Down-regulation of placental mTOR, insulin/IGF-I signaling, and nutrient transporters in response to maternal nutrient restriction in the baboon

Abstract

The mechanisms by which maternal nutrient restriction (MNR) causes reduced fetal growth are poorly understood. We hypothesized that MNR inhibits placental mechanistic target of rapamycin (mTOR) and insulin/IGF-I signaling, down-regulates placental nutrient transporters, and decreases fetal amino acid levels. Pregnant baboons were fed control (ad libitum, n=11) or an MNR diet (70% of controls, n=11) from gestational day (GD) 30. Placenta and umbilical blood were collected at GD 165. Western blot was used to determine the phosphorylation of proteins in the mTOR, insulin/IGF-I, ERK1/2, and GSK-3 signaling pathways in placental homogenates and expression of glucose transporter 1 (GLUT-1), taurine transporter (TAUT), sodium-dependent neutral amino acid transporter (SNAT), and large neutral amino acid transporter (LAT) isoforms in syncytiotrophoblast microvillous membranes (MVMs). MNR reduced fetal weights by 13%, lowered fetal plasma concentrations of essential amino acids, and decreased the phosphorylation of placental S6K, S6 ribosomal protein, 4E-BP1, IRS-1, Akt, ERK-1/2, and GSK-3. MVM protein expression of GLUT-1, TAUT, SNAT-2 and LAT-1/2 was reduced in MNR. This is the first study in primates exploring placental responses to maternal undernutrition. Inhibition of placental mTOR and insulin/IGF-I signaling resulting in down-regulation of placental nutrient transporters may link maternal undernutrition to restricted fetal growth.

Keywords: fetal growth restriction; nonhuman primate; trophoblast.

Figure 1.

Phosphorylation of placental S6 kinase, RPS6, and 4E-BP1 in control and MNR animals. A–C) Representative Western blots for P-S6K (Thr-389; A) and S6K P-RPS6 (Ser-235/236; B) and RPS6 P-4E-BP1 (Thr-37/46 or Thr-70; C) and 4E-BP1 in homogenates of control (C) and MNR (M) baboon placentas at GD 165. D) Histogram summarizes the Western blotting data. Equal loading was performed. After normalization to β-actin, the mean density of C samples was assigned an arbitrary value of 1. Values are given as means + sem. *P < 0.05 vs. control; unpaired Student's t test.

Figure 2.

Placental GSK-3 and ERK1/2 signaling in control and MNR animals. A, B) Representative Western blots for P-GSK-3 (Ser-21/9) and GSK-3 (A) and P-ERK1/2 (Thr-202/Tyr-204) and ERK1/2 (B) in homogenates of control (C) and MNR (M) baboon placenta at GD 165. C) Histogram summarizes the Western blotting data. Equal loading was performed. After normalization to β-actin, the mean density of C samples was assigned an arbitrary value of 1. Values are given as means + sem. *P < 0.05 vs. control; unpaired Student's t test.

Figure 3.

Placental insulin and IGF-I signaling in control and MNR animals. A) Representative Western blots for P-IRS-1 (Tyr-612), IRS-1, P-Akt (Thr-308), and P-Akt in homogenates of control (C) and MNR (M) baboon placenta at GD 165. B) Histogram summarizes the Western blotting data. Equal loading was performed. After normalization to β-actin, the mean density of C samples was assigned an arbitrary value of 1. Values are given as means + sem. *P < 0.05 vs. control; unpaired Student's t test.

Figure 4.

Phosphorylation of placental AMPK and LKB-1 in control and MNR animals. A) Representative Western blots for P-AMPK (Thr-172), AMPK, P-LKB-1 (Ser-428), and LKB in homogenates of control (C) and MNR (M) baboon placenta at GD 165. B) Histogram summarizes the Western blotting data. Equal loading was performed. After normalization to β-actin, the mean density of C samples was assigned an arbitrary value of 1. Values are given as means + sem; unpaired Student's t test.

Figure 5.

Phosphorylation of placental raptor and tuberin/TSC2 in control and MNR animals. A) Representative Western blots for P-raptor (Ser-792), raptor, P-tuberin/TSC2 (Ser-1387), and tuberin/TSC2 in homogenates of control (C) and MNR (M) baboon placenta at GD 165. B) Histogram summarizes the Western blotting data. Equal loading was performed. After normalization to β-actin, the mean density of C samples was assigned an arbitrary value of 1. Values are given as means + sem; unpaired Student's t test.

Figure 6.

Subcellular localization of nutrient transporter isoforms in baboon placenta. Subcellular localization of GLUT-1 (A), SNAT-2 (C), and LAT-1 (E) in placentas of control-fed baboons at 165 d gestation, with corresponding negative controls (B, D, F, respectively).

Figure 7.

Protein expression of system A amino acid transporter isoforms in MVMs. A) Representative Western blots for SNAT-1, SNAT-2, and SNAT-4 in MVM isolated from control (C) and MNR (M) placenta at GD 165. B) Histogram summarizes the Western blotting data. Equal loading was performed. After normalization to β-actin, the mean density of C samples was assigned an arbitrary value of 1. Values are given as means + sem. *P < 0.05 vs. control; unpaired Student's t test.

Figure 8.

Protein expression of system L amino acid transporter isoforms GLUT-1 and TAUT in MVMs. A) Representative Western blots for LAT-1, LAT-2, GLUT-1, and TAUT in MVMs isolated from control (C) and MNR (M) baboon placenta at GD 165. B) Histogram summarizes the Western blotting data. Equal loading was performed. After normalization to β-actin, the mean density of C samples was assigned an arbitrary value of 1. Values are given as means + sem. *P < 0.05 vs. control; unpaired Student's t test.