Regulation of Endothelial Constitutive Nitric Oxide Synthase

Gene Expression in Endothelial Cells and In Vivo

A Specific Vascular Action of Insulin
Koji Kuboki, MD; Zhen Y. Jiang, MD, PhD; Noriko Takahara, MD, PhD; Sung Woo Ha, MD, PhD;
Masahiko Igarashi, MD, PhD; Teruaki Yamauchi, MD, PhD; Edward P. Feener, PhD;
Terrance P. Herbert, MD, PhD; Christopher J. Rhodes, PhD; George L. King, MD

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BackgroundThe vasodilatory effect of insulin can be acute or increase with time from 1 to 7 hours, suggesting that
insulin may enhance the expression of endothelial nitric oxide synthase (eNOS) in endothelial cells. The objective of
the present study was to characterize the extent and signaling pathways by which insulin regulates the expression of
eNOS in endothelial cells and vascular tissues.
Methods and ResultsPhysiological concentrations of insulin (1010 to 107 mmol/L) increased the levels of eNOS
mRNA, protein, and activity by 2-fold after 2 to 8 hours of incubation in cultured bovine aortic endothelial cells. Insulin
enhanced eNOS gene expression in microvessels isolated from Zucker lean rats but not from insulin-resistant Zucker
fatty rats. Inhibitors of phosphatidylinositol-3 kinase (PI-3 kinase) decreased the effect of insulin on eNOS gene
expression, but a general protein kinase C (PKC) inhibitor, GF109203X or PKC isoform inhibitor, LY333531
enhanced eNOS expression. In contrast, PKC activators inhibited both the activation by insulin of PI-3 kinase and eNOS
mRNA levels. Overexpression of PKC isoform in endothelial cells inhibited the stimulation by insulin of eNOS
expression and PI-3 kinase activities in parallel.
ConclusionsInsulin can regulate the expression of eNOS gene, mediated by the activation of PI-3 kinase, in endothelial
cells and microvessels. Thus, insulin may chronically modulate vascular tone. The activation of PKC in the vascular
tissues as in insulin resistance and diabetes may inhibit PI-3 kinase activity and eNOS expression and may lead to
endothelial dysfunctions in these pathological states. (Circulation. 2000;101:676-681.)
Key Words: insulin nitric oxide RNA endothelium cells diabetes mellitus proteins

endothelial cells, eNOS is the most abundant form8 and can

be regulated by acetylcholine, hypoxia, sheer stress, lysophosphatidylcholine (LPC), and cytokines.8 11
In the present study, we characterized the effect of insulin
on the expression of eNOS both in cultured endothelial cells
and in microvessels from lean and insulin-resistant rats. In
addition, the signaling pathway and regulation of the action of
insulin on eNOS gene expression were studied.

nsulin has multiple physiological effects on the vascular

tissues such as vasodilation,1 4 which may be endothelial
cell dependent and can be inhibited by inhibitors of nitric
oxide synthase (NOS).2 6 Zeng and Quon7 suggested that
insulin can increase the production of NO in cultured endothelial cells within a few minutes, indicating an activation of
NOS via the insulin receptors. However, the vasodilatory
effect of insulin in vivo may have both acute and prolonged
actions; Utriainen et al6 and other researchers25 showed that
the effect of insulin on forearm blood flow continued to
increase even after 6 to 7 hours of infusion. In addition, the
concentrations of insulin required to rapidly activate NOS in
cultured endothelial cells were much higher than physiological levels.7 These results suggest that insulin could also be
increasing the production of NO in vivo by inducing both the
expression and the activation of NOS in the endothelial cells.
We investigated the possibility that one mechanism of the
vasodilatory effect of insulin is to increase the expression of
endothelial NOS (eNOS) in endothelial cells. Although all 3
types of NOS have been reported to be expressed by

Methods
Cell Culture
Bovine aortic endothelial cells (BAECs) from passages 4 to 10 were
isolated and used as described previously.12 Confluent cells were
placed in DMEM containing 1% platelet-deprived horse serum
(PDHS) for 24 hours before being studied and pretreated with the
following inhibitors: phosphatidylinositol-3 (PI-3) kinaseselective
inhibitors wortmannin (Sigma Chemical Co)13 and LY294002
(BIOMOL Research Laboratories),14 protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) (Sigma Chemical Co),
general PKC inhibitor GF109203X (GFX) (CalbiochemNovabiochem Corp),15 and PKC isoformselective inhibitor

Received May 26, 1999; revision received July 30, 1999; accepted August 13, 1999.
From the Research Division, Joslin Diabetes Center, and Harvard Medical School, Boston, Mass.
Correspondence to George L. King, MD, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. E-mail kingg@joslab.harvard.edu
2000 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org

676

Kuboki et al
LY333531 (Lilly Inc).16 Cells were then stimulated with insulin
(Sigma Chemical Co), recombinant insulin-like growth factor-1
(IGF-1) (Upstate Biotechnology), and LPC (Avanti Polar Lipid) or
-IR3 antibodies (Sigma).

Construction of Replication-Deficient Recombinant

Adenovirus Containing PKC1 cDNA
The construction of a replication-deficient recombinant adenovirus
for PKC1 expression was performed as described previously.17
Adenovirus-mediated gene transfer to confluent BAECs was performed through a 1-hour adenoviral infection of 109 pfu/mL at 37C
in DMEM containing 10% PDHS as described previously.17 The
infected BAECs were then incubated in DMEM containing 1%
PDHS for 24 hours, incubated with or without insulin (100 nmol/L)
for an additional 6 hours, and harvested. AdV-CMV-PKC1 or
-galactosidase (-Gal)infected BAECs were assessed for PKC
activity and protein expression as previously described.12

Isolation of Vascular Stroma From Epididymal

Fat Pads of Zucker Rats
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Vascular stromas were obtained from the epididymal fat pads of

12-week-old Zucker lean and fatty rats (Harlan Sprague Dawley,
Inc). Epididymal fat pads were isolated, minced, and incubated with
0.2% collagenase I for 30 minutes at 37C. Then, they were
fractionated with the use of a Dounce homogenizer and centrifuged
at 3000g for 20 minutes to isolate vessels from adipocytes. Vascular
stroma were washed with DMEM containing 0.2% BSA and incubated with DMEM containing 0.2% BSA with or without insulin for
6 hours at 37C. The purity of the isolated vascular stroma was
quantified through immunohistochemical staining with antifactor
VIII antibody and through immunoblotting of the stroma with
antibodies to smooth muscle cell -actin. Only preparations that
were stained positively in 90% of the vessels were used.

RNA Isolation and Northern Blot Analysis

Total RNA from cultured BAECs, PKC1-overexpressed BAECs,
and vascular stroma from the epididymal fat pads of Zucker rats were
isolated according to the guanidinium thiocyanate-phenolchloroform method with TRI Reagent (Molecular Research Center)
and solution D containing 4 mol/L guanidinium thiocyanate,
25 mmol/L sodium citrate, pH 7.0, 0.5% sarcosyl, and 0.1 mol/L
2-mercaptoethanol. Total RNA (20 g) was fractionated and hybridized to 650-bp cDNA fragments of rat eNOS (kindly provided by Dr
Mark A. Perella and Arthur M.E. Lee, Harvard School of Public
Health, Boston, Mass), which were labeled with the use of a DNA
labeling system (Multiprime; Amersham Corp).18 The quantification
of eNOS mRNA levels was performed with a PhosphorImager
(Molecular Dynamics) and normalized to 36B4 mRNA.19

Immunoblot Analysis of eNOS

Cells were washed 3 times with ice-cold PBS, pH 7.4, lysed in
50 mmol/L Tris, pH 7.5, 2 mmol/L EDTA, 0.5 mmol/L EGTA,
2 mmol/L PMSF, 25 g/mL leupeptin, 0.1 mg/mL aprotinin,
1 mmol/L dithiothreitol, 50 mmol/L NaF, and 1% Triton X-100
(Sigma Chemical Co); scraped from the dish; rotated for 1 hour at
4C; and centrifuged for 15 minutes at 14 000g. Protein concentrations of the supernatant were measured according to the method of
Bradford20 and separated with the use of 6% SDS-PAGE as
described previously.14 The membrane was incubated for 1 hour with
polyclonal anti-human eNOS antibody (Transduction Laboratories)
diluted in PBS containing 0.1% Tween-20 and 1% BSA, washed 3
times for 10 minutes with PBS containing 0.1% Tween-20, and
incubated with 0.1 Ci/mL 125I-protein A (Amersham Life Science,
Inc). Protein levels of eNOS were quantified with a PhosphorImager.

Assay of PI-3 Kinase Activity

After preincubation with or without 100 nmol/L PMA for 30
minutes, BAECs were stimulated with insulin (100 nmol/L) for 5
minutes. Cells were processed as described previously for this

Effect of Insulin on eNOS Gene Expression

677

assay.14 Aliquots of proteins from the supernatant were immunoprecipitated with 10 L/ml anti-insulin receptor substrates (IRS)-2
antibodies (kindly provided by Dr Morris F. White, Joslin Diabetes
Center, Boston, Mass) for 2 hours and bound to protein ASepharose
beads at 4C as described previously.14 The lipids were quantified
with a PhosphorImager.

Assay of NOS Activity

The amount of NOS activity produced by BAECs was measured by
using an NOS Detect assay kit (Transduction Laboratories) according to the manufacturers instructions. Briefly, BAECs were harvested in PBS containing 1 mmol/L EDTA and centrifuged at
12 000g. The pellets were lysed in homogenization buffer containing
25 mmol/L Tris, pH 7.4, 1 mmol/L EDTA, and 1 mmol/L EGTA and
centrifuged at 12 000g. Aliquots from the supernatant were used for
the measurement of NOS activity through the conversion of [3H]Larginine to [3H]L-citrulline. Data were normalized by the amount of
protein and reaction time.

PKC Activity Assay and Immunoblotting Studies

After adenoviral infection, confluent BAECs were harvested and
PKC activity were measured as previously described.12,19 Briefly,
PKC activities were measured according to 32P labeling of
100 mol/L PKC-specific peptide substrate RKRTLRRL. For immunoblotting studies, total cell lysate (75 g/lane) was fractionated
with the use of PAGE and detected with the use of antibodies to the
PKC1 isoform (Santa Cruz Biotechnology, Inc). A detailed description of the method was reported previously.14,21

Statistical Analysis
Data are expressed as meanSEM and were analyzed with the use
of the Newman-Keuls test for ANOVA for multiple comparisons. A
value of P0.05 was considered statistically significant.

Results
Effect of Insulin on mRNA and Protein Levels
of eNOS
Insulin (100 nmol/L) significantly augmented the eNOS
mRNA expression of eNOS at 1 hour by 339%, reached
7121% at 6 hours, and attained a maximum of 2-fold at 12
hours, which was maintained for 24 hours (Figure 1A).
Expression of eNOS mRNA responded to insulin (Figure 1B)
with a significant increase even at 0.1 nmol/L. At 10 nmol/L
insulin, eNOS mRNA level was significantly increased by
5012%, and a maximum effect of 2-fold was attained at 100
nmol/L. Therefore, stimulation with 100 nmol/L insulin with
an incubation time of 6 hours IGF-1 (25 nmol/L) also
increased the eNOS mRNA level by 4710% in human
umbilical endothelial cells. The addition of IR3, an IGF-1
receptorspecific antibody (1 g/mL), inhibited the effect of
IGF-1 by 60% but did not decrease the effect of insulin.
Insulin also increased eNOS protein levels at 6 hours by
4316% and reached a maximum of 2-fold at 24 hours,
which was maintained for 36 hours.

Effect of PI-3 Kinase Inhibitors Wortmannin and

LY294002 on Expression of eNOS
The acute effect of insulin on NO production in endothelial
cells was reported to be inhibited by wortmannin, a PI-3
kinase inhibitor.7 To determine whether PI-3 kinase activation could be increasing mRNA expression and protein levels
of eNOS, 2 structurally different PI-3 kinase inhibitors,
wortmannin (100 nmol/L) and LY294002 (50 nmol/L), were
preincubated with BAECs before the addition of insulin (100

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Figure 1. Effect of insulin on expression of eNOS mRNA in

BAECs. A, Time course of BAECs cultured to 80% confluence,
starved for 24 hours, and stimulated with insulin (100 nmol/L) for
times indicated. RNA was isolated from BAECs, and Northern
blot analysis was performed with 20 g of total RNA per lane as
described (*P0.05 vs 0 hours, **P0.01 vs 0 hours). B, Dose
response of insulin. Cultured BAECs were incubated with indicated concentrations (Conc.) of insulin for 6 hours, and then
eNOS mRNA expression was measured by Northern blot analysis (*P0.05 vs 0 nmol/L, **P0.01 vs 0 nmol/L). Results were
quantified with a PhosphorImager and normalized by levels of
36B4 mRNA expression. Data are meanSEM of 3 different
experiments and expressed as percentage of control.

nmol/L) (Figure 2). Insulin increased the mRNA level of

eNOS by 5820% compared with control, but the effect of
insulin was inhibited by preincubation with wortmannin
(Figure 2A). Similar to eNOS mRNA levels, insulin significantly increased the eNOS protein level by 749%, which

Figure 3. Effect of PMA on eNOS mRNA expression stimulated

with or without insulin in BAECs. A, Time course of PMAinduced eNOS mRNA expression. After BAECs were starved for
24 hours, cells were treated with 100 nmol/L PMA for indicated
times (*P0.05 vs 0 hours). B, Effect of PMA on insulin-induced
eNOS mRNA expression. Cultured BAECs were starved for 24
hours and treated with 100 nmol/L insulin for 6 hours after pretreatment with or without 100 nmol/L PMA for 30 minutes. Then,
20 g of total RNA extracted from BAECs was used for Northern blot analysis with 32P-CTP-labeled eNOS probes as
described in Methods (*P0.01 vs control [Con.], #P0.01 vs
insulin). Results are expressed as a percentage of control and
shown as meanSEM of 3 different experiments (A) and of 4
different experiments (B).

was completely inhibited by the addition of wortmannin

(Figure 2B).
The pretreatment of BAECs with another PI-3 kinase
inhibitor, LY294002 (50 nmol/L), completely inhibited the
induction of eNOS mRNA expression by insulin. Unlike
wortmannin, LY294002 significantly decreased the basal
mRNA expression of eNOS without insulin treatment by
304%. Correspondingly, LY294002 inhibited the increases
in eNOS protein levels stimulated by insulin and decreased
the basal eNOS protein level by 725%.
Insulin (100 nmol/L) significantly increased NOS activity
from 1159 to 1767 pmol mg protein1 min1 after 24
hours (P0.01, n6). Preincubation with wortmannin (100
nmol/L) for 15 minutes significantly decreased insulininduced NOS activity to 12313 pmol mg protein1 min1,
but the basal levels of NOS activity were unchanged.

Effect of PMA on Insulin-Induced eNOS mRNA

Expression and PI-3 Kinase Activities
Figure 2. Effect of wortmannin on insulin-induced eNOS
expression in BAECs. A, Effect of eNOS mRNA in BAECs. Cultured BAECs were starved in 1% PDHS for 24 hours, preincubated without (Con.) or with 100 nmol/L wortmannin (Wort.) for
15 minutes, and stimulated with 100 nmol/L insulin for 6 hours.
RNA was extracted from BAECs, and expression of eNOS
mRNA was analyzed by Northern blot analysis as described.
Data were visualized with a PhosphorImager and normalized by
levels of 36B4 mRNA expression (*P0.01 vs Con., #P0.01 vs
insulin). B, Effect on protein levels of eNOS. After starvation for
24 hours, cells were pretreated with 100 nmol/L wortmannin for
15 minutes and then stimulated with 100 nmol/L insulin for 24
hours. Aliquots of proteins extracted from BAECs were analyzed
by 6% SDS-PAGE, and membranes were blotted with antieNOS antibodies, followed by 125I-labeled protein A (*P0.01 vs
Con., #P0.01 vs insulin). Results are quantified with a PhosphorImager, expressed as a percentage of control, and shown
as meanSEM of 6 different experiments.

Because PKC activation is observed in the vascular tissue in

diabetes and may regulate eNOS in BAECs,12,16,21 the actions
of PMA, a PKC agonist, on eNOS expression were studied
(Figure 3). In time course experiments, PMA (100 nmol/L)
did not change the eNOS mRNA level for the initial 6 hours
but significantly increased the expression of eNOS mRNA
after 12 and 24 hours of incubation by 6611% and
10514%, respectively (Figure 3A). In contrast, when
BAECs were preincubated with PMA for 30 minutes, the
effect of insulin on eNOS mRNA levels was inhibited
(1413%) (Figure 3B).
Because insulin may increase NO production via activation
of PI-3 kinase through the tyrosine phosphorylation of its
receptors and IRS,22 the effects of PKC activation on the of
insulin induction of eNOS expression and PI-3 kinase activity
were examined in parallel. Insulin significantly increased

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Figure 4. Effect of a PKC inhibitor, GFX, and a PKC isoform

specific inhibitor, LY333531, on insulin-induced eNOS mRNA
expression in BAECs. Cultured BAECs starved for 24 hours
were pretreated with 5 mol/L GFX or 20 nmol/L LY333531 for
30 minutes and stimulated with or without 100 nmol/L insulin for
6 hours. RNA was extracted from cells, and Northern blot analysis was performed with 20 g of total RNA per lane. Results are
normalized by levels of 36B4 mRNA expression and expressed
as a percentage of control and as meanSEM of 4 different
experiments (*P0.05 vs control, **P0.01 vs control [Con.]).

IRS-2associated PI-3 kinase activity by 5.40.4-fold. When

BAECs were preincubated with PMA (100 nmol/L) for 30
minutes, insulin-induced IRS-2associated PI-3 kinase activity was mostly inhibited. However, the basal PI-3 kinase
activity was not changed with PMA treatment.

Effect of PKC Inhibitors on eNOS

mRNA Expression
The exposure of BAECs to the PKC inhibitor GFX
(5 mol/L) without insulin for 6 hours increased the expression of eNOS mRNA by 3810% (Figure 4). The expression
of eNOS mRNA was greater in cells exposed to both insulin
and GFX (by 7620% compared with control cells or those
incubated with either insulin or GFX alone). We have
reported that hyperglycemia may preferentially activate
PKC isoforms in the vascular cells.16 To explore the
possibility that the PKC isoform could also have a role in
regulation of the activation by insulin of PI-3 kinase and
eNOS expression, the effect of LY333531 (20 nmol/L), a
PKC isoform inhibitor, was characterized.16 The addition of
LY333531 also increased eNOS mRNA expression by
6014%, which is similar to insulin or GFX alone.
LY333531 and insulin together did not have a significant
additive effect.

Effect of Overexpression of PKC Isoform on

Insulin-Induced eNOS mRNA Level
To determine directly whether the PKC isoform can regulate the effect of insulin on eNOS expression, we overexpressed the PKC1 isoform in BAECs through the use of
replication-deficient adenovirus containing cDNA of the
PKC1 isoform. Compared with control cells infected with
adenovirus containing the -Gal gene, cells infected with
adenovirus containing the PKC1 gene had a 50-fold increase
in the protein for the PKC1 isoform. Total PKC activities
were also increased by 11- and 7-fold in the cytosol and
membrane fractions, respectively.

Effect of Insulin on eNOS Gene Expression

679

Figure 5. Effect of adenovirus PKC1 infection on eNOS mRNA

expression in BAECs. BAECs were cultured to 80% confluence
and infected with no virus (Con.), adenovirus containing -Gal
cDNA (-gal), and adenovirus containing PKC1 isoform cDNA
(PKC1) as described in Methods. Cells were starved for 24
hours and stimulated with or without insulin for 6 hours. Total
RNA were extracted, and Northern blot analysis was performed
as described in Methods. Results are expressed as percentage
of control and shown as meanSEM of 4 different experiments
(*P0.01 vs control, #P0.01 vs -Gal, ##P0.1 vs
-Galinsulin).

Insulin (100 nmol/L) enhanced eNOS mRNA expression in

BAECs with or without infection with adenovirus containing
only -Gal by as much as 2-fold (Figure 5). In contrast,
insulin did not increase eNOS mRNA levels in cells infected
with adenovirus containing the PKC1 isoform. The expression of eNOS was not changed by overexpression of the
PKC1 isoform (Figure 5) at the basal level. In contrast, LPC
(100 mol/L) increased eNOS mRNA levels by 5- and
4.5-fold in control and adenovirus-containing -Gal cells,
respectively. In BAECs infected with the adenoviral-PKC1
isoform, LPC increased eNOS mRNA by 4-fold, which was
not significantly different from controls.

Effect of Insulin on eNOS mRNA Level in

Vascular Stroma Isolated From Epididymal Fat
Pads of Zucker Fatty and Lean Rats
To determine whether insulin can also change eNOS expression in vascular tissue, we characterized eNOS mRNA levels
in vascular stroma isolated from Zucker lean and fatty
insulin-resistant rats, a model of insulin resistance.23 The
expression of eNOS mRNA with or without insulin (100
nmol/L) for 6 hours in the vascular stroma isolated from
insulin-resistant models (Zucker fatty rats) showed that the
basal levels of eNOS mRNA expression were significantly
decreased to 295% of vascular stroma derived from Zucker
lean rats (Figure 6). The contents of vascular stroma in both
preparations were found to be similar through the use of
immunostaining with factor VIII antibodies and immunoblotting with antibodies to smooth muscle cell -actin. Moreover,
insulin increased eNOS mRNA levels by 5016% in the
vascular stroma from the Zucker lean rats but was ineffective
in vascular stroma isolated from the insulin-resistant rats.

Discussion
One of the important vascular actions of insulin is its
vasodilatory effect, which is associated with NO production,
either from endothelial cells or from perivascular neuronal
cells.1 6 The possibility that insulin can enhance the production of NO is supported by the findings that insulin and IGF-1
increased NO production in endothelial cells in 1

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Figure 6. Effect of insulin on eNOS mRNA expression in vascular stroma from epididymal fat pads of Zucker fatty rats. Vascular stroma was isolated from epididymal fat pads of Zucker fatty
and lean rats as described and incubated with or without 100
nmol/L insulin for 6 hours. Total RNA (20 g) was used for
Northern blot analysis. Results are expressed as a percentage
of control and shown as meanSEM of 5 different experiments
(*P0.05 vs control [Con.] in Zucker lean rats, **P0.01 vs control in Zucker lean rats).

minute.7,24 This suggested that insulin can directly activate

eNOS because protein and mRNA levels of eNOS could not
increase so rapidly. However, the acute effect of insulin on
NO production cannot account for all of the vasodilatory
effects of insulin in vivo, because some physiological studies
have reported that the vasodilatory effect of insulin continues
to increase even after 7 hours of infusion.6 These results
suggest that the vasodilatory effect of insulin has a sustained
component that requires several hours of stimulation when
near-physiological concentrations of insulin are used. Our
findings that physiological concentrations of insulin enhanced the expression of eNOS in the endothelial cells only
after 4 hours of incubation, with maximum effects observed
at 8 hours, are consistent with the sustained component of the
vasodilatory effects of insulin.6
The effect of insulin on eNOS mRNA levels was observed
between 0.1 and 100 nmol/L, which corresponded closely to
the range of binding and activation of insulin receptors in the
endothelial cells and to the physiological levels of insulin in
the plasma. It is interesting that the rapid effect of insulin on
NO production in cultured human umbilical endothelial cells
required pharmacological insulin concentrations of 10 to
10 000 nmol/L.7 This suggests that the acute effect of insulin
on NO production may not be significant under physiological
conditions and may be mediated in part by IGF-1
receptors.7,24
The parallel increases in the mRNA, protein, and enzymatic activities of eNOS indicate that the effect of insulin is
primarily due to the elevation of steady state levels of eNOS
mRNA. It is not possible from these results to determine
whether the effect of insulin on eNOS mRNA levels was due
to increases in the transcription rate or the half-life of eNOS
mRNA. However, it is likely that the changes are due to
increases in the transcription rate, which is the most common
mechanism via which insulin regulates the mRNA levels of
many genes in cells.25
The signaling pathways of insulin action on eNOS mRNA
appear to involve mainly the insulin receptors, because the
maximal effect on eNOS mRNA level was attained with

100 nmol/L, a concentration of insulin shown to bind

minimally to IGF-1 receptors in endothelial cells.1 In addition, the effect of insulin on eNOS mRNA was not prevented
by inhibitory antibodies to IGF-1 receptors. The effects of the
PI-3 kinase inhibitors wortmannin and LY294002 suggest
that the activation of PI-3 kinase is involved. Interestingly,
the rapid effect of insulin on NO production from endothelial
cells was also inhibited by wortmannin, although the direct
effect of insulin on PI-3 kinase was not measured in the
previous study.7
PKC activation appears to modulate the effect of insulin on
eNOS mRNA expression; rapid PKC activation induced by
phorbol esters caused inhibition of insulin-stimulated PI-3
kinase activity and eNOS mRNA expression. The findings
that eNOS expression was increased by the long-term incubation of PMA and by PKC inhibitors are consistent because
both maneuvers will reduce PKC activities in endothelial
cells. These findings confirmed previous reports that PKC
inhibition increased eNOS mRNA in BAECs.21
The findings that both general PKC inhibitor GFX and
specific PKC isoform inhibitor LY333531 increased basal
eNOS levels suggest that PKC activities may regulate eNOS
mRNA levels in endothelial cells. The use of the PKC
isoform inhibitor LY333531 (20 nmol/L, a concentration that
selectively inhibited the PKC isoform) indicated that the
activation of PKC isoform may have a selective effect on
eNOS expression.18 This finding was surprising in that the
PKC isoform is expressed to a lesser extent than other PKC
isoforms in the endothelial cells.26 The inhibitory effect of the
PKC isoform on eNOS mRNA level is directly confirmed
through the overexpression of the PKC isoform in endothelial cells with the use of adenoviral vectors containing
full-length DNA of the PKC1 isoform. The inhibitory effect
of PKC activation on eNOS expression may be specific to
insulin because the stimulating effect of LPC was not
affected. The finding that the activation of the PKC isoform
can inhibit eNOS expression in endothelial cells is of particular interest in the area of diabetic vascular complications
because the activation of PKC isoforms has been associated
with various vascular dysfunctions in the retina, renal glomeruli, and cardiovascular systems of diabetic animals1,27 and
with cardiac hypertrophy in transgenic mice.19
The results obtained for the microvessels isolated from the
Zucker fatty and lean rats support the likelihood that our
findings in cultured endothelial cells have physiological
meaning and that this action of insulin is blunted in insulinresistant states. These in vivo findings are consistent with
previous reports that showed the total NOS activities were
decreased in the skeletal muscle and neuronal tissues of
Zucker fatty rats.28 The basal expression of eNOS was also
much lower in insulin-resistant Zucker fatty rats than in lean
animals, suggesting that insulin may also modulate eNOS
levels in the vessels at the basal state. However, specific
measurements of the signaling steps of insulin must be
determined to document the extent of insulin resistance in the
vascular tissues in insulin-resistant animals or humans.
In summary, insulin can modulate eNOS expression chronically both in vitro and in vivo, which may enhance the NO
production induced by other agonists such as acetylcholine.

Kuboki et al
Because the enhancement of NO production causes vasodilatation and inhibits smooth muscle growth, it is possible that
at physiological levels in an insulin-sensitive state, insulin
can indirectly have antiatherosclerotic effects. In the presence
of hyperglycemia and insulin resistance, which are known to
activate PKC and induce the inhibition of PI-3 kinase
activities in the vasculatures, the effect of insulin on eNOS
expression is blunted, resulting in the loss of its vasodilatory
effects. Further studies are necessary to determine whether
PKC activities are increased in the insulin-resistant state and
whether the use of the inhibitor of the PKC isoform or of
specific insulin sensitizers can improve the vascular actions
of insulin and endothelial cell dysfunctions.

Acknowledgments
This work was supported by grant NIH DK-53105 and by Diabetes
Endocrinology Research Center grant NIH 5-P30-DK-6836.

Effect of Insulin on eNOS Gene Expression

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Regulation of Endothelial Constitutive Nitric Oxide Synthase Gene Expression in

Endothelial Cells and In Vivo: A Specific Vascular Action of Insulin
Koji Kuboki, Zhen Y. Jiang, Noriko Takahara, Sung Woo Ha, Masahiko Igarashi, Teruaki
Yamauchi, Edward P. Feener, Terrance P. Herbert, Christopher J. Rhodes and George L. King
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Circulation. 2000;101:676-681
doi: 10.1161/01.CIR.101.6.676
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