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Acyl-CoA thioesterase activity in human placental choriocarcinoma
(BeWo), cells: effects of fatty acids
Asim K. Duttaroya,*, Delphine Crozetb, Jonathon Taylorb, Margaret J. Gordonb
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Institute for Nutrition Research, University of Oslo, POB 1046 Blindern, N-0316 Oslo, Norway
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Rowett Research Institute, Aberdeen, Scotland, UK
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Received 15 August 2002; accepted 26 September 2002
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Abstract
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The effects of fatty acids on acyl-CoA thioesterase activity and peroxisome proliferator-activated receptor g (PPARg), a regulator
of lipid metabolism, were investigated in placental choriocarcinoma (BeWo) cells. Substrate preference for acyl-CoA thioesterase
was in the following order; g-linolenoyol-CoAXarachidonoyol-CoAcpalmitoyl-CoAXlinoleyol-CoA. However, when these cells
were incubated with fatty acids, acyl-CoA thioesterase activity was increased by both conjugated linoleic and g linolenic acids, but
not by docosahexaenoic and eicosapentaenoic acids. In addition, these fatty acids also increased expression of PPARg in these cells,
suggesting a putative relationship between free fatty acid generated by acyl-CoA thioesterase and expression of PPARg. Since
expression of PPARg is critical for feto-placental growth, these fatty acids may be important during pregnancy.
r 2002 Elsevier Science Ltd. All rights reserved.
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Delivery of fatty acids from the maternal circulation
to the fetus occurs via the placenta, which acts as a
physical barrier between the maternal and fetal blood.
Essential fatty acids (EFAs) and their long-chain
polyunsaturated fatty acids (LCPUFAs) derivatives
are very important for the development of the fetus
[1,2]. They play a crucial role in the structure and
function of membranes, with LCPUFA being especially
important to fetal brain and retina development [2,3].
Although the mechanism involved in the transfer of
these fatty acids across the placenta is now largely
known [1,3], very little information is available on the
fatty acid metabolism during their transit across the
placenta syncytiotrophoblasts. Critical regulators in
cellular lipid homeostasis are lipid-sensing nuclear
transcription factors known as peroxisomal proliferator-activated receptors (PPARs) [4]. There are several
types of PPAR such as PPARa, PPARb (also called d or
NUC1) and PPARg (PPARg-1 and PPARg-2) [4–6].
PPARg found in adipose tissue as well as in placenta
[6,7] is thought to be involved in the stimulation of lipid
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accumulation by controlling expression of several lipid
metabolic genes such as fatty acid transporter/binding
proteins, lipase, the acyl-CoA synthetase, etc. [8,9].
PPARs are also involved in the regulation of cellular
proliferation, differentiation and apoptosis [4–6].
PPARg gene knockout in mice results in severe placental
and fetal abnormalities [7]. Recent studies have demonstrated that PPARg is responsible for the synthesis of
hCG, a hormone essential for human pregnancy, and
also human placental lactogen, human placental growth
hormone, and leptin secretion [10]. Despite its critical
importance in feto-placental development, the natural
ligands for PPARg are still not known. PPARs are
transcriptionally activated by fatty acids (or other
ligands, hypolipidamic agents such as thiazolidinediones, prostaglandins, and eicosanoids) and then form
a heterodimer with the retinoid X receptor (RXR) [8].
LCPUFAs and their derivatives which may serve as
potential natural ligands for PPARg can be obtained
from the maternal circulation by the placenta via the
fatty acid transport system [1,3]; however, their role as
natural ligands are yet to be confirmed. Besides being
potential ligand, fatty acids may also modulate PPARg
expression [8,11,12]. Fatty acids and fatty acid derivatives, in particular, products of the cyclooxygenase and
lipoxygenase pathways, are known to activate members
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1. Introduction
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*Corresponding author. Tel.: +47-22-85-15-47; fax: +47-22-85-1341.
E-mail address: a.k.duttaroy@basalmed.uio.no (A.K. Duttaroy).
0952-3278/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 5 2 - 3 2 7 8 ( 0 2 ) 0 0 2 3 4 - X
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2. Materials and methods
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2.1. Materials
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15-deoxy-D12,14 prostaglandin J2 (15-deoxyPGJ2) was
obtained from Biomol, USA. 3-(4,5-dimethyl thiazol-2yl)-2,5 diphenyl tetrazolium bromide (MTT), 5,50 dithiobis(2-nitrobenzoic acid) (DTNB), g-linoleoylCoA, arachidonoyl-CoA, linoleoyl-CoA, palmitoylCoA and acetyl-CoA were obtained from Sigma, Poole,
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BeWo cells were obtained from European Collection
of Animal cell cultures and were grown in Ham’s F12
medium containing 10% fetal bovine serum (FBS),
6 mM glutamine and 100 IU/ml penicillin–streptomycin.
Cells were maintained as monolayers in 25 cm2 tissue
culture flasks at 371C with a 5% CO2-balanced air and
100% humidity atmosphere [24]. At confluence, they
were subcultured using a Trypsin–EDTA solution to
suspend the cells. Media were renewed three times
weekly. Cell viability was routinely tested as the ability
to exclude trypan blue. For experiments, cells from early
confluent monolayers were dispersed and replated in
58 mm 115 mm culture dishes. When the cells reached
90–95% confluence, the monolayer was detached from
the flask bottom and either subcultured into new flasks
or seeded onto 100 mm2 culture dishes for experimentation.
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2.2. Cell culture
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UK. Proetase inhibitor cocktail set III was obtained
from Novo biochem, UK. Trypsin-EDTA solution,
penicillin–streptomycin solution, Nutrient Ham’s MixF-12 with glutamax-I and William’s medium E with
glutamax-I were obtained from Gibco Life Technologies, Scotland. BeWo cells were from the European
Collection of Animal Cell Cultures (Salisbury, UK).
Polyclonal antibody (goat) raised against PPARg was
from Santa Cruz Biotechnology, USA. Horse radish
peroxidase (HRP) linked anti-sheep/goat IgG was from
Scottish antibody production unit, Carluke. Other
reagents used were of the highest purity available from
Sigma Chemical Co., Poole, UK.
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of the PPAR family whereas the convincing evidence
still remains to be shown that CoA esters can act on
PPARs [13]. PPARg binds free fatty acids (FFAs) not its
fatty acyl-CoA; therefore, it is important to investigate
how the levels of FFAs are regulated in the cells. AcylCoA thiosterases cleave acyl-CoA to produce FFA and
CoASH. They exist in various tissues of many organisms and are widely distributed in the cell (rat liver
mitochondria, peroxisomes, cytosol and microsomes
[14,15], guinea-pig brown adipose tissue mitochondria
[16], Candida rugosa [17], lactating-rabbit mammary
gland cytosol [18].
The acyl-CoA thioesterase activity of cellular compartments is the result of several enzymes [16,17]. There
are three types of acyl-CoA thioesterases, according to
their substrate specificity: (i) short-chain acyl-CoA
thioesterases (acetyl-CoA hydrolases, EC. 3.1.2.1;
short-chain acyl-CoA thioesterase, EC. 3.1.2.3–5 and
EC. 3.1.2.11 [19]); (ii) medium-chain acyl-CoA thioesterases [20]; (iii) long-chain acyl-CoA thioesterases
(palmitoyl-CoA hydrolases, EC. 3.1.2.2 [14]. Several
lines of evidence suggest that PPARs could be involved
in the regulation of the expression of these enzymes
[12,16,21]. So far, very little information is available on
acyl-CoA thioesterase in human placental trophoblast
cells. Nevertheless, the transcript of an adrenocorticotropin-regulated phosphoprotein and intermediary in
steroids synthesis, which is similar to an acyl-CoA
thioesterase, has been detected in placenta [22].
Therefore, the main aims of the present study were to
investigate the presence of acyl-CoA thioesterase in the
placental choriocarcinoma (BeWo) cells, and their
expression by several dietary fatty acids. In addition,
we investigated expression of PPARg by different fatty
acids in BeWo cells. BeWo cells are a good experimental
model for placental trophoblasts to study lipid metabolism as these cells express almost all lipid metabolic
genes [23].
In the present paper, we report the presence of acylCoA thioesterase activity in placental cells and their
regulation by different dietary fatty acids. Our data
suggest that dietary LCPUFAs, specially g-linolenic acid
(GLA) may have important role in the growth and
development of feto-placental unit.
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2.3. Preparation of fatty acids
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The preparation of the fatty acids was done according
to the method described by Campbell et al. [24]. The
appropriate amount of fatty acid was added to this BSA
culture medium to obtain a 25 mM incubation media,
with a fatty acid/albumin ratio of 1:3. The BeWo cells
were grown to a level of confluence between 90% and
95% confluence in the cell culture dishes. Prior to the
addition of the incubation medium, cell monolayers
were incubated in 1% FBS culture medium for 2 h at
371C. Cells were incubated for 16 h at 371C in the
presence and absence of fatty acids. On removal from
the incubator, the monolayers were first rinsed in a
BSA–PBS solution and then ice-cold PBS. The monolayer was then scraped from each dish into 900 ml of the
cell-removal buffer and transferred into a 1.5 ml
microcentrifuge tube. The cells were then homogenised
in PBS containing 1% protease inhibitor cocktail. The
cell homogenate was then used either for acyl-CoA
thioesterase assay or Western Blot analysis for PPARg.
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2.4. Assay of acyl–CoA thioesterase activity
3
Acyl-CoA thioesterase activity was routinely followed
spectrophotometrically by measuring the appearance of
the free thiol groups of CoASH with DTNB, as
described earlier [19]. The reaction mixture contained
50 mM KCl, 10 mM Hepes (pH 7.4) and 0.1 mM
DTNB. The appropriate acyl-CoA was preincubated
and the reaction was started by the addition of enzyme.
The reaction was followed at 412 nm using Unicam 8700
UV/VIS spectrophotometer. The amount of thiol
groups was calculated from molar extinction coefficient
E=13 600 l/mol/cm. Protein was determined according
to Bradford method [25] using BSA as a standard.
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Monospecific polyclonal antisera raised against the
human PPARg was used. Western Blot analysis of
BeWo cell homogenates were carried out as described
before [23,24]. Polyacrylamide gel electrophoresis of
cellular proteins in the presence of SDS was carried out
under reducing conditions on SDS–PAGE gel homogenous 20 (Phast system. Pharmacia, UK). After
electrophoresis, proteins were transferred onto a nitrocellulose membrane by diffusion blotting at 701C for 1 h.
The membranes were probed for the presence of PPARg
by incubating with polyclonal antiserum to this protein.
Antibody–antigen complexes were then detected with
HRP-anti-IgG fraction of donkey polyclonal antiserum
(Scottish Antibody Production Unit).
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2.5. Western Blot analysis of PPARg of BeWo cells
2.6. Cell death analysis
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2.7. Statistical analysis
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Data are presented as mean 7 SEM. Statistical
significance was determined using a two-tailed Student’s
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Mitochondrial variability, as an index of cell viability,
was measured using the MTT method [26]. BeWo cells
were grown in a 96-well microtitre plate until 90–95%
confluent. The cells were then incubated with fatty acids
and 15-deoxyPGJ2. After the 16-h incubation period the
medium was removed and replaced with 100 ml of fresh
serum-free culture medium. Ten microlitres of the stock
MTT solution was then added to each well. A negative
control of 10 ml MTT solution and culture medium was
also included. The plate was then incubated further for
4 h at 371C. At the end of this incubation period, 100 ml
of the SDS solution was added to each well and mixed
thoroughly with the pipette. The microplate was then
incubated overnight at 371C. The next day each sample
was again mixed with MTT reagent and the absorbance
was read at 570 nm.
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Acyl-CoA thioesterase activity of BeWo cell extract
was measured spectrophotometrically, following the
appearance of CoASH. Acyl-CoA thioesterase activity
was measured using different substrates with different
chain lengths such as arachidonoyl-CoA, g-linoleoylCoA, g-linoleoyl-CoA, palmitoyl-CoA and acetyl-CoA
(Fig. 1). Acyl-CoA thioesterase activity of BeWo cell
was mainly specific to g-linoleoyl-CoA and arachidonoyl-CoA. At 30 mM followed by substrate concentration, 6579 nmol CoASH/min/mg was released when glinoleoyl-CoA was used, followed by arachidonoyl-CoA
(4976 nmol CoASH/min/mg of protein) and palmitoylCoA(3178 nmol HSCoA/min/mg of protein) and linoleoyl-CoA (2775 nmol HSCoA/min/mg of protein). No
short-chain acyl-CoA thioesterase activity was observed
in BeWo cells. No short chain acyl-CoA thioesterase
activity was observed in BeWo cells. Acyl-CoA thioesterase activity was also measured in a fraction containing only heavy mitochondria, solubilised with Triton X100. Different concentrations of linoleoyl-CoA were
tested (data not shown). The measured activity was very
low, and a rate was observed only after the first minute
of incubation. Nevertheless, a very low level of acylCoA thioesterase activity exists in heavy mitochondria.
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3.1. Acyl-CoA thioesterase activity in BeWo cells
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3. Results
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t-test; P values were considered significant when equal
to or less than 0.05.
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3.2. Effects of fatty acids on Acyl-CoA thioesterase
activity
Effect of incubation of BeWo cells with fatty acid
solutions on expression of acyl-CoA thioesterase activity
was then investigated. Acyl-CoA thioesterase activity
was measured after incubation of BeWo cells with fatty
acid bound to BSA solutions (oleic acid (OA), eicosapentaenoic acid (EPA), docosahexaneoic acid (DHA),
arachidonic acid (AA), conjugated linoleic acid (CLA),
(GLA), and albumin alone as a control). Hydrolysis
rates were significantly different, depending on the fatty
acid present in the incubation medium. EPA and DHA
and OA had no effect on acylCoA thioesterase activity
compared with controls. But both CLA and GLA
increased
acylCoA
thioesterase
activity
(2573.4 CoASH nmol/min/mg
protein
and
2874.6 CoASH nm/min/mg of protein, respectively)
compared with control (1774.3 nmol CoASH/min/mg
of protein, Po0.05) (Fig. 2). AA also increased the
activity but did not reach the significance (P>0.05).
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nmol/min/mg protein
ArachidonoylCoA
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PalmitoylCoA
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LinoleoylCoA
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AcetylCoA
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EPA DHA ARA
GLA CLA
Fig. 2. Effect of incubation of BeWo cells on acyl-CoA thioesterase
activity. BeWo cells were incubated with 25 mM fatty acid bound to
FFA solutions (fatty acid:BSA ratio of 1:3, oleic acid, OA; g-linolenic
acid, GLA; conjugated linoleic acid, CLA; eicosapentaenoic acid,
EPA; docosahexaenoic acid, DHA) or with BSA alone. The acyl-CoA
thioesterase activity was then measured spectrophotometrically with
DTNB, in a fraction containing cytosol, light mitochondria, peroxisomes and microsomes. The reaction mixture, final volume 1 ml,
contained, amongst other things, 0.1 mM DTNB and 150 mg proteins.
The reaction was initiated by addition of 10 mM palmitoyl-CoA, after
warming up of the proteins for 3 min outside the reaction mixture.
Each value is the mean of experiments performed in duplicate.
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3.4. Effects of fatty acids and other PPARg ligands on
cell death
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MTT assay (a measure of mitochondrial activity) was
performed to monitor cell death in the presence of fatty
acids and 15-deoxyPGJ2, a ligand for PPARg [27].
Concentration response study with the compounds
above revealed that only 15-deoxyPGJ2 was capable of
killing the cells after 16 h incubation. The reduction in
MTT activity in response to 15-deoxyPGJ2 was 50% at
10 mM, whereas only modest loss (20%) of MTT activity
was observed in the case of other fatty acids (GLA,
EPA, DHA, AA, and PA (up to 20 mM).
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4. Discussion
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alone as a control), on PPARg expression was investigated by Western Blotting, using anti-PPARg antibody,
which reacts with both PPARg1 and PPARg2. Fig. 3
shows the expression of PPARg expression in these cells
incubated with different fatty acids. Although all
LCPUFAs used in this experiment increased the
expression of PPARg in these cells compared with
control, CLA and GLA were much stronger inducers of
PPARg expression (Fig. 3).
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Fig. 1. Acyl-CoA thioesterase activity, reaction rate dependence on the concentration of different chain length acyl-CoA. The acyl-CoA thioesterase
activity of BeWo cell extracts was performed spectrophotometrically with DTNB. The reaction mixture, final volume 1 ml, contained, amongst other
things, 0.1 mM DTNB and 200 mg proteins. The reaction was initiated by addition of acyl-CoA, after warming up of the proteins for 3 min outside the
reaction mixture. The data in figure represent the mean+SEM of three experiments performed in triplicate.
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3.3. Effects of fatty acids on PPARg expression in BeWo
cells
The effect of incubation with fatty acid bound to BSA
(GLA, CLA, DHA, AA, linoleic acid (LA) and BSA
Long-chain fatty acyl-CoA esters are important
intermediates in degradation and synthesis of fatty
acids, as well as in having important function in
regulation of intermediary metabolism and gene expression. Although the physiological functions for most
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Expression of PPARγ in BeWo cells.
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increased PPARg expression, it had minimal effects.
While 15-deoxyPGJ2 has been linked with the induction
of cell cycle arrest and apoptosis, the ligand functions
and preference of the PPARg are relatively distinct.
Recently, it has been shown that PPARg modulate
human trophoblast function in a ligand-specific manner
[33]. Exposure of cultured primary human trophoblasts
to troglitazone enhances biochemical and morphological trophoblast differentiation, and hCG synthesis,
whereas 15-deoxyPGJ2 diminishes trophoblast differentiation, Furthermore, 15-deoxyPGJ2 up-regulates p53
expression and promotes apoptosis [33], as we observed
in BeWo cells. Since 15-deoxyPGJ2, the putative natural
PPARg ligand is responsible for placental apoptosis, this
raises the possibility of existence of other hitherto
unknown natural ligands that are responsible for
placental growth and differentiation. Derivatives of
GLA and other LCPUFA may act as such type of
ligands. Our observation may have profound implications in human placental biology. Dysfunction of the
human placenta may result in fetal growth restriction, a
condition associated with abnormal trophoblast differentiation, and enhanced apoptosis may be triggered by
15-deoxyPGJ2. In these conditions, inhibition of 15deoxyPGJ2 production and/or supplementation of GLA
and other LCPUFAs might reverse the apopoptic
pathway and support placental growth and development. These fatty acids may thus also allow placental
development by increasing PPARg levels which in turn
increases expression of several lipid metabolic genes
such as FAT, FATP, FABPpm, FABPs, hCG and other
placental hormones.
In conclusion, placental trophoblast cells express acylCoA thioesterase activity which prefers LCPUFA, GLA
and AA. Incubation of placental cells with GLA and
AA increased acyl-CoA thioesterase activity and PPARg
expression, but had very little effect on cell death. These
fatty acids may thus play a critical role in feto-placental
growth and development, as PPARg is critically
associated with fetal and placental growth and development.
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acyl-CoA thioesterases have not yet been elucidated,
several data suggest that these enzymes may involve in
lipid metabolism by modulation of cellular concentration of acyl-CoA and FFA. In this paper, we report that
placental trophoblast cell line (BeWo) expresses acylCoA thioesterase activity. Substrate preference for acylCoA thioesterase was in the following order: glinolenoyol-CoAXarachidonoyol-CoAcpalmitoylCoAXlinoleyol-CoA. This indicates that these acylCoA derivatives of LCPUFA are preferentially hydrolysed to produce their respective FFA. It has been
shown that acyl-CoA thioesterase hydrolysed preferentially arachidonoyl-CoA to AA in the cytosol of rabbit
kidney medulla [28]. Thus, this enzyme can supply AA
for prostaglandin synthesis in this region. Therefore,
thioesterase activity in these cells may play specific
important role by supplying LCPUFA as FFA for
cellular metabolism. In fact, the fate of fatty acids in
cellular metabolism depends on several physiological or
pathological factors. Under normal conditions, fatty
acids are mainly beta-oxidised in mitochondria and
peroxisomes or incorporated into triacylglycerols and
phospholipids. A prerequisite for these reactions to
occur is the activation of the fatty acids to their
corresponding CoA-thioesters by acyl-CoA synthetases
which are present in several cellular compartments [29–
31]. Long-chain acyl-CoA thioesterase, in theory a
counteracting enzyme activity, has also been described
in several cellular compartments [30,31]. The regulation
of the thioesterase activity by treatment of rats with
peroxisome proliferators has been studied in mitochondria, peroxisomes, microsomes and cytosol. Although
only a few long-chain acyl-CoA thioesterases have been
isolated and characterised, the strong induction of some
of these enzymes by peroxisome proliferators suggests
that their functions may be related to lipid metabolism
[16,21,32].
In this study, we also demonstrate that certain fatty
acids, such as GLA also up-regulated acyl-CoA
thioesterase activity in placental cells, possibly through
the increased expression of PPARg. In these cells, 15deoxyPGJ2 induced apoptosis as determined by MTT
assay, whereas GLA and other fatty acid although
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Fig. 3. Effect of incubation of fatty acids on PPARg expression in BeWo cell. BeWo cells were incubated for 6 h with 25 mM fatty acid bound to
albumin (FA:BSA ratio of 1:3) or with BSA alone. Gel obtained by electrophoresis SDS–PAGE, and stained by Coomassie brilliant blue. The gel was
run with 20 mg of proteins. Westerns Blots obtained using anti-PPARg antibodies (dilution 1/500). Proteins were transferred from gel to PVDF
membrane (oleic acid, OA; g-linolenic acid, GLA; docosahexaenoic acid, DHA, conjugated linoleic acid, CLA).
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