Beyond the brain: disrupted in schizophrenia 1 regulates pancreatic ß-cell function via glycogen synthase kinase-3ß.

Individuals with schizophrenia and their first-degree relatives have higher rates of type 2 diabetes (T2D) than the general population (18-30 vs. 1.2-6.3%), independent of body mass index and antipsychotic medication, suggesting shared genetic components may contribute to both diseases. The cause of this association remains unknown. Mutations in disrupted in schizophrenia 1 (DISC1) increase the risk of developing psychiatric disorders [logarithm (base 10) of odds = 7.1]. Here, we identified DISC1 as a major player controlling pancreatic ß-cell proliferation and insulin secretion via regulation of glycogen synthase kinase-3ß (GSK3ß). DISC1 expression was enriched in developing mouse and human pancreas and adult ß- and ductal cells. Loss of DISC1 function, through siRNA-mediated depletion or expression of a dominant-negative truncation that models the chromosomal translocation of human DISC1 in schizophrenia, resulted in decreased ß-cell proliferation (3 vs. 1%; P < 0.01), increased apoptosis (0.1 vs. 0.6%; P < 0.01), and glucose intolerance in transgenic mice. Insulin secretion was reduced (0.5 vs. 0.1 ng/ml; P < 0.05), and critical ß-cell transcription factors Pdx1 and Nkx6.1 were significantly decreased. Impaired DISC1 allowed inappropriate activation of GSK3ß in ß cells, and antagonizing GSK3ß (SB216763; IC50 = 34.3 nM) rescued the ß-cell defects. These results uncover an unexpected role for DISC1 in normal ß-cell physiology and suggest that DISC1 dysregulation contributes to T2D independently of its importance for cognition.

Deviant development of pancreatic beta cells from embryonic exposure to PCB-126 in zebrafish.

Exposures to co-planar PCBs and dioxins have been associated with diabetes in epidemiologic studies. Individuals may be predisposed to diseases such as diabetes as a result of exposure to environmental contaminants during early life, resulting in dysmorphic pancreatic islets or metabolically fragile ß-cells. We tested the hypothesis that embryonic exposure to a model Ahr-ligand, PCB-126 would cause structural and/or functional alterations to the developing primary pancreatic islet in the zebrafish (Danio rerio). To assess ß-cell development, transgenic zebrafish embryos (Tg(ins:GFP) and Tg(ins:mcherry) were exposed to nominal concentrations of 2 or 5nM PCB-126 or DMSO from 24-48h post fertilization (hpf), and imaged via time-lapse microscopy from 80-102hpf. We identified defects including hypomorphic islets, altered islet migration, islet fragmentation, and formation of ectopic ß-cells. As we recently showed the transcription factor Nrf2a is protective in PCB-126 embryotoxicity, we then assessed the transcriptional function of the islets in wildtype and nrf2a(fh318/fh318) mutant embryos. We measured gene expression of preproinsulin a, somatostatin2, pdx1, ghrelin, and glucagon. Expression of preproinsulin a increased with PCB treatment in wildtype embryos, while expression of all measured pancreas genes was altered by the nrf2a mutant genotype, suggesting misregulation of the glucose homeostasis axis in those embryos, independent of PCB treatment. This study shows that embryonic exposure to PCB-126 can result in deviant development of the pancreatic islet and suggests that Nrf2a plays a role in regulating glucose homeostasis during development.

Novel Observations From Next-Generation RNA Sequencing of Highly Purified Human Adult and Fetal Islet Cell Subsets.

Understanding distinct gene expression patterns of normal adult and developing fetal human pancreatic α- and ß-cells is crucial for developing stem cell therapies, islet regeneration strategies, and therapies designed to increase ß-cell function in patients with diabetes (type 1 or 2). Toward that end, we have developed methods to highly purify α-, ß-, and δ-cells from human fetal and adult pancreata by intracellular staining for the cell-specific hormone content, sorting the subpopulations by flow cytometry, and, using next-generation RNA sequencing, we report the detailed transcriptomes of fetal and adult α- and ß-cells. We observed that human islet composition was not influenced by age, sex, or BMI, and transcripts for inflammatory gene products were noted in fetal ß-cells. In addition, within highly purified adult glucagon-expressing α-cells, we observed surprisingly high insulin mRNA expression, but not insulin protein expression. This transcriptome analysis from highly purified islet α- and ß-cell subsets from fetal and adult pancreata offers clear implications for strategies that seek to increase insulin expression in type 1 and type 2 diabetes.

Role of cilia in normal pancreas function and in diseased states.

Primary cilia play an essential role in modulating signaling cascades that shape cellular responses to environmental cues to maintain proper tissue development. Mutations in primary cilium proteins have been linked to several rare developmental disorders, collectively known as ciliopathies. Together with other disorders associated with dysfunctional cilia/centrosomes, affected individuals have increased risk of developing metabolic syndrome, neurologic disorders, and diabetes. In pancreatic tissues, cilia are found exclusively in islet and ductal cells where they play an essential role in pancreatic tissue organization. Their absence or disorganization leads to pancreatic duct abnormalities, acinar cell loss, polarity defects, and dysregulated insulin secretion. Cilia in pancreatic tissues are hubs for cellular signaling. Many signaling components, such as Hh, Notch, and Wnt, localize to pancreatic primary cilia and are necessary for proper development of pancreatic epithelium and ß-cell morphogenesis. Receptors for neuroendocrine hormones, such as Somatostatin Receptor 3, also localize to the cilium and may play a more direct role in controlling insulin secretion due to somatostatin's inhibitory function. Finally, unique calcium signaling, which is at the heart of ß-cell function, also occurs in primary cilia. Whereas voltage-gated calcium channels trigger insulin secretion and serve a variety of homeostatic functions in ß-cells, transient receptor potential channels regulate calcium levels within the cilium that may serve as a feedback mechanism, regulating insulin secretion. This review article summarizes our current understanding of the role of primary cilia in normal pancreas function and in the diseased state.

Differentiated human stem cells resemble fetal, not adult, ß cells.

Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic ß cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal ß cells than adult ß cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true ß cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional ß cells.

Improved function and proliferation of adult human beta cells engrafted in diabetic immunodeficient NOD-scid IL2rγ(null) mice treated with alogliptin.

PURPOSE: Dipeptidyl-peptidase-4 (DPP-4) inhibitors are known to increase insulin secretion and beta cell proliferation in rodents. To investigate the effects on human beta cells in vivo, we utilize immunodeficient mice transplanted with human islets. The study goal was to determine the efficacy of alogliptin, a DPP-4 inhibitor, to enhance human beta cell function and proliferation in an in vivo context using diabetic immunodeficient mice engrafted with human pancreatic islets. METHODS: Streptozotocin-induced diabetic NOD-scid IL2rγ(null) (NSG) mice were transplanted with adult human islets in three separate trials. Transplanted mice were treated daily by gavage with alogliptin (30 mg/kg/day) or vehicle control. Islet graft function was compared using glucose tolerance tests and non-fasting plasma levels of human insulin and C-peptide; beta cell proliferation was determined by bromodeoxyuridine (BrdU) incorporation. RESULTS: Glucose tolerance tests were significantly improved by alogliptin treatment for mice transplanted with islets from two of the three human islet donors. Islet-engrafted mice treated with alogliptin also had significantly higher plasma levels of human insulin and C-peptide compared to vehicle controls. The percentage of insulin+BrdU+ cells in human islet grafts from alogliptin-treated mice was approximately 10-fold more than from vehicle control mice, consistent with a significant increase in human beta cell proliferation. CONCLUSION: Human islet-engrafted immunodeficient mice treated with alogliptin show improved human insulin secretion and beta cell proliferation compared to control mice engrafted with the same donor islets. Immunodeficient mice transplanted with human islets provide a useful model to interrogate potential therapies to improve human islet function and survival in vivo.

Pathological endoplasmic reticulum stress mediated by the IRE1 pathway contributes to pre-insulitic beta cell apoptosis in a virus-induced rat model of type 1 diabetes.

AIMS/HYPOTHESIS: We hypothesised that pathological endoplasmic reticulum (ER) stress contributes to beta cell death during development of type 1 diabetes. In this study, we investigated the occurrence of beta cell ER stress and the signalling pathways involved during discrete stages of autoimmune diabetes progression. The virus-inducible BBDR rat model was used to systematically interrogate the three main ER stress signalling pathways (IRE1 [inositol-requiring protein-1], PERK [double-stranded RNA-dependent protein kinase (PKR)-like ER kinase] and ATF6 [activating transcription factor 6]) in pancreatic beta cells during type 1 diabetes development. METHODS: ER stress and apoptotic markers were assessed by immunoblot analyses of isolated pancreatic islets and immunofluorescence staining of pancreas sections from control and virus-induced rats. Various time points were analysed: (1) early stages preceding the development of insulitis and (2) a late stage during onset and progression of insulitis, which precedes overt hyperglycaemia. RESULTS: The IRE1 pathway, including its downstream component X-box-binding protein 1, was specifically activated in pancreatic beta cells of virus-induced rats at early stages preceding the development of insulitis. Furthermore, ER stress-specific pro-apoptotic caspase 12 and effector caspase 3 were also activated at this stage. Activation of PERK and its downstream effector pro-apoptotic CHOP (CCAAT/-enhancer-binding-protein homologous protein), only occurred during late stages of diabetes induction concurrent with insulitis, whereas ATF6 activation in pancreatic beta cells was similar in control and virus-induced rats. CONCLUSIONS/INTERPRETATION: Activation of the IRE1 pathway and ER stress-specific pro-apoptotic caspase 12, before the development of insulitis, are indicative of ER stress-mediated beta cell damage. The early occurrence of pathological ER stress and death in pancreatic beta cells may contribute to the initiation and/or progression of virus-induced autoimmune diabetes.

Salicylate prevents virus-induced type 1 diabetes in the BBDR rat.

Epidemiologic and clinical evidence suggests that virus infection plays an important role in human type 1 diabetes pathogenesis. We used the virus-inducible BioBreeding Diabetes Resistant (BBDR) rat to investigate the ability of sodium salicylate, a non-steroidal anti-inflammatory drug (NSAID), to modulate development of type 1 diabetes. BBDR rats treated with Kilham rat virus (KRV) and polyinosinic:polycytidylic acid (pIC, a TLR3 agonist) develop diabetes at nearly 100% incidence by ~2 weeks. We found distinct temporal profiles of the proinflammatory serum cytokines, IL-1ß, IL-6, IFN-γ, IL-12, and haptoglobin (an acute phase protein) in KRV+pIC treated rats. Significant elevations of IL-1ß and IL-12, coupled with sustained elevations of haptoglobin, were specific to KRV+pIC and not found in rats co-treated with pIC and H1, a non-diabetogenic virus. Salicylate administered concurrently with KRV+pIC inhibited the elevations in IL-1ß, IL-6, IFN-γ and haptoglobin almost completely, and reduced IL-12 levels significantly. Salicylate prevented diabetes in a dose-dependent manner, and diabetes-free animals had no evidence of insulitis. Our data support an important role for innate immunity in virus-induced type 1 diabetes pathogenesis. The ability of salicylate to prevent diabetes in this robust animal model demonstrates its potential use to prevent or attenuate human autoimmune diabetes.

Dynamic glucoregulation and mammalian-like responses to metabolic and developmental disruption in zebrafish.

Zebrafish embryos are emerging as models of glucose metabolism. However, patterns of endogenous glucose levels, and the role of the islet in glucoregulation, are unknown. We measured absolute glucose levels in zebrafish and mouse embryos, and demonstrate similar, dynamic glucose fluctuations in both species. Further, we show that chemical and genetic perturbations elicit mammalian-like glycemic responses in zebrafish embryos. We show that glucose is undetectable in early zebrafish and mouse embryos, but increases in parallel with pancreatic islet formation in both species. In zebrafish, increasing glucose is associated with activation of gluconeogenic phosphoenolpyruvate carboxykinase1 (pck1) transcription. Non-hepatic Pck1 protein is expressed in mouse embryos. We show using RNA in situ hybridization, that zebrafish pck1 mRNA is similarly expressed in multiple cell types prior to hepatogenesis. Further, we demonstrate that the Pck1 inhibitor 3-mercaptopicolinic acid suppresses normal glucose accumulation in early zebrafish embryos. This shows that pre- and extra-hepatic pck1 is functional, and provides glucose locally to rapidly developing tissues. To determine if the primary islet is glucoregulatory in early fish embryos, we injected pdx1-specific morpholinos into transgenic embryos expressing GFP in beta cells. Most morphant islets were hypomorphic, not a genetic, but embryos still exhibited persistent hyperglycemia. We conclude from these data that the early zebrafish islet is functional, and regulates endogenous glucose. In summary, we identify mechanisms of glucoregulation in zebrafish embryos that are conserved with embryonic and adult mammals. These observations justify use of this model in mechanistic studies of human metabolic disease.

A novel role for the centrosomal protein, pericentrin, in regulation of insulin secretory vesicle docking in mouse pancreatic beta-cells.

The centrosome is important for microtubule organization and cell cycle progression in animal cells. Recently, mutations in the centrosomal protein, pericentrin, have been linked to human microcephalic osteodysplastic primordial dwarfism (MOPD II), a rare genetic disease characterized by severe growth retardation and early onset of type 2 diabetes among other clinical manifestations. While the link between centrosomal and cell cycle defects may account for growth deficiencies, the mechanism linking pericentrin mutations with dysregulated glucose homeostasis and pre-pubertal onset of diabetes is unknown. In this report we observed abundant expression of pericentrin in quiescent pancreatic beta-cells of normal animals which led us to hypothesize that pericentrin may have a critical function in beta-cells distinct from its known role in regulating cell cycle progression. In addition to the typical centrosome localization, pericentrin was also enriched with secretory vesicles in the cytoplasm. Pericentrin overexpression in beta-cells resulted in aggregation of insulin-containing secretory vesicles with cytoplasmic, but not centrosomal, pericentriolar material and an increase in total levels of intracellular insulin. RNAi- mediated silencing of pericentrin in secretory beta-cells caused dysregulated secretory vesicle hypersecretion of insulin into the media. Together, these data suggest that pericentrin may regulate the intracellular distribution and secretion of insulin. Mice transplanted with pericentrin-depleted islets exhibited abnormal fasting hypoglycemia and inability to regulate blood glucose normally during a glucose challenge, which is consistent with our in vitro data. This previously unrecognized function for a centrosomal protein to mediate vesicle docking in secretory endocrine cells emphasizes the adaptability of these scaffolding proteins to regulate diverse cellular processes and identifies a novel target for modulating regulated protein secretion in disorders such as diabetes.

Human immune system development and rejection of human islet allografts in spontaneously diabetic NOD-Rag1null IL2rgammanull Ins2Akita mice.

OBJECTIVE: To create an immunodeficient mouse model that spontaneously develops hyperglycemia to serve as a diabetic host for human islets and stem cell-derived beta-cells in the absence or presence of a functional human immune system. RESEARCH DESIGN AND METHODS: We backcrossed the Ins2(Akita) mutation onto the NOD-Rag1(null) IL2rgamma(null) strain and determined 1) the spontaneous development of hyperglycemia, 2) the ability of human islets, mouse islets, and dissociated mouse islet cells to restore euglycemia, 3) the generation of a human immune system following engraftment of human hematopoietic stem cells, and 4) the ability of the humanized mice to reject human islet allografts. RESULTS: We confirmed the defects in innate and adaptive immunity and the spontaneous development of hyperglycemia conferred by the IL2rgamma(null), Rag1(null), and Ins2(Akita) genes in NOD-Rag1(null) IL2rgamma(null) Ins2(Akita) (NRG-Akita) mice. Mouse and human islets restored NRG-Akita mice to normoglycemia. Insulin-positive cells in dissociated mouse islets, required to restore euglycemia in chemically diabetic NOD-scid IL2rgamma(null) and spontaneously diabetic NRG-Akita mice, were quantified following transplantation via the intrapancreatic and subrenal routes. Engraftment of human hematopoietic stem cells in newborn NRG-Akita and NRG mice resulted in equivalent human immune system development in a normoglycemic or chronically hyperglycemic environment, with >50% of engrafted NRG-Akita mice capable of rejecting human islet allografts. CONCLUSIONS: NRG-Akita mice provide a model system for validation of the function of human islets and human adult stem cell, embryonic stem cell, or induced pluripotent stem cell-derived beta-cells in the absence or presence of an alloreactive human immune system.

Parameters for establishing humanized mouse models to study human immunity: analysis of human hematopoietic stem cell engraftment in three immunodeficient strains of mice bearing the IL2rgamma(null) mutation.

"Humanized" mouse models created by engraftment of immunodeficient mice with human hematolymphoid cells or tissues are an emerging technology with broad appeal across multiple biomedical disciplines. However, investigators wishing to utilize humanized mice with engrafted functional human immune systems are faced with a myriad of variables to consider. In this study, we analyze HSC engraftment methodologies using three immunodeficient mouse strains harboring the IL2rgamma(null) mutation; NOD-scid IL2rgamma(null), NOD-Rag1(null) IL2rgamma(null), and BALB/c-Rag1(null) IL2rgamma(null) mice. Strategies compared engraftment of human HSC derived from umbilical cord blood following intravenous injection into adult mice and intracardiac and intrahepatic injection into newborn mice. We observed that newborn recipients exhibited enhanced engraftment as compared to adult recipients. Irrespective of the protocol or age of recipient, both immunodeficient NOD strains support enhanced hematopoietic cell engraftment as compared to the BALB/c strain. Our data define key parameters for establishing humanized mouse models to study human immunity.

CHOP mediates endoplasmic reticulum stress-induced apoptosis in Gimap5-deficient T cells.

Gimap5 (GTPase of the immunity-associated protein 5) has been linked to the regulation of T cell survival, and polymorphisms in the human GIMAP5 gene associate with autoimmune disorders. The BioBreeding diabetes-prone (BBDP) rat has a mutation in the Gimap5 gene that leads to spontaneous apoptosis of peripheral T cells by an unknown mechanism. Because Gimap5 localizes to the endoplasmic reticulum (ER), we hypothesized that absence of functional Gimap5 protein initiates T cell death through disruptions in ER homeostasis. We observed increases in ER stress-associated chaperones in T cells but not thymocytes or B cells from Gimap5(-/-) BBDP rats. We then discovered that ER stress-induced apoptotic signaling through C/EBP-homologous protein (CHOP) occurs in Gimap5(-/-) T cells. Knockdown of CHOP by siRNA protected Gimap5(-/-) T cells from ER stress-induced apoptosis, thereby identifying a role for this cellular pathway in the T cell lymphopenia of the BBDP rat. These findings indicate a direct relationship between Gimap5 and the maintenance of ER homeostasis in the survival of T cells.

Failure of alpha-galactosylceramide to prevent diabetes in virus-inducible models of type 1 diabetes in the rat.

BACKGROUND: Alpha-galactosylceramide (alpha-GalCer) is an invariant natural killer T (iNKT) cell ligand that prevents type 1 diabetes in NOD mice. However, alpha-GalCer can activate or suppress immune responses, raising concern about its potential use in human diabetes. MATERIALS AND METHODS: To evaluate this therapeutic issue further, BBDR and LEW.1WR1 rats were treated with Kilham rat virus (KRV) plus polyinosinic-polycytidylic acid, with or without alpha-GalCer, and followed for onset of diabetes. RESULTS: alpha-GalCer did not prevent diabetes in inducible rat models. To investigate this discrepancy, we analyzed iNKT cell function. Splenocytes stimulated with alpha-GalCer produced similar levels of IFNgamma in all rat strains, but less than mouse splenocytes. Rat splenocytes stimulated with alpha-GalCer preferentially produced IL-12, whereas mouse splenocytes preferentially produced IL-4. CONCLUSION: alpha-GalCer elicits species-specific cytokine responses in iNKT cells. In humans with type 1 diabetes, differences in iNKT cell responses to stimulation with alpha-GalCer due to age, genetic variability and other factors may influence its therapeutic potential.

A zebrafish unc-45-related gene expressed during muscle development.

We report the isolation and expression pattern of zebrafish unc45r, a gene related to Caenorhabditis elegans unc-45. UNC-45 is a muscle-specific protein thought to interact with myosin and promote the assembly of muscle thick filaments during C. elegans development. Zebrafish Unc45r shares sequence features with C. elegans UNC-45, including three tetratricopeptide repeats and a CRO1/She4p homology domain. unc45r is expressed in mesoderm adjacent to the dorsal midline during late gastrula stages and is coexpressed with muscle specific genes in somitic mesoderm during development of trunk skeletal muscle. unc45r is also expressed in cranial skeletal muscle as well as in cardiac and smooth muscle. The isolation of a muscle-specific unc-45 related gene from zebrafish suggests a common mechanism for muscle filament assembly between vertebrates and invertebrates.

Sonic hedgehog is required early in pancreatic islet development.

Pancreatic organogenesis relies on a complex interplay of cell-autonomous and extracellular signals. We demonstrate that the morphogen sonic hedgehog (Shh) is required for pancreatic development in zebrafish. Genetic mutants of Shh and its signaling pathway establish this dependence as specific to endocrine, but not exocrine, pancreas. Using cyclopamine to inhibit hedgehog signaling, we show that transient Shh signaling is necessary during gastrulation for subsequent differentiation of endoderm into islet tissue. A second hedgehog-dependent activity occurring later in development was also identified and may be analogous to the known action of Shh in gut endoderm to direct localization of pancreatic development. The early action of Shh may be part of a more general process allowing neuroendocrine cells to originate in nonneuroectodermally derived tissues.