Department of Medicine, Division of Cardiology, Harborview Medical Center, University of Washington, Seattle, Washington 98104
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ABSTRACT |
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Endothelial cells release nitric oxide
(NO) acutely in response to increased "flow" or fluid shear stress
(FSS), and the increase in NO production is correlated with enhanced
phosphorylation and activation of endothelial nitric oxide synthase
(eNOS). Both vascular endothelial growth factor and FSS activate
endothelial protein kinase B (PKB) by way of incompletely understood
pathway(s), and, in turn, PKB phosphorylates eNOS at Ser-1179, causing
its activation. In this study, we found that either FSS or insulin
stimulated insulin receptor substrate-1 (IRS-1) tyrosine and serine
phosphorylation and increased IRS-1-associated phosphatidylinositol
3-kinase activity, phosphorylation of PKB Ser-473, phosphorylation of
eNOS Ser-1179, and NO production. Brief pretreatment of bovine aortic
endothelial cells with tumor necrosis factor- (TNF-
) inhibited
the above described FSS- or insulin-stimulated protein phosphorylation
events and almost totally inhibited FSS- or insulin-stimulated NO
production. These data indicate that FSS and insulin regulate eNOS
phosphorylation and NO production by overlapping mechanisms. This study
suggests one potential mechanism for the development of endothelial
dysfunction in disease states with alterations in insulin regulation
and increased TNF-
levels.
insulin receptor substrate-1; tyrosine phosphorylation; phosphatidylinositol 3-kinase; endothelial nitric oxide synthase
phosphorylation; tumor necrosis factor-; nitric oxide
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INTRODUCTION |
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ONE OF THE MAJOR
ROLES of the endothelium is to mediate the vasodilatory response
by regulating the production of nitric oxide (NO). Endothelium-derived
NO contributes to blood vessel homeostasis by regulating vessel tone
(33), cell growth (18), platelet aggregation
(26), and leukocyte binding to endothelium
(37). Endothelial cells synthesize NO tonically and
increase NO in response to physiological stimuli, such as vascular
endothelial growth factor (VEGF) (21) and fluid shear
stress (FSS) (10, 11). Endothelial nitric oxide synthase
(eNOS or type III NOS) is one of three isoenzymes that converts
L-arginine to L-citrulline and NO. VEGF or an
increase in FSS activates phosphatidylinositol 3-kinase (PI 3-kinase)
in endothelial cells, leading to activation of protein kinase B (PKB).
PKB, in turn, phosphorylates eNOS at Ser-1179 and activates the enzyme,
leading to release of NO (14, 17). Insulin also stimulates
NO production in human umbilical vein endothelial cells (HUVEC) by a
mechanism that is sensitive to wortmannin (a PI 3-kinase inhibitor) or
N-nitro-L-arginine methyl ester,
an inhibitor of eNOS (46). Likewise, NG-monomethyl-L-arginine, an
alternate eNOS inhibitor, prevents insulin-induced vasodilation in
humans (6, 39). In mice lacking insulin receptor
substrate-1 (IRS-1), a major substrate for the insulin receptor (IR)
tyrosine kinase, there is impaired endothelium-dependent vascular
relaxation (1). Together, these studies suggest that insulin, VEGF, and FSS may use elements of the same pathway(s) to
regulate NO production in endothelial cells.
The molecular basis of insulin receptor signal transduction has been well characterized due to its importance in insulin-sensitive target tissues (42, 43). Insulin binding to IR activates receptor tyrosine kinases, which phosphorylates numerous substrates, including IRS-1. Many other ligands signal through IRS-1, including integrins (20), interferon (7), interleukins (7, 27), and gastrin (29). In each of these cases, IRS-1 appears to be specifically regulated according to receptor type and cell context. However, little is known about the function of IRS-1 in vascular endothelial cells. On the basis of our previous observations that FSS activates eNOS through a PI 3-kinase-dependent mechanism, we hypothesized that IRS-1 could participate in the mechanotransduction of FSS to NO production.
Numerous clinical observations demonstrate that elevated tumor necrosis
factor- (TNF-
) levels induce insulin resistance in a variety of
catabolic states, including cancer, sepsis, and trauma (4,
30). Neutralization of TNF-
with a soluble TNF receptor
immunoglobulin fusion protein was found to improve insulin receptor
signaling and insulin sensitivity in obese and insulin-resistant rats
(22). Because of the inflammatory characteristics of
coronary atherosclerosis (38) and studies demonstrating a
negative modulatory effect of TNF-
on insulin signaling (12,
16, 23, 24, 28, 34), we hypothesized that TNF-
could impair
aspects of signaling to eNOS and NO production.
This study shows that both insulin and FSS signal through IRS-1 in
bovine aortic endothelial cells (BAEC) to cause activation of PI
3-kinase by association with IRS-1, and both stimuli enhance PKB and
eNOS phosphorylation as well as NO production. We also demonstrate that
brief pretreatment of BAEC with TNF- before FSS or insulin
stimulation profoundly decreases NO synthesis while impairing many of
the above signaling events.
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METHODS |
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Materials.
The monoclonal antibody (H32) to eNOS was purchased from Biomol
(Plymouth Meeting, MA). Anti-phospho (Ser-473) PKB and anti-PKB polyclonal antibodies were purchased from New England Biolabs (Beverly,
MA). Anti-IRS-1 rabbit polyclonal antibody and anti-phosphotyrosine monoclonal antibody (4G10) were purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal antiserum to IRS-1 was a kind gift
from Dr. Hagit Eldar-Finkelman (Harvard Medical School). Recombinant
human TNF- was obtained from R&D Systems (Minneapolis, MN).
Recombinant human insulin was obtained from Calbiochem (La Jolla, CA).
[32P]orthophosphoric acid (185 MBq) and
[
-32P]ATP (37 MBq) were purchased from NEN Life
Science (Boston, MA). Sequencing-grade modified trypsin was obtained
from Promega (Madison, WI). Phosphoserine, phosphothreonine, and
phosphotyrosine were purchased from Sigma (St. Louis, MO). Thin-layer
cellulose chromatography plates were purchased from EM Science
(Gibbstown, NJ). Bradford protein assay reagents were from Bio-Rad
Laboratories (Hercules, CA). Phosphatidylinositol was purchased from
Avanti Polar Lipid (Alabaster, AL).
BAEC culture and exposure to FSS. BAEC cultures were established and maintained in medium 199 (GIBCO BRL) supplemented with 10% fetal bovine serum as described (10). Cells from passages 3-8 were seeded in 100-mm culture dishes and used at 60-80% confluency for labeling and shear stress experiments. Cells were exposed to laminar FSS in a cone and plate viscometer. Before shear stress or incubation with insulin, BAEC were starved for 3-5 h in serum-free endothelial cell basal medium (Clonetics, San Diego, CA). FSS stimulation of BAEC was for 2 min at 12 dyn/cm2 unless otherwise indicated.
Cell labeling, lysis, and immunoprecipitation of eNOS and IRS-1. These procedures were performed as described (17). All immunoprecipitations and Western blots were carried out using equal amounts of total protein for each condition for each experiment. Protein concentrations were determined by the Bradford assay.
SDS gel electrophoresis, Western blotting, and transfer of proteins to membranes. These procedures were performed as described (17), except that IRS-1 was transferred to nitrocellulose for 1 h at 100 V in transfer buffer (24 mM Tris base and 181 mM glycine) containing 0.1% SDS and 20% methanol.
Phosphopeptide mapping and phosphoamino acid analysis. These procedures were performed as described (17).
PI 3-kinase assay.
After immunoprecipitation with anti-IRS-1 antibody, the immune
complexes were washed, resuspended in 50 µl of 10 mM
Tris · HCl (pH 7.4), 150 mM NaCl, and 5 mM EDTA, and then
incubated with 20 µg of phosphatidylinositol and 5 µl of a stock
solution [30 µCi of [-32P]ATP (3,000 Ci/mmol), 0.88 mM ATP, and 20 mM MgCl2] for 12 min at 30°C. The
reaction was stopped with 20 µl of 6N HCl, and 160 µl of
CHCl3:methanol (1:1) was added. The mixture was
centrifuged, and the lower organic phase was removed and applied to a
silica gel thin-layer chromatography (TLC) plate (Merck) that was
precoated with 1% potassium oxalate. The plates were developed by
ascending chromatography in
CHCl3:MeOH:H2O:NH4OH (60:47:11.3:2)
and dried, and the radiolabeled lipids were visualized and quantified
by PhosphorImager analysis.
Measurement of nitrogen oxide concentrations. NO released by BAEC was measured as its nitrogen oxide (NOx) metabolites, using a chemiluminescence detector as described in detail (10).
Statistics. Statistical analysis was performed using Stata version 6.0 (Stata). Group differences were determined using the standard Student's t-test. All variables are expressed as means ± SE. P < 0.05 was considered statistically significant.
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RESULTS |
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Effects of FSS and insulin on tyrosine phosphorylation of
endothelial IRS-1; inhibition by TNF-.
One of the eNOS serine residues phosphorylated in response to increased
FSS is contained within the PKB substrate consensus phosphorylation
sequence RXRXXSH (in which H is a hydrophobic residue and X is any
amino acid) (17). Since PI 3-kinase is an important
upstream activator of PKB (3) and is itself activated in
some cell contexts through interaction with tyrosine phosphorylated IRS-1, we hypothesized that IRS-1 might be phosphorylated in response to FSS. BAEC were subjected to FSS (12 dyn/cm2) for 2 min
in a cone and plate viscometer. Whole cell lysates were
immunoprecipitated with an anti-IRS-1 antibody, and Western blot
analysis was performed with an anti-phosphotyrosine antibody (4G10).
FSS increased IRS-1 tyrosine phosphorylation by 5.4 ± 1.6-fold (n = 3, Fig.
1A). In view of the known
antagonism of IRS-1 signal transduction by TNF-
in insulin-sensitive
cells, we next determined whether such a mechanism is operative in
endothelial cells. After pretreatment with TNF-
(10 ng/ml for 10 min), FSS stimulated IRS-1 tyrosine phosphorylation by 2.6 ± 0.5-fold, which represented a 48% inhibition. IRS-1 total protein
content was not altered after FSS or TNF-
pretreatment, suggesting
that FSS and TNF-
modify IRS-1 tyrosine phosphorylation through
dynamic, likely posttranslational mechanisms.
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Phosphoamino acid analysis of IRS-1 from BAEC subjected to FSS
without or with TNF- pretreatment.
Inhibition of insulin signaling by TNF-
has been associated with
TNF-induced serine phosphorylation of IRS-1 in adipocytes and Fao
hepatoma cells (23, 34). To determine whether TNF-
also
increases serine phosphorylation of IRS-1 in endothelial cells, BAEC
were incubated for 3 h with [32P]orthophosphate. The
cells were maintained in static culture or exposed to 12 dyn/cm2 FSS for 2 min without or with pretreatment with
TNF-
(10 ng/ml for 10 min). IRS-1 was immunoprecipitated, size
fractionated, and transferred to polyvinylidene difluoride. Figure
2A shows by autoradiography
that compared with the control static condition, exposure to FSS,
TNF-
pretreatment followed by FSS, or TNF-
alone, all increased
32P incorporation into IRS-1. IRS-1 was hydrolyzed, and the
labeled phosphoamino acids were separated by high-voltage
electrophoresis (HVE; Fig. 2B). Compared with cells
maintained in static culture, all treatments were associated with
enhanced serine phosphorylation of IRS-1. Similar increases in serine
phosphorylation of IRS-1 were observed when BAEC were stimulated with
insulin rather than FSS (data not shown). After exposure to FSS or
insulin, phosphoamino acid analysis did not demonstrate changes in the
phosphotyrosine content of IRS-1, in contrast to the results obtained
through anti-phosphotyrosine Western blotting (Fig. 1, A and
B). In fact, by phosphoamino acid analysis, insulin
enhancement of IRS-1 phosphotyrosine content in Chinese hamster ovary
cells transfected with IRS-1 cDNA was very modest (41).
These findings likely reflect the relative lability of tyrosine
phosphate bonds to acid hydrolysis (15).
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Effects of FSS and insulin on IRS-1 association with, and
activation of, PI 3-kinase; inhibition by TNF-.
Since FSS increased tyrosine phosphorylation of IRS-1, we determined
directly whether FSS would cause the association of IRS-1 with PI
3-kinase and activation of the latter. We have previously obtained data
consistent with FSS activation of PI 3-kinase-dependent pathway(s),
since pretreatment of BAEC with the PI 3-kinase inhibitor LY-294002
attenuated NO production in response to FSS (17). After
exposure to FSS, whole cell lysates were immunoprecipitated with an
anti-IRS-1 antibody. PI 3-kinase assays were performed as described in
METHODS. FSS increased the PI 3-kinase activity associated
with IRS-1 by 5.5 ± 1.3-fold (n = 3) over the
control static condition (Fig. 3).
Insulin stimulation increased PI 3-kinase association and activity
levels (Fig. 3) by 4.9 ± 1.3-fold (n = 3). In
C2C12 muscle cells (12) or Fao
hepatoma cells (16), TNF-
pretreatment decreased
insulin-mediated PI 3-kinase association with IRS-1 and PI 3-kinase
activation. TNF-
treatment alone did not significantly affect basal
IRS-1-associated PI 3-kinase activity, but did decrease
insulin-stimulated PI 3-kinase activation by 96% (P < 0.05). The inhibitory effect of TNF-
treatment before FSS was
quantitatively less, causing only a 38% decrease in FSS-induced PI
3-kinase activation; however, this inhibitory effect was not statistically significant (P = 0.27). These
coimmunoprecipitation experiments demonstrate that either FSS or
insulin increases IRS-1-associated PI 3-kinase activity. TNF-
pretreatment inhibits IRS-1-mediated activation of PI 3-kinase after
insulin stimulation.
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Effects of TNF- on FSS and insulin-mediated PKB phosphorylation.
FSS activates both PI 3-kinase (19) and PKB in endothelial
cells, and FSS-induced PKB phosphorylation can be inhibited by wortmannin (13, 14) or LY-294002 (17).
Similarly, activation of PI 3-kinase activity and PKB phosphorylation
by insulin have been shown previously (3). An antibody to
phosphorylated Ser-473 of PKB was employed to examine the effects of
FSS, insulin, and TNF-
on PKB phosphorylation. After FSS or insulin
treatment of BAEC, Western blot analysis using anti-phosphoserine-473
PKB or anti-PKB antibodies was performed. Figure
4A demonstrates that FSS
increased phosphorylation of PKB by 2.1 ± 0.2-fold and that pretreatment with TNF-
for 10 min before FSS reduced FSS-induced phosphorylation by 70% (n = 3). Figure 4B
shows that insulin increased PKB Ser-473 phosphorylation by 2.6 ± 0.4-fold (n = 4) and that pretreatment with 10 min of
TNF-
before insulin stimulation decreased insulin-induced PKB
phosphorylation almost entirely. These results demonstrate that brief
pretreatment with TNF-
before FSS or insulin stimulation leads to
decreased levels of phosphorylation of Ser-473 in PKB.
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FSS stimulates eNOS phosphorylation, which is inhibited by TNF-.
eNOS is basally phosphorylated, its phosphorylation is enhanced in
response to FSS (10, 14, 17), and FSS enhances
phosphorylation of two peptides, designated F1 and F2
(17). PKB phosphorylates eNOS at the F1 site in the cell
and in vitro, increasing eNOS enzymatic activity (17).
BAEC were labeled for 3 h with [32P]orthophosphate
and maintained in static condition or subjected to FSS without or with
TNF-
pretreatment. Exposure to FSS increased eNOS phosphorylation
nearly twofold, as shown previously (17) and in Fig.
5. There was no change in eNOS
phosphorylation relative to the static condition following 10 min of
treatment with TNF-
, but TNF-
pretreatment did inhibit
FSS-dependent phosphorylation of eNOS. Since TNF-
inhibits
flow-dependent PKB Ser-473 phosphorylation, it was determined whether
TNF-
also inhibited flow-dependent phosphorylation of peptide F1.
eNOS was transferred to nitrocellulose and subjected to tryptic
cleavage, and the tryptic peptides were separated by TLC and HVE.
Autoradiograms of the thin-layer plates are shown in Fig. 5
(top). The relative volumes of the phosphopeptide F1 in each
condition were quantified with a PhosphorImager. Compared with static
conditions, the phosphopeptide F1 was increased by 2.4 ± 0.5-fold
(n = 3) by FSS. Although incubation with TNF-
alone
caused no change in the basal level of phosphorylation of eNOS tryptic
peptides, preincubation with TNF-
before flow inhibited F1
phosphorylation by 40% (n = 3).
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Insulin stimulates eNOS phosphorylation, which is inhibited by
TNF-.
Insulin causes vasodilation in humans by way of an eNOS-dependent
mechanism (6, 39). Insulin-dependent production of NO in
endothelial cells can be inhibited by wortmannin (46). These findings suggest that insulin and FSS may activate eNOS by
similar pathways. We therefore determined whether insulin could stimulate eNOS F1 site phosphorylation in BAEC. In Fig. 6, insulin stimulated the phosphorylation of peptide F1 by 2.1 ± 0.1-fold (n = 3) compared with control cells. Preincubation with
TNF-
for 10 min before the addition of insulin inhibited F1
phosphorylation by 71% (n = 3). Thus both insulin and
FSS induced phosphorylation of peptide F1, which is known to activate
eNOS (14, 17), and TNF-
pretreatment inhibited
phosphorylation of F1 in response to FSS or insulin.
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Pretreatment of BAEC with TNF- inhibits both FSS- and
insulin-induced NO production.
FSS is a well-recognized activator of NO production in endothelial
cells (10, 11). Zeng and Quon (46) have
previously shown that insulin stimulation can increase endothelial NO
production, but the effect of TNF-
on NO production in response to
FSS or insulin stimulation has not been previously reported. We
assessed NO directly released by endothelial cells during FSS
stimulation by measuring NOx metabolites using ozone chemiluminescence
as previously described
(10). Figure
7A demonstrates that NO
production increases upon exposure of static BAEC to FSS. The
FSS-dependent increase was inhibited nearly 80% following TNF-
pretreatment. Figure 7B demonstrates that stimulation of
BAEC with insulin also resulted in an increase in NO production that
was inhibited ~70% by pretreatment with TNF-
. Thus a brief
pretreatment of BAEC with TNF-
inhibited both FSS- and
insulin-mediated NO production.
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DISCUSSION |
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The major findings of this study are that either insulin or FSS
stimulates IRS-1 tyrosine and serine phosphorylation with a subsequent
increase in IRS-1-associated PI 3-kinase activity, phosphorylation of
PKB Ser-473, phosphorylation of eNOS at Ser-1179, and NO production in
endothelial cells. Brief pretreatment with TNF- inhibits the above
described FSS- or insulin-stimulated protein phosphorylation events and
FSS- or insulin-stimulated NO production. IRS-1 is tyrosine
phosphorylated and activated upon insulin binding to the IR and in
response to activation by FSS by an unknown tyrosine kinase. As in
other cell types, exposure to TNF-
is associated with increased
IRS-1 serine phosphorylation and concomitant inhibition of its tyrosine
phosphorylation and activation, implying a form of cross-talk between
serine- and tyrosine-specific protein kinases. IRS-1 appears to be a
focal point for the agonists we have utilized, from the perspectives of
both activation and inhibition in being independently regulated by
insulin, FSS, and TNF-
. A logical extension is that disease states
characterized by alterations in insulin function and increased TNF-
may manifest endothelial dysfunction and insulin resistance due to
alteration of the normal balance between these modulators. The model
system utilized for these studies should be helpful for identifying the
TNF-
-dependent protein kinase(s) that phosphorylate IRS-1. It can be
used as a basis for determining whether the form of "insulin
resistance" suggested by these studies is physiologically relevant in vivo.
Recently, Zeng and colleagues (45) have shown that insulin requires insulin receptor tyrosine kinase, PI 3-kinase, and PKB for insulin-induced NO production in HUVEC. Similarly, we report signaling events induced by FSS and insulin in endothelial cells that have commonly been described for insulin in "insulin-responsive cells," namely, increased tyrosine and serine phosphorylation of IRS-1 (41), increased association of IRS-1 with PI 3-kinase (5, 32, 41), and increased PKB phosphorylation (3). The similarities between signaling events induced by either insulin or FSS leading to eNOS phosphorylation and NO production are striking and suggest that insulin and FSS use elements of overlapping pathways to generate NO in BAEC. However, this study has not proven that insulin and FSS use the same pathway, or only one pathway, to stimulate eNOS phosphorylation and NO production.
A key question in vascular biology remains: the identity of the mechanotransducers of FSS to biochemical signals that regulate vessel physiology. FSS could produce a deformation of the endothelial cytoskeleton, changes in membrane physical chemistry, and/or activation of receptors or sensors that trigger intracellular signaling pathways. Growth factor receptors are appropriate candidate mediators, because they may be activated by an array of biological signals independent of hormone/receptor binding (8). Recently, FSS has been shown in BAEC to increase the tyrosine phosphorylation and clustering of the fetal liver kinase 1 (FLK-1) VEGF receptor and to induce its association with the adapter protein shc (9). Although insulin causes tyrosine phosphorylation of the IR in BAEC, FSS does not (Kim, unpublished observations). Thus FLK-1 remains a potential candidate to be the FSS-dependent tyrosine kinase "upstream" of IRS-1. The "downstream" coupling of IRS-1 to PI 3-kinase also appears to be a stimulus-specific response, since NO production was not increased after exposure of endothelial cells to platelet-derived growth factor, a known stimulus of PI 3-kinase in endothelial cells (46).
This study demonstrates that, similar to TNF-'s effects in Fao
hepatoma cells (16), C2C12
myotubes (12) and adipocytes (23, 24), this
cytokine suppresses insulin-induced tyrosine phosphorylation of IRS-1,
increases the serine phosphorylation of IRS-1 (24, 34),
and decreases insulin-stimulated association of IRS-1 with PI 3-kinase
(12). Studies in endothelial (31) and other
cell types show that TNF-
activates PI 3-kinase in the cell
globally, in contrast to our and other investigations (2, 12,
28) that focus on the TNF-
inhibition of insulin-activated PI
3-kinase associated with IRS-1. In these studies, TNF-
induced serine phosphorylation of IRS-1 correlated with decreased IR tyrosine kinase activity and tyrosine phosphorylation of IRS-1 (23, 24, 28). TNF-
-induced serine phosphorylation of IRS-1 has been proposed to alter IRS-1 conformation and thereby impair its ability to
bind PI 3-kinase (2). Increased adipocyte TNF-
mRNA
levels observed in rodent models of obesity and diabetes have led to the suggestion that insulin resistance is mediated by the changes observed in IRS-1 phosphorylation and protein kinase activity observed
in cell cultures in the presence of TNF-
(23-25).
Interestingly, eNOS knockout mice exhibit insulin resistance in the
liver and peripheral tissues (40); it is speculated that
reduced blood flow may impair delivery of insulin and/or glucose to
target tissues.
TNF- pretreatment significantly inhibits FSS-dependent IRS-1
tyrosine phosphorylation, phosphorylation of PKB Ser-473, eNOS phosphorylation at Ser-1179, and, finally, FSS-dependent NO production. TNF-
pretreatment, however, did not significantly alter
IRS-1-associated PI 3-kinase activity following FSS. The reasons for
this modest TNF-
effect is unclear; however, differences in insulin-
and FSS-dependent serine and tyrosine phosphorylation of IRS-1 as well
as differences in FSS- and insulin-dependent phosphatases may play a
role in the observed differences. Nevertheless, the effects of
short-term pretreatment with TNF-
on FSS-dependent NO production has
been clearly demonstrated in our cell culture model. This investigation
examines the acute effects of TNF-
on FSS and insulin signaling
following a brief (10-min) pretreatment of endothelial cells. Long-term
elevated levels of TNF-
may also contribute to decreased NO
production, since chronic incubation of endothelial cells with TNF-
reduces eNOS mRNA half-life by >90% (44).
Important questions that remain include the vascular origins of
TNF-, the identity of the TNF-
receptor(s) stimulated, and the
precise pathway(s) through which TNF-
receptor stimulation is linked
to serine phosphorylation of IRS-1. TNF-
is likely produced in an
autocrine or paracrine manner and may be closely cell associated before
stimulation of TNF receptors. The known p55 and p75 TNF-
receptors
are devoid of intrinsic or associated catalytic activity, but in some
cell types couple to sphingomyelinases, liberating fatty acid
autocoids, and they can activate the protein kinase C and
mitogen-activated protein kinase superfamilies (35, 36). A
set of IRS-1 serine residues (612, 632, 662, and 731) has been
identified as being dually regulated by these kinases, but the
relevance of these sites to the events described in the current report
is unclear. Subsequent work will address these questions, with a
particular focus on determining the identity of the TNF-
-dependent
IRS-1 phosphorylation sites. Identifying these sites should provide the
molecular tools to dissect these pathways in vivo and should suggest
targets for therapeutic agents to treat insulin resistance syndromes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Edwin G. Krebs for careful review of this manuscript.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute National Research Service Awards 1F32-HL-09878-01 (to F. Kim) and 1R21-HL-62885 and 1RO1-HL-64228 (both to B. Gallis and M. A. Corson).
Address for reprint requests and other correspondence: F. Kim, Dept. of Medicine, Div. of Cardiology, Box 359748, Harborview Medical Center, Univ. of Washington, 325 Ninth Ave., Seattle, WA 98104 (E-mail: fkim{at}u.washington.edu).
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. Section 1734 solely to indicate this fact.
Received 10 August 2000; accepted in final form 15 December 2000.
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