Epoxyeicosatrienoic Acid Agonist Regulates
Human Mesenchymal Stem Cell–Derived Adipocytes
Through Activation of HO-1-pAKT Signaling
and a Decrease in PPARg
Dong Hyun Kim,1,*Luca Vanella,1,*Kazuyoshi Inoue,2Angela Burgess,1Katherine Gotlinger,2
Vijaya Lingam Manthati,3Sreenivasulu Reddy Koduru,3Darryl C. Zeldin,4
John R. Falck,3Michal L. Schwartzman,2and Nader G. Abraham1
Human mesenchymal stem cells (MSCs) expressed substantial levels of CYP2J2, a major CYP450 involved in
epoxyeicosatrienoic acid (EET) formation. MSCs synthesized significant levels of EETs (65.8?5.8pg=mg protein)
and dihydroxyeicosatrienoic acids (DHETs) (15.83?1.62pg=mg protein), suggesting the presence of soluble ep-
oxide hydrolase (sEH). The addition of an sEH inhibitor to MSC culture decreased adipogenesis. EETs decreased
MSC-derived adipocytes in a concentration-dependent manner, 8,9- and 14,15-EET having the maximum reductive
effect on adipogenesis. We examined the effect of 12-(3-hexylureido)dodec-8(Z)-enoic acid, an EET agonist, on
MSC-derived adipocytes and demonstrated an increased number of healthy small adipocytes, attenuated fatty acid
synthase (FAS) levels (P<0.01), and reduced PPARg, C=EBPa, FAS, and lipid accumulation (P<0.05). These effects
were accompanied by increased levels of heme oxygenase (HO)-1 and adiponectin (P<0.05), and increased glucose
uptake (P<0.05). Inhibition of HO activity or AKT by tin mesoporphyrin (SnMP) and LY2940002, respectively,
reversed EET-induced inhibition of adipogenesis, suggesting that activation of the HO-1-adiponectin axis underlies
EET effect in MSCs. These findings indicate that EETs decrease MSC-derived adipocyte stem cell differentiation by
upregulation of HO-1-adiponectin-AKT signaling and play essential roles in the regulation of adipocyte differ-
entiation by inhibiting PPARg, C=EBPa, and FAS and in stem cell development. These novel observations highlight
the seminal role of arachidonic acid metabolism in MSCs and suggest that an EET agonist may have potential
therapeutic use in the treatment of dyslipidemia, diabetes, and the metabolic syndrome.
rise to cells of diverse lineages and also modulate the in-
flammatory response through downregulation of proin-
flammatory molecules and upregulation of prosurvival and
antiinflammatory molecules simultaneously . Under con-
ditions of increased levels of oxidative stress and increased
levels of reactive oxygen species (ROS), MSCs exhibit in-
creased senescence, poor differentiation, and a reduced ca-
pacity for tissue repair [2,3]. An increase in ROS occurs,
resulting in a progressive deterioration in adipocyte and
vascular function with a reduced number and function of
esenchymal stem cells (MSCs) are self-renewable
multipotent stromal cells that have the ability to give
circulating endothelial progenitor cells and elevation of in-
flammatory cytokines from adipose tissue [4,5]. Oxidative
stress has been considered a major factor impairing MSC
function leading to a decreased osteogenesis in favor of adi-
pogenesis [6,7]. Due to these characteristics, MSCs have been
studied for the use as potential cell-based transplantation
therapy for treatment of diabetes and obesity [8,9].
Heme oxygenase (HO)-1 has been identified as a primary
antioxidative defense system for hematopoietic stem cells
. We have shown that upregulation of HO-1 gene ex-
pression decreases adipokines such as IL-1 and IL-6 and in-
creases levels of adiponectin, which is produced solely by
adipocyte . Further, an increase in HO-1 protein levels
is associated with a parallel increase in the AMP-activated
1Department of Physiology and Pharmacology, University of Toledo College of Medicine, Toledo, Ohio.
2Department of Pharmacology, New York Medical College, Valhalla, New York.
3Department of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center of Dallas, Dallas, Texas.
4Division of Intramural Research, NIH=NIEHS, Research Triangle Park, North Carolina.
*These authors contributed equally to this work.
STEM CELLS AND DEVELOPMENT
Volume 19, Number 12, 2010
ª Mary Ann Liebert, Inc.
kinase (AMPK) and AKT signaling pathway [12–14]. In-
creased HO-1 expression in obesity and type 2 diabetes re-
sults in a decrease in visceral and subcutaneous fat content,
improved insulin sensitivity, and increased insulin receptor
phosphorylation [15–18]. Further, MRI studies showed that
upregulation of HO-1 decreased adiposity and adipocytes
hypertrophy [16,17,19]. A recent study from our lab also
suggests that decreasing oxidative stress via induction of
HO-1 shifts MSC differentiation in favor of osteogenic rather
than adipogenic lineage .
The cytochrome P450 (P450)-derived epoxyeicosatrienoic
acids (EETs) represent a class of lipid mediators with cyto-
protective properties. Recently, we have shown that EETs
decrease adiposity and insulin resistance in an animal model
of obesity and diabetes via an increase in HO-1 gene ex-
pression and signaling cascade  including activation of
AMPK and phosphorylated AKT (pAKT). Sacerdoti and
coworkers [21,22] studied the interactions between HO-1
gene expression and EETs in vitro and showed that EETs act
as inducers of HO-1 protein and HO activity. EETs are de-
rived from arachidonic acid by the action of different CYP450
enzyme isoforms. These isoforms demonstrate tissue-specific
expression and exhibit relative regioselectivity and stereo-
specificity. Members of the CYP2C and CYP2J families are
the predominant epoxygenases in liver, kidney, brain, and
blood vessels of rodents and humans [23,24]. Human stromal
MSCs express CYP450 monooxygenase and form 20-HETE
, but their ability to form EET has not been investigated.
Moreover, nothing is known about the effect of EET on MSC-
derived adipocyte HO-1, adiponectin, and downstream sig-
naling cascade, including AMPK and pAKT. In this study,
we report that the effect of EETs on MSCs suggests that
MSC-derived adipocytes express CYP-epoxygenase 2J2 and
its product activity, EETs. Treatment with EETs decreased
adipocyte differentiation via an increase in HO-1 and de-
creased in PPARg, C=EBPa, and FAS, suggesting that EETs
are inhibitors of MSC-derived adipocyte stem cell progeni-
tors and lipid homeostasis.
Materials and Methods
Human bone-marrow-derived MSC differentiation
into adipocytes with treatment of EET and EET
agonist and HO activity
Frozen bone marrow mononuclear cells were purchased
from Allcells. After thawing, mononuclear cells were re-
suspended in an a-minimal essential medium (a-MEM; In-
vitrogen) supplemented with 10% heat-inactivated fetal
bovine serum(FBS; Invitrogen)
antimycotic solution (Invitrogen). The cells were plated at a
density of 1–5?106cells per 100cm2dish. The cultures were
maintained at 378C in a 5% CO2incubator, and the medium
was changed after 48h and every 3–4 days thereafter. When
the MSCs were confluent, the cells were recovered by the
addition of 0.25% trypsin–ethylenediaminetetraacetic acid
(Invitrogen). MSCs (Passage 2–3) were plated in a 75-cm2
flask at a density of 1–2?104cells and cultured in a-MEM
with 10% FBS for 7 days. The medium was replaced with the
adipogenic medium, and the cells were cultured for an ad-
ditional 21 days. The adipogenic medium consisted of a
complete culture medium supplemented with Dulbecco’s
modified Eagle medium (DMEM)–high glucose, 10% (v=v)
FBS, 10mg=mL insulin, 0.5mM dexamethasone (Sigma-
Aldrich), 0.5mM isobutylmethylxanthine (Sigma-Aldrich),
and 0.1mM indomethacin (Sigma-Aldrich).
Human MSCs were cultured to characterize the functional
relevance of a numbers of EET agonists to HO-1-adiponectin
and lipid content in adipocytes in the presence and absence
of a soluble epoxide hydrolase (sEH) inhibitor, 12-(3-
adamantan-1-yl-ureido)-dodecanoic acid (AUDA). During
the adipogenesis, 5,6-, 8,9-, 11,12-, and 14,15-EET were added
to human MSC cultures with or without an inhibitor of sEH
to measure the efficiency of 11,12-EET and 14,15-EET to in-
hibit adipogenesis. EETs and AUDA were added every 3 days
at a dose of 1mM. Media were changed every 2 days. MSC-
derived adipocytes were cultured in the adipogenic differen-
tiation medium, and EET agonist was added every 3 days at a
dose of 1mM. After treatment with EET agonist, HO activity
was determined by measuring the amount of CO generated in
the culture medium during adipogenesis at day 14.
Detection of MSC cell markers by fluorescence-
activated cell sorting analysis
Human MSCs are defined by an array of positive and
negative markers. MSCs are normally plastic-adherent under
standard culture conditions, expressing CD105, CD73, and
CD90. MSCs must lack expression of CD45, CD34, CD14 or
CD11b, CD79 or CD19, and HLA-DR. Also, MSCs must be
able to differentiate into osteoblasts, adipocytes, and chon-
droblasts in vitro .
Human MSC phenotype was confirmed by flow cytome-
try (Elite ESP 2358; Beckman-Coulter) using several known
MSC markers. The negative markers used were anti-CD34
and anti-CD45 (BD-Pharmingen), also known to be ex-
pressed as hematopoietic stem cell marker and common
lymphocyte antigen. CD90, CD105, and CD166 were used as
positive markers for MSCs. The data were analyzed using
WinMDI 2.8 software.
Oil Red O staining
For Oil Red O staining, 0.21% Oil Red O in 100% iso-
propanol (Sigma-Aldrich) was used. Briefly, adipocytes were
fixed in 10% formaldehyde, washed in Oil Red O for 10min,
and rinsed with 60% isopropanol (Sigma-Aldrich), and the Oil
Red O was eluted by adding 100% isopropanol for 10min and
OD measured at 490nm, for 0.5-s reading. MSC-derived adi-
pocytes were measured by Oil Red O staining (OD¼490nm)
after day 14. Each values of Oil Red O staining were nor-
malized by cell numbers (values at OD¼490nm=106cells
ratio). Harris and coworkers reported that cytochrome P450
arachidonic acid metabolites are potent mitogens for epithelial
cells , so we normalized adipogenesis level by cell number.
Effect of EETs on lipid droplet size
After induction of adipogenesis, lipid droplets were
stained with 2mM BODIPY 493=503 (Molecular Probes), a
specific stain for cellular lipid droplets . Cell size was
measured using an ImagePro Analyzer (Media Cybernetics).
The classification of the size of lipid droplets was based on
size by area (pixels).
1864 KIM ET AL.
Western blot analysis
Western blot analysis of adipocyte cell lysate was carried
out as described previously [16,17,19]. Levels of the P450
epoxygenase CYP2J2, HO-1, and HO-2 were determined as
described previously [20,29]. The phosphorylation of AKT
and AMPK was analyzed by immunoblots with antibodies
against phospho Ser473 AKT and phospho-Thr172 AMPK.
Total AKT, AMPKa2 or -1, and b-actin were used as loading
controls. Phosphorylation levels were quantified by scanning
densitometry using an imaging densitometer, normalized to
the levels of total protein. The relative phosphorylation in
each signaling molecule was calculated relative to basal
and=or control levels. FAS was measured by immunoblot-
ting with the corresponding antibody and the level was
normalized to loading b-actin and the results are presented
as relative to the basal or to the control levels.
MSCs were cultured in the adipogenic medium containing
10mg=mL insulin, 0.1mM dexamethasone, 0.2mM indo-
methacin, 10% FBS, and 1% antibiotic–antimycotic solution.
At 50% confluence, EET agonist or vehicle solutions were
added every 2 days for 14 days and glucose uptake was
glucose transporter 4 (GLUT4). The assays were performed
3H--deoxyglucose by the methods of
3H-2-deoxyglucose was used as the substrate for
in a 96-well plate with 100nM insulin and 10mM cytocha-
lasin B used as the positive and negative controls, respec-
tively. Samples were examined in triplicate.
Statistical significance (P<0.05) between experimental
groups was determined by the Fisher method of analysis of
multiple comparisons. For comparison between treatment
groups, the null hypothesis was tested by either a single-
factor analysis of variance for multiple groups or un-
paired t-test for 2 groups, and the data are presented as
Determination of MSC phenotypes
MSCs were examined for both positive and negative
markers by flow cytometry to characterize the MSCs. Con-
firmation of MSC phenotype was made by the presence of
the positive markers, CD105 (86.7%), CD90 (99.8%), and
CD166 (98.8%). The absence of CD3 (0%), a hematopoietic
stem cell marker, and CD45 (0.02%), a lymphocytic marker,
confirmed that MSC was not contaminated (Fig. 1). Our
population of MSCs was found to be 86.7% positive for
CD105, 99.8% positive for CD90, and 98.8% positive for
CD166. There was <0.02% contamination by the negative
markers, CD45 and CD34 (Fig. 1).
fluorescence-activated cell sorting analysis. Percent of cells expressing positive markers CD90 (Thy-1), CD105 (endoglin and
SH2), and CD166 (activated leukocyte cell adhesion molecule) were 99.8%, 86.7%, and 98.8%, respectively. (B) CD45 and
CD34 were shown to be expressed in <0.2% of cells, the same as unstained isotypes. CD45 (common lymphocytes antigen)
and CD34 (hematopoietic stem cell marker) were used as negative markers.
(A) Surface expression of CD90, CD105, and CD166 on human mesenchymal stem cells was analyzed by
EET REGULATES ADIPOCYTE STEM CELLS VIA HO-1-PAKT
Ability of MSC-derived adipocyte on manufacture
EET, dihydroxyeicosatrienoic acid,
and CYP2J2 expression
MSCs express higher levels of EETs compared to MSC-
derived adipocyte. The basal levels of all 4 EET region iso-
mers, including 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET,
in human MSCs were decreased in MSC-derived adipocyte.
As shown in Fig. 2A, levels of EETs were 2 times lower in
adipocytes (21.26?2.109pg=mg protein) compared with
MSCs (65.8?5.8pg=mg protein). The dihydroxyeicosatrienoic
acid (DHET) levels (Fig. 2B), which are a determinant for
sEH activity, are higher in adipocytes (41.8?3.24pg=mg
protein) than in MSCs (15.83? 1.62pg=mg protein). Western
blot analysis showed that MSCs display a substantial level
of epoxygenase CYP2J2. MSC-derived adipocytes decreased
epoxygenase CYP2J2 expression (P<0.05), suggesting that
epoxygenase activity decreased during MSC-derived adipo-
cyte differentiation (Fig. 2C).
Effect of 5,6-, 8,9-, 11,12-,14,15-EETs
and AUDA on adipogenesis
Four different epoxide regioisomers—5,6-, 8,9-, 11,12-, and
14,15-EET—are generated by epoxygenase activity. Daily
supplementation of 5,6-, 8,9-, 11,12-, and 14,15-EET was ef-
fective in adipogenesis suppression at 7 or 14 days. Addition
of the sEH inhibitor AUDA increased the inhibitory effect of
11,12-EET and 14,15-EET on adipogenesis (Fig. 3A). These
results are quantified (Fig. 3B) and show that 8,9-EET and
14,15-EET (P<0.01) are more effective in suppressing adi-
pogenesis than 5,6-EET and 11,12-EET, and that AUDA had
a synergistic effect on adipogenesis suppression.
Effect of the 12-(3-hexylureido) dodec-8(Z)-enoic
acid on adipogenesis
We investigated the effect of the EET agonist 12-(3-
hexylureido)dodec-8(Z)-enoic acid on adipogenesis using
standard culture conditions. The effect of the EET agonist
on adipogenesis was determined by counting cells with
lipid droplets in the cytoplasm and cells positive for the
lipid-specific dye, BODIPY (Fig. 4A). The percentage of
cells with morphological lipid droplets was decreased
as EET concentration increased. In untreated MSC–
adipocytes grown in the adipogenesis differentiation me-
dium, formation of lipid droplets was detected: 16.9%?
3.1% of the cells by either morphological or BODIPY
analysis (n¼6). EET agonist decreased adipocyte differ-
entiation in a concentration-dependent manner (Fig. 4A).
BODIPY staining was barely detectable at 10mM, although
the cells became loaded with lipid droplets in untreated
MSC-derived adipocyte progenitor cells. Quantification of
BODIPY-stained cells showed an increase in adipocytes
with the absence of the EET agonist (250?2) compared
with the presence of the EET agonist (100?2.1 and 50?
1.8 pixels) at 1 and 10mM, respectively. In agreement with
this observation, the average size of lipid droplet in the
presence of EET agonist decreased in a dose-dependent
manner (Fig. 4A).
Effect of EET agonist on HO-1 protein, HO activity,
and lipid content in MSC-derived adipocytes
The induction of adipocytes by culturing cells in
the adipogenic medium resulted in a significant (P<0.01)
decrease in HO-1 protein levels (Fig. 5A). This decrease
times lower in adipocytes (21.26?
2.109pg=mg protein) compared with
MSCs (65.8?5.8). (B) The dihydrox-
yeicosatrienoic acid (DHET) levels are
compared to MSCs (15.83?1.62). (C)
protein in MSCs and adipocytes. Va-
lues are means? standard error
(n¼3; *P<0.05). EET, epoxyeicosa-
trienoic acids; MSCs, mesenchymal
(A) Levels of EETs were 2
1866KIM ET AL.
was partially restored by culturing the cells in the pres-
ence of the EET agonist 12-(3-hexylureido)dodec-8(Z)-enoic
acid . HO-1 protein levels were significantly (P<0.05)
increased in MSC-derived adipocytes by treatment with
the EET agonist and displayed a 2-fold increase in HO-1
protein levels (Fig. 5A). In addition, HO activity, as mea-
sured by CO release, was increased (P<0.05) in the pres-
ence of the EETagonist.
activity were 152?11 (pmol CO formed=mg=h) com-
pared to 256?21 (pmol CO formed=mg=h) (Fig. 5B). We
further examined the effect of SnMP on adipogenesis and
lipid content. SnMP caused an increase in adipogenesis
(P<0.01) that was reversed by the presence of the
EET agonist. SnMP and EET agonist together caused
an increase in adipogenesis (P<0.01), indicating that
the effect of SnMP superseded that of the EET agonist
(Fig. 5C). Similar results were seen with lipid content
The basal levels of HO
Effect of EET agonist on the adipogenesis markers
PPARg and C/EBPa in MSC-derived adipocytes
We examined the effect of an EET agonist on PPARg and
C=EBPa expression as adipogenic differentiation markers at
days 5 and 10. Densitometry analysis showed that the levels
of PPARg and C=EBPa were increased on both day 5
(P<0.05) and day 10 (P<0.01) compared to MSCs. EET-
agonist-treated adipocytes displayed a decrease in PPARg
and C=EBPa levels, whereas PPARg and C=EBPa levels were
increased during adipogenesis (Fig. 4A, B).
Effect of EET agonist on adiponectin, pAKT, pAMPK,
FAS, and glucose uptake
As shown in Fig. 6A adiponectin levels were increased
(P<0.05) by the EET agonist and this effect was reversed by
SnMP (Fig. 6A). Phosphorylation of AKT was increased by
content in adipocytes in the presence and absence of a soluble epoxide hydrolase inhibitor (AUDA). n¼4; *P<0.05 versus
5,6- and 11,12-EET; **P<0.001 versus 8,9-, 14,15-, 11,12-EETþAUDA, and 14,15-EETþAUDA. AUDA, 12-(3-adamantan-1-yl-
ureido)-dodecanoic acid; EET, epoxyeicosatrienoic acids.
(A, B) Mesenchymal stem cells were cultured to characterize the functional relevance of a number of EETs to lipid
EET REGULATES ADIPOCYTE STEM CELLS VIA HO-1-PAKT
culturing adipocytes with the EET agonist, while no effect on
AKT was seen (Fig. 6B). The presence of EET reversed the
effect of the adipogenic medium and increased activation of
pAKT. The changes in protein expression of pAKT mirrored
those seen with HO-1 protein expression (Fig. 5A). Thus, EET
increased pAKT and HO-1 [22,31–33]. Densitometry analysis
showed that pAMPK levels were no different between MSC-
derived adipocytes and those treated with EET agonist (Fig.
6B). To further examine if EET agonist-dependent HO-1-pAKT
regulates glucose-induced lipid accumulation and decreased
fatty acid synthesis in adipocytes, the effect of the EET agonist
on protein levels was determined in MSC-derived adipocytes.
As seen in Fig. 6B, untreated adipocytes displayed a marked
increase in FAS levels, while HO-1 levels were decreased
during adipogenesis. The increase in FAS was prevented by
the EET agonist at concentrations ranging from 1 to 2mM,
reaching a level comparable to that in either MSC. Glucose
uptake in MSCs treated with 1mM EET agonist was signifi-
cantly (P<0.05) increased compared to untreated MSCs after
adipogenesis (day 14), indicating an increase in the function of
GLUT4. This was reserved by the HO inhibitor, SnMP, in a
significant (P<0.01) manner (Fig. 6C).
Pharmacological inhibition of EET-mediated
adipogenesis by LY294002
We examined the effect of LY2940002 on adipogenesis,
which was measured as the relative absorbance of Oil Red O
at day 14. EET agonist decreased the levels of Oil Red O
staining compared with control (P<0.02), whereas LY294002
increased Oil Red O staining in MSC-derived adipocytes
(P<0.05). Inhibition of AKT by treatment with LY2940002
(5mM) strongly induced adipogenesis on day 14 (P<0.05)
(Fig. 7). Additionally, as seen in Fig. 7A, LY294002 prevented
the decrease of adipogenesis in EET-treated cells. EET ago-
nist treatment during adipogenesis decreases lipid droplets
compared to control.
This study shows that MSC-derived adipocytes exhibited
decreased activity of the arachidonic acid metabolic pathway
that yields EETs, that is, epoxygenase levels. The inability of
MSC-derived adipocytes to sustain normal levels of EETs
may be the result of the increased levels of sEH coupled with
decreased expression of P450 epoxygenases. We further re-
port that expression of CYP2J2 was significantly decreased in
adipocytes. Further, we demonstrate that MSCs stay in a
pluripotent condition. We have previously shown that MSCs
are pleiotrophic cells that can differentiate to other lineage
such as osteoblasts as a result of crosstalk by specific sig-
naling pathways, including HO-1=-2 expression [7,34].
In this report, we show that epoxygenase product ac-
tivities, EETs, are effective in suppression of adipogenesis.
8,9-EET is more effective in suppression of adipogenesis
compared to 5,6- and 11,12-EET but equally effective as
14,15-EET. Further, inhibition of sEH potentiated the EET-
mediated decrease of adipogenesis. Adipocyte stem cells in
EET agonist on phase-contrast image under the fluorescence microscopy. Lipid droplets were stained with BODIPY and size
of droplets were measured using ImagePro Analyzer (version 6.2, Media Cybernetics; *P<0.05 vs. 1 and 2mM; **P<0.01 vs.
10mM). (B) Effect of EET agonist on PPARg and C=EBPa expression in MSC-derived adipocytes. (*P<0.005 vs. day 5;
**P<0.01 vs. day 10). EET, epoxyeicosatrienoic acids; MSCs, mesenchymal stem cells.
Pharmacological effect of EET agonist on MSC-derived adipocyte cell differentiation. (A) Dose–response effect of
1868KIM ET AL.
culture treated with AUDA, an inhibitor of sEH, caused
an increase in effectiveness of both 11,12- and 14,15-EET in
suppression of adipocyte stem cell differentiation. The anti-
adipogenic effect of an EET agonist, when taken together
with the inhibition of sEH, highlights the therapeutic po-
tential of EET in the management of cardiovascular disease
[35,36]. An association of sEH gene polymorphism with in-
sulin resistance has been reported, implying that sEH plays
an important role in the pathogenesis of insulin resistance
. EET agonist decreased O2?production and prevented
the rapid degradation of EET and subsequent activation of
pAKT [32,38]. Thus, EETs appear capable of reprogramming
adipocyte stem cells, resulting in expression of a new phe-
notype that contains adipocytes of reduced cell size, that are
associated with an increase in adiponectin and a decrease in
inflammatory cytokines. In this report, we also demonstrate
in vitro that the EET agonist 12-(3-hexylureido)dodec-8(Z)-
enoic acid is very effective in suppression of adipogenesis
and that suppression occurs in a dose-dependent manner.
Several reports show that oxidative stress and O2?reduce
EET levels [33,39,40]. EETs are rapidly degraded by O2?to
DHET , and EETs are also inactivated by sEH to DHETre
. Since EET agonists induce HO-1 and vice versa, HO-1
induction increases EET levels . HO-1-mediated induction
of EETs may be related to the ability of HO-1 to decrease
oxidative stress and O2?. Previously, it was shown that
overexpression of HO-1 attenuated and AngII mediated oxi-
dative stress  and vascular injury and dysfunction in hy-
pertensive rats . The mechanism by which HO-1
attenuated ROS involves an increase in extracellular super-
oxide dismutase (EC-SOD) and the restoration of mitochon-
drial function . In addition, a lack of HO-2 creates a setting
that promotes oxidative-stress-related disturbances, including
increases in O2?and decreases in EC-SOD . The fact that
HO-1 and ?2 serve an antioxidative function and preserve
EET levels suggests that the activation of this system in adi-
pose tissue in obesity, a condition of high oxidative stress,
represents an adaptive mechanism that confers MSCs resis-
tance against oxidative stress and inhibits adipogenesis.
The role of EETs in decreasing oxidative stress via an in-
crease in HO-1-mediated adiponectin and pAMPK is in
agreement with the report that EETs increase ERK 1=2 MAP
EETs have been shown to mediate MAP kinase activation
and ERK 1=2 MAP kinase phosphorylation . To date, the
effect of EET on adipocyte MAP kinase has not been inves-
tigated. In addition, the crosstalk between AMPK–AKT and
activation of AMPK is essential for the cellular processes that
are controlled by the energy state of the cell. AMPK is acti-
vated by a decrease in ATP and rise in cellular AMP [45,46],
which leads to the phosphorylation of eNOS and key en-
zymes that subsequently inhibit the synthesis of cholesterol
agonist on HO-1 expression
(**P<0.001 vs. MSC *P<0.05
vs. EET agonist). (B) HO ac-
tivity was measured by CO
formation (*P<0.05 vs. EET
agonist). (C, D) Effect of SnMP
or EET agonist on lipid drop-
lets. Data are expressed as
mean?standard error (n¼4;
*P<0.0001 vs. SnMP, EET
agonistþSnMP). EET, epox-
heme oxygenase; MSCs, mes-
enchymal stem cells; SnMP,
(A) Effect of EET
EET REGULATES ADIPOCYTE STEM CELLS VIA HO-1-PAKT
and increase glucose uptake. Activation of AMPK in skeletal
muscle leads to an enhancement of glucose transport medi-
ated by the translocation of GLUT4 to the membrane and
this appears to be additive to the stimulation in response to
insulin . In addition, pharmacological activation of
AMPK by AICAR in obese Zucker rats improves glucose
tolerance and reduces systolic blood pressure .
This study provides direct evidence that the EET-agonist-
mediated inhibition of adipogenesis is accompanied by the
decrease of FAS, PPARg, and C=EBPa in MSC-derived
adipocyte stem cells. Additionally, these perturbations occur
in sequence, commencing with increased levels of HO-1
expression and decreased lipid accumulation. The lipid-
lowering effect of the EET agonist was completely blocked by
pharmacological suppression of HO activity. In addition,
PPARg and C=EBPa are known to increase adipogenesis .
The ability of the EET agonist to stimulate pAKT and decrease
FAS, PPARg, and C=EBPa supports this hypothesis and that
EETs have a negative effect on adipogenesis. These effects can
be duplicated by inducers of HO-1 such as CoPP and=or L-4F
[7,18], suggesting that HO-1 plays a key role in lipid metab-
olism. These novel observations underscore the importance of
EETs in regulation of MSCs to adipocyte lineages.
Since pharmacological activation of AKT and AMPK in
obese Zucker rats improves obesity and glucose tolerance
and reduces systolic blood pressure , our finding of the
effect of the EET-HO-1 module on AKT activation in adi-
pocytes may be crucial to increase glucose uptake, lipid ho-
meostasis, and inhibition of PPARg and C=EBPa.
FAS mRNA levels were shown to be increased dramatically
during 3T3-L1 adipocyte differentiation . In our experi-
ments, expression of FAS, PPARg, and C=EBPa increased
during adipogenesis; however, FAS, PPARg, and C=EBPa
expression decreased after EET agonist treatment. The action
of EET agonist treatment as manifest by increased levels of
HO-1 and pAKT is associated in an improvement in glucose
uptake. Further, EET agonist effectively restored expression
of adiponectin, which was accompanied with a significant
increase in cellular glucose uptake. In agreement with our
results, adiponectin-deficient cells showed marked down-
regulation of GLUT4, and adipose triglyceride lipase . As
seen in Fig. 7, inhibition of pAKT by LY294002 increased
adipogenesis. In agreement with this, LY294002 was shown to
inhibit GLUT4 translocation . This suggests that EET ag-
onist treatment may increases translocation of GLUT4.
We have presented novel results that indicate the existence
of epoxygenase-mediated generation of EETs in MSCs and a
molecular crosstalk between EETs and HO-1 that regulates
MSC–adipocyte stem cell differentiation and development to
mature adipocytes. This novel action of EETs provides a
mechanistic basis for the EET-mediated control of adipogen-
presentative immunoblotting analysis with antibodies against phosphorylated AKT (pAKT) and pAMPK (*P<0.05 vs. EET
agonist). (C) Effect of EET agonist on glucose uptake in MSC-derived adipocytes (n¼3; *P<0.05 vs. EET agonist;#P<0.01 vs.
EETþSnMP). EET, epoxyeicosatrienoic acids; FAS, fatty acid synthase; MSCs, mesenchymal stem cells; SnMP, tin meso-
porphyrin. pAMPK, phosphorylated AMP-activated kinase.
(A) Effect of SnMP and EET agonist on adiponectin levels (n¼3; *P<0.0001 vs. control and SnMP). (B) Re-
1870 KIM ET AL.
bance of Oil Red O at day 14 after inducing adipogenesis as described in the Materials and Methods section. Bars represent
the mean?standard error of the mean of 4 independent experiments (*P<0.02 vs. EET, LY2940002þEET; **P<0.05 vs.
LY2940002). EET, epoxyeicosatrienoic acids.
(A) Effect of EET agonist and LY2940002 on adipogenesis. (B) Adipogenesis was measured as the relative absor-
lipid accumulation. EET agonist-activating HO-1 expression increase phosphorylation of AMPK and AKT which in turn
decrease FAS, thereby leading to decrease in lipid droplets.
Proposed mechanism for the EET agonist-mediated suppression of MSCs-derived adipocyte differentiation and
EET REGULATES ADIPOCYTE STEM CELLS VIA HO-1-PAKT
esis via HO-1 and adiponectin (Fig. 8). In support of this
conclusion, EET agonist administration has been shown to
inhibit adiposity, increase insulin sensitivity, and improve
vascular function in obese animal model . Thus, targeting
MSCs to increase EET levels could be employed therapeuti-
cally to address the metabolic impairment in MSC-derived
adipocyte function associated with vascular diseases, includ-
ing obesity, diabetes, and hypertension at levels of MSCs.
This work was supported by NIH grants DK068134,
HL55601 (N.G.A.), and HL34300 (M.L.S.), and The Robert A.
Welch Foundation and GM31278 (J.R.F.). This research was
also supported, in part, by the Intramural Research Program of
the NIH, National Institute of Environmental Health Sciences
(Z01 ES025034)(DZ). The authors are indebted to Dr. Attallah
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Dr. Nader G. Abraham
Department of Physiology and Pharmacology
University of Toledo College of Medicine
Toledo, OH 43614
Received for publication March 3, 2010
Accepted after revision April 19, 2010
Prepublished on Liebert Instant Online April 22, 2010
EET REGULATES ADIPOCYTE STEM CELLS VIA HO-1-PAKT
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