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 protected]Download 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 ms) passive transport through the lipid phase of a membrane followed by much slower dissociation from the membrane into the aqueous phase (14, 26, 27, 58, 62, 67). The basis for this interpretation was the finding of apparently extremely rapid flip-flop of FFA across pure lipid vesicles leading to the conclusion that protein-mediated transporters would be irrelevant for FFA transport across biological membranes. At the other end of the spectrum are reports that identify four different proteins that in some way facilitate FFA transport in mammalian cells: FABPpm (53), CD36/FAT (1), FATP (52), and caveolin (63). These studies also report that, in addition to the protein-mediated mechanism, FFA are transported in parallel via a slower, nonspecific, presumably lipid phase mechanism. In addition, it has been proposed that FFA transport occurs through a combination of caveolin and CD36/FAT within lipid rafts (46). However, the only membrane protein that has been clearly demonstrated to be an FFA transporter is FadL, an E. coli outer membrane protein (16). The exceptionally strong evidence supporting this finding includes genetic, structural, and transport characteristics. This protein, which transports long-chain FFA through the polar lipopolysaccharide outer membrane, has no eukaryotic homolog.Our view of FFA membrane transport lies somewhere between these opposing views and is based on our studies with lipid vesicles (13, 28, 30, 31, 35, 59), erythrocyte ghosts (36), and cultured adipocytes (29, 32). Unlike the first view, our studies in lipid vesicles indicate that flip-flop, not dissociation, is the rate-limiting step for transport across lipid membranes, suggesting a potential need for a membrane transport protein in those cells with significant FFA metabolic demand. The results of our adipocyte studies are consistent with a facilitated transport mechanism, which is presumably protein mediated as in the second view (29, 32). However, our results are not consistent with a parallel lipid-mediated pathway or the reported characteristics of the putative transport proteins. In fact, we propose that actual FFA transport proteins have not yet been identified in mammalian cells because appropriate experimental methods have not been used in most studies. In the following Point/Counterpoint, we will identify key issues in the controversies surrounding FFA transport, focusing on our own studies to illustrate these issues.Definition of FFA Membrane TransportThe disagreement concerning the mechanism of FFA transport arises in part because there is no consensus on the definition of FFA membrane transport (for example is it between aqueous phases on either side of the membrane or is it from the outer aqueous phase to an esterified product). This ambiguity has led to disagreement regarding the appropriate experimental methods for investigating the transport mechanism. We therefore begin our discussion with a definition of FFA membrane transport, which will be the basis for a critique of the methods used to study FFA transport across lipid vesicles and cell membranes.We define transport of FFA across membranes as the movement of FFAu from the aqueous phase on one side to the aqueous phase on the other side of the membrane (FIGURE 2). This definition implies that FFA are initially transported across the membrane and into the cytoplasm. They may thereafter be activated to acyl coenzyme A (acylCoA) or, as FFA, partition into intracellular membranes and lipid droplets and bind to intracellular proteins. This process is distinct from transport in which activation of the FFA to acylCoA occurs concurrently with translocation across the membrane (16, 17, 47), generating no significant amount of nonactivated FFAu in the cytosol. The studies by DiRusso et al. in bacteria and yeast (16, 17) and recently by Richards et al. in 3T3-L1 adipocytes (47) have shown clearly that the physiologically important step of FFA activation to acylCoA involves the interaction of long-chain acylCoA synthetases (ACSLs) with membrane-bound FATP. In these studies, virtually all the transported FFA was found to be irreversibly associated with the cells (20), as would be expected if activation of FFA to acylCoA was tightly coupled to the transport step. This process in which activation of FFA to acylCoA is tightly coupled to transport is termed vectorial acylation. FFA activation to acylCoA is of course a necessary step for storage and metabolism of FFA. Nevertheless, as we discuss later, our results in adipocytes indicate that the major portion of FFA transported into the cells remains unesterified (29, 32), and we will explain how methodological issues account for these discrepant conclusions.The Overall Transport Process Can be Decomposed into Discrete Steps.Using the above definition of transport, we can enumerate discrete kinetic steps that constitute aqueous-to-aqueous transport of FFA (FIGURE 1). Influx of FFA, from the extracellular to the intracellular aqueous phases, involves five separate kinetic steps: 1) dissociation [koff(A)] from albumin, 2) diffusion through the outer aqueous phase, 3) insertion into the outer hemileaflet of the membrane (kon), 4) flip-flop from the outer to the inner leaflet (kff), and 5) dissociation from the inner leaflet (koff). If FFA transport is facilitated by a membrane protein, then step 4 (and possibly steps 3 and 5) will be determined by binding to, translocation by, and dissociation from the protein transporter rather than a passive flip-flop step through the lipid bilayer phase of the membrane. Flip-flop denotes the process by which the FFA, with its carboxyl head group at the lipid-water interface and its acyl tail inserted into the hydrocarbon region of one hemileaflet of the lipid bilayer, is translocated across the membrane so that it winds up with the same orientation with respect to the opposite hemileaflet (FIGURE 1). Passive flip-flop may occur in the lipid bilayer phase of a membrane without protein assistance. The molecular details of the passive flip-flop mechanism are only beginning to emerge from work in lipid vesicles (e.g., see Ref. 28), and it is possible that this process might be facilitated by the activity of a transport protein in cellular membranes, as discussed with reference to the models of FIGURE 5. Our studies (29, 32) indicate that the kinetic steps 1–5 are reversible; adding fatty acid-free albumin initiates rapid efflux and completely extracts nonactivated FFA from the cell, which, as discussed later, is most of the FFA transported into the cell. In our view, monitoring intra- and extracellular FFAu levels provides the foundation for studying membrane transport of FFA.Transport of FFA Across Lipid VesiclesA fundamental question in FFA transport is whether a biological membrane needs a protein transport pathway given the prediction by Overton's rule that FFA should readily permeate the lipid bilayer phase of the membrane (65). Determination of the transport characteristics of pure lipid vesicles would seem to be the best way to address this issue. However, considerable controversy has characterized such studies (13, 14, 19, 25, 27, 31, 35, 54, 58, 59, 62). The origin of virtually all the controversies that arise in lipid vesicle or cellular transport of FFA stems from the highly insoluble nature of long-chain FFA. During transport, the overwhelming amount of FFA is bound to lipid and protein phases; generally <1 part in 105 is present in the aqueous phase. Therefore, most methods have inferred transport characteristics indirectly by monitoring the appearance or disappearance of the bound phase FFA and/or FFA metabolites (1, 16, 46, 52, 53, 63). Only in the last decade or so have measurements of the aqueous phase FFA been reported (11, 29).Flip-flop is Rate Limiting for Transport of FFA Across Lipid Membranes.Early studies of FFA membrane transport utilized fluorescently labeled FFA (19, 31, 35, 54, 59), but because the fluorescent label alters transport rates, we will focus on more recent studies that use native FFA. The transport of native FFA across lipid vesicles has been studied by monitoring in the internal aqueous phase of the vesicles the FFA-mediated change in pH using pyranine fluorescence (27) or FFAu concentration using ADIFAB (30). Under the same experimental conditions, virtually identical rates are observed using these two techniques (13). Yet, two opposite outcomes were reported from vesicle transport studies. Most research groups concluded that dissociation from the vesicles is the rate-limiting step in FFA transport and that flip-flop is too fast to measure (14, 27, 54, 62). Our studies concluded that flipflop is rate limiting and that dissociation is faster than flip-flop (13, 30).This discrepancy arises because evidence for extremely rapid flip-flop was derived from influx measurements. There are essentially two methods for delivering FFA to vesicles for an influx experiment: 1) as a complex with a water soluble carrier such as bovine serum albumin (BSA) (FIGURE 3A) or 2) in the absence of a carrier (FIGURE 3B). This distinction is critical because the influx rate constants determined using FFA without a carrier depend on the vesicle concentration (Figure 6 of Ref. 13). This surprising result means that these measured rate constants cannot reflect flipflop, which must be independent of vesicle concentration. In fact, our results reveal that the rate constants obtained with uncomplexed FFA depend linearly on vesicle concentration and therefore reflect binding rather than flip-flop (13). Our results suggest that the reason for this anomalous behavior is that, during influx, exposure to high (>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