INTERLEUKIN-1-BETA INDUCED CHANGES IN THE PROTEIN EXPRESSION OF RAT ISLETS - A COMPUTERIZED DATABASE

Insulin-dependent diabetes mellitus is caused by an autoimmune destruc tion of the beta-cells in the islets of Langerhans. The cytokine inter leukin 1 inhibits insulin release and is selectively cytotoxic to beta -cells in isolated pancreatic rat islets. The antigen(s) triggering th e immune response as well as the intracellular mechanisms of action of interleukin 1-mediated beta-cell cytotoxicity are unknown. However, p revious studies have found an association of beta-cell destruction wit h alterations in protein synthesis. Thus, two-dimensional (2-D) gel el ectrophoresis of pancreatic islet proteins may be an important tool fa cilitating studies of the molecular pathogenesis of insulin-dependent diabetes mellitus. 2-D gel electrophoresis of islet proteins may lead to (i) the determination of qualitative and quantitative changes in sp ecific islet proteins induced by cytokines, (ii) the determination of the effects of agents modulating cytokine action, and (iii) the identi fication of primary islet protein antigen(s) initiating the immune des truction of the beta-cells. Therefore, the aim of this study was to cr eate databases (DB) of all reproducibly detectable protein spots on 10 % and 15% acrylamide 2-D gels of neonatal rat islets (10% and 15% DB), labeled under standardized culture conditions. 1235 and 557 spots wer e present in 5 of 5 gels in the 15% isoelectric focusing (IEF) and non equilibrium pH gradient electrophoresis (NEPHGE) DB, respectively, whe reas 995 and 378 spots were present in 5 of 5 gels in the 10% IEF and NEPHGE DB, respectively, yielding a reproducibility of spot detection between 75.2% and 91.7%. In both DBs, the average coefficient of varia tion of the percentage of integrated optical density (CV% of %IOD) for spots present in all gels was between 42.4% and 45.7%. When the same sample was analyzed in consecutive sets of gels on different days (int erassay analysis), the average CV% of %IOD was 35.5%-36.1%. When the s ame sample was analyzed repeatedly in one set of gels (intra-assay ana lysis), the average CV% of %IOD was 30.2% in the IEF gels, while the a verage CV% of %IOD was 45.7% in the NEPHGE gels. Addition of interlenk in-1 beta (IL-1 beta) to the cultures resulted in statistically signif icant modulation or de novo synthesis of 105 proteins in the 10% gels. In conclusion, we present the first 10% and 15% acrylamide 2-D gel pr otein databases of neonatal rat islets of Langerhans and demonstrate i ts usage to identify proteins altered in expression by IL-1 beta.