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.

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.

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.

Decreased placental folate transporter expression and activity in first and second trimester in obese mothers.

Obese women have an approximately twofold higher risk to deliver an infant with neural tube defects (NTDs) despite folate supplementation. Placental transfer of folate is mediated by folate receptor alpha (FR-α), proton coupled folate transporter (PCFT), and reduced folate carrier (RFC). Decreased placental transport may contribute to NTDs in obese women. Serum folate levels were measured and placental tissue was collected from 13 women with normal BMI (21.9±1.9) and 11 obese women (BMI 33.1±2.8) undergoing elective termination at 8-22 weeks of gestation. The syncytiotrophoblast microvillous plasma membranes (MVM) were isolated using homogenization, magnesium precipitation, and differential centrifugation. MVM expression of FR-α, PCFT and RFC was determined by western blot. Folate transport capacity was assessed using radiolabeled methyl-tetrahydrofolate and rapid filtration techniques. Differences in expression and transport capacity were adjusted for gestational age and maternal age in multivariable regression models. P<.05 was considered statistically significant. Serum folate levels were not significantly different between groups. Placental MVM folate transporter expression did not change with gestational age. MVM RFC (-19%) and FR-α (-17%) expression was significantly reduced in placentas from obese women (P<.05). MVM folate transporter activity was reduced by-52% (P<.05) in obese women. These differences remained after adjustment for gestational age. There was no difference in mTOR signaling between groups. In conclusion, RFC and FR alpha expression and transporter activity in the placental MVM are significantly reduced in obese women in early pregnancy. These results may explain the higher incidence of NTDs in infants of obese women with adequate serum folate.

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

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

Adiponectin receptor agonist AdipoRon induces apoptotic cell death and suppresses proliferation in human ovarian cancer cells.

We tested the hypothesis that stimulation of adiponectin receptors with the synthetic agonist AdipoRon suppresses proliferation and induces apoptotic death in human high grade serous ovarian tumor cell lines and in ex vivo primary tumors, mediated by activation of 5' AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR). We determined the effect of AdipoRon on high grade serous ovarian tumor cells lines (OVCAR3, OVCAR4, A2780) and ex vivo primary tumor tissue. Western blotting analysis was performed to examine changes in activation of AMPK and mTOR signaling and flow cytometry was utilized to examine changes in cell cycle progression. Immunofluorescence of cleaved caspase-3 positive cells and flow cytometry of annexin V positive cells were used to determine changes in apoptotic response. The CyQUANT proliferation assay was used to assess cell proliferation. AdipoRon treatment increased AMPK phosphorylation (OVCAR3 P = 0.01; A2780 P = 0.02) but did not significantly alter mTOR activity. AdipoRon induced G1 cell cycle arrest in OVCAR3 (+ 12.1%, P = 0.03) and A2780 (+ 12.0%, P = 0.002) cells. OVCAR3 and OVCAR4 cells treated with AdipoRon underwent apoptosis based on cleaved caspase-3 and annexin V staining. AdipoRon treatment resulted in a dose dependent decrease in cell number versus vehicle treatment in OVCAR3 (-61.2%, P < 0.001), OVCAR4 (-79%, P < 0.001), and A2780 (-56.9%, P < 0.001). Ex vivo culture of primary tumors treated with AdipoRon resulted in an increase in apoptosis measured with cleaved caspase-3 immunohistochemistry. AdipoRon induces activation of AMPK and exhibits an anti-tumor effect in ovarian cancer cell lines and primary tumor via a mTOR-independent pathway.

Down-regulation of placental folate transporters in intrauterine growth restriction.

Folate deficiency in pregnancy is associated with neural tube defects, restricted fetal growth and fetal programming of diseases later in life. Fetal folate availability is dependent on maternal folate levels and placental folate transport capacity, mediated by two key transporters, Folate Receptor-α and Reduced Folate Carrier (RFC). We tested the hypothesis that intrauterine growth restriction (IUGR) is associated with decreased folate transporter expression and activity in isolated syncytiotrophoblast microvillous plasma membranes (MVM). Women with pregnancies complicated by IUGR (birth weight <3rd percentile, mean birth weight 1804±110 g, gestational age 35.7±0.61 weeks, n=25) and women delivering an appropriately-for gestational age infant (control group, birth weight 25th-75th centile, mean birth weight 2493±216 g, gestational age 33.9±0.95 weeks, n=19) were recruited and placentas were collected at delivery. MVM was isolated and folate transporter protein expression was measured using Western blot and transporter activity was determined using radiolabelled methyltetrahydrofolic acid and rapid filtration. Whereas the expression of FR-α was unaffected, MVM RFC protein expression was significantly decreased in the IUGR group (-34%, P<.05). IUGR MVM had a significantly lower folate uptake compared to the control group (-38%, P<.05). In conclusion, placental folate transport capacity is decreased in IUGR, which may contribute to the restricted fetal growth and intrauterine programming of childhood and adult disease. These findings suggest that continuation of folate supplementation in the second and third trimester is of particular importance in pregnancies complicated by IUGR.

Diet Enriched with Olive Oil Attenuates Placental Dysfunction in Rats with Gestational Diabetes Induced by Intrauterine Programming.

SCOPE: Offspring from rats with mild diabetes develop gestational diabetes mellitus (GDM). We tested the hypothesis that an olive oil-supplemented diet attenuates placental oxidative stress/inflammation, activation of mTOR signaling, and inhibition of peroxisome proliferator-activated receptor γ (PPARγ) and fetal overgrowth in GDM offspring from mild diabetic rats. METHODS AND RESULTS: Female offspring from rats with mild diabetes (group that developed GDM) and controls were fed with either a standard diet or a 6% olive oil-supplemented diet during pregnancy. On day 21 of pregnancy, plasma glucose levels in mothers and fetuses were increased in the GDM group independently of the diet. Fetal overgrowth and activation of placental mTOR signaling were partially prevented in the olive oil-treated GDM group. Placental PPARγ protein expression was decreased in GDM rats, independently of the diet. However, increases in placental lipoperoxidation, connective tissue growth factor, and matrix metalloproteinase 2 levels were prevented by the olive oil-enriched diet. CONCLUSION: Diets enriched with olive oil attenuate placental dysfunction and fetal overgrowth in rats with GDM induced by intrauterine programming.

Mechanistic Target of Rapamycin Is a Novel Molecular Mechanism Linking Folate Availability and Cell Function.

Folate deficiency has been linked to a wide range of disorders, including cancer, neural tube defects, and fetal growth restriction. Folate regulates cellular function mediated by its involvement in the synthesis of nucleotides, which are needed for DNA synthesis, and its function as a methyl donor, which is critical for DNA methylation. Here we review current data showing that folate sensing by mechanistic target of rapamycin (mTOR) constitutes a novel and distinct pathway by which folate modulates cell functions such as nutrient transport, protein synthesis, and mitochondrial respiration. The mTOR signaling pathway responds to growth factors and changes in nutrient availability to control cell growth, proliferation, and metabolism. mTOR exists in 2 complexes, mTOR complex (mTORC) 1 and mTORC2, which have distinct upstream regulators and downstream targets. Folate deficiency in pregnant mice caused a marked inhibition of mTORC1 and mTORC2 signaling in multiple maternal and fetal tissues, downregulation of placental amino acid transporters, and fetal growth restriction. In addition, folate deficiency in primary human trophoblast (PHT) cells resulted in inhibition of mTORC1 and mTORC2 signaling and decreased the activity of key amino acid transporters. Folate sensing by mTOR in PHT cells is independent of the accumulation of homocysteine and requires the proton-coupled folate transporter (PCFT; solute carrier 46A1). Furthermore, mTORC1 and mTORC2 regulate trophoblast folate uptake by modulating the cell surface expression of folate receptor α and the reduced folate carrier. These findings, which provide a novel link between folate availability and cell function, growth, and proliferation, may have broad biological significance given the critical role of folate in normal cell function and the multiple diseases that have been associated with decreased or excessive folate availability. Low maternal folate concentrations are linked to restricted fetal growth, and we propose that the underlying mechanisms involve trophoblast mTOR folate sensing resulting in inhibition of mTORC1 and mTORC2 and downregulation of placental amino acid transporters.

Maternal folate deficiency causes inhibition of mTOR signaling, down-regulation of placental amino acid transporters and fetal growth restriction in mice.

Maternal folate deficiency is linked to restricted fetal growth, however the underlying mechanisms remain to be established. Here we tested the hypothesis that mTOR functions as a folate sensor in vivo in mice and that maternal folate deficiency inhibits placental mTOR signaling and amino acid transporter activity and causes fetal growth restriction. Folate deficient mice had lower serum folate (-60%). In late pregnancy, fetal weight in the folate deficient group was decreased (-17%, p < 0.05), whereas placental weight, litter size and crown rump length were unaltered. Maternal folate deficiency inhibited placental mTORC1 and mTORC2 signaling and decreased trophoblast plasma membrane System A and L amino acid transporter activities and transporter isoform expression. Folate deficiency also caused a decrease in phosphorylation of specific functional readouts of mTORC1 and mTORC2 signaling in multiple maternal and fetal tissues. We have identified a novel specific molecular link between maternal folate availability and fetal growth, involving regulation of placental mTOR signaling by folate, resulting in changes in placental nutrient transport. mTOR folate sensing may have broad biological significance because of the critical role of folate in normal cell function and the wide range of disorders, including cancer, that have been linked to folate availability.

mTOR folate sensing links folate availability to trophoblast cell function.

KEY POINTS: Folate deficiency during pregnancy is associated with restricted fetal growth, although the underlying mechanisms are poorly understood. Here we show that mechanistic target of rapamycin (mTOR) functions as a folate sensor in primary human trophoblast (PHT) cells. Folate sensing by mTOR in PHT cells involves both mTOR Complex 1 and 2 and requires the proton-coupled folate transporter. We report a previously unknown molecular mechanism by which folate regulates trophoblast cell function. Because mTOR is a positive regulator of placental amino acid transport and mitochondrial function, placental mTOR folate sensing may constitute the mechanistic link between maternal folate status and fetal growth. These findings provide new insight into how folate influences human cell physiology and may have implications for our understanding of how altered folate availability causes diseases such as fetal growth restriction, fetal malformations and cancer. ABSTRACT: Folate is a water-soluble B vitamin that is essential for cellular methylation reactions and DNA synthesis and repair. Low maternal folate levels in pregnancy are associated with fetal growth restriction, but the underlying mechanisms are poorly understood. Mechanistic target of rapamycin (mTOR) links nutrient availability to cell growth and function by regulating gene expression and protein translation. Here we show that mTOR functions as a folate sensor in primary human trophoblast (PHT) cells. Folate deficiency in PHT cells caused inhibition of mTOR signalling and decreased the activity of key amino acid transporters. Folate sensing by mTOR in PHT cells involves both mTOR Complex 1 and 2 and requires the proton-coupled folate transporter (PCFT, SLC46A1). The involvement of PCFT in mTOR folate sensing is not dependent on its function as a plasma membrane folate transporter. Increasing levels of homocysteine had no effect on PHT mTOR signalling, suggesting that mTOR senses low folate rather than high homocysteine. In addition, we demonstrate that maternal serum folate is positively correlated to placental mTORC1 and mTORC2 signalling activity in human pregnancy. We have identified a previously unknown molecular link between folate availability and cell function involving PCFT and mTOR signalling. We propose that mTOR folate sensing in trophoblast cells matches placental nutrient transport, and therefore fetal growth, to folate availability. These findings may have implications for our understanding of how altered folate availability causes human diseases such as fetal growth restriction, fetal malformations and cancer.

Effects of maternal obesity on placental function and fetal development.

Obesity has reached epidemic proportions, and pregnancies in obese mothers have increased risk for complications including gestational diabetes, hypertensive disorders, pre-term birth and caesarian section. Children born to obese mothers are at increased risk of obesity and metabolic disease and are susceptible to develop neuropsychiatric and cognitive disorders. Changes in placental function not only play a critical role in the development of pregnancy complications but may also be involved in linking maternal obesity to long-term health risks in the infant. Maternal adipokines, i.e., interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), leptin and adiponectin link maternal nutritional status and adipose tissue metabolism to placental function. Adipokines and metabolic hormones have direct impact on placental function by modulating placental nutrient transport. Nutrient delivery to the fetus is regulated by a complex interaction including insulin signaling, cytokine profile and insulin responsiveness, which is modulated by adiponectin and IL-1ß. In addition, obese pregnant women are at risk for hypertension and preeclampsia with reduced placental vascularity and blood flow, which would restrict placental nutrient delivery to the developing fetus. These sometimes opposing signals regulating placental function may contribute to the diversity of short and long-term outcomes observed in pregnant obese women. This review focuses on the changes in adipokines and obesity-related metabolic hormones, how these factors influence placental function and fetal development to contribute to long-term metabolic and behavioral consequences of children born to obese mothers.

Mechanistic target of rapamycin (mTOR) regulates trophoblast folate uptake by modulating the cell surface expression of FR-α and the RFC.

Folate deficiency in fetal life is strongly associated with structural malformations and linked to intrauterine growth restriction. In addition, limited availability of methyl donors, such as folate, during pregnancy may result in abnormal gene methylation patterns and contribute to developmental programming. The fetus is dependent on placental transfer of folate, however the molecular mechanisms regulating placental folate transport are unknown. We used cultured primary human trophoblast cells to test the hypothesis that mechanistic target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) regulate folate transport by post-translational mechanisms. Silencing raptor (inhibits mTORC1) or rictor (inhibits mTORC2) markedly decreased basal folate uptake. Folate uptake stimulated by insulin + IGF-1 was mediated by mTORC2 but did not involve mTORC1. mTORC1 or mTORC2 silencing markedly decreased the plasma membrane expression of FR-α and RFC transporter isoforms without affecting global protein expression. Inhibition of the ubiquitin ligase Nedd4-2 had no effect on folate transport. In conclusion, we report for the first time that mTORC1/C2 are positive regulators of cellular folate uptake by modulating the cell surface abundance of specific transporter isoforms. We propose that regulation of placental folate transport by mTOR signaling provide a direct link between placental function, gene methylation and fetal programming.

Adiponectin and IGFBP-1 in the development of gestational diabetes in obese mothers.

OBJECTIVE: Gestational diabetes mellitus (GDM) is more common in pregnancies complicated by obesity and both diseases increase the risk for fetal overgrowth and long-term adverse health consequences for the mother and child. Previous studies have linked low maternal serum adiponectin to GDM in normal and overweight women. We hypothesized that lower adiponectin, in particular the high-molecular-weight form, and insulin-like growth factor I (IGF-I) and its binding protein (IGFBP-1) are associated with GDM in pregnant obese Hispanic women. METHODS: 72 obese, predominantly Hispanic (92%), women were recruited at 24-28 weeks of gestation. Adiposity was assessed, fasting serum samples were collected, and glucose, insulin, triglyceride, cholesterol levels, adipokines, and hormones associated with obesity and insulin resistance were measured. 30 women had been recently diagnosed with GDM. RESULTS: Gestational weeks, body mass index, triceps skinfold thickness, mid-arm circumference, serum leptin, IGF-I, tumor necrosis factor α, and interleukin-6 did not differ in the two groups. Obese women with GDM had significantly higher fasting glucose, A1C, triglycerides, very-low-density lipoprotein cholesterol and lower high-density lipoprotein cholesterol, adiponectin, and IGFBP-1 compared to obese women without GDM. Homeostasis model assessment of insulin resistance was positively correlated to IGF-I and negatively correlated to adiponectin. CONCLUSIONS: Obese pregnant women with recently diagnosed GDM had a significantly exacerbated metabolic profile, low serum adiponectin and IGFBP-1 levels at 24-28 weeks of gestation, as compared to women with obesity alone. Because low adiponectin is well established to cause insulin resistance and decreased IGFBP-1 indicates increased IGF-I bioavailability, we propose that these changes are mechanistically linked to the development of GDM in obese Hispanic women.

Fetal serum folate concentrations and placental folate transport in obese women.

OBJECTIVE: We hypothesized that fetal folate serum concentrations are lower and placental folate transport is impaired in pregnancies of obese women. STUDY DESIGN: Umbilical vein serum and placental tissue were collected from normal weight and obese pregnant women at term. Cellular localization (immunohistochemistry) of folate receptor-α (FR-α), proton coupled folate transporter (PCFT), and reduced folate carrier (RFC) was established. Protein expression (Western blot) and transporter activity (isotope labeled methyltetrahydrofolate) were determined in syncytiotrophoblast microvillus membranes (MVM). RESULTS: Fetal folate concentrations were similar in obese women as compared with normal weight women. Protein expression of FR-α in microvillus membranes was increased (+173%), in RFC was decreased (-41%), and in PCFT was unchanged. However, activity of FR-α, PCFT, and RFC was unaltered in obesity. CONCLUSION: Fetal serum folate concentrations and placental folate transport activity are not altered in obesity at term, which suggests that limited availability of folate does not contribute to abnormal gene methylation and developmental programming.

Maternal protein restriction in the rat inhibits placental insulin, mTOR, and STAT3 signaling and down-regulates placental amino acid transporters.

The mechanisms underlying reduced fetal growth in response to maternal protein restriction are not well established. Maternal levels of insulin, IGF-I, and leptin are decreased in rats fed a low protein (LP) diet. Because these hormones stimulate placental amino acid transporters in vitro, we hypothesized that maternal protein restriction inhibits placental leptin, insulin/IGF-I, and mammalian target of rapamycin signaling and down-regulates the expression and activity of placental amino acid transporters. Pregnant rats were fed either an isocaloric low protein (LP, 4% protein) or control diet (18% protein) and studied at gestational day (GD)15, GD19, or GD21 (term 23). At GD19 and GD21, placental expression of phosphorylated eukaryotic initiation factor 4E binding protein 1 (Thr-36/46 or Thr-70) and phosphorylated S6 ribosomal protein (Ser-235/236) was decreased in the LP group. In addition, placental expression of phosphorylated S6 kinase 1 (Thr-389), phosphorylated Akt (Thr-308), and phosphorylated signal transducer and activator of transcription 3 (Tyr-705) was reduced at GD21. In microvillous plasma membranes (MVM) isolated from placentas of LP animals, protein expression of the sodium-coupled neutral amino acid transporter (SNAT)2 and the large neutral amino acid transporters 1 and 2 was reduced at GD19 and GD21. MVM SNAT1 protein expression was reduced at GD21 in LP rats. SNAT4 and 4F2 heavy chain expression in MVM was unaltered. System A and L amino acid transporter activity was decreased in MVM from LP animals at GD19 and GD21. In conclusion, maternal protein restriction inhibits placental insulin, mammalian target of rapamycin signaling, and signal transducer and activator of transcription 3 signaling, which is associated with a down-regulation of placental amino acid transporters. We speculate that maternal endocrine and metabolic control of placental nutrient transport reduces fetal growth in response to protein restriction.

Mammalian target of rapamycin in the human placenta regulates leucine transport and is down-regulated in restricted fetal growth.

Pathological fetal growth is associated with perinatal morbidity and the development of diabetes and cardiovascular disease later in life. Placental nutrient transport is a primary determinant of fetal growth. In human intrauterine growth restriction (IUGR) the activity of key placental amino acid transporters, such as systems A and L, is decreased. However the mechanisms regulating placental nutrient transporters are poorly understood. We tested the hypothesis that the mammalian target of rapamycin (mTOR) signalling pathway regulates amino acid transport in the human placenta and that the activity of the placental mTOR pathway is reduced in IUGR. Using immunohistochemistry and culture of trophoblast cells, we show for the first time that the mTOR protein is expressed in the transporting epithelium of the human placenta. We further demonstrate that placental mTOR regulates activity of the l-amino acid transporter, but not system A or taurine transporters, by determining the mediated uptake of isotope-labelled leucine, methylaminoisobutyric acid and taurine in primary villous fragments after inhibition of mTOR using rapamycin. The protein expression of placental phospho-S6K1 (Thr-389), a measure of the activity of the mTOR signalling pathway, was markedly reduced in placentas obtained from pregnancies complicated by IUGR. These data identify mTOR as an important regulator of placental amino acid transport, and provide a mechanism for the changes in placental leucine transport in IUGR previously demonstrated in humans. We propose that mTOR functions as a placental nutrient sensor, matching fetal growth with maternal nutrient availability by regulating placental nutrient transport.