Study of Energy Homeostasis in a Genetic Model System

描述（由申请人提供）：肥胖的流行，以及由此产生的合并症，估计每年导致750亿美元的直接医疗费用（美国），然而，很少有有效的治疗方法。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。对小鼠单基因肥胖突变体和候选基因方法的研究已经鉴定出几十个在能量稳态方面起重要作用的基因。考虑到这个过程的复杂性，可能会有数百个。最近发现的脂肪激素瘦素（1994年）和假定的饥饿因子胃饥饿素（1999年）强调了该领域发现过程的早期阶段。脊椎动物系统中与肥胖相关的突变的全基因组前向遗传筛查将是一种非常有价值的方法，因为可以以一种无偏倚的方式识别涉及能量稳态的整个基因集合，从而可以识别现有途径中先前未知的步骤以及全新的途径。硬骨鱼体内脂肪的某些成分在功能上似乎是保守的，因为斑马鱼同源的硬骨鱼相关蛋白（AgRP）是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摄食，在禁食时上调，并在鱼体内过度表达时导致肥胖。因此，有可能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稳态有关。为了使用斑马鱼识别和表征这些基因，本基金提出的工作将集中在两个基本组成部分：1)通过筛选霍普金斯收集的斑马鱼早期发育突变，确定了一系列影响下丘脑发育的独特突变，以改变斑马鱼POMC和AgRP基因的表达。2)完成了斑马鱼随机逆转录病毒插入突变的大规模前向遗传筛选，以改变下丘脑POMC和AgRP基因的表达。在这两种筛选中发现的基因可能包括参与下丘脑发育、POMC和AgRP表达的新基因，甚至可能包括参与代谢状态调节这些蛋白质的基因。脂肪体的某些元素，在哺乳动物中具有特征，在硬骨鱼（如实验室斑马鱼）中似乎在功能上是保守的，因此，有可能利用斑马鱼筛选影响能量稳态控制的突变，通过筛选系统中保守元素的缺陷，如中央黑素皮质素系统。在这个应用中，我们建议筛选斑马鱼随机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。在这两种筛选中发现的基因可能包括参与下丘脑发育、POMC和AgRP表达的新基因，甚至可能包括参与代谢状态调节这些蛋白质的基因。

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.