Monocarboxylic acid transporters, MCT1 and MCT2, in cortical
astrocytes in vitro and in vivo
Rekha
Hanu1,
Mary
McKenna2,
Andrea
O'Neill1,
Wendy G.
Resneck1, and
Robert J.
Bloch1
Departments of 1 Physiology and
2 Pediatrics, School of Medicine, University
of Maryland, Baltimore, Maryland 21201
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ABSTRACT |
We used sequence-specific antibodies to
characterize two monocarboxylic acid transporters, MCT1 and MCT2, in
astrocytes. Both proteins are expressed in primary cultures of cortical
astrocytes, as indicated by immunoblotting and immunofluorescence. Both
MCT1 and MCT2 are present in small, punctate structures in the
cytoplasm and at the cell membrane. Cells showing very low levels of
labeling for glial fibrillary acidic protein (GFAP) also label more
dimly for MCT2, but not for MCT1. In vivo, double-label
immunofluorescence studies coupled with confocal microscopy indicate
that MCT1 and MCT2 are rare in astrocytes in the cortex. However, they
are specifically labeled in astrocytes of the glial limiting membrane
and in white matter tracts. Both transporters are also present in the
microvasculature. Comparison of labeling for MCT1 and MCT2 with markers
of the blood-brain barrier shows that the transporters are not always
limited to the astrocytic endfeet in vivo. Our results suggest
that the level of expression of monocarboxylic acid transporters MCT1
and MCT2 by cortical astrocytes in vivo is significantly lower
than in vitro but that astrocytes in some other regions of the
brain can express one or both proteins in significant amounts.
blood-brain barrier; immunofluorescence; plasma membrane; lactate
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INTRODUCTION |
THE METABOLISM OF DIFFERENT compounds by cells in the
brain requires that each of these compounds, or their precursors, be transported across the plasma membranes of neurons or glial cells. Glucose, the most important source of metabolic energy in the adult
brain, is transported into the brain and then into individual cells by
glucose transporters. Developing brain, however, can effectively
utilize monocarboxylic acids such as 3-hydroxybutyrate, acetoacetate,
lactate, and pyruvate for energy (7, 10, 23, 24, 39, 40). Indeed, there
is strong evidence that these substrates, rather than glucose, are
preferentially utilized for energy and the biosynthesis of lipids and
amino acids during the early neonatal period (7, 33, 40). The role of
these molecules in brain metabolism is generally thought to decrease as
the brain matures. Nevertheless, cells in the adult brain continue to
produce large amounts of monocarboxylic acids through normal metabolism (8, 17, 23, 34, 37). Furthermore, lactate and ketone bodies released by
astrocytes (3) may serve as important carbon sources for other brain
cells, especially neurons (2, 8, 27, 34, 38). These observations
suggest that cells in the adult brain express significant levels of the
transporters that mediate the uptake and release of monocarboxylic acids.
This study focuses on the monocarboxylic acid transporters (MCT) in
astrocytes in vitro and in vivo. There is now
considerable evidence that the carrier-mediated transport of lactate by
astrocytes (4, 15, 27, 31, 38), synaptic terminals (28), and neurons
(9, 31) is mediated by multiple transporters that are subject to
differential regulation. The kinetics and susceptibility to mercurials
of lactate uptake in vitro are consistent with a possible role for MCT1
and MCT2 (13, 14, 20, 28, 38). MCT1 and MCT2 are the first members to
be cloned and sequenced of what has recently been recognized as a
family of at least seven homologous proteins that mediate the transport
of monocarboxylic acids in a wide variety of cells (36). The proteins
in this family have molecular masses of 40-60 kDa and contain 12 putative transmembrane domains that, together with large regions of the NH2-terminal domain, share a high degree of homology.
Significantly, however, the COOH-terminal sequences of each of the MCT
are different, allowing the preparation of sequence-specific antibodies
that can distinguish among the different family members. Using
sequence-specific antibodies to MCT1 and MCT2 and ultrastructural
approaches, Gerhart et al. (15, 16) showed that in the brain these
transporters are selectively enriched in astrocytes in the glial
limitans, in ependymocytes of the lateral ventricle, and in the
microvasculature. With the exception of the localization of MCT1 in
astrocytic endfeet, however, these in vivo studies did not
unambiguously identify these or other labeled structures as astrocytes.
These immunocytochemical results have recently been complemented by in
situ hybridization studies (33, 34). Koehler-Stec et al. (22) also
found evidence of high levels of expression of MCT1 in the
microvasculature and in the ependymal lining of the cerebral ventricles
in mouse brain. In addition, they reported the presence of MCT1 mRNA in
white matter tracts, such as the corpus callosum. Using RT-PCR and
Northern blotting, Broer et al. (4) showed that cultures of astroglial
cells contain mRNA encoding MCT1, but little mRNA encoding MCT2. Thus
studies of cells in vitro and in vivo have yielded somewhat different results.
We have now reinvestigated the question of the localization of MCT1 and
MCT2 in astrocytes both in vitro and in vivo, using double-label
immunofluorescence techniques coupled with confocal laser scanning
microscopy. Here we report that neonatal rat astrocytes in primary
culture express significant levels of both MCT1 and MCT2. Using the
same techniques, we confirm previous results indicating that the
expression of the two transporters is restricted to limited populations
of astrocytes in the mature brain (15, 16). However, we also
demonstrate that astrocytes in specific regions of the brain can indeed
express MCT1 or MCT2 at their cell surfaces, where they do not appear
to be restricted to glial endfeet.
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MATERIALS AND METHODS |
Materials.
Tissue-culture dishes (Nunc) were purchased from Vanguard International
(Neptune, NJ). Culture media (MEM with Earle's salts and nonessential
amino acids), and Dulbecco's PBS were purchased from BioWhittaker
(Walkersville, MD). Fetal bovine serum was from HyClone (Logan, UT).
Nylon screening cloth (Nitex) was from Tetko (Elmsford, NY).
Timed-pregnant female rats were purchased from Zivic Miller
Laboratories (Zelienople, PA).
Affinity-purified chicken antibodies to the COOH-terminal sequences of
MCT1 and MCT2 were from Chemicon International (Temecula, CA). The
antigenic peptides were a gift from Dr. L. Drewes (Univ. of Minnesota,
Duluth, MN). Mouse monoclonal antibodies to a blood-brain barrier
antigen (SMI 71) and to glial fibrillary acidic protein (GFAP) were
from Sternberger Monoclonals (Baltimore, MD). Rabbit antibodies to GFAP
and to the erythroid isoform of the glucose transporter, GLUT1, were
from East Acres Biologicals (Southbridge, MA). A rabbit antibody
against S-100 (S2644) and a mouse antibody to a 58-kDa protein of the
Golgi apparatus (G2404) were from Sigma Chemical (St. Louis, MO). Mouse
antibody against vimentin was from ICN Biomedicals (Costa Mesa, CA).
Cell culture.
Primary cultures of cortical astrocytes were prepared as described (27,
29). Briefly, cerebral hemispheres were removed, placed in medium,
cleaned of meninges, and trimmed to retain the neopallium. The isolated
neopallia were dissected from cerebral hemispheres from newborn rat
brain, minced, mechanically disrupted by vortexing, and filtered
through sterile nylon screening cloth. The cell suspension, enriched in
astrocytes, was seeded in medium (MEM with Earle's salts and
nonessential amino acids and 10% fetal bovine serum, 1 ml/brain) in
plastic culture flasks for Western blotting, or on glass coverslips for
immunofluorescence studies. The cells were incubated at 37°C in an
atmosphere of 95% air-5% CO2 with 90-95% humidity.
The culture medium was replaced after 3 days and twice weekly
thereafter. All experiments were done after 10-11 days, when
cultures were usually ~50% confluent.
Western blotting.
Cultures in tissue-culture flasks were tapped sharply three times and
then rinsed twice with PBS to loosen and remove oligodendrocytes (29).
The remaining astrocytes were scraped with a rubber policeman into a
minimal volume of PBS supplemented with a mixture of protease inhibitors (0.15 mM phenylmethylsulfonyl fluoride, 0.22 U/ml aprotinin, 1 mM benzamide, 10 µg/ml leupeptin, 10 µg/ml antipain, and 200 µg/ml soybean trypsin inhibitor). The samples were then homogenized briefly with a Dounce homogenizer in hypotonic buffer (20 mM
Tris · HCl, 40 mM NaCl, and 1 mM dithiothreitol, pH
7.4) (15). Protein concentrations were determined with the Bio-Rad
protein assay reagent (Hercules, CA). Equal amounts of protein were
incubated for 15 min at 37°C in SDS-PAGE sample buffer and loaded
onto 4-15% polyacrylamide gradient gels. After electrophoresis,
samples were transferred to nitrocellulose membranes (Hybond enhanced
chemiluminescence, Amersham, Arlington Heights, IL). The membranes were
blocked in 3% milk-PTA (PTA consists of the following: 10 mM sodium
phosphate, 10 mM sodium azide, 145 mM sodium chloride, and 0.5%
Tween-20, pH 7.2) for 3 h. Samples were incubated overnight
with the primary antibody to MCT1 or MCT2, or with a nonimmune chicken
IgY, diluted to 100-400 ng/ml in 3% milk-PTA. The membranes were
washed repeatedly with 3% milk-PTA for 1 h and then incubated with
goat anti-chicken IgY conjugated to alkaline phosphate (Jackson
Immunoresearch, West Grove, PA), diluted 1:10,000 in 3% milk-PTA.
After repeated washes with 3% milk-PTA, bound antibody was visualized
by chemiluminescence (Hyperfilm ECL, Amersham).
Specificity of the signals was confirmed by probing blots with
antibodies to MCT1 or MCT2 that had been preabsorbed with the appropriate and inappropriate MCT peptide. Antibodies were preincubated with 20 µg/ml of the peptide, diluted in 3% milk-PTA, and the antibody-peptide mixture was then added to the blot. Secondary antibodies and visualization of bound antibodies were as above.
The molecular masses of the bands that labeled specifically with
antibodies to MCT1 and MCT2 were determined by comparison with a
mixture of prestained standard proteins, purchased from GIBCO
(Gaithersberg, MD).
Immunocytochemistry.
Astrocytes plated on coverslips were fixed with 2% paraformaldehyde in
PBS for 15 min at room temperature. Cultures were rinsed three times
with PBS/azide (PBS containing 10 mM sodium azide) and permeabilized
with 0.25% Triton X-100 in PBS/azide for 5 min. The cultures were then
incubated with 10% BSA in PBS/azide for 30 min, followed by incubation
for 1 h with the primary antibodies diluted in the same solution. After
several washes in PBS/azide, bound antibodies were labeled for 1 h with
the appropriate fluorescein- or tetramethylrhodamine-conjugated goat
antibodies to rabbit anti-IgG, mouse anti-IgG, or chicken IgY (Jackson
Immunoresearch). After being washed, the coverslips were mounted in 9 parts glycerol, 1 part 1 M Tris · HCl, pH 8.0, containing 1 mg/ml p-phenylenediamine, to reduce photobleaching
(21).
Antibodies to MCT1 and MCT2 were used at dilutions of 1:100, rabbit
polyclonal anti-GFAP at 1:200, and nonimmune IgY at 2 µg/ml. All
dilutions were in 3% BSA in PBS. Peptide blocking studies were done
using 50 µg/ml of the haptenic peptides.
For studies of the MCT in frozen sections, adult Sprague-Dawley rats
(Zivic Miller) were anesthetized with ketamine and Rompun and perfused
with PBS. The brains were removed, fixed in 4% paraformaldehyde in PBS
for 30 min, placed in a solution of 18% sucrose in PBS, and incubated
overnight at 4°C. The next day the brains were frozen in isopentane
on dry ice. Sagittal sections, 10 µm in thickness, were prepared on a
Reichert-Jung model 2800 Frigocut E cryostat (Reichert-Jung, Cambridge
Instruments, Deerfield, IL), collected on gelatin-coated glass slides,
and stored with dessicant at
70°C. Before immunolabeling,
sections were rehydrated for 5 min in PBS containing 0.1 M glycine,
fixed and permeabilized in methanol at
20°C for 5 min, and
then incubated for 30 min in PBS containing 1 mg/ml BSA (PBS/BSA).
Sections were labeled with primary antibodies overnight at 4°C,
rinsed three times with PBS/BSA, and incubated with secondary
antibodies and mounted, as above. In some experiments, we used
identical procedures to examine sections of brains from younger animals
(postnatal days 1 and 7).
Some frozen sections were double labeled with antibodies to other
astroglial (S-100, vimentin) markers (30) together with anti-GFAP,
anti-MCT1, or anti-MCT2. Antibodies to the latter proteins were used at
the same concentrations mentioned above. When necessary for
double-labeling protocols, we also used rabbit antibodies to GFAP.
Antibodies to S-100 and vimentin were used at dilutions of 1:200
(40 µg/ml) and 1:500, respectively.
Microscopy and imaging.
Samples were observed and imaged with a Zeiss 410 confocal laser
scanning microscope (Carl Zeiss, Tarrytown, NY), with pinholes set for
maximum resolution in both tetramethylrhodamine and fluorescein channels. For comparisons of different samples labeled with the same
antibodies, the contrast and brightness scales were first established
for the brightest positive control, and the same settings were then
used for all other samples. Images were obtained with software provided
by Zeiss and were assembled with CorelDraw (Corel, Ottawa, Canada).
Quantitative comparisons of labeling intensities were made with
MetaMorph software (Universal Imaging, West Chester, PA). Intensities
of MCT1 and MCT2 immunofluorescence were the average of measurements
from three to six astrocytes from each group. The area of the cell body
sampled each time was constant for all measurements. Background
immunofluorescence was subtracted from each of the experimental readings.
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RESULTS |
We used immunoblotting and immunofluorescence to study the presence and
distribution of MCT1 and MCT2 in rat astrocytes grown in vitro and in
astrocytes in frozen sections of adult rat brain. Antibodies generated
in chickens against the unique COOH-terminal sequences of MCT1 and MCT2
were used in all experiments. As controls, we used nonimmune chicken
IgY antibodies. We also routinely used the peptide antigens as
competitive inhibitors of the anti-MCT antibodies. Our experiments
demonstrate that all astrocytes in vitro, but only limited populations
of astrocytes in the brain, express significant levels of MCT1 and MCT2.
MCT1 and MCT2 in astrocytes in vitro.
We grew neonatal rat astrocytes in tissue culture under
well-established conditions (27, 29) and used them as sources of
protein and cells for immunoblotting and immunofluorescence studies.
Immunoblotting tended to give variable results, depending on the method
used to prepare and process the samples. In particular, nonspecific
labeling increased significantly when rat astrocyte proteins were
boiled in SDS-PAGE sample buffer (data not shown). We therefore
used sample buffer at 37°C, which we found in other experiments to
be more useful for immunoblotting of integral membrane proteins. We
found that antibodies to MCT1 generated a single, major band
with an apparent molecular mass of ~40 kDa (Fig.
1, lane A1). Similarly, anti-MCT2
labeled a single band at ~50 kDa (Fig. 1, lane
B1). Nonimmune chicken IgY failed to label a band in this range
(Fig. 1, lane C), suggesting that the labeling by anti-MCT1 and
anti-MCT2 was specific. As an additional control, we used the antigenic
peptides as competitive inhibitors. The COOH-terminal peptide antigen
of MCT1 blocked the labeling by anti-MCT1 antibodies of the ~40-kDa
band (Fig. 1, lane A2) but had no effect on labeling by
anti-MCT2 (Fig. 1, lane B2). Similarly, the MCT2 peptide
blocked the ability of anti-MCT2 antibodies to label the
~50-kDa band (Fig. 1, lane B3) but had no
effect on labeling by anti-MCT1 (Fig. 1, lane A3). These
results suggest that the antibodies to MCT1 and MCT2 react specifically
with polypeptides with apparent polypeptide masses of ~40 and ~50
kDa, respectively. These values are in reasonable agreement with the
values of Gerhart et al. (15, 16), who reported values of 48 and 46 kDa
for MCT1 and MCT2, respectively. The small differences in apparent molecular mass are probably due to differences in sample processing and, in particular, to our use of SDS-PAGE sample buffer at 37°C, rather than 100°C, to dissolve astrocyte proteins.

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Fig. 1.
Immunoblots of proteins of cultured cortical astrocytes with
anti-monocarboxylic acid transporter (MCT)1 and anti-MCT2. Proteins
from cultures of rat astrocytes were subjected to SDS-PAGE and
immunoblotting (see MATERIALS AND METHODS). Anti-MCT1
labeled a band at ~40 kDa (lane A1); this labeling was
blocked by the MCT1 antigenic peptide (lane A2) but not by the
MCT2 antigenic peptide (lane A3). Anti-MCT2 identified a
~50-kDa band (lane B1), labeling of which was blocked by the
MCT2 (lane B3) but not by the MCT1 (lane B2) antigenic
peptide. Nonimmune chicken IgY did not react with any bands in this
region (lane C). These results indicate that anti-MCT1 and
anti-MCT2 specifically recognize proteins of ~40 and ~50 kDa,
respectively, in cultured rat astrocytes.
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In immunofluorescence studies, antibodies to both MCT1 and MCT2 labeled
every astrocyte in our primary cultures at moderate-to-bright intensities (Fig. 2, a and
e). In double-label experiments with antibodies to GFAP,
astrocytes showed strong, punctate MCT1 immunolabeling throughout the
cell. In most cells, anti-MCT1 antibodies also labeled bright clusters
of small spots that presented as asterlike structures near the nuclei
(Fig. 3C, arrow). Although much of the labeling appeared to be intracellular, the anti-MCT1 antibody also
labeled punctate structures at the cell membrane (e.g., Fig. 3A, arrow). All labeling was specific, because it was not
mimicked by nonimmune chicken antibodies (Fig. 2d), and
it was blocked by the appropriate (Fig. 2b) but not by the
inappropriate antigenic peptide (Fig. 2c).

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Fig. 2.
Rat cortical astrocytes in culture express MCT1 and MCT2. Rat
astrocytes in tissue culture were fixed and immunolabeled with
antibodies to MCT1 or MCT2, followed by a fluorescein-conjugated
secondary antibody. Confocal images show labeling for MCT1 (a)
and MCT2 (d). This labeling is specific, since it is abolished
in the presence of the appropriate peptides (b and f,
respectively) but not in the presence of the inappropriate peptides
(c and e, respectively). Nonimmune IgY did not show any
labeling (g). Bar, 25 µm.
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Fig. 3.
Surface and intracellular labeling of MCT in rat astrocytes in culture.
Anti-MCT1 (A) and anti-MCT2 (B) labeling in the
cytoplasm and at the plasma membrane is punctate (arrows). Anti-MCT1
also labels clusters near the nucleus (C, arrow). Bars:
A and B, 10 µm; C, 25 µm.
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Immunofluorescence also showed that all cultured cortical astrocytes
express MCT2. As with MCT1, antibodies to MCT2 labeled punctate
structures in the cytoplasm and at the plasma membrane (Fig.
3B, arrow). In addition, anti-MCT2 antibodies gave dim staining of the nuclei and brighter staining of long, linear structures surrounding nuclei, which we identified as the trans-Golgi
network with appropriate antibodies (data not shown). The
immunolabeling of all these structures by anti-MCT2 was specific, by
the same criteria used for anti-MCT1 (Fig. 2,
e-g).
In the course of our studies, we observed that some cells in the
culture labeled with MCT1 or MCT2 more intensely than others, consistent with previous observations on the microheterogeneity of
astrocytes in primary culture (6, 19, 26). All of the labeled cells
were astrocytes, because they also reacted with antibodies to GFAP in
double-label protocols (see below). In the case of MCT2, the intensity
of labeling tended to correlate with that of GFAP. In 59 of 81 cell
pairs in which anti-GFAP antibodies labeled one cell of the pair
brightly and the other dimly, the former tended to display more MCT2
(P < 0.00001 by the rank sum test). This correlation did not
extend to MCT1, however. In 24 cell pairs examined similarly, only 3 of
the cells that labeled brightly for GFAP displayed significantly higher
levels of MCT1 than the dimly labeled cell (P > 0.1 by the
rank sum test). Thus the level of expression of MCT2, but not of MCT1,
may be governed in part by the same factors that control the expression
of GFAP by cortical astrocytes in culture.
We used digital imaging software to estimate the relative intensities
of immunofluorescence labeling for MCT1 and MCT2 in the cultured
astrocytes (see MATERIALS AND METHODS). All confocal images
were collected identically so that the intensities could be compared
reliably. All readings for anti-MCT1 and anti-MCT2 labeling were
significantly higher than those obtained with nonimmune chicken IgY
(P < 0.007) and with the specific antibodies in the presence
of their appropriate peptide antigens (P < 0.005),
which generated 6- to 10-fold lower values than the mean values
obtained with anti-MCT1 and anti-MCT2 (data not shown). We conclude
from these studies and our immunoblotting experiments that cortical astrocytes in culture express significant amounts of both MCT1 and MCT2.
MCT1 and MCT2 in astrocytes in vivo.
We used the anti-MCT1 and anti-MCT2 antibodies to study the
distribution of these transporters in the brains of adult rats. Labeling by anti-MCT1 and anti-MCT2 was reproducible and specific by
the same criteria summarized above; it was not generated by nonimmune
chicken IgY (data not shown), and it was blocked by the appropriate but
not by the inappropriate peptide (e.g., Fig. 4) for white matter tracts. Similar results
were obtained in all other areas of the brain discussed below. Much of
the labeling by anti-MCT1 and anti-MCT2 was in the cerebrovasculature
and neuropil, but labeling of individual astrocytes was seen in some
regions of the brain. Labeling of neuronal populations by
anti-MCT1 and anti-MCT2 will be discussed elsewhere
(unpublished data).

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Fig. 4.
Immunolabeling of cryosections of brain with antibodies to MCT1 and
MCT2. Frozen sections from adult rat brain were labeled with anti-MCT1
(A-C) and ant-MCT2 (D-F) in
the absence of peptide antigens (A and D) or in the
presence of peptide 1 (B and E) or peptide 2 (C
and F). After labeling with secondary antibodies to chicken
IgY, confocal images of astrocytes in the white matter were collected.
Antibodies to both of the MCT labeled structures in the white matter in
a manner that was blocked by the appropriate but not by the
inappropriate peptide. Bars, 50 µm.
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Labeling of astrocytes in situ by anti-MCT1 was apparent in the glial
limiting membrane of the cerebral cortex (Fig.
5a, arrowheads), in astrocytes in
the corpus callosum (Fig. 5m), and in glial processes radiating
off the surface of the brain stem (Fig. 5g). Antibodies to MCT1
also labeled intensely ependymocytes lining the lateral ventricle (data
not shown), and, somewhat less intensely, cerebral microvessels (see
below). The intensity of immunolabeling of different structures
in the brain, from brightest to dimmest, was ependymocytes > microvessels > glial limiting membrane > astrocytes > neuropil.

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Fig. 5.
Double immunofluorescence labeling with antibodies to MCT1 or MCT2 and
to glial fibrillary acidic protein (GFAP). Frozen sections of adult rat
brain were double labeled with antibodies to GFAP and either anti-MCT1
(a-c, g-i,
m-o) or anti-MCT2 (d-f,
j-l, p-r). Anti-MCT1
(a, g, m) and anti-MCT2 (d, j,
p) were visualized with fluoresceinated donkey anti-chicken
IgY; anti-GFAP (b, e, h, k, n, q)
labeling was visualized with tetramethylrhodamine-conjugated goat
anti-rabbit IgG. In the color composite images (c, f, i, l, o,
r), MCT labeling is shown in green, GFAP labeling in red, and
structures labeled with both antibodies in yellow.
a-f: MCT1 and MCT2 in the cortex. Anti-MCT1 and
anti-MCT2 label the glial limiting membrane (a and d,
arrowheads), which also labels with anti-GFAP (b and
e). Microvessels are positive for both MCT1 and MCT2 (a
and d, short arrows) but are negative for GFAP (b and
e). Astrocytes below the glial limiting membrane are positive
for GFAP (b and e, long arrows) but are negative for
both MCT1 and MCT2, respectively. g-l: Radial
processes near the ventral surface of the brain stem immunolabel for
MCT1 (g), MCT2 (j), and GFAP (h and k).
m-r: Most astrocytes in the corpus callosum that
label with either anti-MCT1 (m) or anti-MCT2 (p)
antibodies also label with anti-GFAP (n and q). Please
note that, due to low levels of label in m, the brightness of
this panel was increased, accounting for the higher than normal green
background that appears in o. Bars: a-f,
250 µm; g-l, 50 µm; m-r,
25 µm.
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We obtained similar but distinct results with antibodies to MCT2. Like
anti-MCT1, antibodies to MCT2 labeled the glial limiting membrane (Fig.
5d, arrowheads), and glial processes radiating off the surface
of the brain stem (Fig. 5j), as well as cerebral microvessels
(see below). While anti-MCT1 labeled scattered astrocytes in white
matter (Fig. 5, m-o), anti-MCT2 labeled more cells
in these regions (Fig. 5, p-r). All these labeled
cells were identified as astrocytes, because they could be
immunolabeled with antibodies to GFAP (Figs. 5 and
6) but not with antibodies to a neuronal marker, calcium/calmodulin-dependent protein kinase II (data not shown). The order of intensity of labeling by anti-MCT2, from brightest
to dimmest, was microvessels > glial limiting membrane > neuropil > astrocytes.

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Fig. 6.
MCT expression in astrocytes outside the endfeet and in individual
capillary endothelial cells. Cryosections were double labeled as in
Fig. 5, with antibodies to MCT1 (a, d, g) or
MCT2 (a', d', g') and to
either GFAP (b, b'), a blood-brain barrier
antigen (e, e'), or GLUT1 (h,
h') to mark the capillary endothelium. In the color
composite images (c, f, c',
f'), anti-MCT labeling is shown in green, the
other antibodies in red, and structures containing both labels in
yellow. Anti-MCT labeling is found in cells that also label with GFAP
but not with antibodies to the blood-brain barrier antigen, even when
the MCT is expressed in astrocytes that are in close association with
blood vessels (e.g., f', arrow). Both MCT are
present in capillaries (g, h, g',
h'). Bars: a-f,
a'-f', 10 µm; g,
h, g', h', 100 µm.
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Given the proximity of many astrocytes to blood vessels and the
presence of MCT in the cerebrovasculature (Fig. 6, g and
g'; see below), we were concerned that many of the
structures we showed to contain the MCT might in fact be blood vessels
and not astrocytes. This is possible if much of the labeling obtained
with anti-GFAP is concentrated in glial endfeet, which cover most of
the surface of the cerebrovasculature (18). We therefore carried out
several double-labeling experiments with anti-MCT and antibodies to
GLUT1, which is highly enriched in the cerebrovasculature (32).
Antibodies to MCT1 and MCT2 labeled cerebral blood vessels (Fig. 6,
g and g', respectively). Occasionally, small
differences in labeling could be detected between anti-MCT and
anti-GLUT1 antibodies (e.g., Fig. 6, g' and
h'). This may reflect the asymmetric distribution of
GLUT1 between abluminal and luminal surfaces, since the amount of GLUT1
is fourfold greater on the abluminal than on the luminal membrane (11),
whereas MCT1 is present on both membranes of endothelial cells (15).
Occasionally, capillary-like structures also appeared to label for GFAP
(data not shown). This is probably due to the presence of MCT in
astrocytic endfeet (16), which are closely apposed to and enclose most
of the capillary surface (18).
We also observed labeling for the MCT in GFAP-positive cells that were
in close association with blood vessels in the region proximal to the
lateral ventricle (e.g., Fig. 6,
d'-f'). These cells were identified as
astrocytes, because they were double labeled by antibodies to the MCT
and to GFAP, but not to GLUT1 (data not shown) or the blood-brain
barrier antigen (e.g., Fig. 6, e' and
f'). In some astrocytic processes (e.g., Fig. 6,
a, a', and d', arrowheads),
labeling by antibodies to the MCT was clearly punctate, as we also
noted for astrocytes in vitro, but this pattern was hard to capture,
even under confocal optics.
Although these experiments showed relatively high levels of labeling
for MCT1 and MCT2 in some astrocytes, especially those in the glial
limiting membrane and in white matter tracts, MCT1 and MCT2 were
difficult to identify in astrocytes in other areas of the brain. In
particular, neither MCT1 nor MCT2 could be detected by double-label
immunofluorescence methods in astrocytes in the cortex (Fig. 5, long
arrows), the hippocampus, or the cerebellum (data not shown). However,
the microvessels in these regions were positive for both transporters
(Fig. 5, short arrows), as shown by double-labeling protocols with an
antibody against GLUT1 (data not shown).
Our observations using double-label immunofluorescence definitively
identified populations of astrocytes in the adult brain that express
MCT1 and MCT2 through the use of antibodies to GFAP. We were concerned,
however, about the apparently low densities of astrocytes that were
labeled for GFAP in the cortex. We therefore used antibodies to two
other astroglial markers, vimentin and S-100, and obtained similar
results (data not shown). These results suggest that astrocytes in
large areas of the adult rat brain, recognized by a variety of
markers, do not express significant levels of MCT1 or MCT2.
Thus, although astrocytes in some brain regions can express high
levels of MCT1, MCT2, or both transporters, astrocytes in many regions
of the adult brain fail to express these proteins at detectable levels.
This is in sharp contrast to the results of our studies with
astrocytes in vitro (see above). Because our cultures are
prepared from neonatal rat brains, we considered the possibility that
MCT1 and MCT2 may be expressed at higher levels immediately after birth and at more limited levels in adulthood. We therefore examined frozen
sections of rat brains collected at 1 and 7 days after birth. Labeling
by MCT1 was present at significant levels at 1 day after birth (Fig.
7A) but did not
codistribute with vimentin (Fig 7B), a
glial marker that is expressed early in development (35). Labeling by
MCT2 was extremely dim in postnatal day 1 brain (Fig.
7). A preliminary examination of early developmental stages
suggests that the expression of MCT2 lags behind the expression of
MCT1. Strong signals for MCT2 are not visible until after day 7 (data not shown). These results suggest that the high levels of
expression of MCT1 and MCT2 by astrocytes in culture are not matched by
the astrocyte populations in neonatal brain.

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|
Fig. 7.
Double labeling of neonatal rat brain sections with anti-MCT antibodies
and anti-vimentin. Frozen sections from newborn (P1) rat brain were
labeled as described in previous figures. In color composite images
(C and F), MCT labeling is shown in green and vimentin
labeling in red, and structures containing both labels are shown in
yellow. A-C: glial processes in P1 cortex that are
positive for vimentin (B and E) do not label with
anti-MCT1 (A) or anti-MCT2 (D). Color composites
(C and F) show that none of the labeled structures
contains both vimentin and MCT. Bars: A-C, 50 µm; D-F, 25 µm.
|
|
 |
DISCUSSION |
We used immunological approaches to study the cellular expression of
two monocarboxylic acid transporters, MCT1 and MCT2, in astrocytes in
vivo and in vitro. Our results establish that astrocytes express
significant levels of both transporters in vitro but that the
expression of these proteins in vivo is restricted to
astrocytic populations in only specific areas of the brain.
Astrocytes in vitro.
The major new finding of the present study is that primary cortical
astrocytes in vitro express significant amounts of both MCT1 and MCT2,
whether assayed by immunofluorescence or by immunoblotting. Both
transporters are readily apparent in primary astrocytes both in the
cytoplasm and at the astrocytic cell membrane. These data contrast
with studies of cortical rat astrocytes in situ (15, 16; this
paper) and with those of Broer et al. (4), who found high levels of
mRNA for MCT1 but not for MCT2 in astroglial cells cultured from mouse brain.
The presence of both MCT1 and MCT2 at the cell membrane is consistent
with earlier studies that suggested that rat astrocytes in vitro use at
least two distinct carrier mechanisms to transport lactate (38). The
activities of these transporters, which share several features with
MCT1 and MCT2, including substrate affinities and susceptibility to
mercurials (28, 38), are also consistent with the rates of lactate
oxidation in astrocytes cultured from brain (27, 28, 40). Thus MCT1 and
MCT2, or closely related transporters, are likely to mediate the uptake
of lactate into astrocytes in vitro.
The labeling of intracellular vesicles and other intracellular
structures with the anti-MCT antibodies was specific by our two
criteria: inhibition by the appropriate peptide and lack of labeling by
nonimmune IgY. Some of this labeling may have been due to the presence
of other proteins that share sequence homology with the antigenic
peptides of MCT1 and MCT2. In this case, however, antibodies should
have reacted specifically with multiple bands in immunoblots. In our
hands, each anti-MCT antibody labeled only one band specifically. We
therefore favor the idea that MCT1 and MCT2 are present in significant
amounts in other structures, including intracellular vesicles.
These results therefore raise the possibility that MCT1 and MCT2 are
involved in the transport of monocarboxylic acids across the
intracellular membranes of astrocytes. Studies by our laboratory (25)
and others (1), demonstrating the presence of two discrete compartments
of pyruvate in the astrocytic cytosol, are consistent with such a role.
Because the transport of monocarboxylic acids such as lactate and
ketone bodies can facilitate the net movement of protons across
membranes, the present data suggest that MCT1 and MCT2 may also
participate in the maintenance of the pH gradients that exist between
the cytoplasm and the lumen of the Golgi apparatus and secretory
vesicles (5). We are currently examining different subcellular
fractions of astrocytes in an attempt to identify the intracellular
vesicles that are labeled by antibodies to MCT1 and MCT2.
Astrocytes in the brain.
Our results in vivo agree in many respects with those of
Gerhart et al. (15, 16), who used similar antibodies and
immunohistochemical approaches to study the distribution of MCT1 and
MCT2 in rat brain. These authors observed significant levels of
labeling of MCT1 in the glial limiting membrane of the cerebral cortex,
in ependymocytes lining the lateral ventricle, in thalamic astrocytes,
and in the prepositus hypoglossal nucleus of the medulla, as well as in
the microvasculature. However, in contrast to our results, which
suggest higher levels of labeling in ependymocytes and cerebral
microvessels than in glial cells, Gerhart et al. (15) reported the most
prominent staining in the glial limiting membrane. These authors (16) also report significant labeling of MCT2 in the glial limiting membrane, ependymocytes, and the astrocytes of the white matter, and
the innermost layers of cerebral cortex. Unlike our findings, however,
they did not detect MCT2 in the cerebrovasculature. These differences
are probably attributable to differences in the techniques used.
The conclusions of the immunological studies described here and by
Gerhart et al. (15, 16) agree substantially with results obtained with
Northern blotting and in situ hybridization (22). In that study, MCT1
expression was seen in the ependymal lining of the ventricles, in the
choroid plexus, and in white matter tracts, as well as in cerebral
microvessels. In contrast, there was no significant expression of mRNA
encoding MCT2 in the white matter, ependyma, or microvessels,
suggesting that MCT2 was primarily expressed by neurons. This is in
agreement with the data of Pellerin et al. (33, 34), who reported
significant levels of expression of both MCT in the microvasculature in
developing but not adult brain. In view of the well-documented
differences in resolution, sensitivity, and interpretation between
immunohistochemistry and in situ hybridization, further studies to
compare these techniques in the same samples will be required to
resolve the apparent discrepancy.
Nevertheless, all the currently available results suggest that some
astrocytes in vivo are capable of expressing MCT1 and probably MCT2 but
that astrocytes in wide areas of the brain fail to do so (e.g.,
cerebral cortex, Fig. 5, long arrows). We speculate that the
predominant transporter of monocarboxylic acids in cortical astrocytes
in vivo is one of the recently identified but as yet uncharacterized MCT that are found in the brain (e.g., MCT5 or MCT6,
Ref. 36).
Differences in vitro and in vivo.
The discrepancies between the results we obtain in vitro and in vivo
suggest that the expression of MCT1 and MCT2 is upregulated in primary
cultures of cortical astrocytes. There are several possible
explanations for this upregulation. 1) Developmental regulation: primary cultures of astrocytes are prepared from neonatal rat brain, and although they differentiate in culture, they may never
reach the state of maturity of astrocytes in vivo. Our results with
neonatal brain (Fig. 7) argue against this possibility. 2) Metabolic regulation: our culture medium contains 5 mM glucose, which
is significantly higher than the concentration in vivo (<1 mM, Ref.
11). The increased production of lactate from glucose may regulate MCT1
and MCT2 synthesis by specific feedback mechanisms, possibly similar to
that which governs the expression of the glucose transporter, GLUT1, in
astrocytes (39). 3) Communication with other cells: cultured
astrocytes are deprived of interactions with the neurons and capillary
endothelial cells with which they interact closely in the adult brain.
Communication, either through direct cell-cell contact or paracrine
mechanisms, may be required to downregulate the expression of MCT1 and
MCT2 in most astrocytes in vivo. Currently ongoing experiments will
test these and other hypotheses.
 |
ACKNOWLEDGEMENTS |
We are grateful to Shirley Huang and Irene Hopkins for the
preparation of the astrocyte cultures, to Dr. L. Drewes (Univ. of
Minnesota, Duluth, MN) for generously providing the antigenic peptides,
and to Drs. R. Schwarcz and H. R. Zielke for their comments on the
paper. We also thank the reviewers for their insightful comments and
helpful suggestions.
 |
FOOTNOTES |
This research was supported by National Institute of Child Health and
Human Development Grant PO1-HD-16596.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. J. Bloch, 660 W. Redwood St., Baltimore, MD 21201 (E-mail:
rbloch{at}umaryland.edu).
Received 18 February 1999; accepted in final form 22 November
1999.
 |
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