The long term goal of the proposed research is to determine the mechanism of free fatty acid (FFA) transport across adipocyte membranes. Circulating FFA are predominantly derived from adipose tissue. Regulation of FFA is critical because FFA provide a major portion of energy needs and elevated levels adversely affect health. Even relatively modest elevations may play an important role in reducing insulin sensitivity in type II diabetes and significantly increase the risk of sudden death. Much greater increases occur acutely, for example in cardiac ischemia and stroke, and these elevations may adversely affect prognosis by inducing arrhythmias, promoting myocyte apoptosis and increasing the risk of severe hemorrhagic events. These increases in circulating FFA are due to net efflux from adipocytes. Therefore understanding whether the adipocyte plasma membrane plays a role in regulating net influx is critical in human health and disease. There is however no consensus about this issue and the key question is whether FFA transport occurs rapidly through the lipid phase or whether transport is regulated by a membrane carrier protein. During the first 4 years of this grant we have demonstrated that transport across lipid bilayers is rate limited by slow flip-flop and that previous reports of rapid flip-flop are invalid. We have obtained strong evidence for a carrier protein in whole adipocytes and consistent results in plasma membrane vesicles. However, the carrier revealed by our studies has characteristics that are unprecedented, including: 1) an ATP dependent FFA pump, 2) influx/efflux asymmetry, 3) an efflux gate, 4) a plasma membrane whose lipid phase is highly refractory to FFA flip-flop, and 5) saturable transport without a """"""""diffusive"""""""" component. In the continuation of this project we will: 1) determine if lipid vesicles can be formed which reproduce the extremely slow FFA transport across the lipid phase of the adipocyte plasma membrane, 2) determine the mechanism of transport across adipocyte plasma membrane vesicles and 3) determine if the transport properties of the whole adipocytes are only consistent with a transport protein. In that event we will begin studies to identify the carrier in adipocytes and their plasma membranes. These studies will be done using RNA interference, multi imaging mass spectroscopy and new forms of ADIFAB the fluorescent probe of FFA to monitor FFA transport. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK058762-05A1
Application #
6985896
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Haft, Carol R
Project Start
2001-01-01
Project End
2009-07-31
Budget Start
2005-08-01
Budget End
2006-07-31
Support Year
5
Fiscal Year
2005
Total Cost
$350,350
Indirect Cost
Name
Torrey Pines Institute for Molecular Studies
Department
Type
DUNS #
605758754
City
Port St. Lucie
State
FL
Country
United States
Zip Code
34987
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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