High glucose and insulin inhibit VSMC MKP-1 expression by
blocking iNOS via p38 MAPK activation
Najma
Begum1,2 and
Louis
Ragolia1
1 Diabetes Research Laboratory,
Winthrop University Hospital, Mineola 11501; and
2 School of Medicine, State
University of New York at Stony Brook, Stony Brook, New York 11794
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ABSTRACT |
Our laboratory has recently demonstrated a
role for the phosphatidylinositol 3-kinase-mediated
inducible NO synthase (iNOS) signaling pathway in acute regulation of
insulin-induced mitogen-activated protein phosphatase-1 (MKP-1)
expression in primary cultures of rat aortic vascular smooth muscle
cells (VSMCs) (N. Begum, L. Ragolia, M. McCarthy, and N. Duddy.
J. Biol. Chem. 273: 25164-25170, 1998). We now show that prolonged treatment of VSMCs with 100 nM
insulin and high glucose (25 mM) for 12-24 h, to mimic
hyperinsulinemia and hyperglycemia, completely blocked MKP-1 mRNA and
protein expression in response to subsequent acute insulin treatment.
To understand the mechanism of insulin resistance induced by high
glucose and insulin, we studied the regulation of iNOS protein
induction in these cells. Both high glucose and chronic insulin
treatment caused a marked impairment of iNOS induction in response to
acute insulin. Blocking of signaling via the p38 mitogen-activated
protein kinase (MAPK) pathway by prior treatment for 1 h with
SB-203580, a synthetic p38 MAPK inhibitor, completely prevented the
inhibition of iNOS induced by high glucose and insulin and restored
MKP-1 induction to levels observed with acute insulin treatment. In
contrast, PD-98059, a MEK inhibitor, had no effect. Furthermore, high
glucose and chronic insulin treatment caused sustained p38 MAPK
activation. We conclude 1) that
chronic insulin and high glucose-induced insulin resistance is
accompanied by marked reductions in both iNOS and MKP-1 inductions due
to p38 MAPK activation that leads to excessive cell growth and
2) that p38 MAPK/extracellular
signal-regulated kinase pathways regulate iNOS induction, thereby
controlling MKP-1 expression, which in turn inactivates MAPKs as a
feedback mechanism and inhibits cell growth.
hyperglycemia; insulin resistance; cell growth; extracellular
signal-regulated kinase signaling; inducible nitric oxide synthase; mitogen-activated protein phosphatase-1; mitogen-activated protein
kinase; vascular smooth muscle cells
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INTRODUCTION |
INSULIN RESISTANCE, hyperinsulinemia, and diabetes are
closely associated with cardiovascular complications such as
atherosclerosis and hypertension (8, 25, 33). The mechanisms linking
hyperinsulinemia and hyperglycemia with these cardiovascular
complications are poorly understood (8, 25, 33). Vascular smooth muscle
cells (VSMCs) are a major constituent of blood vessel walls responsible for the maintenance of vascular tone (26). Accelerated VSMC growth,
hypertrophy, and abnormal vascular tone play a central role in the
development of atherosclerosis (30). Although alterations in insulin
action of the vasculature due to hyperglycemia and hyperinsulinemia
have been proposed to contribute to atherosclerosis and the regulation
of vascular tone, little is known about the specific cellular signaling
pathways that mediate the detrimental hyperinsulinemic and
hyperglycemic effects in VSMCs.
Increasing evidence suggests that mitogen-activated protein kinase
(MAPK) family members play a major role in the regulation of cell
growth and differentiation in VSMCs (7, 22, 24, 27, 36). MAPKs are
activated in response to growth factors and stress signals and have
been implicated in VSMC proliferation, hypertrophy, and migration, all
key processes in the pathology of vascular diseases such as
atherosclerosis and hypertension. Four groups of MAPKs have been
identified in mammalian cells: the extracellular signal-regulated
kinases 1 and 2 (ERK1/ERK2, also known as p42/44 MAPK), the c-Jun
NH2-terminal kinases (JNKs, also
known as stress-activated protein kinase or SAPK), p38 MAPK, and Big
MAPK (ERK5) (24). Although MAPK family members are structurally related, they are generally activated via multistep phosphorylation cascades by distinct extracellular stimuli and phosphorylate different molecular substrates (27). The classic ERKs, ERK1 and ERK2, are
activated through Ras-dependent signal transduction pathways by
hormones and growth factors, whereas JNKs and p38 MAPKs are activated
by environmental stress, oxidants, lipopolysaccharides, osmotic stress,
heat shock, and cytokines (i.e., tumor necrosis factor-
and interleukin-1), leading to alterations in cell growth, prostanoid
production, and other cellular dysfunctions (35).
The activities of all four members of MAPK family are regulated by the
reversible phosphorylation of tyrosine and threonine residues,
indicating that protein phosphatases play a critical role in regulating
the activation status of these enzymes. Inactivation of MAPK signaling
is mediated by a class of dual-specificity protein phosphatases (17,
31). These include mitogen-activated protein phosphatase-1 (MKP-1; also
termed CL100, Erp, and hVH-1), which is encoded by the murine gene
3ch134 (17), MKP-2, MKP-3, PAC-1, and
B23 (17, 31). MKP-1, the most ubiquitously expressed and best studied
of these phosphatases, has dual catalytic activity toward
phosphotyrosine and phosphothreonine and is known to inactivate ERKs,
JNK, and high-osmolarity glycerol p38
(p38HOG) in vivo as well as in
vitro (36). MKP-1 and the other family members are principally
regulated at the transcriptional level, as evidenced by very low to
undetectable mRNA expression in quiescent cells and a rapid mRNA
induction after treatment of cells with growth factors or with agents
that cause oxidative stress and heat shock (36). MKP-1 has
been implicated in a feedback loop serving to inactivate MAPKs after
stimulation by mitogens as well as during the cellular response to
stress (36).
We have recently shown that physiological concentrations of insulin
rapidly induce MKP-1 expression in primary cultures of VSMCs (2,
4). Blocking of NO synthase (NOS) and cGMP (a downstream
effector of NOS) signaling with
N
-monomethyl-L-arginine
(L-NMMA) and
Rp-8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate
(Rp-cGMP), two specific inhibitors of
NOS and cGMP, respectively, as well as with wortmannin, an inhibitor of
phosphatidylinositol 3-kinase (PI 3-kinase), completely abolished
insulin-mediated induction of MKP-1. Moreover, VSMCs isolated from
spontaneously hypertensive rats exhibited resistance to insulin with
respect to MKP-1 expression because of defective signaling via the NOS
signaling pathway, leading to sustained MAPK activation and excessive
cell growth. These observations, together with the fact that the
induction of inducible NOS (iNOS) by insulin precedes MKP-1 expression
and the fact that induction of MKP-1 could be mimicked by sodium
nitroprusside (an NO generator) and dibutyryl guanosine
3',5'-cyclic monophosphate (a cGMP agonist), suggested that
insulin regulates the induction of MKP-1 via the PI 3-kinase-NO-cGMP
signaling pathway (2).
In this study, we tested the hypothesis that high glucose
(hyperglycemia) and chronic insulin treatment inhibit vasorelaxation and promote excessive cell growth by blocking the induction of iNOS and
MKP-1. In addition, we characterized the signaling mechanism by which
sustained insulin level and elevated glucose level exert their
growth-stimulatory effects in VSMCs. Because stress-related signals
mediate hypertrophy in VSMCs and because many stress factors [such as hyperosmolarity, glycation end products, oxidant
formation, and diacylglycerol protein kinase C (PKC) activation]
have been shown to be present in diabetes and insulin-resistant states
(6, 10, 18, 34), we examined the contribution of the stress signaling
pathway in the regulation of iNOS and MKP-1 induction under conditions
of high glucose and insulin.
The results of the present study indicate that prolonged treatment of
VSMCs with insulin and high glucose to simulate hyperinsulinemia and
hyperglycemia completely blocked the induction of iNOS protein and
inhibited MKP-1 mRNA and protein expression due to elevations in p38
MAPK activity. Blocking of the signaling via p38 MAPK with SB-203580, a
p38 MAPK inhibitor, restored cellular responsiveness of iNOS expression
and MKP-1 induction.
 |
METHODS |
Materials.
Fetal bovine serum, antibiotics, trypsin,
L-glutamine, freezing medium,
-MEM, and DMEM containing high glucose were obtained from Life
Technologies (Grand Island, NY).
[
-32P]dCTP (sp act
3,000 Ci/mmol), and
[
-32P]ATP were
purchased from DuPont NEN (Boston, MA). Type I collagenase was from
Worthington Biochemical (Freehold, NJ). The antibodies against MKP-1
and iNOS and the activating transcription factor-2 (ATF-2) substrate
(1-96) were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Phosphospecific antibodies against p38 MAPK and ERKs were obtained
from New England Biolabs. Protein A/G-agarose was from Oncogene Science
(Cambridge, MA). PD-98059 and SB-203580 were from Biomol (Plymouth
Meeting, PA). SDS-PAGE supplies and reagents for Western blot analyses
were from Bio-Rad (Hercules, CA). Rat MKP-1 cDNA was a kind gift of Dr.
Jyotirmoy Kusari (Tulane University, New Orleans, LA). FITC-conjugated
-actin antibody, mannitol, and all other chemicals and reagents were
purchased from Sigma Chemical (St. Louis, MO).
Culture of VSMCs and treatment with high glucose and insulin.
VSMCs in primary culture were obtained by enzymatic digestion of the
aortic media of male normotensive Wistar Kyoto (WKY) rats (body wt
200-220 g), as described in our recent publications (2, 4).
Subcultures of VSMCs at passages
3-5
were used in all the experiments. VSMCs prepared from these rats were
not contaminated with fibroblasts or endothelial cells as evidenced by
a >99% positive immunostaining of smooth muscle
-actin with
FITC-conjugated
-actin antibody (data not shown). All experiments on
MKP-1 induction, iNOS, p38 MAPK, and DNA synthesis were performed on
highly confluent cells (9-11 days in culture) at
passage
5. Before each experiment, cells were
serum starved for 24 h in
-MEM containing 5.5 mM glucose and
antibiotics. The next day, cells were exposed to either normal glucose
(5.5 mM) or high glucose (25 mM) in the presence and the absence of
insulin (100 nM) for 12-24 h, followed by acute insulin treatment
for 30 min. Before acute insulin treatment, cells that were exposed to
chronic insulin for 12 and 24 h were rinsed exhaustively with
serum-free
-MEM containing 5.5 mM glucose to completely remove
insulin and were left in this medium for 1 h. In some experiments, VSMCs were pretreated with various inhibitors for 30 min, followed by
chronic exposure to insulin or high glucose. To prevent glucose and
insulin depletion, the medium was changed to fresh medium containing
high glucose or insulin every 7 h. The cells were used 24 h after
exposure to high glucose or insulin. In some experiments, mannitol
(19.5 mM) was used to control variations due to osmotic pressure.
Northern blot analysis of MKP-1 mRNA expression.
Serum-starved VSMCs exposed to normal glucose, high glucose, and
insulin for 24 h were incubated in the presence or absence of insulin
(0-100 nM) for 30 min. Total RNA was extracted with guanidinium
isothiocyanate using a Qiagen RNAeasy kit as per the manufacturer's
instructions and quantitated by measurement of the ratio of absorbance
at wavelengths of 260 and 280 nm. Equal amounts of RNA (5 µg/lane)
were separated on a 1.2% agarose-formaldehyde denaturing gel,
transferred by capillary action overnight to a nitrocellulose membrane,
hybridized with 32P-labeled MKP-1
cDNA, and detected by autoradiography with standard protocols (2, 4,
16). The membrane was stripped by boiling for 5 min in 1% SDS and
reprobed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The
MKP-1 mRNA and GAPDH expressions were quantitated by densitometric
analyses of the autoradiograms. The MKP-1 mRNA was normalized with
respect to GAPDH.
Immunoblot analysis of MKP-1 and iNOS protein expression.
Immunodetection of MKP-1 and iNOS proteins in control VSMCs and in
VSMCs treated with normal glucose, high glucose, and insulin were
performed by Western blot analyses as described in our recent publication (2). Briefly, 50-100 µg cell lysate proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were probed with anti-MKP-1 antibody and anti-iNOS antibody according to the manufacturer's protocols. Visualization of the primary antibody was with horseradish peroxidase (HRP)-conjugated secondary antibodies, followed by enhanced
chemiluminescence (ECL). Autoradiograms with linear signal were
quantitated by densitometric scanning. In the initial studies,
linearity of the ECL signal was established by blotting various
dilutions of the second antibody conjugated to HRP.
Detection of p38 MAPK and ERK1/ERK2 phosphorylation by Western blot
analyses.
Serum-starved VSMCs were stimulated with insulin (100 nM) for 30 min
(acute treatment) or 12-24 h in the presence and the absence of
high glucose. The dishes were quickly rinsed with ice-cold PBS
containing 2 mM vanadate and dropped into liquid nitrogen. The frozen
dishes were thawed on ice, and the cells were lysed with buffer
containing 20 mM HEPES (pH 7.5), 137 mM NaCl, 1 mM MgCl2, 1 mM
CaCl2, 1 mM sodium orthovanadate,
10% glycerol, 1% Nonidet P-40, and a cocktail of protease and
phosphatase inhibitors (3). Insoluble material was removed by
centrifugation for 15 min at 12,000 g
at 4°C. Cell lysates normalized to 100 µg protein were separated
on 10% SDS-polyacrylamide gels and transferred to PVDF membrane
(2-4). The membranes were probed with phosphospecific p38 MAPK
antibodies and phosphospecific ERK1/ERK2 antibodies, followed by
detection with HRP-conjugated secondary antibody using an ECL detection
kit supplied by Amersham.
Immunoprecipitation and assay of p38 MAPK activity.
p38 MAPK activity was measured by immune complex kinase assay using p38
MAPK antibody with ATF-2 as a substrate. Briefly, equal amounts of cell
lysate proteins (500 µg) from above were immunoprecipitated overnight
at 4°C with 2 µg of anti-p38 MAPK antibody. The next day, the
immunoprecipitates were captured by incubation with 100 µl (50%
vol/vol) of protein A-Sepharose at 4°C with gentle shaking. The
beads were washed four times with lysis buffer and twice with kinase
buffer containing 25 mM Tris (pH 7.5), 5 mM
-glycerophosphate, 2 mM
dithiothreitol, 0.1 mM sodium orthovanadate, 10 mM
MgCl2, and a cocktail of protease inhibitors. The beads were resuspended in 50 µl of kinase buffer containing 0.1 mg/ml ATF-2 as a substrate (21) and 50 µg/ml IP20, a
peptide inhibitor of cAMP-dependent protein kinase. The reaction was
initiated by the addition of 10 µl of a mixture of Mg2+-ATP containing 10 µCi of
[
-32P]ATP. After 10 min of incubation at 30°C, the reaction was terminated by spotting
25 µl of reaction mixture on 2 × 2-cm phosphocellulose Whatman
P-81 discs. The discs were washed four times with 0.75% phosphoric
acid. The radioactivity bound to the filter paper was quantitated by
liquid scintillation counting as described in our earlier publication
(3).
Immunoprecipitation and assay of IRS-1-associated PI 3-kinase
activity.
Immunoprecipitation of cell lysates normalized to 200 µg protein was
performed overnight at 4°C with 2 µg of anti-rabbit IRS-1 antibody directed against the pleckstrin homology domain (United Biotecnology). For negative control, 200 µg of lysate protein were
immunoprecipitated with 2 µg of anti-rabbit IgG. The immunocomplexes were precipitated the next day by incubation with 50 µl of protein G
plus/protein A-agarose beads (50% vol/vol; Calbiochem) for 2 h at
4°C with constant shaking. The immunoprecipitates were washed exhaustively with buffers, and PI 3-kinase activity was assayed in the
immunoprecipitates as described previously (2). The reaction products
were separated by TLC on oxalate-treated silica gel 60 plates in a
solvent of chloroform-methanol-water-ammonia (60:47:12.5:2). Unlabeled
phosphatidylinositol 3-phosphate was used as a standard and visualized
by iodine vapor. The 33P-labeled
phosphatidylinositol 3-phosphate was identified by autoradiography and
quantitated by the cut-and-count technique.
Protein assay.
Proteins in the cellular extracts and lysates were quantitated by the
bicinchoninic acid method (29) or by the Bradford technique (5).
Statistics.
The results are presented as means ± SE of four to six independent
experiments, each performed in triplicate at different times. Unpaired
Student's t-test or ANOVA was used to
compare the mean values among different treatments.
P < 0.05 was considered statistically significant.
 |
RESULTS |
Effect of chronic insulin and high glucose on MKP-1 mRNA induction
in VSMCs.
To understand the exact mechanism whereby high glucose and prolonged
insulin treatment cause sustained MAPK activation and excessive growth
of VSMCs, we examined the effect of high glucose and chronic insulin
treatment on MKP-1 mRNA induction. MKP-1, a dual-specificity
tyrosine/threonine phosphatase, dephosphorylates MAPKs and inactivates
the MAPK signaling pathway. Acute treatment of serum-starved VSMCs with
insulin or sodium nitroprusside (SNP; a nitric oxide donor) for 30 min
caused a three- to fourfold increase in MKP-1 mRNA expression over
basal levels (Fig. 1, top,
compare lanes 2 and 3 with lane
1; quantitation in bottom).
Prolonged exposure to insulin (100 nM for 24 h, to mimic
hyperinsulinemia) completely blocked MKP-1 mRNA expression in response
to subsequent acute insulin treatment (Fig. 1,
top, compare
lane
4 with
lane 2) and decreased the
MKP-1 mRNA expression to below basal levels. Furthermore, chronic
exposure of serum-starved VSMCs to high glucose (25 mM) for 24 h also
blocked the subsequent acute effects of insulin and SNP on MKP-1 mRNA
induction (Fig. 1, top, compare lanes
6 and
7 with
lanes
2 and
3). High glucose alone caused a 30%
decrease in basal MKP-1 mRNA expression compared with cells exposed to
normal glucose (Fig. 1, top, compare
lane
5 with
lane 1).


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Fig. 1.
Treatment with chronic insulin and high glucose (HG) inhibit
mitogen-activated protein phosphatase-1 (MKP-1) mRNA expression in
response to subsequent acute dose of insulin (Ins) and sodium
nitroprusside (SNP) in vascular smooth muscle cells (VSMCs).
Serum-starved VSMCs in normal glucose (NG) were treated with insulin
(100 nM) or SNP for 30 min for acute effects or treated with 100 nM
insulin and 25 mM glucose (HG) for 24 h (for chronic effects), followed
by a subsequent acute treatment with or without insulin or SNP for 30 min. Equal amounts of RNA (5 µg/well) were separated on
agarose-formaldehyde gel, followed by overnight transfer to
nitrocellulose membrane. Membranes were hybridized with
32P-labeled MKP-1 cDNA probe,
stripped, and reprobed for glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). Top: representative
autoradiogram. Bottom: quantitation of
MKP-1 mRNA levels from multiple experiments by densitometric analyses.
Relative mRNA levels were determined by laser densitometric scanning of
autoradiograms. To correct for variations in RNA loading, intensity of
MKP-1 signal was divided by intensity of GAPDH signal. Results are
expressed as arbitrary densitometric units (ADU). For comparison of
results from different experiments, mRNA from acute NG control was
assigned a value of 1 ADU, and rest of data were normalized to NG
control value. Results are means ± SE of 4 separate RNA blots from
different experiments. * P < 0.05 vs. NG control; ** P < 0.05 vs. acute insulin.
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We next examined the effects of high glucose and prolonged insulin
treatment on MKP-1 protein induction. As reported earlier (2, 4), acute
insulin treatment resulted in a threefold increase in MKP-1 protein
accumulation in VSMCs exposed to normal glucose (Fig.
2, top,
compare lane
2 with
lane
1; quantitation in
bottom). A 12-h exposure to insulin
resulted in a significant decrease in MKP-1 protein levels (Fig. 2,
top, compare
lane
3 with
lane
2). A subsequent acute insulin
treatment at the end of 12 or 24 h did not further increase MKP-1
protein expression (Fig. 2, top,
compare lanes
4 and
5 with
lane
2). Exposure to high glucose for 12 and 24 h caused a 75% decrease in basal MKP-1 protein levels compared
with cells exposed to normal glucose (Fig. 2,
top, compare
lanes
6 and
8 with
lane
1). In addition, high-glucose treatment for 12 and 24 h completely abolished the acute stimulatory effects of insulin on MKP-1 protein expression (Fig. 2,
top, compare lanes
7 and
9 with
lane
2). The inhibitory effects of high
glucose on MKP-1 protein induction were more marked than the inhibition observed with chronic insulin treatment under normal glucose conditions (shown in Fig. 2, lanes
3-5).
Combined addition of insulin and high glucose for 24 h did not further
decrease MKP-1 induction (Fig. 2, top,
lane
8).


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Fig. 2.
Western blot analysis of MKP-1 protein expression in VSMCs treated with
NG, HG, and chronic insulin. Serum-starved VSMCs in NG were treated
with insulin for 30 min in acute experiments or treated with HG and
insulin for 12 and 24 h, followed by a subsequent acute insulin
treatment (Ins TX) as detailed under
METHODS. Equal amounts of cell lysate
proteins were subjected to SDS-PAGE and immunoblotting with MKP-1
antibody (Ab). Top: representative
autoradiogram. Similar results were obtained in 3 separate experiments.
Lanes
1, 6,
and 8, NG and HG controls;
lane
2, acute insulin treatment for 30 min;
lane
3, chronic insulin treatment for 12 h;
lane
4, 12-h insulin treatment followed by
30-min acute insulin; lane
5, 24-h insulin followed by subsequent
30-min insulin; lane
7, 12-h HG followed by 30-min insulin;
lane
9, 24-h HG followed by 30-min insulin;
lane
10, 24-h HG plus 24-h insulin
combined. Bottom: data from different
experiments were quantitated by densitometric scanning and plotted as
% of NG controls. Ins TX, insulin treatment.
** P < 0.05 vs. acute insulin;
*** P < 0.05 vs. NG control;
and **** P < 0.05 vs. acute
insulin.
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To examine whether the inhibitory effects of high glucose on MKP-1
expression are due to an increase in glucose-induced osmolarity, we
exposed VSMCs to 19.5 mM mannitol and 5.5 mM glucose for 24 h and
examined the effect of acute and chronic insulin treatment on MKP-1
induction. Acute insulin treatment resulted in a more than twofold
increase in MKP-1 protein expression over basal levels in
mannitol-treated cells (Fig. 3,
top, compare
lane
2 with
lane 1; quantitation in
bottom). The extent of MKP-1
induction by acute insulin in mannitol-treated cells was more or less
comparable to induction in cells exposed to normal glucose (Fig. 2).
Chronic treatment with insulin for 24 h decreased MKP-1 protein
expression to below basal values in response to a subsequent acute dose
(Fig. 3, top, compare
lane
3 with
lane
2). Furthermore, the presence of 25 mM glucose in addition to 19.5 mM mannitol for 12 and 24 h also
inhibited MKP-1 protein induction in response to a subsequent acute
insulin dose (Fig. 3, top, compare
lanes
4 and
5 with
lane 2).


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Fig. 3.
Effect of mannitol on insulin-mediated MKP-1 protein expression.
Serum-starved VSMCs in NG were incubated with 19.5 mM mannitol + NG
with and without insulin for 24 h or with 19.5 mM mannitol + HG for
12-24 h followed by an acute insulin treatment for 30 min. Equal
amounts of cell lysate proteins were subjected to immunoblot analyses.
Top: representative autoradiogram.
Bottom: data from different
experiments were quantitated by densitometric scanning and plotted as
% of NG controls. * P < 0.05 vs. osmotic control; ** P < 0.05 vs. acute insulin.
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High glucose and chronic insulin block iNOS induction.
Results from our recent studies indicated that insulin rapidly induces
the expression of iNOS protein in VSMCs (2). Blocking the signaling via
the NOS/cGMP pathway with synthetic inhibitors, L-NMMA and
Rp-cGMP, respectively, abolished the
effects of insulin on MKP-1 induction, suggesting that the NOS/cGMP
signaling pathway may play a major role in insulin-mediated MKP-1
induction (2). To further understand the molecular basis of the
inhibition of MKP-1 induction observed with high glucose and chronic
insulin treatment, we examined the induction of iNOS protein under
high-glucose and hyperinsulinemic conditions.
As shown in Fig. 4, acute exposure of
serum-starved VSMCs to insulin for 30 min results in a rapid threefold
induction of iNOS protein (Fig. 4,
top, compare
lane
2 with
lane
1; quantitation in
bottom). Chronic exposure to insulin
for 12 and 24 h, respectively, abolished the subsequent effects of
acute insulin treatment on iNOS induction (Fig. 4,
top, compare
lanes
3 and
4 with
lane
2). Chronic exposure to high glucose
for 12 and 24 h, respectively, also abolished the effect of insulin on
iNOS protein induction (Fig. 4, compare
lanes
6 and
7 with
lane
2). The inhibitory effects of high
glucose and chronic insulin were observed only after 12 and 24 h.
Shorter periods of 2-6 h caused a very small decrease in
insulin-induced iNOS induction or MKP-1 expression (results not shown).


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Fig. 4.
HG and chronic insulin treatment inhibit inducible NO synthase (iNOS)
induction. Serum-starved VSMCs were exposed either to NG with or
without insulin for 12-24 h or to HG for 12-24 h, followed by
treatment with acute insulin for 30 min. Equal amounts of cell lysate
proteins were subjected to SDS-PAGE, followed by Western blot analysis
with anti-iNOS Ab. Top: representative
autoradiogram. Similar results were obtained in multiple experiments.
Lanes
1 and
5, NG and HG controls;
lane
2, acute insulin treatment;
lanes
3 and
4, chronic insulin treatment for 12 and 24 h; lanes
6 and
7, HG treatment for 12 and 24 h
followed by insulin for 30 min.
Bottom: data from different
experiments were quantitated by densitometric scanning and plotted as
% of NG controls. * P < 0.05 vs. NG control; ** P < 0.05 vs. acute insulin.
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Effect of high glucose and chronic insulin treatment on insulin
receptor content and PI 3-kinase activation.
The observed inhibitory effects of high glucose and chronic insulin on
iNOS and MKP-1 induction may be due to the downregulation of insulin
receptor and/or desensitization of the downstream signaling molecules.
Therefore, we examined the insulin receptor content and
IRS-1-associated PI 3-kinase activity in these cells. Western blot
analyses of equal amounts of cell lysate proteins with anti-insulin receptor antibodies detected a 95-kDa band corresponding to the
-subunit of the insulin receptor. High glucose and chronic insulin treatment did not alter insulin receptor content in VSMCs (Fig. 5). Furthermore, prolonged exposure to high
glucose did not inhibit PI 3-kinase activation by insulin, as evidenced
by comparable PI 3-kinase activity in IRS-1 immunoprecipitates (Fig.
6). However, chronic exposure
to insulin for 24 h did result in a 40% decrease in insulin-stimulated
PI 3-kinase activity in the IRS-1 immunoprecipitates (Fig. 6).

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Fig. 5.
Effect of HG and chronic insulin treatment on insulin receptor content.
VSMCs were treated as detailed in Fig. 4. Equal amounts of lysate
proteins (300 µg) were subjected to SDS-PAGE, followed by Western
blot analysis with rat anti-insulin receptor Ab and detection with
125I-labeled protein A. Intensity
of signal for 95-kDa insulin receptor -subunit was quantitated by
densitometric analyses of autoradiograms. Intensity of 95-kDa band from
NG controls was assigned a value of 1 ADU and rest of data were
normalized to NG control and expressed as ADU. Results are means ± SE of 3 experiments.
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Fig. 6.
Effect of HG and chronic insulin on phosphatidylinositol 3-kinase (PI
3-kinase) activation. VSMCs were treated as detailed in Fig. 4 and
stimulated acutely with insulin for 5 min. Equal amounts of proteins
(200 µg) were precleared with rat IgG prebound to protein
A-Sepharose. Precleared supernatants were treated with 2 µg of
pleckstrin homology domain anti-rabbit IRS-1 Ab, followed by assay of
PI 3-kinase activity. A representative autoradiogram is shown. Similar
results were obtained in 2 independent experiments.
Lane
1, NG control;
lane
2, acute insulin treatment for 5 min;
lane
3, chronic insulin treatment for 24 h
followed by acute 5 min insulin; lane
4, 24-h HG treatment;
lane
5, 24-h HG plus acute insulin
treatment for 5 min. For IgG negative control
(right
lane), samples from acute insulin
treatment immunoprecipitated with IgG instead of IRS-1 Ab. IP,
immunoprecipitate; PIP, phosphatidylinositol phosphate.
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Inhibition of p38 MAPK signaling with SB-203580 prevents the
inhibitory effects of high glucose and chronic insulin on iNOS and
MKP-1 protein induction.
Our previous studies suggested a potential cross talk between MAPKs and
iNOS signaling pathways, since inhibition of ERKs with PD-98059
completely blocked insulin-mediated iNOS induction and MKP-1 protein
expression (2). In addition, a number of recent studies indicated that
oxidative stress leads to p38 MAPK and/or ERK1/ERK2 activation (12, 19,
23, 32). To further explore the possibility that inhibition of iNOS
induction observed by chronic incubation with high glucose and insulin
may be due to activation of ERKs and/or p38 MAPKs, we examined the
effect of the inhibitors of these signaling pathways on iNOS and MKP-1 protein induction in cells exposed to high glucose and chronic insulin.
As seen in Fig. 7, pretreatment of VSMCs
with 0.3 µM SB-203580, a specific p38 MAPK inhibitor, for 30 min
before chronic insulin exposure completely abolished the inhibitory
effects of chronic insulin on iNOS induction (Fig. 7,
top
left, compare
lane 5 with
lane
3; quantitation in
bottom) and restored insulin
responsiveness to levels comparable to those seen with the acute
insulin treatment of cells (Fig. 7,
top
left, compare
lanes
5 with
lane
2). SB-203580 by itself did not
alter basal iNOS protein levels when present for 24 h (Fig. 7,
top
left, compare
lane
4 with
lane
1). In our earlier studies, we
demonstrated that SB-203580 did not affect the acute stimulatory
effects of insulin on iNOS induction when added 30 min before acute
insulin treatment (2). In contrast to inhibition by SB-203580,
inhibition of MEK with PD-98059 did not prevent the inhibitory effects
of high glucose and insulin on iNOS induction (Fig. 7,
top
left,
lane
7) but decreased iNOS protein levels
below the basal values (Fig. 7, top
left, compare lanes
6 and
7 with
lane
1). In separate experiments, we
observed that SB-203580 also partially prevented the inhibitory effects of high glucose on insulin-mediated iNOS induction (Fig. 7,
top right, compare
lane
5 with
lane
3), whereas PD-98059 was ineffective (Fig. 7, top
right, compare
lane
6 with
lane
3). It should be noted that the
presence of SB-203580 together with high glucose did decrease basal
iNOS protein levels by 40% compared with normal glucose controls (Fig.
7, top
right, compare
lane
4 with
lane 1).



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Fig. 7.
Inhibition of p38 mitogen-activated protein kinase (MAPK) prevents
chronic insulin and HG inhibitory effects on iNOS induction.
Serum-starved VSMCs were pretreated with SB-203580 (SB; 0.2 µM) or
PD-98059 (PD; 50 µM) for 30 min, followed by addition of medium
containing NG or HG. Cells in NG were incubated with insulin for 24 h,
followed by a stimulation with insulin for 30 min. Cells in HG were
incubated for 24 h, followed by acute exposure to insulin.
Top: representative autoradiograms for
NG (left) and HG
(right). Similar results were
obtained in 3 separate experiments.
Lane
1, control;
lane
2, acute insulin treatment;
lane
3, chronic 24-h insulin treatment
followed by a subsequent acute insulin dose;
lane
4, treatment with SB-203580 for 24 h;
lane
5, pretreatment with SB-203580 for 30 min followed by insulin for 24 h; lane
6 (top
left), PD-98059 alone;
lane
6 (top
right) and lane
7 (top
left), pretreatment with PD-98059
followed by insulin for 24 h. Bottom:
data from multiple experiments were quantitated by densitometric
scanning and plotted as % of NG controls.
* P < 0.05 vs. NG control;
** P < 0.05 vs. acute insulin;
*** P < 0.05 vs. chronic
insulin; **** P < 0.05 vs.
SB-203580 + chronic insulin/HG.
|
|
SB-203580 also prevented the inhibitory effects of chronic insulin
treatment (Fig. 8,
top
left, compare
lane
5 with
lane
3; quantitation in
bottom), as well as the effect of
high glucose on MKP-1 induction (Fig. 8,
top
right, compare
lane
5 with
lane 3), whereas PD-98059 was ineffective
(Fig. 8, top
left, compare lane
6 with
lane
3; Fig. 8,
top
right, compare
lane
7 with
lane 3). Thus inhibition of p38 MAPK
signaling with SB-203580 abrogates the deleterious effects of chronic
insulin and restores insulin sensitivity of VSMCs in terms of iNOS and
MKP-1 induction.



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Fig. 8.
SB-203580 prevents inhibitory effects of chronic insulin and HG on
MKP-1 protein induction. Serum-starved VSMCs were pretreated with and
without SB-203580 or PD-98059 for 30 min, followed by treatment with
insulin or HG for 24 h. At end of 24 h, cells were treated with insulin
for 30 min. Cell lysates were examined for MKP-1 protein expression as
detailed in Fig. 2. Top:
representative autoradiograms for NG
(left) and HG
(right). Similar results were
obtained in 4 separate experiments. Lane order is similar to that of
Fig. 7. Bottom: data from multiple
experiments were quantitated by densitometric scanning and plotted as
% of NG controls. * P < 0.05 vs. NG control; ** P < 0.05 vs. acute insulin; *** P < 0.05 vs. chronic insulin; **** P < 0.05 vs. SB-203580 + chronic insulin/HG.
|
|
High glucose and chronic insulin treatment activate p38 MAPK and
ERK1/ERK2.
To further confirm whether high glucose and chronic insulin treatment
results in sustained activation of p38 MAPK and/or ERKs, we examined
the phosphorylation status of p38 MAPK and ERK1/ERK2 using
phosphospecific antibodies. Initial studies were performed to examine
the dose-response and kinetics of the acute effects of insulin on p38
MAPK phosphorylation in cells maintained in normal glucose.
In unstimulated cells, a small amount of p38 MAPK was phosphorylated in
the basal state (Fig. 9). Acute insulin
treatment for 30 min caused a twofold increase in p38 MAPK
phosphorylation compared with control cells (Fig. 9, compare
lane
2 with
lane 1; quantitation in
bottom). The level of p38 MAPK
phosphorylation 12 h after exposure to insulin was comparable to the
increase observed with acute insulin treatment for 30 min (Fig. 9,
compare lanes
3 and
4 with
lane
2). More important, exposure to
chronic insulin for 24 h further increased the phosphorylation in
response to a subsequent acute insulin treatment (Fig. 9, compare
lane 5 with
lanes
2-4).
Exposure to high glucose alone for 12 and 24 h, respectively, resulted
in a time-dependent twofold increase in basal p38 MAPK phosphorylation
compared with cells exposed to normal glucose (Fig. 9, compare
lanes
6 and
8 with
lane
1). Subsequent acute insulin
treatment of these cells (Fig. 9, lane 7 and
lane
9), as well as combined addition of
insulin and high glucose for 24 h (Fig. 9,
lane
10), did not further increase p38 MAPK phosphorylation but rather caused a small reduction.

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Fig. 9.
Chronic insulin and HG treatment increase p38 MAPK phosphorylation in
VSMCs. Serum-starved VSMCs were incubated with NG with or without
insulin for 12 and 24 h or with HG for 12 and 24 h, followed by a
subsequent acute treatment with insulin for 30 min. Equal amounts of
cell lysate proteins were subjected to SDS-PAGE, followed by immunoblot
analysis with phosphospecific p38 MAPK antibodies. Once phosphorylation
status was known, blots were stripped and reprobed with p38 MAPK
antibodies to normalize for variations in amount of p38 MAPK protein.
Top: representative autoradiogram.
Similar results were obtained in 3 or 4 experiments.
Lanes
1, 6,
and 8, NG and HG controls;
lane
2, acute insulin treatment;
lane
3, chronic insulin treatment for 12 h;
lane
4, 12-h insulin treatment followed by
acute insulin for 30 min; lane
5, 24-h insulin treatment followed by
a subsequent acute insulin dose; lane
7, HG 12 h followed by 30-min insulin
treatment; lane
9, HG 12 h followed by insulin 30 min;
lane
10, HG 24 h plus insulin 24 h.
Bottom: data from multiple experiments
were quantitated by densitometric scanning and plotted as % of NG
controls. * P < 0.05 vs. NG
control.
|
|
Quantitation of p38 MAPK activation by the in vitro kinase assay in the
immunocomplexes confirmed the results shown in Fig. 9. With ATF-2 as a
substrate, acute insulin treatment caused an approximately threefold
increase in p38 MAPK activity compared with controls (Fig.
10). The stimulation persisted in cells
exposed to chronic insulin for 24 h. Moreover, SB-203580 blocked p38
MAPK activity stimulated by insulin (Fig. 10). High-glucose exposure for 24 h also increased p38 MAPK activity by 200% compared with cells
exposed to normal glucose. Insulin treatment of VSMCs exposed to high
glucose did not further increase p38 MAPK activity in the
immunoprecipitates (Fig. 10). The glucose effect was not entirely due
to hyperosmolarity, since mannitol at 19.5 mM caused only a small
increase in p38 MAPK activity compared with cells grown under normal
glucose conditions (Fig. 10).

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Fig. 10.
Assay of p38 MAPK activity in immunoprecipitates. VSMCs exposed to
mannitol, NG, acute and chronic insulin, and HG in presence and absence
of SB-203580 and PD-98059 were extracted in lysis buffer containing
phosphatase and protease inhibitors. Equal amounts of cell lysates were
immunoprecipitated with a polyclonal anti-p38 MAPK Ab at 4°C
overnight. Immunocomplexes were sedimented with protein A-Sepharose,
washed extensively, and resuspended in kinase assay buffer. Kinase
activity was then measured using activating transcription factor-2 as a
substrate. Details are given in
METHODS. Results are means ± SE of
4 different experiments, expressed as % of normal control.
* P < 0.05 vs. NG;
** P < 0.05 vs. chronic
insulin and HG.
|
|
In contrast to its effect on p38 MAPK, acute insulin treatment caused
only a small increase (45% over basal) in the levels of phospho-ERKs
(Fig. 11, compare
lane
2 with
lane
1; quantitation in
bottom). However, high glucose and
chronic insulin treatment for 12 and 24 h, respectively, did result in
a twofold increase in the phosphorylation status of ERKs (Fig. 11,
compare lanes
3 and
4 and
lanes
6 and
7 with
lanes
1 and
2). Pretreatment with PD-98059
blocked chronic effects of insulin and high glucose, whereas SB-203580
was ineffective.

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Fig. 11.
HG and chronic insulin treatment increase phosphorylation status of
extracellular signal-regulated kinase (ERKs). VSMCs exposed to NG,
acute and chronic insulin, and HG in presence and absence of SB-203580
and PD-98059 were extracted in lysis buffer containing phosphatase and
protease inhibitors. Equal amounts of cell lysates were subjected to
SDS-PAGE followed by Western blot analyses with phosphospecific ERK Ab.
Once linear signal had been obtained, blots were stripped and reprobed
with anti-ERK antibodies to normalize results for variations in ERK
protein levels. Top: representative
autoradiogram. Bottom: data from
multiple experiments were quantitated by densitometric scanning and
plotted as % of NG controls.
* P < 0.05 vs. NG control;
** P < 0.05 vs. acute insulin;
*** P < 0.05 vs. chronic
insulin.
|
|
High glucose and chronic insulin treatment increase DNA synthesis in
VSMCs via p38 MAPK/ERK1/ERK2 activation.
To examine the impact of sustained p38 MAPK activation on cell
proliferation, we measured DNA synthesis in cells exposed to chronic
insulin and high glucose. As seen in Fig.
12, chronic treatment of VSMCs with
insulin for 24 h caused a 70% increase in
[3H]thymidine
incorporation into DNA compared with basal levels. Pretreatment with
SB-203580 partially abolished the stimulatory effects of insulin on DNA
synthesis. SB-203580 by itself did not significantly alter basal
[3H]thymidine
incorporation into DNA (Fig. 12). Exposure to high glucose resulted in
a fourfold increase in
[3H]thymidine
incorporation into DNA. The presence of insulin together with high
glucose further increased
[3H]thymidine
incorporation by 25% above high-glucose controls. Pretreatment with
SB-203580 decreased the stimulatory effects of high glucose by 50%.
Combined addition of PD-98059 completely inhibited the effect of
insulin on DNA synthesis. PD-98059 alone blocks the effects of insulin
on DNA synthesis (see Ref. 4).

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Fig. 12.
Inhibition of p38 MAPK abolishes chronic insulin and HG-mediated DNA
synthesis. Confluent serum-starved VSMCs were pretreated with SB-203580
(0.2 µM) for 30 min, followed by addition of NG or HG with or without
insulin for an additional 24 h.
[3H]thymidine (1 µCi/ml) was added during last 3 h of incubation. Reaction was stopped
by removing medium and rinsing cells four times with ice-cold PBS and
once with 10% ice-cold TCA. Pellet was rinsed once with ether to
remove TCA and solubilized in 1% SDS containing 0.1 N NaOH. An aliquot
of cell lysate was counted in a scintillation counter. Results are
means ± SE of 3 separate experiments performed in duplicate. DPM,
disintegrations/min. * P < 0.05 vs. NG control; ** P < 0.05 vs. insulin; *** P < 0.05 vs. NG controls; **** P < 0.05 vs. HG control; ***** P < 0.05 vs. HG controls and HG + insulin.
|
|
 |
DISCUSSION |
The results of the present study clearly indicate that the simulation
of hyperinsulinemia and hyperglycemia by chronic insulin and
high-glucose treatment of VSMCs markedly inhibits the induction of
MKP-1 mRNA and protein expression in response to a subsequent acute
insulin stimulus. As expected, the high glucose- and chronic insulin-induced inhibition of MKP-1 induction was accompanied by a
marked impairment in iNOS protein expression. The results of this study
confirm our earlier observations that the iNOS/cGMP signaling pathway
plays a major role in the acute stimulatory effects of insulin on the
induction of MKP-1 expression (2). Our earlier studies also indicated a
potential interaction between MAPK family members and iNOS (2). Thus
blocking MAPKs by pretreatment with PD-98059 completely abolished the
effect of insulin on iNOS induction (2). The inhibition of iNOS protein
induction observed in this study in response to high glucose and
chronic insulin treatment appears to be due mainly to sustained p38
MAPK activation. Thus it appears that a stress-related MAPK pathway
such as p38 MAPK may represent the additional pathway necessary to link
the high glucose- and chronic insulin-induced increase in intracellular oxidative stress to hypertrophy via inhibition of MKP-1, the
phosphatase that turns off MAPK signaling by causing dephosphorylation
and inactivation of MAPK family members. In support of our
observations, recent studies by Igarashi et al. (13) showed
PKC-dependent elevations in p38 MAPK activity in VSMCs isolated from
diabetic rat aortae as well as those exposed to 16.5 mM glucose. The
results of this study add a new dimension to the above observations by documenting that high glucose- and insulin-induced elevations in p38
MAPK result in inhibition of the stimulatory effects of insulin on
MKP-1 induction via iNOS inhibition. Thus our study has identified
MKP-1 as a possible target in vascular cells that can be inhibited by
high glucose and sustained hyperinsulinemia, leading to excessive VSMC growth.
Several lines of evidence presented in this study suggest that the p38
MAPK signaling pathway mediates the inhibitory effects of high glucose
and chronic insulin on iNOS induction, leading to an inhibition of
MKP-1 protein expression. First, blocking p38 MAPK signaling by prior
treatment with SB-203580, a selective p38 MAPK inhibitor, prevents the
inhibitory effects of high glucose and chronic insulin on iNOS protein
induction and restores the acute stimulatory effects of insulin on iNOS
as well as MKP-1 protein induction. The effect is observed only with
SB-203580; PD-98059, a MEK inhibitor that blocks MAPK signaling, does
not prevent the inhibitory effects of high glucose and chronic insulin on iNOS and MKP-1 protein expression even though it blocks high glucose- and chronic insulin-induced ERK phosphorylation. Second, insulin rapidly and dose dependently increases p38 MAPK phosphorylation and its activity, and these elevations in the enzyme activity are
maintained under conditions of high glucose and chronic insulin. Most
important, p38 MAPK can be further stimulated in chronic insulin-treated cells by a subsequent acute insulin treatment. In
contrast, ERK1/ERK2 are phosphorylated only after prolonged 24-h
treatment with insulin and high glucose. Finally, a prolonged incubation period of 12-24 h with high glucose and insulin is needed to observe the inhibitory effects on iNOS induction and MKP-1
expression, suggesting that the p38 MAPK stress response pathway may
downregulate the induction of the above proteins at the transcriptional
level. Given that iNOS protein levels are regulated by transcription,
mRNA stability, translation, and protein turnover, it is hard to
determine exactly how elevations in p38 MAPK downregulate iNOS protein induction.
Further studies with constitutively active as well as dominant negative
mutants of p38 MAPK will help in understanding the exact role of p38
MAPK in iNOS activation and MKP-1 induction. It should be noted that
the NOS signaling pathway does not directly control MAPK activation in
VSMCs, since inhibition of NOS with L-NMMA did not prevent ERK
activation but increased its activation status, presumably due to
inhibition of MKP-1 expression.
Our observations on the inhibitory effects of the p38 MAPK signaling
pathway on iNOS induction coincide with the results of Guan et al. (11)
reporting inhibition of NO synthesis by p38 MAPK pathway in renal
mesangial cells stimulated by interleukin-1
. In contrast, studies by
Da Silva et al. (7a) and LaPointe and Isenovic (20) in mouse astrocytes
and cardiac myocytes, respectively, indicate that blockade of p38 MAPK
signaling results in inhibition of iNOS expression. The most likely
explanation for these seemingly inconsistent results is that the
regulation of iNOS induction is tissue specific and complex, involving
both ERKs and various isoforms of p38 MAPKs with different
sensitivities to the inhibitor SB-203580.
The presence of detectable levels of p38 MAPK phosphorylation and
activity in unstimulated VSMCs maintained under normal glucose conditions suggests that this enzyme or one of its isoforms may be
needed to suppress MKP-1 expression in the basal state. When subjected
to acute insulin treatment, VSMCs may use the ERK-mediated NOS
signaling pathway to cause MKP-1 expression. Thus it appears that the
acute stimulatory effects of insulin on iNOS and MKP-1 protein
induction are likely to be mediated via the MEK/ERK signaling pathway,
whereas the chronic inhibitory effects of insulin and high glucose are
mediated via the p38 MAPK stress-response pathway. Thus chronic
activation of p38 MAPK provides a crucial signaling mechanism, which
may negatively regulate iNOS induction, NO biosynthesis, and MKP-1
expression by a desensitizing mechanism, thereby resulting in the
sustained MAPK activation and excessive cell growth commonly observed
in VSMCs exposed to high glucose and chronic insulin (1, 9, 15).
 |
ACKNOWLEDGEMENTS |
This work was supported in part by a grant-in-aid from the American
Heart Association (New York State Affiliate) and medical education
funds from Winthrop University Hospital.
 |
FOOTNOTES |
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: N. Begum,
Diabetes Research Laboratory, Winthrop University Hospital, 259 First
St., Mineola, NY 11501 (E-mail: nbegum{at}winthrop.org).
Received 4 June 1999; accepted in final form 31 August 1999.
 |
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