Folic acid uptake by the human syncytiotrophoblast is affected by gestational diabetes, hyperleptinemia, and TNF-α

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Pediatric Research
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The mechanisms whereby gestational diabetes mellitus (GDM) increases the risk of fetal overgrowth and development of metabolic diseases later in life are likely to involve changes in nutrient supply to the fetus. Hence, in this work, we hypothesize that GDM may affect folic acid (FA) supply to the placenta and fetus.


We compared 3H-FA uptake by human cytotrophoblasts isolated from normal pregnancies (normal trophoblasts; NTB cells) and GDM pregnancies (diabetic trophoblasts; DTB cells) and investigated the effect of GDM hallmarks on 3H-FA uptake by BeWo cells.


3H-FA uptake by NTB and DTB cells was time dependent and acidic pH stimulated. When compared with NTB, 3H-FA uptake by DTB cells was more sensitive to acidic pH changes and to 5-methyltetrahydrofolate and pemetrexed (PTX) inhibition, indicating a proportionally greater involvement of the proton-coupled folate transporter (PCFT). A 4-h exposure of BeWo cells to lipopolysaccharide (LPS, 1–10μg/ml) or to high levels of tumor necrosis factor-α (TNF-α, 300ng/l) significantly reduced 3H-FA uptake. Moreover, hyperleptinemic conditions (100ng/ml leptin) decreased 3H-FA uptake by BeWo cells in a time-dependent manner when compared with normoleptinemic conditions (1ng/ml leptin).


GDM modulates 3H-FA uptake by the syncytiotrophoblast, and leptin as well as TNF-α downregulate it.

At a glance


  1. Figure 1:

    Time-course of 3H-FA apical uptake by NTB (open circles) and DTB (solid circles) cells. Cells were incubated at pH 5.5 with 20nmol/l 3H-FA for various periods of time (n = 6–8, from three or four distinct placentas). Values shown are arithmetic means ± SEM. DTB, diabetic trophoblasts; FA, folic acid; NTB, normal trophoblasts.

  2. Figure 2:

    pH-dependence of 3H-FA apical uptake by NTB (open circles) and DTB (solid circles) cells. Initial rates of uptake were determined in cells incubated with 20nmol/l 3H-FA for 6min at extracellular pH ranging from 5.0 to 8.0. Values shown are arithmetic means ± SEM (n = 4–8, from two to three distinct placentas). Two-way ANOVA retrieved significance values for the pH (P < 0.0001) and the GDM (P = 0.0026) effects. FA, folic acid; GDM, gestational diabetes mellitus.

  3. Figure 3:

    Effect of folic acid (FA; 100μmol/l), 5-methyltetrahydrofolate (MTHF; 10μmol/l), methotrexate (MTX; 10μmol/l), pemetrexed (PTX; 20μmol/l), 4-acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid disodium salt hydrate (SITS; 500μmol/l), and thiamine pyrophosphate (TPP; 100μmol/l) on 3H-FA apical uptake by NTB (white bars) and DTB (black bars) cells. Cells were incubated with 20nmol/l 3H-FA for 6min at pH 5.5 in the presence of the compound (n = 6–11, from 2 to 4 distinct placentas) or the respective solvent (control; n = 5–13). Values shown are arithmetic means ± SEM. *Significantly different from control (P < 0.05); significantly different from NTB cells (P < 0.05).

  4. Figure 4:

    Effect of GDM-associated conditions on 3H-FA apical uptake by BeWo cells. Cells were exposed to (a) D-glucose at 10 or 30mmol/l (white and black bars respectively; n = 8–9) or 5.6mmol/l (supplemented with mannitol up to 10 or 30mmol/l for isosmotic control, corresponding to normoglycemia (100%), n = 9) for the indicated periods of time; (b) insulin at 1 or 50nmol/l (white and black bars respectively; n = 8–12) or at 0.01nmol/l (normoinsulinemia (100%), n = 9–12) for the indicated periods of time; (c) leptin at 100ng/ml (white bars; n = 8–10) or 1ng/ml (normoleptinemia (100%), n = 9–11) for the indicated periods of time; and (d) LPS 1–50μg/ml (white bars n = 12–17), tumor necrosis factor-α at 10–1,000ng/l (black bars; n = 10–11), or the respective solvents (control (100%); n = 11–14) for 4h. Initial rates of uptake were determined in cells incubated with 50nmol/l 3H-FA for 6min at pH 7.5. Values shown are arithmetic means ± SEM. *Significantly different from control (P < 0.05); Significantly different from normoleptinemia (P < 0.05). FA, folic acid; GDM, gestational diabetes mellitus; LPS, lipopolysaccharide.

  5. Figure 5:

    Effect of inhibitors of signaling pathways on the apical uptake of 3H-FA and on leptin-induced inhibition of 3H-FA uptake by BeWo cells. Initial rates of uptake were determined in cells incubated for 6min with 50nmol/l 3H-FA after treatment for 24h with 100ng/ml leptin, 5 µmol/l AG490, 100ng/ml leptin + 5 µmol/l AG490 (leptin + AG490), 1 µmol/l LY294002, 100ng/ml leptin + 1 µmol/l LY294002 (leptin + LY294002), 1 µmol/l H-89, 100ng/ml leptin + 1 µmol/l H-89 (leptin + H-89), 0.1 µmol/l chelerythrine, 100ng/ml leptin + 0.1 µmol/l chelerythrine (leptin + chelerythrine), 2.5 µmol/l PD98059, 100ng/ml leptin + 2.5 µmol/l PD98059 (leptin + PD98059), 9.6 µmol/l SB203580, 100ng/ml leptin + 9.6 µmol/l SB203580 (leptin + SB203580), 5 µmol/l SP600125, and 100ng/ml leptin + 5 µmol/l SP600125 (leptin + SP600125) (n = 6–11). Values shown are arithmetic means ± SEM. *Significantly different from control (P < 0.05); significantly different from leptin (P < 0.05). FA, folic acid; NS, not significant.


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  1. Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto, Porto, Portugal

    • João R. Araújo,
    • Ana Correia-Branco,
    • Liliana Moreira,
    • Fátima Martel &
    • Elisa Keating
  2. Department of Obstetrics and Gynecology, Centro Hospitalar S. João, Porto, Portugal

    • Carla Ramalho

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