PLIN2, VLDL Biogenesis and Lipid Homeostasis

描述（由申请人提供）：脂肪分化相关蛋白（ADFP）、VLDL生物发生和三酰甘油稳态非酒精性脂肪肝（NAFLD）通常是慢性肝病的前兆，也是心血管疾病的预测因子。NAFLD的标志是肝细胞中存在大量脂滴（LD）。脂肪分化相关蛋白（ADFP）是与这些LD表面结合的最丰富的胞浆蛋白。其表达在脂质浸润的肝细胞中上调（在NAFLD中）。我产生了ADFP缺陷小鼠，发现这些小鼠的肝脏三酰甘油（TG）比WT小鼠减少了60%。TG降低是明显的胞质溶胶，但矛盾的是，TG增加的微粒体部分ADFP缺陷相比，野生型小鼠。同时，在蛋白质水平上，微粒体甘油三酯转移蛋白（MTP）的量增加，但在mRNA水平上，ADFP缺陷小鼠的VLDL分泌增加。肝脏脂质的摄取、产生和利用没有改变。在本申请中，我将追求三个具体目标：（1）确定ADFP缺乏相关的肝MTP蛋白上调的分子机制，（2）确定MTP和ADFP在调节肝TG稳态中的相互作用。（3）研究心脏Adfp缺乏症。我将使用肝细胞系、心肌细胞、原代肝细胞、转化的Adfp缺陷肝细胞、ADFP缺陷和MTP条件性敲除小鼠作为模型。我还将产生条件性Adfp靶向小鼠。我将应用药理学和分子工具的组合，如MTP抑制剂，siRNA和重组辅助依赖性腺病毒来抑制或增强这些模型细胞和动物中的基因表达。我想要实现的最终目标是（i）确定ADFP缺乏时MTP蛋白上调的机制，（ii）找到ADFP缺乏相关的肝脏TG降低和心脏TG升高的机制，（iii）检验ADFP，它参与LD的形成和调节，可能是一种与MTP竞争TG并负调节VLDL分泌的胞浆蛋白，当其过表达时，研究肝脂肪变性，和（iv）鉴定在Adfp缺乏中影响肝脏和心脏TG储存的因素。我的长期目标是了解ADFP和其他LD相关蛋白在LD形成和成熟中的功能，以及这如何影响肝脏和心脏TG动力学的调节，如脂蛋白组装和分泌，以及肝脏、心脏和其他器官中的脂质稳态。

DESCRIPTION (provided by applicant): Adipose Differentiation Related Protein (ADFP), VLDL Biogenesis and Triacylglycerol Homeostasis Nonalcoholic fatty liver disease (NAFLD) is often a precursor to chronic liver diseases, and a predictor of cardiovascular disease. The hallmark of NAFLD is the presence of numerous lipid droplets (LDs) in the hepatocytes. Adipose differentiation related protein (ADFP) is the most abundant cytosolic protein that binds to the surface of these LDs. Its expression is upregulated in lipid infiltrated hepatocytes (in NAFLD). I generated ADFP deficient mice and found a 60% reduction in hepatic triacylglycerol (TG) in these mice compared to WT mice. The TG reduction is pronounced in the cytosol, but, paradoxically, TG is increased in the microsome fraction in ADFP deficient as compared with wildtype mice. Concomitantly, there is an increase in the amount of microsomal triglyceride transfer protein (MTP) at the protein level, but not mRNA level, and a increase in VLDL secretion in the ADFP deficient mice. The hepatic lipid uptake, production and utilization are not altered. In this application, I will pursue three specific aims: (1) to identify the molecular mechanism of ADFP deficiency-associated hepatic MTP protein upregulation, (2) to determine the interplay between MTP and ADFP in modulating hepatic TG homeostasis., and (3) to study Adfp deficiency in the heart. I will use hepatic cell lines, cardiomyocytes, primary hepatocytes, transformed Adfp deficient hepatic cells, ADFP deficient and MTP conditional knockout mice as models. I will also generate conditional Adfp targeting mice. I will apply a combination of pharmacological and molecular tools such as MTP inhibitor, siRNA and recombinant helper dependent adenovirus to suppress or augment gene expression in these model cells and animals. The ultimate objectives I want to achieve are (i) to determine the mechanism of MTP protein upregulation in ADFP deficiency, (ii) to find the mechanism of ADFP deficiency-associated hepatic TG reduction and cardiac TG elevation, (iii) to test the hypothesis that ADFP, with its involvement in LD formation and regulation may represent a cytosolic protein which compete with MTP for TG and negatively regulate VLDL secretion while causing hepatic steatosis when it is overexpressed, and (iv) to identify factors that affect liver and heart TG storage in Adfp deficiency. My long term goal is to understand the function of ADFP and other LD- associated proteins in the formation and maturation of LD and how this affects the regulation of hepatic and cardiac TG dynamics such as lipoprotein assembly and secretion, and lipid homeostasis in liver heart and other organs.