Using BRET to measure insulin resistance and receptor cross-talk

描述(由申请人提供)： 胰岛素抵抗存在于所有II型糖尿病患者中，了解胰岛素抵抗的原因将导致治疗这种疾病的新方法。为了更好地了解胰岛素抵抗的发生机制，需要在细胞培养模型中研究胰岛素抵抗的新技术。本文概述的研究将通过使用一种新的成像技术--生物发光共振能量转移(BRET)来实现这一目标。BRET使用荧光细胞内记者实时测量活细胞中的酶活性和蛋白质-蛋白质相互作用，并有可能极大地促进我们对胰岛素信号转导的理解。 细胞代谢信号的一个重要但被低估的方面是受体串扰的作用。例如，在脂肪细胞中，胰岛素信号和儿茶酚胺信号之间存在平衡，前者有利于能量储存(脂肪生成)，后者有利于能量释放(脂肪分解)。这些激素受体之间的串扰在维持正常代谢稳态方面起着关键作用。由于串扰，胰岛素信号的异常不仅影响胰岛素信号通路，还影响儿茶酚胺信号通路。我们已经证明，这两个激素信号系统之间的一个重要的串扰位置是在受体调节蛋白β-arrestin的水平上。使用BRET，我们将测量胰岛素受体(和IGF-1受体)和β-arrestin之间相互作用的几个方面。 BRET还为我们提供了一种前所未有的能力，可以实时测量活细胞中胰岛素信号的动力学。胰岛素信号动力学异常显然是胰岛素抵抗状态的一部分，可能是胰岛素抵抗最早的表现之一。我们将使用BRET来探索胰岛素信号动力学异常是胰岛素抵抗最早可检测到的表现这一假说，并描述这种异常背后的机制。 公开声明：胰岛素抵抗在肥胖和II型糖尿病患者中普遍存在，是美国医疗保健支出的主要原因。开发研究胰岛素抵抗的新技术是糖尿病研究的主要目标。这项建议使用了一种新的技术，即生物发光共振能量转移，来实时研究活培养细胞的胰岛素抵抗。

DESCRIPTION (provided by applicant): 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.