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.
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