MECHANISMS OF PLASMA MEMBRANE TRANSPORT OF FATTY ACIDS

脂肪酸的质膜转运机制

• 批准号: 6985896
• 负责人: Alan M Kleinfeld
• 金额: $ 35.04万
• 依托单位: TORREY PINES INST FOR MOLECULAR STUDIES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6985896
• 关键词: RNA interference; adipocytes; adipose tissue; cell membrane; fatty acid binding protein; fatty acid transport; fluorescence microscopy; fluorescent dye /probe; free fatty acids; laboratory rat; lipid bilayer membrane; mass spectrometry; membrane activity; membrane permeability; membrane transport proteins; molecular /cellular imaging; stop flow technique; tissue /cell culture; vesicle /vacuole

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.

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 的监管至关重要，因为 FFA 满足了能源需求的主要部分，而游离脂肪酸水平升高会对健康产生不利影响。即使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。例如在心肌缺血和中风时，会出现急剧的大幅升高，并且这些升高可能会诱发心律失常、促进心肌细胞凋亡和增加严重出血事件的风险，从而对预后产生不利影响。循环 FFA 的增加是由于脂肪细胞的净流出所致。因此，了解脂肪细胞质膜是否在调节净流入中发挥作用对于人类健康和疾病至关重要。然而，关于这个问题尚未达成共识，关键问题是 FFA 转运是否快速通过脂相发生，或者转运是否受到膜载体蛋白的调节。在这项资助的前 4 年里，我们已经证明，跨脂质双层的运输受到慢速翻转的速率限制，并且之前关于快速翻转的报告是无效的。我们已经获得了整个脂肪细胞中存在载体蛋白的有力证据，并且在质膜囊泡中获得了一致的结果。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）其脂相对FFA触发器高度难以抵抗的质膜，以及5）没有“扩散”成分的饱和运输。在该项目的后续工作中，我们将：1) 确定是否可以形成脂质囊泡，从而重现穿过脂肪细胞质膜脂质相的极其缓慢的 FFA 运输，2) 确定穿过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特性是否仅与运输蛋白一致。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、多成像质谱和新型 ADIFAB（FFA 荧光探针）来监测 FFA 转运。