Genetic and Proteomic Studies of Lipid Metabolism in Zebrafish

描述（由申请人提供）： 作为一名内分泌学家，我每天都看到肥胖、高脂血症和糖尿病对患者的破坏性影响。这三种疾病正在美国达到大流行状态，需要新的治疗候选物来对抗这些对公共健康的严重风险。为了确定这三种相关疾病中的易受攻击的目标，我建议使用斑马鱼在脂质吸收，运输和代谢的正向遗传和无偏见的蛋白质组学研究。在本项目的第一部分，我将开发一种简单可靠的方法，染色和跟踪透明斑马鱼胚胎中蛋黄衍生和膳食脂质。使用这种方法，我将通过检查进化中心和生理学关键脂蛋白颗粒包装酶微粒体甘油三酯转移蛋白（Mtp）的靶向敲除效果，确认模型生物体用于研究脂质代谢的适用性。应开发生物化学、显微镜和生理学测定，以比较降低斑马鱼Mtp对发育和膳食脂质吸收的影响与无β脂蛋白血症（Mtp突变的人类遗传性疾病）的影响。在该项目的第二部分，将进行无偏的正向遗传筛选，以鉴定在脂质摄取、运输和储存方面发生改变的突变体。遗传筛查应依赖于项目第一部分开发的方法，以识别和充分表征脂质代谢缺陷。模拟人类疾病状态的突变体[原文如此]的特征是膳食脂质吸收不良、脂质消耗动力学改变和脂质在器官中的不适当沉积（即，肝脂肪变性和肌细胞内脂质体），并且产生这些突变表型的基因应使用标准定位克隆方法来鉴定。研究的最后一个分支将检查高脂肪喂养后蛋白质表达的总体变化。我将使用蛋白质组学的方法来寻找蛋白质的表达被改变的高脂肪喂养，并采用质谱方法来确定差异调节的蛋白质。与正向遗传筛选中鉴定的基因一样，应研究这些蛋白质组学研究中鉴定的蛋白质，以开发治疗肥胖症、高脂血症和糖尿病的新策略。这些研究非常适合我作为一名学术物理学家和科学家的职业发展，它们将有助于应对迫在眉睫的公共卫生危机。

DESCRIPTION (provided by applicant): As an Endocrinologist, I see daily the devastating impacts obesity, hyperlipidemia, and diabetes mellitus have on patients. These three diseases are reaching pandemic status in the United States, and novel therapeutic candidates are needed to comabt [sic] these grave risks to the public health. In order to identify pharmacologically amenable targets in these three related diseases, I propose to use zebrafish in forward genetic and unbiased proteomic studies of lipid absorption, transport, and metabolism. In the first part of this project, I shall develop a simple and reliable method for staining and tracking yolk-derived and dietary lipids in the transparent zebrafish embryo. With this method, I shall confirm the suitability of the model organism for studying lipid metabolism by examining the effect of targeted knock-down of the evolutionary central and physiologically critical lipoprotein particle-packaging enzyme microsomal triglcyeride [sic] transfer protein (Mtp). Biochemical, microscopic, and physiologic assays shall be developed in order to compare the effects of decreasing zebrafish Mtp on development and dietary lipid absorption with those of abetalipoproteinemia, the human hereditary disease in which Mtp is mutated. In the second part of the project, an unbiased, forward genetic screen shall be performed to identify mutants that have alterations in lipid uptake, transport, and storage. The genetic screen shall rely on the methods developed in the first part of the project to identify and fully characterize the defects in lipid metabolism. Mutants that model human diseas [sic] states characterized by malabsorption of dietary lipid, altered kinetics of lipid consumption, and inappropriate deposition of lipids in organs (i.e., hepatic steatosis and intramyocellular liposis) shall be identified, and the genes giving rise to these mutant phenotypes shall be identified using standard positional cloning methods. The final arm of the study shall examine global changes in expression of proteins following high fat feeding. I shall use proteomic methods to find proteins whose expression is altered by high fat feeding, and employ mass spectrometric methods to identify the differentially regulated proteins. As with the genes identified in the forward genetic screen, the proteins identified in these proteomic studies shall be studied in order to develop novel strategies to treat obesity, hyperlipidemia and diabetes mellitus. These studies are well suited to the development of my career as an academic physician-scientist and they shall help combat the looming public health crisis.