Unchanged pancreatic beta-cell phenotype, function and sensitivity to cytokine-induced apoptosis in GAD-65 autoantigen-suppressed INS-1 cells

Abstract

The enzyme glutamic acid decarboxylase (GAD) represents a major autoantigen in the early immunopathogenesis of T-cell-mediated destruction of pancreatic beta-cells in type 1 diabetes mellitus. Transgenic suppression of GAD expression in beta-cells of the non obese diabetic (NOD) mouse completely prevents development of the disease (1). Suppression of GAD-expression may thus provide a tool to prevent recurrence of autoimmune beta-cell destruction in transplanted pancreatic beta-cells in type 1 diabetic patients. Recent studies have shown that apoptosis may contribute to pancreatic beta-cell death in autoimmune diabetes. The aim of this study was the functional and phenotypic characterization of insulin producing pancreatic beta-cells with suppressed autoantigenic GAD-65 mRNA expression. GAD-65 expression in the rat pancreatic beta-cell line INS-1 was suppressed by stable transfection of vectors, that allow the overexpression of antisense mRNA for the rat isoform GAD-65 constitutively under the control of the CMV promoter. By reverse transcription polymerase chain reaction significant suppression of GAD-65 mRNA expression by 90% can be demonstrated. In INS-1 cell lines that stably express antisense GAD-65 mRNA, glucose-dependent insulin secretion (insulin RIA), beta-cell specific gene expression (RT-PCR for insulin, BETA2, glucokinase, GLUT2, PDX-1, Pax-4) and cytokine (IL-1beta, TNFalpha, IFNgamma, Mix) induced-apoptosis was tested. No significant alterations of these parameters were observed upon suppression of GAD-65 expression as compared to control cells. In summary these results demonstrate, that forced reduction of expression of the potential autoantigen GAD does not alter phenotype and function of insulin producing cells. Protection from pancreatic beta-cell destruction after GAD-suppression does not seem to be due to altered sensitivity to cytokine induced apoptosis.