Glucose uptake by muscle and fat tissue is a primary mechanism for energy storage and represents a main control mechanism for glucose concentration in the circulation. A detailed understanding of the intracellular signaling system that mediates insulin-triggered translocation of the glucose transporter (GLUT4) will likely provide new findings for attacking diseases such as diabetes and obesity. The main goal of the proposal is to develop a kinetic screen to identify adipocyte regulatory proteins that control dynamic parameters for PIP3 signaling and for GLUT4 translocation and endocytosis. We will establish a wide-field TIRF system to measure the translocation and endocytosis rates of GLUT4 transporters, as well as the generation and degradation rate of PI3P lipids, in individual cells under 12 different perturbation conditions in parallel. We will create an RNAi library to target the approximately 800 adipocyte signaling/secretory proteins and a library of 800 expressed dominant negative and constitutively active expression constructs for complementary pertubation studies. In contrast to earlier approaches in which screens were based on end-point assays in fixed cells, the live-cell strategy proposed here focuses instead on the identification of adipocyte signaling and secretory proteins that control important kinetic parameters in insulin triggered PIP3 signaling as well as in GLUT4 translocation. In the second part, we will execute, analyze and interpret a screen of the insulin-PIP3-GLUT4 signaling network with this new approach and will also perform a screen of cross-talk between insulin and TNF-alpha signaling. These screens will be used to identify new players in the insulin-GLUT4 signaling network and to gain a better understanding of kinetic control mechanisms. The project will also provide a test case for a kinetic screening strategy and a test case for a combined screen using RNAi, as well as DN and CA expression constructs.
