Transport and Metabolism of Fatty Acids in Adipocytes

描述(从申请者的描述中扫描出来)：肥胖是 美国普遍存在的无法治愈或普遍存在的健康问题 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 研究运输和运输方面的策略和分子生物学 游离脂肪酸在分离的脂肪细胞中的代谢。这些是体外培养的 研究允许控制细胞的环境并评估 可能影响脂肪储存的个别因素。我们假设，在此基础上 以前的工作和新的支持性数据，通过 质膜是游离脂肪酸进入和离开的主要途径 在牢房里。FFA的摄取不受运输蛋白的调节，而取决于 游离脂肪酸结合的胞外和胞内浓度及结合亲和力 部分(如白蛋白、膜和细胞内FFA结合蛋白)和 对游离脂肪酸代谢的影响。我们的FFA通过膜扩散的模型 (“触发器”)假定细胞内的pH变化可以通过 荧光pH探头。这种方法将被用来检验我们的假设 FFA通过扩散高效、快速地进入和离开脂肪细胞， 细胞外FFA的增加会导致细胞内FFA的增加。我们有 最近还开发了一种13C核磁共振方法来研究 外源~(13)C标记的游离脂肪酸转化为酰化的脂类最终产物。为了补充这些内容 FFA在脂类中储存的核磁共振研究，我们将使用新的13C核磁共振方法作为 以及~(14)C放射性核素法直接测量FFA的程度 氧化成13C02或14C02。我们的目标始于一个问题，即如何确定 脂肪细胞的结构特征、质膜成分、 小凹的存在，以及不同水平的 细胞内游离脂肪酸结合蛋白aP2影响磷脂酰胆碱的转运和代谢 FFA。然后我们研究外在条件，如胰岛素的存在 和/或葡萄糖，FFA的外部供应，以及激素和 抑制剂影响FFA的转运和代谢的命运。最后，我们自然地使用 作为肥胖和糖尿病来源的动物模型的发生基因变异 用于平行研究的细胞数量，以确定这些病理对 游离脂肪酸的转运和代谢。然后，我们可以解决一个关键问题 肥胖症新疗法的制定：游离脂肪酸的运输是否影响 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?