MECHANISMS OF PLASMA MAMBRANE TRANSPORT OF FATTY ACIDS

描述(申请人提供)：拟议研究的长期目标是确定游离脂肪酸(FFA)跨脂肪细胞膜的转运机制。循环FFA主要来源于脂肪组织。对FFA的监管至关重要，因为FFA提供了能源需求的主要部分，而水平过高会对健康造成不利影响。即使是相对温和的升高，也可能在降低II型糖尿病患者的胰岛素敏感性方面发挥重要作用，并显著增加猝死的风险。更严重的升高发生在心脏缺血和中风中，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。循环中FFA的这些增加是由于脂肪细胞的净外流。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，关键的问题是FFA的转运是否通过脂类快速发生，或者转运是否受膜载体蛋白的调节。在这项资助的前4年中，我们已经证明了跨脂双层的转运受到慢触发的速率限制，并且先前关于快速触发的报道是无效的。我们已经获得了在整个脂肪细胞中存在载体蛋白的强有力证据，并在质膜囊泡中获得了一致的结果。然而，我们的研究揭示了这种载体具有前所未有的特征，包括：1)依赖于ATP的FFA泵，2)流入/外流不对称，3)外流门，4)其脂相对FFA触发器高度阻挡的质膜，以及5)没有“扩散”成分的饱和运输。在这个项目的继续过程中，我们将：1)确定是否可以形成脂泡，从而复制极慢的FFA跨脂肪细胞质膜的运输；2)确定脂肪细胞质膜囊泡的运输机制；3)确定整个脂肪细胞的运输性质是否只与一种运输蛋白一致。在这种情况下，我们将开始研究，以确定脂肪细胞及其质膜中的载体。这些研究将使用RNA干扰、多成像质谱学和新形式的ADIFAB荧光探针FFA来监测FFA的传输。

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.