Third Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan
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ABSTRACT |
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To examine the
effects of chronic hyperinsulinemia on vascular tissues, we examined
the production of superoxide anion
(O2) in the aortic tissues of control
and exogenously hyperinsulinemic rats performed by the implantation of
an insulin pellet for 4 wk. O
2
production by aortic segments from hyperinsulinemic rats was 2.4-fold
(lucigenin chemiluminescence method) and 1.7-fold (cytochrome
c method) of that of control rats
without any differences in O
2
degrading activities in aortic tissues, respectively
(P < 0.025). The increment was
completely abolished in the presence of either 100 µmol/l apocynin
(an inhibitor of NADPH oxidase) or 10 µmol/l diphenyleneiodonium (an
inhibitor of flavin-containing enzyme) and was exclusively endothelium
dependent. Consistently, NAD(P)H oxidase activities in endothelial
homogenate in hyperinsulinemic rats were dose dependently stimulated
above the values of control rats, although these activities in
nonendothelial homogenate were not significantly stimulated by insulin.
Furthermore, an insulin effect was also demonstrated 1 h after exposing
aortic tissues to insulin. These results indicate that
O
2 production specifically increases
in endothelium of aortic tissues in chronic hyperinsulinemic rats
through the activation of NAD(P)H oxidase.
oxidative stress; free radicals; endothelial cells
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INTRODUCTION |
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PREVIOUS EPIDEMIOLOGICAL studies indicate a close relationship between plasma insulin levels and the incidence of cardiovascular disease, supporting the idea that hyperinsulinemia is involved as an atherogenic factor (25, 32). Stout (30) and other researchers (20) have raised the possibility that chronic hyperinsulinemia may contribute to the development of atherosclerosis via the direct effect of insulin on arteries. The following studies also show that insulin increases both the synthesis and release of vasoactive substances related to atherogenesis (6, 19, 23).
In contrast, based on the recent epidemiological studies, the role of
hyperinsulinemia itself as an atherogenic factor has been questioned
(9) because hyperinsulinemia as a result of insulin resistance in
patients is strongly linked to the coexistence of multiple
cardiovascular risk factors, including essential hypertension, obesity,
impaired glucose tolerance, and dyslipidemia (27). In terms of the
biological effects of insulin on vascular cells, insulin has been shown
to have an acute vasodilatory effect through endothelial nitric oxide
(NO) production, and therefore insulin produces an anti-atherogenic
effect (1). On the other hand, insulin stimulates NADPH-dependent
H2O2
generation in human adipocyte plasma membrane (10). Although there is
no study concerning insulin effect on superoxide anion
(O2)-producing in vascular tissues, it
also has been reported that vasoactive peptides can release
O
2 through various
O
2-producing systems such as NAD(P)H
oxidase (7, 26, 31) and lipoxygenase (21), resulting in an atherogenic
insult on vascular tissues. Thus increased
O
2 production is one of possible mechanisms involved in impairing vascular dilatation through scavenging NO (22), as well as the impaired synthesis release of NO (24, 29).
Therefore, it is worthwhile to evaluate the significance of
hyperinsulinemia itself on O
2
production in vascular tissues in experimental animals. To study the
direct effect of high-insulin concentrations on vascular cell function, we created a hyperinsulinemic condition in rats that were not insulin
resistant by the subcutaneous infusion of insulin.
Thus the purpose of this study was to determine whether
O2 production increased in aortic
segments of exogenously hyperinsulinemic rats without insulin
resistance. Furthermore, we tried to characterize the specific vascular
cell type that released O
2 and further
attempted to identify the activated oxidase species in aortic
homogenates in the presence or absence of endothelium of both control
and hyperinsulinemic rats that were induced by the subcutaneous
implantation of an insulin pellet.
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MATERIALS AND METHODS |
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Materials. Insulin pellets were obtained from Linshin Canada. Concanavalin A-sepharose was obtained from Amersham Pharmacia Biotech. All other materials were purchased from Sigma Chemical (St. Louis, MO).
Animals. Six-week-old male Sprague-Dawley rats (Japan SLC, Shizuoka, Japan) weighing 150 g were housed in an environmentally controlled room with 12:12-h light-dark cycle and free access to laboratory chow and water. The animals were divided into two groups; 1) control and 2) hyperinsulinemic rats induced by implantation of an insulin pellet (insulin treated). Those animals were fed on a diet (Oriental Yeast, Tokyo, Japan) consisting of 58% carbohydrate (no fructose), 12% fat, and 30% protein (calorie %). For the continuous delivery of insulin, an incision was made in the midscapular region under pentobarbital anesthesia (50 mg/kg body wt) and either one-half or one piece of insulin pellet was implanted (release rate of 0.5 U/day and 1.0 U/day, respectively) into the back of the rats for 2 and 4 wk, respectively. Blood pressure was measured the day before the experiment, and the rats had been trained with the apparatus three times before measurement. Both systolic blood pressure and diastolic blood pressure in the tail region were measured using an electrosphygmomanometer after the rat was prewarmed for 15 min (2).
Assessment of in vivo insulin action.
Insulin sensitivity was measured by the steady-state plasma glucose
method using somatostatin, originally described by Harano et al. (8).
After an overnight fast, rats were anesthetized with an intraperitoneal
injection of pentobarbital (50 mg/kg body wt) and the right jugular
vein was exposed and cannulated with a polyethylene tube for the
administration of the infusate. Rats were administered with the
infusate containing somatostatin (120 mg · kg1 · h
1),
glucose (1.0 g · kg
1 · h
1),
and insulin (2.0 U · kg
1 · h
1,
human actrapid, NovoNordisk, Denmark) at a flow rate of 2.8 ml/h for
120 min. Blood samples for determining plasma glucose levels were
obtained from the tail vein at 0, 30, 60, 90, and 120 min after the
initiation of the infusion. The steady-state plasma glucose and insulin
levels were measured at 120 min after the initiation of infusion.
Preparation of aortic tissues. After 2- or 4-wk insulin infusion, control and insulin-treated rats were killed under pentobarbital anesthesia. The thoracic aortas (0.6-0.8 cm outside diameter) were isolated and carefully cut into strips to preserve the endothelium. In some strips, the endothelium was removed by gentle rubbing of the intimal surface with a cotton ball. Blood samples for measurements of plasma glucose, lipid, and insulin concentrations were obtained from the tail vein. Blood glucose concentration was determined by glucose oxidase method, and serum total cholesterol, triglyceride, and free fatty acid concentrations were measured by standard enzymatic methods. Plasma insulin concentration was measured by radioimmunoassay using the rat insulin RIA kit (DiaSorin, Stillwater, MN).
Measurements of ex vivo aortic
O2 production.
O
2 production in aortic segments was
measured using lucigenin-enhanced chemiluminescence method as described previously (22). This methodology has been proven to be quite specific
for O
2 detection (18) and useful in
studies of vascular O
2 production.
Segments of thoracic aortas (20 mm) were isolated, placed in a modified Krebs-HEPES buffer (pH 7.4) containing (in mmol/l) 99.0 NaCl, 4.69 KCl,
1.87 CaCl2, 1.20 MgSO4, 1.03 K2HPO4,
25 NaHCO3, 20 Na-HEPES, and 11.1 glucose, and then allowed to equilibrate for 30 min at 37°C. After
5 min of dark adaptation, scintillation vials containing 2 ml
Krebs-HEPES buffer with 50 µmol/l lucigenin were placed into a
scintillation counter (Tri-Carb 1500; Packard Instrument, Meriden, CT)
switched to the out-of-coincidence mode. Chemiluminescence values were
obtained at 30-s intervals over 15 min, and readings in each of the
last 10 min were averaged. The vessel was dried at 90°C for 24 h,
and then the dry weight was measured. Lucigenin count was expressed as
counts per minute per milligram of the dry weight of vessel. Background
counts were determined by vessel-free incubations and subtracted from
vessel readings.
Estimation of enzyme sources for
O2 production.
Aortic segments (10-15 mm) were placed in chilled Krebs-HEPES
buffer. Periadventitial tissues were carefully removed, and the
endothelium in some aortic rings was removed by gentle rubbing of the
internal surface. To study acute insulin effects on
O
2 production through activation of
NAD(P)H oxidase, some aortic rings obtained from control rats were
incubated for 1 h in the absence or presence of 10 nmol/l insulin in
the incubation medium. Those aortic vessels were repeatedly washed and
then cut into small pieces. The vessel homogenate was prepared by
homogenizing in a glass homogenizer after the addition of a 400-µl
homogenizing buffer (pH 7.8; consisting of 50 mmol/l phosphate buffer
and 0.01 mmol/l EDTA). The homogenate was subjected to low-speed
centrifugation (1,000 g) for 10 min
to remove unbroken cells and debris. A 20-µl aliquot of the
supernatant was then added to glass scintillation vials containing
lucigenin (50 mmol/l) in a 2-ml homogenizing buffer. The
chemiluminescence value was recorded at 30-s intervals over 10 min, and
readings in each of the last 5 min were averaged. The background was
determined by measurement in the absence of homogenate and then
subtracted from the readings obtained in the presence of vessel homogenate.
Vascular O2 scavenging
activity.
Vascular O
2-scavenging activity was
spectrophotometrically measured by modification of the method
originally described by Salin and McCord (28). Segments of the thoracic aortas were harvested as previously described and homogenized in PBS (2 ml) with a homogenizer, the homogenates were centrifuged at 13,600 g for 15 min, and the supernatant was
kept on ice until analysis. The aliquot (0.1 ml) was incubated with 100 µmol/l xanthine, 15 µmol/l cytochrome
c (horse heart type IV), 20 mmol/l
NaHCO3, 1 mmol/l
NaN3 , and 0.1 mmol/l EDTA. The
assay was initiated by the addition of 0.025 ml xanthine oxidase (1.0 U/ml), and the resultant superoxide generation was measured by the
increment of absorbance at 550 nm over the 20-s reaction time. The
enzyme activity was calculated from a linear dose-response curve
obtained using 0.1-10 U/ml bovine erythrocyte Cu,Zn-SOD and
expressed as units of SOD normalized based on its protein content. The
residual Cu,Zn-SOD-independent superoxide-scavenging activity was
determined from identically prepared vessels that were incubated with
10 mmol/l diethyldithiocarbamate (an inhibitor of Cu,Zn-SOD) for 30 min.
Statistical analysis. All values were presented as means ± SE. Differences between the two groups were evaluated by Student's t-test, and comparison among three groups were performed using ANOVA with a post hoc Scheffé's comparison. P < 0.05 was considered significant.
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RESULTS |
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Metabolic characteristics and blood pressure of the rats.
As shown in Table 1, control and
insulin-treated (0.5 and 1.0 U/day) rats gained weight to a similar
degree over the study period. Animals receiving insulin infusion (0.5 and 1.0 U/day) for 4 wk demonstrated significant decreases in plasma
glucose levels and significant increases in plasma insulin levels
compared with control rats. However, there were no differences in blood pressure and serum total cholesterol, triglyceride, and free fatty acid
levels between control and insulin-treated rats. Furthermore, chronic
exogenous hyperinsulinemia induced by subcutaneous infusion of insulin
did not impair the insulin effect on in vivo glucose utilization
because the steady-state plasma glucose concentration in
insulin-treated (1.0 U/day for 4 wk) rats measured by the glucose, somatostatin, and insulin infusion method was similar to that of the
control rats (Table 1).
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O2 generation from aortas
with or without endothelium.
We measured the basal O
2 generation
from aortas with or without endothelium in ex vivo conditions using the
lucigenin chemiluminescence method. As shown in Table
2, O
2
production by aortic segments from insulin-treated (1.0 U/day) rats was
2.4-fold that in control rats (P < 0.0001). Endothelial removal produced about a 40% reduction
(P < 0.01) of
O
2 production in vessels from control
rats, whereas a 73% reduction of the
O
2 production was found in vessels
without endothelium in insulin-treated rats. Thus, after removal of the
endothelium, a difference in the O
2 production rate between the two groups was no longer apparent. Furthermore, incubation of intact aortic segments from the two groups
with either 100 µmol/l apocynin (an inhibitor of NADPH oxidase) or 10 µmol/l diphenyleneiodonium [an inhibitor of NAD(P)H oxidase] for 30 min abolished the increment of
O
2 production by insulin treatment. In
contrast, neither oxypurinol (a xanthine oxidase inhibitor), rotenone
(an inhibitor of mitochondrial respiration), indomethacin (an inhibitor
of cyclooxygenase), NDGA (an inhibitor of lipoxygenase), nor
L-NNA (an inhibitor of NO synthase) affected the lucigenin signal in intact aortic segments (Table 2). These results indicate that basal
O
2 production in aortic endothelium
increased in insulin-treated rats at least through the activation of
NAD(P)H oxidase.
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Enzyme source for production of
O2 in vascular homogenates.
O
2 production in response to the
addition of a variety of substrates was examined in vascular
homogenates. As shown in
Fig.2A,
O
2 production derived from either
NADPH or NADH oxidases was greater than that of the other potential
substrates: arachidonic acid, succinate, or xanthine, respectively. Furthermore, the difference in the
O
2 production in the presence or
absence of the endothelium was not significant in control rats,
although activities of both NADPH and NADH oxidases in the presence of
endothelium tended to be greater than in the absence of endothelium.
These results indicate that both activities in vessel homogenates of
control rats were mainly derived from nonendothelial cells. However, in
insulin-treated (1.0 U/day for 4 wk) rat vessels,
O
2 production was from not only NADH
oxidase, but also NADPH oxidase, in the presence of endothelium was
significantly greater than that in the absence of endothelium,
respectively (Fig. 2C). Similarly, chronic insulin treatment for 2 wk also significantly
(P < 0.025) stimulated NADH oxidase
activity (+insulin 10,669 ± 1,837 vs. control 4,035 ± 840 counts · min
1 · mg
protein
1,
n = 4 for each group) and NADPH
oxidases activity (+insulin 5,642 ± 692 vs. control 3,615 ± 723 counts · min
1 · mg
protein
1,
n = 4 for each group) by 2.6-fold and
1.6-fold compared with control rat aortas, respectively. Furthermore,
O
2 generation derived from NADPH and
NADH oxidases in the absence of endothelium was not significantly
different between control and 4-wk insulin-treated rats, respectively
(Fig. 2, A and
C). These results indicate that
chronic insulin infusion for 4 wk results in enhanced NADPH and NADH
oxidase activities only in the presence of endothelium. As shown in
Fig. 3, consistent with increased
O
2 generation in chronic
hyperinsulinemic rat aortas in the ex vivo condition, NAD(P)H oxidase
activities in aortic homogenates were also increased, depending on
exogenous insulin doses, and those activities in endothelium of
insulin-treated (1.0 U/day) rats were about fourfold greater than that
of control rats, respectively. Consistently, the addition of NAD(P)H
oxidase inhibitors (either 100 µmol/l apocynin or 100 µmol/l
diphenyleneiodonium) significantly reduced lucigenin chemiluminescence
in the presence of either 500 µmol/l NADH or 500 µmol/l NADPH in
both control and insulin-treated (1.0 U/day) rats (data not shown). In
contrast, O
2 generation from the
vessel homogenates was not significantly affected in the presence of
either arachidonic acid, succinic acid, or xanthine (Fig. 2). These
results indicate that an increment in
O
2 production was induced by insulin
only through the activation of either NADH or NADPH oxidases.
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Vascular scavenging activity of
O2.
The net vascular production of O
2 is
modified by local O
2-scavenging
activity. Arterial tissue contains considerable amounts of SOD that
effectively converts superoxide to
H2O2.
In the present study, the activity of Cu,Zn-SOD, a major source of
superoxide catabolism in arterial tissue, was not different between the
two groups [control 7.69 ± 0.43 and insulin treated (1.0 U/day) 8.00 ± 0.18 IU/mg protein,
n = 4 for each group]. After
inhibiting Cu,Zn-SOD activity with 10 mmol/l diethyldithiocarbamate,
the residual water-soluble superoxide scavenging activity represented
less than 25% of total activity and this also showed no difference
between control and insulin-treated (1.0 U/day) rats (control 1.84 ± 0.46 and insulin treated 1.80 ± 0.13 IU SOD/mg protein,
n = 4 for each group).
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DISCUSSION |
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In the present study, despite the fact that several types of
O2-scavenging activities were similar
between control and insulin-treated (1.0 U/day) rats, we found
endothelial specific increments of O
2
production through NAD(P)H oxidases in aortic segments of the
insulin-treated rats in both ex vivo and in vitro homogenate
conditions. For the measurement of O
2
production through activation of NAD(P)H oxidase in ex vivo aortic
segments and homogenates, we used the lucigenin-enhanced
chemiluminescence method. Although one previous report discussed a
methodological problem in measuring O
2 using this method (14), several recent reports have confirmed its
usefulness (18, 22). Furthermore, O
2 generation released from aortic segments in ex vivo conditions is
significantly inhibited in the presence of either NADPH or NADH oxidase
inhibitors. To confirm the increased
O
2 production in vessels of
insulin-treated rats, we also measured O
2 using the standard cytochrome
c method (12) and obtained similar
results as found by using the lucigenin chemiluminescence method.
NAD(P)H oxidase activities in vascular homogenate were measured in the
presence of either NADPH or NADH in the incubation medium. To evaluate
those results, it should be noted that cyclooxygenase-prostaglandin hydroperoxidase (PGH synthase) and lipoxygenase can oxidize NAD(P)H and
result in the generation of O2 (11).
However, a significant increase in O
2
production could not be detected when the homogenate was exposed to
arachidonic acid (Fig. 2), and neither 10 µmol/l indomethacin nor 10 µmol/l NDGA (an inhibitor of lipoxygenase) affected
O
2 production in the presence of
either NADPH or NADH (data not shown). We noted that neither
indomethacin nor NDGA affected vascular O
2 production in aortic segments of
insulin-treated rats (Table 2). In addition, 1 µmol/l
L-NNA (an inhibitor of endothelial NO synthase) did not affect
O
2 production in both aortic segments
and vascular homogenate in insulin-treated rats. These results indicate
that O
2 production through either
arachidonic acid metabolism or endothelial NO synthase activity in ex
vivo conditions and in vascular homogenate was not stimulated in
insulin-treated hyperinsulinemic rats. Furthermore, the
addition of either apocynin (an inhibitor of activity assembly of the
components of NADPH oxidase) or diphenyleneiodonium (an inhibitor of
flavin-containing enzymes) significantly decreased the lucigenin
chemiluminescence in the presence of either NADH or NADPH. These
results also confirmed increased activities of NAD(P)H oxidase in
insulin-treated rats. The significance of the membrane-associated NADH
oxidoreductase as the main source of O
2 has been reported in smooth muscle
cells of calf pulmonary artery by Mohazzab and Wolin (18). Thus in the
present study we first demonstrated exogenous hyperinsulinemia without
insulin resistance specifically activated endothelial NAD(P)H oxidase
and then stimulated the generation of
O
2 in aortas of hyperinsulinemic rats
in ex vivo conditions. Interestingly, nonendothelial NAD(P)H oxidase
activities in aortic homogenate prepared in the absence of endothelium
were not significantly stimulated in insulin-treated rats. These
results suggest that insulin differentially regulates vascular NAD(P)H
oxidase between the endothelium and smooth muscle cells. In the present
study, we were not able to further explain the reason for the tissue specificity of this insulin action.
NADPH oxidase in granulocytes is a multicomponent plasma membrane-bound
enzyme that produces O2 in response to
stimuli from microbial infections (4). This enzyme consists of
transmembrane flavocytochrome b558 (two subunits,
gp91phox and
p22phox), and regulatory
cytosolic proteins, p47phox,
p67phox, and small GTPase Rac1 or
Rac2, and p40phox (5, 13).
Although molecular cloning of this multiple component enzyme has not
yet been possible in nongranulocytes, the biological significance of
those enzymes in the regulation of vascular function has been
recognized (7, 26, 31). Recently, it has been reported that apocynin, a
NADPH oxidase inhibitor is effective in tumor necrosis factor-induced
vascular cell adhesion molecule 1 mRNA expression in human umbilical
vein endothelial cells (31). Furthermore,
p22phox is a critical component of
the O
2-generating NAD(P)H oxidase
system and contributes to angiotensin II-induced hypertrophy of
vascular smooth muscle cells (7). However, this NAD(P)H oxidase in
smooth muscle cells preferentially uses NADH as opposed to NADPH as a
substrate (18, 31). Molecular mechanisms of this difference need to be
studied further. In endothelium, the activation of NAD(P)H oxidase is
more likely to be associated with the function of quenching NO or
contributing to a direct intracellular signaling of hormone action that
is found in the effects of angiotensin II on smooth muscle cells (26).
Concerning the activating mechanism for
O
2 generation in aortic vessels
exposed to insulin, we found acute as well as chronic insulin effects
on these enzyme activities. However, in contrast to clearly described
mechanisms for the activation of NADPH oxidase in granulocytes (4, 13),
molecular mechanisms for both acute and chronic activation of NAD(P)H
oxidases by insulin in tissues other than granulocytes have not yet
been well documented.
We found that the aortas obtained from insulin-treated hyperinsulinemic
rats enhanced the production of O2 through the activation of endothelial NAD(P)H oxidase. It is also well
known that insulin stimulates NO production in endothelial cells. This
insulin-stimulated production in both endothelial O
2 and NO may interact and produce
peroxynitrite in vascular tissues. Peroxynitrite at high concentration
is cytotoxic and injures various tissues (2). On the other hand, it
mediates a number of physiological processes that are beneficial in the protection of cellular function such as coronary arterial relaxation and inhibition of platelet aggregation and leukocyte adhesion (15).
Thus peroxynitrite may also be a candidate for modulating vascular
function in in vivo conditions.
In conclusion, we first showed that physiological hyperinsulinemia
could overproduce O2 exclusively in
endothelial cells. The pathophysiological implication for this
endothelial cell-specific activation of NADH-NADPH oxidase by insulin
is now being further studied in our laboratory.
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ACKNOWLEDGEMENTS |
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This study was supported in part by a research grant-in-aid from the Japan Society for the Promotion of Science (JSPS) Fellows and the Ministry of Education, Science, and Culture of Japan.
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FOOTNOTES |
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This study was presented in part at the 57th Annual Meeting and Scientific Sessions of the American Diabetes Association, Boston, 1997.
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: A. Kashiwagi, Third Dept. of Medicine, Shiga Univ. of Medical Science, Tsukinowa-cho, Seta, Otsu, Shiga 520-2192, Japan (E-mail: kasiwagi{at}belle.shiga-med.ac.jp).
Received 1 February 1999; accepted in final form 12 July 1999.
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