Abstract
Background: The placentas of obese women accumulate lipids that may alter fetal lipid exposure. The long-chain omega-3 fatty acids (n–3 FAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) alter FA metabolism in hepatocytes, although their effect on the placenta is poorly understood.
Objective: We aimed to investigate whether n–3 supplementation during pregnancy affects lipid metabolism in the placentas of overweight and obese women at term.
Design: A secondary analysis of a double-blind randomized controlled trial was conducted in healthy overweight and obese pregnant women who were randomly assigned to DHA plus EPA (2 g/d) or placebo twice a day from early pregnancy to term. Placental FA uptake, esterification, and oxidation pathways were studied by measuring the expression of key genes in the placental tissue of women supplemented with placebo and n–3 and in vitro in isolated trophoblast cells in response to DHA and EPA treatment.
Results: Total lipid content was significantly lower in the placentas of overweight and obese women supplemented with n–3 FAs than in those supplemented with placebo (14.14 ± 1.03 compared with 19.63 ± 1.45 mg lipid/g tissue; P < 0.05). The messenger RNA expression of placental FA synthase (FAS) and diacylglycerol O-acyltransferase 1 (DGAT1) was negatively correlated with maternal plasma enrichment in DHA and EPA (P < 0.05). The expression of placental peroxisome proliferator–activated receptor γ (r = −0.39, P = 0.04) and its target genes DGAT1 (r = −0.37, P = 0.02) and PLIN2 (r = −0.38, P = 0.04) significantly decreased, with an increasing maternal n–3:n–6 ratio (representing the n–3 status) near the end of pregnancy. The expression of genes that regulate FA oxidation or uptake was not changed. Birth weight and length were significantly higher in the offspring of n–3-supplemented women than in those in the placebo group (P < 0.05), but no differences in the ponderal index were observed. Supplementation of n–3 significantly decreased FA esterification in isolated trophoblasts without affecting FA oxidation.
Conclusion: Supplementing overweight and obese women with n–3 FAs during pregnancy inhibited the ability of the placenta to esterify and store lipids. This trial was registered at clinicaltrials.gov as NCT00957476.
Keywords: lipid metabolism, ω-3 fatty acids, placenta, pregnancy, obesity
INTRODUCTION
More than 40% of women who become pregnant in the United States are currently overweight or obese [BMI (in kg/m2) >25] (1). Obese women are predisposed to complications during pregnancy, and their offspring have generally higher adiposity at birth and are more likely to develop cardiovascular and metabolic disease later in life (2). Many of these poor outcomes have been associated with an altered lipid exposure in utero such as decreased ω-3 (n–3) long-chain PUFAs (LCPUFAs)5 during fetal development (3, 4). Indeed, a high maternal BMI is associated with lower circulating n–3 LCPUFA concentrations in pregnant women and their offspring (5, 6). n–3 Supplementation during pregnancy has been shown to ameliorate some of the poor outcomes associated with low n–3 supply during development (e.g., hypertension, visual acuity) (7–9), but the effect of n–3 supplementation on overweight or obese women and their offspring is poorly understood.
The placentas of obese women have greater lipid accumulation than those in lean women (10). This lipid accumulation may be caused by changes in placental FA handling, such as fatty acid (FA) uptake, decreased FA oxidation (FAO) (11), or increased esterification, which can ultimately affect the lipid supply to the fetus and therefore fetal adiposity. Decreases in placental unsaturated FA uptake reported in obese women (12, 13) may drive some of these changes. n–3 LCPUFAs have beneficial effects against inflammation, cardiovascular disease, and diabetes by regulating the expression of key genes pertinent to lipid metabolism and energy utilization (FA synthesis and oxidation) and decreasing tissue lipid storage (14, 15). In the liver, n–3 supplementation induces lipid oxidation and decreases the synthesis and secretion of triglycerides (16). However, the effect of n–3 LCPUFAs on placental lipid metabolism, storage, and their further impact on fetal growth and development is largely unknown.
We found that the placental inflammation associated with maternal obesity (10, 17, 18) was significantly reduced after n–3 supplementation for >25 wk during pregnancy (18). Because placental inflammation is associated with an increase in lipid storage (lipotoxicity) (10, 19, 20), we hypothesized that the decrease in inflammation observed in placental tissue after n–3 supplementation may be linked to impaired lipid storage. To test this hypothesis, we performed 1) a secondary analysis of a randomized controlled trial of n–3 supplementation during pregnancy in overweight and obese women to measure placental FAO and esterification and 2) in vitro studies of lipid metabolism in response to n–3 LCPUFAs in trophoblasts isolated from obese women.
METHODS
Study design
This study (NCT00957476) was a secondary analysis of a double-blind randomized controlled trial that enrolled obese and overweight women in early gestation (14.3 ± 1.7 wk) to receive oral supplements containing 800 mg DHA (22:6 n–3) and 1200 mg eicosapentaenoic acid (EPA; 20:5 n–3) for a total of 2000 mg n–3 LCPUFAs (n = 17). The supplements were divided into 4 capsules or matching placebo capsules that contained wheat germ oil (n = 16) for the remainder of pregnancy. Subjects, other than having a BMI ≥25, were generally healthy. Details of the study design and inclusion criteria are reported in Haghiac et al. (18). Briefly, 72 women were randomly assigned in the trial, and 22 were lost to follow-up. Of the 49 women that completed the trial (24 placebo- and 25 n–3-supplemented), placental tissue was collected from 67% of the participants (16 placebo- and 17 n–3-supplemented). Maternal blood and body weight were collected before randomization (visit 1 between 8 and 16 wk) and near the end of pregnancy (visit 2 between 34 and 36 wk). Placental tissue was collected at the time of delivery from the maternal face of the placenta, avoiding calcified or underperfused cotyledons. Several full-depth samples were collected randomly across the surface of the placenta from multiple cotyledons. Chorionic membranes and maternal decidua layers were removed, and large samples were further dissected into small pieces that were blotted for the removal of blood and separately snap-frozen in liquid nitrogen within 5 min of biopsy. Birth weight and length were recorded at delivery. Written and informed consent was obtained before participation, and the study was approved by the Institutional Review Board of MetroHealth Medical Center, Case Western Reserve University.
Placental total lipid analysis
Total lipids were extracted from 80 to 100 mg frozen placental tissue with chloroform:methanol (2:1 volume:volume) (21), allowed to dry under SpeedVac (Thermo Scientific), and normalized to tissue weight. Data were expressed as total extractable lipids/g tissue.
Placental fatty acid profile analysis
Total placental lipids were extracted from frozen tissues and hydrolyzed in ethanolic potassium hydroxide at 70°C for 3 h as described previously (22). Briefly, 2 mL H2O were added, and the solution was acidified to pH 1 by adding 6 M HCl. Diethyl ether was used to extract the free FAs, and the pH of the aqueous solution was adjusted to ∼7 with NaOH. Deuterated free FAs were added to samples before extraction as internal standards. After hydrolysis and extraction, FAs were derivatized to the pentafluorobenzyl esters. The FA pentafluorobenzyl esters were analyzed by gas chromatography–mass spectroscopy.
Placental gene expression analysis by qualitative polymerase chain reaction
Total RNA was obtained after homogenizing ∼50 mg placental tissue in Trizol reagent (Invitrogen) following the manufacturer’s guidelines. RNA integrity was assessed for each sample by visualizing ribosomal RNA via gel electrophoresis. Reverse transcription of 1 μg RNA to cDNA was performed with the use of MultiScribe Reverse Transcriptase with random primers following the manufacturer’s guidelines and cycling conditions (Applied Biosystems high-capacity cDNA reverse transcriptase kit). Gene expression was monitored by real-time polymerase chain reaction with the use of a Roche Applied Science thermal cycler with Lightcycler FastStart DNA Sybr Green 1 master mix. Gene-specific primers were designed to analyze the expression of genes involved in 1) FA transport and/or uptake: endothelial lipase, lipoprotein lipase, FA translocase (CD36), plasma membrane FA-binding protein (FABP), and FABP-4; 2) FAO: carnitine palmitoyltransferase 1b (CPT1b), peroxisome proliferator–activated receptor α (PPAR-α), and AMP-activated protein kinase α and 3) FA accumulation/esterification: acetyl-CoA carboxylase, FA synthase (FAS), steroyl-CoA desaturase, PPAR-γ, diacylglycerol O-acyltransferases 1 and 2 (DGAT1 and DGAT2), peripilin 2 (PLIN2), and sterol regulatory element binding transcription factor 1. Primer sequences are shown in Supplemental Table 1. The cycling conditions for the real-time polymerase chain reaction were the same for all primer pairs: 1) 95°C for 10 min; 2) 40 cycles of 95°C for 20 s, 55°C for 30 s, and 72°C for 30 s; and 3) final elongation at 72°C for 10 min. For each primer pair, a standard curve without template control and unknowns were run in triplicate. The melting curve of the resulting amplicon was analyzed to ensure that a single product was detected for each replicate, as described previously (13). The quantification cycle was calculated for each replicate with the use of Roche LightCycler 480 software version 1.5.1. Comparative quantification (corrected for the efficiency of the respective standard curve) was used to generate values for each replicate based on the comparative quantification with the use of Roche software. The same sample was used as the calibrator in all assays. L19 was used as a reference gene because no association between n–3 supplementation and L19 expression within the placenta was observed. Values were expressed as a ratio of the gene of interest:reference gene in each sample.
Western blotting
To further confirm the association between the maternal plasma n–3:n–6 ratio and PPAR-γ expression, Western blotting was performed in a subset of samples (n = 14). Briefly, 20 μg total protein were loaded into a 10% SDS polyacrylamide gel and electrotransferred onto a nitrocellulose membrane. After blocking the membrane with 5% milk phosphate-buffered saline with Tween 20, it was incubated with primary antibody rabbit anti-PPAR-γ (1:2000; Santa Cruz) for 1 h at room temperature. After washing with phosphate-buffered saline with Tween 20, the membrane was exposed to the anti-rabbit secondary antibody (1:3000; Cell Signaling) for 1 h at room temperature. β-Actin was the housekeeping protein used to normalize the data. Values are expressed as the ratio between PPAR-γ and β-actin expression. Image was detected with the use of enhanced chemiluminescence (Amersham) and edited with NIH Image J software (v 1.48).
Isolation of human trophoblast cells
To determine the effect of DHA and EPA at the cellular level in placental tissue, human trophoblast cells were freshly isolated from 12 obese women with a singleton pregnancy recruited at term (38–40 wk) before an elective cesarean delivery. This separate study was approved by the Institutional Review Board of MetroHealth Medical Center, Case Western Reserve University, and written informed consent was obtained before collecting placental tissue. Trophoblasts were isolated by sequential trypsin, and DNase digestion followed by gradient centrifugation as described previously (23). Cells were seeded into 6-well plates at a density of 3 × 106 cells/well and cultured overnight in Iscove’s modified DMEM culture medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and maintained at 37°C under 5% CO2.
FAO assay in human trophoblast cells
Mitochondrial FAO assays were performed ex vivo in isolated placental trophoblast cells as described previously, with some modifications (24). Trophoblast cells were incubated in fresh culture medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in the presence of 1.25% bovine serum albumin (BSA), 0.1 mmol unlabeled palmitate/L, and 18,500 Bq/mL [3H]palmitate (100 μmol/L) with or without 50 μmol DHA/L, 50 μmol EPA/L, or 50 μmol stearic acid/L as a control for 18 h at 37°C under 5% CO2. BSA + FA mixture was incubated before addition to the cells to allow for FA-BSA conjugates to form. The FA concentrations were used within the physiologic range (18, 25). At the end of the treatment period, the medium was collected, and tritiated water (3H2O), representing the oxidized palmitate, was determined by the phase equilibration method (26). Data were calculated as nmol palmitate ∙ mg protein–1 ∙ h–1 and expressed as a percentage of palmitate control. The [9, 10-3H]palmitic acid was from Movarek Biochemicals; DHA was from Cayman Chem; and unlabeled palmitate, EPA, stearic acid, and FA-free BSA were from Sigma-Aldrich.
Esterification into total lipids in trophoblast cells
The FA esterification in isolated placental trophoblast cells was determined as described previously, with some modifications (24). After incubating in the conditions described for FAO [1.25% BSA, 0.1 mmol unlabeled palmitate/L, and 18,500 Bq/mL [3H]palmitate (100 μmol/L) in the presence or absence of 50 μmol DHA/L, 50 μmol EPA/L, or 50 μmol stearic acid/L for 18 h], trophoblast cells were washed 4 times with 2 mL ice-cold phosphate-buffered saline and homogenized in 200 μL HPLC-grade acetone. After incubation with agitation at room temperature overnight, radioactivity in a 100-μL aliquot, representing esterified palmitate, was counted on a Beckman Coulter LS3801 Liquid Scintillation Counter. An additional aliquot was used to determine total proteins with the use of the bicinchoninic acid method (Sigma-Aldrich). Esterification was calculated as nmol palmitate · mg protein−1 · h−1 and expressed as a percentage of palmitate control.
De novo lipogenesis in trophoblast cells
As described previously, isolated trophoblasts were incubated with BSA alone or unlabeled FAs in media supplemented with 10% D2O (Sigma-Aldrich) for 18 h. This dose was chosen in preliminary experiments because of its low cytotoxicity and its ability to detect 2H-FAs, products of de novo lipogenesis, in the tissue. Total FAs and glycerol were isolated from cells by chemical hydrolysis and extraction. The percentage of 2H-labeled glycerol and 2H-labeled FAs were analyzed by gas chromatography–mass spectroscopy. The contribution of de novo lipogenesis to the lipid pool was calculated as previously described (22). Data were expressed as percentage newly synthesized.
Statistical analysis
All data are presented as means ± SEMs unless noted otherwise. Results of the messenger RNA (mRNA) quantification were expressed in arbitrary units. Data were normalized (by natural log transformation) before analysis. Differences between treatment groups were analyzed as intention to treat with the use of Student’s t test. Maternal plasma enrichment in DHA or EPA was analyzed as the difference of visit 2 minus visit 1. Correlations between mRNA expression, PPAR-γ protein levels, and maternal plasma measurements were assessed with the use of Pearson’s correlation coefficients. Trophoblast in vitro data were analyzed with the use of a 1-factor ANOVA with Dunnett’s multiple comparison test. Statistical analysis was performed with the use of GraphPad Prism version 6. P < 0.05 was considered statistically significant.
RESULTS
Demographic data of all participants are summarized in Table 1. There were no significant differences in maternal age, gestational age at delivery, or BMI at visit 1 and visit 2 between the placebo- and n–3-supplemented group. As expected, at the time of visit 2 (near the end of pregnancy), the maternal plasma n–3:n–6 ratio was significantly higher in the n–3-treated group than in the placebo group (P = 0.02). In addition, the maternal plasma enrichment (expressed as the difference between visit 2 and visit 1) EPA and DHA was significantly higher in the n–3-supplemented group than in the placebo group (P < 0.05). Birth weight and length were significantly higher in the offspring of n–3-supplemented women than in the placebo group (P < 0.05), but no differences were observed in the neonatal ponderal index (P = 0.22).
TABLE 1.
Maternal and neonatal characteristics of the study population1
| Placebo (n = 16) | n–3 (n = 17) | Treatment effect (P value) | |
| Maternal | |||
| Age, y | 27.9 ± 3.5 | 26.8 ± 5.3 | 0.46 |
| Gestational age at delivery, wk | 38.6 ± 1.2 | 39.0 ± 1.8 | 0.51 |
| BMI, kg/m2 | |||
| Visit 1 | 32.5 ± 6.3 | 34.3 ± 7.0 | 0.45 |
| Visit 2 | 35.4 ± 5.3 | 37.2 ± 6.6 | 0.41 |
| Plasma n–3:n–6 ratio | |||
| Visit 1 | 0.09 ± 0.01 | 0.09 ± 0.01 | 0.98 |
| Visit 2 | 0.09 ± 0.02 | 0.12 ± 0.04 | 0.022 |
| Plasma EPA visit 1, % | 0.22 ± 0.07 | 0.21 ± 0.08 | 0.73 |
| Plasma Δ EPA | −0.04 ± 0.12 | 0.39 ± 0.45 | 0.001 |
| Plasma DHA visit 1, % | 2.97 ± 0.60 | 2.86 ± 0.55 | 0.59 |
| Plasma Δ DHA | −0.47 ± 0.28 | 0.12 ± 0.65 | 0.002 |
| Neonatal | |||
| Birth weight, kg | 2.86 ± 0.39 | 3.30 ± 0.46 | 0.006 |
| Length, cm | 47.79 ± 2.19 | 49.43 ± 2.05 | 0.033 |
| Ponderal index, g/cm3 × 100 | 2.61 ± 0.21 | 2.73 ± 0.31 | 0.22 |
Values are means ± SDs for placebo (n = 16) and n–3 (n = 17). P < 0.05 compared with placebo by Student’s t test. Δ, visit 2 − visit 1 plasma fatty acids.
Reduced lipid content in the placentas of obese n–3-supplemented women
To determine the effect of n–3 supplementation on placental lipid content, total placental lipids were extracted. The placentas of obese and overweight women supplemented with n–3 had 28% less lipids than the placentas of the placebo group (19.63 ± 1.45 compared with 14.14 ± 1.03 mg lipid/g tissue; P = 0.004) (Figure 1). Placental FA profiles were measured with the use of mass spectrophotometry (Table 2). n–3 Supplementation during pregnancy in obese and overweight women was associated with a 30% reduction in palmitic acid (16:0) (a major component of lipid droplets) compared with the placebo group (P < 0.001). The abundance of other saturated FAs [stearic (18:0), arachidic (20:0), and behenic (22:0)] was also significantly reduced in the placentas of obese and overweight women supplemented with n–3 compared with the placebo group (P < 0.05). However, placental levels of the medium-chain myristic acid (14:0) were not significantly different between treatment groups, suggesting no differences in the peroxisomal oxidative pathway (27, 28). Of the PUFAs analyzed, the concentration of oleic acid (18:1) (elongation product of palmitoleic acid), gondoic acid (20:1), and linoleic acid (18:2) (precursor of the n–6 FAs) was significantly reduced compared with the placebo group (P < 0.05). EPA and DHA percentages in placental tissue were measured previously, and no significant differences between the placebo and n–3 treatment groups were observed [total n–3 FAs: 8.7% ± 1.2% compared with 8.2% ± 1.5%; P > 0.05 (18)]. These results suggest that n–3 supplementation during pregnancy in obese and overweight women significantly reduced placental accumulation of both saturated and unsaturated FAs in the lipid droplets.
FIGURE 1.
Quantification of total lipid in the placentas of women supplemented with placebo or n–3 FAs. Total lipid was extracted with the use of the Folch method and normalized to tissue weight (placebo: n = 16; n–3 FAs: n = 17). Data are means ± SEMs. **P < 0.01 compared with placebo by Student’s t test. FA, fatty acid.
TABLE 2.
Placental fatty acid composition1
| Placebo (n = 16) | n–3 (n = 17) | Treatment effect (P value) | |
| Saturated fatty acids, μmol/g | |||
| Myristic (14:0) | 0.17 ± 0.03 | 0.19 ± 0.02 | 0.23 |
| Palmitate (16:0) | 14.50 ± 0.98 | 9.78 ± 0.49 | <0.0001 |
| Stearic (18:0) | 23.20 ± 3.16 | 16.60 ± 1.28 | 0.048 |
| Arachidic (20:0) | 0.18 ± 0.02 | 0.13 ± 0.01 | 0.049 |
| Behenic (22:0) | 0.023 ± 0.002 | 0.015 ± 0.002 | 0.010 |
| Unsaturated fatty acids, μmol/g | |||
| Oleic (18:1, n–9) | 4.63 ± 0.33 | 3.27 ± 0.21 | 0.001 |
| Gondoic (20:1, n–9) | 0.054 ± 0.005 | 0.039 ± 0.003 | 0.013 |
| Linoleic (18:2, n–6) | 2.93 ± 0.19 | 1.99 ± 0.10 | <0.0001 |
Values are means ± SEMs for placebo (n = 16) and n–3 (n = 17). P < 0.05 compared with placebo by Student’s t test.
Effect of n–3 supplementation on placental lipid metabolism gene and protein expression
When data were analyzed by intention-to-treat analysis, expression of placental genes involved in FA transport/uptake (endothelial lipase, lipoprotein lipase, CD36, plasma membrane FABP, and FABP-4) (Figure 2A); FAO (CPT1b, PPAR-α, and AMP-activated protein kinase α) (Figure 2B); and FA accumulation/esterification (acetyl-CoA carboxylase, FAS, steroyl-CoA desaturase, PPAR-γ, DGAT1, DGAT2, PLIN2, and sterol regulatory element binding transcription factor 1) (Figure 2C) did not differ significantly between the groups after dietary supplementation. However, we observed a significant negative correlation between maternal plasma enrichment of DHA and EPA from visit 1 to visit 2 and placental messenger RNA expression of FAS and DGAT1 (Figure 3).
FIGURE 2.
Effect of maternal n–3 supplementation during pregnancy on mRNA expression of placental genes involved in FA transport/uptake (A), FA oxidation (B), and FA esterification (C). Data (means ± SEMs) are expressed as the ratio of gene of interest:reference gene (L19). None of the genes studied was significantly different between groups by Student’s t test (placebo: n = 16; n–3 FAs: n = 17). ACC, acetyl-CoA carboxylase; AMPK-α, AMP-activated protein α AU, arbitrary unit; CD36, fatty acid translocase; CPT1b, carnitine palmitoyltransferase 1b; DGAT, diacylglycerol O-acyltransferase; EL, endothelial lipase; FA, fatty acid; FABPpm, plasma membrane fatty acid–binding protein; FABP-4, fatty acid–binding protein 4; FAS, fatty acid synthase; LPL, lipoprotein lipase; mRNA, messenger RNA; PLIN2, peripilin 2; PPAR, peroxisome proliferator-activated receptor; SCD, steroyl-CoA desaturase; SREBP1c, sterol regulatory element binding transcription factor 1.
FIGURE 3.
Correlations between placental FAS, DGAT1 mRNA expression, and maternal plasma enrichment (Δ, calculated as visit 2 – visit 1) of DHA (A and B) and EPA (C and D) (placebo: n = 16; n–3 FAs: n = 17). Pearson’s correlation coefficients (r) are shown. P < 0.05 was considered statistically significant. AU, arbitrary unit; DGAT1, diacylglycerol O-acyltransferase 1; FA, fatty acid; FAS, fatty acid synthase; mRNA, messenger RNA.
In addition, we observed significant negative correlations between the placental mRNA expression of PPAR-γ, DGAT1, and PLIN2, with an increasing maternal plasma n–3:n–6 ratio at visit 2 (representing the n–3 status near the end of pregnancy) (Figure 4A–C). A similar significant negative correlation was observed in the placental PPAR-γ protein concentrations and increasing maternal plasma n–3:n–6 ratio at visit 2 (Figure 5A, B).
FIGURE 4.
Correlations between the maternal plasma n–3:n–6 ratio after supplementation (visit 2) and placental mRNA expression of PPAR-γ (A), DGAT1 (B), and PLIN2 (C) (placebo: n = 16; n–3 FAs: n = 17). Pearson’s correlation coefficients (r) are shown. P < 0.05 was considered statistically significant. AU, arbitrary unit; DGAT1, diacylglycerol O-acyltransferase 1; FA, fatty acid; mRNA, messenger RNA; PLIN2, peripilin 2; PPAR-γ, peroxisome proliferator-activated receptor-γ.
FIGURE 5.
Representative Western blot of PPAR-γ in the placentas of obese and overweight women supplemented with n–3 FAs or placebo during pregnancy (A). Placental PPAR-γ/β-actin protein expression was negatively correlated with a maternal plasma n–3:n–6 ratio after supplementation (visit 2) (B) (placebo: n = 7; n–3 FAs: n = 7). Pearson’s correlation coefficient (r) is shown. P < 0.05 was considered statistically significant. FA, fatty acid; PPAR-γ, peroxisome proliferator-activated receptor-γ.
Effect of in vitro n–3 treatment on lipid esterification and FAO
To assess the effect of n–3 treatment on placental lipid metabolism, we conducted in vitro experiments in freshly isolated trophoblast cells from healthy obese women at the scheduled cesarean delivery. Maternal metabolic characteristics (Table 3) were similar in women who consented to the double-blind controlled trial. Incubation of trophoblast cells in the presence of DHA and EPA significantly reduced the esterification of [3H]palmitate by a mean of 25% compared with treatment with palmitate alone (P < 0.05) (Figure 6A). In addition, the combined treatment of DHA + EPA for 18 h significantly reduced the esterification of [3H]palmitate by 35% compared with treatment with palmitate alone (P < 0.05). However, incubation with the saturated stearic acid (n = 3) had no effect on [3H]palmitate esterification into total lipids (data not shown). n–3 Treatment did not have a significant effect on FAOs in isolated trophoblast cells (Figure 6B). De novo glycerol synthesis was increased in trophoblasts treated with either palmitate alone or palmitate with DHA + EPA (P < 0.05 compared with BSA-only control), reflecting the cellular uptake and utilization of FAs, although we did not detect an effect of n–3 treatment compared with palmitate alone (Table 4). De novo FAS was not significantly altered by n–3 treatment compared with palmitate alone.
TABLE 3.
Maternal and neonatal characteristics of the study population used for isolating trophoblast cells1
| Value | |
| Maternal | |
| Age, y | 27.2 ± 4.1 |
| Gestational age at delivery, wk | 38.9 ± 0.3 |
| BMI, kg/m2 | |
| Prepregnancy | 37.2 ± 7.3 |
| Late pregnancy | 41.1 ± 6.3 |
| Neonatal | |
| Birth weight, kg | 3.3 ± 0.4 |
| Ponderal index, g/cm3 × 100 | 2.6 ± 0.3 |
Values are means ± SDs. n = 12.
FIGURE 6.
Fatty acid esterification and oxidation in trophoblasts isolated from obese women. [3H]PA esterification was significantly reduced in isolated trophoblast cells after treatment with 50 μM DHA, 50 μM EPA, and 50 μM DHA + 50 μM EPA for 18 h (A). [3H]PA oxidation was not significantly affected by any of the treatments (B). Data were calculated as a percentage of control ([3H]PA alone) for each independent experiment in triplicate and are expressed as means ± SEMs of 12 experiments (DHA + EPA, n = 5). *P < 0.05 compared with PA control by 1-factor ANOVA with Dunnett’s multiple comparison test. PA, palmitate.
TABLE 4.
De novo lipogenesis in trophoblast cells in vitro1
| Newly synthesized, % | Control | PA | PA + DHA + EPA |
| Glycerol | 7.6 ± 1.6 | 14.3 ± 5.2* | 13.2 ± 3.0* |
| Saturated FAs | |||
| Myristic (14:0) | 1.9 ± 0.9 | 0.7 ± 0.2 | 1.0 ± 1.0 |
| Palmitate (16:0) | 0.29 ± 0.26 | 0.12 ± 0.06 | 0.16 ± 0.12 |
| Stearic (18:0) | 0.68 ± 0.11 | 0.73 ± 0.17 | 0.75 ± 0.13 |
| Arachidic (20:0) | 4.1 ± 0.4 | 3.9 ± 0.4 | 3.5 ± 0.6 |
| Unsaturated FAs | |||
| Oleic (18:1, n–9) | 0.29 ± 0.17 | 0.23 ± 0.14 | 0.22 ± 0.04 |
| Linoleic (18:2, n–6) | 0.09 ± 0.05 | 0.03 ± 0.01 | 0.06 ± 0.02 |
Values are means ± SDs. n = 5–10/group. *P < 0.05 compared with control by 1-factor ANOVA with Dunnett’s multiple comparison test. FA, fatty acid; PA, palmitic acid.
DISCUSSION
The key finding of this study was that overweight and obese women supplemented with n–3 FAs during pregnancy had lower placental lipid concentrations than those randomly assigned to placebo. Furthermore, high maternal plasma n–3 concentrations, secondary to supplementation, were associated with a lower expression of genes that regulate FA esterification and storage. Last, we showed that n–3 FAs inhibited isotope-labeled palmitate esterification in placental trophoblasts isolated from obese women. Our results support an important role for the n–3 FAs EPA and DHA in placental lipid metabolism and storage.
Saben et al. (10) reported that the placentas of obese women have a higher lipid content than those of lean mothers. This may be because of greater fat consumption in obese women and/or to increased esterification and storage of FAs taken up by the placenta from the maternal circulation. In our study, n–3 supplementation of overweight and obese women for >20 wk of pregnancy was associated with a 30% reduction in placental lipid content, and because <5% of placental lipids are nonesterified (29), this was largely caused by decreases in esterified lipids (i.e., glycerides). These results are consistent with previous reports that showed that both EPA and DHA decrease lipid storage in hepatocytes (16, 30). It is unclear how placental lipid stores affect fetal FA delivery and adiposity, but we speculate that lipid storage may help to protect the fetus from excessive maternal lipids by diverting FAs toward esterification and away from transport, although this can eventually be overwhelmed, leading to spillover to the fetus and placental lipotoxicity. Interestingly, we found that birth weight and length were greater with n–3 supplementation, suggesting that nutrient availability was increased. Consistent with our findings (31–34), observational studies of populations that consume high concentrations of n–3 FA in their diet have shown an association between n–3 FA intake and birth weight after accounting for gestational age. Alternatively, maternal metabolism may have been altered by n–3 supplementation, affecting placental nutrient supply. However, maternal triglycerides—the primary FA source for placental uptake (35)—were not altered by n–3 supplementation (P Catalano, unpublished data), suggesting the placental lipid supply was similar between groups. Although the effect of placental lipid storage on fetal adiposity remains unclear, it is known that tissue accumulation of lipids leads to oxidative stress and increased inflammation, a condition known as lipotoxicity (10, 36, 37). Indeed, our group (18) has reported lower concentrations of inflammation in the placental tissue of n–3-supplemented women. It is possible that the lipid-lowering effects of n–3 FAs in the placenta underlie some of their anti-inflammatory effects in this tissue.
DHA and EPA may lower tissue lipid concentrations through several mechanisms: increased FAO, decreased FA synthesis or esterification, or increased lipolysis of stored glycerides (16, 30, 36). Our results suggest that n–3 supplementation lowered placental lipid concentrations by inhibiting the lipid esterification pathway. Maternal n–3 enrichment and a high circulating n–3:n–6 ratio late in pregnancy were associated with a lower expression of genes involved in the first committed step in triglyceride synthesis (DGAT1) (38) and lipid droplet formation (PLIN2) (39), consistent with the effects of DHA and EPA in hepatocytes (16, 40). Gene and protein concentrations of PPAR-γ, an FA-sensitive nuclear transcription factor that regulates lipid esterification and storage (41, 42), were lower in the placentas of women with a high n–3:n–6 ratio (low n–6 FAs). Metabolites of the n–6 fatty acid arachidonate stimulate PPAR-γ in the placenta and other tissues (43, 44), suggesting that low n–6 may contribute to these effects. Although FAS expression was negatively correlated with maternal n–3 enrichment, FAS activity is very modest in the human placenta (45). This was consistent with our in vitro data; de novo lipogenesis contributed to <1% of most long-chain FAs measured and was not altered by the n–3 treatment of trophoblasts. Interestingly, we did not see a difference in the expression of genes from the FAO pathway (i.e., PPAR -α, CPT1b), FA transporters, or lipases in response to n–3 supplementation, suggesting that in the placenta n–3 FAs have targeted effects on lipid esterification and storage and not lipid oxidation or uptake. Although our initial intention-to-treat analysis did not show differences in placental gene expression between groups, we found significant correlations between placental lipid metabolism genes and maternal n–3 FA concentrations that were largely driven by supplemented mothers. As seen in Figure 3, maternal DHA and EPA concentrations (percentage of total FAs) normally decrease over gestation because these long-chain FAs, essential for fetal brain and cardiovascular development, are increasingly transferred to the fetus (46). In most n–3-supplemented women, DHA and EPA concentrations increased slightly, and it is these women that showed the decreases in FAS and DGAT1 gene expression. Thus, maternal compliance had a strong effect on placental gene expression changes in this study. Consistent with the importance of maternal n-3 FA concentrations for placenta gene expression, Haghiac et al. (18) did not detect changes in the placental n–3 FA composition of supplemented women, suggesting that the lipid metabolism differences reported herein were caused by increases in systemic DHA and EPA and transit through the placenta rather than tissue accumulation.
To further investigate the associations found between n–3 FAs and placental lipid metabolism pathways in vivo, we quantified the effect of DHA and EPA treatment on the metabolism of the isotope-labeled palmitate in primary trophoblast cells isolated from obese women at term. Trophoblasts are the cells responsible for the bulk of placental nutrient metabolism and transport. Consistent with our in vivo findings, treatment with DHA and EPA, separately and in combination, inhibited the esterification of [3H]palmitate in isolated trophoblasts compared with treatment with [3H]palmitate alone. The observation that co-incubation with another saturated FA, stearate, did not inhibit palmitate esterification suggests that the effects of the n–3 FAs were not caused by nonspecific competitive inhibition. Furthermore, in support of our findings that n–3 supplementation did not alter placental FAO gene pathways, we did not detect a significant effect of DHA or EPA on [3H]palmitate oxidation in vitro. Together, these data suggest that n–3 FAs have a specific inhibitory effect on placental FA esterification pathways possibly via changes in gene expression, resulting in lower FA esterification and storage.
Randomly assigning women to either an n–3 supplement or placebo to determine the effect of n–3 supplementation on pregnancy and placental outcomes is a strength of this study. The small number of women who completed the trial (n = 16–17/group) is a study limitation and may have left us underpowered to detect smaller differences in gene expression. However, we were, by definition, well-powered to detect a 30% decrease in placental lipid levels. Our lack of data on placental FA fluxes is another limitation. Future studies will be needed to determine the effects of changes in placental lipid metabolism on fetal FA acquisition and long-term outcomes.
In summary, we found that n–3 supplementation of overweight and obese women during mid- to late pregnancy was associated with an inhibition of placental lipid esterification pathways and a significant reduction in placental lipid accumulation. In vitro experiments on isolated trophoblasts further support that n–3 FAs inhibit placental FA esterification in obese women. Thus, the effect of n–3 supplementation during pregnancy inhibits the ability of the placenta to esterify and store lipids, possibly reducing lipotoxicity and inflammation. The effect of these changes to placental lipid metabolism pathways on fetal lipid acquisition and adiposity remains to be determined in future studies.
Acknowledgments
The authors’ responsibilities were as follows—VC-N: conducted the molecular analyses and in vitro studies, analyzed the data, and drafted the manuscript; MP: performed the placental FA profiles and de novo lipogenesis assays; PG: assisted with the sample analysis; MH: conducted the primary analysis of the n–3 supplementation study designed by PC and SHM; JM: recruited volunteers and isolated trophoblast cells; PO-G: conceived and designed the study aims and drafted the manuscript; and all authors: read and approved the final manuscript. None of the authors reported a conflict of interest related to this study.
Footnotes
Abbreviations used: BSA, bovine serum albumin; CD36, FA translocase; CPT1b, carnitine palmitoyltransferase 1b; DGAT, diacylglycerol O-acyltransferase; FA, fatty acid; FABP, fatty acid–binding protein; FAO, fatty acid oxidation; FAS, fatty acid synthase; LCPUFA, long-chain PUFA; mRNA, messenger RNA; PLIN2, peripilin 2; PPAR, peroxisome proliferator–activated receptor.
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