Docosahexaenoic Acid Supplementation in Pregnancy Modulates Placental Cellular Signaling and Nutrient Transport Capacity in Obese Women

Abstract

Context:

Maternal obesity in pregnancy has profound impacts on maternal metabolism and promotes placental nutrient transport, which may contribute to fetal overgrowth in these pregnancies. The fatty acid docosahexaenoic acid (DHA) has bioactive properties that may improve outcomes in obese pregnant women by modulating placental function.

Objective:

To determine the effects of DHA supplementation in obese pregnant women on maternal metabolism and placental function.

Design:

Pregnant women were supplemented with DHA or placebo. Maternal fasting blood was collected at 26 and 36 weeks’ gestation, and placentas were collected at term.

Setting:

Academic health care institution.

Subjects:

Thirty-eight pregnant women with pregravid body mass index ≥30 kg/m².

Intervention:

DHA (800 mg, algal oil) or placebo (corn/soy oil) daily from 26 weeks to term.

Main Outcomes:

DHA content of maternal erythrocyte and placental membranes, maternal fasting blood glucose, cytokines, metabolic hormones, and circulating lipids were determined. Insulin, mTOR, and inflammatory signaling were assessed in placental homogenates, and nutrient transport capacity was determined in isolated syncytiotrophoblast plasma membranes.

Results:

DHA supplementation increased erythrocyte (P < 0.0001) and placental membrane DHA levels (P < 0.0001) but did not influence maternal inflammatory status, insulin sensitivity, or lipids. DHA supplementation decreased placental inflammation, amino acid transporter expression, and activity (P < 0.01) and increased placental protein expression of fatty acid transporting protein 4 (P < 0.05).

Conclusions:

Maternal DHA supplementation in pregnancy decreases placental inflammation and differentially modulates placental nutrient transport capacity and may mitigate adverse effects of maternal obesity on placental function.

Obese women supplemented with 800 mg/d DHA compared with placebo show changes in placental inflammation and nutrient transport related to DHA status of the placenta.

As populations in developed countries have become progressively heavier, an increasing number of women are obese when they enter pregnancy (1–3). Pregravid obesity is associated with an increased risk of adverse pregnancy complications, including gestational diabetes mellitus (GDM), preeclampsia, delivery by caesarean section, and fetal overgrowth (4, 5). The consequences of pregravid obesity are not limited to pregnancy alone because children of high body mass index (BMI) mothers are at increased risk for metabolic and cardiovascular disease, mental health disorders, and some types of cancer (6–9). Ample evidence suggests that changes in maternal nutrition/metabolism alter fetal development and growth mediated by modulation of placental function (10, 11).

Fetal overgrowth in obese women is associated with increased placental macronutrient transport capacity. Specifically, amino acid, fatty acid, and glucose transporter activity/expression have been reported to be increased in isolated syncytiotrophoblast plasma membranes in obese women (12–14). Obese, pregnant women are characterized by endocrine and metabolic perturbations, including insulin resistance, hyperlipidemia, and systemic inflammation (15–18). Such metabolic perturbations may affect placental function because the placenta expresses multiple cytokine and hormone receptors on the maternally facing microvillous membrane (MVM), which interacts with maternal signals. Recent studies have shown that maternal obesity increases placental inflammation, oxidative stress, and both mechanistic target of rapamycin (mTOR) and signal transducer and activator of transcription (STAT) signaling (12, 16–19). Some of these factors are established regulators of placental macronutrient transport capacity in vitro (20–26).

Given the dramatic increase in the number of pregnancies complicated by obesity, there is an urgent need to develop intervention strategies to break the vicious cycle of intrauterine transmission of metabolic and cardiovascular disease from mother to the next generation. Docosahexaenoic acid (DHA) supplementation during pregnancy reduces circulating and placental inflammatory markers in overweight/obese women (27). In addition, in vitro experiments suggest that DHA has a potential for reducing adverse effects of maternal obesity on the placental function (28–31). For instance, DHA attenuates mTOR signaling and reduces amino acid transport in cultured primary human trophoblast cells (32), which may help mitigate the activation of placental mTOR and amino acid transport in obese women delivering large babies (12).

Multiple studies have investigated the effects of maternal supplementation with DHA/fish oil during pregnancy on maternal and child outcomes (33). However, few studies have explored the effects of DHA supplementation on the placenta in human pregnancy (27, 34–37). Apart from a recent report suggesting alterations in placental fatty acid metabolism in DHA-supplemented mothers (37), the effects of maternal DHA supplementation on placental nutrient transport capacity and cellular signaling in obese women are largely unknown. Therefore, we tested the hypothesis that DHA supplementation in the second half of pregnancy in obese women modulates placental cellular signaling (insulin, inflammation, and mTOR) and nutrient transport capacity.

We determined circulatory, inflammatory, and metabolic markers at enrollment (26 weeks’ gestation) and after 10 weeks of DHA supplementation in a cohort of obese pregnant women. At term delivery, placental membrane DHA levels were assessed and analysis was performed on the relationship between placental DHA levels and cellular signaling and expression and activity of amino acid, glucose, and fatty acid transport proteins.

Methods

Ethics

Informed, written consent was obtained from all participating women after careful explanation of the study. The study protocol was approved by the Institutional Review Board at the University of Texas Health Science Center, San Antonio (IRB HCS20090506H).

Study participants

Obese women (pregravid BMI ≥30 kg/m²; n = 51) were enrolled at 25 to 29 weeks’ gestation (mean, 26.6 weeks) and randomized to either placebo (corn/soy oil) or DHA (800 mg/d) supplementation (DSM Nutritional Products, Heerlen, The Netherlands) for the remainder of their pregnancy. Inclusion and exclusion criteria are presented in Supplemental Table 1 ^{(35.9KB, docx)} . Fasting maternal blood samples were collected at enrollment and at 36 weeks’ gestation from 38 women who completed the study. All women were also seen at 30 weeks’ gestation for a compliance check. Placentas were collected from 35 subjects who delivered at term (≥37 weeks’ gestation). Thirteen participants were not included in the maternal circulatory factor analysis (nine women developed GDM, and four women chose to not complete the study), and three participants were not included in the placenta analysis study (one woman delivered preterm, one placenta was not collected at delivery, and one woman declined placenta collection). Hence, the final number of participants was 38 for the maternal blood analysis and 35 for the placental analysis [n = 31 for MVM and basal plasma membrane (BM) parameters, due to membranes not successfully isolated from four placentas]. Data were obtained from the participants’ medical records, including medications taken during pregnancy, medical diagnoses, gestational age at delivery, and weight, length, and head circumference of the neonate.

Measurement of fatty acids

Fatty acid content (expressed as a percentage of total phospholipid fatty acids) was measured in maternal erythrocytes and total placental membrane preparations by gas chromatography/mass spectrometry (GC/MS), using well-established protocols (38). Briefly, samples were saponified with methanolic sodium hydroxide and the fatty acids converted to methyl esters using boron trifluoride in methanol. After addition of saturated sodium chloride solution and hexane, the hexane fraction was transferred into a vial that contained sodium sulfate for drying, then transferred to a second vial for analysis by GC/MS on a Thermo Fisher DSQ using electron impact ionization. Fatty acid identification was made by comparison of the electron ionization mass spectra and GC retention times with those of known standards. Peak areas were determined by manual integration using the DSQ Xcalibur software.

Maternal blood collection and analysis

At enrollment and at 36 weeks’ gestation, fasting maternal venous blood samples were obtained and processed immediately for glucose and lipid analysis by standard clinical assays. In addition, erythrocytes and plasma were collected. Fatty acid composition was measured in erythrocyte membranes. Maternal cytokine and hormone levels [insulin, leptin, total adiponectin, interleukin-6 (IL-6), and tumor necrosis factor α] were determined by enzyme-linked immunosorbent assay according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Homeostasis model assessment of insulin resistance was calculated according to the following equation: [insulin (mIU/L) × glucose (mg/dL)] / 405 (39).

Placenta collection

Placentas were obtained immediately after delivery, and dissections were carried out on ice. After removing the decidua basalis and chorionic plate, ∼100 g of villous tissue was rinsed in ice-cold physiological saline. The villous tissue was transferred to ice-cold buffer D (250 mM sucrose, 10 mM Hepes, pH 7.4) containing protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO), then homogenized on ice. The placental homogenates were frozen in liquid nitrogen and stored at −80°C until processed further. Separate aliquots were used for isolation of syncytiotrophoblast plasma membranes, protein expression analysis, and isolation of nuclei.

Isolation of syncytiotrophoblast plasma membranes

The isolation of syncytiotrophoblast BM and MVM was performed according to a previously described protocol (40). All procedures were performed on ice. After an initial isolation of all cellular membranes (used for determining placental membrane fatty acid levels), the MVM was purified by magnesium chloride precipitation, and BM was purified by sucrose gradient centrifugation. Alkaline phosphatase activity was, on average, almost 18-fold greater in isolated MVM vesicles, and ferroportin expression was >30-fold higher in BM vesicles compared with activity/expression in placental homogenates. There were no differences in enrichment in DHA- vs placebo-treated groups.

Western blotting

Western blot was performed as previously described (41). Commercially obtained primary antibodies used are listed in Supplemental Table 2 ^{(35.9KB, docx)} . The l-type amino acid transporter (LAT) 1 and LAT2 antibodies were the kind gifts of Dr. Y. Kanai (Osaka University, Japan), and the sodium-dependent neutral amino acid transporter (SNAT) 2 antibody was from Dr. P. D. Prasad (University of Georgia). The SNAT4 antibody was generated by Eurogentec (peptide sequence: YGEVEDELLHAYSKV; Seraing, Belgium).

Nuclear factor–κB p65 and peroxisome proliferator-activated receptor γ activity

Nuclei were isolated from homogenates of placental villous tissue using a nuclear isolation kit (Active Motif, Carlsbad, CA). DNA binding activity of nuclear factor–κB (NF-κB) p65 (20 µg) and peroxisome proliferator-activated receptor γ (PPARγ) (10 µg) was measured in nuclear extracts with transcription factor assay kits (Active Motif, Carlsbad, CA). Each sample was measured in duplicate.

Amino acid and glucose transporter activities

System A and system L amino acid transporter activities in MVM vesicles, as well as glucose transporter activity in BM and MVM vesicles, were assessed using radioisotope-labeled substrates and protocols described in detail previously (12, 13). Each condition was studied in triplicate (amino acid uptakes) or quadruplicate (glucose uptakes) for all the uptake experiments.

Lipase activity

Placental lipase activity in MVM vesicles was measured with a commercial kit (LPL activity assay; Roar Biomedicals, Inc., New York, NY), and 20 μg of MVM vesicles was incubated with the fluorescent substrate solution for 60 minutes according to the manufacturer’s instructions.

Oxidative stress

Placental free radical and hydrogen peroxide levels and total antioxidant capacity were measured by a commercially available kit (Oxiselect; Cell Laboratories Inc., San Diego, CA).

Data presentation and statistics

Differences between DHA-supplemented and placebo control groups were evaluated by t test. Spearman correlation was used to identify associations between all continuous variables. To evaluate relationships between key variables, including DHA status (maternal erythrocyte DHA content, placental membrane DHA content, and treatment assignment), variables were grouped into nine nonredundant major categories (presented in Table 1). For each category the first principal component of the scaled variables was computed to capture the major axis of variation. These first principal components were correlated to each of the key variables and outcomes using the t test for discrete factors (treatment assignment), with Spearman correlation for the continuous outcomes (placental membrane DHA content). We applied the Benjamini-Hochberg false discovery rate adjustment to account for multiple testing. All tests were two-sided, and P values <0.05 and false discovery rate values <0.2 were considered significant. Statistical calculations were performed in R v2.18 (Vienna, Austria) or GraphPad Prism 5 (version 5.04; GraphPad Software, La Jolla, CA).

Table 1.

Correlations Between Placental Membrane DHA Levels and Groups of Placental Factors (Principal Component Analysis)

Characteristic	Variables Included	Spearman R Value	P Value	FDR
Amino acid transport	System A activity, system L activity, SNAT1, SNAT2, SNAT4, LAT1, LAT2	−0.511	0.003	0.02
Fatty acid transport	Lipase activity, FAT/CD36, FATP2, FATP4	0.432	0.02	0.05
Fetal factors	Birth weight, head circumference, length, placental weight, ponderal index	−0.077	0.66	0.74
Glucose transport	Glucose uptake, GLUT1, GLUT9	0.257	0.16	0.24
Inflammation	COX-2, GPR120 (MVM), IκBα, JNK, NF-κB p65 activation, p38α	−0.585	0.0002	0.002
Insulin signaling	AKT, ERK, FOXO 1/3, GRB2, GSK3α, insulin receptor β, IRS1, PI3K p85	0.256	0.14	0.24
mTOR signaling	4EBP1, AMPK, mTOR, REDD1, rpS6	0.201	0.25	0.32
Oxidative stress	Free radicals, H2O2, total antioxidant capacity	−0.040	0.82	0.82
STAT signaling	SOCS3, STAT3, STAT5	0.289	0.09	0.21

Abbreviations: 4EBP1, eukaryotic translation initiation factor 4E-binding protein 1; AMPK, AMP-activated protein kinase; COX-2, cyclooxygenase 2; ERK, extracellular signal-regulated kinase; FAT, fatty acid translocase; FATP, fatty acid transport protein; FDR, false discovery rate; FOXO 1/3, forkhead box protein 1/3; GLUT, glucose transporter; GRB2, growth factor receptor-binding protein 2; GSK3, glycogen synthase kinase 3; IRS, insulin receptor substrate; PI3K, phosphatidylinositide 3-kinase; REDD1, regulated in development and DNA damage responses 1; rpS6, ribosomal protein S6; SOCS3, suppressor of cytokine signaling 3.

Results

Clinical characteristics

Maternal age, prepregnancy BMI, and erythrocyte DHA levels at enrollment did not differ between the placebo (n = 19) and DHA-supplemented (n = 19) groups (Table 2). After 10 weeks of supplementation, maternal erythrocyte DHA levels were significantly higher in the DHA-supplemented group (P < 0.0001; Table 2). Fetal growth (measured by birth weight, length, ponderal index, and head circumference) did not differ between the placebo and DHA-supplemented groups (Table 2). In both groups, there were more male than female fetuses. Placental weight tended to be heavier in the DHA-supplemented group (P = 0.07; Table 2).

Table 2.

Clinical Characteristics of Study Subjects

Characteristic	Placebo	DHA Supplemented	P Value
Mother
n	19	19
Age (y)	28.5 ± 1.2	27.8 ± 1.2	0.72
BMI (kg/m2)	32.9 ± 1.0	31.5 ± 1.1	0.35
Ethnicity (Hispanic/not Hispanic), n	16/3	18/1	0.60
RBC DHA (%), baseline	5.8 ± 0.5	6.0 ± 0.5	0.82
RBC DHA (%), 36 weeks	6.1 ± 0.5	10.0 ± 0.6	<0.0001
Gestational length (d)	275.4 ± 2.2	277.9 ± 1.7	0.37
Delivery mode (caesarean section/vaginal), n	6/13	11/8	0.19
Newborn
n	19	19
Sex (female/male), n	6/13	6/13	>0.99
Birth weight (g)	3503 ± 94	3452 ± 94	0.70
Length (cm)	51.1 ± 0.5	51.2 ± 0.4	0.87
Ponderal index (g × 100/cm3)	2.62 ± 0.04	2.57 ± 0.05	0.46
Head circumference (cm)	34.7 ± 0.2	34.6 ± 0.3	0.69
Placenta
n	17	18
Weight (g)	708 ± 26	791 ± 35	0.07
Membrane DHA (%)	6.8 ± 0.5	9.8 ± 0.9	0.006
Membrane AA (%)	38.4 ± 1.0	34.9 ± 1.0	0.02
Fetal/placental weight ratio	5.1 ± 0.2	4.5 ± 0.1	0.01

Data are presented as mean ± standard error of the mean unless otherwise noted. Differences between groups were evaluated by t test or Fisher’s exact test.

Abbreviations: AA, arachidonic acid; RBC, red blood cell.

Maternal circulatory factors

Maternal circulating levels of adiponectin, glucose, IL-6, insulin, leptin, and tumor necrosis factor α did not differ between the two groups either at the time of enrollment or after 10 weeks of supplementation (Supplemental Table 3 ^{(35.9KB, docx)} ). Similarly, the homeostasis model assessment of insulin resistance index was not affected by DHA supplementation (Supplemental Table 3 ^{(35.9KB, docx)} ). At enrollment, maternal levels of cholesterol, high-density lipoprotein, and low-density lipoprotein were similar between the two groups. However, triglyceride and very low-density lipoprotein levels at the start of the study were significantly higher in the DHA-supplemented group (P < 0.05; Supplemental Table 3 ^{(35.9KB, docx)} ). This was not anticipated and is likely due to small sample sizes. Importantly, after 10 weeks of supplementation, there was no major difference in any of the maternal lipid levels between the two groups (Supplemental Table 3 ^{(35.9KB, docx)} ).

Placental membrane DHA

Placental membrane DHA levels were significantly higher in the DHA-supplemented group compared with the placebo group (P < 0.01; Table 2); placental membrane DHA levels correlated with maternal erythrocyte DHA levels (P < 0.05; Fig. 1). Analysis of the outcomes by treatment group (t test, DHA- vs placebo-supplemented women) indicated no statistically significant differences in placental signaling or transport between the two groups (data not shown).

Figure 1.

Placental membrane DHA level correlates with maternal red blood cell (RBC) DHA levels. Maternal RBC DHA levels were measured at 36 weeks’ gestation and placental membrane DHA levels at term. Spearman r = 0.372, P < 0.05, best-fit line, n = 35.

DHA status is dependent on several factors, including initial levels, compliance with study protocol in taking supplements, diet, genetics, and other variations in lipid metabolism. Therefore, we found large variation in the DHA levels in both the placebo and supplemented groups at 36 weeks. It has been suggested that it is more appropriate to report outcomes of nutritional supplementation studies based on acquired nutritional status (i.e., DHA levels at study completion) rather than by group assignment (42). Therefore, in our secondary analysis, fetal growth, placental cellular signaling, and transport capacity were analyzed by correlation with the continuous variable placental DHA levels at delivery, irrespective of supplementation group assignment.

Fetal growth

Birth weight, length, ponderal index, and head circumference did not correlate with placental membrane DHA levels (Supplemental Table 4 ^{(35.9KB, docx)} ). Principal component analysis (PCA) confirmed the lack of association between fetal growth parameters and DHA levels in the placental membranes (Table 1).

Inflammatory signals

The PCA indicated an inverse association between placental inflammation and DHA levels in the placental membranes (P < 0.001, Table 1). The inflammatory signaling pathways in placental homogenates included expression of IκBα, which was inversely correlated (P < 0.01; Table 3), and NF-κB p65 DNA binding activity, which tended to be positively correlated (P = 0.055; Table 3) with placental membrane DHA levels. Furthermore, there was a trend toward an inverse correlation between placental membrane DHA levels and p38α mitogen-activated protein kinase (MAPK) phosphorylation (P = 0.059; Table 3) and a significant inverse correlation with total p38 MAPK expression (P < 0.05; Table 3). The expression of cyclooxygenase 2 correlated positively with placental membrane DHA levels (P < 0.01; Table 3). There was no significant association between placental membrane DHA levels and JNK expression or phosphorylation, or MVM expression of GPR120 (Supplemental Table 4 ^{(35.9KB, docx)} ).

Table 3.

Correlations Between Placental Membrane DHA Levels and Placental Cellular Signaling and Nutrient Transport Capacity

Characteristic	Spearman R Value	P Value	FDR
Amino acid transporter expression
LAT1 (MVM)	−0.636	0.0001	0.01
Inflammation
IκBα	−0.488	0.003	0.04
COX-2	0.463	0.01	0.05
p38α (total)	−0.389	0.02	0.10
NF-κB p65 activation	0.328	0.05	0.17
p38α (phospho; T180/Y182)	−0.322	0.06	0.17
Insulin signaling
IRS1 (total)	−0.404	0.02	0.10
GRB2	−0.385	0.02	0.10
AMPK (total)	−0.359	0.03	0.13
ERK (total)	0.342	0.04	0.16
AKT (total)	0.310	0.07	0.19
STAT signaling
STAT5 (total)	−0.525	0.001	0.02
mTOR signaling
mTOR (total)	−0.403	0.02	0.10

Abbreviations: AMPK, AMP-activated protein kinase; COX-2, cyclooxygenase 2; ERK, extracellular signal-regulated kinase; FDR, false discovery rate; IRS1, insulin receptor substrate 1.

Insulin signaling

Insulin signaling has been implicated as a key regulator of amino acid transport activity in trophoblast cells. To assess insulin signaling, we studied the protein expression of several insulin pathway intermediates. Expression of insulin receptor substrate 1 (P < 0.05; Table 3) and growth factor receptor-binding protein 2 (GRB2) (P < 0.05; Table 3) was inversely correlated with placental membrane DHA levels. There was a positive correlation between extracellular signal-regulated kinase 1/2 expression and placental membrane DHA levels (P < 0.05; Table 3), and AKT expression tended toward a positive association with placental membrane DHA levels (P = 0.07; Table 3). AMP-activated protein kinase expression was negatively associated with placental membrane DHA levels (P < 0.05; Table 3). Phosphorylation of AMP-activated protein kinase, AKT, extracellular signal-regulated kinase 1/2, forkhead box protein 1/3, glycogen synthase kinase 3, and insulin receptor substrate 1 and expression of glycogen synthase kinase and phosphatidylinositide 3-kinase p85 were not related to placental membrane DHA levels (Supplemental Table 4 ^{(35.9KB, docx)} ). Expression of insulin receptor β in isolated MVM and BM vesicles did not correlate with placental membrane DHA levels (Supplemental Table 4 ^{(35.9KB, docx)} ). PCA indicated a lack of association between insulin signaling and placental membrane DHA levels (Table 1).

mTOR signaling

There was an inverse correlation between mTOR expression and membrane DHA levels in the placentas (P < 0.05; Table 3). Components of the mTOR signaling pathway (eukaryotic translation initiation factor 4E-binding protein 1, regulated in development and DNA damage responses 1, and ribosomal protein S6) showed no association between expression/phosphorylation and placental membrane DHA levels (Supplemental Table 4 ^{(35.9KB, docx)} ). No association between mTOR signaling and placental membrane DHA levels was found with PCA (Table 1).

Oxidative stress

Indicators of oxidative stress (free radicals and hydrogen peroxide levels) and total antioxidant capacity were not associated with placental membrane DHA levels (Table 1 and Supplemental Table 4 ^{(35.9KB, docx)} ).

PPARγ activity

PPARγ DNA binding activity did not correlate with placental membrane DHA levels (Supplemental Table 4 ^{(35.9KB, docx)} ).

STAT signaling

STAT5 expression in placental homogenates correlated inversely with placental membrane DHA levels (P < 0.01; Table 3). There was no association between DHA levels in the placental membranes and expression of suppressor of cytokine signaling 3, phosphorylation of STAT3 or STAT5, or total STAT3 (Supplemental Table 4 ^{(35.9KB, docx)} ). The PCA analysis revealed no association between STAT signaling and placental membrane DHA levels (Table 1).

Amino acid transport

The PCA suggested an inverse association between placental amino acid transport capacity and placental membrane DHA levels (P = 0.003, Table 1). System A amino acid transporter activity in the isolated MVM inversely correlated with placental membrane DHA levels [P < 0.05; Fig. 2(a)]. Similarly, MVM system L activity [P < 0.001; Fig. 2(b)] and the MVM expression of the system L transporter LAT1 (P < 0.0001; Table 3) were also inversely correlated with placental membrane DHA levels. MVM expression of the system A transporters SNAT1, SNAT2, and SNAT4, as well as BM expression of the system L transporter LAT2, did not correlate with placental membrane DHA levels (Supplemental Table 4 ^{(35.9KB, docx)} ).

Figure 2.

Higher placental DHA levels are associated with reduced placental amino acid transport. In isolated MVM vesicles, (a) system A (R = −0.413, P < 0.05, false discovery rate < 0.099) and (b) system L (R = −0.576, P < 0.001, false discovery rate < 0.02) amino acid transport capacities were inversely correlated with placental membrane DHA levels. Spearman, best-fit line, n = 31.

Fatty acid transport

Placental capacity to transport fatty acids correlated with placental membrane DHA levels (PCA, P = 0.02, Table 1). MVM expression of fatty acid transport protein (FATP) 4 [P < 0.01; Fig. 3(a)] and BM FATP4 expression [P = 0.059; Fig. 3(b)] correlated positively with placental membrane DHA levels. Expression of CD36/fatty acid translocase, FATP2, and lipase activity were, however, not related to placental membrane DHA levels (Supplemental Table 4 ^{(35.9KB, docx)} ).

Figure 3.

Placental membrane DHA levels and fatty acid and glucose transporter expression. Expression of FATP4 in isolated (a) MVM vesicles [R = 0.488, P < 0.01, false discovery rate (FDR) < 0.05] and (b) BM vesicles (R = 0.343, P = 0.059, FDR < 0.18) correlated positively with placental membrane DHA levels. Expression of glucose transporter (c) GLUT1 (R = 0.434, P < 0.05, FDR < 0.096) and (d) GLUT9 (R = −0.396, P < 0.05, FDR < 0.11) in isolated BM vesicles correlated with placental membrane DHA levels. Spearman, best-fit line, n = 31.

Glucose transport

In the BM, expression of glucose transporter (GLUT) 1 correlated positively with placental membrane DHA levels [P < 0.05; Fig. 3(c)], whereas GLUT9 BM expression was inversely correlated [P < 0.05; Fig. 3(d)]. MVM expression of GLUT1 and GLUT9, as well as glucose uptake in either plasma membrane, was not related to placental membrane DHA levels (Supplemental Table 4 ^{(35.9KB, docx)} ). The PCA analysis suggests no association between placental glucose transport capacity and placental membrane DHA levels (Table 1).

Discussion

To our knowledge, this is the first detailed report on the effects of DHA supplementation in obese pregnant women on placental functions such as amino acid, glucose, and fatty acid transport capacity. We demonstrate that increasing maternal DHA results in higher placental membrane DHA content. Moreover, the increase in placental membrane DHA was associated with a reduction in placental amino acid transport and inflammatory markers, along with an increase in fatty acid transport capacity. We propose that DHA supplementation in obese women with low DHA intake may mitigate some of the adverse effects of obesity on placental function by decreasing placental inflammation and modulating the composition of nutrients transported to the fetus. Furthermore, some of these effects may represent direct cause-and-effect relationships because we have previously reported similar responses to physiological levels of DHA in cultured human trophoblast cells (32).

As expected, DHA supplementation resulted in higher maternal erythrocyte DHA levels. Obesity in pregnancy without diagnosis of GDM has been suggested to result in a metabolic profile of chronic inflammation, insulin resistance, and hyperlipidemia (43). The ω-3 long-chain polyunsaturated fatty acids have been reported as beneficial for metabolic syndrome disorders such as dyslipidemia and inflammation (44). However, in general agreement with a previous study (27), DHA supplementation in the second half of pregnancy did not affect maternal circulating inflammatory or metabolic markers or insulin sensitivity. At enrollment, triglyceride and very-low-density lipoprotein levels were significantly higher in the group assigned to DHA supplementation. This difference was inadvertent and likely due to the relatively low number of women enrolled in this study. Importantly, however, these differences had disappeared following 10 weeks of DHA supplementation, suggesting that DHA, if anything, may improve maternal lipid profiles.

With the lack of significant change in maternal metabolic parameters, it was not surprising that a groupwise comparison of placental variables resulted in no significant changes. Maternal DHA supplementation significantly increased placental membrane DHA levels, confirming previously reported findings (34, 35). Together, these observations strongly suggest that maternal intake of DHA is reflected in the placental membrane DHA content. This is further supported by the positive correlation between maternal and placental DHA levels. Analyzing cellular signaling and functional characteristics based on the placental membrane DHA content is important because DHA status depends on not only DHA intake but also other factors, such as diet and genetic variation related to lipid metabolism (45, 46). Supplementation studies, such as ours, are further complicated by variable compliance and potentially modified dietary intake in both the placebo and supplemented groups. Indeed, the overall outcome of our study at 36 weeks was a continuum of maternal erythrocyte DHA levels rather than a clear distinction between the two groups (as shown in Fig. 1). Therefore, as in a previous study correlating Na⁺K⁺ATPase activity to cell membrane DHA levels (47), we conducted correlation analyses to determine the impact of variation in placental DHA levels on functional transport capacity and cellular signaling. This study provides a comprehensive evaluation of placental cellular signaling and nutrient transport capacity in a cohort of obese women with a variation in placental DHA content.

In this study, placental membrane DHA content was measured in samples containing a pool of all membranes, rather than in the isolated syncytiotrophoblast cellular membranes. This approach was selected due to the low yield of purified BM, which restricted what studies could be performed. Another limitation in the current study is that the effect of DHA supplementation on fetal endothelial cells has not been specifically addressed. Further studies are required to delineate the effects of maternal DHA supplementation on endothelial cell function, fetal adiposity, and net transport of DHA and other nutrients to the fetus.

Maternal obesity during pregnancy is associated with increased placental inflammation (16–18) such as increased p38 MAPK phosphorylation (18). With increased DHA content, we found reduced p38 MAPK phosphorylation, indicating that DHA mitigates obesity-related placental inflammation. This observation may represent a cause-and-effect relationship because we have previously demonstrated that DHA reduces p38 MAPK phosphorylation in vitro using cultured primary trophoblast (32). We also found a nonsignificant trend toward increased NF-κB nuclear translocation, reduced IκBα expression, and increased cyclooxygenase 2 expression with higher DHA levels, suggesting that not all inflammatory pathways are improved with increasing placental DHA content.

Maternal obesity is linked with impairment in placental insulin signaling (48). In the current study, activation of the insulin signaling pathway (measured by phosphorylation of pathway intermediaries) was not significantly correlated with placental DHA levels. However, total expressions of critical signaling nodes were modified in association to changes in placental DHA. What the consequences are of these altered expression levels for overall cellular insulin signaling remain to be established. In myotubes, decreased expression of GRB2 increases insulin sensitivity (49). This suggests that the lower GRB2 expression associated with higher placental DHA levels may improve placental insulin sensitivity.

DHA levels were not associated with alterations in phosphorylation of placental STAT3 or downstream functional readouts of mTOR (eukaryotic translation initiation factor 4E-binding protein 1, ribosomal protein S6). Total mTOR expression was negatively correlated with placental DHA content. In a recent study, we demonstrated that DHA reduces signaling through mTOR and STAT3 pathways in cultured primary human trophoblast cells (32). mTOR and STAT3 are known positive regulators of placental amino acid transport (20, 24, 25). However, the lack significant change in mTOR and STAT3 in response to increased placental DHA content in this study suggests other cellular signaling systems may link DHA and placental amino acid transport in vivo. It should also be noted that placental mTOR (but not STAT3) signaling is reduced in response to labor (41). Therefore, we cannot exclude that any effects of DHA may have been obscured in this cohort where a majority of deliveries were vaginal.

A concern of DHA supplementation in pregnancy is that it may cause increased oxidative stress (31). In vitro placental models have suggested exposure to high levels of DHA increases cellular oxidative stress (31, 50). Because maternal obesity has been associated with elevated oxidative stress in the placenta (19), further exaggerating this condition could have detrimental effects on placental function. However, we did not find any evidence of increased oxidative stress in response to DHA supplementation, suggesting that increasing placental DHA levels do not aggravate placental oxidative stress in pregnancies of obese women.

Placental amino acid transporter activity is closely associated with alterations in fetal growth (11); for instance, in the syncytiotrophoblast MVM system A, amino acid transport activity correlates positively with birth weight, and SNAT2 expression is positively associated with maternal BMI (12). We have previously demonstrated that a diverse array of maternal metabolic parameters associated with obesity (insulin, leptin, IL-6, hyperlipidemia) can activate amino acid transporter system A in cultured primary human trophoblast cells (20, 22, 51). In the MVM isolated from placentas in our study cohort, amino acid transport was reduced in association with increasing placental DHA levels. We have previously shown that DHA treatment reduces amino acid transport in cultured primary human trophoblast cells (32). If enhanced transport of amino acids contributes to fetal overgrowth in pregnancies complicated by maternal obesity, increasing placental DHA levels represents a potential mechanism to prevent overgrowth. Fetal overgrowth is linked to adverse medical complications at birth, including operative delivery, stillbirth (52), and long-term metabolic disease in the offspring (6, 7). It is important to note that birth weights were not affected in the study subjects. However, due to the small sample size, we could not address the frequency of large for gestational age infants. Although the sample is small, the extensive characterization of the placentas accomplished in this study would not have been possible in a larger cohort. These data suggest that the impact of DHA on amino acid transport capacity in women of varying BMIs should be carefully evaluated in a larger cohort of both normal-weight and obese mothers.

Contrary to amino acid transport, placental capacity to transfer fatty acids was increased with elevated placental DHA levels. In support of our finding, DHA has been reported to stimulate fatty acid uptake in BeWo choriocarcinoma cells (53). Transfer of fatty acids across the placental barrier is a complex process, involving multiple steps. It has previously been shown that FATP4 messenger RNA expression correlates positively with placental phospholipid DHA levels and that the FATP4 may facilitate ω-3 fatty acid uptake (34). In general agreement with this report, we demonstrate a positive correlation between placental DHA concentrations and FATP4 protein expression in the plasma membranes of the syncytiotrophoblast.

Glucose transfer across the BM of the syncytiotrophoblast is believed to be the rate-limiting step for transplacental glucose transport, and GLUT1 is likely the major glucose transporter at term in human placenta (54). BM expression of GLUT1 was positively associated with placental DHA content. The higher GLUT1 expression with increasing placental DHA levels implies that glucose delivery to the fetus may be enhanced in response to increased DHA levels, but we could not demonstrate an increased activity in the isolated BM vesicles. Currently, preferred substrates for GLUT9 are in debate. Transport capacity for glucose, fructose, and urate has been described (55). Recently, the ability of GLUT9 to transport glucose has been challenged (56). Further studies are needed to elucidate the importance and function of GLUT9 in human placenta.

In conclusion, maternal DHA supplementation in pregnancies complicated by obesity did not modulate maternal circulatory markers of inflammation or insulin sensitivity. Maternal DHA supplementation increased placental membrane DHA levels, which was associated with modulation of placental nutrient transport capacity and decreased placental inflammatory markers. Increasing placental DHA may be beneficial in obese pregnancies by attenuating excessive placental amino acid transport, which otherwise contributes to fetal overgrowth. In contrast, increased placental DHA was associated with upregulation of fatty acid transporters that are believed to be responsible for delivering critically needed ω-3 fatty acids to the fetus. We speculate that maternal DHA supplementation in obese pregnant women with low DHA intake may mitigate some of the adverse effect of obesity on placental function.