Antisense GLUT-1 protects mesangial cells from
glucose induction of GLUT-1 and fibronectin
expression
Charles W.
Heilig1,2,3,
Jeffrey I.
Kreisberg4,
Svend
Freytag3,
Takashi
Murakami5,
Yousuke
Ebina5,
Lirong
Guo2,
Kathleen
Heilig1,2,
Robert
Loberg6,
Xuan
Qu6,
Ying
Jin1,
Douglas
Henry7, and
Frank C.
Brosius III6
1 Division of Nephrology, Johns Hopkins University School of
Medicine, Baltimore, Maryland 21205; 2 Nephrology Unit,
University of Rochester Medical Center, Rochester, New York 14642;
3 Division of Molecular Biology, Henry Ford Hospital, Detroit
48202; 6 Division of Nephrology, Department of Medicine and
Veterans Affairs Medical Center, University of Michigan, Ann Arbor
48109; 7 Department of Physiology, College of Human Medicine,
Michigan State University, East Lansing, Michigan 48824;
4 Department of Surgery, University of Texas Health Science
Center and the Audie Murphy Veterans Administration Hospital, San
Antonio, Texas 78284; and 5 School of Medicine, Tokushima
University, Tokushima 770, Japan
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ABSTRACT |
A stable clone of rat
mesangial cells expressing antisense GLUT-1 (i.e., MCGT1AS cells) was
developed to protect them from high glucose exposure. GLUT-1 protein
was reduced 50%, and the 2-deoxy-[3H]glucose uptake rate
was reduced 33% in MCGT1AS. MCLacZ control cells and MCGT1
GLUT-1-overexpressing cells were used for comparisons. In MCLacZ, 20 mM
D-glucose increased GLUT-1 transcription 90% vs. no
increase in MCGT1AS. Glucose (8 mM) and 12 mM xylitol [a hexose
monophosphate (HMP) shunt substrate] did not stimulate GLUT-1
transcription. An 87% replacement of the standard 8 mM D-glucose with 3-O-methylglucose reduced GLUT-1
transcription 80%. D-Glucose (20 mM) increased fibronectin
mRNA and protein by 47 and 100%, respectively, in MCLacZ vs. no
increases in MCGT1AS. Fibronectin synthesis was elevated 48% in MCGT1
and reduced 44% in MCGT1AS. We conclude that 1)
transcription of GLUT-1 in response to D-glucose depends on
glucose metabolism, although not through the HMP shunt, and
2) antisense GLUT-1 treatment of mesangial cells blocks
D-glucose-induced GLUT-1 and fibronectin expression, thereby demonstrating a protective effect that could be beneficial in
the setting of diabetes.
glucose; GLUT-1; antisense; chloramphenicol acetyltransferase
assay; fibronectin
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INTRODUCTION |
SUBSTANTIAL PROGRESS
has been made over the last 10 years toward understanding the processes
that mediate diabetic nephropathy. A strong case has developed for
hyperglycemia per se as the major factor responsible for development of
diabetic tissue complications, including diabetic nephropathy (2,
7). The key process mediating diabetic renal disease is believed
to be excessive mesangial synthesis and release of extracellular matrix
(ECM) molecules that accumulate in and expand the mesangium, thereby
scarring the glomerulus and impairing renal function (25,
28). The hypothesis that hyperglycemia plays a key role in
development of renal disease was greatly strengthened by results from
the Diabetes Control and Complications Research Group (7)
in 1993 that demonstrated tight control of blood glucose concentrations
prevented or delayed diabetic renal failure in humans. The precise
cellular mechanisms involved in mediating glucose-induced mesangial
cell ECM synthesis are now the focus of intense research.
In 1992, Dimitrakoudis et al. (8) reported increased
translocation of GLUT-1 to the sarcolemma of diabetic muscle. GLUT-4 levels were decreased and caused overall GLUT expression in skeletal muscle to be decreased; however, GLUT-1 increased in the sarcolemma. This led the authors to speculate that tissues that do not require insulin for glucose uptake might have substantial quantities of GLUT-1
and that, in diabetes, the increased availability of GLUT-1 could be
expected to lead to excessive glucose uptake and tissue damage in the
eye, kidney, and nerves (8). This theory went untested,
however, until our discovery of D-glucose-induced GLUT-1 expression in mesangial cells (12, 13, 15) and the
description by Kumagai et al. (21) of the GLUT-1 response
in retinal endothelial cells. Both of these cell types are from classic
diabetes target organs.
The in vivo correlates of excess mesangial cell ECM synthesis are
glomerulosclerosis and renal failure (25, 28). Previous studies have focused heavily on the aldose reductase (or polyol) pathway (3, 19, 26, 31), the protein kinase C pathway (10, 11, 29), and growth factors such as transforming
growth factor-
1, insulin-like growth factor I, and
platelet-derived growth factor (3, 20, 24, 30, 33, 34) in
addition to advanced glycosylation end products (4, 9,
30). Common to all four pathways is their stimulation by high
extracellular glucose concentrations (4, 5, 12, 13, 15, 20, 35, 36). We have previously demonstrated that GLUT-1 is a major glucose transporter of the glomerulus (14) and of
mesangial cells in vitro (12, 13). Our recent work in
mesangial cells demonstrated that high glucose in the diabetic range
(20 mM) is a potent stimulus to increased mesangial cell GLUT-1
expression and glucose uptake (13). Furthermore,
overexpression of GLUT-1 in mesangial cells stimulates ECM production
(12). The Michaelis constant for GLUT-1 is low
(0.5-3.7 mM) in mesangial cells, as it is in most tissues, and it
is at or near saturation at physiological glucose concentrations
(12, 13, 15). Therefore, GLUT-1 represents a site for
regulation of ECM synthesis by factors that regulate GLUT-1 expression,
translocation, and/or activity (13, 16, 18). Rates of ECM
synthesis may be directly proportional to levels of plasmalemmal GLUT-1
expression. Subsequently, in the present study, we investigated the
potential protective effect of antisense GLUT-1 against
D-glucose-induced GLUT-1 and ECM (fibronectin, FN) expression.
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MATERIALS AND METHODS |
DNA Construction, Preparation of Infective Virus, and
Transduction of Rat Mesangial Cells
We previously published the basic method for development of the
-galactosidase-expressing MCLacZ control cells and the
GLUT-1-overexpressing MCGT1 cells (12). The same method
was used here for development of a GLUT-1-underexpressing MCGT1AS cell
clone. The pWZLneo MoMuLV retrovirus vector (Ariad, Cambridge, MA) was
employed for all gene transductions (12). Transcription of
the dicistronic proviral RNA is driven from the MoMuLV long terminal
repeat. pWZLneoLacZ (control), pWZLneoGT1 (GLUT-1 sense), and
pWZLneoGT1(AS) (GLUT-1 antisense) DNA constructs were used to transfect
packaging cells, produce live virus, and transduce subconfluent
mesangial cells. Mesangial cell clones surviving G418 selection were
initially screened by GLUT-1 Western analysis, by
2-deoxy-[3H]glucose (2-DG) uptake rates, or by
immunostaining with X-gal to confirm LacZ gene expression in the
control MCLacZ cells. Subsequent characterization was carried out as
described in RESULTS.
The antisense GLUT-1 DNA expression construct, pWZLneoGT1(AS), was
produced by splicing the full-length human GLUT-1 cDNA (gift from M. Mueckler, Washington University, St. Louis, MO) into the
BamH I restriction site of the polycloning region of the
MoMuLV expression vector pWZLneo (Ariad), in the 3'-5' orientation (Fig. 1). This purified construct was
then linearized and transfected into the ecotropic Psi Cre packaging
cell line by calcium phosphate precipitation. The transfected packaging
cells were then cultured to collect active retrovirus from the medium.
These retroviruses contained the RNA coding sequence for antisense
GLUT-1 and the selectable gene neo. They were subsequently used to
transduce cultured mesangial cells.

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Fig. 1.
pWZLneoGT1AS DNA expression construct used for stable
transduction of mesangial cells to persistently express antisense
GLUT-1 mRNA at a high level. The internal ribosomal entry site (IRES)
in the construct allows GLUT-1 and neo to be expressed in a single
transcript of size 5.6 kb. This 5.6-kb band is far above the native
2.8-kb GLUT-1 band that allows both to be visualized in the same lane
on Northern blots.
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Culture of Rat Mesangial Cells
Cells were cultured in a 37°C, 5% CO2 humidified
incubator. The media used for culture of the three cloned mesangial
cell lines, MCLacZ
-galactosidase-expressing control cells, MCGT1 GLUT-1-overexpressing cells, and MCGT1AS GLUT-1-underexpressing cells,
was based on standard RPMI 1640 medium lacking glucose. D-glucose was added to the medium to achieve the desired
concentrations. NuSerum IV (Collaborative Biomedical Research, Bedford,
MA), a supplemented FCS, was added to the media to achieve a final
concentration of 20% by volume. The concentration of insulin in the
serum was 25 µg/ml. The final concentration of insulin in our
standard 20% serum medium is 0.88 µmol/l. Our previous work has
demonstrated that alterations in the medium insulin concentration up to
10 µmol/l do not alter total GLUT-1 protein levels in the mesangial cells (13). Insulin concentration in the medium was not
altered in the experiments, except when low serum medium (1%) was
required. The final concentration of myo-inositol in the
medium was ~60 µM. Cells were fed fresh media every other day
unless otherwise noted, and they did not significantly deplete their
medium of glucose between feedings (12).
Northern Analyses for GLUT-1, FN, and
-Tubulin
A standard method was employed for Northern analysis as
previously described (12, 13). Cells were seeded at a
density of 5 × 105 cells/150-mm culture plate and
were grown until 90-100% confluent (7 days). On the final day of
growth, total RNA was harvested using a commercial preparation of
guanidinium and phenol (RNA STAT-60; Tel-Test, Friendswood, TX). Total
RNA from each sample was isolated by following the manufacturer's
instructions (Tel-Test). RNA was resuspended in diethyl
pyrocarbonate-treated double-distilled water and was stored at
80°C
until use. RNA samples (20 µg each) were denatured in glyoxal/DMSO at
55°C for 1 h and then loaded to individual lanes of a 10 mM
sodium phosphate-1% agarose gel inside a circulating buffer
electrophoresis gel box (Hoeffer Super Sub; Hoeffer Scientific
Instruments, San Francisco, CA). Gels were run at 90 volts overnight.
Subsequently, they were stained with ethidium bromide, destained, and
photographed. Integrity of the RNA was confirmed by inspection of
ribosomal RNA bands. Gels were blotted to Genescreen hybridization
membranes (NEN, Boston, MA) for 36 h with 10× saline-sodium
citrate using a standard method (27a). Blots were then ultravioletly
fixed with a Stratalinker (Stratagene, La Jolla, CA), prehybridized,
and probed for GLUT-1, FN, and
-tubulin using the respective cDNAs.
The specific cDNAs used here were as follows: 1) a 2.4-kb
human GLUT-1 cDNA (gift of M. Mueckler, Washington University); 2) a rat
-tubulin cDNA (gift of P. Marsden, University of
Toronto); and 3) a 0.5-kb human FN cDNA (ATCC). Each of
these cDNAs has been used successfully for Northern analyses in the
past, confirming their sensitivity and specificity for the individual
messages (12, 13). In the present investigation, we
confirmed their ability to detect the respective transcripts in rat
mesangial cells. The [32P]cDNA probes were produced by
the Random Hexamer Priming method (PRIME-1 kit; Sigma, St. Louis, MO).
Northern blots were exposed to Kodak XAR-5 film for periods of
3-14 days, and autoradiograms were analyzed by optical scanning
densitometry, followed by analysis with the National Institutes of
Health (NIH) Image gel-plotting software (NIH Image, version 1.52;
National Technical Information Service, Springfield, VA). Changes in
cell transcript levels were compared after normalization to the 2.1-kb
mRNA for the
-tubulin housekeeping gene.
Immunoblotting of GLUT-1 and FN
A chemiluminescent immunoblot assay (ECL Western Blot kit;
Amersham Life Sciences, Buckinghamshire, UK) was used in which luminol
was employed to detect GLUT-1 and FN proteins using specific antibodies. The GLUT-1 antibody was obtained from East Acres
Biologicals (Southbridge, MA). It was directed against a unique
13-amino acid carboxy terminal sequence of the protein. The FN antibody
was obtained from Chemicon International (Temecula, CA). This
monoclonal antibody recognizes rat FN protein on immunoblots. SDS-PAGE
(10% gels for GLUT-1 and 6% gels for FN) was used to electrophorese 50 µg of total protein/lane by the method of Laemmli
(22). Equal lane loading was further confirmed with
Ponceau-S staining or antibody to
-tubulin. GLUT-1 was detected in
mesangial cells as a protein of ~48 kDa. Total protein was measured
for each sample by the assay of Lowry et al. (23), and
exactly 50 µg were loaded to each lane of the SDS-PAGE. FN protein
was identified by the size of the single detected band at ~210 kDa.
GLUT-1 protein was identified by the 48-kDa size of the single band and
was confirmed by preadsorption of GLUT-1 antibody with the specific
carboxy terminal antigen.
Immunoprecipitation of FN Protein to Measure Changes in its
Synthesis by Mesangial Cells
Determination of FN protein synthesis was performed using a
modification of the procedure described by Ayo et al. (1). Mesangial cells were first grown to confluence and then were assigned to different treatment groups for specified periods of time (up to 4 days). At the end of the incubations under the different treatment
regimens, the medium was aspirated, and the cells were washed three
times with Hanks' balanced salt solution without Ca2+ or
Mg2+ and incubated in methionine- and cysteine-deficient
RPMI 1640 medium containing 50 mCi/ml [35S]methionine and
[35S]cysteine (ICN Biomedicals, Irvine, CA) for 2 h.
The cell layers were solubilized in electrophoresis sample buffer
[2.5% SDS, 0.0625 M Tris (pH 6.8), and 10% glycerol] and then were
sheered 20 times through a 21-gauge needle. The samples were then
diluted 1:1 with immunoprecipitation buffer [20 mM PBS (pH 7.2), 680 mM sucrose, 2% CA-630, 1 mM EDTA, and 2 mM phenylmethylsulfonyl
fluoride] and incubated with 20 µg of rabbit IgG (Sigma) for 1 h at
4°C with gentle shaking. Protein G agarose (20 µl; Santa Cruz
Biotechnology) was added, and the solution was incubated for 4 h
at 4°C with gentle shaking. The protein G agarose was then pelleted
by centrifugation in a microfuge at 14,000 rpm for 2 min. The
supernatants were then incubated overnight with 20 mg of a rabbit
polyclonal antiserum to human FN (Cappel, West Chester, PA) at 4°C.
After incubation with 20 µl of protein G agarose for 4 h at
4°C, the immunoprecipitates were pelleted by spinning at 14,000 rpm
for 1 min. The pellets were washed two times with immunoprecipitation
buffer and repelleted. The final pellet was dissolved in 35 µl of
electrophoresis buffer plus 5 ml loading buffer [50 mM
Tris · HCl (pH 6.8), 3% SDS, 50% glycerol, and 5%
-mercaptoethanol] and was heated at 100°C for 5 min. The samples
were separated on 6% SDS-PAGE and then were transferred to modified
nitrocellulose membranes. The membranes were exposed in a
PhosphorImager, and the FN band intensities were quantitated.
D-Glucose (20 mM, High Concentration),
3-O-Methylglucose, or Xylitol Treatment of MCLacZ, MCGT1, and
MCGT1AS Cells
MCLacZ, MCGT1, and MCGT1AS cells were chronically adapted to
standard 8 mM glucose RPMI 1640 medium with 20% NuSerum plus penicillin/streptomycin. For the majority of experiments described here, the cells were grown in this 8 mM glucose medium. However, some
experiments required the use of high extracellular glucose in the
diabetic range (e.g., 20 mM glucose = 360 mg/dl), and for these
experiments the glucose concentration in the medium was changed from 8 to 20 mM high glucose for periods up to 14 days. We have previously
grown mesangial cells in 20 mM glucose media with every-other-day
replenishment of the media and regular passaging for periods of 3 mo
and longer without apparent loss of viability (13). This
was done without excessive depletion of medium glucose concentrations
in either 8 or 20 mM glucose-treated groups (12, 13).
Experiments with 3-O-methylglucose.
The majority (87.5%) of the standard culture medium 8 mM
D-glucose was replaced by nonmetabolizable
3-O-methylglucose (3OMG), and cells were exposed to 3OMG for
the same number of days as was used in 20 mM glucose experiments,
before GLUT-1-chloramphenicol acetyltransferase (CAT) assay analysis.
Replacement of D-glucose was achieved by preparing media
with 1 mM D-glucose plus 7 mM 3OMG.
Experiments with xylitol.
Culture media was prepared with the standard 8 mM
D-glucose, plus 12 mM xylitol, and its effect on
mesangial cell GLUT-1 transcription was compared with the effects of 8 and 20 mM D-glucose media. Cells were exposed to xylitol
for the same duration as was used for 20mM glucose exposure,
before GLUT-1-CAT assay analysis.
Transient Transfection of Three Different Transduced Rat
Mesangial Cell Lines for CAT Assay Detection of GLUT-1 Promoter
Activation
The Lipofectamine reagent (Bethesda Research Laboratories) was
used for transfection of cultured, transduced rat mesangial cells with
a GLUT-1-CAT reporter construct containing a 1.3-kb fragment (promoter)
of the murine GLUT-1 gene. The cloned, transduced mesangial cell lines
undergoing transient transfection for the CAT assay were as follows:
MCLacZ cells, which express the control reporter gene
-galactosidase; MCGT1 cells, which overexpress GLUT-1 10-fold; and
MCGT1AS cells, which underexpress GLUT-1 by 50%. The effects of
altered GLUT-1 expression or altered media composition on the cells
were determined by CAT assay analysis with the Fast CAT system from
Molecular Probes (Eugene, OR). This assay produces a single acetylated
product that is detected and semiquantitated by fluorescence at 545 nm
(Bio-Image-Analyzer BAS-2000 fluorescence detector). Cells (7 × 105) were seeded to 90-mm-diameter culture dishes 48 h
before transfection. Transfections were carried out by lipofection
(Lipofectamine reagent; Bethesda Research Laboratories; see Ref.
32). One microgram of either the GLUT-1-CAT gene
recombinant plasmid DNA or pCAT3 control DNA plasmid (GIBCO-BRL) was
diluted in 100 µl of water, and 80 µg of Lipofectamine reagent were
also diluted to 100 µl in water. Next, 100 µl of DNA sample and 100 µl of the Lipofectamine reagent dilution were combined in 15-ml
polystyrene tubes, mixed, and left to stand at room temperature for 15 min. In addition, 6.5 ml of serum-free 8 mM glucose RPMI 1640 medium
were added, and the preparation was mixed. Three milliliters of the
medium containing the DNA-Lipofectamine complex were added to two
dishes of mesangial cells previously washed with serum-free 8 mM
glucose RPMI 1640 medium. The cells were incubated for 7 h, and
then 1 ml of medium supplemented with 2% NuSerum was added. This
preparation was incubated for 10 h, and the old medium was
replaced with new medium containing 0.5% NuSerum. Incubation was then
allowed to proceed for 38 h. Cells were scraped off, and the
products were isolated. Semiquantitation of the single band acetylated
substrate on the TLC gels was performed using a Bio-Image Analyzer
BAS-2000 fluorescence detector. CAT activity was normalized for
transfection efficiency, dividing GLUT-1-CAT activity by the pCAT3
control CAT activity from parallel wells of identically treated cells.
Statistical Analyses
The Student's t-test was used for analysis of
differences between two groups. ANOVA was employed to determine
F-statistics in experiments that involved more than two
groups. The Bonferroni t-test correction was also used in
circumstances where multiple comparisons were made. Means ± SE
were calculated for individual groups in each experiment. P
values of < 0.05 were considered significant for group comparisons.
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RESULTS |
Development of a Clone of Antisense GLUT-1-Transduced Mesangial
Cells (MCGT1AS Cells)
The pWZLneoGT1(AS) antisense GLUT-1 expression construct (Fig. 1)
was assembled and packaged into retroviruses as described in
MATERIALS AND METHODS. These retroviruses were then used to transduce cultured rat mesangial cells (MCs) (16KC2
line) and express proviral RNA of ~5.6 kb containing the antisense
GLUT-1 and neo sequences. We then isolated multiple G418-resistant
mesangial cell clones that, by definition, also expressed the antisense GLUT-1 RNA. Next, 2DOG-uptake rates were performed on individual cell
clones to identify a clone we labeled MCGT1AS, exhibiting a stable 33%
reduction in the 2DOG uptake rate (Fig.
2). This clone had an approximately 50%
reduction in native GLUT-1 mRNA and protein levels, as determined by
Northern analysis and Western analysis, respectively (Fig.
3). It also grew slower than the MCGT1
and MCLacZ cells (Fig. 4). This clone
maintained its stable phenotype even after many months in culture.

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Fig. 2.
2-Deoxy-[3H]glucose (2DOG) uptake rates in
MCLacZ control cells and in MCGT1AS antisense GLUT-1 cells. The MCGT1AS
cells have a persistent 33% reduction in glucose uptake rate. Uptake
rates were measured at 5 min on the linear portion of the 2DOG uptake
curve. *P < 0.01 for MCGT1AS vs. MCLacZ;
n = 6 experiments in each group.
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Fig. 3.
A: Northern blot of native (2.8 kb) and
antisense (5.6 kb) GLUT-1 transcripts in MCLacZ control cells and in
MCGT1AS antisense GLUT-1 cells. This exposure allows one to visualize
the native 2.8-kb GLUT-1 transcript, whereas darker exposures have a an
intense signal from the antisense GLUT-1 5.6-kb RNA, which begins to
overshadow the lighter band at 2.8 kb. MCLacZ cells are represented in
lanes 1, 3, and 5, and MCGT1AS cells
are represented in lanes 2, 4, and 6.
MCGT1AS cells have the expected 5.6-kb band, whereas, simultaneously,
the native GLUT-1 mRNA at 2.8 kb is clearly suppressed. In contrast are
the MCLacZ control cells, which have only the native GLUT-1 transcript
at 2.8 kb. MCLacZ cells make -galactosidase, which allows the cells
to turn blue on exposure to the substrate X-gal. B: Western
blot of GLUT-1 protein in MCLacZ control cells (C), MCGT1
GLUT-1-overexpressing cells (GT1), and MCGT1AS antisense GLUT-1 cells
(AS). Total protein (50 µg) was loaded to each lane. GLUT-1 protein
was suppressed ~50% in the MCGT1AS cells as shown.
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Fig. 4.
Growth curves are shown for MCLacZ control cells,
MCGT1 GLUT-1-overexpressing cells, and MCGT1AS GLUT-1-underexpressing
cells. MCGT1AS cells grew slower than either the control cells or the
GLUT-1-overexpressing cells. *P < 0.05 for MCGT1 vs.
MCLacZ at days 7-15. **P < 0.02 for
MCGT1AS vs. MCLacZ at day 9.
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GLUT-1-CAT Assays in Mesangial Cell Clones
The effect of GLUT-1 overexpression on GLUT-1 gene transcription
was assessed in mesangial cells by CAT assay analysis. MCLacZ and MCGT1
cells were grown in their standard 8 mM glucose RPMI 1640 medium with
20% NuSerum for 3 days and then were switched to 8 or 20 mM glucose
medium for another 4 days. At that time, the cells were trypsinized and
transferred to 35-mm wells of six-well culture plates for the CAT
assay. The GLUT-1-CAT construct consisted of the murine GLUT-1 promoter
(1.3 kb) connected to the bacterial CAT gene in the low background
pSVOOCAT vector (27; Fig. 5). This GLUT-1-CAT construct was then transfected into mesangial cells with
Lipofectamine, as described in MATERIALS AND METHODS. We found that GLUT-1-CAT activity was detectable in MCLacZ control cells
at a relatively low level and that GLUT-1-CAT activity was 280% higher
in MCGT1 cells (P < 0.001; Fig.
6), indicating a much higher
transcriptional rate in the latter cells. When MCLacZ control cells
were switched from 8 to 20 mM high-glucose medium for 96 h, we
observed a 90% increase in GLUT-1-CAT activity in the 20 mM
glucose-treated cells (P < 0.05; Fig. 6), consistent
with the response observed in MCGT1 cells, although of lesser
magnitude. In contrast, GLUT-1-CAT activity in MCGT1AS cells treated
with 20 mM high glucose medium for the same duration was not increased above GLUT-1-CAT activity in 8 mM glucose-treated MCLacZ control cells
(Fig. 7), i.e., the response was
completely blocked by antisense GLUT-1.

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Fig. 5.
Map of the pGT1-1.3CAT reporter construct carrying
the mouse GLUT-1 promoter (black) in a 1.3-kb 5'-flank sequence. This
construct was transfected into mesangial cells to investigate the
GLUT-1 promoter response to altered glucose concentration and GLUT-1
expression.
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Fig. 6.
CAT assay for GLUT-1 transcription (A) in
MCLacZ control cells and in MCGT1 GLUT-1-overexpressing cells.
Lanes 1, 3, and 5 are from MCLacZ
cells transfected with the pGT1-1.3CAT reporter construct.
Lanes 2, 4, and 6 are from MCGT1
GLUT-1-overexpressing cells transfected with this same construct. Data
from experiments with MCLacZ cells in standard 8 mM glucose, MCLacZ
cells treated with 20 mM high glucose, and MCGT1 cells in the standard
8 mM glucose medium are summarized (B). *P < 0.01 for 20 mM glucose MCLacZ vs. 8 mM glucose MCLacZ.
* P < 0.02 for 8 mM glucose MCGT1 vs. 20 mM glucose
MCLacZ.
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Fig. 7.
CAT assay for GLUT-1 transcription (A) in MCLacZ control
cells with 8 mM glucose medium, MCLacZ cells with 20 mM high glucose
medium, and MCGT1AS (antisense GLUT-1) cells with 20 mM high glucose
medium. Glucose (20 mM) exposures were of 4 days' duration. Glucose
(20 mM) stimulated GLUT-1 transcription in the MCLacZ control cells,
whereas 20 mM glucose was unable to stimulate GLUT-1 transcription when
GLUT-1 expression was suppressed by antisense GLUT-1 (i.e., MCGT1AS
cells). Data from experiments are summarized in B.
*P < 0.05 for 20 vs. 8 mM glucose in MCLacZ cells.
**P < 0.02 for 20 mM glucose MCGT1AS cells vs. 8 mM
glucose MCLacZ cells.
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The near-total (87%) replacement of 8 mM D-glucose
in the culture medium of MCLacZ control cells with nonmetabolizable
3OMG led to an 80% reduction in baseline GLUT-1-CAT activity (Fig. 8). This result suggested that metabolism
of D-glucose was required to maintain baseline GLUT-1
transcription. However, GLUT-1-CAT activity was maintained in MCGT1
cells by a similar replacement of D-glucose with 3OMG,
presumably due to their persistent fivefold higher glucose uptake rate.
When the MCLacZ control cells were treated with 8 mM glucose, 20 mM
glucose, or 8 mM glucose plus 12 mM xylitol [precursor to xylulose
5-phosphate via the hexose monophosphate (HMP) shunt], there was no
stimulation of GLUT-1-CAT activity by the supplemental xylitol
[P < 0.1 (not significant); Fig. 8]. This suggested
that metabolism of D-glucose via the HMP shunt was not a
likely pathway for 20 mM D-glucose stimulation of
GLUT-1-CAT activity in the mesangial cells and, in addition, that
hyperosmolality was not a stimulus for increased GLUT-1 transcription.

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Fig. 8.
CAT activity for GLUT-1 transcription in MCLacZ control
cells. In some experiments (left), 87% of 8 mM
D-glucose media was replaced with the nonmetabolizable
3-O-methylglucose (3OMG). In other experiments, 12 mM
xylitol, a hexose monophosphate (HMP) shunt substrate, was added to 8 mM glucose medium, as opposed to treating with 20 mM glucose. The 3OMG
and xylitol exposures were of 4 days duration.
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FN mRNA Levels, Protein Levels, and Protein Synthesis in Mesangial
Cells With Altered GLUT-1 Expression
We previously reported that the FN mRNA level is increased in
MCGT1 cells vs. MCLacZ cells (12). Here, the potential for D-glucose to stimulate FN mRNA levels in MCLacZ control
cells and MCGT1AS cells was assessed in response to 3 and 14 days of 8 vs. 20 mM D-glucose exposure (Fig.
9). By 2 wk of 20 mM glucose treatment,
the FN mRNA level in MCLacZ cells was 47% higher than it was at 3 days
(P < 0.05). In contrast, the FN mRNA level in MCGT1AS
cells did not increase. We next investigated the response of FN protein
levels to 20 mM high glucose in MCLacZ, MCGT1, and MCGT1AS cells. Cells
were seeded and grown for 3 days in 8 mM glucose medium and then were
switched to 8 or 20 mM glucose medium for 14 more days. Total protein
was then harvested from each sample. FN protein levels were assessed on
immunoblots by optical scanning densitometry. FN protein increased
100% in MCLacZ cells to a level significantly higher than that of
MCGT1AS cells after exposure to 20 mM high glucose (P < 0.0001; Fig. 10). In fact, the FN
protein level in MCGT1AS cells was not significantly increased by 20 mM high glucose exposure (P > 0.7). The FN protein level
in MCGT1 cells in 8 mM glucose was 161% higher than the FN level in
MCLacZ cells in 8 mM glucose (P < 0.004). In addition,
when MCGT1 cells were exposed to 20 mM high glucose, the FN protein
level was 285% higher than in similarly treated MCGT1AS cells
(P < 0.0001).

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Fig. 9.
A: fibronectin (FN) mRNA in MCLacZ control
cells and in MCGT1AS antisense GLUT-1 cells in response to 20 mM
high-glucose exposure for 3 days and 2 wk. The FN mRNA level increased
47% in MCLacZ cells at 2 wk of high glucose treatment, whereas it did
not increase in the MCGT1AS cells. B: FN mRNA levels in
MCLacZ and MCGT1AS cells in response to 2-wk treatment with 8 or 20 mM
glucose. Glucose (20 mM) stimulation of FN mRNA was inhibited in the
MCGT1AS cells. O.D., optical density. * P < 0.05 for 20 vs. 8 mM glucose in MCLacZ cells. **P < 0.01 for 20 mM
glucose in MCGT1AS vs. 20 mM glucose in MCLacZ cells.
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Fig. 10.
A: Western blots of FN protein in MCLacZ, MCGT1, and
MCGT1AS cells under 8 and 20 mM glucose conditions. FN protein band is
shown at ~210 kDa. B: results of 8 vs. 20 mM glucose
treatment of all three cell types; n = 11 for MCLacZ 8 and 20 mM glucose groups; n = 4 for MCGT1 8 and 20 mM
glucose groups; and n = 8 and 11 for MCGT1AS 8 and 20 mM glucose groups, respectively. Note that 20 mM high glucose increases
expression of FN protein to a significantly higher level in the MCLacZ
cells than in the MCGT1AS cells, with P < 0.0001 for
MCLacZ 20 mM glucose vs. MCGT1AS 20 mM glucose. High glucose
stimulation of FN protein expression was completely blocked in MCGT1AS
cells, thereby demonstrating a protective effect of antisense GLUT-1
treatment (P > 0.7).
|
|
We subsequently investigated the potential for GLUT-1 expression to
regulate FN protein synthesis in MCLacZ, MCGT1, and MCGT1AS cells. FN
synthesis was analyzed by immunoprecipitation of the 35S-labeled protein and quantitation of the radiolabel as
described in MATERIALS AND METHODS. All three cell lines
were grown in 8 mM glucose medium. We found that FN synthesis was
elevated 48% in MCGT1 cells (P < 0.025) and reduced
44% in MCGT1AS cells (P < 0.01, Fig.
11). Therefore, FN synthesis was
tightly regulated by GLUT-1 expression in mesangial cells, analogous to
the GLUT-1 regulation of GLUT-1 transcription, and not by extracellular
glucose concentration per se or by osmolality.

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Fig. 11.
A: immunoprecipitation blot of the
extracellular matrix protein FN synthesized by MCLacZ control cells and
MCGT1AS GLUT-1-underexpressing cells. Cells were grown in the standard
8 mM glucose RPMI 1640 medium with 20% NuSerum. B: elevated
FN synthesis observed in MCGT1 GLUT-1-overexpressing cells and reduced
FN synthesis observed in MCGT1AS GLUT-1-underexpressing cells when
compared with FN synthesis in MCLacZ control cells. Both MCGT1 and
MCGT1AS cells have significantly (*) altered FN synthesis when
compared with the MCLacZ cells. These FN changes parallel the changes
in GLUT-1 expression and glucose uptake rate in these cells.
P < 0.025 for MCGT1 vs. MCLacZ. P < 0.01 for MCGT1AS vs. MCLacZ.
|
|
 |
DISCUSSION |
We have previously demonstrated that D-glucose is a
stimulus for GLUT-1 expression in rat mesangial cells
(13). Here we investigated the roles of GLUT-1 and of high
extracellular glucose exposure in regulating GLUT-1 gene transcription,
by CAT assay analysis. We found that both overexpression of GLUT-1 and
exposure to high extracellular glucose in the diabetic range led to
stimulation of GLUT-1 transcription in mesangial cells. Overexpression
of GLUT-1 in mesangial cells grown in our normal glucose medium (8 mM)
was more potent in stimulating the GLUT-1 promoter than was 20 mM high
glucose exposure of MCLacZ control cells. Furthermore, we found that,
by suppressing GLUT-1 with an antisense method (i.e., MCGT1AS cell
line), we could protect the mesangial cells from 20 mM high glucose
induction of GLUT-1 transcription, which would otherwise lead to
enhanced glucose uptake and metabolism, with potential adverse
consequences such as excess ECM production (12, 13).
The baseline level of GLUT-1 transcription was impaired by partial
replacement of culture medium D-glucose with
nonmetabolizable 3OMG. This suggested that glucose uptake and
metabolism were important to the maintenance of GLUT-1 gene
transcription in the control MCLacZ cells grown in their normal 8 mM
glucose medium. In contrast, the baseline GLUT-1-CAT activity in the
MCGT1 cells, which overexpress GLUT-1 protein 10-fold and which exhibit
a 5-fold higher glucose uptake rate than MCLacZ control cells, was not
suppressed by the partial (87%) replacement of culture medium
D-glucose with 3OMG. Therefore, overexpression of GLUT-1
allowed the mesangial cells to maintain GLUT-1 gene transcription in
the face of a low extracellular D-glucose concentration.
This is consistent with our previous report of MCGT1 cells, which
demonstrated that they develop the diabetic mesangial cell phenotype in
the absence of high extracellular glucose (12).
The mechanism by which diabetes leads to glomerulosclerosis and renal
failure in approximately one-third of the patients is the subject of
intense investigation. We recently described a new mechanism by which
elevated glucose concentrations in the diabetic range could stimulate
mesangial cell ECM synthesis, i.e., by glucose-induced expression of
the GLUT-1 facilitative glucose transporter in these cells (12,
13). We also demonstrated that isolated overexpression of GLUT-1
in mesangial cells to mimic the effect of high glucose reproduces the
diabetic mesangial cell phenotype in the absence of high extracellular
glucose exposure. In the current study, we described a mechanism by
which D-glucose stimulates expression of its own
transporter (i.e., increased transcription). We demonstrated that
either enhanced glucose transport alone or exposure to high glucose
stimulated GLUT-1 transcription via the promoter, suggesting the
presence of glucose response elements (17) in the
promoter. Suppression of glucose uptake by antisense GLUT-1 treatment
was effective in blocking high D-glucose induction of
GLUT-1 transcription, indicating glucose uptake was important for the
response. Treatment of mesangial cells with xylitol in place of high
extracellular D-glucose did not stimulate GLUT-1
transcription, suggesting glucose metabolism via the HMP shunt was not
the pathway by which glucose mediated its effect. Antisense GLUT-1
protection of mesangial cells from the adverse effects of
D-glucose was also demonstrated by prevention of 20 mM high
glucose stimulation of FN mRNA and protein levels in the MCGT1AS cells.
In addition, we found FN synthesis to be suppressed in these cells. The
expression of other ECM genes in MCGT1AS cells, such as collagens I and
IV and laminin, may be examined in future studies. In summary, we have
demonstrated that D-glucose and GLUT-1 expression regulate
GLUT-1 gene transcription in a positive feedback mechanism. In
addition, antisense GLUT-1 treatment prevented high glucose stimulation
of GLUT-1 transcription and FN expression, indicating a protective
effect. In future studies we may examine potential mechanisms by which
D-glucose mediates GLUT-1 transcription, including an
investigation for transcription factors and examination of the GLUT-1
gene for potential glucose response elements.
 |
ACKNOWLEDGEMENTS |
Experiments for these studies were supported by National Institute
of Diabetes and Digestive and Kidney Diseases Grants 1-K08-DK-01953 (C. W. Heilig), 1 RO1 DK-54507-01 (C. W. Heilig), and
Physician Scientist Award K11-DK-02193 (D. Henry); the American
Diabetes Association (C. W. Heilig); the National Kidney
Foundation of Upstate New York (C. W. Heilig); Juvenile Diabetes
Foundation Grants 1-1998-174 (F. C. Brosius) and 195044 (D. Henry); the National Kidney Foundation of Michigan (F. C. Brosius); and a Veterans Affairs (VA) Merit Review Award (J. I. Kreisberg) and a VA Career Scientist Award (J. I. Kreisberg) from
the South Texas Veterans Health Care Division, Research Division, Audie
Murphy VA Hospital.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: C. W. Heilig, Div. of Nephrology, Johns Hopkins Univ. School of Medicine, Ross Bldg. 965 S., 720 Rutland Ave., Baltimore, MD 21205 (E-mail: cheilig{at}welch.jhu.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 1 March 2000; accepted in final form 6 December 2000.
 |
REFERENCES |
1.
Ayo, S,
Radnik R,
Garoni W,
Glass W,
and
Kreisberg J.
High glucose causes an increase in extracellular matrix proteins in cultured mesangial cells.
Am J Pathol
136:
1339-1348,
1990[Abstract].
2.
Ayo, S,
Radnick R,
Glass W,
Rampt E,
Appling D,
and
Kreisberg J.
Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high glucose medium.
Am J Physiol Renal Fluid Electrolyte Physiol
260:
F185-F191,
1991[Abstract/Free Full Text].
3.
Bleyer, A,
Fumo P,
Snipes E,
Goldfarb S,
Simmons D,
and
Ziyadeh F.
Polyol pathway mediates high glucose-induced collagen synthesis in proximal tubule.
Kidney Int
45:
659-666,
1994[ISI][Medline].
4.
Cohen, M,
Sharma K,
Jin Y,
Hud E,
Wu V,
Tomaszewski J,
and
Ziyadeh F.
Prevention of diabetic nephropathy in db/db mice with glycated albumin antagonists. A novel treatment strategy.
J Clin Invest
95:
2338-2345,
1995[ISI][Medline].
5.
DeRubertis, F,
and
Craven P.
Activation of protein kinase C in glomerular cells in diabetes: mechanisms and potential links to the pathogenesis of diabetic glomerulopathy.
Diabetes
43:
1-8,
1994[Abstract].
7.
Diabetes Control and Complications Research Group.
The effects of intensive treatment of diabetes on the development and progression of long term complications in insulin-dependent diabetes mellitus.
N Engl J Med
329:
977-986,
1993[Abstract/Free Full Text].
8.
Dimitrakoudis, D,
Vranic M,
and
Klip A.
Effects of hyperglycemia on glucose transporters of the muscle: use of the renal glucose reabsorption inhibitor phlorizin to control glycemia.
J Am Soc Nephrol
3:
1078-1091,
1992[Abstract].
9.
Doi, T,
Vlassara H,
Kirstein M,
Yamada Y,
Striker G,
and
Striker L.
Receptor-specific increase in extracellular matrix production in mouse mesangial cells by advanced glycosylation end products is mediated via platelet-derived growth factor.
Proc Natl Acad Sci USA
89:
2873-2877,
1992[Abstract].
10.
Fumo, P,
Kuncio G,
and
Ziyadeh F.
PKC and high glucose stimulate collagen alpha(1) IV transcriptional activity in a reporter mesangial cell line.
Am J Physiol Renal Fluid Electrolyte Physiol
267:
F632-F638,
1994[Abstract/Free Full Text].
11.
Haneda, M,
Kikkawa R,
Sugimoto T,
Koya D,
Araki S,
Togawa M,
and
Shigeta Y.
Abnormalities in protein kinase C and MAP kinase cascade in mesangial cells cultured under high glucose conditions.
J Diabetes Complications
9:
246-248,
1995[ISI][Medline].
12.
Heilig, C,
Concepcion L,
Riser B,
and
Freytag S.
Overexpression of GLUT1 in rat mesangial cells cultured in a normal glucose milieu mimics the diabetic phenotype.
J Clin Invest
9:
1802-1814,
1995.
13.
Heilig, C,
Liu Y,
England R,
Freytag S,
Gilbert J,
Zhu M,
Concepcion L,
and
Brosius F.
D-Glucose stimulates mesangial cell GLUT1 expression, and basal and IGF1-sensitive glucose uptake in rat mesangial cells: implications for diabetic nephropathy.
Diabetes
46:
1030-1039,
1997[Abstract].
14.
Heilig, C,
Zaloga C,
Lee M,
Zhao X,
Riser B,
and
Cortes P.
Immunogold localization of high affinity GLUT isoforms in normal rat kidney.
Lab Invest
73:
674-684,
1995[ISI][Medline].
15.
Henry, DN,
Busik JV,
Brosius FC, III,
and
Heilig CW.
Glucose transporters control gene expression of aldose reductase, PKCalpha, and GLUT-1 in mesangial cells in vitro.
Am J Physiol Renal Physiol
277:
F97-F104,
1999[Abstract/Free Full Text].
16.
Inoki, K,
Haneda M,
Maeda S,
Koya D,
and
Kikkawa R.
TGF-beta 1 stimulates glucose uptake by enhancing GLUT1 expression in mesangial cells.
Kidney Int
55:
1704-1712,
1999[ISI][Medline].
17.
Kahn, A.
Transcriptional regulation by glucose in the liver.
Biochimie
79:
113-118,
1997[ISI][Medline].
18.
Kahn, B,
and
Flier J.
Regulation of glucose transporter gene regulation in vitro and in vivo.
Diabetes Care
13:
548-564,
1990[Abstract].
19.
Kikkawa, R,
Umemura K,
Haneda M,
Arimura T,
Ebata K,
and
Shigeta Y.
Evidence for existence of polyol pathway in cultured rat mesangial cells.
Diabetes
36:
240-243,
1987[Abstract].
20.
Kreisberg, J,
Garoni J,
Radnik R,
and
Ayo S.
High glucose and TGF
stimulate fibronectin gene expression through a cAMP response element.
Kidney Int
46:
1019-1024,
1994[ISI][Medline].
21.
Kumagai, A,
Vinores S,
and
Pardridge W.
Pathological upregulation of inner blood-retinal barrier GLUT1 glucose transporter expression in diabetes mellitus.
Brain Res
706:
313-317,
1995[ISI].
22.
Laemmli, U.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
23.
Lowry, O,
Rosebrough N,
Farr A,
and
Randall R.
Protein measurement with the folin phenol reagent.
J Biol Chem
193:
265-275,
1951[Free Full Text].
24.
Matejka, G,
Erikson P,
Carlson B,
and
Jennische E.
Distribution of IGF1 mRNA and IHG1 binding sites in the rat kidney.
Histochemistry
97:
173-180,
1992[ISI][Medline].
25.
Mauer, S,
Steffes M,
Ellis E,
Sutherland D,
Brown D,
and
Goetz F.
Structural-functional relationships in diabetic nephropathy.
J Clin Invest
74:
1143-1155,
1984[ISI][Medline].
26.
Moriyama, T,
Garcia-Perez A,
and
Burg M.
Osmotic regulation of aldose reductase protein synthesis in renal medullary cells.
J Biol Chem
264:
16810-16814,
1989[Abstract/Free Full Text].
27.
Murakami, T,
Nishiyama T,
Shirotani T,
Shinohara Y,
Kan M,
Ishii K,
Kanai F,
Nakazuru S,
and
Ebina Y.
Identification of two enhancer elements in the gene encoding the type I glucose transporter from the mouse which are responsive to serum, growth factor, and oncogenes.
J Biol Chem
13:
9300-9306,
1992.
27a.
Sambrook, J,
Fritsch EF,
and
Maniatis T.
Molecular Cloning. A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.
28.
Steffes, M,
Osterby R,
Chavers B,
and
Mauer S.
Mesangial expansion as a central mechanism for loss of kidney function in diabetic patients.
Diabetes
38:
1077-1081,
1989[Abstract].
29.
Studer, R,
Craven P,
and
DeRubertis F.
Role for protein kinase C in the mediation of increased fibronectin accumulation by mesangial cells grown in high glucose medium.
Diabetes
42:
118-126,
1993[Abstract].
30.
Throckmorton, D,
Brogden A,
Min B,
Rasmussen H,
and
Kahgarian M.
PDGF and TGF
mediate collagen production by mesangial cells exposed to advanced glycosylation end products.
Kidney Int
48:
111-117,
1995[ISI][Medline].
31.
Wirthensohn, G,
Lefrank S,
Schmolke M,
and
Guder WG.
Regulation of organic osmolyte concentrations in tubules from rat renal inner medulla.
Am J Physiol Renal Fluid Electrolyte Physiol
256:
F128-F135,
1989[Abstract/Free Full Text].
32.
Yamamoto, M,
Okumura S,
Schwencke C,
Sadoshima J,
and
Ishikawa Y.
High efficiency gene transfer by multiple transfection protocol.
Histochem J
31:
241-243,
1999[ISI][Medline].
33.
Yamamoto, T,
Nakamura T,
Noble NA,
Ruoslahti E,
and
Border WA.
Expression of transforming growth factor beta is elevated in human and experimental diabetic nephropathy.
Proc Natl Acad Sci USA
90:
1814-1818,
1993[Abstract].
34.
Yamamoto, T,
Noble N,
Miller D,
and
Border W.
Sustained expression of TGF
1 underlies development of progressive kidney fibrosis.
Kidney Int
45:
916-927,
1994[ISI][Medline].
35.
Ziyadeh, F.
Mediators of hyperglycemia and the pathogenesis of matrix accumulation in renal disease.
Miner Electrolyte Metab
21:
292-302,
1995[ISI][Medline].
36.
Ziyadeh, FN,
Sharma K,
Ericksen M,
and
Wolf G.
Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by autocrine activation of transforming growth factor-beta.
J Clin Invest
93:
536-542,
1994[ISI][Medline].
Am J Physiol Renal Fluid Electrolyte Physiol 280(4):F657-F666
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