Genetic Analysis of Feeding Behavior and Fat Deposition

描述（由申请人提供）： 果蝇和人类的摄食和脂肪沉积的重要控制机制是保守的。这验证了果蝇作为遗传模型系统，用于识别和表征介导代谢过程的新基因。在初步研究中，我们已经证明血清素（5-羟色胺，5 HT）通过激活一种或多种5 HT受体作为苍蝇摄食行为的有效抑制剂。这些发现提供了与哺乳动物的直接平行，对于哺乳动物，已经确定5 HT 2C受体是摄食行为的重要介体和公认的治疗肥胖的靶点。本申请的目的1提出利用我们实验室目前正在产生的现有突变体和RNA干扰果蝇的组合，进一步探索果蝇摄食的β-胡萝卜素能控制的分子机制。这些菌株将提供研究不同5 HT受体亚型及其组织特异性表达（例如CMS与肠）在多大程度上构成摄食的多巴胺能控制的基础的工具。我们已经建立的用于评估5 HT介导的食物摄入的高灵敏度食物摄入测定也已用于开始正向遗传筛选（目的2）。到目前为止，1315株突变果蝇，每一个携带一个精确定位的插入，已经筛选出异常的喂养。已选出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，包括生物化学（例如脂肪酸分析）、组织学（例如脂肪组织的扫描电子显微镜）和遗传学（例如转基因拯救）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（Aim 2B）。该后续努力的目标是定义（i）基础条件下的食物摄入量（ii）存在5 HT时的食物摄入量（以确定5 HT和交叉途径内的摄食调节剂）和（iii）脂肪沉积的调节剂。为了提高在人类中发现表型和鉴定具有生理相关性的基因的可能性，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，和（ii）存在对应于靶向果蝇基因的哺乳动物同源物。到目前为止，已经确定了约2，500个符合这些标准的插入轴承菌株。将评估这些品系中的每一种的摄食行为和脂肪沉积的改变。离群值将如上所述进行表征。我们预期这些研究将发现与人类肥胖病理生理学相关的新基因

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