L-methionine placental uptake: characterization and modulation in gestational diabetes mellitus

Abstract

Our aim was to investigate the influence of gestational diabetes mellitus (GDM) and GDM-associated conditions upon the placental uptake of (14)C-l-methionine ((14)C-l-Met). The (14)C-l-Met uptake by human trophoblasts (TBs) obtained from normal pregnancies (normal trophoblast [NTB] cells) is mainly system l-type amino acid transporter 1 (LAT1 [L])-mediated, although a small contribution of system y(+)LAT2 is also present. Comparison of (14)C-l-Met uptake by NTB and by human TBs obtained from GDM pregnancies (diabetic trophoblast [DTB] cells) reveals similar kinetics, but a contribution of systems A, LAT2, and b(0+) and a greater contribution of system y(+)LAT1 appears to exist in DTB cells. Short-term exposure to insulin and long-term exposure to high glucose, tumor necrosis factor-α, and leptin decrease (14)C-l-Met uptake in a human TB (Bewo) cell line. The effect of leptin was dependent upon phosphoinositide 3-kinase, extracellular-signal-regulated kinase 1/2 (ERK/MEK 1/2), and p38 mitogen-activated protein kinase. In conclusion, GDM does not quantitatively alter (14)C-l-Met placental uptake, although it changes the nature of transporters involved in that process.

Keywords: l-methionine; gestational diabetes; placenta; transport.

Figure 1.

Time course (A) and kinetics (B) of ¹⁴C-l-methionine (¹⁴C-l-Met) uptake by normal trophoblast (NTB) and diabetic trophoblast (DTB) cells. For time course experiments, cells were incubated for various periods of time at 37°C with 250 nmol/L ¹⁴C-l-Met, pH 7.5 (n = 6-7, from 3 distinct placenta). For kinetic experiments, initial rates of uptake were determined in cells incubated at 37°C with increasing concentrations of ¹⁴C-l-Met (0.25-50 μmol/L) for 6 minutes (n = 9-12, from 3 to 4 distinct placenta). Shown is arithmetic mean ± standard error of the mean.

Figure 2.

Extracellular Na⁺ dependence of ¹⁴C-l-methionine (¹⁴C-l-Met) uptake in normal trophoblast (NTB) and diabetic trophoblast (DTB) cells incubated at 37°C with 250 nmol/L ¹⁴C-l-Met for 6 minutes, at pH 7.5. NaCl in the preincubation and incubation buffer was isotonically replaced by either LiCl or choline chloride (ChCl) (n = 6-11, from 2 to 3 distinct placenta). Shown is arithmetic mean ± standard error of the mean. *Significantly different from control (NaCl; P < .05).

Figure 3.

Pharmacological characterization of ¹⁴C-l-methionine (¹⁴C-l-Met) uptake in normal trophoblast (NTB) and diabetic trophoblast (DTB) cells. Initial rates of uptake were determined in cells incubated at 37°C with 250 nmol/L ¹⁴C-l-Met for 6 minutes in the absence (control; corresponding to 100%) or in the presence of (A) 1 mmol/L 2-amino-2-norbornanecarboxylic acid (BCH), 100 μmol/L l-phenylalanine (l-Phe), or 100 μmol/L l-tryptophan (l-Trp), (B) 100 μmol/L d-leucine (d-Leu), 100 μmol/L d-phenylalanine (d-Phe), 100 μmol/L l-serine (l-Ser), or 100 μmol/L l-alanine (l-Ala), and (C) 100 μmol/L l-arginine (l-Arg), 100 μmol/L l-lysine (l-Lys), or 1 mmol/L α-(methylamino)isobutyric acid (MeAIB). Shown is arithmetic mean ± standard error of the mean (n = 5-9 from 2 to 3 distinct placenta). *Significantly different from control (P < .05) and ^#significantly different from uptake by NTB cells (P < .05).

Figure 4.

Time course (A) and characterization (B) of ¹⁴C-l-methionine (¹⁴C-l-Met) uptake by Bewo cells. For time course experiments, the cells were incubated for various periods of time at 37°C with 250 nmol/L ¹⁴C-l-Met, at pH 7.5 (n = 8). Analysis of the time course allowed the determination of the steady state accumulation (A _max) and the rate constant for inward (k _in) and outward (k _out) transport. For the characterization experiments, initial rates of uptake were determined in cells incubated at 37°C with 250 nmol/L ¹⁴C-l-Met for 6 minutes in the absence (control; corresponding to 100%) or in the presence of 1 mmol/L 2-amino-2-norbornanecarboxylic acid (BCH), 100 µmol/L l-Phe, 100 µmol/L l-Trp, 100 µmol/L d-Leu, 100 µmol/L d-Phe, 100 µmol/L l-Ser, 100 µmol/L l-Ala, 100 µmol/L l-Lys, 100 µmol/L l-Arg, or 1 mmol/L α-(methylamino)isobutyric acid (MeAIB; n = 6-12). Shown is arithmetic mean ± standard error of the mean. *Significantly different from control (P < .05).

Figure 5.

Effect of hyperglycemia upon ¹⁴C-l-methionine (¹⁴C-l-Met) uptake by Bewo cells. Cells were exposed to 10 or 30 mmol/L d-glucose (n = 6-13) or mannitol (control; corresponding to 100%) for 1 to 72 hours, and initial rates of uptake were then determined by incubating cells for 6 minutes at 37°C in buffer with 250 nmol/L ¹⁴C-l-Met. Shown are arithmetic mean ± standard error of the mean (n = 6-13). *Significantly different from control (P < .05).

Figure 6.

Effect of insulin upon ¹⁴C-l-methionine (¹⁴C-l-Met) uptake by Bewo cells. (A) Cells were exposed to 0.01, 1, or 50 nmol/L insulin or the respective solvent (control; corresponding to 100%) for 1 to 48 hours, and initial rates of uptake were then determined by incubating cells for 6 minutes at 37°C in buffer with 250 nmol/L ¹⁴C-l-Met (n = 9-14); (B) cells were exposed to 0.01, 1, or 50 nmol/L insulin or the respective solvent (control, corresponding to 100%) for 1 or 4 hours, and initial rates of uptake were then determined by incubating cells for 6 minutes at 37°C in fetal calf serum (FCS)-free culture medium with 250 nmol/L ¹⁴C-l-Met (n = 8-13). Shown is arithmetic mean ± standard error of the mean. *Significantly different from control (P < .05) and ^#significantly different from insulin 0.01 nmol/L (P < .05).

Figure 7.

Effect of leptin and tumor necrosis factor-α (TNF-α) upon ¹⁴C-l-methionine (¹⁴C-l-Met) uptake by Bewo cells. (A) Cells were exposed to 1, 100, 300, or 1000 ng/mL leptin or the respective solvent (control; corresponding to 100%) for 1 to 72 hours, and initial rates of uptake were then determined by incubating cells for 6 minutes at 37°C with 250 nmol/L ¹⁴C-l-Met (n = 5-13); (B) cells were exposed to 100, 300, or 1000 ng/L TNF-α or the respective solvent (control; corresponding to 100%) for 1 to 48 hours, and initial rates of uptake were then determined by incubating cells for 6 minutes at 37°C with 250 nmol/L ¹⁴C-l-Met (n = 5-16). Shown is arithmetic mean ± standard error of the mean. *Significantly different from control (P < .05) and ^#significantly different from leptin (1 ng/mL; P < .05).

Figure 8.

Effect of the inhibitors of intracellular signaling pathways upon hyperleptinemia-induced inhibition of ¹⁴C-l-methionine (¹⁴C-l-Met) uptake by Bewo cells. Initial rates of uptake were determined in cells incubated for 6 minutes with ¹⁴C-l-Met 250 nmol/L, after treatment for 48 hours with leptin 100 ng/mL (leptin), AG 490 5 µmol/L (AG 490), leptin 100 ng/mL + AG 490 5 µmol/L (leptin + AG 490), LY-294002 1 µmol/L (LY-294002), leptin 100 ng/mL + LY-294002 1 µmol/L (leptin + LY-294002), chelerythrine 0.1 µmol/L (chelerythrine), leptin 100 ng/mL + chelerythrine 0.1 µmol/L (leptin + chelerythrine), H-89 1 µmol/L (H-89), leptin 100 ng/mL + H-89 1 µmol/L (leptin + H-89), PD 98058 2.5 µmol/L (PD 98058), leptin 100 ng/mL + PD 98058 2.5 µmol/L (leptin + PD 98058), SB 203580 9.6 µmol/L (SB 203580), leptin 100 ng/mL + SB 203580 9.6 µmol/L (leptin + SB 203580), SP 600125 5 µmol/L (SP 600125), and leptin 100 ng/mL + SP 600125 5 µmol/L (leptin + SP 600125; n = 9-13). Shown is arithmetic mean ± standard error of the mean. *Significantly different from the respective control (P < .05) and ^#significantly different from leptin (100 ng/mL; P < .05).