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.—Kavitha, J. V., Rosario, F. J., Nijland, M. J., McDonald, T. J., Wu, G., Kanai, Y., Powell, T. L., Nathanielsz, P. W., Jansson, T. Down-regulation of placental mTOR, insulin/IGF-I signaling, and nutrient transporters in response to maternal nutrient restriction in the baboon.

Maternal undernutrition during pregnancy remains a serious problem worldwide and constitutes a significant problem also in the United States, because more than 50 million Americans live in households experiencing food insecurity or hunger at least some time during the year (1). Maternal undernutrition is the most common cause of intrauterine growth restriction (IUGR) in developing countries. IUGR is associated with perinatal morbidity and mortality (2) and increases the risk for diabetes and cardiovascular disease in adult life (3–5). However, the mechanisms linking maternal nutrient restriction to reduced fetal growth and programming of adult disease remain to be fully established.

Previous studies in experimental animals have implicated changes in placental growth, structure, and function as critical mediators of adverse pregnancy outcomes in response to altered maternal nutrient availability (6–11). In human IUGR due to placental insufficiency, the activity of the placental system A and system L amino acid transporters is decreased (12–15), consistent with the possibility that changes in the activity of placental nutrient transporters may directly contribute to abnormal fetal growth (16–18). System A is a sodium-dependent transporter mediating the uptake of nonessential neutral amino acids into the cell (19). All three known isoforms of system A, sodium-dependent neutral amino acid transporter (SNAT)-1 (SLC38A1), SNAT-2 (SLC38A2), and SNAT-4 (SLC38A4) are expressed in the placenta (20). System A activity establishes the high intracellular concentration of amino acids like glycine, which is used to exchange for extracellular essential amino acids via system L. Thus, system A activity is critical for placental transport of both nonessential and essential amino acids. System L is a sodium-independent amino acid exchanger mediating cellular uptake of essential amino acids, including leucine (21). The system L amino acid transporter is a heterodimer, consisting of a light chain, typically large neutral amino acid transporter (LAT)-1 (SLC7A5) or LAT-2 (SLC7A8), and a heavy chain, 4F2hc/CD98 (SLC3A2).

Maternal hormones, such as insulin and IGF-I, as well as trophoblast mechanistic target of rapamycin (mTOR) signaling, are important regulators of placental amino acid transport (22–28). Calorie restriction in humans and animals typically decreases circulating levels of IGF-I and insulin, (29, 30), and maternal serum concentrations of insulin and IGF-I are reduced in pregnant rats fed a low-protein diet (8). mTOR is a serine/threonine kinase and represents an important nutrient-sensing pathway in mammalian cells, which controls cell growth, proliferation, and metabolism in response to nutrient availability and growth factor signaling. mTOR exists in two complexes, mTOR complex 1 (mTORC1) and mTORC2. The downstream effects of mTORC1 are mediated by phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and p70 S6 kinase (S6K). mTORC2 phosphorylates Akt, PKCα, and serum and glucocorticoid-regulated kinase 1 (SGK1) and influences the actin skeleton. Placental mTOR activity is decreased in human IUGR (26, 31) as well as in animal models of IUGR, such as low-protein diet in rats (11). In addition, maternal protein restriction in rats inhibits placental insulin/IGF-I signaling (11).

The effects of maternal nutrient restriction on the placenta have been studied experimentally in sheep (32–35) and rodents (6, 8, 11, 36–38). However, findings in the rodent and sheep placentas may not be representative of the human. Studies exploring the effect of nutrition on pregnancy outcome in pregnant women are difficult to perform due to poor sensitivity of methods assessing dietary intake, poor compliance, high biological variability, and inability to administer strict dietary regimens. Studies in pregnant nonhuman primates, with reproductive physiology that in many respects is more similar to human than laboratory rodent species and sheep, can be used to fill this gap of knowledge. However, no data are available on the effect of maternal nutrient restriction on placental signaling and transport functions in nonhuman primates. Here we studied the effect of maternal nutrient restriction on placental signaling and nutrient transporter expression in pregnant baboons, a nonhuman primate with a placental structure and development very similar to that of humans (39). We utilized a well-established model of global maternal nutrient restriction (MNR; animals given 70% of the control diet) associated with moderate IUGR and low maternal circulating levels of IGF-I. This degree of maternal, and subsequent fetal, nutrient restriction results in IUGR accompanied by major changes in the fetal brain frontal cortex (40), liver (41), and kidney (42). Fetal cortisol is also elevated (41), and offspring show an altered postnatal phenotype with decreased peripheral glucose disposal and elevated fasting glucose (43) and behavior (44). We hypothesized that MNR in baboons inhibits placental mTOR and insulin/IGF-I signaling, down-regulates placental nutrient transporters, and decreases fetal circulating levels of amino acids.

MATERIALS AND METHODS

Animals and diets

All procedures were approved by the Texas Biomedical Research Institute Institutional Animal Care and Use Committee and conducted in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Baboons (Papio species) were housed in outdoor metal and concrete gang cages, each containing 10–16 females and 1 male. Details of housing and environmental enrichment have been described elsewhere (45).

System for controlling and recording individual feeding

The feeding system used has been described in detail (45). Briefly, once a day prior to feeding, all baboons were placed in individual feeding cages. Baboons passed along a chute, over a scale, and into an individual feeding cage. The weight of each baboon was obtained as it crossed an electronic scale system (GSE 665; GSE Scale Systems, Livonia, MI, USA). The weight recorded was the mean of 50 individual measurements over 3 s. If the sd of the weight measurement was >1% of the mean weight, the weight was automatically discarded, and the weighing procedure was repeated.

Once housed in an individual cage, each animal was fed between either 07:00 and 09:00 or 11:00 and 13:00. Water was available continuously in the individual feeding cage and the group cages. Animals were fed Purina Monkey Diet 5038 (Purina, St. Louis, MO, USA), described by the vendor as “a complete life-cycle diet for all Old World Primates.” The biscuit contains stabilized vitamin C as well as all other required vitamins. Its basic composition is crude protein ≥15%, crude fat ≥5%, crude fiber ≤6%, ash ≤5%, and added minerals ≤3%. At the start of the feeding period, each baboon was given 60 biscuits in the feeding tray of the individual cage. At the end of the 2 h feeding period, the baboons were returned to the group cage. Biscuits remaining in the tray, on the floor of the cage, and in the pan beneath the cage were counted. Food consumption of animals, weights, and health status were recorded each day.

Study design

Fertile female baboons were selected to participate in this study on the basis of their reproductive age (8–15 yr old), body weight (10–15 kg), and absence of genital and extragenital pathological signs. Initially, animals were placed into two group cages with a vasectomized male to establish a stable social group. Assignment to each group was random. At the end of the acclimation period to the group and to feeding in the individual nutritional cages (30 d), a fertile male was introduced into each breeding cage. All baboons were observed twice daily for well-being and 3 times/wk for turgescence (swelling), color of sex skin, and signs of vaginal bleeding to enable timing of ovulation and subsequent conception.

Pregnancy was dated initially by timing of ovulation and changes in sex skin color and confirmed at gestational day (GD) 30 by ultrasonography when the experimental feeding period was started. Ad libitum-fed control baboons were given 60 biscuits in their individual cage. Biscuits remaining were counted after baboons returned to their group cage. Animals subjected to MNR were fed 70% of the total food intake of contemporaneous controls on a per-kilogram basis.

Collection of tissue and blood samples

Cesarean sections were performed under isoflurane anesthesia at GD 165 (term 184). Briefly, animals were tranquilized with ketamine hydrochloride (10 mg/kg), intubated, and anesthetized using isoflurane (starting rate 2% with oxygen: 2.0 L/min). Conventional cesarean sections using standard sterile technique were performed as described previously (45). At cesarean section, fetuses and placentas were towel dried and weighed. Postoperative analgesia was provided using buprenorphine (0.015 mg/kg/d as 2 doses) for 3 d (45).

Trophoblast villous tissue was obtained from 8 different locations according to a standardized protocol, and either immediately snap-frozen in liquid nitrogen and stored at −80°C or fixed in formalin (10% buffered formalin) and embedded in paraffin for immunohistochemistry. Maternal blood was collected from the femoral vein at cesarean section, and fetal blood was obtained by umbilical venous blood sampling (45). Plasma was prepared and frozen at −80°C until analysis.

Amino acid analysis

Plasma samples (0.1 ml) were deproteinized with 0.1 ml of 1.5 M HClO₄ and neutralized with 0.05 ml of 2 M K₂CO₃. The solution was centrifuged at 12,000 g at 4°C for 1 min, and the supernatant was used for analysis. Amino acids were determined by HPLC methods involving precolumn derivatization with o-phthaldialdehyde, as described previously (46). All amino acids were quantified on the basis of authentic standards (Sigma Chemicals, St. Louis, MO, USA) using Millenium-32 Software (Waters, Milford, MA, USA). Maternal and fetal plasma samples were available from all 11 control animals but only for 6 of the 11 MNR animals from which placental tissue was collected. To increase statistical power, plasma samples from 11 additional control animals were analyzed, resulting in n = 22 in this group.

Isolation of trophoblast plasma microvillous membranes (MVMs)

Approximately 0.5–1 g frozen trophoblast tissue was thawed on ice and homogenized using a Polytron homogenizer (Kinematica, Bohemia, NY, USA) in 1.5–3 ml of buffer D (250 mM sucrose, 10 mM HEPES-Tris, and 1 mM EDTA, pH 7.4, at 4°C) with protease and phosphatase inhibitors. Syncytiotrophoblast plasma MVMs were prepared as described previously (47, 48), with the exception that the preparation was scaled down to fit the small amount of starting tissue (11). Briefly, after initial centrifugation steps, MVMs were separated by Mg²⁺ precipitation and further purified with differential centrifugation. Samples were frozen in liquid nitrogen and stored at −80°C. MVM purity was determined as the enrichment of alkaline phosphatase activity compared to homogenates (49, 50) and was assessed using standard activity assays for alkaline phosphatase. MVM enrichment of alkaline phosphatase activity in the control (4.9±0.4-fold, n=8) and MNR groups (5.0±0.5-fold, n=8) was not significantly different. As an additional enrichment marker, the expression of the insulin receptor, previously shown to be highly expressed in the MVM of human placenta (51), was determined using Western blot. MVM enrichment of the insulin receptor in the control (9.4±1.0 fold, n=8) and MNR groups (9.1±0.9 fold, n=8) was not significantly different (Supplemental Fig. S1). Protein content of the vesicles was determined by the method of Bradford.

Western blot analysis

Protein expression of total and phosphorylated mTOR (S-2448), S6K (Thr-389), 4E-BP1 (Thr-37/46 or Thr-70), S6 ribosomal protein (Ser-235/236), glycogen synthase kinase 3 (GSK-3; Ser-21/9), AMPKα (Thr-172), extracellular signal-regulated kinase ½ (ERK1/2; Thr-202/Tyr-204), Akt (Thr-308), raptor (Ser-792), tuberin/TSC-2 (Ser-1387) and LKB-1 (Ser-248) was determined in placental homogenates using commercial antibodies (Cell Signaling Technology, Boston, MA, USA). Antibodies recognizing insulin receptor β were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-β-actin and IRS-1 (Tyr-612) antibodies were obtained from Sigma-Aldrich.

Protein expression of the system A amino acid transporter isoforms SNAT-1, SNAT-2, and SNAT-4 and the system L amino acid transporter isoforms LAT-1, LAT-2, glucose transporter (GLUT)-1, and taurine transporter (TAUT), was analyzed in MVMs. The justification for determining protein expression of transporters in MVMs rather than in homogenates is that trophoblast nutrient transporters mediate cellular uptake and transfer across the placental barrier only if localized in the syncytiotrophoblast plasma membranes. Thus, data on amino acid transporter and GLUT-1 protein expression in MVMs is more informative than determination of protein expression in placental homogenates. The SNAT-1 antibody was received as a generous gift from Dr. Jean Jiang (University of Texas Health Science Center, San Antonio, TX, USA). A polyclonal SNAT-2 antibody generated in rabbits (52), was generously provided by Dr. Puttur Prasad (University of Georgia, Athens, GA, USA). Affinity-purified polyclonal anti-SNAT-4 antibodies were produced in rabbits using the epitope YGEVEDELLHAYSKV in human SNAT-4 (Eurogentec, Seraing, Belgium). Antibodies targeting LAT-1 and LAT-2 were produced in rabbits as described previously (53). GLUT-1 and TAUT antibodies were purchased from Millipore (Billerica, MA, USA).

Western blot analysis was performed as described previously (11). In brief, 20 μg of total protein was loaded onto a NuPAGE Novex (Invitrogen, Carlsbad, CA, USA) precast 4–12% Bis–Tris gels and electrophoresis was performed at a constant 200 V for 40 min. Proteins were transferred onto nitrocellulose membranes at a constant 40 V. After transfer, membranes were blocked in 5% milk in Tris-buffered saline (w/v) plus 0.1% Tween 20 (v/v) for 1 h at room temperature. Membranes were incubated with primary antibodies overnight at 4°C. Subsequently, membranes were incubated with the appropriate peroxidase-labeled secondary antibodies for 1 h. After washing, bands were visualized using enhanced chemiluminescence detection reagents (Pierce Biotechnology, Rockford, IL, USA). Blots were stripped using β-mercaptoethanol and reprobed for β-actin as a loading control. Analysis of the blots was performed by densitometry using an αImager (Alpha Innotech Corporation, San Leandro, CA, USA). For each protein target, the mean density of the control sample bands was assigned an arbitrary value of 1. Subsequently, all individual control and MNR density values were expressed relative to this mean.

Immunohistochemistry

Sections (5 μm) of formalin-fixed trophoblast tissue were deparaffinized in xylene, rehydrated in descending grades of alcohol (100, 70, and 45%) to water, immersed in citrate buffer (0.01 M, pH 6.0), and heated to boiling for 10–15 min for antigen retrieval. After cooling for 15 min, the sections were rinsed in PBS, washed for 10 min in a solution of 1.5% H₂O₂ and methanol and then for 5 min in PBS. Sections were placed in diluted (10%) normal serum for 20 min and then incubated in primary antibody overnight at 4°C using a humidified chamber. Subsequently, sections were rinsed in PBS and incubated for 1 h at room temperature with goat anti-rabbit biotinylated secondary antibody. Antigens were localized using 3,3′-diaminobenzidine in PBS for 1–5 min. Finally, tissues were stained with hematoxylin and mounted. Negative controls consisted of parallel incubations of sections after preabsorbation of the primary antibody using antigen peptide (where available) or after replacement of the primary antibody with normal horse serum.

Data presentation and statistics

The number of male and female fetuses was similar in the control (6 males/5 females) and MNR (5 males/6 females) groups. Because of the limited number of observations in each of the 4 groups, data were not analyzed for males and females separately. Data are presented as means ± sem or + sem. If not stated otherwise, 11 control and 11 MNR animals were studied. Statistical significance of differences between control and MNR diet groups was assessed using Student's t test. Values of P < 0.05 were considered significant. With the number of statistical tests performed in this study, it is expected that ∼4 of the statistically significant differences (P<0.05) identified have occurred only by chance, i.e., represent false positives (type I error). However, with the following justification, we elected not to adjust for multiple tests. Many outcome variables in our study are highly correlated, and adjusting for multiple testing is therefore not critical (54). Because we identified 24 differences that were statistically significant, it is clear that the overwhelming majority of these are, in fact, true differences. Many of the identified statistically significant differences were highly significant. Thus, adjustment for multiple testing, taking the highly correlated outcome variables into account, would have small effects. In this article, we do not discuss individual outcome variables but rather groups of outcome variables changing in the same direction.

RESULTS

Fetal and placental weights

At GD 165, fetal weights in the MNR group were reduced by 13% (control, 812.6±36.8; MNR, 706.5±26.0 g, n=11/group, P=0.03). Placental weights (control, 207.2±14.2; MNR, 171.3±11.9 g, P=0.06) were comparable between the groups. Furthermore, fetal/placental weight ratio (4.0±0.16 vs. 4.2±0.25, P=0.4) did not differ between the groups.

Maternal and fetal plasma amino acid concentrations

Maternal plasma concentrations of aspartic acid (−55%, P=0.02), glutamic acid (−30%, P=0.05), tyrosine (−38%, P=0.001), tryptophan (−36%, P=0.01), phenylalanine (−52%, P=0.002), leucine (−31%, P=0.01) and ornithine (−76%, P=0.001) were significantly decreased in the MNR animals compared to control. In contrast, maternal plasma concentrations of glycine (20%, P=0.04) were increased in the MNR group (n=6, Table 1) compared to control (n=22, Table 1). Fetal plasma concentrations of taurine (−32%, P=0.006), tyrosine (−34%, P=0.001), phenylalanine (−36%, P=0.003), leucine (−32%, P=0.01) and ornithine (−49%, P=0.005) were lower in MNR animals (n=6, Table 2) compared to control (n=22, Table 2).

Table 1.

Maternal plasma amino acid concentrations in control and MNR animals

Amino acid	Control	MNR	P
Aspartic acid	16 ± 1.6	6.3 ± 1.2	0.02
Glutamic acid	81.6 ± 6.2	57.1 ± 9.4	0.05
Asparagine	24.1 ± 1.4	27.1 ± 2.8	0.36
Serine	68.9 ± 2.8	79.9 ± 8.5	0.16
Glutamine	353.3 ± 17.6	345.6 ± 34.2	0.81
Histidine	81.8 ± 3.9	83.1 ± 8.5	0.92
Glycine	230.8 ± 9.4	276.4 ± 20.1	0.04
Threonine	80.3 ± 3.7	68.5 ± 2.1	0.13
Citrulline	13.3 ± 0.9	13.2 ± 2.0	0.98
Arginine	34.4 ± 1.9	41.4 ± 3.4	0.09
β-Alanine	4.6 ± 0.4	5.4 ± 0.7	0.34
Taurine	151.5 ± 10.3	122.3 ± 8.3	0.19
Alanine	177.8 ± 13.3	212.6 ± 19.7	0.12
Tyrosine	36.8 ± 1.9	22.7 ± 2.1	0.001
Tryptophan	28.3 ± 2.0	18.0 ± 1.8	0.01
Methionine	22.3 ± 1.1	18.7 ± 1.6	0.10
Valine	89.6 ± 3.9	89.6 ± 5.6	0.92
Phenylalanine	70.5 ± 5.6	33.8 ± 1.9	0.002
Isoleucine	52.3 ± 3.5	40.3 ± 4.6	0.08
Leucine	78.3 ± 4.8	54.2 ± 7.3	0.01
Ornithine	26.8 ± 3.2	6.5 ± 0.9	0.001
Lysine	140.6 ± 8.0	149.9 ± 11.3	0.46

Amino acid concentrations (μM) were measured at GD 165. Values are given as means ± sem; n = 22 (control) and 6 (MNR).

^*P < 0.05 vs. control; unpaired Student's t test.

Table 2.

Fetal plasma amino acid concentrations in control and MNR animals

Amino acid	Control	MNR	P
Aspartic acid	10.1 ± 1.9	3.7 ± 0.2	0.08
Glutamic acid	81.7 ± 21.6	38.9 ± 5.9	0.22
Asparagine	55.9 ± 4.5	45.2 ± 4.0	0.18
Serine	147.0 ± 7.1	147 ± 9.8	0.90
Glutamine	766.8 ± 40.6	663.6 ± 32.1	0.19
Histidine	144.0 ± 6.5	139.0 ± 10.1	0.76
Glycine	394.0 ± 19.5	442.0 ± 24.7	0.20
Threonine	139.0 ± 8.5	125.0 ± 13.8	0.39
Citrulline	26.8 ± 1.8	32.2 ± 4.0	0.23
Arginine	93.0 ± 5.3	111.0 ± 13.0	0.20
β-Alanine	8.0 ± 1.8	7.0 ± 1.1	0.78
Taurine	206.3 ± 12.1	140.6 ± 17.5	0.006
Alanine	360.5 ± 32.2	403.5 ± 42.5	0.32
Tyrosine	63.0 ± 4	42.0 ± 2.3	0.001
Tryptophan	46.8 ± 1.6	45.9 ± 2.0	0.88
Methionine	45.1 ± 1.8	38.4 ± 2.4	0.06
Valine	169.0 ± 8.0	162.0 ± 10.4	0.71
Phenylalanine	98.6 ± 6.1	63.2 ± 3.5	0.003
Isoleucine	77.7 ± 5.4	63.2 ± 2.8	0.16
Leucine	108.0 ± 8.6	73.6 ± 2.2	0.01
Ornithine	54.8 ± 5.9	27.7 ± 3.0	0.005
Lysine	369.3 ± 23.2	394.5 ± 23.3	0.45

Amino acid concentrations (μM) were measured at GD 165. Values are given as means ± sem; n = 22 (control) and 6 (MNR), unpaired Student's t test.

Inhibition of placental mTORC1 signaling in response to MNR

We determined the total expression and phosphorylation of S6K1 and 4E-BP1 in placental homogenates as functional readouts for mTORC1 activity. Phosphorylation of S6K1 (−78%, P=0.03) and ribosomal protein S6 (RPS6; −36%, P=0.04) was decreased in MNR placentas compared with control (Fig. 1A, B, D). However, total and phosphorylated mTOR (Ser-2448) was not significantly different in control and MNR groups (Supplemental Fig. S2A, B). Total expression of S6K1 and S6 ribosomal protein was unaffected by MNR (Fig. 1A, B, D).

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 1C shows representative Western blots using antibodies directed against 4E-BP1 phosphorylated at Thr-37/46 or at Thr-70. Phosphorylation of 4E-BP1 is hierarchical, in that phosphorylation of Thr-37/46 is required for further phosphorylation at Thr-70. Both phosphorylation at Thr-37/46 (−31%, P=0.007) and at Thr-70 (−30%, P=0.01) was decreased in MNR placentas compared to control (Fig. 1C, D). In contrast, total placental 4E-BP1 expression was comparable between control and MNR groups (Fig. 1C, D).

Inhibition of placental GSK-3 signaling in response to MNR

GSK-3 is regulated by insulin/IGF-I signaling via Akt and phosphorylates glycogen synthase. In the MNR placenta, phosphorylation of GSK-3 at Ser-21/9 was decreased by 42% (P=0.01) compared to control (Fig. 2A, C). However, total GSK-3 expression was comparable between the control and MNR groups (Fig. 2A, C).

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.

Inhibition of placental ERK1/2 signaling in response to MNR

ERK1/2 is the activated mitogen-activated protein kinase (MAPK) in mammals, and activation of ERK1/2 predominantly occurs through mitogenic stimuli, such as growth factors and hormones. In the MNR placenta, phosphorylation of ERK1/2 at (Thr-202/Tyr-204) was decreased by 83% (P=0.005) as compared to control (Fig. 2B, C). In contrast, total ERK1/2 expression was similar in control and MNR groups (Fig. 2A, B).

Inhibition of placental insulin/IGF-I signaling in response to MNR

Placental insulin/IGF-I signaling activity was assessed by determining phosphorylation of IRS-1 at Tyr-612 and Akt at Thr-308. In the MNR placenta, phosphorylation of IRS-1 at Tyr-612 (−68%, P=0.006) and Akt at Thr-308 (−57%, P=0.0005) was decreased compared to control Fig. 3A, B). However, total IRS-1 and Akt expression was similar in control and MNR groups (Fig. 3A, B).

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.

MNR does not alter placental AMPK, LKB, raptor, and tuberin/TSC2 phosphorylation

To test the hypothesis that MNR activates the placental AMPK/LKB/raptor/tuberin-TSC2 signaling pathway, total expression and phosphorylation of AMPK (Thr-172), LKB (Ser-428), raptor (Ser-792), and tuberin-TSC2 (Ser-1387) was assessed by Western blot. AMPK, LKB, raptor, or tuberin-TSC2 phosphorylation was not significantly different in control and MNR groups. Similarly, total AMPK, LKB, raptor, and tuberin-TSC2 expression was unaltered by MNR (Figs. 4 and 5A, B).

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.

Cellular localization of nutrient transporters in the baboon placenta

Using immunohistochemistry, we demonstrate that GLUT-1 is expressed in the syncytiotrophoblast MVM and, to a lesser extent, plasma basal membrane (BM) of baboon placentas from control animals (Fig. 6A). In addition, SNAT-2 (Fig. 6C) and LAT-1 (Fig. 6E) are predominantly expressed in MVMs. No significant staining could be detected in negative control sections (Fig. 6B, D, F).

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).

Down-regulation of placental nutrient transporter expression in response to MNR

Protein expression of system A transporter isoforms SNAT-1, SNAT-2, and SNAT-4 were detected in isolated placental MVMs at ∼50–52 kDa. Protein expression of SNAT-2 in MVMs was reduced (−22%, P=0.03) in MNR placentas compared to control (Fig. 7A, B); however, no changes could be observed in SNAT-1 and SNAT-4 expression levels (Fig. 7A, B). Feeding baboons a MNR diet significantly reduced the expression of the system L transporter isoforms LAT-1 (−28%, P=0.002) and LAT-2 (−53%, P=0.0002) in placental MVMs (Fig. 8A, B). Furthermore, protein expression of GLUT-1 and TAUT in MVMs was decreased (GLUT-1, −46%, P=0.0001, n=8/group; TAUT, −90%, n=8/group; P=0.02) in MNR placentas compared to control (Fig. 8A, B).

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.

DISCUSSION

The effect of maternal undernutrition on placental function has not been studied in pregnant women, and results from previous studies in rodents cannot directly be extrapolated to humans. This is the first study exploring changes in placental signaling and nutrient transporter expression in response to maternal nutrient restriction in a nonhuman primate. We report that maternal nutrient restriction during pregnancy (GD 30 to GD 165) in baboons is associated with an inhibition of insulin/IGF-I and mTOR signaling pathways, decreased expression of key amino acid and glucose transporter isoforms in the placenta, and lower fetal levels of essential amino acids. Because placental insulin/IGF-I and mTOR signaling pathways are known to be positive regulators of placental amino acid transporters, we speculate that the observed changes contribute to the decreased fetal circulating levels of amino acids and reduced fetal growth.

MNR decreased maternal plasma concentrations of aspartate, glutamate, tyrosine, tryptophan, leucine, and phenylalanine at GD 165. The mTOR signaling pathway is especially sensitive to changes in the concentrations of essential amino acids, in particular leucine (55–57). It is, therefore, possible that lower maternal leucine concentrations could contribute to mTOR inhibition in MNR placentas. MNR decreased the fetal plasma levels of the essential amino acids tyrosine, taurine, leucine, and phenylalanine, which may be a result of lower maternal amino acid levels and down-regulation of key placental amino acid transporters.

Phosphorylation is the most significant post-translational modification that reversibly regulates protein function and ultimately cell function. In the current study, we used immunoblotting targeting specific phosphoproteins, which is a powerful approach to assess steady-state protein phosphorylation in tissue. However, protein phosphorylation is a dynamic process regulated by the activity of upstream kinases and phosphatases, and it is recognized that Western blotting in a tissue sample does not provide detailed information on dynamics of protein phosphorylation. Placental signaling pathways linking maternal nutrition and metabolism to changes in nutrient transport may include mTOR, which is regulated by a wide range of factors, including amino acids, glucose, oxygen and energy status, and insulin/IGF-I, leptin, and TNF-α signaling (58). We show that phosphorylation of S6K1, RPS6, and 4E-BP1, representing well-established functional readouts of the mTORC1 signaling pathways, was decreased in placental homogenates of MNR placentas compared to control, consistent with an inhibition of placental mTOR signaling in MNR. In contrast, phosphorylation of mTOR at Ser-2448 was not altered in MNR. The functional significance of mTOR phosphorylation remains to be fully established, and whether mTOR phosphorylation is a positive, negative, or nonconsequential modification is controversial (55, 58). Furthermore, it has been reported that it is S6K1, rather than kinases upstream of mTOR that phosphorylates mTOR at Thr-2446 and Ser-2448 (59, 60). The unchanged phosphorylation at Ser-2448-mTOR despite inhibited S6K1 activity in MNR may be due to other kinases targeting this particular residue.

Placental mTOR signaling is a positive regulator of system A and L activity in cultured primary human trophoblast cells (11, 26, 61, 62). We recently demonstrated that mTOR regulates system A and L amino acid transport activity by modulating cell surface abundance of SNAT-2 and LAT-1 isoforms in cultured primary human trophoblast cells (61), providing one possible mechanism underlying the decrease in MVM system A and L isoform expression in MNR. AMPK is a well-established inhibitor of mTOR signaling. However, AMPK activation is unlikely to explain the observed inhibition in placental mTOR in association with MNR because phosphorylation of both LKB, an upstream regulator of AMPK, and AMPK was not significantly altered in MNR. Further, we report that phospho-raptor (Ser-722/792) and phospho-tuberin TSC2 (Ser-1387) (63), which are targets directly phosphorylated by AMPK, were unaltered in MNR. ERK1/2 has been reported to be a positive regulator of mTOR signaling by inactivation of TSC2 (64) and to directly phosphorylate 4E-BP1 (65). We suggest that the inhibition of the ERK1/2 and insulin/IGF-I signaling pathways that we observed in MNR placentas contribute to decreased mTOR activity and down-regulation of placental nutrient transporters.

We have reported that serum concentrations of IGF-I in pregnant MNR baboons are significantly decreased at GD 90 compared to controls (66). IGF-I (25, 67) and insulin (23, 24) stimulate the activity of trophoblast amino acid transporters (22), in part mediated by mTOR signaling (61). Nutrient restriction in humans and animal models decreases circulating levels of IGF-I and insulin (8, 29, 30). In addition, receptors for most of these hormones are highly expressed on the maternal-facing plasma membrane of the trophoblast cell (51, 68–70). Collectively, these data are consistent with the possibility that the down-regulation of placental amino acid transporters is caused by decreased maternal levels of growth factors such as IGF-I and insulin. In agreement with this hypothesis, we observed a decreased phosphorylation of GSK-3, an insulin/IGF-I target, in MNR placentas. Insulin promotes phosphorylation of GSK-3 at two serine residues, causing inhibition of the enzyme. Consequently, decreased phosphorylation at these sites activates GSK-3, which stimulates glycogen breakdown.

The similarity in subcellular localization of nutrient transporters for glucose and amino acids between the human (20, 71) and baboon placentas suggest that the mechanisms for nutrient delivery to the fetus are similar in the two species. SNAT-2 expression in MVMs, but not that of SNAT-1 or SNAT-4, was down-regulated in MNR. These findings are in agreement with other studies of SNAT isoform regulation in the placenta, suggesting that SNAT-2 is a highly regulated isoform (11, 28). In addition, system L amino acid transporter isoforms LAT-1 and LAT-2 expression in placental MVMs was reduced in MNR. GLUT-1 protein was found to be highly expressed in baboon MVMs, in agreement with the human placenta (71). Interestingly, MVM GLUT-1 protein expression was significantly decreased in MNR. ERK1/2 plays a central role in up-regulating GLUT-1 expression, thereby augmenting glucose transport (72, 73). We suggest that the inhibition of the ERK1/2 signaling pathways observed in MNR placentas contributes to down-regulation of MVM GLUT1 transporter expression. Although we did not measure fetal glucose concentrations in this study, these changes could contribute to decreased placental glucose transport in MNR. We observed a marked decrease in MVM TAUT expression in MNR placentas, and we propose that these changes contribute to the lower plasma taurine levels in the MNR fetus.

Fetal growth is intimately linked to placental nutrient transport. A significant body of evidence demonstrates that the activity of key placental amino acid transporters is decreased in human IUGR due to placental insufficiency (12–15), and both placental amino acid and glucose transporters have been reported to be up-regulated in fetal overgrowth (48, 74, 75). Furthermore, Malandro et al. (6) demonstrated that maternal protein malnutrition in rats results in down-regulation of placental amino acid transporters and IUGR in late gestation. In a more detailed analysis using the same experimental model, we demonstrated that placental amino acid transporters were down-regulated several days before IUGR could be observed, without any sign of compensatory up-regulation earlier in gestation (8, 11). Collectively, these reports and the current study show that the expression of placental nutrient transporters is positively correlated with fetal growth, compatible with the possibility that changes in placental nutrient transport directly contribute to the altered fetal growth. To explain the down-regulation of placental nutrient transport in response to maternal undernutrition and decreased uteroplacental blood flow, we have proposed a model (17) that the placenta responds to maternal nutritional cues, resulting in down-regulation of placental nutrient transporters in response to maternal undernutrition or restricted uteroplacental blood flow. In some cases, these signals dominate over fetal demand signals, fetal nutrient availability becomes limited, and fetal growth decreases. We have proposed that this mechanism matches fetal growth to the ability of the maternal supply line to allocate resources to the fetus. In this model, changes in placental growth and nutrient transport directly contribute to, or cause, alterations in fetal growth. We have proposed that these mechanisms have evolved due to the evolutionary pressures of maternal undernutrition. Matching fetal growth to maternal resources in response to maternal undernutrition will produce an offspring that is smaller in size but which, in most instances, will survive and be able to reproduce (76).

In this study we used a well-established nonhuman primate model in which pregnant baboons are fed 70% of the control diet on a per-kilogram basis to achieve global MNR. These studies are not possible to perform in humans because pregnant women cannot be subjected to experimental MNR. The striking similarities in reproductive physiology and placental structure and the close evolutionary relationship between nonhuman primates and humans contribute to the relevance of these studies. Maternal undernutrition is, together with infections, the most common cause of IUGR in developing countries. In addition, maternal undernutrition is a serious public health problem not only in the developing world, because more than 50 million Americans live in households experiencing food insecurity or hunger at least some time during the year (1). Thus, our nonhuman primate model, involving a 30% maternal calorie reduction throughout most of pregnancy, is highly relevant for human health and disease. Babies who were in utero during the wartime famine in the winter of 1944–1945 in Holland (the Dutch famine cohort) were moderately growth restricted and showed increased incidence of obesity and metabolic and cardiovascular disease in adulthood (77), demonstrating that maternal nutrition during gestation has important effects on health in later life in humans. Although most cases of IUGR in Western societies are related to impaired placental function (e.g., reduced placental blood flow, leading to “placental insufficiency”) rather than maternal undernutrition, the effects of these two distinct perturbations on placental signaling and function are strikingly similar (8, 11, 18, 26). Studies exploring the mechanisms underlying changes in placental function in response to maternal nutrient restriction may, therefore, be relevant also for placental insufficiency. In summary, we propose that inhibition of placental insulin/IGF-I, ERK1/2, and mTOR signaling and down-regulation of the expression of key placental nutrient transporters contribute to decreased fetal nutrient availability and reduced fetal growth in response to MNR in nonhuman primates (Supplemental Fig. S3).