Conserved Mechanisms of Lipid Homeostasis in C. elegans

线虫脂质稳态的保守机制

• 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
• 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

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球死亡的主要原因。不幸的是，只有冒险的手术程序才能有效的长期治疗。更好地理解肥胖的分子机制对于急需的治疗剂的发展是必要的。人类代谢的重要遗传调节剂，包括胰岛素信号通路的关键组成部分，首先是在the虫秀丽隐杆线虫中发现的。尽管秀丽隐杆线虫具有巨大的潜力，可以通过自动高通量（HT）筛选来发现基因和药物发现，但尚未开发所需的方法。为了填补这一空白，我一直在开发技术，该技术将允许自动化的HT C.秀丽隐杆线虫屏幕（O'Rourke等，2009）。同时，在研究秀丽隐杆线虫对禁食的反应的过程中，我发现，最常用的测定法评估了秀丽隐杆线虫中脂肪的积累，尼罗河红色染色，只染了一小部分iysososys中的脂肪。因此，我开发了一种替代方法，可以读出秀丽隐杆线虫主要的脂肪储存，即油红色O染色。我建议将油红色O染色与HT方法结合使用，我建议对调节秀丽隐杆线虫中脂肪积累的基因进行自动RNAi筛选。由于先前的屏幕使用尼罗红色作为其读数，因此提出的屏幕将构成第一个调节整个生命动物主要脂肪存储的基因的屏幕。同时，我发现，当秀丽隐杆线虫和[sic]小鼠被禁食的情况下，在秀丽隐杆线虫的情况下，脂肪分解（脂肪分解）的转录激活发生在脂肪储存的情况下（O'Rourke等人，降低了30％）。在蠕虫中，这种脂解的转录激活是通过转录阻遏物mx/-3的失活而介导的。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在很大程度上没有探索。为了探索小型RNA在能量稳态中的作用，我建议对禁食和喂养良好的蠕虫的所有小rNA进行测序。完成HT筛选方法的优化将使我能够在自动图像分析和大规模数据处理中获取知识。调节脂肪积累的基因的屏幕将为我提供功能基因组学的经验。通过采矿和表征小RNA，我将熟悉最先进的深层测序技术。在此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代谢领域成功独立职业的过渡。拟议的HT筛查方法的开发完成不仅构成了整个C.秀丽隐杆线社区的启示技术，而且最初将为我提供独特的优势。在指导阶段进行的试验基因搜索将构成奖励证明，将在奖励的独立阶段进行利用，当时将对基因和试点筛查进行大规模筛选，用于改变脂肪积累的药物。同样在独立阶段，我将对在指导阶段发现的代谢的基因和小的RNA调节剂进行深度遗传和生化分析。我预计我的研究将发现调节能量平衡的分子和生物学途径。这将加快药物的设计和测试，以预防和治疗肥胖的主要健康问题。 公共卫生相关性：肥胖已成为全球死亡的主要原因。遗传成分以及环境有助于综合征的严重程度。秀丽隐杆线虫是一种简单的模型生物体，一种round虫，与人类共享50％的基因。秀丽隐杆线虫过去已成功地用于发现与人类疾病有关的基因和药物。我将致力于开发一种有能力的技术，该技术将加速发现可能容易缓解人类疾病的基因。我将特别专注于寻找控制脂肪积累和促进脂解的化合物的基因（分解脂肪的过程）。了解有关导致脂解激活的分子机制的更多信息，可以使我们能够操纵脂肪储存和消耗之间的平衡。激活脂质分解的药物可以用作治疗肥胖症的治疗剂。