Glucose transporters control gene expression of aldose
reductase, PKC
, and GLUT1 in mesangial cells in vitro
Douglas N.
Henry1,2,
Julia V.
Busik1,
Frank C.
Brosius III3, and
Charles W.
Heilig4
1 Department of Physiology,
2 Department of Pediatrics and
Human Development, Division of Pediatric Endocrinology, College of
Human Medicine, Michigan State University, East Lansing 48824-1101;
3 Department of Medicine, Division
of Nephrology, University of Michigan Medical School and Ann Arbor
Veterans Affairs Hospital, Ann Arbor, Michigan 48109; and
4 Department of Medicine, Division
of Nephrology, University of Rochester School of Medicine,
Rochester, New York 14642
 |
ABSTRACT |
The process linking increased glucose utilization
and activation of metabolic pathways leading to end-organ damage from
diabetes is not known. We have previously described rat mesangial cells that were transduced to constitutively express the
facilitative glucose transporter 1 (GLUT1, MCGT1 cells) or bacterial
-galactosidase (MCLacZ, control cells). Glucose transport was rate
limiting for extracellular matrix production in the MCGT1 cells. In the
present work, we investigated the effect of GLUT1 overexpression in
mesangial cells on aldose reductase (AR), protein kinase C
(PKC
),
and native GLUT1 transcript levels, to determine whether changes in GLUT1 alone could regulate their expression in the absence of high
extracellular glucose concentrations. MCGT1 cells grown in normal (8 mM) or elevated (20 mM) glucose had elevated abundance of AR, PKC
,
and the native GLUT1 transcripts compared with control cells. AR
protein levels, AR activity, sorbitol production, and PKC
protein
content were also greater in the MCGT1 cells than in control cells
grown in the same media. This is the first report of the concomitant
activation of AR, PKC
, and GLUT1 genes by enhanced GLUT1 expression.
We conclude that increased GLUT1 expression leads to a positive
feedback of greater GLUT1 expression, increased AR expression and
activity with polyol accumulation, and increased total and
active PKC
protein levels, which leads to detrimental stimulation of
matrix protein synthesis by diabetic mesangial cells.
gene regulation; facilitative glucose transport; diabetic
nephropathy; genetics; hyperglycemia
 |
INTRODUCTION |
OVER THE PAST SEVERAL YEARS, reports have documented
the process linking increased glucose utilization and stimulating of extracellular matrix (ECM) production by renal mesangial cells (RMC).
These findings have reinforced the important role of glucose in
stimulating mesangial cell ECM production, which in vivo would lead to
glomerulosclerosis in diabetes. It has long been assumed that by
unknown mechanisms, high extracellular glucose concentrations lead to
increased glucose uptake into the mesangial cell to stimulate a
processes that culminate in increased ECM accumulation and
glomerulosclerosis. Recently, three facilitative glucose transporters
were identified in RMC (8, 9, 30, 59). All three
transporters are
low-Km (Km of 1-7
mM), high-affinity transporters for glucose, which would be at or near
saturation at physiological glucose concentrations. Therefore, we
recently investigated the mechanism by which high extracellular glucose
leads to increased intracellular glucose availability in RMC (29, 35).
We found that extracellular glucose in the diabetic range (20 mM) is a
potent stimulus to increase mesangial cell facilitative glucose
transporter 1 (GLUT1) expression (29). Furthermore, it leads to
increased glucose uptake and enhances insulin-like growth factor I
(IGF-I)-induced glucose uptake in RMC (29). The increase in mesangial
cell GLUT1 is maintained even after 3 mo in 20 mM high glucose (29).
Therefore, a detrimental positive feedback mechanism exists in RMC,
whereby high extracellular glucose may stimulate its own uptake and
induce the diabetic mesangial cell phenotype. The potential for GLUT1 to regulate mesangial cell glucose uptake, and by implication the
downstream pathways leading to ECM synthesis, was thereby recognized
(31).
To examine the role of GLUT1 in regulating mesangial cell ECM
synthesis, we subsequently overexpressed GLUT1 in rat RMC (MCGT1) or
-galactosidase as a control (MCLacZ) (35). MCGT1 cells demonstrated a fivefold increase in glucose uptake and greater synthesis of individual ECM components than the MCLacZ control cell (35). This work
clearly demonstrated a role for GLUT1 in regulating mesangial cell ECM
gene expression and synthesis. By developing a stable transduced
mesangial cell line overexpressing the human GLUT1 facilitative
transporter, we demonstrated that high extracellular glucose was not
required for increased glucose uptake or for accumulation of sorbitol,
lactate, and ECM synthesis (35). It was also clear that hyperosmolality
was not required for these events to take place.
Previous work by other investigators demonstrated what was believed to
be hyperosmolar induction of the aldose reductase (AR) gene by high
extracellular NaCl or glucose concentrations (33, 50). A specific role
for glucose transport or metabolism was not examined. Currently, much
effort is being expended to identify osmotic response elements in the
AR gene (6, 21); however, no data exist concerning the potential
existence of a glucose responsive element (GlRE) in this gene. Protein
kinase C (PKC) is activated in diabetic glomeruli and glucose-treated
mesangial cells, which may direct the transcription of ECM genes (4, 16, 37). No work to our knowledge has examined the PKC
or GLUT1
genes for GlREs.
Therefore, in this work we investigated the effect of GLUT1
overexpression in RMC on AR, PKC
, and GLUT1 transcript levels to
determine whether changes in GLUT1 alone could regulate their expression in the absence of high extracellular glucose concentrations. We found the transcript levels for AR, PKC
, and GLUT1 were all substantially increased in MCGT1 cells vs. the MCLacZ control cells.
Furthermore, AR activity and protein were significantly elevated under
normal and elevated glucose conditions, corresponding with increased
sorbitol levels. MCGT1 cells grown in normal (8 mM) or
pathophysiological (20 mM) levels of glucose had high expression of AR.
These results confirmed an important role for GLUT1 in regulating the
expression of these genes, separate from high extracellular glucose
exposure and hyperosmolality. The results further imply the presence of
a GlRE or transcript stabilization mechanisms activated by increased
expression of GLUT1. The GLUT1-regulated pathway of glucose
transporters, polyol pathway, and the PKC
pathway activation likely
contribute to the enhanced ECM synthesis characteristic of
GLUT1-overexpressing RMC and potentially to the development of
mesangial scarring in diabetes.
 |
METHODS |
Reagents and supplies. Unless
otherwise noted, culture media, trypsin, antibiotics, and Hanks' PBS
were obtained from GIBCO-BRL (Life Technologies, Gaithersburg, MD);
culture dishes, wells, and flasks were from Falcon (Becton-Dickinson,
Lincoln Park, NJ); chemicals and reagents (highest purity available)
were from Sigma Chemical (St. Louis, MO); radioisotopes were from New
England Nuclear Research Products (DuPont, Wilmington, DE); and NuSerum was from Collaborative Research (Bedford, MA).
Tissue culture. Rat RMC transduced to
overexpress human GLUT1 transporter (MCGT1 cells) or
-galactosidase
(MCLacZ cells) were used in these experiments (35). Transduction of
these cells was previously described (35) using a pWZLneo retroviral
vector (Ariad Pharmaceutical, Cambridge, MA) that contains an internal ribosomal entry site from the encephalomyelocarditis
virus, which allows for translation of the GLUT1 and
neomycin phosphotransferase (neor) gene products from the
same RNA transcript. A control construct containing the bacterial
-galactosidase gene (LacZ) but lacking the GLUT1 gene was used to
create the MCLacZ control cells (35). The cells were grown in RPMI-1640
media with 8 mM glucose and 2.05 mM glutamine, supplemented with folic
acid (225 µM), 1% penicillin/streptomycin, 20% NuSerum, and 250 µg/ml of G418 (Geneticin, GIBCO-BRL). Cells were grown in 95%
humidified air with 5% CO2 at
37°C. MCLacZ and MCGT1 cells were seeded at 10,000 cells/cm2 in 10-cm
plastic tissue culture dishes in media containing 8 mM
glucose and allowed to grow for 48 h prior to changing media to
experimental conditions (8 mM normal glucose and 20 mM elevated glucose). Media was changed every other day without a significant decline in glucose concentration (data not shown). Experiments were
terminated after 7 days of growth in experimental conditions when the
cells reached confluence.
Northern analysis. Total RNA was
isolated using a modification of the acid phenol extraction method
(13). This procedure yields ~100 µg of total RNA from confluent
monolayers of RMC grown in 10-cm plates. A quantity of 10 µg of total
RNA was resolved on denaturing 2.2 M formaldehyde-1% agarose gels and
transferred to ZetaBind (Cuno, Meriden, CN) nylon filters by capillary
blotting. The filter was stained with methylene blue to examine the
integrity of the RNA and to assess the uniformity of loading and
transfer. The filters were fixed by ultraviolet cross-linking and
prehybridized at 55°C in 0.5 M
NaH2PO4,
pH 7.0, 1 M EDTA, 7% SDS, and 1% BSA for 4-6 h before adding the
denatured cDNA probes according to the protocol of Church and Gilbert
(15). Probes were labeled with
[
-32P]dCTP (3,000 Ci/mmol) using random primers to a specific activity of
109 dpm/mg and separated from
unincorporated nucleotides by gel filtration (20). After 18 h, filters
were washed twice with 40 mM
NaH2PO4, pH 7.0, 1 mM EDTA, and 1% SDS at 55°C. Autoradiograms were
obtained using multiple exposures to remain within the linear range of the film (X-Omat; Eastman-Kodak, Rochester, NY). Each blot was hybridized concomitantly with a human AR (33), GLUT1 (45), and rat
PKC
(48) cDNA probes. Filters were stripped until free of
radioactivity and were checked by autoradiography prior to rehybridization with a rat
-tubulin cDNA probe (53).
Optical-scanning densitometry (AGFA; AGFA-Gevnert, Mortsel, Belgium),
high-resolution optical scanner, and NIH Image gel plotting software
(46) were used to analyze the autoradiograms.
Measurement of AR activity and sorbitol
content. AR protein activity was measured
spectrophotometrically at 340 nm using 10 mM
DL-glyceraldehyde as substrate
and expressed as the amount of NADPH that was oxidized per minute per
milligram protein (51) at 37°C using a temperature-controlled
spectrophotometer (Beckman DU model 640) with a modification of the
method of Das and Srivastava (17). Sorbitol levels were determined
spectrophotometrically at 340 nm, at room temperature, using
NAD+ as substrate and sorbitol
dehydrogenase (7). Samples were deproteinized using 6%
perchloric acid prior to assay. Standard curves of sorbitol
concentrations in the sorbitol dehydrogenase reaction were performed
for each determination, and results normalized to total cellular
protein content (51).
Immunoblotting of AR. Immunoblots of
AR protein were performed according to the methods previously described
(1). We mixed 300-µl aliquots of RMC cell homogenate with 150 µl of
SDS-containing sample buffer, heated at 95°C for 5 min, mixed with
50 µl of 20%
-mercaptoethanol, and stored at
20°C.
Following thawing, samples corresponding to 10 µg of cell protein per
lane were electrophoresed on 7.5% linear polyacrylamide minigels. The
separated proteins were then transferred to nitrocellulose sheets by
conventional procedures. The transferred AR protein was visualized by
incubation for 2 h at 23°C in PBS containing a 1:400 dilution of
the primary rat anti-AR antibody, followed by a 90-min incubation at
23°C in PBS containing a 1:400 dilution of a commercial
immunoglobulin-peroxidase conjugate (Sigma Chemical). Following
development for peroxidase reactivity, scanning densitometry with a
high-resolution AGFA optical scanner and NIH Image software were used
to quantitate blots. Identification of AR was confirmed
by analysis of the molecular weights and comparison with previously
reported molecular weights (1).
Immunoblotting of PKC
.
Immunoblot analysis was carried out according to methods previously
described with minor modification (43). Solubilized protein samples of
50 µg were separated by SDS-PAGE and
electrophoretically transferred to Hybond-ECL nitrocellulose membranes
(Amersham). As primary antibodies, PKC
, PKC
1, PKC
, and PKC
polyclonal antibodies were used (43). The secondary antibody was a
horseradish peroxidase anti-rabbit Ig conjugate (Amersham).
Immunoblotting of
-tubulin or Ponceau-S staining was used as a
confirmatory method to assure equal sample loading between gel lanes.
Identification of PKC isoforms was confirmed by analysis of the
molecular weights and comparison with previously reported molecular weights.
Statistical analysis. Results are
expressed as means ± SE of at least four experiments except where
noted. Statistical significance of differences between experimental
groups was determined using ANOVA, Student's
t-test, or the Bonferroni correction
of t-test. Other statistical methods
were used where noted.
 |
RESULTS |
Induction of AR, PKC
, and native
GLUT1 mRNAs in RMCs in vitro.
AR is the rate-limiting enzyme of polyol metabolism and has been
implicated in the development of diabetic complications in retinal,
renal, nerve, and vascular smooth muscle tissue. As noted above, both
facilitative glucose transporters and PKC have been reported to play a
key role in the development of mesangial cell ECM synthesis in
diabetes. Transcripts for each of these proteins can be resolved by
northern analysis because of their differences in size (Fig.
1; AR, 1.8 kb; native GLUT1, 2.8 kb; and
PKC
, 4.3 kb). The proviral human GLUT1 transcript was constitutively
expressed at a high level in the MCGT1 cells, which can be identified
by the larger size of the proviral transcript (5.6 kb) compared with the native GLUT1 transcript (2.8 kb). Concomitant increases in the
abundance of AR, native (rat) GLUT1, and PKC
mRNA were conferred by
increased (human) GLUT1 mRNA expression in the MCGT1 cells grown in all
glucose concentrations vs. the MCLacZ control cells grown in the same
conditions.


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Fig. 1.
Concomitant activation of aldose reductase (AR), protein kinase C
(PKC ), and native facilitative glucose transporter 1 (GLUT1) in
renal mesangial cell (RMC) that overexpress the GLUT1 transporter
(MCGT1 cells). A: Northern blot of 10 µg of total RMC RNA. Blot was then concomitantly hybridized with
cDNAs for GLUT1, PKC , and AR. Blot was then stripped and reprobed
with -tubulin to determine relative loading of total RNA. Control
cells are RMCs transduced to overexpress the bacterial
-galactosidase gene (MCLacZ). Cells were grown for 4 days in media
containing 8 mM and 20 mM glucose. B:
quantitation of AR mRNA expression in RMC from Northern blots. Data for
AR (solid bar), GLUT1 (hatched bar), and PKC (open bar) were
normalized to -tubulin mRNA expression.
* P < 0.05 for MCGT1
vs. MCLacZ in 8 mM glucose.
# P < 0.05 for MCGT1 vs. MCLacZ in 20 mM glucose. Measured transcripts
were significantly (P < 0.05)
greater for MCGT1 vs. MCLacZ in all growth conditions. Results are
means ± SE of 4 experiments.
|
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Although high glucose transport concomitantly increased AR, PKC
, and
native GLUT1 transcript levels in the MCGT1 cells, the control MCLacZ
cells also demonstrated increases in each of these transcripts when
exposed to increases in glucose concentration in the growth media.
MCLacZ AR mRNA increased 2.1-fold when grown in 20 mM vs. 8 mM glucose.
GLUT1 transcript abundance also increased in MCLacZ cells grown in
media containing 20 mM vs. 8 mM glucose. The PKC
transcript was
detected at the limits of sensitivity by Northern analysis when the
MCLacZ cells were grown in 8 mM glucose.
Effect of increased glucose transport on AR protein
expression, AR enzyme activity, and sorbitol content.
To further understand the effects of increased glucose transport on
activation of polyol metabolism in the RMC cells, we quantitated
changes in AR protein content and activity in MCGT1 and MCLacZ cells
grown in media containing 8 or 20 mM glucose. The increase in the
abundance of AR mRNA was accompanied by increased AR protein expression
(Fig. 2) and enzyme activity (Fig.
3) in the RMC cells.

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Fig. 2.
Expression of AR protein in RMC cells.
A: immunoblot of AR protein from
mesangial cells grown in 8 mM and 20 mM glucose. MCLacZ cells
(left) and MCGT1 cells
(right). AR protein was detected at
~39 kDa. B: quantitation of AR
protein expression from 3 experiments. MCLacZ cells (open bars),
MCGT1 cells (solid bars). * P < 0.05 for 20 mM vs. 8 mM glucose. AR protein was significantly
(P < 0.05) greater for MCGT1 vs.
MCLacZ in all growth conditions. Single statistic
t-test,
n = 4 experiments.
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Fig. 3.
AR activity in RMC. AR enzyme protein activities were measured in
MCLacZ (open bars) and MCGT1 (solid bars) cells grown in 8 mM or 20 mM
glucose. * P < 0.05 for 20 mM
vs. 8 mM glucose. Measured AR activities were significantly
(P < 0.05) greater for MCGT1 vs.
MCLacZ in all growth conditions. Results are means ± SE of 4 experiments.
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The metabolic correlates of AR gene expression were assessed by
comparing the dose-dependent changes in sorbitol content after exposure
to normal (8 mM) or hyperglycemic concentrations of glucose (20 mM) in
MCGT1 and MCLacZ cells (Fig. 4). MCGT1
cells had greater abundance of sorbitol than the MCLacZ cells when
grown in both 8 mM glucose (68% sorbitol increase) and 20 mM glucose
(60% sorbitol increase). Although both RMC lines demonstrated
increased AR protein content and activity in high glucose, the MCGT1
cells had greater levels of AR protein and higher AR activity than did
MCLacZ cells grown in the same glucose concentrations.

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Fig. 4.
Sorbitol content in RMC. Sorbitol levels (nmol/mg protein) were
determined in MCLacZ (open bars) and MCGT1 (solid bars) after 4 days of
growth in media containing 8 mM or 20 mM glucose.
* P < 0.05 for 20 mM vs. 8 mM
glucose. Measured sorbitol contents were significantly
(P < 0.05) greater for MCGT1 vs.
MCLacZ in all growth conditions. Results are means ± SE of 4 experiments.
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Effect of increased glucose transport on PKC
expression. Quantitative immunoblotting of PKC isoforms
was conducted to determine whether enhanced glucose transport alone
enhanced PKC expression. Total PKC
protein was 40% more abundant in
the MCGT1 cells than in the MCLacZ cells (Fig.
5). Total PKC
and PKC
protein levels were not different in the MCGT1 and MCLacZ cells (Fig. 5). Immunoblot analyses of PKC
protein in MCLacZ and MCGT1 cells were also
performed after separation of proteins into their cytosolic and
particulate membrane fractions (Fig. 6).
Quantitation of PKC
cytosolic and particulate (active) membrane
fractions demonstrated a 30% greater cytosolic and 60% particulate
(active) PKC
fraction in the MCGT1 than in the MCLacZ cells when RMC
were grown in media containing 8 mM glucose (Fig.
7).

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Fig. 5.
Expression of PKC , - , and - proteins in transduced RMC (MCLacZ
and MCGT1 cells) grown in 8 mM glucose medium. Total PKC , - , and
- isoform proteins are shown in MCGT1 cells (solid bars with SE)
compared with their levels in control MCLacZ cells (note line at
100%). Levels of individual PKC proteins determined by densitometric
analysis are shown for MCGT1 cells (solid bars) plotted as percent of
control (MCLacZ) (error lines are SE).
* P < 0.05 for PKC in MCGT1
vs. MCLacZ cells, single statistic
t-test,
n = 4 experiments.
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Fig. 6.
Immunoblot of PKC protein in MCLacZ and MCGT1 cells grown in 8 mM
glucose. Cytosolic (Cyto) and particulate membrane (PM) fractions were
isolated from the 2 cell types. PKC protein was identified on
immunoblots with specific antibody, at ~80 kDa.
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Fig. 7.
Semiquantitation of PKC protein in cytosolic and particulate
membrane fractions of MCLacZ and MCGT1 cells. MCLacZ and MCGT1 cells
were both grown in 8 mM glucose medium. PKC protein was detected at
~80 kDa with specific antibody. Levels of PKC protein in each cell
fraction were determined by densitometric analysis, and levels in MCGT1
cells were plotted as percent control (+1 SE) vs. levels in control
MCLacZ cells (100%). * P < 0.05 (single statistic t-test) for
MCGT1 vs. MCLacZ cells in both the cytosolic (hatched bar) and
particulate membrane (solid bar) fractions;
n = 5 experiments.
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 |
DISCUSSION |
This is the first report of concomitant activation of AR, PKC
, and
GLUT1 genes from excessive transport of glucose in the absence of high
extracellular glucose or osmolality. RMCs overutilizing glucose
concomitantly activate genes believed to contribute to the metabolic
disturbances that promote the development of diabetic nephropathy.
Excessive ECM synthesis and release by mesangial cells is believed to
be the key event leading to glomerulosclerosis and renal failure in
diabetes (4). Consistent with the finding in clinical studies that
hyperglycemia plays a key role in the development of diabetic kidney
disease (17a), investigators have confirmed an important role for high
extracellular glucose concentrations in stimulating mesangial cell ECM
synthesis in vitro (5, 22, 25, 37, 38, 43). Facilitative diffusion of
glucose into RMC has previously been reported (37). GLUT1 is the
predominant facilitative glucose transporter in cultured mesangial
cells (30) and is a high-affinity, low-capacitance transporter
with a
Km in the range
of 1-7 mM for 2-deoxyglucose (30). These kinetic properties
suggest that the capacity for elevations in extracellular glucose
concentration to increase glucose uptake in the mesangial cell may be
limited by saturation of glucose transport at or near the physiological
glucose concentrations. Early increases in cellular glucose uptake by
RMC in response to high extracellular glucose may occur before GLUT1
upregulation, i.e., glucose transport capacity may not be fully
saturated at physiological glucose concentrations.
Glucose transporters are membrane-embedded proteins that mediate the
facilitative uptake of glucose from the surrounding medium into the
cell. The regulation of glucose transporters by glucose itself is
poorly understood, although it appears to be tissue specific (23, 34,
36). Many cell types exposed to hyperglycemic conditions limit their
increase in glucose metabolism by reducing the activity or expression
of facilitative glucose transporters, mainly in tissues that are not
the target of end-organ damage from diabetes (i.e., adipose and
skeletal muscle) (34). Recent reports have described the upregulation
of facilitative glucose transporters by pathophysiological
concentrations of glucose in the retina (40, 52, 55), erythrocytes
(27), and mesangial cells (29). In addition, troglitazone (54) and
tumor necrosis factor-
(44) have been reported to stimulate glucose
transport. The clinical relevance of enhanced glucose utilization and
diabetic nephropathy has recently been reported by Christian et al.
(14). The findings of Christian et al. (14) of increased renal glucose uptake and metabolism in human subjects with diabetes would suggest that altered glucose transport and/or metabolism is a part of the
kidney response to elevations in ambient glucose in
diabetes. These findings suggest that end-organs may be subject to
increased risk of diabetic complications by failing to reduce glucose
transporter activity or expression in the face of elevated
extracellular glucose concentrations.
How increased glucose utilization activates AR, PKC
, and the native
GLUT1 genes is not known, although preliminary data suggest that
glucose increased transcription of the GLUT1 gene through putative GlRE
(32). High extracellular glucose was not necessary to activate AR and
increase sorbitol content in the RMCs cells. Pathophysiological levels
of glucose increased AR mRNA abundance, protein activity, and sorbitol
production in both the MCGT1 and MCLacZ cell. AR has classically been
described as an osmoprotective gene protecting the renal medullary
cells from high external osmolality (24, 35), but high extracellular
glucose is not necessary to activate AR in the RMCs. Standard growth
media containing 12 mM mannitol as an osmotic control had no
appreciable effects on the expression of AR, PKC
, or GLUT1 mRNAs in
the MCGT1 or MCLacZ cells (data not shown). These findings are similar
to glucose-specific, nonosmotic activation of AR in the human
pancreatic duct epithelial cell line CAPAN-1, as reported by our
laboratory (11). CAPAN-1 human pancreatic duct cells also
demonstrate an AR inhibitor reversible 29% decrease in Na-K-ATPase and
a glucose-dependent increase in AR protein and AR activity
(11).
Much of our present understanding of AR gene regulation has been
derived from studies examining the highly conserved adaptive response
of renal medullary cells to hyperosmolality (osmoregulation/compatible osmolyte hypothesis) (10, 12, 60). Although these discoveries have
contributed greatly to our understanding of hyperosmotic regulation of
AR in the renal medulla, little is known about AR gene regulation by
pathophysiological levels of glucose seen in diabetes. In diabetes,
hyperosmolarity would not appear to be sustained long or high enough to
account for the increase in AR seen in end-organs affected by diabetes.
Although the physiological role of AR outside of the renal medulla is
not known, pathophysiological concentrations of glucose levels may
provide the basis for the detrimental activation of AR in RMC.
Activation of AR and the metabolic consequences of AR activation were
greater in the MCGT1 cell than in the MCLacZ cells and may reflect the
43-fold greater glucose utilization by the MCGT1 cells
for glucose metabolism (35). It is not known whether the increased rate of glucose transport per se or increased glucose utilization is responsible for activation of the AR gene; however, previous reports of GlREs in the liver (LPK and S14 genes) indicate a
requirement for glucose metabolism (49).
Central to the metabolism of glucose is the requirement for its
phosphorylation to glucose-6-phosphate by glucokinase or hexokinase (23). It is not yet known whether phosphorylation is necessary for
activation of AR, PKC
, or GLUT1. An as yet unidentified GlRE could
be responsible for activation of AR, GLUT1, or PKC
in the RMCs.
Increases in PKC induced by glucose in RMC rather that high glucose per
se may signal increases in GLUT1 or AR expression. In preliminary
studies (32), we have identified the mesangial cell GLUT1 gene as being
glucose responsive and have identified two putative GlREs in the GLUT1
gene. GlREs have been studied mostly in the context of
glucose-dependent regulation of gene expression such as the L-type
pyruvate kinase gene (42), fatty acid synthetase (23), or the S14 gene
as reviewed by Towle (58). There are common consensus sequence and
transacting factors (CACGTG motifs) that are known to confer
a glucose-specific response in mammalian tissues (49,
59). In a recent report, the pentose phosphate shunt intermediate
xylulose-5-phosphate conferred glucose responsiveness in the L-type
pyruvate kinase gene (18); hence, an intermediate of glucose metabolism
and not glucose transport per se may confer glucose-specific
responsiveness of these genes.
The metabolic consequences of increased glucose flux in MCGT1 cells
have been partially characterized and include 1.9-, 2.5-, and 2.1-fold
increase in the production of
myo-inositol, sorbitol, and lactate
content, respectively, as well as increases in glucose uptake (5-fold)
and in net utilization of glucose (43-fold). The findings in this
report also demonstrate a persistent increase in PKC
mRNA and total
protein in the MCGT1 cells. These findings suggest increased
transcription and or increased PKC
transcript stability and
increased synthesis of PKC
protein in response to enhanced glucose
transport. Increased PKC
could be the result of de novo synthesis of
diacylglycerol (3) leading to production of
jun and
fos protooncogenes that form the
transcription activator protein 1 complex (AP-1), which binds to the
cis-acting sequences in the promoter
of ECM genes (2). High glucose has been demonstrated to increase PCK
in RMC (16, 25, 39).
We have recently reported that glucose stimulates RMCs GLUT1 expression
and uptake and IGF-I-sensitive glucose uptake (29). IGF-I
has previously been reported to stimulate glucose uptake in cultured
RMCs (26). RMCs increase IGF-I production (19), expression of IGF-I
receptor (47), and IGF-I stimulated fibronectin accumulation in
diabetic mesangial cells (57). In that report, medium containing 20 mM
glucose increased GLUT1 mRNA (134%), GLUT1 protein (68%), and the
Vmax for glucose
uptake by 50% in RMCs (29). The enhanced glucose uptake was further
enhanced by the addition of IGF-I to the growth media (29). Thus IGF-I
may have an autocrine effect to increase GLUT-1 activity or
translocation in RMCs exposed to elevated glucose. Posttranscriptional
changes in AR protein stability or activity were not determined in the MCGT1 cell but could have accounted for the differences in AR mRNA, AR
protein, and AR activity between MCGT1 and MCLacZ cells.
Although the findings of the Diabetes Control and Complications Trial
(17a) have removed any doubt regarding the role of elevated blood
glucose and the risk for long-term complications from diabetes, the
mechanism(s) of tissue injury in diabetes is poorly understood. There
is presently no unified biochemical or molecular theory that explains
the development of long-term complications in all target organs
affected by diabetes; however, the metabolism of glucose is central to
all proposed theories. The rate of glucose entry or metabolism may be a
key proximal event in the development of glucose-mediated tissue damage
from diabetes. Our findings in RMCs suggest a detrimental positive
feedback relationship may exist when the cell is unable to limit the
rate of glucose uptake in the face of elevated ambient glucose
concentrations. Increased transport and metabolism of glucose augmented
AR activity and polyol metabolism, PKC
expression, increased ECM
accumulation (31, 35), and native GLUT1 expression and glucose
transport. Many of our present therapies to prevent the long-term
complications of diabetes are directed at the normalization of blood
glucose often at the expense of increased glucose transport into cells. New treatments aimed at limiting the rate of glucose transport into
end-organs affected by diabetes may provide another means of preventing
diabetic complications.
 |
ACKNOWLEDGEMENTS |
The anti-AR antibody was a generous gift from Peter F. Kador, Phd,
of the Laboratory of Ocular Therapeutics, National Eye Institute,
Bethesda, MD. We thank Seth R. Hootman, Phd, for technical assistance
in immunoblotting of aldose reductase and Cameron Deyarmon Henry for
assistance in the preparation of the manuscript.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases (NIDDK) Physician Scientist Award K11-DK-02193 and
Juvenile Diabetes Foundation International Research Grant 195044 (to D. N. Henry), NIDDK Grant K08-DK-01953 (to C. W. Heilig), and a National
Kidney Foundation of Michigan Grant. F. C. Brosius III is
an established investigator of the American Heart Association.
Portions of this work were presented in abstract form at the 29th
Annual Meeting, American Society of Nephrology, Nov. 3-6, 1996, in
New Orleans, LA.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. N. Henry, 113 Giltner Hall, Dept. of Physiology, Michigan State Univ., East Lansing,
MI 48824-1101 (E-mail: henry{at}psl.msu.edu).
Received 25 June 1998; accepted in final form 29 March 1999.
 |
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