? Insulin resistance is present in all persons with type II diabetes, and understanding the causes of insulin resistance will lead to new treatments for this disease. To better understand the mechanisms involved in the development of insulin resistance, new techniques for studying insulin resistance in cell culture models are needed. The studies outlined herein will accomplish this objective through the use of a novel imaging technique, bioluminescence resonance energy transfer (BRET). BRET uses fluorescent intracellular reporters to measure enzyme activity and protein-protein interactions in real time in living cells, and has the potential to greatly advance our understanding of insulin signaling. ? One important yet undervalued aspect of cellular metabolic signaling is the role of receptor cross-talk. For example, in adipocytes, a balance exists between insulin signaling, favoring energy storage (lipogenesis), and catecholamine signaling, favoring energy release (lipolysis). Cross-talk between the receptors for these hormones plays a critical role in maintaining normal metabolic homeostasis. Because of cross-talk, abnormalities in insulin signaling affect not only insulin signaling pathways, but also catecholamine signaling pathways. We have shown that one important site of cross-talk between these two hormonal signaling systems is at the level of the receptor regulatory protein beta-arrestin. Using BRET, we will measure several aspects of the interaction between the insulin receptor (and the IGF-1 receptor) and beta-arrestin. ? BRET also gives us an unprecedented ability to measure the kinetics of insulin signaling in real time in live cells. Abnormal kinetics of insulin signaling are clearly a part of the insulin resistant state, and may represent one of the earliest manifestations of insulin resistance. We will use BRET to explore the hypothesis that abnormal insulin signaling kinetics is the earliest detectable manifestation of insulin resistance, and to describe the mechanism behind this abnormality. ? PUBLIC STATEMENT : Insulin resistance, universal among persons with obesity and type II diabetes mellitus, is a major cause of health care expenditures in the U.S. Developing new techniques to study insulin resistance is a major goal of diabetes research. This proposal uses a novel technology, bioluminescence resonance energy transfer, to study insulin resistance in real time in live cultured cells. ? ?
