Genomic determinants of leptin expression

描述（由申请人提供）：肥胖和2型糖尿病的巨大影响要求我们更好地理解代谢活跃组织如何解释和整合营养信号。特别是，有新的认识到，脂肪组织作为一个内分泌器官，负责协调范围广泛的稳态机制。第一个被发现的脂肪衍生激素（所谓的“脂肪因子”）是瘦素，由瘦素基因以或多或少的脂肪选择性方式编码。目前已知瘦素通过调节食欲起到脂肪代谢的作用，并且在葡萄糖稳态、免疫功能、生殖和骨密度方面也起着重要作用。提高瘦素水平对于脂肪营养不良和某些遗传形式的肥胖是一种有用的治疗策略，并且可能对普通肥胖的一个子集有帮助。尽管有这些临床意义和对瘦素的大量基础科学兴趣，但实际上对调节瘦素mRNA合成的转录途径一无所知。在这里，我们提出了一些创新的方法来解决这个长期存在的问题。例如，我们已经使用BAC转基因技术对脂肪细胞选择性Lep表达所需的区域进行了粗略的定位，并取得了巨大的成功。第一个Aim使用额外的BAC转基因来进一步缩小目标区域。第二个Aim允许通过高通量dna酶超敏作图、修饰组蛋白的染色质免疫沉淀和电泳迁移转移测定来精细绘制关键顺式区域。第三个目标是通过有限的RNAi筛选在Lep报告细胞中识别影响Lep表达的反式作用因子。综上所述，这些研究将为Lep脂肪细胞表达的转录基础提供巨大的见解。瘦素是一种脂肪细胞衍生的激素，对食欲和葡萄糖耐量有深远的影响，此外还影响其他生理过程，包括骨密度、生殖和免疫功能。尽管经过了十年的深入研究，但实际上没有关于调节瘦素合成的关键转录途径的数据。该提案使用各种创新技术来解决这一缺陷，包括BAC转基因，高通量dna酶超敏分析和shRNA筛选。

DESCRIPTION (provided by applicant): The enormous impact of obesity and type 2 diabetes demands that we develop a better understanding of how nutritional signals are interpreted and integrated by metabolically active tissues. In particular, there is newfound appreciation that adipose tissue serves as an endocrine organ with responsibility for coordinating a wide range of homeostatic mechanisms. The first of the adipose-derived hormones (the so-called 'adipokines') to be discovered was leptin, encoded by the Lep gene in a more or less adipose-selective manner. Leptin is now known to serve an adipostatic role by regulating appetite, and also plays a major role in glucose homeostasis, immune function, reproduction, and bone density. Raising leptin levels is a useful therapeutic strategy in lipodystrophy and in some genetic forms of obesity, and may be helpful in a subset of common obesity. Despite these clinical implications and the enormous amount of basic scientific interest in leptin, virtually nothing is known about the transcriptional pathways that regulate leptin mRNA synthesis. Here we propose several innovative approaches to solving this longstanding problem. For example, we have used BAC transgenesis to perform gross mapping of the regions required for adipocyte-selective Lep expression with great success. The first Aim uses additional rounds of BAC transgenesis to further reduce the target area. The second Aim allows fine mapping of critical cis regions through high- throughput DNase hypersensitivity mapping, chromatin immunoprecipitation of modified histone, and electrophoretic mobility shift assays. The third Aim is directed at identifying trans-acting factors that affect Lep expression through a limited RNAi screen in Lep reporter cells. Taken together, these studies will provide enormous insight into the transcriptional basis of Lep adipocyte expression. Leptin is an adipocyte-derived hormone with profound effects on appetite and glucose tolerance, in addition to effects on other physiological processes including bone density, reproduction, and immune function. Despite a decade of intensive study, there are virtually no data on the critical transcriptional pathways that regulate leptin synthesis. This proposal uses a variety of innovative technologies to address this deficit, including BAC transgenesis, high-throughput DNase hypersensitivity analysis, and shRNA screening.