Development of definitive therapy for both type 1 and type 2 diabetes depends on a thorough understanding of the molecular events involved in the production of insulin within pancreatic islets. Our lab has cloned and characterized a highly conserved oxidoreductase, NCB5OR, that is localized in the endoplasmic reticulum (ER). The targeted inactivation of this gene in mice results in a diabetic phenotype. By 7 weeks of age Ncb5or-/- mice develop severe hyperglycemia with markedly decreased serum insulin levels. Pancreatic islets show marked deficiency in beta-cells but normal numbers of alpha, delta and PP cells. Our Research Plan is predicated on the hypothesis that NCB5OR protects the pancreatic beta-cell against oxidant-induced damage in the ER. In the first Specific Aim we plan studies that further characterize the impact of NCB5OR deficiency in the intact mouse. We will address the important question of whether NCB5OR plays a biologically important role outside the pancreatic beta-cell by transplanting the knockout mouse with normal beta-cells. We will also study Ncb5or -/- and +/+ mice expressing a transgene that reports ER stress in different organs and tissues. Finally, we will prepare mice homozygous for deficiencies in both NCB5OR and CHOP, a transcription factor required for ER stress induced apoptosis.
Specific Aim 2 focuses on the impact of NCB5OR on responses to ER and oxidative stress. Pancreatic beta-cells are particularly prone to ER stress. Therefore we have designed experiments to determine whether NCB5OR-deficient pancreatic islets, insulinoma cell lines and mouse embryonic fibroblasts (MEFs) evince changes in gene expression and signal transduction characteristic of the ER stress response. We will also assess the production of reactive oxygen species and the ratio of reduced to oxidized glutathione in the ER of Ncb5or-/- tissues and MEFs as well as in NCB5OR depleted insulinoma cells. The last Specific Aim entails a comprehensive assessment of the biochemical function of NCB5OR. Cellular and cell-free pull-down experiments will be employed to test and confirm potential partner proteins. We will also develop a cell-free system to identify the biologic substrate(s) and product(s) of NCB5OR. We will test the hypothesis that NCB5OR mediates fatty acid desaturation in the ER membrane by analyzing lipid profiles in ER preparations from livers of Ncb5or-/- and +/+ mice. The experiments planned in these three Specific Aims are closely inter-related and, collectively, should advance our understanding of beta-cell's defense against oxidant stress. These studies may provide new insights into the pathogenesis and treatment of diabetes.
