Placental mTOR Research Articles

Methionine supply during mid-gestation modulates the bovine placental mTOR pathway, nutrient transporters, and offspring birth weight in a sex-specific manner.

The mechanistic target of rapamycin (mTOR) predominantly regulates the expression and activity of placental nutrient transporters. The mTOR pathway can be activated by several nutrients, including the essential amino acid methionine. Additionally, previous research in nonruminant animals suggests that mTOR is influenced in a sexually dimorphic manner. In bovine, there is limited understanding of how maternal nutrition and offspring sexual dimorphism affect the placental transfer of nutrients. Thus, we investigated the effects of increasing the supply of dietary methionine to beef cattle heifers during mid-gestation on mTOR signaling, placental nutrient transporters, and fetal growth in male and female offspring. Forty purebred Angus heifers were used in a randomized complete block design experiment. From day 90 to 180 of gestation, heifers received a basal diet with no added methionine (CON, n = 20), or the basal diet plus 8.3g of rumen-protected methionine (MET, n = 20) per animal daily. All animals received a basal diet in the first and third trimesters of gestation. Cotyledonary tissue samples were collected at parturition and utilized to examine the mTOR pathway and nutrient transporters through protein and gene expression analysis. The offspring's body weight was measured at birth. Data were analyzed using a mixed model that included the fixed effect of treatment, offspring sex, their interactions, and the random effect of block. At day 170 of gestation, MET-supplemented heifers showed higher plasma concentrations of methionine and glutamate (P < 0.01) and lower glycine and proline levels (P ≤ 0.01) compared to the CON group. A treatment × sex interaction was observed for calf birth weight (P = 0.03). In heifers that delivered male calves, MET supplementation increased the birth weight of the calves (P < 0.01). However, the dietary treatments had no effect on the birth weight of female calves (P = 0.32). The increase in birth weight of male calves from MET-fed heifers resembles with the upregulation of placental mTOR and phosphorylated mTOR (P ≤ 0.03), as well as the amino acid transporters SLC1A5, SLC7A5, SLC38A6, and SLC38A11, and the glucose transporters SLC2A1 and SLC2A8 (P ≤ 0.05). Our findings suggest that increasing the supply of methionine to beef heifers during mid-gestation can modulate placental nutrient transport and fetal growth in a sex-dependent manner and that these effects are mediated, at least in part, by the mTOR pathway.

Maternal Docosahexaenoic Acid Supplementation Alters Maternal and Fetal Docosahexaenoic Acid Status and Placenta Phospholipids in Pregnancies Complicated by High Body Mass Index.

An optimal fetal supply of docosahexaenoic acid (DHA) is critical for normal brain development. The relationship between maternal DHA intake and DHA delivery to the fetus is complex and is dependent on placental handling of DHA. Little data exist on placental DHA levels in pregnancies supplemented with the recommended dose of 200 mg/d. Our objective was to determine how prenatal DHA at the recommended 200 mg/d impacts maternal, placental, and fetal DHA status in both normal-weight and high-BMI women compared to women taking no supplements. Maternal blood, placenta, and cord blood were collected from 30 healthy pregnant women (BMI 18.9-43.26 kg/m2) giving birth at term. Red blood cells (RBCs) and villous tissue were isolated, and lipids were extracted to determine DHA content by LC-MS/MS. Data were analyzed by supplement group (0 vs. 200 mg/d) and maternal BMI (normal weight or high BMI) using two-way ANOVA. We measured maternal choline levels in maternal and cord plasma samples. Supplementation with 200 mg/d DHA significantly increased (p < 0.05) maternal and cord RBC DHA content only in pregnancies complicated by high BMI. We did not find any impact of choline levels on maternal or cord RBC phospholipids. There were no significant differences in total placental DHA content by supplementation or maternal BMI (p > 0.05). Placental levels of phosphatidylinositol (PI) and phosphatidic acid containing DHA species were higher (p < 0.05) in high-BMI women without DHA supplementation compared to both normal-BMI and high-BMI women taking DHA supplements. Maternal DHA supplementation at recommended doses cord increased RBC DHA content only in pregnancies complicated by higher BMI. Surprisingly, we found that obesity was related to an increase in placental PI and phosphatidic acid species, which was ameliorated by DHA supplementation. Phosphatidic acid activates placental mTOR, which regulates amino acid transport and may explain previous findings of the impact of DHA on placental function. Current recommendations for DHA supplementation may not be achieving the goal of improving fetal DHA levels in normal-weight women.

Fetal growth restriction exhibits various mTOR signaling in different regions of mouse placentas with altered lipid metabolism

Fetal growth restriction (FGR) is a common complication of pregnancy and can have significant impact on obstetric and neonatal outcomes. Increasing evidence has shown that the inhibited mechanistic target of rapamycin (mTOR) signaling in placenta is associated with FGR. However, interpretation of existing research is limited due to inconsistent methodologies and varying understanding of the mechanism by which mTOR activity contributes to FGR. Hereby, we have demonstrated that different anatomic regions of human and mouse placentas exhibited different levels of mTOR activity in normal compared to FGR pregnancies. When using the rapamycin-induced FGR mouse model, we found that placentas of FGR pregnancies exhibited abnormal morphological changes and reduced mTOR activity in the decidual-junctional layer. Using transcriptomics and lipidomics, we revealed that lipid and energy metabolism was significantly disrupted in the placentas of FGR mice. Finally, we demonstrated that maternal physical exercise during gestation in our FGR mouse model was associated with increased fetal and placental weight as well as increased placental mTOR activity and lipid metabolism. Collectively, our data indicate that the inhibited placental mTOR signaling contributes to FGR with altered lipid metabolism in mouse placentas, and maternal exercise could be an effective method to reduce the occurrence of FGR or alleviate the adverse outcomes associated with FGR.Graphical Human and mouse placentas have different mTOR signaling activities in different anatomic regions in normal and FGR pregnancies.Pregnant mice with FGR induced by rapamycin show smaller placentas, decreased mTOR activity in DJ layer of placenta and altered lipid metabolism.Maternal exercise partially alleviates the abnormal outcomes of FGR model.

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.

CHOP upregulation and dysregulation of the mature form of the SNAT2 amino acid transporter in the placentas from small for gestational age newborns.

The placentas from newborns that are small for gestational age (SGA; birth weight < -2 SD for gestational age) may display multiple pathological characteristics. A key determinant of fetal growth and, therefore, birth weight is placental amino acid transport, which is under the control of the serine/threonine kinase mechanistic target of rapamycin (mTOR). The effects of endoplasmic reticulum (ER) stress on the mTOR pathway and the levels of amino acid transporters are not well established. Placentas from SGA and appropriate for gestational age (AGA) newborns and the human placental BeWo cell line exposed to the ER stressor tunicamycin were used. We detected a significant increase in the levels of C/EBP homologous protein (CHOP) in the placentas from SGA newborns compared with those from AGA newborns, while the levels of other ER stress markers were barely affected. In addition, placental mTOR Complex 1 (mTORC1) activity and the levels of the mature form of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) were also reduced in the SGA group. Interestingly, CHOP has been reported to upregulate growth arrest and DNA damage-inducible protein 34 (GADD34), which in turn suppresses mTORC1 activity. The GADD34 inhibitor guanabenz attenuated the increase in CHOP protein levels and the reduction in mTORC1 activity caused by the ER stressor tunicamycin in the human placental cell line BeWo, but it did not recover mature SNAT2 protein levels, which might be reduced as a result of defective glycosylation. Collectively, these data reveal that GADD34A activity and glycosylation are key factors controlling mTORC1 signaling and mature SNAT2 levels in trophoblasts, respectively, and might contribute to the SGA condition. Video Abstract.

Impact of placental mTOR deficiency on peripheral insulin signaling in adult mice offspring.

Suboptimal in utero environments such as poor maternal nutrition and gestational diabetes can impact fetal birth weight and the metabolic health trajectory of the adult offspring. Fetal growth is associated with alterations in placental mechanistic target of rapamycin (mTOR) signaling; it is reduced in fetal growth restriction and increased in fetal overgrowth. We previously reported that when metabolically challenged by a high-fat diet, placental mTORKO (mTORKOpl) adult female offspring develop obesity and insulin resistance, whereas placental TSC2KO (TSC2KOpl) female offspring are protected from diet-induced obesity and maintain proper glucose homeostasis. In the present study, we sought to investigate whether reducing or increasing placental mTOR signaling in utero alters the programming of adult offspring metabolic tissues preceding a metabolic challenge. Adult male and female mTORKOpl, TSC2KOpl, and respective controls on a normal chow diet were subjected to an acute intraperitoneal insulin injection. Upon insulin stimulation, insulin signaling via phosphorylation of Akt and nutrient sensing via phosphorylation of mTOR target ribosomal S6 were evaluated in the offspring liver, white adipose tissue, and skeletal muscle. Among tested tissues, we observed significant changes only in the liver signaling. In the male mTORKOpl adult offspring liver, insulin-stimulated phospho-Akt was enhanced compared to littermate controls. Basal phospho-S6 level was increased in the mTORKOpl female offspring liver compared to littermate controls and did not increase further in response to insulin. RNA sequencing of offspring liver identified placental mTORC1 programming-mediated differentially expressed genes. The expression of major urinary protein 1 (Mup1) was differentially altered in female mTORKOpl and TSC2KOpl offspring livers and we show that MUP1 level is dependent on overnutrition and fasting status. In summary, deletion of placental mTOR nutrient sensing in utero programs hepatic response to insulin action in a sexually dimorphic manner. Additionally, we highlight a possible role for hepatic and circulating MUP1 in glucose homeostasis that warrants further investigation.

175-LB: Altered Glycogen Cell Number in Placental mTOR Signaling Deficiency Model in Mice

Fetal Growth Restriction (FGR) is a determinant for the development of chronic diseases, such as type II diabetes in adulthood. FGR is caused by the failure of the placenta to provide adequate nutrients and oxygen for fetal development. mTOR regulates cell metabolism and growth, and it is reduced in the FGR placenta. We previously reported that placental mTOR ablation induces insulin resistance and glucose intolerance in adult mice. We hypothesize that mTOR signaling regulates glucose utilization in the placenta, which stores glucose reservoirs as glycogen. Appropriate localization and breakdown of glycogen cells is crucial for normal fetal growth. We leveraged our mouse model lacking placental mTOR signaling to ask whether glycogen cell number was altered. First, we found that critical glycogenesis and glycogenolysis enzymes, as well as the glucagon receptor, were expressed in placental tissue, indicating the capability for glycogen cell synthesis and breakdown, like glycogen-rich tissues such as the liver. Next, we show increased glycogen cell counts in the junctional zone of the mTORKO placenta compared to littermate controls by IHC. However, whole placental glycogen content, measured by ELISA, demonstrates no changes between KO and control. These data suggest that mTOR signaling modulates the localization and migration of glycogen cells during high metabolic needs. Glucagon mRNA is expressed in the placenta, with significant increases in the male KO placenta, indicating the placenta may produce its own glucagon. However, IHC staining indicates increased glucagon levels in female KO placenta in the labyrinth zone. Together, these data suggest that the placenta may have the capability to regulate its own glycogen breakdown by altering glucagon expression. Our results indicate that the FGR placenta induced by mTOR deletion has altered glycogen cell content and mislocalization, which mimics the phenotypes of placenta impacted by gestational diabetes, suggesting a shared mechanistic pathway. Disclosure B. Clifton: None. M. Beetch: None. E. Alejandro: None. Funding National Institutes of Health (R01DK115720)

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.

Decreased Placental mTOR Signaling And Increased Placental Apoptosis Are Associated With Secondhand Smoke (SHS)‐Induced Intrauterine Growth Restriction (IUGR) In Mice

Intrauterine growth restriction (IUGR) is a disease affecting 10% of all pregnancies. IUGR is associated with maternal, fetal, and placental abnormalities, but studies investigating the inducibility of IUGR by secondhand smoke (SHS) are limited. The mTOR pathway regulates protein expression and cell growth. Disrupted mTOR signaling and apoptosis are associated with the development of several obstetric complications including IUGR. We tested the hypothesis that SHS exposure disrupts the mTOR pathway and apoptosis in mice. C57Bl6 mice were exposed to SHS for 4 days from day 14 to day 17 of gestation (dGA). At the time of necropsy (18 dGA) placental and fetal weights were recorded and tissues were immediately frozen for immunoblot analysis. Mice exposed to SHS demonstrated a significant reduction in fetal weight (7.35‐fold; p≤0.0001) and placental weight (1.13‐fold; p≤0.0001) compared to controls. Significant decreases were observed for placental activation of mTOR (2.1‐fold; p&lt;0.03), p70 (1.9‐fold; p,0.03) and 4EBP1 (1.3‐fold (p&lt;0.03) in IUGR placentas compared to controls. Increased placental active caspase 3 (1.1‐fold; p&lt;0.04) and the antiapoptotic placental XIAP protein (2.2‐fold; p&lt;0.03) were also detected during SHS treatment compared to controls. We conclude that decreased mTOR pathway is associated with lessened fetal and placental size during SHS induced IUGR. This decrease was associated with increased caspase 3 and that elevated XIAP protein may signify an attempted protective mechanism that counters increased apoptosis observed in the SHS‐induced IUGR placenta. These studies provide insight into tobacco‐mediated IUGR development and clarify avenues that may be helpful in the alleviation of placental complications.

Tadalafil Treatment of Mice with Fetal Growth Restriction and Preeclampsia Improves Placental mTOR Signaling.

Fetal growth restriction (FGR) is a major cause of poor perinatal outcomes. Although several studies have been conducted to improve the prognosis of FGR in infants, no effective intrauterine treatment method has been established. This study aimed to use tadalafil, a phosphodiesterase 5 inhibitor (PDE5) inhibitor, as a novel intrauterine treatment and conducted several basic and clinical studies. The study investigated the effects of tadalafil on placental mTOR signaling. Tadalafil was administered to mice with L-NG-nitroarginine methyl ester (L-NAME)-induced FGR and associated preeclampsia (PE). Placental phosphorylated mTOR (p-mTOR) signaling was assessed by fluorescent immunohistochemical staining and Western blotting. The expression of p-mTOR was significantly decreased in mice with FGR on 13 days post coitum (d.p.c.) but recovered to the same level as that of the control on 17 d.p.c. following tadalafil treatment. The results were similar for 4E-binding protein 1 (4E-BP1) and S6 ribosomal (S6R) protein, which act downstream in the mTOR signaling pathway. We demonstrate that the tadalafil treatment of FGR in mice improved placental mTOR signaling to facilitate fetal growth. Our study provides the key mechanistic detail about the mode of action of tadalafil and thus would be helpful for future clinical studies on FGR.

Placental mTOR Signaling and Sexual Dimorphism in Metabolic Health across the Lifespan of Offspring

Robust evidence of fetal programming of adult disease has surfaced in the last several decades. Human and preclinical investigations of intrauterine insults report perturbations in placental nutrient sensing by the mechanistic target of rapamycin (mTOR). This review focuses on pregnancy complications associated with placental mTOR regulation, such as fetal growth restriction (FGR), fetal overgrowth, gestational diabetes mellitus (GDM), polycystic ovarian syndrome (PCOS), maternal nutrient restriction (MNR), preeclampsia (PE), maternal smoking, and related effects on offspring birthweight. The link between mTOR-associated birthweight outcomes and offspring metabolic health trajectory with a focus on sexual dimorphism are discussed. Both human physiology and animal models are summarized to facilitate in depth understanding. GDM, PCOS and fetal overgrowth are associated with increased placental mTOR, whereas FGR, MNR and maternal smoking are linked to decreased placental mTOR activity. Generally, birth weight is reduced in complications with decreased mTOR (i.e., FGR, MNR, maternal smoking) and higher with increased mTOR (GDM, PCOS). Offspring display obesity or a higher body mass index in childhood and adulthood, impaired glucose and insulin tolerance in adulthood, and deficiencies in pancreatic beta-cell mass and function compared to offspring from uncomplicated pregnancies. Defining causal players in the fetal programming of offspring metabolic health across the lifespan will aid in stopping the vicious cycle of obesity and type II diabetes.

Placental mTOR complex 1 regulates fetal programming of obesity and insulin resistance in mice.

Fetal growth restriction, or low birth weight, is a strong determinant for eventual obesity and type 2 diabetes. Clinical studies suggest placental mechanistic target of rapamycin (mTOR) signaling regulates fetal birth weight and the metabolic health trajectory of the offspring. In the current study, we used a genetic model with loss of placental mTOR function (mTOR-KOPlacenta) to test the direct role of mTOR signaling on birth weight and metabolic health in the adult offspring. mTOR-KOPlacenta animals displayed reduced placental area and total weight, as well as fetal body weight at embryonic day (E) 17.5. Birth weight and serum insulin levels were reduced; however, β cell mass was normal in mTOR-KOPlacenta newborns. Adult mTOR-KOPlacenta offspring, under a metabolic high-fat challenge, displayed exacerbated obesity and metabolic dysfunction compared with littermate controls. Subsequently, we tested whether enhancing placental mTOR complex 1 (mTORC1) signaling, via genetic ablation of TSC2, in utero would improve glucose homeostasis in the offspring. Indeed, increased placental mTORC1 conferred protection from diet-induced obesity in the offspring. In conclusion, placental mTORC1 serves as a mechanistic link between placental function and programming of obesity and insulin resistance in the adult offspring.

Preconceptional Lipid-Based Nutrient Supplementation in 2 Low-Resource Countries Results in Distinctly Different IGF-1/mTOR Placental Responses

BackgroundPreconceptional maternal small-quantity lipid-based nutrient supplementation (SQLNS) improved intrauterine linear growth in low-resource countries as demonstrated by the Women First Preconception Maternal Nutrition Trial (WF). Fetal growth is dependent on nutrient availability and regulated by insulin-like growth factor 1 (IGF-1) through changes in placental transfer capacity, mediated by the mechanistic target of rapamycin (mTOR) pathway. ObjectivesOur objective was to evaluate the role of placental mTOR and IGF-1 signaling on fetal growth in women from 2 low-resource countries with high rates of stunting after they received preconceptional SQLNS. MethodsWe studied 48 women from preconception through delivery who were from Guatemala and Pakistan and received SQLNS or not, as part of the WF study. Placental samples were obtained at delivery (control, n = 24; SQLNS, n = 24). Placental protein or mRNA expression of eukaryotic translation initiation factor binding protein-1 (4E-BP1), ribosomal protein S6 (rpS6), AMP-activated protein kinase α (AMPKA), IGF-1, insulin-like growth factor receptor (IGF-1R), and pregnancy associated plasma protein (PAPP)-A, and DNA methylation of the IGF1 promoter were determined. Maternal serum IGF-1, insulin-like growth factor binding protein (IGFBP)-3, IGFBP-4, IGFBP-5, PAPP-A, PAPP-A2, and zinc were measured. ResultsMean ± SEM maternal prepregnancy BMI differed between participants in Guatemala (26.5 ± 1.3) and Pakistan (19.8 ± 0.7) (P < 0.001). In Pakistani participants, SQLNS increased the placental rpS6(T37/46):rpS6 ratio (1.5-fold) and decreased the AMPKA(T172):AMPKA ratio. Placental IGF1 mRNA expression was positively correlated with birth length and birth weight z-scores. Placental PAPP-A (30-fold) and maternal serum zinc (1.2-fold) increased with SQLNS. In Guatemalan participants SQLNS did not influence placental mTOR signaling. Placental IGF-1R protein expression was positively associated with birth length and birth weight z-scores. SQLNS increased placental PAPP-A (40-fold) and maternal serum IGFBP-4 (1.6-fold). ConclusionsIn Pakistani pregnant women with poor nutritional status, preconceptional SQLNS activated placental mTOR and IGF-1 signaling and was associated with improved fetal growth. In contrast, in Guatemalan women SQLNS did not activate placental nutrient-sensing pathways. In populations experiencing childhood stunting, preconceptional SQLNS improves placental function and fetal growth only in the context of poor maternal nutrition. This trial was registered at clinicaltrials.gov as NCT01883193.

Is there a definite relationship between placental mTOR signaling and fetal growth?

Fetal growth restriction and overgrowth are common obstetrical complications that result in adverse perinatal outcomes and long-term health risks later in life, including neurodevelopmental dysfunction and adult metabolic syndrome. The placenta plays a critical role in the nutrition transfer from mother to fetus and even exerts adaptive mechanism when the fetus is under poor developmental conditions. The mammalian/mechanistic target of rapamycin (mTOR) signaling serves as a critical hub of cell growth, survival, and metabolism in response to nutrients, growth factors, energy, and stress signals. Placental mTOR signaling regulates placental function, including oxygen and nutrient transport. Therefore, placental mTOR signaling is hypothesized to have a positive relationship with fetal growth. In this review, we summarize that most studies support the current evidence that there is connection between placental mTOR signaling and abnormal fetal growth; however, but more studies should be performed following a vigorous and unanimous method for assessment to determine placental mTOR activity.

SUN-635 Profiling of Activation Patterns of Placental mTOR in Pregnancies Complicated by Gestational Diabetes Mellitus

The mammalian target of rapamycin (mTOR) couples’ energy and nutrient abundance to cell growth and is critically involved in the onset and progression of diabetes, cancer and ageing. Placental mTOR is involved in nutrient sensing and transfer to the fetus; animal models suggest that placental mTOR is upregulated in pregnancies complicated by hyperglycaemia (1). In this study we investigated expression patterns and activation of placental mTOR and possible effects of gestational diabetes (GDM). Our study consisted of GDM-mothers (n=28) and their offspring and ii) mothers (n=33) with normal pregnancies (non-GDM) and their infants. Total and phospho-mTOR (Ser2448) expression were determined in placental biopsies using either immunoblotting and immunohistochemistry (IHC) analysis. Newborn anthropometric parameters were also determined at delivery. GDM pregnant women presented with higher fasting glucose levels than non-GDM (98.12±22.82mg/dL; 73.61±9.89mg/dL; p<0.001). No significant difference was found in birth weight or baby length between GDM and non-GDM infants. IHC analysis showed that both total and activated mTOR were predominantly expressed in trophoblasts and to a lesser extend in syncytiotrophoblasts, in both GDM and non-GDM placentas. GDM placentas exhibited a higher mTOR H-score (2) compared to non-GDM (p<0.012), and WB analysis showed a higher phosphor-mTOR signal intensity (p=0.047) in the same group, most likely due to increased total mTOR expression. mTOR expression was also increased in both GDM syncytiotrophoblasts and endothelial cells compared to non-GDM (p<0.001) whereas a reduced signal was detected in stromal phospho-mTOR (p=0.004). No difference was found in trophoblasts or endothelial cells between the 2 study groups suggesting that activation of this kinase is tightly regulated and is relatively independent of changes in total kinase levels. Interestingly bivariate correlation analysis identified an extensive network of significant associations in the expression levels of total, phosphor-mTOR and P/T mTOR between trophoblasts, stroma, endothelial and syncytiotrophoblastsin control placental biopsies; this network was significantly disrupted in GDM placentas, identifying a disheveled regulation of placental mTOR activity. In conclusion, placental mTOR/PmTOR expression is differentially regulated across different placental cell types and is sensitive to hyperglycaemia associated with gestational diabetes mellitus.(1)M. Castillo-Castrejon and TL. Powell. Front Endocrinol (Lausanne). 2017; 8: 306. (2) E. Lakiotaki, et al., Scientific Reports 2016; 6, 21252.

Maternal Immune Activation Alters Placental and Fetal Brain Amino Acid Transport in Rats

ProblemMaternal immune activation (MIA) during pregnancy is an environmental risk factor for neurodevelopmental disorders such as autism and schizophrenia. However, the exact mechanism through which this occurs is unknown. Due to the important roles that amino acids play in neurodevelopment, altered transport of amino acids across the placenta and throughout the developing fetal brain could play a role in neurodevelopmental disorders. Our objectives were to determine whether virally mediated MIA alters amino acid transporter expression in the placenta and fetal brain, and to explore the underlying mechanism(s) associated with these changes.MethodsPregnant rats received poly(I:C) on gestational day 14 and placental and fetal tissues were collected 6, 24, and 48 hours later. Amino acid transporter expression in the placenta and fetal brain was measured using qPCR and immunoblotting. Free amino acid concentrations in the fetal brain were quantified using HPLC. To explore a possible mechanism, the expression and phosphorylation of components of the placental mTOR and AMPK signaling pathways were investigated using immunoblotting.ResultsPoly(I:C) induced the mRNA expression of multiple amino acid transporters in the placenta and fetal brain over all three timepoints. Conversely, poly(I:C) significantly decreased protein expression of ASCT1 and EAAT2 in the placenta, and SNAT5, EAAT1, and GLYT1 in the fetal brain. Changes in transporter expression were accompanied by functional changes in the concentrations of numerous amino acids in the fetal brain. We observed activation of AMPK and inhibition of mTOR signaling in the placentas of poly(I:C)‐treated dams.ConclusionsMIA induced by poly(I:C) significantly alters placental and fetal brain amino acid transporter expression as well as amino acid concentrations in the fetal brain. Due to the importance of amino acids in neurodevelopment, changes in amino acid transporters could contribute to the development of a neurodevelopmental disorder. AMPK and mTOR have been previously shown to regulate amino acid transporter expression. Since poly(I:C) altered the activation of placental and AMPK and mTOR, these could provide novel treatment targets to alleviate MIA‐induced changes in amino acid transport and investigate the implications of these changes in neurodevelopment.Support or Funding InformationThis work was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) awarded to Dr. Piquette‐Miller [MOP 13346]. Eliza McColl was a recipient of a CIHR Canadian Graduate Scholarship (Master’s) and an Ontario Graduate Scholarship, and is currently funded by a CIHR Banting and Best Canadian Graduate Scholarship (Doctoral) [GSD‐164238].

Impact of lipid-based nutrient supplementation on placental mTOR signaling and IGF-1 gene methylation and fetal growth

To describe oncological and functional outcomes in patients treated with reconstructive organ-sparing surgery (OSS) for squamous cell carcinoma of the penis. Plastic reconstructive OSS of the penis with a split thickness skin graft has been proposed as a treatment option for penile cancer, with the objective being preservation of physiological voiding and sexual function without comprising oncological control.Multicenter study reporting clinicopathological data of 57 patients with malignant lesions of the penis treated with OSS and plastic reconstructive surgery with split thickness skin graft from 2007 to 2019. Health related quality of life (HRQoL) was assessed with EuroQoL-5D-3L, urinary symptoms with the International Consultation on Incontinence Modular Questionnaire for Male Lower Urinary Tract Symptoms, and erectile function with the International Index of erectile function (IIEF)-5.Fifty-seven patients underwent OSS reconstructive surgery. Twenty underwent glans resurfacing, 23 partial penectomy, and 14 glansectomy. Median age was 55.1 years (interquartile range [IQR] 29-90), median follow-up 55.7 months (3-149). At the time of data analysis, 6 patients had died of Squamous Cell Carcinoma (SCC) (12.5%) and 10 (17.8%) had progressed. Kaplan-Meier estimates showed a 5-year survival rate of 87.5% and a 5-year progression-free survival of 83%. We assessed HRQoL and functional outcomes in 32 patients. EuroQol 5D-3L showed a mean health status of 82.5%, median Voiding score of the ICIQ-MLTUS was 4 (IQR 1-15), and median IIEF-5 19 (IQR 10.75-25).OSS of the penis remains a safe and viable option for the treatment of SCC, ensuring a favorable appearance of the penis, preserving urinary and sexual function, with good HRQoL and without comprising oncological safety in selected cases.

Placental mTOR insufficiency during pregnancy predisposes female offspring to obesity-induced insulin resistance

Placental mTOR insufficiency during pregnancy predisposes female offspring to obesity-induced insulin resistance

Effect of a High-Fat Diet and Metformin on Placental mTOR Signaling in Mice.

Objective This study was aimed to measure the effects of a high-fat diet and metformin on placental mechanistic target of rapamycin (mTOR) signaling in mice. Study Design Pregnant friend virus B (FVB)-strain mice were allocated on embryonic day (e) 0.5 to one of four groups; group 1: control diet (CD, 10% fat) + control treatment (CT), group 2: CD + metformin treatment (MT), group 3: high-fat diet (HFD, 60% fat) + CT, and group 4: HFD + MT. Metformin (2.5 mg/mL) was provided in water; CT mice received water. Fetuses and placentas were collected. Western blot measured placental p-Akt and p-S6 expression. Results 20 dams (five/group) and 192 fetuses were studied. Compared with CD-fed, HFD-fed dams had higher placental p-Akt protein expression ( p < 0.0001). Among HFD-dams, placental p-Akt was higher in metformin-treated compared with control-treated ( p < 0.001). Among CD-fed dams, there was no significant difference in placental p-S6 expression in MT versus CT groups. Among HFD-fed dams placental p-S6 expression was lower in those exposed to metformin-treated versus controls ( p = 0.001). Conclusion Increased placental mTOR signaling and metformin inhibition of placental mTOR signaling only occurred in the presence of an HFD exposure. These findings suggest that metformin may modulate placental mTOR signaling in the presence of metabolic exposures during pregnancy.

Exercise initiated during pregnancy in rats born growth restricted alters placental mTOR and nutrient transporter expression.

Fetal growth is dependent on effective placental nutrient transportation, which is regulated by mammalian target of rapamycin (mTOR) complex 1 modulation of nutrient transporter expression. These transporters are dysregulated in pregnancies affected by uteroplacental insufficiency and maternal obesity. Nutrient transporters and mTOR were altered in placentae of mothers born growth restricted compared to normal birth weight dams, with maternal diet- and fetal sex-specific responses. Exercise initiated during pregnancy downregulated mTOR protein expression, despite an increase in mTOR activation in male associated placentae, and reduced nutrient transporter gene abundance, which was also dependent on maternal diet and fetal sex. Limited changes were characterized with exercise initiated before and continued throughout pregnancy in nutrient transporter and mTOR expression. Maternal exercise during pregnancy differentially regulated mTOR and nutrient transporters in a diet- and sex-specific manner, which likely aimed to improve late gestational placental growth and neonatal survival. Adequate transplacental nutrient delivery is essential for fetoplacental development. Intrauterine growth restriction and maternal obesity independently alter placental nutrient transporter expression. Although exercise is beneficial for maternal health, limited studies have characterized how the timing of exercise initiation influences placental nutrient transport. Therefore, this study investigated the impact of maternal exercise on placental mechanistic target of rapamycin (mTOR) and nutrient transporter expression in growth restricted mothers and whether these outcomes were dependent on maternal diet or fetal sex. Uteroplacental insufficiency or sham surgery was induced on embryonic day (E) 18 in Wistar-Kyoto rats. F1 offspring were fed a chow or high-fat diet from weaning and at 16 weeks were randomly allocated to an exercise protocol: sedentary, exercised prior to and during pregnancy, or exercised during pregnancy only. Females were mated with normal males (20 weeks) and F2 placentae collected at E20. Exercise during pregnancy only, reduced mTOR protein expression in all groups and increased mTOR activation in male associated placentae. Exercise during pregnancy only, decreased the expression of amino acid transporters in a diet- and sex-specific manner. Maternal growth restriction altered mTOR and system A amino acid transporter expression in a sex- and diet-specific manner. These data highlight that maternal exercise initiated during pregnancy alters placental mTOR expression, which may directly regulate amino acid transporter expression, to a greater extent than exercise initiated prior to and continued during pregnancy, in a diet- and fetal sex-dependent manner. These findings highlight that the timing of exercise initiation is important for optimal placental function.