MECHANISM OF PLASMA MEMBRANE TRANSPORT OF FATTY ACIDS

描述：拟议研究的长期目标是确定 脂肪酸（FA）跨膜转运的机制。脂肪酸是 对维持生理平衡至关重要为了让FA 利用它们必须通过质膜运输出来的 脂肪细胞，在那里他们产生，并进入细胞，如肌肉，在那里他们 都被代谢了因此，了解FA运输及其如何调节是 对理解正常生理至关重要此外，由于FA升高， 糖尿病、冠状动脉疾病和癌症都与此有关， 运输系统或其监管的缺陷可能会 导致这些疾病的发病率。因此，新的治疗策略 这些疾病可能是可以预见的，如果特定的运输机制， 鉴定关于是否存在这种具体机制的问题， 有争议的;证据已经提供了两个非特异性 （脂质介导的）和膜蛋白介导的FA转运机制 穿过生物膜如本申请中所述， 这场争论的一个组成部分是FA的实际运输，即在 膜两侧的水相尚未测量 先前，因为不可能检测水相FA， 足够的精度和时间分辨率。这一限制已被克服 随着游离脂肪酸（FFA）荧光探针ADIFAB的开发，期间 在过去的几年里，我们已经将ADIFAB捕获在膜囊泡内， 将其显微注射到细胞中，以确定FFA运动的时间过程 在通过膜分离的水相之间。这些方法构成了 建议的研究。这些研究的目的是确定是否有特定的 蛋白质介导跨脂肪细胞和肌肉细胞膜的转运。做 我们将：1）确定脂质相中的相互作用如何产生 运输障碍，2）决定了整个运输的特点 脂肪细胞和肌肉细胞，和3）确定是否运输性能的 全细胞是这些细胞分离的质膜的特征 细胞为了进行这些研究，我们将利用荧光停流 我们开发的脂质囊泡和红细胞的动力学方法 问题研究此外，我们将研究整个细胞运输使用一个强大的新 通过荧光比率成像细胞内FFA水平的方法 显微镜下观察用ADIFAB显微注射的单个活细胞。

DESCRIPTION: The long-term goal of the proposed research is to determine the mechanism of fatty acid (FA) transport across membranes. Fatty acids are critical to the maintenance of physiological homeostasis. In order for FA to be utilized they must be transported out across the plasma membrane of the adipocyte, where they are generated, and into cells, such as muscle, where they are metabolized. Thus understanding FA transport and how it may be regulated is critical for understanding normal physiology. Moreover, because elevated FA levels have been implicated in diabetes, coronary artery disease, and cancer, it is possible that defects in the transport system, or its regulation, may contribute to the morbidity of these diseases. Thus new strategies for treating these diseases might be envisioned if specific transport mechanisms could be identified. The issue of whether such specific mechanisms exist is quite controversial; evidence has been provided for both non-specific (lipid-mediated) and membrane protein-mediated mechanisms for FA transport across biological membranes. As described in the application, an essential component of this controversy is that actual transport of FA, that is between the aqueous phases on either side of a membrane, has not been measured previously because it was not possible to detect the aqueous phase FA with sufficient accuracy and temporal resolution. This limitation has been overcome with our development of ADIFAB, the fluorescent probe of free FA (FFA). During the past several years we have ADIFAB trapped within membrane vesicles and microinjected it into cells to determine the time course of FFA movement between aqueous phases separated by membranes. These methods form the basis for the proposed studies. The goal of these studies is to determine if specific proteins mediate transport across adipocyte and muscle cell membranes. To do this we will, 1) determine how interactions in the lipid phase can generate barriers to transport, 2) determine the transport characteristics of whole adipocyte and muscle cells, and 3) determine if the transport properties of whole cells are characteristic of the isolated plasma membranes from these cells. To carry out these studies we will utilize the fluorescent stopped-flow kinetic methods that we developed for the lipid vesicle and red blood cell studies. In addition, we will study whole cell transport using a powerful new method that allows imaging of intracellular FFA levels by fluorescence ratio microscopy of single living cells microinjected with ADIFAB.