Defining mechanisms for lipid transport across capillary endothelial cells

 描述（由申请人提供）：项目名称：“确定脂质跨毛细血管内皮细胞转运的机制”我们的目标是了解脂质如何从富含甘油三酯的脂蛋白（TRL）跨毛细血管内皮细胞向实质细胞移动。我们对这个主题的兴趣-血浆甘油三酯代谢中最不了解的领域-源于我们对TRL血管内处理的分子机制的理解。在过去的几年中，我们发现GPIHBP 1，一个GPI锚定的蛋白质的毛细血管内皮细胞，是唯一负责穿梭脂蛋白脂肪酶（LPL）从间隙空间到其在毛细血管腔中的作用位点。最近，我们发现LPL-GPIHBP 1复合物对于TRL沿着毛细血管的边缘化是至关重要的（因此LPL介导的TRL加工可以进行）。脂质营养物穿过毛细血管到实质细胞的运动对于将燃料输送到重要器官和脂质储存在脂肪组织中至关重要。不幸的是，对这一过程的了解很少。没有人理解：（1）脂肪分解的脂肪酸产物是否简单地扩散穿过内皮细胞;（2）脂质是否在穿梭GPIHBP 1和LPL的相同囊泡中穿过内皮细胞;（3）完整的TRL是否穿过内皮细胞移动到内皮下空间;（4）脂质穿过毛细血管运输是否需要内皮细胞上的CD 36结合脂肪酸。此外，没有人了解脂质如何在其他脊椎动物的毛细血管中移动（例如，鸟、鱼）。在这些生物体中，GPIHBP 1不存在，并且LPL似乎大部分（如果不是唯一的话）位于血管外空间（即，不 与毛细血管相关）。在其他脊椎动物中，我们怀疑TRL可能通过毛细血管运输到内皮下空间--LPL所在的地方。对其他脊椎动物中TRL代谢的进一步了解可能会深入了解哺乳动物中TRL加工的辅助机制。了解脂质跨毛细血管转运进展缓慢的主要原因之一是没有办法可视化脂质转运。“观察”脂质如何在毛细血管中移动对于破译分子机制和设计可验证的假设至关重要。幸运的是，我们已经克服了“成像路障”。“在过去的两年中，我们使用NanoSIMS和背散射电子（BSE）成像来创建TRL的高分辨率图像，因为它们沿着沿着毛细血管边缘和TRL脂质穿过内皮细胞移动到实质细胞。这些研究表明，一些TRL脂质在囊泡中穿过内皮细胞，但需要额外的高分辨率成像研究来确定是否涉及脂肪酸沿着质膜扩散或脂质穿过胞质溶胶的运输。相同的方法可用于确定特定蛋白质的作用（例如，CD 36）在脂质跨内皮细胞转运中的作用。在接下来的五年里，我们将使用NanoSIMS和BSE成像来定义脂质通过毛细血管运输的细胞和分子机制。我们还将确定在体内的功能相关的CD 36的TRL衍生的脂质跨毛细血管内皮细胞的运输。

 DESCRIPTION (provided by applicant): Title of Project: "Defining mechanisms for lipid transport across capillary endothelial cells" Our objective is to understand how lipids from triglyceride-rich lipoproteins (TRLs) move across capillary endothelial cells towards parenchymal cells. Our interest in this topic-the least understood area within plasma triglyceride metabolism-arose from our efforts to understand molecular mechanisms for the intravascular processing of TRLs. During the past few years, we showed that GPIHBP1, a GPI-anchored protein of capillary endothelial cells, is solely responsible for shuttling lipoprotein lipase (LPL) from the interstitial spaces to its site of action in the capillary lumen. More recenty, we showed that the LPL-GPIHBP1 complex is critical for the margination of TRLs along capillaries (so that the LPL-mediated processing of TRLs can proceed). The movement of lipid nutrients across capillaries to parenchymal cells is crucial for delivering fuel to vital organs an lipids for storage in adipose tissue. Unfortunately, there are few insights into this process. No one understands: (1) whether the fatty acid products of lipolysis simply diffuse across endothelial cells; (2) whether lipids move across endothelial in the very same vesicles that shuttle GPIHBP1 and LPL; (3) whether intact TRLs move across endothelial cells to the subendothelial spaces; and (4) whether binding of fatty acids by CD36 on endothelial cells is required for lipid transport across capillaries. Also, no one understands how lipids move across capillaries in other vertebrates (e.g., birds, fish). In those organisms, GPIHBP1 is absent and LPL appears to be located largely, if not exclusively, in the extravascular spaces (i.e., it is not associated with capillaries). In other vertebrates, we suspect that the TRLs might be transported across capillaries to the subendothelial spaces-to where the LPL is located. An improved understanding of TRL metabolism in other vertebrates will likely yield insights into accessory mechanisms for TRL processing in mammals. One of the main reasons for the slow progress in understanding lipid transport across capillaries is that there has been no way to visualize lipid transport. "Seeing" how lipids move across capillaries is crucial for deciphering molecular mechanisms and designing testable hypotheses. Fortunately, we have overcome the "imaging roadblock." During the past two years, we have used NanoSIMS and backscattered electron (BSE) imaging to create high-resolution images of TRLs as they marginate along capillaries and as the TRL lipids move across endothelial cells to parenchymal cells. These studies have demonstrated that some TRL lipids move across endothelial cells in vesicles, but additional high-resolution imaging studies are required to determine if diffusion of fatty acids along plasma membranes-or transport of lipids across the cytosol-is involved. The same methods can be used to define the role of specific proteins (e.g., CD36) in lipid transport across endothelial cels. For the next five years, we will use NanoSIMS and BSE imaging to define the cellular and molecular mechanisms for lipid transport across capillaries. We will also define the in vivo functional relevance of CD36 for the transport of TRL-derived lipids across capillary endothelial cells.