Subcellular modeling of lipid trafficking proteins in living Zebrafish intestine

活体斑马鱼肠道脂质运输蛋白的亚细胞模型

• 批准号: 8080409
• 负责人: James William Walters
• 金额: $ 5.13万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8080409
• 关键词: Address; Adipocytes; Affect; American; Animals; Apical; BODIPY; Bile fluid; Biochemical; Biological Assay; Biological Models; Blood; Brush Border; Caveolae; Cell Culture Techniques; Cells; Chimeric Proteins; Chylomicrons; Confocal Microscopy; Coronary heart disease; Coupled; Cultured Cells; Data; Development; Diet; Dietary Cholesterol; Dietary Fats; Diffusion; Dyes; Electron Microscopy; Embryo; Endoplasmic Reticulum; Enterocytes; Epidemic; Fatty Acids; Fatty acid glycerol esters; Feeding Methods; Fluorescent Dyes; Goals; Golgi Apparatus; Human; Image; Imagery; In Vitro; Intestines; Intracellular Accumulation of Lipids; Label; Larva; Life; Lipids; Location; Lymphatic System; Mediating; Membrane; Metabolism; Modeling; Nature; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Obesity associated disease; Optics; Organelles; Organism; Pathway interactions; Pharmaceutical Preparations; Physiology; Process; Proteins; Reporter Genes; Role; Route; Sorting - Cell Movement; Specimen; Subcellular structure; System; Testing; Time; Transgenes; Vertebrates; Villus; Work; Zebrafish; absorption; base; cell type; cholesterol absorption; ezetimibe; feeding; hypercholesterolemia; in vivo; in vivo Model; insight; lipid metabolism; lipid transport; new therapeutic target; public health relevance; receptor; research study; small molecule; tissue fixing; tool; trafficking; uptake

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.

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，这一功能可以实现活体动物脂质吸收的可视化。该提案的目的是：（1）可视化活体斑马鱼幼虫的肠道脂质吸收。我将开发用高脂肪饮食喂养斑马鱼幼虫并对肠道脂质吸收进行成像的方法。所提出的实验将使用特定的亲脂性荧光染料结合共聚焦显微镜来确定活肠细胞中 LA 的形成和利用率。 (2)确定参与肠上皮细胞脂质吸收的亚细胞细胞器/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 随着时间的推移形成，确定 LA 是否由位于顶端肠细胞室附近的 ER 形成 创建荧光融合蛋白，以确定 ER 是否以及何时与喂养后产生的 LD 共定位。 (3) 确定 LA 是否利用 Niemann-Pick C1 Like 1 (Npc1l1)/ezetimibe 途径。依泽替米贝是一种目前用于治疗人类高胆固醇血症的药物，推测其靶向肠道 NPC1I1。然而，NPC1L1 的时间、位置以及依折麦布治疗期间发生的脂质定位/运输的亚细胞变化尚不清楚。人类 NPC1L1-YFP 转基因将用于测试喂养改变 NPC1L1 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。在美国，肥胖症已达到流行病水平，到 2015 年将困扰 41% 的美国人。 公共卫生相关性：冠心病和 2 型糖尿病等与肥胖相关的疾病是美国的主要杀手。了解脊椎动物肠道如何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与肥胖相关的疾病及其影响 治疗。