Stimulation of GLUT-1 glucose transporter expression in
response to hyperosmolarity
Daw-Yang
Hwang and
Faramarz
Ismail-Beigi
Departments of Physiology and Biophysics and Medicine, Case
Western Reserve University, Cleveland, Ohio 44106
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ABSTRACT |
Glucose transporter isoform-1
(GLUT-1) expression is stimulated in response to stressful
conditions. Here we examined the mechanisms mediating the enhanced
expression of GLUT-1 by hyperosmolarity. GLUT-1 mRNA, GLUT-1 protein,
and glucose transport increased after exposure of Clone 9 cells to 600 mosmol/l (produced by addition of mannitol). The stimulation of glucose
transport was biphasic: in the early phase (0-6 h) a ~2.5-fold
stimulation of glucose uptake was associated with no change in the
content of GLUT-1 mRNA, GLUT-1 protein, or GLUT-1 in the plasma
membrane, whereas the ~17-fold stimulation of glucose transport
during the late phase (12-24 h) was associated with increases in
both GLUT-1 mRNA (~7.5-fold) and GLUT-1 protein content. Cell
sorbitol increased after 3 h of exposure to hyperosmolarity. The
increase in GLUT-1 mRNA content was associated with an increase in the
half-life of the mRNA from 2 to 8 h. A 44-bp region in the
proximal GLUT-1 promoter was necessary for basal activity and for the
two- to threefold increases in expression by hyperosmolarity. It is
concluded that the increase in GLUT-1 mRNA content is mediated by both
enhanced transcription and stabilization of GLUT-1 mRNA and is
associated with increases in GLUT-1 content and glucose transport activity.
glucose transporter isoform-1; sorbitol; aldose reductase; GLUT-1
messenger ribonucleic acid; GLUT-1 promoter; GLUT-1 messenger
ribonucleic acid half-life
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INTRODUCTION |
THE ADAPTIVE RESPONSE
OF CELLS to stressful conditions is mediated by a variety of
specific transcriptional and posttranscriptional mechanisms. GLUT-1, a
member of the facilitative glucose transporter family of proteins, is
among a subset of genes and gene products that respond to stress
(21, 23). GLUT-1-mediated glucose transport is augmented
by a variety of stressful stimuli and conditions, including hypoxia, a
rise in intracellular calcium concentration, inhibition of oxidative
phosphorylation, transformation, and incubation in medium low in
glucose or high in pH (15, 34). Detailed study of the
glucose transport response to some of the above stimuli demonstrates
that the enhancement of glucose transport is biphasic with the early
phase being mediated by posttranslational mechanisms and the late phase
involving enhancement of GLUT-1 gene expression (2, 27).
The generalized cellular response to hyperosmolar stress has been
examined in considerable detail, and the specific pathways and
mechanisms underlying this response continue to be under active study.
After the acute phase of the response, which is mediated predominantly
by changes in the ionic fluxes across the plasma membrane (and hence in
the ionic composition of cells), there is a chronic adaptive phase that
involves net cellular accumulation of osmolytes and a further
adjustment of the internal ionic composition (10, 18).
These osmolytes are either accumulated from the external medium after
the induction of their specific transporters (e.g., betaine,
myo-inositol) or are synthesized within the cells (e.g., sorbitol).
In the present study, we describe and examine the mechanisms underlying
the stimulation of GLUT-1-mediated glucose transport after exposure to
hyperosmolar stress. Our interest to study this regulation stems from
suggestions that GLUT-1 is considered to be a stress-response protein
(32, 34). An increase in the content of GLUT-1 in response
to prolonged (24 h) exposure to hyperosmolarity has been described in
L6 cells (30), but mechanisms underlying the response are
not known. We chose Clone 9 cells (a nontransformed rat liver cell
line) for investigation because this cell line has been used in other
studies focused on hyperosmolarity (16) and because
previous results have shown that glucose transport is rate limiting for
glucose metabolism in these cells (7, 20). In addition,
GLUT-1 appears to be the only isoform of the GLUT family that is
expressed in these cells, which serves to simplify the analysis
(26). A preliminary report of some of these findings has
been presented (14).
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MATERIALS AND METHODS |
Materials.
Clone 9 cells were obtained from American Type Culture Collection
(Rockville, MD). [
-32P]dCTP (3,000 Ci/mmol) and
3-O-methyl-D-[3H]glucose
(3-[3H]OMG; 3.4 mCi/mmol) were purchased from NEN Life
Science Products (Boston, MA) and Amersham Pharmacia Biotech
(Piscataway, NJ), respectively. Random primed DNA labeling kit and
FuGENE 6 were from Roche Molecular Biochemicals (Indianapolis, IN).
Nitrocellulose paper (BA-S 85) was obtained from Schleicher and Schuell
(Keene, NH). Quickhyb was obtained from Stratagene (La Jolla, CA).
Dulbecco's modified Eagle's medium (DMEM), Hanks' balanced salt
solution, calf serum, horseradish peroxidase-conjugated anti-rabbit
antibody, and streptavidin-agarose bead were obtained from Life
Technologies (Grand Island, NY). Culture dishes were obtained from
Corning (Acton, MA). Qiagen Plasmid Maxi Kit was obtained from Qiagen (Valencia, CA). Dual luciferase reporter assay system, pGL2-Basic plasmid, pRL-TK plasmid, Wizard Plus Minipreps system, and restriction endonucleases were purchased from Promega (Madison, WI).
Sulfo-NHS-SS-biotin was from Pierce (Rockford, IL). Standard chemicals
were obtained from Sigma (St. Louis, MO).
Reporter constructs.
The ~6.3 kbp rat GLUT-1 promoter region was deleted serially from its
5'-end to prepare constructs containing different segments of the
GLUT-1 promoter, as previously described (3). Some
reporter constructs were prepared by PCR amplification of specific
regions of the promoter that were then subcloned into pGL2-Basic
luciferase reporter vector. In all instances the products were verified
by sequencing of both DNA strands.
Cell culture, transfection, and luciferase reporter assays.
Clone 9 cells were passed and maintained in DMEM containing 10% calf
serum at 37°C with 8% CO2 (20). Cells were
employed between passages 29 and 45. The medium
was changed to serum-free DMEM for all experiments. Culture media were
made hyperosmolar by the addition of various osmolytes, usually
mannitol at 300 mosmol/l. Cells (in duplicate or triplicate culture
dishes) were cotransfected (at 60% confluence) with 2 µg of
pGL2-Basic plasmid constructs containing various segments of the GLUT-1
promoter and expressing the firefly luciferase and 0.2 µg of pRL-TK
plasmid (expressing Renilla luciferase) employing FuGENE 6. After 48 h, the media were changed to serum-free isotonic or
hypertonic medium, and the incubation continued for an additional
24 h before measurement of luciferase activity. In experiments
designed to measure basal GLUT-1 promoter activity, the activity of
firefly luciferase was corrected against Renilla luciferase
activity. In experiments testing the effect of hyperosmolarity, firefly
luciferase activity was normalized against the protein content of
control and experimental culture plates, because Renilla
luciferase activity increased dramatically in response to hyperosmolarity.
Measurement of 3-[3H]OMG uptake.
Culture plates in triplicate were incubated for 60 s in glucose
uptake medium, as described (20). The uptake medium
consisted of 1.0 ml DMEM containing 5 µCi 3-[3H]OMG and
1 µl of either DMSO alone or DMSO containing cytochalasin B (CB) such
that the final concentration of the latter was 50 µM. In cells
incubated in hyperosmolar medium, the osmolarity of the uptake medium
was adjusted to hyperosmolar levels before use. Cells were harvested in
1 ml of H2O, and the radioactivity was determined by
scintillation spectrometry. CB-inhibitable 3-OMG transport was
calculated as the difference between the uptake in the absence and
presence of CB. Uptakes in control and treated cells were performed in parallel.
Measurement of sorbitol.
Sorbitol was measured by enzymatic assay using sorbitol dehydrogenase
(19). In short, cells were washed with ice-cold
phosphate-buffered saline (PBS) twice and collected in 1 ml PBS, and an
aliquot was used for measurement of protein. Two milliliters of
ice-cold 6% perchloric acid was added, and tubes were incubated on ice
for 10 min before centrifugation for 10 min at 14,000 g. The
supernatant was neutralized with a solution of 3 M potassium carbonate
and 0.5 M triethanolamine to pH 7-9 followed by centrifugation at 14,000 g for 10 min at 4°C. Five-hundred-microliter
samples were mixed with 1 ml of a solution containing 50 mM glycine,
1.2 mM
-nicotinamide adenine dinucleotide, and 2.5 U of sorbitol
dehydrogenase for 30 min at 25°C followed by measurement of the
optical density at 340 nm.
Cell surface biotinylation and isolation of plasma membrane.
Previously described methods were employed without modification
(28) except sulfo-NHS-SS-biotin was used. Briefly, cells on two or three 100-mm culture dishes were rinsed with ice-cold PBS
followed by incubation on a shaking platform for 30 min with 1.5 ml
biotinylation buffer containing sulfo-NHS-SS-biotin. After washing and
lysis of cells, streptavidin-agarose beads were added, and the tubes
were rotated for 30 min at 4°C. Pellets were washed, and proteins
were eluted from the beads with Laemmli loading buffer devoid of
2-mercaptoethanol and bromphenol blue (for measurement of protein using
a Bio-Rad kit); this was followed by SDS-PAGE and Western blot analysis.
SDS-PAGE and Western blotting.
Whole cell lysates were prepared by scraping the cells into 100 µl of
lysis buffer (150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.1 mM
phenylmethylsulfonyl fluoride; pH 7.4). Lysates were cleared of nuclei
by centrifugation at 14,000 g for 1 min. Protein samples were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked by 5% nonfat milk and incubated with rabbit anti-GLUT-1 IgG (Chemicon International) at 1:3,000 dilution in Tris-buffered saline-Tween 20 (TBST: 50 mM Tris, 150 mM
NaCl, and 0.05% Tween 20, vol/vol). The secondary antibody was
1:10,000 dilution of horseradish peroxidase-conjugated anti-rabbit antibody diluted in TBST. Immunoreactive bands were visualized on the
Kodak X-Omat film with Western blot luminol reagent (Santa Cruz) and
quantified by densitometry.
Northern blot analysis.
Clone 9 cells were exposed to isotonic or hypertonic medium for various
times as indicated in different experiments. Cytoplasmic RNA was
isolated and fractionated as described previously (26). Blots were probed using rat GLUT-1 (26) or aldose
reductase cDNA (11) labeled with
[
-32P]dCTP. A 915-bp aldose reductase cDNA fragment
was prepared by RT-PCR of Clone 9 cell cytoplasmic RNA employing
upstream and downstream oligonucleotides with the following sequences:
5'-atggctagccatctggaactc and 5'-tcagacttctgcgtggaagg,
respectively (11). Membranes were probed at 68°C
overnight using Quick-Hyb and washed four times for 15 min each with a
solution containing 0.1% SDS and 1× standard saline citrate (SSC) at
58°C. The blots were autoradiographed using X-ray film or by
employing a Phosphorimager (Molecular Dynamics). Relative intensities
of the specific mRNA bands were normalized against the 28S rRNA band
measured by ethidium bromide staining of the membrane.
Statistical analysis.
Results are expressed as means ± SE. Unpaired Student's
t-test was used, and P < 0.05 was
considered significant (29).
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RESULTS |
Effect of hyperosmolarity on GLUT-1-mediated glucose transport,
GLUT-1 protein and mRNA content, and cell surface GLUT-1 content.
Figure 1 shows the time course of the
effect of hyperosmolarity (produced by the addition of 300 mosmol/l
mannitol to the medium) on the rate of cytochalasin B (CB)-inhibitable
glucose transport in Clone 9 cells. In repeated experiments it was
found that the rate of CB-inhibitable 3-OMG uptake increased ~2.5
fold (P < 0.05) at 3 and 6 h after exposure to
hyperosmolarity, and thereafter the rate increased markedly, reaching
15- and 25-fold of control levels by 24 and 48 h, respectively.

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Fig. 1.
Time course of stimulation of glucose uptake by
hyperosmolarity. Clone 9 cells were incubated in serum-free medium
overnight before changing to either isotonic (300 mosmol/l) or
hypertonic medium (600 mosmol/l) by addition of mannitol. Glucose
uptake, measured as cytochalasin B (CB)-inhibitable
3-O-methyl-D-[3H]glucose
(3-[3H]OMG) transport at the times indicated, is
expressed as fold effect compared with value at time 0. The
rate of glucose uptake in the control cells remained constant. This
experiment is representative of 2 experiments performed in triplicate
plates; values are means ± SE.
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We next determined whether the stimulation of glucose transport in
response to hyperosmolarity is associated with an increase in the
content of GLUT-1 mRNA and GLUT-1 transporter. In initial experiments
the effect of different levels of hyperosmolarity on the expression of
GLUT-1 mRNA was examined (Fig. 2). After 24 h of incubation, GLUT-1 mRNA content increased by the addition of mannitol at either 200 or 300 mosmol/l (but not 50 or 100 mosmol/l) to the culture medium. In all subsequent experiments mannitol added at
300 mosmol/l was employed. After the addition of mannitol, the content
of GLUT-1 mRNA remained unchanged up to 9 h, increased significantly by 12 h, and reached near-plateau levels at
24-48 h (Fig. 3A). The
time course of changes in GLUT-1 protein after exposure of cells to
hyperosmolarity is shown in Fig. 3B. The content of GLUT-1
was unchanged at 3 h, increased significantly at 6 h
(2.4 ± 0.7-fold), and increased to 9- and 11-fold of the control level at 12 and 24 h, respectively. The time point
of 6 h is shown in Fig. 3B because the increase
in cell GLUT-1 content occurred well before any increase in cell
content of GLUT-1 mRNA.

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Fig. 2.
Induction of GLUT-1 mRNA in the presence of hyperosmotic
mannitol. Northern blot analysis of GLUT-1 mRNA in Clone 9 cells
cultured in the presence of different concentrations of mannitol for
24 h is shown. Similar results were obtained in a repeat
experiment.
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Fig. 3.
Time course of induction of GLUT-1 mRNA (A)
and GLUT-1 protein (B) by hyperosmolarity. Clone 9 cells
were incubated in hyperosmotic medium (600 mosmol/l) for the times
indicated. Cytoplasmic RNA and protein were collected as described in
MATERIALS AND METHODS. A: Northern blot analysis
of GLUT-1 mRNA. B: Western blot analysis of cell lysates
using an anti-GLUT-1 polyclonal antibody. Blots are representative of 3 independent experiments.
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Results of several experiments that were performed to quantitatively
measure the effect of hyperosmolarity on the rate of glucose
transport, the content of GLUT-1 mRNA, and GLUT-1 protein after 3 and
24 h of exposure are summarized in Table
1. As is apparent from data in Table 1
(and Figs. 1 and 3), the glucose transport response to hyperosmolarity
is biphasic with the early phase occurring with no change in the
content of GLUT-1 mRNA or GLUT-1 protein; in contrast, the stimulation
of glucose transport during the late phase is associated with
significant increases in the contents of both GLUT-1 mRNA and GLUT-1.
To test whether the observed increase in GLUT-1 mRNA expression in
response to hyperosmolarity is specific to mannitol, or occurs with the
use of other osmolytes, Clone 9 cells were exposed to dextrose,
mannitol, NaCl, and urea (all added to a final osmolarity of 600 mosmol/l) (Fig. 4). GLUT-1 mRNA content
was significantly increased after exposure to dextrose, mannitol, and
NaCl but was only slightly increased after exposure to urea; the
smaller response to urea presumably reflects the fact that this reagent
is not a perfect osmolyte. The interpretation of results employing NaCl may be difficult because of potential secondary changes in other intracellular ions.

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Fig. 4.
Induction of GLUT-1 mRNA by various osmolytes. Clone 9 cells were exposed for 24 h to media containing different
osmolytes at a final osmolarity of 600 mosmol/l, while control cells
were exposed to medium containing 300 mosmol/l. C, control; D,
dextrose; M, mannitol; N, sodium chloride; U, urea. Similar results
were obtained in a repeat experiment.
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We next determined whether the stimulation of glucose transport during
the early phase of the response to hyperosmolarity is associated with
an increase in the content of GLUT-1 in the plasma membrane. A
previously described procedure for cell surface biotinylation followed
by isolation of plasma membrane was employed (1, 17, 28).
Cells treated with 250 µM CoCl2 for 24 h were used
in these studies as a control, since this treatment is associated with
an increase in cell and plasma membrane GLUT-1 content (1, 3,
17). The results showed no significant changes in plasma membrane GLUT-1 protein content after 3 h of incubation in
hyperosmotic vs. control medium (1.1 ± 0.3-fold,
n = 4; Fig. 5). This
suggests the increased glucose uptake at the 3-h time point is not due to increased membrane GLUT-1 protein and is consistent with an activation mechanism.

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Fig. 5.
Effect of exposure to hyperosmolarity for 3 h on the
content of GLUT-1 in Clone 9 cell lysate and plasma membrane. Cell
lysate and plasma membrane were collected after surface biotinylation
(as described in MATERIALS AND METHODS) after incubation in
control or 600 mosmol/l medium for 3 h. To perform Western blot
analysis, equal protein amounts of cell lysate and plasma membrane were
loaded per lane. Cells incubated with 250 µM cobalt chloride for
24 h were used as a positive control. C, control; H, 3-h
hyperosmotic incubation; Co, overnight incubation with cobalt
chloride.
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Effect of hyperosmolarity on aldose reductase mRNA expression and
cell sorbitol content.
Aldose reductase, an enzyme that is induced in response to
hyperosmolarity, catalyzes the production of sorbitol from glucose. The
effect of hyperosmolarity on aldose reductase mRNA expression was hence
determined (Fig. 6). In accordance with
previous reports that the content of aldose reductase mRNA is enhanced
in these and other cells (10, 16), we found a significant
increase in aldose reductase mRNA (~2.5-fold) as early as 3 h
after exposure to hyperosmolarity. Higher degrees of stimulation were
evident at later time points.

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Fig. 6.
Induction of aldose reductase (AR) mRNA by
hyperosmolarity. Northern blot analysis of cytoplasmic RNA isolated
from Clone 9 cells exposed to hypertonic medium (600 mosmol/l) for the
different periods indicated is shown. Similar results were obtained in
repeated experiments.
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We next determined whether the stimulatory effect of hyperosmolarity on
glucose transport is associated with an increase in cellular content of
sorbitol. Table 2 summarizes the cellular levels of sorbitol after 3 and 24 h of incubation in hyperosmolar medium. The increased expression of aldose reductase mRNA noted above
(and presumably aldose reductase protein) is associated with
significant increases in cellular content of sorbitol.
Mechanism of induction of GLUT-1 mRNA in response to
hyperosmolarity.
The increase in the content of GLUT-1 mRNA can be mediated by increased
GLUT-1 gene transcription, decreased GLUT-1 mRNA degradation, or a
combination of both mechanisms. To dissect the effects of hyperosmolarity on GLUT-1 mRNA expression, the RNA synthesis inhibitor, actinomycin D, was employed to determine whether changes in the rate of
GLUT-1 mRNA degradation play a role in the observed induction of the
mRNA. Cells were pretreated with isotonic or hypertonic media for
24 h before addition of actinomycin D, and cytoplasmic RNA was
collected after 0, 2, 4, and 6 h of exposure to the inhibitor. (The time of addition of actinomycin D was varied to enable harvesting of the cells at the same time.) Preexposure of cells to hyperosmolarity increased the relative content of GLUT-1 mRNA by ~9-fold compared with cells incubated under isotonic conditions (Fig.
7, A and B). The
half-life of GLUT-1 mRNA incubated in isotonic medium was 2.2 ± 0.1 h (n = 3), which is comparable to previous
observations in these cells (26). In these studies, the
half-life of GLUT-1 mRNA from each of three experiments was plotted,
and the resulting half-lives were averaged. After 24 h of
incubation in hypertonic medium, the half-life of GLUT-1 mRNA increased
to 7.9 ± 1.8 h (n = 3; P < 0.05).
These results suggest that the increase in the content of GLUT-1 mRNA
in response to hyperosmolarity is mediated in part by an increase in
the stability of the mRNA.

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Fig. 7.
Effect of hyperosmolarity in GLUT-1 mRNA stability. Clone
9 cells were preincubated in isotonic or hypertonic medium for 24 h before addition of actinomycin D (AD) at a final concentration of 10 µM. Cytoplasmic RNA was collected at indicated times after exposure
to AD. A: representative Northern blot of GLUT-1 mRNA after
addition of AD to cells preincubated in isotonic or hypertonic medium.
B: GLUT-1 mRNA content after addition of AD to cells
preincubated in isotonic ( ) or hypertonic medium
( ). Multiple exposures were taken to ensure that the
exposure was in the linear range of the film. Figure is average of 3 experiments performed in duplicate at each time point; values are
means ± SE.
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To elucidate the potential role of enhanced transcription of the GLUT-1
gene in the response to hyperosmolarity, we employed reporter
constructs containing different regions of the ~6 kbp rat GLUT-1
promoter (3); cells were cotransfected with pRL-TK plasmid
expressing the Renilla luciferase to control for
transfection efficiency. Clone 9 cells were transiently transfected and
assayed for luciferase activity after 24-h exposure to either isotonic or hypertonic medium. Luciferase expression measured under basal (isotonic) condition showed some change with 5'-truncations of the
promoter from ~6 kbp to ~104 bp upstream of the major
transcription start-site of the gene (33)
(constructs A-D), but the promoter remained active
(Fig. 8, A and B).
Since these constructs included 133 bp of the 5'-untranslated region of
GLUT-1 mRNA (which contains an AP-1 site and a glucocorticoid response
element), additional constructs devoid of this region were also
prepared and tested (constructs E and F). These
latter constructs were nearly as active as their respective controls
under basal conditions. However, further 5'-truncation of the promoter
to
85,
66, and
60 or to
11 (constructs
G-J) resulted in a marked decrease in the basal rate
of transcription.

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Fig. 8.
Effect of hyperosmolarity on GLUT-1 promoter activities.
A: nucleotide sequence of GLUT-1 proximal promoter region
and 133 nt of GLUT-1 5'-untranslated region. B: lengths and
positions of the GLUT-1 promoter used in the constructs are shown at
left, where +1 refers to the major transcription start site
of the gene. Clone 9 cells were transiently transfected. After 48 h, the medium was changed to isotonic or hypertonic and the incubation
continued for an additional 18 h before measurement of luciferase
activity. Data represent at least 4 independent experiments performed
in triplicate; values are means ± SE. Luciferase activity in
cells under basal conditions was normalized against Renilla
luciferase to control for transfection efficiency. Because expression
of Renilla luciferase was enhanced in response to
hyperosmolarity, the effect of hyperosmolarity on firefly
luciferase activity was normalized against protein content of cells
exposed to isotonic and hypertonic medium. Open bars, isotonic; filled
bars, hypertonic. * P < 0.05 compared with cells
maintained in isotonic media; ns, not significant.
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The effect of hypertonic media on luciferase expression in transfected
cells was next examined. In these experiments, firefly luciferase
activity was normalized against cell protein since the expression of
Renilla was stimulated by hyperosmolarity. All constructs
demonstrating significant expression of luciferase under basal
conditions showed a significant (and nearly equivalent) response to
exposure to the hypertonic medium. This included a modification of
construct C in which we mutated the consensus CAT box
located in the proximal region of the GLUT-1 promoter (data not shown).
Inclusion of proximal promoter up to
104 bp was associated
with significant increases in luciferase activities after exposure to
hyperosmolarity. Constructs G and H exhibited some basal activity and responded significantly to hyperosmolarity. In
contrast, GLUT-1 promoter constructs I and J
(containing less of the promoter) showed a small amount of basal
expression but failed to show any increase in luciferase expression in
response to hypertonic medium. Cells transfected with pGL2-Basic
(devoid of GLUT-1 promoter) showed a small amount of luciferase
expression, which was not stimulated in response to exposure to
hyperosmolarity (data not shown).
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DISCUSSION |
The present study focused on the stimulation of glucose transport
in response to exposure to hyperosmolar conditions and yielded new
insights on the mechanisms underlying this response. Results showed
that the stimulation of GLUT-1-mediated glucose transport is biphasic
with the early phase (0-3 h) being mediated entirely by
posttranslational mechanisms, while the late phase (12-48 h) is
associated with a dramatic enhancement of GLUT-1 gene expression. We
demonstrated that sorbitol levels increased in cells as early as 3 h and that the stimulation of glucose transport during the early phase
is not associated with an increase in the content of GLUT-1 in the
plasma membrane. We made the further novel observation that the
increase in cellular content of GLUT-1 mRNA in response to
hyperosmolarity is mediated by both increased transcription and
decreased degradation of GLUT-1 mRNA. Finally, we localized the region
of the GLUT-1 promoter necessary for its basal expression and for the
positive transcriptional response to hyperosmolarity to a 44-bp segment
in the proximal 5'-flanking region of the gene.
The chronic phase of the adaptive response to hyperosmolarity is
characterized by enhanced expression of several genes encoding proteins
that mediate the accumulation of organic osmolytes; the specific genes
are those encoding betaine, myo-inositol and taurine transporters, and the enzyme aldose reductase (10, 12).
The transporters serve to increase the intracellular concentration of
organic osmolytes by facilitating their transport from the external
medium, while, in contrast, aldose reductase functions to convert
intracellular glucose to sorbitol. The expression of these genes has
been shown to be stimulated at the transcriptional level, and osmotic
response elements have been identified in their promoter regions
(8, 9); posttranscriptional mechanisms have also been
demonstrated in some instances (31). In addition to the
above, tissues differ in their usage of organic osmolytes during the
adaptive response. For example, the concentration of all of the above
osmolytes is upregulated in kidney medullary cells in response to
hyperosmolarity, while all except sorbitol are accumulated in the rat
brain during adaptation to acute and chronic hypernatremia (5,
18).
The increase in the content of GLUT-1 mRNA in response to
hyperosmolarity occurred after a significant delay period (>9 h). This
finding suggests that the measured changes in rates of synthesis and
degradation of the mRNA also occurred after a significant delay period.
The reason for this delay is not known. It is possible, for example,
that changes occurring during the acute phase of the response, such as
alterations in cell volume, ionic composition, or actin skeleton
(24), help initiate the subsequent responses. In this
context, the induction of aldose reductase mRNA and increased cellular
content of sorbitol (a product of glucose metabolism), which was
observed as early as 3 h, occurred significantly before the
increase in the abundance of GLUT-1 mRNA. It is hence possible that the
enhanced synthesis of sorbitol plays an important role in the induction
of GLUT-1 mRNA. This issue requires further study.
The region of GLUT-1 promoter necessary for the response to
hyperosmolarity was demarcated to a 44-bp region of the proximal promoter (from
104 to
60). Moreover, this 44-bp region does not
contain a classical osmotic response element nor is there such an
element present in the published 600-bp segment of the proximal GLUT-1
promoter (33). The lack of a previously described consensus osmotic response element raises the possibility that the
induction of the GLUT-1 gene may occur secondary to the induction of
one or more genes. It is alternatively possible that exposure to
hyperosmolarity results in a stimulation of the transcriptional protein
complex of the GLUT-1 gene. Such a mechanism would help explain why the
promoter region mediating the hyperosmolar response appears to
correspond to the region necessary for basal transcription. A similar
finding concerning basal and stimulated expression was recently
reported in the transcriptional response of the aldose reductase gene
to hyperosmolarity in Clone 9 cells (16). Finally, the
44-bp segment located within the proximal promoter region of the GLUT-1
gene mentioned above is highly GC rich (~75%) and contains a typical
Sp1 binding site (and other Sp1-like binding sites) that has been
reported to be of importance in the developmental regulation of the
GLUT-1 gene in neonatal rat heart (25). In addition, an
Sp1 binding site has also been implicated as being responsible for the
induction of serum- and glucocorticoid-inducible protein kinase under
hyperosmotic stress (4). Whether this transcription factor
plays an important role in the response to hyperosmolarity is not
known. Further studies are necessary to determine the molecular basis
of the stimulation of GLUT-1 gene transcription as well as the
posttranscriptional stabilization of GLUT-1 mRNA in response to hyperosmolarity.
The small but significant increase in glucose uptake in the early phase
(0 to 3 h) cannot be attributed to altered GLUT-1 gene expression
or to increased translation of GLUT-1 and hence represents
posttranslational regulation of GLUT-1-mediated glucose transport.
Moreover, we found no evidence of an increase in the content of GLUT-1
in the plasma membrane at this early time point, suggesting an
activation rather than a translocation mechanism. Acute stimulation of
glucose uptake in the presence of a constant amount of cell GLUT-1 has
also been observed in response to other stimuli (such as inhibition of
oxidative phosphorylation) that can be classified as cellular stress
(34). Stimulation of p38 mitogen-activated protein kinase
(MAPK) has been implicated in the acute glucose transport response to
stress (2), and p38 activation has been implicated in the
increase in GLUT-1 content in response to hyperosmolarity after 24 h of exposure (30). Whether the early phase of the
response to hyperosmolarity involves the p38 MAPK pathway needs to be
verified. In addition, hyperosmolarity has been recently reported to
acutely stimulate AMP-activated protein kinase (AMPK)
(13), and we have observed that stimulation of the kinase
is associated with an increase in GLUT-1-mediated glucose transport
through an activation mechanism (1). It remains to be
determined, however, whether the stimulation of AMPK mediates the acute
glucose transport response to hyperosmolarity. In addition, we did
observe a small but significant increase in cell GLUT-1 protein content
6 h after exposure to hyperosmolarity, a time at which no increase
in the content of GLUT-1 mRNA had yet occurred. This finding suggests
that the observed induction of GLUT-1 at this time point could be in
part mediated at the translational level, in support of the possibility
that the regulation of GLUT-1 expression and function can be mediated
at multiple levels.
Previous results have shown that the transcription of GLUT-1 gene is
enhanced in response to hypoxia (or by exposure to cobalt chloride as a
surrogate of hypoxia), to inhibition of oxidation phosphorylation
by azide, to an ionophore-induced increase in the concentration of
cytosolic calcium, and after transformation (15, 34). The
response to these stimuli, in addition to the response to
hyperosmolarity shown here, may represent a generalized response of the
GLUT-1 gene to cellular stress. Comparison of the findings of the
present study with previous results identifying regions of the GLUT-1
promoter necessary for stimulation by hypoxia and azide demonstrates
that different regions of the promoter mediate each of these responses.
Specifically, a 666-bp region some ~6 kbp upstream to the
transcriptional start site mediates the transcriptional response of the
promoter to azide, while a ~480-bp region located ~2,500 bp
upstream of the start site (and which contains a hypoxia-response
element) is necessary for the stimulation by hypoxia or cobalt chloride
(3, 6). Still another element in the mentioned ~480-bp
region partly mediates the response to transformation
(22). Finally, results of the present study showed that
the region mediating the response to hyperosmolarity is in close
proximity to the transcriptional start site. These findings suggest
that the regions of the GLUT-1 promoter that mediate the
transcriptional response of the gene to each of the above stressful
stimuli (azide, hypoxia, transformation, and hyperosmolarity) are
markedly different. The results hence imply that the above stimuli do
not share a common transcriptional stress response pathway, and that
different trans-acting factors are likely involved in the induction of
the GLUT-1 gene in response to each of these stimuli.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-45945.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: F. Ismail-Beigi, Clinical and Molecular Endocrinology, Case Western Reserve Univ., Cleveland, OH 44106-4951 (E-mail:
fxi2{at}po.cwru.edu).
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.
Received 26 September 2000; accepted in final form 8 June 2001.
 |
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