Fellowship/Mosley: Function and Regulation of the Pancreatic Homeodomain Protein Pdx-1

Glucose homeostasis is essential to maintain proper metabolic function. Defects in the pathways that control glucose homeostasis can lead to the pathogenesis of type II diabetes mellitus, one of the six major risk factors for cardiovascular disease. The major goal of this proposal is to characterize the signal transduction pathway triggered by glucose that leads to the activation of insulin gene transcription in pancreatic beta-cells. The beta-cell specific transcription factor Pdx-1 is known to be the target of the glucose signal. Pdx-1 is a homeodomain transcription factor that is known to activate gene expression of many beta cell specific genes involved in maintaining glucose homeostasis such as insulin, GLUT-2, and glucokinase. The principal hypothesis is that Pdx-1 up-regulates insulin gene transcription in response to increases in extracellular glucose by a yet unknown signal transduction pathway. Studies on Pdx-1 function and regulation by glucose may reveal novel target proteins for diabetes therapy, as well as increase the knowledge of how transcription factors are regulated in response to environmental stimuli. The specific aims of this proposal will address three different aspects of Pdx-1 function and regulation. The first aim focuses on the characterization of Pdx-1 point mutations found in type II diabetic patients. These mutations will be introduced into the mouse Pdx-1 gene to analyze their effects on DNA binding and transactivation. The second aim concentrates on the analysis of the mechanisms involved in glucose regulation of Pdx-1. This will be done by following the changes in the subcellular localization of Pdx-1 in response to high extracellular glucose levels. The third aim is to analyze the binding kinetics of Pdx-1 in vivo. For this purpose we will use an in vivo chromatin immunoprecipitation assay to measure the amount of Pdx-1 binding to various promoters under different conditions. The proposed experiments will contribute to our understanding of Pdx-1 function and development and will provide novel insights which may lead to the prevention of type II diabetes and its secondary complications such as cardiovascular disease.