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.

Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

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 (Amax) and the rate constant for inward (kin) and outward (kout) 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).