Study of Energy Homeostasis in a Genetic Model System

描述(由申请人提供)：肥胖症的流行以及由此导致的共病，估计会导致直接的医疗费用(美国)每年750亿美元，然而，有效的治疗方法很少。因此，识别肥胖相关基因对于开发有效的治疗方法可能是必不可少的。在小鼠中对单基因肥胖突变的研究和候选基因方法已经导致了几十个在能量稳态方面发挥重要作用的基因的鉴定。考虑到这一过程的复杂性，很可能会有数百人。最近发现的脂肪激素瘦素(1994)和假定的饥饿因子Ghrelin(1999)强调了这一领域发现过程的早期阶段。脊椎动物系统中肥胖相关突变的全基因组正向基因筛查将是一种非常有价值的方法，因为参与能量稳态的整个基因集合可以无偏见地识别，从而既可以识别现有途径中以前未知的步骤，也可以识别全新的途径。在硬骨鱼中，脂肪的某些成分似乎在功能上是保守的，因为斑马鱼的刺鼠相关蛋白(AgRP)的同源基因，下丘脑黑素皮质素系统的一个组成部分，在硬骨鱼大脑中给药时刺激进食，因禁食而上调，当在鱼类中过度表达时会导致肥胖。因此，可以利用斑马鱼来筛选影响中枢黑素皮质素系统的突变，其中许多突变可能与哺乳动物的能量动态平衡有关。为了利用斑马鱼鉴定和鉴定这些基因，本赠款中建议的工作将集中在两个基本组成部分上：1)通过筛选Hopkins斑马鱼早期发育突变体的集合来鉴定影响下丘脑发育的独特突变的集合，以确定是否有改变斑马鱼POMC和AgRP基因表达的基因；2)完成对斑马鱼POMC和AgRP基因表达改变基因的斑马鱼随机逆转录病毒插入突变的大规模遗传正向筛选。在这两个筛选中发现的基因可能包括参与下丘脑发育的新基因，参与POMC和AgRP表达的基因，甚至可能涉及通过代谢状态调节这些蛋白的基因。在哺乳动物中，脂肪的某些元件似乎在功能上是保守的，在硬骨鱼中，例如实验室的斑马鱼，因此，通过筛选系统保守元件中的缺陷，例如中央黑素皮质素系统，可以使用斑马鱼来筛选影响能量稳态控制的突变。在这一应用中，我们建议在斑马鱼中筛选随机的逆转录病毒插入突变体，以确定改变下丘脑POMC和AgRP表达的基因。在这两个筛选中发现的基因可能包括参与下丘脑发育的新基因，参与POMC和AgRP表达的基因，甚至可能涉及通过代谢状态调节这些蛋白的基因。

DESCRIPTION (provided by applicant): The obesity epidemic, and resulting co-morbidities, are estimated to result in direct medical costs (U.S.) of $75 billion per year, however there are few effective treatments available. Consequently, identification of obesity-related genes may be indispensable for the development of effective therapeutics. Study of monogenic obesity mutants in the mouse and candidate gene approaches have led to the identification of a few dozen genes that play important roles in aspects of energy homeostasis. Given the complexity of the process, there are likely to be hundreds. The recent discovery of the adipostatic hormone leptin (1994), and the putative hunger factor ghrelin (1999) underscore the early stage of the discovery process in this field. Whole genome forward genetic screening for obesity-related mutations in a vertebrate system would be a highly valuable approach since entire collections of genes involved in energy homeostasis could be identified in an unbiased fashion, allowing identification both of previously unknown steps in existing pathways as well as entirely new pathways. Certain elements of the adipostat appear functionally conserved in teleosts, since the zebrafish ortholog of agouti-related protein (AgRP), a component of the hypothalamic melanocortin system, stimulates feeding when administered in the teleost brain, is upregulated by fasting, and causes obesity when overexpressed in fish. Thus, it is possible to use the zebrafish to screen for mutations that effect the central melanocortin system, and many of these mutations are likely to be relevant to mammalian energy homeostasis. In order to identify and characterize these genes using zebrafish the work proposed in this grant will focus on two essential components: 1) characterization of a collection of unique mutations affecting hypothalamic development, identified by screening the Hopkins collection of zebrafish early developmental mutants for genes that alter the expression of zebrafish POMC and AgRP and 2) completion of a large scale forward genetic screen of random retroviral insertional mutations in the zebrafish for genes that alter expression of hypothalamic POMC and AgRP genes. The genes identified in these two screens are likely to include novel genes involved in hypothalamic development, in the expression of POMC and AgRP, and possibly even genes involved in the regulation of these proteins by metabolic state. Certain elements of the adipostat, characterized in mammals, appear functionally conserved in teleosts, such as the laboratory zebrafish, and thus, it is possible to use the zebrafish to screen for mutations that effect the control of energy homeostasis, by screening for defects in conserved elements of the system, such as central melanocortin system. In this application, we propose to screen random retroviral insertional mutants in the zebrafish to identify genes that alter expression of hypothalamic POMC and AgRP. The genes identified in these two screens are likely to include novel genes involved in hypothalamic development, in the expression of POMC and AgRP, and possibly even genes involved in the regulation of these proteins by metabolic state.