Genetic analysis of a truncated insulin receptor.

项目摘要 胰岛素抵抗发生的分子机制仍不清楚，但 更好地理解这一过程是至关重要的，因为肥胖、代谢疾病和糖尿病 在美国达到流行状态。我们发现蠕虫体内胰岛素受体的选择性剪接， 小鼠和人产生截短的同种型，其能够通过抑制胰岛素信号传导来减弱胰岛素信号传导， 通过全长受体传递信号。确定这些新亚型的生理学意义 在确定它们在胰岛素抵抗的病理生理学中的作用中是至关重要的。然而，这些研究在 哺乳动物是昂贵的、耗时的和危险的。而线虫C. elegans提供了一个 经济的系统，其中快速确定其生理相关性。为此，我们已开始 表征线虫C中胰岛素受体的截短同种型。elegans，被称为E1 B-2B，即 直接类似于哺乳动物截短的受体，称为IR-C和IR-D。 初步分析表明，daf-2b的表达在不同组织和发育过程中存在差异。 重要的是，我们发现截短的daf-2b亚型的基因缺失赋予胰岛素敏感性，而过度的daf-2b亚型的基因缺失赋予胰岛素敏感性。 daf-2b cDNA在蠕虫中的表达产生与减弱的胰岛素信号传导一致的表型。我们 现在集中于确定DAF-2b介导其对胰岛素敏感性的作用的机制， 使用新的报告基因的组合，其告知daf-2b动力学，以及组织特异性遗传学， DAF-2B的操作。使用一种新的报告菌株，允许可视化之间的差异剪接 全长和截短的daf-2同种型在体内，我们确定了特定的剪接因子，改变 daf-2b的表达。剪接因子活性调控表达机制的探讨 DAF-2B的研究将确定这一途径中的新的调节和干预点。这些发现在C. 线虫现在正在指导哺乳动物截短IR的研究，包括表征作用模式 的IR-C和IR-D，以及检查导致这些截短的表达的调节因子。 哺乳动物的受体最后，对小鼠IR-C和IR-D在体内的表达进行了全面的调查， 相对于全长胰岛素受体，将建立其生理和病理生理调节。 我们假设，在正常人中，截短的IR-C和IR-D亚型的异常或错误调节的表达可能是由于在正常人中，IR-C和IR-D亚型的异常或错误调节。 哺乳动物可能与胰岛素抵抗、糖尿病和其他形式的糖尿病的发病机制有因果关系。 代谢性疾病在这方面，确定遗传靶点和调控机制， 影响截短的胰岛素受体亚型的表达将加速治疗策略的发现 靶向这种新型胰岛素调节机制。

PROJECT SUMMARY The molecular mechanisms that underpin the development of insulin resistance remain poorly defined, yet a better understanding of this process is critical, given the fact that obesity, metabolic disease and diabetes have reached epidemic status in the US. We discovered that alternative splicing of the insulin receptor, in worms, mice and humans, generates truncated isoforms that are able to attenuate insulin signaling, by inhibiting signaling through the full length receptor. Determination of the physiological significance of these new isoforms is critical in establishing their role in the pathophysiology of insulin resistance. However, such studies in mammals are expensive, time consuming and risky. In contrast, the nematode C. elegans provides an economical system in which to rapidly determine their physiological relevance. To this end, we have started to characterize the truncated isoform of the insulin receptor in the nematode C. elegans, termed DAF-2B, that is directly analogous to mammalian truncated receptors, termed IR-C and IR-D. Preliminary analysis indicates that daf-2b expression varies across tissues and through development. Importantly, we find that genetic deletion of the truncated daf-2b isoform confers insulin sensitivity, while over- expression of a daf-2b cDNA in worms produces phenotypes consistent with attenuated insulin signaling. We are now focused on determining the mechanism(s) by which daf-2b mediates its effects on insulin sensitivity, using a combination of novel reporters that inform on daf-2b dynamics, as well as tissue-specific genetic manipulation of daf-2b. Using a novel reporter strain that permits visualization of differential splicing between the full length and truncated daf-2 isoforms in vivo, we identified specific splicing factors that alter the expression of daf-2b. Exploration of the mechanism by which splicing factor activity regulates the expression of daf-2b will identify novel points of regulation and intervention in this pathway. These discoveries in C. elegans are now guiding studies of the mammalian truncated IRs, including characterization the mode of action of IR-C and IR-D, as well as examining the regulatory factors that lead to expression of these truncated receptors in mammals. Finally, a comprehensive survey of the expression of murine IR-C and IR-D in vivo, relative to full length insulin receptors, will establish their physiological and pathophysiological regulation. We hypothesize that aberrant or mis-regulated expression of truncated IR-C and IR-D isoforms in mammals could be causally involved in the pathogenesis of insulin resistance, diabetes and other forms of metabolic disease. In this respect, the identification of genetic targets and regulatory mechanisms that influence expression of truncated insulin receptor isoforms will expedite the discovery of therapeutic strategies that target this novel insulin regulatory mechanism.