Elucidating the mechanism of transendothelial fatty acid transport

脂肪酸及其所产生的脂类是每一种细胞形式生命的基本成分。外源脂肪酸通过质膜运输被吸收，这通常紧随其后的是它们与辅酶A的酯化作用。这种脂肪酸的“激活”对于它们的进一步新陈代谢是必不可少的。在我们已发表的初步工作中，我们已经确定了几种与细胞脂肪酸摄取有关的膜蛋白，特别是假定的转运蛋白(FATPs和CD36)和脂肪酰辅酶A连接酶。现在，我们想利用已开发的分子工具来解决这个仍然非常有争议的研究领域中最紧迫的开放问题，特别是脂肪酸运输和新陈代谢之间的关系。模型系统将是外周微血管内皮细胞，其特点是具有较高的脂肪酸摄取和运输能力。这一提议的总体关键问题是：相对于内皮细胞对脂肪酸的代谢，哪一种分子机制有利于脂肪酸通过内皮细胞转运？脂肪酸运输的驱动力原则上是：脂肪酸转运蛋白的调节，腔内皮细胞膜和腔内皮细胞膜之间的浓度差异，与新陈代谢相比更快的运输速度，以及非酯化脂肪酸对细胞溶质导向的辅酶A连接酶的保护。这些假设将由工作计划进行检验，并相互关联。候选蛋白的表达水平将分别通过逆转录病毒介导的RNA干扰或过表达来改变。永生化的内皮细胞和原代培养的内皮细胞将在可渗透的过滤器上进行区分，以便能够同时测量脂肪酸的运输和新陈代谢。根据这些假设得出的重点项目目标是：1.阐明FATP转运体的作用机制。确定与内皮代谢相比，脂肪酸运输速度高的潜在显性决定因素。研究胞浆蛋白或小窝是否保护未酯化的脂肪酸免受代谢酶的影响。综上所述，内皮细胞是一个独特的模型系统，非常适合回答关于细胞脂肪酸运输的基本和重要问题。我们认为，我们的结果也将与血脑屏障的运输相关，以及对广泛存在的脂质相关代谢性疾病的理解。

Fatty acids and the lipids made from them are essential components of every cellular form of life. Exogenous fatty acids are taken up by transport across the plasma membrane, and this is usually followed very soon by their esterification with coenzyme A. This "activation" of fatty acids is indispensable for their further metabolism. In our published preliminary work we have characterized several membrane proteins involved in cellular fatty acid uptake, especially putative transporter proteins (FATPs and CD36) and fatty acyl-CoA ligases. Now we would like to use the developed molecular tools thereby to address the most urgent open questions in this still very controversial field of research, in particular the relationship between fatty acid transport and metabolism. The model system is going to be peripheral microvascular endothelial cells which are distinguished by a high capacity for fatty acid uptake and transport.The overall key question of this proposal is: Which is the molecular mechanism for the favored transport of fatty acids across the endothelium as opposed to their metabolism by the endothelial cells? Driving forces for the transport of fatty acids are in principle: regulation by fatty acid transport proteins, concentration differences between luminal and abluminal endothelial plasma membrane, higher velocity of transport as compared to metabolism, and the protection of non-esterified fatty acids against the cytosolically oriented CoA ligases. These hypotheses will be examined by the work program, and correlated to each other. The expression level of the candidate proteins is going to be modified by retrovirus mediated RNA interference or overexpression respectively. Immortalized as well as primary cultures of endothelial cells will be differentiated on permeable filters to be able to measure fatty acid transport and metabolism simultaneously. The focused project aims derived from the hypotheses are:1. Elucidation of the mechanism of action of the FATP transporters.2. Identification of the underlying dominant determinants for the high velocity of fatty acid transport as compared to endothelial metabolism.3. Investigation if cytosolic proteins or caveolae protect non-esterified fatty acids from the metabolic enzymes.Summarizing, endothelial cells are a unique model system which are excellently suited to answer basic and important questions regarding cellular fatty acid transport. We assume that our results will also be relevant for the transport across the blood brain barrier as well as for the understanding of widespread lipid associated metabolic diseases.