Subcellular modeling of lipid trafficking proteins in living Zebrafish intestine

描述（由申请人提供）：我的长期目标是了解肠道脂质摄取的细胞机制。我的工作重点是开发工具来利用斑马鱼胚胎的光学清晰度，这一特性使活体动物的脂质摄取可视化。本提案的目的是：(1)可视化活斑马鱼幼虫肠道脂质吸收。我将开发喂幼斑马鱼高脂肪饮食和成像肠道脂质吸收的方法。提出的实验将利用特异性亲脂性荧光染料和共聚焦显微镜确定LA在活肠细胞中的形成和利用速率。(2)确定参与肠细胞脂质吸收的亚细胞器/蛋白质。目前尚不清楚哪些细胞器被肠细胞用来对吸收过程中吸收的大量混合脂质进行分类。通过检查LAs的形成随时间的推移，确定LAs是否由位于肠细胞顶层室附近的内质网形成，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ld共定位。(3)确定LAs是否利用Niemann-Pick C1 Like 1 (Npc1l1)/ezetimibe通路。依zetimibe是一种目前用于治疗人类高胆固醇血症的药物，并假设针对肠道NPC1I1。然而，在依zetimibe治疗期间发生的NPC1L1的时间、位置和脂质定位/运输的亚细胞变化尚未被充分描述。一个人类NPC1L1- yfp转基因将被用来验证摄食改变NPC1L1定位的假设。本研究旨在阐明依可替米贝的作用机制和NPCIH在脂质吸收中的作用。美国的肥胖已经达到了流行病的程度，到2015年将有41%的美国人患有肥胖症。公共健康相关性：与肥胖相关的疾病，如冠心病和2型糖尿病是美国的主要杀手。了解脊椎动物肠道如何吸收脂质以及小分子如何调节这些过程将有助于了解与肥胖相关的疾病及其治疗方法。

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.