Genetic Analysis of Feeding Behavior and Fat Deposition

摄食行为和脂肪沉积的遗传分析

• 批准号: 7037357
• 负责人: ALAN S KOPIN
• 金额: $ 36.68万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7037357
• 关键词: Drosophilidae; RNA interference; behavior test; bioinformatics; colorimetry; eating; eating disorders; enzyme activity; fat body; genetic screening; genetic strain; lipid metabolism; metamorphosis; neural transmission; neurogenetics; neuropharmacology; neuroregulation; nucleic acid quantitation /detection; phenotype; receptor binding; receptor expression; scanning electron microscopy; serotonin; serotonin inhibitor; serotonin receptor; site directed mutagenesis

DESCRIPTION (provided by applicant): Important mechanisms underlying the control of feeding and fat deposition have been conserved between Drosophila melanogaster and human. This validates the fruit fly as a genetic model system for identifying and characterizing novel genes that mediate metabolic processes. In Preliminary Studies, we have demonstrated that serotonin (5-hydroxytryptamine, 5HT) acts as a potent inhibitor of fly feeding behavior through activation of one or more 5HT receptors. These findings provide a direct parallel with mammals, for which it has been established that the 5HT2C receptor is an important mediator of feeding behavior and a well recognized target for the treatment of obesity. Aim 1 of this application proposes to further explore the molecular mechanisms underlying the serotonergic control of feeding in Drosophila, utilizing a combination of existing mutants and RNA interference flies which are currently being generated in our laboratory. These strains will provide tools to investigate the extent to which different 5HT receptor subtypes as well as their tissue specific expression (e.g. CMS vs. intestine) underlie the serotonergic control of feeding. The highly sensitive food intake assay that we have established for assessing 5HT mediated food intake has also been utilized to begin a forward genetic screen (Aim 2). To date, 1315 strains of mutant Drosophila, each carrying a single precisely mapped insertion, have been screened for abnormalities in feeding. Twelve of the most promising candidates have been selected for detailed analysis (Aim 2A). To explore the role of the , candidate gene (and its corresponding mammalian homolog) in modulating feeding and/or fat deposition, a combination of approaches will be used, including biochemical (e.g. fatty acid profiling), histologic (e.g. scanning electron microscopy of fat tissue), and genetic (e.g. transgenic rescue) methodologies. Based on our initial success in identifying candidate genes, we propose to continue the forward genetic screen (Aim 2B). The goal of this subsequent effort is to define modifiers of (i) food intake under basal conditions (ii) food intake in the presence of 5HT (to identify modifiers of feeding within the 5HT and intersecting pathways) and (iii) fat deposition. To enhance the likelihood of finding a phenotype and of identifying a gene with physiologic relevance in humans, we will utilize bioinformatic tools to pre-select insertion strains for screening. The selection criteria will include (i) disruption of the transcription unit, and (ii) existence of a mammalian homolog corresponding to the targeted fly gene. To date, -2,500 insertion bearing strains which meet these criteria have been identified. Each of these strains will be assessed for alterations in feeding behavior and fat deposition. Outliers will be characterized as described above. We anticipate that the proposed studies will identify novel genes relevant to the pathophysiology of human obesity

描述（由申请人提供）： 控制摄食和脂肪沉积的重要机制在果蝇和人类之间是保守的。这验证了果蝇作为识别和表征介导代谢过程的新基因的遗传模型系统。在初步研究中，我们已经证明，血清素（5-羟色胺，5HT）通过激活一种或多种 5HT 受体，成为果蝇摄食行为的有效抑制剂。这些发现与哺乳动物提供了直接的相似之处，已经确定 5HT2C 受体是进食行为的重要介质，也是公认的肥胖治疗靶点。本申请的目标 1 提议利用现有突变体和我们实验室目前正在产生的 RNA 干扰果蝇的组合，进一步探索果蝇摄食的血清素控制的分子机制。这些菌株将提供工具来研究不同 5HT 受体亚型及其组织特异性表达（例如 CMS 与肠道）在进食过程中血清素控制的程度。我们为评估 5HT 介导的食物摄入而建立的高度敏感的食物摄入测定也已用于开始正向遗传筛选（目标 2）。迄今为止，已经对 1315 株突变果蝇进行了喂养异常筛查，每株都携带一个精确定位的插入片段。已选择 12 个最有希望的候选者进行详细分析（目标 2A）。为了探索候选基因（及其相应的哺乳动物同源物）在调节进食和/或脂肪沉积中的作用，将使用多种方法的组合，包括生化（例如脂肪酸分析）、组织学（例如脂肪的扫描电子显微镜）组织）和遗传（例如转基因救援）方法。基于我们在识别候选基因方面的初步成功，我们建议继续进行正向遗传筛选（目标 2B）。随后努力的目标是定义 (i) 基础条件下的食物摄入量 (ii) 5HT 存在下的食物摄入量的调节因素（以确定 5HT 和交叉途径内喂养的调节因素）和 (iii) 脂肪沉积。为了提高发现表型和鉴定与人类生理相关的基因的可能性，我们将利用生物信息学工具预先选择插入菌株进行筛选。选择标准将包括（i）转录单位的破坏，以及（ii）与目标果蝇基因相对应的哺乳动物同源物的存在。迄今为止，已确定-2,500 个符合这些标准的插入轴承应变。将评估每种菌株的摄食行为和脂肪沉积的变化。异常值的特征将如上所述。我们预计拟议的研究将鉴定与人类肥胖病理生理学相关的新基因