Elucidating the mechanism of transendothelial fatty acid transport

脂肪酸和由它们构成的脂质是每一种细胞形式生命的基本组成部分。外源脂肪酸通过跨质膜的运输被吸收，通常很快就会与辅酶a发生酯化反应。脂肪酸的这种“活化”对其进一步代谢是必不可少的。在我们发表的初步工作中，我们描述了几种参与细胞脂肪酸摄取的膜蛋白，特别是推定的转运蛋白（FATPs和CD36）和脂肪酰基-辅酶a连接酶。现在，我们希望利用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性问题，特别是脂肪酸运输与代谢之间的关系。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。这个提议的关键问题是：哪一种分子机制是脂肪酸在内皮细胞间的转运，而不是在内皮细胞内的代谢？脂肪酸转运的驱动力原则上有：脂肪酸转运蛋白的调节、管腔和管腔内皮质膜的浓度差异、相对于代谢的更高的转运速度以及非酯化脂肪酸对细胞胞质导向的辅酶a连接酶的保护。这些假设将通过工作程序进行检验，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干扰或过表达进行修饰。内皮细胞的永生化和原代培养将在可渗透过滤器上分化，以便能够同时测量脂肪酸的运输和代谢。从假设中得出的重点项目目标是：1。阐明FATP转运蛋白的作用机制。与内皮代谢相比，确定脂肪酸高速运输的潜在显性决定因素。研究胞质蛋白或小泡是否保护非酯化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。我们假设我们的结果也将与血脑屏障的运输以及对广泛的脂质相关代谢疾病的理解有关。

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.