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Nonesterified long-chain fatty acids may enter cells by free diffusion or by membrane protein transporters. A requirement for proteins to transport fatty acids across the plasma membrane would imply low partitioning of fatty acids into the membrane lipids, and/or a slower rate of diffusion (flip-flop) through the lipid domains compared to the rates of intracellular metabolism of fatty acids. We used both vesicles of the plasma membrane of adipocytes and intact adipocytes to study transmembrane fluxes of externally added oleic acid at concentrations below its solubility limit at pH 7.4. Binding of oleic acid to the plasma membrane was determined by measuring the fluorescent fatty acid-binding protein ADIFAB added to the external medium. Changes in internal pH caused by flip-flop and metabolism were measured by trapping a fluorescent pH indicator in the cells. The metabolic end products of oleic acid were evaluated over the time interval required for the return of intracellular pH to its initial value. The primary findings were that (i) oleic acid rapidly binds with high avidity in the lipid domains of the plasma membrane with an apparent partition coefficient similar to that of protein-free phospholipid bilayers; (ii) oleic acid rapidly crosses the plasma membrane by the flip-flop mechanism (both events occur within 5 s); and (iii) the kinetics of esterification of oleic acid closely follow the time dependence of the recovery of intracellular pH. Any postulated transport mechanism for facilitating translocation of fatty acid across the plasma membrane of adipocytes, including a protein transporter, would have to compete with the highly effective flip-flop mechanism.Adipocytes are highly differentiated cells specialized in handling large quantities of un-esterified long-chain fatty acids (FA).1 During lipid storage in the fed state, FA are released in the blood from chylomicrons by lipolysis or from albumin, move through the endothelium, bind to the outer leaflet of the plasma membrane, and cross the membrane bilayer. FA are trapped in the cytoplasm by conversion to acyl-CoA and stored primarily as triglycerides in lipid droplets (reviewed in Glatz et al. (1)). During lipolysis of stored triglycerides in the fasting state, FA are released from intracellular lipid droplets, move to and cross the plasma membrane, and are released into the interstitial space, where they bind to albumin. Subsequently, the FA pass through the endothelial cells or diffuse through the spaces between them to reach the blood. These large bidirectional fluxes could occur by several postulated mechanisms, both complex and simple. Cytological changes observed in adipocytes during release and deposition of intracellular lipid led to the complex model that large intracellular fluxes of FA occur by formation of vesicles from the plasma membrane of adipocytes and endothelial cells (2, 3). It has also been postulated that caveolin, a protein present in invaginations of the plasma membrane (caveolae), plays a role in FA uptake (4). There are severa...

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Adipocytes play an important role in the insulin-dependent regulation of organismal fuel metabolism and express caveolae at levels as high or higher than any other cell type. Recently, a link between insulin signaling and caveolae has been suggested; nevertheless, adipocyte caveolae have been the subject of relatively few studies, and their contents have been minimally characterized. With the aid of a new monoclonal antibody, we developed a rapid procedure for the immunoisolation of caveolae derived from the plasma membrane of adipocytes, and we characterized their protein content. We find that immunopurified adipocyte caveolae have a relatively limited protein composition, and they lack the raft protein, flotillin, and insulin receptors. Immunogold labeling and electron microscopy of the adipocyte plasma membrane confirmed the lack of insulin receptors in caveolae. In addition to caveolins, the structural components of caveolae, their major protein constituents, are the semicarbazide-sensitive amine oxidase and the scavenger lipoprotein receptor CD36. The results are consistent with a role for caveolae in lipid flux in and of adipocytes.

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