Subcellular modeling of lipid trafficking proteins in living Zebrafish intestine

描述(申请人提供)：我的长期目标是了解肠道脂肪摄取的细胞机制。我的工作重点是开发工具，以利用斑马鱼胚胎的光学透明度，这一特征使活体动物的脂肪摄取可视化。这项建议的目的是：(1)可视化活体斑马鱼幼体的肠道脂肪吸收。我将研究用高脂肪饲料喂养斑马鱼幼体和成像肠道脂肪吸收的方法。建议的实验将使用特定的亲脂荧光染料与共聚焦显微镜相结合来确定活的肠细胞中LA的形成和利用速度。(2)确定参与肠细胞脂质吸收的亚细胞器/蛋白质。目前尚不清楚肠细胞使用哪些细胞器来分类吸收过程中摄取的大量混合脂质。通过研究LAS随时间的形成，确定位于顶端肠细胞室附近的内质网形成的LAS是否产生荧光融合蛋白，以确定内质网是否以及何时与喂养后产生的LDS共定位。(3)确定LAS是否利用Niemann-Pick C1like 1(NPC1L1)/ezetimibe途径。依折麦布是一种目前用于治疗人类高胆固醇血症的药物，假设靶向是肠道NPC1I1。然而，在依折麦布治疗期间发生的NPC1L1的时间、位置以及脂质定位/转运的亚细胞变化的特征很差。一个人的NPC1L1-YFP转基因将被用来检验喂养改变NPC1L1定位的假设。这一目的将阐明Ezetimibe的作用机制和NPCIH在脂质吸收中的作用。肥胖症在美国已经达到流行水平，到2015年将困扰41%的美国人。公共卫生相关性：冠心病和2型糖尿病等肥胖相关疾病是美国的头号杀手。了解脊椎动物肠道如何吸收脂肪的机制以及小分子如何调节这些过程将有助于深入了解肥胖相关疾病及其治疗。

DESCRIPTION (provided by applicant): My long-term goal is to understand the cellular mechanisms of intestinal lipid uptake. My work focuses on developing tools to exploit the optical clarity of the zebrafish embryo, a feature that enables visualization of lipid uptake in live animals. The aims of this proposal are: (1) Visualize intestinal lipid absorption in live zebrafish larvae. I will develop methods for feeding larval zebrafish a high-fat diet and imaging intestinal lipid absorption. Experiments proposed will determine the rates of LA formation and utilization in live enterocytes using specific lipophilic fluorescent dyes coupled with confocal microscopy. (2) Determine the subcelluar organelles/proteins involved in enterocyte lipid absorption. It is unknown which organelles are used by enterocytes to sort the large amounts of mixed lipids taken in during absorption. By examining the formation of LAs over time, determine if LAs form from ER that is localized near the apical enterocyte compartment Create fluorescent fusion proteins, to determine if and when ER co-localizes with LDs produced following feeding. (3) Determine if LAs utilize a Niemann-Pick C1 Like 1 (Npc1l1)/ezetimibe pathway. Ezetimibe is a drug currently used to treat human hypercholesterolemia and hypothesized to target intestinal NPC1I1. However the timing, location of NPC1L1, and subcellular changes in lipid localization/trafficking that occur during ezetimibe treatment are poorly characterized. A human NPC1L1-YFP transgene will be used to test the hypothesis that feeding alters NPC1L1 localization. This Aim will clarify ezetimibe's mechanism of action and NPCIH's role in lipid absorption. Obesity in the U.S. has reached epidemic levels and will afflict 41% of Americans by 2015. PUBLIC HEALTH RELEVANCE: Obesity-related diseases such as coronary heart disease and type-2 diabetes are leading killers in the U.S. Understanding the mechanisms involved in how the vertebrate intestine absorbs lipids and how small molecules can modulate these processes will help provide insights into obesity-associated diseases and their treatments.