MECHANISMS OF PLASMA MAMBRANE 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）跨脂肪细胞膜运输的机制。循环游离脂肪酸主要来源于脂肪组织。对游离脂肪酸的调节至关重要，因为游离脂肪酸提供了能量需求的主要部分，其水平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对降低II型糖尿病患者的胰岛素敏感性起重要作用，并显著增加猝死的风险。更大的增加发生在急性，例如心脏缺血和中风，这些升高可能通过诱发心律失常，促进心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。循环游离脂肪酸的增加是由于脂肪细胞的净外排。因此，了解脂肪细胞膜是否在调节净内流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转运是通过脂质期快速发生，还是由膜载体蛋白调节。在这项资助的前4年，我们已经证明了脂质双分子层的转运受到缓慢翻转的速率限制，而之前关于快速翻转的报道是无效的。我们已经获得了在整个脂肪细胞中存在载体蛋白的有力证据，并且在质膜囊泡中得到了一致的结果。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依赖的FFA泵，2)流入/流出不对称，3)流出门，4)质膜的脂相对FFA的翻转具有高度难耐性，5)无“扩散”成分的饱和运输。在本项目的后续研究中，我们将：1)确定是否可以形成脂质囊泡，使FFA在脂肪质膜的脂质相上进行极慢的转运；2)确定脂肪质膜囊泡间的转运机制；3)确定整个脂肪细胞的转运性质是否只与转运蛋白一致。在这种情况下，我们将开始研究识别脂肪细胞及其质膜中的载体。这些研究将利用RNA干扰、多成像质谱和新型的FFA荧光探针ADIFAB来监测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.