1 Department of Medicine, University of Toronto, Toronto, and 2 Department of Medicine, McMaster University, Hamilton, Ontario M5G 2C4, Canada
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ABSTRACT |
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Expression of glutamine:fructose-6-phosphate amidotransferase
(GFAT), the rate-limiting enzyme for glucose entry into the hexosamine
pathway, is transcriptionally regulated. Immunohistochemical studies of
human kidney biopsies demonstrate increased GFAT expression in diabetic
glomeruli, but the mechanism responsible for this overexpression is
unknown. Given the role of ANG II in diabetic kidney disease, we chose
to study the effect of ANG II on GFAT promoter activity in mesangial
cells (MC). Exposure of MC to ANG II (107 M) increased
GFAT promoter activity (2.5-fold), mRNA (3-fold), and protein
(1.6-fold). ANG II-mediated GFAT promoter activation was inhibited by
the ANG II type I receptor antagonist candesartan (10
8 M)
but was unaffected by the ANG II type II receptor antagonist PD-123319
(10
8 M). The intracellular calcium chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(10
6 M), protein kinase C (PKC) inhibitors
bisindoylmaleimide-4 (10
6 M) and calphostin C
(10
7 M), protein tyrosine kinase (PTK) inhibitor
genistein (10
4 M), Src family kinase inhibitor PP2
(2.5 × 10
7 M), p42/44 mitogen-activated protein
kinase (MAPK) inhibitor PD-98059 (10
5 M), and the
epidermal growth factor (EGF) inhibitor AG-1478 all attenuated GFAT
promoter activation by ANG II. We conclude that the GFAT promoter is
activated by ANG II via the AT1 receptor. Promoter
activation is calcium dependent and PKC dependent but also involves PTK
signaling pathways including Src, the EGF receptor, and p42/44 MAPK.
angiotensin II; signaling; glomerulus; mesangial cells; glutamine:fructose-6-phosphate amidotransferase
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INTRODUCTION |
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THE HEXOSAMINE
PATHWAY has been implicated in some of the adverse effects of
glucose (6, 8, 20, 40), and glucose flux through
the hexosamine pathway may contribute to the development of
diabetic kidney disease. Under physiological conditions, a small percentage (1-3%) of glucose entering cells is
shunted through the hexosamine pathway (40). In
the first step of the pathway (Fig. 1),
fructose-6-phosphate is converted to glucosamine-6-phosphate by the
rate-limiting enzyme glutamine:fructose-6-phosphate
amidotransferase (GFAT) (40). Glucose flux through
the hexosamine pathway plays an important role in the development of
insulin resistance in adipocytes (8, 40). Cultured cells
that overexpress GFAT develop insulin resistance in the absence of
hyperglycemia (6, 8), and transgenic mice that overexpress
GFAT in skeletal muscle and adipose tissue are insulin resistant
(20). In mesangial cells (MC), flux through the hexosamine
pathway has been implicated in glucose-induced increases in
transforming growth factor-1 (TGF-
1) expression
(31).
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Despite these experimental observations, the regulation of expression
of GFAT has not been studied extensively. In vitro, GFAT mRNA levels
are transcriptionally regulated. Epidermal growth factor- (EGF-
)
activates the GFAT promoter in cells that overexpress the EGF receptor,
but glucose does not activate the promoter in these cells
(49). Immunohistochemical studies of human kidney biopsies
have revealed increased GFAT expression in diabetic glomeruli (47), but the mechanism responsible for this effect is
unknown. ANG II plays an important role in the pathogenesis of diabetic kidney injury (18, 29, 41). Blockade of the
renin-angiotensin system activity by angiotensin-converting enzyme
(ACE) inhibition or ANG II type I (AT1) receptor antagonism
slows progression of clinical and experimental diabetic nephropathy
(18, 29, 36, 37, 62). We hypothesized that ANG II
increases GFAT mRNA levels in glomerular MC and sought to determine
whether ANG II would activate the GFAT promoter in cultured MC.
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EXPERIMENTAL PROCEDURES |
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Materials.
D-Glucose, ANG II, phorbol 12-myristate 13-acetate (PMA),
bisindoylmaleimide-4 (BIM4), calphostin C,
o-diazoacetyl-L-serine (azaserine),
6-diazo-5-oxonorleucine (DON),
o-nitrophenyl--D-galactopyranoside (ONGP), and
benzyl-2-acetamido-2-deoxy-
-D-galactopyranoside (BADGP) were obtained from Sigma-Aldrich (Mississauga, Ontario, Canada). A23187, PD-98059, PP2, and AG-1478 were purchased from Calbiochem (La
Jolla, CA). Genistein and daidzein were purchased from Alexis (San Diego, CA), whereas candesartan was generously provided by AstraZeneca (Molndal, Sweden). Dulbecco's modified Eagle's medium (DMEM), FBS, and Trizol reagent were purchased from GIBCO-BRL (Life
Technologies, Grand Island, NY). Reporter cell lysis buffer was
obtained from Promega (Madison, WI), and Effectene transfection reagent
was from Qiagen (Mississauga, Ontario, Canada).
Preparation and culture of mesangial cells. MC were obtained from male Sprague-Dawley rats as described (27, 60). The cells were cultured (37°C, 5% CO2) in DMEM supplemented with FBS (20%), penicillin (100 U/ml), streptomycin (100 µg/ml), and glutamine (2 mmol/l). Cells were used between passages 14 and 20.
Plasmids.
Plasmid pGFAT, created by the ligation of the GFAT promoter
(1822/+88, GenBank accession no. U39442) upstream of the luciferase gene in plasmid pGL2, was generously provided by Dr. Jeff Kudlow and
has been previously described (58). Vascular cell adhesion molecule-1 (VCAM-1) promoter-luciferase construct pVCAM
(9) was generously provided by Dr. J. M. Redondo,
Universidad Autonoma, Madrid, Spain. pCMV-
gal (Promega) was
used to control for variation in transfection efficiency.
Transient transfection of mesangial cells.
MC (1.5 × 105 cells/well) were plated onto 6-well
plastic plates (Sarstedt), and transfection was carried out
24 h later using Effectene (Qiagen) as described by the
manufacturer. Briefly, MC (70-80% confluent) were cotransfected
with 0.35 µg pGFAT or pVCAM and 0.05 µg pCMV-gal and then
cultured for 6 h in DMEM containing FBS (20%) and 5.6 mmol/l
D-glucose. Subsequently, the media were changed to DMEM
with 0.5% FBS and glucose (5.6 to 30 mmol/l). ANG II
(10
10 to 10
7 M) or inhibitors were added as
required. At various times, the cells were harvested and used for
analyses as described below.
Assay of luciferase and -galactosidase activity.
Media were aspirated, wells washed twice with PBS, and lysis was
performed using 0.2 ml/well of Reporter lysis buffer (Qiagen). MC were
incubated for 15 min at 4°C, then transferred to microcentrifuge tubes using a rubber policeman. Cell debris was pelleted by
centrifugation (12,000 g, 4°C, 1.0 min), and the
supernatant was used to assay for luciferase (0.02 ml) and
-galactosidase (0.05 ml) activities using commercially available
reagents. Luciferase was measured in a luminometer (EG&G, Berthold,
TN), and
-galactosidase activity was based on the absorbance at 405 nm. Luciferase activity was normalized to the
-galactosidase
activity and cell protein. Protein was determined on an aliquot of the
supernatant obtained from cell lysis using Bio-Rad protein assay dye
reagent (Bio-Rad Laboratories).
RNA isolation and semiquantitative RT-PCR.
Total RNA from MC was isolated by the single-step method of Chomczynski
and Sacchi (7), as we have published (27, 28, 60). Isolated RNA was stored in diethyl pyrocarbonate-treated water at 80°C The purity and concentration were determined by measuring the optical density at 260 nm and 280 nm prior to use. The
absorbance ratio, A260/A280, ranged from
1.75-1.95.
Western blotting of GFAT.
Nontransfected MC were exposed to ANG II (107 M) for
16 h, media were removed, and the cells were washed once with
ice-cold PBS. Total cellular protein was obtained and used for Western blotting as we have described (27). GFAT antibody
(47) was generously provided by Dr. E. D. Schleicher,
University of Tübingen, Germany.
Statistical analysis. Statistical analyses were performed with the INSTAT statistical package (GraphPad Software, San Diego, CA). The difference between means was analyzed using the Bonferroni multiple comparison test. Significance was defined as P < 0.05.
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RESULTS |
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ANG II increases GFAT mRNA and protein levels in MC.
GFAT expression is transcriptionally regulated (49), and
immunohistochemical studies of human diabetic kidney biopsies have revealed increased GFAT expression (47). However, the
mechanism(s) responsible for the increased in GFAT observed in diabetic
glomeruli remains unclear. Since ANG II is known to play an important
role in the progression of diabetic kidney disease (18, 36,
41), we sought to determine the effect of ANG II on GFAT mRNA
and protein levels in nontransfected MC. Nontransfected MC were growth
arrested in DMEM/0.5% FBS, then exposed to ANG II (107
M). At the end of the incubation period, total RNA was extracted and
used for RT-PCR analysis (7, 15, 16). RT-PCR analysis (Fig. 2) indicated that after 8 h of
exposure to ANG II (10
7 M), there was a threefold
increase in mRNA for GFAT (P < 0.02, n = 4).
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ANG II activates the GFAT promoter in a time- and
concentration-dependent manner.
Subsequently, we determined whether ANG II would activate a transiently
transfected GFAT promoter-reporter construct in rat MC. Six hours after
transfection with pGFAT and pCMV-gal, MC maintained in DMEM/FBS
(0.5%)/5.6 mM glucose were exposed to ANG II (10
7 M) for
an additional 2-48 h. Concurrent control MC, maintained in
DMEM/FBS (0.5%)/5.6 mM glucose, were not exposed to ANG II. GFAT
promoter activity was assessed by measuring luciferase activity, normalized to
-galactosidase activity and total cellular protein. GFAT promoter activity increased 1.5-fold after 4 h of exposure to
ANG II (P < 0.01, n = 5) and by
2.5-fold after 24 h of ANG II exposure (Fig.
3A).
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ANG II activation of the GFAT promoter is mediated through the AT1 receptor. Given the central role of the AT1 receptor in most pathological effects of ANG II (2, 37), we sought to clarify whether the observed increase in GFAT expression in MC by ANG II was mediated through this receptor.
MC were transiently transfected with pGFAT and pCMV-
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ANG II activation of the GFAT promoter is calcium dependent.
Intracellular signaling in response to ANG II after binding to
the AT1 receptor is complex. First, ANG II increases
intracellular calcium (3, 25, 43, 44, 51), which appears
to play an important role in propagation of the ANG II signal (3,
25, 43, 44). To examine the role of calcium in ANG II activation of the GFAT promoter, MC transiently transfected with pGFAT exactly as
above were exposed to the intracellular calcium chelator,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA, 106 M) for 24 h.
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ANG II activation of the GFAT promoter is protein kinase C
dependent.
Many observed intracellular effects of ANG II are responsive to protein
kinase C (PKC) inhibition, implicating PKC in ANG II signaling
(3, 23, 25, 26, 43). To study the role of PKC, we examined
the effect of PMA on GFAT promoter activity in transiently transfected
MC. Acute exposure to PMA leads to PKC activation in many cells,
whereas protracted exposure (greater than 24-48 h) leads to PKC
depletion (42, 66). Consequently, MC transiently
transfected with pGFAT as above were exposed to PMA (5 × 107 M) or vehicle [dimethyl sulfoxide (DMSO)] for 30 min. PMA or vehicle was removed, and cells were washed with PBS, then
exposed to PKC inhibitors BIM4 (10
6 M) and calphostin C
(10
7 M), or vehicle (DMSO) for 1 h. Media were
removed, and cells were washed with PBS, then cultured for an
additional 24 h in DMEM/0.5% FBS/5.6 mM glucose.
ANG II activation of the GFAT promoter depends on protein tyrosine kinase. Total phosphotyrosine increase markedly in cells exposed to ANG II (25, 39, 54, 59), and many ANG II effects are sensitive to protein tyrosine kinase (PTK) inhibition (25, 39, 59). Accordingly, we tested whether inhibition of these signaling components affected the GFAT induction observed in MC exposed to ANG II. Following transfection of MC, all experiments were conducted in DMEM/0.5% FBS containing physiological (5.6 mM) glucose concentrations.
To determine the role of tyrosine phosphorylation in the intracellular signaling in response to ANG II that ultimately results in GFAT induction, transiently transfected MC were cultured in DMEM/0.5% FBS/5.6 mM glucose with and with ANG II (10
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The hexosamine pathway participates in ANG II-induced increases in
mRNA for TGF-1 and VCAM-1.
ANG II activates the genes for TGF-
1 and VCAM-1, and both
TGF-
1 and VCAM-1 have been implicated in glomerular injury
(46, 61, 63). Therefore, to determine whether activation
of GFAT is important in the downstream effects of ANG II on genes that have been implicated in glomerular injury, we examined whether the
effect of ANG II on TGF-
1 and VCAM-1 mRNA levels in MC was dependent
on flux through the hexosamine pathway.
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High glucose increases GFAT mRNA and activates the GFAT promoter in
MC.
A final series of experiments were designed to determine whether
glucose had any effect on the mRNA levels for GFAT and on GFAT promoter
activity in MC. Six hours after transfection with pGFAT and
pCMV-gal, MC were exposed to ANG II (10
8 M) for
24 h in medium (DMEM/0.5% FBS) containing either physiological (5.6 mM) or high (30 mM) glucose concentrations, and promoter activity
was assessed by measuring luciferase activity normalized to
-galactosidase activity. Control experiments with MC cultured in
DMEM/0.5% FBS containing physiological glucose concentration (5.6 mM)
were performed concurrently.
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DISCUSSION |
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One-third of diabetic patients will develop kidney disease
characterized by progressive mesangial matrix expansion, proteinuria, and declining glomerular filtration rate, but the mechanisms
responsible for diabetic glomerular injury remain poorly understood.
ANG II plays a central role in the progression of diabetic glomerular disease (5, 18, 36, 41, 69). ACE inhibition or the use of
an AT1 receptor antagonist abrogates the ANG
II-mediated increase in TGF-1 and attenuates the development of
glomerular sclerosis in both diabetic and nondiabetic models (5,
18, 36, 37, 41, 70).
Recently, a new metabolic pathway for intracellular glucose, the hexosamine pathway, has been described, and it may play a role in diabetic injury. Kolm-Litty and co-workers (31) have suggested that glucose flux through the hexosamine pathway (Fig. 1) is important in the pathogenesis of diabetic glomerulopathy. In the hexosamine pathway, fructose-6-phosphate is first converted to glucosamine-6-phosphate by the rate-limiting enzyme GFAT (40). Under normal physiological conditions, only a small percentage (1-3%) of glucose entering cells is shunted through this pathway (40). The end product of the hexosamine pathway, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), is a substrate for O-glycosylation of intracellular proteins (19, 32, 40, 52, 56). O-glycosylation is important for posttranslational modification of transcription factors and may affect gene expression (19, 32, 56). The hexosamine pathway is believed to allow cells to sense the level of glucose in the extracellular environment (19, 65).
Immunohistochemical studies of human kidney biopsies have revealed increased GFAT expression in diabetic glomeruli (47), but the mechanism(s) responsible for this effect is unknown. We hypothesized that ANG II, in view of its important role in development of glomerular injury and diabetic kidney disease, might regulate GFAT expression in MC. To test our hypothesis, we first chose to determine whether ANG II increased MC GFAT mRNA levels. Subsequently, we sought to fully characterize the effect of ANG II on GFAT promoter activity by transiently transfecting MC with a GFAT promoter-reporter construct (pGFAT).
Our first major finding was that ANG II increased GFAT mRNA and protein levels in our primary cultured MC. In accord with previous studies showing the GFAT is transcriptionally regulated (50), ANG II also activated the GFAT promoter in a dose- and time-dependent fashion. Most of the actions of ANG II are mediated by binding to the AT1 receptor at the cell surface (4, 27, 39). ANG II-induced activation of the GFAT promoter was mediated by the AT1 receptor, because candesartan, the specific AT1 receptor antagonist, completely prevented GFAT promoter activation by ANG II, whereas the AT2 receptor antagonist was without effect.
The AT1 receptor is a seven-transmembrane protein
that lacks inherent PTK activity, and the activity of this receptor is
coupled to G proteins at the plasma membrane (2, 26, 38,
59). Following the ligation of the AT1 receptor by
ANG II, intracellular signaling may proceed by three possible pathways
(2, 26, 38). ANG II binding to the AT1
receptor leads to activation of phospholipase C- and the generation
of inositol trisphosphate (IP3) and diacylglycerol (DAG).
IP3 promotes calcium release from the endoplasmic reticulum
and consequently increases intracellular calcium levels that may lead
to activation of gene transcription (43, 44). On the other
hand, DAG activates PKC, and the latter may then influence target gene
expression (23, 38, 39). Finally, when ANG II binds to the
AT1 receptor, intracellular tyrosine kinase signaling
pathways, including MAPK, are activated, leading to changes in gene
transcription (24, 39).
Our data indicate that ANG II regulates GFAT promoter activity by modulating signaling pathways that include calcium, PKC, and tyrosine kinase cascades. A direct role for PKC in ANG II-mediated GFAT promoter activation is suggested by the observations that PMA was sufficient to activate the promoter and, second, that inhibitors of PKC prevented GFAT promoter activation by ANG II. On the other hand, our observation that an intracellular calcium chelating agent, BAPTA, attenuated the activation of the GFAT promoter by ANG II suggests calcium was necessary for ANG II-mediated activation of the GFAT promoter. However, the inability of the calcium ionophore A23187 to activate the promoter suggests calcium did not have an independent effect on GFAT promoter activation. The effect of ANG II on isolated glomeruli may depend on interactions between PKC and calcium signaling (12), and calcium may enhance PKC activity (48). Therefore, it is possible that calcium-PKC interactions may play a role in GFAT promoter activation by ANG II. Similarly, calcium-dependent tyrosine phosphorylation of intracellular protein has been described (22); therefore, it is also possible that calcium may participate in GFAT promoter activation via this latter mechanism.
Some of the actions of ANG II are dependent on activation of tyrosine
kinase signaling cascades (39, 54). We first looked at the
effect of a nonspecific PTK inhibitor (genistein) and its inactive
analog on ANG II-induced GFAT promoter activity. The activation of the
GFAT promoter by ANG II was abolished by the tyrosine kinase inhibitor
genistein but not its inactive analog, implicating tyrosine kinase
signaling in the promoter response to ANG II. To further characterize
the effect of PTK inhibition on ANG II-induced GFAT promoter
activation, we studied the Src kinase inhibitor PP2 and the
EGF-specific receptor tyrphostin AG-1478, because src kinases
and transactivation of the EGF have been implicated in ANG II signaling
(50, 55). Finally, we studied the effect of PD-98059, an
inhibitor of p42/44 MAPK, on activation of the GFAT promoter by ANG II.
The src kinase inhibitor, the EGF receptor tyrphostin, and
PD-98059 inhibited the response to ANG II. Taken together, these
results suggest that one important pathway linking ANG II and
activation of the GFAT promoter is via transactivation of the EGF
receptor with the subsequent downstream activation of p42/44 MAPK.
Transactivation of the EGF receptor and MAPK activation has been
implicated in the signaling response to ANG II (4, 11, 17, 45,
67, 68). Furthermore, signaling pathways including src,
calcium and PKC have been implicated in the response to ANG II by
vascular smooth muscle cells (55) and in the response to
neuropeptides by Swiss 3T3 cells (53). The regulatory
elements in the GFAT promoter (58) (GenBank accession no.
U39442) that are responsible for ANG II-induced activation were not
identified in the current study. However, the PKC dependence, taken
together with the observation that PMA is sufficient to activate the
promoter, suggests that two AP1 sites, located at positions 542 and
309 of the promoter, may be responsible for the effects of ANG II
(4, 34, 35).
ANG II is known to activate transcription of TGF-1 and other
profibrotic and proinflammatory cytokines (30, 46, 61, 62, 69,
71). In addition, ANG II induces VCAM-1 expression (63), and ACE inhibition decreases the level of soluble
VCAM-1 in type II diabetics (13, 14). We have also shown
that GFAT overexpression leads to activation of genes that are
important in fibrosis (28). Thus we sought to determine
whether flux through the hexosamine pathway mediated some of the
downstream effects of ANG II. Inhibition of GFAT, the rate-limiting
enzyme for flux through the hexosamine pathway, with azaserine or DON,
attenuated but did not abolish the effect of ANG II on VCAM-1 and
TGF-
1 mRNA levels in MC. Furthermore, using BADGP, an inhibitor of
O-glycosylation (21, 33), also prevented the
ANG II-mediated increase in GFAT promoter activity. These results
suggest that part of the MC response to ANG II is dependent on flux
through the hexosamine pathway.
Both the Diabetes Control and Complications Trial Research Group and the UK Prospective Diabetes Study Group trials (10, 64) have also shown that glycemic control is a key determinant of diabetic microvascular injury. Accordingly, we further sought to determine whether ANG II and high glucose concentrations would have independent, additive, or synergistic effects on GFAT promoter activity in MC. In a previous study, Paterson and Kudlow (49) found that elevated glucose concentrations alone had no impact on GFAT promoter activity in a breast cell cancer line, but when combined with EGF, glucose (25 mM) attenuated activation of the GFAT promoter by EGF. In contrast to this report, we observed that high glucose levels (20 and 30 mM) activated the GFAT promoter in cultured MC. Thus there are cell-specific differences in the regulation of the GFAT promoter. High glucose concentrations have been shown to downregulate AT1 receptor density in MC and to increase PKC isoform expression (1). The sum of these effects on ANG II-dependent signaling responses in the MC is difficult to predict, so we looked at the activation of the GFAT promoter by ANG II in MC maintained in 30 mM glucose. Although each condition led to an increase in promoter activity, there was no additive or synergistic effect between glucose and ANG II.
We believe that our finding that ANG II activates the promoter for GFAT
has important implications in view of the role of ANG II in the
progression of renal disease, particularly because ANG II is known to
activate transcription of TGF-1 and other profibrotic and
pro-inflammatory cytokines (30, 46, 61, 62, 69, 71). In
addition, we have also shown that GFAT overexpression leads to
activation of genes for plasminogen activator inhibitor-1 (PAI-1),
TGF-
1, and TGF-
receptors in MC (28).
Therefore, ANG II may also affect expression of genes that promote
glomerulosclerosis by influencing glucose flux through the hexosamine pathway.
Although the regulation of GFAT promoter activity by ANG II has been studied with a mouse promoter, we believe that these results are relevant to humans. The cDNA for mouse GFAT has 91% homology with the human sequence, and the amino acid sequence of mouse GFAT shows 98.6% homology with human GFAT (57). Given that mice and rats have been extensively used as models for studying diabetes mellitus and its complications (including glomerular diseases), we believe that our model system will have relevance to understanding how GFAT is regulated in human cells and will provide insights into the role that the hexosamine pathway may play in the pathogenesis of diabetic complications such as nephropathy.
In summary, our findings support the hypothesis that ANG II, acting via the AT1 receptor, increases expression of GFAT, the rate-limiting enzyme for glucose entry into the hexosamine pathway. ANG II regulates GFAT promoter activity in cultured MC by modulating signaling pathways that include calcium, PKC, src kinases, the EGF receptor, and p42/44 MAPK. These findings suggest that a link between ANG II and the metabolic fate of glucose may contribute to the development of diabetic complications such as vascular and glomerular injury.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jeff Kudlow (University of Alabama, Birmingham, AL) for generously providing the GFAT promoter-luciferase reporter plasmid (pGFAT) and AstraZeneca for supplying candesartan. We are grateful to Dr. J. M. Redondo (Centro de Biologia Molecular, Universidad Autonoma, Madrid, Spain), who provided the VCAM-1 promoter construct, and Dr. E. D. Schleicher (University of Tübingen, Germany) for GFAT antiserum.
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FOOTNOTES |
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This work was supported by a grant from the Juvenile Diabetes Foundation and the Medical Research Council of Canada to J. W. Scholey. L. R. James is the recipient of a Research Fellowship from the Kidney Foundation of Canada-Medical Research Council Partnership Program.
Address for reprint requests and other correspondence: L. R. James, 13 EN-243, Toronto General Hospital, Univ. Health Network, 200 Elizabeth St., Toronto, Ontario M5G 2C4, Canada (E-mail: leighton_james{at}med.unc.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 11 September 2000; accepted in final form 22 February 2001.
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