The role of cell membranes in regulating the flux of long chain free fatty acids (FFA) into and out of adipocytes is intensely debated. Four different membrane proteins including, FABPpm, CD36/FAT, caveolin-1, and FATP have been identified as facilitating FFA transport. Moreover, CD36 and caveolin-1 are also reported to mediate transport in conjunction with lipid rafts. The principal evidence for these findings is a correlation of the level of FFA uptake with the expression level of these proteins and with the integrity of lipid rafts. The 3T3-L1 and 3T3-F442A cell lines in their preadipocyte states reveal little or no expression of these proteins and correspondingly low levels of uptake. Here we have microinjected the adipocyte and preadipocyte cell lines with ADIFAB, the fluorescent indicator of FFA. The ADIFAB fluorescence allowed us to monitor the intracellular unbound FFA concentration during FFA influx and efflux. We show that these measurements of transport, in contrast to FFA uptake measurements, correlate neither with expression of these proteins nor with lipid raft integrity in preadipocytes and adipocytes. Transport characteristics, including the generation of an ATP-dependent FFA concentration gradient, are virtually identical in adipocytes and preadipocytes. We suggest that the origin of the discrepancy between uptake and our measurements is that most of the FFA transported into the cells is lost during the uptake but not in the transport protocols. We conclude that long chain fatty acid transport in adipocytes is very likely mediated by an as-yet-unidentified membrane protein pump.
- intracellular unbound free fatty acids
- fatty acid pump
- monitoring live cells
- ratio fluorescence microscopy
transport of free fatty acids (FFA) across cell membranes is central to physiological homeostasis. The mechanism by which FFA are transported between the aqueous phases on opposite sides of cell membranes is not well understood. An understanding of how FFA are transported across membranes has been an area of intense focus, because of the possibility that transport of FFA across membranes may require facilitation by membrane proteins and might therefore be subject to specific regulatory mechanisms. Proteins that have been associated with FFA transport across adipocyte plasma membranes include FAT/CD36 (1), FABPpm (30, 34), FATP1 (28), and caveolin-1 (31). That several distinct proteins have been identified as FFA transporters in the adipocyte raises the possibility that different processes are being measured in these different studies.
Virtually all studies that identified specific membrane proteins as transporters in adipocytes measured FFA uptake into whole cells (1, 28, 30, 31, 34). These measurements involved the determination of the amount of radioactive or fluorescent fatty acid associated with the whole cell. Our previous results, as discussed in Ref. 18 and the discussion, raise the possibility that features of uptake measurements may limit their ability to determine the rate-limiting step for FFA translocation across the membrane. Most important, our results suggest that, in uptake measurements, much of the FFA transported into the adipocytes may have been removed in the wash step before measurement of the amount of cell-associated FFA (17, 19).
To overcome the limitations imposed by uptake measurements, we previously developed two methods to investigate FFA transport across adipocyte plasma membranes (17, 19). These methods allow FFA influx to be monitored without having to remove extracellular FFA by washing, as is required in the uptake measurements. This is important, because most of the intracellular FFA can be rapidly extracted by removal of extracellular FFA (17, 19). In the first method, we monitored in living cells the changes in the cytosolic unbound FFA concentration in response to changes in the extracellular unbound FFA concentration using adipocytes microinjected with acrylodan-labeled rat intestinal fatty acid binding protein (ADIFAB) (17). In the second method, we measured in adipocytes the lipid droplet concentrations of [13C]oleate using multi-isotope imaging mass spectrometry (MIMS) (19). Results of both studies were consistent with a membrane protein-mediated FFA transport mechanism (17, 19). However, the characteristics we observed were not the same as those reported in the uptake measurements in which the specific membrane proteins were identified (1, 28, 30, 31, 34).
The proteins that have been identified as adipocyte FFA transporters in uptake measurements are all upregulated on preadipocyte-to-adipocyte differentiation. Protein expression in adipocytes correlates with FFA uptake (2, 28, 29, 34); compared with adipocytes, little or no uptake is observed in preadipocytes. To evaluate the role of these proteins, we have in this study applied the methods we developed for measuring FFA transport in living 3T3-F442A adipocytes to determine FFA transport characteristics in 3T3-F442A preadipocytes and in 3T3-L1 adipocytes and preadipocytes. Moreover, uptake by CD36 and caveolin-1 has been reported to involve lipid rafts, and disruption of lipid rafts by cholesterol removal was found to reduce uptake in 3T3-L1 adipocytes but had no effect on uptake in 3T3-L1 preadipocytes (23, 24). We therefore also carried out transport measurements in preadipocytes and adipocytes depleted of cholesterol. Our results reveal virtually identical FFA transport characteristics in adipocytes and preadipocytes and in cholesterol-depleted cells. We conclude that FFA transport across the adipocyte plasma membrane is not mediated by CD36, FATP1, FABPpm, caveolin-1, or lipid rafts. Instead, our results suggest that FFA transport is mediated by an as-yet-unidentified membrane protein pump.
MATERIALS AND METHODS
Buffering the extracellular unbound FFA concentration.
An essential feature of our approach to measuring FFA transport is that the extracellular unbound FFA is clamped at fixed values throughout the measured time courses using complexes of FFA and BSA as described previously (17). In the present study, all measurements were performed using sodium oleate (OA) (Nu-Chek Prep). OA-BSA complexes were formed using 600 μM BSA (Sigma) in a buffer consisting of 20 mM HEPES, 140 mM NaCl, 5.5 mM glucose, 5 mM KCl, 1 mM NaH2PO4, 1 mM CaCl2, and 1 mM MgSO4 at pH 7.4 (C-HEPES). The unbound OA concentration ([OAu]) for each complex was determined using the fluorescent probe ADIFAB (FFA Sciences) as described previously (26, 27). Measuring [OAu] directly is critical for correctly interpreting transport results, especially at high extracellular OAu concentrations ([OAo]), where saturation is most apparent and where buffering of [OAo] by BSA is weakest. Under these conditions, the actual [OAo] may be smaller than calculated using BSA binding constants, thereby giving the appearance of saturation.
3T3-F442A and 3T3-L1 preadipocytes and adipocytes were prepared as described by Green and Kehinde (11). 3T3-F442A preadipocytes were grown to confluence in calf serum and DMEM and were either harvested for experiments as preadipocytes or converted to adipocytes with FCS and DMEM plus insulin (11, 17). 3T3-L1 preadipocytes were also grown to confluence in calf serum plus DMEM and were converted by replacing the media with FCS, DMEM, insulin, 3-isobutyl-1-methylxanthine and dexamethasone. Preadipocytes were harvested for transport measurements at ∼1 wk after seeding. Adipocyte conversion required 1–2 wk, at which time they were prepared for transport measurements. Cells to be used for transport measurements were harvested in phosphate-buffered saline and seeded at ∼105 cells onto 24 × 40-mm microscope coverslips.
Fluorescence microscopy and microinjection.
Microscopy and microinjection of cells with ADIFAB were performed as described previously (17). Briefly, cell-seeded coverslips were mounted in a heated (37°C) perfusion chamber (Warner) and placed on the stage of a Nikon inverted fluorescence microscope. Cells were microinjected (Eppendorf) with 400 μM ADIFAB in C-HEPES, and fluorescence images were recorded with a charge-coupled device camera (Princeton Instruments) at 435 and 505 nm on excitation at 380 nm. Ratios of the 505/435-nm images in the cytosol of the cell, with background subtracted, were averaged, and these values were used to determine the average intracellular OAu concentration. The region selected for averaging was an ellipse that included virtually the entire area of the cell made fluorescent by intracellular ADIFAB.
Measurements of OA influx and efflux were performed as described previously (17). Cells microinjected with ADIFAB were first incubated for ∼5 min with medium composed of C-HEPES plus 600 μM fatty acid-free BSA (FAFBSA). The cells were washed twice with C-HEPES to remove the FAFBSA, and the C-HEPES was replaced with C-HEPES plus OA-BSA (600 μM BSA) to initiate influx. Each medium exchange and mixing required ∼10 s. After the intracellular OAu concentration ([OAi]) reached steady state, the OA-BSA medium was changed to FAFBSA, and this triggered efflux. The C-HEPES with OA-BSA used in each transport cycle was saved, and [OAo] was determined in these samples with ADIFAB (27). Rate constants for influx (kin) and efflux (kout) were determined by fitting single exponential functions to, respectively, the increasing and decreasing portions of the [OAi] time course. Fitting was performed using Origin 7.5 (OriginLab), which yielded R2 values within 4% of unity in virtually all cases. Initial influx rates were determined as kin* [OAi](steady state), where [OAi](steady state) is the value of [OAi] when steady state is reached. Student's t-test was used to assess significance in comparing rate constants.
ATP, intracellular pH, and cholesterol depletion.
ATP was depleted by incubation of cells in 10 μg/ml oligomycin (Sigma) and 37 mM deoxyglucose (Sigma) in C-HEPES without glucose (D-HEPES) for ∼60 min. In our previous study (17), we microinjected ADIFAB in a group of cells, measured transport, added oligomycin and deoxyglucose, incubated for 60 min, and then remeasured transport on the same cells. We have since found that results were more consistent if cells were depleted of ATP before microinjection, possibly because of a modification of ADIFAB during the 60-min incubation in the presence of oligomycin and deoxyglucose. In the present study, we used different cells (on separate coverslips) from the same culture to measure transport in ATP-replete and -depleted cells. Intracellular pH was measured using BCECF (Invitrogen) as described previously (17).
For studies of the effect of cholesterol depletion, oleate transport was measured first in cholesterol-replete cells. The medium from these same cells was removed from the coverslip and replaced with 10 mM methyl-β-cyclodextrin (Sigma) in C-HEPES. After incubation for 25 min at 37°C, the methyl-β-cyclodextrin-containing medium was removed, the cells were washed with C-HEPES, and oleate transport was measured as described above. Cholesterol levels were assayed using cells adhered to culture flasks either treated or not with 10 mM methyl-β-cyclodextrin for 25 min at 37°C. After treatment, cells were assayed for cholesterol levels using the Amplex Red Cholesterol Assay (Molecular Probes).
Western blots and immunofluorescence.
Western blots were generated essentially as described previously (5). Briefly, samples subjected to PAGE consisted of whole cell lysate from ∼106 cells. Following electrophoresis, proteins were transferred to nitrocellulose and incubated overnight at 4°C with the following primary antibodies: CD36 (monoclonal, from Cascade Biosciences), caveolin-1 (monoclonal, from Transduction Laboratories), FATP1 (polyclonal antibodies were obtained as a kind gift from Dr. David Bernlohr of the Univ. of Minnesota, Minneapolis, MN, and from Santa Cruz Biotechnology, Santa Cruz, CA). Blots were incubated for 1 h at 22°C with secondary antibodies conjugated to alkaline phosphatase (Sigma and Jackson Laboratories) and developed within 5 min. The stained blots were scanned and analyzed using Image J (Rasband WS, National Institutes of Health, Bethesda, MD).
Plasma membrane expression of CD36 in F442A adipocytes and preadipocytes was investigated using immunofluorescence. Intact cells were incubated with anti-CD36 monoclonal antibodies (BD Biosciences) at 1.4 μg/ml for 1 h at 22°C. The cells were washed and incubated with FITC-labeled monoclonal anti-mouse IgA (BD Biosciences), also at 1.4 μg/ml for 1 h at 22°C. Fluorescence and phase images were obtained after the washing.
Similar FFA transport characteristics in preadipocyte and adipocyte cell lines.
We monitored transport of FFA into (influx) and out of (efflux) adipocytes and preadipocytes using ratio fluorescence microscopy of cells microinjected with ADIFAB, as described previously for 3T3-F442A adipocytes (17). Phase and fluorescence images from a typical experiment in which oleate influx and efflux were monitored in 3T3-L1 preadipocytes are shown in Fig. 1. These images are representative of all cell types (3T3-F442A and 3T3-L1 adipocytes and preadipocytes) we studied and are similar to the transport results we obtained previously for 3T3-F442A adipocytes (Fig. 1 of Ref. 17). In Fig. 1, the phase image reveals the typical fibroblastic morphology of these preadipocytes, which lack significant lipid droplets, in contrast to the differentiated adipocyte (as seen, for example, in Fig. 1 of Ref. 17). The false color fluorescence ratio images in Fig. 1 represent snapshots of the time course of oleate influx and efflux. These images (Fig. 1, panels 2–5) reveal the increase (blue to red) in [OAi] on oleate influx and the decrease back to blue on oleate efflux.
Time courses for FFA influx and efflux were obtained from these images by averaging the [OAi] values in the cytosol (materials and methods). Transport characteristics were determined from the change in the cytosol averaged [OAi] in response to changes in [OAo]. Figure 2 is a representative example of the influx and efflux time courses for the 3T3-L1 and 3T3-F442A adipocytes and preadipocytes. Figure 2 reveals the influx time course as [OAi] increases from baseline to a steady-state level in ∼200 s, followed by the efflux time course as [OAi] returns to baseline after reducing [OAo] to zero.
Rate constants for influx and efflux were obtained from these time courses by fitting, respectively, rising and falling portions of the time courses with single exponential functions (materials and methods). Influx rate constants were similar (P > 0.1) for both 3T3-L1 and 3T3-F442A preadipocyte and adipocyte pairs (Table 1). Efflux rate constants were also essentially the same except for a 20% larger kout value for the 3T3-F442A preadipocytes than the adipocytes. In contrast, FFA uptake was found to be >10-fold lower in preadipocytes compared with adipocytes (2, 28, 29, 34).
The time courses also illustrate that, at steady state, [OAi] is larger than [OAo] by between 30 and 300% (Fig. 2 and Table 2). The average gradient is similar for all cell types. As we discussed previously for 3T3-F442A adipocytes, the existence of this gradient requires an energy source, and indeed we found previously that depletion of ATP abolishes this gradient (17, 19). Also as reported previously (17) for the 3T3-F442A adipocytes, we found that the steady-state [OAi] levels and therefore the inside-outside gradients exhibit substantial cell-to-cell heterogeneity for all cell types (Fig. 2). In Ref. 17, we provided evidence that this [OAi] heterogeneity was correlated with [ATPi] heterogeneity. In the present study, we also investigated transport in 3T3-L1 and 3T3-F442A preadipocytes that were depleted of ATP. The results reveal similar influx and efflux rate constants as for ATP-replete cells, except that, in the ATP-depleted cells, the [OAi] > [OAo] gradient and the cell-to-cell heterogeneity of steady-state levels were abolished (Fig. 3). This indicates that the ATP-dependent pump we described previously for 3T3-F442A adipocytes also functions in 3T3-L1 and 3T3-F442A preadipocytes and, presumably, in the 3T3-L1 adipocytes.
The features of plasma membrane transport of FFA that we described previously for 3T3-F442A adipocytes were consistent with transport being a facilitated, presumably protein-mediated process without a parallel lipid phase mechanism. An important feature of a facilitated or carrier mechanism is that the initial rate of transport should reveal saturation at sufficiently high [OAo], as we demonstrated previously for 3T3-F442A adipocytes. As shown in Fig. 4, the 3T3-L1 preadipocytes also reveal saturation of the initial influx rate with increasing [OAo]. We described previously (17, 19) the modification of a model for FFA transport based on a four-state carrier model of membrane transport, described by Weiss (33). In this model, binding to the carrier at the water-membrane interface is assumed to be fast compared with translocation, and therefore the binding reaction can be assumed to be at equilibrium. At the outer surface, binding is described by the equilibrium dissociation constant Ko (Ko = [FFAo][carrier empty]/[carrier bound to FFA]). The carrier model (17, 19)-fit parameters of maximum inward velocity (Roi) and Ko for the 3T3-L1 preadipocytes yield values of 9 ± 1 nM/s and 150 ± 40 nM, respectively. These values are quite similar to those (12 ± 2 nM/s and 180 ± 100 nM, respectively) that we found previously for 3T3-F442A adipocytes.
Expression of CD36, FATP1, and caveolin-1 in preadipocytes is lower than in adipocytes.
The plasma membrane proteins CD36, FATP1, and caveolin-1 have been identified as adipocyte FFA transport proteins from measurements of FFA uptake (1, 28, 31). Important support for this identification is that FFA uptake is larger (>10-fold) in adipocytes compared with preadipocytes, and adipocytes express much larger (>5-fold) amounts of CD36, FATP1, and caveolin-1 than preadipocytes (2, 13, 25, 29, 34). To confirm similar differences in expression of these proteins in our cell preparations, we determined the expression of these proteins in the 3T3-L1 and 3T3-F442A adipocytes and preadipocytes by Western blotting and, in addition, by immunostaining for CD36. The immunoblotting revealed expression levels in the preadipocytes that were between 4 and 26% of the adipocyte levels for the three proteins (Fig. 5 and Table 3). These results are in reasonable agreement with previous estimates for CD36 and FATP1 (1, 28), whereas previous results for caveolin-1 suggest a >20-fold difference (29) compared with the 5- to 10-fold difference in the present study. Immunostaining with anti-CD36 antibodies revealed virtually no staining of preadipocyte plasma membranes compared with adipocytes (Fig. 6).
Cholesterol depletion does not reduce transport.
Lipid rafts have been reported to mediate fatty acid uptake/transport in 3T3-L1 cells (23, 24). Disruption of the lipid rafts by extraction of cholesterol, using methyl-β-cyclodextrin, was found to reduce oleate uptake in 3T3-L1 adipocytes by ∼50% but had little or no effect in 3T3-L1 preadipocytes. To determine the effect of cholesterol depletion, we measured oleate transport in 3T3-L1 and 3T3-F442A adipocytes and preadipocytes depleted of cholesterol by methyl-β-cyclodextrin treatment. For all cell types investigated, the methyl-β-cyclodextrin treatment reduced cellular levels of cholesterol by between 50 and 90%. As is apparent in Fig. 7, cholesterol depletion did not reduce oleate transport. In fact, in some cases, steady-state levels revealed a modest (<20%) increase. This increase was not due to a decrease in intracellular pH, which could give the appearance (17) of increased [OAi], or to increased ATP levels (data not shown).
Our results demonstrate that plasma membrane transport of FFA is virtually identical in 3T3-F442A and 3T3-L1 adipocytes and preadipocytes, and that cholesterol depletion of these cells does not reduce transport. Because levels of FAT/CD36, FATP1, caveolin-1, and FABPpm are between at least 4- and >25-fold lower in preadipocytes compared with adipocytes (Refs. 1, 28, 29, 34 and Fig. 5), our results suggest that these proteins are not involved in the transport of FFA across adipocyte or preadipocyte plasma membranes. Although we did not determine expression levels of FABPpm in this study, FABPpm levels were found by Zhou et al. (34) to be undetectable in 3T3-L1 preadipocytes but present at high levels in 3T3-L1 adipocytes. Our results also suggest that lipid rafts do not mediate membrane transport of FFA, because transport is independent of CD36 and caveolin-1 expression, and cholesterol depletion does not reduce FFA transport.
These negative results might be viewed as evidence that membrane transport of FFA is not protein mediated. Indeed, a proposed mechanism suggests that FFA undergo rapid (<2 s) diffusion through the lipid phase of membranes (12, 14, 22). Measurements reporting rapid transport through the lipid phase were based on methods and interpretations that were used for assessing transport across lipid vesicles (15). These studies reported virtually instantaneous flip-flop of FFA across lipid vesicles. Recently, we have shown that these vesicle findings are based on incorrect interpretations of the kinetics of both FFA influx into and efflux out of the vesicles (8, 16). Rapid influx was observed when, as in Ref. 15, the added FFA were not complexed with albumin. This gives rise to a vesicle concentration dependence of the influx rate constant and is therefore not due to flip-flop, which must be independent of the vesicle concentration (8, 16). Addition of FFA as a complex with BSA yields flip-flop rate constants that are independent of the vesicle concentration and are rate limiting for FFA transport (8, 16). In fact, our results indicate that the lipid phase of even a pure lipid vesicle can represent a significant barrier to flip-flop. Moreover, our present (Fig. 4) and previous (17) results suggest that the lipid phase of the adipocyte and preadipocyte membranes is refractory to FFA flip-flop (see below).
Thus it is likely that FFA transport in adipocytes is protein mediated. However, our results are not consistent with the uptake measurements that have been the basis for identifying FAT/CD36, FABPpm, FATP1, and caveolin-1 as transport proteins in adipocytes. In addition to finding a lack of correlation with expression levels of FAT/CD36, FATP1, and caveolin-1, the characteristics of FFA transport determined in the present and previous studies (17, 19) are different from those reported in studies of FFA uptake. First, [FFAi] values at steady state are larger (2- to 5-fold) than for [FFAo], and maintenance of this [FFAi] > [FFAo] gradient involves ATP. These results indicate that FFA transport is assisted by a pump that is affected by intracellular ATP levels. This ATP dependence is unrelated to covalent modification of the FFA through ATP-dependent acyl-CoA formation, because FFA are rapidly extracted (17, 19), in contrast to acyl-CoA (7, 9, 25).
Second, our results are consistent with transport being mediated by a simple saturable mechanism (Figs. 4 and 4 of Ref. 17), with no additional contribution required from, for example, a diffusive component through the membrane's lipid phase. Third, in our previous study (17), we provided evidence that the transporter possesses an [FFAo] sensing gate that modulates efflux so that, at elevated [FFAo] levels, the efflux rate constant decreases and becomes equal to or less than the influx rate constant. Presumably, this mechanism reduces FFAi leakage and thereby helps to generate the [FFAi] > [FFAo] gradient. Fourth, transport across the plasma membrane is asymmetric; efflux rate constants, when [FFAo] = 0, are approximately twice as fast as for influx. Fifth, FFA transport properties of the adipocyte plasma membrane are distinct from lipid vesicles; transport rate constants in adipocyte are ∼100-fold slower and have different dependencies on FFA type compared with lipid vesicles (8, 16). Last, we observed no effect of reagents that have been shown to modulate uptake, either at the metabolic level (insulin, triacsin C) (10, 25) or through inhibition (phloretin, DIDS, and proteases) (3, 4).
It should be emphasized that the present results are relevant specifically to preadipocytes and adipocytes. Because FFA influx and efflux are the major functions of adipocytes, different transport mechanisms might be expected in nonadipose cells such as cardiac and skeletal muscle, where, for example, FFA efflux may not be a physiological function. Moreover, muscle cells reveal more complex and acute regulation of FFA uptake and protein expression than adipocytes (6). This includes the redistribution of FAT/CD36 and FABPpm between intracellular and plasma membrane sites and FFA uptake rates that depend on muscle contraction. In contrast, these proteins are not expressed in preadipocytes and are expressed only in the plasma membranes of adipocytes (2, 34).
Because our results contrast with those observed in FFA uptake studies, we considered in our previous study of FFA transport in adipocytes (17) a number of factors relating to our ADIFAB methodology that might have generated erroneous results. Although no such factors were identified, we nevertheless previously developed an independent method to determine [FFAi] values in FFA transport (19). This method involves a new technology, MIMS, that allows for the quantitative imaging of the distribution of [13C]oleate in 3T3-F442A adipocytes (20).
Samples in our previous MIMS experiments were prepared in the same way as in the present studies, except that the 3T3-F442A adipocytes were grown on silicon chips, and after incubation with [13C]oleate-BSA, the cells were dried under argon (19). We then used MIMS to obtain 13C/12C ratio images of the cells, which, at a resolution of 40 nm, clearly delineated the intracellular lipid droplets. The 13C/12C ratio within the lipid droplets, together with the oleate lipid-water partition coefficient, allowed us to calculate the steady-state [FFAi]. Two important features of these previous MIMS results support the conclusions obtained from our previous (17) and present ADIFAB experiments. First, steady-state [FFAi] was found to be four- to fivefold greater than [FFAo], in agreement with the concentration gradients observed using ADIFAB. Second, the 13C/12C images revealed that virtually all of the 13C was rapidly extracted on reducing [FFAo] to zero. This removal of all the FFA from the cell is consistent with the ADIFAB results demonstrating the rapid return to baseline of [FFAi] on reducing [FFAo] to zero (Figs. 2 and 7).
Although our results indicate that FAT/CD36, FATP1, caveolin-1, and FABPpm do not mediate the membrane translocation step in preadipocyte and adipocyte FFA transport, these proteins have been shown to facilitate FFA uptake in adipocytes (1, 2, 23, 28, 31, 34). We suggest that this discrepancy arises because of the difference in the experimental configuration of uptake and our measurements. Uptake measurements determine the quantity of labeled (generally radioactive or fluorescent) fatty acid associated with the whole cell. In the uptake experiments, labeled FFA is added to the cells, generally as a complex with BSA, and then the cells are washed to remove nontransported FFA (for example, see Refs. 4, 21, 32). To remove the extracellular FFA without disturbing cell-associated FFA, most uptake studies use specific reagents, such as phloretin (4), to inhibit FFA efflux. However, neither our measurements (17) nor those of Faergeman et al. (9) revealed any effect of phloretin on FFA transport. Moreover, recent studies of FFA uptake reveal that virtually all of the (labeled) FFA that remain after washing are irreversibly associated with cells and are probably activated to acyl-CoA, consistent with a vectorial acylation mechanism for transport of FFA (9, 25).
In contrast to uptake experiments, our measurements involve no wash step; the FFA-BSA complexes are present continuously during the measurement of the influx time course (for example, Fig. 2). Under these conditions and in the MIMS studies (19), we observe that virtually all the FFA transported into the cells can be extracted rapidly from the cells by washing. The MIMS results indicate that very little (<3%) of the cell-associated FFA were esterified during the 20-min incubation with [13C]oleate. These results suggest that uptake measurements detect the small fraction of FFA transported into the cells that become activated to acyl-CoA and are therefore irreversibly associated with the cells, but that most of the transported FFA remain unesterified and partition reversibly into hydrophobic phases such as lipid droplets. Thus our results suggest that uptake measurements are not sensitive to the actual FFA transport step, and that the actual membrane FFA transporter is an ATP-dependent pump that has yet to be identified.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-058762.
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- Copyright © 2007 by American Physiological Society