THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 6, pp.

3763–3774, February 7, 2014

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Apelin Attenuates Oxidative Stress in Human Adipocytes*

Received for publication, October 9, 2013, and in revised form, December 13, 2013 Published, JBC Papers in Press, December 20, 2013, DOI 10.1074/jbc.M113.526210
Aung Than‡, Xiaohong Zhang‡, Melvin Khee-Shing Leow§¶储, Chueh Loo Poh‡, Seow Khoon Chong‡,
and Peng Chen‡1
From the ‡Division of Bioengineering, Nanyang Technological University, 70 Nanyang Drive, Singapore 637457, the §Singapore
Institute for Clinical Sciences, Brenner Center for Molecular Medicine, 30 Medical Drive, Singapore 117609, ¶Endocrine and
Diabetes, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore 308433, and the 储Duke-NUS Graduate Medical School, 8
College Road, Singapore 169857
Background: Antioxidant effects of apelin on adipocytes are unknown.
Results: Apelin not only suppresses production and release of reactive oxygen species, but also relieves oxidative-stress induced
cellular dysfunctions in adipocytes.
Conclusion: Apelin-APJ signaling acts as the negative autocrine feedbacks against oxidative stress in adipocytes.
Significance: Apelin signaling may serve as a potential therapeutic target for metabolic disorders.

It has been recently recognized that the increased oxidative
stress (ROS overproduction) in obese condition is a key contrib-
utor to the pathogenesis of obesity-associated metabolic dis-
eases. Apelin is an adipocytokine secreted by adipocytes, and
known for its anti-obesity and anti-diabetic properties. In obe-
sity, both oxidative stress and plasma level of apelin are
increased. However, the regulatory roles of apelin on oxidative
stress in adipocytes remain unknown. In the present study, we
provide evidence that apelin, through its interaction with apelin
receptor (APJ), suppresses production and release of reactive
oxygen species (ROS) in adipocytes. This is further supported by
the observations that apelin promotes the expression of anti-
oxidant enzymes via MAPK kinase/ERK and AMPK pathways,
and suppresses the expression of pro-oxidant enzyme via
AMPK pathway. We further demonstrate that apelin is able to
relieve oxidative stress-induced dysregulations of the expres-
sion of anti- and pro-oxidant enzymes, mitochondrial biogene-
sis and function, as well as release of pro- and anti-inflammatory
adipocytokines. This study, for the first time, reveals the antiox-
idant properties of apelin in adipocytes, and suggests its poten-
tial as a novel therapeutic target for metabolic diseases.
Oxidative stress is a condition in which antioxidant defenses
are overwhelmed by reactive oxygen species (ROS).2 The
* This research was supported by the Singapore Ministry of Health’s National
Medical Research Council under its EDG Grant (NMRC/EDG/1048/2011)
and Singapore Ministry of Education under its AcRF Tier 1 Grant (RGT7/13).
1
To whom correspondence should be addressed: Division of Bioengineering,
Nanyang Technological University, Singapore 637457. Tel.: ⫹65-6514-
1086; E-mail: chenpeng@ntu.edu.sg.
2
The abbreviations used are: ROS, reactive oxygen species; Akt, protein
kinase B; AMPK, 5⬘-adenosine monophosphate-activated protein kinase;
APJ, apelin receptor; carboxy-H2DCFDA, 6-carboxy-2⬘,7⬘-dichlorodihydro
fluorescein diacetate; COX1, cytochrome c oxidase subunit I; ERK, extracel-
lular signal-regulated kinase; GPx, glutathione peroxidase; NADPH oxi-
dase, nicotinamide adenine dinucleotide phosphate oxidase; PGC-1␣, per-
oxisome proliferator-activated receptor gamma coactivator 1-alpha; PI3K,
phosphatidylinositol-3 kinase; PMA, phorbol 12-myristate 13-acetate;
SDHA, succinate dehydrogenase subunit A; SOD-1, superoxide dismu-
tase-1; TNF␣, tumor necrosis factor alpha; ABTS, 2,2-azino-bis-3-ethylbenz-
thiazoline-6-sulfonic acid; DHE, dihydroethidim.
resulting excess free radicals cause damages to cells and tissues,
and are implicated in the pathogenesis of various diseases, such
as diabetes, cancer, hypertension, and atherosclerosis (1). Oxi-
dative stress is increased in obesity (2, 3). Many studies have
demonstrated it is an important link factor between obesity and
other metabolic disorders (e.g. diabetes) (4, 5). For example,
oxidative stress suppresses insulin secretion from pancreatic ␤
cells (6), and also disrupts glucose uptake in muscle and fat
tissue (7, 8). And the accompanying inflammation in tissues
worsens the problems (e.g. insulin resistance) caused by oxida-
tive stress (9).
It is increasingly recognized that adipose tissue critically reg-
ulate metabolic homeostasis via secretion of various adipocy-
tokines and metabolic factors (10). Adipose tissue is also a
major source of the plasma ROS (3). Studies have showed that
ROS production increases in parallel with fat accumulation in
adipocytes (3, 11). On the other hand, excess ROS disrupts adi-
pocyte functions. It, for examples, impairs insulin-induced glu-
cose uptake in adipocytes (12), promotes adipogenesis and
lipolysis leading to over-release of free fatty acids (11, 13), and
jeopardizes mitochondrial biogenesis and functions which, in
turn, further exacerbates the oxidative stress (14, 15). In addi-
tion, ROS overproduction in adipose tissue leads to decreased
expression of anti-oxidative enzymes, increased expression of
NADPH oxidase, and dysregulation of adipocytokine expres-
sion (e.g. adiponectin and IL-6) (3).
Apelin, a novel peptide hormone and relatively recent addi-
tion of adipocytokine family, is abundantly secreted by adi-
pocytes (16, 17). Through interaction with G-protein coupled
APJ receptors, apelin plays important roles in regulating energy
metabolism, and has been recognized with its anti-obesity and
anti-diabetic properties (18). Apelin, for examples, promotes
glucose utilization and fatty acid oxidation in muscles of insu-
lin-resistant mice (19, 20), stimulates insulin signaling path-
ways and glycogen synthesis in hepatocytes and liver tissues of
mice (21), and suppresses adipogenesis and lipolysis (22). Inter-
estingly, apelin has also been shown to alleviate oxidative stress
in cardiomyocytes and vascular smooth muscle cells (23, 24).
However, its regulations of oxidative stress in adipocytes have
never been investigated. Considering that apelin expression in

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Apelin Suppresses Oxidative Stress
adipocytes is up-regulated in obesity (17) and its plasma level is
increased in obese subjects (25, 26), we speculate that apelin
may act as the feedback control to limit obesity-associated oxi-
dative stress.
Here, we aim for the first time to reveal the effects of apelin
on ROS production and oxidative stress in adipocytes, using
human primary adipocyte as the in vitro cell model. Our results
show that apelin/APJ suppresses ROS production and release
by regulating anti- and pro-oxidant enzyme expressions via
MAPK kinase/ERK and AMPK signaling pathways. In addition,
we provide evidence that apelin can prevent oxidative-stress-
induced dysregulations of ROS-related enzyme expressions,
release of anti-inflammatory adipocytokine (adiponectin) and
pro-inflammatory adipocytokines (IL-6, TNF␣), as well as
mitochondrial biogenesis and function. Our experiments also
show that oxidative stress stimulates apelin release from adi-
pocytes. This study highlights apelin as a promising therapeutic
target for oxidative stress-related metabolic disorders.
EXPERIMENTAL PROCEDURES
Cell Culture and Differentiation—Human pre-adipocytes
(SP-F-2; Zen-Bio Inc.), collected from subcutaneous adipose
tissue of non-diabetic male subjects (with body-mass-index
25–29.9), were cultured in pre-adipocyte growth medium
(DMEM/Nutrient mixture F-12 medium (1:1, v/v) containing
15 mM HEPES, 2 mM L-glutamine, 10% fetal bovine serum
(FBS), 100 units/ml penicillin, and 100 mg/ml streptomycin), at
37 °C and in a humidified atmosphere containing 5% CO2. Two
days after reaching confluence (defined as day 0), the cells were
induced to differentiate into mature adipocytes similarly as the
previously reported (27, 28). Specifically, the cells were incu-
bated with adipocyte growth medium (serum-free DMEM/Ham’s
F-12 medium containing 15 mM HEPES, 2 mM L-glutamine, 33 ␮M
biotin, 17 ␮M pantothenate, 10 ␮g/ml transferrin, 0.2 nM triiodo-
thyronine, 100 units/ml penicillin, and 100 mg/ml streptomy-
cin) supplemented with 0.5 ␮g/ml insulin, 1 ␮M dexametha-
sone, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 10
␮M rosiglitazone. On day 2 of differentiation, the cells were
treated for 2 days with the growth medium containing insulin
and dexamethasone. The medium was thereafter replaced with
the growth medium alone, every 2 days in the following 8 –10
days. Adipocytes differentiation was confirmed by Oil-red-O
staining and visual appearance of intracellular lipid droplets.
For knockdown of apelin (APLN) or APJ receptor (APLNR),
cells (day 6 –7 after induction of differentiation) were trans-
fected with control siRNA or apelin siRNA or APLNR-siRNA
(25 nM) (SantaCruz Biotech) using Lipofectamine-RNAiMAX
transfection reagent (Invitrogen) for 2 days. Adipocytes were
then maintained in the growth medium for 2–3 days before the
experiments. Knocking down of apelin or APJ receptor expres-
sion was confirmed by immunoblot analyses. All culture media,
supplements and sera were purchased from Invitrogen. Unless
otherwise stated, all reagents and chemicals were obtained
from Sigma-Aldrich.
Confocal Microscopy—Adipocytes grown on the glass-cover-
slips were placed in an imaging-chamber filled with the growth
medium before imaging with a confocal laser scanning micro-
scope (LSM 510 Meta, Carl Zeiss GMbH, Germany). For detec-
3764 JOURNAL OF BIOLOGICAL CHEMISTRY
tion of intracellular ROS, the cells were loaded with 6-carboxy-
2⬘,7⬘-dichlorodihydro fluorescein diacetate (carboxy-H2DCFDA,
20 ␮M, Invitrogen), a ROS indicator which gives green fluores-
cence while being oxidized by ROS. After incubation with the
dye for 30 min at 37 °C with 5% CO2 in a humidified incubator,
adipocytes were then washed with the growth medium, fol-
lowed by 200 ␮M H2O2 treatment for 60 min (or without the
treatment as control). Excited at 488 nm, the fluorescence of
the dye was monitored with a 505–550 nm filter. For co-local-
ization studies of cellular and mitochondrial ROS, cells were
loaded for 30 min with carboxy-H2DCFDA and 0.2 ␮M Mito-
Tracker Red CM-H2Xros (Invitrogen). The latter, which is a
reduced non-fluorescent version of MitoTracker Red, accumu-
lates in mitochondria and fluoresces upon oxidation by ROS gen-
erated in mitochondria. To reveal the mitochondrial morphology,
the cells were loaded with 0.2 ␮M MitoTracker Red-FM (Invitro-
gen). The MitoTracker fluorescence was measured above 580 nm
using 543 nm as the excitation wavelength. For detecting superox-
ide anions (O2. ), the cells were incubated for 30 min with 10 ␮M
dihydroethidium dye (DHE, Sigma-Aldrich) and then washed
with the cell medium. DHE oxidation by (O2. ) produces red fluo-
rescent 2-hydroxyethidium (29, 30). In some experiments, the cells
were pre-treated with or without pyr-aplein13 (1 ␮M) and/or
TNF␣ (50 ng/ml) before dye loading and imaging. ROS levels were
quantified using a fluorescent plate reader (Victor3, PerkinElmer).
Western Blot Analyses—The adipocytes were washed twice
with ice-cold phosphate-buffered saline (PBS), and scraped in
M-PER Mammalian Protein Extraction Reagent (Thermo Sci-
entific) containing freshly added protease/phosphatase inhibi-
tor mixture (Cell Signaling Technology). After centrifugation at
4 °C, the supernatant of the sample was collected and its protein
content was determined using a protein assay kit (Bio-Rad)
based on Bradford method. Each sample with equal amount of
proteins (as the loading control) was mixed with 5x SDS sample
buffer, boiled for 5 min, and separated on 12% sodium dodecyl
sulfate-polyacrylamide electrophoresis (SDS-PAGE) before
transferring the proteins onto a nitrocellulose membrane. The
membranes were then blocked at room temperature for 2–3 h
with 5% bovine serum albumin (BSA) in Tris-buffered saline-
Tween (TBST) (10 mM Tris, 150 mM NaCl and 0.05% Tween-
20, pH 7.4), followed by subsequent incubation at 4 °C over-
night in TBST containing the different primary antibodies
(1:200 – 400 dilution). After washing three times (15 min each)
with TBST, the membranes were incubated for 6 – 8 h in TBST
containing the horseradish peroxidase-conjugated secondary
antibodies (1:2000 –3000 dilution), followed by washing again
with TBST for three times. The protein bands were detected in
a G:BOX Chemi XT4 imaging system (Syngene) using Super-
Signal West Pico Chemiluminescent Substrate (Thermo Scien-
tific). Antibodies against apelin (sc-33469), APJ receptor (APLNR,
sc-33823), superoxide dismutase-1 (SOD-1, sc-8637), catalase (sc-
34280), glutathione peroxidase-1/2 (GPx-1/2, sc-30147), p47-
phox (sc-14015), PPAR␥ coactivator-1 (PGC-1␣, sc-13067),
cytochrome c oxidase subunit I (COX1, sc-23982), succinate
dehydrogenase subunit A (SDHA, sc-27992), Akt (sc8312), phos-
pho-Akt (Ser-473, sc135651), AMP-activated protein kinase
(AMPK␣1/2, sc-25792), and phospho-AMPK␣1/2 (Thr-172,
sc-33524) were purchased from Santa Cruz Biotechnology. Anti-
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 Apelin Suppresses Oxidative Stress

FIGURE 1. Apelin suppresses intracellular ROS production and release, and increases the antioxidant activity in adipocytes. A, bright-field image of a
human adipocyte and its confocal image of intracellular ROS level (reported by fluorescent carboxy-H2DCF-DA). Scale bars, 10 ␮m. B, representative confocal
images of intracellular ROS in adipocytes, without (control) or with exposure to pyr-apelin13 (1 ␮M, 24 h), before (upper lane) or after (lower lane) 30-min
stimulation with 200 ␮M H2O2. Some adipocytes were transfected with APLNR-siRNA to knock-down expression of APJ receptors. Scale bars, 50 ␮m. C,
intracellular ROS levels indicated by the fluorescence intensity of carboxy-H2DCF-DA. The values are normalized to the intensity in control cells (mean ⫾ S.E.,
n ⫽ 4). D and E, real time-responses of ROS level after H2O2 exposure. The statistics (mean ⫾ S.E.) in E is obtained from 11 cells (three independent experiments).
F, H2O2 release from adipocytes without (control) or with exposure to 1 ␮M pyr-apelin13 for 24 h (mean ⫾ S.E., n ⫽ 4). G, antioxidant activities in adipocytes after
24 h treatment with various doses of pyr-apelin13 (0 –5 ␮M). The results are expressed in terms of Trolox-equivalent antioxidant-capacity (mean ⫾ S.E., n ⫽ 5).
Student’s t test was used: *, p ⬍ 0.05; **, p ⬍ 0.01 versus control; #, p ⬍ 0.05; ##, p ⬍ 0.01 between indicated pairs.

bodies against extracellular signal-regulated kinase (ERK1/2,
cat.no. 3085-100), and phospho-ERK1/2 (cat.no. 3441-100) were
purchased from Biovision.
Enzyme Immunoassay (EIA) and Enzyme-linked Immunosor-
bent Assay (ELISA)—The concentration of IL-6, TNF␣, adi-
ponectin, and apelin in the medium was determined using a
human IL-6 ELISA kit (RAB0306, Sigma), or a human TNF␣
ELISA kit (RAB0476, Sigma), or a human adiponectin EIA kit
(RAB0004, Sigma), or a human apelin EIA kit (RAB0018,
Sigma), respectively. Each sample contains the same amount of
proteins (determined by Bradford assay as the internal control
of the total cell number).
Assays of Antioxidant Activity, H2O2 Release, and ATP
Production—Total cell antioxidant capacity was measured
using the antioxidant assay kit (Sigma) according to the manu-
facturer’s instructions. In brief, cell lysates with equal amount
of proteins were mixed (5 min at room temperature) with myo-
globin, hydrogen peroxide, and 2,2-azino-bis-3-ethylbenz-thi-
azoline-6-sulfonic acid (ABTS), before measuring the absorb-
ance at 405 nm using a plate reader. The cell antioxidant activity
is quantitatively calibrated to the activity of a defined concen-
tration of trolox, which is a water-soluble vitamin E analog.
H2O2 released from adipocytes (total amount of cellular pro-
teins was used as the internal control) was detected using the
AmplexRed hydrogen peroxide assay kit (Invitrogen) according
to manufacturer’s instructions. The absorbance is measured at
⬃560 nm and plotted against a standard curve containing serial
dilutions of known concentration of H2O2.
The cellular ATP content (total amount of proteins was used
as the internal control) was determined using ATP colorimet-
ric/fluorometric assay kit (Biovision) according to the manu-
facturer’s instructions. Optical density at 570 nm was measured
as the reporter.
Statistical Analysis—The variability between data sets was
determined by unpaired Student’s t test. All results were
expressed as mean ⫾ S.E. based on the measurements from at
least three independent experiments. p ⬍ 0.05 was considered
statistically significant.

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Apelin Suppresses Oxidative Stress

FIGURE 2. Apelin modulates the expression of enzymes related to ROS in adipocytes. A–E, Western blot analyses of the expression of SOD1 (⬃23 kDa),
catalase (⬃64 kDa), GPx (⬃23 kDa), p47 phox (⬃47 kDa), and actin (⬃43 kDa) in adipocytes treated with various doses of pyr-apelin (0 nM, 100 nM, 1 ␮M, 5 ␮M,
24 h). F–J, Western blot analyses of the expression of SOD1, catalase, GPx, p47 phox, and actin in adipocytes transfected with control-siRNA, apelin-siRNA, or
APLNR-siRNA. Representative immunoblots are shown in A and F, respectively. The statistics of the blot intensity in B–E and G–J (mean ⫾ S.E., n ⫽ 3) is
normalized to that of actin (as the internal control). Each sample contains the same amount of total proteins (as the loading control). Student’s t test was used:
*, p ⬍ 0.05; **, p ⬍ 0.01 versus control (0 nM apelin, or control-siRNA).

RESULTS duction and release in adipocytes. This suggests the beneficial

Apelin Decreases ROS Production and Release in Adipocytes
via APJ Receptor—In adipocytes, preproapelin (77-amino acid
peptide) is expressed by APLN gene and cleaved into shorter
active peptides including pyr-apelin13 (pyroglutamated 13-a-
mino acid peptide which is the major apelin isoform in human
plasma) (31, 32). Apelin’s functional effects are mediated by G
protein-coupled APJ receptor, which is expressed by APLNR
gene (33). We have previously demonstrated that apelin sup-
presses the adipocyte release of free fatty acids-one of the main
contributors to the development of insulin resistance (22).
Since excess ROS such as hydrogen peroxide (H2O2) and super-
oxide radical (O2. ) in the condition of oxidative stress also cause
insulin resistance (4), we sought to investigate whether apelin
has any effects on ROS production and release in adipocytes.
ROS level in live adipocytes was reported by a cell-permeable
ROS indicator dye (carboxy-H2DCF-DA) under confocal
microscopy (Fig. 1A). As shown in Fig. 1, B and C, the basal ROS
level was significantly reduced in the cells pre-treated with
apelin (1 ␮M pyr-apelin13 for 24 h). In addition, the surge of
ROS level after H2O2 stimulation (200 ␮M for 30 min) was also
largely obliterated by apelin. The real-time fluorescence mea-
surement indicates that H2O2 causes acute cellular increase of
ROS level (within 5 min) and it can be suppressed by apelin
treatment (Fig. 1, D and E). To elucidate whether APJ receptor
is involved in the apelin effect on ROS level, APJ receptors were
knockdown using APLNR-siRNA. As expected, apelin failed to
reduce basal and induced ROS levels in APLNR-gene knock-
down adipocytes (Fig. 1, B and C). Furthermore, as shown in
Fig. 1F, apelin treatment also suppressed H2O2 release from
adipocytes. Taken together, our observations establish that,
mediated by APJ receptor, apelin is able to suppress ROS pro-
role of apelin to reduce oxidative stress in adipocytes as well as
stress in other tissues stimulated by the released ROS from
adipose.
Apelin Increases Anti-oxidant Enzyme Activity and Expres-
sion, and Suppresses Pro-oxidant Enzyme Expression in
Adipocytes—Previous studies have shown that ROS level in
adipocytes was closely related to the activities and expression
levels of antioxidant enzymes such as Cu/Zn superoxide
dismutase (Cu/Zn SOD or SOD1), catalase, and glutathione
peroxidase (GPx) (3, 34), which are essential to prevent high
ROS level caused oxidative stress and cellular damage (35). As
shown in Fig. 1G, antioxidant activity in adipocytes was greatly
augmented by apelin treatment in dose-dependent manner.
Consistently, the expression of antioxidant enzymes (SOD1,
catalase, and GPx) was markedly increased in apelin-treated
adipocytes (Fig. 2, A–D). Contrarily, apelin significantly sup-
pressed the expression of p47phox, which is a cytosolic subunit
of the superoxide-producing enzyme complex-nicotinamide
adenine dinucleotide phosphate oxidase (NADPH oxidase) (36)
(Fig. 2E). On the other hand, siRNA knockdown of apelin or
APJ expression in adipocytes produced opposite effects, con-
firming the autocrine regulation of apelin on the expression of
ROS-related enzymes in adipocytes (Fig. 2, F–J). Therefore, it
may be concluded that apelin-APJ can refrain ROS level, at least
in part, by regulating the expression and activities of both anti-
and pro-oxidant enzymes.
TNF␣-induced ROS Production and TNF␣-impaired Antiox-
idant Enzyme Expression Are Relieved by Apelin—It has been
increasingly recognized that ROS overproduction critically
contributes to the development of insulin resistance that char-
acterizes type 2 diabetes (4, 8). It has been shown that TNF␣ can

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 Apelin Suppresses Oxidative Stress

FIGURE 3. A–F, apelin suppresses TNF␣-induced ROS production. A, confocal images of intracellular ROS (detected by carboxy-H2DCF-DA, upper left) and
mitochondrial ROS (detected by MitoTracker Red CM-H2XRos, lower left). The merged fluorescence image (upper right) and bright-field image (lower right) are
also shown. Scale bars, 10 ␮m. B–D, representative confocal images of intracellular ROS, mitochondrial ROS, and intracellular superoxide levels (detected by
DHE), without (control) or with exposure to pyr-apelin13 (1 ␮M) and/or TNF␣ (10 ng/ml) for 24 h. Scale bars, 50 ␮m. E and F, the statistics (mean ⫾ S.E., n ⫽ 4)
of intracellular (E) and mitochondrial (F) ROS level based on the fluorescence intensity normalized to that in control cells. G–K, apelin remedies TNF␣- or
ROS-impaired expression of ROS-relevant enzymes. Cells were pre-incubated without (⫺) (control) or with (⫹) 1 ␮M pyr-apelin13 for 24 h, followed by 12 h
exposure to 10 ng/ml TNF␣ or 200 ␮M H2O2. The representative immunoblots of SOD1, catalase, GPx, p47 phox, and actin are shown in G. The statistics of the
blot intensity in H–K (mean ⫾ S.E., n ⫽ 4 - 5) is normalized to that of actin (as the internal control). Each sample contains the same amount of total proteins.
Student’s t test: *, p ⬍ 0.05; **, p ⬍ 0.01 versus control; #, p ⬍ 0.05; ##, p ⬍ 0.01 between indicated pairs.

decrease insulin sensitivity in adipocytes by inducing ROS over-
production (37, 38), and apelin is able to improve TNF␣-in-
duced insulin resistance in adipocytes (39). It was also recently
discovered that apelin acts oppositely to TNF␣ in regulating the
trafficking of insulin receptors in adipocytes (40). We thus
hypothesize that apelin may suppress TNF␣-induced ROS gen-
eration in adipocytes. To test this hypothesis, adipocytes were
simultaneously loaded with the carboxy-H2DCF-DA and Mito-
Tracker Red CM-H2XRos, which are fluorescent reporters for
intracellular ROS and mitochondrial ROS levels, respectively
(Fig. 3A). As shown in Fig. 3, B–F, we observed that apelin not
only reduces intracellular ROS level, but also decreases ROS
generation in mitochondria (an important source of cellular
ROS). In agreement with the previous findings (38, 41), TNF␣
stimulated both the intracellular and mitochondrial ROS levels.
As we anticipated, apelin treatment suppressed the TNF␣-in-
duced ROS overproduction (Fig. 3, B–F).
Since the primary ROS generated by mitochondria is super-
oxide anion (O2. ) (42), we also investigated the intracellular level
of O2. , which can be detected by dihydroethidim (DHE), a
widely used superoxide fluorescent indicator (29, 43). Similarly,
apelin treatment resulted in decreased O2. level, and also abol-
ished the TNF␣-stimulated O2. generation in adipocytes (Fig.
3D). These results indicate that apelin is able to ameliorate the
TNF␣-induced ROS overproduction (hence oxidative stress
and associated insulin resistance in adipocytes).
It is known that oxidative stress in adipose tissue is accom-
panied by increase of mRNA expression of NADPH oxidase
subunits, and decrease of mRNA expression of antioxidant
enzymes (3, 44). Consistently as shown in Fig. 3, G–K, TNF␣-
treated adipocytes exhibited a significant decrease in antioxi-
dant enzyme contents (SOD1, catalase, GPx); and decreased
expression of the antioxidant enzymes was also observed after
exposure to excessive ROS (200 ␮M H2O2), which did not
apparently affect cell viability (data not shown).The decrease of
antioxidant enzyme expression was essentially rectified by ape-
lin pre-treatment (Fig. 3, G–K). On the other hand, TNF␣ or
H2O2 treatment caused increase of intracellular NADPH oxi-
dase subunit (p47phox), which can be attenuated by apelin.
These results further support the assertion that apelin is pro-
tective against the deleterious effects of oxidative stress by reg-
ulating expression of anti- and pro-oxidant enzymes.
The Mitochondrial Biogenesis and Function Impaired TNF␣
or H2O2 Are Ameliorated by Apelin—It is well established that
enhanced mitochondrial biogenesis and function can improve
insulin sensitivity while impaired mitochondrial biogenesis and
function are observed in adipose tissue of obese and diabetic
subjects (45, 46). Studies also demonstrated that mitochondrial

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Apelin Suppresses Oxidative Stress

FIGURE 4. Apelin improves TNF␣- or ROS-impaired mitochondrial biogenesis and function in adipocytes. Cells were incubated without (⫺) (control) or
with (⫹) 1 ␮M pyr-apelin13 for 24 h, followed by 12 h exposure to without or with 10 ng/ml TNF␣/200 ␮M H2O2. A, confocal images of mitochondria (detected
by MitoTracker) in adipocytes, and their corresponding bright-field images and 3⫻ magnification images are shown in upper right and lower right, respectively.
Scale bars, 10 ␮m. B, ATP content in adipocytes, as indicated by various treatment, was determined by using ATP colorimetric assay kit (mean ⫾ S.E., n ⫽ 4). C–F,
Western blot analyses of the expression of PGC-1␣ (⬃90 kDa), COX1 (⬃57 kDa), SDHA (⬃70 kDa), and actin in adipocytes as indicated by various treatment. The
representative immunoblots are shown in C; the statistics of the blot intensity in D–F (mean ⫾ S.E., n ⫽ 4 - 5) is normalized to that of actin (as the internal
control). G, Western blot analyses of the expression of PGC-1␣ in adipocytes transfected with control-siRNA, apelin-siRNA, or APLNR-siRNA. The upper panel
shows the representative immunoblots, and the lower panel shows the statistics of the blot intensity normalized to that of actin (mean ⫾ S.E., n ⫽ 3). Each
sample contains the same amount of total proteins. Student’s t test: *, p ⬍ 0.05; **, p ⬍ 0.01 versus control; #, p ⬍ 0.05 between indicated pairs.

morphology is abnormal in adipocytes from diabetic and obese
mice (47, 48). In addition, factors that lead to ROS overproduc-
tion (such as FFA, TNF␣, etc.) is usually accompanied by
reduced mitochondrial biogenesis and function, and increased
insulin resistance in adipocytes (38, 49, 50). Mitochondrial dys-
function, in turn, leads to produce more ROS (15). Here, we
investigated the apelin’s effects on ROS impaired mitochon-
drial morphology, biogenesis, and function.
Mitochondria morphology in live adipocytes can be resolved
by confocal microscopy using the specific mitochondria-
staining dye (MitoTracker) (51–53). As shown in Fig. 4,
mitochondria are short and densely packed in mature adi-
pocytes; and apelin treatment increased the mitochondria
density (Fig. 4A). In comparison, mitochondria in TNF␣-
treated adipocytes assume tubular morphology while those in
H2O2 treated cells appear swollen. Interestingly, the aberrant
morphologies observed in the pro-inflammatory factor or ROS-
treated cells were largely remedied by apelin (Fig. 4A).
To investigate the effects of apelin on mitochondrial biogen-
esis, we examined the expression of PGC1␣ (a master regulator
of mitochondrial biogenesis) (54) and the mitochondrial pro-
teins including cytochrome c oxidase subunit 1 (COX1) and
succinate dehydrogenase complex subunit A (SDHA). As
shown in Fig. 4, B–E, apelin is able to enhance the basal expres-
sion of these markers for mitochondrial biogenesis and partially
recover the expression impaired by TNF␣ or H2O2. On the
contrary, PGC1␣ expression was largely decreased in the ape-
lin- or APLNR- gene knockdown adipocytes (Fig. 4G). This
further ratifies the autocrine stimulation of apelin on the
expression of PGC1␣. Cellular ATP production is an indicator
of mitochondrial function (45, 46). We further demonstrate
that apelin can abrogate TNF␣ or H2O2 impairment on ATP

3768 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 6 • FEBRUARY 7, 2014

 Apelin Suppresses Oxidative Stress

FIGURE 5. Signaling pathways underlying the apelin-mediated expression of the proteins related to mitochondrial biogenesis and ROS level. A–C,
Western blot analyses of the phosphorylation and total protein levels of A, AMPK (⬃62 kDa), B, Akt (⬃60 kDa), and C, ERK1/2 (⬃42– 44 kDa) in adipocytes (15
min, 30 min, 2 h), without (⫺) (control) or with (⫹) exposure to 1 ␮M pyr-apelin13. The top panels show the representative immunoblots. The lower panels show
the statistics (mean ⫾ S.E., n ⫽ 3) of the optical density ratio between the blots: pAMPK and AMPK, or pAkt and Akt, or pERK1/2 and ERK1/2. D–F, representative
immunoblots of the phosphorylation and total protein levels of AMPK, Akt, and ERK1/2 in adipocytes, after 30 min incubation without (⫺) or with (⫹) 1 ␮M
pyr-apelin13 in the absence (⫺) or presence (⫹; added 1 h before apelin treatment) of 50 ␮M PD98059, 10 ␮M LY294002, or 1 ␮M dorsomorphin (n ⫽ 3). G–-N,
Western blot analyses of the expression of p47phox, PGC-1␣, COX1, SDHA, SOD1, Catalase, GPx, and actin in adipocytes, after 24 h incubated without (⫺)
(control) or with (⫹) 1 ␮M pyr-apelin13 in the absence (⫺) or presence (⫹; added 1 h before apelin treatment) of 50 ␮M PD98059, 10 ␮M LY294002, or 1 ␮M
dorsomorphin. The representative immunoblots are shown in G. The statistics of the blot intensity in H–N (mean ⫾ S.E., n ⫽ 4) is normalized to that of actin. Each
sample contains the same amount of total proteins. Student’s t test: *, p ⬍ 0.05; **, p ⬍ 0.01 versus control; #, p ⬍ 0.05, ##, p ⬍ 0.01 between indicated pairs.

production (Fig. 4F). Taken together, we provide evidence that
the impaired mitochondrial biogenesis and function in oxida-
tive stress can be remedied by apelin.
Signaling Pathways Underlying the Apelin-mediated Expres-
sion of the Proteins Related to Mitochondrial Biogenesis and
ROS Generation—Mitochondrial biogenesis depends on at
least two separate pathways: AMP-activated protein kinase
(AMPK) and phosphatidylinositol 3 kinase/Akt (PI3K/Akt)
(55). In agreement with the previous reports (39, 56, 57) that
apelin increased activation (phosphorylation) of AMPK Akt,
and ERK1/2 in adipocytes (Fig. 5, A–C), in a dose-dependent
manner (data not shown). As shown in Fig. 5D, G, and I, inhib-
iting AMPK phosphorylation by dorsomorphin (a potent and
selective AMPK inhibitor) (58) abolished the apelin-stimulated
expression of PGC1␣ (a nodal regulator of mitochondrial
biogenesis). Dorsomorphin treatment also effectively blocked
the apelin-induced expression of the mitochondrial proteins
(COX1 and SDHA). Blocking the PI3K/Akt pathway by
LY294002 (a potent and selective PI3K inhibitor) (59) similarly
abolished the apelin-induced expression of COX1 and SDHA
(Fig. 5E, J, and K). In contrast, blocking the MAPK kinase by its
specific inhibitor (PD98059) (60) had no effect on the expres-
sion of PGC1␣, COX1, and SDHA (Fig. 5F, I–K). These obser-
vations suggest that apelin stimulates mitochondrial biogenesis
through two distinct routes: 1) activating the AMPK signaling,
which leads to increase of PGC1␣ expression; and 2) activating
the PI3K/Akt signaling, which enhances expression of mito-
chondrial proteins.
Studies have shown that AMPK signaling (likely via PGC1␣)
stimulates the expression of anti-oxidant enzymes (61, 62)
whereas PI3K/Akt signaling impairs ROS scavenging (63, 64).
Consistently, we observed that blocking the AMPK pathway

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Apelin Suppresses Oxidative Stress

FIGURE 6. A–C, apelin modulates ROS-induced dysregulation of IL-6, TNF␣, and adiponectin release from adipocytes. After 24 h exposure to without (⫺) or with
(⫹) 1 ␮M pyr-apelin13, the media were changed, and adipocytes were treated without (⫺) (control) or with (⫹) pyr-apelin and/or 200 ␮M H2O2 for 12 h. IL6,
TNF␣, and adiponectin release from adipocytes were measured with ELIZA or EIA (A–C). D–E, expression of APJ (⬃40 kDa) and actin in adipocytes were analyzed
by Western blot. Representative immunoblots are shown in D; the statistics of the blot intensity in E is normalized to that of actin (as the internal control). F, ROS
stimulate apelin secretion from adipocytes. Apelin release from adipocytes (2 h, 12 h), without (⫺) (control) or with exposure to (⫹) 200 ␮M H2O2, were
measured with EIA. The results are shown as mean ⫾ S.E. (n ⫽ 4 – 6) of 3– 4 independent samples (Each sample contains the same amount of total proteins).
Student’s t test: *, p ⬍ 0.05; **, p ⬍ 0.01 versus control; #, p ⬍ 0.05 between indicated pairs.

largely eliminated the apelin-stimulated expression of anti-ox-
idant enzymes (catalase, GPx) and apelin-suppressed expres-
sion of pro-oxidant enzyme (P47phox subunit of NADPH
oxidase) and inhibiting PI3K/Akt led to up-regulation of anti-
oxidant enzymes (SOD1 and catalase) (Fig. 5, G, L–N). In addi-
tion, apelin stimulation on antioxidant enzyme expression is
also dependent on MAPK kinase/ERK pathway, since PD98059
treatment effectively eliminated the apelin-induced expression
of SOD1, catalase, and GPx (Fig. 5, G, L–N). Taken together,
our results show that apelin-mediated expression of anti- or
pro-oxidant enzymes are, at least in part, dependent on AMPK
and MAPK kinase/ERK signaling pathways.
Apelin Attenuates the ROS-stimulated Release of Pro-inflam-
matory Adipocytokines—ROS overproduction (oxidative stress) is
closely associated with inflammation in adipose tissue (9). Spe-
cifically, pro-inflammatory adipocytokines secreted from adi-
pocytes increase ROS generation in adipocytes; ROS overpro-
duction, on the other hand, stimulates adipocytic release of
pro-inflammatory factors (38, 44). Thus, oxidative stress and
inflammation in adipose form a vicious positive cycle. Figs. 3
and 4 demonstrate that apelin is able to suppress TNF␣-in-
duced ROS production. Here, we further sought to investigate
the apelin effects on ROS-stimulated release of pro-inflamma-
tory (IL6 and TNF␣) and anti-inflammatory (adiponectin)
adipocytokines.
As shown in Fig. 6, A–C, ROS (H2O2) stimulated adipocytic
release of pro-inflammatory factors (IL6 and TNF␣) and
reduced release of anti-inflammatory factor (adiponectin),
indicating that ROS can cause or worsen local inflammation in
adipose tissue. Interestingly, apelin treatment largely rectified
these effects (Fig. 6, A–C), suggesting its ability to attenuate the
ROS-induced inflammation in adipose tissue. ROS also sup-
pressed the expression of APJ receptor, while apelin treatment
induced it (Fig. 6, D and E). On the other hand, it was observed

3770 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 6 • FEBRUARY 7, 2014


FIGURE 7. The apelin-APJ signaling pathways on regulating oxidative
stress.
that H2O2 enhances apelin release from adipocytes (Fig. 6F).
This is consistent with the previous report that phorbol myris-
tate acetate (PMA, a potent ROS inducer) can acutely stimulate
adipocytic release of apelin (16). Therefore, it is likely that the
excess release of apelin from adipocytes (instead of increased
APJ receptor expression) acts as a negative autocrine control to
the oxidative stress and inflammation in adipose.
DISCUSSION
Adipocytes (fat cells), the major depots for excess energy, are
also active endocrine cells producing a variety of secretory fac-
tors (adipocytokines) that play critical roles in regulating met-
abolic homeostasis (10, 65, 66). Obesity (excess fat accumula-
tion) is closely associated with various metabolic disorders (67).
It has been recently recognized that the increased oxidative
stress (ROS overproduction) in obese condition is a key con-
tributor to the pathogenesis of obesity-associated metabolic
diseases, particularly, insulin resistance (2, 5, 68). Adipose tis-
sue is a major source of plasma ROS level (3). Oxidative stress in
adipose tissue, in turn, impairs adipocyte functions and accel-
erates ROS overproduction (69, 70). In addition, the local
inflammation induced by excess ROS further exacerbates the
oxidative stress (9). Such vicious positive cycles in adipose tis-
sue lead to systematic oxidative stress and inflammation which
causes development of insulin resistance in peripheral tissues as
well as other diseases (e.g. atherosclerosis and hypertension) (1,
4).
In this study, we provide evidences that apelin (secreted by
adipocytes as an adipocytokine, and other cells) is able to halt
the abovementioned adverse chain reactions. Based on our
experiments, the following scenario may be proposed (Fig. 7).
As shown, excess ROS inhibits release of anti-inflammatory
factors (adiponectin) and promotes release of pro-inflamma-
tory factors (TNF␣ and IL6) from adipocytes (Fig. 6, A–C). Such
inflammatory responses, in turn, further stimulate the produc-
tion of ROS, because, for example, the autocrine interaction
between the release TNF␣ and its receptor on adipocyte mem-
FEBRUARY 7, 2014 • VOLUME 289 • NUMBER 6
Apelin Suppresses Oxidative Stress
brane inhibits the expression of anti-oxidant enzymes (SOD,
GPx, and catalase) and increases the expression of pro-oxidant
enzymes (e.g. NADPH oxidase) (Fig. 3). In addition, excess ROS
impairs the mitochondrial biogenesis and function (Fig. 4). It
has been shown that mitochondrial malfunction leads to ROS
overproduction (15, 69). Excess ROS together with the trig-
gered vicious positive feedbacks promote the development of
insulin resistance. For example, it is known that oxidative stress
impairs the function of insulin receptor substrate-1(IRS1)
whereby the translocation of glucose transporter (GLUT4) and
thus glucose uptake are reduced (8).
On the other hand, excess ROS enhances the release of apelin
from adipocyte to initiate the negative controls to the oxidative
stress (Figs. 1 and 6F). Specifically, the autocrine interaction
between the released apelin and APJ receptors on adipocyte
membrane stimulates the expression of anti-oxidant enzymes
via AMPK and MAPK kinase/ERK signaling pathways, and
inhibits the expression of pro-oxidant enzymes via AMPK sig-
naling (Figs. 1 and 5). The resulting suppression of ROS rectifies
the positive echo between oxidative stress and inflammation
(Figs. 3 and 6, A–C). Furthermore, the apelin-APJ activation
also remedies ROS-impaired mitochondrial biogenesis and
function by, at least in part, stimulating the expression of
PGC1␣ (a nodal transcription factor of mitochondrial biogen-
esis) through AMPK pathway (Figs. 4 and 5). The notion that
apelin signaling is a beneficial physiological response to oxida-
tive stress in adipose is supported by the previous observation
that the plasma apelin level is elevated in obesity (25, 26). It also
explains the previous observations in mice that apelin treat-
ment leads to a significant decrease in plasma lipid hydroper-
oxide (an index of systemic oxidative stress) (23), and improves
glucose uptake in adipose tissue (18). Dray et al. has demon-
strated that intravenous injection of apelin has a powerful effect
to enhance glucose utilization in skeletal muscle and adipose
tissue in high-fat fed obese and insulin-resistant mice (20). As
reported in this study, endothelial NO synthase (eNOS) activa-
tion is essential to apelin’s effect on skeletal muscle (20). eNOS
activity is also important for glucose uptake in adipocytes (71,
72). It is conceivable that eNOS activation may be partly attrib-
uted to apelin inhibition on ROS level (73, 74).
Oxidative stress can increase fat tissue mass since ROS facil-
itates adipogenesis via accelerating mitotic clonal expansion
(11, 75). It is also known that mitochondrial oxidative stress
triggered by ROS inhibits aconitase in the tricarboxylic acid
cycle (TCA cycle) leading to increased biosynthesis of fatty
acids (76, 77). Moreover, ROS stimulates lipolysis by recruiting
the hormone sensitive lipase to the lipid droplets (13, 78),
resulting in increased release of free fatty acids which, in turn,
leads to increase of ROS generation and insulin resistance (79,
80). We have previously shown that apelin suppresses adipo-
genesis and lipolysis (22). Our current study implies that the
antioxidant effects of apelin can attenuate ROS-induced adipo-
genesis, lipogenesis, lipolysis, and release of free fatty acids.
Lipoic acid (an antioxidant) is a commercial dietary supple-
ment, which has been shown to be beneficial for reducing body
adiposity and insulin resistance (81, 82). This study and our
previous study suggest that apelin-APJ signaling pathways may
JOURNAL OF BIOLOGICAL CHEMISTRY 3771
Apelin Suppresses Oxidative Stress
serve as the novel therapeutic targets for obesity and obesity-
associated metabolic diseases.
REFERENCES
1. Roberts, C. K., and Sindhu, K. K. (2009) Oxidative stress and metabolic
syndrome. Life Sci. 84, 705–712
2. Sankhla, M., Sharma, T. K., Mathur, K., Rathor, J. S., Butolia, V., Gadhok,
A. K., Vardey, S. K., Sinha, M., and Kaushik, G. G. (2012) Relationship of
oxidative stress with obesity and its role in obesity induced metabolic
syndrome. Clin. Lab. 58, 385–392
3. Furukawa, S., Fujita, T., Shimabukuro, M., Iwaki, M., Yamada, Y., Naka-
jima, Y., Nakayama, O., Makishima, M., Matsuda, M., and Shimomura, I.
(2004) Increased oxidative stress in obesity and its impact on metabolic
syndrome. J. Clin. Invest. 114, 1752–1761
4. Maritim, A. C., Sanders, R. A., and Watkins, J. B., 3rd. (2003) Diabetes,
oxidative stress, and antioxidants: a review. J. Biochem. Mol. Toxicol. 17,
24 –38
5. Whaley-Connell, A., McCullough, P. A., and Sowers, J. R. (2011) The role
of oxidative stress in the metabolic syndrome. Rev. Cardiovasc. Med. 12,
21–29
6. Matsuoka, T., Kajimoto, Y., Watada, H., Kaneto, H., Kishimoto, M.,
Umayahara, Y., Fujitani, Y., Kamada, T., Kawamori, R., and Yamasaki, Y.
(1997) Glycation-dependent, reactive oxygen species-mediated suppres-
sion of the insulin gene promoter activity in HIT cells. J. Clin. Invest. 99,
144 –150
7. Maddux, B. A., See, W., Lawrence, J. C., Jr., Goldfine, A. L., Goldfine, I. D.,
and Evans, J. L. (2001) Protection against oxidative stress-induced insulin
resistance in rat L6 muscle cells by micromolar concentrations of ␣-lipoic
acid. Diabetes 50, 404 – 410
8. Rudich, A., Tirosh, A., Potashnik, R., Hemi, R., Kanety, H., and Bashan, N.
(1998) Prolonged oxidative stress impairs insulin-induced GLUT4 trans-
location in 3T3-L1 adipocytes. Diabetes 47, 1562–1569
9. Fernández-Sánchez, A., Madrigal-Santillán, E., Bautista, M., Esquivel-
Soto, J., Morales-González, A., Esquivel-Chirino, C., Durante-Montiel, I.,
Sánchez-Rivera, G., Valadez-Vega, C., and Morales-González, J. A. (2011)
Inflammation, oxidative stress, and obesity. Int. J. Mol. Sci. 12, 3117–3132
10. Trayhurn, P., Bing, C., and Wood, I. S. (2006) Adipose tissue and
adipokines– energy regulation from the human perspective. J. Nutr. 136,
1935S-1939S
11. Lee, H., Lee, Y. J., Choi, H., Ko, E. H., and Kim, J. W. (2009) Reactive oxygen
species facilitate adipocyte differentiation by accelerating mitotic clonal
expansion. J. Biol. Chem. 284, 10601–10609
12. Shibata, M., Hakuno, F., Yamanaka, D., Okajima, H., Fukushima, T., Hase-
gawa, T., Ogata, T., Toyoshima, Y., Chida, K., Kimura, K., Sakoda, H.,
Takenaka, A., Asano, T., and Takahashi, S. (2010) Paraquat-induced oxi-
dative stress represses phosphatidylinositol 3-kinase activities leading to
impaired glucose uptake in 3T3-L1 adipocytes. J. Biol. Chem. 285,
20915–20925
13. Krawczyk, S. A., Haller, J. F., Ferrante, T., Zoeller, R. A., and Corkey, B. E.
(2012) Reactive oxygen species facilitate translocation of hormone sensi-
tive lipase to the lipid droplet during lipolysis in human differentiated
adipocytes. PLoS One 7, e34904
14. Kirkinezos, I. G., and Moraes, C. T. (2001) Reactive oxygen species and
mitochondrial diseases. Semin Cell Dev. Biol. 12, 449 – 457
15. Kim, J. A., Wei, Y., and Sowers, J. R. (2008) Role of mitochondrial dysfunc-
tion in insulin resistance. Circ. Res. 102, 401– 414
16. Than, A., Tee, W. T., and Chen, P. (2012) Apelin secretion and expression
of apelin receptors in 3T3-L1 adipocytes are differentially regulated by
angiotensin type 1 and type 2 receptors. Mol. Cell. Endocrinol. 351,
296 –305
17. Boucher, J., Masri, B., Daviaud, D., Gesta, S., Guigné, C., Mazzucotelli, A.,
Castan-Laurell, I., Tack, I., Knibiehler, B., Carpéné, C., Audigier, Y.,
Saulnier-Blache, J. S., and Valet, P. (2005) Apelin, a newly identified adi-
pokine up-regulated by insulin and obesity. Endocrinology 146,
1764 –1771
18. Castan-Laurell, I., Dray, C., Attané, C., Duparc, T., Knauf, C., and Valet, P.
(2011) Apelin, diabetes, and obesity. Endocrine 40, 1–9
3772 JOURNAL OF BIOLOGICAL CHEMISTRY
19. Attané, C., Foussal, C., Le Gonidec, S., Benani, A., Daviaud, D., Wanecq, E.,
Guzmán-Ruiz, R., Dray, C., Bezaire, V., Rancoule, C., Kuba, K., Ruiz-Gayo,
M., Levade, T., Penninger, J., Burcelin, R., Pénicaud, L., Valet, P., and
Castan-Laurell, I. (2012) Apelin treatment increases complete Fatty Acid
oxidation, mitochondrial oxidative capacity, and biogenesis in muscle of
insulin-resistant mice. Diabetes 61, 310 –320
20. Dray, C., Knauf, C., Daviaud, D., Waget, A., Boucher, J., Buléon, M., Cani,
P. D., Attané, C., Guigné, C., Carpéné, C., Burcelin, R., Castan-Laurell, I.,
and Valet, P. (2008) Apelin stimulates glucose utilization in normal and
obese insulin-resistant mice. Cell Metab. 8, 437– 445
21. Chu, J., Zhang, H., Huang, X., Lin, Y., Shen, T., Chen, B., Man, Y., Wang, S.,
and Li, J. (2013) Apelin ameliorates TNF-␣-induced reduction of glycogen
synthesis in the hepatocytes through G protein-coupled receptor APJ.
PLoS One. 8, e57231
22. Than, A., Cheng, Y., Foh, L. C., Leow, M. K., Lim, S. C., Chuah, Y. J., Kang,
Y., and Chen, P. (2012) Apelin inhibits adipogenesis and lipolysis through
distinct molecular pathways. Mol. Cell. Endocrinol. 362, 227–241
23. Foussal, C., Lairez, O., Calise, D., Pathak, A., Guilbeau-Frugier, C., Valet,
P., Parini, A., and Kunduzova, O. (2010) Activation of catalase by apelin
prevents oxidative stress-linked cardiac hypertrophy. FEBS Lett. 584,
2363–2370
24. Chun, H. J., Ali, Z. A., Kojima, Y., Kundu, R. K., Sheikh, A. Y., Agrawal, R.,
Zheng, L., Leeper, N. J., Pearl, N. E., Patterson, A. J., Anderson, J. P., Tsao,
P. S., Lenardo, M. J., Ashley, E. A., and Quertermous, T. (2008) Apelin
signaling antagonizes Ang II effects in mouse models of atherosclerosis.
J. Clin. Invest. 118, 3343–3354
25. Castan-Laurell, I., Vítkova, M., Daviaud, D., Dray, C., Kováciková, M.,
Kovacova, Z., Hejnova, J., Stich, V., and Valet, P. (2008) Effect of hypoca-
loric diet-induced weight loss in obese women on plasma apelin and adi-
pose tissue expression of apelin and APJ. Eur. J. Endocrinol. 158, 905–910
26. Soriguer, F., Garrido-Sanchez, L., Garcia-Serrano, S., Garcia-Almeida,
J. M., Garcia-Arnes, J., Tinahones, F. J., and Garcia-Fuentes, E. (2009)
Apelin levels are increased in morbidly obese subjects with type 2 diabetes
mellitus. Obes. Surg. 19, 1574 –1580
27. Than, A., Leow, M. K., and Chen, P. (2013) Control of adipogenesis by the
autocrine interplays between angiotensin 1–7/Mas receptor and angio-
tensin II/AT1 receptor signaling pathways. J. Biol. Chem. 288,
15520 –15531
28. Hemmrich, K., von Heimburg, D., Cierpka, K., Haydarlioglu, S., and Pal-
lua, N. (2005) Optimization of the differentiation of human preadipocytes
in vitro. Differentiation 73, 28 –35
29. Peshavariya, H. M., Dusting, G. J., and Selemidis, S. (2007) Analysis of
dihydroethidium fluorescence for the detection of intracellular and extra-
cellular superoxide produced by NADPH oxidase. Free Radic. Res. 41,
699 –712
30. Zhao, H., Joseph, J., Fales, H. M., Sokoloski, E. A., Levine, R. L., Vasquez-
Vivar, J., and Kalyanaraman, B. (2005) Detection and characterization of
the product of hydroethidine and intracellular superoxide by HPLC and
limitations of fluorescence. Proc. Natl. Acad. Sci. U.S.A. 102, 5727–5732
31. Kleinz, M. J., and Davenport, A. P. (2005) Emerging roles of apelin in
biology and medicine. Pharmacol. Ther. 107, 198 –211
32. Zhen, E. Y., Higgs, R. E., and Gutierrez, J. A. (2013) Pyroglutamyl apelin-13
identified as the major apelin isoform in human plasma. Anal. Biochem.
442, 1–9
33. Pitkin, S. L., Maguire, J. J., Bonner, T. I., and Davenport, A. P. (2010)
International Union of Basic and Clinical Pharmacology. LXXIV. Apelin
receptor nomenclature, distribution, pharmacology, and function. Phar-
macol. Rev. 62, 331–342
34. Kobayashi, H., Matsuda, M., Fukuhara, A., Komuro, R., and Shimomura, I.
(2009) Dysregulated glutathione metabolism links to impaired insulin ac-
tion in adipocytes. Am. J. Physiol. Endocrinol. Metab. 296, E1326 –E1334
35. Ray, G., and Husain, S. A. (2002) Oxidants, antioxidants and carcinogen-
esis. Indian J. Exp. Biol. 40, 1213–1232
36. Babior, B. M. (1999) NADPH oxidase: an update. Blood 93, 1464 –1476
37. Cawthorn, W. P., and Sethi, J. K. (2008) TNF-␣ and adipocyte biology.
FEBS Lett. 582, 117–131
38. Chen, X. H., Zhao, Y. P., Xue, M., Ji, C. B., Gao, C. L., Zhu, J. G., Qin, D. N.,
Kou, C. Z., Qin, X. H., Tong, M. L., and Guo, X. R. (2010) TNF-␣ induces
VOLUME 289 • NUMBER 6 • FEBRUARY 7, 2014

mitochondrial dysfunction in 3T3-L1 adipocytes. Mol. Cell. Endocrinol.
328, 63– 69
39. Zhu, S., Sun, F., Li, W., Cao, Y., Wang, C., Wang, Y., Liang, D., Zhang, R.,
Zhang, S., Wang, H., and Cao, F. (2011) Apelin stimulates glucose uptake
through the PI3K/Akt pathway and improves insulin resistance in 3T3-L1
adipocytes. Mol. Cell Biochem. 353, 305–313
40. Zheng, X. T., Than, A., Ananthanaraya, A., Kim, D. H., and Chen, P. (2013)
Graphene quantum dots as universal fluorophores and their use in reveal-
ing regulated trafficking of insulin receptors in adipocytes. ACS Nano. 7,
6278 – 6286
41. Woo, C. H., Eom, Y. W., Yoo, M. H., You, H. J., Han, H. J., Song, W. K., Yoo,
Y. J., Chun, J. S., and Kim, J. H. (2000) Tumor necrosis factor-␣ generates
reactive oxygen species via a cytosolic phospholipase A2-linked cascade.
J. Biol. Chem. 275, 32357–32362
42. Turrens, J. F. (2003) Mitochondrial formation of reactive oxygen species.
J. Physiol. 552, 335–344
43. Bindokas, V. P., Jordán, J., Lee, C. C., and Miller, R. J. (1996) Superoxide
production in rat hippocampal neurons: selective imaging with hydroethi-
dine. J. Neurosci. 16, 1324 –1336
44. Park, J., Choe, S. S., Choi, A. H., Kim, K. H., Yoon, M. J., Suganami, T.,
Ogawa, Y., and Kim, J. B. (2006) Increase in glucose-6-phosphate dehy-
drogenase in adipocytes stimulates oxidative stress and inflammatory sig-
nals. Diabetes 55, 2939 –2949
45. Kusminski, C. M., and Scherer, P. E. (2012) Mitochondrial dysfunction in
white adipose tissue. Trends Endocrinol. Metab. 23, 435– 443
46. Bournat, J. C., and Brown, C. W. (2010) Mitochondrial dysfunction in
obesity. Curr. Opin. Endocrinol. Diabetes Obes. 17, 446 – 452
47. Choo, H. J., Kim, J. H., Kwon, O. B., Lee, C. S., Mun, J. Y., Han, S. S., Yoon,
Y. S., Yoon, G., Choi, K. M., and Ko, Y. G. (2006) Mitochondria are im-
paired in the adipocytes of type 2 diabetic mice. Diabetologia. 49, 784 –791
48. Wilson-Fritch, L., Nicoloro, S., Chouinard, M., Lazar, M. A., Chui, P. C.,
Leszyk, J., Straubhaar, J., Czech, M. P., and Corvera, S. (2004) Mitochon-
drial remodeling in adipose tissue associated with obesity and treatment
with rosiglitazone. J. Clin. Invest. 114, 1281–1289
49. Gao, C. L., Zhu, C., Zhao, Y. P., Chen, X. H., Ji, C. B., Zhang, C. M., Zhu,
J. G., Xia, Z. K., Tong, M. L., and Guo, X. R. (2010) Mitochondrial dysfunc-
tion is induced by high levels of glucose and free fatty acids in 3T3-L1
adipocytes. Mol. Cell. Endocrinol. 320, 25–33
50. Valerio, A., Cardile, A., Cozzi, V., Bracale, R., Tedesco, L., Pisconti, A.,
Palomba, L., Cantoni, O., Clementi, E., Moncada, S., Carruba, M. O., and
Nisoli, E. (2006) TNF-alpha downregulates eNOS expression and mito-
chondrial biogenesis in fat and muscle of obese rodents. J. Clin. Invest. 116,
2791–2798
51. Zhu, M., Li, W., and Lu, C. (2012) Role of -synuclein protein levels in
mitochondrial morphology and cell survival in cell lines. PLoS One. 7,
e36377
52. Wilson-Fritch, L., Burkart, A., Bell, G., Mendelson, K., Leszyk, J., Nicoloro,
S., Czech, M., and Corvera, S. (2003) Mitochondrial biogenesis and re-
modeling during adipogenesis and in response to the insulin sensitizer
rosiglitazone. Mol. Cell. Biol. 23, 1085–1094
53. Xu, S., Pi, H., Chen, Y., Zhang, N., Guo, P., Lu, Y., He, M., Xie, J., Zhong, M.,
Zhang, Y., Yu, Z., and Zhou, Z. (2013) Cadmium induced Drp1-dependent
mitochondrial fragmentation by disturbing calcium homeostasis in its
hepatotoxicity. Cell Death Dis. 4, e540
54. Fernandez-Marcos, P. J., and Auwerx, J. (2011) Regulation of PGC-1alpha,
a nodal regulator of mitochondrial biogenesis. Am. J. Clin. Nutr. 93,
884S– 890S
55. Wenz, T. (2013) Regulation of mitochondrial biogenesis and PGC-1␣ un-
der cellular stress. Mitochondrion 13, 134 –142
56. Yue, P., Jin, H., Xu, S., Aillaud, M., Deng, A. C., Azuma, J., Kundu, R. K.,
Reaven, G. M., Quertermous, T., and Tsao, P. S. (2011) Apelin decreases
lipolysis via G(q), G(i), and AMPK-Dependent Mechanisms. Endocrinol-
ogy 152, 59 – 68
57. Masri, B., Morin, N., Pedebernade, L., Knibiehler, B., and Audigier, Y.
(2006) The apelin receptor is coupled to Gi1 or Gi2 protein and is differ-
entially desensitized by apelin fragments. J. Biol. Chem. 281, 18317–18326
58. McCullough, L. D., Zeng, Z., Li, H., Landree, L. E., McFadden, J., and
Ronnett, G. V. (2005) Pharmacological inhibition of AMP-activated pro-
FEBRUARY 7, 2014 • VOLUME 289 • NUMBER 6
Apelin Suppresses Oxidative Stress
tein kinase provides neuroprotection in stroke. J. Biol. Chem. 280,
20493–20502
59. Vlahos, C. J., Matter, W. F., Hui, K. Y., and Brown, R. F. (1994) A specific
inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-
4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269, 5241–5248
60. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995)
PD 098059 is a specific inhibitor of the activation of mitogen-activated
protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270, 27489 –27494
61. Colombo, S. L., and Moncada, S. (2009) AMPKalpha1 regulates the anti-
oxidant status of vascular endothelial cells. Biochem. J. 421, 163–169
62. Olmos, Y., Valle, I., Borniquel, S., Tierrez, A., Soria, E., Lamas, S., and
Monsalve, M. (2009) Mutual dependence of Foxo3a and PGC-1alpha in
the induction of oxidative stress genes. J. Biol. Chem. 284, 14476 –14484
63. Nogueira, V., Park, Y., Chen, C. C., Xu, P. Z., Chen, M. L., Tonic, I.,
Unterman, T., and Hay, N. (2008) Akt determines replicative senescence
and oxidative or oncogenic premature senescence and sensitizes cells to
oxidative apoptosis. Cancer Cell. 14, 458 – 470
64. Los, M., Maddika, S., Erb, B., and Schulze-Osthoff, K. (2009) Switching
Akt: from survival signaling to deadly response. Bioessays 31, 492– 495
65. Than, A., Ye, F., Xue, R., Ong, J. W., Poh, C. L., and Chen, P. (2011) The
crosstalks between adipokines and catecholamines. Mol. Cell. Endocrinol.
332, 261–270
66. Ye, F., Than, A., Zhao, Y., Goh, K. H., and Chen, P. (2010) Vesicular
storage, vesicle trafficking, and secretion of leptin and resistin: the simi-
larities, differences, and interplays. J. Endocrinol. 206, 27–36
67. Grundy, S. M. (2004) Obesity, metabolic syndrome, and cardiovascular
disease. J. Clin. Endocrinol. Metab. 89, 2595–2600
68. Meigs, J. B., Larson, M. G., Fox, C. S., Keaney, J. F., Jr., Vasan, R. S., and
Benjamin, E. J. (2007) Association of oxidative stress, insulin resistance,
and diabetes risk phenotypes: the Framingham Offspring Study. Diabetes
Care 30, 2529 –2535
69. Wang, C. H., Wang, C. C., Huang, H. C., and Wei, Y. H. (2013) Mitochon-
drial dysfunction leads to impairment of insulin sensitivity and adiponec-
tin secretion in adipocytes. Febs. J. 280, 1039 –1050
70. Crujeiras, A. B., Díaz-Lagares, A., Carreira, M. C., Amil, M., and Casan-
ueva, F. F. (2013) Oxidative stress associated to dysfunctional adipose
tissue: a potential link between obesity, type 2 diabetes mellitus and breast
cancer. Free Radic. Res. 47, 243–256
71. Tanaka, T., Nakatani, K., Morioka, K., Urakawa, H., Maruyama, N.,
Kitagawa, N., Katsuki, A., Araki-Sasaki, R., Hori, Y., Gabazza, E. C., Yano,
Y., Wada, H., Nobori, T., Sumida, Y., and Adachi, Y. (2003) Nitric oxide
stimulates glucose transport through insulin-independent GLUT4 trans-
location in 3T3-L1 adipocytes. Eur. J. Endocrinol. 149, 61– 67
72. Beard, K. M., Lu, H., Ho, K., and Fantus, I. G. (2006) Bradykinin augments
insulin-stimulated glucose transport in rat adipocytes via endothelial ni-
tric oxide synthase-mediated inhibition of Jun NH2-terminal kinase. Di-
abetes 55, 2678 –2687
73. Rafikov, R., Fonseca, F. V., Kumar, S., Pardo, D., Darragh, C., Elms, S.,
Fulton, D., and Black, S. M. (2011) eNOS activation and NO function:
structural motifs responsible for the posttranslational control of endothe-
lial nitric oxide synthase activity. J. Endocrinol. 210, 271–284
74. Kumar, S., Sun, X., Wedgwood, S., and Black, S. M. (2008) Hydrogen
peroxide decreases endothelial nitric oxide synthase promoter activity
through the inhibition of AP-1 activity. Am. J. Physiol. Lung Cell Mol.
Physiol. 295, L370 –L377
75. Kanda, Y., Hinata, T., Kang, S. W., and Watanabe, Y. (2011) Reactive
oxygen species mediate adipocyte differentiation in mesenchymal stem
cells. Life Sci. 89, 250 –258
76. Tong, W. H., and Rouault, T. A. (2007) Metabolic regulation of citrate and
iron by aconitases: role of iron-sulfur cluster biogenesis. Biometals 20,
549 –564
77. Yan, L. J., Levine, R. L., and Sohal, R. S. (1997) Oxidative damage during
aging targets mitochondrial aconitase. Proc. Natl. Acad. Sci. U.S.A. 94,
11168 –11172
78. Zhang, X., Wang, Z., Li, J., Gu, D., Li, S., Shen, C., and Song, Z. (2013)
Increased 4-hydroxynonenal formation contributes to obesity-related
lipolytic activation in adipocytes. PLoS One. 8, e70663
79. Subauste, A. R., and Burant, C. F. (2007) Role of FoxO1 in FFA-induced
JOURNAL OF BIOLOGICAL CHEMISTRY 3773
Apelin Suppresses Oxidative Stress
oxidative stress in adipocytes. Am. J. Physiol. Endocrinol. Metab. 293,
E159 –E164
80. Roden, M., Price, T. B., Perseghin, G., Petersen, K. F., Rothman, D. L.,
Cline, G. W., and Shulman, G. I. (1996) Mechanism of free fatty acid-
induced insulin resistance in humans. J. Clin. Invest. 97, 2859 –2865
81. Evans, J. L., and Goldfine, I. D. (2000) ␣-lipoic acid: a multifunctional
3774 JOURNAL OF BIOLOGICAL CHEMISTRY
antioxidant that improves insulin sensitivity in patients with type 2 diabe-
tes. Diabetes Technol. Ther. 2, 401– 413
82. Prieto-Hontoria, P. L., Pérez-Matute, P., Fernández-Galilea, M., Barber,
A., Martínez, J. A., and Moreno-Aliaga, M. J. (2009) Lipoic acid prevents
body weight gain induced by a high fat diet in rats: effects on intestinal
sugar transport. J. Physiol. Biochem. 65, 43–50
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