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J. Biol. Chem., Vol. 275, Issue 51, 40042-40047, December 22, 2000
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From the Renal Division and Department of Medicine, Joslin Diabetes
Center, Beth Israel Deaconess Medical Center, and Harvard Medical
School, Boston, Massachusetts 02215 and the
Received for publication, August 17, 2000, and in revised form, September 26, 2000
Recent studies have shown that hyperglycemia is a
principal cause of cellular damage in patients with diabetes mellitus.
A major consequence of hyperglycemia is increased oxidative stress. Glucose-6-phosphate dehydrogenase (G6PD) plays an essential role in the
regulation of oxidative stress by primarily regulating NADPH, the main
intracellular reductant. In this paper we show that increased glucose
(10-25 mM) caused inhibition of G6PD resulting in
decreased NADPH levels in bovine aortic endothelial cells (BAEC). Inhibition was seen within 15 min. High glucose-induced inhibition of
G6PD predisposed cells to cell death. High glucose via increased activity of adenylate cyclase also stimulated an increase in cAMP levels in BAEC. Agents that increased cAMP caused a decrease in G6PD
activity. Inhibition of cAMP-dependent protein kinase A
ameliorated the high glucose-induced inhibition of G6PD. Finally, high
glucose stimulated phosphorylation of G6PD. These results suggest that, in BAEC, high glucose stimulated increased cAMP, which led to increased
protein kinase A activity, phosphorylation of G6PD, and inhibition of
G6PD activity. We conclude that these changes in G6PD activity play an
important role in high glucose-induced cell damage/death.
Diabetes mellitus can lead to complications in the eye, heart,
kidney, and nerves. Hyperglycemia has been shown to play a principal
role in the pathogenesis of diabetic complications in both type 1 and
type 2 diabetes mellitus (1, 2). Thus it is critical to understand how
hyperglycemia leads to cellular damage. Hyperglycemia-induced increases
in oxidative stress have been suggested to be of central pathogenic
importance (3, 4). An increase in oxidative stress may be due to an
increase in processes that produce oxidants or due to a decrease in
antioxidant defenses. Glucose-6-phosphate dehydrogenase
(G6PD),1 the rate-limiting
enzyme of the pentose phosphate pathway, is required for the
antioxidant defense system because it produces NADPH, the cells'
principal reductant (5, 6). Previous research from our laboratory has
shown that G6PD, an enzyme traditionally thought to be under little
post-translational control, is regulated both in its activity and
intracellular location by specific signal transduction molecules
(7-9). Importantly, our laboratory has also shown that modest changes
in G6PD activity itself have significant effects on cell growth and
cell death in a variety of cell types (5, 6). Thus proper activity of
G6PD is required for adequate defense against oxidative stress and
prevention of cell damage/death. Previous researchers have shown that
the pentose phosphate pathway (as measured by release of radioactively
labeled CO2) was impaired after exposure to high glucose.
Asahina et al. (10) showed that when human umbilical
endothelial cells in high glucose (33 mM) were exposed to
H2O2 the expected increase in pentose phosphate pathway activity was almost completely inhibited as compared
with cells incubated in normal glucose conditions (5 mM).
The data suggested that high glucose predisposes cells to oxidative
damage because of inadequate activation of the pentose phosphate
pathway after exposure to an oxidant. G6PD per se was not
evaluated in this study, nor was the mechanism of high glucose effect
on G6PD reported. Thus we hypothesized that under high glucose
conditions impaired activity of G6PD would predispose cells to oxidant
damage and cell death. The data reported in this paper support the
previous studies (10) and provide mechanistic information by showing that inhibition of G6PD occurs at least in part by high
glucose-stimulated increases in cAMP levels.
Materials--
Chemicals were obtained from Sigma. G6PD antibody
was a generous gift from Rolf Kletzien (Upjohn).
Cell Culture--
Bovine aortic endothelial cells (BAEC) were
grown in DMEM + 10% calf serum. Cells were used between passages 5 and
10. Cells were harvested by scraping and then lysed in a buffer
containing phosphate-buffered saline + 5% Nonidet P-40, and in
protease inhibitors.
Measurement of G6PD Activity--
G6PD activity of the lysates
was measured as described previously except that substrate
concentrations were 500 µM glucose 6-phosphate and 250 µM NADP+ (7).
Measurement of Cell Death--
Cell death was measured by trypan
blue exclusion as has been done previously (6).
Measurement of Apoptosis--
Apoptosis was measured by
4',6'-diamidine-2'-phenylindole dihydrochloride as described previously
(6).
Measurement of NADPH--
NADPH was measured by a new method
designed by us (11). In brief, the method is based on the fact that
only NADH and NADPH (and not NAD+ and NADP+)
affect absorbance at 340 nm. Cell extracts are separated into 3 aliquots (A1, A2, and A3).
A1 is untreated and the absorbance at 340 nm is measured.
A2 is treated with an enzyme that converts all of the
NADP+ to NADPH, and then the absorbance at 340 nm is
measured. A3 is treated with an enzyme that converts all of
the NADPH to NADP+, and then the absorbance at 340 nm is
measured. A1 Measurement of GSH--
BAEC, which were 80-90% confluent,
were incubated with 5.5 and 25 mM glucose for 3 h. GSH
was measured using the GSH kit BIOXYTECH GSH-400 from OXIS
International, Inc.
Measurement of Reactive Oxygen Species (ROS)--
ROS were
measured as described previously using the dye
2',7'-dichlorofluorescein diacetate (6). Dichlorofluorescein
fluorescence was measured using a microplate fluorometer (Cambridge Technology).
Measurement of cAMP and Adenylate Cyclase Activity--
BAEC
were incubated with 5.5 and 25 mM glucose for 3 h.
Adenylate cyclase activity was measured by a new method designed by us.
The method is based on relative differences between two cell lysates
rather than absolute activities. Cell lysates either from cells exposed
to 5 mM glucose or lysates from cells exposed to 25 mM glucose were prepared. Then each lysate was separated
into two aliquots (A and B). To be certain there was no limitation on
substrate availability, ATP (the substrate for adenylate cyclase) was
added to each aliquot, and then aliquot A was immediately boiled for 10 min. Aliquot B was incubated at 37 °C for 15 min and then boiled for
10 min. Then cAMP was measured using cAMP kit TRK432 from Amersham
Pharmacia Biotech. Isobutylmethylxanthine (1 mM) was
included in all solutions to inhibit cAMP-dependent phosphodiesterase activity. The amount of cAMP from aliquot A represents the base-line cAMP level. The difference between the cAMP
amount in aliquot B and in aliquot A (i.e. B Measurement of G6PD Phosphorylation--
BAEC that were 80%
confluent were exposed to 1 mCi of 32P/P100 culture plate
in phosphate-free buffer for 2 h. Then the glucose concentration
was increased by adding glucose to the plate. After 3 h cells were
harvested and lysed, and G6PD was immunoprecipitated using antibody to
G6PD as described previously (5). Following immunoprecipitation, G6PD
protein was resolved by SDS-polyacrylamide gel electrophoresis and then
exposed to x-ray film.
Antisense Experiment--
Antisense experiments were done with
the excellent assistance of Drs. Jane Alexander and Joseph Loscalzo of
the Boston University Medical Center, who provided both the antisense
sequence and suggestions on transfection techniques. Transfection was
carried out using the method described by the Sequitur, Inc.
kit. In brief, BAEC, which were 80-90% confluent, were washed once
with Opti-MEM without serum or antibiotics. Then 13.2 µl of
Oligofectin I (stock solution, 2 mg/ml) was added in 1 ml of Opti-MEM
in a polystyrene tube. In another tube 4.0 µl of antisense oligomer
was added to 1 ml of Opti-MEM. The two solutions were mixed together
and allowed to sit for 15 min. The mixture was then added to cells for
5 h. 0.5 ml of mixture was added to each well of a 24-well plate.
After 5 h of incubation, the medium was removed, and
Opti-MEM with 10% serum was added to the cells for 24 h.
Experiments were then started at this time. The antisense sequence we
used was 5'-AGUAGUACCCACGUAGCCCACUGGGA-3'. After transfection, BAEC
were incubated with 5.5 and 25 mM glucose for 24 h.
Then, cell death and G6PD activity were measured as described above.
Vascular endothelial cells are known targets of hyperglycemia.
Thus we have used BAEC, a commonly used system, for the studies. BAEC
were incubated with various concentrations of glucose. Cells were
harvested, and cell lysates were obtained as described previously (7,
8). G6PD activity was measured as described previously (7, 8). Fig.
1A shows that glucose
concentrations as low as 10 mM were sufficient to inhibit
G6PD activity. As an osmotic control another sugar, raffinose, was
added to medium containing 5.5 mM glucose to achieve the
same concentrations as seen with the high glucose conditions. Raffinose
had no effect on G6PD activity (data not shown). Also note that a
raffinose control was done for all the remaining experiments and in all
cases did not cause any of the effects seen with increased glucose.
High glucose has been shown to increase cell death in BAEC (12). As
seen in Fig. 1B, increased glucose led to cell death. To
further determine an association between G6PD activity and BAEC cell
death, two inhibitors of G6PD were used. Both dehydroepiandrosterone (5, 6) and 6-aminonicotinamide (5, 6) worsened cell death at all
glucose levels. Importantly there was a close correlation with impaired
G6PD activity and cell death (Fig. 1, compare A and
B). If high glucose-induced cell death is due, at least in part, to impaired activity of G6PD, then cells exposed to high glucose
should be more susceptible to oxidant-induced cell death. Fig.
1C shows that H2O2-induced cell
death was significantly enhanced in cells exposed to high glucose.
We have recently published that G6PD activity can affect apoptosis in a
variety of cell types including BAEC (6). Because high glucose led to
an increase in cell death we explored whether there was an increase in
apoptosis. Fig. 1D shows that high glucose increased
apoptosis in BAEC.
Considering the relative lack of specificity of the G6PD inhibitor,
DHEA, we more specifically addressed the role of G6PD by using an
antisense oligonucleotide to reduce G6PD activity. Fig.
2A shows that both in normal
and high glucose conditions transfection with an antisense sequence led
to a highly significant decrease in G6PD activity. Note that the
decrease in G6PD activity was greater in the BAEC exposed to high
glucose. Also the transfection efficiency for the Sequitur
Oligofectin system is about 30%. As is clear from the results in Fig.
2A, there is almost a 60% decrease in G6PD activity
following transfection. Although we have no specific answer as to why
there is such a profound decrease, it is our belief that there is
on-going cell death, which leads to release of the oligonucleotide and
subsequent transfection of other cells over time. This observation and
idea as to the reason for the profound effect on G6PD activity was also
seen and suggested by a
collaborator.2 Fig.
2B shows that the antisense sequence also led to a
significant enhancement of the cell death especially under high glucose
conditions. These results strongly support an important role for G6PD
in high glucose-induced cell death.
High Glucose Inhibits Glucose-6-phosphate Dehydrogenase via cAMP
in Aortic Endothelial Cells*
, and Chemistry Department, Southern Connecticut State
University, New Haven, Connecticut 06515
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A3 is the NADPH content, and
A2 A1 is the NADP+ content of
the extract.
A) represents the adenylate cyclase-produced cAMP. In the
figures, the results are expressed as the increase in cAMP (aliquot B
cAMP aliquot A cAMP level).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (59K):
[in a new window]
Fig. 1.
A, high glucose causes inhibition
of G6PD activity. Concentrations as low as 10 mM glucose as
early as 15 min after exposure are sufficient to suppress G6PD
activity. BAEC were initially grown in DMEM (5.5 mM
glucose) + 10% serum that were about 80% confluent and then
switched to DMEM with 2% serum plus various concentrations of glucose
ranging from 5.5 to 25 mM for 15 min to 24 h. The same
experiment done on confluent cells yielded similar results as shown
above. All subsequent figures use the same approach with slight changes
in either percent confluence or incubation time. The data were
normalized by protein and presented as average ± S.E. from three
separate experiments, each run in at least triplicate (*,
p < 0.01; **, p < 0.005 compared with
control). B, high glucose and G6PD inhibitors increase cell
death as measured by trypan blue exclusion. High glucose increased cell
death. Inhibition of G6PD enhanced BAEC cell death. BAEC were treated
as in Fig. 1A (except used at 60% confluent), switched to
DMEM + 2% serum and glucose for 24 h, and then exposed to an
inhibitor of G6PD (either 100 µM DHEA or 5 mM
6-aminonicotinamide (ANAD) for 3 h. Data were
normalized by cell number and expressed as means ± S.E. of 10 separate experiments, each run in triplicate (***, p < 0.001 compared with control). C,
H2O2 enhances cell death in the presence of
high glucose. BAEC were prepared as described in B. Then
cells were treated with 400 µM
H2O2 for 3 h. Cell death was determined by
trypan blue exclusion. Data were normalized by cell number and
expressed as means ± S.E. of five separate experiments, each run
in triplicate (***, p < 0.001; ****, p < 0.0001 compared with control). D, high glucose increases
apoptosis. Apoptosis was measured by 4',6'-diamidine-2'-phenylindole
dihydrochloride after 12 h of incubation to glucose ± 100 µM DHEA and 5 mM
6-aminonicotinamide (ANAD). Data were normalized by cell
number and expressed as means ± S.E. of three separate
experiments, each run in triplicate (**, p < 0.005;
****, p < 0.0001 compared with control).
View larger version (27K):
[in a new window]
Fig. 2.
A, antisense oligonucleotide to G6PD
leads to inhibition of G6PD activity. BAEC were grown in DMEM (5.5 mM glucose) + 10% serum until they were about 80-90%
confluent, and then cells were transfected with an antisense
oligonucleotide. Following transfection, BAEC were incubated with
either 5 or 25 mM glucose medium for 24 h. G6PD
activity was measured as described under "Experimental Procedures."
The data were normalized by protein and presented as average ± S.E. from three separate experiments, each run in at least triplicate
(***, p < 0.001 compared with control). B,
antisense oligonucleotide to G6PD enhances high glucose-induced cell
death as measured by trypan blue exclusion. High glucose increased cell
death. Inhibition of G6PD enhanced BAEC cell death. BAEC were treated
as described in A, and cell death was determined as
described in Fig. 1B. Data were normalized by cell number
and expressed as means ± S.E. of three separate experiments, each
run in triplicate (***, p < 0.001 compared with
control).
We have previously shown that inhibition of G6PD by pharmacologic
agents led to a decrease in NADPH levels and an increase in ROS (5, 6).
Fig. 3, A and B,
shows that a high glucose-induced inhibition of G6PD also led to a
decrease in NADPH and an increase in ROS. Because NADPH is the critical
substrate required to maintain reduced levels of glutathione, we also
measured GSH levels under normal and high glucose conditions. Fig.
3C shows that high glucose also led to a decrease in GSH
levels consistent with our findings on NADPH.
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Published reports led us to hypothesize that high glucose-induced increase in cAMP may mediate the inhibition of G6PD, at least in part. First, increased glucose leads to an increase in cAMP levels (13). Second, increased cAMP has led to enhancement of cell death in certain cell types (13). Third, Costa Rosa et al. (14) showed that compounds that increase cAMP levels led to a decrease in G6PD activity from rat peritoneal macrophages.
Fig. 4A shows that high
glucose caused highly significant increases in cAMP. The high
glucose-induced increase in cAMP in BAEC could be due to either
increased adenylate cyclase activity and/or to decreased
phosphodiesterase activity. Fig. 4B shows that high glucose
led to an increase in adenylate cyclase activity. Considering the
significant increase in adenylate cyclase activity in Fig.
4B and the more modest increase in cAMP in Fig.
4A, it is likely that phosphodiesterase activity is
increased as well. If increased cAMP leads to inhibition of G6PD, then
pharmacologically induced increases in cAMP should lead to inhibition
of G6PD activity. Fig. 4C shows that
isobutylmethylxanthine, a phosphodiesterase inhibitor, led to
inhibition of G6PD activity. Fig. 4D shows that an analog of
cAMP, 8-bromo-cAMP, also led to inhibition of G6PD activity. These
results confirm that increased cAMP levels cause inhibition of G6PD
activity in BAEC.
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cAMP is believed to cause most of its effects via
cAMP-dependent protein kinase A (PKA). H89 is an inhibitor
of PKA activity. If PKA is inhibiting G6PD then inhibition of PKA
should lead to an increase in G6PD activity. Fig.
5A shows that H89 led to an increase in G6PD activity. To determine whether PKA acts directly on
G6PD, PKA was incubated with G6PD in cell lysates. Fig. 5B shows that incubation of the catalytic subunit of PKA with G6PD in vitro led to inhibition of G6PD. If PKA is acting
directly on G6PD, then high glucose should cause phosphorylation of
G6PD. Fig. 5C shows that high glucose led to a highly
significant increase in phosphorylation.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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G6PD Is the Principal Source of NADPH and Is Critical for the Defense against Oxidative Stress-- There is compelling evidence that G6PD is the principal source of NADPH utilized in redox regulation. First, under oxidative stress conditions many studies have shown that G6PD and the pentose phosphate pathway are routinely elevated (15-17). Second, Pandolfi et al. (24) used homologous recombination in mouse embryonic stem cells to produce a cell line totally deficient in G6PD. The G6PD null cells were exquisitely sensitive to oxidative stress. In addition the null cells had significantly decreased growth rates as compared with cells expressing G6PD and significantly reduced cloning efficiencies. The authors concluded that G6PD was critical for NADPH production and was the principal source of NADPH. Earlier work by Rosenstraus and Chaisin (18) using a G6PD-deficient Chinese hamster ovary cell line, which was produced through classic mutagenesis techniques, also showed that the G6PD-deficient cells were more susceptible to oxidative stress. Recently Ursini and colleagues (19) overexpressed G6PD in Hl-60 cells and showed that the glutathione levels in the cell were elevated and the cells were more resistant to oxidative stress. Taken together, these results show that G6PD activity is of central importance to cellular redox regulation.
Thus Fig. 3A shows that the decrease in NADPH caused by high glucose is consistent with the fact that decreased activity of G6PD is the mechanistic reason for this decrease.
G6PD Plays an Essential Role in the Regulation of Cellular
Redox--
The redox level in the cell is a balance between reductants
and oxidants. Intracellular increases in ROS can be due to exposing cells to external oxidants such as H2O2 and
diamide (20-22) or due to the intracellular production of ROS in the
form of H2O2, O
2, ·OH, and
singlet oxygen (20-22). ROS can be formed by normal biologic reactions
via the actions of a variety of intracellular oxidases such as xanthine
oxidase, monoamine oxidase, NADPH oxidase, and urate oxidase (20, 21).
Nonenzymatic production of ROS can also occur. Superoxide may be formed
from the auto-oxidation by molecular oxygen (21, 22) of such chemical
groups as thiols, quinones, catechols, etc. (20, 21). ROS can also be
produced in cellular compartments such as mitochondria, peroxisomes,
and microsomes (20, 21). The consequences of increased oxidative stress
include oxidation of lipids, proteins, carbohydrates, and nucleic acids
that lead to defects in metabolism, cell growth, and other cell
physiologic processes (20, 21). Proteins involved in redox regulation
are glutathione, catalase, and superoxide dismutase. All of these
enzymes require a reductant for their operation. The main intracellular
reductant is NADPH, which is principally produced by G6PD (23-26). The
next enzyme in the pentose phosphate pathway is also a dehydrogenase,
6-phosphogluconate dehydrogenase, which also produces NADPH. However,
the activity of this enzyme is completely dependent on G6PD as G6PD is
the sole source of the substrate for this enzyme (6-phosphogluconate). There is one other enzyme that produces NADPH,
NADP+-dependent malate dehydrogenase. In most
cells this enzyme cannot replace G6PD as the principal source of NADPH.
However, in liver, adipose tissue, pancreatic 
-cells, and
macrophages NADP+-dependent malate
dehydrogenase may play a significant role in NADPH production. Thus in
most cells, proper activity of G6PD is required for the cell to
properly defend against oxidative stress.
Thus the results in Fig. 3B showing increased ROS following exposure to high glucose are likely due, at least in part, to decreased G6PD activity.
Oxidative Stress Plays an Important Role in the Complications of Diabetes-- The evidence that oxidative stress plays a role in the complications of diabetes mellitus is as follows (3, 4, 10, 27). 1) Cells exposed to high glucose have increased levels of ROS. 2) Cellular antioxidant mechanisms are altered in cells exposed to high glucose and in cells from patients with diabetes. 3) Studies have shown increased levels of lipid peroxidation and increased levels of reactive oxygen species (4, 27). 4) In cell culture systems protection against various effects of high glucose can be obtained by adding a variety of intracellular antioxidants such as glutathione, superoxide dismutase, or catalase to the cells (4). 5) Many of the proposed mechanisms, e.g. increased protein kinase C (28), increased advanced glycation end products (4, 29), and increased aldose reductase, are all associated with increased oxidative stress. Increased oxidative stress may be a unifying factor for all of these mechanisms. Thus there is strong evidence that increased oxidative stress is of major importance in the etiology of diabetic complications.
G6PD Is Important for Cell Survival-- Recent work by our laboratory has shown that G6PD activity per se is important for proper cell growth (5) and preventing cell death under various conditions (6). In particular cell death caused by oxidative stress was dependent on G6PD levels. Thus the results seen in this paper and our previously published studies are consistent with the hypothesis that altered G6PD activity (caused by high glucose) would predispose cells to cell damage/death. In particular, the studies using the G6PD inhibitors and the antisense oligonucleotide to G6PD strongly support an important role for G6PD in high glucose cell death.
High Glucose Stimulates cAMP in Certain Cell Types-- In 1975, Zawalich et al. (30) showed that increased glucose led to an increase in cAMP in pancreatic islets. In 1978, Jackowski et al. (31) showed that rats exposed to high glucose had increased cAMP levels in their adipose tissue. More recently, Kamal et al. (13) showed that increased cAMP likely played an antiproliferative role in human dermal microvascular endothelial cell line. Thus there is published evidence for high glucose causing an increase in cAMP levels. The results shown in Figs. 3 and 4 are consistent with these previous observations. Fig. 4B suggests that a mechanism underlying this effect is at least in part due to increased activation of adenylate cyclase. Interestingly, the effect may differ depending on cell type. For example human corneal cells exposed to increased glucose showed a decrease in cAMP levels (32). The different responses of cAMP levels may truly reflect cell-specific responses or they may reflect different experimental conditions (e.g. incubation time may play a role). Further studies are required to clarify this.
Regulation of G6PD by cAMP-- As previously noted, Costa Rosa et al. (14) showed that cAMP could inhibit macrophage G6PD. However, they suggested that this was true for only macrophage G6PD. Costa Rosa et al. (14) compared their findings on macrophage G6PD to only one other source of G6PD. Thus they did not do an extensive study of various sources of G6PD. The research reported here suggests that cAMP inhibition of G6PD is not limited to macrophages. Thus it is possible that cAMP might regulate G6PD in a variety of cell types.
Taken together, our results strongly suggest that in BAEC, high glucose
led to an increase in cAMP that, via cAMP-dependent PKA,
caused phosphorylation and inhibition of G6PD activity. This led to
decreased NADPH, increased ROS, and then cell damage/death. This
impaired activity of G6PD likely plays a critical role in the
pathogenesis of endothelial cell damage observed in patients with diabetes.
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ACKNOWLEDGEMENTS |
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We are very grateful to Chun-Rui Zhao for excellent technical assistance in the phosphorylation experiments and to Jia Yu for excellent technical assistance and critical suggestions. We are also very grateful to Drs. Joseph Loscalzo and Jane Leopold of the Boston University Medical Center for their assistance in the antisense experiments.
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FOOTNOTES |
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* This work was supported in part by Grant 1-1998-147 from the Juvenile Diabetes Foundation (to R. C. S.) and a grant from the Adler Foundation (to Z. Z.).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.
§ To whom correspondence and reprint requests should be addressed: Renal Division, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel.: 617-732-2477; Fax: 617-732-2467; E-mail: robert.stanton@joslin.harvard.edu.
Published, JBC Papers in Press, September 27, 2000, DOI 10.1074/jbc.M007505200
2 J. Loscalzo, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: G6PD, glucose-6-phosphate dehydrogenase; BAEC, bovine aortic endothelial cells; DMEM, Dulbecco's modified Eagle's medium; ROS, reactive oxygen species; PKA, protein kinase A; DHEA, dehydroepiandrosterone.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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