Faculty Bibliography

Type 1 diabetes (T1D) results from progressive loss of pancreatic islet mass through autoimmunity targeted at a diverse, yet limited, series of molecules that are expressed in the pancreatic beta cell. Identification of these molecular targets provides insight into the pathogenic process, diagnostic assays, and potential therapeutic agents. Autoantigen candidates were identified from microarray expression profiling of human and rodent pancreas and islet cells and screened with radioimmunoprecipitation assays using new-onset T1D and prediabetic sera. A high-ranking candidate, the zinc transporter ZnT8 (Slc30A8), was targeted by autoantibodies in 60-80% of new-onset T1D compared with <2% of controls and <3% type 2 diabetic and in up to 30% of patients with other autoimmune disorders with a T1D association. ZnT8 antibodies (ZnTA) were found in 26% of T1D subjects classified as autoantibody-negative on the basis of existing markers [glutamate decarboxylase (GADA), protein tyrosine phosphatase IA2 (IA2A), antibodies to insulin (IAA), and islet cytoplasmic autoantibodies (ICA)]. Individuals followed from birth to T1D showed ZnT8A as early as 2 years of age and increasing levels and prevalence persisting to disease onset. ZnT8A generally emerged later than GADA and IAA in prediabetes, although not in a strict order. The combined measurement of ZnT8A, GADA, IA2A, and IAA raised autoimmunity detection rates to 98% at disease onset, a level that approaches that needed to detect prediabetes in a general pediatric population. The combination of bioinformatics and molecular engineering used here will potentially generate other diabetes autoimmunity markers and is also broadly applicable to other autoimmune disorders.

In liver, glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate, the final step in the gluconeogenic and glycogenolytic pathways. Mutations in the glucose-6-phosphatase catalytic subunit (G6Pase) give rise to glycogen storage disease (GSD) type 1a, which is characterized in part by hypoglycemia, growth retardation, hypertriglyceridemia, hypercholesterolemia, and hepatic glycogen accumulation. Recently, a novel G6Pase isoform was identified, designated UGRP/G6Pase-beta. The activity of UGRP relative to G6Pase in vitro is disputed, raising the question as to whether G6P is a physiologically important substrate for this protein. To address this issue we have characterized the phenotype of UGRP knock-out mice. G6P hydrolytic activity was decreased by approximately 50% in homogenates of UGRP(-/-) mouse brain relative to wild type tissue, consistent with the ability of UGRP to hydrolyze G6P. In addition, female, but not male, UGRP(-/-) mice exhibit growth retardation as do G6Pase(-/-) mice and patients with GSD type 1a. However, in contrast to G6Pase(-/-) mice and patients with GSD type 1a, UGRP(-/-) mice exhibit no change in hepatic glycogen content, blood glucose, or triglyceride levels. Although UGRP(-/-) mice are not hypoglycemic, female UGRP(-/-) mice have elevated ( approximately 60%) plasma glucagon and reduced ( approximately 20%) plasma cholesterol. We hypothesize that the hyperglucagonemia prevents hypoglycemia and that the hypocholesterolemia is secondary to the hyperglucagonemia. As such, the phenotype of UGRP(-/-) mice is mild, indicating that G6Pase is the major glucose-6-phosphatase of physiological importance for glucose homeostasis in vivo.

The response to exposure of all-trans-retinoic acid (RA) during development varies from physiologic to severe teratogenic outcomes and is dependent upon the dose and the stage of development in all species. Effects of RA-mediated teratogenesis may be due to its ability to cause apoptosis. We have recently reported the modulation of p53 in murine stem cells by RA. The aim of this study was to characterize the temporal and spatial pattern of p53 expression in Swiss Webster mouse fetuses following maternal treatment with a single oral dose of 100 mg/kg body weight of RA during organogenesis. RA treatment resulted in a decreased p53 mRNA level in fetuses 24, 48, and 72 h after maternal treatment as detected by semiquantitative reverse transcriptase polymerase chain reaction. Western blot analysis showed a decrease in p53 protein at 24 and 48 h. Immunohistochemistry revealed decreased localization of p53 in the neuroepithelium of fetuses exposed to RA in utero. RA treatment also resulted in decreased nuclear p21 and decreased expression of cytosolic as well as nuclear p27 at 72 h in the fetuses. These results demonstrated that RA-mediated teratogenesis is accompanied by a reduction in the temporal and spatial pattern of p53 gene and protein expression in addition to the disruption of the cell cycle by modulation of p21 and p27.

The Myc family of genes regulates proliferation, differentiation, and apoptosis. Temporal expression of Myc family genes and several pro-apoptotic genes were investigated during Swiss Webster mice organogenesis after maternal treatment with an oral dose of 100 mg/kg trans-retinoic acid (RA) or vehicle on day 10 post-coitum. Reverse transcriptase-polymerase chain reaction and ribonuclease protection assay revealed decreased c-myc expression at 48 h followed by an increase at 72 h in fetuses from RA-treated dams. Increased c-Myc protein was detected at 72 h in the RA-treated group. In utero RA-treatment resulted in decreased expression of max, mad, caspases, bax, and bad genes at 48 h. Terminal uridinetriphosphate nick end-labeling (TUNEL) analysis revealed increased apoptosis at 24-48 h, followed by decreased apoptosis 72 h after in utero RA-exposure, which correlated with the decreased expression of pro-apoptotic genes noted at 48 h. Further investigations are needed to understand the role of Myc family genes during RA-mediated teratogenesis.

Apoptosis plays an important role during embryonic development. Apoptotic cell death is executed by caspases and can be regulated by the Bcl-2 family of genes. Ribonuclease protection assay was used to investigate the expression of selected apoptosis-related genes of the Bcl-2 family, pro-apoptotic Bax, Bad and anti-apoptotic Bcl-2, during differentiation of murine embryonic stem cells (ES) mediated by all-trans-retinoic acid. The mRNA expression of caspase 3, caspase 6 and certain pro-inflammatory cytokines was also investigated simultaneously. ES cells exposed to 1 microM all-trans-retinoic acid on day 8, 9 and 10 of differentiation revealed increased expression of Bax and Bad compared to the vehicle-treated cells. No effect on Bcl-2 mRNA was noted after all-trans-retinoic acid treatment. Increased mRNA expression of caspase 3 and caspase 6 in all-trans-retinoic acid-exposed ES cells suggested that caspases play an important role in retinoic acid-mediated apoptosis during ES differentiation. Increase in the expression of TNF alpha and macrophage migration inhibitory factor (MIF) was noted in retinoic acid-treated cells on day 14. Significant increase observed in interferon gamma inducing factor (IGIF/IL-18) mRNA expression in all-trans-retinoic acid-treated cells on day 14 and 17 did not translate to increased INF gamma expression. No change in the expression of other pro-inflammatory cytokines was noted with all-trans-retinoic acid treatment. The function of TNF alpha, IGIF/IL-18 and MIF in all-trans-retinoic acid-treated cells during ES differentiation and apoptosis is still speculatory. Results suggested that RA-mediated apoptosis during neural differentiation of ES cells involves up-regulation of caspase 3, caspase 6, Bad, and Bax.