Conserved Mechanisms of Lipid Homeostasis in C. elegans

线虫脂质稳态的保守机制

基本信息

批准号：
8053802
负责人：
Eyleen Jorgelina O'Rourke
金额：
$ 9万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8053802
关键词：
Animal Model Animals Area Award Biochemical Biochemical Genetics Biological Biological Assay Caenorhabditis elegans Cause of Death Cell Culture Techniques Cloning Collaborations Communities Complex Computer software Consumption Data Development Disease Ensure Environment Equilibrium Fasting Fatty acid glycerol esters Gene Silencing Gene Targeting Genes Genetic Health Heart Diseases Homeostasis Human Image Image Analysis Institutes Insulin Signaling Pathway Knowledge Laboratories Lead Learning Life Lipids Lipolysis Malignant Neoplasms Measures Mediating Mentors Mentorship Metabolism Methodology Methods Mining Modeling Molecular Mus Nematoda Non-Insulin-Dependent Diabetes Mellitus Obesity Operative Surgical Procedures Pathway interactions Pharmaceutical Preparations Phase Physiological Preclinical Drug Evaluation Prevention Process Publications RNA Interference Reading Reagent Regulation Research Research Personnel Role Science Screening procedure Severities Small RNA Software Tools Staining method Stains Syndrome Technology Therapeutic Therapeutic Agents Time Training Transcription Repressor/Corepressor Transcriptional Activation Work bariatric surgery base career computerized data processing design drug discovery drug testing energy balance experience feeding functional genomics gene discovery genetic analysis high throughput screening human disease nile red novel obesity treatment oil red O prevent public health relevance response technology development tool

项目摘要

DESCRIPTION (provided by applicant): Obesity and its associated diseases, type 2 diabetes, heart disease, and cancer have become a leading cause of death worldwide. Unfortunately, only risky surgical procedures constitute an effective long-term treatment. Better understanding of the molecular mechanisms underlying obesity is necessary for the development of urgently needed therapeutics. Important genetic regulators of human metabolism, including key components of the insulin signaling pathway, were first discovered in the roundworm Caenorhabditis elegans. Despite the great potential of C. elegans for gene and drug discovery through automated high-throughput (HT) screens, the required methodology has not yet been developed. To fill this void, I have been developing technology that will allow automated HT C. elegans-based RNAi screens (O'Rourke et al, 2009). At the same time, in the course of studying the C. elegans response to fasting, I uncovered that the most commonly used assay to assess fat accumulation in C. elegans, Nile Red staining, only stains a small subset of fats contained in Iysosomes. I therefore developed an alternative methodology that reads out C. elegans major fat stores, Oil-Red-O staining. Using Oil-Red-O staining in combination with the HT methodology, I am proposing to perform an automated RNAi screen for genes that regulate fat accumulation in C. elegans. As previous screens used Nile Red as their readout, the proposed screen will constitute the first screen for genes that regulate major fat stores in a whole-living animal. In parallel, I discovered that there is transcriptional activation of lipolysis (fat breakdown) that occurs when either C. elegans and [sic] mice are fasted, leading, in the case of C. elegans, to a 30% decrease in fat stores (O'Rourke et al, submitted to Science). In worms, this transcriptional activation of lipolysis is mediated by the inactivation of the transcriptional repressor mx/-3. In turn, preliminary data suggest that mx/-3 inactivation is mediated by small RNAs. The role of small RNAs in the control of energy balance is largely unexplored. In order to explore the role of small RNAs in energy homeostasis I propose to sequence all small RNAs from fasted and well-fed worms. Finishing the optimization of the HT screening methodology will allow me to acquire knowledge in automated image analysis and large-scale data processing. The screen for genes that regulate fat accumulation will provide me with experience in functional genomics. By mining and characterizing small RNAs, I will familiarize with cutting-edge deep sequencing technologies. The knowledge, reagents, publications, and collaborations gained and generated during this training period will greatly facilitate my transition to a successful independent career in the field of metabolism. The completion of the development of the proposed HT screening methodology not only constitutes an enabling technology for the whole C. elegans community, but it will initially provide me with a unique advantage as an independent researcher. The pilot gene search performed during the mentored phase will constitute a proof-of-principle to be exploited during the independent phase of the award when large-scale screens for genes and pilot screens for drugs that alter fat accumulation will be performed. Also during the independent phase, I will perform an in depth genetic and biochemical analysis of the genes and small RNA regulators of metabolism uncovered during the mentored phase. I anticipate that my research will uncover molecules and biological pathways that regulate energy balance. This will accelerate the design and testing of drugs to prevent and treat the major human health problem of obesity. PUBLIC HEALTH RELEVANCE: Obesity has become a leading cause of death worldwide. Genetic components, as well as the environment contribute to the severity of the syndrome. Caenorhabditis elegans is a simple model organism, a roundworm, sharing 50% of their genes with humans. C. elegans has been successfully used to uncover genes and drugs relevant to human disease in the past. I am going to work on developing an enabling technology that will accelerate the discovery of genes that could predispose and compounds that could relief human disease. I will particularly focus on finding genes that control fat accumulation and compounds that promote lipolysis (the process of breaking down fats). Learning more about the molecular mechanisms that lead to the activation of lipolysis could allow us to manipulate the balance between fat storage and consumption. Drugs that activate the breakdown of lipids could be used as therapeutic agents for the treatment of obesity.

描述（由申请人提供）：肥胖及其相关疾病、2 型糖尿病、心脏病和癌症已成为全世界死亡的主要原因。不幸的是，只有有风险的外科手术才能构成有效的长期治疗。更好地了解肥胖背后的分子机制对于开发急需的治疗方法至关重要。人类新陈代谢的重要遗传调节因子，包括胰岛素信号通路的关键成分，首先是在秀丽隐杆线虫中发现的。尽管线虫通过自动化高通量（HT）筛选在基因和药物发现方面具有巨大潜力，但所需的方法尚未开发出来。为了填补这一空白，我一直在开发能够实现基于 HT 线虫的自动化 RNAi 筛选的技术（O'Rourke 等，2009）。与此同时，在研究线虫对禁食反应的过程中，我发现评估线虫脂肪积累最常用的检测方法尼罗红染色只能对溶酶体中所含脂肪的一小部分进行染色。因此，我开发了一种替代方法，即油红 O 染色，可以读取秀丽隐杆线虫的主要脂肪储备。我建议使用油红 O 染色与 HT 方法相结合，对秀丽隐杆线虫中调节脂肪积累的基因进行自动 RNAi 筛选。由于之前的筛选使用尼罗红作为读数，因此拟议的筛选将构成对调节整体动物主要脂肪储存的基因的首次筛选。与此同时，我发现当线虫和[原文如此]小鼠禁食时，会发生脂肪分解（脂肪分解）的转录激活，导致线虫的脂肪储存减少 30%（ O'Rourke 等人，提交给《科学》杂志）。在线虫中，这种脂肪分解的转录激活是由转录抑制因子 mx/-3 的失活介导的。反过来，初步数据表明 mx/-3 失活是由小 RNA 介导的。小RNA在控制能量平衡中的作用很大程度上尚未被探索。为了探索小 RNA 在能量稳态中的作用，我建议对来自禁食和喂养良好的线虫的所有小 RNA 进行测序。完成 HT 筛选方法的优化将使我能够获得自动图像分析和大规模数据处理方面的知识。调节脂肪积累的基因的筛选将为我提供功能基因组学的经验。通过挖掘和表征小RNA，我将熟悉尖端的深度测序技术。在培训期间获得和产生的知识、试剂、出版物和合作将极大地促进我向新陈代谢领域成功的独立职业生涯过渡。所提出的 HT 筛选方法的开发完成不仅为整个线虫社区提供了一项支持技术，而且最初将为我作为独立研究人员提供独特的优势。在指导阶段进行的试点基因搜索将构成在奖励的独立阶段进行的原理验证，届时将进行大规模的基因筛选和改变脂肪积累的药物的试点筛选。同样在独立阶段，我将对指导阶段发现的基因和代谢小 RNA 调节因子进行深入的遗传和生化分析。我预计我的研究将揭示调节能量平衡的分子和生物途径。这将加速药物的设计和测试，以预防和治疗肥胖这一人类主要健康问题。公共健康相关性：肥胖已成为全球死亡的主要原因。遗传成分以及环境都会导致该综合征的严重程度。秀丽隐杆线虫是一种简单的模式生物，是一种蛔虫，与人类有 50% 的基因相同。过去，秀丽隐杆线虫已成功用于发现与人类疾病相关的基因和药物。我将致力于开发一种使能技术，以加速发现可能诱发人类疾病的基因和可以缓解人类疾病的化合物。我将特别关注寻找控制脂肪积累的基因和促进脂肪分解（分解脂肪的过程）的化合物。更多地了解导致脂肪分解激活的分子机制可以让我们控制脂肪储存和消耗之间的平衡。激活脂质分解的药物可用作治疗肥胖症的治疗剂。