Departments of 1 Medicine and 2 Molecular Physiology and Biophysics, University of Vermont College of Medicine, Burlington, Vermont 05446
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ABSTRACT |
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Obesity is associated with hyperinsulinemia and elevated
concentrations of tumor necrosis factor- (TNF-
) in
adipose tissue. TNF-
has been implicated as an inducer of the
synthesis of plasminogen activator inhibitor-1 (PAI-1), the primary
physiological inhibitor of fibrinolysis, mediated by plasminogen
activators in cultured adipocytes. To identify mechanism(s) through
which TNF-
induces PAI-1, 3T3-L1 preadipocytes were differentiated
into adipocytes and exposed to TNF-
for 24 h. TNF-
selectively
increased the synthesis of PAI-1 without increasing activity of
plasminogen activators. Both superoxide (generated by xanthine oxidase
plus hypoxanthine) and hydrogen peroxide were potent inducers of PAI-1, and hydroxyl radical scavengers completely abolished the TNF-
induction of PAI-1. Exposure of adipocytes to TNF-
or insulin alone
over 5 days increased PAI-1 production. These agonists exert synergistic effects. Results obtained suggest that TNF-
stimulates PAI-1 production by adipocytes, an effect potentiated by insulin, and
that adipocyte generation of reactive oxygen centered radicals mediates
the induction of PAI-1 production by TNF-
. Because induction of
PAI-1 by TNF-
is potentiated synergistically by insulin, both agonists appear likely to contribute to the impairment of fibrinolytic system capacity typical in obese, hyperinsulinemic patients.
cytokine; insulin resistance; obesity; tumor necrosis factor-
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INTRODUCTION |
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OBESITY OFTEN IS ASSOCIATED with hyperinsulinemia and
dysfunction of the fibrinolytic system. These factors have been
implicated in the pathogenesis of thromboembolic phenomena and
vasculopathy (9). The fibrinolytic system is highly regulated at the
transcriptional, translational, and posttranslational levels by diverse
growth factors and cytokines (18). This study was performed to
determine whether tumor necrosis factor- (TNF-
), an inflammatory
cytokine produced by activated immune system cells among others (30), can modulate expression by adipocytes of proteins involved in the
fibrinolytic system and whether TNF-
effects are potentiated by
insulin, known to be elevated in the blood of patients with insulin-resistant states such as obesity.
Adipocytes elaborate plasminogen activator inhibitor-1 (PAI-1), the
primary physiological inhibitor of tissue type and urokinase type
plasminogen activators (t-PA and u-PA, respectively) (18, 23). We have
shown previously that adipocytes in vitro and adipose tissue in vivo
elaborate PAI-1 in response to transforming growth factor-, a
multifunctional growth factor released by activated platelets (20).
Others have found that PAI-1 mRNA is increased in murine adipose tissue
and adipocytes in response to TNF-
(25), a cytokine that can alter
expression of t-PA, PAI-1, PAI-2, and collagen in some cell types (6,
31, 34). However, mechanisms underlying the effects of TNF-
on the
synthesis of fibrinolytic system proteins by adipocytes have not yet
been elucidated.
TNF- expression is increased in adipose tissue from human subjects
with obesity and insulin resistance (12, 15). Because increased TNF-
in adipose tissue, coupled with the hyperinsulinemia associated with
obesity, may predispose to thromboembolic phenomenon and vasculopathy,
we sought to determine whether TNF-
and insulin alter production of
fibrinolytic system components in vitro in cultured adipocytes and to
elucidate possible mechanisms for these effects.
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METHODS |
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Materials.
3T3-L1 mouse preadipocytes were obtained from the American Type Culture
Collection. Penicillin, streptomycin, DMEM, trypsin, lipopolysaccharide
(LPS; Escherichia
coli O111:B4), and human insulin were
purchased from Sigma Chemical (St. Louis, MO). Cosmic calf serum was
from HyClone (Logan, UT), six-well culture plates were from Becton
Dickinson Labware (Lincoln Park, NJ), human insulin ELISA kits were
from Diagnostic Products (Los Angeles, CA), and recombinant mouse
TNF- and mouse TNF-
ELISA kits were from R&D Systems
(Minneapolis, MN). Sheep anti-mouse PAI-1 IgG, rabbit anti-mouse u-PA
antibody and anti-mouse t-PA antibody were acquired from American
Diagnostica (Greenwich, CT), Western Exposure chemiluminescent detection system was from Clontech Laboratories (Palo Alto, CA), X-ray
films were from Kodak (Rochester, NY), bicinchoninic acid (BCA) protein
assay reagent was from Pierce (Rockford, IL), and kits to assay free
fatty acids (Wako NEFA C) were from Biochemical Diagnostics (Edgewood,
NY). All chemicals were of the highest available commercial grade.
Cell culture procedures. Preadipocytes, incubated with DMEM containing 10% calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin, were grown to confluence on six-well plates and differentiated into adipocytes with dexamethasone and isobutylxanthine (ISBX) as described previously (20). In brief, confluent preadipocytes were exposed to dexamethasone (0.25 µM) and ISBX (0.5 mM) for 48 h. The cells were then placed in standard medium (without dexamethasone or ISBX), in which they accumulated small lipid droplets that grew to occupy a large fraction of total cell volume within 5 days. Seven days after initiation of differentiation (assessed by this criterion), 85-90% of the cells were judged to be differentiated.
Differentiated cells (7 days after exposure to dexamethasone) were incubated overnight in six-well plates in fresh DMEM containing 10% calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin, followed by incubation in fresh medium containing TNF-Assays for PAI-1 activity, plasminogen activator activity by
zymography, and total protein.
PAI-1 activity was measured spectrophotometrically (20) and verified to
be PAI-1 as judged by elimination of activity with inclusion of a
PAI-1-neutralizing antibody in the medium. Samples were incubated with
exogenous t-PA at room temperature for 10 min under conditions in which
PAI-1 but not other low-affinity plasminogen activator inhibitors would
bind to the t-PA. Subsequently, samples were acidified to eliminate
nonspecific inhibition such as that attributable to
2-antiplasmin activity.
Residual t-PA activity was assayed by incubation with plasminogen and a
chromogenic substrate, S-2251.
Assay of PAI-1 protein.
Concentrations of PAI-1 were determined by Western blotting with
antibody specific for the antigen. Equivalent amounts of protein from
conditioned media were diluted 1:1 with reduced sample buffer (0.5 mol/l Tris · HCl, pH 6.8, 10% glycerol, 2% SDS, 5% -mercaptoethanol, and 0.04% bromphenol blue), heated at 100°C for 5 min, cooled, and loaded on 10% polyacrylamide gels. Proteins were electrophoresed for 60 min and transferred to polyvinylidene difluoride membranes that were then blocked with 1% BSA and 0.1% Tween 20 in PBS (pH 7.4). Membranes were washed with 0.5% BSA and
0.1% Tween 20 in PBS several times and incubated with 1% BSA and
0.1% Tween 20 in PBS with 2 µg/ml sheep anti-mouse PAI-1 IgG containing 0.02% sodium azide for detection of PAI-1 by Western blotting. Membranes were washed with 0.5% BSA and 0.1% Tween 20 in
PBS several times and incubated with alkaline phosphatase-conjugated goat anti-sheep IgG diluted 1:5,000 with 1% BSA and 0.1% Tween 20 in
PBS containing 0.02% sodium azide. Membranes were incubated with
chemiluminescent enhancer followed by 0.25 mM chemiluminescent substrate and exposed to X-ray film. Bands on developed film were quantified with the use of a densitometer (Bio-Rad model GS-700). Correlation between different doses of recombinant mouse PAI-1 and
measured density on the X-ray films was linear up to 5 ng PAI-1 per lane.
Statistics. Data are expressed as means ± SE. Differences were assessed by ANOVA with Bonferroni least significant difference post hoc tests for comparisons within multiple groups. When appropriate, data were evaluated by ANOVA for repeated measures. Significance was defined as P < 0.05.
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RESULTS |
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Effects of TNF- on adipocyte PAI-1 activity.
The proinflammatory cytokine TNF-
increased PAI-1 activity in the
conditioned media [2.6 ± 1.0 arbitrary units (AU)/ml in control cultures compared with 8.5 ± 1.1 AU/ml with 10 ng/ml
TNF-
; n = 7; Fig.
1A].
TNF-
increased PAI-1 protein accumulation (2.6 ± 0.2-fold over
control with 10 ng/ml, n = 6; Fig.
1B). At 5 ng/ml TNF-
, there were
no significant differences in PAI-1 protein or activity compared with
control conditions in media lacking TNF-
. Accumulation of PAI-1 was
evident within 6 h of exposure of cells to TNF-
, with further
increases over 24 h. Cycloheximide (25 µg/ml) inhibited
TNF-
-induced PAI-1 accumulation by 96 ± 10%
(n = 9). LPS, in concentrations up to
100 µg/ml, did not induce PAI-1 accumulation
(n = 3), indicating that endotoxin was
not inducing PAI-1 in the adipocytes in vitro.
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Effects of reactive oxygen species.
The hydroxyl radical scavenger tetramethylthiourea (TMTU; 20 mM) almost
completely inhibited both the increase in PAI-1 activity (n = 6; Fig.
2A) and
the accumulation of PAI-1 protein induced by TNF-
(n = 6; Fig.
2B). Equimolar urea, used as a
control, had no effect. Another hydroxyl radical scavenger, DMSO,
similarly inhibited the increase of PAI-1 activity induced by TNF-
(1.5 ± 0.7 AU/ml in control media; 5.1 ± 0.6 with 10 ng/ml
TNF-
; 2.2 ± 0.9 with 10 ng/ml TNF-
plus 1.0% DMSO) and PAI-1
protein (100.0 ± 7.2% in control media; 186.3 ± 13.2 with 10 ng/ml TNF-
; 116.7 ± 3.5 with 10 ng/ml TNF-
plus 1.0% DMSO).
When cells were incubated with hydrogen peroxide (100 µM) or a
superoxide-generating system (10 mU/ml xanthine oxidase plus 0.6 mM
hypoxanthine), PAI-1 activity increased (12.3 ± 1.1 AU/ml with
hydrogen peroxide and 21.1 ± 0.9 with xanthine oxidase plus
hypoxanthine, n = 6; Fig.
3A), and
PAI-1 protein accumulation increased as well
(n = 6; Fig. 3B).
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Effects of exposure of adipocytes to TNF-, insulin,
or both for 5 days.
Exposure of adipocytes to TNF-
(5 and 10 ng/ml;
n = 6; Fig.
4A) or
insulin (20 and 100 nM; n = 6; Fig.
4B) in the media over 5 days
increased production of PAI-1 protein. We also determined the levels of
agent (TNF-
or insulin) left in the media during the experiment.
After initial addition of 20 nM insulin to the media, levels of insulin
in media had declined to 84 ± 5.3% by 24 h, to 56 ± 3.2% by 3 days, and to 33% by 5 days. After initial addition of 100 nM insulin to media, the levels of insulin in media declined to 61.7 ± 9.9% by 3 days, and insulin levels in media remained the same at
5 days. For TNF-
added at 5 ng/ml, values of TNF-
in media
increased somewhat after addition of the agent, indicating that the
cells themselves may have been producing some TNF-
. After addition
of 10 ng/ml TNF-
, values of TNF-
in media were 2,259 ± 269 pg/ml at 24 h, 2,937 ± 310 pg/ml at 3 days, and 2,472 ± 213 pg/ml at 5 days.
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DISCUSSION |
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Results of this study suggest a mechanism that may contribute
to exacerbation of cardiovascular disease in obese diabetic human
subjects. They indicate that TNF- alters production of PAI-1 in
adipocytes, consistent with its effects in endothelial cells, smooth
muscle cells, HepG2 hepatoma cells, and human mesangial cells (5, 6,
21, 31) and in marked contrast to its effects in chondrocytes and
synovial fibroblasts (4, 8). Analogous results were obtained in studies
of mouse adipose tissue, with TNF-
shown to induce PAI-1 protein
(20) and mRNA (25) expression. In contrast, Alessi et al. (1) did not
observe an effect of TNF-
on PAI-1 protein production in human
adipose tissue. However, it appears unlikely that species specificity
is the explanation for the difference in results, in view of the strong
association between insulin resistance and elevated PAI-1 in blood in
human subjects and between TNF-
and insulin resistance observed in adipocytes (12, 14, 21, 26).
The TNF--induced PAI-1 expression was diminished by cycloheximide,
suggesting that the increase requires protein synthesis. It is possible
that the state of differentiation of the cells affected rates of
protein synthesis. Lipid breakdown is one of the effects of TNF-
on
adipocytes. To determine whether the state of differentiation of the
cells had changed with treatment, we assessed lipid droplet
accumulation, which was comparable in cells exposed to the agonists or
to control media, consistent with a lack of change in differentiation
between control and agonist-treated cells.
The effects of TNF- on PAI-1 protein content were both time and
concentration dependent, with a maximal effect at 10 ng/ml. A lag time
of several hours was evident, suggesting that autocrine factor(s) may
be involved.
Second messengers involved in signal transduction pathways leading to
induction of PAI-1 are not yet well characterized. Reactive oxygen
intermediates, commonly produced by activated inflammatory cells (32),
may function as autacoids (2). TNF- can induce both superoxide and
hydrogen peroxide production (24). In the present study, the hydroxyl
radical scavengers TMTU and DMSO inhibited TNF-
-induced PAI-1
expression. Conversely, hydrogen peroxide as well as a
superoxide-generating system induced PAI-1. Hydrogen peroxide is likely
to have been rapidly depleted from the media. Accordingly, metabolites
such as superoxide anion and hydroxyl radical may have been responsible
for effects induced by exposure of the cells to agents and may have
been the direct effector rather than hydrogen peroxide per se. Although
both TNF-
and hydrogen peroxide induced PAI-1 production by
adipocytes, TNF-
had a greater effect on PAI-1 protein relative to
PAI-1 activity than did hydrogen peroxide. One explanation is that
TNF-
can stimulate production of plasminogen activators as well and
that this may have reduced apparent PAI-1 activity by neutralizing it.
Our results indicate that the expression of non-NADPH oxidase-dependent
reactive oxygen species (3) may modify PAI-1 production by adipocytes
and that TNF- may act via reactive oxygen species that act as
autacoids and may therefore contribute to impaired fibrinolysis
associated with obesity. PAI-1, the primary inhibitor of t-PA and u-PA,
can be induced by TNF-
, known to be increased in blood of obese
subjects and those with insulin resistance (16). It appears likely that
the TNF-
-induced expression of PAI-1 contributes to the exacerbation
of vasculopathy in obese and insulin-resistant subjects (33).
We have shown previously that insulin and its precursors augment PAI-1
synthesis (22). The synergistic induction of PAI-1 by insulin and
TNF- demonstrated in this study is likely to impair fibrinolysis not
only in obese patients but also in others with insulin resistance.
Although mechanisms contributing to the apparent synergy have not yet
been determined, TNF-
inhibits insulin signaling and induces insulin
resistance (29). Therefore, our results suggest that TNF-
-induced
adipocyte resistance to insulin will not obviate augmented adipocyte
PAI-1 production. Indeed, they are consistent with the observation that
PAI-1 expression in visceral fat is increased in obese rats and that
concentrations of PAI-1 in blood correlate closely with visceral fat
mass in human subjects (28).
Insulin resistance has been implicated strongly in the pathogenesis of
atherosclerosis (27). Obesity and insulin resistance are associated
with endothelial cell dysfunction (29). The susceptibility of diabetic
patients to vascular complications may be a function, in part, of an
impaired endogenous antioxidant status (7). Our results suggest that
antioxidants may limit PAI-1 induction otherwise associated with high
concentrations of insulin and TNF- in blood and in adipose tissue
typical of insulin-resistant states.
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ACKNOWLEDGEMENTS |
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We thank Amy Guala for technical assistance and Elizabeth Commons, Amy Prue, and Lori Dales for secretarial support.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-17646 and by the American Heart Association (Missouri Affiliate). T. Sakamoto was supported by a fellowship from the Japan Heart Foundation (Tokyo, Japan).
Preliminary results from this study were presented at the 46th Annual Scientific Sessions of the American College of Cardiology (Anaheim, CA; 16-19 March 1997) and at the 70th Scientific Sessions of the American Heart Association (Orlando, FL; 9-12 November 1997).
Address for reprint requests and other correspondence: S. Fujii, c/o J. Woodcock-Mitchell, University of Vermont College of Medicine, Colchester Research Facility, 55A S. Park Dr., Colchester, VT 05446 (E-mail: jjmitche{at}zoo.uvm.edu).
Received 19 August 1997; accepted in final form 8 March 1999.
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