Philip diIorio

Differentiated human stem cells resemble fetal, not adult, β cells

Significance Human pluripotent stem cells (hPSCs) can be produced from any person and have the potential to differentiate into any cell type in the body. This study focuses on the generation of insulin-expressing cells from hPSCs and compares their gene expression, as assayed by transcriptional gene products, to that of insulin-expressing β cells from human fetal and adult samples. We employ a new method to isolate and profile insulin-expressing cells and conclude that several different hPSC lines generate very similar insulin-expressing cells, cells whose transcripts resemble fetal rather than adult β cells. This study advances the possibility of directing the differentiation of stem cells into functional β cells by comparing and cataloging differences between hPSC-derived insulin-expressing cells and human β cells. Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic β 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 β cells than adult β cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS+ cells and true β cells and provides a catalog of genes whose manipulation may convert hPSC-INS+ cells into functional β cells.

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 β-cells is crucial for developing stem cell therapies, islet regeneration strategies, and therapies designed to increase β-cell function in patients with diabetes (type 1 or 2). Toward that end, we have developed methods to highly purify α-, β-, 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 β-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 β-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 β-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.

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.

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.

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.

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/hypothesisWe 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.MethodsER 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.ResultsThe 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/interpretationActivation 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.

A Novel Role for the Centrosomal Protein, Pericentrin, in Regulation of Insulin Secretory Vesicle Docking in Mouse Pancreatic β-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 β-cells of normal animals which led us to hypothesize that pericentrin may have a critical function in β-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 β-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 β-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.

Hyperglycemia-induced proliferation of adult human beta cells engrafted into spontaneously diabetic immunodeficient NOD-Rag1null IL2rγnull Ins2Akita mice.

Impaired insulin secretion by the pancreatic beta cell and a reduction in beta cell mass are central to the pathogenesis of type 1 and type 2 diabetes. An ideal approach for treating individuals with diabetes is to develop modalities that increase beta cell numbers by inducing their proliferation in situ. Much evidence demonstrates that, in rodents, beta cells exhibit robust proliferative capacity both in vitro and in vivo. Induction of mouse beta cell

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

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 β‐cell proliferation and insulin secretion via regulation of glycogen synthase kinase‐3β (GSK3β). DISC1 expression was enriched in developing mouse and human pancreas and adult β‐ 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 β‐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 β‐cell transcription factors Pdx1 and Nkx6.1 were significantly decreased. Impaired DISC1 allowed inappropriate activation of GSK3β in β cells, and antagonizing GSK3β (SB216763; IC50 = 34.3 nM) rescued the β‐cell defects. These results uncover an unexpected role for DISC1 in normal β‐cell physiology and suggest that DISC1 dysregulation contributes to T2D independently of its importance for cognition.—Jurczyk, A., Nowosielska, A., Przewozniak, N., Aryee, K.‐E., DiIorio, P., Blodgett, D., Yang, C., Campbell‐Thompson, M., Atkinson, M., Shultz, L., Rittenhouse, A., Harlan, D., Greiner, D., Bortell, R. Beyond the brain: disrupted in schizophrenia 1 regulates pancreatic β‐cell function via glycogen synthase kinase‐3β. FASEB J. 30, 983–993 (2016). www.fasebj.org

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-1β, IL-6, IFN-γ, IL-12, and haptoglobin (an acute phase protein) in KRV+pIC treated rats. Significant elevations of IL-1β 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-1β, 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.