MECHANISMS OF PLASMA MEMBRANE TRANSPORT OF FATTY ACIDS

描述（由申请人提供）：拟议研究的长期目标是确定游离脂肪酸（FFA）跨脂肪细胞膜转运的机制。循环FFA主要来源于脂肪组织。FFA的监管是至关重要的，因为FFA提供了能源需求的主要部分和水平的升高会对健康产生不利影响。即使是相对适度的升高也可能在降低II型糖尿病的胰岛素敏感性方面发挥重要作用，并显着增加猝死的风险。更大的增加发生在急性，例如在心脏缺血和中风中，并且这些升高可能通过诱导心律失常、促进肌细胞凋亡和增加严重出血事件的风险而对预后产生不利影响。这些循环FFA的增加是由于脂肪细胞的净流出。因此，了解脂肪细胞质膜是否在调节净流入中起作用对人类健康和疾病至关重要。然而，关于这个问题还没有达成共识，关键问题是FFA转运是否通过脂质相快速发生，或者转运是否受膜载体蛋白的调节。在第一个4年的补助金，我们已经证明，跨脂质双层的运输速度受到缓慢的触发器和快速触发器以前的报告是无效的。我们已经获得了强有力的证据，载体蛋白在整个脂肪细胞和质膜囊泡一致的结果。然而，我们的研究揭示的载体具有前所未有的特征，包括：1）ATP依赖性FFA泵，2）流入/流出不对称性，3）流出门，4）其脂质相对FFA触发器高度不敏感的质膜，以及5）没有“扩散”组分的可饱和转运。在本项目的继续中，我们将：1）确定是否可以形成脂质囊泡，其再现了脂肪细胞质膜脂质相的极慢FFA转运，2）确定跨脂肪细胞质膜囊泡的转运机制，3）确定整个脂肪细胞的转运特性是否仅与转运蛋白一致。在这种情况下，我们将开始研究，以确定在脂肪细胞及其质膜的载体。这些研究将使用RNA干扰，多成像质谱和新形式的ADIFAB荧光探针FFA监测FFA的运输。

DESCRIPTION (provided by applicant): The long term goal of the proposed research is to determine the mechanism of free fatty acid (FFA) transport across adipocyte membranes. Circulating FFA are predominantly derived from adipose tissue. Regulation of FFA is critical because FFA provide a major portion of energy needs and elevated levels adversely affect health. Even relatively modest elevations may play an important role in reducing insulin sensitivity in type II diabetes and significantly increase the risk of sudden death. Much greater increases occur acutely, for example in cardiac ischemia and stroke, and these elevations may adversely affect prognosis by inducing arrhythmias, promoting myocyte apoptosis and increasing the risk of severe hemorrhagic events. These increases in circulating FFA are due to net efflux from adipocytes. Therefore understanding whether the adipocyte plasma membrane plays a role in regulating net influx is critical in human health and disease. There is however no consensus about this issue and the key question is whether FFA transport occurs rapidly through the lipid phase or whether transport is regulated by a membrane carrier protein. During the first 4 years of this grant we have demonstrated that transport across lipid bilayers is rate limited by slow flip-flop and that previous reports of rapid flip-flop are invalid. We have obtained strong evidence for a carrier protein in whole adipocytes and consistent results in plasma membrane vesicles. However, the carrier revealed by our studies has characteristics that are unprecedented, including: 1) an ATP dependent FFA pump, 2) influx/efflux asymmetry, 3) an efflux gate, 4) a plasma membrane whose lipid phase is highly refractory to FFA flip-flop, and 5) saturable transport without a "diffusive" component. In the continuation of this project we will: 1) determine if lipid vesicles can be formed which reproduce the extremely slow FFA transport across the lipid phase of the adipocyte plasma membrane, 2) determine the mechanism of transport across adipocyte plasma membrane vesicles and 3) determine if the transport properties of the whole adipocytes are only consistent with a transport protein. In that event we will begin studies to identify the carrier in adipocytes and their plasma membranes. These studies will be done using RNA interference, multi imaging mass spectroscopy and new forms of ADIFAB the fluorescent probe of FFA to monitor FFA transport.