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.

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.

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.

Supplementation with polyunsaturated fatty acids in pregnant rats with mild diabetes normalizes placental PPARγ and mTOR signaling in female offspring developing gestational diabetes.

Maternal diabetes impairs fetoplacental development and programs metabolic diseases in the offspring. We have previously reported that female offspring of pregnant rats with mild diabetes develop gestational diabetes mellitus (GDM) when they become pregnant. Here, we studied the effects of supplementation with polyunsaturated fatty acids (PUFAs) in pregnant mild diabetic rats (F0) by feeding a 6% safflower-oil-enriched diet from day 1 to 14 followed by a 6% chia-oil-enriched diet from day 14 of pregnancy to term. We analyzed maternal metabolic parameters and placental signaling at term in pregnant offspring (F1). The offspring of both PUFAs-treated and untreated mild diabetic rats developed GDM. Although gestational hyperglycemia was not prevented by dietary PUFAs treatment in F0, triglyceridemia and cholesterolemia in F1 mothers were normalized by F0 PUFAs dietary treatment. In the placenta of F1 GDM rats, PPARγ levels were reduced and lipoperoxidation was increased, changes that were prevented by the maternal diets enriched in PUFAs in the F0 generation. Moreover, fetal overgrowth and placental activation of mTOR signaling pathways were reduced in F1 GDM rats whose mothers were treated with PUFAs diets. These results suggest that F0 PUFAs dietary treatment in pregnancies with mild diabetes improves maternal dyslipidemia, fetal overgrowth and placental signaling in female offspring when they become pregnant. We speculate that an increased PUFAs intake in pregnancies complicated by diabetes may prove effective to ameliorate metabolic programming in the offspring, thereby improving the health of future generations.

Maternal testosterone and placental function: Effect of electroacupuncture on placental expression of angiogenic markers and fetal growth.

Women with polycystic ovary syndrome (PCOS) have elevated circulating androgens during pregnancy and are at an increased risk of adverse pregnancy outcomes. Here we tested the hypotheses that maternal androgen excess decrease placental and fetal growth, and placental expression of markers of steroidogenesis, angiogenesis and sympathetic activity, and that acupuncture with low-frequency electrical stimulation prevents these changes. Pregnant rats were exposed to vehicle or testosterone on gestational day (GD)15-19. Low-frequency electroacupuncture (EA) or handling, as a control for the EA procedure, was given to control or testosterone exposed dams on GD16-20. On GD21, blood pressure was measured and maternal blood, fetuses and placentas collected. Placental steroid receptor expression and proteins involved in angiogenic, neurotrophic and adrenergic signaling were analyzed. EA did not affect any variables in control rats except maternal serum corticosterone, which was reduced. EA in testosterone exposed dams compared with controls increased systolic pressure by 30%, decreased circulating norepinephrine and corticosterone, fetal and placental weight and placental VEGFR1 and proNGF protein expression, and increased the VEGFA/VEGFR1 ratio, mature NGF (mNGF) and the mNGF/proNGF ratio. In conclusion, low-frequency EA in control animals did not have any negative influence on any of the studied variables. In contrast, EA in pregnant dams exposed to testosterone increased blood pressure and impaired placental growth and function, leading to decreased fetal growth.

Maternal taurine supplementation in the late pregnant rat stimulates postnatal growth and induces obesity and insulin resistance in adult offspring.

An adequate supply of taurine during fetal life is important for normal beta-cell development and insulin action. An altered availability of taurine may programme glucose metabolism in utero and result in type 2 diabetes in adult age. We examined whether maternal taurine supplementation in late pregnant rats affects postnatal growth, adult body composition, insulin sensitivity and endogenous insulin secretion in intrauterine growth restricted (IUGR) and normal offspring. Uterine artery ligation or sham operations were performed on gestational day (GD) 19. Taurine supplementation was given to half of the dams from GD 18 until term, resulting in four groups of offspring: sham (n = 22), sham/taurine (n = 22), IUGR (n = 22) and IUGR/taurine (n = 24). The offspring were studied at 12 weeks of age. In offspring with normal birth weight, fetal taurine supplementation markedly stimulated postnatal growth. In sham/taurine females, fat depots, plasma free fatty acid and leptin concentrations were increased, and insulin sensitivity was reduced. Insulin sensitivity was unaltered in IUGR and IUGR/taurine offspring. However, whereas IUGR offspring showed little catch-up growth, 50% of IUGR/taurine animals displayed complete catch-up at 12 weeks of age, and these animals had increased fat depots and reduced insulin sensitivity. In conclusion, taurine supplementation in late gestation resulted in accelerated postnatal growth, which was associated with adult obesity and insulin resistance in both IUGR and normal offspring. This effect was particularly evident in females. These data suggest that fetal taurine availability is an important determinant for postnatal growth, insulin sensitivity and fat accumulation.