Palmitate transport and fatty acid transporters in red and
white muscles
A.
Bonen1,
J. J. F. P.
Luiken1,
S.
Liu1,
D. J.
Dyck1,
B.
Kiens2,
S.
Kristiansen2,
L. P.
Turcotte3,
G. J.
Van Der
Vusse4, and
J. F. C.
Glatz4
1 Department of Kinesiology,
University of Waterloo, Waterloo, Ontario, Canada N2L 3G1;
2 The Copenhagen Muscle Research
Centre, August Krogh Institute, University of Copenhagen, DK-2100
Copenhagen, Denmark; 3 Department
of Exercise Sciences, University of Southern California, Los Angeles,
California 90089; and 4 Department
of Physiology, Cardiovascular Research Institute Maastricht,
Maastricht University, 6200 MD Maastricht, The Netherlands
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ABSTRACT |
We performed studies
1) to investigate the kinetics of
palmitate transport into giant sarcolemmal vesicles,
2) to determine whether the
transport capacity is greater in red muscles than in white muscles, and
3) to determine whether putative
long-chain fatty acid (LCFA) transporters are more abundant in red than
in white muscles. For these studies we used giant sarcolemmal vesicles, which contained cytoplasmic fatty acid binding protein
(FABPc), an intravesicular fatty
acid sink. Intravesicular FABPc
concentrations were sufficiently high so as not to limit the uptake of
palmitate under conditions of maximal palmitate uptake (i.e., 4.5-fold
excess in white and 31.3-fold excess in red muscle vesicles). All of the palmitate taken up was recovered as unesterified palmitate. Palmitate uptake was reduced by phloretin (
50%),
sulfo-N-succinimidyl oleate
(
43%), anti-plasma membrane-bound FABP
(FABPpm,
30%), trypsin
(
45%), and when incubation temperature was lowered to 0°C
(
70%). Palmitate uptake was also reduced by excess oleate (
65%), but not by excess octanoate or by glucose. Kinetic
studies showed that maximal transport was 1.8-fold greater in red
vesicles than in white vesicles. The Michaelis-Menten constant in both types of vesicles was ~6 nM. Fatty acid transport protein mRNA and
fatty acid translocase (FAT) mRNA were about fivefold greater in red
muscles than in white muscles. FAT/CD36 and
FABPpm proteins in red vesicles or
in homogenates were greater than in white vesicles or homogenates
(P < 0.05). These studies provide
the first evidence of a protein-mediated LCFA transport system in
skeletal muscle. In this tissue, palmitate transport rates are greater
in red than in white muscles because more LCFA transporters are
available.
fatty acid translocase; fatty acid transport protein; plasma
membrane-bound fatty acid binding protein; giant sarcolemmal vesicles
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INTRODUCTION |
CIRCULATING SUBSTRATES provide important fuels for cell
metabolism. Glucose and lactate are known to cross the cell membrane via a facilitated transport system for which a number of transport proteins have been identified (7, 10, 14, 15). Because of the
hydrophobic nature of long-chain fatty acids (LCFAs), it has generally
been assumed that they can rapidly traverse the lipid bilayer of the
cell membrane. This has been demonstrated using purified protein-free
phospholipid bilayers, suggesting that a specific transport system is
unnecessary (24). However, it has been argued that spontaneous
dissociation of LCFAs from their albumin carrier is insufficient to
account for uptake observed in various tissues, as well as the fact
that, at physiological pH, LCFAs bound to albumin exist in an ionized
form, which could hinder their unassisted diffusion across a charged
membrane (6, 35, 45). Recently, evidence has begun to accumulate
showing that LCFA may, in part, enter the cell via a carrier-mediated process in a variety of physiologically important cells, including cardiac myocytes (26, 37), adipocytes (2, 3), and hepatocytes (36, 39).
Unfortunately, in none of these studies has it been possible to divorce
LCFA uptake from LCFA metabolism.
Skeletal muscle is dependent on the oxidation of LCFAs to sustain its
ATP production. Indeed, LCFAs are the major substrate for this tissue,
both at rest and during moderate exercise (18, 33). In resting muscles
we have shown that 90% of the lipid metabolism is provided by
exogenous fatty acids, and only 10% is derived from the endogenous
triacylglycerols (13). With the onset of contraction, the LCFA uptake
by muscle is greatly increased, either in vivo (17) or in vitro (12).
Although it is believed that the increased LCFA uptake into skeletal
muscles is dependent on the increased delivery of LCFAs (18), it has
been reported that LCFA uptake by perfused skeletal muscle is saturable
(41), possibly involving a carrier-mediated process. However, no direct evidence of an LCFA transport system in this tissue has been
demonstrated.
Skeletal muscles are metabolically heterogeneous, with some muscles
being much more dependent on oxidative metabolism than other types of
muscles (4, 13, 30). We have shown that oxidative muscles exhibit much
greater lactate transport (27) and insulin-stimulated glucose transport
capacities (30) than glycolytic muscles. Facilitated transport of these
substrates into muscle is highly correlated with the available number
of transporter proteins for lactate (monocarboxylate transporter 1)
(27, 28) and glucose (GLUT-4) (22, 30). Because red muscles exhibit a
greater capacity for LCFA metabolism than white muscles (13), it is
also possible that the LCFA transport rate in oxidative (red) muscles
will be greater than in glycolytic (white) muscles. Therefore, we
performed studies 1) to investigate the kinetics of palmitate transport into skeletal muscle vesicles, to
determine whether the LCFA transport capacity is greater in red muscles
than in white muscles, and 2) if so,
whether putative LCFA transporters are more abundant in red than in
white muscles. We have developed a procedure to measure palmitate
transport in giant sarcolemmal vesicles, derived from red and white rat
skeletal muscles. Such giant vesicles have previously been used to
examine the transport systems for glucose (31) and lactate (27). An advantage of these vesicles is that they are are oriented right side
out and are devoid of mitochondria (31). Therefore, we were able to
examine LCFA transport in the absence of LCFA metabolism.
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METHODS |
Animals.
Sprague-Dawley rats (male) weighing ~300 g were used in these
studies. They were kept on a reverse 12:12-h light-dark cycle and were
fed rat chow ad libitum. Water was freely accessible. All procedures
were approved by the Committee on Animal Care at the University of
Waterloo.
Giant sarcolemmal vesicles.
Vesicles were prepared as we (27) and others (23, 31) have described
previously. Briefly, rat hindlimb muscles from both legs were divided
into pools of red muscles (vastus intermedius, red vastus lateralis,
soleus, red gastrocnemius, red tibialis anterior) and white muscles
(plantaris, white vastus lateralis, white gastrocnemius, white tibialis
anterior) on the basis of the fiber composition of these muscles as we
have determined it in previous studies (27, 30). The muscle samples
were cut into thin layers (~1-3 mm thick) and incubated for 1 h
at 34°C in 140 mM KCl-10 mM MOPS (pH 7.4), collagenase (150 U/ml),
and aprotinin (0.01 g/ml). The muscle was then washed with KCl/MOPS and
10 mM EDTA, and the supernatant was collected. Percoll (final concentration 16%) and aprotinin were added to the supernatant. This
supernatant was placed at the bottom of a density gradient consisting
of a 3-ml middle layer of 4% Nycodenz (wt/vol) and a 1-ml KCl-MOPS
upper layer. The samples were spun at 60 g for 45 min at room temperature.
After centrifugation, the vesicles were harvested from the interface of
the two upper solutions. The vesicles were diluted in KCl-MOPS and
recentrifuged at 800 g for 30 min.
Vesicles were immediately used for transport experiments. Some of the
vesicles were placed in a blood cell counting chamber and examined with
a phase contrast microscope. Vesicles from a pool of mixed fibers were
photographed and sized from the photomicrographs. Vesicles were also
prepared and stored at
80°C for protein and marker enzyme
analysis.
Fatty acid transport.
Palmitate uptake was measured by addition of unlabeled palmitate
(Sigma) and radiolabeled
[3H]palmitate (0.3 µCi, Amersham) and
[14C]mannitol (0.06 µCi, Amersham) in a 0.1% BSA-KCl-MOPS solution to 40 µl of
vesicles (~80 µg protein). The reaction was carried out at room
temperature for 10 s, unless otherwise specified. Palmitate uptake was
terminated by addition of 1.4 ml ice-cold KCl-MOPS, 2.5 mM
HgCl, and 0.1% BSA. The sample was quickly centrifuged at
maximal speed in a microfuge for 1 min. The supernatant was discarded,
and radioactivity was determined in the tip of the tube. Nonspecific
uptake was measured by adding the stop solution to the membrane before
the addition of the isotopes. To calculate palmitate transport, the
contribution of palmitate diffusion was subtracted from the palmitate
uptake as we have done previously for determining lactate transport
(29) and as has been done by Abumrad et al. (3) for determining LCFA
transport. In experiments in which the inhibition of palmitate uptake
was examined, the inhibitors anti-plasma membrane-bound fatty acid
binding protein (FABPpm; 8.1 µg/µl), phloretin (200 µM), and
sulfo-N-succinimidyl oleate (SSO; 50 µM) were added 30 min before the transport studies were conducted.
Vesicles were also exposed to trypsin (0.5%) for 30 min, after which
the trypsin was washed from the vesicles before palmitate transport was
determined. Competition for palmitate uptake was examined by adding
excess oleate and octanoate during the transport measurements.
Specificity of fatty acid uptake was also examined by comparing the
uptake of palmitate and oleate into the vesicles.
Western blots.
The putative LCFA transporters fatty acid translocase (FAT/CD36) and
FABPpm were measured in giant
vesicles as well as in muscle homogenates, which we (22, 27, 28) have
used for the measurement of other transport proteins. The monoclonal
antibody to CD36 (Cedarlane Laboratories, Hornby, ON, Canada) was used to detect FAT/CD36. In other studies CD36 has been shown to be the
human analog to rat FAT (1). Antibodies against
FABPpm were those used in previous
studies (41). Plasma membranes (80 µg) and prestained molecular
weight markers (Bio-Rad) were separated on 12% SDS-polyacrylamide gels
(150 V for 1 h). Proteins were then transferred from the gel to
Immobilon polyvinylidene difluoride membranes (100 V, 90 min).
Membranes were incubated on a shaker overnight (16 h) in
buffer A [20 mM
Tris · base, 137 mM NaCl, 0.1 M HCl (adjusted to pH
7.5), 0.1% (vol/vol) Tween 20, and 10% (wt/vol) nonfat dried
milk] at room temperature. Vesicle membranes and muscle
homogenate were then incubated with FAT/CD36 in buffer A for 2 h, followed by three washes in
buffer B (i.e.,
buffer A without dried milk; a 15-min
wash and two 5-min washes) followed by incubation for 1 h with donkey
anti-rabbit immunoglobulin G horseradish peroxidase-conjugated
secondary antibody (1:3,000; Amersham, NA 934) in
buffer B. Membranes were washed as
before with buffer B, and then
FAT/CD36 was detected using an enhanced chemiluminescence detection
method by exposing the membranes to film (Hyperfilm-ECL; Amersham,
Oakville, ON, Canada) at room temperature according to the instructions
of the manufacturer. Film was developed and fixed in GBX
fixer/replenisher (Kodak). FAT/CD36 protein band densities were
obtained by scanning the blots on a densitometer connected to a
Macintosh LC computer with appropriate software.
Northern blotting.
Procedures for Northern blotting have been described in detail
elsewhere (44). Briefly, muscles were rapidly excised and frozen in
liquid N2. RNA was extracted from
100-150 mg of muscle by use of the method of Chomczynski and
Sacchi (11). RNA integrity was determined on a 1% formaldehyde
denaturing gel, separated on an agarose gel, and transferred overnight
to an uncharged nylon membrane. To check that RNA (10 µg/lane) was
intact and evenly loaded and to check transfer to the nylon membrane,
RNA was stained with methylene blue to visualize 28S and 18S ribosomal
bands (corrections for total RNA loading errors were made using the 18S
signal RNA). RNA was probed with cDNAs for FAT (1) and fatty acid
transport protein (FATP) (34) labeled with a randomized priming kit.
Filters were prehybridized (1 h) and hybridized overnight (58°C,
6× standard sodium citrate containing 0.5 g/l each of Ficoll,
polyvinylpyrrolidone, and BSA, 0.5% SDS, and 100 g/ml salmon sperm
DNA). After filters were washed, they were exposed to X-ray film at
80°C and to imaging screens for scanning and quantitation.
Determination of maximal enzyme activities and cytoplasmic FABP
content.
For the purposes of enzyme activity determinations, frozen muscle
samples were homogenized in a
K2HPO4
buffer (pH = 8.1) as previously described (5). Muscle homogenates were
subsequently analyzed for maximal
-hydroxybutyrate dehydrogenase
(
-HAD) and citrate synthase (CS) activities as previously described
(13). Potassium-stimulated
p-nitrophenylphosphatase activity
(K+-pNPPase) was assayed in muscle
homogenates and giant sarcolemmal vesicles as described by Ploug et al.
(31). The content of heart type cytoplasmic FABP
(FABPc) in muscles was
determined by a sandwich-type ELISA as previously described (46).
Protein concentrations were determined by the bicinchoninic acid assay
with BSA as a standard.
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RESULTS |
Descriptive characteristics of red and white muscles.
Red [e.g., soleus (SOL) and red gastrocnemius (RG)]
and white muscles [e.g., flexor digitorum brevis (FDB) and white
gastrocnemius (WG)] differ considerably in their capacities for
oxidative and LCFA metabolism. We characterized red and white muscles
on the basis of their enzyme activities and
FABPc content. Red muscles exhibited a greater maximal CS activity than white muscles (Fig. 1). With respect to the potential for lipid
metabolism, red muscles 1) contained
a greater sink for palmitate that is taken up, because the
FABPc content was much higher in
red than in white muscles (Fig. 1), and
2) also demonstrated a greater
capacity for
-oxidation, because the
-HAD activities were more
than twofold greater in red muscles than in white muscles (Fig. 1).

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Fig. 1.
Comparison of citrate synthase activity
(A), -hydroxybutyrate
dehydrogenase ( -HAD) activity
(B), and cytoplasmic fatty acid
binding protein (FABP) concentrations
(C) in selected white and red
skeletal muscles. White muscles: white gastrocnemius (WG) and flexor
digitorum brevis (FDB); red muscles: soleus (SOL) and red gastrocnemius
(RG). Values are means ± SE for 5 experiments of each type.
* P < 0.05, SOL or RG vs.
white muscles (FDB, WG). ** P < 0.05, SOL vs. RG.
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Giant sarcolemmal vesicle characteristics.
To determine carrier-mediated fatty acid transport, it is important to
divorce transport from its metabolism. For this purpose giant
sarcolemmal vesicles obtained from rat skeletal muscles were used. The
giant vesicles were spherical in appearance and averaged 9.8 ± 0.2 µm in diameter. Total vesicular protein consisted of ~90%
nonmembrane-bound protein (Ref. 31 and Bonen, unpublished data).
The vesicles provide an enriched fraction of muscle plasma membranes,
as evidenced by the 27-fold enrichment in
K+pNPPase in plasma membranes (8.2 ± 1.3 µmol · mg
protein
1 · h
1)
compared with muscle homogenates (0.3 ± 0.2 µmol · mg
protein
1 · h
1).
In other studies using the identical vesicle preparation, the characteristics of the vesicles were very similar (31) and were not
contaminated by the sarcoplasmic reticulum or T tubule membranes (31).
Palmitate transport by giant sarcolemmal vesicles.
In preliminary work, palmitate uptake at the highest concentration used
in our studies was linear for up to 25 s in vesicles from red and white
muscles. A linear increase in palmitate uptake also occurred with
increasing quantities of protein (i.e., vesicle quantity; data not
shown). In the present set of experiments, palmitate uptake was
determined over a 10-s period with the use of 80 µg of total
vesicular protein.
Palmitate uptake was determined over a range of fatty acid-to-BSA
ratios designed to yield varying amounts of unbound palmitate. For these purposes we used the calculations of Richieri et al. (32).
The uptake of palmitate into vesicles from pools of red and white rat
hindlimb muscles occurred at a rapid rate, and when corrected for the
diffusible component, palmitate uptake was saturable in both the red
and white vesicles (Fig. 2), suggesting
that palmitate was being transported into these vesicles. Maximal
transport
(Vmax) into the
red vesicles was 1.8 times greater than in the white vesicles (Fig. 2).
The Michaelis-Menten constant
(Km) for
palmitate was ~6 nM in both red and white vesicles. This corresponds
closely to the Km
(6 nM) that can be calculated from the palmitate uptake in perfused rat
hindlimb muscles (41).

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Fig. 2.
Palmitate transport in giant sarcolemmal vesicles obtained from red
(A) and white
(B) muscles (means ± SE;
n = 5 separate experiments).
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It was important to establish that there was an appropriate
intravesicular sink for palmitate. Without this sink palmitate uptake
would be be severely limited, and the plateau in palmitate uptake could
then be due to its limited solubility in an aqueous medium. For this
reason we determined the FABPc
content in vesicles derived from red and white muscle.
FABPc content was 7.6-fold greater
in vesicles from the red muscles than in those from white muscles (Fig.
3). In both types of vesicles, however,
FABPc is calculated to be present
in excess, even at maximal rates of palmitate uptake, because only 3.2 and 22% of the intravesicular
FABPc in red and white vesicles,
respectively, would be occupied with palmitate.

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Fig. 3.
Cytoplasmic FABP content in giant sarcolemmal vesicles obtained from
red and white skeletal muscles (means ± SE of 4 separate red muscle
and 4 separate white muscle vesicle preparations).
* P < 0.05, red vs. white
giant sarcolemmal vesicles.
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To establish that palmitate was indeed transported into the vesicles,
additional experiments were performed. Palmitate uptake was inhibited
by anti-FABPpm, SSO, and trypsin
and was reduced when transport was examined at 0°C (Fig.
4).
Anti-FABPpm also inhibits fatty
acid uptake in hepatocytes (39) and cardiac myocytes (38). The
reduction by phloretin (
50%) is similar to the
phloretin-induced inhibition of fatty acid uptake in adipocytes (3).
Similarly, the inhibition of palmitate uptake by 50 µM SSO
(
43%), a nonpermeable sulfo-N-succinimidyl LCFA derivative
that specifically inhibits LCFA transport in adipocytes (19), was
similar to the inhibition of LCFA uptake (
65%) by 200 µM SSO
in adipocytes (19). It was also observed that palmitate uptake was
lowered in the presence of excess (100 µM) oleate but not in the
presence of octanoate (100 µM) or glucose (1 mM) (Fig. 4). When the
osmolarity was increased from 300 to 600 mosM, glucose transport was
reduced (data not shown) as has been reported previously for glucose
(25), and palmitate transport was increased by 35 ± 5%
(n = 3 experiments). Thus both
substrates enter an osmotically reactive space rather than binding to
the plasma membrane. Differences between glucose and palmitate
responses to the increase in extravesicular osmolarity presumably
reflect a reduction in the intravesicular sink for glucose (i.e., cell
water) and an increase in the intravesicular sink for palmitate, namely
an increase in FABPc
concentrations. The increased
FABPc concentration would reduce
the intravesicular diffusion distance for palmitate once it had been
taken across the sarcolemmal membrane.

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Fig. 4.
Inhibition of palmitate uptake into giant sarcolemmal vesicles by
phloretin (200 µM),
sulfo-N-succinimidyl oleate (SSO, 50 µM), anti-plasma membrane-bound FABP
(FABPpm), trypsin (0.5%),
temperature at 0°C, oleate (100 µM), octanoate (100 µM), and
glucose (1 mM). Values are means ± SE for 3 separate control and
experimental treatments for each condition; data are expressed relative
to untreated control (100%).
* P < 0.05.
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To further examine the fate of the palmitate taken up by the vesicles,
we lysed the vesicles immediately at the end of the transport period.
From these lysed vesicles we were able to recover 100% of the
3H label in the cytosol
compartment (Table 1). It was also found that the intravesicular radiolabel was present in the vesicles as
unesterified
[3H]palmitate (100%)
(Table 1). These data clearly indicate that palmitate sequestration by
these vesicles is due to the traversal of palmitate across the
sarcolemmal membrane rather than to a physical partitioning in the
membranes.
Fatty acid transporters.
Because palmitate was being transported into red and white vesicles at
different maximal rates, it was important to determine whether putative
LCFA transporters were present in greater abundance in red muscles
compared with white muscles. Both FAT mRNA and FATP mRNA were present
in red and white muscles (Fig. 5). The abundance of these transcripts was five- to sixfold greater in red
(SOL) than in white (FDB) muscle (P < 0.05). The FAT/CD36 protein content in the plasma membranes from
red muscles was 1.4-fold greater than in plasma membranes from white
muscles (Fig. 6), whereas
FABPpm was 1.2-fold greater in red
vesicles than in white vesicles (Fig. 6).

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Fig. 5.
Fatty acid translocase (FAT) mRNA and fatty acid transport protein
(FATP) mRNA in red (SOL) and white (FDB) skeletal muscles. Data are
expressed relative to rat heart FAT mRNA (100%) and rat heart FATP
mRNA (100%). Values are means ± SE of 5 SOL and 5 FDB muscles; 10 µg/lane of total RNA were loaded.
* P < 0.05, SOL vs. FDB.
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Fig. 6.
FAT/CD36 and FABPpm proteins in
giant sarcolemmal vesicles obtained from red and white muscles. Values
are means ± SE from 3 experiments; results are based on 80 µg
protein/lane. * P < 0.05, red
vs. white.
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DISCUSSION |
This study is the first to demonstrate
1) that palmitate is transported
across the sarcolemmal membrane of skeletal muscles and
2) that transport into giant
vesicles from red muscles is greater than that into vesicles obtained
from white muscles. In addition, we showed
3) that the transcripts of two
putative LCFA transporters (FAT and FATP) are present in greater
quantities in a red muscle (SOL) than in a white muscle (FDB), and
4) we found a greater content of the
FAT/CD36 and FABPpm protein in homogenates and in membranes obtained from red muscles than in those
obtained from white muscles. A major advantage of the giant sarcolemmal
vesicle preparation is that LCFA transport occurred in the absence of
any LCFA metabolism and esterification. Thus we are now able to examine
LCFA transport into vesicles obtained from a metabolically important
tissue in which LCFA metabolism may be altered by physiological
(exercise), pathological (diabetes), and dietary (fasting) conditions.
Palmitate uptake by red and white muscles.
Skeletal muscles in the rat have a wide range of metabolic capacities.
These can be roughly divided into red and white muscles, reflecting a
great capacity for oxidative and glycolytic metabolism, respectively.
The diversity for LCFA metabolism in red and white muscles has been
shown in recent studies (13). Differences in LCFA uptake in these
studies between red and white muscles could not be attributed to
differences either in the delivery of palmitate or in the
vascularization of these two muscles, because red and white muscles
were incubated with identical palmitate concentrations (13). Therefore,
the differences in palmitate uptake, oxidation, and esterification in
the intact red and white muscles reflect differences in their
biochemical machinery for these processes. One of these processes is
the uptake of palmitate by these muscles. The saturable nature of LCFA
uptake by perfused rat muscle (41) pointed toward a potentially
important role for a protein-mediated transport process. Alternatively,
the saturation of LCFA uptake could also have been related to a
saturation of LCFA metabolism. To establish conclusively whether
cellular LCFA uptake occurs via a carrier-mediated process, a vesicle
preparation was used to examine the transsarcolemmal movement of
palmitate.
The giant sarcolemmal vesicle preparation in the present study offers a
number of advantages over other systems.
1) Metabolism is divorced from
transport in the vesicles, thereby avoiding the potential problems of
esterification and oxidation that have occurred in other preparations
used to date [adipocytes, cardiac myocytes, and hepatocytes (3,
8, 9)] in which LCFA transport has been investigated.
2) The giant sarcolemmal vesicles
(~10 µm in diameter) are more suited for transport studies than
small sarcolemmal vesicles (<1 µm diameter), because the volume of
the larger vesicles avoids the problem of backflux of the substrate
that can occur in small vesicles, making it difficult to obtain initial
rates of transport (31). 3) Finally,
the metabolic distinction, with respect to the uptake of LCFA between
intact red and white muscles, is preserved when LCFA transport is
examined in vesicles derived from red and white muscles.
A number of observations lead us to the conclusion that palmitate
uptake by giant sarcolemmal vesicles occurs via a facilitated transport
system. First, we showed that palmitate uptake was a saturable process
in the giant sarcolemmal vesicles. Second, palmitate uptake was
inhibited by antibodies to FABPpm,
by phloretin [an inhibitor of many transport processes, including
LCFA transport (3, 37)], and by SSO, a specific inhibitor of LCFA
transport in adipocytes (19). Palmitate uptake was also reduced when
the temperature was lowered from 20°C to 0°C, which is largely
taken to be due to a reduction in palmitate transport rather than
diffusion. That palmitate uptake is protein mediated is also shown by
the selectivity of fatty acid transport (i.e., palmitate uptake rate >> octanoate uptake rate) and the competition for transport between oleate and palmitate but not between octanoate and
palmitate. Our experiments also demonstrate that the palmitate was not
esterified, because 1) 95% of the
palmitate was able to efflux from the vesicles when not impeded by
phloretin (data not shown), and 2)
we recovered 100% of the radiolabeled palmitate in the cytosol from
lysed vesicles. Importantly, we also found that all of the radiolabeled
material was palmitate, thereby confirming the complete absence of LCFA metabolism in these vesicles. Collectively, these experiments indicate
very strongly that palmitate was taken up by the vesicles via a
carrier-mediated transport mechanism similar to that described for
adipocytes (3, 8), hepatocytes (8), and cardiac myocytes (8, 26, 37).
However, unlike these latter preparations, in which LCFA metabolism
confounds the determination of transport, the absence of LCFA
metabolism in the giant sarcolemmal vesicles provides an excellent
model for examining LCFA transport in a metabolically important tissue.
The sarcolemmal vesicles used in these experiments contained
FABPc (5.4 µg FABP/mg protein in
red vesicles and 0.7 µg FABP/mg protein in white vesicles) that was
somewhat higher than has been found in intact red skeletal muscles,
such as the SOL (~2 µg FABP/mg protein) and RG (0.75 µg FABP/mg
protein), and in a white muscle, such as the FDB (~0.1 µg FABP/mg
protein) and WG (~0.2 µg FABP/mg protein) (13). The cytosolic FABP
content of the vesicles is critical, because this sink is required to
bind palmitate once it has traversed the sarcolemmal membrane.
Inadequate concentrations of FABPc
could limit the uptake of transport into the vesicles. However, we can
calculate that the giant sarcolemmal vesicles contained more than
adequate amounts of FABPc to
provide the necessary intravesicular sink for palmitate. In the course
of our 10-s experiments to measure palmitate uptake, only 3.2 and 22%
of the FABPc is complexed with
palmitate in red and white vesicles, respectively, at the maximal
palmitate concentrations used in our studies. Alternatively stated, it
appears that FABPc is present in
4.5-fold and 31.3-fold excess in red and white vesicles, respectively.
Thus, as in vivo, most of the intravesicular FABP is not complexed with
fatty acids (47).
Presumably, the LCFA transport system in muscle is functionally
coordinated with the different metabolic capacities for lipid metabolism in red and white muscles. We (13) have clearly established that the capacity for LCFA metabolism is greater in red than in white
muscles. Therefore, we would expect that LCFA transport should also be
greater in red than in white muscles. This was indeed observed: the
Vmax for
palmitate transport was greater in red muscle vesicles than in white
muscle vesicles, whereas the
Km was ~6 nM in
vesicles from both types of muscles. This greater maximal transport
capacity in red muscles appears to reflect the greater number of LCFA
transport proteins in the plasma membranes obtained from these
oxidative muscles.
Sarcolemmal fatty acid transporters.
Recently, Abumrad et al. (1) and Schaffer and Lodish (34) cloned
distinct LCFA transporters. FAT migrates at 88 kDa (1), and FATP
migrates at 63 kDa (34). Previously, Stremmel et al. (40) implicated a
40- to 43-kDa FABPpm with the
transport of fatty acids in liver.
FABPpm is also present in muscle
(42). There is evidence that each of these proteins stimulates fatty acid transport when their respective cDNAs are transfected into various
cell types (16, 20, 21, 34, 43). In our studies, transcripts for the
putative LCFA transporters FATP and FAT were present in red and white
muscles, with red muscle (SOL) containing five- to sixfold more FAT
mRNA and FATP mRNA than the white muscles (FDB). A similar difference
between red and white rodent skeletal muscle FAT mRNA has recently been
shown in other studies (44). Unfortunately, we cannot resolve the
debate as to which of the putative LCFA transporters is more important
in muscle. Thus, although all putative LCFA transporters are
coexpressed in muscle, it is not known whether they are functionally
independent or interdependent. However, studies by Berk et al. (8)
showed that putative fatty acid transporters are regulated in a
tissue-specific manner in diabetic animals and that the three LCFA
transporters (FAT, FATP, and
FABPpm) appear to be selectively
regulated in diabetic animals (8).
In summary, we have provided evidence that palmitate is transported
into skeletal muscle by a mechanism involving membrane-associated fatty
acid transport proteins. Palmitate transport studies were performed
with giant sarcolemmal vesicles obtained from rat hindlimb skeletal
muscles. In these vesicles palmitate was not metabolized and
esterified, and thus transport was divorced from metabolism. Palmitate
transport was greater in red than in white muscle vesicles, consistent
with differences in palmitate uptake and metabolism in isolated, intact
red and white muscles. Skeletal muscles contained FAT mRNA and FATP
mRNA, with five- to sixfold more of these transcripts in red compared
with white muscles. The putative LCFA transporters FAT/CD36 and
FABPpm were greater in vesicles from red muscles compared
with those derived from white muscles.
 |
ACKNOWLEDGEMENTS |
We thank P. M. H. Willemsen, Y. F. de Jong, and Y. Arumugam for
expert technical assistance. We also thank Dr. N. Abumrad, SUNY (Stony
Brook, NY), for providing us with the FAT cDNA and SSO and Dr. J. Schaffer, Washington Unversity School of Medicine (St. Louis, MO), for
providing us with the FATP cDNA.
 |
FOOTNOTES |
This study was supported by a collaborative grant from the Canadian
Natural Sciences and Engineering Research Council, the Medical Research
Council of Canada, the Danish National Research Foundation, National
Institute of Arthritis and Musculoskeletal and Skin Diseases Grant
AR-45168-01, and The Netherlands Heart Foundation.
Address for reprint requests: A. Bonen, Dept. of Kinesiology, Univ. of
Waterloo, Waterloo, ON, Canada N2L 3G1.
Received 6 October 1997; accepted in final form 22 April 1998.
 |
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