Placental Amino Acid Transport Research Articles

Regulation of placental amino acid transporter activity by mammalian target of rapamycin

The activity of placental amino acid transporters is decreased in intrauterine growth restriction (IUGR), but the underlying regulatory mechanisms have not been established. Inhibition of the mammalian target of rapamycin (mTOR) signaling pathway has been shown to decrease the activity of the system L amino acid transporter in human placental villous fragments, and placental mTOR activity is decreased in IUGR. In the present study, we used cultured primary trophoblast cells to study mTOR regulation of placental amino acid transporters in more detail and to test the hypothesis that mTOR alters amino acid transport activity by changes in transporter expression. Inhibition of mTOR by rapamycin significantly reduced the activity of system A (-17%), system L (-28%), and taurine (-40%) amino acid transporters. mRNA expression of isoforms of the three amino acid transporter systems in response to mTOR inhibition was measured using quantitative real-time PCR. mRNA expression of l-type amino acid transporter 1 (LAT1; a system L isoform) and taurine transporter was reduced by 13% and 50%, respectively; however, mTOR inhibition did not alter the mRNA expression of system A isoforms (sodium-coupled neutral amino acid transporter-1, -2, and -4), LAT2, or 4F2hc. Rapamycin treatment did not significantly affect the protein expression of any of the transporter isoforms. We conclude that mTOR signaling regulates the activity of key placental amino acid transporters and that this effect is not due to a decrease in total protein expression. These data suggest that mTOR regulates placental amino acid transporters by posttranslational modifications or by affecting transporter translocation to the plasma membrane.

Altered Placental Function of 11β-Hydroxysteroid Dehydrogenase 2 Knockout Mice

Fetal glucocorticoid exposure is a key mechanism proposed to underlie prenatal "programming" of adult cardiometabolic and neuropsychiatric disorders. Regulation of fetal glucocorticoid exposure is achieved by the placental glucocorticoid "barrier," which involves glucocorticoid inactivation within the labyrinth zone of the murine placenta by 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2). Thus, the absence of placental 11beta-HSD2 may impact on fetal and placental development. The current study investigated transport of amino acids and glucose, key factors required for fetal growth, and vascular development in placentas from 11beta-HSD2(+/+), (+/-), and (-/-) fetuses derived from 11beta-HSD2(+/-) matings. At embryonic d 15 (E15) (term = E19), 11beta-HSD2(-/-) fetal weight was maintained in comparison to 11beta-HSD2(+/+) fetuses. The maintenance of 11beta-HSD2(-/-) fetal weight occurred despite a reduction in placental weight, suggesting that compensatory changes occur in the placenta to maintain function. However, by E18, 11beta-HSD2(-/-) fetal and placental weights were both reduced. Transport studies revealed up-regulation of placental amino acid transport to 11beta-HSD2(-/-) offspring at E15, coinciding with an increase in the expression of the amino acid transporters. Furthermore, at E18, placental glucose transport to 11beta-HSD2(-/-) offspring was markedly reduced, correlating with lower fetal weight and a decrease in glucose transporter 3 expression. Stereological analyses of the labyrinth zone of the placenta revealed that the reduction in placental weight at E18 was associated with restriction of the normal increase in fetal vessel density over the final third of pregnancy. Our data suggest that restriction of fetal growth in 11beta-HSD2(-/-) mice is mediated, at least in part, via altered placental transport of nutrients and reduction in placental vascularization.

Placental Amino Acid transport is decreased after exposure to hypoxia

Placental Amino Acid transport is decreased after exposure to hypoxia

Leptin alters placental amino acid transport

Maternal leptin is elevated during pregnancy and preeclampsia, and is postulated to be an important mediator of reproductive function and metabolic regulation. Amino acids serve as one of the main nutrient sources for the fetus and the activity of placental amino acid transporters is crucial in the regulation of fetal growth. However, the role of leptin in placental function is not fully understood. Objective: To investigate the role of leptin on system A amino acid transport in human placenta. Methods: The uptake of C14-MeAIB was investigated after incubation of primary villous fragments isolated from placentas of primiparous women with uncomplicated pregnancies in leptin (n=22), insulin (n=14) and angiotensin II (n=14). Western Blotting was performed to identify the leptin receptor and STAT3 phosphorylation as a measure of leptin receptor activation (n=6). Leptin induced matrix metalloproteinase (MMP) activity of villous fragments was measured by zymography (n=5). Data are expressed as mean±SEM of the fold change compared to control. Statistical analyzes was done by Wilcoxon/Kruskal Wallis test, probability values were considered significant at p<0.05. Results: Leptin increased system A amino acid transporter activity by 33% within 1 hr of incubation. However, this increase in activity was lost after 2 hr. Placental villous fragments expressed both the membrane bound leptin receptor and additionally released a soluble leptin receptor. Leptin activated JAK-STAT signaling pathway and increased MMP–9 activity in placental villous fragments. Conclusions: These data support the role of leptin as a signal at the maternal-fetal interface of the human placenta.

The Mechanisms and Regulation of Placental Amino Acid Transport to the Human Foetus

The mechanisms by which amino acids are transferred across the human placenta are fundamental to our understanding of foetal nutrition. Amino acid transfer across the human placenta is dependent on transport across both the microvillous and basal plasma membranes of the placental syncytiotrophoblast, and on metabolism within the syncytiotrophoblast. Although the principles underlying uptake of amino acids across the microvillous plasma membrane are well understood, the extent to which amino acids are metabolised within human placenta and the mechanisms by which amino acids are transported out of the placenta across the basal plasma membrane are not well understood. Understanding the mechanisms and regulation of amino acid transport is necessary to understand the causes of intrauterine growth restriction in human pregnancy.

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.

Regulation of Placental Nutrient Transport – A Review

Fetal growth is primarily determined by nutrient availability, which is intimately related to placental nutrient transport. Detailed information on the regulation of placental nutrient transporters is therefore critical in order to understand the mechanisms underlying altered fetal growth and fetal programming. After briefly summarizing the cellular mechanisms for placental transport of glucose, amino acids and free fatty acids, we will discuss factors shown to regulate placental nutrient transporters and review the data describing how these factors are altered in pregnancy complications associated with abnormal fetal growth. We propose an integrated model of regulation of placental nutrient transport by maternal and placental factors in IUGR.

Placental transport: a function of permeability and perfusion

Studies in ovine fetus and placenta have pointed to an interaction between the fetal liver and the placenta. The supply of amino acids and carbohydrates depends on this interaction. These studies have led to clinical studies in normal and high-risk pregnancies. The objective of the present review was to compare changes in fetal circulation, in terms of both velocimetry and actual blood flow measurements, and to couple such data with data on the placental transport of amino acids. Flow studies were carried out on the umbilical vein with measurements of time-averaged velocity and venous diameter. A similar approach was used for measurements of ductus venosus flow. Stable-isotope-labeled amino acids were used to study placental transport by the non–steady state approach. The studies of flow showed a marked reduction in umbilical blood flow even when expressed per kilogram fetal body weight in fetal-growth-restricted pregnancies. This may be coupled with an increased ductus venosus shunt, the combination leading to a marked reduction in fetal hepatic blood flow. The placental transport of some amino acids is reduced in fetal-growth-restricted pregnancies. Furthermore, nonglucose carbohydrates and polyols are found in fetal blood, some in concentrations higher than maternal concentrations. There is significant uptake of several polyols and of mannose across the umbilical circulation in normal pregnancies. In conclusion, both perfusion and permeability can now be studied in both normal and high-risk pregnancies.

Decreased placental amino acid transport and intrauterine growth restriction: which is the chicken and which is the egg?

Decreased placental amino acid transport and intrauterine growth restriction: which is the chicken and which is the egg?

Down‐regulation of placental transport of amino acids precedes the development of intrauterine growth restriction in rats fed a low protein diet

Intrauterine growth restriction (IUGR) represents an important risk factor for perinatal complications and for adult disease. IUGR is associated with a down‐regulation of placental amino acid transporters; however, whether these changes are primary events directly contributing to IUGR or a secondary consequence is unknown. We investigated the time course of changes in placental and fetal growth, placental nutrient transport in vivo and the expression of placental nutrient transporters in pregnant rats subjected to protein malnutrition, a model for IUGR. Pregnant rats were given either a low protein (LP) diet (n= 64) or an isocaloric control diet (n= 66) throughout pregnancy. Maternal insulin, leptin and IGF‐I levels decreased, whereas maternal amino acid concentrations increased moderately in response to the LP diet. Fetal and placental weights in the LP group were unaltered compared to control diet at gestational day (GD) 15, 18 and 19 but significantly reduced at GD 21. Placental system A transport activity was reduced at GD 19 and 21 in response to a low protein diet. Placental protein expression of SNAT2 was decreased at GD 21. In conclusion, placental amino acid transport is down‐regulated prior to the development of IUGR, suggesting that these placental transport changes are a cause, rather than a consequence, of IUGR. Reduced maternal levels of insulin, leptin and IGF‐1 may link maternal protein malnutrition to reduced fetal growth by down‐regulation of key placental amino acid transporters.

Down-regulation of placental transport of amino acids precedes the development of intrauterine growth restriction in rats fed a low protein diet

Intrauterine growth restriction (IUGR) represents an important risk factor for perinatal complications and for adult disease. IUGR is associated with a down-regulation of placental amino acid transporters; however, whether these changes are primary events directly contributing to IUGR or a secondary consequence is unknown. We investigated the time course of changes in placental and fetal growth, placental nutrient transport in vivo and the expression of placental nutrient transporters in pregnant rats subjected to protein malnutrition, a model for IUGR. Pregnant rats were given either a low protein (LP) diet (n = 64) or an isocaloric control diet (n = 66) throughout pregnancy. Maternal insulin, leptin and IGF-I levels decreased, whereas maternal amino acid concentrations increased moderately in response to the LP diet. Fetal and placental weights in the LP group were unaltered compared to control diet at gestational day (GD) 15, 18 and 19 but significantly reduced at GD 21. Placental system A transport activity was reduced at GD 19 and 21 in response to a low protein diet. Placental protein expression of SNAT2 was decreased at GD 21. In conclusion, placental amino acid transport is down-regulated prior to the development of IUGR, suggesting that these placental transport changes are a cause, rather than a consequence, of IUGR. Reduced maternal levels of insulin, leptin and IGF-1 may link maternal protein malnutrition to reduced fetal growth by down-regulation of key placental amino acid transporters.

新規胎盤塩基性アミノ酸トランスポーターの分子同定と機能解析(博士(医学),杏林大学学位論文要旨および審査要旨)

新規胎盤塩基性アミノ酸トランスポーターの分子同定と機能解析(博士(医学),杏林大学学位論文要旨および審査要旨)

Adaptation of nutrient supply to fetal demand in the mouse involves interaction between the Igf2 gene and placental transporter systems

The mammalian fetus is unique in its dependence during gestation on the supply of maternal nutrients through the placenta. Maternal supply and fetal demand for nutrients need to be fine tuned for healthy growth and development of the fetus along its genetic trajectory. An altered balance between supply and demand can lead to deviations from this trajectory with long-term consequences for health. We have previously shown that in a knockout lacking the imprinted placental-specific Igf2 transcript (P0), growth of the placenta is compromised from early gestation but fetal growth is normal until late gestation, suggesting functional adaptation of the placenta to meet the fetal demands. Here, we show that placental transport of glucose and amino acids are increased in the Igf2 P0(+/-) null and that this up-regulation of transport occurs, at least in part, through increased expression of the transporter genes Slc2a3 and Slc38a4, the imprinted member of the System A amino acid transporter gene family. Decreasing fetal demand genetically by removal of fetal Igf2 abolished up-regulation of both transport systems and reduced placental System A amino acid transport activity and expression of Slc38a2 in late gestation. Our results provide direct evidence that the placenta can respond to fetal demand signals through regulation of expression of specific placental transport systems. Thus, crosstalk between an imprinted growth demand gene (Igf2) and placental supply transporter genes (Slc38a4, Slc38a2, and Slc2a3) may be a component of the genetic control of nutrient supply and demand during mammalian development.

Placental amino acid transport systems and fetal growth restriction – A workshop report

Keywords: Placental amino acid transport; Fetal growth restriction; Amino acid transportersINTRODUCTIONTwo circulations perfuse the placental barrier, the maternal oruterine on one side and the fetal or umbilical on the other.This barrier consists of two specific membranes, the maternalfacing, microvillous (MVM) or apical membrane and the fetalfacing or basal membrane (BM). As human gestation advancesthe surface area of the microvillous membrane increases.Though late in pregnancy, this increase is not of comparablerate to the growth rate of the fetus, supporting the concept thatduring later pregnancy the increasing physiological needs ofthe fetus are achieved by multiple functional changes in thetotal placental exchange capacity. One component of thiscapacity is amino acid transport. Both placental membraneshave a range of active energy dependent amino acid transportsystems [1]. These systems act in a coordinated manner totransport amino acids from both circulations into thetrophoblast, as well as out of the trophoblast, into circulation.In addition to surface area, many other factors also influenceoverall activity. Maternal circulating concentrations of aminoacids also play a major role in regulating umbilical uptake,where the transport of any given amino acid from placenta tofetus is considered a function of both its own maternalconcentration and the maternal concentration of inhibitoryamino acids [2]. Additionally, electrical gradients exist over theplacenta, across which transport systems operate, indicative ofthe energy requirement of these systems [3]. It is important toremember that the trophoblast acts not only as a conduit foramino acids between the two circulations, but also has a highmetabolic rate, utilizing amino acids for its own metabolicfunctions, which include hormone and steroid production aswell as waste removal from fetal circulation [4].To understand the overall functional capacity of theplacental barrier, attempts to consider all of the abovecomponents need to be made, especially when investigatinghow adverse situations may impact upon the developmentaland functional capacity of the placenta. Specifically, in thesituation of fetal growth restriction (FGR), there is in vivo andin vitro data to demonstrate significant changes in the placentalsurface area/permeability constants [5], its amino acid trans-port activity in vitro [6e8], and in vivo [9e12]. The FGRplacenta has decreased total villous surface area, indicating thatmorphometric changes in vascularization may contribute to theoverall reduction in placental diffusional transport capacity.Thus, both decreased surface area for diffusional exchange forcompounds such as oxygen and reductions in the number ofspecific nutrient transporters and their activities may contrib-ute to a global reduction in nutrient transport in FGRpregnancies.The purpose of this workshop was to deal with a range ofwork representing aspects of amino acid transport systems,including their locations, their substrates and their activities,from both in vitro and in vivo settings. However, rather thanact as a forum for mini reviews, this workshop was designedfor presenters to put forward experimental inconsistencies insome of these aspects for general discussion and input from thegathered group. The aim, at the end of the workshop, was tohave a consensus of where specific placenta amino acidtransporters may be located, what their substrates and theirindividual activities may be, how they might interact with one

Placental uptake and transport of ACP, a neutral nonmetabolizable amino acid, in an ovine model of fetal growth restriction.

Reductions in fetal plasma concentrations of certain amino acids and reduced amino acid transport in vesicle studies suggest impaired placental amino acid transport in human fetal growth restriction (FGR). In the present study, we tested the hypothesis of an impairment in amino acid transport in the ovine model of hyperthermia-induced FGR by determining transplacental and placental retention and total placental clearance of a branched-chain amino acid (BCAA) analog, the nonmetabolizable neutral amino acid aminocyclopentane-1-carboxylic acid (ACP), in singleton control (C) and FGR pregnancies at 135 days gestation age (dGA; term 147 dGA). At study, based on the severity of the placental dysfunction, FGR fetuses were allocated to severe (sFGR, n = 6) and moderate FGR (mFGR, n = 4) groups. Fetal (C, 3,801.91 +/- 156.83; mFGR, 2,911.33 +/- 181.35; sFGR, 1,795.99 +/- 238.85 g; P < 0.05) and placental weights (C, 414.38 +/- 38.35; mFGR, 306.23 +/- 32.41; sFGR, 165.64 +/- 28.25 g; P < 0.05) were reduced. Transplacental and total placental clearances of ACP per 100 g placenta were significantly reduced in the sFGR but not in the mFGR group, whereas placental retention clearances were unaltered. These data indicate that both entry of ACP into the placenta and movement from the placenta into fetal circulation are impaired in severe ovine FGR and support the hypothesis of impaired placental BCAA transport in severe human FGR.

Placental transport of leucine in a porcine model of low birth weight.

Low birth weight is a major factor in neonatal morbidity and mortality in humans and domestic species and is a predictor of physiological disorders in adulthood. This study utilised the naturally occurring variation in pig fetal size within a uterus to test the hypothesis that placental amino acid transport capability is associated with fetal growth. Leucine uptake by trophoblast vesicles prepared from placentas supplying an average-sized fetus and the smallest fetus in the uterus was assessed. On days 45 and 65 of gestation, uptake of leucine by the porcine placenta was predominantly sodium independent and was inhibited by the non-metabolised leucine analogue 2-amino-2-norbornane-carboxylic acid, indicating that uptake occurs via system L. By day 100 the uptake of leucine by placentas supplying average-sized fetuses had changed from being predominantly sodium independent to involving both sodium-dependent (system B0) and -independent (system L) pathways. This change was not seen in placentas supplying the smallest fetus, which continued to display predominantly sodium-independent uptake. In conclusion, these data show gestational- and fetal size-dependent changes in the transport of leucine across the porcine placenta.

Placental transport of amino acids in normal and growth-restricted pregnancies

In human pregnancies placental amino acid transport has been studied at the time of delivery and also by in utero fetal blood sampling (FBS). A significant reduction in amino acid fetal–maternal gradients and in umbilical veno-arterial differences has been demonstrated in intrauterine growth-restricted (IUGR) pregnancies. Fetal–maternal transfer rates have been further investigated in vivo by stable isotope methodologies. Following a maternal bolus infusion of [1- 13 C ]-glycine and [1- 13 C ]-leucine performed at fetal blood sampling, the transfer rate of the non-essential amino acid glycine is significantly lower than that for the essential amino acid leucine, suggesting that glycine can be newly synthesized in the feto-placental unit. Moreover, in growth-restricted pregnancies the fetal/maternal ratio of [1- 13 C ]-leucine is significantly lower, and proportional to the degree of severity. In vitro studies have described a variety of transport systems for amino acids within the microvillous membrane (MVM) and the basal membrane (BM) of the placenta and significant differences have been reported in growth-restricted pregnancies for system A, system L, and taurine transporters. These changes are significantly associated to both biophysical and biochemical parameters of severity. Moreover, significant relationships can be found in arginine transport system and uterine oxygenation, suggesting a role in nitric oxide (NO) synthesis.

Manipulation of CD98 expression affects both trophoblast cell fusion and amino acid transport activity during syncytialization of human placental BeWo cells.

The physiological importance of CD98 surface antigen in regulating placental trophoblast cell fusion and amino acid transport activity has been studied in parallel in a cell model of syncytialization (the cytotrophoblast cell line BeWo following increased intracellular cAMP by forskolin treatment) using antisense oligonucleotides. CD98 protein abundance (determined by Western blot) was decreased by 50 % following antisense oligonucleotide transfection. Transfection with antisense oligonucleotide altered the responses of BeWo to forskolin. Cell fusion (determined by a quantitative flow cytometry assay) was inhibited by 57 %, and both human chorionic gonadotropin secretion and L-leucine influx through system L were suppressed. These findings show that CD98 is involved in the process of cell fusion necessary for syncytiotrophoblast formation and that during this physiologically important event, amino acid transport activity is also regulated through expression of this membrane protein.

Maternal and fetal amino acid concentrations and fetal outcomes during pre-eclampsia.

The pathophysiology of pre-eclampsia is contested, but one hypothesis indicates that it is a heterogeneous condition in which only a subset of affected women bear small-for-gestational age (SGA) babies. In intrauterine growth-restricted (IUGR) infants, placental transport of amino acids is diminished and the resulting decrease in cord-blood amino acid concentrations is thought to contribute to their stunted growth. In contrast, the metabolic syndrome (dyslipidaemia, hyperinsulinaemia, hyperglycaemia, hypertension and obesity) which is associated with high amino acid concentrations is more prevalent in women with pre-eclampsia. The focus of this study was to compare maternal and fetal serum amino acid concentrations during normal pregnancy and pre-eclampsia and to evaluate the associations between the amino acid concentrations and fetal growth. The results indicate that maternal and cord-blood amino acid concentrations were significantly higher in women with pre-eclampsia compared with normal pregnant women and the concentrations were inversely associated with measures of infant growth. Maternal and cord-blood amino acid concentrations were also significantly higher in pre-eclamptic mothers with SGA infants compared with pre-eclamptic mothers whose babies were not SGA. These data indicate that, in contrast to IUGR, pre-eclampsia is associated with enhanced placental amino acid transport or reduced fetal amino acid utilization. Furthermore, the data are consistent with the hypothesis that pre-eclampsia is a heterogeneous disease associated with the metabolic syndrome.

Human placental amino acid transporter genes: expression and function

Recent findings on amino acid transporter genes are reviewed with particular focus on matching previously described transport systems to individual genes. Functional studies using cloned and expressed transporters are considered as the critical tool allowing identification of the functional properties of individual genes. Specifically, these experiments allow identification of the transported substrate amino acids and of the transport mechanism. We focus on the very recent discovery and properties of the heterodimeric family of amino acid transport proteins where two subunits encoded in different genes are required. For these transporters, co-expression of both subunits is mandatory for functional studies. The field of placental amino acid transport is further complicated by complexities arising from both gestational age-specific and species-specific gene expression. The function of the transporter also depends on its cellular localization in the trophoblast. In addition, for transporters that are coupled to ion gradients, both membrane potential and ion pumping will contribute to the rate of amino acid delivery to the fetus. Regulation of function is important not only for fetal nutrition but also for specific additional aspects of placental biology.