© 2000 American Heart Association, Inc.
Vascular Biology |
Insulin-Mediated Stimulation of Protein Kinase Akt
A Potent Survival Signaling Cascade for Endothelial Cells
From Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Frankfurt, Germany.
Correspondence to Stefanie Dimmeler, PhD, Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. E-mail Dimmeler{at}em.uni-frankfurt.de
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Abstract—Insulin exerts potent antiapoptotic effects in neuronal cells and has been suggested to promote angiogenesis. Therefore, we investigated whether insulin inhibits tumor necrosis factor-
(TNF-)–induced apoptosis
in human umbilical vein endothelial cells (HUVECs).
Because insulin has been shown to stimulate the protein kinase Akt, we
investigated whether activation of Akt contributes to the
apoptosis-suppressive effect of insulin and characterized the
downstream signaling pathway. Incubation with insulin dose-dependently
prevented apoptosis induced by TNF- (50 ng/mL). The extent
of apoptosis suppression by insulin was similar to the effect
of vascular endothelial growth factor. Pharmacological
inhibition of Akt activation or overexpression of a dominant-negative
Akt mutant prevented the antiapoptotic effect of insulin.
Furthermore, we investigated the effect of TNF- on Akt
phosphorylation by Western blot analysis with
the use of a phosphospecific Akt antibody. Incubation of HUVECs with
TNF- induced a marked dephosphorylation of Akt.
Insulin counteracted this TNF-–induced
dephosphorylation of Akt. Furthermore, we investigated
the downstream signaling events. Akt has been shown to mediate its
apoptosis-suppressive effects via
phosphorylation of Bad or caspase-9. However,
incubation with insulin did not lead to enhanced
phosphorylation of Bad at Ser 136 or Ser 112. In
contrast, insulin inhibited caspase-9 activity and prevented
caspase-9–induced apoptosis. Mutation of the Akt site within
caspase-9 significantly reduced the apoptosis-suppressive
effect of insulin. The present study demonstrates an
important role for insulin-mediated Akt activation in the prevention of
endothelial cell apoptosis, which may
importantly contribute to cell homeostasis and the integrity of the
endothelium. In endothelial cells, Akt
seems to mediate its antiapoptotic effect, at least in part,
via phosphorylation of caspase-9 rather than Bad.
Key Words: endothelial cells • insulin • Akt kinase • atherosclerosis
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Introduction |
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The survival of endothelial cells critically determines vessel growth and inflammatory processes in the vessel wall. Survival of the cells is counterbalanced by the induction of physiological cell death, apoptosis. Besides the contribution of apoptosis to endothelial injury, apoptotic cell death regulates vessel growth. Thus, recent studies have provided evidence that inhibition of endothelial cell apoptosis in combination with enhanced proliferation is implicated as the basis for angiogenesis associated with diabetic retinopathy, tumor growth, and metastasis. Interestingly, potent endothelial mitogenic stimuli, such as vascular endothelial growth factor (VEGF) as well as basic fibroblast growth factor, protect endothelial cells against apoptosis.1 2 3 Moreover, angiostatin, an inhibitor of neoangiogenesis, induces endothelial cell apoptosis.4 Thus, the balance between positive and negative regulators of endothelial cell death may determine vascular development.
Recent studies also provide evidence that activation of the serine/threonine protein kinase Akt, also known as protein kinase B or Rac kinase, is involved in embryonic vascular development and neoangiogenesis.5 Stimulation of the endothelium-specific receptor tyrosine kinase Tie2 triggers the activation of Akt.5 Moreover, hypoxia-induced expression of VEGF involves the Akt signaling pathway.6 Furthermore, activation of Akt kinase potently prevents apoptotic cell death.7 8 9 10 Stimulation of the protein kinase Akt was shown to be dependent on phosphoinositide 3-OH kinase (PI3K), which mediates growth factors and hypoxia-induced Akt activation.11 12 13 14
The hormone insulin exerts various biological effects. For example, insulin is mitogenic in certain cell types15 16 and potently suppresses apoptotic cell death in fibroblasts and neuronal cells.17 However, the effects of insulin on endothelial cell survival are poorly defined. Therefore, the aim of the present study was to investigate the effect of insulin on endothelial cell apoptosis and to elucidate the underlying signaling events. Because the Akt kinase pathway has been shown to be involved in apoptosis suppression as well as angiogenesis, we determined the role of protein kinase Akt in the insulin-mediated inhibition of endothelial cell apoptosis.
The results of the present study demonstrate that insulin prevents
TNF-–induced apoptosis of endothelial
cells. The apoptosis-suppressive effects of insulin are
mediated by PI3K-dependent activation of protein kinase Akt, as
demonstrated by the pharmacological inhibition of PI3K and the
overexpression of a dominant-negative Akt mutant. Insulin thereby
stimulated the phosphorylation of Akt and, moreover,
counteracted the TNF-–induced dephosphorylation of
Akt. Furthermore, the effects of Akt on endothelial
cell survival are independent of p70 S6 kinase and the
phosphorylation of the proapoptotic Bad
protein.
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Methods |
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Cell Culture
Human umbilical vein endothelial cells (HUVECs) were purchased from Cell Systems/Clonetics, Solingen, Germany, and were cultured in endothelial basal medium (EGM-1) supplemented with hydrocortisone (1 µg/mL), bovine brain extract (3 µg/mL), gentamicin (50 µg/mL), amphotericin B (50 µg/mL), epidermal growth factor (10 µg/mL), and 10% FCS until the third passage. After detachment with trypsin, cells (3.5x105) were grown in 6-cm cell-culture dishes for at least 18 hours in EGM-1 (Clonetics) with 10% FCS. Insulin or sphingosine 1-phosphate (SPP) was preincubated for 15 minutes before addition of TNF-
. Wortmannin and Ly-294002 were
preincubated for 30 minutes before insulin addition. For serum
starvation, complete EGM-1 with 10% FCS was replaced by EGM-1 without
supplements.
Determination of Akt Activation and Western Blot Analysis
For determination of the phosphorylated form of
Akt, HUVECs were washed twice with ice-cold PBS followed by incubation
of the wells with 200 µL of cell lysis buffer (20 mmol/L Tris
[pH 7.4], 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L
EGTA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L
ß-glycerophosphate, 1 mmol/L
Na3VO4, 1 µg/mL
leupeptin, and 1 mmol/L PMSF) for 5 minutes on ice. Then the cells
were scraped off the plates and sonified with a Branson Sonifier (3x 5
seconds, output control 2, duty cycle 100%) on ice. After
centrifugation for 10 minutes at 20 000g at
4°C, the protein concentration was determined in the supernatant by
using the Bio-Rad reagent (Bio-Rad).
Proteins (50 µg per lane) were loaded onto 8% SDS-polyacrylamide gels and blotted onto polyvinylidenedifluoride membranes. After they were blocked with 5% milk powder at room temperature for 2 hours, the antibodies were incubated as follows: for Akt or phospho-Akt (Biolabs) 1:500, 4°C overnight in Tris-buffered saline (TBS; 50 mmol/L Tris-HCl [pH 8], 150 mmol/L NaCl, and 2.5 mmol/L KCl), 0.1% Tween-20, and 3% BSA. After incubation with the second antibody (anti-rabbit, 1:4000) for 1 hour, enhanced chemiluminescence was performed according to the instructions of the manufacturer (Amersham). Fifteen microliters of phosphorylated or unphosphorylated Akt (Biolabs) was loaded as a positive or negative control, respectively. Then the blots were reprobed with actin (1:2000 in TBS, 0.1% Tween-20, and 3% BSA for 2 hours; Boehringer Mannheim).
For detection of Bad phosphorylation, phosphospecific Ser 112 antibodies (1:500 in TBS, Tween-20, and 3% BSA) or phosphospecific Ser 136 antibodies (1:500 in TBS, Tween-20, 3% milk powder, and 0.6% FCS) were used. The antibodies and the control protein were supplied by New England Biolabs. The autoradiograms were scanned and semiquantitatively analyzed.
Caspase-9 Activity
For detection of caspase-9 activity,
4x105 cells were lysed in 100 µL of buffer
(1% Triton X-100, 0.32 mol/L sucrose, 5 mmol/L EDTA, 1
mmol/L PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 2 mmol/L
DTT, and 10 mmol/L Tris-HCl [pH 8]) for 20 minutes at 4°C,
followed by centrifugation (20 000g for 15
minutes). Caspase-9 activity was detected in the resulting supernatants
by measuring the proteolytic cleavage of the fluorogenic substrate
7-amino-4-trifluoromethyl coumarin (AFC)–LEHD with AFC as the standard
and by using an excitation wavelength of 400 nm and an emission
wavelength of 505 nm. Then 70 µL of supernatant was incubated with
630 µL of assay buffer (100 mmol/L HEPES, 10% sucrose, 0.1%
CHAPS [pH 7.5], 2 mmol/L DTT, 200 µmol/L PMSF, 10 µg/mL
aprotinin, and 10 µg/mL leupeptin) for 5 minutes before starting the
enzymatic reaction with 48 µmol/L AFC-LEHD. Protein content was
analyzed with the Bio-Rad assay (Bio-Rad), and enzyme activity
was calculated as moles of AFC released per milligram protein per
second.
Detection of Apoptosis
For morphological staining of nuclei, cells were
centrifuged (10 minutes, 700g), fixed in 4%
formaldehyde, and stained with 4',6-diamidino-2-phenylindole (DAPI; 0.2
µg/mL in 10 mmol/L Tris-HCl [pH 7], 10 mmol/L EDTA, and
100 mmol/L NaCl) for 20 minutes. Five hundred cells were counted
by 2 independent investigators blinded to the nature of the
experiment, and the percentage of apoptotic cells per
total number of cells was determined.
For internucleosomal DNA laddering, 106 cells were removed from the culture flask, washed with PBS, and incubated in lysis buffer (5 mmol/L Tris-HCl [pH 8], 20 mmol/L EDTA, and 0.5% Triton X-100) for 15 minutes at 4°C. Then the samples were incubated with RNAse A for 1 hour at 37°C, followed by addition of a final concentration of 0.5 mg/mL proteinase K and 1% SDS; afterward, the samples were incubated overnight at 65°C. After isolation of DNA by phenol-chloroform extraction, the DNA was precipitated with 70% isopropanol and 0.1 mol/L NaCl. The resulting pellet was resolved in Tris-EDTA buffer (10 mmol/L Tris-HCl [pH 8] and 1 mmol/L EDTA), and the DNA samples were incubated with 5 U of Klenow polymerase and 0.5 µCi of [32P]dCTP in the presence of 10 mmol/L Tris-HCl (pH 7.5) and 5 mmol/L MgCl2 for 10 minutes at room temperature according to Rösl.18 The reaction was terminated by addition of 10 mmol/L EDTA, and the unincorporated nucleotides were removed with Sephadex G-50 spin columns. Labeled DNA fragments were separated on a 1.0% agarose gel, transferred to nitrocellulose membranes, and exposed to x-ray film.
Transfection
The plasmid encoding the dominant-negative Akt mutant (Aktmt;
truncated form) was kindly donated by Dr Julian Downward
(Imperial Cancer Research Fund, London, UK).19 After
digestion with HincII/EcoRI, Aktmt was subcloned
into the respective sites (EcoRV/EcoRI) of
pcDNA3.1 (InVitrogen). Caspase-9 was cloned by polymerase chain
reaction (PCR) into the EcoRV/BamHI sites of the
pcDNA3.1 vector. Ser 196 was mutated by PCR-directed mutagenesis into
alanine. HUVECs were cotransfected with pcDNA3.1-LacZ and either
pcDNA3.1-Aktmt or pcDNA3.1 control vector lacking an insert as
described.7 For this purpose, 150 µL of EGM-1
without FCS was mixed with 3 µg of plasmids (1 µg of pcDNA3.1-LacZ
and 2 µg of pcDNA3.1-Aktmt or pcDNA3.1) and 30 µL of Superfect and
incubated for 10 minutes at room temperature. During the incubation
time, EGM-1 with 10% FCS was removed from the cell-culture plates, and
HUVECs were washed once in EGM-1 without FCS. Complete medium (1 mL)
with 10% FCS was added to the plasmid-Superfect mixture, and HUVECs
were incubated with this mixture for 3 hours at 37°C. After
incubation, the culture medium was removed, 3 mL of fresh EGM-1 with
10% FCS was added, and HUVECs were incubated for 24 hours to allow for
protein expression. The transfected cells were identified by
ß-galactosidase staining. Then the plates were centrifuged to
pellet the detached cells. The cells were subsequently fixed in 2%
formalin–0.2% glutaraldehyde, and ß-galactosidase
activity was determined by incubation with 40 µg/mL
5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside for 6
hours at 37°C. The transfection efficiency in this study was
25±0.2% in pcDNA3.1- and pcDNA3.1-LacZ–cotransfected cells.
Transfection with 3 µg of pcDNA3.1-LacZ results in an 
53±16%
transfection efficiency. Viable versus dead (stained) cells were
counted by 2 blinded investigators, and results were expressed as
dead/total cellsx100. In addition, necrotic cell death was
excluded by measuring lactate dehydrogenase release, thus indicating
that death of the transfected cells was caused by apoptosis.
Statistical Analysis
Data are expressed as mean±SEM from at least 3 independent
experiments. Statistical analysis was performed with ANOVA
followed by a modified least significant difference test (SPSS
Software).
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Results |
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Effects of Insulin on TNF-
–Induced Apoptosis
in HUVECsTo investigate the effect of insulin on endothelial cell survival, apoptosis of HUVECs was induced by TNF-
as previously
described.20 HUVECs were cultivated in FCS-containing
medium and incubated with TNF- (50 ng/mL) for 18 hours.
Apoptosis was detected by morphological analysis of
DAPI-stained cells as shown in Figure 1A
.
TNF- stimulated apoptosis in 7.5±2.3% of the cells
compared with 2.9±1% in unstimulated control cells. Incubation with
insulin dose-dependently inhibited TNF-–induced apoptosis
(Figure 1B). Inhibition of TNF-–induced apoptosis by
100 nmol/L insulin was further confirmed by prevention of the typical
DNA laddering (Figure 1C). These findings indicate that insulin
protects endothelial cells against apoptosis
induced by TNF-. In addition, incubation of
endothelial cells with insulin reduced the basal levels
of apoptosis by 21%. Moreover, apoptosis induced by
angiotensin II (Ang II) was completely prevented by insulin
(Ang II 5.2±0.25% compared with Ang II plus insulin 2.6±0.1%,
P<0.05), indicating a general antiapoptotic action
of insulin.
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Furthermore, we compared the apoptosis-suppressive effect of
insulin with that of VEGF.1 2 VEGF dose-dependently
prevented TNF-–induced apoptosis with maximal effects at
100 ng/mL (with TNF-, 7.5±2.3% apoptotic cells compared
with TNF- plus VEGF 2.2±0.7%, P<0.05). Moreover,
similar results were obtained when apoptosis was induced by
growth factor withdrawal. Incubation of HUVECs for 24 hours in EGM-1
without supplements induced apoptosis in 57±7% of the cells.
Coincubation with VEGF (100 ng/mL) or insulin (100 nmol/L)
significantly reduced apoptosis to 35±7% and 27±9%,
respectively (P<0.001). Thus, the
apoptosis-suppressive effect of insulin is essentially
equipotent to the effect of VEGF.
Effects of Insulin on the PI3K/Akt Pathway in HUVECs
Insulin has been shown to stimulate the protein kinase Akt via
activation of PI3K.21 To elucidate the effects of insulin
on Akt in endothelial cells, Akt
phosphorylation was determined by Western blotting with
a phosphospecific Akt antibody, which has been shown to correlate with
enzyme activity.7 Therefore, HUVECs were serum-starved for
12 hours and then incubated with insulin. Insulin dose- and
time-dependently increased Akt phosphorylation (Figures 2A and 2B), with a maximal increase of
3-fold after incubation with 100 nmol/L insulin for 1 hour. In the
presence of serum, insulin also induced a 2-fold increase in Akt
phosphorylation. Western blot analysis with
antibodies against total Akt revealed equal protein levels in all
samples, thus demonstrating that the increased amount of
phosphorylated Akt after insulin stimulation was not
due to increased expression of Akt (data not shown). In addition,
reprobing of the Western blot with an anti-actin antibody demonstrated
equal loading (Figures 2A and 2B).
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To verify the contribution of PI3K in mediating Akt
phosphorylation, the effect of PI3K
inhibitors was investigated. Preincubation of HUVECs with
the PI3K inhibitors wortmannin (20 nmol/L) or Ly-294002
(10 µmol/L) completely prevented the insulin-induced Akt
phosphorylation, as illustrated in Figure 2C
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Insulin Inhibits TNF-–Induced Apoptosis via the
PI3K/Akt Pathway
Having demonstrated that insulin stimulates Akt
phosphorylation via PI3K in HUVECs, we tested the
contribution of Akt in the apoptosis-suppressive effect of
insulin. Inhibition of PI3K by wortmannin (20 nmol/L) or Ly-294002
(10 µmol/L) completely abrogated the
apoptosis-suppressive effect of insulin (100 nmol/L) on
TNF-–induced apoptosis (Figure 3A and data not shown). The compounds did
not significantly enhance TNF-–induced apoptosis (Figure 3A) and further elicited only a minor effect on basal
apoptosis (Ly-294002 171±31% and wortmannin 106±1.5%). To
test whether the activation of Akt is involved in the
antiapoptotic effect of insulin, we transfected HUVECs with
Aktmt, a dominant-negative Akt mutant. Overexpression of the
dominant-negative Aktmt inhibited insulin-stimulated Akt activation
(data not shown). Moreover, the antiapoptotic effect of insulin
on TNF-–induced apoptosis was completely abolished in cells
transfected with dominant-negative Aktmt, whereas insulin still exerted
protective effects in cells transfected with a control vector (Figure 3B). Transfection of the cells with dominant-negative Aktmt did
not enhance basal or TNF-–induced apoptosis (Figure 3B).
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TNF- Induces Dephosphorylation of Akt
To further test whether TNF- affects basal Akt activity, HUVECs
were cultured in FCS-containing medium to allow for basal Akt
activation (see the first lane in Figure 4A) and then incubated with TNF- for
various times. TNF- (50 ng/mL) induced a time-dependent decrease in
basal Akt phosphorylation (Figure 4A). Insulin
prevented the dephosphorylation of Akt induced by
TNF- (Figure 4A), thus supporting the important role of Akt
in preventing apoptosis. Recently, it has been shown that the
protease family of caspases, which are activated by TNF-,
specifically cleave the protein kinase Akt, thereby lowering the amount
of Akt.22 However, Western blotting with antibodies
against phosphorylated and
unphosphorylated forms of Akt did not reveal a decrease
of Akt protein levels after TNF- stimulation (Figure 4B),
thus suggesting that TNF- induces the
dephosphorylation of Akt but not the cleavage of Akt. A
recent study showed that ceramides trigger the
dephosphorylation of Akt.23 Because
TNF- is known to stimulate ceramide release,24 we
investigated whether SPP, a metabolite of ceramide that is known to
inhibit ceramide-induced apoptosis,25 can prevent
TNF-–induced Akt dephosphorylation. As shown in
Figure 4C, coincubation of HUVECs with TNF- (50 ng/mL) and
SPP (1 µmol/L) completely blocked the TNF-–induced
dephosphorylation of Akt and moreover enhanced the
phosphorylation of Akt compared with unstimulated
controls. In addition, SPP inhibited TNF-–induced apoptosis
(Figure 4D), suggesting an important role for ceramide in
TNF-–induced Akt dephosphorylation.
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Downstream Effects of Akt
Having demonstrated that insulin counteracts TNF-–induced
dephosphorylation of Akt, thereby preventing
TNF-–induced apoptosis, we investigated the role of
potential downstream targets of Akt, p70 S6 kinase26 and
the Bcl-2–like proapoptotic protein Bad. However, the
antiapoptotic effect of insulin was not prevented by the
p70 S6 kinase inhibitor rapamycin (TNF- 257±40%,
TNF- plus insulin 157±26%, TNF- plus insulin plus rapamycin
159±37%, and rapamycin alone 121±30%). Furthermore, insulin did not
stimulate the phosphorylation of Bad as detected by
Western blotting with phosphospecific antibodies against the Akt sites
Ser 112 and Ser 136, although the control proteins were detected
(Figure 5A). Moreover, incubation of
HUVECs with insulin did not induce a mobility shift of Bad protein on
the SDS–polyacrylamide gel electrophoresis gels (data not
shown).
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Insulin has been shown to stimulate the production of NO in
endothelial cells in a PI3K-dependent
manner.27 Moreover, we have recently shown that the
phosphorylation of endothelial NO
synthase by Akt enhances the enzymatic activity of NO
synthase.28 However, inhibition of NO synthase by
treatment of the cells with
NG-monomethyl-L-arginine
(LNMMA, 1 mmol/L) did not reduce the antiapoptotic effect
of insulin (Figure 5B), suggesting that the Akt-mediated
antiapoptotic effect of insulin is independent of the
generation of NO.
Recent studies provide evidence that Akt phosphorylates
caspase-9 at Ser 196 and thereby inhibits its activity.29
Indeed, insulin inhibited the TNF-–stimulated caspase-9 activity in
a wortmannin-sensitive manner (TNF- 155±36%, TNF- with or
without insulin 118+28%, and TNF- plus insulin plus wortmannin
172±54%; P<0.05). These results were confirmed by
detection of caspase-9 cleavage in Western blots. Thus, inhibition of
TNF- as mediated by insulin was reversed by wortmannin coincubation
(data not shown). Moreover, apoptosis directly induced by
transfection of HUVECs with caspase-9 was inhibited by insulin (Figure 5C). In contrast, the insulin effect was significantly reduced
when apoptosis was induced by overexpression of a caspase-9
mutant, wherein the Akt site Ser 196 was mutated to alanine (Figure 5C). These results suggest that the Akt-mediated
antiapoptotic effects of insulin are at least in part mediated
by phosphorylation of Ser 196 within caspase-9.
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Discussion |
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Angiogenesis, the development of new blood vessels, is a highly regulated process that is important for the neovascularization of new tissue and revascularization after ischemic infarction and counteracts the vascular regression after injury of the blood vessel. Angiogenesis is also involved in several pathological processes, like vascularization of tumor tissue or proliferative diabetic retinopathy.30 Angiogenesis is mainly determined by cell proliferation and the inhibition of cell death combined, processes mediated by growth factors and hormones. Here, we demonstrate that insulin protects endothelial cells from induction of apoptosis induced by TNF-
or Ang II.
The antiapoptotic effect of insulin is thereby equipotent to
the potent angiogenic factor VEGF. Furthermore, the present study
demonstrates that the apoptosis-suppressive effects of insulin
are mediated by activation of the protein kinase Akt.
The effects of insulin on protein synthesis, glucose uptake, and
cellular growth are known to be mediated via the insulin receptor and
the subsequent stimulation of insulin receptor substrate-1–mediated
activation of PI3K.31 In line with these general
considerations, the insulin-mediated activation of Akt in HUVECs was
PI3K dependent, as confirmed by our use of the pharmacological PI3K
inhibitors wortmannin and Ly-294002. In
nonendothelial cells, the apoptosis-suppressive
effect of insulin has been shown to be mediated by PI3K-dependent
activation of Akt.17 32 Indeed, similar results were
obtained in the present study, wherein TNF-–induced
apoptosis of endothelial cells was inhibited by
insulin in a PI3K-dependent fashion. Moreover, overexpression of the
dominant-negative Akt mutant, Aktmt, completely abolished the
apoptosis-suppressive effect of insulin, indicating that the
insulin-induced, PI3K-dependent activation of Akt is indeed necessary
for the antiapoptotic function of insulin in HUVECs. In
addition, we excluded a potential role for p70 S6 kinase, which is
known to be activated by PI3K/Akt,26 33 in the
antiapoptotic effect of insulin. Inhibition of p70 S6 kinase by
rapamycin did not affect the inhibition of apoptosis by
insulin. This result is consistent with recent data showing
that PI3K-dependent activation of Akt, but not of p70 S6 kinase, is
necessary for the prevention of apoptosis in rat-1
cells10 and neuronal cells.17
Taken together, these findings indicate that insulin exerts
antiapoptotic effects via the protein kinase Akt in a variety
of cellular models, suggesting a general antiapoptotic
principle. The present study shows, for the first time, the
inhibition of endothelial cell apoptosis by
insulin and links this cell-protective effect of insulin to the
activation of Akt. In addition, our findings demonstrate that insulin
not only counteracts apoptosis due to growth factor withdrawal
but also prevents TNF- receptor–mediated cell death, which occurs
in the presence of growth factors. TNF- has been shown to mediate
its proapoptotic effect via specific signaling pathways,
leading to activation of the caspase cascade.20 Our data
now extend these findings, giving evidence that TNF-
dephosphorylates and deactivates Akt kinase.
The dephosphorylation of Akt kinase therefore precedes
the activation of caspases and cell death. Two to 3 hours after TNF-
stimulation, Akt was maximally dephosphorylated,
whereas apoptosis induction and caspase activation were
observed between 12 and 18 hours afterward under our experimental
setting.20 A recent study suggests that caspase-induced
cleavage of Akt may contribute to cell death.22 However,
our data demonstrate that TNF- treatment does not affect the protein
level of Akt but significantly reduces its
phosphorylation. These data are in accordance with
those of Kennedy et al34 or Cardone et al,29
who demonstrated that the protein kinase Akt is not a substrate for
caspases but, in contrast, directly inhibits caspase activation. Thus,
inhibition of Akt by TNF- might have a major impact on the
TNF-–induced activation of caspases and the induction of
apoptosis. The TNF-–induced
dephosphorylation of Akt is therefore likely mediated
via the ceramide pathway. In fact, the ceramide metabolite SPP, which
antagonizes ceramide actions,25 prevented
dephosphorylation of Akt, thus correlating with the
inhibition of TNF-–induced apoptosis.
The TNF-–induced dephosphorylation of Akt was
abrogated by coincubation with insulin. Thus, insulin not only
stimulates basal Akt activity but also reverses the
dephosphorylation of Akt induced by TNF-. The
interference of insulin in TNF-–induced Akt
dephosphorylation could be explained by enhanced
activation of Akt, thus compensating for the induced
dephosphorylation, or by inhibition of the signaling
cascade leading to Akt dephosphorylation. Whereas
preincubation of TNF- and C2-ceramide has been
shown to inhibit insulin signaling,35 36 the present
study now demonstrates that insulin, when added before or
simultaneously (data not shown), can prevent
TNF-–induced signaling in endothelial cells.
Having demonstrated that the activity of Akt is an important survival signal for endothelial cells, we further investigated the downstream effector pathways. A recent study gives evidence that the apoptosis-suppressive effect of Akt is mediated via the proapoptotic Bcl-2 family member Bad, which is inactivated by Akt-dependent phosphorylation.12 Bad, in its unphosphorylated form, is thought to induce cell death via heterodimerization or homodimerization. Growth factors as well as interleukin-3 and interleukin-4 have been shown to suppress apoptosis by inducing phosphorylation of Bad at Ser 112 and Ser 136, which can be confirmed by its slower migration on SDS-polyacrylamide gel electrophoresis (PAGE) gels.12 37 Hence, phosphorylation of Bad at Ser 136, which has been shown to be Akt dependent, seems to be the basic requirement for the antiapoptotic action of Bad. However, we could not detect a modification of Bad migration on SDS-PAGE after incubation with insulin. Furthermore, no phosphorylation of Ser 112 or Ser 136 was detectable in insulin-stimulated HUVECs on Western blots treated with phosphospecific antibodies, although the control protein was recognized. These data suggest that Akt may affect other targets besides Bad. Indeed, this suggestion is supported by a recent publication demonstrating that Akt-mediated cell survival is not causally related to phosphorylation of Bad protein.37
Recent studies provide evidence that Akt stimulates the
phosphorylation of endothelial NO
synthase and thereby activates the enzyme.28
However, blocking the insulin-stimulated generation of NO by LNMMA, an
inhibitor of NO synthase, did not reduce the
apoptosis-suppressive effect of insulin on TNF-–induced
apoptosis of HUVECs.
Finally, we analyzed the contribution of Akt-stimulated
phosphorylation of Ser 196 within caspase-9, which
inhibits caspase-9 activity.29 Indeed, TNF-–induced
caspase-9 activity was reduced by insulin in a PI3K-dependent manner.
Moreover, caspase-9–induced apoptosis was significantly
prevented by insulin. Most important, apoptosis induced by
overexpression of a caspase-9 mutant lacking the Akt
phosphorylation site Ser 196 was only partially
reversed by insulin. Taken together, the suppression of
apoptosis by insulin appears to be at least in part mediated by
Akt-stimulated caspase-9 phosphorylation and
inactivation, whereas the other Akt substrates Bad,
endothelial NO synthase, or p70 S6 kinase do not seem
to be involved. A very recent study demonstrated that the inhibition of
glycogen synthase kinase-3 by Akt-dependent
phosphorylation prevented growth factor
withdrawal–induced apoptosis.38 Further studies
are required to elucidate the role of glycogen synthase kinase-3 in
insulin-mediated inhibition of endothelial cell
apoptosis.
To summarize, this study reveals an important role for Akt in the
survival of endothelial cells. The proposed signal
transduction pathways suggested by the data of the present study
are illustrated in Figure 6. The
antiapoptotic effect of insulin and VEGF (data not shown) is
mediated via activation of Akt in a PI3K-dependent manner. The
cell-protective effects of insulin in endothelial cells
are mediated not only by enhancing basal activity of the protein kinase
Akt but also by abrogating the TNF-–induced Akt
dephosphorylation.
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Beyond that, this study now indicates that insulin prevents apoptotic death of endothelial cells induced by inflammatory stimuli and the proatherosclerotic factor Ang II. This effect may importantly contribute to cell homeostasis and protect the integrity of the endothelium. This may be of general importance for angiogenesis, for which an undisturbed proliferation of cells is a prerequisite. In fact, angiogenesis occurs as a natural reaction to chronic ischemia. Because the time course of spontaneous neovascularization under pathophysiological conditions is too slow, selective stimulation of the Akt pathway may provide a novel therapeutic approach to enhance the neovascularization process.
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Acknowledgments |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft Di 600/2–3 and SFB-553. C.H. has a fellowship from Boehringer Ingelheim Fond.
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Footnotes |
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The first 2 authors contributed equally to this work.
Received March 26, 1999; accepted August 30, 1999.
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