Amino Acid Transport Activity Research Articles

SNAT3-mediated glutamine transport in perisynaptic astrocytes in situ is regulated by intracellular sodium.

The release of glutamine from astrocytes adjacent to synapses in the central nervous system is thought to play a vital role in the mechanism of glutamate recycling and is therefore important for maintaining excitatory neurotransmission. Here we investigate the nature of astrocytic membrane transport of glutamine in rat brainstem slices, using electrophysiological recording and fluorescent imaging of pHi and Nai+. Glutamine application to perisynaptic astrocytes induced a membrane current, caused by activation of system A (SA) family transporters. A significant electroneutral component was also observed, which was mediated by the system N (SN) family transporters. This response was stimulated by glutamine (KM of 1.57 mM), histidine, and asparagine, but not by leucine or serine, indicating activation of the SNAT3 isoform of SN. We hypothesized that increasing the [Na+ ]i would alter the SNAT3 transporter equilibrium, thereby stimulating glutamine release. In support of this hypothesis, we show that SNAT3 transport can be driven by changing cation concentration and that manipulations to raise [Na+ ]i (activation of excitatory amino acid transporters (EAATs), SA transporters or AMPA receptors) all directly influence SNAT3 transport rate. A kinetic model of glutamine fluxes is presented, which shows that EAAT activation causes the release of glutamine, driven mainly by the increased [Na+ ]i . These data demonstrate that SNAT3 is functionally active in perisynaptic astrocytes in situ. As a result, astrocytic Nai+ signaling, as would be stimulated by neighboring synaptic activity, has the capacity to stimulate astrocytic glutamine release to support glutamate recycling.

The gene expression of numerous SLC transporters is altered in the immortalized hypothalamic cell line N25/2 following amino acid starvation.

Amino acids are known to play a key role in gene expression regulation, and in mammalian cells, amino acid signaling is mainly mediated via two pathways, the mammalian target of rapamycin complex 1 (mTORC1) pathway and the amino acid responsive (AAR) pathway. It is vital for cells to have a system to sense amino acid levels, in order to control protein and amino acid synthesis and catabolism. Amino acid transporters are crucial in these pathways, due to both their sensing and transport functions. In this large‐scale study, an immortalized mouse hypothalamic cell line (N25/2) was used to study the gene expression changes following 1, 2, 3, 5 or 16 h of amino acid starvation. We focused on genes encoding solute carriers (SLCs) and putative SLCs, more specifically on amino acid transporters. The microarray contained 28 270 genes and 86.2% of the genes were expressed in the cell line. At 5 h of starvation, 1001 genes were upregulated and 848 genes were downregulated, and among these, 47 genes from the SLC superfamily or atypical SLCs were found. Of these, 15 were genes encoding amino acid transporters and 32 were genes encoding other SLCs or atypical SLCs. Increased expression was detected for genes encoding amino acid transporters from system A, ASC, L, N, T, xc‐, and y+. Using GO annotations, genes involved in amino acid transport and amino acid transmembrane transporter activity were found to be most upregulated at 3 h and 5 h of starvation.

Microenvironmental autophagy promotes tumour growth.

As malignant tumours develop, they interact intimately with their microenvironment and can activate autophagy, a catabolic process which provides nutrients during starvation. How tumours regulate autophagy in vivo and whether autophagy affects tumour growth is controversial. Here we demonstrate, using a well characterized Drosophila melanogaster malignant tumour model, that non-cell-autonomous autophagy is induced both in the tumour microenvironment and systemically in distant tissues. Tumour growth can be pharmacologically restrained using autophagy inhibitors, and early-stage tumour growth and invasion are genetically dependent on autophagy within the local tumour microenvironment. Induction of autophagy is mediated by Drosophila tumour necrosis factor and interleukin-6-like signalling from metabolically stressed tumour cells, whereas tumour growth depends on active amino acid transport. We show that dormant growth-impaired tumours from autophagy-deficient animals reactivate tumorous growth when transplanted into autophagy-proficient hosts. We conclude that transformed cells engage surrounding normal cells as active and essential microenvironmental contributors to early tumour growth through nutrient-generating autophagy.

1,25-Dihydroxy vitamin D3 stimulates system A amino acid transport in primary human trophoblast cells

Vitamin D deficiency during pregnancy is linked to adverse perinatal outcomes such as small for gestational age infants. Recent evidence suggests that changes in placental amino acid transport contribute to altered fetal growth. We tested the hypothesis that 1,25-dihydroxy vitamin D3 increases the gene expression of System A and L amino acid transporter isoforms and stimulates placental amino acid transport activity in cultured primary human trophoblast cells mediated by mTOR signaling. Treatment with 1,25-dihydroxy vitamin D3 significantly increased mRNA expression of the System A isoform SNAT2 and System A activity, but had no effect on System L and did not affect mTOR signaling. siRNA silencing of the vitamin D receptor prevented 1,25-dihydroxy vitamin D3-stimulated System A transport. In conclusion, 1,25-dihydroxy vitamin D3 regulates System A activity through increased mRNA expression of SNAT2 transporters. Effects on placental amino acid transport may be the mechanism underlying the association between maternal vitamin D status and fetal growth.

Down-Regulation of Placental Transport of Amino Acids Precedes the Development of Intrauterine Growth Restriction in Maternal Nutrient Restricted Baboons

Intrauterine growth restriction (IUGR) is an important risk factor for perinatal complications and adult disease. IUGR is associated with down-regulation of placental amino acid transporter expression and activity at birth. It is unknown whether these changes are a cause or a consequence of human IUGR. We hypothesized that placental amino acid transport capacity is reduced prior to onset of reduced fetal growth in baboons with maternal nutrient restriction (MNR). Pregnant baboons were fed either a control (n = 8) or MNR diet (70% of control diet, n = 9) from Gestational Day 30. At Gestational Day 120 (0.65 of gestation), fetuses and placentas were collected. Microvillous (MVM) and basal (BM) plasma membrane vesicles were isolated. System A and system L transport activity was determined in MVM, and leucine transporter activity was assessed in BM using radiolabeled substrates. MVM amino acid transporter isoform expression (SNAT1, SNAT2, and SNAT4 and LAT1 and LAT2) was measured using Western blots. LAT1 and LAT2 expression were also determined in BM. Maternal and fetal plasma amino acids concentrations were determined using mass spectrometry. Fetal and placental weights were unaffected by MNR. MVM system A activity was decreased by 37% in MNR baboon placentas (P = 0.03); however MVM system A amino acid transporter protein expression was unchanged. MVM system L activity and BM leucine transporter activity were not altered by MNR. Fetal plasma concentrations of essential amino acids isoleucine and leucine were reduced, while citrulline increased (P < 0.05) in MNR fetuses compared to controls. In this primate model of IUGR, placental MVM system A amino acid transporter activity is decreased prior to the onset of reduction in the fetal growth trajectory. The reduction in plasma leucine and isoleucine in MNR fetuses may be caused by reduced activity of MVM system A, which is strongly coupled with system L essential amino acid uptake. Our findings indicate that reduced placental amino acid transport may be a cause rather than a consequence of IUGR due to inadequate maternal nutrition.

Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of VBNC behaviour using a transcriptomic approach

The spoilage potential of Brettanomyces bruxellensis in wine is strongly connected with the aptitude of this yeast to enter in a Viable But Non Culturable (VBNC) state when exposed to the harsh wine conditions. In this work, we characterized the VBNC behaviour of seven strains of B. bruxellensis representing a regional intraspecific biodiversity, reporting conclusive evidence for the assessment of VBNC as a strain-dependent character. The VBNC behaviour was monitored by fluorescein diacetate staining/flow cytometry for eleven days after addition of 0.4, 0.6, 0.8, 1 and 1.2 mg/L of molecular SO2 (entrance in the VBNC state) and after SO2 removal (exit from the VBNC state). Furthermore, one representative strain was selected and RNA-seq analysis performed after exposure to 1.2 mg/L SO2 and during the recovery phase. 30 and 1634 genes were identified as differentially expressed following VBNC entrance and ‘resuscitation’, respectively. The results reported strongly suggested that the entrance in the SO2-induced VBNC state in B. bruxellensis is associated with both, sulfite toxicity and oxidative stress response, confirming the crucial role of genes/proteins involved in redox cell homeostasis. Among the genes induced during recovery, the expression of genes involved in carbohydrate metabolism and encoding heat shock proteins, as well as enriched categories including amino acid transport and transporter activity was observed. The evidences of a general repression of genes involved in DNA replication suggest the occurrence of a true resuscitation of cell rather than a simple regrowth.

Expression of apical Na(+)-L-glutamine co-transport activity, B(0)-system neutral amino acid co-transporter (B(0)AT1) and angiotensin-converting enzyme 2 along the jejunal crypt-villus axis in young pigs fed a liquid formula.

Gut apical amino acid (AA) transport activity is high at birth and during suckling, thus being essential to maintain luminal nutrient-dependent mucosal growth through providing AA as essential metabolic fuel, substrates and nutrient stimuli for cellular growth. Because system-B(0) Na(+)-neutral AA co-transporter (B(0)AT1, encoded by the SLC6A19 gene) plays a dominant role for apical uptake of large neutral AA including L-Gln, we hypothesized that high apical Na(+)-Gln co-transport activity, and B(0)AT1 (SLC6A19) in co-expression with angiotensin-converting enzyme 2 (ACE2) were expressed along the entire small intestinal crypt-villus axis in young animals via unique control mechanisms. Kinetics of Na(+)-Gln co-transport activity in the apical membrane vesicles, prepared from epithelial cells sequentially isolated along the jejunal crypt-villus axis from liquid formula-fed young pigs, were measured with the membrane potential being clamped to zero using thiocyanate. Apical maximal Na(+)-Gln co-transport activity was much higher (p<0.05) in the upper villus cells than in the middle villus (by 29%) and the crypt (by 30%) cells, whereas Na(+)-Gln co-transport affinity was lower (p<0.05) in the upper villus cells than in the middle villus and the crypt cells. The B(0)AT1 (SLC6A19) mRNA abundance was lower (p<0.05) in the crypt (by 40-47%) than in the villus cells. There were no significant differences in B(0)AT1 and ACE2 protein abundances on the apical membrane among the upper villus, the middle villus and the crypt cells. Our study suggests that piglet fast growth is associated with very high intestinal apical Na(+)-neutral AA uptake activities via abundantly co-expressing B(0)AT1 and ACE2 proteins in the apical membrane and by transcribing the B(0)AT1 (SLC6A19) gene in the epithelia along the entire crypt-villus axis.

Characterization of vacuolar amino acid transporter from Fusarium oxysporum in Saccharomyces cerevisiae

Fusarium oxysporum causes wilt disease in many plant families, and many genes are involved in its development or growth in host plants. A recent study revealed that vacuolar amino acid transporters play an important role in spore formation in Schizosaccharomyces pombe and Saccharomyces cerevisiae. To investigate the role of vacuolar amino acid transporters of this phytopathogenic fungus, the FOXG_11334 (FoAVT3) gene from F. oxysporum was isolated and its function was characterized. Transcription of FoAVT3 was upregulated after rapamycin treatment. A green fluorescent protein fusion of FoAvt3p was localized to vacuolar membranes in both S. cerevisiae and F. oxysporum. Analysis of the amino acid content of the vacuolar fraction and amino acid transport activities using vacuolar membrane vesicles from S. cerevisiae cells heterologously expressing FoAVT3 revealed that FoAvt3p functions as a vacuolar amino acid transporter, exporting neutral amino acids. We conclude that the FoAVT3 gene encodes a vacuolar neutral amino acid transporter.

The impact of a human IGF-II analog ([Leu27]IGF-II) on fetal growth in a mouse model of fetal growth restriction.

Enhancing placental insulin-like growth factor (IGF) availability appears to be an attractive strategy for improving outcomes in fetal growth restriction (FGR). Our approach was the novel use of [Leu27]IGF-II, a human IGF-II analog that binds the IGF-II clearance receptor IGF-IIR in fetal growth-restricted (FGR) mice. We hypothesized that the impact of [Leu27]IGF-II infusion in C57BL/6J (wild-type) and endothelial nitric oxide synthase knockout (eNOS−/−; FGR) mice would be to enhance fetal growth and investigated this from mid- to late gestation; 1 mg·kg−1·day−1 [Leu27]IGF-II was delivered via a subcutaneous miniosmotic pump from E12.5 to E18.5. Fetal and placental weights recorded at E18.5 were used to generate frequency distribution curves; fetuses <5th centile were deemed growth restricted. Placentas were harvested for immunohistochemical analysis of the IGF system, and maternal serum was collected for measurement of exogenously administered IGF-II. In WT pregnancies, [Leu27]IGF-II treatment halved the number of FGR fetuses, reduced fetal(P = 0.028) and placental weight variations (P = 0.0032), and increased the numbers of pups close to the mean fetal weight (131 vs. 112 pups within 1 SD). Mixed-model analysis confirmed litter size to be negatively correlated with fetal and placental weight and showed that [Leu27]IGF-II preferentially improved fetal weight in the largest litters, as defined by number. Unidirectional 14CMeAIB transfer per gram placenta (System A amino acid transporter activity) was inversely correlated with fetal weight in [Leu27]IGF-II-treated WT animals (P < 0.01). In eNOS−/− mice, [Leu27]IGF-II reduced the number of FGR fetuses(1 vs. 5 in the untreated group). The observed reduction in FGR pup numbers in both C57 and eNOS−/− litters suggests the use of this analog as a means of standardizing and rescuing fetal growth, preferentially in the smallest offspring.

Increased ubiquitination and reduced plasma membrane trafficking of placental amino acid transporter SNAT-2 in human IUGR.

Placental amino acid transport is decreased in intrauterine growth restriction (IUGR); however, the underlying mechanisms remain largely unknown. We have shown that mechanistic target of rapamycin (mTOR) signalling regulates system A amino acid transport by modulating the ubiquitination and plasma membrane trafficking of sodium-coupled neutral amino acid transporter 2 (SNAT-2) in cultured primary human trophoblast cells. We hypothesize that IUGR is associated with (1) inhibition of placental mTORC1 and mTORC2 signalling pathways, (2) increased amino acid transporter ubiquitination in placental homogenates and (3) decreased protein expression of SNAT-2 in the syncytiotrophoblast microvillous plasma membrane (MVM). To test this hypothesis, we collected placental tissue and isolated MVM from women with pregnancies complicated by IUGR (n=25) and gestational age-matched women with appropriately grown control infants (n=19, birth weights between the twenty-fifth to seventy-fifth percentiles). The activity of mTORC1 and mTORC2 was decreased whereas the protein expression of the ubiquitin ligase NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2; +72%, P<0.0001) and the ubiquitination of SNAT-2 (+180%, P<0.05) were increased in homogenates of IUGR placentas. Furthermore, IUGR was associated with decreased system A amino acid transport activity (-72%, P<0.0001) and SNAT-1 (-42%, P<0.05) and SNAT-2 (-31%, P<0.05) protein expression in MVM. In summary, these findings are consistent with the possibility that decreased placental mTOR activity causes down-regulation of placental system A activity by shifting SNAT-2 trafficking towards proteasomal degradation, thereby contributing to decreased fetal amino acid availability and restricted fetal growth in IUGR.

Reduced placental amino acid transport in response to maternal nutrient restriction in the baboon.

Intrauterine growth restriction increases the risk of perinatal complications and predisposes the infant to diabetes and cardiovascular disease in later life. Mechanisms by which maternal nutrient restriction (MNR) reduces fetal growth are poorly understood. We hypothesized that MNR decreases placental amino acid (AA) transporter activity, leading to reduced transplacental transfer of AAs. Pregnant baboons were fed either a control (ad libitum, n = 7), or MNR diet (70% of control diet, n = 7) from gestational day (GD) 30. At GD 165 (0.9 gestation), placentas (n = 7 in each group) were collected, and microvillous plasma membrane vesicles (MVM) isolated. MVM system A and system L AA transport was determined in vitro using radiolabeled substrates and rapid filtration techniques. In vivo transplacental AA transport was assessed by infusing nine (13)C- or (2)H-labeled essential AA as a bolus into the maternal circulation (n = 5 control, n = 4 MNR) at cesarean section. A fetal vein-to-maternal artery mole percent excess ratio for each essential AA was calculated. Fetal and placental weights were significantly reduced in the MNR group compared with controls (P < 0.01). The activity of system A and system L was markedly reduced by 73 and 84%, respectively, in MVM isolated from baboon placentas at GD 165 following MNR (P < 0.01). In vivo, the fetal vein-to-maternal artery mole percent excess ratio was significantly reduced for leucine, isoleucine, methionine, phenylalanine, threonine, and tryptophan in MNR baboons (P < 0.05). This is the first study to investigate placental AA transport in a nonhuman primate model of MNR. We demonstrate that the downregulation of system A and system L activity in syncytiotrophoblast MVM in MNR leads to decreased transplacental AA transport and, consequently, reduced circulating fetal AA concentrations, a potential mechanism linking maternal undernutrition to reduced fetal growth.

Increased placental nutrient transport in a novel mouse model of maternal obesity with fetal overgrowth.

OBJECTIVETo identify possible mechanisms linking obesity in pregnancy to increased fetal adiposity and growth, we developed a unique mouse model of maternal obesity associated with fetal overgrowth and tested the hypothesis that maternal obesity causes up-regulation of placental nutrient transporter expression and activity.METHODSC57BL/6J female mice were fed a control (C) or a high fat/high sugar (HF/HS) pelleted diet supplemented by ad libitum access to sucrose (20%) solution, mated and studied at embryonic day 18.5.RESULTSHF/HS diet increased maternal fat mass by 2.2-fold (p<0.01) and resulted in glucose intolerance with normal fasting glucose. Maternal circulating insulin, leptin, and cholesterol were increased (p<0.05) whereas total and high molecular weight (HMW) adiponectin were decreased (p<0.05). HF/HS diet increased fetal weight (+18%, p=0.0005). In trophoblast plasma membrane (TPM) isolated from placentas of HF/HS fed animals, protein expression of glucose transporter (GLUT) 1 and 3, sodium-coupled neutral amino acid transporter (SNAT) 2 and large neutral amino acid transporters 1 (LAT1) was increased. TPM System A and L amino acid transporter activity was increased in the HF/HS group.CONCLUSIONUp-regulation of specific placental nutrient transporter isoforms may constitute a mechanism underlying fetal overgrowth in maternal obesity.

Influx mechanisms in the embryonic and adult rat choroid plexus: a transcriptome study.

The transcriptome of embryonic and adult rat lateral ventricular choroid plexus, using a combination of RNA-Sequencing and microarray data, was analyzed by functional groups of influx transporters, particularly solute carrier (SLC) transporters. RNA-Seq was performed at embryonic day (E) 15 and adult with additional data obtained at intermediate ages from microarray analysis. The largest represented functional group in the embryo was amino acid transporters (twelve) with expression levels 2–98 times greater than in the adult. In contrast, in the adult only six amino acid transporters were up-regulated compared to the embryo and at more modest enrichment levels (<5-fold enrichment above E15). In E15 plexus five glucose transporters, in particular Glut-1, and only one monocarboxylate transporter were enriched compared to the adult, whereas only two glucose transporters but six monocarboxylate transporters in the adult plexus were expressed at higher levels than in embryos. These results are compared with earlier published physiological studies of amino acid and monocarboxylate transport in developing rodents. This comparison shows correlation of high expression of some transporters in the developing brain with higher amino acid transport activity reported previously. Data for divalent metal transporters are also considered. Immunohistochemistry of several transporters (e.g., Slc16a10, a thyroid hormone transporter) gene products was carried out to confirm translational activity and to define cellular distribution of the proteins. Overall the results show that there is substantial expression of numerous influx transporters in the embryonic choroid plexus, many at higher levels than in the adult. This, together with immunohistochemical evidence and data from published physiological transport studies suggests that the choroid plexus in embryonic brain plays a major role in supplying the developing brain with essential nutrients.

Reply to “Letter to the editor: ‘Fatty acids and placental transport: insight or in vitro artifact?’”

reply: In our recent paper (2) we reported on the effects of fatty acids on trophoblast cellular signaling and amino acid transport activity in vitro. As with any other cell culture experiments, we are aware that this is an artificial environment on multiple levels. No matter how well designed the experiment, cell cultures in and of themselves cannot completely reproduce in vivo physiological conditions. However, despite their inherent limitations, cell culture experiments do provide an important tool for studying cell physiology. In vivo, cells are exposed to a multitude of effectors at variable concentrations and we concur with Dr. Keelan (1) that it is important to study effectors in combination. However, a problem arises with respect to which effectors should be included. For example, when studying effects of fatty acids on trophoblast cell function, should all effectors known to affect mTOR signaling and amino acid transport be included in the cell culture media for the experiment to be considered valid? By analogy, in studies of insulin effects on cell function, do all known effectors impinging on the insulin signaling pathway (adiponectin, leptin, and cytokines, just to mention a few) have to be included for the experiment to be physiologically relevant? If so, Dr. Keelan's argument invalidates most of the published studies in cell physiology. We believe that a reductionist approach in which single effector molecules are studied remains valid and we disagree with the argument that fatty acids should only be studied in combination. We reported that, in the presence of oleic acid, the effects of docosahexaenoic acid (DHA) alone were no longer predominant. Dr. Keelan suggests that this finding should be interpreted that DHA responses were an in vitro artifact. However, such an interpretation fails to appreciate that DHA counteracted the observed effects of oleic acid alone. Clearly, both DHA and oleic acid have the capacity to influence trophoblast signaling and function, both in isolation and when combined. In our paper we demonstrate that DHA reduces the activity of the amino transporters System A and System L in cultured primary human trophoblast cells. In a separate study following pregnant women supplemented with DHA, we found that placental DHA levels measured at term inversely correlated with System A and System L amino acid transport capacity (3). Such data from clinical samples strongly support the assertion that the reported in vitro effects are physiologically relevant and not an experimental artifact. As already discussed in our paper, we believe that these results merit further exploration of the effects of fatty acids on trophoblast function, both singularly and in combination.

Effects of Strain and Production-Cycle on Indices of Lysine Catabolism in Turkeys

In typical turkey production diets, lysine is frequently the second limiting amino acid. Understanding its catabolism could provide opportunities to increase the efficiency of lysine use for protein synthesis. We hypothesize that indices of lysine catabolism in turkey liver vary throughout the production cycle. Two commercial strains of turkey, Strain A and B, were analyzed 8 times over a period of 17 weeks (n = 8 birds/sampling time/strain) for lysine alpha-ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) activities and mRNA abundance, in vitro lysine oxidation (LOX), L-amino acid oxidase (LAAO) activity, lysyl oxidase (LO) activity and mRNA expression of cationic amino acid transporters (CAT) 1, 2 and 3. We found differences in hepatic LKR activity (p = 0.007), LKR mRNA (p = 0.0004), SDH activity (p = 0.008), LOX (p<0.0001), lysyl oxidase activity (p<0.01), LAAO (p<0.0001) and CAT1 (p = 0.005) and 2 mRNA abundance (p = 0.0022) throughout the production cycle. We also found an effect of strain on SDH (p = 0.046) and CAT-2 mRNA abundance (p = 0.02); while no age or strain effects were detected for CAT3 mRNA abundance. Interestingly, the average LKR and SDH activities across strains and weeks was 240 and 420 nmol per minute per gram of liver, respectively, as opposed to the average LAAO activity, lysyl oxidase activity and L OX which were 0.70, 0.10 and 13.5 nmol per minute per gram liver, respectively. These data indicate that the saccharopine-dependent pathway is the predominant pathway of lysine degradation in turkey liver and that indices of hepatic lysine catabolism vary throughout the production cycle.

Placental, Lipid, and Glucidic Effects of Mammalian Target of Rapamycin Inhibitors: Impact on Fetal Growth and Metabolic Disorders During Pregnancy After Solid Organ Transplantation

BackgroundMammalian target of rapamycin inhibitors (mTORi) are a promising new family of immunosuppressive drugs. No teratogenic effects have been reported to date. Their lipid and glucidic effects should not be underestimated, however, especially during pregnancy. Moreover, mTORi may affect fetal growth by mTOR placental activity. ObjectiveOur purpose was to highlight mTORi placental impact and metabolic implications to detect possible maternal or fetal effects and define management guidelines in pregnant women after solid organ transplantation. MethodsA literature search was performed for articles from the Medline and Pubmed databases with the use of the following keywords: mTOR inhibitors, pregnancy, placental transport, lipid metabolism, glucose metabolism. ResultsmTOR works as a positive regulator of system A, system L, and taurine placental amino acid transporter activity, which are critical for the transport of amino acids to the fetus. Exposing trophoblast cells to rapamycin reduces system L activity; therefore, treatment with rapamycin in human pregnancies could alter fetal growth with intrauterine growth restriction (IUGR). Regarding the metabolic effects mTORi increase lipolysis, impair insulin's antilipolytic effect and reduce lipid storage, which may potentially contribute to dyslipidemia. Chronic rapamycin treatment reduces adipose tissue size and β-cell mass/function, causes hyperlipidemia, severe insulin resistance, and glucose intolerance, and promotes hepatic gluconeogenesis. ConclusionsThe studies on mTORi treatment in transplanted pregnant women have not focused to date on the potential metabolic and placental effects. Selection of women at high risk for metabolic disorders could be needed and consideration of switching to another immunosuppressive drug required if diabetes and abnormal blood lipids have been diagnosed in prepregnancy counseling. It seems to be mandatory to encourage prompt reporting of any additional cases of pregnancy during mTORi exposure to provide a better understanding of the placental effects and safety profile of these immunosuppressive drugs.

Placental amino acid transporter function adapts to maternal and fetal nutrient conditions

s / Placenta 35 (2014) A1eA23 A10 O-026. PLACENTAL AMINO ACID TRANSPORTER FUNCTION ADAPTS TO MATERNAL AND FETAL NUTRIENT CONDITIONS Eiji Shibata David, J. Askew, Yukiyo Aiko, Chiharu Tomonaga, Satoshi Aramaki, Hirohide Inagaki, Toru Hachisuga. Department of Obstetrics and Gynecology, University of Occupational and Environmental Health, Japan Background: Studies suggest that placental nutrient uptake activity is responsive to both maternal and fetal nutrient demands. We hypothesize that under conditions of limited nutrient availability to the fetus, as often present in preeclampsia (PE), IUGR, and insufficient weight-gain during pregnancy, a general adaptive response aimed to increase amino acid transport activity may be observed in the placenta. Method: A total of 40 placentas from full-term (n1⁄410) and pre-term (n1⁄410) normal pregnancies, IUGR (n1⁄410), and PE (n1⁄410) associated pregnancies were looked at by immunohistochemistry followed by relative qualitative scoring to compare expression levels and localization of System L, ASCT2, and mTOR proteins. Result: Syncytiotrophoblast (ST) in placenta of pregnancies complicated by IUGR or PE showed significant increases in the levels of 4F2hc and LAT1 compared to both full-term control and pre-term pregnancies seperately. Elevated mTOR protein was uniquely higher in IUGR placentas compared to full-term controls. Total cellular ASCT2 transporter protein levels were similar in all groups, however, levels of ASCT2 protein localized to the ST microvillous membrane (MVM) were significantly lower in IUGR compared to both full-term and pre-term pregnancies. Additionally, ASCT2 and mTOR protein levels were positively associated with maternal prepregnancy BMI. Conclusion: First, in conditions of limited nutrient availability, such as PE or IUGR, there is an overall increase in the level of System L and mTOR protein expression in the ST, suggestive of an adaptive response. Second, a decrease in ASCT2 protein at the ST MVM suggests a post-translational event that may decrease AAT activity in IUGR placentas. Third, a physiological link between transporter expression and pre-pregnancy BMI is suggested based upon a positive association observed with ASCT2 and mTOR expression values. O-027. ENDOMETRIAL PERIVASCULAR CELLS ESTABLISH UNIQUE CYTOKINE GRADIENTS IN DECIDUAL PROCESS Keisuke Murakami , Keiji Kuroda , Shintaro Makino , Atsuo Itakura , Satoru Takeda , Jan J. Brosens . Department of Obstetrics and Gynaecology, Juntendo University Faculty of Medicine, Japan; Division of Reproductive Health, Clinical Science Research Laboratories, Warwick Medical School, University of Warwick, Japan Aim: The endometrial perivascular microenvironment is rich in mesenchymal stem-like cells that express type 1 integral membrane protein Sushi domain containing 2 (SUSD2) but the role of these cells in the decidual transformation of this tissue in pregnancy is unknown. We used an antibody directed against SUSD2 to isolate perivascular (SUSD2+) and non-perivascular (SUSD2-) fibroblasts from mid-luteal biopsies. Method: SUSD2+ and SUSD2cells were purified from mid-luteal endometrial biopsies by magnetic bead separation and each cell population were expanded in vitro, then, subjected to RNA-seq analysis or differentiation experiments induced by cAMP andMPA to assess secretome profile. Result: On average, positive cells comprised 6.8% of endometrial stromal cells and 6-fold enriched in clonogenic cells. RNA sequencing revealed that cultures derived from SUSD2+ progenitor cells remain phenotypically distinct by the enrichment of novel and established endometrial perivascular signature genes including AOC3, NOTCH3, ELN, MYH11, DES, ACTA2, MCAM, and CSPG4. Decidualization transformed perivascular SUSD2+ -derived cells into the dominant source of a range of chemokines and cytokines, including CCL7, LIF and LTA. Conclusion: Our findings confirmed that SUSD2+ -derived cells remain the expression of perivascular signature genes even after in-vitro culture. Decidual response is spatially organized at the feto-maternal interface with differentiating perivascular cells establishing distinct cytokine and chemokine gradients that could have the key role in decidual process. O-028. LOCALIZATION OF TRPV5 (TRANSIENT RECEPTOR POTENTIAL VANILLOID RECEPTOR 5) PROTEIN IN HUMAN PLACENTAL TROPHOBLASTS Yoko Aoyama, David J. Askew, Yukiyo Aiko, Chiharu Tomonaga, Satoshi Aramaki, Hirohide Inagaki, Toru Hachisuga, Eiji Shibata. Department of Obstetrics and Gynecology, University of Occupational and Environmental

Ex vivo dual perfusion of a human placental cotelydon – modifications of access to the intervillous space

Ex vivo dual perfusion of a human placental cotelydon – modifications of access to the intervillous space

Inhibition of placental mTORC1 signalling may explain decreased placental nutrient transport and contribute to restricted fetal growth in placental malaria

Inhibition of placental mTORC1 signalling may explain decreased placental nutrient transport and contribute to restricted fetal growth in placental malaria

Synthetic glucocorticoid reduces human placental system a transport in women treated with antenatal therapy.

Synthetic glucocorticoids (sGCs) are routinely given to women with threatened preterm labor and have been linked to fetal growth restriction and developmental programming. Reductions in fetal growth are likely to be mediated by placental dysfunction, including altered nutrient transport. sGCs modify the system A neutral amino acid transporter in vitro, but there are no in vivo comparable data in human placenta. Because ∼ 30% of women who receive sGCs carry to term, our objective was to examine the short- and longer-term consequences of antenatal sGCs on placental system A transport. Placental tissue was collected from women treated with sGCs between 24 hours and 14 days before delivery (24h-14d), 14 days after treatment but before term (14d-term), or at term, compared with healthy term (control) deliveries to measure system A-mediated activity (Na(+)-dependent [(14)C]methylaminoisobutyric acid uptake per gram placenta) and mRNA expression. After sGC treatment, system A activity was significantly reduced at term compared with both sGC placentas delivered 24h-14d and compared with controls. Placentae from women treated with sGCs who delivered between 14d-term also had significantly reduced system A activity compared with 24h-14d placentas. SLC38A1 and SLC38A2 mRNA expression was unaffected. However, SLC38A4 was significantly reduced by sGCs at term compared with placentas delivered between 14d-term. We conclude that women who are at risk of preterm labor and receive sGCs but deliver at term have significantly reduced placental system A amino acid transporter activity. Altered placental transporter function could affect fetal growth and may contribute to developmental programming reported in both animal and clinical studies.