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Arachidonic acid stimulates internalisation of leptin by human
placental choriocarcinoma (BeWo) cells
Asim K. Duttaroy,a,* Jonathon Taylor,b Margaret J. Gordon,b
Nigel Hoggard,b and Fiona M. Campbellc
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Institute for Nutrition Research, University of Oslo, P.O. Box 1046, Blinern, N-0316 Oslo, Norway
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Rowett Research Institute, Aberdeen AB21 9SB, Scotland, UK
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Cardiovascular Research Group, University of Alberta, Canada
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Received 24 October 2002
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Arachidonic acid at 100 nM stimulated internalisation of 125 I-leptin in human placental choriocarcinoma (BeWo) cells by 3-fold
compared with controls. In contrast, eicosapentaenoic acid at similar concentration decreased internalisation of leptin by 2-fold. Use
of ibuprofen and indomethacin (inhibitors of prostaglandin synthesis) inhibited the stimulatory effect of arachidonic acid. Prostaglandin E2 , a cyclooxygenase metabolite of arachidonic acid, stimulated internalisation of leptin by these cells. All these data
demonstrate that stimulation of leptin internalisation by arachidonic acid in placental trophoblasts may be mediated via prostaglandin E2 .
Ó 2002 Elsevier Science (USA). All rights reserved.
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Leptin has a role in haematopoiesis, angiogenesis,
immune system, and various other metabolic functions
such as bone formation, insulin regulation, brain development, and in reproduction [1–3]. Leptin is mainly
produced by adipocyte tissue and controls body composition mostly via hypothalamic receptors that regulate
food intake and body weight [1–3]. Expression of leptin
and its receptors was also observed in the placenta and
fetal tissues [4], suggesting that leptin may also be involved in the regulation of reproductive processes [5].
Leptin has been demonstrated to mediate these effects
by interaction with its cognate receptor, the leptin receptor (OB-R) [6–8]. To date, only a single gene encoding multiple forms of the leptin receptor has been
identified [6,9]. Two major isoforms of the leptin receptor are the result of alternative splicing from a single
gene. These include a long form, thought to be the major
signalling form of the receptor, a soluble form lacking a
trans-membrane domain, and multiple short forms,
varying in the length of their cytoplasmic domains [8].
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Corresponding author. Fax: +47-22-85-13-41.
E-mail address: a.k.duttaroy@basalmed.uio.no (A.K. Duttaroy).
Short forms of the receptor are expressed throughout
the body, whereas the strongest expression of the long
form has been localised to particular nuclei within the
hypothalamus, with lesser amounts observed in other
tissues [6,9–13]. The short form is thought to be responsible for leptin transport in tissues. Once leptin
binds to its cell surface receptor, the receptor–ligand
complex is internalised. The role of two isoforms (long
and short) of leptin receptors in leptin internalisation
has been recently investigated [14,15]. Although the exact fate of the internalised receptor–ligand complex is
unknown, nevertheless this process appears to be important in cellular homeostasis. Our previous study
suggested that plasma-free fatty acids may regulate
leptin receptor–ligand interaction through their binding
with leptin [16]. Recent study also suggests that eicosapentaenoic acid stimulates both expression and secretion
of leptin by mouse 3T3-L1 adipocytes [17]. In order to
understand the role of fatty acids on the uptake of leptin
by human placental choriocarcinoma (BeWo) cells, we
have studied the internalisation of 125 I-leptin by these
cells in the presence of various fatty acids.
We report for the first time that internalisation of
leptin by placental trophoblast cells (BeWo) is regulated
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.
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69 Materials and methods
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Results
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Internalisation of leptin receptors: effect of fatty acids
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Equilibrium of binding of 125 I-leptin to BeWo cells
was attained after 30 min of incubation at 4 °C.
Scatchard analysis of leptin binding data indicates the
presence of single binding sites with a dissociation
constant (Kd ) of 8:1 2:3 pM and capacity (n) of
15,000 1000 receptor sites per cell.
After the incubation of 125 I-leptin with BeWo cells for
30 min at 4 °C, internalisation of bound 125 I-leptin by
BeWo cells was examined for various times at 37 °C.
Fig. 1 shows representative experiments demonstrating
the internalisation 125 I-leptin as a function of time.
Maximum internalisation of leptin occurred after 30 min
at 37 °C. Internalisation of leptin appears to be temperature-dependant as incubation at 4 °C abolished 125 Ileptin internalisation by 90% (0:79 0:01 fmol 125 I-leptin/mg protein at 37 °C vs. 0:075 0:002 fmol 125 I-leptin/
mg of protein at 4 °C). In order to understand the effects
of fatty acids on the internalisation of 125 I-leptin, confluent cells were washed with serum-free medium and
internalisation of 125 I-leptin in the presence of n 3 or
n 6 long chain polyunsaturated fatty acids (such as
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Materials. Unlabelled human recombinant leptin was obtained
from Pepro Tech, UK. 125 I-leptin was obtained from Amersham, UK.
Penicillin-streptomycin solution, Nutrient HamÕs Mix-F-12, Immobilon-P, polyvinylidene difluoride (PVDF) membranes, molecular weight
markers, fatty acids, trypsin–EDTA solution, prostaglandins, indomethacin, and ibuprofen were obtained from Sigma, Poole, UK.
Trypsin–EDTA solution was obtained from Gibco Life Technologies,
Scotland, UK. Leptin receptor (Ob-R) antisera were obtained from
Santa Cruz Biotechnology, Germany. This Ob-R antibody recognises
all splice variants of the leptin receptor. Affinity-purified goat polyclonal antibody was raised against a peptide corresponding to amino
acids 32–51 mapping at the amino terminus of Ob-R of human origin.
All other chemicals and solvents were of high purity obtained from
either Sigma, or Aldrich chemical, UK.
Cell culture. BeWo cells were obtained from European Collection
of Animal Cell Cultures and grown in HamÕs F12 medium containing
10% fetal bovine serum, 0.4 M glutamine and 100 IU/ml penicillin, and
streptomycin (0.1 mg/ml) as described [18]. Cells were maintained as
monolayer in 75 cm2 tissue culture flask at 37 °C with a 5% CO2 -balanced air and 100% atmospheric humidity. At confluence they were
sub-cultured using a Trypsin–EDTA solution to suspend the cells.
Medium was renewed every 24–48 h. Cell viability was routinely tested
through the exclusion of trypan blue. For experiments, cells from early
confluent monolayers were dispersed and replated in 35 mm 6-well
plate (95 cm2 ) culture dishes.
Binding and internalisation of 125 I-leptin. Because of the internalisation of significant amounts of leptin into BeWo cells at 37 °C, the
binding of radiolabelled leptin was carried out at 4 °C. Confluent cells
were washed with serum-free media. The cells were then incubated in
PBS with 11 pM 125 I-leptin in the absence and presence of unlabelled
leptin for 30 min, unless otherwise mentioned. The binding reactions
were terminated and cell associated 125 I-leptin was determined as described [19]. All incubations were performed in triplicates. The interaction of leptin with cell surface receptors was analysed by the
Scatchard method [20]. The dissociation constant (Kd ) and the binding
capacity (n) were obtained from non-linear regression analysis of the
equilibrium binding using a computer program (Enzfitter, Biosoft,
UK).
In order to determine the internalisation of bound 125 I-leptin by
BeWo cells, cell surface associated radioactivity was extracted with
acid wash by a modification of the method of Golden et al. [21]. Cells
were initially incubated with 125 I-leptin (11 pM) for 30 min at 4 °C.
After 30 min incubation cells were then warmed to 37 °C for period up
to 60 min. At each time point, cells were washed and pelleted, and
internalisation of 125 I-leptin was measured after removing cell surfacebound leptin. To remove cell surface-bound ligand, cell suspensions
were incubated with 500 ll barbital sodium acetate buffer, pH 3.0,
containing 28 mM Na acetate, 20 mM Na barbital, and 117 mM NaCl
at 4 °C for 6 min. Control studies demonstrated that this procedure
resulted in maximum and complete release of surface-bound radioactivity. Following the extraction period, the cells were washed twice
with ice-cold PBS, 1 ml of 1 M NaOH was added to each well, and the
plates were left overnight at 4 °C. Radioactivity associated with solubilised cells was then measured.
Effects of fatty acids and prostaglandins on of 125 I-leptin internalisation to BeWo cells. The effects of fatty acids on internalisation of
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leptin were investigated by incubating BeWo cells with 125 I-leptin in the
presence of various concentrations of arachidonic acid, 20:4n 6, eicosapentaenoic acid, 20:5n 3, docosahexaenoic acid, 22:6n 3, eicosatetrayonic acid (ETYA), various prostaglandins (PGE2 , PGF2a ,
PGE1 , PGA1 , PGD2 , PGA2 , and 6ketoPGF1a ), indomethacin, and
ibuprofen. Internalisation of 125 I-leptin by BeWo cells was then determined, as described above.
Western blot analysis of leptin receptor: effects of fatty acids. Western blot analysis of leptin receptors of BeWo cell extract was carried
out as described previously [22]. Cells were plated into 100 mm plates
and grown to 90% confluency. They were then rinsed twice with serumfree media before the incubations were carried out. The cells were incubated with various concentrations of arachidonic acid. Three plates
were set up for each of the incubation mixtures and a control was
included without fatty acid. Incubations were carried out for 30 min at
37 °C and then the plates were rinsed twice with ice-cold PBS. Cells
were scraped in a minimum volume of TES buffer containing 2 mM
EDTA and Protease Inhibitor Cocktail III (Calbiochem) diluted 1 in
100. Cells were centrifuged at 10,000g for 10 min. at 4 °C to pellet cells,
then resuspended in above buffer, and homogenised using a hand-held
glass homogeniser. Polyacrylamide gel electrophoresis of cell extracts
(100 ug) in the presence of SDS was carried out under reducing conditions on using Tris–HCl 7.5% ready gels (Bio-Rad). After electrophoresis, proteins were transferred onto a polyvinylidene difluoride
(PVDF) membrane using wet blotting, 30 V for two and a half hours.
The membrane was probed for the presence of leptin receptor by incubating with rabbit polyclonal antiserum to leptin receptor. Antibody–antigen complex was then detected with horseradish peroxidase
(HRP)-anti-rabbit IgG fraction of donkey polyclonal antiserum
(Scottish Antibody production unit).
Statistical analysis. Results are given as means standard error of
the mean (SEM). Each experimental condition was carried out in
triplicate and all experiments were performed at least three times. The
statistical significance was determined using a two-tailed StudentÕs t
test; P values equal to or less than 0.05 were considered significant.
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by the n 3 and n 6 long chain polyunsaturated fatty
acids (LCPUFA) but in an opposite manner, and the
stimulatory effect of arachidonic acid was mediated, in
part, via its cyclooxygenase metabolite, prostaglandin
E2 .
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Fig. 2. Effect of various concentrations of arachidonic acid (0–300 nM)
on the internalisation of 125 I-leptin by BeWo cells. After incubation,
cells were washed with ice-cold PBS buffer and subsequently cells were
acid washed to remove surface-bound leptin. The internalised leptin
was then determined as described, in Materials and methods.
ulatory effect of arachidonic acid on leptin internalisation (Table 1). This suggests that that among other
mechanisms, arachidonic acid-derived eicosanoids may
be involved in stimulating leptin internalisation by these
cells. The degree of internalisation of leptin depended on
the prostaglandin structures. Among all the prostaglandins (PGE2 , PGF2a , PGE1 , PGA1 , PGD2 , PGA2 ,
6ketoPGF1a ), tested, only PGE2 stimulated internalisation of leptin in these cells (Table 2). PGE2 at 10 lM
concentration stimulated internalisation of leptin
(2:08 0:06 fmol/mg of protein) compared with the
control, whereas PGF2a had no effect. PGE2 and PGF2a
are the main prostaglandins produced by placental
syncytiotrophoblasts, including human choriocarcinoma trophoblastic cells [23,24]. The stimulation of
leptin internalisation by PGE2 was dose dependent and
maximum stimulation occurred at or above 30 concentration of PGE2 . The EC50 for PGE2 -dependent
leptin internalisation was around 12 nM (Fig. 3).
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arachidonic acid, 20:4n 6, eicosapentaenoic acid,
20:5n 3, and docosahexaenoic acid, 22:6n 3), ETYA,
prostaglandins, and their synthesis inhibitors (indomethacin and ibuprofen) was investigated. Among all
these fatty acids tested, arachidonic acid stimulated
internalisation of 125 I-leptin by 3-fold (from 0:79
0:01 fmol 125 I-leptin/mg protein to 2:62 0:07 fmol 125 Ileptin/mg protein, p < 0:001, n ¼ 6, mean SD) (Table
1). Maximum stimulatory effect of arachidonic acid
on leptin internalisation was observed at or above
50–100 nM concentration (Fig. 2).
Incubation of these cells with eicosapentaenoic acid
(100 nM) and docosahexaenoic acid (100 nM) decreased
internalisation of leptin to 0:41 0:03 and 0:56
0:02 fmol/mg of protein, respectively, compared with
control (0:79 0:01 fmol/mg protein) (p < 0:05). ETYA,
an inactive homologue of arachidonic acid, also affected
arachidonic acid-stimulated internalisation of leptin.
Pre-treatment of BeWo cells with ibuprofen (10–20 lM)
and indomethacin (5–10 lM) partly abolished the stim-
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Fig. 1. Internalisation of 125 I-leptin to BeWo cells with time. BeWo
cells were incubated with 11 pM 125 I-leptin for 30 min at 37 °C. After
the incubation internalisation of leptin was determined, as described in
Materials and methods.
Table 1
Effect of various fatty acids/compounds on internalisation of
Addition
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Control at 4 °C
Control at 37 °C
Arachidonic acid, 20:4n 6 at 4 °C
Arachidonic acid, 20:4n 6 at 37 °C
Eicosapentaenoic acid, 20:5n 3 at 37 °C
Docosahexaenoic acid, 22:6n 3 at 37 °C
Arachidonic acid, 20:4n 6 þ ETYA at 37 °C
Arachidonic acid, 20:4n 6 þ Ibuprofen (20 lM) at 37 °C
Arachidonic acid, 20:4n 6 þ Indomethacin (10 lM) at 37 °C
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I-leptin by Bewo cells
Internalisation of
125
I-leptin (fmol/mg protein)
0:075 0:002
0:79 0:01
0:09 0:003
2:62 0:07**
0:41 0:04*
0:56 :02*
1:05 0:02*
1:08 0:04*
1:05 0:07*
Experiments were carried out in triplicates (n ¼ 9). BeWo cells were incubated with various fatty acids (50 nM) and in the presence and absence of
ETYA, ibuprofen, and indomethacin, for 30 min, and the internalisation of 125 I-leptin was then followed as described in Materials and methods.
Significantly different from control, **P < 0:001, *P < 0:05.
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Addition
Internalisation of 125 I-leptin
(fmol/mg protein)
Control at 37 °C
Prostaglandin E2 (10 lM)
Prostaglandin F2a (10 lM)
Prostaglandin E1 (10 lM)
Prostaglandin A2 (10 lM)
Prostaglandin D2 (10 lM)
Prostaglandin A1 (10 lM)
6keto Prostaglandin F2a (10 lM)
0:79 0:01
2:08 0:06*
0:87 0:02
1:08 0:06
0:69 0:04
0:86 0:08
0:71 0:05
0:75 0:04
Experiments were carried out in triplicates (n ¼ 9), as described in
Table 1. BeWo cells were incubated with various prostaglandins
(10 lM) for 30 min and the internalisation of 125 I-leptin was then followed as described in Materials and methods. Significantly different
from control, *P < 0:05.
223 Presence of leptin receptors in BeWo cells
232
In this paper we report the regulation of leptin internalisation by n 3 and n 6 LCPUFA in placental
cells. Arachidonic acid, 20:4n 6, and PGE2 stimulated
whereas eicosapentaenoic acid, 20:5n 3, or docosahexaenoic acid, 2:6n 3 decreased internalisation of
leptin by these cells. Arachidonic acid stimulated the
degradation of internalised leptin receptors as evidenced
by the Western blot analysis of cell extract.
The leptin receptors are found in many tissues, including the human placenta syncytiotrophoblasts. Receptor mediated endocytosis is a well-characterised
mechanism for selectively transporting nutrients, hormones, and growth factors into cells. Often receptors
are concentrated in clathrin-coated pits and then internalised in clathrin-coated vesicles, although non-clathrin-mediated uptake of receptors also occurs [25–28].
Recently, it has been shown that that large numbers of
leptin receptors in intracellular compartments and no
visible leptin receptors in the classical recycling pathways were defined by transferrin [25–28].
Locally formed arachidonic acid metabolites are
important as modulators of many aspects of regulation
of the activity of membrane receptors/enzymes [29].
Arachidonic acid is either released within the cytoplasm
from cell membranes or taken up by the cells from
circulation and has three possible destinations, diffusion
outside the cells, re-incorporation into membrane
phospholipids, and further metabolism. It can be metabolised by three distinct enzyme pathways, cyclooxygenase, lipoxygenase or epoxygenase which produces,
respectively, prostaglandins and thromboxanes, leukotrienes, and hydroxyeicosatetraenoic acids and epoxides
[30]. Several products of these pathways and arachidonic acid itself are known to affect cell metabolism.
Eicosatetraynoic acid (ETYA), an inhibitor of all arachidonic acid-metabolising pathways, had also affected
the internalisation. ETYA is commonly used an inhibitor of the arachidonic acid pathway [31,32]. It has also
been demonstrated that ETYA inactivates cylcooxygenase, probably acting as a suicide substrate [30].
Arachidonic acid-induced stimulation of internalisation
of leptin receptors was blocked by the ibuprofen and
indomethacin, suggesting the possible involvement of
arachidonic acid metabolites via cyclooxygenase pathways. Antagonising roles of arachidonic acid and
eicosapentaenoic acid are well known both in terms of
prostaglandin synthesis and several other functions
[29,33]. Therefore, opposing effect of eicosapentaneoic
acid on leptin internalisation in BeWo cells is not unexpected. Negative effect of eicosapentaenoic acid on
receptor function was also demonstrated previously, as
enrichment of immune cells in vivo or in vitro with this
fatty acid decreased the number of IFN-c receptors
[34].
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In order to determine the presence of leptin receptors
in BeWo cells, we investigated the expression of leptin
receptors (Ob-R) in BeWo cell extract using leptin antibody. Western blot analysis demonstrated the presence
of leptin receptors in BeWo cells. Arachidonic acid (50–
100 nM) decreased significantly (more than 50%) leptin
receptors in BeWo cells compared with the control (data
not shown).
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Discussion
I-leptin by
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Table 2
Effect of various prostaglandins on internalisation of
BeWo cells
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Fig. 3. Effect of various concentrations of prostaglandin E2 (0–140 nM)
on the internalisation of 125 I-leptin by BeWo cells. After incubation,
cells were washed with ice-cold PBS buffer and subsequently cells were
acid washed to remove surface-bound leptin. The internalised leptin
was then determined as described, in Materials and methods.
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PGE2 , a cylcooxygenase metabolite of arachidonic
acid, also stimulated leptin internalisation in these cells
at nM concentrations. This indicates that the stimulatory action of arachidonic acid is most possibly mediated through the production of PGE2 . The Kd values for
PGE2 receptors are in nM range [35] therefore the
stimulatory action of PGE2 is probably mediated
through the interaction with their cell surface receptors.
PGE2 has pleiotropic actions in a range of tissues, including the feto-placental unit [36]. In fact, PGE2 elicits
a wide array of biological responses due to the presence
of at least four subclasses of EP receptors (EP1, EP2,
EP3, and EP4) [37]. PGE2 acts through these four G
protein-coupled receptors that display different tissue
distributions and deliver distinct intracellular signals.
The EP receptor isoforms have unique expression patterns and they couple to distinct signalling pathways.
The EP1 receptor is coupled to intracellular calcium,
while the EP2 and EP4 receptors are coupled to G
proteins and signal by stimulating adenylyl cyclase.
Signalling by the EP3 receptor is more complex [37].
Multiple EP3 receptor isoforms are generated by alternative splicing from a single EP3 receptor gene and these
EP3 receptor isoforms couple to different signalling
pathways including Gi , Gs , and calcium [37]. The existence of this family of EP receptors coupled to distinct
intracellular signals provides a molecular basis for the
diverse physiological actions of PGE2 . At the moment
we do not know which receptor subtype is involved.
Since PGE1 did not stimulate leptin internalisation in
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be involved in this process. Further work is in progress
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To the best of our knowledge the stimulatory action
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[39], however, the effects of fatty acids and/or their metabolites on these receptors were not reported before.
The importance of n 6 and n 3 long chain polyunsaturated fatty acids (LCPUFA) in the feto-placental
growth and development has been related to their
structural action, their specific interaction with membrane proteins, or their ability to serve as precursors
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In conclusion, we for the first time report that arachidonic acid, 20:4n 6, stimulates whereas eicosapentaenoic acid, 20:5n 3, decreases leptin uptake by
placental trophoblast cells and arachidonic acid-induced
stimulatory effect is mediated in part through prostaglandin E2 . This may have important implications in
LCPUFA metabolism and leptin function in the fetoplacental unit.
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