Regulation of placental amino acid transport in health and disease.

Abnormal fetal growth, i.e., intrauterine growth restriction (IUGR) or fetal growth restriction (FGR) and fetal overgrowth, is associated with increased perinatal morbidity and mortality and is strongly linked to the development of metabolic and cardiovascular disease in childhood and later in life. Emerging evidence suggests that changes in placental amino acid transport may contribute to abnormal fetal growth. This review is focused on amino acid transport in the human placenta, however, relevant animal models will be discussed to add mechanistic insights. At least 25 distinct amino acid transporters with different characteristics and substrate preferences have been identified in the human placenta. Of these, System A, transporting neutral nonessential amino acids, and System L, mediating the transport of essential amino acids, have been studied in some detail. Importantly, decreased placental Systems A and L transporter activity is strongly associated with IUGR and increased placental activity of these two amino acid transporters has been linked to fetal overgrowth in human pregnancy. An array of factors in the maternal circulation, including insulin, IGF-1, and adiponectin, and placental signaling pathways such as mTOR, have been identified as key regulators of placental Systems A and L. Studies using trophoblast-specific gene targeting in mice have provided compelling evidence that changes in placental Systems A and L are mechanistically linked to altered fetal growth. It is possible that targeting specific placental amino acid transporters or their upstream regulators represents a novel intervention to alleviate the short- and long-term consequences of abnormal fetal growth in the future.

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.

Oleic acid stimulation of amino acid uptake in primary human trophoblast cells is mediated by phosphatidic acid and mTOR signaling.

Normal fetal development is critically dependent on optimal nutrient supply by the placenta, and placental amino acid transport has been demonstrated to be positively associated with fetal growth. Mechanistic target of rapamycin (mTOR) is a positive regulator of placental amino acid transporters, such as System A. Oleic acid (OA) has been previously shown to have a stimulatory role on placental mTOR signaling and System A amino acid uptake in primary human trophoblast (PHT) cells. We investigated the mechanistic link between OA and System A activity in PHT. We found that inhibition of mTOR complex 1 or 2, using small interfering RNA to knock down raptor or rictor, prevented OA-stimulated System A amino acid transport indicating the interaction of OA with mTOR. Phosphatidic acid (PA) is a key intermediary for phospholipid biosynthesis and a known regulator of the mTOR pathway; however, phospholipid biosynthetic pathways have not been extensively studied in placenta. We identified placental isoforms of acyl transferase enzymes involved in de novo phospholipid synthesis. Silencing of 1-acylglycerol-3-phosphate-O-acyltransferase-4, an enzyme in this pathway, prevented OA mediated stimulation of mTOR and System A amino acid transport. These data indicate that OA stimulates mTOR and amino acid transport in PHT cells mediated through de novo synthesis of PA. We speculate that fatty acids in the maternal circulation, such as OA, regulate placental functions critical for fetal growth by interaction with mTOR and that late pregnancy hyperlipidemia may be critical for increasing nutrient transfer to the fetus.

Cord blood DNA methylation of immune and lipid metabolism genes is associated with maternal triglycerides and child adiposity.

OBJECTIVE: Fetal exposures may impact offspring epigenetic signatures and adiposity. The authors hypothesized that maternal metabolic traits associate with cord blood DNA methylation, which, in turn, associates with child adiposity. METHODS: Fasting serum was obtained in 588 pregnant women (27-34 weeks' gestation), and insulin, glucose, high-density lipoprotein cholesterol, triglycerides, and free fatty acids were measured. Cord blood DNA methylation and child adiposity were measured at birth, 4-6 months, and 4-6 years. The association of maternal metabolic traits with DNA methylation (429,246 CpGs) for differentially methylated probes (DMPs) and regions (DMRs) was tested. The association of the first principal component of each DMR with child adiposity was tested, and mediation analysis was performed. RESULTS: Maternal triglycerides were associated with the most DMPs and DMRs of all traits tested (261 and 198, respectively, false discovery rate < 0.05). DMRs were near genes involved in immune function and lipid metabolism. Triglyceride-associated CpGs were associated with child adiposity at 4-6 months (32 CpGs) and 4-6 years (2 CpGs). One, near CD226, was observed at both timepoints, mediating 10% and 22% of the relationship between maternal triglycerides and child adiposity at 4-6 months and 4-6 years, respectively. CONCLUSIONS: DNA methylation may play a role in the association of maternal triglycerides and child adiposity.

Knockdown of Placental Major Facilitator Superfamily Domain Containing 2a in Pregnant Mice Reduces Fetal Brain Growth and Phospholipid Docosahexaenoic Acid Content.

INTRODUCTION: Docosahexaenoic acid (DHA) is an n-3 long chain polyunsaturated fatty acid critical for fetal brain development that is transported to the fetus from the mother by the placenta. The lysophosphatidylcholine (LPC) transporter, Major Facilitator Superfamily Domain Containing 2a (MFSD2a), is localized in the basal plasma membrane of the syncytiotrophoblast of the human placenta, and MFSD2a expression correlates with umbilical cord blood LPC-DHA levels in human pregnancy. We hypothesized that placenta-specific knockdown of MFSD2a in pregnant mice reduces phospholipid DHA accumulation in the fetal brain. METHODS: Mouse blastocysts (E3.5) were transduced with an EGFP-expressing lentivirus containing either an shRNA targeting MFSD2a or a non-coding sequence (SCR), then transferred to pseudopregnant females. At E18.5, fetuses were weighed and their placenta, brain, liver and plasma were collected. MFSD2a mRNA expression was determined by qPCR in the brain, liver and placenta and phospholipid DHA was quantified by LC-MS/MS. RESULTS: MFSD2a-targeting shRNA reduced placental mRNA MFSD2a expression by 38% at E18.5 (n = 45, p < 0.008) compared with SCR controls. MFSD2a expression in the fetal brain and liver were unchanged. Fetal brain weight was reduced by 13% (p = 0.006). Body weight, placenta and liver weights were unaffected. Fetal brain phosphatidyl choline and phosphatidyl ethanolamine DHA content was lower in fetuses with placenta-specific MFSD2a knockdown. CONCLUSIONS: Placenta-specific reduction in expression of the LPC-DHA transporter MFSD2a resulted in reduced fetal brain weight and lower phospholipid DHA content in the fetal brain. These data provide mechanistic evidence that placental MFSD2a mediates maternal-fetal transfer of LPC-DHA, which is critical for brain growth.

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-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.

Fetal sex differences in placental LCPUFA ether and plasmalogen phosphatidylethanolamine and phosphatidylcholine contents in pregnancies complicated by obesity.

BACKGROUND: We have previously reported that maternal obesity reduces placental transport capacity for lysophosphatidylcholine-docosahexaenoic acid (LPC-DHA), a preferred form for transfer of DHA (omega 3) to the fetal brain, but only in male fetuses. Phosphatidylethanolamine (PE) and phosphatidylcholine (PC), have either sn-1 ester, ether or vinyl ether (plasmalogen) linkages to primarily unsaturated and monounsaturated fatty acids and DHA or arachidonic acid (ARA, omega 6) in the sn-2 position. Whether ether and plasmalogen PC and PE metabolism in placenta impacts transfer to the fetus is unexplored. We hypothesized that ether and plasmalogen PC and PE containing DHA and ARA are reduced in maternal-fetal unit in pregnancies complicated by obesity and these differences are dependent on fetal sex. METHODS: In maternal, umbilical cord plasma and placentas from obese women (11 female/5 male infants) and normal weight women (9 female/7 male infants), all PC and PE species containing DHA and ARA were analyzed by LC-MS/MS. Placental protein expression of enzymes involved in phospholipid synthesis, were determined by immunoblotting. All variables were compared between control vs obese groups and separated by fetal sex, in each sample using the Benjamini-Hochberg false discovery rate adjustment to account for multiple testing. RESULTS: Levels of ester PC containing DHA and ARA were profoundly reduced by 60-92% in male placentas of obese mothers, while levels of ether and plasmalogen PE containing DHA and ARA were decreased by 51-84% in female placentas. PLA2G4C abundance was lower in male placentas and LPCAT4 abundance was lower solely in females in obesity. In umbilical cord, levels of ester, ether and plasmalogen PC and PE with DHA were reduced by 43-61% in male, but not female, fetuses of obese mothers. CONCLUSIONS: We found a fetal sex effect in placental PE and PC ester, ether and plasmalogen PE and PC containing DHA in response to maternal obesity which appears to reflect an ability of female placentas to adapt to maintain optimal fetal DHA transfer in maternal obesity.

Docosahexaenoic acid (DHA) is a critical omega 3 long chain polyunsaturated fatty acid (LCPUFA) for fetal brain development. We have recently reported that maternal obesity reduces placental transport capacity for LysophosPhatidylCholine-DHA (LPC-DHA), a preferred form for transfer of DHA to the fetal brain, but only in male fetuses. Other important lipids, the plasmalogen phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are considered DHA reservoirs, but its roles in the maternal­fetal unit are largely unexplored. We examined these lipid species in maternal and fetal circulation and in placental tissue to uncover potential novel roles for ether and plasmalogen lipids in the regulation of placenta delivery of these vital nutrients in pregnancies complicated by obesity depending of fetal sex. We demonstrated for the first time, that female fetuses of obese mothers decrease placental ether and plasmalogen PE containing DHA and arachidonic acid (ARA, omega 6), and show a high fetal­placental adaptability and placental reserve capacity that can maintain the PC-LCPUFA synthesis and the transfer of these crucial species to the fetus to preserve brain development. Our study also demonstrated that male fetuses, in response to maternal obesity, reduce the placental ester PC species containing DHA and ARA and reduce the ether and plasmalogen PE reservoir of DHA and ARA in fetal circulation. Our findings support a fetal sex effect in placental ester, ether and plasmalogen PE and PC containing DHA in response to maternal obesity which appears to reflect an ability of female placentas to adapt to maintain optimal fetal DHA transfer in maternal obesity.

Maternal obesity alters the placental transcriptome in a fetal sex-dependent manner.

Infants born to obese mothers have an increased risk of developing obesity and metabolic diseases in childhood and adulthood. Although the molecular mechanisms linking maternal obesity during pregnancy to the development of metabolic diseases in offspring are poorly understood, evidence suggests that changes in the placental function may play a role. Using a mouse model of diet-induced obesity with fetal overgrowth, we performed RNA-seq analysis at embryonic day 18.5 to identify genes differentially expressed in the placentas of obese and normal-weight dams (controls). In male placentas, 511 genes were upregulated and 791 genes were downregulated in response to maternal obesity. In female placentas, 722 genes were downregulated and 474 genes were upregulated in response to maternal obesity. The top canonical pathway downregulated in maternal obesity in male placentas was oxidative phosphorylation. In contrast, sirtuin signaling, NF-kB signaling, phosphatidylinositol, and fatty acid degradation were upregulated. In female placentas, the top canonical pathways downregulated in maternal obesity were triacylglycerol biosynthesis, glycerophospholipid metabolism, and endocytosis. In contrast, bone morphogenetic protein, TNF, and MAPK signaling were upregulated in the female placentas of the obese group. In agreement with RNA-seq data, the expression of proteins associated with oxidative phosphorylation was downregulated in male but not female placentas of obese mice. Similarly, sex-specific changes in the protein expression of mitochondrial complexes were found in placentas collected from obese women delivering large-for-gestational-age (LGA) babies. In conclusion, maternal obesity with fetal overgrowth differentially regulates the placental transcriptome in male and female placentas, including genes involved in oxidative phosphorylation.

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.

Novel Metabolic Subtypes in Pregnant Women and Risk of Early Childhood Obesity in Offspring.

Importance: The in utero metabolic milieu is associated with offspring adiposity. Standard definitions of maternal obesity (according to prepregnancy body mass index [BMI]) and gestational diabetes (GDM) may not be adequate to capture subtle yet important differences in the intrauterine environment that could be involved in programming. Objectives: To identify maternal metabolic subgroups during pregnancy and to examine associations of subgroup classification with adiposity traits in their children. Design, Setting, and Participants: This cohort study included mother-offspring pairs in the Healthy Start prebirth cohort (enrollment: 2010-2014) recruited from University of Colorado Hospital obstetrics clinics in Aurora, Colorado. Follow-up of women and children is ongoing. Data were analyzed from March to December 2022. Exposures: Metabolic subtypes of pregnant women ascertained by applying k-means clustering on 7 biomarkers and 2 biomarker indices measured at approximately 17 gestational weeks: glucose, insulin, Homeostatic Model Assessment for Insulin Resistance, total cholesterol, high-density lipoprotein cholesterol (HDL-C), triglycerides, free fatty acids (FFA), HDL-C:triglycerides ratio, and tumor necrosis factor α. Main Outcomes and Measures: Offspring birthweight z score and neonatal fat mass percentage (FM%). In childhood at approximately 5 years of age, offspring BMI percentile, FM%, BMI in the 95th percentile or higher, and FM% in the 95th percentile or higher. Results: A total of 1325 pregnant women (mean [SD] age, 27.8 [6.2 years]; 322 [24.3%] Hispanic, 207 non-Hispanic Black [15.6%], and 713 [53.8%] non-Hispanic White), and 727 offspring with anthropometric data measured in childhood (mean [SD] age 4.81 [0.72] years, 48% female) were included. We identified the following 5 maternal metabolic subgroups: reference (438 participants), high HDL-C (355 participants), dyslipidemic-high triglycerides (182 participants), dyslipidemic-high FFA (234 participants), and insulin resistant (IR)-hyperglycemic (116 participants). Compared with the reference subgroup, women in the IR-hyperglycemic and dyslipidemic-high FFA subgroups had offspring with 4.27% (95% CI, 1.94-6.59) and 1.96% (95% CI, 0.45-3.47) greater FM% during childhood, respectively. There was a higher risk of high FM% among offspring of the IR-hyperglycemic (relative risk, 8.7; 95% CI, 2.7-27.8) and dyslipidemic-high FFA (relative risk, 3.4; 95% CI, 1.0-11.3) subgroups; this risk was of greater magnitude compared with prepregnancy obesity alone, GDM alone, or both conditions. Conclusions and Relevance: In this cohort study, an unsupervised clustering approach revealed distinct metabolic subgroups of pregnant women. These subgroups exhibited differences in risk of offspring adiposity in early childhood. Such approaches have the potential to refine understanding of the in utero metabolic milieu, with utility for capturing variation in sociocultural, anthropometric, and biochemical risk factors for offspring adiposity.

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.

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.

Increased Colocalization and Interaction Between Decidual Protein Kinase A and Insulin-like Growth Factor-Binding Protein-1 in Intrauterine Growth Restriction.

Increased phosphorylation of decidual insulin-like growth factor-binding protein-1 (IGFBP-1) can contribute to intrauterine growth restriction (IUGR) by decreasing the bioavailability of insulin-like growth factor-1 (IGF-1). However, the molecular mechanisms regulating IGFBP-1 phosphorylation at the maternal-fetal interface are poorly understood. Protein kinase A (PKA) is required for normal decidualization. Consensus sequences for PKA are present in IGFBP-1. We hypothesized that the expression/interaction of PKA with decidual IGFBP-1 is increased in IUGR. Parallel reaction monitoring-mass spectrometry (PRM-MS) identified multiple PKA peptides (n=>30) co-immunoprecipitating with IGFBP-1 in decidualized primary human endometrial stromal cells (HESC). PRM-MS also detected active PKApThr197 and greater site-specific IGFBP-1 phosphorylation(pSer119), (pSer98+pSer101) (pSer169+pSer174) in response to hypoxia. Hypoxia promoted colocalization [dual immunofluorescence (IF)] of PKA with IGFBP-1 in decidualized HESC. Colocalization (IF) and interaction (proximity ligation assay) of PKA and IGFBP-1 were increased in decidua collected from placenta of human IUGR pregnancies (n=8) compared with decidua from pregnancies with normal fetal growth. Similar changes were detected in decidual PKA/IGFBP-1 using placenta from baboons subjected to maternal nutrient reduction (MNR) vs controls (n=3 each). In baboons, these effects were evident in MNR at gestational day 120 prior to IUGR onset. Increased PKA-mediated phosphorylation of decidual IGFBP-1 may contribute to decreased IGF-1 bioavailability in the maternal-fetal interface in IUGR.

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.

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.

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.

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.

Insulin Increases Adipose Adiponectin in Pregnancy by Inhibiting Ubiquitination and Degradation: Impact of Obesity.

CONTEXT: Circulating adiponectin levels are decreased in pregnant women with obesity or gestational diabetes, and this is believed to contribute to the insulin resistance and increased risk of fetal overgrowth associated with these conditions. However, the molecular mechanisms regulating adiponectin secretion from maternal adipose tissues in pregnancy are poorly understood. OBJECTIVE: We tested the hypothesis that obesity in pregnancy is associated with adipose tissue insulin resistance and increased adiponectin ubiquitination and degradation, caused by inflammation and endoplasmic reticulum (ER) stress. METHODS: Visceral adipose tissues were collected from lean and obese pregnant humans and mice. Total and ubiquitinated adiponectin, and markers of inflammation, ER stress, and insulin resistance were examined in adipose tissues. The role of insulin, inflammation, and ER stress in mediating adiponectin ubiquitination and degradation was examined using 3T3L-1 adipocytes. RESULTS: Obesity in pregnancy is associated with adipose tissue inflammation, ER stress, insulin resistance, increased adiponectin ubiquitination, and decreased total abundance of adiponectin. Adiponectin ubiquitination was increased in visceral fat of obese pregnant women as compared to lean pregnant women. We further observed that insulin prevents, whereas ER stress and inflammation promote, adiponectin ubiquitination and degradation in differentiated 3T3-L1 adipocytes. CONCLUSION: We have identified adiponectin ubiquitination as a key mechanism by which obesity diminishes adiponectin secretion in pregnancy. This information will help us better understand the mechanisms controlling maternal insulin resistance and fetal growth in pregnancy and may provide a foundation for the development of strategies aimed at improving adiponectin production in pregnant women with obesity or gestational diabetes.