Genetic Analysis of Feeding Behavior and Fat Deposition

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。这证实了果蝇是一种遗传模型系统，用于识别和表征调节新陈代谢过程的新基因。在初步研究中，我们已经证明5-羟色胺(5-羟色胺，5-羟色胺)通过激活一个或多个5-羟色胺受体而有效地抑制苍蝇的取食行为。这些发现提供了与哺乳动物的直接相似之处，对哺乳动物来说，5HT2C受体是摄食行为的重要媒介，也是公认的肥胖症治疗靶点。本申请的目的1建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5-羟色胺能控制的分子机制。这些菌株将提供工具，以研究不同的5-羟色胺受体亚型及其组织特异性表达(例如，CMS与肠道)在多大程度上影响摄食的5-羟色胺能控制。我们建立的用于评估5-羟色胺介导的食物摄入量的高度敏感的食物摄入量试验也被用于开始正向遗传筛查(目标2)。到目前为止，已经对1315个突变果蝇品系进行了摄食异常筛查，每个品系都带有一个精确定位的插入片段。挑选了12名最有希望的候选人进行详细分析(目标2 A)。为了探索候选基因(及其相应的哺乳动物同源基因)在调节摄食和/或脂肪沉积方面的作用，将结合使用多种方法，包括生化方法(例如脂肪酸图谱)、组织学方法(例如脂肪组织的扫描电子显微镜)和遗传方法(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查(目标2B)。这项后续工作的目标是定义(I)基础条件下的食物摄入量(Ii)在5HT存在的情况下的食物摄入量(以确定5HT和交叉通路内的摄食调节因子)和(Iii)脂肪沉积的调节因子。为了提高在人类中发现表型和识别与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)转录单位的中断，以及(Ii)是否存在与目标苍蝇基因相对应的哺乳动物同源基因。到目前为止，已鉴定出-2,500个符合这些标准的插入轴承菌株。将评估这些菌株中的每一种在取食行为和脂肪沉积方面的变化。异常值的特征将如上所述。我们预计，拟议的研究将确定与人类肥胖的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