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Activation of Placental mTOR Signaling and Amino
Acid Transporters in Obese Women Giving Birth to
Large Babies
Nina Jansson,* Fredrick J. Rosario,* Francesca Gaccioli, Susanne Lager,
Helen N. Jones, Sara Roos, Thomas Jansson, and Theresa L. Powell
Center for Pregnancy and Newborn Research (F.J.R., F.G., S.L., T.J., T.L.P.), Department of
Obstetrics/Gynecology, University of Texas Health Science Center, San Antonio, Texas 78229; Division of
Pediatric Surgery (H.N.J.), Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229; and
Institutes of Neuroscience and Physiology (N.J.) and Biomedicine (S.R.), Sahlgrenska Academy at University of
Gothenburg, S-40530 Gothenburg, Sweden
Context: Babies of obese women are often large at birth, which is associated with perinatal com-
plications and metabolic syndrome later in life. The mechanisms linking maternal obesity to fetal
overgrowth are largely unknown.
Objective: We tested the hypothesis that placental insulin/IGF-I and mammalian target of rapamycin
(mTOR) signaling is activated and amino acid transporter activity is increased in large babies of obese
women.
Design and Setting: Pregnant women were recruited prospectively for collection of placental tissue
at a university hospital and academic biomedical center.
Patients or Other Participants: Twenty-three Swedish pregnant women with first trimester body mass
index ranging from 18.5 to 44.9 kg/m
2
and with uncomplicated pregnancies participated in the study.
Interventions: There were no interventions.
Main Outcome Measures: We determined the phosphorylation of key signaling molecules (in-
cluding Akt, IRS-1, S6K1, 4EBP-1, RPS6, and AMPK) in the placental insulin/IGF-I, AMPK, and mTOR
signaling pathways. The activity and protein expression of the amino acid transporter systems A
and L were measured in syncytiotrophoblast microvillous plasma membranes.
Results: Birth weights (range, 3025– 4235 g) were positively correlated to maternal body mass index
(P⬍0.05). The activity of placental insulin/IGF-I and mTOR signaling was positively correlated (P⬍
0.001), whereas AMPK phosphorylation was inversely (P⬍0.05) correlated to birth weight. Mi-
crovillous plasma membrane system A, but not system L, activity and protein expression of the
system A isoform SNAT2 were positively correlated to birth weight (P⬍0.001).
Conclusions: Up-regulation of specific placental amino acid transporter isoforms may contribute
to fetal overgrowth in maternal obesity. This effect may be mediated by activation of insulin/IGF-I
and mTOR signaling pathways, which are positive regulators of placental amino acid transporters.
(J Clin Endocrinol Metab 98: 105–113, 2013)
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2013 by The Endocrine Society
doi: 10.1210/jc.2012–2667 Received July 3, 2012. Accepted October 9, 2012.
First Published Online November 12, 2012
* N.J. and F.J.R. contributed equally to this work.
Abbreviations: AMPK, AMP-activated kinase; BMI, body mass index; 4E-BP1, eukaryotic
initiation factor 4E-binding protein 1; GDM, gestational diabetes mellitus; IRS-1, insulin
receptor substrate 1; IUGR, intrauterine growth restriction; LGA, large for gestational age;
MeAIB,
14
C-methyl-aminoisobutyric acid; mTOR, mammalian target of rapamycin;
mTORC, mTOR complex; MVM, microvillous plasma membrane; PKC
␣
, protein kinase C-
␣
;
RPS6, ribosomal protein S6; SGK1, serum and glucocorticoid-regulated kinase 1; S6K1, S6
kinase 1; SNAT, sodium-dependent neutral amino acid transporter.
ORIGINAL ARTICLE
Endocrine Care
J Clin Endocrinol Metab, January 2013, 98(1):105–113 jcem.endojournals.org 105
Women entering pregnancy overweight [body mass
index (BMI), 25–29.9 kg/m
2
] or obese (BMI ⱖ30
kg/m
2
) have an increased risk to deliver a large for gesta-
tional age (LGA) baby, often defined as a birth weight
above the 90th centile (1–3). Large babies have an in-
creased risk for shoulder dystocia and plexus injury at
delivery (1, 2) and are susceptible to develop obesity, di-
abetes, and hypertension in childhood and later in life (4).
The mechanisms underlying the relationship between ex-
cess maternal adiposity and fetal overgrowth are not well
established.
To grow appropriately, the fetus is critically dependent
on nutrient supply across the placenta, which is deter-
mined by numerous factors including placental and um-
bilical blood flows, transplacental concentration gradi-
ents, and placental metabolism. In addition, the type,
number, and activity of transporter proteins in the syncy-
tiotrophoblast plasma membranes constitute an impor-
tant determinant for the transplacental transport of nu-
trients such as glucose and amino acids. In pregnancies
complicated by intrauterine growth restriction (IUGR),
placental nutrient transporters for amino acids, such as the
sodium-dependent system A (5, 6), are down-regulated.
Women having type 1 diabetes or developing gestational
diabetes mellitus (GDM) are more likely to give birth to a
LGA baby (7), and an increased placental nutrient trans-
port capacity may be one important factor contributing to
fetal overgrowth in these pregnancy complications. For
example, placental leucine transport has been shown to be
increased in GDM/LGA (8), and system A activity was
increased in microvillous plasma membrane (MVM) iso-
lated from placentas obtained from pregnancies compli-
cated by diabetes (8). In contrast to these findings, a pre-
vious study showed that system A amino acid transporter
activity is reduced, and the activity of system L is unaltered
in MVM vesicles isolated from type 1 diabetes pregnancies
with LGA babies (9). Placental glucose transport and glu-
cose transporter protein 1 protein expression were re-
ported to be increased in type 1 diabetes (10, 11). In ad-
dition, emerging evidence suggests that fatty acid
transport to the fetus may be increased in diabetes with or
without obesity, providing one possible explanation for
the increased adiposity observed in babies of mothers with
diabetes. For example, the activity of placental lipoprotein
lipase has been shown to be increased in pregnancies with
type 1 diabetes and fetal overgrowth (12), and placental
expression of the fatty acid binding protein 4 (13) and
endothelial lipase (14) is elevated in pregnancies of obese
women with diabetes. Although it is well established that
high prepregnancy BMI is strongly associated to fetal
overgrowth (1–3), the effect of maternal overweight and
obesity on placental function in women without diabetes
remains largely unknown (15, 16).
Placental nutrient transport is controlled by fetal, placen-
tal, and maternal factors. Placental mammalian target of
rapamycin (mTOR) constitutes a positive regulator of tro-
phoblast amino acid transporters (17, 18). In addition, in
vitro studies have demonstrated that hormones such as in-
sulin, IGF-I, and leptin, which are upstream regulators of
mTOR, stimulate placental transporters for amino acids
(19–22). Thus, placental growth and nutrient transport are
under the regulation of metabolic hormones (23–25). Obe-
sity in pregnancy is associated with perturbed maternal me-
tabolism and circulating hormone levels. For example, obese
pregnant women have higher serum levels of leptin, insulin,
and IL-6 in late pregnancy compared with pregnant women
with normal prepregnancy BMI (26). We recently extended
these observations and reported increased circulating levels
of leptin and insulin already in first trimester among over-
weight and obese women (27). Thus, it is possible that in-
creased levels of maternal hormones such as insulin, leptin,
and IGF-I provide a link between maternal obesity and fetal
overgrowth by up-regulation of placental nutrient transport
capacity. In the current study, we tested the hypothesis that
placental insulin/IGF-I and mTOR signaling is activated and
amino acid transporter activity is increased in large babies of
obese women.
Patients and Methods
Ethical approval
These studies conformed to the standards set by the latest
revision of the Declaration of Helsinki and were approved by the
Committee for Research Ethics at the University of Gothenburg.
Informed consent was obtained from subjects at recruitment.
After obtaining all the relevant clinical information, samples
were coded and deidentified. Some analyses were performed at
the University of Gothenburg, and deidentified samples were
subsequently transferred to the University of Texas Health Sci-
ence Center San Antonio for further studies.
Subjects
Pregnant women with an early pregnancy BMI [weight (ki-
lograms)/height (meters)
2
] ranging from 18.5 to 44.9 kg/m
2
were
enrolled in Gothenburg, Sweden, and placentas were collected
after term delivery. BMI was determined based on length and
weight measurements at the first prenatal visit at 8–12 wk ges-
tation. Estimated date of delivery was determined from the last
menstrual period and confirmed by ultrasound at 16–18 wk
gestation. When a large fetus was suspected based on clinical
signs, repeated ultrasounds were carried out to confirm acceler-
ation of fetal growth. Study subjects were recruited either im-
mediately before delivery (n ⫽7) at the Sahlgrenska University
Hospital or in gestational wk 8–12 at the Lundby Prenatal Care
Center (n ⫽16). Subjects recruited in early pregnancy were part
of a prospective cohort of 49 pregnant women described in detail
106 Jansson et al. Maternal Obesity and Placental Function J Clin Endocrinol Metab, January 2013, 98(1):105–113
elsewhere (27). The 16 subjects from this cohort included in the
current study represented the cases in which the placenta was ob-
tained immediately after delivery and therefore were a random sam-
ple of the larger cohort. The same inclusion and exclusion criteria
were used for all study subjects. The inclusion criteria were Scan-
dinavian heritage, good health, and age of at least 20 yr. The ex-
clusion criteria were smoking, vegetarianism, assisted reproduc-
tion, concurrent disease such as eating disorder, chronic
hypertension and diabetes, and development of pregnancy compli-
cations such as gestational diabetes, preeclampsia, or IUGR.
Preparation of placental homogenates and
syncytiotrophoblast microvillous membranes
Placentas were collected and weighed before trimming of the
cord and membranes. MVM vesicles were prepared as described
previously (8, 28). Briefly, placentas were immediately placed on
ice after delivery and dissected. The chorionic plate, amniotic
sac, and decidua were removed. Approximately 50 g of villous
tissue was cut into small pieces and rinsed with ice-cold physi-
ological saline. Tissue was placed in ice-cold buffer D [250 mM
sucrose, 0.7
Mpepstatin A, 1.6
Mantipain, 80
Maprotinin,
1mMEDTA, 10 mMHEPES-Tris (pH 7.4)] at 4 C and homog-
enized on ice using a polytron (Kinematika AG, Lucerne, Swit-
zerland). The homogenate was snap-frozen in liquid nitrogen
and stored at ⫺80 C until analysis or further processing. To
prepare MVM vesicles, homogenates were thawed on ice and
then centrifuged twice at 10,000 ⫻gfor 15 min, and the resulting
supernatants were combined and centrifuged at 125,000 ⫻gfor
30 min. The pelleted crude membrane fraction was resuspended
in buffer D, and 12 mMMgCl
2
was added. The resulting sus-
pension was subjected to slow stirring on ice for 20 min. Sub-
sequently, the suspension was centrifuged for 10 min at 2500 ⫻
g. The supernatant, which contained the MVM, was centrifuged
two times for 30 min at 125,000 ⫻g. Vesicles were aliquoted,
snap-frozen in liquid nitrogen, and stored at ⫺80 C until use.
MVM enrichment was determined as the MVM/homogenate
ratio of alkaline phosphatase activity, which was assessed using
standard activity assays. Enrichment of alkaline phosphatase ac-
tivity in MVM was 30 ⫾6-fold and was independent of maternal
BMI. Protein content of the homogenates and MVM was deter-
mined by the method of Bradford.
Western blot
Protein expression of total and phosphorylated Akt (Thr-308
or Ser-473), insulin receptor substrate 1 (IRS-1; Tyr-612), AMP-
activated kinase (AMPK
␣
; Thr-172), serum and glucocorticoid-
regulated kinase 1 (SGK1; Ser-422), protein kinase C-
␣
(PKC
␣
;
Ser-657), S6 kinase 1 (S6K1; Thr-389), eukaryotic initiation fac-
tor 4E-binding protein 1 (4E-BP1; Thr-37/46 or Thr-70), and
ribosomal protein S6 (RPS6; Ser-235/236) was analyzed in pla-
cental homogenates. The IRS-1 and PKC
␣
antibodies were pur-
chased from Millipore (Billerica, MA), the SGK1 antibody was
obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and
the remaining antibodies were purchased from Cell Signaling
Technology (Boston, MA). Protein expression of the three sys-
tem A amino acid transporter isoforms SNAT (sodium-depen-
dent neutral amino acid transporter) 1, -2, and -4 was determined
in MVM vesicles using Western blotting. A polyclonal SNAT2
antibody generated in rabbits (29) was a generous gift from Dr.
P. D. Prasad at the University of Georgia. Affinity-purified poly-
clonal anti-SNAT1 (raised against the peptide sequence
VPEDDNISNDSNDFT) and anti-SNAT4 antibodies (raised
against the peptide sequence YGEVEDELLHAYSKV) were gen-
erated in rabbits by Eurogentec (Seraing, Belgium). For negative
controls, the purified antigenic peptide was used in 15-fold ex-
cess to preabsorb antibody overnight at 4 C. Western blotting
was performed as described previously (30). Briefly, total protein
(10–20
g) from placental homogenate/MVM was loaded and
separated on Bis-Tris gels (7–12% acrylamide) and transferred
onto nitrocellulose membranes. Membrane blocking and anti-
body incubations were performed as described in the protocol
provided by the manufacturer. Subsequently, membranes were
incubated with the appropriate secondary peroxidase-labeled
antibodies for 1 h. After washing, bands were visualized using
enhanced chemiluminescence detection reagents (GE Health-
care, Piscataway, NJ). Because protein expression of

-actin in
placental samples was independent of BMI (Supplemental Fig. 1,
published on The Endocrine Society’s Journals Online web site
at http://jcem.endojournals.org), all blots were stripped and re-
probed for

-actin. Analysis of the blots was performed by den-
sitometry using
␣
Imager (Alpha Innotech Corporation, Santa
Clara, CA). To account for variation in loading, the density of the
target band was divided by the corresponding

-actin band. For
each target, all values were expressed in relation to the highest tar-
get/

-actin ratio, which was arbitrarily assigned a value of 1.0.
Measurements of amino acid transporter activity
in MVM
The activity of the amino acid transporter systems A and L was
measured as previously described (8). In brief, MVM vesicles were
preloaded by incubation in 300 mMmannitol and 10 mMHEPES-
Tris (pH 7.4) overnight at 4 C. At time zero, 30
l of vesicles were
rapidly mixed (1:2) with the appropriate incubation buffer con-
taining
14
C-methyl-aminoisobutyric acid (MeAIB; 150
M)or
3
H-
L-leucine (0.375
M) at 37 C. Uptake of radiolabeled substrate was
terminated by the addition of 2 ml of ice-cold PBS after 30 sec
(MeAIB) or 8 sec (leucine) (8). Subsequently, vesicles were rapidly
separated from the substrate medium by filtration on mixed ester
filters (0.45-
m pore size; Millipore Corporation, Billerica, MA)
and washed with 3 ⫻2 ml of PBS. In studies of MeAIB transport,
150 mMNaCl and 150 mMKCl were used in incubation buffers to
assess total and sodium-independent uptake, respectively. In leucine
transport experiments, nonmediated flux was studied in the pres-
ence of 30 mMunlabeled L-leucine.
In all uptake experiments, each condition was studied in trip-
licate for each vesicle preparation. Filters were dissolved in 2 ml
of liquid scintillation fluid and counted, and uptakes were ex-
pressed as picomoles per milligram of protein. Na
⫹
-dependent
uptake of MeAIB (corresponding to system A activity) was cal-
culated by subtracting Na
⫹
-independent from total uptakes.
Mediated leucine uptake, which in isolated MVM almost en-
tirely represents system L activity (31), was calculated by sub-
tracting nonmediated transport from total uptake. Uptakes were
expressed as picomoles per minute per 30 sec (system A) or pi-
comoles per minute per 8 sec (system L).
Data presentation and statistics
Summary data are presented as means ⫾SEM. Variables were
analyzed as continuous across the range of BMI and birth
weights, and linear relationships between variables were deter-
mined using bivariate analysis and Pearson’s correlation coeffi-
cients. A P⬍0.05 value (two-tailed) was considered significant.
J Clin Endocrinol Metab, January 2013, 98(1):105–113 jcem.endojournals.org 107
Results
Clinical data
Table 1 shows selected clinical data for the study sub-
jects, divided into Normal BMI (18.5–24.9 kg/m
2
) and
High BMI (25– 44.9 kg/m
2
) groups according to measure-
ments of body weight and length in early pregnancy. There
were no statistical differences between BMI groups with
regard to gestational weight gain or gestational age. There
was a trend toward higher birth weights and placental
weights in the High BMI group; however, these differences
failed to reach statistical significance. However, when an-
alyzed across the BMI range of all subjects, placental
weights (r ⫽0.43; P⬍0.05) and birth weights (r ⫽0.46;
P⬍0.05) were positively correlated to maternal BMI.
Activity of placental insulin/IGF-I signaling
Placental insulin/IGF-I signaling activity was assessed
by determining phosphorylation of IRS-1 at Tyr-612 and
Akt at Thr-308 (Fig. 1). IRS-1 phosphorylation was pos-
itively correlated to BMI (P⬍0.05; Fig. 1B). In addition,
phosphorylation of both IRS-1 and Akt was positively
correlated to birth weight (P⬍0.01; Fig. 1, C and D).
There was no significant correlation between BMI or birth
weight and total IRS-1 or Akt expression.
Activity of placental AMPK signaling
As shown in Fig. 2, placental AMPK activity, as deter-
mined by Thr-172 phosphorylation, was inversely corre-
lated to maternal BMI (P⬍0.05) and birth weight (P⬍
0.05). There was no significant correlation between BMI
or birth weight and total AMPK expression.
Activity of placental mTOR complex (mTORC)-1
signaling
mTOR is a ubiquitously expressed serine/threonine ki-
nase that exists as two complexes, mTORC1 and -2, with
distinct regulation and function (32). S6K1, RPS6, and
FIG. 1. Placental insulin/IGF-I signaling in relation to BMI and birth
weight. A, Representative Western blots for total and
phosphorylated IRS-1 (Tyr-612) and Akt (Thr-308) in homogenates
of placentas from pregnancies with varying maternal BMI and birth
weights. There was no significant correlation between BMI or birth
weight and total IRS-1 or Akt. B, Relationship between BMI and
phosphorylation of placental IRS-1. C and D, Relationship between
birth weight and phosphorylation of placental IRS-1 (C) or Akt (D).
n⫽17; r ⫽Pearson’s correlation coefficient.
TABLE 1. Selected clinical data
Normal BMI
(18.5–24.9)
High BMI
(25.0–44.9)
n1112
BMI (kg/m
2
) 21.7 ⫾0.6 38.8 ⫾2.3
a
Gestational weight gain (kg) 11.3 ⫾1.1 13.1 ⫾1.4
Gestational age (wk) 40.6 ⫾0.4 40.4 ⫾0.5
Birth weight (g) 3423 ⫾67 3635 ⫾151
Placental weight (g) 599 ⫾45 679 ⫾28
Data are expressed as means ⫾SEM.
a
P⬍0.0001 vs. Normal BMI, Student’s ttest.
FIG. 2. Placental AMPK signaling in relation to BMI and birth weight. A,
Representative Western blots for total and phosphorylated AMPK
␣
(Thr-
172) in homogenates of placentas from pregnancies with varying BMI and
birth weights. There was no significant correlation between BMI or birth
weight and total AMPK
␣
. B and C, Relationship between BMI (B) or birth
weight (C) and phosphorylation of placental AMPK
␣
.n⫽17; r ⫽
Pearson’s correlation coefficient.
108 Jansson et al. Maternal Obesity and Placental Function J Clin Endocrinol Metab, January 2013, 98(1):105–113
4E-BP1 are key downstream targets of mTORC1. Placen-
tal 4E-BP1 phosphorylation (both at Thr-37/46 and Thr-
70) was positively correlated to early pregnancy BMI (P⬍
0.01; Fig. 3, B and C). Phosphorylation of S6K1 (Thr-389;
P⬍0.01), RPS6 (Ser235/236; P⬍0.05), and 4E-BP1 (Thr
37/46, P⬍0.05; and Thr-70, P⬍0.001) was positively
correlated to birth weight (Fig. 3, D–G). There was no
significant correlation between BMI or birth weight and
total S6K1, RPS6, or 4E-BP1 expression.
Activity of placental mTORC2 signaling
mTORC2 phosphorylates Akt (Ser-473), SGK1 (Ser-
422), and PKC
␣
(Ser-657). Phosphorylation of placental
SGK1 was positively correlated to maternal early preg-
FIG. 3. Placental mTORC1 signaling in relation to BMI and birth weight. A, Representative Western blots for total and phosphorylated S6K1 (Thr-
389), 4E-BP1 (Thr-37/46 or Thr-70), and RPS6 (Ser-235/236) in homogenates of placentas from pregnancies with varying BMI and birth weights.
There was no significant correlation between BMI or birth weight and total S6K1, 4E-BP1, or RPS6. B and C, Relationship between BMI and
phosphorylation of placental 4EBP-1 (Thr-37/46) (A) or 4E-BP1 (Thr-70) (B). D–G, Relationship between birth weight and phosphorylation of
placental S6K1 (D), RPS6 (E), 4E-BP1 (Thr-37/46) (F), or 4EBP-1 (Thr-70) (G). n ⫽17; r ⫽Pearson’s correlation coefficient.
J Clin Endocrinol Metab, January 2013, 98(1):105–113 jcem.endojournals.org 109
nancy BMI (P⬍0.05; Fig. 4B). In addition, phosphory-
lation of all three mTORC2 targets was positively corre-
lated to birth weight (Fig. 4, C–E). The expression of total
SGK1 and PKC
␣
was not influenced by maternal BMI or
birth weight.
MVM system A and L transport activity
The activity of system A and system L in MVM isolated
from women with normal BMI and appropriate fetal
growth were similar to what we have reported previously
(8). MVM system A activity in the High BMI group
(69.7 ⫾9.3 pmol/mg ⫻30 sec) was not significantly dif-
ferent from the normal BMI group (57.5 ⫾9.4 pmol/mg ⫻
30 sec). Similarly, MVM system L activity was not altered
in the High BMI group (0.055 ⫾0.013 pmol/mg ⫻8 sec)
compared with the Normal BMI group (0.069 ⫾0.013
pmol/mg ⫻8 sec). However, MVM system A transport
activity (Fig. 5A), but not system L activity (data not
shown), showed a strong positive correlation to birth
weight (r ⫽0.60; P⬍0.01), but not to maternal BMI (data
not shown).
Protein expression of system A amino acid
transporter isoforms in MVM
MVM SNAT1 or SNAT4 protein expression was not
significantly correlated to birth weight or BMI (data not
shown). In contrast, SNAT2 expression was positively
correlated to maternal early pregnancy BMI (P⬍0.05;
Fig. 5C) and birth weight (P⬍0.01; Fig. 5D).
Discussion
To the best of our knowledge, this is the first report to
study placental signaling and amino acid transport in
women with high BMI without pregnancy complications
such as gestational diabetes. We demonstrate that the ac-
tivity of the insulin/IGF-I and mTOR signaling pathways,
system A amino acid transporter activity, and protein ex-
pression of the SNAT2 isoform are increased in placentas
of obese women giving birth to large babies. We propose
that up-regulation of specific placental amino acid trans-
porter isoforms may contribute to fetal overgrowth in
obese women. This effect may be mediated by activation
of insulin and mTOR signaling pathways, which are pos-
itive regulators of placental amino acid transporters.
The microvillous plasma membrane of the syncytiotro-
phoblast, which is bathed in maternal blood, expresses a
number of hormone receptors, including receptors for in-
sulin (33) and IGF-I (34), suggesting that trophoblast
function is regulated by maternal hormones. Maternal cir-
culating IGF-I concentrations are positively correlated to
fetal growth in normal pregnancy (35), and maternal se-
rum concentrations of IGF-I have consistently been shown
to be decreased in IUGR (36). These findings are in agree-
ment with reports of inhibition of placental insulin/IGF-I
signaling in IUGR (37, 38). In addition, fasting insulin is
increased in obese pregnant women (26, 27). These ob-
servations are consistent with the increased phosphoryla-
tion of IRS-1 and Akt that we found in the placenta of high
BMI women giving birth to large babies, indicating acti-
vation of insulin/IGF-I signaling.
mTOR signaling constitutes a master regulator of pro-
tein translation, thereby controlling cell growth and me-
tabolism in response to a large number of upstream reg-
ulators, including growth factors, nutrient, oxygen, and
energy levels (32). We found that both placental mTORC1
and mTORC2 signaling pathways were activated in as-
sociation to high BMI and increased fetal growth. AMPK
FIG. 4. Placental mTORC2 signaling in relation to BMI and birth
weight. A, Representative Western blots for phosphorylated Akt (Ser-
473), total and phosphorylated SGK1 (Ser-422), and PKC
␣
(Ser-657) in
homogenates of placentas from pregnancies with varying BMI and
birth weights. There was no significant correlation between BMI or
birth weight and total SGK1 or PKC
␣
. B, Relationship between BMI
and phosphorylation of placental SGK1. C–E, Relationship between
birth weight and phosphorylation of placental Akt (Ser-473) (C), SGK1
(D), or PKC
␣
(E). n ⫽17; r ⫽Pearson’s correlation coefficient.
110 Jansson et al. Maternal Obesity and Placental Function J Clin Endocrinol Metab, January 2013, 98(1):105–113
is the primary cellular energy sensor and is phosphorylated
at Thr-172 in response to increased AMP/ATP ratio as-
sociated with energy deprivation. In this study we dem-
onstrated that the activity of placental AMPK, which in-
hibits mTORC1, was decreased and that IGF-I/insulin
signaling, which stimulates mTORC1 and -2, was acti-
vated in association to increased BMI and fetal growth.
The observed changes in AMPK and insulin/IGF-I signal-
ing are therefore likely to contribute to mTOR activation
in placentas of obese women giving birth to large babies.
System A is a sodium-dependent transporter mediating
the uptake of nonessential neutral amino acids into the
cell. System A activity establishes the high intracellular
concentration of amino acids like glycine, which is used to
exchange for extracellular essential amino acids via system
L. Thus, system A activity is critical for placental transport
of both nonessential and essential amino acids. System L
is a sodium-independent exchanger mediating cellular up-
take of essential amino acids including leucine. We found
that system A activity is increased in MVM isolated from
large babies of obese women, which may contribute to
increased fetal amino acid availability and fetal growth.
We reported previously that MVM system A activity was
increased in pregnancies complicated by GDM or type 1
diabetes independently of fetal overgrowth. However,
MVM system A activity was unaffected in lean nondia-
betic women giving birth to LGA fe-
tuses (8). This is consistent with the
possibility that obesity and diabetes in
pregnancy have common underlying
metabolic disturbances that can result
in increased placental nutrient trans-
port and fetal growth.
It is well established that IGF-I, in-
sulin, and mTOR signaling stimulate
placental amino acid transport (17–19,
21, 22). The activation of insulin/IGF-I
and mTOR signaling that we observed
in placentas of obese women giving
birth to large babies is likely to contrib-
ute to the observed increase in system A
activity. However, system A amino acid
transporter activity is also regulated by
other signaling pathways, such as leptin,
that we have not directly addressed in the
present study. This is relevant because
leptin stimulates trophoblast system A
amino acid transport in vitro (19) and ma-
ternal leptin levels are elevated in pregnant
women with high BMI (27). All three
known isoforms of system A, SNAT1
(SLC38A1), SNAT2 (SLC38A2), and
SNAT4 (SLC38A4), are expressed in the
placenta (39). The effect of obesity on system A was isoform-
specific because protein expression of SNAT2, but not
SNAT1 and SNAT4, was up-regulated in MVM isolated
from placentas of large babies of obese women. Little is
known with respect to regulation of placental SNATs; how-
ever, SNAT4 appears to be gestationally regulated in the
human placenta (39), and SNAT1 and -2 expression has been
reported to be differentially regulated in response to amino
acid deprivation in BeWo cells (40). Furthermore, placental
IGF-II has been shown to specifically regulate SNAT4 gene
expression in mice (41).
Despite a small sample size, birth weight was positively
correlated to maternal BMI, in agreement with the liter-
ature (1–3). Some of the outcome variables in our study
(phosphorylation of IRS-1, 4EBP-1, AMPK and SGK1,
and SNAT2 protein expression) were significantly corre-
lated to both birth weight and maternal early pregnancy
BMI, whereas others (phosphorylation of Akt, S6K1,
RPS6, and PKC
␣
, and system A activity) were significantly
correlated to birth weight only. This may reflect that fac-
tors unrelated to BMI regulate placental signaling and
amino acid transport and contribute to increased fetal
growth. Alternatively, these findings may be explained by
the small sample size, which is a limitation of our study.
Our results should be confirmed in larger studies, which
FIG. 5. MVM system A activity and SNAT 2 expression in relation to BMI and birth weight. A,
Relationship between birth weight and system A amino acid transport activity was measured
in vitro in MVM isolated from placentas of pregnancies with varying BMI and birth weights
(n ⫽23). B, Representative Western blots for MVM expression of SNAT1, -2, and -4 isoforms
of the system A amino acid transporter. C and D, Relationship between BMI (C) or birth
weight (D) and MVM SNAT 2 expression. n ⫽22; r ⫽Pearson’s correlation coefficient.
J Clin Endocrinol Metab, January 2013, 98(1):105–113 jcem.endojournals.org 111
will have the power to allow multiple regression modeling.
Furthermore, our sample included a wide range of BMIs,
and it cannot be excluded that a few subjects with a BMI
over 40 kg/m
2
may have skewed results in the obese group.
Future studies may therefore benefit from stratifying study
subjects by BMI category. Placental system A and L trans-
port activity in pregnancies complicated by diabetes and
fetal overgrowth has been reported to be different in a
Swedish (8) and a British population (9). This suggests that
there may be ethnic or population differences in the pla-
cental response to maternal metabolic disease. Thus, ex-
ploration of the impact of maternal obesity on placental
signaling and transport in populations other than the one
studied in the current report appears warranted. This in-
formation will increase our understanding of the mecha-
nisms linking maternal obesity to large size at birth and
may facilitate the development of novel intervention strat-
egies to alleviate fetal overgrowth.
Acknowledgments
In memory of Margareta Wennergren (1948–2011), to whom
we are profoundly indebted for her support and inspiration over
many years.
We thank the midwives at Lundby Prenatal Care Center for
helping us to recruit pregnant women to our study and Ellen
Samuelsson and staff at O
¨stra KKO
¨and Mo¨ lndals hospitals who
made it possible for us to collect the placentas.
Address all correspondence and requests for reprints to:
Theresa L. Powell, Ph.D., Center for Pregnancy and Newborn
Research, Department of Obstetrics and Gynecology, Univer-
sity of Texas Health Science Center San Antonio, Mail Code
7836, 7703 Floyd Curl Drive, San Antonio, Texas 78229-
3900. E-mail: powellt3@uthscsa.edu.
This work was supported by grants from the Swedish Re-
search Council (10838 to T.J. and 14555 to T.L.P.) and the
National Institutes of Health (HD68370 to T.J. and DK89989 to
T.L.P.).
Disclosure Summary: The authors declare that there are no
conflicts of interest in relation to this study.
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