Cell Biology/Signaling
Peroxisome Proliferator-Activated Receptor-␥ Coactivator
1-␣ Overexpression Prevents Endothelial Apoptosis by
Increasing ATP/ADP Translocase Activity
Jong Chul Won; Joong-Yeol Park; Yun Mi Kim; Eun Hee Koh; Somi Seol; Byeong Hwan Jeon;
Jin Han; Jung Ran Kim; Tae-Sik Park; Cheol Soo Choi; Woo Je Lee; Min-Seon Kim; In-Kyu Lee;
Jang Hyun Youn; Ki-Up Lee
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Objective—Fatty acids increase reactive oxygen species generation and cell apoptosis in endothelial cells. The peroxisome
proliferator-activated receptor-␥ coactivator 1-␣ (PGC-1␣) is a transcriptional coactivator that increases mitochondrial
biogenesis and fatty acid oxidation in various cells. This study was undertaken to investigate the possible preventive
effect of PGC-1␣ on endothelial apoptosis and its molecular mechanism.
Methods and Results—Treatment with linoleic acid in cultured human aortic endothelial cells increased reactive oxygen
species generation and cell apoptosis. These effects appeared to be mediated by increases in cytosolic fat metabolites,
ie, fatty acyl CoA, diacylglycerol, and ceramide, and consequent decreases in ATP/ADP translocase activity of adenine
nucleotide translocator. Adenoviral overexpression of PGC-1␣ prevented linoleic acid-induced increases in reactive
oxygen species generation and cell apoptosis in human aortic endothelial cells by increasing fatty acid oxidation,
decreasing diacylglycerol and ceramide, and increasing ATP/ADP translocase activity. In isolated aorta, PGC-1␣
overexpression prevented linoleic acid-induced decrease in endothelium-dependent vasorelaxation, and this effect was
abolished by adenine nucleotide translocator1 shRNA.
Conclusions—PGC-1␣ regulates reactive oxygen species generation and apoptosis in endothelial cells by increasing fatty
acid oxidation and enhancing ATP/ADP translocase activity. Measures to increase PGC-1␣ expression or ATP/ADP
translocase activity in vascular cells may aid in the prevention or treatment of atherosclerosis. (Arterioscler Thromb
Vasc Biol. 2010;30:290-297.)
Key Words: adenine nucleotide translocator 䡲 peroxisome proliferator-actived receptor-␥ coactivator 1-␣
䡲 endothelial apoptosis 䡲 mitochondrial membrane potential 䡲 reactive oxygen species
C
entral obesity is associated with increased cardiovascular
morbidity and mortality.1 Endothelial cell apoptosis and
consequent impairment of endothelium-dependent vascular
relaxation (endothelial dysfunction) are important early
events in the pathogenesis of atherosclerosis.2 Increased
levels of plasma free fatty acids in obesity may lead to
endothelial cell apoptosis by increasing the accumulation of
lipid metabolites and the generation of reactive oxygen
species (ROS).3,4
Major sites of intracellular ROS generation are mitochondria and cell membrane NAD(P)H oxidase.4 The mitochondrial respiratory chain generates ROS when the electrochemical gradient between the mitochondrial inner membrane is
high and the rate of electron transport is limited. In oxidative
phosphorylation, electrons are transferred from electron donors, NADH or FADH, to electron acceptors in the mitochondrial electron transport chain. This transfer releases energy,
and most of the energy is captured by proton pumps that build
a proton gradient across the mitochondrial inner membrane
(⌬␺m), which is the driving force for the phosphorylation of
ADP to ATP by ATP synthase.5,6 However, sustained increase in ⌬␺m impairs the flow of electrons through the ETC
and increases the accidental transfer of electrons to oxygen to
form superoxide.6,7
⌬␺m is determined by the balance between the export of
protons from the mitochondrial matrix into the intermembra-
Received January 9, 2009; revision accepted November 23, 2009.
From Department of Internal Medicine (J.C.W., E.H.K., W.J.L., M.-S.K., J.-Y.P., K.-U.L.), University of Ulsan College of Medicine, Seoul, Korea;
Asan Institute for Life Sciences (Y.M.K., S.S., B.H.J.), Seoul, Korea; Mitochondrial Signaling Laboratory (J.H.), Department of Physiology and
Biophysics, College of Medicine, Inje University, Pusan, Korea; Lee Gil Ya Cancer and Diabetes Institute (J.R.K., T.-S.P., C.S.C), Gachon University
of Medicine and Science, Incheon, Korea; Department of Internal Medicine and Biochemistry and Cell Biology (I.-K.L.), Kyungpook National University
School of Medicine, Daegu, Korea; Department of Physiology and Biophysics (J.H.Y.), University of Southern California Keck School of Medicine, Los
Angeles, Calif; Department of Internal Medicine, Mitochondrial Research Group, Inje University College of Medicine, Seoul, Korea (J.C.W.).
J.C.W. and J.-Y.P. contributed equally contributed to this work.
Correspondence to Ki-Up Lee, MD, PhD, Department of Internal Medicine, University of Ulsan College of Medicine, Song-Pa P.O. Box 145, Seoul
138-736, Korea. E-mail [email protected]
© 2010 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
290
DOI: 10.1161/ATVBAHA.109.198721
Won et al
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nous space and the import of protons into the matrix through
ATP synthase and uncoupling proteins (UCP).6 The ATP
synthesized in the mitochondria is exchanged for cytosolic
ADP by adenine nucleotide translocator (ANT) to provide a
continuous supply of ADP. Intramitochondrial ADP deficiency resulting from reduced ATP/ADP translocase activity
of ANT slows the rate of ATP synthesis and increases ⌬␺m.8
Thus, ATP/ADP exchange by ANT is essential for the
maintenance of ATP synthase activity and normal levels of
⌬␺m.8 In addition, ANT is responsible for a significant
portion of basal uncoupling or proton leak5,9 independent of
its function in ATP/ADP translocase.10 The proton conductance of mitochondria depends on ANT content,11 and ANT
and UCP constitute 2 major molecules that determine mitochondrial uncoupling.5
The peroxisome proliferator-activated receptor-␥ coactivator 1-␣ (PGC-1␣) is a transcriptional coactivator of nuclear
receptors involved in cellular energy metabolism.12 PGC-1␣
increases mitochondrial biogenesis and fatty acid oxidation
(FAO) in various cells, including endothelial cells.13 In
addition, recent studies have indicated that PGC-1␣ is a major
regulator of intracellular ROS generation; PGC-1␣ was
shown to increase the expression of ROS-detoxifying enzymes.14,15 Here, we show an additional novel mechanism by
which PGC-1␣ reduces cell apoptosis and ROS generation in
endothelial cells; PGC-1␣ overexpression normalizes fatty
acid-induced increases in mitochondrial membrane potential
(⌬␺m) and ROS generation by increasing ATP/ADP translocase activity of ANT.
Materials and Methods
Expanded methods are available in the online data supplement.
Cell Culture
Human aortic endothelial cells (HAEC; BioWhittaker) were cultured
in endothelial growth medium-2 (BioWhittaker) supplemented with
specific growth factors and 2% fetal bovine serum.
Transfection of ANT siRNA, Ad-PGC-1␣, and
Linoleic Acid Treatment
Fifty percent to 60% confluent HAEC were infected with adenoviruses carrying ␤-gal (Ad-␤-gal) or PGC-1␣ (Ad-PGC-1␣) at a titer
of 5⫻106 pfu/mL for 1 hour at 37°C in DMEM without serum.
Infected HAEC were then incubated in endothelial growth medium-2
with growth factors and 2% fetal bovine serum for 48 hours. After
that, the media were replaced with M199 (BioWhittaker) without
growth factors and with 1% fetal bovine serum for 1 hour, and then
the cells were treated with 60 ␮mol/L of linoleic acid (LA) or same
amounts of vehicle for indicated times. For the experiments using
ANT1 siRNA, 50 nM of ANT1 siRNA or control siRNA was
transfected into HAEC using LipofectAMINE 2000 (Invitrogen) 24
hours before Ad-PGC-1␣ infection.
Analysis of Apoptosis
Apoptosis was measured by various methods, including a cell death
enzyme-linked immunosorbent assay kit examining cytoplasmic
histone-associated DNA fragmentation (Roche Diagnostics), ApoAlert
caspase 3 fluorescence assay kit (Clontech), and Western blots for
cleaved caspase 3 and poly (ADP-ribose) polymerase.16,17
Measurement of ROS Levels
Intracellular ROS levels and mitochondrial-specific ROS generation
was measured by flow cytometry using DCFH2-DA (Molecular
PGC-1␣ Overexpression
291
Probes) and MitoSOX Red fluorescent dye (Molecular Probes),
respectively (see online Methods).
Measurement of ⌬␺m
The ⌬␺m was measured by flow cytometry using JC-1 (Molecular
Probes; see online Methods).
Measurement of ATP/ADP Translocase Activity
of ANT
ATP/ADP translocase activity was measured as previously described18 (see online Methods).
Western Blot Analysis
Protein expression in cells was measured by Western blot analysis as
previously described.19
Real-Time Polymerase Chain Reaction Analysis
The mRNA expression in cells was measured by real-time polymerase chain reaction analysis (see online Methods).
Animals
Eight-week-old male Sprague-Dawley rats (Orient, Sungnam,
Korea) weighing 250 to 300 g were housed in cages containing 4 rats
per cage and allowed ad libitum access to water and food. All
experimental procedures were approved by the Institutional Animal
Care and Use Committee of the Asan Institute for Life Sciences.
Ex Vivo Measurement of
Endothelium-Dependent Vasorelaxation
Endothelium-dependent vascular relaxation was measured as previously described.20 The thoracic aorta was excised from SpragueDawley rats after euthanization with pentobarbital, and then cleaned
to remove fat and adhering tissue. The vessel was cut into several
individual ring segments 2 to 3 mm in width. Adenoviral overexpression of PGC-1␣ or knockdown of ANT1 were achieved by
infection of aortic rings with Ad-PGC-1␣ or Ad-ANT1 shRNA
(6⫻106 pfu/mL each) for 30 minutes at 37°C in DMEM without
serum. The tissues were subsequently incubated in medium containing 5% bovine serum albumin for 24 hours. After that, the aortic
rings were pre-exposed to 60 ␮mol/L of LA for 2 hours, and
contraction was induced by treatment with 300 ␮mol/L phenylephrine. Acetylcholine (from 10⫺9–10⫺5 mol/L) was then added serially
to the bath to induce endothelium-dependent vasorelaxation. The
tension was measured by an isotonic force displacement transducer
(Hugo Sachs Elektronik KG D-7806) and recorded using a polygraph
(Graphtec Linerecorder mark 8 WR3500).
Statistical Analysis
All data are shown as means⫾SEM. Comparisons between 2 groups
were performed using unpaired Student t tests and comparisons
between multiple groups were performed by ANOVA. Timedependent changes of ROS and ⌬␺m were assessed by 2-way
ANOVA. The significance of differences in vascular relaxation
between the 4 experimental groups was assessed by ANOVA with
repeated measures. Differences were classified as significant at
P⬍0.05.
Results
Expanded results are available in the online data supplement
at http://atvb.ahajournals.org.
PGC-1␣ Overexpression Prevents LA-Induced
ROS Generation and Apoptosis
Previous studies established that fatty acids increase ROS
generation and cell apoptosis in endothelial cells.21,22 In
accordance with these studies, incubation of HAEC with LA
significantly increased intracellular and mitochondrial ROS
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February 2010
enzymes and UCP-2 were also significantly increased by
Ad-PGC-1␣ (Figure 2B, supplemental Figure III)14,15 and
reduced by PGC-1␣–specific siRNA (supplemental Figure
IV). This result suggests that endogenous PGC-1␣ plays a
role in regulating the expression of antioxidant enzymes and
UCP-2.14,15 However, in the presence of LA or glucose
oxidase, Ad-PGC-1␣ did not further increase the expression
of antioxidant enzymes or UCP-2 (Figure 2B, supplemental
Figure III). This result suggests that the ability of Ad-PGC-1␣
to normalize LA-induced increases in intracellular ROS
production and cell apoptosis cannot be explained by differences in the expression of antioxidant enzymes or UCP-2.
LA Decreases and PGC-1␣ Overexpression
Increases ATP/ADP Translocase Activity by
Affecting Intracellular Lipid Metabolites
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Figure 1. Time-dependent changes in intracellular (A) and mitochondrial ROS (B) generation in response to LA treatment
(60 ␮mol/L). *P⬍0.05 vs baseline; **P⬍0.05 vs cells infected
with adenoviruses carrying ␤-gal (Ad-␤-gal). C, Apoptosis measured at 16 hours by the cell death enzyme-linked immunosorbent assay kit. *P⬍0.05 vs control; **P⬍0.05 vs Ad-␤-gal (n⫽5,
each).
generation and apoptosis (Figure 1, supplemental Figures I,
II, available online at http://atvb.ahajournals.org). Adenoviral
overexpression of PGC-1␣ (Ad-PGC-1␣) prevented LAinduced increases in ROS generation and apoptosis (Figure 1,
Figure I).
PGC-1␣ Overexpression Increases the Expression
of Antioxidant Genes
LA had a slight but significant effect of increasing endogenous PGC-1␣ protein (Figure 2A). LA or glucose oxidase23
increased the mRNA and protein expression of antioxidant
enzymes, including manganese superoxide dismutase,
copper-zinc superoxide dismutase, catalase, glutathione peroxidase, and uncoupling protein 2 (UCP-2; Figure 2B, Figure
III). This finding is consistent with the notion that oxidative
stress can induce cellular antioxidant responses.24,25 The
mRNA and protein expressions of the same antioxidant
Recent studies have shown that a significant increase in ⌬␺m
(hyperpolarization), which induces ROS production,26 is an
earlier prerequisite for apoptosis.27,28 In our study, LA treatment significantly increased ⌬␺m during the initial 4 hours,
which was followed by depolarization after 20 hours (Figure
3A). Because intramitochondrial ADP deficiency, resulting
from reduced ATP/ADP exchange, is known to slow the rate
of ATP synthesis and to increase ⌬␺m,8 we reasoned that
decreased ATP/ADP translocase activity might be responsible
for the hyperpolarization. In accordance with a previous study,29
ATP/ADP translocase activity, measured by 14C-ADP import,
was significantly decreased after 4 hours of LA treatment
(Figure 3B).
We next examined the mechanism underlying the LAinduced inhibition of ATP/ADP translocase activity. LA
decreased FAO and increased intracellular levels of triglycerides, ceramide, and DAG (Figure 3C, D). In the basal state
(ie, without LA), etomoxir, a carnitine palmitoyl transferase
inhibitor, significantly decreased ATP/ADP translocase activity (data not shown). However, triacsin and myriocin,
inhibitors of fatty acyl CoA synthase and ceramide synthase,
respectively, significantly reduced LA-induced effects on
ATP/ADP translocase activity (Figure 3E). Collectively,
these results suggest that increased levels of lipid metabolites,
ie, fatty acyl CoA, DAG, and ceramide, may be involved in
LA-induced decreases in ATP/ADP translocase activity.
PGC-1␣ overexpression significantly increased FAO and
decreased intracellular triglycerides, ceramide, and DAG
levels (Figure 3C, D). PGC-1␣ overexpression completely
reversed LA-dependent decreases in ATP/ADP translocase
activity (Figure 3B), which was associated with a complete
prevention of LA-induced time-dependent changes in ⌬␺m
(Figure 3A).
Role of ANT1 in Cell Apoptosis and
ROS Generation
Among the known ANT isoforms, ANT1 is predominantly
expressed in the heart, skeletal muscle, and brain.30 We
examined the effects of LA and PGC-1␣ on ANT1 expression
in endothelial cells. LA significantly increased ANT1 expression (Figure 4A), and this effect was prevented by the
antioxidant N-acetylcysteine (Figure 4B). However,
N-acetylcysteine treatment did not affect ATP/ADP translo-
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Figure 2. Effect of LA and
Ad-PGC-1␣ on the expression
of PGC-1␣ and antioxidant
genes. HAEC were infected with
either Ad-␤-gal or Ad-PGC-1␣.
After 48 hours, cells were
treated with control vehicle,
60 ␮mol/L LA, or 1.5 mU/mL
glucose oxidase for 4 hours. A,
Representative Western blots of
PGC-1␣ protein expression. B,
Real-time polymerase chain
reaction analysis of the mRNA
expression of manganese
superoxide dismutase (MnSOD),
copper-zinc superoxide dismutase (Cu/Zn SOD1), catalase,
glutathione peroxidase, and
UCP-2. *P⬍0.05 vs Ad-␤-gal;
**P⬍0.05 vs control (cells not
treated with LA or glucose oxidase) (n⫽5, each).
case activity with or without LA treatment (Figure 4C),
suggesting that increased ROS generation with LA may be
responsible for the increase in ANT1 expression and, more
importantly, that changes in ANT1 expression cannot account
for the decrease in ATP/ADP translocase activity with LA.
PGC-1␣ also increased ANT1 expression but did not further
increase ANT1 expression in the presence of LA (Figure 4A).
We next examined the effects of ANT1 siRNA (Figure 4D)
on ROS generation and apoptosis. ANT1 siRNA significantly
increased cell apoptosis and ROS generation (Figure 4E, F).
This was associated with increased expression of antioxidant
enzymes, ie, manganese superoxide dismutase, copper-zinc
superoxide dismutase, catalase, glutathione peroxidase, and
UCP-2 (supplemental Figure V), and changes in mitochondrial morphology (supplemental Figure VI). Taken together,
these data suggest that reduced ANT1 expression/activity can
cause increased cell apoptosis, ROS generation, and antioxidant enzyme expression, as seen with LA, and support the
notion that LA increases cell apoptosis and ROS generation
by reducing ATP/ADP translocase activity of ANT. Increased
ANT1 expression observed with LA may be a compensatory
response to increased intracellular ROS.
Increased ATP/ADP Translocase Activity Is
Required for Effects of PGC-1␣ to Prevent
LA-Induced Cell Apoptosis and ROS Generation
As already described, PGC-1␣ overexpression completely
prevented LA-induced decreases in ATP/ADP translocase
activity (Figure 3B). We tested whether this effect is critically
required for the beneficial effects of PGC-1␣ to prevent
LA-induced increases in cellular apoptosis and ROS generaFigure 3. PGC-1␣ prevents
LA-induced mitochondrial membrane hyperpolarization by
increasing ATP/ADP translocase
activity. A, Effect of Ad-PGC-1␣
on LA-induced changes in ⌬␺m.
*P⬍0.05 vs baseline; **P⬍0.05 vs
Ad-␤-gal. B–D, Effect of
Ad-PGC-1␣ on ADP/ATP translocase activity, fatty acid oxidation,
and intracellular levels of triglycerides (TG), ceramide and DAG.
HAEC preinfected with Ad-␤-gal
or Ad-PGC-1␣ were treated with
control vehicle or 60 ␮mol/L LA
for 4 hours. *P⬍0.05 vs Ad-␤-gal;
**P⬍0.05 vs control. E, Effect of
500 nM triacsin and 500 nM
myriocin on LA-induced change
in ADP/ATP translocase activity
measured at 4 hours. *P⬍0.05 vs
untreated cells; **P⬍0.05 vs
LA-treated cells (n⫽5, each).
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Figure 4. ANT1 expression is
induced by ROS and reduces it.
A, Effects of LA and PGC-1␣ on
ANT1 mRNA expression. HAEC
preinfected with Ad-␤-gal or
Ad-PGC-1␣ were treated with
control vehicle or 60 ␮mol/L LA
for 4 hours. *P⬍0.05 vs Ad-␤gal. B and C, Effect of
N-acetylcysteine (10 mmol/L) on
ANT1 mRNA expression and
ATP/ADP translocase activity.
*P⬍0.05 vs untreated cells.
**P⬍0.05 vs LA-treated cells.
D–F, Effect of ANT1 siRNA on
ANT1 protein expression, cellular apoptosis, and ROS generation. HAEC were treated with 50
nM of ANT1 siRNA or control
siRNA. ANT1 protein and ROS
generation were measured at 24
hours. Apoptosis was measured
at 48 hours. *P⬍0.05 vs control
siRNA (n⫽5, each).
tion. For this, we examined the effect of ANT1 siRNA on
PGC-1␣– dependent changes in ATP/ADP translocase activity, ROS generation, and apoptosis in the presence of LA.
ANT1 siRNA almost completely abolished PGC-1␣–induced
changes in ATP/ADP translocase activity, cell apoptosis,
ROS generation, and ⌬␺m with LA (Figure 5A–E). These
findings indicate that PGC-1␣ prevents LA-induced ROS
production and apoptosis in HAEC by improving ATP/ADP
translocase activity of ANT.
ANT Mediates the Effects of PGC-1␣ on
LA-Induced Endothelial Dysfunction
To investigate whether PGC-1␣ overexpression in aortic
tissue improves endothelial dysfunction, we infected the
aortic ring of Sprague-Dawley rat with Ad-PGC-1␣ or
Ad-ANT1 shRNA ex vivo (Figure 6A). As reported previously,31 LA treatment significantly decreased endotheliumdependent vascular relaxation in the adenoviruses carrying
␤-gal–infected aortic rings (Figure 6B). Administration of
Figure 5. Effect of ANT1
siRNA on ATP/ADP translocase activity (A), apoptosis (B,
C), intracellular ROS (D), and
time-dependent changes in
⌬␺m (E) (n⫽5, each); 50 nM of
ANT1 siRNA or control siRNA
was transfected into HAEC.
After 24 hours, cells were
infected with Ad-␤-gal or
Ad-PGC-1␣ (5⫻106 pfu/mL).
After 48 hours, cells were
treated with 60 ␮mol/L of LA
or vehicle. ATP/ADP translocase activity and ROS generation were measured at 4
hours, and apoptosis was
measured at 16 hours. A–D,
*P⬍0.05 vs control (without
LA); **P⬍0.05 vs Ad-␤-gal;
#P⬍0.05 vs control siRNA. E,
*P⬍0.05 vs baseline; **P⬍0.05
vs Ad-␤-gal; #P⬍0.05 vs control siRNA.
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Figure 6. Effect of Ad-PGC-1␣
or Ad-ANT1 shRNA on
LA-induced impairment in
vasorelaxation. Adenoviral
overexpression of rat aortic
ring was achieved by infection
with Ad-PGC-1␣ aor Ad-ANT1
shRNA. Endotheliumdependent vasorelaxation was
measured by the method
described in Materials and
Methods. A, Western blots
showing changes in PGC-1␣
and ANT1 expressions. B,
Effect of Ad-PGC-1␣ on
LA-induced (60 ␮mol/L)
impairment of vascular dilatory
response. *P⬍0.05 vs control
(Ad-␤-gal alone); **P⬍0.05 vs
LA plus Ad-␤-gal. C, Effect of
Ad-ANT1 shRNA on Ad-PGC1␣–induced changes in the
vasodilatory response.
*P⬍0.05 vs LA plus Ad-␤-gal
plus Ad-control shRNA;
**P⬍0.05 vs LA plus
Ad-PGC-1␣ plus Ad-control
shRNA (n⫽5, each).
nitric oxide synthase inhibitor L-NAME nearly completely
inhibited acetylcholine-induced vasorelaxation, both in LAtreated and untreated aorta. Because LA treatment was
associated with reduced vasorelaxation in response to acetylcholine, the net difference between L-NAME–treated and
L-NAME– untreated vessels was less in the LA-treated than in
LA-untreated vessels. These results suggest that vascular
dysfunction by LA treatment is attributable to reduced NO
bioavailability (supplemental Figure VIII). Ad-PGC-1␣ significantly inhibited LA-induced decreases in endotheliumdependent vasorelaxation compared to adenoviruses carrying
␤-gal (Figure 6B, C). This effect was significantly reduced by
Ad-ANT1 siRNA (Figure 6C).
Discussion
In this study, we confirmed that fatty acids (ie, LA) increase
ROS production and apoptosis in cultured HAEC. These
effects were accompanied by decreases in FAO and ATP/
ADP translocase activity. In addition, we found that PGC-1␣
overexpression prevented fatty acid-induced increases in
ROS production and apoptosis. These PGC-1␣ effects were
accompanied by normalization of FAO and ATP/ADP translocase activity. Furthermore, a knockdown of ATP/ADP
translocase activity (via ANT1 siRNA) led to an increase in
ROS production and apoptosis, similar to the changes with
LA, and abolished the effects of PGC-1␣ to prevent LAinduced increases in ROS production and apoptosis. Taken
together, these data indicate ATP/ADP translocase activity of
ANT as a major regulatory site of ROS production and
apoptosis in endothelial cells affected by LA and PGC-1␣
overexpression.
Treatment of HAEC with LA caused significant increases in intracellular triglycerides, ceramide, and DAG
concentrations, which were all normalized by PGC-1␣
overexpression. This is consistent with the well-known
effect of PGC-1␣ to increase mitochondrial biogenesis and
FAO, which would increase the clearance of cytosolic fat
moieties. These data increase the possibility that fat metabolites may be responsible for the changes in ATP/ADP
translocase activity induced by LA or PGC-1␣ overexpression.
To support this idea, triacsin, a fatty acyl-CoA synthase inhibitor, prevented LA-induced decreases in ATP/ADP translocase
activity. In addition, myriocin, which inhibits ceramide synthesis, also prevented LA-induced decreases in ATP/ADP translocase activity.
ANT is considered a dual-edged sword. The prime function
of ANT is the exchange of ATP and ADP across the inner
mitochondrial membrane, which is rate-limiting for oxidative
phosphorylation in the resting state. However, in states
promoting apoptosis, ANT plays an important regulatory role
in mitochondrial permeability transition pore opening,32 even
though recent studies cast doubt on the central role of the
ANT as a leading contender for the membrane component
that forms the transmembrane channel of the mitochondrial
permeability transition pore.33 In addition, ANT is responsible for a significant portion of basal uncoupling or proton
leak5,9 independent of its function in ATP/ADP exchange. In
fact, ANT and UCP are considered 2 major molecules that
determine mitochondrial uncoupling.5
Whereas LA decreased ATP/ADP translocase activity, LA
increased ANT1 expression. Fatty acids have long been
known to increase mitochondrial uncoupling,34 and LAinduced increases in ANT1 and UCP expression may be a
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compensatory response to an increase in intracellular ROS.
To support this, the present data show that LA-induced
increases in ANT1 expression were completely prevented by
N-acetylcysteine, a well-established thiol antioxidant. However, N-acetylcysteine treatment did not affect ATP/ADP
translocase activity with or without LA treatment, suggesting
that LA-induced decrease in ATP/ADP translocase activity is
ROS-independent. A previous study suggested that PGC-1␣
may be involved in cold-induced upregulation of UCP and
ANT in skeletal muscle.35 In agreement with this study,
forced expression of PGC-1␣ increased the expression of
ANT1 in endothelial cells (without LA treatment). However,
this effect did not appear to be the major mechanism by which
PGC-1␣ improves ROS generation and apoptosis in cells
treated with LA because PGC-1␣ did not increase ANT1 or
UCP-2 expression above the levels induced by LA. Despite a
lack of effect on ANT1 expression, PGC-1␣ normalized the
ATP/ADP translocase activity of ANT in cells treated with
LA, and this was associated with the reversal of all of the
changes induced by LA, including time-dependent changes in
⌬␺m, ROS production, and apoptosis. In addition, knockdown of ATP/ADP translocase activity of ANT via ANT1
siRNA abolished all of these beneficial effects of PGC-1␣.
Taken together, these results suggest that PGC-1␣– dependent
enhancement of ATP/ADP translocase activity of ANT,
which may be mediated by cytosolic fat metabolites as
discussed, is critically required for the beneficial effects of
PGC-1␣ on endothelial function.
Impaired endothelium-dependent vascular relaxation is
generally considered a prerequisite for atherosclerosis.36 In
this study, we examined the effect of ex vivo adenoviral
overexpression of PGC-1␣ on endothelium-dependent vasorelaxation. Consistent with in vitro study, LA significantly
impaired endothelium-dependent vasorelaxation. This inhibitory effect of LA treatment on vasorelaxation may be
through reduction in the activity of endothelial nitric oxide
synthase (supplemental Figure VIII). Ad-PGC-1␣ significantly inhibited LA-induced decreases in endotheliumdependent vasorelaxation compared to adenoviruses carrying
␤-gal, and this effect was significantly reduced by Ad-ANT1
shRNA. Collectively, these ex vivo results confirm antiatherogenic effects of PGC-1␣ in vascular endothelial cells.
Conclusion
In conclusion, we propose that fatty acids increase ROS
generation and apoptosis in endothelial cells by reducing
ATP/ADP translocase activity of ANT, which may be mediated by increased cytosolic fat metabolites. PGC-1␣ overexpression prevented fatty acid-induced changes in apoptosis,
ROS generation, FAO, and ⌬␺m by normalizing ATP/ADP
translocase activity, enabling endothelial cells to better cope
with a high lipid load. These findings indicate ATP/ADP
exchange activity as a crucial regulatory site for the effects of
fatty acids or PGC-1␣ on fuel metabolism and cell apoptosis
in endothelial cells. Measures to increase PGC-1␣ expression
or ATP/ADP translocase activity in vascular endothelial cells
may be useful in the prevention or treatment of
atherosclerosis.
Acknowledgments
The authors thank Professor Shey-Shing Sheu (University of Rochester Medical Center) for insightful comments and discussion.
Sources of Funding
This work was supported by the Korea Science and Engineering
Foundation (KOSEF) grants funded by the Ministry of Science and
Technology (M10642140004-06N4214-00410 to K.U.L. and NRL
M1040000000804J000000810 to J.Y.P.), and a grant (2008-122)
from Asan Institute for Life Sciences, Seoul, Korea.
Disclosure
None.
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Peroxisome Proliferator-Activated Receptor-γ Coactivator 1-α Overexpression Prevents
Endothelial Apoptosis by Increasing ATP/ADP Translocase Activity
Jong Chul Won, Joong-Yeol Park, Yun Mi Kim, Eun Hee Koh, Somi Seol, Byeong Hwan Jeon,
Jin Han, Jung Ran Kim, Tae-Sik Park, Cheol Soo Choi, Woo Je Lee, Min-Seon Kim, In-Kyu
Lee, Jang Hyun Youn and Ki-Up Lee
Arterioscler Thromb Vasc Biol. 2010;30:290-297; originally published online December 3,
2009;
doi: 10.1161/ATVBAHA.109.198721
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2009 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://atvb.ahajournals.org/content/30/2/290
An erratum has been published regarding this article. Please see the attached page for:
/content/30/9/e175.full.pdf
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Correction
In the article, “Peroxisome Proliferator-Activated Receptor-␥ Coactivator 1-␣ Overexpression
Prevents Endothelial Apoptosis by Increasing ATP/ADP Translocase Activity” by Won et al,
which appeared in the February 2010 issue of the journal (Arterioscler Thromb Vasc Biol.
2010;30:290 –297; DOI: 10.1161/ATVBAHA.109.198721), the publisher omitted several important corrections from the final, published version:
1. Page 292, right column, 2nd full paragraph, line 7, “triasin” should have appeared as
“triacsin.”
2. Page 296, Conclusion, lines 4 through 8, the sentence should have appeared as “PGC-1␣
overexpression prevented fatty acid-induced changes in apoptosis, ROS generation, FAO,
and ⌬⌿m by normalizing ATP/ADP translocase activity, enabling endothelial cells to better
cope with a high lipid load.”
The online version has been corrected.
The publisher sincerely regrets the errors.
DOI: 10.1161/ATV.0b013e3181f596b6
(Arterioscler Thromb Vasc Biol. 2010;30:e175.)
© 2010 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
e175
Online data supplement
Methods
Reagents
For Western blot analysis, antibodies against caspases-3, -9, poly (ADP) ribose polymerase
(PARP), VDAC and CuZnSOD were purchased from Cell Signaling. Anti-ANT1, PGC-1α,
cyclophilin D, catalase and mitochondrial creatinine kinase antibodies were from Santa Cruz
Biotechnology. Anti-β-actin antibody was from Calbiochem. MnSOD and UCP2 antibodies
were R&D systems. Myriocin, N-acetylcysteine, triacsin, NG-nitro-L-arginine methyl ester (LNAME), cyclosporine A and etomoxir were purchased from Sigma.
Construction of small interfering RNA (siRNA)
We designed siRNA targeted against human PGC-1α, ANT1 and rat ANT1 mRNAs using the
design algorithm developed by GenScript. The sequences for human PGC-1α siRNA are 5’CCA AGA CUC UAG ACA ACU ATT-3’, human ANT1 siRNA are 5´-GUG UUC AUU AAA
CCA CAC ATT-3´, control non-targeting siRNA are 5´-GUU CAG CGU GUC CGG CGA
GTT-3´ (Samchunlly Pharm). The sequences for rat ANT1 siRNA are 5´- GUG CUU GGU
1
CCA ACG UAC U-3´(Bioneer). We also made control siRNAs, which have the same GC
content as the target sequences and have no effect on gene expression (Bioneer).
Preparation of recombinant PGC-1α adenovirus
Adenoviruses containing PGC-1α (Ad-PGC-1α) and β-galactosidase (Ad-β-gal) were prepared
as described previously.1 In brief, the cDNAs for PGC-1α and β-galactosidase were cloned into
a pAd-YC2 shuttle vector, which has the cytomegalovirus promoter and the bovine growth
hormone polyadenylation sequence. For homologous recombination, pAd-YC2-PGC1α and
pAd-YC2-β-galactosidase shuttle vector (5 g) and a rescue vector, pJM17 (5 g), were cotransfected into human embryonic kidney 293 cells. To purify pure plaques, cell culture
supernatant was serially diluted into serum-free media and incubated with 293 cells at 37°C for
1 h. An equal volume mixture of 2 × medium and 1% agarose was overlaid on the 293 cells.
After 7 days, plaques that were well isolated were purified further and propagated in 293 cells
and screened by PCR, using upstream primers derived from the cytomegalovirus promoter, and
downstream primers from the bovine growth hormone polyadenylation sequence. Then, the
recombinants were amplified in 293 cells and were purified and isolated using CsCl 2 (Sigma).
The preparations were collected and desalted, and titers were determined by counting the
2
number of plaques.
Preparation of recombinant ANT1 shRNA adenovirus
ANT1 shRNA adenovirus was prepared as previously described.2 The oligonucleotides of ANT1
shRNA were cloned into a pRNATin-H1.2 shuttle vector (Genescript) that contain inducible H1
promoter following the manufacturer’s instructions.
Measurement of ROS levels
For measurement of intracellular ROS levels, HAECs were incubated for 15 min with 2.5 μmol/
ml DCFH2-DA (Molecular Probes) at 37°C for 30 min. The increase in DCFH2-DA oxidation
was measured by a flow cytometry (FACSCaliber). Fluorescence was measured at an excitation
wavelength of 488 nm and an emission wavelength of 530 nm at 2, 4, and 8 h after treatment.
Mitochondrial-specific ROS generation was measured by using the MitoSOX Red fluorescent
dye (Molecular Probes) as described by the manufacturer. HAECs were loaded with MitoSOX
Red (5 μmol/L) in Dulbecco’s modified Eagle’s medium for 15 min at room temperature,
followed by wash out. Confocal images were obtained after treatment of Ad-β-gal- or Ad-PGC1α-infected cells by LA, and fluorescence was measured by flow cytometry using excitation at
3
488 nm and emission at 530 nm.
Measurement of ∆ψm
To measure ∆ψm, HAECs were resusupended in 5 μg/ml solution of the cationic voltagedependent dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1,
Molecular Probes) in growth media.3 After 15 min incubation at 37C, cells were washed twice
and then were immediately analyzed by flow cytometry. JC-1 aggregates were detectable in the
propidium iodide channel (red fluorescence, emission at 590 nm), and JC-1 monomers were
detectable in the fluorescein isothiocyanate channel (green fluorescence, emission at 527 nm). 4
The ratio between green and red depends on Δψm5 and was normalized by comparison with the
control red/green ratio.
Measurement of ATP/ADP translocase activity of ANT
HAECs were immediately placed in ice-cold mitochondrial isolation buffer (200 mM mannitol,
70 mM sucrose, 1 mM EDTA, 10 mM HEPES). Homogenized cells were centrifuged at 1,000
xg for 10 min and the supernatant was centrifuged at 15,000 xg for 10 min at 4C. The
mitochondrial pellet was resuspended in 260 μL of ADP import buffer (250 mM sucrose, 20
4
mM HEPES, 10 mM KCl, 5 mM succinate, 3 mM KH2PO4, 1.5 mM MgCl2, 1 mM EGTA, 5
μM rotenone [pH 7.2]). Samples were incubated for 10 min with 1 μCi of 14C-ADP (NEN Life
Sciences) in the ADP import buffer on ice. To inhibit ADP import, 140 μL of ADP import
buffer containing 50 μM atractyloside (Sigma) was included. The samples were washed twice in
ADP import buffer, mixed with liquid scintillation cocktail, and quantified by a beta counter
(PerkinElmer Wallace).
Measurement of TG level and FAO
Intracellular TG level was measured by using the GPO Trinder Kit (Sigma) and FAO was
assessed by measuring
14
CO2 generation from 1-[14C]palmitate (NEN Life Sciences) as
described previously.6
Measurement of diacylglycerol and ceramide
For diacylglycerol (DAG) measurement, lipids from HAECs were extracted with
chloroform/methanol (2:1, v/v) containing 0.01% butylated hydroxytoluene. The mass
spectroscopy system with an Atmospheric Pressure Chemical Ionization (APCI) ion source at a
positive ion mode was used as described previously.7 1,3-dinonadecanoin (671 → 355 m/z) was
added as an internal standard for quantification. DAGs were isolated from triglycerides using a
5
diol bonded-phase SPE column (Waters, Inc.) and 10 μl of sample was injected into LC/MS/MS
to quantify individual DAGs, i.e., 16:0 DAG, 18:1 DAG, 16:0-18:1 DAG, 18:0-18:2 DAG, and
18:0-20:4 DAG (Avanti Polar Lipids).
For analysis of ceramides, total lipids were extracted by chloroform/methanol (2:1, v/v) and
phospholipids were saponified by adding NaOH. An Applied Biosystems (Foster city, CA) 4000
QTrap triple quadrupole/linear ion trap mass spectrometer (MS/MS) equipped with an Agilent
1200 binary pump inlet, with a Turbo V ESI source set to positive ionization mode, and Analyst
4.0 operating software, was used for all quantitative determinations. Ceramides, i.e., C14:0,
C16:0, C18:0, C18:1, C20:0, C24:0, C24:1 (Avanti Polar Lipids), were analyzed as reported
previously.8 N-heptadecanoyl-D-erythro-sphingosine (C17 ceramide) was used as an internal
standard. Precursor-to-product ion transitions were established via direct infusion of each
compound into the mass spectrometer. A sample volume of 10 μl was injected into the liquid
chromatography LC/MS/MS system and ceramides were separated using a Phenomex
(Torrance) Gemini C18 column (2 mm  150 mm with 5μm particles).
Real-time PCR analysis
2 g of total RNA was reverse-transcribed with oligo (dt) using M-MuLV reverse transcriptase
(Roche Diagnostics). In 96-well optical plates, 12.5 μl SYBR Green master mix was added to
6
12.5 μl cDNA (corresponding to 50 ng of total RNA input) and 200 nM of forward and reverse
primers in water. Plates were heated for 10 min at 95°C followed by 40 PCR cycles of 15
seconds at 95°C and 60 seconds at 60°C. Amplification of 18S rRNA was used as the internal
control. Ratios of target gene and 18S rRNA expression levels were calculated by subtracting
the Ct (threshold cycle) of the target gene from the Ct of 18S and raising 2 to the power of this
difference. The parameter Ct is defined as the fractional cycle number at which the fluorescence
passes the fixed threshold. A plot of the log of initial target copy number for a set of standards
versus Ct is a straight line. Quantification of the amount of target in unknown samples is
accomplished by measuring Ct and using the standard curve to determine starting copy number.
The entire process of calculating Ct, preparing a standard curve, and determining starting copy
number for unknowns was performed by the software of the 7700 system. The primers were
designed on the basis of nucleotide sequences in the GenBank database. The relative amounts of
mRNAs were calculated using the relative cycle threshold method (PerkinElmer Wallace).
7
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Yoon M, Lee KU, Park JY. AMPK activation increases fatty acid oxidation in skeletal muscle
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9
Supplemental Table 1. Primer pairs used in real-time PCR to quantify changes in gene
expression
10
Supplemental Table 2. Changes in individual DAG and ceramide in response to LA and/or AdPGC-1α. At 48 h after transfection with Ad-PGC-1α or Ad-β-gal, HAECs were treated for 4 h
with either control vehicle (dextrin) or 60 mol/L LA. Values are mean ± SEM of 5 independent
experiments. P < 0.05 vs Ad-β-gal-infected cells, P < 0.05 vs control cells.
11
Supplemental Figure 1. PGC-1α decreases intracellular ROS production and prevents LAinduced endothelial apoptosis
HAECs were infected with either Ad--gal or Ad-PGC-1α. After 48 h, cells were treated with
control vehicle or 60 μmol/L LA. A-B. Intracellular (A) and mitochondrial (B) ROS generation.
After 4 h, HAECs were processed for confocal microscopy by using the oxidant sensitive probe
DCFH2-DA (A) and MitoSOX Red (B), respectively. C. Western blots showing cleaved caspase
3 and PARP. Apoptosis was measured at 16 h after incubation with LA or vehicle.
12
Supplemental Figure 2. LA increases and PGC-1α decreases cyclophilin D expression
The effects of LA and PGC-1α on the expression of VDAC, CK and cyclophilin D (A-C). Both
LA and PGC-1α increased the levels of VDAC and CK. On the other hand, LA increased, but
PGC-1α decreased cyclophilin D expression. P < 0.05 vs Ad--gal without LA, P < 0.05 vs
Ad--gal with LA. (D) Effect of cyclosprin A, an inhibitor of cyclophilin D, on LA-induced
endothelial cell apoptosis (n =5, each). P < 0.05 vs control, #P < 0.05 vs LA-treated cells.
13
The mitochondrial membrane permeability transition (MPT) pore complex, which is a key
participant in the machinery that controls cellular apoptosis and necrosis,1 putatively consists of
the voltage–dependent anion channel (VDAC), ANT, cyclophilin D and mitochondrial
creatinine kinase (CK), etc.2 As cyclophilin D increases the susceptibility for mitochondria
permeability transition pore opening,2 our data suggest that PGC-1α’s protective effect on cell
apoptosis may be partly through decreases of cyclophilin D expression.
14
Supplemental Figure 3. Effect of LA and Ad-PGC-1α on the protein expression levels of
antioxidant genes
HAECs were infected with either Ad--gal or Ad-PGC-1α. After 48 h, cells were treated with
control vehicle or 60 μmol/L LA for 4 h. The left panel shows representative Western blots of
antioxidant genes. The right panel shows quantification of proteins under the experimental
conditions. The values are means ± SEM of 4 independent experiments. P < 0.05 vs Ad-β-gal
without LA.
15
Supplemental Figure 4. Effect of endogenous suppression of PGC-1α on the mRNA
expression of antioxidant enzymes
A. Validation of PGC-1α siRNA. HAECs were treated with 50 nM of PGC-1α siRNA (PGC-1α)
or control siRNA (Control) and proteins were detected by Western blot analysis. B. The mRNA
expression of MnSOD, catalase, glutathione peroxidase and UCP2 was analyzed by real-time
PCR. RNA quantity was normalized by using amplification of 18S rRNA as an internal control.
16
HAECs were treated with 50 nM of PGC-1α siRNA or control siRNA for 48 h. Levels obtained
from the control, untreated values were assigned the arbitrary value of 1.0. *P < 0.05 vs control
siRNA-transfected cells. The values are mean ± SEM of 4 independent experiments.
17
Supplemental Figure 5. Effect of ANT1 siRNA on the expression of antioxidant enzyme
genes
HAECs were transfected with control or ANT1 siRNA for 24 h. Real-time PCR analysis was
used to show the effect of ANT1 siRNA on mRNA levels of MnSOD, CuZnSOD, catalase,
glutathione peroxidase and UCP2. The values are mean ± SEM of 5 independent experiments.
*P < 0.05 vs control siRNA-transfected cells.
18
Supplemental Figure 6. Effect of ANT1 siRNA on mitochondrial morphology
HAECs were incubated with or without 50 nM ANT1 siRNA for 24 h, and were examined by
transmission electron microscopy. 12,000 x original magnification. Compared with control
siRNA-treated cells, ANT1 siRNA-treated cells show increased dysmorphism of mitochondria.
19
Supplemental Figure 7. Effects of Ad-PGC-1α and/or ANT1 siRNA on ANT1 protein
expression
HAECs were transfected with 50 nM of ANT1 siRNA or control siRNA. After 24 h, cells were
infected with Ad-β-gal or Ad-PGC-1α. After 48 h, cells were treated with LA or vehicle for 4 h.
n = 4 each. *P < 0.05 vs Ad--gal, **P < 0.05 vs control siRNA.
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Supplemental Figure 8. Effect of NO inhibition on LA-induced decrease in vasorelaxation
A. After a preincubation with or without 50 μmol/L L-NAME for 2 h, the aorta was incubated
with 60 μmol/L LA for 2 h. The values are mean ± SEM of 5 independent experiments. *P <
0.05 vs LA-untreated control aorta, #P< 0.05 vs NAME-untreated aorta. B. The effect of LA
treatment on net difference between vasorelaxation of L-NAME-treated and –untreated aorta.
*P < 0.05 vs LA-untreated aorta.
Administration of NO synthase inhibitor L-NAME nearly completely inhibited acetylcholineinduced vasorelaxation, both in LA-treated and untreated aorta. As LA treatment was associated
with reduced vasorelaxation in response to acetylcholine, the net difference between L-NAMEtreated and -untreated vessels was less in the LA-treated than in -untreated vessels. These results
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support the concept that the inhibitory effect of LA treatment on vasorelaxation is through a
reduction in the activity of eNOS.
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References
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identity and regulation of the mitochondrial permeability transition pore: where the known
meets the unknown. Ann N Y Acad Sci. 2008;1123:197-212.
2. Tsujimoto Y, Shimizu S. Role of the mitochondrial membrane permeability transition in cell
death. Apoptosis. 2007;12:835-840.
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