged β-cells would rejuvenate proliferation. Induction of an activated form of FoxM1 was sufficient to increase β-cell mass and proliferation in 12-month-old male mice after just 2 weeks. Unexpectedly, at 2 months of age, induction of activated FoxM1 in male mice improved glucose homeostasis with unchanged β-cell mass. Cells expressing activated FoxM1 demonstrated enhanced glucose-stimulated Ca2+ influx, which resulted in improved glucose tolerance through enhanced β-cell function. Conversely, our laboratory has previously demonstrated that mice lacking FoxM1 in the pancreas display glucose intolerance or diabetes with only a 60% reduction in β-cell mass, suggesting that the loss of FoxM1 is detrimental to β-cell function. Ex vivo insulin secretion was therefore examined in size-matched islets from young mice lacking FoxM1 in β-cells. Foxm1-deficient islets indeed displayed reduced insulin secretion. Our studies reveal that activated FoxM1 increases β-cell replication while simultaneously enhancing insulin secretion and improving glucose homeostasis, making FoxM1 an attractive therapeutic target for diabetes. [ABSTRACT FROM AUTHOR]

β-Cell mass is a parameter commonly measured in studies of islet biology and diabetes. However, the rigorous quantification of pancreatic β-cell mass using conventional histological methods is a time-consuming process. Rapidly evolving virtual slide technology with high-resolution slide scanners and newly developed image analysis tools has the potential to transform β-cell mass measurement. To test the effectiveness and accuracy of this new approach, we assessed pancreata from normal C57Bl/6J mice and from mouse models of β-cell ablation (streptozotocin-treated mice) and β-cell hyperplasia (leptin-deficient mice), using a standardized systematic sampling of pancreatic specimens. Our data indicate that automated analysis of virtual pancreatic slides is highly reliable and yields results consistent with those obtained by conventional morphometric analysis. This new methodology will allow investigators to dramatically reduce the time required for β-cell mass measurement by automating high-resolution image capture and analysis of entire pancreatic sections. [ABSTRACT FROM AUTHOR]

Enzymatic activity of glucokinase was demonstrated, quantitated, and characterized kinetically in rat and mouse pituitary extracts using a highly specific and sensitive spectrometric assay. A previously proposed hypothesis that the glucokinase gene might be expressed in the pituitary corticotrophic cells was therefore reexamined using mRNA in situ hybridization and immunohistochemical techniques. No evidence was found that corticotrophs are glucokinase positive, and the identity of glucokinase-expressing cells remains to be determined. The findings do, however, suggest a novel hypothesis that a critical subgroup of anterior pituitary cells might function as glucose sensor cells and that direct fuel regulation of such cells may modify the classical indirect neuroendocrine pathways that are known to control hormone secretion from anterior pituitary cells. Diabetes 55:1923-1929, 2006 [ABSTRACT FROM AUTHOR]

Background: Acute RT-induced damage to the lung is characterized by inflammatory changes, which proceed to the development of fibrotic lesions in the late phase of injury. Ultimately, complete structural ablation will ensue, if the source of inflammatory/fibrogenic mediators and oxidative stress is not removed or attenuated. Therefore, the purpose of this study is to determine whether overexpression of extracellular superoxide dismutase (EC-SOD) in mice ameliorates acute radiation induced injury by inhibiting activation of TGFβ1 and downregulating the Smad 3 arm of its signal transduction pathway. Methods: Whole thorax radiation (single dose, 15 Gy) was delivered to EC-SOD overexpressing transgenic (XRT-TG) and wild-type (XRT-WT) animals. Mice were sacrificed at 1 day, 1 week, 3, 6, 10 and 14 weeks. Breathing rates, right lung weights, total/differential leukocyte count, activated TGFβ1 and components of its signal transduction pathway (Smad 3 and p-Smad 2/3) were assessed to determine lung injury. Results: Irradiated wild-type (XRT-WT) animals exhibited time dependent increase in breathing rates and right lung weights, whereas these parameters were significantly less increased (p < 0.05) at 3, 6, 10 and 14 weeks in irradiated transgenic (XRT-TG) mice. An inflammatory response characterized predominantly by macrophage infiltration was pronounced in XRT-WT mice. This acute inflammation was significantly attenuated (p < 0.05) in XRT-TG animals at 1, 3, 6 and 14 weeks. Expression of activated TGFβ1 and components of its signal transduction pathway were significantly reduced (p < 0.05) at later time-points in XRT-TG vs. XRT-WT. Conclusion: This study shows that overexpression of EC-SOD confers protection against RT-induced acute lung injury. EC-SOD appears to work, in part, via an attenuation of the macrophage response and also decreases TGFβ1 activation with a subsequent downregulation of the profibrotic TGFβ pathway. [ABSTRACT FROM AUTHOR]