(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?
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