Pancreatic beta cells are essential for glucose homeostasis and therefore are subject to precise control of both number and their secretory function. Apoptosis of beta cells is a hallmark of diabetic diseases and preventing apoptosis is a major practical problem in islet transplantation. Much is known about mitogenic and apoptotic signaling in beta cells and the various signaling pathways are known to interact in a highly complex network. A given cytokine or growth factor often initiates multiple signaling pathways, and it is only the integration of signaling which determines the outcome for the cell. Because of this complexity, a method that enables screening of multiple signaling cascades to determine which ones are operating under a particular set of conditions is much needed. We propose to create method through a combination of antibody arrays and mass spectrometry. Suitable antibodies will be selected and used to isolate their nominal antigens plus any associated proteins, using a SELDI mass spectrometer as a method. We will prepare an array of antibodies that report on various signaling pathways involved in beta cell death validate its usefulness by testing known components in the IGF-II pathway. Once a validated array is available it will be used to identify signaling complexes in beta cells undergoing cell death by the novel pro-inflammatory cytokine PANDER, as an example of an apoptotic agent whose receptor and molecular effects are unknown. The advantage to this method is that it does not presume prior knowledge about the sample tested, it is fast, can be adapted to high throughput formats, and allows not only identification but also isolation of signaling complexes, so that new information can be discovered. After both the known and unknown signaling pathways are successfully investigated, the method will be applied to human islets. Signaling complexes from pre-transplant human islets will be analyzed retrospectively and correlated with the clinical outcome in transplant recipients, to test the predictive power of the method.
