Study of Energy Homeostasis in a Genetic Model System

描述（由申请人提供）：肥胖流行病和由此产生的合并症估计会导致直接医疗费用（美国）每年750亿美元，但有效的治疗方法很少。因此，肥胖相关基因的鉴定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的研究和候选基因方法已经鉴定出几十个在能量稳态方面发挥重要作用的基因。考虑到这一过程的复杂性，可能有数百人。最近发现的脂肪抑制激素瘦素（1994年）和假定的饥饿因子生长激素释放肽（1999年）强调了在这一领域的发现过程的早期阶段。在脊椎动物系统中进行肥胖相关突变的全基因组正向遗传筛查将是一种非常有价值的方法，因为可以以无偏的方式鉴定参与能量稳态的基因的整个集合，从而允许鉴定现有途径中先前未知的步骤以及全新的途径。adipostat的某些元素似乎在硬骨鱼中功能保守，因为下丘脑黑皮质素系统的一个组成部分，即刺鼠相关蛋白（AgRP）的斑马鱼直系同源物，在硬骨鱼脑中给药时刺激进食，通过禁食上调，并在鱼类中过度表达时导致肥胖。因此，有可能使用斑马鱼来筛选影响中枢黑皮质素系统的突变，并且这些突变中的许多可能与哺乳动物能量稳态有关。为了使用斑马鱼识别和表征这些基因，这项资助中提出的工作将集中在两个基本组成部分：1）表征影响下丘脑发育的独特突变的集合，通过筛选霍普金斯收集的斑马鱼早期发育突变体中改变斑马鱼POMC和AgRP表达的基因来鉴定，和2）完成了对斑马鱼中随机逆转录病毒插入突变的大规模正向遗传筛选，以寻找改变下丘脑POMC和AgRP基因表达的基因。在这两个屏幕中确定的基因可能包括新的基因参与下丘脑发育，POMC和AgRP的表达，甚至可能参与这些蛋白质的代谢状态的调节基因。在哺乳动物中表征的adipostat的某些元件在硬骨鱼（例如实验室斑马鱼）中出现功能保守，因此，可以通过筛选系统（例如中央黑皮质素系统）的保守元件中的缺陷，使用斑马鱼来筛选影响能量稳态控制的突变。在这个应用中，我们建议在斑马鱼中筛选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。在这两个屏幕中确定的基因可能包括新的基因参与下丘脑发育，POMC和AgRP的表达，甚至可能参与这些蛋白质的代谢状态的调节基因。

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.