Maternal-fetal cross-talk via the placenta: influence on offspring development and metabolism.

Compelling epidemiological and animal experimental data demonstrate that cardiometabolic and neuropsychiatric diseases originate in a suboptimal intrauterine environment. Here, we review evidence suggesting that altered placental function may, at least in part, mediate the link between the maternal environment and changes in fetal growth and development. Emerging evidence indicates that the placenta controls the development and function of several fetal tissues through nutrient sensing, modulation of trophoblast nutrient transporters and by altering the number and cargo of released extracellular vesicles. In this Review, we discuss the development and functions of the maternal-placental-fetal interface (in humans and mice) and how cross-talk between these compartments may be a mechanism for in utero programming, focusing on mechanistic target of rapamycin (mTOR), adiponectin and O-GlcNac transferase (OGT) signaling. We also discuss how maternal diet and stress influences fetal development and metabolism and how fetal growth restriction can result in susceptibility to developing chronic disease later in life. Finally, we speculate how interventions targeting placental function may offer unprecedented opportunities to prevent cardiometabolic disease in future generations.

Long-term impact of maternal obesity on the gliovascular unit and ephrin signaling in the hippocampus of adult offspring.

BACKGROUND: Children born to obese mothers are at increased risk of developing mood disorders and cognitive impairment. Experimental studies have reported structural changes in the brain such as the gliovascular unit as well as activation of neuroinflammatory cells as a part of neuroinflammation processing in aged offspring of obese mothers. However, the molecular mechanisms linking maternal obesity to poor neurodevelopmental outcomes are not well established. The ephrin system plays a major role in a variety of cellular processes including cell-cell interaction, synaptic plasticity, and long-term potentiation. Therefore, in this study we determined the impact of maternal obesity in pregnancy on cortical, hippocampal development, vasculature and ephrin-A3/EphA4-signaling, in the adult offspring in mice. METHODS: Maternal obesity was induced in mice by a high fat/high sugar Western type of diet (HF/HS). We collected brain tissue (prefrontal cortex and hippocampus) from 6-month-old offspring of obese and lean (control) dams. Hippocampal volume, cortical thickness, myelination of white matter, density of astrocytes and microglia in relation to their activity were analyzed using 3-D stereological quantification. mRNA expression of ephrin-A3, EphA4 and synaptic markers were measured by qPCR in the brain tissue. Moreover, expression of gap junction protein connexin-43, lipocalin-2, and vascular CD31/Aquaporin 4 were determined in the hippocampus by immunohistochemistry. RESULTS: Volume of hippocampus and cortical thickness were significantly smaller, and myelination impaired, while mRNA levels of hippocampal EphA4 and post-synaptic density (PSD) 95 were significantly lower in the hippocampus in the offspring of obese dams as compared to offspring of controls. Further analysis of the hippocampal gliovascular unit indicated higher coverage of capillaries by astrocytic end-feet, expression of connexin-43 and lipocalin-2 in endothelial cells in the offspring of obese dams. In addition, offspring of obese dams demonstrated activation of microglia together with higher density of cells, while astrocyte cell density was lower. CONCLUSION: Maternal obesity affects brain size, impairs myelination, disrupts the hippocampal gliovascular unit and decreases the mRNA expression of EphA4 and PSD-95 in the hippocampus of adult offspring. These results indicate that the vasculature-glia cross-talk may be an important mediator of altered synaptic plasticity, which could be a link between maternal obesity and neurodevelopmental/neuropsychiatric disorders in the offspring.

Overexpression of the LAT1 in primary human trophoblast cells increases the uptake of essential amino acids and activates mTOR signaling.

The System L amino acid transporter, particularly the isoform Large Neutral Amino Acid Transporter Small Subunit 1 (LAT1) encoded by SLC7A5, is believed to mediate the transfer of essential amino acids in the human placenta. Placental System L amino acid transporter expression and activity is decreased in pregnancies complicated by IUGR and increased in fetal overgrowth. However, it remains unknown if changes in the expression of LAT1 are mechanistically linked to System L amino acid transport activity. Here, we combined overexpression approaches with protein analysis and functional studies in cultured primary human trophoblast (PHT) cells to test the hypothesis that SLC7A5 overexpression increases the uptake of essential amino acids and activates mTOR signaling in PHT cells. Overexpression of SLC7A5 resulted in a marked increase in protein expression of LAT1 in the PHT cells microvillous plasma membrane and System L amino acid transporter activity. Moreover, mTOR signaling was activated, and System A amino acid transporter activity increased following SLC7A5 overexpression, suggesting coordination of trophoblast amino transporter expression and activity to ensure balanced nutrient flux to the fetus. This is the first report showing that overexpression of LAT1 is sufficient to increase the uptake of essential amino acids in PHT cells, which activates mTOR, a master regulator of placental function. The decreased placental System L activity in human IUGR and the increased placental activity of this transporter system in some cases of fetal overgrowth may directly contribute to changes in fetal amino acid availability and altered fetal growth in these pregnancy complications.

Maternal glucagon-like peptide-1 is positively associated with fetal growth in pregnancies complicated with obesity.

Pregnant women with obesity are more likely to deliver infants who are large for gestational age (LGA). LGA is associated with increased perinatal morbidity and risk of developing metabolic disease later in life. However, the mechanisms underpinning fetal overgrowth remain to be fully established. Here, we identified maternal, placental, and fetal factors that are associated with fetal overgrowth in pregnant women with obesity. Maternal and umbilical cord plasma and placentas were collected from women with obesity delivering infants who were LGA (n=30) or appropriate for gestational age (AGA, n=21) at term. Maternal and umbilical cord plasma analytes were measured using multiplex sandwich assay and ELISA. Insulin/mechanistic target of rapamycin (mTOR) signaling activity was determined in placental homogenates. Amino acid transporter activity was measured in isolated syncytiotrophoblast microvillous membrane (MVM) and basal membrane (BM). Glucagon-like peptide-1 receptor (GLP-1R) protein expression and signaling were measured in cultured primary human trophoblast (PHT) cells. Maternal plasma glucagon-like peptide-1 (GLP-1) was higher in LGA pregnancies and positively correlated to birthweight. Umbilical cord plasma insulin, C-peptide, and GLP-1 were increased in obese-large for gestational age (OB-LGA) infants. LGA placentas were larger but showed no change in insulin/mTOR signaling or amino acid transport activity. GLP-1R protein was expressed in the MVM isolated from human placenta. GLP-1R activation stimulated protein kinase alpha (PKA), extracellular signal-regulated kinase-1 and-2 (ERK1/2), and mTOR pathways in PHT cells. Our results suggest elevated maternal GLP-1 may drive fetal overgrowth in obese pregnant women. We speculate that maternal GLP-1 acts as a novel regulator of fetal growth by promoting placental growth and function.

Normalization of maternal adiponectin in obese pregnant mice prevents programming of impaired glucose metabolism in adult offspring.

Infants born to obese mothers have a greater risk for childhood obesity and insulin resistance. However, the underlying biological mechanism remains elusive, which constitutes a significant roadblock for developing specific prevention strategies. Maternal adiponectin levels are lower in obese pregnant women, which is linked with increased placental nutrient transport and fetal overgrowth. We have previously reported that adiponectin supplementation to obese dams during the last four days of pregnancy prevented the development of obesity, glucose intolerance, muscle insulin resistance, and fatty liver in three months old offspring. In the present study, we tested the hypothesis that 6-9-month-old offspring of obese dams show glucose intolerance associated with muscle insulin resistance and mitochondrial dysfunction and that normalization of maternal adiponectin in obese pregnant mice prevents the development of this phenotype in the offspring. Male and female offspring of obese mice exhibited in vivo glucose intolerance and insulin resistance at 6 and 9 months of age. In gastrocnemius muscles ex vivo, male and female offspring of obese dams showed reduced phosphorylation of insulin receptor substrate 1Tyr-608 , AktThr-308 , and decreased Glut4 plasma membrane translocation upon insulin stimulation. These metabolic abnormalities in offspring born to obese mice were largely prevented by normalization of maternal adiponectin levels in late pregnancy. We provide evidence that low circulating maternal adiponectin is a critical mechanistic link between maternal obesity and the development of metabolic disease in offspring. Strategies aimed at improving maternal adiponectin levels may prevent long-term metabolic dysfunction in offspring of obese mothers.

Placental Remote Control of Fetal Metabolism: Trophoblast mTOR Signaling Regulates Liver IGFBP-1 Phosphorylation and IGF-1 Bioavailability.

The mechanisms mediating the restricted growth in intrauterine growth restriction (IUGR) remain to be fully established. Mechanistic target of rapamycin (mTOR) signaling functions as a placental nutrient sensor, indirectly influencing fetal growth by regulating placental function. Increased secretion and the phosphorylation of fetal liver IGFBP-1 are known to markedly decrease the bioavailability of IGF-1, a major fetal growth factor. We hypothesized that an inhibition of trophoblast mTOR increases liver IGFBP-1 secretion and phosphorylation. We collected conditioned media (CM) from cultured primary human trophoblast (PHT) cells with a silenced RAPTOR (specific inhibition of mTOR Complex 1), RICTOR (inhibition of mTOR Complex 2), or DEPTOR (activates both mTOR Complexes). Subsequently, HepG2 cells, a well-established model for human fetal hepatocytes, were cultured in CM from PHT cells, and IGFBP-1 secretion and phosphorylation were determined. CM from PHT cells with either mTORC1 or mTORC2 inhibition caused the marked hyperphosphorylation of IGFBP-1 in HepG2 cells as determined by 2D-immunoblotting while Parallel Reaction Monitoring-Mass Spectrometry (PRM-MS) identified increased dually phosphorylated Ser169 + Ser174. Furthermore, using the same samples, PRM-MS identified multiple CK2 peptides coimmunoprecipitated with IGFBP-1 and greater CK2 autophosphorylation, indicating the activation of CK2, a key enzyme mediating IGFBP-1 phosphorylation. Increased IGFBP-1 phosphorylation inhibited IGF-1 function, as determined by the reduced IGF-1R autophosphorylation. Conversely, CM from PHT cells with mTOR activation decreased IGFBP-1 phosphorylation. CM from non-trophoblast cells with mTORC1 or mTORC2 inhibition had no effect on HepG2 IGFBP-1 phosphorylation. Placental mTOR signaling may regulate fetal growth by the remote control of fetal liver IGFBP-1 phosphorylation.

Maternal obesity causes fetal cardiac hypertrophy and alters adult offspring myocardial metabolism in mice.

Obesity in pregnant women causes fetal cardiac dysfunction and increases offspring cardiovascular disease risk, but its effect on myocardial metabolism is unknown. We hypothesized that maternal obesity alters fetal cardiac expression of metabolism-related genes and shifts offspring myocardial substrate preference from glucose towards lipids. Female mice were fed control or obesogenic diets before and during pregnancy. Fetal hearts were studied in late gestation (embryonic day (E) 18.5; term ≈ E21), and offspring were studied at 3, 6, 9 or 24 months postnatally. Maternal obesity increased heart weight and peroxisome proliferator activated receptor gamma (Pparg) expression in female and male fetuses and caused left ventricular diastolic dysfunction in the adult offspring. Cardiac dysfunction worsened progressively with age in female, but not male, offspring of obese dams, in comparison to age-matched control animals. In 6-month-old offspring, exposure to maternal obesity increased cardiac palmitoyl carnitine-supported mitochondrial respiration in males and reduced myocardial 18 F-fluorodeoxyglucose uptake in females. Cardiac Pparg expression remained higher in adult offspring of obese dams than control dams and was correlated with contractile and metabolic function. Maternal obesity did not affect cardiac palmitoyl carnitine respiration in females or 18 F-fluorodeoxyglucose uptake in males and did not alter cardiac 3 H-oleic acid uptake, pyruvate respiration, lipid content or fatty acid/glucose transporter abundance in offspring of either sex. The results support our hypothesis and show that maternal obesity affects offspring cardiac metabolism in a sex-dependent manner. Persistent upregulation of Pparg expression in response to overnutrition in utero might underpin programmed cardiac impairments mechanistically and contribute to cardiovascular disease risk in children of women with obesity. KEY POINTS: Obesity in pregnant women causes cardiac dysfunction in the fetus and increases lifelong cardiovascular disease risk in the offspring. In this study, we showed that maternal obesity in mice induces hypertrophy of the fetal heart in association with altered expression of genes related to nutrient metabolism. Maternal obesity also alters cardiac metabolism of carbohydrates and lipids in the adult offspring. The results suggest that overnutrition in utero might contribute to increased cardiovascular disease risk in children of women with obesity.

Transcriptomic responses are sex-dependent in the skeletal muscle and liver in offspring of obese mice.

Infants born to obese mothers are more likely to develop metabolic disease, including glucose intolerance and hepatic steatosis, in adult life. We examined the effects of maternal obesity on the transcriptome of skeletal muscle and liver tissues of the near-term fetus and 3-mo-old offspring in mice born to dams fed a high-fat and -sugar diet. Previously, we have shown that male, but not female, offspring develop glucose intolerance, insulin resistance, and liver steatosis at 3 mo old. Female C57BL6/J mice were fed normal chow or an obesogenic high-calorie diet before mating and throughout pregnancy. RNAseq was performed on the liver and gastrocnemius muscle following collection from fetuses on embryonic day 18.5 (E18.5) as well as from 3-mo-old offspring from obese dams and control dams. Significant genes were generated for each sex, queried for enrichment, and modeled to canonical pathways. RNAseq was corroborated by protein quantification in offspring. The transcriptomic response to maternal obesity in the liver was more marked in males than females. However, in both male and female offspring of obese dams, we found significant enrichment for fatty acid metabolism, mitochondrial transport, and oxidative stress in the liver transcriptomes as well as decreased protein concentrations of electron transport chain members. In skeletal muscle, pathway analysis of gene expression revealed sexual dimorphic patterns, including metabolic processes of fatty acids and glucose, as well as PPAR, AMPK, and PI3K-Akt signaling pathways. Transcriptomic responses to maternal obesity in skeletal muscle were more marked in female offspring than males. Female offspring had greater expression of genes associated with glucose uptake, and protein abundance reflected greater activation of mTOR signaling. Skeletal muscle and livers in mice born to obese dams had sexually dimorphic transcriptomic responses that changed from the fetus to the adult offspring. These data provide insights into mechanisms underpinning metabolic programming in maternal obesity.NEW & NOTEWORTHY Transcriptomic data support that fetuses of obese mothers modulate metabolism in both muscle and liver. These changes were strikingly sexually dimorphic in agreement with published findings that male offspring of obese dams exhibit pronounced metabolic disease earlier. In both males and females, the transcriptomic responses in the fetus were different than those at 3 mo, implicating adaptive mechanisms throughout adulthood.

Placenta-derived proteins across gestation in healthy pregnancies-a novel approach to assess placental function?

BACKGROUND: Placenta-derived proteins in the systemic maternal circulation are suggested as potential biomarkers for placental function. However, the identity and longitudinal patterns of such proteins are largely unknown due to the inaccessibility of the human placenta and limitations in assay technologies. We aimed to identify proteins derived from and taken up by the placenta in the maternal circulation. Furthermore, we aimed to describe the longitudinal patterns across gestation of placenta-derived proteins as well as identify placenta-derived proteins that can serve as reference curves for placental function. METHODS: We analyzed proteins in plasma samples collected in two cohorts using the Somalogic 5000-plex platform. Antecubital vein samples were collected at three time points (gestational weeks 14-16, 22-24, and 30-32) across gestation in 70 healthy pregnancies in the longitudinal STORK cohort. In the cross sectional 4-vessel cohort, blood samples were collected simultaneously from the maternal antecubital vein (AV), radial artery (RA), and uterine vein (UV) during cesarean section in 75 healthy pregnancies. Placenta-derived proteins and proteins taken up by the placenta were identified using venoarterial differences (UV-RA). Placenta-derived proteins were defined as placenta-specific by comparison to the venoarterial difference in the antecubital vein-radial artery (AV-RA). These proteins were described longitudinally based on the STORK cohort samples using a linear mixed effects model per protein. Using a machine learning algorithm, we identified placenta-derived proteins that could predict gestational age, meaning that they closely tracked gestation, and were potential read-outs of placental function. RESULTS: Among the nearly 5000 measured proteins, we identified 256 placenta-derived proteins and 101 proteins taken up by the placenta (FDR < 0.05). Among the 256 placenta-derived proteins released to maternal circulation, 101 proteins were defined as placenta-specific. These proteins formed two clusters with distinct developmental patterns across gestation. We identified five placenta-derived proteins that closely tracked gestational age when measured in the systemic maternal circulation, termed a "placental proteomic clock." CONCLUSIONS: Together, these data may serve as a first step towards a reference for the healthy placenta-derived proteome that can be measured in the systemic maternal circulation and potentially serve as biomarkers of placental function. The "placental proteomic clock" represents a novel concept that warrants further investigation. Deviations in the proteomic pattern across gestation of such proteomic clock proteins may serve as an indication of placental dysfunction.

Maternal Diet Quality Is Associated with Placental Proteins in the Placental Insulin/Growth Factor, Environmental Stress, Inflammation, and mTOR Signaling Pathways: The Healthy Start ECHO Cohort.

BACKGROUND: Maternal nutritional status affects placental function, which may underlie the intrauterine origins of obesity and diabetes. The extent to which diet quality is associated with placental signaling and which specific pathways are impacted is unknown. OBJECTIVES: To examine sex-specific associations of maternal diet quality according to the Healthy Eating Index (HEI)-developed to align with recommendations from the Dietary Guidelines for Americans-with placental proteins involved in metabolism and mediators of environmental stress, inflammation, and growth factors. METHODS: Among 108 women from the Healthy Start cohort with a mean ± SD age of 29.0 ± 6.1 y and a prepregnancy BMI (in kg/m2) of 24.8 ± 5.3, we conducted multivariable linear regression analysis stratified by offspring sex. We adjusted for maternal race or ethnicity, age, education, prenatal smoking habits, and physical activity and tested for an association of maternal HEI >57 compared with ≤57 and the abundance and phosphorylation of key proteins involved in insulin/growth factor signaling; mediators of environmental stress, inflammation, and growth factors; mechanistic target of rapamycin signaling proteins; and energy sensing in placental villus samples. HEI >57 was chosen given its prior relevance among Healthy Start mother-child dyads. RESULTS: In adjusted models, HEI >57 was associated with greater abundance of insulin receptor ß (0.80; 95% CI: 0.11, 1.49) in placentas of females. In males, maternal HEI >57 was associated with greater activation and abundance of select placental nutrient-sensing proteins and environmental stress, inflammation, and growth factor proteins (S6K1Thr389/S6K1: 0.81; 95% CI: 0.21, 1.41; JNK1Thr183/Tyr185/JNK1: 0.82; 95% CI: 0.27, 1.37; JNK2Thr183/Tyr185/JNK2: 0.57; 95% CI: 0.02, 1.11). CONCLUSIONS: Higher-quality diet had sex-specific associations with placental protein abundance/phosphorylation. Given that these proteins have been correlated with neonatal anthropometry, our findings provide insight into modifiable factors and placental pathways that should be examined in future studies as potential links between maternal diet and offspring metabolic health. This trial was registered at clinicaltrials.gov as NCT02273297.

A novel technique using chronic infusion of small extracellular vesicles from gestational diabetes mellitus causes glucose intolerance in pregnant mice.

Small extracellular vesicles (sEVs) play a central role in cell-to-cell communication in normal physiology and in disease, including gestational diabetes mellitus (GDM). The goal of the present study was to test the hypothesis that chronic administration of sEVs isolated from GDM causes glucose intolerance in healthy pregnant mice. Small EVs were isolated from plasma between 24 and 28 weeks gestation from healthy pregnant women (controls) and GDM, and infused intravenously for 4 days in late pregnant mice using a mini-osmotic pump. Subsequently in vivo glucose tolerance was assessed, and muscle and adipose tissue insulin sensitivity and islet glucose stimulated insulin secretion (GSIS) were determined in vitro. Mice infused with sEVs from GDM developed glucose intolerance. Administration of sEVs from controls, but not sEVs from GDM women, stimulated islet GSIS and increased fasting insulin levels in pregnant mice. Neither infusion of sEVs from controls nor from GDM women affected muscle insulin sensitivity, placental insulin or mTOR signaling, placental and fetal weight. Moreover, these results were not associated with immunomodulatory effects as human sEVs did not activate mouse T cells in vitro. We suggest that circulating sEVs regulate maternal glucose homeostasis in pregnancy and may contribute to the attenuated islet insulin secretion and more pronounced glucose intolerance in GDM as compared with healthy pregnancy.

Mechanisms linking hypoxia to phosphorylation of insulin-like growth factor binding protein-1 in baboon fetuses with intrauterine growth restriction and in cell culture.

Hypoxia increases fetal hepatic insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation mediated by mechanistic target of rapamycin (mTOR) inhibition. Whether maternal nutrient restriction (MNR) causes fetal hypoxia remains unclear. We used fetal liver from a baboon (Papio sp.) model of intrauterine growth restriction due to MNR (70% global diet of Control) and liver hepatocellular carcinoma (HepG2) cells as a model for human fetal hepatocytes and tested the hypothesis that mTOR-mediated IGFBP-1 hyperphosphorylation in response to hypoxia requires hypoxia-inducible factor-1α (HIF-1α) and regulated in development and DNA-damage responses-1 (REDD-1) signaling. Western blotting (n = 6) and immunohistochemistry (n = 3) using fetal liver indicated greater expression of HIF-1α, REDD-1 as well as erythropoietin and its receptor, and vascular endothelial growth factor at GD120 (GD185 term) in MNR versus Control. Moreover, treatment of HepG2 cells with hypoxia (1% pO2 ) (n = 3) induced REDD-1, inhibited mTOR complex-1 (mTORC1) activity and increased IGFBP-1 secretion/phosphorylation (Ser101/Ser119/Ser169). HIF-1α inhibition by echinomycin or small interfering RNA silencing prevented the hypoxia-mediated inhibition of mTORC1 and induction of IGFBP-1 secretion/phosphorylation. dimethyloxaloylglycine (DMOG) induced HIF-1α and also REDD-1 expression, inhibited mTORC1 and increased IGFBP-1 secretion/phosphorylation. Induction of HIF-1α (DMOG) and REDD-1 by Compound 3 inhibited mTORC1, increased IGFBP-1 secretion/ phosphorylation and protein kinase PKCα expression. Together, our data demonstrate that HIF-1α induction, increased REDD-1 expression and mTORC1 inhibition represent the mechanistic link between hypoxia and increased IGFBP-1 secretion/phosphorylation. We propose that maternal undernutrition limits fetal oxygen delivery, as demonstrated by increased fetal liver expression of hypoxia-responsive proteins in baboon MNR. These findings have important implications for our understanding of the pathophysiology of restricted fetal growth.

Placental proteins with predicted roles in fetal development decrease in premature infants.

BACKGROUND: Emerging evidence from animal experiments indicate that factors secreted by the placenta are critical for normal fetal organ development. Our objective was to characterize the umbilical vein and artery proteome in preterm infants and identify proteins that decrease in the neonatal circulation following delivery. METHODS: Cord blood at delivery and neonatal blood at 48-72 h of life was collected in 25 preterm infants. Plasma protein abundance was determined using the SomaLogic platform. RESULTS: When comparing protein levels of umbilical venous to arterial cord blood, 434 proteins were significantly higher indicating placental secretion into the fetal circulation. Moreover, when comparing neonatal blood to umbilical vein levels, 142 proteins were significantly lower. These proteins included Endoplasmic reticulum resident protein 29, CD59, Fibroblast growth factor 2 and Dynactin subunit 2, which are involved in brain development and prevention of brain damage as well as Fibroblast growth factor 1 which prevents lung fibrosis. CONCLUSIONS: The late second trimester human placenta secretes proteins into the fetal circulation which decrease following delivery. Many of these proteins are predicted to be important in the development of fetal organs. Further studies are needed to directly link placental proteins to organ development and poor outcomes in preterm infants. IMPACT: Prematurity remains a leading cause of morbidity and mortality requiring the development of novel treatments. Emerging evidence from animal studies suggest that factors secreted from the placenta may be critical in the development of the fetus. We report that the preterm human placenta secretes an array of proteins into the fetal circulation. Some of these proteins are predicted to be involved in the development of the brain and the lung. When born prematurely, infants are deprived of these placental proteins, which may contribute to their poor outcomes.

Inhibition of MTOR signaling impairs rat embryo organogenesis by affecting folate availability.

Mechanistic target of rapamycin (MTOR) is essential for embryo development by acting as a nutrient sensor to regulate cell growth, proliferation and metabolism. Folate is required for normal embryonic development and it was recently reported that MTOR functions as a folate sensor. In this work, we tested the hypothesis that MTOR functions as a folate sensor in the embryo and its inhibition result in embryonic developmental delay affecting neural tube closure and that these effects can be rescued by folate supplementation. Administration of rapamycin (0.5 mg/kg) to rats during early organogenesis inhibited embryonic ribosomal protein S6, a downstream target of MTOR Complex1, markedly reduced embryonic folate incorporation (-84%, P < 0.01) and induced embryo developmental impairments, as shown by an increased resorption rate, reduced embryo somite number and delayed neural tube closure. These alterations were prevented by folic acid administered to the dams. Differently, although an increased rate of embryonic rotation defects was observed in the rapamycin-treated dams, this alteration was not prevented by maternal folic acid supplementation. In conclusion, MTOR inhibition during organogenesis in the rat resulted in decreased folate levels in the embryo, increased embryo resorption rate and impaired embryo development. These data suggest that MTOR signaling influences embryo folate availability, possibly by regulating the transfer of folate across the maternal-embryonic interface.

Placenta-specific Slc38a2/SNAT2 knockdown causes fetal growth restriction in mice.

Fetal growth restriction (FGR) is a complication of pregnancy that reduces birth weight, markedly increases infant mortality and morbidity and is associated with later-life cardiometabolic disease. No specific treatment is available for FGR. Placentas of human FGR infants have low abundance of sodium-coupled neutral amino acid transporter 2 (Slc38a2/SNAT2), which supplies the fetus with amino acids required for growth. We determined the mechanistic role of placental Slc38a2/SNAT2 deficiency in the development of restricted fetal growth, hypothesizing that placenta-specific Slc38a2 knockdown causes FGR in mice. Using lentiviral transduction of blastocysts with a small hairpin RNA (shRNA), we achieved 59% knockdown of placental Slc38a2, without altering fetal Slc38a2 expression. Placenta-specific Slc38a2 knockdown reduced near-term fetal and placental weight, fetal viability, trophoblast plasma membrane (TPM) SNAT2 protein abundance, and both absolute and weight-specific placental uptake of the amino acid transport System A tracer, 14C-methylaminoisobutyric acid (MeAIB). We also measured human placental SLC38A2 gene expression in a well-defined term clinical cohort and found that SLC38A2 expression was decreased in late-onset, but not early-onset FGR, compared with appropriate for gestational age (AGA) control placentas. The results demonstrate that low placental Slc38a2/SNAT2 causes FGR and could be a target for clinical therapies for late-onset FGR.

Regulation of glucose homeostasis by small extracellular vesicles in normal pregnancy and in gestational diabetes.

The mechanisms underpinning maternal metabolic adaptations to a healthy pregnancy and in gestational diabetes mellitus (GDM) remain poorly understood. We hypothesized that small extracellular vesicles (sEVs) isolated from healthy pregnant women promote islet glucose-stimulated insulin secretion (GSIS) and peripheral insulin resistance in nonpregnant mice and that sEVs from GDM women fail to stimulate insulin secretion and cause exacerbated insulin resistance. Small EVs were isolated from plasma of nonpregnant, healthy pregnant, and GDM women at 24-28 weeks of gestation. We developed a novel approach in nonpregnant mice involving a mini-osmotic pump for continuous 4-day jugular venous infusion of sEVs and determined their effects on glucose tolerance in vivo and islets and skeletal muscle in vitro. Fasting insulin was elevated in mice infused with pregnant sEVs as compared to sEVs from nonpregnant and GDM women. Mice infused with sEVs from GDM women developed glucose intolerance. GSIS was increased in mice infused with healthy pregnancy sEVs compared to mice receiving nonpregnant sEVs. GSIS and muscle basal insulin signaling, and insulin responsiveness were attenuated in mice infused with GDM sEVs. sEVs represent a novel mechanism regulating maternal glucose homeostasis in pregnancy and we speculate that altered sEV content contributes to the development of GDM.

Reduced Na+ K+ -ATPase activity may reduce amino acid uptake in intrauterine growth restricted fetal sheep muscle despite unchanged ex vivo amino acid transporter activity.

KEY POINTS: Fetuses with intrauterine growth restriction (IUGR) have reduced muscle mass that persists postnatally, which may contribute to their increased risk for adult onset metabolic diseases, such as diabetes and obesity. Amino acid transporter-mediated histidine uptake and system L amino acid transporter activity were similar in sarcolemmal membranes isolated from control and IUGR hindlimb skeletal muscle. Activity of Na+ K+ -ATPase, which is responsible for establishing the sodium gradient necessary for system A and N amino acid transporter function, was significantly reduced in IUGR skeletal muscle sarcolemma compared to control. ATP content was lower in IUGR skeletal muscle. Expression and phosphorylation of proteins in the mechanistic target of rapamycin pathway were similar in control and IUGR skeletal muscle homogenate. Our data suggest that lower Na+ K+ -ATPase activity, which reduces the driving force for active amino acid transport, and lower ATP availability contribute to reduced amino acid uptake and protein synthesis in IUGR fetal skeletal muscle. ABSTRACT: Fetuses with intrauterine growth restriction (IUGR) have lower muscle mass that persists postnatally. Using a sheep model of placental insufficiency and IUGR, we have previously demonstrated lower net total uptake of amino acids by the fetal hindlimb and lower skeletal muscle protein synthesis rates. To investigate the mechanisms underlying these changes, we tested the hypothesis that ex vivo amino acid transporter and Na+ K+ -ATPase activity is reduced, and ex vivo ATP levels are lower in hindlimb skeletal muscle of the IUGR fetus. We developed a novel protocol to measure transporter-mediated histidine uptake, system L amino acid transporter activity and Na+ K+ -ATPase activity using sarcolemmal membranes isolated from hindlimb muscle of control (CON, n = 11-12) and IUGR (n = 12) late gestation fetal sheep. We also determined ATP content and the activity of insulin and mechanistic target of rapamycin (mTOR) signalling, which are involved in regulating cellular amino acid uptake and protein synthesis, by measuring the expression and phosphorylation of AKT, 4E-BP1, eIF2α, AMPKα, p70 S6 kinase and rpS6 in muscle homogenates. Transporter-mediated histidine uptake and system L activity were similar in control and IUGR sarcolemma, although ex vivo Na+ K+ -ATPase activity was lower by 64% (P = 0.019) in IUGR sarcolemma. ATP content was lower by 25% (P = 0.007) in IUGR muscle. Insulin, AMPK, and mTOR signalling activity was similar in control and IUGR muscle. We speculate that reduced muscle sarcolemmal Na+ K+ -ATPase activity and lower ATP content diminishes the sodium gradient in vivo, resulting in a reduced driving force for sodium-dependent transporters and subsequently lower muscle amino acid uptake.

Placental fatty acid transport across late gestation in a baboon model of intrauterine growth restriction.

KEY POINTS: Intrauterine growth restriction (IUGR) is associated with perinatal morbidity and increased risk of lifelong disease, including neurodevelopmental impairment. Fatty acids (FA) are critical for normal brain development, although their transport across the placenta in IUGR pregnancies is poorly understood. The present study used a baboon model of IUGR (maternal nutrient restriction, MNR) to investigate placental expression of FA transport and binding proteins, and to determine gestational age-related changes in maternal and fetal plasma FA concentrations. We found MNR to be associated with increased placental expression of FA binding and transport proteins in late gestation, with fetal plasma FA concentrations that were similar to those of control animals. The present study is the first to report a profile of fetal and maternal plasma FA concentrations in a baboon model of growth restriction with data that suggest adaptation of placental transport to maintain delivery of critically needed FA. ABSTRACT: Intrauterine growth restriction (IUGR) is associated with specific changes in placental transport of amino acids, folate and ions. However, little is known about placental fatty acid (FA) transport in IUGR. We hypothesized that placental FA transport proteins (FATP) and FA binding proteins (FABP) are up-regulated and fetal plasma FA concentrations are decreased at term in a baboon model of IUGR. Pregnant baboons were fed control or maternal nutrient restricted (MNR) diet (70% of control calories) from gestation day (GD) 30 (term 184 days). Plasma and placental samples were collected at GD120 (control n = 8, MNR n = 9), GD140 (control n = 6, MNR n = 7) and GD170 (control n = 6, MNR n = 6). Placentas were homogenized, and syncytiotrophoblast microvillous plasma membrane (MVM) and basal plasma membranes (BM) were isolated. Protein expression of FABP1, 3, 4 and 5 (homogenate) and FATP2, 4, and 6 (MVM, BM) was determined by Western blotting. FA content in maternal and umbilical vein plasma was measured by gas chromatography-mass spectrometry. Placental FABP1 and FABP5 expression was increased in MNR compared to controls at GD170, as was MVM FATP2 and FATP6 expression at GD140 and FATP2 expression at GD170. BM FATP4 and FATP6 expression was increased in MNR at GD140. Fetal plasma FA concentrations were similar in controls and MNR. These data suggest the adaptation of placental transport when aiming to maintain delivery of critically needed FAs for fetal growth and brain development.

Hyperphosphorylation of fetal liver IGFBP-1 precedes slowing of fetal growth in nutrient-restricted baboons and may be a mechanism underlying IUGR.

In cultured fetal liver cells, insulin-like growth factor (IGF) binding protein (IGFBP)-1 hyperphosphorylation in response to hypoxia and amino acid deprivation is mediated by inhibition of mechanistic target of rapamycin (mTOR) and activation of amino acid response (AAR) signaling and casein kinase (CK)2. We hypothesized that fetal liver mTOR inhibition, activation of AAR and CK2, and IGFBP-1 hyperphosphorylation occur before development of intrauterine growth restriction (IUGR). Pregnant baboons were fed a control (C) or a maternal nutrient restriction (MNR; 70% calories of control) diet starting at gestational day (GD) 30 (term GD 185). Umbilical blood and fetal liver tissue were obtained at GD 120 (C, n = 7; MNR, n = 10) and 165 (C, n = 7; MNR, n = 8). Fetal weights were unchanged at GD 120 but decreased at GD 165 in the MNR group (-13%, P = 0.03). IGFBP-1 phosphorylation, as determined by parallel reaction monitoring mass spectrometry (PRM-MS), immunohistochemistry, and/or Western blot, was enhanced in MNR fetal liver and umbilical plasma at GD 120 and 165. IGF-I receptor autophosphorylationTyr1135 (-64%, P = 0.05) was reduced in MNR fetal liver at GD 120. Furthermore, fetal liver CK2 (α/α'/ß) expression, CK2ß colocalization, proximity with IGFBP-1, and CK2 autophosphorylationTyr182 were greater at GD 120 and 165 in MNR vs. C. Additionally, mTOR complex (mTORC)1 (p-P70S6KThr389, -52%, P = 0.05) and mTORC2 (p-AktSer473, -56%, P < 0.001) activity were decreased and AAR was activated (p-GCN2Thr898, +117%, P = 0.02; p-eIF2αSer51, +294%, P = 0.002; p-ERKThr202, +111%, P = 0.03) in MNR liver at GD 120. Our data suggest that fetal liver IGFBP-1 hyperphosphorylation, mediated by mTOR inhibition and both AAR and CK2 activation, is a key link between restricted nutrient and oxygen availability and the development of IUGR.

Normalisation of circulating adiponectin levels in obese pregnant mice prevents cardiac dysfunction in adult offspring.

BACKGROUND/OBJECTIVES: Adiponectin concentrations are low in obese pregnant women. Restoring normal adiponectin concentrations by infusion in obese pregnant mice prevents placental dysfunction, foetal overgrowth and metabolic syndrome in the offspring. We hypothesised that normalising maternal adiponectin in obese late pregnant dams prevents cardiac dysfunction in the adult offspring. SUBJECTS/METHODS: Pregnant female mice with diet-induced obesity were infused with adiponectin (0.62 µg g-1 day-1, n = 24) or saline (n = 22) over days 14.5-18.5 of pregnancy (term = day 19.5). Control dams ate standard chow and received saline (n = 22). Offspring were studied at 3 and 6 months of age. RESULTS: Maternal obesity impaired ventricular diastolic function, increased cardiomyocyte cross-sectional area and upregulated cardiac brain natriuretic peptide (Nppb) and α-skeletal actin (Acta1) gene expression in adult male offspring, compared to control offspring. In adult female offspring, maternal obesity increased Nppb expression, decreased end-diastolic volume and caused age-dependent diastolic dysfunction but not cardiomyocyte hypertrophy. Maternal obesity also activated cardiac Akt and mechanistic target of rapamycin (mTOR) signalling in male, but not in female, offspring and inhibited cardiac extracellular signal-regulated kinase 1/2 (ERK1/2) in both sexes. Normalising maternal circulating adiponectin concentrations by infusing obese dams with adiponectin prevented offspring diastolic dysfunction and ventricular dilation and normalised cardiac Akt-mTOR signalling irrespective of sex. Maternal adiponectin infusion also reduced cardiac Nppb expression and increased ERK1/2 signalling in offspring of obese dams. Adiponectin infusion did not prevent cardiomyocyte hypertrophy but reduced ventricular wall thickness in male offspring and increased collagen content in female offspring of obese dams, compared to controls. CONCLUSIONS: Low maternal adiponectin levels in obese mice in late pregnancy are mechanistically linked to in utero programming of cardiac dysfunction in their offspring. Interventions enhancing endogenous adiponectin secretion or signalling in obese pregnant women could prevent the development of cardiac dysfunction in their children.