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.

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IL-6 stimulates system A amino acid transporter activity in trophoblast cells through STAT3 and increased expression of SNAT2

AJP Cell Physiology ◽

10.1152/ajpcell.00195.2009 ◽

2009 ◽

Vol 297 (5) ◽

pp. C1228-C1235 ◽

Cited By ~ 108

Author(s):

H. N. Jones ◽

T. Jansson ◽

T. L. Powell

Keyword(s):

Amino Acid ◽

Protein Expression ◽

Nutrient Transport ◽

Amino Acid Transporter ◽

Amino Acid Transporters ◽

Trophoblast Cells ◽

System A ◽

Tnf Α ◽

Gene And Protein Expression ◽

Acid Transporter

Changes in placental nutrient transport are closely associated with abnormal fetal growth. However, the molecular mechanisms underlying the regulation of placental amino acid transporters are unknown. We demonstrate that physiological concentrations of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α stimulate the activity of amino acid transporter system A, but not system L, in cultured human primary trophoblast cells. Both cytokines increased the gene and protein expression of the Na+-coupled neutral amino acid transporter (SNAT)2 isoform and upregulated SNAT1 protein expression. IL-6 increased Tyr705 phosphorylation of signal transducer and activator of transcription 3 (STAT3). In cells transfected with small interfering RNA (siRNA) targeting STAT3, the RNA and protein expression of SNAT2, but not SNAT1, was reduced and the stimulating effect of IL-6 on system A activity was abolished. Despite eliciting similar responses in amino acid transport activity and transporter expression, TNF-α effects on system A activity were not mediated through the JAK/STAT pathway. In conclusion, we have identified a novel regulatory pathway involving increased gene expression of the SNAT2 isoform mediated by a STAT-dependent pathway, which links IL-6 to increased activity of system A, a ubiquitously expressed transporter of neutral amino acids. From these new findings, we propose that upregulation of amino acid transporters by cytokines may contribute to increased placental nutrient transport and fetal overgrowth, which are commonly found in pregnancies complicated by maternal diabetes and obesity.

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Downregulation of Placental Amino Acid Transporter Expression and mTORC1 Signaling Activity Contributes to Fetal Growth Retardation in Diabetic Rats

International Journal of Molecular Sciences ◽

10.3390/ijms21051849 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1849

Author(s):

Jie Xu ◽

Jiao Wang ◽

Yang Cao ◽

Xiaotong Jia ◽

Yujia Huang ◽

...

Keyword(s):

Amino Acid ◽

Rat Model ◽

Amino Acid Transporter ◽

Amino Acid Transporters ◽

Amino Acid Transport System ◽

Intrauterine Growth ◽

System L ◽

Acid Transporter ◽

Transporter Expression

Alterations in placental transport may contribute to abnormal fetal intrauterine growth in pregnancies complicated by diabetes, but it is not clear whether the placental amino acid transport system is altered in diabetic pregnancies. We therefore studied the changes in the expressions of placental amino acid transporters in a rat model of diabetes induced by streptozotocin, and tested the effects of hyperglycemia on trophoblast amino acid transporter in vitro. Our results showed that the expressions for key isoforms of system L amino acid transporters were significantly reduced in the placentas of streptozotocin-induced diabetic pregnant rats, which was associated with the decreased birthweight in the rats. A decreased placental efficiency and decreased placental mammalian target of rapamycin (mTOR) complex 1 (mTORC1) activity were also found in the rat model. In addition, hyperglycemia in vitro could inhibit amino acid transporter expression and mTORC1 activity in human trophoblast. Inhibition of mTORC1 activity led to reduced amino acid transporter expression in placental trophoblast. We concluded that reduced placental mTORC1 activity during pregnancy resulted in decreased placental amino acid transporter expression and, subsequently, contributed to fetal intrauterine growth restriction in pregnancies complicated with diabetes.

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SNAT4 isoform of system A amino acid transporter is expressed in human placenta

AJP Cell Physiology ◽

10.1152/ajpcell.00258.2005 ◽

2006 ◽

Vol 290 (1) ◽

pp. C305-C312 ◽

Cited By ~ 78

Author(s):

M. Desforges ◽

H. A. Lacey ◽

J. D. Glazier ◽

S. L. Greenwood ◽

K. J. Mynett ◽

...

Keyword(s):

Amino Acid ◽

Protein Expression ◽

Western Blot ◽

Human Placenta ◽

Blot Analysis ◽

First Trimester ◽

Amino Acid Transporter ◽

Full Term ◽

System A ◽

Acid Transporter

The system A amino acid transporter is encoded by three members of the Slc38 gene family, giving rise to three subtypes: Na+-coupled neutral amino acid transporter (SNAT)1, SNAT2, and SNAT4. SNAT2 is expressed ubiquitously in mammalian tissues; SNAT1 is predominantly expressed in heart, brain, and placenta; and SNAT4 is reported to be expressed solely by the liver. In the placenta, system A has an essential role in the supply of neutral amino acids needed for fetal growth. In the present study, we examined expression and localization of SNAT1, SNAT2, and SNAT4 in human placenta during gestation. Real-time quantitative PCR was used to examine steady-state levels of system A subtype mRNA in early (6–10 wk) and late (10–13 wk) first-trimester and full-term (38–40 wk) placentas. We detected mRNA for all three isoforms from early gestation onward. There were no differences in SNAT1 and SNAT2 mRNA expression with gestation. However, SNAT4 mRNA expression was significantly higher early in the first trimester compared with the full-term placenta ( P < 0.01). We next investigated SNAT4 protein expression in human placenta. In contrast to the observation for gene expression, Western blot analysis revealed that SNAT4 protein expression was significantly higher at term compared with the first trimester ( P < 0.05). Immunohistochemistry and Western blot analysis showed that SNAT4 is localized to the microvillous and basal plasma membranes of the syncytiotrophoblast, suggesting a role for this isoform of system A in amino acid transport across the placenta. This study therefore provides the first evidence of SNAT4 mRNA and protein expression in the human placenta, both at the first trimester and at full term.

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Amino acid transporters: éminences grises of nutrient signalling mechanisms?

Biochemical Society Transactions ◽

10.1042/bst0370237 ◽

2009 ◽

Vol 37 (1) ◽

pp. 237-241 ◽

Cited By ~ 27

Author(s):

Peter M. Taylor

Keyword(s):

Amino Acid ◽

Binding Sites ◽

Mammalian Target Of Rapamycin ◽

Amino Acid Transporters ◽

System A ◽

System L ◽

Underlying Mechanisms ◽

Substrate Binding Sites ◽

Amino Acid Concentrations ◽

Mtor Signalling

Nutrient signalling by the mTOR (mammalian target of rapamycin) pathway involves upstream sensing of free AA (amino acid) concentrations. Several AA-regulated kinases have recently been identified as putative intracellular AA sensors. Their activity will reflect the balance between AA flows through underlying mechanisms which together determine the size of the intracellular free AA pool. For indispensable AAs, these mechanisms are primarily (i) AA transport across the cell membrane, and (ii) protein synthesis/breakdown. The System L AA transporter is the primary conduit for cellular entry of indispensable neutral AAs (including leucine and phenylalanine) and potentially a key modulator of AA-sensitive mTOR signalling. Coupling of substrate flows through System L and other AA transporters (e.g. System A) may extend the scope for sensing nutrient abundance. Factors influencing AA transporter activity (e.g. hormones) may affect intracellular AA concentrations and hence indirectly mTOR pathway activity. Several AA transporters are themselves regulated by AA availability through ‘adaptive regulation’, which may help to adjust the gain of AA sensing. The substrate-binding sites of AA transporters are potentially direct sensors of AA availability at both faces of the cell surface, and there is growing evidence that AA transporters of the SNAT (sodium-coupled neutral AA transporter) and PAT (proton-assisted AA transporter) families may operate, at least under some circumstances, as transporter-like sensors (or ‘transceptors’) upstream of mTOR.

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Identification of a Novel System L Amino Acid Transporter Structurally Distinct from Heterodimeric Amino Acid Transporters

Journal of Biological Chemistry ◽

10.1074/jbc.m305221200 ◽

2003 ◽

Vol 278 (44) ◽

pp. 43838-43845 ◽

Cited By ~ 167

Author(s):

Ellappan Babu ◽

Yoshikatsu Kanai ◽

Arthit Chairoungdua ◽

Do Kyung Kim ◽

Yuji Iribe ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Transporter ◽

Amino Acid Transporters ◽

System L ◽

Acid Transporter

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The amino acid transporter Slc7a5 regulates the mTOR pathway and is required for granule cell development

Human Molecular Genetics ◽

10.1093/hmg/ddaa186 ◽

2020 ◽

Vol 29 (18) ◽

pp. 3003-3013 ◽

Cited By ~ 1

Author(s):

Aidan M Sokolov ◽

Jennie C Holmberg ◽

David M Feliciano

Keyword(s):

Amino Acid ◽

Granule Cell ◽

Neural Development ◽

Mammalian Target Of Rapamycin ◽

Amino Acid Transporter ◽

Mtor Pathway ◽

Normal Brain ◽

Mtor Inhibition ◽

Pathway Activity ◽

Acid Transporter

Abstract Pathogenic mutations in the solute carrier family 7 member 5 (SLC7A5) gene, which encodes an amino acid transporter cause microcephaly and seizures, yet the mechanisms responsible for these phenotypes are unclear. Models have demonstrated that Slc7a5 deletion is embryonic lethal and that these embryos lack a fully formed telencephalon. This phenotype is similar to that of mammalian target of rapamycin (mTOR) protein kinase deletion or mTOR inhibition. Notably, in many cells, Slc7a5 import of amino acids is required to maintain mTOR activity. Slc7a5 is present within neurogenic regions during embryogenesis, is found in cultured neurons and can modulate neuronal electrophysiological properties. However, Slc7a5 is also highly expressed within endothelial cells of the blood–brain barrier where removal in conditional mice leads to severe behavioral defects and non-cell autonomous changes in neurons. Therefore, the extent that neural Slc7a5 is required for development is unclear. Here, subventricular zone neural stem cells that generate olfactory bulb granule cell neurons were electroporated with SLC7A5 or Slc7a5 short hairpin RNA encoding plasmids. Although early phases of neural development were unaltered, Slc7a5 knockdown effected late phases of GC dendrite maturation and survival. Slc7a5 knockdown also decreased mTOR pathway activity. Ras homolog enriched in brain, an mTOR activator, rescued the effect of Slc7a5 knockdown on mTOR pathway activity and dendrite arbors. The data presented here demonstrate that Slc7a5 is required for GC mTOR pathway activity, maturation and survival, which may help explain why Slc7a5 mutations prevent normal brain development and function.

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Synthetic Glucocorticoid Reduces Human Placental System A Transport in Women Treated With Antenatal Therapy

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2014-2157 ◽

2014 ◽

Vol 99 (11) ◽

pp. E2226-E2233 ◽

Cited By ~ 18

Author(s):

Melanie C. Audette ◽

John R. G. Challis ◽

Rebecca L. Jones ◽

Colin P. Sibley ◽

Stephen G. Matthews

Keyword(s):

Amino Acid ◽

Mrna Expression ◽

Fetal Growth ◽

Preterm Labor ◽

Developmental Programming ◽

Amino Acid Transporter ◽

Acid Uptake ◽

System A ◽

Acid Transporter

Context: 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. Objective: 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. Methods and Patients: 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 [14C]methylaminoisobutyric acid uptake per gram placenta) and mRNA expression. Results: 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. Conclusion: 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.

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Expression and Role of the System L Amino Acid Transporter in FOB Human Osteoblast Cells

Journal of the Korean Society of Food Science and Nutrition ◽

10.3746/jkfn.2005.34.9.1367 ◽

2005 ◽

Vol 34 (9) ◽

pp. 1367-1374

Keyword(s):

Amino Acid ◽

Amino Acid Transporter ◽

Human Osteoblast ◽

Osteoblast Cells ◽

System L ◽

Acid Transporter

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Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein

Methods and Protocols ◽

10.3390/mps4030051 ◽

2021 ◽

Vol 4 (3) ◽

pp. 51

Author(s):

Satish Kantipudi ◽

Daniel Harder ◽

Sara Bonetti ◽

Dimitrios Fotiadis ◽

Jean-Marc Jeckelmann

Keyword(s):

Amino Acid ◽

Pichia Pastoris ◽

Protein Complexes ◽

Amino Acid Transporter ◽

Amino Acid Transporters ◽

Expression Host ◽

Amino Acids And Derivatives ◽

Heterodimeric Complex ◽

Acid Transporter ◽

And Function

Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 is composed of the type-II membrane N-glycoprotein 4F2hc (SLC3A2) and the L-type amino acid transporter LAT1 (SLC7A5). 4F2hc-LAT1 is medically relevant, and its dysfunction and overexpression are associated with autism and tumor progression. Here, we provide a general applicable protocol on how to screen for the best membrane transport protein-expressing clone in terms of protein amount and function using Pichia pastoris as expression host. Furthermore, we describe an overexpression and purification procedure for the production of the HAT 4F2hc-LAT1. The isolated heterodimeric complex is pure, correctly assembled, stable, binds the substrate L-leucine, and is thus properly folded. Therefore, this Pichia pastoris-derived recombinant human 4F2hc-LAT1 sample can be used for downstream biochemical and biophysical characterizations.

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