Type 2 diabetes susceptibility gene GRK5 regulates physiological pancreatic ß-cell proliferation via phosphorylation of HDAC5.

Restoring functional ß cell mass is a potential therapy for those with diabetes. However, the pathways regulating ß cell mass are not fully understood. Previously, we demonstrated that Sox4 is required for ß cell proliferation during prediabetes. Here, we report that Sox4 regulates ß cell mass through modulating expression of the type 2 diabetes (T2D) susceptibility gene GRK5. ß cell-specific Grk5 knockout mice showed impaired glucose tolerance with reduced ß cell mass, which was accompanied by upregulation of cell cycle inhibitor gene Cdkn1a. Furthermore, we found that Grk5 may drive ß cell proliferation through a pathway that includes phosphorylation of HDAC5 and subsequent transcription of immediate-early genes (IEGs) such as Nr4a1, Fosb, Junb, Arc, Egr1, and Srf. Together, these studies suggest GRK5 is linked to T2D through regulation of ß cell growth and that it may be a target to preserve ß cells during the development of T2D.

Methylation of histone H3 lysine 4 is required for maintenance of beta cell function in adult mice.

AIMS/HYPOTHESIS: Beta cells control glucose homeostasis via regulated production and secretion of insulin. This function arises from a highly specialised gene expression programme that is established during development and then sustained, with limited flexibility, in terminally differentiated cells. Dysregulation of this programme is seen in type 2 diabetes but mechanisms that preserve gene expression or underlie its dysregulation in mature cells are not well resolved. This study investigated whether methylation of histone H3 lysine 4 (H3K4), a marker of gene promoters with unresolved functional importance, is necessary for the maintenance of mature beta cell function. METHODS: Beta cell function, gene expression and chromatin modifications were analysed in conditional Dpy30 knockout mice, in which H3K4 methyltransferase activity is impaired, and in a mouse model of diabetes. RESULTS: H3K4 methylation maintains expression of genes that are important for insulin biosynthesis and glucose responsiveness. Deficient methylation of H3K4 leads to a less active and more repressed epigenome profile that locally correlates with gene expression deficits but does not globally reduce gene expression. Instead, developmentally regulated genes and genes in weakly active or suppressed states particularly rely on H3K4 methylation. We further show that H3K4 trimethylation (H3K4me3) is reorganised in islets from the Leprdb/db mouse model of diabetes in favour of weakly active and disallowed genes at the expense of terminal beta cell markers with broad H3K4me3 peaks. CONCLUSIONS/INTERPRETATION: Sustained methylation of H3K4 is critical for the maintenance of beta cell function. Redistribution of H3K4me3 is linked to gene expression changes that are implicated in diabetes pathology.

Beta-cell specific Insr deletion promotes insulin hypersecretion and improves glucose tolerance prior to global insulin resistance.

Insulin receptor (Insr) protein is present at higher levels in pancreatic ß-cells than in most other tissues, but the consequences of ß-cell insulin resistance remain enigmatic. Here, we use an Ins1cre knock-in allele to delete Insr specifically in ß-cells of both female and male mice. We compare experimental mice to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined ß-cells reveals transcriptomic consequences of Insr loss, which differ between female and male mice. Action potential and calcium oscillation frequencies are increased in Insr knockout ß-cells from female, but not male mice, whereas only male ßInsrKO islets have reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female ßInsrKO and ßInsrHET mice exhibit elevated insulin release in ex vivo perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr does not alter ß-cell area up to 9 months of age, nor does it impair hyperglycemia-induced proliferation. Based on our data, we adapt a mathematical model to include ß-cell insulin resistance, which predicts that ß-cell Insr knockout improves glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance is significantly improved in female ßInsrKO and ßInsrHET mice compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We observe no improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of ß-cell specific insulin resistance. The propensity for hyperinsulinemia is associated with mildly reduced fasting glucose and increased body weight. We further validate our main in vivo findings using an Ins1-CreERT transgenic line and find that male mice have improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that ß-cell insulin resistance in the form of reduced ß-cell Insr contributes to hyperinsulinemia in the context of glucose stimulation, thereby improving glucose homeostasis in otherwise insulin sensitive sex, dietary and age contexts.

The role of mitochondrial apoptotic pathway in islet amyloid-induced ß-cell death.

Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to ß-cell death in type 2 diabetes. We previously showed that extracellular hIAPP aggregates promote Fas-mediated ß-cell apoptosis. Here, we tested if hIAPP aggregates can trigger the mitochondrial apoptotic pathway (MAP). hIAPP aggregation in Ad-hIAPP transduced INS-1 and human islet ß-cells promoted cytochrome c release, caspase-9 activation and apoptosis, which were reduced by Bax inhibitor. Amyloid formation in hIAPP-expressing mouse islets during culture increased caspase-9 activation in ß-cells. Ad-hIAPP transduced islets from CytcKA/KA and BaxBak ßDKO mice (models of blocked MAP), had lower caspase-9-positive and apoptotic ß-cells than transduced wild-type islets, despite comparable amyloid formation. Blocking Fas (markedly) and Bax or caspase-9 (modestly) reduced ß-cell death induced by extracellular hIAPP aggregates. These findings suggest a role for MAP in amyloid-induced ß-cell death and a potential strategy to reduce intracellular amyloid ß-cell toxicity by blocking cytochrome c apoptotic function.

Bax and Bak jointly control survival and dampen the early unfolded protein response in pancreatic ß-cells under glucolipotoxic stress.

ER stress and apoptosis contribute to the loss of pancreatic ß-cells under pro-diabetic conditions of glucolipotoxicity. Although activation of canonical intrinsic apoptosis is known to require pro-apoptotic Bcl-2 family proteins Bax and Bak, their individual and combined involvement in glucolipotoxic ß-cell death are not known. It has also remained an open question if Bax and Bak in ß-cells have non-apoptotic roles in mitochondrial function and ER stress signaling, as suggested in other cell types. Using mice with individual or combined ß-cell deletion of Bax and Bak, we demonstrated that glucolipotoxic ß-cell death in vitro occurs by both non-apoptotic and apoptotic mechanisms, and the apoptosis could be triggered by either Bax or Bak alone. In contrast, they had non-redundant roles in mediating staurosporine-induced apoptosis. We further established that Bax and Bak do not affect normal glucose-stimulated ß-cell Ca2+ responses, insulin secretion, or in vivo glucose tolerance. Finally, our experiments revealed that combined deletion of Bax and Bak amplified the unfolded protein response in islets during the early stages of chemical- or glucolipotoxicity-induced ER stress. These findings shed new light on roles of the core apoptosis machinery in ß-cell survival and stress signals of importance for the pathobiology of diabetes.

A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic ß-cells.

Live-cell imaging of mitochondrial function and dynamics can provide vital insights into both physiology and pathophysiology, including of metabolic diseases like type 2 diabetes. However, without super-resolution microscopy and commercial analysis software, it is challenging to accurately extract features from dense multilayered mitochondrial networks, such as those in insulin-secreting pancreatic ß-cells. Motivated by this, we developed a comprehensive pipeline and associated ImageJ plugin that enables 2D/3D quantification of mitochondrial network morphology and dynamics in mouse ß-cells and by extension other similarly challenging cell types. The approach is based on standard confocal microscopy and shareware, making it widely accessible. The pipeline was validated using mitochondrial photolabeling and unsupervised cluster analysis and is capable of morphological and functional analyses on a per-organelle basis, including in 4D (xyzt). Overall, this tool offers a powerful framework for multiplexed analysis of mitochondrial state/function and provides a valuable resource to accelerate mitochondrial research in health and disease.

Cellular metabolism constrains innate immune responses in early human ontogeny.

Pathogen immune responses are profoundly attenuated in fetuses and premature infants, yet the mechanisms underlying this developmental immaturity remain unclear. Here we show transcriptomic, metabolic and polysome profiling and find that monocytes isolated from infants born early in gestation display perturbations in PPAR-γ-regulated metabolic pathways, limited glycolytic capacity and reduced ribosomal activity. These metabolic changes are linked to a lack of translation of most cytokines and of MALT1 signalosome genes essential to respond to the neonatal pathogen Candida. In contrast, they have little impact on house-keeping phagocytosis functions. Transcriptome analyses further indicate a role for mTOR and its putative negative regulator DNA Damage Inducible Transcript 4-Like in regulating these metabolic constraints. Our results provide a molecular basis for the broad susceptibility to multiple pathogens in these infants, and suggest that the fetal immune system is metabolically programmed to avoid energetically costly, dispensable and potentially harmful immune responses during ontogeny.

The p300 and CBP Transcriptional Coactivators Are Required for ß-Cell and α-Cell Proliferation.

p300 (EP300) and CBP (CREBBP) are transcriptional coactivators with histone acetyltransferase activity. Various ß-cell transcription factors can recruit p300/CBP, and thus the coactivators could be important for ß-cell function and health in vivo. We hypothesized that p300/CBP contribute to the development and proper function of pancreatic islets. To test this, we bred and studied mice lacking p300/CBP in their islets. Mice lacking either p300 or CBP in islets developed glucose intolerance attributable to impaired insulin secretion, together with reduced α- and ß-cell area and islet insulin content. These phenotypes were exacerbated in mice with only a single copy of p300 or CBP expressed in islets. Removing p300 in pancreatic endocrine progenitors impaired proliferation of neonatal α- and ß-cells. Mice lacking all four copies of p300/CBP in pancreatic endocrine progenitors failed to establish α- and ß-cell mass postnatally. Transcriptomic analyses revealed significant overlaps between p300/CBP-downregulated genes and genes downregulated in Hnf1α-null islets and Nkx2.2-null islets, among others. Furthermore, p300/CBP are important for the acetylation of H3K27 at loci downregulated in Hnf1α-null islets. We conclude that p300 and CBP are limiting cofactors for islet development, and hence for postnatal glucose homeostasis, with some functional redundancy.

Bcl-2 Regulates Reactive Oxygen Species Signaling and a Redox-Sensitive Mitochondrial Proton Leak in Mouse Pancreatic ß-Cells.

In pancreatic ß-cells, controlling the levels of reactive oxygen species (ROS) is critical to counter oxidative stress, dysfunction and death under nutrient excess. Moreover, the fine-tuning of ROS and redox balance is important in the regulation of normal ß-cell physiology. We recently demonstrated that Bcl-2 and Bcl-xL, in addition to promoting survival, suppress ß-cell glucose metabolism and insulin secretion. Here, we tested the hypothesis that the nonapoptotic roles of endogenous Bcl-2 extend to the regulation of ß-cell ROS and redox balance. We exposed mouse islet cells and MIN6 cells to the Bcl-2/Bcl-xL antagonist Compound 6 and the Bcl-2-specific antagonist ABT-199 and evaluated ROS levels, Ca(2+) responses, respiratory control, superoxide dismutase activity and cell death. Both acute glucose stimulation and the inhibition of endogenous Bcl-2 progressively increased peroxides and stimulated superoxide dismutase activity in mouse islets. Importantly, conditional ß-cell knockout of Bcl-2 amplified glucose-induced formation of peroxides. Bcl-2 antagonism also induced a mitochondrial proton leak that was prevented by the antioxidant N-acetyl-L-cysteine and, therefore, secondary to redox changes. We further established that the proton leak was independent of uncoupling protein 2 but partly mediated by the mitochondrial permeability transition pore. Acutely, inhibitor-induced peroxides promoted Ca(2+) influx, whereas under prolonged Bcl inhibition, the elevated ROS was required for induction of ß-cell apoptosis. In conclusion, our data reveal that endogenous Bcl-2 modulates moment-to-moment ROS signaling and suppresses a redox-regulated mitochondrial proton leak in ß-cells. These noncanonical roles of Bcl-2 may be important for ß-cell function and survival under conditions of high metabolic demand.

Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor.

Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases.

Myt3 Mediates Laminin-V/Integrin-ß1-Induced Islet-Cell Migration via Tgfbi.

Myt3 is a prosurvival factor in pancreatic islets; however, its role in islet-cell development is not known. Here, we demonstrate that myelin transcription factor 3 (Myt3) is expressed in migrating islet cells in the developing and neonatal pancreas and thus sought to determine whether Myt3 plays a role in this process. Using an ex vivo model of islet-cell migration, we demonstrate that Myt3 suppression significantly inhibits laminin-V/integrin-ß1-dependent α- and ß-cell migration onto 804G, and impaired 804G-induced F-actin and E-cadherin redistribution. Exposure of islets to proinflammatory cytokines, which suppress Myt3 expression, had a similar effect, whereas Myt3 overexpression partially rescued the migratory ability of the islet cells. We show that loss of islet-cell migration, due to Myt3 suppression or cytokine exposure, is independent of effects on islet-cell survival or proliferation. Myt3 suppression also had no effect on glucose-induced calcium influx, F-actin remodeling or insulin secretion by ß-cells. RNA-sequencing (RNA-seq) analysis of transduced islets showed that Myt3 suppression results in the up-regulation of Tgfbi, a secreted diabetogenic factor thought to impair cellular adhesion. Exposure of islets to exogenous transforming growth factor ß-induced (Tgfbi) impaired islet-cell migration similar to Myt3 suppression. Taken together, these data suggest a model by which cytokine-induced Myt3 suppression leads to Tgfbi de-repression and subsequently to impaired islet-cell migration, revealing a novel role for Myt3 in regulating islet-cell migration.

B7-H4 as a protective shield for pancreatic islet beta cells.

Auto- and alloreactive T cells are major culprits that damage ß-cells in type 1 diabetes (T1D) and islet transplantation. Current immunosuppressive drugs can alleviate immune-mediated attacks on islets. T cell co-stimulation blockade has shown great promise in autoimmunity and transplantation as it solely targets activated T cells, and therefore avoids toxicity of current immunosuppressive drugs. An attractive approach is offered by the newly-identified negative T cell co-signaling molecule B7-H4 which is expressed in normal human islets, and its expression co-localizes with insulin. A concomitant decrease in B7-H4/insulin co-localization is observed in human type 1 diabetic islets. B7-H4 may play protective roles in the pancreatic islets, preserving their function and survival. In this review we outline the protective effect of B7-H4 in the contexts of T1D, islet cell transplantation, and potentially type 2 diabetes. Current evidence offers encouraging data regarding the role of B7-H4 in reversal of autoimmune diabetes and donor-specific islet allograft tolerance. Additionally, unique expression of B7-H4 may serve as a potential biomarker for the development of T1D. Future studies should continue to focus on the islet-specific effects of B7-H4 with emphasis on mechanistic pathways in order to promote B7-H4 as a potential therapy and cure for T1D.

Control of insulin secretion by cytochrome C and calcium signaling in islets with impaired metabolism.

The aim of the study was to assess the relative control of insulin secretion rate (ISR) by calcium influx and signaling from cytochrome c in islets where, as in diabetes, the metabolic pathways are impaired. This was achieved either by culturing isolated islets at low (3 mm) glucose or by fasting rats prior to the isolation of the islets. Culture in low glucose greatly reduced the glucose response of cytochrome c reduction and translocation and ISR, but did not affect the response to the mitochondrial fuel α-ketoisocaproate. Unexpectedly, glucose-stimulated calcium influx was only slightly reduced in low glucose-cultured islets and was not responsible for the impairment in glucose-stimulated ISR. A glucokinase activator acutely restored cytochrome c reduction and translocation and ISR, independent of effects on calcium influx. Islets from fasted rats had reduced ISR and cytochrome c reduction in response to both glucose and α-ketoisocaproate despite normal responses of calcium. Our data are consistent with the scenario where cytochrome c reduction and translocation are essential signals in the stimulation of ISR, the loss of which can result in impaired ISR even when calcium response is normal.

Complex patterns of metabolic and Ca²âº entrainment in pancreatic islets by oscillatory glucose.

Glucose-stimulated insulin secretion is pulsatile and driven by intrinsic oscillations in metabolism, electrical activity, and Ca(2+) in pancreatic islets. Periodic variations in glucose can entrain islet Ca(2+) and insulin secretion, possibly promoting interislet synchronization. Here, we used fluorescence microscopy to demonstrate that glucose oscillations can induce distinct 1:1 and 1:2 entrainment of oscillations (one and two oscillations for each period of exogenous stimulus, respectively) in islet Ca(2+), NAD(P)H, and mitochondrial membrane potential. To our knowledge, this is the first demonstration of metabolic entrainment in islets, and we found that entrainment of metabolic oscillations requires voltage-gated Ca(2+) influx. We identified diverse patterns of 1:2 entrainment and showed that islet synchronization during entrainment involves adjustments of both oscillatory phase and period. All experimental findings could be recapitulated by our recently developed mathematical model, and simulations suggested that interislet variability in 1:2 entrainment patterns reflects differences in their glucose sensitivity. Finally, our simulations and recordings showed that a heterogeneous group of islets synchronized during 1:2 entrainment, resulting in a clear oscillatory response from the collective. In summary, we demonstrate that oscillatory glucose can induce complex modes of entrainment of metabolically driven oscillations in islets, and provide additional support for the notion that entrainment promotes interislet synchrony in the pancreas.

Cardiomyocyte ATP production, metabolic flexibility, and survival require calcium flux through cardiac ryanodine receptors in vivo.

Ca(2+) fluxes between adjacent organelles are thought to control many cellular processes, including metabolism and cell survival. In vitro evidence has been presented that constitutive Ca(2+) flux from intracellular stores into mitochondria is required for basal cellular metabolism, but these observations have not been made in vivo. We report that controlled in vivo depletion of cardiac RYR2, using a conditional gene knock-out strategy (cRyr2KO mice), is sufficient to reduce mitochondrial Ca(2+) and oxidative metabolism, and to establish a pseudohypoxic state with increased autophagy. Dramatic metabolic reprogramming was evident at the transcriptional level via Sirt1/Foxo1/Pgc1α, Atf3, and Klf15 gene networks. Ryr2 loss also induced a non-apoptotic form of programmed cell death associated with increased calpain-10 but not caspase-3 activation or endoplasmic reticulum stress. Remarkably, cRyr2KO mice rapidly exhibited many of the structural, metabolic, and molecular characteristics of heart failure at a time when RYR2 protein was reduced 50%, a similar degree to that which has been reported in heart failure. RYR2-mediated Ca(2+) fluxes are therefore proximal controllers of mitochondrial Ca(2+), ATP levels, and a cascade of transcription factors controlling metabolism and survival.

Bcl-2 and Bcl-xL suppress glucose signaling in pancreatic ß-cells.

B-cell lymphoma 2 (Bcl-2) family proteins are established regulators of cell survival, but their involvement in the normal function of primary cells has only recently begun to receive attention. In this study, we demonstrate that chemical and genetic loss-of-function of antiapoptotic Bcl-2 and Bcl-x(L) significantly augments glucose-dependent metabolic and Ca(2+) signals in primary pancreatic ß-cells. Antagonism of Bcl-2/Bcl-x(L) by two distinct small-molecule compounds rapidly hyperpolarized ß-cell mitochondria, increased cytosolic Ca(2+), and stimulated insulin release via the ATP-dependent pathway in ß-cell under substimulatory glucose conditions. Experiments with single and double Bax-Bak knockout ß-cells established that this occurred independently of these proapoptotic binding partners. Pancreatic ß-cells from Bcl-2(-/-) mice responded to glucose with significantly increased NAD(P)H levels and cytosolic Ca(2+) signals, as well as significantly augmented insulin secretion. Inducible deletion of Bcl-x(L) in adult mouse ß-cells also increased glucose-stimulated NAD(P)H and Ca(2+) responses and resulted in an improvement of in vivo glucose tolerance in the conditional Bcl-x(L) knockout animals. Our work suggests that prosurvival Bcl proteins normally dampen the ß-cell response to glucose and thus reveals these core apoptosis proteins as integrators of cell death and physiology in pancreatic ß-cells.

Nanospaces between endoplasmic reticulum and mitochondria as control centres of pancreatic ß-cell metabolism and survival.

Nanometre-scale spaces between organelles represent focused nodes for signal transduction and the control of cellular decisions. The endoplasmic reticulum (ER) and the mitochondria form dynamic quasi-synaptic interaction nanodomains in all cell types examined, but the functional role of these junctions in cellular metabolism and cell survival remains to be fully understood. In this paper, we review recent evidence that ER Ca(2+) channels, such as the RyR and IP(3)R, can signal specifically across this nanodomain to the adjacent mitochondria to pace basal metabolism, with focus on the pancreatic ß-cell. Blocking these signals in the basal state leads to a form of programmed cell death associated with reduced ATP and the induction of calpain-10 and hypoxia-inducible factors. On the other hand, the hyperactivity of this signalling domain plays a deleterious role during classical forms of apoptosis. Thus, the nanospace between ER and mitochondria represents a critical rheostat controlling both metabolism and programmed cell death. Many aspects of the mechanisms underlying this control system remain to be uncovered, and new nanotechnologies are required understand these domains at a molecular level.

MISC-1/OGC links mitochondrial metabolism, apoptosis and insulin secretion.

We identified MISC-1 (Mitochondrial Solute Carrier) as the C. elegans orthologue of mammalian OGC (2-oxoglutarate carrier). OGC was originally identified for its ability to transfer α-ketoglutarate across the inner mitochondrial membrane. However, we found that MISC-1 and OGC are not solely involved in metabolic control. Our data show that these orthologous proteins participate in phylogenetically conserved cellular processes, like control of mitochondrial morphology and induction of apoptosis. We show that MISC-1/OGC is required for proper mitochondrial fusion and fission events in both C. elegans and human cells. Transmission electron microscopy reveals that loss of MISC-1 results in a decreased number of mitochondrial cristae, which have a blebbed appearance. Furthermore, our pull-down experiments show that MISC-1 and OGC interact with the anti-apoptotic proteins CED-9 and Bcl-x(L), respectively, and with the pro-apoptotic protein ANT. Knock-down of misc-1 in C. elegans and OGC in mouse cells induces apoptosis through the caspase cascade. Genetic analysis suggests that MISC-1 controls apoptosis through the physiological pathway mediated by the LIN-35/Rb-like protein. We provide genetic and molecular evidence that absence of MISC-1 increases insulin secretion and enhances germline stem cell proliferation in C. elegans. Our study suggests that the mitochondrial metabolic protein MISC-1/OGC integrates metabolic, apoptotic and insulin secretion functions. We propose a novel mechanism by which mitochondria integrate metabolic and cell survival signals. Our data suggest that MISC-1/OGC functions by sensing the metabolic status of mitochondria and directly activate the apoptotic program when required. Our results suggest that controlling MISC-1/OGC function allows regulation of mitochondrial morphology and cell survival decisions by the metabolic needs of the cell.

A multi-parameter, high-content, high-throughput screening platform to identify natural compounds that modulate insulin and Pdx1 expression.

Diabetes is a devastating disease that is ultimately caused by the malfunction or loss of insulin-producing pancreatic beta-cells. Drugs capable of inducing the development of new beta-cells or improving the function or survival of existing beta-cells could conceivably cure this disease. We report a novel high-throughput screening platform that exploits multi-parameter high-content analysis to determine the effect of compounds on beta-cell survival, as well as the promoter activity of two key beta-cell genes, insulin and pdx1. Dispersed human pancreatic islets and MIN6 beta-cells were infected with a dual reporter lentivirus containing both eGFP driven by the insulin promoter and mRFP driven by the pdx1 promoter. B-score statistical transformation was used to correct systemic row and column biases. Using this approach and 5 replicate screens, we identified 7 extracts that reproducibly changed insulin and/or pdx1 promoter activity from a library of 1319 marine invertebrate extracts. The ability of compounds purified from these extracts to significantly modulate insulin mRNA levels was confirmed with real-time PCR. Insulin secretion was analyzed by RIA. Follow-up studies focused on two lead compounds, one that stimulates insulin gene expression and one that inhibits insulin gene expression. Thus, we demonstrate that multi-parameter, high-content screening can identify novel regulators of beta-cell gene expression, such as bivittoside D. This work represents an important step towards the development of drugs to increase insulin expression in diabetes and during in vitro differentiation of beta-cell replacements.

Mechanisms of pancreatic beta-cell apoptosis in diabetes and its therapies.

Diabetes occurs when beta-cells no longer function properly or have been destroyed. Pancreatic beta-cell death by apoptosis contributes significantly in both autoimmune type 1 diabetes and type 2 diabetes. Pancreatic beta-cell death can be induced by multiple stresses in both major types of diabetes. There are also several rare forms of diabetes, including Wolcott-Rallison syndrome, Wolfram syndrome, as well as some forms of maturity onset diabetes of the young that are caused by mutations in genes that may play important roles in beta-cell survival. The use of islet transplantation as a treatment for diabetes is also limited by excessive beta-cell apoptosis. Mechanistic insights into the control of pancreatic beta-cell apoptosis are therefore important for the prevention and treatment of diabetes. Indeed, a substantial quantity of research has been dedicated to this area over the past decade. In this chapter, we review the factors that influence the propensity of beta-cells to undergo apoptosis and the mechanisms of this programmed cell death in the initiation and progression of diabetes.