Hyperandrogenism diminishes maternal-fetal fatty acid transport by increasing FABP4-mediated placental lipid accumulation†

Abstract

Long-chain polyunsaturated fatty acids (LCPUFAs) are critical for fetal brain development. Infants born to preeclamptic mothers or those born growth restricted due to placental insufficiency have reduced LCPUFA and are at higher risk for developing neurodevelopmental disorders. Since plasma levels of testosterone (T) and fatty acid-binding protein 4 (FABP4) are elevated in preeclampsia, we hypothesized that elevated T induces the expression of FABP4 in the placenta leading to compromised transplacental transport of LCPUFAs. Increased maternal T in pregnant rats significantly decreased n-3 and n-6 LCPUFA levels in maternal and fetal circulation, but increased their placental accumulation. Dietary LCPUFAs supplementation in T dams increased LCPUFA levels in the maternal circulation and further augmented placental storage, while failing to increase fetal levels. The placenta in T dams exhibited increased FABP4 mRNA and protein levels. In vitro, T dose-dependently upregulated FABP4 transcription in trophoblasts. Testosterone stimulated androgen receptor (AR) recruitment to the androgen response element and trans-activated FABP4 promoter activity, both of which were abolished by AR antagonist. Testosterone in pregnant rats and cultured trophoblasts significantly reduced transplacental transport of C14-docosahexaenoic acid (DHA) and increased C14-DHA accumulation in the placenta. Importantly, FABP4 overexpression by itself in pregnant rats and trophoblasts increased transplacental transport of C14-DHA with no significant placental accumulation. Testosterone exposure, in contrast, inhibited this FABP4-mediated effect by promoting C14-DHA placental accumulation.

Keywords: FABP4; androgens; fatty acids; fetal growth; placenta; preeclampsia; pregnancy.

Graphical Abstract

Figure 1

Absolute amounts of total n3 and n6 fatty acids in (A) maternal serum, (B) fetal serum, and (C) placenta of control and T dams with and without dietary LCPUFA supplementation. Fatty acids were quantified using LC–MS/MS. Data presented as mean ± SEM of six rats in each group. ^*P < 0.05 vs respective control group, ^#P < 0.05 vs respective group without LCPUFA supplementation.

Figure 2

Effect of elevated T on mRNA expression of genes involved in (A) fatty acid synthesis, (B) FATP, and (C) FABPs in the placenta. (D) Effect of elevated T on FABP4 protein expression in the placenta. Representative Western blots for FABP4 and β-actin are shown at top; blot density obtained from densitometric scanning of FABP4 normalized to β-actin is shown at bottom. (E) FABP4 mRNA expression in the liver and adipose tissue of control and T dams. mRNA expression was normalized to β-actin. Data presented as mean ± SEM of six rats per group. ^*P < 0.05 vs control. Acac, Acetyl-CoA Carboxylase Alpha; Fasn, Fatty Acid Synthase; Lipe, Lipase E, Hormone Sensitive Type; Fads2, Delta-6-Desaturase; Fads1, Delta-5 Desaturase; Elovl5, ELOVL Fatty Acid Elongase 5; Lpl, Lipoprotein Lipase; Lipg, Lipase G, Gastric Type.

Figure 3

Effect of T on FABP4 (A) mRNA and (B) protein expression in cultured trophoblast cells (BeWo) in vitro. (C) Effect of T on FABP4 mRNA expression in cultured hepatocytes (HepG2) and adipocytes (3T3-L1). Representative Western blots for FABP4 and β-actin are shown at top; blot density obtained from densitometric scanning of FABP4 normalized to β-actin is shown at bottom. mRNA expression was normalized with β-actin. Data presented as mean ± SEM from four independent experiments. ^*P < 0.05 vs vehicle control, ^#P < 0.05 vs 10 nM T treatment. HF, hydroxyflutamide (100 nM).

Figure 4

Bioinformatic analysis of FABP4 gene promoter and functional validation of androgen receptor binding motifs. (A) Schematics showing the location of AREs on FABP4 promoter, (B) ChIP assay showing T-induced AR interaction with a putative ARE (C) reporter assay showing luciferase activity in BeWo cells transfected with the reporter plasmid containing ARE 1, 2 and 3 in the presence of T (10 nM) with and without hydroxylflutamide (HF, 100 nM). Data presented as mean ± SEM from four independent experiments. ^*P < 0.05 vs Vehicle, ^#P < 0.05 vs T treatment.

Figure 5

Effect of T and FABP4 overexpression on transplacental DHA transport in pregnant rats in vivo. Radiotracer transport studies using C¹⁴-DHA were performed and placental transport to the fetus (fetal dpm per milliliter serum) and placental accumulation (placental dpm per gram placenta) were measured. Data presented as mean ± SEM from n = 6 dams in each group. ^*P < 0.05 vs respective control, ^#P < 0.05 vs wild type. FABP O/E, FABP4 overexpressed.

Figure 6

Effect of T and FABP4 overexpression on transcellular DHA transport in cultured BeWo cells in vitro. (A) T-induced dose- and time-dependent changes in transcellular C¹⁴-DHA transport from apical to the basal reservoir in normal trophoblasts, (B) dose-dependent changes in intracellular C¹⁴-DHA accumulation in normal trophoblasts after 24 h of T treatment, (C) Time-dependent changes in transcellular C¹⁴-DHA transport in normal and FABP4-overexpressed trophoblasts in the presence and absence of T (10 nM) treatment. (D) Changes in intracellular C¹⁴-DHA accumulation in normal and FABP4-overexpressed trophoblasts in the presence and absence of 24 h T (10 nM) treatment. Data presented as mean ± SEM of four independent experiments. ^*P < 0.05 vs control. FABP O/E, FABP4 overexpressed.