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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK058762-03
Application #
6626997
Study Section
Special Emphasis Panel (ZRG1-SSS-T (02))
Program Officer
Haft, Carol R
Project Start
2001-01-01
Project End
2004-12-31
Budget Start
2003-01-01
Budget End
2003-12-31
Support Year
3
Fiscal Year
2003
Total Cost
$315,000
Indirect Cost
Name
Torrey Pines Institute for Molecular Studies
Department
Type
DUNS #
605758754
City
San Diego
State
CA
Country
United States
Zip Code
Huber, Andrew H; Kampf, J Patrick; Kwan, Thomas et al. (2014) Usefulness of serum unbound free fatty acid levels to predict death early in patients with ST-segment elevation myocardial infarction (from the Thrombolysis In Myocardial Infarction [TIMI] II trial). Am J Cardiol 113:279-84
Zhang, Duan-Sun; Piazza, Valeria; Perrin, Benjamin J et al. (2012) Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia. Nature 481:520-4
Carley, Andrew N; Kleinfeld, Alan M (2011) Fatty acid (FFA) transport in cardiomyocytes revealed by imaging unbound FFA is mediated by an FFA pump modulated by the CD36 protein. J Biol Chem 286:4589-97
Carley, Andrew N; Kleinfeld, Alan M (2009) Flip-flop is the rate-limiting step for transport of free fatty acids across lipid vesicle membranes. Biochemistry 48:10437-45
Kampf, J Patrick; Kleinfeld, Alan M (2007) Is membrane transport of FFA mediated by lipid, protein, or both? An unknown protein mediates free fatty acid transport across the adipocyte plasma membrane. Physiology (Bethesda) 22:7-14
Kampf, J Patrick; Parmley, Danielle; Kleinfeld, Alan M (2007) Free fatty acid transport across adipocytes is mediated by an unknown membrane protein pump. Am J Physiol Endocrinol Metab 293:E1207-14
Kampf, J Patrick; Cupp, David; Kleinfeld, Alan M (2006) Different mechanisms of free fatty acid flip-flop and dissociation revealed by temperature and molecular species dependence of transport across lipid vesicles. J Biol Chem 281:21566-74
Lechene, Claude; Hillion, Francois; McMahon, Greg et al. (2006) High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry. J Biol 5:20
Kampf, J Patrick; Kleinfeld, Alan M (2004) Fatty acid transport in adipocytes monitored by imaging intracellular free fatty acid levels. J Biol Chem 279:35775-80
Cupp, David; Kampf, J Patrick; Kleinfeld, Alan M (2004) Fatty acid-albumin complexes and the determination of the transport of long chain free fatty acids across membranes. Biochemistry 43:4473-81

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