From the Obesity Research Center, Boston Medical
Center, Boston, Massachusetts 02118 and the Departments of
¶ Physiology and Biophysics,
Medicine and
** Biochemistry, Boston University School of Medicine,
Boston, Massachusetts 02118
Received for publication, July 3, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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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 several other candidate
proteins for enhancement of FA fluxes into adipocytes and other cells;
for a detailed review see Ref. 5. These include the adipocyte fatty
acid-binding protein (aP2) located in the cytosol (1, 6, 7) and
membrane-bound "transport proteins" (8-10), such as "FA
translocase" (FAT (FATP)/CD36) (11, 12), adipose
differentiation related protein (ADRP (13)), and "FA transport
protein" (14, 15). It is not clear whether these proteins enhance the
transmembrane movement of FA or catalyze some other step in the overall
process of FA uptake (16-19).
The simplest mechanism is that FA diffuse freely and rapidly as
monomers from the blood plasma to the cytoplasm (and the reverse) and
cross cell membranes by permeation of their un-ionized form. For
decades, this has been the paradigm for short-chain FA and other weak
electrolytes (20-25). Membrane permeability coefficients for short-
(e.g. acetic acid) and medium-chain (e.g.
octanoic acid) FA are high and increase with increasing chain length
(hydrophobicity), in accord with the Overton rule, up to a chain length
of 12 carbons (24, 26, 27). Long-chain fatty acids (LCFA) have much
lower water solubility (~6 µM for oleic acid (28)) but
bind to albumin in a transitory manner to enable large fluxes.
Therefore, their permeability coefficients cannot be measured in the
same way as those for short- and medium-chain FA (24). However, because LCFA bind more avidly than short-chain FA to phospholipid membranes, the Overton rule would predict that the membrane permeability of
long-chain FA should be higher than that of medium-chain FA, as long as
association, flip-flop, and desorption are not dramatically hindered by
the longer alkyl-chain lengths (16, 25, 29-39). In cell membranes,
transmembrane fluxes of long-chain FA would be limited primarily by
diffusion of FA monomers through the unstirred water layers adjacent to
the membrane, a limitation that might be overcome by albumin (24,
31).
Previously, we developed a method to monitor the transbilayer movement
of FA that uses a fluorescent pH probe (pyranin) trapped inside
phospholipid vesicles (32, 33). This method was designed to test the
hypothesis that fatty acids in their un-ionized form will cross the
phospholipid bilayer, reach ionization equilibrium in the membrane
interface, and release protons that are detected by a water-soluble pH
dye. Binding of FA at one side of the membrane is not sufficient to
cause pH changes at the opposite site of the membrane, and dissociation
of the proton from the FA (but not the desorption of FA from the
membrane) is measured. Furthermore, natural FA instead of synthetic
derivatives can be studied by this method.
The changes in pH measured for several FA by stopped-flow fluorescence
were extremely rapid (t1/2 <50 ms (34)) and matched
quantitatively the expected changes based on the vesicle diameter and
buffer strength of the internal volume of the vesicles (33, 34). We
concluded that LCFA flip-flop across model phospholipid bilayers
readily in their un-ionized form (32-34). The desorption step from the
vesicular membrane is slower than flip-flop but still fast
(koff >1 s Despite these new approaches and results from other laboratories
supportive of rapid spontaneous flip-flop of FA in membranes (39,
42-47), several areas of contention remain. Some investigators have
proposed that the unfacilitated transmembrane movement (flip-flop) of
FA is the rate-limiting step for FA transport in model membranes (30,
48, 49). Moreover, some have argued that the flip-flop of FA in model
membranes might be much faster than in biological membranes, where the
different curvature, lipid composition, and the presence of membrane
proteins might slow down FA diffusion (9, 10, 30, 50). Specific
FA-binding proteins in the membrane might then catalyze the slow
diffusion of FA across the membrane. Although mechanisms have not been
established for the candidate transporters, one postulated mechanism is
transport of the FA anion, as described in detail recently (51). It has been variously suggested that free diffusion does not compete well with
protein-mediated transport of FA anions at low FA concentrations (9,
10, 51, 52), or alternatively at high concentrations (50), or plays
no significant role in FA uptake at any concentration (14, 15).
In this study we focus on the adsorption and transmembrane movement
steps of FA transport in the plasma membrane of adipocytes. We use a
dual fluorescence approach to monitor the adsorption step at the same
time as the flux across the membrane is measured. We present new
evidence favoring fast adsorption and flip-flop of FA in vesicles
prepared from the plasma membrane of adipocytes and in the plasma
membrane of intact adipocytes.
Preparation of Small Unilamellar Vesicles
(SUV)--
Protein-free SUV with trapped
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) (1 mM) were prepared by sonication as previously described
(32).
Preparation of Adipocytes--
Intact adipocytes were prepared
by collagenase digestion of the epididymal fat pads of 2 to 4 male rats
(180-200 g) as described (40). BCECF was trapped by incubating for 30 min with 1 µM BCECF-AM at 37 °C under gentle shaking
and washing the cells with albumin-free Krebs buffer (40).
Preparation of Plasma Membrane Vesicles (PMV)--
Epididymal
fat cells were isolated as above from 20 male rats as described (40)
and subsequently homogenized in buffer (20 mM HEPES-NaOH, 5 mM EDTA, 250 mM sucrose, pH 7.40) containing 0.5 mM of the fluorescent probe pyranin. The plasma
membrane fraction was isolated by differential centrifugation according
to Simpson et al. (53) as modified by Kublaoui et
al. (54), and the untrapped pyranin was removed by gel filtration
(Sephadex G-25). The phospholipid and protein contents of the obtained
PMV and adipocytes were determined with standard assays (55, 56). The
protein/phospholipid ratio was high (1:1, w/w). Homogenization of the
vesicles by extrusion was not successful, possibly because of the high
protein content. Electron micrographs (negative stain) showed that the
vesicle population of the preparation was heterogeneous in size and
lamellarity yet included a significant fraction of large unilamellar
vesicles with a diameter of 100 to 500 nm. Adipocyte PMV made using
this protocol have the right-side-out orientation, according to
independent analyses of insulin receptor orientation (57).
Fluorescence Measurements--
Pyranin, BCECF, and its
acetoxymethylester (BCECF-AM) were purchased from Molecular Probes,
Eugene, OR. Vesicles (SUV or plasma membrane vesicles) were suspended
in a stirred polystyrene cuvette containing 2.5 ml of buffer at pH 7.4 and 20 °C with, in some cases, 0.2 µM of the
engineered fatty acid-binding protein ADIFAB (Molecular Probes).
Measurements were done with a SPEX Fluoromax fluorimeter with the
sampling time set to 1.0 s (the mixing time in the cuvette). The
fluorescence of pyranin was measured using excitation at 455 nm and
emission at 509 nm with a 2-nm bandpass. For ADIFAB, excitation was at
390 nm and the emission at 505 nm; 432 nm was sampled alternately with
2.0-s time intervals, and the fluorescence ratio (R) was calculated
(bandpass 3-5 nm). For BCECF, excitation wavelengths 439 (pH-dependent) and 505 nm (pH-independent) were used and
emission at 535 nm was measured (sampling time, 2.0 s; bandpass 3 nm). Intact adipocytes (106 cells/ml) were suspended in a
well stirred cuvette with 2 ml of albumin-free Krebs buffer (pH 7.4)
with 0.2 µM ADIFAB at 37 °C. The fluorescent signals
of BCECF and ADIFAB were sampled simultaneously, with 4.0-s time
intervals, and their fluorescence ratios were evaluated (3-nm
bandpass). There was no interference between the signals of BCECF and
ADIFAB at the chosen wavelengths.
Calibration of BCECF--
The relationship between internal pH
and the BCECF fluorescence ratio was calibrated by permeabilizing the
cells or SUV to H+ with 1 µM nigericin (a
K+/H+ exchanger) and to K+ with 1 µM valinomycin. The external pH was adjusted with
aliquots of KOH (33), and the pH was measured with a mini-pH electrode. pH equilibration was ensured by the two ionophores and the relatively high K+ concentration on both sides of the membrane.
Measurement of Aqueous Oleic Acid Concentrations--
The
fluorescent FA indicator ADIFAB (FFA Sciences, San Diego, CA) was
dissolved (0.2 µM) in the buffer used for measurements with PMV or cells. The concentration of unbound aqueous oleic acid
([OA]a) was calculated from [OA]a = Kd · 19.5 · (R Measurement of Metabolic Fate of
FA--
[9,10-3H]Oleic acid was used to monitor
incorporation of exogenous oleic acid into esterified products. 50 nmol
of OA* (i.e. OA with a fraction of labeled OA, 1000 dpm/nmol) was added to 2.0 ml of adipocytes in suspension
(106 cells/ml) at 37 °C. All reactions were stopped by
mixing the cell suspension with 10 ml of chloroform/methanol (2:1, v/v)
at chosen time intervals after addition of OA* (10 s, 1, 5, and 20 min). The lipid phase was extracted according to the method of Folch
et al. (59) and concentrated by evaporating the organic solvent under a stream of nitrogen gas. The mixture of products was
subsequently redissolved in 100 µl of chloroform and separated by
thin-layer chromatography (TLC; hexane/ethyl ether/acetic acid, 80:20:1). Because the lipid extract was overwhelmed with triglycerides, unlabeled lipids (diglycerides, phospholipids, and oleic acid) were
added as indicators of the minor components. The developed TLC plate
was visualized in iodine vapor, and the corresponding lipid components
were scraped off and quantified by scintillation counting.
Rapid Flip-flop of Oleic Acid in the Plasma Membrane--
Changes
in internal pH upon addition of external FA have been observed both in
protein-free model membranes and in cells. The rapid flip-flop of
un-ionized FA in model membranes does not guarantee that FA
redistribute quickly across the plasma membrane of cells by the same
mechanism (30, 60). In addition, the observed internal acidification
upon the addition of FA to cells might be attributed to an indirect
effect on cell metabolism (9). Furthermore, the cytosol contains an
abundance of intracellular membranes and FABP that could affect pH
changes following exposure to FA.
To address such questions, we applied our flip-flop assay (32, 33) to
vesicles prepared from the plasma membrane of rat epididymal fat cells.
The isolated PMV contain proteins (including putative FA
transport proteins) but do not metabolize FA in the absence of
essential co-factors and reactants in the cytosolic compartment. PMV
with trapped pyranin were suspended in buffer, and an aliquot of oleic
acid (10 nmol, corresponding to a final concentration in the cuvette of
4 µM) was added to the buffer (Fig.
1). The pyranine fluorescence intensity
dropped, corresponding to a drop in internal pH. This change occurred
within a few seconds, the time resolution (mixing time) of the
experiment. Furthermore, as with protein-free phospholipid vesicles,
the pH gradient persisted because of very slow leakage of
H+. We attribute the pH change to fast translocation of
un-ionized FA across the lipid bilayer of the plasma membrane followed
by ionization of FA in the inner leaflet, as observed previously in
protein-free vesicles (29, 32, 33). Because ionized FA translocate very
slowly, as shown previously in experiments with valinomycin (33),
transport of cyclic H+ is limited and the generated pH
gradient dissipates very slowly.
Avid Partition of Oleic Acid in the Lipid Phase of the Plasma
Membrane--
To test further our hypothesis that the movement of
un-ionized FA through the plasma membrane of adipocytes occurred
through the lipid domains (phospholipid bilayer) of the plasma
membrane, we addressed whether the added FA bind primarily to the lipid domains or to membrane proteins. The partitioning of FA between the
membrane and adjacent water phases could be qualitatively and
quantitatively different for plasma membranes compared to protein-free
model membranes because of the high protein content of the former.
Using the fluorescent FA indicator ADIFAB (58, 61), we investigated
whether a single partition coefficient can describe FA distribution
between the aqueous phase and PMV in the same way as for protein-free
model membranes (58). Plasma membrane vesicles were suspended in buffer
containing 0.2 µM ADIFAB. The concentration of unbound
oleic acid in the water phase outside the vesicles ([OA]a)
was evaluated ("Experimental Procedures") using
R0 = 0.33. Before oleic acid was added to the
suspension of PMV, [OA]a was already 0.24 µM
(Fig. 2), most likely because of FA
present in the plasma membrane, some of which might have been produced
by hydrolysis during the preparation of PMV. When oleic acid was added
in aliquots of 2 µM, the ADIFAB fluorescence ratio
increased, and the calculated [OA]a increased linearly with
the total concentration of OA. Because the solubility limit of oleic
acid is about 6 µM (28), we assumed that after each addition the added oleic acid bound to the PMV, and that no oleic acid
precipitated or bound to the walls of the cuvette (58). The
concentration of oleic acid in the membrane phase, [OA]m, is
then equal to the difference between the total added oleic and the
unbound oleic acid in the water phase after equilibration. Independently, we determined the amount of phospholipid in the PMV
preparation (76 µM phospholipid for each experiment
illustrated in Figs. 2 and 3) by chemical
analysis ("Experimental Procedures"). The volume of the
phospholipid phase (Vm) was calculated assuming
Vm/Va = 10 Dual Fluorescence Measurements in Adipocytes--
In our previous
studies, the pH changes measured with fluorescent pH probes were faster
in model membranes (t1/2 <1 s) than in cells
(t1/2 = s to 1-2 min). It was necessary to add
higher concentrations of FA to the cells to observe changes in
pHin, at least in part because of the lower
surface-to-internal volume ratio in cells compared with vesicles. In
our experiments adding FA without albumin, if some of the added FA
precipitated, the association of the FA to the cells would be limited
in part by desorption of FA from the precipitates. The resultant
complex kinetics could have produced the biphasic changes noted in some
of our previous studies (40). In the experiments reported herein, we
overcame such potential artifacts by using a more sensitive fluorimeter
that yielded reliable measurements with a 10-fold lower concentration
of FA and permitted simultaneous measurement of changes in unbound
external FA and pHin.
Our previous studies also used pyranin for phospholipid vesicles and
BCECF for cells, because intact cells cannot internalize pyranin. To
ensure that the BCECF provides a quantitative measure of protons
transported into cells, we calibrated the pHin changes as
measured by BCECF. The relationship between the ratio of the pH-dependent and pH-independent fluorescence of BCECF was
the same whether the probe was dissolved in buffer, trapped in SUV, or
trapped in cells (Fig. 3).
Applying our improved methodology to cells, we added
K+-oleate at final concentrations below the solubility
limit of oleic acid at pH 7.4 (6 µM) to a suspension of
adipocytes. We also performed dual fluorescence measurements to
discriminate the adsorption step of FA transport (ADIFAB) from the
combined steps of adsorption and transmembrane movement (BCECF). BCECF
was trapped inside the adipocytes, and ADIFAB was present in the
external buffer. Changes in pHin and external
[OA]a were evaluated simultaneously in 4.0-s time intervals
(see "Experimental Procedures"). When 10 nmol of oleic acid (4 µM final concentration) was added to a suspension of
adipocytes (106 cells/ml), the concentration of unbound
oleic acid, [OA]a, increased only to 20 nM in the
first 4-s time interval (Fig. 4). This
result from ADIFAB fluorescence indicates that >99% of the added
oleic acid immediately partitioned to the plasma membrane but did not
provide information about whether the added oleic acid crossed the
bilayer. The latter was monitored by the BCECF fluorescence, which
showed a drop in pHin of 0.01 units immediately after the
addition of oleic acid and the completion of this drop within the time
resolution (4 s) of the experiment. We increased the time resolution to
1.0 s by repeating the experiment in the absence of ADIFAB and
measured only the fluorescence of BCECF. The decrease in
pHin was complete within 1 to 2 s (the time required for mixing the cell suspension) after the addition of oleic acid (not
shown). Although the fast initial pHin drop after addition of FA to cells is probably because of flip-flop of un-ionized FA as in
protein-free model membranes and in PMV, it may also be because of some
indirect effect of the added FA on cellular metabolism. To exclude the
latter, we performed additional quantitative studies, repeating the
experiment of Fig. 4 for different amounts of oleic acid and examining
the metabolic fate of oleic acid, as described below.
Concentration Dependence of pH Drop--
Establishment of the
mechanism of FA movement through membranes requires quantitation of the
relationship between the concentration of FA added to cells and the
amount actually taken up by the cells. In most studies this has been
done by using albumin as vehicle for FA delivery and calculating the
concentration of unbound FA in equilibrium with albumin. The amount of
FA taken up by the cells is measured by stopping the incubation at
chosen times after the addition of the albumin/FA aliquot and
separating the cells from the incubation medium. Unless the cell
contains high levels of endogenous FA, the actual amount of FA
delivered to the cells is not equal to but exceeds the unbound pool of
FA, because some of the FA bound to albumin is also transferred to the
cells. In fact, the FA/albumin ratio changes continuously until
equilibrium is reached, and, depending on the experimental conditions,
the concentration of unbound FA can change as well (37). In experiments without albumin, the concentration of unbound FA added to cells is
fixed and known precisely. Furthermore, essentially all of the added FA
binds to the cell, so that there is always a net flux of FA into the
cell. In experiments using the protocol of Fig. 4 and aliquots of oleic
acid below its solubility limit, the drop in pHin was
proportional to the amount of oleic acid added (Fig.
5A) within the error of the
measurements. With the addition of ~10-fold lower concentrations of
oleic acid, the pH decrease was ~10-fold lower than in our previous
study (40).
Partitioning of Oleic Acid into the Plasma Membrane of Intact
Cells--
As with PMV, we addressed whether the added FA binds mainly
to the phospholipid domains of the plasma membrane or to proteins in
the plasma membrane. From the ADIFAB fluorescence we evaluated the
immediate rise (i.e. after 4 s, the first reading) in
unbound [OA]a after adding different concentrations of oleic acid. An aliquot of 10 nmol of oleic acid is the equivalent of a total
OA concentration of 4 µM. We found that the increase in ADIFAB fluorescence, corresponding to the increase in unbound oleic
acid, was proportional to the amount of oleic acid added (Fig.
5B). Assuming that none of the added OA became esterified in
the first 4 s (see below) and that the plasma membrane provided the major pool of cellular fatty acid-binding sites, at least initially
(e.g. Ref. 25, 62, and 63), we calculated the equilibrium
distribution (Kp) between the amount of unbound oleic acid in the external water phase ([OA]a, measured with
ADIFAB) and the amount bound to the plasma membrane ([OA]m, calculated from the total added [OA] minus unbound [OA]a). As with PMV, the volume ratio between the aqueous phase and membrane phase was estimated by quantifying the phospholipid content of fat
cells (150 nmol of phospholipid/106 cells). The
concentration of oleic acid in the plasma membrane was calculated for
each addition of oleic acid and the apparent partition coefficient from
the slope ([OA]m/[OA]a) (Fig. 5B). We
found Kp = 1.4 × 106, close to the
Kp found for model phospholipid membranes (0.5 × 106 (61)) and for plasma membrane vesicles (1.0 × 106). The quantitative similarity of these results to those
for the PMV is additional evidence that the initial drop in
pHin in adipocytes upon addition of FA is probably because
of the flip-flop mechanism.
Esterification of FA--
In contrast to PMV, initial
acidification following the addition of oleic acid to adipocytes
returned to the basal value within 2 min (Fig. 4). This recovery might
be because of proton leak across the membrane by the
Na+/H+ exchanger, by any other H+
leakage, or by a pumping pathway. It is also possible that the recovery
of pHin is due, at least in part, to esterification of FA,
which offsets the drop in pHin caused by diffusion of
un-ionized FA across the plasma membrane, as we postulated previously
(40). The net reaction of the esterification of OA is as follows.
To monitor both pHin changes and metabolism we added 50 nmol of oleic acid plus a trace of 3H-labeled OA
(i.e. OA*) to five separate 2-ml aliquots of an adipocyte suspension with trapped BCECF. The fluorescence experiment (Fig. 6, top) was terminated by
mixing the content of each cuvette with 10 ml of stop agent at chosen
times of 10 s, 1, 5, or 20 min (see "Experimental
Procedures"). In the control experiment (zero time point), cells were
mixed with stop agent and OA* was added. The lipids were then extracted
and different end products (unesterified oleic acid, diglycerides, and
triglycerides) of OA* determined ("Experimental Procedures"). The
adipocytes readily esterified the added FA, with kinetics that
corresponded closely with the kinetics of recovery of the pH gradient.
Fig. 6 (lower panel) shows that at 10 s, the majority
of oleic acid (80%) was unesterified, whereas at 1 min, 40% of the
added oleic acid remained unesterified. The esterification kinetics
were slightly faster when smaller amounts of OA* were used (as low as 5 nmol), but the distribution of the end products after 1 min was the
same (not shown). We found the same general trend for the kinetics of
metabolism of low concentrations of oleic acid added to adipocytes as
reported previously (51).
Our current data support the hypothesis that FA cross the plasma
membrane of adipocytes rapidly by free diffusion. The adipocyte has
been at the center of research and discussion about mechanisms of
transport of FA through membranes in general. Our new results show that
some of the differences in interpretation of uptake of FA into
adipocytes may be a result of differences in methodologies and in the
duration of measurements. In "classic" uptake studies, cells are
incubated with FA-albumin complexes and initial uptake rates are
evaluated by measuring traces of radiolabeled FA trapped in the cells
within the first few minutes after the incubation. In these assays,
separation of the FA-albumin complexes and cells, followed by washing
with FA-free albumin, is necessary (16, 51). FA uptake depends on the
FA/albumin ratio, i.e. the buffered [FA]a at
equilibrium with the albumin (33, 64). The initial rate of FA uptake
has been described as saturable when plotted against [FA]a
and as diminished by certain metabolic inhibitors (8-10). Thus,
several authors have argued that diffusion is a feasible mechanism at
high FA concentrations in the medium but that FA are transported
primarily by proteins at the low "physiological" concentrations of
[OA]a in media containing albumin (i.e. [FA]a = 2-20 nM (9) or We previously developed and employed a new fluorescence assay for
uptake of FA into cells (38) that eliminated the need to separate FA
donors (e.g. FA-albumin complexes) and cells and used
natural FA instead of probe molecules. We carried out our present
experiments without albumin as a donor of FA (i) to avoid the
complication of not knowing precisely what proportion of added FA
partitions from albumin to the cells and (ii) to focus on the membrane
events of FA transport rather than on issues of partitioning of FA from
albumin. The concentrations of added oleic acid were in the low
micromolar range, and essentially all of the oleic acid partitioned
into the cells.
In contrast, in the presence of albumin, FA partition according to
their relative affinities for binding sites on albumin and on the
membrane, and the amount delivered to cells is neither the amount on
the aqueous phase nor the amount bound to albumin. As an example (16),
incubation of FA-free and protein-free model membranes with 0.5 mM bovine serum albumin complexed with 1.0 mM
palmitic acid would result in net transfer of only ~0.5% of the
bound palmitic acid. However, the equivalent of 5 µM
unbound palmitic acid is transferred to the membrane (16), and a very similar partitioning will occur for oleic acid. Clearly, this is a
large excess relative to the concentration of unbound FA (25 nM) in the presence of albumin and yields about 2.5 mol % FA with respect to phospholipid.
In the experiments reported herein, the added concentrations of oleic
acid were in the same physiological range as discussed above;
i.e. the binding of oleic acid to cells is similar to the transfer achieved with low ratios of LCFA/albumin at physiological concentrations of albumin. In our studies, the lowest amount of oleic
acid added to cells (5 nmol) corresponded to a concentration of 2.0 µM before binding to the cells and 3.2 mol % with
respect to phospholipid in the cells. In addition to measurement
of oleic uptake at low concentrations by the pH assay, other novel
aspects of our study included the use of two fluorescent probes to
monitor the adsorption step simultaneously with the transmembrane
movement of FA, and evaluation of the effects of metabolism on
intracellular pH.
Several new observations support the hypothesis of membrane transport
of FA by free diffusion on a time scale faster than metabolism. First,
in PMV of adipocytes, we showed rapid changes in pHin upon
addition of FA (t1/2 <2 s) (Fig. 1). Therefore, as
in protein-free model membranes, flip-flop of un-ionized FA is rapid in
the plasma membrane of adipocytes. (We should emphasize that this is an
upper limit for the t1/2 of flip-flop, and an exact rate constant cannot be derived from this measurement.) Adipocyte membranes contain a large proportion of proteins, including the putative FA transport proteins FATP and FAT/CD36 (5, 9). If the added
FA had been transported primarily as anions by these proteins, the
changes in pHin would have been in the opposite (alkaline)
direction. In our studies with intact adipocytes, key findings
were that (i) oleic acid binds immediately to the cells (Fig. 4), (ii)
changes in pHin are complete within seconds upon the
binding of oleic acid to the cells (Fig. 4), and (iii) these changes
increase proportionally with the addition of larger amounts of oleic
acid to the outer medium (Fig. 5A).
A fourth key finding was that oleic acid binds avidly in the lipid
phase of the plasma membrane in vesicles and cells (Figs. 2 and
5B). We quantified, for the first time, the partitioning of
oleic acid into PMV and adipocytes and determined that the apparent
Kp of PMV is very similar to the
Kp of protein-free vesicles (Fig. 2). If most of the
oleic acid had bound to proteins in the plasma membrane, competing with
the binding to the phospholipids, the evaluated Kp
of PMV would have been orders of magnitude larger. Furthermore, the
binding curve would have been complex, and we would not have found a
single Kp if binding of oleic acid to proteins was
significantly greater at lower concentrations of oleic acid than at
higher concentrations.
In addition, we determined that the Kp for binding
of oleic acid to intact cells (Fig. 5B) is the same as for
PMV. Recent models of enhancement of the movement of FA through
membranes by membrane-bound proteins have proposed that FA exposed to a phospholipid bilayer with protein "transporters" bind only to the
proteins (8, 15). To compensate for their lower abundance and surface
coverage as compared with phospholipids, such proteins would have to
bind FA with considerably higher affinities than the lipid bilayer.
This would be reflected in a higher partition coefficient for cell
membranes compared with protein-free bilayers, which is not supported
by our data.
We performed new experiments to investigate the possibility that
FA-induced decreases in pHin in cells with active
metabolism and membrane transport of other metabolites and ions are
because of some secondary pH change caused by a FA-induced metabolic
event, as with glucose (66). The observed fast decreases in pH in PMV indicate that the cytosolic compartment and its participation in
metabolism of FA are not essential for the decrease in pH after addition of FA. In intact cells we found that changes in
pHin were proportional to the amount of OA added (Fig.
5A) and that very little FA had been metabolized within the
time course of the pH drop (Fig. 6). These observations support our
conclusion that the immediate pHin changes upon addition of
FA to cells are caused by FA flip-flop. These results are consistent
with previous observations that metabolically inert FA analogues such
as a FA dimer and alkylamine promote H+ transport in cells
by flip-flop and not by a secondary effect (40, 45).
The fluorescence of both BCECF and ADIFAB showed rapid changes followed
by a slow recovery (Fig. 4). After addition of oleic acid, the ADIFAB
fluorescence increased immediately and subsequently recovered with
kinetics similar to the drop in pHin (e.g. Fig. 4). The cells rapidly esterified oleic acid, with kinetics similar to
those of the recovery of pHin (Fig. 6). The ADIFAB signal
reflects the FA in outer leaflet of the plasma membrane in equilibrium with the external water phase at a given time point. Because flip-flop occurs within 1 to 2 s, the maximal signal (at 4 s)
represents oleic acid in the outer leaflet in equilibrium with oleic
acid on the inner leaflet that has undergone flip-flop immediately after addition of oleic acid. As oleic acid becomes activated to
acyl-CoA, oleic acid in the plasma membrane flip-flop rapidly across
the plasma membrane to replace the metabolized oleic acid, and the
concentration of oleic acid on both sides of the membrane decreases.
The decrease in the ADIFAB signal reveals that the added oleic acid in
the membrane was activated (and probably esterified) within 2 min (Fig.
4). Because the pHin recovered with about the same rate as
the ADIFAB signal, the pHin recovery probably reflects only
FA activation and not H+ leakage. This interpretation is
supported by our previous finding that addition of FA-free albumin
after the recovery of the pHin did not cause any changes in
the BCECF fluorescence (40), implying that all the FA had been metabolized.
There are several lines of additional evidence for the interpretation
that the pH recovery we observe on the time scale of seconds to minutes
is primarily a result of metabolism rather than of proton leakage. (i)
The return of pH in PMV was very slow (Fig. 1). (ii) Replacing the
medium containing Na+ with Mg2+ did not affect
the pH change in adipocytes, suggesting the
Na+/H+ exchanger does not play a significant
role in the observed intracellular pH changes, particularly the return
of the pH to the initial level (40). (iii) Addition of metabolically
inert amphipathic molecules that cause intracellular pH changes (FA
dimer and alkylamine) result in sustained changes in pH with
essentially no recovery (40) over the time monitored (minutes). (iv)
The long-chain FA cis-parinaric, a polyunsaturated FA that
is very slowly metabolized, causes a pH drop in adipocytes, and the
recovery is very slow (67). (v). In 3T3L1 preadipocytes, which
metabolize exogenously added fatty acids very slowly, there is a
sustained pH drop.2 This
interpretation of the pH recovery after addition of exogenous FA to
adipocytes can be considered a working hypothesis worthy of additional
investigation. If our interpretation is correct, the fluorescence
approaches offer a powerful real-time visual indicator of membrane
transport and metabolism of FA in living cells.
Our previous studies with adipocytes demonstrated the mechanism of
flip-flop and showed that it is fast, arguing against the notion that
diffusion of LCFA across the plasma membrane is intrinsically slow
(40). Our present study demonstrates the flip-flop mechanism at lower
concentrations of FA, which we argue represent a range of relevant
physiological concentrations. The correspondence of the intracellular
pH changes with the partitioning of FA into the plasma membrane
indicates either that the mechanism is solely diffusion or that a
parallel mechanism makes a constant contribution. The latter
interpretation contradicts the prevalent ideas that transporters are
the only uptake mechanism at low FA concentrations and that this
mechanism becomes progressively less important with increasing FA
concentration (8, 50, 51). Specifically, our kinetic data showing fast
rates in this study do not support the concept that diffusion of FA at
low concentrations is too slow to compete with (putative) transport by
anion transporters (51).
FA transport through membranes must be interpreted in the context of
partitioning of FA between membranes, albumin, and the water phase (16,
25). The high avidity with which FA partition into phospholipid
bilayers membranes makes it unlikely that "transport" proteins
could compete with the fast flip-flop of FA at low concentrations. Even
at [OA]a as low as 2 nM in equilibrium with oleic acid bound to albumin (a mole ratio of oleic acid/albumin of 0.5 (64)), the oleic acid concentration in the lipid domains of the
plasma membrane could be as high as 2 mM (0.2 mol % relative to phospholipid), and flip-flop would permit a transmembrane
flux with which membrane proteins could not compete by any known
mechanism (25).
Our new and previous data with adipocytes lead to the conclusion
that it is unlikely that putative FA transporters facilitate the
transmembrane movement of FA in a direct manner, for example by acting
as a flippase or by forming pores in the membrane (8). Nevertheless,
there is much evidence that FA transporters are involved in the overall
process of FA uptake and metabolism (9, 16). Our new data on diffusion
of FA into adipocytes is not necessarily incompatible with other data
on FA uptake based on conventional assays. Uptake has generally been
monitored over longer time periods than we now show are required for
adsorption and transmembrane diffusion (<5 s). Changes in uptake over
longer intervals, which include slower events, might involve putative membrane transporters. Such proteins could facilitate the overall transport of FA into cells by involvement in the activation of FA, as
proposed for members of the FA transport protein family (50, 68).
INTRODUCTION
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DISCUSSION
SUMMARY
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1) for FA with an
acyl-chain length of up to 18 carbons (35, 36). On the basis of these
properties in model membranes, the diffusion mechanism can be
considered a viable mechanism for transport of FA across cell membranes
(16, 25, 29, 37). We also extended our measurements of the
transmembrane movement of FA to cells with a trapped pH dye (BCECF),
both in suspension (38, 40), and as single cells (41). Decreases in
cytosolic pH occurred immediately after the addition of FA to the
external medium, supporting the hypothesis that FA diffuse across the
plasma membrane by the flip-flop mechanism.
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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DISCUSSION
SUMMARY
REFERENCES
R0)/(11.5-R), where
Kd = 0.28 µM for OA, and R
is the fluorescence ratio of ADIFAB. The value of
R0 was provided by FFA Sciences, or was measured
when [OA]a was assumed to be zero (58).
RESULTS
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DISCUSSION
SUMMARY
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Fig. 1.
Oleic acid flip-flops rapidly in the
adipocyte PMV. Addition of OA to buffer containing PMV causes a
rapid acidification of the internal volume. PMV (100 µl) with trapped
pyranin were suspended in 2.5 ml of buffer (see "Experimental
Procedures"), and the fluorescence of pyranin was measured. At
t = 120 s, 10 nmol of OA (1 µl of a 10 mM K+-oleate stock solution of pH > 10)
was added.
3/(mM phospholipid), where
Va is the volume (2.5 ml) of the water phase (61).
The data in Fig. 2 encompassed a wide range of concentrations of OA, up
to 25 mol % with respect to phospholipid. Partitioning was quantitated
from the linear relationship between the membrane-bound oleic acid
calculated after every addition of oleic acid (Fig. 2) and
[OA]a (not shown). The apparent coefficient
(Kp = [OA]m/[OA]a) evaluated from the slope was 1.0 × 106, similar to the
Kp for protein-free vesicles (58, 61). When the
experiment was repeated with PMV prepared from adipocytes preincubated
with insulin, we obtained results (not shown) very similar to those
shown in Fig. 2.
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Fig. 2.
Partitioning of OA into PMV is a linear
function of OA added to the external buffer and is described by a
single partition coefficient. Vesicles (100 µl; without trapped
pyranin; final phospholipid concentration 76 µM) were
suspended in 2.5 ml of buffer (see "Experimental Procedures")
containing 0.2 µM ADIFAB. Aliquots of 2 µM
OA were added to the stirred cuvette, and the increases in
[OA]a were evaluated from the ADIFAB fluorescence. Most
(>99%) of the added OA bound to the membrane. The linear least
squares fit of the data is shown (r = 0.99).
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Fig. 3.
Calibration curves of the fluorescence of
BCECF versus pH in protein-free phospholipid vesicles
and adipocytes are identical. Fluorescence is expressed as the
ratio (R) between the pH-dependent and
pH-independent fluorescence of BCECF (see "Experimental
Procedures"): , 1 µM BCECF in 2.5 ml of 100 mM HEPES;
, 1 mM BCECF trapped in SUV
suspended in 2.5 ml of 100 mM HEPES buffer;
, BCECF
trapped in adipocytes (106 cells/ml) suspended in 2.5 ml of
Krebs buffer. pH was varied with aliquots of 1 M KOH and 1 M HCl. Vesicles and cells were permeabilized with 1 µM valinomycin and 1 µM nigericin.
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Fig. 4.
The binding of oleic acid to adipocytes and
diffusion of oleic acid across the plasma membrane are rapid
events. Intracellular pH (pHin) in adipocytes and the
extracellular OA concentration ([OA]a) were monitored
simultaneously in real time. OA was added as an aliquot of a 10 mM K+ oleate stock solution (pH > 10) to
a suspension (2.5 ml) of adipocytes (106 cells/ml) in Krebs
buffer without albumin. The fluorescence ratios of BCECF and ADIFAB
were sampled in 4-s time intervals, and the corresponding
pHin and [OA]a were calculated.
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Fig. 5.
The drop in pHin in adipocytes
and the partitioning of OA into the plasma membrane of adipocytes are a
linear function of the concentration of exogenously added OA.
A, the indicated nanomolar amounts of oleic acid were added
to adipocytes in suspension, with conditions as described in the legend
to Fig. 4. B, partitioning of OA between the adipocyte
plasma membrane ([OA]m, calculated as under "Experimental
Procedures") and the outer water phase ([OA]a). The
concentration of unbound OA [OA]a was measured by ADIFAB
fluorescence immediately after different aliquots of OA were added to
intact adipocytes with conditions as described in the legend to Fig. 4.
Results are representative of adipocytes from other rats. The straight
line in each plot (r = 0.98 for A;
r = 0.97 for B) is the linear least squares
fit of the data with the zero point included.
With this simplified overview of the contributing pathways of FA
esterification, we can predict the return to initial pHin in a straightforward way. According to our hypothesis, each oleic acid
that diffuses across the plasma membrane (adsorption, flip-flop, and
desorption) releases one H+ in the cytosol. For every oleic
acid that is esterified, one H+ is removed from the
cytosol, thereby offsetting the drop in pHin caused by
transmembrane FA transport.
(Eq. 1)
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Fig. 6.
Following addition of exogenous OA to
adipocytes, changes in pHin and metabolism show a similar
time course. Adipocytes (106 cells/ml) with entrapped
BCECF were suspended in 2.5 ml of Krebs buffer and 50 nmol of OA
containing a trace amount of 3H-labeled OA (OA*) was added.
Top, the change in pHin upon the addition of OA;
recovery occurs within a few minutes. Bottom, the metabolic
fates of the added OA when the experiment was terminated at various
time points after addition of OA.
DISCUSSION
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SUMMARY
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2 µM
(10), see also Refs. 6 and 65). According to a recent detailed analysis
of uptake of oleic acid into adipocytes, the diffusion mechanism is not significant below molar ratios of oleic acid/albumin of 3:1 (51).
SUMMARY
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL26335 (to J. A. H.) and DK30425 and DK56935 (to P. F. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Institute of Physiological Chemistry, University of Munich, Schillerstrasse 44, D-80336 Munich, Germany.
To whom correspondence should be addressed: Dept. of Physiology
and Biophysics, Boston University School of Medicine, 715 Albany St.,
Boston, MA 02118. Tel.: 617-638-5048; Fax: 617-638-4041; E-mail:
jhamilt@bu.edu.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M206648200
2 W. Guo and J. A. Hamilton, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: FA, fatty acids; LCFA, long-chain fatty acids; FABP, fatty acid-binding proteins; PMV, plasma membrane vesicles; SUV, small unilamellar vesicles; BCECF, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; OA, oleic acid; ADIFAB, acrylodan-modified intestinal fatty acid-binding protein.
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