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Insulin increases placental triglyceride as a potential mechanism for fetal adiposity in maternal obesity

Objective Maternal obesity increases the incidence of excess adiposity in newborns, resulting in lifelong diabetes risk. Elevated intrauterine fetal adiposity has been attributed to maternal hyperglycemia; however, this hypothesis does not account for the increased adiposity seen in newborns of mothers with obesity who have euglycemia. We aimed to explore the placental response to maternal hyperinsulinemia and the effect of insulin-like growth factor 2 (IGF-2) in promoting fetal adiposity by increasing storage and availability of nutrients to the fetus. Methods We used placental villous explants and isolated trophoblasts from normal weight and obese women to assess the effect of insulin and IGF-2 on triglyceride content and insulin receptor signaling. Stable isotope tracer methods were used ex vivo to determine effect of hormone treatment on de novo lipogenesis (DNL), fatty acid uptake, fatty acid oxidation, and esterification in the placenta. Results Here we show that placentae from euglycemic women with normal weight and obesity both have abundant insulin receptor. Placental depth and triglyceride were greater in women with obesity compared with normal weight women. In syncytialized placental trophoblasts and villous explants, insulin and IGF-2 activate insulin receptor, induce expression of lipogenic transcription factor SREBP-1 (sterol regulatory element-binding protein 1), and stimulate triglyceride accumulation. We demonstrate elevated triglyceride is attributable to increased esterification of fatty acids, without contribution from DNL and without an acceleration of fatty acid uptake. Conclusions Our work reveals that obesity-driven aberrations in maternal metabolism, such as hyperinsulinemia, alter placental metabolism in euglycemic conditions, and may explain the higher prevalence of excess adiposity in the newborns of obese women.


INTRODUCTION
Maternal obesity is common. Almost 40% of reproductive-age women in the United States have obesity and more than 20% of reproductiveage women will have obesity globally by 2025 [1]. Maternal obesity is associated with excess adiposity in newborns [2e6]. Excess neonatal adiposity is a risk factor for the development of obesity and type 2 diabetes (T2DM) in childhood and adulthood [7e12]. While there is a clear relationship between higher maternal pre-pregnancy weight and obesity in offspring, the links between a dysfunctional intrauterine environment and excess neonatal adiposity remain poorly understood. Studying how maternal obesity alters the intrauterine environment may be key to understanding the mechanisms underlying the intergenerational transmission of obesity and diabetes. Insulin is an anabolic hormone that drives the storage of excess energy, mainly triglyceride (TG), in white adipose tissue. Six decades ago, large newborns were documented in women with insulin deficiency or type 1 diabetes (T1DM). To explain this phenomenon, Pedersen hypothesized that high levels of maternal glucose pass through the placenta to the fetus and stimulate the fetal pancreas to secrete more insulin [13]. With the influx of glucose and fetal insulin, the fetal liver converts glucose to TG by de novo lipogenesis (DNL), and TG accumulates in fetal adipose. This glucose-centric model is successfully applied in pregnant women with T1DM, T2DM and gestational diabetes, in whom glucose-lowering techniques prevent neonatal overgrowth and excess adiposity [14]. However, this model has several weaknesses and does not adequately address excess neonatal adiposity in offspring of obese women who have normoglycemia or only intermittent mild hyperglycemia. It discounts any role of maternal insulin, which increases in the third trimester of pregnancy and is further elevated in obesity [15]. Additionally, DNL is minimal in the fetal liver, and fetal mitochondrial capacity for fatty acid oxidation is limited [16,17]. Thus, lipids are more likely to provide substrate for fetal fat accretion than glucose. TG levels increase with each trimester of 1 pregnancy, with normative ranges of 40e159 mg/dL in the first trimester, 75e382 mg/dL in the second trimester, and 131e453 mg/ dL in the third trimester [18]. By the third trimester when fetal fat accretion is at its highest, maternal TG levels increase by two-fold [19]. Finally, the Pedersen hypothesis interprets the placenta as a passive player in nutrient flux, ignoring the placenta's capability to regulate nutrient metabolism and to respond to maternal and fetal signals. A myriad of maternal and placental metabolic signals is present throughout pregnancy, including insulin, insulin-like growth factor 2 (IGF-2), and insulin-like growth factor 1 (IGF-1). The effect of maternal insulin on the placenta is poorly defined since most glucose uptake is through insulin-independent glucose transporters (GLUT) and there are conflicting reports on the location of insulin receptor in the placenta [20]. Maternal IGF-1 has been shown to stimulate fetal growth by increasing the transfer of nutrients to the fetus acting through placental IGF-1 receptor, and is secreted by the cytotrophoblast and syncytiotrophoblast at all stages in gestation [21,22]. IGF-2 is a paracrine hormone secreted by placental syncytiotrophoblasts and encoded by an imprinted gene of paternal origin [23]. Since IGF-2 regulates placenta size and fetal growth, in part by controlling nutrient flux to the fetus, and activates placental IGF-1 receptor and the mitogenic insulin receptor A isoform (IR-A) [24e26], it is an ideal hormone to study alongside insulin and IGF-1. Placental metabolism remains poorly understood, due to challenges posed by different placentation in animal models. Here we use placental villous explants and isolated trophoblasts from normal weight and obese women as models to investigate how the placental response to maternal hyperinsulinemia may promote fetal adiposity by increasing storage and availability of nutrients to the fetus. We examined the effect of insulin and IGF-2 on triglyceride content ex vivo in conjunction with stable isotope and radioisotope techniques to investigate placental lipid metabolism. Our study reveals that insulin and IGF-2 stimulate triglyceride accumulation ex vivo via esterification, and not DNL. These results favor a role of maternal fatty acids and triglyceride over glucose for energy storage in the intrauterine environment.

Study design
We studied two cohorts of pregnant women. In the first cohort, we studied de-identified clinical data and placenta tissue from women enrolled in a pregnancy study, through the Yale University Reproductive Sciences (YURS) Biobank. Collection was approved by the Yale University Human Investigations Committee. We enrolled women into two groups: normal weight (NW, BMI 18.5e24.9 kg/m 2 ) and obese (defined as class 1 obesity, OB, BMI 30e35 kg/m 2 ), as determined from pre-pregnancy BMI. Gestational diabetes (GDM) screening was determined using the two-step approach, in which a 1-h oral glucose tolerance test with 50 g glucose was performed, and if glucose was above the threshold (!135 mg/dL), diagnosis was made per Carpenter and Coustan criteria using a 3-h oral glucose tolerance test (100 g oral glucose solution) [27]. All women included in the study had a negative screening test. Inclusion criteria included delivery by scheduled cesarean section (CS) and gestational age of 38.5e39.9 weeks. Exclusion criteria included any trial of labor prior to CS, history of prepregnancy Type 1 or Type 2 diabetes mellitus, use of glucoselowering or lipid-lowering therapy, hypertension, or pre-eclampsia during pregnancy. The study population for this cohort comprised of 28 NW women of mean age 33.8 AE 0.8 years and mean BMI 22.0 AE 0.3 kg/m 2 and 19 OB women of mean age 32.1 AE 1 years and mean BMI 31.9 AE 0.4 kg/m 2 . In a second cohort, we evaluated maternal levels of hormones and nutrients in NW (n ¼ 9) and OB women (n ¼ 5). Fasting maternal serum was obtained at the third trimester of pregnancy and was drawn on the day of scheduled CS. Clinical data were collected on maternal age, BMI, race/ethnicity, gravidity/parity, and pregnancy complications. Neonatal data included gestational age, gender, birth weight, neonatal hypoglycemia, congenital abnormalities, and medical conditions diagnosed in first 28 days after birth.
2.2. Isolation and culture of placental explants and primary trophoblasts Placentae were processed within 30 min of delivery and placed on ice. Placentae were weighed with the umbilical cord and membranes attached using a digital scale. Placental weight was recorded in grams. Placental thickness was recorded in units of 0.1 cm by piercing the chorionic plate with a wooden toothpick and measuring against a ruler. Chorionic membranes and maternal decidua layers were removed. Placenta were placed in RNAlaterÒ (RL) or flash frozen (FF), then stored at À80 C. For ex vivo experiments including RNA expression analysis, full thickness placental samples from multiple cotyledons were obtained while avoiding calcified or hypoperfused cotyledons. Large villous tissue samples were dissected into small 50 mg (5 mm) pieces and rinsed in cold phosphate buffered saline (PBS). Then, w400 mg of placental villous explants was transferred to a six-well plate containing Dulbecco's Modified Eagles Medium (DMEM) (Life Technologies, Carlsbad, CA) with 5.5 mM glucose, 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% amphotericin B, and incubated at 37 C with 5% CO 2 e95% O 2 , as modified from prior studies [28,29]. For ex vivo and in vitro experiments, the glucose level in the culture media was 5.5 mM (equivalent to 99 mg/dL), which was intentionally selected to reflect normoglycemic conditions in pregnancy. For in vitro experiments, cytotrophoblasts were isolated using the protocol described by Lee et al. [30]. Syncytiotrophoblasts were generated by culturing cytotrophoblasts for 72 h, leading to spontaneous differentiation as initially described by Kliman et al. [31].

Hormone treatment
After 24-h incubation, placental villous explants were starved in serum-free, phenol-free DMEM (with 5.5 mM glucose) for 4 h at 37 C. Placental explants were treated with insulin (100 nM), IGF-2 (100 nM), IGF-1 (100 nM), or vehicle in phenol-free DMEM containing 10% charcoal-stripped serum and 5.5 mM glucose. As determined by mass spectrometry, the treatment medium contained 3 mg/dL triglyceride and 10 mmol/L nonesterified fatty acids. After 30 min, 6 h, or 48 h of hormone stimulation, placental villous explants were collected for triglyceride, protein, or mRNA quantification and analysis. In addition, placental explants from NW women (n ¼ 8e9) were cultured in media containing different levels of glucose (5.5 mM [99 mg/dL], 10 mM [180 mg/dL] or 25 mM [450 mg/dL]) for 24 h, starved, and treated with insulin (100 nM), IGF-2 (100 nM), or vehicle. Separately, a group of placental explants from NW women (n ¼ 7) and OB women (n ¼ 6) were cultured for 24 h, starved, and then treated for 48 h with 0.1 nM, 1 nM, 10 nM, or 100 nM of insulin, or vehicle to assess tissue response to insulin stimulation.

RNA isolation and qRT-PCR analysis
For whole placenta tissues, tissue was homogenized in TRIzolÒ (Life Technologies). RNA was isolated with chloroform, precipitated with isopropanol, washed twice with 75% ethanol, and dissolved in RNasefree water. RNA was treated with RNase-free DNase I and purified via Original Article RNeasy spin columns. For in vitro studies, RNA was extracted using the RNeasyÒ Mini Kit (Qiagen, Valencia, CA). The purity and concentration of the isolated RNA was assessed using a NanodropÔ 2000 Spectrophotometer (Thermo Scientific). RNA was reverse transcribed using the iScriptÔ cDNA Synthesis Kit (Bio-Rad). For each sample, 12.5 ng of cDNA template was amplified in triplicate using iQÔ SYBRÒ Green Supermix on the MyiQÔ Real-Time PCR Detection System (Bio-Rad). PCR primers were designed using Primer Blast (http://www.ncbi.nlm. nih.gov/tools/primer-blast, NCBI, Bethesda, MD), and synthesized at W.M. Keck Foundation Oligo Synthesis Resource (Yale University, New Haven, CT). Optimal primer concentrations were determined by calculation of primer efficiency using Human XpressRefÔ Universal Total RNA (Qiagen), and primer specificity was assessed by melting curve analysis. Supplementary Table 1 summarizes the primer sequences, concentrations, and efficiency for each assay. Each qRT-PCR assay included non-amplification and water controls. Gene expression levels were normalized to b-actin and b-actin levels remained stable across treatment groups.

Protein extraction
Protein was isolated from TRIzolÒ-chloroform fraction following RNA isolation. Protein was isolated with ethanol, precipitated with isopropanol, and washed with 0.3 M guanidine in 95% ethanol. The protein pellets were re-dissolved by sonication in 1:1 solution of 1% SDS and 8 M urea, containing PIC and PMSF. Insoluble material was separated from soluble protein by centrifugation. The protein concentration in each lysate was quantified using the PierceÒ Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Scientific) and the Bio-Rad iMark Microplate Absorbance Reader.

Western blot analysis
Proteins were separated on 4e12% SDS-PAGE gels (Bio-Rad), transferred onto AmershamÔ HybondÔ polyvinylidene fluoride membranes (GE Healthcare), incubated with antibodies, and developed using AmershamÔ ECL Advance (GE Healthcare). Immunoprecipitation for Western blot analysis of phosphorylated IR was performed using Dynabeads Protein A (Invitrogen, Thermo Fisher Scientific, Waltham, MA) for cell lysate pre-clearing, IR total for formation of immunocomplex, and then isolation of the immunocomplex with magnetic bead slurry. Antibodies to phosphorylated and/or total forms of AKT, b-Actin, and GAPDH were from Cell Signaling Technology (Danvers, MA), as previously cited [32]. Antibodies to phosphorylated and total forms of S6, were obtained from Cell Signaling Technology (Danvers, MA). Antibodies to the total form of IR-b C-terminal, SREBP-1, PPARg, and GPAM (GPAT1) were obtained from Santa Cruz Biotechnology (Dallas, TX). Antibodies to DGAT1 and DGAT2 were obtained from Abcam (Waltham, MA). Antibodies to GPAT3 were obtained from Invitrogen e Thermo Fisher Scientific (Waltham, MA) (Supplementary Table 2). Densitometry was performed for immunoblots using ImageJ software, version 1.44 (National Institutes of Health, Bethesda, Maryland). Densitometry was performed for immunoblots using ImageJ software, version 1.44 (National Institutes of Health, Bethesda, Maryland).

Immunohistochemistry
Placenta tissue specimens were fixed in 10% neutral buffered formalin for at least 1 day and embedded in paraffin, after which 5-mm sections were placed on coated glass slides designed for immunohistochemistry (IHC) processing. Hematoxylin and eosin-stained slides were also produced for histological examination of each placenta. Normal rabbit serum (R9133, used at 1 mg IgG/mL; SigmaeAldrich) was used as a negative control. Placenta tissue specimens from 11 NW women and 6 OB women were blindly screened with anti-insulin receptor antibody, and subsequently, one representative from each group was selected for performing additional markers. Antibodies and concentrations used are listed in Supplementary Table 3. Serial sections (5 microns each) were immunohistochemically stained using EnVisionþ horseradish peroxidase Rabbit DABþ (K4011; Dako Agilent, Santa Clara, CA) to mark the sites of antibody binding with a brown deposit as previously described by Kliman et al. [33]. Antibody concentrations were chosen to produce strong staining in the positive cellular structures without background staining.
2.8. Triglyceride assay Total tissue lipid was extracted from 150 to 200 mg placental explant tissue with chloroform:methanol [2:1 volume-to-volume ratio (v/v)] using a modified Bligh and Dyer method [34]. Following a 48h hormone treatment, placental villous tissue was homogenized in chloroform:methanol using a bead mill homogenizer (Tissue Lyser, Qiagen, Germantown, MD). Phase separation was achieved by adding sulfuric acid, and the organic layer was retrieved and dried under a nitrogen stream. Triglyceride content was quantified using the Sekisui Triglyceride-SL Kit (Burlington, MA) and a spectrophotometer (Bio-Rad SmartspecÔ Plus, Hercules, CA) in duplicate. Explant triglyceride content was normalized to explant weight. De novo lipogenesis measurement. Placental villous explants (w400 mg) were transferred to a six-well plate and incubated in DMEM with 10% FBS, 5.5 mM glucose, 1% penicillin/streptomycin, and 1% amphotericin B, and incubated at 37 C with 5% CO 2 e95% O 2 for 24 h. Next, placental villous explants were starved in serum-free, phenol-free DMEM supplemented with 5.5 mM glucose for 4 h at 37 C. Placental explants were treated with insulin (100 nM), IGF-2 (100 nM), IGF-1 (100 nM), or vehicle in phenol-free DMEM supplemented with 5.5 mM glucose and enriched with 5% deuterium ( 2 H 2 O, 99.9%, Cambridge Isotope Laboratories, Tewksbury, MA) and 10% charcoalstripped serum. The 5% 2 H 2 O-enriched media was changed every 12 h for the duration of the 48-h hormone treatments. Placental explants were harvested on dry ice and then stored at À80 C. Aliquots of media were saved from the final time point for all samples. Triglycerides were extracted into chloroform as described above. Fatty acid stable isotope enrichment by GCeMS. Explant lipid samples were spotted onto Silica gel 60 plates, and thin layer chromatography (TLC) was performed with a mobile phase of hexane:diethyl ether:acetic acid (80:20:1). Plates were developed with 0.005% primuline in acetone:water (80:20). Purified samples were collected while absorbed to Silica and eluted with diethyl ester. Triglyceride-fatty acids were analyzed by GCeMS (5975CI, Agilent Technologies, Santa Clara, CA, USA) as fatty acid methyl esters following derivatization with methanolic boron trifluoride.

Plasma deuterium enrichment
The plasma 2 H 2 O pool was assessed by exchange of hydrogens from plasma to acetone in the presence of sodium hydroxide; acetone deuterium enrichment was analyzed by GCeMS.

Calculation of % DNL
The calculation of de novo palmitate synthesis from isotopic data has been previously described [35e37]. The fraction of the placental explant palmitate pool newly synthesized during the experiment (F) was calculated as F ¼ ME O (N Â p), where molar enrichment (ME) equals m1 þ (2 Â m2), where m1 and m2 are the atom percent enrichments (APEs) of singly and doubly deuterium-labeled palmitate; N is the number of exchangeable hydrogen atoms in a palmitate molecule, and p is the plasma APE of deuterium in water.

Measurement of triglyceride synthesis by fatty acid esterification
Triglyceride biosynthesis by esterification of preformed fatty acids was assessed by incorporation of 13 C-16 palmitate into placental villous explant triglyceride. Potassium U-13 C-16 palmitate (Cambridge Isotope Laboratories, Tewksbury, MA) was conjugated to bovine serum albumin (BSA), in a 7% BSA/5 mM 13 C-16 palmitate (w5:1 molar ratio) solution following a previously published protocol [38]. Placental explants were incubated in 5.5 mM glucose DMEM with 10% FBS for 24 h, then starved in serum-free, phenol-free 5.5 mM glucose DMEM for 4 h at 37 C. The explants were placed in phenol-free DMEM (5.5 mM glucose) labeled with 0.1 mM palmitate and treated with insulin (100 nM), IGF-2 (100 nM), or vehicle for 0 (no tracer), 1, 2, 4, and 6 h. Aliquots of media were saved at the end of each time point, and explant tissue was weighed at the end of each experiment and flash frozen. Triglycerides were extracted into chloroform as described above and explant lipid samples were analyzed as indicated in 'Fatty acid stable isotope enrichment by GCeMS.' 2.10. Fatty acid uptake assay in placental explants Fatty acid uptake in placental explants was performed as previously described with the following modifications [39,40]. Briefly, six-well plates containing placental explants were incubated in 5.5 mM glucose DMEM. Then, 1 mL assay buffer containing 200 mM palmitate, 20 mM BSA, 0.5 mCi/mL 1-14 C palmitic acid (Perkin Elmer) and hormone treatment (100 nM vehicle, insulin, or IGF-2) were added to each well and the explants were incubated for nine different timepoints, ranging from 5 min to 24 h at 37 C. Assays were stopped by adding ice cold stop buffer (500 mM phloretin and 0.1% BSA in PBS) and washing the placental explants three times. The villous explants were suspended in water, kept on ice, and homogenized using a bead mill homogenizer (Tissue Lyser, Qiagen, Germantown, MD). Then, tissue homogenate was transferred into scintillation vials and analyzed by scintillation counting. All assays were corrected for the tissue weight per assay.

Fatty acid oxidation assay in placental explants
Ex vivo placental fatty acid oxidation experiments were performed as previously described with minor modifications [41,42]. For each placenta, there were 3e4 experimental replicates which were representative of at least 2 technical replicates per condition. Briefly, fresh placental villous explants were transferred to glass vials containing 2 mL oxygenated KrebseRinger bicarbonate buffer (KRBB) supplemented with 2.6% NaHCO 3 (w/v), 100 mM CaCl 2 (w/v), and 0.5% fatty acid free BSA (w/v), pH 7.4 for 1 h at 35 C, 70 RPM. Following incubation, explants were transferred to vials containing 1.5 mL of the above media supplemented with 100 mM sodium palmitate, 200 mM carnitine, and 0.2 mCi/mL [1-14 C] palmitic acid [Perkin Elmer, Waltham, MA]. Explants were then treated with 100 mM Etomoxir, 100 nM insulin, 100 nM IGF2 or vehicle control and incubated for 2 h at 35 C, 70 RPM. Etomoxir is a commonly used inhibitor of fatty acid oxidation through its irreversible inhibition of carnitine palmitoyl-transferase 1a (CPT1a) and was used as a positive control, since fatty acid oxidation is expected to decrease with etomoxir. Following incubation, placental tissue was removed, blotted (to remove excess media), weighed and snap frozen in liquid nitrogen. Vials were subsequently tightly capped and media was acidified with 0.5 mL of 2 N HCl. 14 CO 2 was trapped in Eppendorf tubes containing 0.3 mL 2 N NaOH for 2 h at 40 C, 120 RPM. The 14 CO 2 absorbed in the NaOH solution was quantified by liquid scintillation counting. Rates of fatty acid oxidation were determined as a function of 14 CO 2 produced over time and normalized to explant weight.

Statistical analysis
Triglyceride content was normalized to tissue/explant weight and analyzed by log transformation and mixed effects analysis with Dunnett's multiple comparisons test, using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Protein densitometry data was normalized to total protein, b-actin, or GAPDH. Gene expression was normalized to b-actin. Data was log-transformed, analyzed by one-way ANOVA or by two-tailed Student's t-test, and represented as fold-change per vehicle. Significance was defined as P < 0.05.

Study approval
De-identified clinical data and placenta tissue from women was collected through the Yale University Reproductive Sciences (YURS) Biobank. Collection was approved by the Yale University Human Investigations Committee (approval number 2000023300). For the second cohort, collection of maternal serum and cord blood was approved by the Yale University Human Investigations Committee (approval number 1301011387). Written informed consent was obtained from all women prior to participation.

Obesity increases placental depth independently of changes in placental weight
To evaluate the effect of insulin on placental lipid metabolism, we recruited a cohort of normal weight (NW, n ¼ 28) and obese (OB, n ¼ 19) women without history of diabetes, undergoing elective cesarean section. Mean maternal age, maternal race/ethnicity, parity, and gestational age at delivery were similar between the two groups ( Table 1). Per study design, the pre-pregnancy BMI and BMI at delivery were significantly higher in the OB group compared to the NW group (P < 0.0001 for both, Table 1). All glucose levels at 1-h during oral glucose tolerance testing were in euglycemic range, and mean glucose was not significantly different between NW and OB women (Table 1). In  (Figure 1). All women in the NW and OB groups had serum TG levels within the expected laboratory range for the 3rd trimester. Finally, birthweight and frequency of neonatal hypoglycemia did not differ in the offspring of NW and OB women (3.5 AE 0.1 vs 3.6 AE 0.1 kg, 0% vs 11%, P ¼ NS) (Table 1). Demographic information about the study cohort is summarized in Table 1. Placenta depth was significantly higher in the OB group (2.2 AE 0.1 vs 2.6 AE 0.1 cm, P < 0.05), but there were no differences in placenta weight between the groups (691 AE 29 vs 739 AE 44 g, P ¼ NS, respectively) (Figure 2AeB). Since IGF-2 directly affects placental size and fetal growth by controlling placental supply of, and genetically driven demand for maternal nutrients [24e26], we examined placental mRNA expression of IGF-2 and IGF2-R. Although we found that placenta depth was significantly increased in the OB group, this was not associated with differences in IGF-2 or IGF-2R in the placentae of NW and OB women (0.675 AE 0.07 vs 0.651 AE 0.07, 0.029 AE 0.009 vs 0.034 AE 0.009, respectively, P ¼ NS) (Figure 2CeD).

Insulin receptor abundance did not differ between NW and OB placentae
To assess placental receptor content, we evaluated it at a morphological level by immunohistochemistry, and quantitated its abundance by qPCR and immunoblotting. Immunohistochemistry was performed in a subset of whole placental tissue from NW (n ¼ 11) and OB (n ¼ 6) women, and the slides were evaluated in a blinded fashion. The trophoblast villous tree is the functional unit of the placenta and is covered by multinucleated syncytiotrophoblasts, which are derived from fused cytotrophoblasts. The hormone-producing epithelium of the placenta, the syncytiotrophoblast, is the main barrier for movement of nutrients from the maternal to fetal circulation. Staining of term placentae from NW and OB women with anti-insulin receptor antibody revealed uniform and abundant syncytiotrophoblast surface staining (Figure 3AeD). Notably, the trophoblast layer appeared considerably thicker in placentae from OB women compared to those from NW women. To distinguish which trophoblast cell type was enlarged, placentae were stained for syncytiotrophoblast and cytotrophoblast specific markers. Staining with human placental lactogen (hPL), a hormone made only in syncytiotrophoblasts, demonstrated a markedly thickened syncytiotrophoblast layer in the placenta from OB women, compared to that from NW women (Figure 3EeF). Conversely, antivestigial like family member 1 (VGLL1), a marker specific for cytotrophoblasts, showed a similar amount of staining in placentae from both groups, confirming that the syncytiotrophoblast layer is hypertrophied in placentae from obese women. Western blot analysis for total insulin receptor (IR) protein in NW and OB placentae did not reveal differences in IR protein levels ( Figure 3I). The two splice variant isoforms of IR are the mitogenic IR-A and the metabolic IR-B, which are expressed with variable abundance in tissues. To evaluate the relative abundance of IR isoforms, we quantified mRNA expression and found a 60:40 ratio of IR-A to IR-B in placenta. We did not find significant differences in the mean mRNA expression of mitogenic IR-A, metabolic IR-B, and insulin-like growth factor 1 receptor (IGF1-R) in term placentae between NW and OB women (Figure 3JeL). In placentae from fasting NW and OB women, phosphorylated IR was not detected.

Women with obesity have higher placental triglyceride content
We observed that mean TG levels were 1.6-fold higher in placentae from OB women, compared with NW women (9.4 AE 0.6 vs 5.7 AE 0.5 mg/mg tissue, P ¼ 0.0001, Figure 4A). Furthermore, we found a strong positive correlation between maternal BMI (kg/m 2 ) and placental TG content (r 2 ¼ 0.53, P ¼ 0.0002) ( Supplementary Fig. 1). As such, placental TG content is a potential link between maternal obesity and dysfunctional placental metabolism.

Insulin and IGF-2 stimulate triglyceride accumulation in placental villous explants
We investigated the effect of different glucose levels on placental TG content in NW women ex vivo. We found that 25 mM glucose (equivalent of 450 mg/dL) was associated with increased TG content in both vehicle and insulin-treated placental explants ( Figure 4B). Since OB women had significantly higher serum insulin levels and placental TG content compared to NW women, we sought to determine whether insulin regulates placental TG metabolism. While insulin has equal affinity for IR-A and IR-B isoforms, we also assessed the effect of IGF-2 because of its known physiological role in the placenta and its high affinity for IR-A and minimal binding to IR-B [43]. After 48-h hormone treatment in normoglycemic conditions (5.5 mM glucose), we found that insulin increased TG content by 2.5-fold in villous explants from NW women, relative to vehicle (20.6 AE 1.6 vs 8.1 AE 0.7 mg/mg tissue, P < 0.0001), indicating that maternal insulin regulates placental TG accumulation ( Figure 4C). Similarly, in OB women, insulin stimulated a 2-fold increase in placental TG content, relative to vehicle (17.9 AE 2.2 vs 9.1 AE 1.3 mg/mg tissue, P < 0.0001) ( Figure 4D). Interestingly, IGF-

Placental sensitivity to insulin is preserved in OB women
During pregnancy, the placenta acts as an endocrine organ and reprograms maternal physiology to an insulin-resistant state to provide adequate nutrients for fetal growth [44]. Insulin resistance occurs in maternal liver, muscle and adipose tissue as a result of placental hormone action, and shunts metabolic substrates toward the placenta and fetus [45]. In women with obesity, insulin resistance is present prior to conception and may compound pregnancy-induced insulin resistance. Importantly, it is unclear whether the placenta becomes resistant to the action of maternal insulin in women with obesityinduced insulin resistance. To assess tissue response to insulin stimulation, we treated placental explants from NW and OB women with a dose curve of insulin (0.1 nMe100 nM) or control for 48 h. We found that TG content peaked with 1 nM insulin treatment of ex vivo   Figure 4E).

Insulin receptor signaling and mRNA expression in syncytialized trophoblasts
We next sought to identify whether insulin or IGF-2 (as a paracrine factor that activates IR) increases TG content in the placenta by activating insulin signaling pathways in primary, syncytialized trophoblasts isolated from the placenta of NW women. We found that acute stimulation with insulin significantly increased phosphorylation of IR (P < 0.01) and AKT (P ¼ 0.0005), relative to vehicle, but no changes were seen in phosphorylated S6 in the mTOR pathway. Similarly, acute stimulation with IGF-2 resulted in increased phosphorylation of IR (P < 0.05) and AKT (P < 0.01), relative to vehicle, albeit less robustly compared to insulin stimulation (Figure 5AeB). SREBP-1 (sterol regulatory elementbinding protein 1), an insulin responsive transcription factor, induces the expression of genes involved in glucose utilization and fatty acid synthesis. Chronic stimulation of placental trophoblasts with either insulin (P < 0.05) or IGF-2 (P < 0.005) significantly increased levels of the active, cleaved form of SREBP-1 protein. In addition, while chronic insulin stimulation resulted in increased PPARg (peroxisome proliferatoractivated receptor g) protein levels (P < 0.05), IGF-2 had no significant effect ( Figure 5CeD). Also, at 48-h, mRNA expression of SREBF1, the gene encoding SREBP-1, was increased by insulin (P < 0.05), but no significant differences were seen in the genes encoding enzymes downstream of SREBP-1, such as acetyl Co-A carboxylase (ACACA) and fatty acid synthase (FASN) ( Figure 5E).

Insulin does not alter fatty acid uptake or fatty acid oxidation in placental explants
Maternal TG carried in lipoproteins are hydrolyzed to non-esterified free fatty acids (NEFA) at the placental microvillous membrane by endothelial lipase (EL) or lipoprotein lipase (LPL) [46]. NEFA are thought to enter cells by multiple mechanisms, including passive diffusion into the placental syncytium, and facilitated transport by fatty acid transporter proteins (FATP1-6) or the fatty acid translocase CD36 [47,48]. We evaluated the effect of insulin and IGF-2 on fatty acid uptake in placental villous explants. Notably, neither insulin nor IGF-2 treatment resulted in significant increases in palmitate uptake, compared with vehicle treated controls ( Figure 6A). Fatty acid (FA) oxidation in the placenta may also affect lipid delivery to the fetus. In FA oxidation assays of fresh placental villous explants, we observed similar amounts of FA oxidation with vehicle, insulin, or IGF-2 treatment ( Figure 6B).
3.8. Insulin and IGF-2 stimulate esterification, but not de novo lipogenesis in the placenta Glucose and other small molecules are converted to fatty acids (de novo lipogenesis) in the postabsorptive state, and the relationship between lipogenic gene expression and insulin is well-established to be mediated by the transcription factor SREBP-1c. On the other hand, esterification of fatty acids to form triglycerides is primarily substratedependent in the liver, with insulin's role in transcription of lipogenic enzymes [49]. To gain insight into the mechanism by which insulin and IGF-2 increase TG content in placental explants, we assessed insulin's effect on rates of DNL and esterification under normoglycemic conditions in explants from NW women. DNL was assessed by incorporation of deuterium from deuterated water-enriched media into palmitate from triglyceride. Over the course of a 48-h study, essentially no deuterium was incorporated into palmitate, and calculated rates of lipogenesis were effectively zero, ranging from 0 to 0.6% ( Figure 6C). In the same placental villous explants used to measure DNL, TG significantly increased with insulin, IGF-2, and IGF-1 treatment, relative to vehicle, confirming that TG accumulated over the same period of time despite negligible rates of lipogenesis ( Figure 6D). In preliminary dose range studies with small groups of ex vivo placental explants, we did not find any increase in DNL in explants treated with insulin (100 nM), IGF-2 (100 nM) or vehicle in the presence of varying glucose levels (10 mM or 25 mM). In addition, we did not detect DNL with additional substrates, such as fructose, glycerol, lactate, or pyruvate in our ex vivo model (data not shown). Next, we assessed whether insulin stimulates esterification in placental villous explants. We found that over time, insulin (P < 0.0005) or IGF-2 (P < 0.0001) treatment of ex vivo placental explants led to significant increases in TG accumulation, relative to vehicle ( Figure 6E). The accelerated rate of TG accumulation in ex vivo placental explants treated with insulin or IGF-2 was Original Article 8 proportional to increased rates of TG production by esterification of fatty acids taken from the media free fatty acid pool. (r 2 ¼ 0.6; P ¼ 0.0005) ( Figure 6F).

Abundance of esterification-catalyzing enzymes is not affected by insulin or IGF-2 in vitro
Since insulin appears to drive TG synthesis via esterification of free fatty acids from the culture media in placental villous explants, we sought to determine whether insulin increased the quantity of enzymes catalyzing the process of fatty acid esterification to glycerol-3phosphate. We did not detect differences in the ubiquitous or mitochondrial enzymes for glycerolipid synthesis, such as GPAT and GPAM (glycerol-3-phosphate acyltransferase) in syncytialized trophoblasts treated with insulin or IGF-2 for 48-h ( Supplementary Fig. 4). DGAT1 and DGAT2 (diacylglycerol O-acyltransferase) enzymes were also not significantly different between treatment groups.

DISCUSSION
Evidence is accumulating that the intrauterine environment is a source of transgenerational programming of metabolic disorders, including obesity and T2DM [3,4,6,10]. Indeed, the placenta may mediate the adverse effects of maternal obesity on fetal development and neonatal adiposity.
Our study demonstrates that insulin regulates lipid metabolism and storage in the placenta, thus raising the potential that maternal hyperinsulinemia may contribute to excess neonatal adiposity (Figure 7). We report that insulin and IGF-2 increase placental TG content via esterification, without contribution from FA uptake or de novo lipogenesis, or changes in FA oxidation under euglycemic conditions in an ex vivo model. From our findings, we propose that the placenta has a much larger role than previously thought in determining substrate flux and nutrient availability for the fetus. Importantly, maternal glucose does not need to be elevated for hyperinsulinemia to sequester lipid in the placenta. (E) SREBF1, ACACA and FASN mRNA expression in primary trophoblasts isolated from NW women (n ¼ 9). Data are mean AE SEM. ***P ¼ 0.005, **P < 0.01, *P < 0.05, relative to vehicle by one-way ANOVA.
We found that pathophysiological levels of glucose results in higher placenta triglyceride, in the presence or absence of insulin, consistent with the Pedersen Hypothesis. However, women with gestational diabetes or even T2DM, rarely have glucose levels approaching 25 nM (450 ng/dL). Instead, clinical studies have found that maternal obesity and lipid levels correlate with excess neonatal fat deposition, particularly liver fat [11,50]. At this time, in vivo studies have not been done to determine whether placenta triglyceride is used directly for fetal nutrient supply and storage. Future studies are needed to assess placental ability to hydrolyze stored triglyceride and provide non-esterified fatty acids (NEFA) to the fetal circulation. Multiple prior investigations suggest this may be an important mechanism by which fetal NEFA supply is regulated. Perazzolo et al. conducted a modeling study that suggests the slow delivery of fatty acids from the maternal circulation to the fetal circulation can be explained at the level of placenta triglyceride assembly [51]. It has been reasoned for years that one explanation for the preferential transfer of polyunsaturated fatty acids to the fetal circulation is due to a placenta esterification step [52].
Our finding that the insulin receptor abundantly lines the microvillous membrane of the syncytiotrophoblast in term placenta is critical to understanding a physiological role for maternal insulin. Since insulin does not cross the placenta [53,54], our study of insulin action using a villous explant model, in which tissue architecture is maintained, further supports receptor localization to the microvillus membrane. Prior findings that insulin receptor expression at term localizes only to placental endothelial cells and fetoplacental vessels [55,56] may be attributable to the use of different antibodies and variations in immunohistochemistry techniques. In our study, the severity of maternal hyperinsulinemia and degree of excess TG accumulation in placentae from women with obesity are consistent with reports by other investigators [15,29,57]. All women in the obese and normalweight groups had serum TG levels within the expected laboratory range for the 3rd trimester, supporting our assertion that insulin's role in placental TG accumulation is not dependent on pathophysiological levels of maternal triglyceride. Importantly, we show that placental sensitivity to insulin is intact in women with obesity with respect to insulin-induced TG synthesis. Placental insulin sensitivity is preserved despite a peak of systemic insulin resistance in the third trimester of pregnancy and background of obesity-induced insulin resistance in peripheral organs. Other investigators also found that the placenta maintains insulin-responsiveness as characterized by AKT signaling activity and amino acid transport [58]. In sum, these experimental findings suggest maternal obesity does not protect the placenta from the effects of hyperinsulinemia, and this may have implications for placental metabolism and function earlier in pregnancy as well.
Notably, we found that IGF-2 also robustly increased placental TG content in our ex vivo model. Placental IGF-2 plays a role in placental size, nutrient delivery, and fetal growth [24,25]. In our study, placental IGF-2 and IGF-2R expression were similar between women with normal weight and obesity, and placental TG correlated significantly with maternal body mass index. In animal models, diet-induced increases in maternal adiposity were associated with changes in placental IGF2 expression independently of methylation, suggesting the role of maternal obesity in transgenerational metabolic programming [59].
Our key findings suggest that insulin and IGF-2 drive TG assembly by shifting intracellular fatty acid flux toward esterification and increasing levels of transcription factors involved in esterification pathways, such as SREBP-1 and PPARg. Our results are consistent with an in vitro study showing placental prioritization of esterification over b-oxidation for fatty acid metabolism, leading to greater lipid droplet formation in trophoblasts [60]. Another study localized lipid droplets, using perilipin-2, within the syncytiotrophoblast layer of term placentae from women with obesity [29]. In a study tracking the movement of a fluorescently labeled long-chain fatty acid analog in placental explants, all cell layers took up long-chain fatty acids [61]. However, in the absence of insulin, investigators found that these fatty acids were rapidly esterified and incorporated into lipid droplets primarily in the cytotrophoblast layer. Insulin may alter the relative metabolic activities of syncytiotrophoblasts and cytotrophoblasts. Nutrient status may also alter differentiation of cytotrophoblasts to syncytiotrophoblasts [60]. On a functional level, the re-esterification of maternal fatty acids into placental TG storage could be either protective or detrimental for the fetus in the setting of maternal obesity. To determine the balance between buffer and reservoir in the setting of obesity, more work is needed to trace placental TG export and lipolysis for fatty acid transport to the fetal circulation. DNL occurred at negligible rates in our euglycemic ex vivo model, a finding which favors a role of maternal fatty acids and triglycerides over glucose for energy storage in the intrauterine environment. However, the placenta expresses enzymes for DNL, such as FASN. FASN and related enzymes may play a role in placental lipid biosynthesis under different conditions or toward a different cellular function. For example, fatty acids synthesized de novo may be funneled toward the synthesis of other molecules containing fatty acid moieties, such as those specialized for signaling, rather than large quantities of energystoring TG. We also found that neither insulin nor IGF-2 significantly affected fatty acid uptake ex vivo, and other reports also suggest that fatty acid transporters in the placenta may not be insulin sensitive [62,63]. In addition, a study using orally ingested stable isotope-labeled fatty acids and computational modeling, materno-fetal NEFA transfer was equal between normal weight and obese women, yet slow in the fasting state [64], indicating that NEFA uptake is highly regulated and not a process of passive diffusion as once thought [65]. Another study provided some evidence that regulation of specific fatty acids may differ, when they found equal enrichment of 13 C-palmitic acid in placentae from women with GDM and women with normoglycemia, yet impaired uptake of 13 C-docosahexaenoic acid in women with GDM [66].
In an unexpected finding, we observed thicker placenta depth and a hypertrophied syncytiotrophoblast layer in term placentae from women with obesity. This finding suggests that obesity may induce changes in placental structure, which in turn, affects placental function. Further investigation is needed to define structural markers of obesity-driven pathophysiology, since gross metrics such as placenta weight are affected by collection techniques and may lack specificity. Strengths of our study include the use of multiple molecular and physiological methods to investigate placental lipid metabolism, and the use of placental tissue from a well-characterized cohort of women. Our conclusions are confined by the limitations of an ex vivo placenta model and a smaller number of biological replicates for assessing the abundance of esterification-catalyzing enzymes. Despite its limitations, the ex vivo placental perfusion model maintains placental function and allows direct assessment of physiology [67], and can be used to support future in vivo studies of maternalefetal placental lipid metabolism in pregnant women.

Conclusions
In conclusion, we propose that elevated maternal insulin and an abundance of placental insulin receptor have consequences for placental lipid metabolism, even in the setting of normoglycemia. Fatty acids are an efficient substrate for TG synthesis and storage in fetal adipose tissue. Our findings suggest that the obesogenic intrauterine environment is characterized by hyperinsulinemia and may impact the transgenerational programming of metabolic disease.

AUTHOR CONTRIBUTIONS
AKA, KMC, DFV, and CAF conceived and designed the studies, interpreted results, and wrote the manuscript. SMG and HST contributed with key insights to study design. JVO and LEP recruited participants and collected clinical data and placentae. AKA, KMC, MBD performed experiments, analyzed data, and interpreted results. JJH contributed clinical data and samples.
LG and HJK contributed to interpreting results. All authors edited and approved the manuscript.

FUNDING
This project was supported by grants from a McKern Scholar Award, the Eunice Kennedy Shriver NICHD K08HD071010 and R01 HD097368, and Yale Diabetes Research Center pilot award from the National Institute of Diabetes and Digestive and Kidney Diseases P30 DK45735 to CAF, R01 DK124272 to DFV, K99 HL150234 to LG, R01 DK113984, R01 DK119968, P30 DK045735 to GIS, and T32 DK007058 that supported AKA.

DATA AVAILABILITY
Data will be made available on request.

ACKNOWLEDGMENTS
We thank our participants for placenta donation, and the Yale University Reproductive Sciences Biobank for the ethical and coordinated biospecimen collection. We thank our longstanding collaborator and mentor Dr. Gerald Shulman (Yale School of Medicine), who shared experimental infrastructure that was critical for the completion of this study.