© 1997 American Heart Association, Inc.
Articles |
Mechanisms of Action of Troglitazone in the Prevention of High Glucose–Induced Migration and Proliferation of Cultured Coronary Smooth Muscle Cells
From The First Department of Internal Medicine, Osaka City (Japan) University Medical School.
Correspondence to Kenichi Yasunari, MD, The First Department of Internal Medicine, Osaka City University Medical School, 1–5-7 Asahi-machi, Abeno-ku, Osaka 545, Japan.
Abstract
Abstract Recent findings suggest that high glucose levels may promote atherosclerosis in coronary vascular smooth muscle cells (VSMCs). To explore the intracellular mechanisms of action by which troglitazone affects this process, we examined the effect of troglitazone on the migration and growth characteristics of cultured rabbit coronary VSMCs. Treatment with chronic high glucose medium (22.2 mmol/L) for 5 days increased VSMC migration by 92%, [3H]thymidine incorporation by 135%, and cell number by 32% compared with VSMCs treated with normal glucose (5.5 mmol/L glucose+16.6 mmol/L mannose) medium. Troglitazone at 100 nmol/L and 1 µmol/L significantly suppressed high glucose–induced VSMC migration by 34% and 42%, respectively, the proliferative effect (as measured by cell number) by 17% and 27%, and [3H]thymidine incorporation by 45% and 60% (n=6, P<.05). The high glucose–induced impairment of insulin-mediated [3H]deoxyglucose uptake was blocked by a protein kinase C (PKC) inhibitor (calphostin C, 1 µmol/L) and was also improved by troglitazone without any change in insulin receptor number and affinity. The high glucose–induced insulin-mediated increase in cell number and in [3H]thymidine incorporation was suppressed by troglitazone. Troglitazone (1 µmol/L) also suppressed high glucose–induced phospholipase D activation, elevation of the cytosolic NADH/NAD+ ratio (as measured by the cytosolic ratio of lactate/pyruvate), and membrane-bound PKC activation. Flow cytometric DNA histogram analysis of cell cycle stage showed that high glucose–induced increase in the percentage of cells in the S phase was suppressed by 1 µmol/L troglitazone. These findings suggest that PKC may be a link between impairment of insulin-mediated glucose uptake and the increase in migration and proliferation induced by high glucose levels and that troglitazone may be clinically useful for the treatment of high glucose–induced coronary atherosclerosis.
Key Words: antioxidant • glucose • vascular smooth muscle • phospholipase D • protein kinase C
Hyperglycemia is probably an important causative factor in the development of macrovascular complications in patients with coronary artery disease both before1 and after2 percutaneous transmural coronary angioplasty. However, the mechanisms participating in accelerated atherosclerotic disease in patients with diabetes are unclear. VSMC migration and proliferation appear to play important roles in the development of atherosclerosis.3 Although hyperglycemia has been suggested to contribute to the development of complications in patients with diabetes, few studies have focused on the direct effect of elevated glucose levels on VSMCs.
It has been reported that elevated glucose-induced increases in the NADH/NAD+ ratio and glyco-oxidation products in diabetic humans and animals are evidence of diabetes-induced oxidative stress.4 However, it is unknown to what extent these parameters of increased oxidative stress reflect increased VSMC migration and proliferation.
Troglitazone, originally introduced for clinical use as an insulin sensitizer, has been shown to possess potent antioxidant properties.5 The finding that active oxygen species stimulate VSMC growth6 suggests that troglitazone might prevent active oxygen species–induced vascular growth. These observations led us to speculate that troglitazone may prevent high glucose–induced migration and hyperproliferation of VSMCs, possibly through its antioxidative effects.
Hyperglycemia7 and insulin resistance8 are also considered independent risk factors for coronary artery disease. However, it is unknown whether insulin resistance exists in coronary VSMCs and whether hyperinsulinemia directly promotes coronary VSMC proliferation.
The present study was, therefore, designed to investigate the effect of troglitazone on high glucose–induced coronary VSMC migration and proliferation and on high glucose and insulin-mediated glucose uptake and proliferation by coronary VSMCs. Possible intracellular mechanisms of action of troglitazone were also studied.
Materials and Methods
Materials
Type II collagenase, Nonidet P-40, and
dithiothreitol were purchased from Sigma Chemical Co. DMEM,
penicillin-streptomycin, trypsin EDTA (Versine), and FCS were purchased
from GIBCO Laboratories. [3H]Thymidine,
[3H]ethanolamine, 125I-insulin, and the PKC
assay system were purchased from Amersham Japan Co. Multiwell pipettes
and flasks were purchased from Becton Dickinson & Co. Troglitazone and
pioglitazone were donated by Sankyo Co. 
-Tocopherol was
donated by Eisai Co.
Cell Culture
VSMCs were grown from explants of 4-week-old male Japanese White
rabbit coronary arteries by the explant method. Cells were
identified as VSMCs on the basis of their morphological and growth
characteristics as previously reported.9,10 Briefly, VSMCs
exhibited a typical "hill and valley" growth pattern and also
exhibited positive fluorescence with antibodies against
-smooth muscle actin but no fluorescence with antibodies
against factor VIII antigen. VSMCs were grown in DMEM supplemented with
10% FCS. Cells from passages 3 to 5 were used and were subcultured
after trypsinization on a weekly basis, since cells became confluent in
1 week. Each plate was replenished twice a week with fresh medium. For
studies of cells under hyperglycemic conditions, cells were allowed to
grow for 5 days in high glucose (22.2 mmol/L) medium before
use in order to more closely simulate chronic hyperglycemia. As a
control for osmolarity, cells were grown for 5 days in normal glucose
(5.6 mmol/L glucose+16.6 mmol/L mannose)
medium.
Experimental Protocol
In the migration experiment, VSMCs were allowed to grow for 5
days in high or normal glucose medium with 10% FCS in the
presence or absence of troglitazone, pioglitazone, or
-tocopherol. VSMCs were trypsinized and suspended at a
concentration of 1.5x105 cells/mL in DMEM without FCS
supplemented with 0.4% BSA. Then VSMCs were used in the migration
experiment described below. In growth experiments, VSMCs were allowed
to grow for 5 days in high or normal glucose medium with 10% FCS in
the presence or absence of troglitazone, pioglitazone, or
-tocopherol. VSMCs were cultured in DMEM without FCS for
48 hours to induce quiescence. Then VSMCs were used in cell count
experiments, in [3H]thymidine incorporation experiments,
and in the measurement of PLD and PKC.
Migration Assay
The migration of VSMCs was assayed by a modified version of
Boyden’s chamber method using microchemotaxis chambers (Neuro Probe
Inc) and polycarbonate filters (Nucleopore Corp) with pores 5.0
µm in diameter, as previously reported.11 In all
experiments, collagen-coated filters were used. Briefly, the membranes
were treated with 0.5N acetic acid and then incubated for 48 to 72
hours at 25°C in a collagen solution (100 µg/mL type I
collagen in 0.5N acetic acid). They were then air-dried. Cultured VSMCs
were trypsinized and suspended at a concentration of
1.5x105 cells/mL in DMEM supplemented with 0.4% BSA.
Cell number was counted with an electronic cell counter (model ZB1,
Coulter Electronics). A 200-µL volume of VSMC suspension
(3.0x104 cells) was placed in the upper chamber, and 40
µL of DMEM/0.4% BSA containing a migration factor was placed in the
lower chamber. The chamber was incubated at 37°C under 5%
CO2 in air for 4 hours. After incubation, the filter was
removed, and the VSMCs on the upper side of the filter were scraped
off. The VSMCs that had migrated to the lower side of the filter were
fixed in methanol, stained with Diff-Quick staining solution, and
counted under a microscope for quantification of VSMC migration.
Migration activity was expressed as the number of cells that had
migrated per high-power field (x400). In experiments to determine the
effects of troglitazone on VSMC migration, these agents were added to
the lower chamber before the incubation.
Growth Curves
For determination of cell numbers, VSMCs were placed in six-well
culture dishes at 2x104/mL and grown in DMEM containing
10% FCS, which was changed every 72 hours. After the medium was
aspirated, the same medium without FCS was applied for 48 hours.
Cultures were washed with a calcium- and magnesium-free PBS and
harvested with trypsin EDTA solution. Counts were performed with a
Coulter counter.12
Determination of DNA Synthesis
Relative rates of DNA synthesis were assessed by
determination of [3H]thymidine incorporation into
trichloroacetic acid–precipitable material.13 Quiescent
VSMCs grown in 24-well culture dishes were pulsed 4 hours with
[3H]thymidine (10 µCi/mL), washed with cold calcium-
and magnesium-free PBS, and incubated with 5% trichloroacetic acid at
4°C for 10 minutes. Cells were dissolved in 1N NaOH at 37°C for 30
minutes and then neutralized. Radioactivity was determined by liquid
scintillation counting.
Flow Cytometric Analysis of Cell Cycle Stage
Quiescent VSMCs grown in flasks were detached with 0.25%
trypsin at 37°C for 5 minutes and then pelleted by
centrifugation (1000 rpm for 5 minutes). The cells were
resuspended in 200 µL of solution A (trypsin, 30 mg/L; citric
acid, 3.4 mmol/L; spermine, 1.5 mmol/L;
Tris-HCl, 0.5 mmol/L; and Nonidet P-40, 2
mL/L). Ten minutes later, 150 µL of solution B (trypsin
inhibitor, 500 mg/L; RNase, 100 mg/L; citric
acid, 3.4 mmol/L; spermine, 1.5 mmol/L;
Tris-HCl, 0.5 mmol/L, and Nonidet P-40, 2 mL/L)
was added, and the mixture was left to stand for >10 minutes. All cell
cycle samples14 were analyzed within 3 hours using
a flow cytometer (Epics Profile). Red blood cells were used as an
internal standard for DNA analysis.
Metabolic and Biochemical Assays
VSMCs were incubated in normal or high glucose medium with or
without troglitazone (1 µmol/L) for 48 hours. Incubations
were terminated by rapidly adding 3N perchloric acid to the culture
medium with shaking. The tubes were then centrifuged, and the
supernatant was removed. The effect of elevated glucose levels on the
cytosolic lactate/pyruvate ratio is a more reliable indicator of the
cytosolic NADH/NAD+ ratio than are measurements of pyridine
nucleotides themselves in tissue extracts.15
Insulin Binding
VSMCs cultured in 24-well dishes were rinsed with assay buffer
(pH 7.8) (HEPES, 100 mmol/L; NaCl, 120 mmol/L;
KCl, 5 mmol/L; MgSO4, 1.2 mmol/L;
and glucose, 8 mmol/L; along with 0.1% bovine serum
albumin) and incubated for 30 minutes at 37°C in 250 µL
buffer containing 25 µL125 I-insulin (25 000 cpm) and 25
µL buffer (for total binding) or 25 µL unlabeled insulin or IGF-1
at specified concentrations. The assay was terminated by cooling plates
on ice. Aliquots of supernatants were counted for free radioactivity.
Medium was removed, and cells were then washed with three times with
fresh ice-cold assay buffer and solubilized in 0.2 mol/L acetic
acid (pH 2.5) containing 0.5 mmol/L NaCl for 6 minutes to
extract only surface-bound ligands.7 Radioactivity was
measured in a gamma counter. Nonspecific binding of
125I-insulin was defined as binding in the presence of
0.1 µmol/L unlabeled insulin. Binding data were
graphically analyzed, and dissociation constants were
determined from Scatchard plots.
Glucose Transport Analysis
For glucose transport studies, VSMCs were grown to confluence
and, on the day of the experiment, were incubated with PSS containing
145 mmol/L NaCl, 5 mmol/L KCl, 10
mmol/L HEPES, 1 mmol/L
Mg2SO4, 0.5 mmol/L
Na2HPO4, and 1.5 mmol/L
CaCl2. Cells were acclimatized in PSS for 1 hour, after
which the buffer was replaced with PSS containing vehicle (0.01
mmol/L HCl) or 100 µU/L porcine insulin. After 20 minutes of
this pretreatment, solutions were replaced with identical solutions
containing tracer amounts (700 pmol/L) of [3H]DOG.
Transport of [3H]DOG was allowed to proceed for 5
minutes. Wells were then aspirated and washed three times with ice-cold
PSS. Cells were solubilized with 0.5 mol/L NaOH and neutralized
with HCl, and the mixture was quantitatively transferred (using three
washes with PSS) to scintillation vials. All experiments were repeated
a minimum of four times using four replicates per group per experiment.
PLD Activity Measured by Ethanolamine Release
VSMCs in 35-mm dishes were cultured in medium containing
[3H]ethanolamine (5 µCi/mL per dish) for 24 hours to
label cellular phosphatidylethanolamine. After removal of the labeling
medium, the cells were washed twice with buffer A (20
mmol/L HEPES [pH 7.4], 120 mmol/L NaCl, 5
mmol/L glucose+16.6 mmol/L mannose, or 22.2
mmol/L glucose). After a 0.5- to 1-hour incubation with buffer
A, the reaction was terminated by removing buffer A and adding 0.75 mL
methanol. The cells were harvested by gentle scraping. The dishes were
then washed again with 0.75 mL methanol. Distilled water (0.6 mL) and
0.75 mL chloroform were added to the cells. The aqueous and lipid
phases were obtained from the cell extracts by adding 0.75 mL
chloroform and 0.75 mL water, followed by
centrifugation (3000g for 10 minutes).
Fractionation of ethanolamine metabolites from the aqueous phases was
performed on Dowex 50w H+-packed columns (1-mL bed volume,
Bio-Rad Econo columns) as previously described.16 The
initial flow-through (1 mL) along with the following 3 mL water
contained glycerophosphoethanolamine. Ethanolamine phosphate was eluted
with 15 mL water. Finally, ethanolamine was eluted with 8 mL of 1
mol/L HCl.
Cell Fractionation and Assay of PKC
VSMCs were washed twice with an ice-cold assay buffer (50
mmol/L Tris-HCl [pH 7.5] buffer containing 2
mmol/L EDTA, 2 mmol/L EGTA, 0.25 mol/L
sucrose, 10 mmol/L 2-mercaptoethanol, 0.21
mmol/L leupeptin, and 0.23 mmol/L
phenylmethylsulfonyl fluoride). Cells were then scraped and
were sonicated with three 10-second bursts. The homogenates
were centrifuged at 100 000g for 60 minutes at
4°C to separate the cytosolic and particulate fractions. The
cytosolic fraction was kept on ice for 1 hour and then
centrifuged at 100 000g for 30 minutes. PKC
activity was measured by a modified version of a method previously
reported17 using the Amersham PKC assay system. In brief, a
sample of the reaction mixture (50 mmol/L Tris-HCl [pH
7.5], 3 mmol/L calcium acetate, 100 µmol/L
-phosphatidyl-L-serine, 1 µmol/L PMA,
225 µmol/L substrate peptide, 7.5 mmol/L
dithiothreitol, and 0.05% [wt/vol] sodium azide) was mixed with
magnesium [32P]ATP and incubated at 25°C for 15
minutes. The substrate peptide interacts with 8% of PKC activity with
cAMP-dependent protein kinase, with 13% of PKC activity with
phosphorylase kinase, and with 38% of PKC activity with
proteolytic PKC fragment but does not interact with hexokinase or
myosin light chain kinase. An acidic reaction-quenching reagent was
added to stop the reaction. Phosphorylated peptide was
separated on binding paper. After the paper was washed, the extent of
phosphorylation was measured by scintillation counting.
Results of the PKC assay were linear for 15 minutes. PKC activity was
determined by subtracting the initial rate of protein kinase activity
(in the absence of activators) from the initial rate of
protein kinase activity in the presence of
phosphatidylserine, calcium acetate, and PMA.
Statistical Methods
Statistical analysis was performed by ANOVA and
Scheffé’s modified t test.18 Values of
P<.05 were considered significant.
Results
Inhibition of High Glucose–Induced Cell Migration by Troglitazone
PDGF-BB (1 ng/mL) stimulated the migration of VSMCs in a
time-dependent manner. VSMC migration induced by PDGF-BB was increased
during the initial 4 hours of incubation, after which VSMC migration
showed a significant decline.11 Therefore, subsequent
studies of VSMC migration were performed with cells incubated for 4
hours.
PDGF-BB stimulated the migration of VSMCs in a dose-dependent (0.1 to 1
ng/mL) manner (Fig 1A
). Therefore,
1 ng/mL PDGF-BB stimulation was used in subsequent studies.
PDGF-BB–stimulated migration of VSMCs was enhanced in high
glucose–treated cells, but this enhancement was completely suppressed
by coincubation with 1 µmol/L troglitazone (Fig 1A).
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The effect of troglitazone on the migration of VSMCs treated with
PDGF-BB (1 ng/mL) is shown in Fig 1B
. Troglitazone,
pioglitazone, and -tocopherol inhibited
glucose-potentiated PDGF-directed VSMC migration. This inhibition was
concentration dependent, and the suppression of migration was greater
for troglitazone-treated cells than for pioglitazone-treated cells.
- Tocopherol (1 µmol/L) completely
inhibited high glucose–potentiated PDGF-directed migration. Although
nonstimulated VSMCs exhibited low migration activity, troglitazone did
not inhibit their basal migration activity.
Inhibition of High Glucose–Induced Cell Proliferation by Troglitazone
As shown in Fig 2, cell
proliferation in high glucose medium was greater than that in normal
glucose medium. Troglitazone inhibited the high glucose–induced cell
proliferation of VSMCs. There was no difference in the numbers of
5.6 mmol/L glucose–treated cells and 5.6
mmol/L glucose+16.6 mol/L mannose–treated cells. In
these experiments, VSMCs on confluence were cultured in FCS-free medium
for 48 hours to induce quiescence. Troglitazone (100 nmol/L and
1 µmol/L) reduced the number of VSMCs cultured in high
glucose medium by 17% and 27%, respectively (Fig 2). Pioglitazone
(100 nmol/L and 1 µmol/L) also reduced the number
of VSMCs cultured in high glucose medium, although the reduction was
smaller than that for cells treated with troglitazone.
-Tocopherol (1 µmol/L) completely
suppressed the high glucose–induced increase in cell number. Cell
viability was checked by trypan blue staining, which confirmed that
>99% of the cells were alive.
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Fig 3 shows the effect of troglitazone on
[3H]thymidine incorporation by postconfluent VSMCs in
FCS-free normal or high glucose medium. Troglitazone (100 nmol/L
and 1 µmol/L) inhibited DNA synthesis by VSMCs by 45%
and 60%, respectively. Pioglitazone (100 µmol/L and
1 µmol/L) also reduced [3H]thymidine
incorporation by VSMCs cultured in high glucose, although the reduction
was smaller than that for cells treated with troglitazone.
-Tocopherol (1 µmol/L) completely
suppressed the high glucose–induced increase in
[3H]thymidine incorporation. Troglitazone did not cause a
loss of cells in the confluent state. Five days after the addition of
troglitazone, <1% of the cells were found to be present in the
supernatant medium. Cell viability was also checked by trypan blue
staining, which confirmed that >99% of the cells were alive.
|
Flow Cytometric Analysis
VSMCs cultured without FCS for 48 hours were in the
G0-G1 stage (100%). Chronic high glucose
treatment itself without growth factors did not change the stage of the
cell in the cell cycle (data not shown). Mean±SD data are shown in the
Table. PDGF-BB (1 ng/mL) treatment for 24 hours changed the
stage of the cell in the cell cycle from G0-G1
to S (18.0%) and G2-M (2.8%). High glucose treatment
changed the stage of the cell in the cell cycle from
G0-G1 to S (28.1%) and G2-M
(4.6%). Troglitazone (1 µmol/L) eliminated these high
glucose–induced changes.
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Effect of Troglitazone on Glucose-Induced Increase in VSMC Cytosolic NADH/NAD+ Ratio
For more reliable assessment of the VSMC cytosolic
NADH/NAD+ ratio, lactate/pyruvate ratios were measured in
VSMCs plus medium. As shown in Fig 4, the
ratio was 91.7% higher with high glucose than with normal glucose
medium after 24 hours of incubation. Troglitazone (1
µmol/L) significantly reduced the extent of this increase.
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Insulin Binding
The specific binding of 125I-insulin
was 0.8±0.1% of total 125I-insulin added. The
concentration needed to obtain half-maximal displacement
(IC50) was estimated to be 160 pmol/L. IGF-1 was
>10 000 times less potent than insulin in displacing
125I-insulin (Fig 5).
Scatchard plots obtained using a model that assumes two independent
binding sites revealed a high-affinity insulin receptor with a
dissociation constant (Kd) of
9.5±1.5x10-9 mol/L and a binding capacity
(Bmax) of 8.9±0.7 x103 sites/cell and a
low-affinity receptor with a Kd of
4.3x10-8 mol/L. Incubation of cells with 22.2
mmol/L glucose for 48 hours before binding changed neither the
Kd (8.1±1.6x10-9 mol/L)
nor the Bmax (9.7±0.6x103 sites/cell) for
specific binding at 37°C for 30 minutes.
|
) and untreated
(
) cells. Values are means of four measurements. SD was 5.0% to
16.0% of the mean value.
Insulin-Stimulated [3H]DOG Uptake
Uptake of [3H]DOG into cells was linear between 0
and 15 minutes of incubation regardless of glucose concentration.
Insulin-stimulated [3H]DOG uptake was observed at a
concentration of 1 pmol/L and was increased at a concentration
of 1 nmol/L (Fig 6A). After 5 days
of incubation with 22.2 mmol/L glucose, insulin-stimulated
[3H]DOG uptake was significantly decreased; this decrease
was prevented by coincubation with 1 µmol/L calphostin C,
a PKC inhibitor, and by coincubation with 100 nmol/L
troglitazone (Fig 6B). Glucose treatment did not change basal
[3H]DOG transport activity. Mannose had no effect on
basal and insulin-stimulated [3H]DOG uptake.
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Effect of High Glucose on Insulin-Stimulated VSMC Proliferation
Cell number and [3H]thymidine incorporation were
measured to study the effect of high glucose on insulin-mediated VSMC
growth.
High glucose (22.2 mmol/L) treatment for 5 days enhanced
insulin-stimulated increase in cell number and
[3H]thymidine incorporation. These changes were
completely eliminated by coincubation with troglitazone (1
µmol/L) (Fig 7).
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PLD Activities With Normal and High Glucose Media
In order to evaluate one possible mechanism of suppression of high
glucose–induced VSMC migration and proliferation, PLD activities in
VSMCs in normal glucose and high glucose media were measured. As shown
in Fig 8, PLD activities in high
glucose–treated cells were greater than those in normal
glucose–treated cells. VSMCs were coincubated with troglitazone
(1 µmol/L) in either normal or high glucose medium for 5
days before measurement of PLD activity. When high glucose medium was
coincubated with troglitazone (1 µmol/L), high
glucose–induced PLD activation was significantly reduced.
-Tocopherol (1 µmol/L) completely
eliminated glucose-induced PLD activation.
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PKC Activities in Normal Glucose and High Glucose Media
To evaluate one possible mechanism of suppression of high
glucose–induced VSMC proliferation, PKC activities in VSMCs in normal
and high glucose media were measured. As shown in Fig 9, membrane-bound (particulate) PKC
activities in high glucose–treated cells were greater than those in
normal glucose–treated cells, with a corresponding decrease in
cytosolic PKC activities. VSMCs were coincubated with troglitazone
(1 µmol/L) in either normal or high glucose medium for 5
days before the determination of PKC activity. With normal glucose
medium, troglitazone did not reduce basal PKC activity. When high
glucose medium was coincubated with troglitazone (1
µmol/L), glucose-induced PKC activation was significantly
reduced. -Tocopherol (1 µmol/L) completely
eliminated high glucose–induced PKC activation (Fig 9).
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Discussion
In the present study, we showed for the first time that troglitazone reduces high glucose–induced migration and hyperproliferation of coronary VSMCs in a concentration-dependent manner. This preventive effect was observed for concentrations of 100 nmol/L to 1 µmol/L troglitazone, which are less than those obtained with oral administration.19 This suggests that troglitazone might prevent VSMC migration and proliferation in vivo. In fact, Law et al20 have reported that troglitazone suppresses intimal hyperplasia in a balloon injury model of rat aorta.
We have also demonstrated for the first time that high glucose medium
increases migration of coronary VSMCs and that troglitazone
dose-dependently inhibits this increase in migration. The precise
mechanisms of this increase in migration by high glucose and inhibition
in migration by troglitazone remain to be determined. However, it has
been reported that high glucose medium increases the production
of active oxygen species21 and that the suppression of
increased active oxygen species by catalase and by the antioxidant
N-acetylcysteine results in suppression of migration of
VSMCs.22 These findings suggest that oxidative stress by
high glucose may cause increased migration of VSMCs and that the
antioxidants troglitazone and -tocopherol may prevent
this increase. We have also shown in this study that PLD activity was
increased in high glucose–treated cells and that this increase was
prevented by troglitazone and -tocopherol. Since PLD
increases the formation of diacylglycerol,23 which may play
a role in migration,24 suppression of PLD by troglitazone
or -tocopherol may suppress the increase in VSMC
migration induced by high glucose.
There are at least three possible mechanisms of the inhibition of high
glucose–induced hyperproliferation of coronary VSMCs by
troglitazone. The first is the blockade of oxidative modification of
LDLs, as already reported.4 The second is suppression of
the sorbitol pathway; we have already reported the importance of this
pathway in high glucose–induced hyperproliferation of
VSMCs.14 In the present study, we showed that the
increase in the NADH/NAD+ ratio, which is an indicator of
sorbitol pathway activity,4 induced by high glucose medium
is suppressed by troglitazone, suggesting that troglitazone may
suppress the activity of this pathway. In fact, the antioxidant vitamin
C is reported to suppress the activity in the sorbitol pathway induced
by high glucose.25 The third possible mechanism is
suppression of the oxidative stress induced by high glucose. It has
been reported that production of active oxygen species can
occur as a result of glyco-oxidation (nonenzymatic
glycation)21 and increased PKC activity induced by
incubation in high glucose medium.26 We also showed in the
present study that -tocopherol suppressed high
glucose–induced migration and proliferation of VSMCs. We have also
observed that increased concentrations of the scavenging enzyme
catalase suppressed high glucose–induced VSMC proliferation (data not
shown). These findings suggest that oxidative stress may play a role in
the high glucose–induced hyperproliferation of VSMCs.
We also showed in the present study that troglitazone and
-tocopherol suppress the high glucose–induced increase
in membrane-bound PKC activity. This finding is explained in part by
the suppression of increase in the NADH/NAD+ ratio induced
by high glucose.14 Since activated PKC produces
active oxygen species27 and active oxygen species stimulate
vascular smooth muscle cell growth,6 it is reasonable to
assume that this suppression of increase in PKC activity by
troglitazone and -tocopherol plays a role in the
suppression of high glucose–mediated VSMC hyperproliferation. We and
others have in fact demonstrated that PKC inhibitors
prevent high glucose–induced hyperproliferation in
vitro16,28 and vascular dysfunction in vivo29
through suppression of PKC. Since PLD is reported to activate
PKC through the formation of diacylglycerol in VSMCs,30
troglitazone may inhibit PLD, resulting in the suppression of PKC
activity.
Cell cycle analysis was also performed to determine the mechanism of high glucose–induced hyperplasia. Glucose (22.2 mmol/L) alone without growth factors did not change the stage of cells in the cell cycle (data not shown). Glucose (22.2 mmol/L) with PDGF-BB (1 ng/mL) significantly increased the percentage of VSMCs in the S and G2-M stages, suggesting that glucose is not a competence growth factor but is a progression growth factor. Troglitazone (100 nmol/L) inhibited this increase. Since troglitazone decreases membrane-bound PKC activity and activation of PKC is required for cell cycle progression and S-phase entry of VSMCs,16 the high glucose–induced change in stage in the cell cycle may be due to PKC activation, and this change may be blocked by troglitazone.
Troglitazone, a thiazolidinedione compound, is presently being
tested as a new oral antidiabetic agent. There is evidence from animal
studies and clinical trials with non–insulin-dependent diabetes
mellitus patients that troglitazone may reduce insulin
resistance,31,32 which is consistent with the
findings of the present study. The molecular mechanism of reducing
insulin resistance is not understood. The possible involvement of PPAR
in insulin responsiveness of adipocytes has been
reported.33 Although little is known concerning PPAR
function in VSMCs, the finding that pioglitazone suppresses
mitogen-induced34 and high glucose–induced VSMC migration
and growth suggests that this receptor may play a role in the mechanism
of action of troglitazone and that the effect of troglitazone is not
solely as an antioxidant. Troglitazone may possess a more potent hybrid
activity as an antioxidizing thiazolidinedione. It has also been
reported that troglitazone interferes with hyperglycemia-induced
PKC-mediated insulin receptor kinase.35 Therefore,
suppression of PKC by troglitazone, possibly as a result of its
antioxidant effect, may play a role in reducing insulin resistance.
Diabetes is associated not only with high glucose levels but also, at
least in patients with non–insulin-dependent diabetes mellitus, with
increased plasma insulin levels.36 Therefore,
characterization of the interaction between high glucose and insulin
would have important clinical implications. Since
hyperglycemia1,37 and insulin2,38 have been
implicated in the genesis of macroangiopathy and coronary
artery disease in patients with diabetes, we also investigated the
effect of high glucose on insulin-mediated [3H]DOG
uptake. Chronic glucose treatment decreased insulin-mediated
[3H]DOG uptake (possibly via insulin resistance) without
any change in insulin receptor number or affinity (Fig 6B),
consistent with the findings of a previous study.39
This result indicates that chronic exposure of VSMCs to glucose induces
an increase in insulin resistance in the glucose transport effector
system. Glucose may impair the efficacy of recruitment of intracellular
glucose carriers to the cell surface by insulin. This is the first
demonstration that troglitazone improves impaired insulin response in
vascular tissue. This impairment of response may be due to increased
PKC activity, since activation of PKC increases insulin
resistance40 and PKC inhibitor prevented this
impairment. Since troglitazone, which was synthesized with an
-tocopherol substitution in an effort to obtain an
antioxidative effect, was shown to prevent the glucose-induced
inhibition of insulin receptor response in cultured
fibroblasts,11 it may be that troglitazone restores high
glucose–induced insulin resistance, possibly as a result of its
antioxidative effect in VSMCs. Normalization of PKC activity or
translocation by troglitazone may play a role in this effect. In fact,
prevention by PKC inhibitors of glucose-induced insulin
resistance in rat fat cells has been reported.40 PKC may
thus be a link between insulin resistance and increased migration and
proliferation of coronary VSMCs induced by high glucose
medium.41 In addition, impaired insulin-mediated glucose
uptake is an indicator of increased PKC activity.
Impairment of insulin-mediated glucose uptake and increase in growth of
VSMCs by high glucose medium may provide evidence for a new hypothesis
concerning the mechanism of insulin resistance at the cellular level.
It has been reported that VSMCs possess receptors for insulin and
IGF-1.42,43 The IGF-1 receptors form a common pathway for
the growth effects of both insulin and IGF-1 in VSMCs.44
The effects on glucose uptake39 and growth44 of
IGF-1 were 10 to 100 times more potent than those of insulin. These
findings suggest that PKC activation in VSMCs may impair glucose uptake
and increase growth of VSMCs through effects on IGF-1 receptors.
However, as shown in the present and another study,45
overlap in signaling due to IGF-1 receptor interaction may be minimal,
since IGF-1 did not displace labeled insulin in VSMCs (Fig 7A).
Therefore, impairment of glucose uptake and increase in growth of VSMCs
through effects on insulin receptors resulting from PKC activation can
be also postulated to occur at least at
physiological concentrations of <100 µU/mL (0.7
nmol/L),46 because insulin-mediated
[3H]DOG uptake increased at concentrations above 1
nmol/L, suggesting the involvement of IGF-1 receptors at
concentrations above this level.
It has recently been reported that troglitazone inhibited PDGF-directed migration and basic fibroblast growth factor–induced proliferation of VSMCs from rat aorta via inhibition of mitogen-activated protein kinase signaling.20 The present study further demonstrated that troglitazone improved insulin resistance and inhibited high glucose–potentiated PDGF-directed migration and high glucose–induced proliferation of rabbit coronary VSMCs via inhibition of PLD and PKC activation, possibly as a result of an antioxidant effect. This finding may be clinically useful, since troglitazone is mainly used in diabetic patients.
In conclusion, our findings suggest that oxidative stress may play a role in the vascular migration and hyperproliferation induced by high glucose stimulation. Our findings suggest that troglitazone might be useful in the treatment of high glucose–induced coronary atherosclerosis. The intracellular mechanism of action of troglitazone may include, in part, the suppression of PLD activation, PKC activation, and an increased percentage of cells in the S phase induced by high glucose, and this suppression may be the result of intracellular antioxidative effects of troglitazone. PKC may be a link between impairment of insulin-mediated glucose uptake and accelerated migration and proliferation induced by high glucose.
Selected Abbreviations and Acronyms
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Acknowledgments
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, by grants from the Osaka City University Medical Research Foundation, Uehara Memorial Foundation, and Kimura Memorial Foundation, and by a Japan Heart Foundation Grant for Research on Hypertension and Vascular Metabolism. We would like to thank Atsumi Ohnishi for excellent technical assistance.
Received December 30, 1996; accepted August 29, 1997.
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