| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Perinatal Center, Department of Physiology, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden; 2Division of Bio-System Pharmacology, Department of Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan; 3Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, Georgia; and 4Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, Cincinnati, Ohio
Submitted 24 June 2008 ; accepted in final form 3 November 2008
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
system A; system L; taurine transporter
The most important determinant of fetal growth is the capacity of the placenta to deliver nutrients from the mother to the fetus, which is dependent on the expression and function of nutrient transporters in the placental barrier. Studies in the isolated microvillous membrane (MVM) and basal plasma membrane (BM) of the syncytiotrophoblast, the transporting epithelium of the placenta, have shown that fetal growth abnormalities are associated with alterations of specific placental amino acid transporters. In the fetal overgrowth MVM, system A and system L activities have been reported to be upregulated (30). However, in one study (42), system A activity has been shown to be reduced in MVM from pregnancies where the mothers had insulin-dependent diabetes. The reasons for these discrepancies are unclear but may be related to differences in the two study populations, and placental weight was increased in one study (30), whereas placental weight was unaffected in the other study (42). In contrast, IUGR is characterized by a decreased activity of placental amino acid transporters. For example, the MVM activity of Na+-dependent transporter system A, which transports small, neutral, nonessential amino acids, is reduced in IUGR (15, 24, 46). The activities of transporters for the essential amino acids leucine (system L) and taurine [taurine transporter (TAUT)] are also reduced in MVM and/or BM isolated from IUGR placentas (31, 50). The downregulation of placental amino acid transporters in IUGR could be the cause of the reduced fetal plasma amino acid concentrations seen in this pregnancy complication (12, 16). As amino acids are the primary stimuli for insulin secretion from the fetal pancreas, there may be a direct link between changes in placental amino acid transporter activity and altered fetal growth. We (28) recently reported in a rodent model of protein deprivation during pregnancy that placental system A activity is downregulated several days before IUGR develops. These data suggest that downregulation of placental system A activity is a cause, rather than a consequence, of IUGR in this model.
To better understand the pathophysiology of abnormal fetal growth, it is critical to identify the factors regulating placental nutrient transporters. System A is subjected to extensive regulation, and, in the placenta, it has been shown to be inhibited by IL-1β (64), hypoxia (48), nitric oxide (NO) formation (37), hyperglycemia (21), and growth hormone (20). Insulin (27, 36), IGF-I (35), EGF (5), amino acid deprivation (34), cortisol (33), and leptin (27) all stimulate system A transport. The regulation of placental system L and TAUT is less well established. Placental TAUT activity is regulated by taurine itself (32), calcium (41), PKC (40, 59), NO (37, 59), and cyclosporin A (57). Increases in intracellular Ca2+ concentrations, PKC, and low extracellular pH (7, 51, 56) all stimulate system L activity.
The mammalian target of rapamycin (mTOR) signaling pathway integrates various extracellular and intracellular signals. Growth factor- and hormone-induced stimulation of mTOR is the best-characterized upstream regulator of mTOR, and it is mediated by the activation of phosphatidylinositol 3-kinase (PI3K) (66). Nutrients represent another major signal input that activates mTOR; however, the exact mechanism of nutrient regulation remains unclear. It has recently been suggested that both the class III PI3K hVps34 (10, 49) and the Ste20-related kinase MAP4K3 (22) play a role in mTOR-mediated nutrient sensing. The best-described function of mTOR is its regulation of translation, which occurs by phosphorylation of the key translation regulators p70 ribosomal S6 kinase (S6K1) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1). There is some evidence indicating that mTOR regulates the expression of nutrient transporters in nonplacental tissues. In FL5.12 cells, growth factors have been shown to not only increase transporter gene transcription but also promote transporter surface expression (17). Treatment of human BJAB B-lymphoma cells and murine CTLL-2 T lymphocytes with rapamycin leads to the downregulation of the expression of genes participating in nutrient transport (53). Liu et al. (44) have shown that the expression of L-type amino acid transporter (LAT)1, an isoform of the L-amino acid transporter light chain, in vascular smooth muscle cells is stimulated by PDGF in a mTOR-dependent manner. We (58) recently reported that rapamycin, a highly specific mTOR inhibitor, reduced system L activity, but not the activities of system A transporters or TAUT, in primary villous fragments from the human placenta. In these experiments, transport measurements were carried out after 4 h of rapamycin incubation, a time period where the regulation of transport mediated by effects on transcription and translation may not be observable. Considering that the most-established and widely reported cellular effects of mTOR signaling are the regulation of protein translation, we tested the hypothesis that mTOR alters amino acid transport activity by changes in transporter protein and mRNA expression. To this effect, mTOR signaling was inhibited for 24 h in cultured primary human trophoblast cells and, subsequently, the activity, mRNA experssion, and protein expression of three key placental amino acid transporters were measured.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Trophoblast Cell Culture
Cytotrophoblast cells were isolated from human placentas and cultured as previously described (38, 45). Cells were plated in either plastic 6-well plates at a density of 
1.5 x 106 cells/well (for amino acid uptake experiments) or 25-cm2 flasks at a density of 10 x 106 cells/flask (for RNA extraction and Western blot analysis) and cultured for 90 h. Between 66 and 90 h of culture, cells were incubated with 100 nM rapamycin (LC Laboratories, Woburn, MA), a specific mTOR inhibitor, or vehicle (0.02% DMSO). After 90 h in culture, cells were either used in amino acid transporter activity assays or collected for Western blot analysis or mRNA extraction.
Assessment of Biochemical Differentiation and Viability To confirm that our cells were undergoing biochemical differentiation, and to assess their viability with time in culture, the release of human chorionic gonadotropin (hCG) by trophoblast cells into the culture medium after 18, 42, 66, and 90 h was measured using a commercial ELISA kit, which detects the β-subunit of hCG (DRG Instruments). To verify the trophoblast nature of isolated cells, an anti-cytokeratin-7 (clone OVTL 12/30) antibody (antibody 9098, Abcam, Cambridge, UK) was used. Vimentin was used as a marker for the identification of contaminating mesenchyme-derived cells (antibody 20346, Abcam).
Lactate dehydrogenase (LDH) release into the medium was assessed using a LDH-based in vitro toxicology assay (Sigma-Aldrich). LDH release measured after cells had been subjected to sonication was used as a positive control.
Antibodies directed against caspase-3 (R&D Systems, Minneapolis, MN) and cleaved poly(ADP-ribose) polymerase (PARP; Affinity BioReagents, Golden, CA) were used to determine the presence of apoptosis in cultured cells.
Measurement of Amino Acid Transporter Activity Amino acid transporter activities were measured according to a method developed for primary villous fragments (27, 58, 59), which we modified for use in cultured trophoblast cells. Briefly, system A [Na+-coupled neutral amino acid transporter (SNAT)] and TAUT activities were measured as Na+-dependent [14C]methylaminoisobuturyric acid (MeAIB) or [3H]taurine uptake, respectively, and system L amino acid transporter (LAT) activity was determined as the 2-amino-2-norbornanecarboxylic acid (BCH)-inhibitable uptake of [3H]leucine. After 24 h of incubation with rapamycin (100 nM) or vehicle (0.02% DMSO), cells were washed twice with 3 ml of Tyrode solution at 37°C with or without Na+ and then incubated for variable times for up to 10 min in 1.5 ml of Tyrode solution (with or without Na+ and 1 mM BCH) containing [14C] MeAIB and [3H]leucine or [3H]taurine in final concentrations of 10 µM, 50 nM, and 25 nM, respectively. Each condition was studied in triplicate. Uptake was terminated by washing three times with 4 ml of ice-cold Tyrode solution without Na+. Cells were lysed in distilled H2O for 1 h and then denaturated in 0.3 M NaOH for 2 h or overnight. The water containing the tracers released from the cells was mixed with scintillation fluid and counted in a β-counter. After denaturation, the protein concentration was measured using the BCA protein assay kit (Pierce, Rockford, IL) or the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Transporter-mediated amino acid transport was calculated by subtracting the uptake in the buffer, representing the nonmediated uptake, from the uptake in the buffer, representing the total uptake. Uptakes were calculated as picomoles of amino acid uptake per milligram of total protein per 8 min. Time courses of taurine, leucine, and MeAIB uptake in cytotrophoblast cells were performed. Control cells were incubated with vehicle only to obtain amino acid uptake under control conditions. Control amino acid uptakes were arbitrarily set to a value of 1 for the purpose of comparison.
Western Blot Analysis Cells were harvested in buffer D (250 mM sucrose, 10 mM HEPES-Tris, 0.7 µM pepstatin A, 1.6 µM antipain, and 80 µM aprotinin; pH 7.4 at 4°C) with freshly added phosphatase inhibitor cocktails 1 and 2 (1:100, Sigma-Alrich) using a cell scraper. Cells were then homogenized by passing the lysate several times through a 20-gauge needle fitted to a syringe. Protein concentrations were determined using the method of Bradford (Bio-Rad). To ensure equal loading of samples, all membranes were reprobed with monoclonal anti-β-actin antibody (Sigma-Aldrich).
S6K1 and 4E-BP1. To confirm that rapamycin does inhibit the mTOR signaling pathway in cultured primary human trophoblast cells, equal amounts of protein [20 µg for phospho-S6K1 (Thr389) and phospho-4E-BP1 (Thr37/46) and 15 µg for phospho-4E-BP1(Thr70)] were separated on NuPAGE Novex (Invitrogen, Lidingö, Sweden) precast 4–12% (phospho-S6K1) or 10% (phospho-4E-BP1) Bis-Tris gels. All antibodies were purchased from Cell Signaling Technology (Danvers, MA), and antibody incubations were carried out according to the protocol provided by the manufacturer except for the phospho-4E-BP1 (Thr37/46) antibody, which was diluted 1:2,000 to 0.04 µg/ml. Duplicate gels were probed with antibodies to total 4E-BP1 and S6K1 (Cell Signaling Technology).
SNAT2 and SNAT4. For SNAT2 Western blots, samples were prepared in Laemmli buffer and heated for 5 min at 95°C. For SNAT4 immunoblots, a 3x DTT sample buffer (8 M urea, 170 mM SDS, 0.04 units of bromphenol blue, and 450 mM DTT in 50 mM Tris·HCl; pH 6.8) was used, and samples were heated for 3 min at 100°C before being loaded. Protein (20 µg) was separated on precast 4–12% Bis-Tris gel (Invitrogen) and transferred onto a nitrocellulose membrane. Membranes were blocked for 1 h in 5% milk in PBS-0.1% Tween (PBST) and then washed in PBST (3 x 5 min). The SNAT2 antibody (43) was diluted 1:4,000 to 0.25 µg/ml and incubated overnight at 4°C. The SNAT4 antibody was raised to the amino acid sequence YGEVEDELLHAYSKV of human SNAT4 (Eurogentec, Seraing, Belgium) and was used at a dilution of 1:4,000 (0.5 µg/ml). The SNAT4 antibody incubation was performed for 1 h at room temperature. After a final wash (3 x 5 min), the signal was visualized using Super Signal Western Dura Substrate (Pierce).
TAUT. Probing for TAUT protein was preformed as previously described (59). Briefly, SNAT2 gels were stripped in stripping solution (2% SDS, 62.5 mM Tris·HCl, and 100 mM β-mercaptoethanol; pH 6.7) for 30 min at 50°C and reprobed with an antibody directed against TAUT (Chemicon) diluted 1:4,000 to 0.25 µg/ml in PBST overnight at 4°C. The signal was then detected using Pierce ECL chemicals.
4F2hc. Protein (20 µg) was separated on NuPAGE Novex (Invitrogen) precast 4–12% gels under reducing conditions. Membranes were probed with goat anti-human 4F2hc polyclonal antibody (diluted 1:400 to 0.5 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA) and diluted in 5% milk in Tris-buffered saline-0.1%Tween 20 for 1 h. The signal was detected with the Pierce ECL system.
LAT2. LAT2 antibody was produced in rabbits using the peptide sequence EEANEDMEEQQQC (52). The antibody was diluted 1:2,000 to 0.3 µg/ml and was used to probe membranes onto which 20 µg of protein had been transferred. Preincubation of the primary antibody with its blocking peptide (Eurogentec) was used to confirm the specificity of the LAT2 band.
The relative density of bands was assessed by densitometry with Multi Gauge Analyses software (version 3.0, Fuji Film). The mean densitometry value of control cells was assigned an arbitrary value of 1 for comparison purposes, and the mean density of rapamycin-treated cells was expressed relative to control cells.
mRNA Isolation and Quantitative Real-Time PCR The relative mRNA expression of amino acid transporter was analyzed by quantitative real-time PCR using a LightCycler (Roche). Total RNA was extracted from trophoblast cells, and first-strand cDNA synthesis was then performed as previously described (21). Oligonucleotide primers for subunit A of succinate dehydrogenase complex (SDHA) and TATA box-binding protein (TBP) were designed using the Primer3 program (version 0.4.0, http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). SNAT1, SNAT2, SNAT4, SDHA, LAT1, LAT2, 4F2hc, and TBP primers were synthesized by Cybergene (Huddinge, Sweden), and primer sequences are shown in Table 1. For detection of TAUT (NM_003043) mRNA, the Hs_SLC6A6 QuantiTect Primer Assay from Qiagen (Solna, Sweden) was used. For Cybergene-synthesized primers, real-time PCRs were performed in 20-µl mixtures containing 2 µl cDNA (diluted 1:4), 2 mM MgCl2 (LAT1, SNAT2, SNAT4, and SDHA) or 3 mM MgCl2 (LAT2, SNAT1, 4F2hc, and TBP), 0.5 µM of each primer, and 2 µl of LightCycler FastStart DNA Master SYBR Green I (Roche) and carried out according to the manufacturer's instructions. For the detection of TAUT, real-time PCR was performed in 20 µl using the QuantiFast SYBR Green PCR Kit (Qiagen) following the manufacturer's instructions. The mitochondrial protein SDHA and TBP served as internal controls (47). All samples were assayed in duplicate, and water was used as a negative template control. A standard curve for each gene product was generated using a dilution series of cDNA (1:2–1:32). The amplification transcripts were quantified using the relative standard curve and normalized against SDHA and TBP by calculating a normalization factor by averaging SDHA and TBP using the geometric mean. The mean mRNA expression value of control cells for each gene was assigned an arbitrary value of 1 for comparison purposes, and the mean mRNA expression of rapamycin-treated cells was expressed relative to control cells.
|
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
Expression of apoptotic markers. Both control and rapamycin-treated trophoblast cells were PARP negative (Fig. 1C), demonstrating that apoptotic activity in our cells is low and that rapamycin does not increase apoptosis. This conclusion was strongly supported by data showing that there were no differences in the expression of active caspase-3 between control and rapamycin-treated cells (Fig. 1C), which is in line with a report (68) demonstrating that trophoblast cells do express active caspases.
Amino Acid Transporter Activity Na+-dependent uptakes of [3H]taurine (25 nM) and [14C]MeAIB (10 µM) as well as the BCH-inhibitable uptake of [3H]leucine (50 nM) were all linear up to at least 10 min [system-β, r = 0.84, P < 0.01, n = 9 (Fig. 2A); system A, r = 0.95, P < 0.01, n = 9 (Fig. 2B); and system L, r = 0.89, P < 0.01, n = 16 (Fig. 2C)]. Based on these time course experiments, an incubation time of 8 min was chosen in subsequent experiments of the effect of mTOR inhibition on trophoblast amino acid uptake.
|
|
|
70 kDa. Two distinct bands at 48 and 55 kDa were observed in cells when the SNAT2 antibody was used; both bands were analyzed, and the sum of the two bands was used as the densitometry value for that sample. SNAT4 was detected at 62 kDa. 4F2hc and LAT2 proteins were identified at 80 and 40 kDa, respectively. A MVM sample from the human placenta was used as a positive control (Fig. 5). The specificity of the SNAT2, SNAT4, and LAT2 bands was verified by incubating the primary antibodies with their respective blocking peptides (data not shown). Trophoblast expression of these amino acid transporter proteins was unaffected by rapamycin treatment (Fig. 5).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Cultured human trophoblast cells have been used extensively in studies of placental nutrient transporters (5, 23, 35, 36, 48). Our cells were cytokeratin positive and vimentin negative and showed an exponential increase in the secretion of the syncytial biochemical marker hCG, verifying that we had syncytializing trophoblast cells in our culture without significant contamination of cells derived from the mesenchyme. We (58) have previously shown that mTOR protein is present in the syncytiotrophoblast, and, in the present study, we verified that rapamycin efficiently inhibited mTOR signaling in cultured trophoblast cells as evidenced by decreased phosphorylation of 4E-BP1 and abolishment of the expression of phospho-Thr389-S6K1 in response to rapamycin. This is a specific downregulation, since the expression of total 4E-BP1 and S6K1 was unaffected. Furthermore, rapamycin did not alter the characteristics of the cells, given that there were no differences in cytokeratin 7 expression, hCG production, LDH release, or protein expression of the apoptotic markers PARP and caspase-3 between rapamycin-treated and control cells.
We demonstrated that inhibition of mTOR signaling decreases the activity of system L, system A, and taurine amino acid transporters in cultured primary trophoblast cells. We suggest that the size of the observed changes (17–40% decrease) in the activity of key amino acid transporters are physiologically relevant. For example, TAUT is the only transporter mediating the uptake of taurine across the MVM, and the only known pathway for MVM uptake of essential amino acids such as leucine is system L. Since the transfer across the MVM is likely to be the rate-limiting step for placental amino acid transport (29), a 40% (taurine) and 28% (leucine) decrease in the uptake of these amino acids in vivo is likely to have significant effects on the fetal amino acid supply. Along these lines, the effects of mTOR inhibition on amino acid transport activity in the present study are of similar magnitude as reported for changes in placental transporter activity in human IUGR. For example, we have previously shown that IUGR is associated with a 34% decrease in TAUT activity in the MVM (50) and a 38% and 46% decrease in system L amino acid transporter activity in the BM and MVM, respectively (31).
The finding that mTOR stimulates system L activity is consistent with our previous finding in primary villous fragments from the human placenta (58). Intriguingly, the effect of mTOR inhibition for 24 h on system A and TAUT activity in cultured trophoblast cells differs from the response to 4 h of inhibition with rapamycin in primary villous fragments in which system A and TAUT activities were unaffected (58). The mechanisms underlying these different findings are currently unknown but may be related to distinct differences in the two experimental systems. Alternatively, the much longer rapamycin incubation time used in the cultured trophoblast cells may explain this discrepancy. It has previously been shown in L6 myotubes that short-term rapamycin incubation (3 h) does not affect system A activity (55), and prolonged exposure to rapamycin has been shown to inhibit mTORC2 function (60), which was previously thought to be rapamycin insensitive. Thus, it is possible that the incubation of our primary trophoblast cells for 24 h in rapamycin also inhibited mTORC2, which may downregulate system A and TAUT activity.
In our experiments of the role of mTOR signaling in the regulation of nutrient transporters, we have used transporter activity as the primary outcome measurement, and the experiments have been undertaken in primary human cells, which we believe contribute to the physiological significance of our findings. In contrast, most previous studies on mTOR regulation of nutrient transporters have focused on the effects on transporter expression in cell lines. For example, in lymphoma cells, rapamycin selectively downregulated the expression of five genes involved in amino acid transport (53). LAT1 mRNA has been shown to be increased in PDGF-treated vascular smooth muscle cells, and this induction was dependent on mTOR (44). In a murine T cell line, cell surface expression of 4F2hc was inhibited by 24-h rapamycin incubation (19). However, there are a few reports in which the effects of mTOR inhibition on transporter activity have been assessed. System A activity in L6 myotubes has been shown to be upregulated by leucine in a mTOR-dependent manner (55), and rapamycin treatment reduced basal gluose uptake in these cells (63). Yeast cells treated with rapamycin have reduced leucine uptake (65), and primary hepatocytes incubated with rapamycin for 18 h have reduced glucose uptake (9). Coexpression of mTOR and Na+-coupled phosphate transporter SLC34A2 and creatine transporter SLC6A8 in Xenopus oocytes stimulated the activity of these transporters (61, 62). Collectively, these findings in the literature indicate that various nutrient transporters are regulated by mTOR signaling, and we extend these observations by showing that mTOR regulates the activity of key amino acid transporters in human primary trophoblast cells. In addition, the regulation of taurine transporter activity by mTOR has not previously been reported, identifying a novel regulatory mechanism for this transporter.
mTOR is a serine/threonine kinase that has been shown to control cell growth through the regulation of translation and transcription. However, the effect of mTOR on system A transporter, system L transporter, and TAUT activity in primary human trophoblast cells does not appear to be mediated by these mechanisms, since we demonstrated that protein expression in whole cell lysates of SNAT2, SNAT4, TAUT, 4F2hc, and LAT2 was unchanged in response to rapamycin. For the SNAT2 and SNAT4 isoforms of the system A transporter, the lack of change in protein expression was in parallel to the unaltered mRNA expression. Similarly, the unchanged protein expression of LAT2 and 4F2hc in response to mTOR inhibition was consistent with the unaltered mRNA expression of these isoforms. However, the mRNA expression of LAT1 was decreased in rapamycin-treated cells. Since LAT1 protein expression was not measured in this study, due to lack of commercial available antibodies, we cannot exclude a contribution of expression changes to the decreased system L transport activity when mTOR signaling was inhibited. With respect to TAUT, the protein expression was not significantly altered in response to rapamycin despite a marked reduction in TAUT mRNA abundance. The mechanism underlying this discrepancy remains to be established. Nevertheless, these data suggest that mTOR regulates trophoblast nutrient transporter activity by posttranslational modifications and/or by affecting transporter translocation to the plasma membrane. We propose that mTOR regulates amino acid transporters by affecting the trafficking of the transporters to the plasma membrane, which would alter the expression and overall activity of the transporter in the plasma membrane but not whole cell protein expression. This suggestion is supported by emerging evidence in the literature (18, 19, 25). Edinger and coworkers (19) demonstrated that the expression of an activated mutant of Akt is sufficient to support cell surface expression of 4F2hc in growth factor-deprived FL5.12 cells, and rapamycin reversed this effect. They also reported that a kinase-inactive mutant of mTOR alters 4F2hc localization in these cells (18). In the fat body of Drosophila, the surface levels of Slimfast, a cationic amino acid importer, were increased by mTOR activation through clonal overexpression of Rheb (25). The possibility that mTOR signaling affects the membrane trafficking of transporters is further supported by recent understanding that prolonged incubation of cells in rapamycin may also inhibit, in addition to mTORC1, mTORC2 (60). mTORC2 regulates the actin cytoskeleton (26), which may constitute a mechanism linking mTOR to nutrient transporter trafficking, which relies on the cytoskeleton. However, further research is required to test this hypothesis.
|
GRANTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
2. Ananth CV, Wen SW. Trends in fetal growth among singleton gestations in the United States and Canada, 1985 through 1998. Semin Perinatol 26: 260–267, 2002.[CrossRef][Web of Science][Medline]
3. Asano S, Kameyama M, Oura A, Morisato A, Sakai H, Tabuchi Y, Chairoungdua A, Endou H, Kanai Y. L-type amino acid transporter-1 expressed in human astrocytomas, U343MGa. Biol Pharm Bull 30: 415–422, 2007.[CrossRef][Web of Science][Medline]
4. Barker DJ. The Wellcome Foundation Lecture, 1994. The fetal origins of adult disease. Proc Biol Sci 262: 37–43, 1995.
5. Bloxam DL, Bax BE, Bax CM. Epidermal growth factor and insulin-like growth factor I differently influence the directional accumulation and transfer of 2-aminoisobutyrate (AIB) by human placental trophoblast in two-sided culture. Biochem Biophys Res Commun 199: 922–929, 1994.[CrossRef][Web of Science][Medline]
6. Boney CM, Verma A, Tucker R, Vohr BR. Metabolic syndrome in childhood: association with birth weight, maternal obesity, and gestational diabetes mellitus. Pediatrics 115: e290–e296, 2005.
7. Brandsch M, Leibach FH, Mahesh VB, Ganapathy V. Calmodulin-dependent modulation of pH sensitivity of the amino acid transport system L in human placental choriocarcinoma cells. Biochim Biophys Acta 1192: 177–184, 1994.[Medline]
8. Brodsky D, Christou H. Current concepts in intrauterine growth restriction. J Intensive Care Med 19: 307–319, 2004.
9. Brown NF, Stefanovic-Racic M, Sipula IJ, Perdomo G. The mammalian target of rapamycin regulates lipid metabolism in primary cultures of rat hepatocytes. Metabolism 56: 1500–1507, 2007.[CrossRef][Web of Science][Medline]
10. Byfield MP, Murray JT, Backer JM. hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6 kinase. J Biol Chem 280: 33076–33082, 2005.
11. Casey BM, Lucas MJ, McIntire DD, Leveno KJ. Pregnancy outcomes in women with gestational diabetes compared with the general obstetric population. Obstet Gynecol 90: 869–873, 1997.[CrossRef][Web of Science][Medline]
12. Cetin I, Corbetta C, Sereni LP, Marconi AM, Bozzetti P, Pardi G, Battaglia FC. Umbilical amino acid concentrations in normal and growth-retarded fetuses sampled in utero by cordocentesis. Am J Obstet Gynecol 162: 253–261, 1990.[Web of Science][Medline]
13. Curhan GC, Willett WC, Rimm EB, Spiegelman D, Ascherio AL, Stampfer MJ. Birth weight and adult hypertension, diabetes mellitus, and obesity in US men. Circulation 94: 3246–3250, 1996.
14. Desforges M, Lacey HA, Glazier JD, Greenwood SL, Mynett KJ, Speake PF, Sibley CP. SNAT4 isoform of system A amino acid transporter is expressed in human placenta. Am J Physiol Cell Physiol 290: C305–C312, 2006.
15. Dicke JM, Henderson GI. Placental amino acid uptake in normal and complicated pregnancies. Am J Med Sci 295: 223–227, 1988.[CrossRef][Web of Science][Medline]
16. Economides DL, Nicolaides KH, Gahl WA, Bernardini I, Evans MI. Plasma amino acids in appropriate- and small-for-gestational-age fetuses. Am J Obstet Gynecol 161: 1219–1227, 1989.[Web of Science][Medline]
17. Edinger AL. Growth factors regulate cell survival by controlling nutrient transporter expression. Biochem Soc Trans 33: 225–227, 2005.[CrossRef][Web of Science][Medline]
18. Edinger AL, Linardic CM, Chiang GG, Thompson CB, Abraham RT. Differential effects of rapamycin on mammalian target of rapamycin signaling functions in mammalian cells. Cancer Res 63: 8451–8460, 2003.
19. Edinger AL, Thompson CB. Akt maintains cell size and survival by increasing mTOR-dependent nutrient uptake. Mol Biol Cell 13: 2276–2288, 2002.
20. Ericsson A, Hamark B, Jansson N, Johansson BR, Powell TL, Jansson T. Hormonal regulation of glucose and system A amino acid transport in first trimester placental villous fragments. Am J Physiol Regul Integr Comp Physiol 288: R656–R662, 2005.
21. Ericsson A, Saljo K, Sjostrand E, Jansson N, Prasad PD, Powell TL, Jansson T. Brief hyperglycaemia in the early pregnant rat increases fetal weight at term by stimulating placental growth and affecting placental nutrient transport. J Physiol 581: 1323–1332, 2007.
22. Findlay GM, Yan L, Procter J, Mieulet V, Lamb RF. A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling. Biochem J 403: 13–20, 2007.[CrossRef][Web of Science][Medline]
23. Furesz TC, Smith CH, Moe AJ. ASC system activity is altered by development of cell polarity in trophoblast from human placenta. Am J Physiol Cell Physiol 265: C212–C217, 1993.
24. Glazier JD, Cetin I, Perugino G, Ronzoni S, Grey AM, Mahendran D, Marconi AM, Pardi G, Sibley CP. Association between the activity of the system A amino acid transporter in the microvillous plasma membrane of the human placenta and severity of fetal compromise in intrauterine growth restriction. Pediatr Res 42: 514–519, 1997.[Web of Science][Medline]
25. Hennig KM, Colombani J, Neufeld TP. TOR coordinates bulk and targeted endocytosis in the Drosophila melanogaster fat body to regulate cell growth. J Cell Biol 173: 963–974, 2006.
26. Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, Hall A, Hall MN. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 6: 1122–1128, 2004.[CrossRef][Web of Science][Medline]
27. Jansson N, Greenwood SL, Johansson BR, Powell TL, Jansson T. Leptin stimulates the activity of the system A amino acid transporter in human placental villous fragments. J Clin Endocrinol Metab 88: 1205–1211, 2003.
28. Jansson N, Pettersson J, Haafiz A, Ericsson A, Palmberg I, Tranberg M, Ganapathy V, Powell TL, Jansson T. Down-regulation of placental transport of amino acids precedes the development of intrauterine growth restriction in rats fed a low protein diet. J Physiol 576: 935–946, 2006.
29. Jansson T. Amino acid transporters in the human placenta. Pediatr Res 49: 141–147, 2001.[Web of Science][Medline]
30. Jansson T, Ekstrand Y, Bjorn C, Wennergren M, Powell TL. Alterations in the activity of placental amino acid transporters in pregnancies complicated by diabetes. Diabetes 51: 2214–2219, 2002.
31. Jansson T, Scholtbach V, Powell TL. Placental transport of leucine and lysine is reduced in intrauterine growth restriction. Pediatr Res 44: 532–537, 1998.[Web of Science][Medline]
32. Jayanthi LD, Ramamoorthy S, Mahesh VB, Leibach FH, Ganapathy V. Substrate-specific regulation of the taurine transporter in human placental choriocarcinoma cells (JAR). Biochim Biophys Acta 1235: 351–360, 1995.[Medline]
33. Jones HN, Ashworth CJ, Page KR, McArdle HJ. Cortisol stimulates system A amino acid transport and SNAT2 expression in a human placental cell line (BeWo). Am J Physiol Endocrinol Metab 291: E596–E603, 2006.
34. Jones HN, Ashworth CJ, Page KR, McArdle HJ. Expression and adaptive regulation of amino acid transport system A in a placental cell line under amino acid restriction. Reproduction 131: 951–960, 2006.
35. Karl PI. Insulin-like growth factor-1 stimulates amino acid uptake by the cultured human placental trophoblast. J Cell Physiol 165: 83–88, 1995.[CrossRef][Web of Science][Medline]
36. Karl PI, Alpy KL, Fisher SE. Amino acid transport by the cultured human placental trophoblast: effect of insulin on AIB transport. Am J Physiol Cell Physiol 262: C834–C839, 1992.
37. Khullar S, Greenwood SL, McCord N, Glazier JD, Ayuk PT. Nitric oxide and superoxide impair human placental amino acid uptake and increase Na+ permeability: implications for fetal growth. Free Radic Biol Med 36: 271–277, 2004.[CrossRef][Web of Science][Medline]
38. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss JF, 3rd. Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118: 1567–1582, 1986.
39. Kudo Y, Boyd CA. Heterodimeric amino acid transporters: expression of heavy but not light chains of CD98 correlates with induction of amino acid transport systems in human placental trophoblast. J Physiol 523: 13–18, 2000.
40. Kulanthaivel P, Cool DR, Ramamoorthy S, Mahesh VB, Leibach FH, Ganapathy V. Transport of taurine and its regulation by protein kinase C in the JAR human placental choriocarcinoma cell line. Biochem J 277: 53–58, 1991.[Web of Science][Medline]
41. Kulanthaivel P, Miyamoto Y, Mahesh VB, Leibach FH, Ganapathy V. Inactivation of taurine transporter by calcium in purified human placental brush border membrane vesicles. Placenta 12: 327–340, 1991.[Web of Science][Medline]
42. Kuruvilla AG, D'Souza SW, Glazier JD, Mahendran D, Maresh MJ, Sibley CP. Altered activity of the system A amino acid transporter in microvillous membrane vesicles from placentas of macrosomic babies born to diabetic women. J Clin Invest 94: 689–695, 1994.[Web of Science][Medline]
43. Ling R, Bridges CC, Sugawara M, Fujita T, Leibach FH, Prasad PD, Ganapathy V. Involvement of transporter recruitment as well as gene expression in the substrate-induced adaptive regulation of amino acid transport system A. Biochim Biophys Acta 1512: 15–21, 2001.[Medline]
44. Liu XM, Reyna SV, Ensenat D, Peyton KJ, Wang H, Schafer AI, Durante W. Platelet-derived growth factor stimulates LAT1 gene expression in vascular smooth muscle: role in cell growth. FASEB J 18: 768–770, 2004.
45. Magnusson-Olsson AL, Lager S, Jacobsson B, Jansson T, Powell TL. Effect of maternal triglycerides and free fatty acids on placental LPL in cultured primary trophoblast cells and in a case of maternal LPL deficiency. Am J Physiol Endocrinol Metab 293: E24–E30, 2007.
46. Mahendran D, Donnai P, Glazier JD, D'Souza SW, Boyd RD, Sibley CP. Amino acid (system A) transporter activity in microvillous membrane vesicles from the placentas of appropriate and small for gestational age babies. Pediatr Res 34: 661–665, 1993.[Web of Science][Medline]
47. Meller M, Vadachkoria S, Luthy DA, Williams MA. Evaluation of housekeeping genes in placental comparative expression studies. Placenta 26: 601–607, 2005.[CrossRef][Web of Science][Medline]
48. Nelson DM, Smith SD, Furesz TC, Sadovsky Y, Ganapathy V, Parvin CA, Smith CH. Hypoxia reduces expression and function of system A amino acid transporters in cultured term human trophoblasts. Am J Physiol Cell Physiol 284: C310–C315, 2003.
49. Nobukuni T, Joaquin M, Roccio M, Dann SG, Kim SY, Gulati P, Byfield MP, Backer JM, Natt F, Bos JL, Zwartkruis FJ, Thomas G. Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proc Natl Acad Sci USA 102: 14238–14243, 2005.
50. Norberg S, Powell TL, Jansson T. Intrauterine growth restriction is associated with a reduced activity of placental taurine transporters. Pediatr Res 44: 233–238, 1998.[Web of Science][Medline]
51. Okamoto Y, Sakata M, Ogura K, Yamamoto T, Yamaguchi M, Tasaka K, Kurachi H, Tsurudome M, Murata Y. Expression and regulation of 4F2hc and hLAT1 in human trophoblasts. Am J Physiol Cell Physiol 282: C196–C204, 2002.
52. Park SY, Kim JK, Kim IJ, Choi BK, Jung KY, Lee S, Park KJ, Chairoungdua A, Kanai Y, Endou H, Kim DK. Reabsorption of neutral amino acids mediated by amino acid transporter LAT2 and TAT1 in the basolateral membrane of proximal tubule. Arch Pharm Res 28: 421–432, 2005.[CrossRef][Web of Science][Medline]
53. Peng T, Golub TR, Sabatini DM. The immunosuppressant rapamycin mimics a starvation-like signal distinct from amino acid and glucose deprivation. Mol Cell Biol 22: 5575–5584, 2002.
54. Pettitt DJ, Bennett PH, Saad MF, Charles MA, Nelson RG, Knowler WC. Abnormal glucose tolerance during pregnancy in Pima Indian women. Long-term effects on offspring. Diabetes 40, Suppl 2: 126–130, 1991.
55. Peyrollier K, Hajduch E, Blair AS, Hyde R, Hundal HS. L-Leucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the involvement of the mammalian target of rapamycin (mTOR) pathway in the L-leucine-induced up-regulation of system A amino acid transport. Biochem J 350: 361–368, 2000.[CrossRef][Web of Science][Medline]
56. Ramamoorthy S, Leibach FH, Mahesh VB, Ganapathy V. Modulation of the activity of amino acid transport system L by phorbol esters and calmodulin antagonists in a human placental choriocarcinoma cell line. Biochim Biophys Acta 1136: 181–188, 1992.[Medline]
57. Ramamoorthy S, Leibach FH, Mahesh VB, Ganapathy V. Selective impairment of taurine transport by cyclosporin A in a human placental cell line. Pediatr Res 32: 125–127, 1992.[Web of Science][Medline]
58. Roos S, Jansson NL, Palmberg I, Saljo K, Powell TL, Jansson T. Mammalian target of rapamycin in the human placenta regulates leucine transport and is down-regulated in restricted foetal growth. J Physiol 582: 449–459, 2007.
59. Roos S, Powell TL, Jansson T. Human placental taurine transporter in uncomplicated and IUGR pregnancies: cellular localization, protein expression, and regulation. Am J Physiol Regul Integr Comp Physiol 287: R886–R893, 2004.
60. Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF, Markhard AL, Sabatini DM. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 22: 159–168, 2006.[CrossRef][Web of Science][Medline]
61. Shojaiefard M, Christie DL, Lang F. Stimulation of the creatine transporter SLC6A8 by the protein kinase mTOR. Biochem Biophys Res Commun 341: 945–949, 2006.[CrossRef][Web of Science][Medline]
62. Shojaiefard M, Lang F. Stimulation of the intestinal phosphate transporter SLC34A2 by the protein kinase mTOR. Biochem Biophys Res Commun 345: 1611–1614, 2006.[CrossRef][Web of Science][Medline]
63. Sipula IJ, Brown NF, Perdomo G. Rapamycin-mediated inhibition of mammalian target of rapamycin in skeletal muscle cells reduces glucose utilization and increases fatty acid oxidation. Metabolism 55: 1637–1644, 2006.[CrossRef][Web of Science][Medline]
64. Thongsong B, Subramanian RK, Ganapathy V, Prasad PD. Inhibition of amino acid transport system a by interleukin-1beta in trophoblasts. J Soc Gynecol Investig 12: 495–503, 2005.[CrossRef][Web of Science][Medline]
65. Weisman R, Roitburg I, Nahari T, Kupiec M. Regulation of leucine uptake by tor1+ in Schizosaccharomyces pombe is sensitive to rapamycin. Genetics 169: 539–550, 2005.
66. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell 124: 471–484, 2006.[CrossRef][Web of Science][Medline]
67. Yung HW, Calabrese S, Hynx D, Hemmings BA, Cetin I, Charnock-Jones DS, Burton GJ. Evidence of placental translation inhibition and endoplasmic reticulum stress in the etiology of human intrauterine growth restriction. Am J Pathol 173: 451–462, 2008.
68. Yusuf K, Smith SD, Sadovsky Y, Nelson DM. Trophoblast differentiation modulates the activity of caspases in primary cultures of term human trophoblasts. Pediatr Res 52: 411–415, 2002.[CrossRef][Web of Science][Medline]
This article has been cited by other articles:
|
|
 |
|
  H. N. Jones, T. Jansson, and T. L. Powell IL-6 stimulates system A amino acid transporter activity in trophoblast cells through STAT3 and increased expression of SNAT2 Am J Physiol Cell Physiol, November 1, 2009; 297(5): C1228 - C1235. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Roos, O. Lagerlof, M. Wennergren, T. L. Powell, and T. Jansson Regulation of amino acid transporters by glucose and growth factors in cultured primary human trophoblast cells is mediated by mTOR signaling Am J Physiol Cell Physiol, January 1, 2009; 297(3): C723 - C731. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
