Transport and Metabolism of Fatty Acids in Adipocytes

描述（从申请人的描述扫描）：肥胖是最 在美国普遍存在的健康问题，没有治愈或一般 有效的治疗。虽然在全身水平上很容易识别，但肥胖是 在细胞水平上知之甚少。该项目将使用新的生物物理 战略和分子生物学研究的运输和 在分离的脂肪细胞中游离脂肪酸（FFA）的代谢。这些体外 研究允许控制细胞的环境和评估细胞的生物学特性。 可能影响脂肪储存的因素。我们假设，基于 以前的工作和新的支持数据，通过自由扩散 质膜是FFA进入和排出的主要途径，如果不是唯一途径的话 在细胞中。FFA的摄取不受转运蛋白的调节，而是依赖于 FFA结合的细胞外和细胞内浓度和结合亲和力 部分（例如白蛋白、膜和细胞内FFA结合蛋白）和 对FFA代谢的影响。我们的FFA膜扩散模型 （“触发器”）假设细胞内pH值的变化，可以通过检测到的 荧光pH探针这种方法将被用来测试我们的假设， FFA通过扩散有效和快速地进入和离开脂肪细胞， 细胞外FFA的增加将导致细胞内FFA的增加。我们有 最近还开发了一种13 C NMR方法来研究 外源性13 C标记的FFA转化为酰化脂质终产物。为了补充这些 在脂质中FFA储存的NMR研究中，我们将使用新的13 C NMR方法， 14 C放射性同位素法直接测量FFA的程度 氧化成13 CO2或14 CO2。我们的目标开始的问题是， 脂肪细胞的结构特征，质膜组成， 小窝的存在，以及不同水平的 细胞内FFA结合蛋白aP 2影响细胞内FFA的转运和代谢， FFA。然后，我们研究如何外在条件，如胰岛素的存在， 和/或葡萄糖，FFA的外部供应，以及激素的作用， 抑制剂影响FFA转运和代谢命运。最后，我们自然地使用 发生的遗传变异作为肥胖和糖尿病的动物模型的来源 进行平行研究，以确定这些病理对 FFA运输和代谢。然后我们可以解决一个关键问题， 肥胖症新疗法的制定：FFA转运是否影响 FFA在储存和用作燃料之间的分配？

DESCRIPTION (Scanned from the Applicant's Description): Obesity is the most pervasive health problem in the United States that has no cure or generally effective therapy. While easily recognized at the whole body level, obesity is poorly understood at the cellular level. This project will use new biophysical strategies and molecular biology to study aspects of the transport and metabolism of free fatty acids (FFA) in isolated adipocytes. These in vitro studies allow control of the environment of the cell and evaluation of individual factors that may influence fat storage. We hypothesize, on the basis of previous work and new supportive data, that free diffusion through the plasma membrane is a major if not exclusive pathway for entry and exit of FFA in cells. Uptake of FFA is not regulated by transport proteins but depends on extra-and intracellular concentrations and binding affinities of FFA binding moieties (e.g. albumin, membranes, and intracellular FFA -binding proteins) and on metabolism of FFA. Our model of FFA diffusion through a membrane ("flip-flop") postulates intracellular pH changes that can be detected by a fluorescent pH probe. This approach will be used to test our hypothesis that FFA enter and leave adipocytes efficiently and rapidly by diffusion, and that increased extracellular FFA will lead to increased intracellular FFA. We have also recently developed a 13C NMR approach to study the incorporation of exogenous 13C-labeled FFA into acylated lipid end products. To complement these NMR studies of FFA storage in lipids, we will use a new 13C NMR approach as well as 14C radioisotope methods to measure directly the extent of FFA oxidation into 13C02 or 14C02. Our aims begin with the question of how certain structural features of the adipocyte, the plasma membrane composition, the presence of caveolae, and the expression of different levels of the intracellular FFA-binding protein, aP2, affect the transport and metabolism of FFA. We then examine how extrinsic conditions such as the presence of insulin and/or glucose, the external supply of FFA, and the action of hormones and inhibitors affect FFA transport and metabolic fate. Finally, we use naturally occurring genetic variants as animal models of obesity and diabetes as sources of cells for parallel studies to determine the effects of these pathologies on FFA transport and metabolism. We can then address a key question for formulation of new therapies for obesity: does FFA transport affect the partitioning of FFA between storage and utilization as fuel?