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，以利用斑马鱼胚胎的光学清晰度，这一特征使活体动物的脂质摄取可视化。本研究的主要目的是：（1）研究斑马鱼幼鱼肠道脂质吸收的可视化过程。我将开发高脂肪饲料喂养斑马鱼幼鱼和成像肠道脂质吸收的方法。实验将确定LA的形成和利用率在活肠上皮细胞使用特定的亲脂性荧光染料加上共聚焦显微镜。 (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入的大量混合脂质。通过检查LA随时间的形成，确定LA是否由位于顶端肠上皮细胞隔室附近的ER形成。创建荧光融合蛋白，以确定ER是否以及何时与喂食后产生的LD共定位。 (3)确定LA是否利用尼曼-匹克C1样1（Npc 1 l1）/依折麦布途径。依折麦布是目前用于治疗人类高胆固醇血症的药物，并假设靶向肠道NPC 1 I1。然而，对依折麦布治疗期间发生的NPC 1 L1的时间、位置和脂质定位/转运的亚细胞变化的表征较差。人NPC 1 L1-YFP转基因将用于检验喂养改变NPC 1 L1定位的假设。该目的将阐明依折麦布的作用机制和NPCIH在脂质吸收中的作用。肥胖在美国已经达到流行病的水平，到2015年将影响41%的美国人。公共卫生相关性：肥胖相关疾病，如冠心病和2型糖尿病是美国的主要杀手，了解脊椎动物肠道吸收脂质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.