Distinct localization of GLUT-1, -3, and -5 in human
monocyte-derived macrophages: effects of cell activation
Daniela
Malide1,
Theresa M.
Davies-Hill1,
Mark
Levine2, and
Ian A.
Simpson1
1 Experimental Diabetes,
Metabolism, and Nutrition and
2 Molecular and Clinical
Nutrition Sections, Diabetes Branch, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, Maryland 20892
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ABSTRACT |
We determined subcellular localization of
GLUT-1, GLUT-3, and GLUT-5 as human monocytes differentiate into
macrophages in culture, and effects of the activating agents
N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA). Western blot analysis demonstrated progressively increased GLUT-1, rapidly decreased GLUT-3,
and a delayed increase of GLUT-5 expression during differentiation. Confocal microscopy revealed that each isoform displayed a unique subcellular distribution and cell-activation response. GLUT-1 was
localized primarily to the cell surface but was also detected in the
perinuclear region in a pattern characteristic of recycling endosomes.
GLUT-3 exhibited predominantly a distinct vesicle-like staining but was
present only in monocytes. GLUT-5 was found primarily at the cell
surface but was detectable intracellularly. Activation with fMLP
induced similar GLUT-1 and GLUT-5 redistributions from intracellular
compartments toward the cell surface. PMA elicited a similar
translocation of GLUT-1, but GLUT-5 was redistributed from the plasma
membrane to a distinct intracellular compartment that appeared
connected to the cell surface. These results suggest specific
subcellular targeting of each transporter isoform and differential
regulation of their trafficking pathways in cultured macrophages.
glucose transporter targeting; recycling pathways; phagocytic
cells; protein kinase C; confocal microscopy
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INTRODUCTION |
THE FACILITATIVE GLUCOSE TRANSPORTERS (GLUT-1 through
GLUT-5) comprise a family of integral membrane proteins that mediate the transport of glucose and structurally related substances across cellular membranes (see reviews in Refs. 7 and 27). GLUT-5, unlike all
other family members, is a very poor transporter of glucose and may
function as a fructose transporter in human and rat small intestine
enterocytes, human spermatozoa, and Chinese hamster ovary cells (2, 5,
11, 31). Other studies have challenged the functional role of GLUT-5 as
solely a fructose transporter. In both human and rodent brain, GLUT-5
has been localized exclusively to the resident macrophages, microglial
cells, where its expression is profoundly upregulated in response to
ischemic injury (15, 26, 39). Because the brain has extremely low concentrations of fructose, the function of GLUT-5 in microglial cells
is unclear.
One means to obtain clues about the function of GLUT-5 in microglia is
to study its localization and expression under different conditions.
However, it is difficult to study glucose transporters in primary human
microglial cells because of their availability. Human peripheral blood
monocytes in culture acquire a macrophage-like phenotype similar to
microglial cells in brain. Both cultured macrophages and microglial
cells are very active phagocytic cells that respond comparably to cell
activation (30, 41). Therefore, we tested whether peripheral monocytes
were a suitable model for study of GLUT-5 transporter expression over
time. To facilitate interpretation of GLUT-5 expression, we also
determined differential expression of other glucose transporters
responsible for glucose uptake as a function of time and cell
activation. Using biochemical analyses and high-resolution confocal
microscopy, we characterized the subcellular localization of each
transporter isoform and the distinct responses of each isoform to cell
activation by the chemotactic peptide
N-formyl-methionyl-leucyl-phenylalanine
(fMLP) and phorbol 12-myristate 13-acetate (PMA). The results
demonstrate that each of these isoforms displays a unique subcellular
distribution in cultured human monocyte-derived macrophages. Phorbol
ester activation reveals a specific GLUT-5-containing intracellular
compartment, which has potentially broader significance in membrane
protein trafficking in these cells.
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MATERIALS AND METHODS |
Isolation and culture of human monocyte-derived macrophages.
Elutriated monocytes were obtained from the Department of Transfusion
Medicine at the Clinical Center of the National Institutes of Health.
The monocytes were pelleted (300 g for
10 min), resuspended in RPMI 1640 (Biofluids, Rockville, MD) containing
13% pooled human AB, heat-inactivated serum (HS;
14-490E; BioWhittaker, Walkersville, MD), and counted in a Coulter
counter by use of a 70-µm aperture (model ZM, Coulter, Hialeah, FL).
Cells were diluted in RPMI 1640 containing 13% HS and plated onto
100-mm dishes (Nunc, Naperville, IL) or 2 chamber slides (Lab Tek no.
177429; Nunc) at a density of 375,000 cells/cm2. After 7 days in
culture, the medium was replaced with fresh RPMI-13% HS. In addition
to the 10 mM glucose present in the initial medium, cells were
supplemented with 10 mM glucose every other day to accommodate the
increased glucose utilization by the differentiating cells and to
prevent an upregulation of GLUT-1 resulting from glucose deprivation
(data not shown). In experiments investigating the effects of the
phorbol ester and chemotactic substance, cells on day
13 of culture were incubated with or without 20 nM PMA and 10 nM fMLP in 0.1% DMSO (Sigma Chemical, St. Louis, MO) for 24 h
at 37°C. Untreated cells were incubated with the same 0.1% concentration of DMSO as the PMA- and fMLP-treated cells in all experiments. All cell cultures and incubations were carried out in a
humidified 37°C incubator maintained with a 5%
CO2-95% air atmosphere. Cells
maintained in culture for various times up to 14 days were used to
perform glucose and fructose uptake assays and immunoblotting and
confocal immunofluorescence analyses of GLUT-1, GLUT-3, and GLUT-5
subcellular distribution.
2-Deoxy-[3H]glucose uptake.
Monocyte-derived macrophages grown in two-chamber slides were washed in
buffer without glucose [(in mM) 100 HEPES, 120 NaCl, 1 MgSO4, 1 CaCl2, and 4 K2HPO4,
pH 7.4], incubated at 37°C in 1 ml of buffer containing
0.1 mM 2-deoxyglucose and 2.5 µCi/ml 2-deoxy-[1,2-3H(N)]glucose
(New England Nuclear, Boston, MA), and subsequently washed four times
with buffer at room temperature. Uptake was linear over 10 min; a 6-min
time point was chosen for the assay. Cells were solubilized with 1 N
NaOH and neutralized with 7% acetic acid, and uptake was
determined by liquid scintillation counting. Protein content was
determined by the bicinchoninic acid assay (Sigma Chemical).
D-[U-14C]fructose
uptake.
Monocyte-derived macrophages were incubated for 6 min in the same HEPES
buffer as previously described, containing 0.9 mM D-fructose and 0.65 µCi/ml
D-[U-14C]fructose
(Amersham, Arlington Heights, IL) with 0.9 mM
L-glucose and 4 µCi/ml
L-[1-3H(N)]glucose
(New England Nuclear, Boston, MA) to correct for nonspecific uptake and
trapping. The cells were processed as described above.
Antibodies.
The following antibodies used in immunoblotting and immunofluorescence
experiments were generously provided as gifts, and all have been
previously characterized: rabbit polyclonal antisera raised against
COOH-terminal peptides of human GLUT-1 and GLUT-3 (of 20 and 16 amino
acid residues, respectively) were provided by Hoffmann-La Roche
(Nutley, NJ) (18); the mouse monoclonal antibody F-18 for human GLUT-1
was provided by Dr. P. N. Jorgensen, Novo Nordisk, Bagsvaerd, Denmark
(29), and the mouse monoclonal antibody F-68 for human GLUT-3 was
provided by Dr. Thorkil Ploug, Panum Institute, Copenhagen, Denmark
(39); and a rabbit polyclonal antiserum and affinity-purified antibody
raised to a 20-amino acid COOH-terminal peptide of human GLUT-5 were
provided by Hoffmann-La Roche and affinity purified by Dr. Peter Davis,
Albert Einstein College of Medicine, New York (26).
The secondary antibodies FITC- and lissamine rhodamine sulfonyl
chloride-conjugated anti-rabbit or anti-mouse immunoglobulins used in
immunofluorescence experiments were obtained from Jackson ImmunoResearch (West Grove, PA).
Western blotting.
Membranes were prepared from differentiating monocytes at various days
in culture by resuspending the cell pellets in PBS, pH 7.4, containing
the protease inhibitors aprotinin, pepstatin, leupeptin, and
4-(2-aminoethyl)-benzenesulfonyl fluoride (all from ICN Biomedicals,
Cosa Mesa, CA) at concentrations of 10 µg/ml. The suspensions were
homogenized by sonication (Bransom 250 Sonifier, Danbury, CT) (2 × 20-s bursts at setting 3). The lysate was centrifuged at
200,000 g for 10 min at 4°C to
obtain a total membrane pellet. The membranes were solubilized in 15 mM
Tris-1.5% SDS-2.3 M urea-100 mM dithiothreitol, pH 6.8, and the
proteins were fractionated on 10% SDS-polyacrylamide gels and
transferred to nitrocellulose. GLUT-1, GLUT-3, and GLUT-5 were detected
with isoform-specific anti-COOH-terminal rabbit polyclonal antisera,
followed by 125I-labeled protein A
(Du Pont-NEN, Boston, MA), as previously described (20, 26). A sample
of membrane prepared from human brain was processed with all blots to
serve as an internal standard to allow quantitative comparison among
sample groups (20, 26).
Immunofluorescence and confocal microscopy.
Single labeling experiments were performed using monocyte-derived
macrophages grown in chamber slides. Cultured cells were rinsed quickly
with 0.15 M PBS, pH 7.4, at room temperature to remove the culture
medium, fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Ft
Washington, PA) in calcium- and magnesium-free PBS for 20 min, rinsed
three times with PBS, and incubated for 10 min with 0.15 M glycine to
quench free aldehyde residues. Subsequently, the cells were
permeabilized, and the nonspecific binding sites were blocked with
0.1% saponin (Sigma Chemical) in PBS containing 3% HS for 45 min. The
cells were then incubated for 2 h with the primary antibodies diluted
in the same solution, washed with PBS containing 0.1% saponin to
remove the unbound immunoglobulins, incubated for 1 h with the
fluorochrome-conjugated secondary antibodies, washed, and mounted using
Vectashield (Vector Laboratories, Burlingame, CA). The specificity of
the staining was assessed through the following control experiments.
Several adsorptions were performed in which the specific antibodies
were preadsorbed with their corresponding COOH-terminal peptides (0.2 mg/ml overnight at 4°C). In other controls the specific antibodies
were omitted or replaced with nonimmune heat-inactivated (50°C, 30 min) rabbit or mouse sera.
Confocal microscopy and image analysis were performed as previously
described (21). Staining was observed with a Nikon Optiphot 2 fluorescence microscope equipped with a Bio-Rad MRC-1024 confocal laser
scanning imaging system from Bio-Rad Labs (Hercules, CA). For each
experimental condition, 8-10 images/cell from
10-15 cells
were collected by Kalman averaging with a planapochromat ×60/1,4NA oil objective at an optical zoom of 1 to 2.5 by use of
488-nm and 568-nm krypton-argon lines and Lasersharp image analysis
software. Instrument settings used to acquire images for each glucose
transporter isoform were recorded and kept constant over the culture
period and with the different reagent treatments to reveal possible
variations of the fluorescent signal intensities. For three-dimensional
reconstruction, series of optical sections were collected at 0.5-µm
intervals along the z-axis. For
presentation, digitized images were cropped and assembled using the
Adobe Photoshop 3.0 program from Adobe Systems (Mountain View, CA) and
printed with a Kodak 8650 PS digital printer (Eastman Kodak, New
Haven, CT).
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RESULTS |
The elutriated monocyte preparations used throughout this study were
all of >90% purity, with the principal contaminant being platelets.
Over 14 days in culture, the platelets disappeared by
days 2-3, and the monocytes
divided twice and underwent differentiation into tissue marcophage-like
cells. To determine which members of the family of glucose transporters
were expressed over this culture period, cell membranes were analyzed
by Western blotting for GLUT-1 through GLUT-5. Only GLUT-1, -3, and -5 were detected (Fig. 1). GLUT-1 was detected
at a molecular mass of 45 kDa throughout the culture period. For the
first 3 days in culture, the GLUT-1 levels were constant, followed by a
marked increase between days 3 and
6-7, which plateaued through
day 14. GLUT-3 levels rapidly declined
to undetectable amounts by day 4.
GLUT-5 immunoreactivity, also detected as 45-kDa species, was apparent
only at days 5-6 and increased
progressively through day 14. For all
the antibodies used for immunoblotting, the signal was completely
abolished by preadsorption of the antibodies with their corresponding
COOH-terminal peptides, confirming the high specificity of the results
(data not shown).

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Fig. 1.
Immunoblots of GLUT-1, -3, and -5 expressed in human cultured
monocyte-derived macrophages over a 14-day differentiation period.
Membrane samples (30 µg) were analyzed for GLUT-1, GLUT-3, and GLUT-5
content at various days in culture by use of specific polyclonal
antisera (as described in MATERIALS AND
METHODS). A sample of membranes prepared from human
brain (HB; right lane) was also
blotted as an internal control. Mr, relative
molecular weight.
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To define the subcellular localization of the different glucose
transporter isoforms during the culture period, we employed confocal
laser scanning microscopy. Single optical sections (0.3 µm in
thickness) collected in the middle of the cells and tangential to the
cell surface at 1, 8, and 14 days of culture are presented for
comparison (Figs. 2 and
3). At early times in culture
(day 1), only GLUT-1 (Fig.
2A) and GLUT-3 (Fig.
2B), but not GLUT-5 (Fig.
2C) immunoreactivities were detected
in the monocytes, consistent with the immunoblotting results. These
monocytes had diameters of ~15 µm, large nuclei, and a thin
cytoplasm containing only few phase-dense granules (Fig.
2D). GLUT-1 displayed a cell surface and juxtanuclear punctate staining pattern (Fig.
2A). In contrast, GLUT-3
immunoreactivity was primarily present in the few contaminating platelets that exhibited very bright immunofluorescence (arrowhead in
Fig. 2B), whereas monocytes showed a
much weaker punctate intracellular staining (Fig.
2B). By day
8 of differentiation in culture, the cells had a
macrophage-like appearance and displayed brighter GLUT-1
immunofluorescence intracellularly, but GLUT-1 was still detectable at
the cell surface (Fig. 2E). Closer
examination revealed very intense clusters of concentrated perinuclear
staining (inset in Fig.
2E). At this time GLUT-3
immunoreactivity was below the level of detection in macrophages. The
platelets completely disappeared within 2 days of culture (Fig.
2F). GLUT-5 immunofluoresence was detectable by day 5 and exhibited a
heterogeneous distribution, both between individual cells and within a
given cell (data not shown). By day 8,
GLUT-5 was clearly observed in ~20% of the cells (Fig.
2G). In most cross-sectional images,
GLUT-5 staining displayed a peripheral rim, suggesting a cell-surface
pattern. Some intracellular fine punctate staining was also present but
appeared distinct from any structure visible in the corresponding phase
image (Fig. 2H).

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Fig. 2.
Immunolocalization of GLUT-1, -3, and -5 in human monocyte-derived
macrophages by confocal microscopy: days
1 (A-D)
and 8 (E-H)
in culture. In cells at day 1 (A) and day
8 (E) in culture,
GLUT-1 exhibits a similar uniform staining of the cell surface and a
punctate staining in the perinuclear region (shown at higher
magnification in inset in
E). In the cells at
day 1 (B) in culture, very intense GLUT-3
staining is detected in the platelets (arrowhead); GLUT-3 is also
detected at lower intensity in punctate intracellular staining in the
monocyte-like cells that becomes undetectable by day
3 (F). In the cells
at day 1 (C) in culture, GLUT-5 is not
detectable but is present in ~20% of cells at day
8 (G) in culture;
note some intracellular staining as well as outlining the cell
periphery (G). Phase-contrast
micrographs (D,
H) corresponding to fluorescence
micrographs (C,
G) show changes in morphology during
differentiation; monocyte-like cells
(D) have large nuclei, thin
cytoplasm, and only few phase-dense granules, whereas the
macrophage-like cells (H) are
larger, become semiconfluent, and contain numerous phase-dense
granules. Bars: 5 µm (C,
D,
inset in
E); 10 µm
(A,
B, G,
H); 20 µm
(E,
F).
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Fig. 3.
Effects of cell activation on GLUT-1 subcellular localization in fully
differentiated human monocyte-derived macrophages. Phase contrast
(left) and corresponding fluorescent
(right) images are presented from
control (A and
B) cells, and cells incubated for 24 h with either 20 nM phorbol 12-myristate 13-acetate (PMA,
C and
D) or 10 nM
N-formyl-methionyl-leucyl-phenylalanine
(fMLP, E and
F). In control
(A and
B) differentiated macrophages,
GLUT-1 exhibits punctate perinuclear staining
(B) in a region devoid of the
numerous phase-dense granules visible on the corresponding phase
contrast image (A). In
phase-contrast image (C) of
PMA-activated cell (arrow), marked degranulation and ruffles are
visible as phase-dense lines at the periphery of the cells. In
fluorescence image (D) corresponding
to the same cell (arrow), GLUT-1 staining redistributes from
perinuclear region toward cell surface. Note in fMLP-incubated cells
similar but less extensive changes of cell morphology in phase-contrast
image (E) and corresponding GLUT-1
staining (F). Bars: 5 µm
(A-C);
10 µm
(D-F).
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In addition to morphological differentiation, the cells divided at
days 6-8. Changes in morphology
comprised an increase in cell size (mean diameter 25 µm) and the
presence of numerous phase-dense granules corresponding to lipid
droplets, dense lysosomes, and secretory granules (Fig.
2H). Fully differentiated
macrophages (day 14) were larger
cells (mean diameter 40 µm) containing numerous phase-dense granules
in their cytoplasm (Figs. 3A and
4A). Immunofluorescence labeling
demonstrated that, although all cells expressed both GLUT-1 and GLUT-5,
there were significant differences in their subcellular distributions.
In these cells most of the GLUT-1 immunoreactivity was present in
crescent-like clusters next to the nucleus in a region devoid of
granules, with a continuing low labeling at the cell surface (Fig.
3B). A similar staining pattern
extended in focal planes from the midsection of the cells through and
above the nucleus, indicating labeled vesicles lateral and dorsal to the nucleus. This distinctive perinuclear pattern resembles the previously described population of vesicles found close to the microtubule organizing center, which corresponds to the recycling endosome compartment in many cell types (4). In marked contrast, optical sections tangential to the cell surface and cross sections of
the cells revealed GLUT-5 staining predominantly in a cell surface
pattern (Fig. 4,
B and
C). Some faint diffuse intracellular GLUT-5 staining, clearly different from that of GLUT-1, was also observed (Fig. 4C). Staining of the
cells with primary antibodies preadsorbed with their antigenic peptides
or with normal nonimmune rabbit or mouse sera instead of the primary
antibodies was not detectable (data not shown).

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Fig. 4.
Effects of cell activation on GLUT-5 subcellular localization in fully
differentiated human monocyte-derived macrophages. Phase-contrast
images of control cells (A),
PMA-treated (D), or fMLP-treated
(F) cells are comparable to those
described in Fig. 3. In confocal optical sections tangential to the
surface (B) and through the middle
(C) of the control cells, GLUT-5
exhibits a cell surface pattern besides some faint intracellular
staining. In PMA-activated cells
(E), GLUT-5 staining is most often
intracellular and decreases at the cell surface. Corresponding cells
are indicated (arrowheads) in the phase-contrast
(D) and fluorescence
(E) images. In fMLP-incubated cells
(G), GLUT-5 displays brighter
staining at the cell surface compared with control cells
(C). Bar: 10 µm
(A-G).
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Because phorbol esters and chemotactic agents are known to activate
macrophages, the effects of PMA and fMLP on the subcellular distributions of GLUT-1 and GLUT-5 were examined. Incubation of mature
cultured macrophages (13 day) for 24 h with 20 nM PMA markedly changed
the cells' morphology compared with control cells. Irregular ruffling
at the cell surface and a significant decrease in phase-dense granules
indicated activation and degranulation of PMA-treated macrophages
(Figs. 3C and
4D). PMA treatment led to a dramatic redistribution of GLUT-1 staining from the perinuclear region toward
the cell periphery (Fig. 3D). In
optical cross sections, most of the juxtanuclear GLUT-1 staining
disappeared, and punctate fluorescence appeared just beneath the cell
surface (Fig. 3D). Conversely, PMA
treatment also resulted in a striking redistribution of GLUT-5
immunofluorescence from the cell surface to an intracellular membrane
compartment (Fig. 4E). In single
optical cross sections, GLUT-5 intracellular staining exhibited a
tubular pattern with a network-like appearance (Fig.
4E). At the light-microscopy level, we could not identify any association with a particular cellular compartment in the corresponding phase-contrast images (Fig. 4, D and
E). The complex three-dimensional
organization of this membrane compartment is revealed in a
reconstructed three-dimensional (3-D) image of GLUT-5 staining in a
whole cell or, alternatively, by analysis of the montage images of
single optical sections taken 0.5 µm apart, starting tangentially to
the cell surface and moving through the cell to the opposite side, as
illustrated in Fig. 5,
A-F.
These images clearly show that GLUT-5 immunofluorescence is almost
completely localized to this very large intracellular compartment and
is hardly visible at the cell surface compared with corresponding
control cells. Further processing of these images was performed using
an algorithm to segment adjacent voxels (in 3-D) by the dual criteria
of intensity and connectivity ("seed filling" feature of
Lasersharp software). This search resulted in a series of images
representing contiguous structures separated from the complex 3-D
volume (Fig. 6,
A-T)
and allowed clear visualization of the complexity of this
surface-connected compartment.

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Fig. 5.
PMA-induced GLUT-5-containing compartment. Montage of single confocal
optical sections of GLUT-5 staining selected from a series of images
collected along the z-axis from the
surface (A) through the middle
(F) of the cells stimulated with
PMA. GLUT-5 staining can be observed in an elongated and tortuous
compartment that extends deep within the cells
(A-F).
Bar: 10 µm
(A-F).
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Fig. 6.
GLUT-5-containing compartment is interconnected and opened to the cell
surface. A stack of consecutive sections (0.4 µm apart) of GLUT-5
whole cell staining was used for the 3-D reconstruction and further
processed in search for connected volumes, with application of "seed
filling" feature of Lasersharp software as described in
RESULTS. Montage illustrates the
GLUT-5-containing compartment outlined from other structures. Note
complex interconnections
(B-S)
and connection to surface of cell (A
and T). Bar: 10 µm
(A-T).
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Macrophages exposed to 10 nM fMLP for 24 h exhibited morphological
changes similar to those seen with PMA treatment. However, the ruffling
and particularly the degree of degranulation appeared less pronounced
in phase-contrast images of fMLP-treated cells (Figs.
3E and
4F). Nevertheless, distinct changes
in GLUT-1 and GLUT-5 subcellular localization were apparent. GLUT-1
redistributed from the compact perinuclear region to a punctate
staining toward the cell periphery; this redistribution was not
complete, as fainter punctate perinuclear staining was seen in some
cells (Fig. 3F). In fMLP-treated
cells, GLUT-5 exhibited much brighter fluorescence than in untreated
cells despite a similar peripheral-rim pattern indicative of cell
surface localization (Fig. 4G).
To establish that the changes observed in the biochemical and
morphological analyses during cell differentiation relate to the
functions of GLUT-1 as a glucose transporter and GLUT-5 as a fructose
transporter, respectively, both glucose and fructose transport
activities were measured in cells at different days in culture (Fig.
7, A and
B, respectively). Consistent with the increased GLUT-1 expression shown by immunoblot analysis, we observed a
50% increase in 2-deoxyglucose uptake early during differentiation, with no further significant changes after day
8. The apparent slight decrease in 2-deoxyglucose
uptake after the latter time point reflects changes in cell size and
protein content. Likewise, the progressive increase in
[U-14C]fructose
transport activity with differentiation reflected the extent of
functional GLUT-5 at the cell surface and correlated with the number of
transporters associated with the plasma membrane (Fig.
7B).

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Fig. 7.
Hexose transport activity in cultured human monocyte-derived
macrophages over a 14-day differentiation period.
2-Deoxy-[3H]glucose
(2-DG) transport activity (A) and
[U-14C]fructose
transport activity (B) were measured
as described in MATERIALS AND METHODS
in cells at various (indicated) days in culture. Results are means ± SD (n = 4). Determinations are
representative of 5 separate experiments.
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To further investigate whether the changes observed in the subcellular
distributions of GLUT-1 and GLUT-5 during macrophage activation
correlate with their transporter function at the cell surface, we
measured glucose and fructose transport activities in PMA- and
fMLP-stimulated cells (Fig. 8). PMA
activation resulted in an ~35% increase in 2-deoxyglucose uptake,
whereas fMLP activation did not appear to induce any changes in
transport activity compared with control cells (Fig.
8A). Similarly, the most significant change in
[U-14C]fructose
uptake, an ~35% decrease, was observed after PMA exposure, whereas
fMLP treatment did not appear to induce any changes compared with
control cells (Fig. 8B). Thus
activation of macrophages by PMA resulted in an increase in glucose
uptake and a decrease in the fructose uptake that paralleled the
morphological redistributions of GLUT-1 and GLUT-5, respectively. Such
a relationship did not hold with fMLP stimulation.

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Fig. 8.
Effects of cell activation on hexose transport activity in fully
differentiated human monocyte-derived macrophages. 2-DG
(A) and
[U-14C]fructose
(B) transport activity was measured
from cells incubated as described in MATERIALS AND
METHODS with either 0.1% DMSO alone
(C), 20 nM PMA, or 10 nM fMLP for 24 h as indicated. Results are means ± SD
(n = 4). Determinations are
representative of 3 separate experiments.
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DISCUSSION |
The present study demonstrates that cultured human
monocyte-derived macrophages are a very suitable in vitro system for
the determination of subcellular distributions, trafficking pathways, and pharmacological modulation of the glucose transporters. These cells
expressed variable levels of GLUT-1, GLUT-3, and GLUT-5 but lacked the
GLUT-2 and GLUT-4 isoforms. Using a combination of immunoblotting,
immunofluorescence confocal microscopy, and measurements of glucose and
fructose transport activities, we demonstrated
differentiation-dependent regulation of the levels and subcellular
distributions of each transporter isoform and, in the mature
macrophages, modulation by agents known to activate these cells.
GLUT-1 was detected throughout the culture period. In the
undifferentiated monocytes, which are characteristically small (15 µm) and possess few vesicles and phase-dense granules, GLUT-1 was
located predominantly in the plasma membrane with little juxtanuclear staining. Over the 14 days in culture, the cells divided, became significantly larger (40 µm), and progressively accumulated lipid droplets, phase-dense lysosomes, and secretory granules. GLUT-1 immunofluorescence in the fully differentiated macrophage-like cell was
found to be in distinct perinuclear vesicles, with relatively less
GLUT-1 in the plasma membrane. In most nonpolarized cells, GLUT-1 is
targeted to the plasma membrane, and in polarized cells, e.g., Caco-2,
Madin-Darby canine kidney (MDCK) cells, and enterocytes, more
specifically to the basolateral membrane (see Refs. 1, 13 for reviews).
An intracellular perinuclear location has been described for GLUT-1 in
3T3-L1 adipocytes and cultured neurons (18, 28). However, in the latter
cell types, the subcellular distribution more closely resembled the
monocytes in this study than the differentiated macrophages, with more
GLUT-1 in the plasma membrane than intracellularly.
In marked contrast to the GLUT-1 distribution, GLUT-3 were found only
early in culture, in the monocyte-like cells, where they showed
predominantly an intracellular localization. At this level of
resolution GLUT-3 pattern was punctate throughout the cytoplasm,
suggesting vesicle-like structures, and appeared clearly distinct from
the perinuclear GLUT-1. This intracellular location of GLUT-3 in the
monocytes is somewhat surprising. Most previous data suggested that
GLUT-3 are exclusively confined to the plasma membrane in neurons and
spermatozoa and are more specifically targeted to the apical cell
surface in polarized cells, such as Caco-2, MDCK, and embryonic stem
cells (3, 8, 17, 24, 25, 39). However, very recent biochemical and
morphological studies in platelets demonstrated not only the typical
plasma membrane location but also a substantial intracellular GLUT-3 compartment (9, 37). In these cells, immuno-electron
microscopy revealed that GLUT-3 are associated with
-granules, indicating targeting to a regulated secretory pathway
(9). In addition, platelet activation by thrombin or agents that
stimulate protein kinase C induces fusion of the
-granules with
the plasma membrane and functional insertion of GLUT-3 into the plasma
membrane, thus enhancing the glucose transport capacity of the cell. In
the present study (Fig. 2B) in
platelets contaminating the monocyte preparation, we observed a
predominantly cell-surface pattern of GLUT-3 immunofluoresence indicative of activation.
In contrast to GLUT-1 and GLUT-3, GLUT-5 was not detectable in the
monocyte-like cells by either immunoblotting or immunofluorescence but
was first detected at days 5-6,
after which their level subsequently increased through
day 14 of culture. The significance of
this observation remains to be established, but this pattern resembles that previously reported for GLUT-5 during differentiation of a human
colonic adenocarcinoma Caco-2 cell line (8). A possible relationship
with cell differentiation is suggested by the concurrence of GLUT-5
expression with cell replication when the cells become semiconfluent.
GLUT-5 was primarily localized at the cell surface in fully
differentiated resting macrophages, although some vesicle-like intracellular staining was also detected. The correlation of increasing fructose transport activity with increasing GLUT-5 expression in the
cell periphery during differentiation suggests that these transporters
are functional and located at least in part on the cell surface. This
is in agreement with previous data showing that GLUT-5 plays a
physiological role in the transport of dietary fructose across the
apical membranes of enterocytes from human and rat small intestine and
in human spermatozoa that are also exposed to high concentrations of
fructose (2, 5, 12, 31). Similarly, in the polarized Caco-2 cell line
and MDCK cells, GLUT-5 is found most often on the apical membrane,
although it is also present on basolateral and intracellular membranes
(8, 25). However, the functional role of GLUT-5 in nonpolarized cells,
such as muscle, and adipose cells, as well as macrophages and
microglia, remains to be further elucidated (10, 35), as the
physiological concentrations of fructose in blood and tissues are very
low and GLUT-5 is an extremely poor glucose transporter. We have
investigated a wide range of potential substrates for GLUT-5, including
ascorbate and dehydroascorbic acid, both of which have fructose-like
structures, but failed to demonstrate transport activity in oocytes
(33). Similarly, ribose, deoxyribose, and membrane glycoprotein
constituents do not compete with fructose for uptake in oocytes.
Activation of fully differentiated monocyte-derived macrophages by the
phorbol ester PMA and the chemotactic substance fMLP results in marked
degranulation and plasma membrane ruffling, as well as specific changes
in the subcellular localization of each transporter. In PMA-activated
macrophages, GLUT-1 redistributed from the perinuclear compartment to
the cell surface and was paralleled by an increase in
2-deoxyglucose uptake, suggesting that protein kinase C is involved
in the GLUT-1 trafficking pathway. Analogous PMA-induced subcellular
redistribution of GLUT-1 is seen in rat adipose cells and 3T3-L1
adipocytes, with a much smaller effect on GLUT-4, and of
GLUT-3 in platelets (6, 23, 34, 37). A difficult question remains as to
what is accomplished by the decrease in GLUT-3 and increase in GLUT-1
or -5 expression during macrophage differentiation. In monocytes, the
GLUT-3 transporter isoform is capable of transporting glucose about
sevenfold more rapidly than GLUT-1, but the latter is more responsive
to induction by stress, hypoxia, and glucose deprivation (19, 39). The absolute levels of each transporter have not been measured; however, with differentiation, the increase in GLUT-1 clearly compensates for
the loss in GLUT-3.
The GLUT-5 trafficking pathway also appears to involve PMA-sensitive
step(s), but this pathway is distinct from that of GLUT-1 recycling. In
contrast to GLUT-1, GLUT-5 redistributes from the cell surface to an
intracellular compartment in response to PMA. However, detailed
analysis and processing of reconstructed 3-D confocal microscopy images
clearly indicated that the complex intracellular GLUT-5-containing
compartment induced by PMA remains cell surface connected. This is
supported by the failure to observe more than an ~35% decrease in
fructose transport activity, despite an almost complete redistribution
of GLUT-5 from the cell surface to the apparent intracellular
compartment. This tortuous network-like compartment has the
localization and morphology of a previously described surface-connected
compartment, which is involved in the sequestration of cholesterol
crystals and acetylated low-density lipoproteins in human
monocyte-derived macrophages and mouse peritoneal macrophages (14, 22).
It has been suggested that lipids traffic toward lysosomes and lipid
droplets through such surface-connected compartments (14). It remains
to be established whether the GLUT-5-containing compartment reported
here is identical to this putative lipid-transporting endocytic
compartment, and what its significance in membrane protein trafficking
might be. In addition, the GLUT-5-containing compartment is
morphologically distinct from previously described tubular lysosomal
compartments that form in murine peritoneal macrophages after
stimulation with phorbol esters (16, 32, 38). Although the exact
mechanism of the PMA effect on GLUT-5 subcellular distribution observed
in the present study in macrophages remains unclear, PMA has been shown to stimulate endocytosis in Caco-2 cells, with the net effect of
redistributing apical membrane proteins to intracellular locations (36). Because in Caco-2 cells the GLUT-5 subcellular localization resembles that seen in macrophages, one would predict similar effects
of PMA on GLUT-5 in these cell types.
fMLP-induced activation results in a pattern of GLUT-1
immunofluorescence similar to that induced by PMA, although the
redistribution of GLUT-1 from their intracellular compartment to the
cell surface is not as extensive with fMLP as with PMA. In contrast,
the effects of PMA and fMLP on the GLUT-5 subcellular distribution are
completely different. Whereas PMA led to a major redistribution of
GLUT-5 from the cell surface to intracellular location, fMLP resulted in an apparent increase of GLUT-5 at the cell surface. These divergent effects on GLUT-5 subcellular distribution may be the result of different signaling pathways activated by PMA and fMLP, as previously reported in neutrophils (40).
In conclusion, this study reveals that
1) the glucose transporter proteins
GLUT-1, GLUT-3, and GLUT-5 are each expressed in a distinct temporal
pattern with characteristic subcellular location during the course of
human monocyte to macrophage differentiation and
2) PMA elicits a profound
subcellular redistribution of the transporters in the mature
macrophages: GLUT-1 is translocated to the cell surface, resulting in
enhanced glucose transport activity, and GLUT-5 is recruited to a large
novel intracellular compartment, which appears to remain connected to
the cell surface.
 |
ACKNOWLEDGEMENTS |
We thank the Department of Transfusion Medicine, Clinical Center,
National Institutes of Health for carrying out monocytopheresis. We are
grateful to Drs. Jan W. Slot and David E. James for making their
manuscript available before publication. We thank Drs. Samuel W. Cushman, Howard S. Kruth, Evelyn Ralston, and Steven C. Rumsey for many
helpful discussions and for critically reading the manuscript.
 |
FOOTNOTES |
Address for reprint requests: Daniela Malide, EDMNS/DB/NIDDK/NIH, Bldg.
10, Rm. 5N102, 10 Center Drive MSC 1420, Bethesda, MD 20892-1420.
Received 28 August 1997; accepted in final form 10 December 1997.
 |
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