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J Biol Chem, Vol. 273, Issue 26, 15920-15926, June 26, 1998
From the The newly cloned proton-linked monocarboxylate
transporter MCT3 was shown by Western blotting and immunofluorescence
confocal microscopy to be expressed in all muscle fibers. In contrast, MCT1 is expressed most abundantly in oxidative fibers but is almost totally absent in fast-twitch glycolytic fibers. Thus MCT3 appears to
be the major MCT isoform responsible for efflux of glycolytically derived lactic acid from white skeletal muscle. MCT3 is also expressed in several other tissues requiring rapid lactic acid efflux. The expression of both MCT3 and MCT1 was decreased by 40-60% 3 weeks after denervation of rat hind limb muscles, whereas chronic stimulation of the muscles for 7 days increased expression of MCT1 2-3-fold but
had no effect on MCT3 expression. The kinetics and substrate and
inhibitor specificities of monocarboxylate transport into cell lines
expressing only MCT3 or MCT1 have been determined. Differences in the
properties of MCT1 and MCT3 are relatively modest, suggesting that the
significance of the two isoforms may be related to their regulation
rather than their intrinsic properties.
Lactic acid is both a major fuel for skeletal muscle ("red"
oxidative fibers) and a major metabolic end product ("white"
glycolytic muscles). Even oxidative fibers become net lactic acid
exporters when oxygen supply cannot meet demand, and glycolysis is
stimulated to maintain ATP supplies. Fatigue occurs when lactic acid
builds up within the myocyte. This causes intracellular pH
(pHi) to drop, inhibiting both glycolysis and contractile
activity (1, 2). In the extreme case further muscle activity is totally prevented, a phenomenon used to advantage by anglers "playing" their fish to exhaustion. The transport of lactic acid out of skeletal
muscle fibers is essential if such intracellular accumulation of lactic
acid is to be prevented. Better removal of lactic acid from the muscle
fibers might improve athletic performance during intense exercise and
enable better muscle function and subsequent recovery under
pathological conditions such as inherited mitochondrial diseases,
hypoxia, and reperfusion following a period of ischemia.
Transport of lactic acid into skeletal muscle fibers for oxidation is
thought to be mediated by the proton-linked monocarboxylate transporter
(MCT)1 isoform MCT1 whose
expression correlates with the oxidative capacity of muscle fibers and
is increased following chronic muscle stimulation (3, 4). However,
sarcolemmal membranes of muscle fibers that are primarily glycolytic do
not contain significant amounts of MCT1 yet transport lactic acid by
means of a saturable carrier that is inhibited by known inhibitors of
MCT1 (3, 5, 6). These data imply the presence of another MCT isoform in
such glycolytic fibers. MCT kinetics in heart (7-9) and liver (10)
cells also imply the existence of other MCT isoforms, and this
conclusion has been confirmed by cloning and sequencing studies.
The first MCT isoform (MCT1) was cloned from Chinese hamster ovary
cells (11) and has since been cloned and sequenced from human (12), rat
(13), and mouse (14). Topology predictions suggest that the transporter
contains 12 transmembrane helical domains in common with many other
substrate transporters, and we have confirmed this prediction
experimentally (15). Another MCT isoform (MCT2) has been cloned from
hamster (16) and rat (17). This is the predominant MCT isoform in
hamster liver, but is not widely distributed in rat tissues and is
absent in skeletal muscle (17). Thus it seems probable that an
additional MCT isoform is responsible for lactic acid efflux from those
muscle fibers lacking MCT1. Recently we have cloned and sequenced four new members of the MCT family and investigated their tissue
distribution by Northern blotting (18). We found that mRNA for one
of these isoforms is present in large amounts in skeletal muscle, and
we termed this isoform MCT3 because of its close homology to chicken MCT3, an MCT isoform found exclusively in the chicken retinal epithelium (19, 20). It remains to be established whether chicken MCT3
and mammalian MCT3 are produced by equivalent genes, perhaps as
alternatively spliced forms (20), or are distinct gene products. Here
we use antipeptide antibodies to confirm that mammalian MCT3 protein is
also strongly expressed in skeletal muscle and in several other
glycolytic cells. We have characterized its kinetics and shown that it
is down-regulated in rat hind limb skeletal muscle by chronic
denervation.
Materials
Ehrlich Lettré tumor cells (ELT), the bovine kidney cell
line NBL1, and COS cells were cultured as described elsewhere (21-23). Isolated rat cardiac myocytes, prepared as described previously (8),
were kindly provided by Dr. Elinor Griffiths and human cardiac
ventricle muscle by Professor Giannni Angelini (Bristol Heart
Institute). Human white cells were obtained from the local blood bank
and human placenta from the local maternity hospital. Antipeptide
antibodies to MCT1 and MCT3 were raised in rabbits against C-terminal
peptides, conjugated to keyhole limpet hemocyanin as described
previously (15). The peptides used, including an N-terminal cysteine
for coupling to keyhole limpet hemocyanin, were CQKDTEGGPKEEESPV for
human MCT1 (cross-reacts with MCT1 from most species),
CPQQNSSGDPAEEESPV for Chinese hamster MCT1 (cross-reacts with rat but
not human MCT1), and CEPEKNGEVVHTPETSV for human MCT3. Antibodies were
purified by affinity purification using the immobilized peptide as
described previously (15). All reagents for immunofluorescence
microscopy were from Sigma.
Methods
Detection of MCT Isoforms in Cell and Tissue Membrane
Fractions--
Preparation of crude plasma membranes from a variety of
tissue homogenates was performed in the presence of a range of protease inhibitors (24) and samples separated by SDS-PAGE and analyzed by
Westen blotting with anti-MCT1 and -MCT3 antibodies using ECL detection
(15, 17). For preparation of plasma membranes from cell lines the
initial cell disruption involved homogenization with a Potter-Elvehjem
homogenizer (50 strokes) followed by 20 passes through a fine
(25-gauge) gauge needle.
Measurement of MCT Isoforms in Individual Muscles--
Where
required, denervation or chronic stimulation of rat hind limb muscles
was performed as described previously (4, 5); the procedures had
received ethical approval by the relevant committees. An untreated
contralateral limb always acted as a control for the treated limb of
the same animal. Muscles were dissected out, frozen in liquid nitrogen,
and stored at Histochemistry and Immunofluorescence Confocal
Microscopy--
Skeletal muscles were dissected from the hind limbs of
freshly killed rats under a relaxing solution (127 mM
KCH3SO4, 13 mM KCl, 5 mM HEPES, pH 7.4) before preparation of frozen sections (10 µm), while isolated cardiac myocytes were added directly to polylysine-coated coverslips. Unless otherwise stated, all subsequent protocols were carried out in phosphate-buffered saline (PBS) at pH
7.3. Staining of sections for succinate dehydrogenase was performed as
described by Spurway et al. (25). For immunohistochemistry fixation was with 4% paraformaldehyde for 30 min and permeabilization with 0.3% (v/v) Triton X-100 for 45 s. Nonspecific binding of antibody was reduced by blocking for 60 min with 10% (v/v) normal swine serum. Incubation with primary antibody (1 in 100) supplemented with 1% (w/v) bovine serum albumin was for 45 min. Following three washes in PBS containing 1% bovine serum albumin, samples were again
blocked with normal swine serum before addition of rhodamine-conjugated anti-rabbit IgG and incubation for 45 min at room temperature. After
washing, samples were mounted in Mowiol (Calbiochem) and examined with
a Leica TCS-NT confocal scanning microscope (63 × 1.32Na oil
immersion objective).
Measurement of Monocarboxylate Transport into Cultured
Cells--
After washing twice with PBS, cells were detatched from the
culture flasks by incubation with trypsin solution (Sigma) for 5 min
(COS) or 30 min (NBL1), followed by inactivation of the trypsin with
PBS containing 10% (w/v) fetal bovine serum. Cells were then washed
twice in PBS and once in transport buffer (150 mM NaCl, 5 mM KCl, 1 mM KH2PO4, 1 mM MgSO4, 0.2 mM CaCl2,
3.3 mM MOPS, 10 mM HEPES, pH 7.4) before
incubating in transport buffer containing 5 µM BCECF/AM
(Boehringer Mannheim) for 20 min at room temperature. Loaded cells were
collected by centrifugation and resuspended to about 5.107
cells/ml in transport buffer. A sample (10 µl) of these cells was
then placed in a 50-µl perspex incubation chamber on
polylysine-coated coverslips, held in a thermostatically controlled
incubation vessel attached to the stage of a Nikon Diaphot inverted
microscope. After allowing cells to fix to the coverslip for 5 min,
unattached cells were washed away by flowing buffer at 2 ml/min through
the incubation chamber. Buffer inflow was regulated by an eight-roller peristaltic pump and outflow by a second pump removing buffer from the
distal end of the incubation chamber so as to maintain a constant
volume. The objective was then focused on a suitable group of cells, of
which four to six cells were selected with a rectangular diaphragm.
Measurement of changes in BCECF fluorescence was made with a Cairn
Instruments spinning wheel fluorescence attachment with excitation at
440 and 490 nm and emission at >510 nm as described previously (8, 9).
The filter wheel was rotated at 32 rpm and data averaged to display 2 data points/s. Changes of substrate and inhibitor concentration were
enabled by rapid switching (solenoid valves) between solutions from
eight thermostatically controlled vessels containing buffer with the required additions. If desired, solutions could be mixed in any combination before delivery to the incubation vessel. Complete change
of substrate or inhibitor concentration within any point of the
incubation vessel took <1 s, allowing accurate measurement of initial
rates of transport.
Tissue Distribution of MCT3--
Antipeptide antibodies to the C
terminus of human MCT3 have been raised and used in Western blotting to
determine the presence of MCT3 in plasma membranes from various rat and
human tissues. Data are shown in Fig. 1.
We subsequently cloned and sequenced rat MCT3
cDNA2 and confirmed that
it exhibited 100% identity over the region against which the antibody
was raised. MCT3 was detected in plasma membranes from rat hind limb
muscle (primarily white muscle), which contained very little MCT1. In
contrast no MCT3 was detected in membranes from rat liver and heart or
from brain, kidney, and testis (data not shown), all of which contained
large amounts of MCT1. In available human tissues we found MCT3 in
membrane preparations from heart, placenta, and white blood cells (Fig. 1), whereas MCT1 was detected only in membranes from heart. In all
cases care was taken to inhibit proteases that might destroy the
epitope detected by the antibody, and thus, the absence of a particular
MCT in the membrane preparations appears to be real. The lack of MCT3
in rat heart is puzzling in view of its presence in human heart, but
the result was confirmed using immunofluorescence confocal microscopy
of isolated rat heart cells. This readily visualized the presence of
MCT1, especially in the intercalated disk and T-tubular regions as
reported previously (11, 26), while detecting no specific binding of
the MCT3 antibody (data not shown).
Lactic Acid Efflux from White Skeletal Muscle Is Catalyzed by the
Monocarboxylate Transporter Isoform MCT3*
,§,,¶,
,,,, and§§
Department of Biochemistry, School of
Medical Sciences, University of Bristol, Bristol BS8 1TD, United
Kingdom, the Copenhagen Muscle Research Centre, August Krogh
Institute, University of Copenhagen DK2100, Copenhagen, Denmark, the
** Department of Kinesiology, University of Waterloo, Waterloo, Ontario
N2L 3G1, Canada, and the Department of
Physiology, University of Glasgow, Glasgow G12 8QQ, Scotland
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
80 °C. Samples of muscle (40 mg) were homogenized
(Polytron 2100, 2 × 30 s on maximum setting) in 1 ml of
buffer (250 mM sucrose, 30 mM HEPES, 2 mM EGTA, 40 mM NaCl, 2 mM
phenylmethylsulfonyl fluoride, pH 7.4). After centrifugation at
200,000 × g for 90 min at 4 °C, the pellet was
resuspended in 500 µl of Tris-SDS (10 mM Tris, 4% (w/v)
SDS, 1 mM EDTA, pH 7.4) and homogenized (2 × 15 s with a Polytron). Following protein determination (Bio-Rad,
detergent-compatible protein assay), samples were diluted to 2 mg of
protein/ml and an equal volume of sample buffer added. Samples (20 µl) were then subjected to SDS-PAGE (8-18% gradient gel) and
Western blotting as above. Quantification was performed by scanning the
film and analysis of band intensities with "Band Leader", a
shareware program. As described previously (3), by using increasing
concentrations of homogenate it was confirmed that in all cases protein
loading was such that the response of the film was linear with respect
to the amount of MCT present. Samples of contralateral control muscle
homogenates were analyzed on the same gel as samples (containing the
same amount of protein) from denervated or chronically contracting muscles.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (48K):
[in a new window]
Fig. 1.
Relative expression of MCT1 and MCT3 in crude
plasma membrane preparations from different cells and tissues.
Membrane preparations from skeletal muscle (Msc), heart
(Hrt), liver (Liv), placenta (Plc),
white blood cells (Wbc), NBL1 cells, Ehrlich Lettré
tumor cells (ELT), and COS cells were separated by SDS-PAGE
and Western blots probed with anti-MCT1 or -MCT3 antibodies.
MCT1 and MCT3 Expression in Different Muscle Types-- Skeletal muscles are often referred to as either white or red depending on their myoglobin and mitochondrial content. More precisely, muscle fibers may be divided into three groups. Fast-twitch glycolytic (FG) fibers have few mitochondria and are almost exclusively glycolytic in their energy metabolism. Fast-twitch oxidative glycolytic fibers (FOG) have more mitochondria and are both oxidative and glycolytic, while slow-twitch oxidative fibers (SO) have the highest mitochondrial content and are specialized for oxidative energy metabolism (2). MCT1 expression in individual muscles correlates with their mitochondrial content, suggesting that its expression may reflect the need to transport lactic acid into the cell for oxidation (3). In Fig. 2 we report the MCT1 and MCT3 content of different muscle types, measured by immunoblotting of homogenates of individual rat muscles. Unlike MCT1, MCT3 was found to be expressed to a similar extent in almost all muscle types irrespective of their fiber content. An exception was soleus muscle, which contains mainly SO fibers, where MCT3 expression was found to be significantly lower. These data were confirmed with immunofluorescence confocal microscopy using specific MCT1 and MCT3 antibodies. Data are shown in Fig. 3 for four rat muscles, white gastrocnemius (16% FOG and 84% FG), semimembranosis (7% SO, 43% FOG, 50% FG), semitendinosis (7% SO, 45% FOG, 48% FG), and soleus (87% SO, 13% FOG). Muscle sections were also stained for succinate dehydrogenase, and data are shown for soleus and semitendinosis. These data confirm that all fibers in the soleus, but only the small fibers in the semitendinosis and semimembranosis, have high oxidative capacity, and these were also the fibers that expressed most MCT1.
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, 
) and are expressed as
means ± S.E. (error bars) of three or six to eight
muscle samples from separate rats respectively. The x axis
represents the percentage of oxidative fibers (slow-twitch oxidative + fast-twitch oxidative glycolytic) in each muscle as reported elsewhere
(
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Regulation of MCT3 Expression-- We have previously shown that chronic electrical stimulation of rat hind limb muscles for 7 days produced a 2-3-fold increase in MCT1 expression in the red and white tibialis anterior and extensor digitorum longus muscles (4). However, with the same experimental treatment we have found no significant change in MCT3 expression (data not shown). In contrast, the data of Fig. 4 show that denervation for 3 weeks gave a significant decrease in MCT3 expression (expressed per mg of total homogenate protein) in soleus (56.1 ± 5.8%), red gastrocnemius (39.8 ± 7.9%), white gastrocnemius (52.6 ± 11.4%), and white tibialis anterior (56.0 ± 7.7%) muscles. A smaller decrease (10-20%, but not statistically significant) was observed after only 3 days denervation. MCT1 expression also decreased significantly after 3 weeks denervation in soleus (52.6 ± 7.0%) and red gastrocnemius (60.8 ± 11.5%) muscles, while expression in white gastrocnemius and white tibialis anterior muscles was too low to detect any further decrease. In earlier experiments we demonstrated that after 3 weeks denervation the rate of lactate transport out of giant sarcolemmal vesicles prepared from white gastrocnemius, red gastrocnemius, and soleus decreased by 36, 41, and 50%, respectively (5).
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Characterization of the Kinetics of MCT3-- In order to characterize the kinetics of MCT3, it is necessary to express the active protein in a cell line that contains no endogenous MCT activity. We have been unable to find such a mammalian cell line; indeed, even the human breast tumor cell line used by Garcia et al. (11) to express MCT1 has significant endogenous MCT activity (data not shown). This is not surprising since all mammalian cells are partially glycolytic and many immortal cells exclusively so, necessitating rapid rates of lactic acid efflux (1). In contrast it has been reported that some insect cell lines do lack MCT activity (16), and we have confirmed this for Spodoptera f21 and Drosophila Schneider cells. However, we have been unsuccessful in establishing a stable MCT3-transfected insect cell line using a variety of expression vectors. Thus we decided to look for a mammalian cell line that expressed MCT3 but no other MCT isoform. For this purpose we screened a range of cell lines using Western blotting with antipeptide antibodies that we have raised against C-terminal peptide sequences specific for each of the different MCT isoforms. As shown in Fig. 1 we have confirmed that mouse ELT tumor cells express only MCT1 as suggested by our previous studies (14), while the bovine kidney cell line NBL1 express only MCT3. Although not shown in Fig. 1 we have also been unable to detect any protein reacting with antipeptide antibodies we have raised to MCT2, MCT4, MCT5, MCT6, or MCT7 in either ELT or NBL1 cells. The COS cell line derived from monkey kidney also appeared to express mainly MCT3. However, the MCT1 antibody did give a faint band of about 43 kDa and also an additional band of about 85 kDa, both of which were abolished in the presence of MCT1 peptide. Thus it is likely that significant amounts of MCT1 are present in COS cells, the 85-kDa band probably reflecting dimeric MCT1, since this protein is known to aggregate in detergent (27).
For each of these three cell lines monocarboxylate transport was measured using the intracellular pH-sensitive fluorescent dye, BCECF, which detects the rapid decrease in pHi that accompanies proton-linked transport (8-10, 21). Data are shown in Fig. 5. Measurement of the true initial rate of fluorescence change was possible by fitting the curve to a first order uptake equation and subsequently converted into a rate of monocarboxylate transport in nanomoles per µl intracellular volume per minute (10, 21). The latter calculation required measurement of the pH gradient across the plasma membrane (0.1 pH units acid inside) determined using butyrate and trimethylamine and the total change in fluorescence ratio induced by increasing L-lactate concentrations once equilibrium had been reached. For each cell type, initial rates of monocarboxylate transport were measured on three or four different fields of cells at 1, 2, 5, 10, 20, and 30 mM L-lactate, D-lactate, and pyruvate. Results are summarized in Fig. 6 and the derived Km and Vmax values presented in Table I.
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), and pyruvate
(
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-cyanocinnamate derivatives, stilbene
disulfonates, and phloretin) has indicated that different MCT isoforms
may have different inhibitor specificities (1, 7-10). Thus we have
determined K0.5 values for these compounds as
inhibitors of the transport of 4 mM L-lactate
into each of the three cell types. Data are shown in Fig.
7 and Table I. For ELT cells
K0.5 values for -cyano-4-hydroxycinnamate
(0.45 mM), DIDS (0.64 mM), and phloretin (4.4 µM) were quite similar to the values (0.17 mM, 0.43 mM, and 5.1 µM,
respectively) measured previously using cell suspensions (21). For NBL1
cells the K0.5 values for
-cyano-4-hydroxycinnamate and phloretin were 1.0 mM and
3.2 µM, respectively, while for DIDS the pattern of
inhibition was more complex. It appeared that there was a
noninhibitable component present (about 20% of the uninhibited
rate) with the inhibitable component having a
K0.5 for DIDS of 28 µM. DIDS has
also been shown to exhibit partial inhibition in rat heart cells, with
a K0.5 of about 79 µM (9). Data
for the inhibition or L-lactate transport into COS cells
are also given in Fig. 7 and Table I. K0.5
values are more similar to the data for NBL1 cells than for ELT cells,
and this is consistent with the large amount of MCT3 in both cell lines
(Fig. 1).
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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MCT3 Is a Major Route for Lactic Acid Efflux from White Skeletal Muscle and Other Cells-- The correlation of MCT1 expression with the oxidative capacity of skeletal muscle fibers and its low levels in fast-twitch glycolytic fibers has led us to propose that this MCT isoform is important for the transport of lactic acid into muscle fibers for use as a respiratory fuel (3, 4). This implies that another MCT isoform must be responsible for lactic acid efflux from fibers that are primarily glycolytic and express little MCT1. The data we present here suggest that MCT3 fulfils this role. It is present in all muscle fibers, but expressed least in totally oxidative fibers such as the soleus (Figs. 2 and 3). The abrupt decrease in MCT3 expression between red gastrocnemius (SO 30%, FOG 62%, FG 8%) and soleus (87% SO, 13% FOG) suggests that it is the slow oxidative fibers that are especially low in MCT3, but more detailed analysis will be required to confirm this. There is likely to be an important role for MCT3 in lactic acid export in other cells; for example MCT3 is the only MCT expressed in human white blood cells, which are also highly glycolytic lactic acid exporters. Its presence in two kidney cell lines (COS and NBL1 cells) may also reflect a requirement of the cells from which they were derived (renal tubular epithelium) to export lactic acid taken up from the tubule lumen across the basolateral membrane and into the blood.
This does not mean that MCT1 has an exclusive role in lactic acid influx and MCT3 in lactic acid efflux. Indeed, the data shown in Fig. 2 demonstrate that the correlation between MCT isoform expression and percentage of oxidative fibers is not that precise. There is extensive evidence to show that the different MCT isoforms can catalyze proton-linked monocarboxylate transport in both directions with the Km and Vmax values for influx and efflux obeying the Haldane equation (see Refs. 1, 2, and 8). Furthermore, the data of Fig. 5 confirm that MCT1 and MCT3 catalyze both lactic acid influx and efflux; removal of lactate from the superfusion medium leads to a rapid return of pHi to basal levels as lactic acid leaves the cell. There are also no major differences in the kinetics of the two isoforms that might make MCT1 more efficient for lactic acid influx and MCT3 for lactic acid efflux. Thus it would seem most likely that the presence of two isoforms allows greater flexibility in the regulation of lactic acid transport. Under conditions that are associated with high rates of glycolytic lactic acid production, MCT3 expression may be favored (FG and FOG fibers), while when high rates of lactic acid oxidation are required, MCT1 expression may predominate (SO fibers).Comparison of the Properties of MCT1 and MCT3 with the Characterisitics of Lactate Transport into Skeletal Muscle-- A comparison of the kinetics of MCT3 with the kinetics of monocarboxylate transport into skeletal muscle fibers is difficult for two reasons. First, the properties of the transporter may vary according to the cellular environment as a comparison of MCT1 kinetics in red blood cells and tumor cells illustrates (21). This may partially reflect the ability of MCT1 to interact specifically with other membrane proteins (27). Second, there are inherent problems in measuring monocarboxylate transport across the sarcolemmal membrane. Radiotracers have been used in the perfused rat hind limb, a cultured myocyte cell line that can form myotubules, and in isolated sarcolemmal vesicle preparations (see Refs. 1-4). However, the first preparation is incapable of giving accurate kinetic data for plasma membrane transport, while sarcolemmal vesicle preparations may contain vesicles of different sizes and are usually derived from more than a single fiber type containing an unknown mixture of MCT1 and MCT3. These factors complicate kinetic analysis, although the problems are minimized by using giant vesicles (2). Km values for L-lactate derived using such vesicles are in the range 12-40 mM and are similar whether vesicles are derived mainly from white or red fibers (28). This is consistent with the similar Km values of MCT1 and MCT3 for L-lactate described here. Myotubules should be capable of giving accurate transport data, and the derived Km value for L-lactate in this system was 12.5 mM, again similar to the values we have derived. However, the profile of MCT isoforms in this cultured myocyte cell line has not been established. In frog sartorius muscle microelectrode measurements of single fibers gave a Km value for L-lactate of 10 mM (29). These data on a single white muscle fiber probably most closely represent MCT3 kinetics. Indeed, the Km is the same as that reported here for NBL1 cells, which also contain only MCT3. Km values for the transport of pyruvate and D-lactate and the Ki values for CHC, DIDS, and phloretin cannot easily be compared with data obtained for sarcolemmal membrane vesicles or the perfused hind limb, since there is a great deal of variation in the literature (2). This may be due to the limitations discussed above.
Regulation of MCT3 Expression in Skeletal Muscle-- We present data in this paper to show that denervation of rat hind limb muscles down-regulates expression of both MCT1 and MCT3 in white gastrocnemius, red gastrocnemius, and soleus muscles. This parallels the decrease in rate of lactate efflux measured in giant sarcolemmal vesicles prepared from the same muscles and reflects the ability of denervation to diminish the activity of both oxidative and glycolytic fibers. The effect is not due to a general decrease in muscle protein, since the control and denervated samples used for SDS-PAGE contained the same amount of protein. Furthermore, we have shown previously that denervation increases GLUT-1 expression, while decreasing GLUT-4 expression (30), and that the decrease in succinate dehydrogenase and lactate dehydrogenase activities is less than MCT1 and MCT3 (5). In contrast to denervation, chronic stimulation of the rat hind limb up-regulates lactate transport and MCT1 expression without affecting MCT3 expression. The rate of stimulation used (10 Hz) induces a rate of contraction that mimics activation of oxidative fibers, and this is reflected in an increase in citrate synthase expression in both SO and FOG fibers (4). Whatever the mechanisms involved, it is clear that the expression of MCT1 and MCT3 can be separately regulated, perhaps in response to increased oxidative and glycolytic metabolism, respectively.
MCT3 and the Heart-- It might be expected that heart myocytes should also contain both MCT1 and MCT3, since like skeletal muscle fibers, they can both oxidize lactic acid and, when hypoxic, produce large quantities of lactic acid by glycolysis. The presence of MCT1 in heart cells is well established, but it is located primarily in the intercalated disk and T-tubule regions (11, 26, 31), whereas transport occurs more rapidly in the center of the cell where MCT1 expression is less (31). This implies the presence of another MCT isoform, and we have provided extensive kinetic evidence for the presence of two distinct MCT isoforms in both rat and guinea pig cardiac myocytes (7-9, 31). The properties of MCT1 and MCT3 described here are consistent with these being the two isoforms present in the heart. Thus in heart cells stilbene disulfonates such as DIDS also give only partial inhibition, and this inhibition appears to be of an isoform with a lower Km for pyruvate and D-lactate than the stilbene disulfonate-insensitive isoform but with a similar Km for L-lactate. Both isoforms have a similar K0.5 for phloretin. However, although we were able to detect large amounts of both MCT1 and MCT3 in sarcolemmal membrane from human heart, we were barely able to detect MCT3 in an equivalent membrane preparation from rat hearts (Fig. 1). We have also been unable to detect MCT3 mRNA in rat heart by either Northern blots or reverse transcription-polymerase chain reaction analysis. Neither did immunofluorescent confocal microscopy reveal any MCT3 in isolated rat heart cells (data not shown). Thus the absence of MCT3 in rat muscle, but its presence in human heart muscle, represents a species difference and implies that another MCT isoform with similar properties to MCT3 may be present in these cells.
In summary, MCT3 appears to be the MCT isoform expressed most in muscle fibers whose energy metabolism is mainly glycolytic, whereas MCT1 predominates in muscles that rely more on oxidative metabolism. Denervation of rat hind limb muscles decreases the expression of both MCT3 and MCT1, whereas chronic stimulation of the muscle increases expression of MCT1 but not MCT3. Differences in the kinetics of MCT1 and MCT3 are relatively modest and suggest that the significance of the two isoforms may be related more to their regulation than to any minor differences in their functional properties.| |
ACKNOWLEDGEMENTS |
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We thank Dr. Mark Jepson, Manager of the School of Medicine Sciences Imaging Facility at the University of Bristol, for his assistance in the confocal microscopy.
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Note Added in Proof |
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Very recently Philp et al. (Philp, N. J., Yoon, H., and Grollman, E. F. (1998) Am. J. Physiol. 274, R1824-R1828) have identified an additional rat MCT isoform (GenBankTM accession number AF059258), which has a similar degree of identity to chicken MCT3 as does our MCT isoform referred to as MCT3 in this paper and Ref. 18. However, the new rat MCT isoform of Philp et al. has the same exclusive retinal pigment epithelial distribution as chicken MCT3. Thus the MCT isoform expressed in mammalian muscle that is referred to as MCT3 in the present paper will in future be termed MCT4. Three further MCT isoforms identified by searching dbEST and subsequently cloned and sequenced (this paper and Ref. 18) will in future be referred to as MCT5, MCT6, and MCT7.
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FOOTNOTES |
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* This work was supported by The Wellcome Trust (United Kingdom), the British Heart Foundation, the Natural Sciences and Engineering Research Council of Canada, The Heart and Stroke Foundation of Ontario, The Danish National Research Foundation, and by Medical Research Council (United Kingdom) Award G4500006 (for providing an infrastructure to establish the School of Medical Sciences Imaging Facility at the University of Bristol).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: Dept. of Biochemistry, University of Glasgow, Glasgow G12 8QQ, Scotland.
¶ Present address: Hannah Research Institute, Ayr KA6 5HL, Scotland.
§§ To whom correspondence should be addressed. Tel.: 44-7-9288592; Fax: 44-7-9288274; E-mail: a.halestrap{at}Bristol.ac.uk.
1
The abbreviations used are: MCT, monocarboxylate
transporter; ELT, Ehrlich Lettré tumor; PAGE, polyacrylamide gel
electrophoresis; PBS, phosphate-buffered saline; MOPS,
4-morpholinepropanesulfonic acid; BCECF,
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; DIDS,
4,4'-diisothiocyanostilbene-2,2'-disulfonate; FG, fast-twitch glycolytic fiber; FOG, fast-twitch oxidative glycolytic fiber; SO,
slow-twitch oxidative fiber; CHC, -cyano-4-hydroxycinnamate.
2 V. N. Jackson, N. T. Price, and A. P. Halestrap, unpublished data (GenBankTM Accession number U87627).
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Abstract
Introduction
Procedures
Results
Discussion
References
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