Is Membrane Transport of FFA Mediated by Lipid, Protein, or Both?

POINT/COUNTERPOINTIs Membrane Transport of FFA Mediated by Lipid, Protein, or Both?J. Patrick Kampf, and Alan M. KleinfeldJ. Patrick KampfTorrey Pines Institute for Molecular Studies, San Diego, California. , and Alan M. KleinfeldTorrey Pines Institute for Molecular Studies, San Diego, California. Published Online:01 Feb 2007https://doi.org/10.1152/physiol.00011.2006This article has been correctedMoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations An Unknown Protein Mediates Free Fatty Acid Transport Across the Adipocyte Plasma Membrane J. Patrick Kampf Torrey Pines Institute for Molecular Studies, San Diego, CaliforniaDownload figureDownload PowerPoint Alan M. Kleinfeld Torrey Pines Institute for Molecular Studies, San Diego, California email protectedDownload figureDownload PowerPoint The mechanism of free fatty acid (FFA) transport between aqueous phases on either side of a biological membrane is intensely debated. Here, we critically analyze features of the methodologies that have contributed to this debate. We discuss our studies indicating that transport across lipid vesicles is limited by the rate of translocation (flip-flop) between the hemileaflets of the lipid bilayer. These results raise the possibility of a significant barrier to transport through the lipid phase of biological membranes. Our results in adipocytes suggest that FFA transport is mediated by an as yet unidentified membrane protein pump.Circulating Free Fatty Acids and Membrane TransportFree fatty acids (FFA), particularly the long-chain FFA, serve a variety of essential functions; they provide a major portion of physiological energy needs (42), are important constituents in the synthesis of complex lipids, and play critical roles in cell signaling (18). Most FFA are obtained from circulating plasma FFA, the levels of which are regulated by the rate at which FFA are generated, predominantly by adipose tissue (43), and the rate at which they are metabolized, predominantly by liver, muscle, and adipose tissue. Regulation of circulating FFA levels is critical because FFA play essential roles in homeostasis; deviations from normal levels reflect pathology and may adversely affect health (5, 6, 23, 31, 33, 34, 37, 40, 44, 45, 55). Although most FFA in circulation are bound to albumin (48, 56), the small fraction of unbound FFA (FFAu) in the aqueous phase mediates physiological activity and is most sensitive to changes in health and disease (5, 49, 66). Moreover, the FFAu, not FFA-albumin complexes, are transported across membranes.In the course of their storage, production, and consumption, FFA must cross the plasma and mitochondrial membranes of many different cells. Because these membranes might be involved in the regulation of FFA trafficking, the mechanism by which FFA are transported across membranes has been an area of considerable interest for over 40 years (57).Why the Mechanism of FFA Transport Across Membranes is ControversialEven after 40 years of study, there remains considerable disagreement about the mechanism by which FFA are transported across membranes. This controversy stems from a lack of agreement about 1) the rate-limiting kinetic step, 2) the magnitude of the transport rate, 3) the need for membrane proteins in cellular transport, and 4) the function of the several membrane proteins that have been found to play a role in the cellular uptake of FFA.Three general views of the mechanism of FFA membrane transport have emerged (FIGURE 1). At one end of the spectrum, it has been proposed that transport of long-chain FFA would involve extremely rapid (5 μM) concentrations of FFAu that are present in the absence of BSA (or other carriers) perturbs the bilayer structure (13, 30, 36). In contrast, delivering FFA in complex with BSA exposes the vesicles to 100-fold lower FFAu concentrations and reveals 100-fold slower influx rate constants compared with uncomplexed FFA (13). In our measurements, FFA dissociation from BSA does not affect the determination of the flip-flop rate constants because the time required to transfer FFA from BSA to vesicles or cells, under the highly buffered conditions of our experiments, is much less than flip-flop (13, 15, 29). This lack of dependence on dissociation from BSA is apparent from the two orders of magnitude difference in influx rates we observe from SUV (3 s−1) to adipocytes (0.02 s−1) using the same FFA-BSA complexes (13, 28, 29, 36).Flip-flop can equally well be measured by monitoring FFA efflux from vesicles to an external FFA sink such as BSA (FIGURE 3A). Two kinetic steps must be resolved because FFA transfer to BSA involves both flip-flop and dissociation. Studies in which only the vesicle-trapped pyranine fluorescence was monitored found a single rate, which was interpreted as dissociation because it was assumed that flip-flop was immeasurably fast (62, 67). However, direct measurements of dissociation, by monitoring FFA binding to BSA, reveal rate constants that are 5- to 10-fold faster than those obtained from the pyranine fluorescence (13, 41). The time course of FFA transfer from the vesicle to BSA reveals two rate constants: a slow one that is equal to that obtained from pyranine measurements (flip-flop) and one that is 5-to 10-fold faster (dissociation) (13). It is important to emphasize that the measured rate constants are remarkably accurate; disparate groups have obtained virtually identical values for the rate constants for efflux (13, 67) and dissociation (13, 41). Thus the findings that FFA transport involves extremely rapid flip-flop and rate-limiting dissociation (26) are based on a misinterpretation of the influx and efflux measurements.Different Mechanisms of Flip-flop and Dissociation.To begin to understand the flip-flop barrier, we recently determined the temperature and FFA size dependence of transport in lipid vesicles (28). Both flip-flop and dissociation rates decreased exponentially with FFA size, but only flip-flop was temperature dependent. The results suggest that the barrier to flip-flop is the work needed to create sufficient free volume to allow the FFA to reorient between the lipid-water interfaces on either side of the bilayer. The free volume model of FFA transport provides a possible explanation for the observed deviations (i.e., slower transport for larger partition coefficients) from Overton's rule. Dissociation is limited primarily by the FFA's aqueous solubility (see, for example, Figure 8 of Ref. 28). In summary, the results of this study reveal distinct mechanisms governing the barriers to flip-flop and dissociation and identify factors that might affect transport of FFA through the lipid phase of biological membranes.Limitations of Lipid Vesicle Studies.The critical finding of the lipid vesicle studies is that the rate-limiting step across simple lipid vesicles is the translocation step (flip-flop) within the bilayer itself. This raises the possibility that more complex lipid phases, as might exist within biological membranes, could generate barriers large enough to prevent significant transport through the membrane's lipid phase. For example, our results imply a virtually impenetrable lipid phase barrier in the adipocyte plasma membrane (29). However, there is little possibility of reproducing in a lipid vesicle the actual lipid phase within a biological membrane given the extremely large number of different lipid species and transverse and lateral asymmetry of such membranes. Nevertheless, if a composition of lipid vesicles can be discovered that generates very slow transport, as observed in adipocytes, it would help support the case for a membrane protein-mediated transport mechanism, and such vesicles could be used for reconstitution studies. Work in progress in our laboratory reveals that specific lipid compositions can significantly reduce rates of FFA transport across lipid vesicles.Transport of FFA Across Cell MembranesAs mentioned above, three views have emerged for how FFA are transported across cell membranes: 1) rapid diffusion through the lipid phase, 2) (known) membrane protein plus lipid phase diffusion, and 3) (unknown) membrane protein only. Because methodology is key to resolving these disparate views, we will mostly focus on our studies in human erythrocytes and adipocytes where the FFAu concentration was monitored.Erythrocyte Ghosts.Our first investigation of cellular FFAu transport was carried out in human erythrocyte ghosts simply because such ghosts have been characterized extensively and because virtually identical stopped-flow mixing methods as used for lipid vesicles could be used for ghosts. In these studies, we trapped ADIFAB or pyranine in resealed ghosts and performed influx studies by adding FFA as complexes with BSA and measured efflux from FFA-loaded ghosts to ADIFAB (36). We found that flipflop was rate limiting, with rate constants similar to those for large lipid vesicles (13). Moreover, we found no effect on FFA transport by a variety of protein-specific reagents. Although we cannot rigorously exclude a protein-mediated process, the simplest mechanism consistent with these results and the uptake measurements of Ref. 10 is a passive lipid phase pathway.Transport Across Adipocyte Membranes.FFA metabolism is not the primary function of human erythrocytes. If regulating FFA transport across membranes is a vital physiological function of other cell types, one would expect adipocytes, whose major function is to store and release FFA, to manifest a high degree of regulation. Not surprisingly, FFA uptake (2–4, 46, 52, 60, 61, 63, 68) and transport (12, 24, 29) have been studied extensively in adipocytes. Uptake studies have linked the four different proteins discussed above to adipocyte transport: FABPpm (53), CD36 (1), FATP (52), and caveolin-1 (63). Support for the identification of these proteins as transporters was obtained by observing that uptake is 1) inhibited by specific reagents (2, 4, 60, 61), 2) correlated with expression levels of the protein (1, 22, 51), and 3) (partially) saturated at high extracellular FFAu concentrations (4, 61). Similar results have been obtained in uptake studies in giant sarcolemmal vesicles of muscle (7, 8).Uptake Measurements Do Not Reveal Transport Kinetics.In our view, the role of these proteins as FFA transporters is uncertain because uptake measurements share several features that may limit their ability to elucidate the membrane transport steps as defined in FIGURE 1. Three criticisms of uptake measurements will be discussed. First, the most important issue relates to the experimental configuration of uptake measurements and whether the quantity measured as transported FFA is reversibly associated with the cell. An uptake involves the determination of the quantity of labeled or fatty acid associated and presumably transported the cell. In contrast, the ADIFAB of FFAu provides a direct measure of the aqueous to aqueous mechanism in FIGURE In the uptake experiments, FFA is to the cells, generally as a complex with BSA, and after a time the cells are with fatty acid free BSA to FFA (for example, see 4, The amount of or fluorescence associated with the cells is as the transported FFA. the extracellular FFA without FFA, most uptake studies use specific such as to FFA However, our measurements those of et al. effect of on FFA to uptake measurements, our experimental configuration for influx involves no the FFA-BSA complexes are present as the intracellular FFAu concentration is monitored in time (29). Under these conditions, we observe that virtually all the FFA transported into the cells can be from the cells by (29, 32). This that very little of the transported FFA is activated to acylCoA because acylCoA is to be from the of the cells Yet, as discussed above, virtually all labeled FFA is irreversibly associated with cells in uptake measurements and is activated to consistent with vectorial These results suggest that, uptake measurements the small fraction of FFA transported into the cells that most of the transported FFA remains unesterified and reversibly into phases such as lipid Thus our results suggest that uptake measurements are not sensitive to the actual FFA transport of the transport rate with extracellular FFAu concentration provides an important of a protein-mediated transport mechanism. Yet, virtually all uptake measurements reveal only after of a that is assumed to be diffusion through the membrane lipid phase. Moreover, without it is at high where is most that the actual is significantly than using albumin binding the appearance of The reason for this is that exponentially with of FFA bound to albumin In the of cells, and therefore will be through FFA transfer to the cells by an amount that on the number and of cells. This in can be large at high because of the and without as in Ref. the of cannot be identification of specific proteins as transporters on the of uptake with protein Uptake studies reveal little or no transport in compared with cells, and this lack of transport has been correlated with the expression of the four proteins identified as the little or of these proteins (1, 22, In to uptake studies, our measurements of FFAu reveal no significant difference in FFA transport characteristics in compared with adipocytes, suggesting that the above identified proteins are not involved in the membrane translocation step Measurements Reveal Transport as we are only three reports of adipocyte transport the for transport measurements we have (11, 29, 32). In our own studies, we used two methods to actual In the we ADIFAB into adipocytes, the concentration to a and then monitored of intracellular FFAu using fluorescence of ADIFAB (29). In the second we used of to provide an independent of our intracellular also in adipocytes (29, 32). The cell studies the first measurements of unbound intracellular FFA concentrations and the time for influx and efflux to be monitored under physiological conditions (29). influx, we from and used ADIFAB to that a in (FIGURE slow influx rates were also observed by monitoring intracellular pH in human and adipocytes from different been was to and efflux was measured by monitoring and found to be as fast as influx in the an important that these studies from uptake measurements is that, during these transport measurements, the extracellular of FFA is only from influx to ability to generate and of has to the characteristics of actual FFA transport in adipocytes, the major features of which the as in Ref. First, and most concentrations at are larger to than the of cellular the concentration which that FFA transport involves an It is important to that this dependence is to the dependence of acylCoA because 1) FFA are (29, 32), 2) acylCoA with has no effect on the transport rates or the and 3) transport occurs with the same rate constants in cells. measurements with reveal a single and transport mechanism, which implies that transport occurs through a membrane protein with no significant transport through the lipid phase (Figure 4 of Ref. 29). the transporter an that efflux so that, at the efflux rate and equal to or less than influx (Figure 3 of Ref. 29). FFA transport of the adipocyte plasma membrane are distinct from lipid In addition to the characteristics above, which not occur in lipid adipocyte rate constants are slower than in lipid vesicles we observed no effect of reagents that have been shown to uptake either at the metabolic or through and which that the effect of these reagents on measurements of uptake are at a different step than is by measurements of the nature of these it was important to key features using an independent one that could provide direct about we used to study FFA transport in adipocytes This is a that to the of with a of 40 within the of the In these studies, cells were as in the complexes were used to FFA. with for of the cells by that most of the was in the lipid The were used to the concentration of in the which in was used to the aqueous concentrations of the The results concentrations that were than consistent with an FFA transport as found in the cell studies. This study also demonstrated that virtually all the label was reversibly associated with the cells, consistent with our ADIFAB that to is to (FIGURE This result that, after the at only a fraction of the FFA is metabolized, suggesting that vectorial may not play a significant role in uptake of FFA, at for adipocytes to most studies with pure lipid we that flip-flop is the rate-limiting step for transport across lipid (13). The flip-flop rate on both FFA structure and vesicle and may be to the free volume within the bilayer (28). Because flip-flop is slow and on the membrane