No evidence of attenuation of placental insulin-stimulated Akt phosphorylation and amino acid transport in maternal obesity and gestational diabetes mellitus

Abstract

Pregnancies complicated by obesity and/or gestational diabetes (GDM) are associated with peripheral insulin resistance; however, the insulin responsiveness of the placenta in these pregnancy complications remains largely unknown. We tested the hypothesis that primary human trophoblast cells and placental villous explants will be insulin responsive, characterized by amino acid transport, Akt and Erk activity with maternal obesity, and/or GDM. We evaluated term placentas from women with normal body mass index (BMI) (normal; n = 15), obesity (OB; n = 11), normal BMI with GDM (N-GDM; n = 11), and obesity with GDM (OB-GDM; n = 11). In a subgroup, primary human trophoblast cells (PHT) were isolated, and in an independent subgroup placental villous explants were exposed to varying concentrations of insulin. Amino acid transport capacity and insulin signaling activity were determined. Insulin significantly increased amino acid transport activity to a similar degree in PHT cells isolated from normal (+21%), N-GDM (+38%), OB (+37%), and OB-GDM (+35%) pregnancies. Insulin increased Akt and Erk phosphorylation in PHT cells (3-fold) and in villous explants (2-fold) in all groups to a similar degree. In contrast to the peripheral maternal insulin resistance commonly associated with obesity and/or GDM, we found that the placenta is insulin sensitive in these pregnancy complications. We suggest that elevated maternal insulin levels in pregnancies complicated by obesity and/or GDM promote critical placental functions, including amino acid transport. Insulin-stimulated placental nutrient delivery may contribute to the increased risk of fetal overgrowth and adiposity in these pregnancies. Moreover, our findings may inform efforts to optimize insulin regimens for women with GDM.

Keywords: human; insulin sensitivity; placental transport; pregnancy; syncytiotrophoblast.

Fig. 1.

Correlations with birth weight and placenta weight. Correlations of placenta weight and birth weight (A), placenta weight and maternal body mass index (BMI; B), and maternal BMI and birth weight (C). Pearson’s correlation coefficient. P < 0.05 and α = 0.05 were considered statistically significant. GDM, gestational diabetes mellitus.

Fig. 2.

Effect of insulin on system A amino acid transport in primary human trophoblast (PHT) cells. Differentiated PHT cells were incubated with insulin for 24 h before the activity assay. Data are presented as Na⁺-dependent uptake of ¹⁴C-methyl-aminoisobutyric acid (MeAIB), representing system A activity. Normal (n = 6; A), normal and gestational diabetes mellitus (N-GDM; n = 6; B), obese (OB; n = 6; C), obese and gestational diabetes mellitus (OB-GDM; n = 6; D) groups. Data are presented as means ± SE. Repeated-measures one-way ANOVA followed by Fisher’s least significant difference test, P < 0.05. Treatment groups with different letters are significantly different.

Fig. 3.

Secretion of human chorionic gonadotropin (hCG) from primary human trophoblast cells in culture. Significantly higher amounts of hCG were secreted into the media after 66 and 92 h in all 4 groups as compared with 18 h of culture [normal: n = 6; normal and gestational diabetes mellitus (N-GDM): n = 6; obese (OB): n = 6; obese and gestational diabetes mellitus (OB-GDM): n = 6]. Data are presented as means ± SE. Repeated-measures one-way ANOVA, followed by Fisher’s least significant difference test, P < 0.05. Time points with different letters are significantly different.

Fig. 4.

Effect of insulin on Akt (T308) phosphorylation in primary human trophoblast (PHT) cells. A: representative Western blots of PHT cells treated with insulin for 3 h. Histogram illustrates relative protein expression of Akt (T308)/Akt. B–E: normal n = 6 (B), normal and gestational diabetes mellitus (N-GDM) n = 6 (C), obese (OB) n = 6 (D), obese and gestational diabetes mellitus (OB-GDM) n = 6 (E). Data are presented as means ± SE. Repeated-measures one-way ANOVA, followed by Fisher’s least significant difference test, P < 0.05. Treatment groups with different letters are significantly different.

Fig. 5.

Effect of insulin on Akt (S473) phosphorylation in primary human trophoblast (PHT) cells. A: representative simple Western images (60) of lysates of PHT cells incubated for 3 h with insulin. Histograms illustrate relative protein expression of Akt (S473)/Akt. B–E: normal n = 6 (B), normal and gestational diabetes mellitus (N-GDM) n = 6 (C), obese (OB) n = 6 (D), obese and gestational diabetes mellitus (OB-GDM) n = 4 (E). Data are presented as means ± SE. Repeated-measures one-way ANOVA, followed by Fisher’s least significant difference test, P < 0.05. Treatment groups with different letters are significantly different.

Fig. 6.

Effect of insulin on Akt (T308) phosphorylation in villous explants. A: representative Western blots of villous explants incubated with insulin for 3 h. Histogram illustrates relative protein expression of Akt (T308)/Akt. B–E: normal n = 9 (B), normal and gestational diabetes mellitus (N-GDM) n = 5 (C), obese (OB) n = 5 (D), obese and gestational diabetes mellitus (OB-GDM) n = 5 (E). Data are presented as means ± SE. Repeated-measures one-way ANOVA, followed by Fisher’s least significant difference test, P < 0.05. Treatment groups with different letters are significantly different.

Fig. 7.

Effect of insulin on Akt (S473) phosphorylation in villous explants. A: representative simple Western images (60) of villous explants incubated with insulin. Histogram illustrates relative protein expression of Akt (S473)/Akt. B–E: normal n = 8 (B), normal and gestational diabetes mellitus (N-GDM) n = 6 (C), obese (OB) n = 7 (D), obese and gestational diabetes mellitus (OB-GDM) n = 6 (E). Data are presented as means ± SE. Repeated-measures one-way ANOVA, followed by Fisher’s least significant difference test, P < 0.05. Treatment groups with different letters are significantly different.

Fig. 8.

Effect of insulin on MAPK ERK1/2 (T202/Y204) phosphorylation in villous explants. A: representative simple Western images (60) of villous explants incubated with insulin. Histogram illustrates relative protein expression of MAPK ERK1/2 (T202/Y204)/MAPK ERK1/2. B–E: normal n = 6 (B), normal and gestational diabetes mellitus (N-GDM) n = 6 (C), obese (OB) n = 6 (D), obese and gestational diabetes mellitus (OB-GDM) n = 6 (E). Data are presented as means ± SE. Paired t-test, P < 0.05. Treatment group with different letters are significantly different.