Maternal obesity results in decreased syncytiotrophoblast synthesis of palmitoleic acid, a fatty acid with anti-inflammatory and insulin-sensitizing properties

Article abstract of DOI:10.1096/fj.201802444R

The fetus is dependent on delivery of fatty acids (FAs) by the syncytiotrophoblast, the transporting epithelium of the human placenta. Obese pregnant women have dyslipidemia; however, whether obesity impacts placental lipid transport and metabolism remains to be fully established. Palmitoleic acid (POA), an FA with antiinflammatory and insulin-sensitizing properties, is synthesized from palmitic acid (PA) catalyzed by stearoyl-coenzyme A desaturase (SCD) activity. We hypothesized that the uptake and incorporation of FAs and POA synthesis are reduced in primary human trophoblasts (PHTs) isolated from pregnancies complicated by maternal obesity. Villous cytotrophoblasts were isolated from 7 placentas of obese [body mass index (BMI) = 37.5 ± 1.9] and 12 normal (BMI = 23.6 ± 0.6) mothers. FA uptake and incorporation were assessed using uniformly labeled (U[¹³C])–FA mixtures of PA, oleic acid (OA), linoleic acid, and docosahexaenoic acid. Cellular [¹³C] FAs were quantified both in total cellular lipids and in lipid classes by GC-MS. Uptake and incorporation of [¹³C] FAs in total cellular lipids were not different in PHTs isolated from obese mothers compared with normal mothers. Only the concentration of OA was increased in the triglyceride fraction (P < 0.05) if the mother was obese. We found an isotopic enrichment of POA after U[¹³C]-PA treatment, demonstrating SCD activity in PHT cells. Labeled POA content and the POA:PA ratio were significantly lower in PHTs isolated from placentas of obese mothers compared with normal, healthy controls. Decreased syncytiotrophoblast POA synthesis may contribute to insulin resistance and low-grade inflammation in the mother, placenta, or fetus (or a combination of the 3) in pregnancies complicated by obesity.—Ferchaud-Roucher, V., Barner, K., Jansson, T., Powell, T. L. Maternal obesity results in decreased syncytiotrophoblast synthesis of palmitoleic acid, a fatty acid with anti-inflammatory and insulin-sensitizing properties. FASEB J. 33, 6643–6654 (2019). www.fasebj.org.

Full text of DOI:10.1096/fj.201802444R

THE
JOURNAL
•
RESEARCH
•
www.fasebj.org
Maternal obesity results in decreased
syncytiotrophoblast synthesis of palmitoleic
acid, a fatty acid with anti-inflammatory and
insulin-sensitizing properties
,1
Veronique Ferchaud-Roucher,* Kelsey Barner,* Thomas Jansson,* and Theresa L. Powell*,†
´
*Division of Reproductive Sciences, Department of Obstetrics and Gynecology, and †Section of Neonatology, Department of Pediatrics,
University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
ABSTRACT: The fetus is dependent on delivery of fatty acids (FAs) by the syncytiotrophoblast, the transporting
epithelium of the human placenta. Obese pregnant women have dyslipidemia; however, whether obesity impacts
placental lipid transport and metabolism remains to be fully established. Palmitoleic acid (POA), an FA with anti-
inflammatory and insulin-sensitizing properties, is synthesized from palmitic acid (PA) catalyzed by stearoyl–
coenzyme A desaturase (SCD) activity. We hypothesized that the uptake and incorporation of FAs and POA
synthesis are reduced in primary human trophoblasts (PHTs) isolated from pregnancies complicated by maternal
obesity. Villous cytotrophoblasts were isolated from 7 placentas of obese [body mass index (BMI) = 37.5 6 1.9] and
12 normal (BMI = 23.6 6 0.6) mothers. FA uptake and incorporation were assessed using uniformly-labeled
(U[13C])–FA mixtures of PA, oleic acid (OA), linoleic acid, and docosahexaenoic acid. Cellular [13C] FAs were
quantified both in total cellular lipids and in lipid classes by GC-MS. Uptake and incorporation of [13C] FAs
in total cellular lipids were not different in PHTs isolated from obese mothers compared with normal
mothers. Only the concentration of OA was increased in the triglyceride fraction (P < 0.05) if the mother was
obese. We found an isotopic enrichment of POA after U[13C]-PA treatment, demonstrating SCD activity in
PHT cells. Labeled POA content and the POA:PA ratio were significantly lower in PHTs isolated from
placentas of obese mothers compared with normal, healthy controls. Decreased syncytiotrophoblast POA
synthesis may contribute to insulin resistance and low-grade inflammation in the mother, placenta, or fetus
(or a combination of the 3) in pregnancies complicated by obesity.—Ferchaud-Roucher, V., Barner, K.,
Jansson, T., Powell, T. L. Maternal obesity results in decreased syncytiotrophoblast synthesis of palmitoleic
acid, a fatty acid with anti-inflammatory and insulin-sensitizing properties. FASEB J. 33, 000–000 (2019).
www.fasebj.org
•
•
•
KEY WORDS: POA synthesis pregnancy placenta lipids
Almost two-thirds of American women now enter preg- women are more likely to deliver an infant who is large for
nancy either overweight or obese (1, 2), conditions which gestational age (4, 5), has increasedadiposity (6), or is insulin
are associated with a range of short- and long-term ad-
verse maternal and fetal outcomes (3). For example, obese
resistant at birth (or a combination of the 3) (7). Babies born
to obese mothers, in particular if they were large at birth,
have an increased risk to develop obesity, diabetes, and
cardiovascular disease in childhood and later in life (8–10).
Lipolytic activity in maternal adipose tissue is increased
in the third trimester, promoting the release of fatty acids
(FAs), which are primarily incorporated into triglycerides
(TGs)bythematernalliver, leadingtothemarkedlyhigher
circulating TG levels toward the end of pregnancy (11).
This physiologic hyperlipidemia is exacerbated in obese
pregnant women and may be associated with lipotoxicity,
leading to maternal endothelial dysfunction (12).
ABBREVIATIONS: BCA, bicinchoninic acid; BMI, body mass index; BSA,
bovine serum albumin; CE, cholesterol ester; CPT, carnitine palmitoyl-
transferase; DHA, docosahexaenoic acid; FA, fatty acid; hCG, human
chorionic gonadotropin; LA, linoleic acid; LCPUFA, long-chain poly-
unsaturated FA; MPE, mole percent excess; MUFA, monounsaturated FA;
NEFA, nonesterified FA; OA, oleic acid; PA, palmitic acid; PHT, primary
human trophoblast; PL, phospholipid; POA, palmitoleic acid; SA, stearic
acid; SCD, stearoyl–coenzyme A desaturase; SFA, saturated FA; TBS, Tris-
buffered saline; TG, triglyceride; U[13C], uniformly-labeled [13C]
1 Correspondence: Department of Obstetrics and Gynecology, University
of Colorado Anschutz Medical Campus, Research Complex-II, Mail Stop
8613, 12700 East 19th Ave., Aurora, CO 80045, USA. E-mail: veronique.
ferchaudroucher@ucdenver.edu
The composition of FAs delivered to the fetus is de-
termined by both the maternal circulating lipid levels and
FA uptake and metabolism by the syncytiotrophoblast,
doi: 10.1096/fj.201802444R
0892-6638/19/0033-0001 © FASEB
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the transporting epithelium of the human placenta. Tro- representative NEFA species in PHT cells using a physio-
phoblasts are believed to take up nonesterified FAs logic mixture of 4 [13C] stable isotope–labeled FAs [PA, OA,
(NEFAs) released from circulating maternal TGs and linoleic acid (LA), and docosahexaenoic acid (DHA)]. We
phospholipids (PLs) through hydrolysis by maternal li- determined the incorporation of these 4 representative [13C]-
poprotein lipase, endothelial lipase (13), and phos- FAs into 3 complex lipid forms: cholesterol esters (CEs), TGs,
pholipase A2. Preferential transfer of some long-chain and PLs in PHT cells isolated from placentas of normal,
polyunsaturated FAs (LCPUFAs) across the syncytio- healthy pregnancies and those complicated by obesity. We
trophoblast has been described and can be attributable to also studied the synthesis of POA from [13C] PA. This study
differences in the affinity of LCPUFAs for specific FA advances our understanding of the mechanisms involved in
transport proteins and FA binding proteins that are re- placental FA uptake and metabolism and how these are
sponsible for their uptake. Once taken up, FAs have 3 altered by maternal obesity and identifies novel factors in the
potential cellular fates: b-oxidation by the mitochondria, delivery of FAs to the fetus.
re-esterification (which occurs in the endoplasmic re-
ticulum), or transfer to the fetus across the basal plasma
membrane of the syncytiotrophoblast. The mechanisms
MATERIALS AND METHODS
underlying uptake (the first step of transplacental de-
livery of lipids) and cellular esterification of FAs by the
Study subjects for placenta collection
syncytiotrophoblast remain poorly understood. More-
Women were recruited to donate their placenta to a data/
over, the impact of maternal obesity on placental lipid
transfer to the fetus remains to be fully established.
Maternal obesity has been reported to be associated with
an increased placental lipid accumulation in the cellular TG
fraction (14). This high placental lipid content was not
explained by an increased FA uptake (14, 15) but rather by an
alteration in lipid esterification and down-regulation in FA
oxidation (16), suggesting that the placenta of obese mothers
may be modulating delivery of lipids to the fetus. In most
studies, only the total placental lipid content has been ana-
lyzed. Because the concentration and composition of FAs in
distinct cellular lipid fractions varies significantly under
changing physiologic conditions and in disease states, it is
important to understand how maternal obesity influences
placental incorporation of FAs in different lipid classes.
Palmitoleic acid (C16:1n-7, POA) and oleic acid (C18:1n-
9, OA), 2 monounsaturated FAs (MUFAs), are synthesized
from palmitic acid (PA) and stearic acid (SA), respectively.
The reaction is catalyzed by the rate-limiting enzyme d-9
desaturase, or stearoyl–coenzyme A desaturase (SCD).
Studies by Ntambi et al. (17) employing global SCD1
biorepository for use in multiple research studies. Informed
written consent was obtained from all participants for collection
of placental tissue and for the use of their protected health in-
formationunder a protocol approved bythe Institutional Review
Board at the University of Colorado–Denver (Colorado Multiple
Institutional Review Board, 14-1073). Inclusion criteria for the
repository included a range of prepregnancy or early pregnancy
body mass index (BMI), ultrasound confirmation of gestational
age at 14–18 wk, singleton pregnancy, and maternal age of
18–45 yr. Exclusion criteria included concurrent inflammatory,
vascular, or metabolic disease; current use of tobacco, street
drugs, or medications; fetal malformations; history of pregnancy
loss; or preterm delivery. Maternal and infant characteristics of
the subjects selected for the current study are summarized in
Table 1 and demonstrate that maternal BMI was the key factor
that distinguished the 2 groups. Anonymized tissue was trans-
ferred to the laboratory for isolation of cytotrophoblast cells im-
mediately after delivery.
Culture of PHT cells and uniformly-labeled
[
13C]-FA uptake and metabolism
Villous cytotrophoblast cells were isolated and purified as pre-
knockout mice and, more recently, by Vazquez-Vela et al. viously described in refs. 25 and 26. Briefly, cells were isolated
´
using DNase and trypsin digestion and purification on a Percoll
gradient. PHTswereplatedata density of 4 3106 per 60-mmdish
and cultured for 90 h in medium containingHam’s F-12and high-
glucose DMEM (1:1, v/v), 10% fetal bovine serum (S11550;
Atlanta Biologicals, Flowery Branch, GA, USA), and antibiotics
(penicillin, gentamicin, and streptomycin) according to a well-
established protocol (27–29).
At 66 h of culture, trophoblast cells were treated
with a combination of uniformlylabeled [13C] containing
(U[13C])–PA (95 mM), U[13C]-OA (95 mM), U[13C]-LA (95 mM),
and U[13C]-DHA (15 mM) (Cambridge Isotope Laboratories,
Tewksbury, MA, USA). Each labeled FA was solubilized in 10%
FA-free bovine serum albumin (BSA) and added to culture me-
dium containing 10% lipoprotein-depleted fetal bovine serum
(Kalen Biomedical, Germantown, MD, USA). These FAs were
selected to represent the 4 main types of FAs: PA, a saturated FA
(SFA);OA, aMUFA;LA, ann-6 essentialpolyunsaturatedFA;and
DHA, an n-3 LCPUFA in physiologic concentrations found in
maternal circulation at the end of normal pregnancy. Control cells
were treated in the same conditions by adding an equal volume of
FA-free BSA to the culture medium. Cells were incubated at 37°C
with 5% CO2/atmosphere air and studied over a time course from
30 min to 36 h. Cells were harvested as previously described (30),
(18) using mice lacking SCD1 specifically in the liver have
indicated that endogenous MUFA synthesis contributes to
whole-body energy homeostasis. Mice lacking SCD1 are
lean and protected against induction of obesity by diet (17)
or leptin deficiency (19). OA and POA are both products of
SCD1 and have distinct metabolic properties. POA is con-
sidered to be a strong indicator for de novo lipogenesis and
exerts lipokine effects by strongly stimulating insulin sen-
sitivity in muscles and suppressing hepatosteatosis in mice
(20, 21). POA also reverses proinflammatory gene expres-
sion in bone marrow cells and macrophage polarization
through the activation of AMPK (22, 23). Finally, POA has
been associated with increased insulin sensitivity and with
a lower incidence of diabetes in humans (24). Currently,
there are no reports about a potential role of POA in lipid
metabolism in the human placenta.
We hypothesized that the uptake and incorporation
of FAs and POA synthesis are reduced in primary hu-
man trophoblast (PHT) cells isolated from pregnancies
complicated by maternal obesity. We developed a novel
methodology to study the uptake and metabolism of 4 and at each time point, cells were rapidly and carefully washed 4
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TABLE 1. Clinical characteristics of subjects
Variable
Time course
Control
Obese
Subjects (n)
5
7
7
Maternal BMI (kg/m2)
Age at delivery (yr)
Gestational age (wk)
Placental weight (g)
Birth weight (g)
Infant sex
23.4 6 1.0
33.6 6 1.4
39.1 6 0.1
727.2 6 70.2
3502.8 6 92.4
5 males
23.7 6 0.8
37.5 6 1.9*
32.3 6 1.7
32.3 6 1.5
39.2 6 0.1
738.7 6 65.5
3483.7 6 108.8
6 males/1 female
39.3 6 0.1
742.6 6 70.2
3517.0 6 180.8
2 males/5 females
Data are presented as means 6 SEM. Time course (normal BMI) = PHT cells used for U[13C]-FA
model. Obese vs. control = PHT cells used for studying the placental lipid changes in pregnancy com-
plicated by obesity. *P , 0.05 (Mann-Whitney U test).
times in ice-cold PBS, scraped using a rubber-tipped spatula in ChemiXL1.4 (SynGene, Cambridge, United Kingdom). Densi-
0.9% NaCl, and stored at 280°C until analysis.
tometry analysis of target protein bands was performed with
GeneTools (SynGene), and target protein expression was ad-
justed for any unequal loading and transfer by staining the
membrane for protein using Amido Black.
After validation of the experimental model using a U[13C]-FA
mixture incubated with PHT cells isolated from placentas of 5
normal, healthy pregnant women (Fig. 1), this method was applied
in PHT cells isolated from placentas from pregnant women who
were obese or who had a normal BMI (n = 7 in each group). Ex-
amination of the uptake and incorporation of individual FAs over
time indicated that 2 h represented the initial uptake phase and 30 h
represented steady-state incorporation of the 4 FAs studied.
Analysis of FAs
Lipidsincelllysates collectedafter different incubationtimes were
extracted and separated into lipid classes according to the method
previously described (30). Briefly, lipids were extracted with a
methanol, water, and dichloromethane solution and fractionated
in 2 aliquots to determine the FA composition in total cellular
lipids and for separation into 4 lipid classes. CEs, TGs, NEFAs,
and PLs were separated by solid-phase extraction on NH2 car-
tridges (Phenomenex, Torrance, CA, USA) (30), using hexane to
elute CEs and 1% diethyl ether:10% dichloromethane in hexane to
collect TGs. A solution of diethyl ether:acetic acid (95:5, v/v)
(MilliporeSigma) was used to collect NEFAs, and finally metha-
nol:chloroform (6:1, v/v) and 100% of methanol to elute the PLs.
All the samples from the total lipid fraction and lipid classes were
dried under nitrogen, resuspended in ethanol, and hydrolyzed
with sodium hydroxide (1 N) at 90°C for 1 h. Free FAs were
extracted with isooctane and derivatized to make penta-
fluorobenzyl FA esters prior to analysis by GC-MS in negative-ion
chemical ionization mode as previously described (30, 33).
Characterization of cultured PHT cells
Release of human chorionic gonadotropin (hCG) from tropho-
blast cells was used as a biochemical marker of differentiation.
Concentrations of hCG in the cell culture medium were mea-
sured (b-hCG ELISA; DRG Instruments, Marburg, Germany) at
18, 42, 66, and 90 h after plating the cells (26). hCG secretion into
the culture medium increased more than 4-fold between 18 and
66 h (unpublished results), confirming differentiation, syncyti-
alization, and cell viability (27), and remained stable for the du-
ration of the incubation with [13C] FAs.
Western blotting
Western blot was performed as previously described (31, 32)
using a precast gel system (Bio-Rad, Hercules, CA, USA). Total
protein content of trophoblast cells collected after 90 h of culture
was determined using a bicinchoninic acid (BCA) assay (Pierce
BCA Protein Assay Kit; Thermo Fisher Scientific, Waltham, MA,
USA). Tenmicrograms of total protein was loadedand separated
on Bis-Tris Gels (4–20%; Bio-Rad). Lysate of human embryonic
kidney 293 cells (HEK293, ab7902; Abcam, Cambridge, MA,
USA) served as a positive control. Electrophoresis was per-
formed at 120 V for 20 min and then at a constant 150 V for 1 h.
Proteins were transferred onto a PVDF membrane (Bio-Rad)
overnight at 4°C and a constant 35 V. The membrane was first
stainedfor totalproteinwithAmidoBlackstain(MilliporeSigma,
Burlington, MA, USA)and then cleared and blocked for 1 h in 5%
BSA in Tris-buffered saline (TBS)–Tween. Incubation with pri-
mary antibodies [anti-SCD1, diluted 1:500, ab39969; anti–
Quantification of labeled FAs in total cellular lipid
fraction and lipid classes
Absolute FA concentrations of U[13C]-PA, U[13C]-OA, U[13C]-LA,
and U[13C]-DHA as well as all unlabeled FAs in the same MW
range were determined using calibration curves built with tar-
geted internal standards. FA content was expressed in nanomoles
of FA normalized to total cellular protein content, determined
using a BCA assay (Pierce BCA Protein Assay Kit) (30). Uptake of
each labeled FA by the PHT cells and their incorporation into the
cellular lipid classes were measured at 2 and 30 h after U[13C]-FA
incubation in PHT cells isolated from placentas of obese pregnant
women and compared with those of normal BMI mothers.
In the total lipid fraction, we determined the presence of a
[
13C] label in other quantifiable FA species to assess whether
carnitine palmitoyltransferase (CPT) 1b, diluted 1:1000, PHTs are able to convert PA, OA, LA, or DHA into any other
ab134988; anti-CPT2, diluted 1:1000, ab181114; all antibodies FA species over 36 h of incubation. The isotopic enrichment of
purchased from Abcam] was carried out overnight at 4°C in 5% PA and POA in the total lipid fraction was calculated using
BSA in TBS-Tween except for CPT2, for which 5% milk in TBS- individually calibrated FA curves ranging from 0 to 100% of
Tween was used. The washed membranes were incubated for each U[13C]-FA enrichment relative to similar [12C]-FA en-
1 h at room temperature with secondary antibody (peroxidase- richment and measured by GC-MS. Isotopic enrichments of
labeled anti-rabbit IgG; Cell Signaling Technology, Danvers, PA and POA were expressed as mole percent excess (MPE)
MA, USA) diluted at 1:10000 forSCD1 and CPT1b and 1:2000for (%) over time (34). This unit expresses the molar ratio of tracer
CPT2. ImmunolabelingwasmadevisiblewithSuperSignalWest ([13C]) to the sum of the tracer ([13C]) + tracee ([12C]) extracted
Pico Plus detection solution (Thermo Fisher Scientific) in a G:Box from the PHT cells. For example, the formula for determination
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Figure 1. U[13C]-FA concentration in total lipid pool in cultured PHT cells. Change over time in absolute concentrations of
labeled PA, OA, LA and DHA expressed as nanomoles of FA per microgram of protein in total lipid pool extracted from
PHT cells (n = 3 to 5 for each time point, means + SEM). A) Uptake of U[13C]-FAs over the first 4 h of incubation. B) Total U[13C]-
FA content over 36 h of incubation.
of PA enrichment was: MPE (%) = [U[13C]-PA/(U[13C]-PA +
RESULTS
[12C]-PA)] 3 100.
PHT cells were cultured and differentiated for 66 h before
Statistical analyses
incubation with uniformly-stable isotope–labeled FAs
(PA, OA, LA, DHA) complexed to FA-free BSA, and cells
were subsequently collected at different times to de-
termine the uptake of FA over time as well as the in-
corporation of different types of FA into cellular lipid
Data are presented as means 6 SEM and means + SEM. All statistical
tests were performed using Prism v.6.07 (GraphPad Software, La
Jolla, CA, USA). The Mann-Whitney U test was used to determine
the statistical significance of differences between obese vs. control
groups at2and 30 h. A valueof P,0.05wasconsideredsignificant. fractions.
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The absolute concentrations of labeled FA
increase in total cellular lipid fraction
over time
FAs are highly concentrated in PL fraction in
PHT cells
FAs taken up into cells are rapidly acted upon by coenzyme
A synthase enzymes and shuttled to specific pools for either
oxidation, re-esterification, or transport to the fetus. To
We determined the absolute concentrations of labeled
FAs expressed in nanomoles of U[13C]-FA per microgram
ofproteinforeachFAinthetotallipidfractioninPHTcells provide novel insights on the mechanisms involved in the
syncytiotrophoblast FA uptake and re-esterification, quan-
tities of the labeled PA, OA, LA, and DHA were determined
in 4 cellular lipid fractions over the initial 4 h of incubation
(Fig. 2) and later times (Fig. 3). All 4 FAs were rapidly taken
upbyPHTcellsandincorporatedintothemajorlipidclasses
starting at 30 min of incubation. For all FAs studied, the
highest concentrations were found in the PL fraction com-
pared with other lipid fractions. This is indicative of a rapid
synthesis and turnover of PLs and incorporation into PLs of
NEFAs taken up from the maternal circulation.
(Fig. 1A). Initially, the absolute concentrations of all FAs
increased linearly over time, reflecting a rapid FA uptake
and transport in these cells (Fig. 1A). As expected, the
concentrations of U[13C]-FAs continued to increase over
time, beyond 4 h, in the total lipid pool of cultured PHTs
and reached a plateau between 24 and 36 h of incubation
time (Fig. 1B), representing FA incorporation. Of the FAs
added at the same concentration to culture medium
(95 mM), PA and OA displayed similar levels over time,
whereas LA incorporation was 2.5-fold less than PA or
OA in PHT cells at equilibrium, reflecting lower levels of
LA in the cells. U[13C]-DHA was present in the culture
medium in a much lower concentration (15 mM) than the
other FAs, reflecting physiologic FA concentration in the
maternal circulation. DHA was rapidly taken up by
the cells, and as expected, intracellular U[13C]-DHA con-
centrations remained lower that the other labeled FAs.
Higher levels of unlabeled FAs in PHT cells
isolated from obese mothers
Next, we determined the impact of maternal obesity
on PHT FA uptake and metabolism using the same ap-
proach ofincubation in uniformly-stable isotope–labeled
Figure 2. Uptake and incorporation of each labeled FA in lipid classes in cultured PHTs after the initial 4 h. Absolute concentrations
of labeled PA, OA, LA, and DHA in 4 lipid classes in PHT cells isolated from normal-term placentas, expressed as nanomoles of FA per
microgram of protein, were determined at 0.5, 1, 2, and 4 h after U[13C]-FA mix was added to culture medium (n = 3, means + SEM).
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Figure 3. Uptake and incorporation of each labeled FA in lipid classes in cultured PHTs after 24 and 30 h of incubation. Absolute
concentrations of labeled PA, OA, LA, and DHA in 4 lipid classes in PHT cells isolated from normal-term placentas, expressed as
nanomoles of FA per microgram of protein, were determined at 24 and 30 h to measure the FA esterification at the plateau (n =
3, means + SEM).
FAs (PA, OA, LA, DHA) complexed to FA-free BSA. Maternal obesity alters FA esterification by
increasing OA incorporation in TGs in PHT cells
PHT cells were collected at 2 h (uptake) and 30 h (in-
corporation), and the accumulation of [13C]-labeled FAs
as well as the unlabeled FA pool were assessed.
To gain insight into cellular lipid metabolism and esterifi-
cation in placentas of obese women, we quantified the la-
beled FAs in 4 main cellular lipid classes. After 30 h of
incubation, the incorporation of [13C] PA, OA, and LA was
not different in PL and CE fractions, whereas a significant
increase was found in the OA content of the TG fraction
(Fig. 5). These data suggest an accumulation of lipid in a
storage form by the placental syncytiotrophoblast in
pregnancies complicated by obesity.
We found a significantly higher concentration of un-
labeled SFAs, MUFAs, and essential FAs (n-6 and n-3
LCPUFA) in the total lipid pool in PHT cells isolated from
obese women when compared with normal BMI placenta at
2 and 30 h of incubation (Fig. 4A). Specifically, after 2 h of
incubation, the concentration of unlabeled OA was signifi-
cantly higher in PHT cells isolated from obese mothers as
compared with PHT cells from normal, healthy mothers. By
30 h of incubation, the unlabeled FA concentrations, al-
though lower than at 2 h, were all significantly greater in
PHT cells from obese pregnancies. These data suggest an
alteration in FA metabolism and storage in PHTs from obese
mothers that persist after 66–90 h of culture and when ex-
posed to a physiologic mixture of FAs in the culture medium.
U[13C]-PA is converted into U[13C]-POA
Incubation with U[13C]-FAs allows for determination
of conversion of labeled FAs to other FA species
through the action of either elongase or desaturase
enzymes. Figure 6 shows the conversion of U[13C]-PA
(16:0) to U[13C]-POA (16:1 n-7) in total lipids in
PHT cells over time. The conversion of PA to POA is
catalyzed by SCD1; therefore, our data are consistent
Maternal obesity does not modify the uptake
of FAs in PHT cells
To determine whether the uptake of [13C]-labeled FAs with the presence of an active D9 desaturase in
in PHT cells isolated from pregnancies complicated by PHT cells. The cellular enrichment of POA in total lipids
maternal obesity was different from normal-term preg- over time is the result of the conversion of PA to POA
nancies, we incubated PHT cells with our labeled FA and represents a lipid metabolic pathway not pre-
mixture for 2 and 30 h and determined uptake into the viously described in PHT cells. U[13C]-POA enrich-
total lipid pool. We found no difference in uptake of ments measured in total lipids mirror the cellular
U[13C]-PA, U[13C]-OA, U[13C]-LA, and U[13C]-DHA in enrichment of PA over time (Fig. 6). Importantly, no
PHT cells isolated from obese mothers compared with other conversions of any of the other U[13C]-FAs were
placentas of normal BMI mothers (Fig. 4B).
observed at any time because [13C] enrichments were
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Figure 4. The effect of maternal obesity on
absolute concentrations of unlabeled FAs and
on the uptake and incorporation of U[13C]-
FAs in PHT cells. A) Absolute concentrations
of unlabeled SFAs (PA), MUFAs (OA), and
essential FAs (n-6 LCPUFA, LA, and n-3
LCPUFA, DHA) in PHT cells isolated from
obese mothers (gray bars) compared with
trophoblasts isolated from normal BMI moth-
ers (Ctr; black bars) after incubation with
U[13C]-FA mixture for 2 and 30 h. B) Uptake
of U[13C]-FAs in PHT cells isolated from obese
and normal BMI mothers after incubation with
U[13C]-FA mixture for 2 and 30 h. Results are
presented for the total cellular lipid pool and
expressed as nanomoles of FA per microgram
of protein for each labeled FA studied (n = 7 in
each group, means + SEM). *P , 0.05 (Mann-
Whitney U test).
not found in any other FA species in total lipids, sug- 2 and 30 h of incubation in PHT cells isolated from
gesting there is no detectable elongase, D5, or D6 desa- placentas of obese mothers. These results are confirmed
turase enzyme activities in PHT cells.
by the decreased ratio of U[13C]-POA to U[13C]-PA,
reflecting a reduced SCD1 activity catalyzing the con-
version of PA to POA (Fig. 7). These findings indicate a
decreased placental syncytiotrophoblast POA synthe-
sis in obese pregnant women, likely due to reduced
SCD activity rather than limitations in substrate
availability.
Decreased synthesis of POA and activity of
SCD1 in PHT cells in maternal obesity
We found a marked decrease in endogenous unlabeled
POA concentration in PHTs from pregnancies compli-
cated by maternal obesity (Fig. 7), whereas unlabeled
PA was unchanged at the 2 h time point (Fig. 4A). De-
creased POA in PHTs isolated from obese mothers was
also observed at 30 h in spite of a significantly increased
Maternal obesity does not alter protein
expression of SCD1 in PHT cells
concentration of unlabeled PA at this time point (Fig. Using Western blot, we found no difference in protein
4A). Similarly, we found a decrease in the synthesis of expression of SCD1 in PHT cells in the obese vs. normal
U[13C]-POA from U[13C]-PA added to the media at both BMI group (Fig. 8A, B). The results are consistent with
Figure 5. The effect of maternal obesity on the
incorporation of U[13C]-FAs in lipid classes in
PHT cells. FA esterification and incorporation
into lipid fractions in PHT cells isolated from
obese (gray bars) and normal BMI (Ctr; black
bars) mothers at 30 h of incubation with
U[13C]-FA mixture. Results are expressed as
nanomoles of FA per microgram of protein
for each labeled FA studied (n = 7 in each
group, means + SEM). *P , 0.05 (Mann-
Whitney U test).
DECREASED PLACENTAL POA IN OBESITY
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syncytiotrophoblast POA synthesis. However, we were
not able to successfully identify SCD5 expression in
PHTs, another isoform of SCD found exclusively in
primates.
Maternal obesity does not affect the
b-oxidation pathway in PHT cells
Increased cellular TG synthesis is believed to be asso-
ciated with a decrease in the b-oxidation pathway. We
measured protein expression of CPT1b and CPT2, 2
enzymes representing the rate-limiting synthesis of the
b-oxidation, in PHT cells from obese and normal BMI
mothers. We found no significant differences in the
protein expression of these 2 specific enzymes in
PHT cells (Fig. 8C, D).
Figure 6. Time course of cellular enrichment of POA (U[13C]-
POA, MUFA) produced from the conversion of U[13C]-PA
(SFA) added to the cultured media in PHT cells isolated from
normal BMI placentas. U[13C]-POA is expressed in mole
percent excess (MPE, %) in the total lipids in the PHT cells
over 36 h (n = 3 to 5 for each time, mean 6 SEM).
DISCUSSION
We developed a novel approach utilizing 4 stable isotope–
the fact that we found no change in the OA:SA ratio labeled FA species to study the uptake and incorporation of
(unpublished results), which is also catalyzed by SCD1. FAs in a cultured primary human syncytiotrophoblast, a
These results are not entirely consistent with the de- well-established and physiologically relevant model for the
creased conversion of U[13C]-PA to U[13C]-POA, sug- study of human placental function. In normal pregnancies,
gesting that other SCD isoforms may be involved in all 4 of the U[13C]-FAs were rapidly taken up and
Figure 7. Decreased synthesis of POA in PHT cells in maternal obesity. Unlabeled POA, newly synthesized U[13C]-POA, and the
respective POA:PA ratios in total cellular lipid fraction in PHT cells isolated from obese (Ob, gray bars) and normal BMI (C,
black bars) mothers at 2 and 30 h of incubation with U[13C]-FA mixture. A, B) Unlabeled POA concentration (A) and unlabeled
POA:PA ratio (B) at 2 and 30 h in obese mothers vs. those with normal BMI. C) U[13C]-POA concentration produced from
desaturation of U[13C]-PA at 2 and 30 h. D) U[13C]-POA:U[13C]-PA ratio in obese mothers vs. control group (n = 7 in each
group, means + SEM). *P , 0.05, **P , 0.01 (Mann-Whitney U test).
x
8
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Figure 8. Maternal obesity does not alter protein expression of SCD1, CPT1, or CPT2 in PHT cells. Effect of maternal obesity on
lipogenesis and b-oxidation pathways in PHT cells. A, C) Relative protein expression of SCD1, CPT1b, and CPT2 in PHT cells
isolated from obese (gray bars) and normal BMI (black bars) mothers. B, D) Representative Western blot of SCD1, CPT1b, and
CPT2 (n = 7 in each group, means + SEM); P , 0.05 was considered significant (Mann-Whitney U test). A.U., arbitrary unit.
differentially incorporated into the total cellular lipid pool
In this study, 4 stable isotope–labeled [13C] FA
in PHT cells over a 36-h time course. Moreover, the syn- species—PA (16:0), OA (18:1 n-9), LA (18:2 n-6), and DHA
cytiotrophoblast displayed a rapid uptake and in- (22:6 n-3)—representing an SFA, a MUFA, an essential
corporation of all 4 U[13C]-FAs studied into the PL polyunsaturated FA, and an n-3 LCPUFA were added to
fraction, reflecting a strong preference for this cellular culture medium in physiologic concentrations to establish
lipid fraction in this transporting epithelium. PLs are a novel methodology for studying FA uptake, esterifica-
critical substrates for plasma membrane production, and tion, and metabolism by the primary human syncytio-
we recently suggested that PL intermediaries, in partic- trophoblast. In PHTs from healthy pregnancy, U[13C]-FA
ular lysophosphatidylcholine, may be transferred to the levels increased rapidly over time, reaching a plateau at
fetus across the basal membrane via major facilitator about 24 h. The data also show that PHT cells prioritize the
superfamily domain containing 2a (Mfsd2a), a specific de novo synthesis and remodeling of PLs over other cellular
lysophosphatidylcholine transporter (35). In addition, lipid forms, consistent with our previous study (30). These
we report for the first time the synthesis of U[13C]-POA, a findings are also in agreement with the study of Perazzolo
lipid signaling molecule with insulin-sensitizing and et al. (36) using perfused human cotyledons and with the in
´
anti-inflammatory properties, resulting from conversion vivo results reported by Gil-Sanchez et al. (37) showing a
of U[13C]-PA by SCD activity. Importantly, POA synthe- higher U[13C]-FA concentration in placental PL than in any
sis was decreased in PHT cells from obese pregnancies, other lipid fractions when labeled PA, OA, LA, and DHA
which may contribute to insulin resistance and low-grade were orally administered to pregnant women 12 h before
inflammation in obese pregnant women.
delivery. The rapid incorporation of the FAs into more
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complex forms suggests that PHTs are converting these with the study of Calabuig-Navarro et al. (42) showing no
FAs into less toxic forms for cellular storage and later uti- changes in the placental protein expression of CPT1 as
lization. However, the mechanisms by which FAs are re- well as no change in the rate of [3H]palmitate oxidation in
leased from intracellular esterified lipid pools for transfer PHT cells isolated from pregnancies complicated by ma-
across the syncytiotrophoblast basal plasma membrane ternal obesity. Our findings are not in completeagreement
and delivery to the fetus remain largely unknown.
with the report by Pathmaperuma et al. (45) in which they
In the total cellular lipid pool, U[13C]-OA and U[13C]- demonstrated a decreased b-oxidation in human tropho-
PA are the FAs in greatest abundance, reflective of their blast cells isolated from normal-term placentas treated
high levels in the incubation media and in maternal cir- with a PA:OA (1:1) mixture tomimic hyperlipidemia. One
culation. These 2 FAs had the greatest overall uptake by key difference that may explain the different findings in
PHT cells over time and were incorporated preferentially the 2 studies is the concentrations of FAs used. We used
into PLs and then TGs followed by CEs; only low levels much lower concentrations of PA and OA (0.095 mM),
remained as NEFAs in PHT cells. OA is the major MUFA reflecting physiologic levels of FA in maternal circulation
in maternal circulation and is found in very high levels in compared with the 0.25 mM PA and OA mixture used in
membrane PLs of red blood cells at term (38) as well as the study by Pathmaperuma et al. (45). In addition, using
being abundantly incorporated into phosphatidylcholine high concentrations of only 2 FA species in cell culture
in placental tissue (39). PA is also present in high abun- conditions may not fully represent in vivo hyperlipidemia
dance in maternal circulation and is considered lipotoxic of pregnancy.
and atherogenic in other tissues (40). Although the uptake
In our model using physiologic U[13C]-LA concentra-
and incorporation of DHA appears to be slower than that tions, we found no detectable [13C] label in any n-6
for the other 3 FAs, in particular when compared with LCPUFA, including arachidonic acid species (20:4 n-6),
U[13C]-LA (an essential LCPUFA), it is important to therefore no arachidonic acid was formed from U[13C]-LA
consider the differences in extracellular concentrations in the total cellular lipid fraction over the 30 h of in-
of the FAs to fully understand the handling of DHA. cubation. Likewise, we found no evidence of conversion of
We aimed to represent physiologic U[13C]-DHA concen- U[13C]-PA (16:0) into SA (18:0). These findings confirm a
trationsandused15mM in the medium, whereas the other very low or absent activity of D6 and D5 desaturases and
3 were used at 95 mM, in accordance with relative circu- elongases in PHT cells and are in agreement with a pre-
lating levels in late-term pregnant women. Considering vious report by Hanebutt et al. (48) of low or nonexistent
the rapid uptake ofDHA fromthe lowextracellularlevels, activity of these enzymes in placental tissue.
preferential handling in uptake and incorporation of
The levels of POA in the diet are low, suggesting that
DHA by the syncytiotrophoblast, as previously re- POA is largely produced by endogenous desaturation of
´
ported by Gil-Sanchez et al. (37), is likely occurring when the most abundant SFA, PA, by SCD. Interestingly, we
the FAs are presented together, but because of low measured an isotopic enrichment of POA derived from U
availability of DHA, the intracellular levels remain low. [13C]-PA conversion, demonstrating an activity of SCD in
In PHT cells isolated from the placenta of obese moth- PHT cells. POA has been called a lipokine and reported to
ers, we found higher concentrations of unlabeled PA, OA, act as an insulin sensitizer that improves glucose metab-
LA and DHA, whereas the uptake of U[13C]-PA, U[13C]- olism (20) and has anti-inflammatory properties (22), and
OA, U[13C]-LA, and U[13C]-DHA into the total lipid pool it is therefore possible that placental POA synthesis may
was not different at 2 and 30 h compared with PHTs from modulate placental, fetal, or maternal insulin sensitivity
mothers with normal BMI. However, we observed a sig- and inflammation (or a combination of all of these).
nificant increase of U[13C]-OA in the TG fraction after 30 h
We report that POA:PA and U[13C]-POA:U[13C]-PA
of incubation, which is consistent with a high cellular ratios are decreased in PHT cells isolated from obese
abundance and the major role of OA to promote the TG mothers, reflecting the decreased conversion of PA to
synthesis (41). These findings are in agreement with pre- POA. We did not find changes in SCD1 protein expression
vious studies showing higher lipid levels in the total lipid in PHT cells isolated from pregnancies complicated by
fraction and storage in cellular TG pools in the placenta of maternal obesity, which is in contrast to a recent study by
obese pregnant women (42–44). Thecurrentstudy extends Calabuig-Navarro et al. (42) reporting an increased pla-
these observations by identifying OA as the predominant cental SCD1 protein expression in obese women. We
FA accumulating in placental TG in maternal obesity.
speculate that the activity of SCD1 in the syncytiotropho-
Increased placental lipid storage has been associated blast is regulated by post-translational modification in
with a decrease in FA oxidation capacity in trophoblast PHTs. Because POA is a known insulin sensitizer and anti-
cells isolated from normal BMI placentas when incubated inflammatory lipokine (49), decreased syncytiotropho-
with a high concentration of PA and OA (45). The blast POA synthesis may contribute to insulin resistance
transport of FAs across the mitochondrial membrane is and low-grade inflammation in obese pregnant women.
mediatedbyCPT1andCPT2(46,47), whichrepresentsthe
In summary, PHT cells take up FAs rapidly and pref-
rate-limiting step in mitochondrial b-oxidation of long- erentially use them to synthesize PLs. In trophoblast cells
chain FAs. Although we did not measure FA oxidation isolated from obese mothers, we found a continued accu-
directly, the protein expression of CPT1b and CPT2 in mulation of unlabeled FAs in PHTs after 30 h of labeled FA
PHT cells was not affected by maternal obesity in our incubation and an increase in the esterification and storage
study. These data suggest that the mitochondrial FA oxi- of OA in the TG fraction but no indication of alterations of
dation capacityremains intact. Our findings are consistent the FA oxidation capacity. We found activity of SCD, a
x
10 Vol. 33 May 2019
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ACKNOWLEDGMENTS
Placental collection was supported by the Perinatal
Clinical Translational Research Center at the University of
Colorado Anschutz Medical Campus. This work was sup-
ported by U.S. National Institutes of Health (NIH) National
Institute of Diabetes and Digestive and Kidney Diseases Grant
DK048520-23, and NIH National Center for Advancing
Translational Sciences Colorado Clinical and Translational
Sciences Institute Clinical and Translational Science Award
Grant UL1 TR002535. The authors declare no conflicts of
interest.
AUTHOR CONTRIBUTIONS
19. Flowers, J. B., Rabaglia, M. E., Schueler, K. L., Flowers, M. T., Lan, H.,
Keller, M. P., Ntambi, J. M., and Attie, A. D. (2007) Loss of stearoyl-
CoA desaturase-1 improves insulin sensitivity in lean mice but worsens
diabetes in leptin-deficient obese mice. Diabetes 56, 1228–1239
20. Cao, H., Gerhold, K., Mayers, J. R., Wiest, M. M., Watkins, S. M., and
Hotamisligil, G. S. (2008)Identification of a lipokine, a lipid hormone
linking adipose tissue to systemic metabolism. Cell 134, 933–944
21. Frigolet, M. E., and Gutie´rrez-Aguilar, R. (2017) The role of the novel
lipokine palmitoleic acid in health and disease. Adv. Nutr. 8, 173S–181S
22. Chan, K. L., Pillon, N. J., Sivaloganathan, D. M., Costford, S. R., Liu, Z.,
The´ret, M., Chazaud, B., and Klip, A. (2015) Palmitoleate reverses
high fat-induced proinflammatorymacrophagepolarizationviaAMP-
activated protein kinase (AMPK). J. Biol. Chem. 290, 16979–16988
23. Talbot, N. A., Wheeler-Jones, C. P., and Cleasby, M. E. (2014)
Palmitoleic acid prevents palmitic acid-induced macrophage activa-
tion and consequent p38 MAPK-mediated skeletal muscle insulin
resistance. Mol. Cell. Endocrinol. 393, 129–142
24. Rise´rus, U., Tan, G. D., Fielding, B. A., Neville, M. J., Currie, J., Savage,
D. B., Chatterjee, V. K., Frayn, K. N., O’Rahilly, S., andKarpe, F. (2005)
Rosiglitazone increases indexes of stearoyl-CoA desaturase activity in
humans: link to insulin sensitization and the role of dominant-
negative mutation in peroxisome proliferator-activated receptor-
gamma. Diabetes 54, 1379–1384
25. Lager, S., Jansson, N., Olsson, A. L., Wennergren, M., Jansson, T., and
Powell, T. L. (2011) Effect of IL-6 and TNF-a on fatty acid uptake in
cultured human primary trophoblast cells. Placenta 32, 121–127
26. Lager, S., Gaccioli, F., Ramirez, V. I., Jones, H. N., Jansson, T., and
Powell, T. L. (2013) Oleic acid stimulates system A amino acid
transport in primary human trophoblast cells mediated by toll-like
receptor 4. J. Lipid Res. 54, 725–733
V. Ferchaud-Roucher, T. Jansson, and T. L. Powell
designed the research studies; V. Ferchaud-Roucher
conducted the experiments, analyzed the results, and
wrote the manuscript; K. Barner performed the Western
blot experiments; and all authors discussedthe results and
edited the manuscript.
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Received for publication November 14, 2018.
Accepted for publication January 28, 2019.
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