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L-type neutral amino acid transporter 1 (LAT1) is a heterodimeric membrane transport protein involved in neutral amino acid transport. LAT1 is highly expressed in various malignant solid tumors and plays an essential role in cell proliferation. However, its role in malignant lymphoma remains unknown. Here, we evaluated LAT1 expression level in tissues from 138 patients with Non-Hodgkin lymphoma (NHL). Overexpression of LAT1 was confirmed in all types of NHL and we found that there is a significant correlation between the level of LAT1 expression and lymphoma grade. The LAT1 expression was higher in aggressive types of lymphomas when compared with static types of lymphomas, suggesting that active tumor proliferation requires nutrient uptake via LAT1. The expression level of LAT1 was inversely correlated with patients’ survival span. Furthermore, pharmacological inhibition of LAT1 by a specific inhibitor JPH203 inhibits lymphoma cell growth. In conclusion, our study demonstrated that LAT1 expression can be used as a prognostic marker for patients with NHL and targeting LAT1 by JPH203 can be a novel therapeutic modality for NHL.

Abnormal epigenetic patterns correlate with effector T cell malfunction in tumours1-4, but the cause of this link is unknown. Here we show that tumour cells disrupt methionine metabolism in CD8+ T cells, thereby lowering intracellular levels of methionine and the methyl donor S-adenosylmethionine (SAM) and resulting in loss of dimethylation at lysine 79 of histone H3 (H3K79me2). Loss of H3K79me2 led to low expression of STAT5 and impaired T cell immunity. Mechanistically, tumour cells avidly consumed methionine and outcompeted T cells for methionine by expressing high levels of the methionine transporter SLC43A2. Genetic and biochemical inhibition of tumour SLC43A2 restored H3K79me2 in T cells, thereby boosting spontaneous and checkpoint-induced tumour immunity. Moreover, methionine supplementation improved the expression of H3K79me2 and STAT5 in T cells, and this was accompanied by increased T cell immunity in tumour-bearing mice and patients with colon cancer. Clinically, tumour SLC43A2 correlated negatively with T cell histone methylation and functional gene signatures. Our results identify a mechanistic connection between methionine metabolism, histone patterns, and T cell immunity in the tumour microenvironment. Thus, cancer methionine consumption is an immune evasion mechanism, and targeting cancer methionine signalling may provide an immunotherapeutic approach.

Introduction: Amino acid transport across the placenta is crucial for fetal growth. In rodent models, the visceral yolk sac (referred to as yolk sac hereafter) is also likely to contribute to fetal amino acid provision. System L amino acid transporters mediate the transport of essential amino acids. System L activity is mediated by light chains LAT1 (Slc7a5) and LAT2 (Slc7a8) which form functional complexes by heterodimeric linkage to CD98 (Slc3a2). LAT4 (Slc43a2) is monomeric, possessing overlapping amino acid substrate specificity with LAT1 and LAT2. Methods: This study investigates the expression of these LAT subtypes in fetus-matched rat placenta and yolk sac. Results: Slc7a5, Slc7a8 and Slc43a2 transcripts were expressed in placenta and yolk sac with similar expression patterns between sexes. LAT1 expression was significantly higher in placenta than yolk sac. Conversely, LAT2 and LAT4 expression was significantly higher in yolk sac than placenta; CD98 expression was comparable. LAT1, LAT2, LAT4 and CD98 were distributed to rat placental labyrinth zone (LZ) and junctional zone (JZ). LAT1 and LAT4 demonstrated higher expression in LZ, whilst LAT2 was more intensely distributed to JZ. LAT1, LAT2, LAT4 and CD98 were expressed in yolk sac, with punctate LAT1 staining to endodermal cell cytoplasm, contrasting with the intense LAT2, LAT4 and CD98 endodermal cell basolateral distribution, accounting for greater LAT2 and LAT4 expression in yolk sac compared to placenta. Conclusion: LAT1, LAT2 and LAT4 are expressed in rat placenta and yolk sac implicating a combined role for these LAT subtypes in supporting fetal growth and development.

Adaptive regulation of epithelial transporters to nutrient intake is essential to decrease energy costs of their synthesis and maintenance, however such regulation is understudied. Previously we demonstrated that the transport function of the basolateral amino acid uniporter LAT4 (Slc43a2) is increased by dephosphorylation of serine 274 (S274) and nearly abolished by dephosphorylation of serine 297 (S297) when expressed in Xenopus oocytes. Phosphorylation changes in the jejunum of food-entrained mice suggested an increase in LAT4 transport function during food expectation. Thus, we investigated further how phosphorylation, expression and localization of mouse intestinal LAT4 respond to food-entrained diurnal rhythm and dietary protein content. In mice entrained with 18% protein diet, LAT4 mRNA was not submitted to diurnal regulation, unlike mRNAs of luminal symporters and antiporters. Only in duodenum, LAT4 protein expression increased during food intake. Concurrently, S274 phosphorylation was decreased in all three small intestinal segments, whereas S297 phosphorylation was increased only in jejunum. Interestingly, during food intake, S274 phosphorylation was nearly absent in ileum and accompanied by strong phosphorylation of mTORC1 target S6. Entraining mice with 8% protein diet provoked a shift in jejunal LAT4 localization from the cell surface to intracellular stores and increased S274 phosphorylation in both jejunum and ileum during food anticipation, suggesting decreased transport function. In contrast, 40% dietary protein content led to increased LAT4 expression in jejunum and its internalization in ileum. Ex vivo treatments of isolated intestinal villi fraction demonstrated that S274 phosphorylation was stimulated by protein kinase A. Rapamycin-sensitive insulin treatment and amino acids increased S297 phosphorylation, suggesting that the response to food intake might be regulated via the insulin-mTORC1 pathway. Ghrelin, an oscillating orexigenic hormone, did not affect phosphorylation of intestinal LAT4. Overall, we show that phosphorylation, expression and localization of intestinal mouse LAT4 responds to diurnal and dietary stimuli in location-specific manner.

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.

Background Testicular germ cell tumors (TGCTs) are the most common malignant cancer in young men. Although TGCTs are generally responsive to platinum-based chemotherapy particularly cisplatin, acquired resistance in patients with metastasis still occurs resulting in poor prognosis. Specifically, differentiation of embryonal carcinoma (EC) cells, the stem cells of TGCTs, can lead to the reduction of cisplatin responsiveness. Therefore, novel therapeutic strategies for TGCTs are needed. System L amino acid transporters have been reported to be up-regulated and to play an important role in tumorigenesis. However, expression and role of system L amino acid transporters in TGCTs remain elusive. Materials and methods Expression of system L amino acid transporters was analyzed in TGCT samples from The Cancer Genome Atlas (TCGA). Expression of LAT1, LAT2, and 4F2hc was examined in human embryonal carcinoma cell line NTERA2. Roles of system L amino acid transporters on NTERA2 cell survival, cell proliferation, pluripotency, and cisplatin sensitivity were evaluated. Results Based upon TCGA datasets, we found that two isoforms of system L (LAT1 and LAT2) and their chaperone protein 4F2hc are highly expressed in EC samples compared with other groups. Treatment with the system L inhibitor BCH significantly suppressed leucine uptake into the pluripotent EC cell line NTERA2. The malignant phenotypes including cell viability, cell proliferation, and clonal ability were decreased following BCH treatment. Nonetheless, system L inhibition did not alter expression of stemness genes in NTERA2 cells. After NTERA2 differentiation, expressions of LAT1 and LAT2 were decreased. Finally, co-administration of BCH enhanced cisplatin sensitivity in both undifferentiated and differentiated cells. These effects were associated with the reduction in p70S6K phosphorylation. Conclusion Taken together, these results shed light on the roles of system L amino acid transporters in TGCTs. Therefore, system L amino acid transporters could provide novel therapeutic targets for treatment against TGCTs.

Apelin is an insulin-sensitizing hormone increased in abundance with obesity. Apelin and its receptor, APJ, are expressed in the human placenta, but whether apelin regulates placental function in normal body mass index (BMI) and obese pregnant women remains unknown. We hypothesized that apelin stimulates amino acid transport in cultured primary human trophoblast (PHT) cells and that maternal circulating apelin levels are elevated in obese pregnant women delivering large babies. Treating PHT cells with physiological concentrations of the pyroglutamated form [Pyr1]apelin-13 (0.1–10.0 ng/ml) for 24 h dose-dependently increased System A amino acid transport ( P < 0.05) but did not affect System L transport activity. Mechanistic target of rapamycin (mTOR), extracellular signal-regulated kinase-1/2 (ERK1/2), and AMP-activated protein kinase-α (AMPKα) signaling were unaffected by apelin ( P > 0.05). Plasma apelin was not different in obese women (BMI 35.8 ± 0.7, n = 21) with large babies compared with normal-BMI women (23.1 ± 0.5, n = 16) delivering normal birth weight infants. Apelin was highly expressed in placental villous tissue (20-fold higher vs. adipose), and APJ was present in syncytiotrophoblast microvillous membrane, but neither differed in abundance between normal-BMI and obese women. Phosphorylation (Thr172) of placental AMPKα strongly correlated with microvillous membrane APJ expression ( P < 0.01, R = 0.63) but negatively correlated with placental apelin abundance ( P < 0.01, R = −0.62). Neither placental APJ nor apelin abundance correlated with maternal BMI, plasma insulin, birth weight, or mTOR or ERK1/2 signaling ( P > 0.05). Hence, apelin stimulates trophoblast amino acid uptake, establishing a novel mechanism regulating placental function. We found no evidence that apelin constitutes an endocrine link between maternal obesity and fetal overgrowth.

Intrauterine growth restriction (IUGR) is associated with reduced placental amino acid transport (AAT). However, it remains to be established if changes in AAT contribute to restricted fetal growth. We hypothesized that reduced in vivo placental AAT precedes the development of IUGR in baboons with maternal nutrient restriction (MNR). Baboons were fed either a control (ad libitum) or MNR diet (70% of control diet) from gestational day (GD) 30. At GD 140, in vivo transplacental AA transport was measured by infusing nine (13)C- or (2)H-labeled essential amino acids (EAAs) as a bolus into the maternal circulation at cesarean section. A fetal vein-to-maternal artery mole percent excess ratio for each EAA was measured. Microvillous plasma membrane (MVM) system A and system L transport activity were determined. Fetal and placental weights were not significantly different between MNR and control. In vivo, the fetal vein-to-maternal artery mole percent excess ratio was significantly decreased for tryptophan in MNR. MVM system A and system L activity was markedly reduced in MNR. Reduction of in vivo placental amino acid transport precedes fetal growth restriction in the non-human primate, suggesting that reduced placental amino acid transfer may contribute to IUGR.

System l amino acid transporter 1 (LAT1) is highly expressed in various types of human cancer, and contributes to cancer growth and survival. Recently, we have shown that LAT1 expression is closely related to the growth and aggressiveness of esophageal cancer, and is an independent marker of poor prognosis. However, it remains unclear whether LAT1 inhibition could suppress esophageal cancer growth. In this study, we investigated the tumor-suppressive effects of the inhibition of LAT1. Both LAT1 and CD98, which covalently associates to LAT1 on the membrane, were expressed in human esophageal cancer cell lines KYSE30 and KYSE150. Quantitative PCR analysis showed that the expression of LAT1 was much higher than other subtypes of LAT. A selective inhibitor of LAT, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), suppressed cellular uptake of l-14 C-leucine and cell proliferation in a dose-dependent manner. It also suppressed phosphorylation of mammalian target of rapamycin, 4E-BP1, and p70S6K protein, and induced cell cycle arrest at G1 phase. These results suggest that suppression of both mammalian target of rapamycin signaling and cell cycle progression is involved in BCH-induced growth inhibition. In tumor-bearing mice, daily treatment with BCH significantly delayed tumor growth and decreased glucose metabolism, indicating that LAT1 inhibition potentially suppresses esophageal cancer growth in vivo. Thus, our results suggest that LAT1 inhibition could be a promising molecular target for the esophageal cancer therapy.

Plasma concentrations of amino acids reflect the intracellular amino acid pool in mammals. However, the regulatory mechanism requires clarification. In this study, we examined the effect of leucine administration on plasma amino acid profiles in mice with and without the treatment of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) or rapamycin as an inhibitor of system L or mammalian target of rapamycin complex 1, respectively. The elevation of plasma leucine concentration after leucine administration was associated with a significant decrease in the plasma concentrations of isoleucine, valine, methionine, phenylalanine, and tyrosine; BCH treatment almost completely blocked the leucine-induced decrease in plasma amino acid concentrations. Rapamycin treatment had much less effects on the actions of leucine than BCH treatment. These results suggest that leucine regulates the plasma concentrations of branched-chain amino acids, methionine, phenylalanine, and tyrosine, and that system L amino acid transporters are involved in the leucine action.

Thyroid hormone (TH) transporters facilitate cellular TH influx and efflux, which is paramount for normal physiology. The L-type amino acid transporters LAT1 and LAT2 are known to facilitate TH transport. However, the role of LAT3, LAT4, and LAT5 is still unclear. Therefore, the aim of this study was to further characterize TH transport by LAT1 and LAT2 and to explore possible TH transport by LAT3, LAT4, and LAT5. FLAG-LAT1–5 constructs were transiently expressed in COS1 cells. LAT1 and LAT2 were cotransfected with the CD98 heavy chain. Cellular transport was measured using 10 nM 125I-labeled T4, T3, rT3, 3,3′-T2, and 10 μM [125I]3′-iodotyrosine (MIT) as substrates. Intracellular metabolism of these substrates was determined in cells cotransfected with either of the LATs with type 1 or type 3 deiodinase. LAT1 facilitated cellular uptake of all substrates and LAT2 showed a net uptake of T3, 3,3′-T2, and MIT. Expression of LAT3 or LAT4 did not affect transport of T4 and T3 but resulted in the decreased cellular accumulation of 3,3′-T2 and MIT. LAT5 did not facilitate the transport of any substrate. Cotransfection with LAT3 or LAT4 strongly diminished the cellular accumulation of 3,3′-T2 and MIT by LAT1 and LAT2. These data were confirmed by metabolism studies. LAT1 and LAT2 show distinct preferences for the uptake of the different iodocompounds, whereas LAT3 and LAT4 specifically facilitate the 3,3′-T2 and MIT efflux. Together our findings suggest that different sets of transporters with specific influx or efflux capacities may cooperate to regulate the cellular thyroid state.

The organic mercury compound methylmercury (MeHg) is able to target the fetal brain. However, the uptake of the toxicant into placental cells is incompletely understood. MeHg strongly binds to thiol-S containing molecules such as cysteine. This MeHg-l-cysteine exhibits some structural similarity to methionine. System L plays a crucial role in placental transport of essential amino acids such as leucine and methionine and thus has been assumed to also transport MeHg-l-cysteine across the placenta. The uptake of methylmercury and tritiated leucine and methionine into the choriocarcinoma cell line BeWo was examined using transwell assay and small interfering (si)RNA mediated gene knockdown. Upon the downregulation of large neutral amino acids transporter (LAT)2 and 4F2 cell-surface antigen heavy chain (4F2hc), respectively, the levels of [3H]leucine in BeWo cells are significantly reduced compared to controls treated with non-targeting siRNA (p < 0.05). The uptake of [3H]methionine was reduced upon LAT2 down-regulation as well as methylmercury uptake after 4F2hc silencing (p < 0.05, respectively). These findings suggest an important role of system L in the placental uptake of the metal. Comparing the cellular accumulation of mercury, leucine, and methionine, it can be assumed that (1) MeHg is transported through system L amino acid transporters and (2) system L is responsible for the uptake of amino acids and MeHg primarily at the apical membrane of the trophoblast. The findings together can explain why mercury in contrast to other heavy metals such as lead or cadmium is efficiently transported to fetal blood.

Polyamine (PA) transport as well as PA biosynthesis, degradation and conjugation plays a vital role in the regulation of intracellular PA levels, which are essential for cell growth. Generally, PA uptake activity is elevated in rapidly proliferating cells. Previous studies showed that PA uptake in plant cells occurred via energy-dependent, protein-mediated transport systems. Numerous lines of evidence suggest that paraquat (PQ), one of the most widely used herbicides, is transported by the PA transport system in diverse organisms including plants. The PA/PQ transport interactions are proposed to be due to specific structural similarities between PA and PQ. The understanding of PA transport mechanisms has progressed in parallel with that of PQ transport, but the molecular identity of the plant PA/PQ transporter has remained an enigma. Recently, independent studies identified the L-type amino acid transporter (LAT) family transmembrane proteins as transporters of both PA and PQ. Arabidopsis LAT family proteins showed different subcellular localization properties, which suggested that these transporters were involved in intracellular PA trafficking and PA uptake across the plasma membrane. The identification of plant PA transporters is an important step in understanding the mechanism of PA homeostasis in plant cells. In this review, we highlight recent advances in the study of PA transport systems that are linked to the understanding of PQ translocation.

Compared to most normal cells that express L-type amino acid transporter 2, L-type amino acid transporter 1 is highly expressed in cancer cells and presumed to support their elevated growth and proliferation. This study examined JPH203, a potent and selective L-type amino acid transporter 1 inhibitor, and its ability to suppress YD-38 human oral cancer cell growth. The YD-38 cells express L-type amino acid transporter 1 with its associating protein 4F2 heavy chain, but not L-type amino acid transporter 2. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, completely inhibited l-leucine uptake in YD-38 cells. As expected, the intrinsic affinity of JPH203 to inhibit l-leucine uptake was far more efficient than BCH. Likewise, JPH203 and BCH inhibited YD-38 cell growth, with JPH203 being superior to BCH. JPH203 up-regulated the population of apoptotic YD-38 cells through the activation of apoptotic factors, including caspases and PARP. These results suggest that the inhibition of L-type amino acid transporter 1 activity via JPH203, which may act as a potential novel anti–oral-cancer agent, leads to apoptosis by inducing the intracellular depletion of the neutral amino acids essential for cancer cell growth in YD-38 human oral cancer cells. Keywords:: JPH203, L-type amino acid transporter 1, apoptosis, oral cancer cell, anti-cancer therapy

Interleukin-1β (IL-1β) promotes insulin resistance in tissues such as liver and skeletal muscle; however the influence of IL-1β on placental insulin signaling is unknown. We recently reported increased IL-1β protein expression in placentas of obese mothers, which could contribute to insulin resistance. In this study, we tested the hypothesis that IL-1β inhibits insulin signaling and prevents insulin-stimulated amino acid transport in cultured primary human trophoblast (PHT) cells. Cultured trophoblasts isolated from term placentas were treated with physiological concentrations of IL-1β (10 pg/ml) for 24 hours. IL-1β increased the phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser307 (inhibitory) and decreased total IRS-1 protein abundance but did not affect insulin receptor β expression. Furthermore, IL-1β inhibited insulin-stimulated phosphorylation of IRS-1 (Tyr612, activation site) and Akt (Thr308) and prevented insulin-stimulated increase in PI3K/p85 and Grb2 protein expression. IL-1β alone stimulated cRaf (Ser338), MEK (Ser221) and Erk1/2 (Thr202/Tyr204) phosphorylation. The inflammatory pathways nuclear factor kappa B and c-Jun N-terminal kinase, which are involved in insulin resistance, were also activated by IL-1β treatment. Moreover, IL-1β inhibited insulin-stimulated System A, but not System L amino acid uptake, indicating functional impairment of insulin signaling. In conclusion, IL-1β inhibited the insulin signaling pathway by inhibiting IRS-1 signaling and prevented insulin-stimulated System A transport, thereby promoting insulin resistance in cultured PHT cells. These findings indicate that conditions which lead to increased systemic maternal or placental IL-1β levels may attenuate the effects of maternal insulin on placental function and consequently fetal growth.

SummaryThe system L neutral amino acid transporter (LAT; LAT1, LAT2, LAT3, or LAT4) has multiple functions in human biology, including the cellular import of S-nitrosothiols (SNOs), biologically active derivatives of nitric oxide (NO). SNO formation by haemoglobin within red blood cells (RBC) has been studied, but the conduit whereby a SNO leaves the RBC remains unidentified. Here we hypothesised that SNO export by RBCs may also depend on LAT activity, and investigated the role of RBC LAT in modulating SNO-sensitive RBC-endothelial cell (EC) adhesion. We used multiple pharmacologic inhibitors of LAT in vitro and in vivo to test the role of LAT in SNO export from RBCs and in thereby modulating RBC-EC adhesion. Inhibition of human RBC LAT by type-1-specific or nonspecific LAT antagonists increased RBC-endothelial adhesivity in vitro, and LAT inhibitors tended to increase post-transfusion RBC sequestration in the lung and decreased oxygenation in vivo. A LAT1-specific inhibitor attenuated SNO export from RBCs, and we demonstrated LAT1 in RBC membranes and LAT1 mRNA in reticulocytes. The proadhesive effects of inhibiting LAT1 could be overcome by supplemental L-CSNO (S-nitroso-L-cysteine), but not D-CSNO or L-Cys, and suggest a basal anti-adhesive role for stereospecific intercellular SNO transport. This study reveals for the first time a novel role of LAT1 in the export of SNOs from RBCs to prevent their adhesion to ECs. The findings have implications for the mechanisms of intercellular SNO signalling, and for thrombosis, sickle cell disease, and post-storage RBC transfusion, when RBC adhesivity is increased.

The branched-chain amino acids (BCAA) leucine, isoleucine and valine, are essential amino acids that play a critical role in cellular signalling and metabolism. They acutely stimulate insulin secretion and activate the regulatory serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1), a kinase that promotes increased β-cell mass and function. The effects of BCAA on cellular function are dependent on their active transport into the mammalian cells via amino acid transporters and thus the expression and activity of these transporters likely influence β-cell signalling and function. In this report, we show that the System-L transporters are required for BCAA uptake into clonal β-cell lines and pancreatic islets, and that these are essential for signalling to mTORC1. Further investigation revealed that the System-L amino acid transporter 1 (LAT1) is abundantly expressed in the islets, and that knockdown of LAT1 using siRNA inhibits mTORC1 signalling, leucine-stimulated insulin secretion and islet cell proliferation. In summary, we show that the LAT1 is required for regulating β-cell signalling and function in islets and thus may be a novel pharmacological/nutritional target for the treatment and prevention of type 2 diabetes.

We investigated the regulation of L-type amino acid transporter 1 (LAT1) in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells) in response to glucose deprivation. The amounts of LAT1 and 4F2 heavy chain (4F2hc) mRNA in TR-iBRB2 cells exposed to glucose-free culture medium for 8 to 24 h were significantly elevated compared with those in control medium. Concomitantly, [³H]L-leucine uptake activity was increased, suggesting that LAT1 transport activity is induced under glucose-deprivation. To determine the transcriptional activity of the LAT1 gene under glucose-free conditions, the promoter activity of the LAT1 gene of approximately 2 kbp (-1958 bp to +70 bp) in TR-iBRB2 cells was assayed using a dual-luciferase reporter assay system. The transcriptional activity of the 2 kbp LAT1 promoter under the glucose-free conditions was 1.7-fold greater than that under normal glucose conditions. The presence of an activator site(s) between -162 bp and -155 bp was indicated by the low activities exhibited by the construct spanning this region and mutagenesis. These results suggest that the glucose deprivation sensitivity of LAT1 expression is transcriptionally regulated, and cis-elements within the LAT1 promoter region from -162 bp to -155 bp mediate this regulation.

BACKGROUND Little is known about metabolic processes in the developing ovarian follicle. Using mouse ovarian follicles, we investigated uptake of L-leucine by follicles at varying stages of maturity in the presence of insulin-like growth factor (IGF)-1. METHODS Mouse ovarian follicles were cultured in vitro for 5 days in increasing concentrations of IGF-1, and follicle diameter and atresia measured as endpoints for growth. Uptake of (3)H-leucine was measured in follicles at different stages of development. In optimal IGF-1-mediated growth conditions, competitive inhibition of (3)H-leucine uptake by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), a non-metabolizable substrate analogue of L-leucine, was performed to demonstrate specificity of influx, via system L transporters. To test whether uptake rates were dependent on intracellular amino acid availability, follicles from in vitro cultures were pre-treated with L-phenylalanine prior to (3)H-leucine uptake. RESULTS Follicle development (P< 0.001) and survival (P< 0.001) increased with IGF-1 treatment. As pre-antral follicles progressed to late antral stage, we observed an increase in L-leucine uptake, which was reduced in pre-ovulatory follicles. BCH decreased L-leucine uptake rates in early antral (P< 0.05), antral (P< 0.001) and pre-ovulatory follicles (P< 0.01). L-leucine influx increased in follicles preloaded with phenylalanine (trans-stimulation). In follicles lacking free intracellular amino acids (zero-trans suppression), uptake rate was reduced (P< 0.05). CONCLUSIONS These results demonstrate, for the first time, evidence of specific system L amino acid transport in intact, mouse ovarian follicles and profile L-leucine uptake during folliculogenesis. A better understanding of ovarian follicle metabolic pathways is necessary for improved in vitro maturation as well as determining the impact of altered metabolism on fertility.

Interest in radiolabeled amino acids for metabolic imaging of cancer and limitations with [(11)C]methionine has prompted the development of a new (18)F-labeled methionine derivative S-(3-[(18)F]fluoropropyl)homocysteine ([(18)F]FPHCys). The L and D enantiomers of [(18)F]FPHCys were prepared from their respective protected S-(3-tosyloxypropyl)homocysteine precursors 1 by [(18)F]fluoride substitution using K(2.2.2) and potassium oxalate, followed by acid hydrolysis on a Tracerlab FX(FN) synthesis module. [(18)F]-L-FPHCys and [(18)F]-D-FPHCys were isolated in 20 ± 5% radiochemical yield and98% radiochemical and enantiomeric purity in 65 min. Competitive uptake studies in A375 and HT29 tumor cells suggest that L- and D-[(18)F]FPHCys are taken up by the L-transporter system. [(18)F]-L-FPHCys and [(18)F]-D-FPHCys displayed good stability In Vivo without incorporation into protein at least 2 h postinjection. Biodistribution studies demonstrate good uptake in A375 tumor-bearing rodents with tumor to blood ratios of 3.5 and 5.0 for [(18)F]-L-FPHCys and [(18)F]-D-FPHCys, respectively, at 2 h postinjection.

The transport systems involved in intestinal methionine (Met) absorption are described as Na+-dependent and Na+-independent mechanisms. However, since recent studies have suggested the importance of the H+gradient as a driving force for intestinal nutrient absorption, the aim of the present work was to test whether Met transport across the apical membrane of Caco-2 cells is affected by extracellular pH. The results show that l- and d-Met uptake was increased by lowering extracellular pH from 7.4 to 5.5, in both the presence and absence of Na+. Cis-inhibition experiments revealed that inhibition of l-Met transport by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) or l-lysine (l-Lys) was higher at a pH of 5.5. Moreover, the BCH-insensitive component was not affected by pH, whereas the l-Lys-insensitive component was increased by lowering extracellular pH, thus suggesting the participation of system L. The contribution of another mechanism, sensitive to both BCH and l-Lys, was also considered. The inhibition obtained with taurine (Tau) was also higher at a pH of 5.5, thus suggesting the involvement of system B0,+on pH-stimulated component. As for d-Met uptake, the results showed higher inhibition with l-Lys and Tau at a pH of 5.5 and no effect on the l-Lys- or Tau-insensitive component. In conclusion, Met transport across the apical membrane of Caco-2 cells is increased by low extracellular pH as the result of the stimulation of two transport systems functionally identified with systems L and B0,+for l-Met and with system B0,+for d-Met.

Nonproteinogenic amino acids that either occur naturally or are synthesized chemically are becoming important tools in modern drug discovery. In this context, fluorinated amino acids have great potential in the development of novel pharmaceuticals and drugs. To assess whether different fluorinated aromatic amino acid analogues of phenylalanine, tyrosine, and tryptophan are potentially interesting as therapeutic drugs, we examined their cytostatic and cytotoxic effects on the growth of the human breast cancer cell line MCF-7. Of all the tested analogues L-4-fluorotryptophan, L-6-fluorotryptophan and L-p-fluorophenylalanine effectively and irreversibly inhibited cell growth with IC(50) values in the low micromolar range (3-15 microM). Additionally, using L-4-[14C]fluorotryptophan, and L-6-[14C]fluorotryptophan, we discovered that the cellular uptake of these fluorinated amino acids occurs through active transport with a 70-fold excess of intracellular over extracellular concentrations. We identified system L as the responsible amino acid transporter. Our findings fully support the idea that fluorinated aromatic amino acid analogues are promising chemotherapeutics with the potential for use in combination with classical cancer therapy, and as new cytotoxic drugs for certain tumor types such as melanoma.

Stem cells are at the forefront of current regenerative and biomedical research. Thus, there exists an imperative and urgent need to understand the mechanisms that drive stem cell function in order to exploit their use as a therapeutic tool. Amino acids are potent inducers of signaling cascades that drive stem cell proliferation and differentiation. With a focus on mouse embryonic stem (mES) cells, Threonine (Thr) is the only amino acid required in culture media for mES cell proliferation. Current research associates this need for Thr with threonine dehydrogenase (TDH), which catabolizes Thr to glycine and acetyl-CoA in mES cells. This theory depends, in part, on the ability of 3- hydroxynorvaline (3-HNV) to inhibit both TDH and mES cell proliferation. However, the concentration of 3-HNV needed to inhibit mES cell proliferation is more than an order of magnitude less than its apparent Ki for TDH inhibition. Additionally, 3-HNV inhibits human embryonic stem (hES) cell proliferation, but hES cells do not express a functional tdh gene. Such findings indicate another mechanism for Thr stimulated mES and hES cell proliferation. Since amino acid transporters may be inducers of signaling cascades, we characterized the Thr transport systems in mES cells. We found that there is a Na(+)-dependent and a Na(+)-independent component of substrate-saturable transport, with the Na(+)-dependent component predominating. We also found that of 20 amino acids tested, the amino acids that were the strongest inhibitors of the Na(+)-dependent component of radiolabeled Thr transport were Ser, Cys, 4-OH-Pro, Asn, Met, and non-radiolabeled Thr itself. Such findings are consistent with characteristics of the ASC transport system, suggesting that this ASC system is responsible for the majority of Thr transport in mES cells. We confirmed expression of mRNA encoding the ASC system transporters, ASCT1 and ASCT2, in mES cells using RT-PCR. In conclusion, mES cells likely express at least three transporters of Thr; at least two Na(+)-dependent transporters and one Na(+)-independent one.

Expression of system L amino acid transporters (LAT) is strongly increased in many types of tumor cells. The purpose of this study was to demonstrate that 18F-labeled amino acids, for example, 3-O-methyl-6- 18 F-fluoro-L-dopa ( 18 F-OMFD), that accumulate in tumors via LAT represent an important class of imaging agents for visualization of tumors in vivo by PET. Methods: 18 F-OMFD uptake kinetics, transport inhibition, and system L messenger RNA expression were studied in vitro in human adenocarcinoma (HT-29), squamous cell carcinoma (FaDu), macrophages (THP1), and primary aortic endothelial cells (HAEC) and in vivo in the corresponding mouse tumor xenograft models. Results: Uptake of 18F-OMFD in all cell lines tested was mediated mainly by the sodium-independent high-capacity LAT. We found higher uptake inFaDu cells (Vmax,10.6 61.1nmol/min ·mg of cellprotein) and in the corresponding FaDu tumor xenografts than in the other cells and corresponding xenograft models studied. Quantitative messenger RNA analysis revealed that tumor cells and xenografts have a higher expression of LAT1 than do HAEC and THP-1 macrophages. However, only in the FaDu tumor model did an increased 18 F-OMFD uptake seem to be explained by increased LAT expression. Furthermore, we demonstrated a high expression of LAT4, a recently identified LAT. Conclusion: Ourfindingssupport thehypothesis that 18F-OMFD is atracer for visualization of tumor cells. 18 F-OMFD particularly seems to be a suitable tracer for diagnostic imaging of amino acid transport in poorly differentiated squamous cell head and neck carcinoma with increased LAT1 and LAT4 expression.

System L amino acid transporters have been shown to be responsible for cellular uptake of S-nitroso-l-cysteine (l-CSNO). In this study, we examined the characteristics of l-CSNO uptake in Xenopus laevis oocytes expressing system L transporters and found that uptake increased only when both 4F2 heavy chain (4F2HC) and either L-type amino acid transporter 1 (LAT1) or LAT2 light chain were coexpressed. The Kmfor transport was 57 ± 8 μM for 4F2HC-LAT1 and 520 ± 52 μM for 4F2HC-LAT2. Vascular endothelial and smooth muscle cells were shown to express transcripts for 4F2HC and for both LAT1 and LAT2. Transport of l-CSNO into red blood cells, endothelial cells, and smooth muscle cells was inhibited by 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid (BCH) and by large neutral amino acids demonstrating functional system L transporters in each cell type. Uptake of l-CSNO led to accumulation of cellular S-nitrosothiols and inhibition of both growth factor-induced ERK phosphorylation and TNF-α-mediated IκB degradation. Similar effects were seen when cells were incubated simultaneously with S-nitrosoalbumin and l-cysteine but not with d-cysteine or with S-nitrosoalbumin alone. In each case, nitrosylation of proteins and cellular responses were blocked by BCH. Together, these data suggest that transmembrane movement of nitric oxide (NO) equivalents from the plasma albumin NO reservoir is mediated by cysteine, which serves as a carrier. The mechanism requires transnitrosylation from S-nitrosoalbumin to free cysteine and activity of system L transporters, thereby providing a unique pathway for cellular responses to S-nitrosoalbumin.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Transport of L-homocysteine into and out of the human vascular endothelium is poorly understood. We hypothesized that cultured human aortic endothelial cells (HAEC) would import L-homocysteine on one or more of the L-cysteine transport systems. Inhibitors of the transporters were used to characterize the uptake of [35S]L-homocysteine, [35S]L-homocystine, and [35S]L-cysteine. We found that L-homocysteine uptake is mediated by the sodium-dependent cysteine transport systems X(AG), ASC, and A, and the sodium-independent transport system L. Thus, HAEC utilize multiple cysteine transporters (X(AG) > or = L > ASC > A) to import L-homocysteine. Kinetic analysis supported the uptake results. Michaelis-Menten constants (Km) for the four systems yielded values of 19.0, 27.1, 112, and 1000 microM for systems L, X(AG), ASC, and A, respectively. The binding and uptake of [35S]L-homocystine, the disulfide homodimer of L-homocysteine, was mediated by systems X(AG), L, and ASC but not by system A. In contrast to [35S]L-homocysteine, system x(c) was active for [35S]L-homocystine uptake. A similar pattern was observed for [35S]L-cysteine. Thus, L-homocysteine and L-homocystine found in hyperhomocysteinemic subjects can gain entry into the vascular endothelium by way of multiple L-cysteine transporters.

Previously, we reported the expression and function of system L amino acid transporter in KB human oral epidermoid carcinoma cells. In the present study, therefore, we investigated the expression and function of system L amino acid transporter in human normal oral keratinocytes (HNOK) and compared the expressions and functions of system L amino acid transporters in HNOK and KB cells. The HNOK expressed L-type amino acid transporter 1 (LAT1) and L-type amino acid transporter 2 (LAT2) with their subunit 4F2hc in the plasma membrane but the expression of LAT1 was very weak, which is in contrast to the KB cells expressing LAT1 but not LAT2 with the 4F2hc in the plasma membrane. The [14C] L-leucine uptake by HNOK, as well as KB cells, was inhibited by the system L selective inhibitor BCH. The majority of [14C] L-leucine uptake was, therefore, mainly mediated by LAT2 in the HNOK and by LAT1 in the KB cells. These results suggest that the transport of neutral amino acids including several essential amino acids into the HNOK and KB cells are mainly mediated by LAT2 and LAT1, respectively. The specific inhibition of LAT1 in oral cancer cells could be a new rationale for anti-cancer therapy.

Pregabalin exhibits robust activity in preclinical assays indicative of potential antiepileptic, anxiolytic, and antihyperalgesic clinical efficacy. It binds with high affinity to the alpha(2)-delta subunit of voltage-gated calcium channels and is a substrate of the system L neutral amino acid transporter. A series of pregabalin analogues were prepared and evaluated for their alpha(2)-delta binding affinity as demonstrated by their ability to inhibit binding of [(3)H]gabapentin to pig brain membranes and for their potency to inhibit the uptake of [(3)H]leucine into CHO cells, a measure of their ability to compete with the endogenous substrate at the system L transporter. Compounds were also assessed in vivo for their ability to promote anxiolytic, analgesic, and anticonvulsant actions. These studies suggest that distinct structure activity relationships exist for alpha(2)-delta binding and system L transport inhibition. However, both interactions appear to play an important role in the in vivo profile of these compounds.

Altered expression of metabolite transporters is observed frequently in tumor cell lines and primary neoplasms. The extent to which these may to contribute to the growth autonomy associated with cancer is not clear. LAT1 is a major L-type amino acid transporter over-expressed in a variety of cancer types and a light chain component of the CD98 heterodimer. We utilized an adenoviral expression system to modulate the level of LAT1 in a hepatic in vitro model to examine phenotypic changes associated with short-term exogenous and blocked expression. LAT1 levels were increased three fold and resulted in increased L-type amino acid transport as a result of adenoviral expression in murine hepatocytes. The protein was expressed on the cell surface and complexed with the CD98 heavy chain known as 4F2. Surprisingly, levels of the total CD98 protein complex were increased 2.4-fold as a result of adenoviral expression of light chain only, suggesting coordinate regulation. Exogenous overexpression was less effective in normal rat liver cells relative to mouse. LAT1 antisense expression in hepatic tumor cells resulted in a modest though statistically significant decrease in cell number, viability and S-phase cells over a 5-day period relative to controls despite the absence of a significant decrease in L-type transport over this period. These studies are preparatory to in vivo efforts focusing on LAT1/CD98 as a potential therapeutic target.

During renal reabsorption, the amino acid transporters b o,+ and y + L have a major role in the apical uptake of cystine and dibasic amino acids and in the basolateral efflux of dibasic amino acids, respectively. In contrast, the transporters responsible for the basolateral efflux of the apically transported cystine are unknown. This study shows the expression of system L and y + L transport activities in the basolateral domain of the proximal tubule-derived cell line OK and the cloning of the corresponding LAT-2 and y + LAT-1 cDNAs. Stable transfection with a LAT-2 antisense sequence demonstrated the specific role of LAT-2 in the basolateral system L amino acid exchange activity in OK cells. This partial reduction of LAT-2 expression decreased apical-to-basolateral trans -epithelial flux of cystine and resulted in a twofold to threefold increase in the intracellular content of cysteine. In contrast, the content of serine, threonine, and alanine showed a tendency to decrease, whereas other LAT-2 substrates were not affected. This demonstrates that LAT-2 plays a major specific role in the net basolateral efflux of cysteine and points to LAT-2 as a candidate gene to modulate cystine reabsorption. E-mail: mpalacin@bio.ub.es

We examined transport of 3-[(125)I]iodo-alpha-methyl-L-tyrosine ([(125)I]IMT) in Xenopus laevis oocytes co-expressing human L-type amino acid transporter 1 (a component of system L) and human 4F2hc. Human LAT1 mediated transport of [(125)I]IMT. [(125)I]IMT uptake was decreased by the presence of L-isomers of Cys, Leu, Ileu, Phe, Met, Tyr, His, Trp and Val and D-isomers of Leu, Phe and Met. Human LAT1-mediated [(125)I]IMT uptake was highly stereoselective for the L-isomers of Tyr, His, Trp, Val and Ileu. To examine the effects of 3-iodination and alpha-methylation on IMT transport, kinetic parameters of IMT were compared with those of mother Tyr and 3-[(125)I]iodo-L-tyrosine (3-I-Tyr). Uptake of Tyr, 3-I-Tyr and [(125)I]IMT followed Michaelis-Menten kinetics, with K(m) values of 29.0 +/- 5.1, 12.6 +/- 6.1 and 22.6 +/- 4.1 microM, respectively. Neither the alpha-methyl group nor the size of the 3-iodinated Tyr residue was an obstacle to transport via hLAT1. Furthermore, affinity of IMT for hLAT1 is higher than that of the natural parent tyrosine. The level of efflux mediated by hLAT1 was highly stimulated by extracellularly applied L-Leu, suggesting exchange of [(125)I]IMT and L-Leu via hLAT1.

The blood-brain barrier (BBB) is formed by the presence of tight junction complexes between brain endothelial cells that restrict paracellular permeability. As a consequence, a number of transport proteins are expressed on cerebral endothelial cells to facilitate the transport of nutrients into the brain. Although the modulation of barrier tight junction properties by glial-conditioned medium and by second messengers is well established, little is known about the effects of these factors on carrier-mediated BBB transport processes. The ECV304 cell line shows an endothelial phenotype and can be induced to upregulate certain BBB features in the presence of glial factors. In the present study, we have examined the effect of conditioned medium derived from rat C6-glioma cells (C6CM) on the function of the L-system amino acid transporter in ECV304 cells, using L-leucine as the model substrate, and have determined whether the changes observed can be mimicked by modulating intracellular cAMP levels. ECV304 cells exposed to C6CM exhibited a significant increase in both the affinity of leucine transport and the diffusional constant (Michaelis-Menten), while the maximal transport capacity remained unchanged. Conversely, acute exposure to modulators of the PKA and PKC second messenger pathways was found to reduce significantly the maximal transport capacity and diffusion constants, while transport affinity remained unchanged. In both cases, the maximal flux of leucine was increased, indicating transport of greater efficiency. This study indicates that exposure of ECV304 cells to C6CM provides an influence inducing L-system transport properties characteristic of brain endothelial cells. Furthermore, it appears that L-system-mediated transport of amino acids can be modulated by several distinct pathways.

syn- and anti-1-amino-3-[18F]fluoromethyl-cyclobutane-1-carboxylic acid (FMACBC, 16 and 17), analogues of anti-1-amino-3-[18F]fluorocyclobutyl-1-carboxylic acid (FACBC), were prepared to evaluate the contributions of C-3 substitution and configuration on the uptake of these radiolabeled amino acids in a rodent model of brain tumors. Radiofluorinated targets [18F]16 and [18F]17 were prepared by no-carrier-added radiofluorination from their corresponding methanesulfonyl esters 12 and 13, respectively, with decay-corrected radiochemical yields of 30% for [18F]16 and 20% for [18F]17. In amino acid transport assays performed in vitro using 9L gliosarcoma cells, both [18F]16 and [18F]17 were substrates for L type amino acid transport, while [18F]17 but not [18F]16 was a substrate for A type transport. Biodistribution studies in normal Fischer rats with [18F]16 and [18F]17 showed high uptake of radioactivity (2.0% dose/g) in the pancreas while other tissues studied, including liver, heart, lung, kidney, blood, muscle, and testis, showed relatively low uptake of radioactivity (1.0% dose/g). In rats implanted intracranially with 9L gliosarcoma cells, the retention of radioactivity in tumor tissue was high at 5, 60, and 120 min after intravenous injection of [18F]16 and [18F]17 while the uptake of radioactivity in brain tissue contralateral to the tumor remained low (0.3% dose/g). Ratios of tumor uptake to normal brain uptake for [18F]16 were 7.5:1, 7:1, and 5:1 at 5, 60, and 120 min, respectively, while for [18F]17 the ratios were 7.5:1, 9:1, and 9:1 at the same time points. This work demonstrates that like anti-[18F]FACBC, [18F]16 and [18F]17 are excellent candidates for imaging brain tumors.

Background: Early theories or hepatic encephalopathy focused on ammonia-driven disruption of the Krebs cycle and cellular energy production. The “false-neurotransmitter” theory directed attention toward the interactions of amino acids, metabolism, the blood-brain barrier and neurotransmission. As they evolved, these studies revealed surprising and subtle effects of ammonia on brain amino acid uptake. Data Sources: Research over a 15-year period in Josef E. Fischer’s laboratory explored many aspects of these interactions. Subsequent studies by others have confirmed and extended them into other areas. Insights from this work continue to stimulate attempts to confirm or disprove the clinical utility of branched chain amino acids. Conclusions: Increased understanding of the factors affecting ammonia, amino acid and neurotransmitter disturbances in chronic liver failure have made a significant and ongoing contribution to the study of metabolism in health and disease.

Background Anticoagulants, e.g. low-molecular weight heparins (LMWHs) and acetylsalicylic acid (ASA) are prescribed to women at risk for pregnancy complications that are associated with impaired placentation and placental hypoxia. Beyond their role as anticoagulants these compounds exhibit direct effects on trophoblast but their impact on placental function is unknown. The amino acid transport systems A and L, which preferably transfer essential amino acids, are well-described models to study placental nutrient transport. We aimed to examine the effect of hypoxia, LMWHs and ASA on the activity of the placental amino acid transport systems A and L and associated signalling mechanisms. Methods The uptake of C14-MeAIB (system A) or H3-leucin (system L) was investigated after incubation of primary villous fragments isolated from term placentas. Villous tissue was incubated at 2% O2 (hypoxia), 8% O2 and standard culture conditions (21% O2) or at 2% O2 and 21% O2 with dalteparin or ASA. Activation of the JAK/STAT or mTOR signalling pathways was determined by Western analysis of total and phosphorylated STAT3 or Raptor. Results Hypoxia decreased system A mediated MeAIB uptake and increased system L mediated leucine uptake compared to standard culture conditions (21% O2). This was accompanied by an impairment of STAT3 and a stimulation of Raptor signalling. System L activity increased at 8% O2. Dalteparin treatment reduced system A and system L activity under normoxic conditions and ASA (1 mM) decreased system A and L transporter activity under normoxic and hypoxic conditions. Conclusions Our data underline the dependency of placental function on oxygen supply. LMWHs and ASA are not able to reverse the effects of hypoxia on placental amino acid transport. These findings and the uncovering of the signalling mechanisms in more detail will help to understand the impact of LMWHs and ASA on placental function and fetal growth.

Introduction: Reduced glutathione is an important antioxidant in red cells whose depletion may contribute to the pathophysiology of sickle cell disease. The current study was designed to examine the availability of reduced glutathione precursors (glutamate, cysteine, glycine and possibly glutamine) together with the activity of the main transport pathways for their uptake (system ASC for cysteine and glycine; system gly for glycine). Materials and methods: Blood samples were obtained from normal (HbAA, HbA cells) and sickle cell disease patients (HbSS, HbS cells); amino acids were measured by HPLC; and transporter activity was measured by radioactive tracer fluxes (using serine and glycine for activity of system ASC; and glycine for that of system gly). Results: Plasma concentrations of cysteine and glycine were increased and concentrations of all amino acids were elevated in HbS cells. The activity of system ASC was increased in HbS cells (both transport capacity and aAnity were elevated for serine transport; transport capacity only for glycine). Activity of system gly was also increased (twofold increase in Vmax for glycine flux), though not significantly. Oxygenation also increased the activity of both transporters in normal and HbS cells. CO prevented deoxy-inhibition of glycine transport. Staurosporine (5 mM) inhibited O2-stimulated glycine transport through system ASC. It also inhibited the absolute magnitude of transport through system gly, but the O2-dependent flux was unaAected. Conclusion: Low reduced glutathione levels in HbS cells were not due to decreased substrate availability and O2 stimulated transport of reduced glutathione percursors in both normal and HbScells,throughamechanismthatislikelytoinvolveHbandpossiblyproteinphosphorylation. The Hematology Journal (2000) 1, 243‐249

Platelet-derived growth factor (PDGF) contributes to vascular disease by stimulating the growth of vascular smooth muscle cells (SMCs). Since amino acids are required for cell growth, the present study examined the effect of PDGF on system L amino acid transport, which is the predominant cellular pathway for the uptake of essential amino acids. System L amino acid transport was monitored by measuring the uptake of L-leucine. Treatment of SMCs with PDGF stimulated L-leucine transport in a concentration- and time-dependent manner, and this was associated with a selective increase in LAT1 mRNA and protein. PDGF failed to induce the expression of the other system L transport proteins, LAT2 and the heavy chain of the 4F2 cell surface antigen. The induction of LAT1 by PDGF was dependent on de novo RNA and protein synthesis and on mTOR activity. Serum, thrombin, and angiotensin II likewise stimulated L-leucine transport by inducing LAT1 expression. Inhibition of system L amino acid transport by the model substrate 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid blocked growth factor-mediated SMC proliferation and induced SMC apoptosis, whereas it had no effect on quiescent cells. These results demonstrate that growth factors stimulate system L amino acid transport by inducing LAT1 gene expression and that system L amino acid transport is essential for SMC proliferation and survival. The capacity of vascular mitogens to induce LAT1 expression may represent a basic mechanism by which tho acid transport * apoptosis

Abnormal fetal growth increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Emerging evidence suggests that changes in placental amino acid transport directly contribute to altered fetal growth. However, the molecular mechanisms regulating placental amino acid transport are largely unknown. Here we combined small interfering (si) RNA-mediated silencing approaches with protein expression/localization and functional studies in cultured primary human trophoblast cells to test the hypothesis that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) regulate amino acid transporters by post-translational mechanisms. Silencing raptor (inhibits mTORC1) or rictor (inhibits mTORC2) markedly decreased basal System A and System L amino acid transport activity but had no effect on growth factor-stimulated amino acid uptake. Simultaneous inhibition of mTORC1 and 2 completely inhibited both basal and growth factor-stimulated amino acid transport activity. In contrast, mTOR inhibition had no effect on serotonin transport. mTORC1 or mTORC2 silencing markedly decreased the plasma membrane expression of specific System A (SNAT2, SLC38A2) and System L (LAT1, SLC7A5) transporter isoforms without affecting global protein expression. In conclusion, mTORC1 and mTORC2 regulate human trophoblast amino acid transporters by modulating the cell surface abundance of specific transporter isoforms. This is the first report showing regulation of amino acid transport by mTORC2. Because placental mTOR activity and amino acid transport are decreased in human intrauterine growth restriction our data are consistent with the possibility that dysregulation of placental mTOR plays an important role in the development of abnormal fetal growth.

UNLABELLED Large neutral l amino acids are substrates of system L amino acid transporters. The level of one of these LAT1 is increased in many tumors. Aromatic l amino acids may also be substrates of aromatic l amino acid decarboxylase (AADC) the level of which is enhanced in endocrine tumors. Increased amino acid uptake and subsequent decarboxylation result in the intracellular accumulation of the amino acid and its decarboxylation product. (18)F and (11)C labeled neutral aromatic amino acids such as l 34 dihydroxy 6 (18)F fluorophenylalanine ((18)F FDOPA) and 5 hydroxy l [ß (11)C]tryptophan are thus successfully used in PET to image endocrine tumors. However 5 hydroxy l [ß (11)C]tryptophan has a relatively short physical half life (20 min). In this work we evaluated the in vitro and in vivo characteristics of the (18)F labeled tryptophan analog 5 (2 (18)F fluoroethoxy) l tryptophan ((18)F l FEHTP) as a PET probe for tumor imaging. METHODS (18)F l FEHTP was synthesized by no carrier added (18)F fluorination of 5 hydroxy l tryptophan. In vitro cell uptake and efflux of (18)F l FEHTP and (18)F FDOPA were studied with NCI H69 endocrine small cell lung cancer cells PC 3 pseudoendocrine prostate cancer cells and MDA MB 231 exocrine breast cancer cells. Small animal PET was performed with the respective xenograft bearing mice. Tissues were analyzed for potential metabolites. RESULTS (18)F l FEHTP specific activity and radiochemical purity were 50 150 GBq/µmol and greater than 95 respectively. In vitro cell uptake of (18)F l FEHTP was between 48 and 113 of added radioactivity per milligram of protein within 60 min at 37°C and was blocked by greater than 95 in all tested cell lines by the LAT1/2 inhibitor 2 amino 2 norboranecarboxylic acid. (18)F FDOPA uptake ranged from 26 to 53/mg. PET studies revealed similar xenograft to reference tissue ratios for (18)F l FEHTP and (18)F FDOPA at 30 45 min after injection. In contrast to the (18)F FDOPA PET results pretreatment with the AADC inhibitor S carbidopa did not affect the (18)F l FEHTP PET results. No decarboxylation products of (18)F l FEHTP were detected in the xenograft homogenates. CONCLUSION (18)F l FEHTP accumulates in endocrine and nonendocrine tumor models via LAT1 transport but is not decarboxylated by AADC. (18)F l FEHTP may thus serve as a PET probe for tumor imaging and quantification of tumor LAT1 activity. These findings are of interest in view of the ongoing evaluation of LAT1 substrates and inhibitors for cancer therapy.

IDO is the rate-limiting enzyme in the kynurenine pathway, catabolizing tryptophan to kynurenine. Tryptophan depletion by IDO-expressing tumors is a common mechanism of immune evasion inducing regulatory T cells and inhibiting effector T cells. Because mammalian cells cannot synthesize tryptophan, it remains unclear how IDO(+) tumor cells overcome the detrimental effects of local tryptophan depletion. We demonstrate that IDO(+) tumor cells express a novel amino acid transporter, which accounts for ∼50% of the tryptophan uptake. The induced transporter is biochemically distinguished from the constitutively expressed tryptophan transporter System L by increased resistance to inhibitors of System L, resistance to inhibition by high concentrations of most amino acids tested, and high substrate specificity for tryptophan. Under conditions of low extracellular tryptophan, expression of this novel transporter significantly increases tryptophan entry into IDO(+) tumors relative to tryptophan uptake through the low-affinity System L alone, and further decreases tryptophan levels in the microenvironment. Targeting this additional tryptophan transporter could be a way of pharmacological inhibition of IDO-mediated tumor escape. These findings highlight the ability of IDO-expressing tumor cells to thrive in a tryptophan-depleted microenvironment by expressing a novel, highly tryptophan-specific transporter, which is resistant to inhibition by most other amino acids. The additional transporter allows tumor cells to strike the ideal balance between supply of tryptophan essential for their own proliferation and survival, and depleting the extracellular milieu of tryptophan to inhibit T cell proliferation.

BACKGROUND: The specificity in discriminating pancreatitis is limited in the positron emission tomography (PET) using Fluorine-18-fluorodeoxyglucose. Furthermore, PET is not widely available compared to the single photon emission computed tomography (SPECT). Since amino acids play a minor role in metabolism of inflammatory cells, the potential of the SPECT tracer, 3-[123I]iodo-L-α-methyltyrosine (123I-IMT), for detecting pancreatic cancer was examined in xenotransplantation models of human pancreatic carcinoma in mice. METHODS: 123I-IMT was injected to eight mice inoculated with subcutaneous or orthotopic pancreatic tumors. Fused high-resolution-micro-SPECT (Hi-SPECT) and magnetic resonance imaging were performed. The gene expression level of L amino acid transport-system 1 (LAT1) was analyzed and correlated with tumor uptake of 123 I-IMT. RESULTS: A high uptake of 123I-IMT was detected in all tumor-bearing mice. The median tumor-to-background ratio (T/B) was 12.1 (2.0-13.2) for orthotopic and 8.4 (1.8-11.1) for subcutaneous xenotransplantation, respectively. Accordingly, the LAT1 expression in transplanted Colo357 cells was increased compared to non-malignant controls. CONCLUSIONS: Our mouse model could show a high 123I-IMT uptake in pancreatic cancer. Fused MRI scans facilitate precise evaluation of uptake in the specific regions of interest. Further studies are required to confirm these findings in tumors derived from other human pancreatic cancer cells. Since amino acids play a minor role in the metabolism of inflammatory cells, the potential for application of 123I-IMT to distinguish pancreatic tumor from inflammatory pancreatitis warrants further investigation.

High-voltage activated Ca(2+) (Ca(V)) channels play a key role in the regulation of numerous physiological events by causing transient changes in the intracellular Ca(2+) concentration. These channels consist of a pore-forming Ca(V)α(1) protein and three auxiliary subunits (Ca(V)β, Ca(V)α(2)δ and Ca(V)γ). Ca(V)α(2)δ is an important component of Ca(V) channels in many tissues and of great interest as a drug target. It is well known that anticonvulsant agent gabapentin (GBP) binds to Ca(V)α(2)δ and reduces Ca(2+) currents by modulating the expression and/or function of the Ca(V)α(1) subunit. Recently, we showed that an adamantane derivative of GABA, AdGABA, has also inhibitory effects on Ca(V) channels. However, the importance of the interaction of AdGABA with the Ca(V)α(2)δ subunit has not been conclusively demonstrated and the mechanism of action of the drug has yet to be elucidated. Here, we describe studies on the mechanism of action of AdGABA. Using a combined approach of patch-clamp recordings and molecular biology we show that AdGABA inhibits Ca(2+) currents acting on Ca(V)α(2)δ only when applied chronically, both in a heterologous expression system and in dorsal root-ganglion neurons. AdGABA seems to require uptake and be acting intracellularly given that its effects are prevented by an inhibitor of the L-amino acid transport system. Interestingly, a mutation in the Ca(V)α(2)δ that abolishes GBP binding did not affect AdGABA actions, revealing that its mechanism of action is similar but not identical to that of GBP. These results indicate that AdGABA is an important Ca(V)α(2)δ ligand that regulates Ca(V) channels.

Elevated maternal plasma concentrations of homocysteine (Hcy) are associated with pregnancy complications and adverse neonatal outcomes. The postulate that we wish to advance here is that placental transport of Hcy, by competing with endogenous amino acids for transporter activity, may account for some of the damaging impacts of Hcy on placental metabolism and function as well as fetal development. In this article, we provide an overview of some recent studies characterising the transport mechanisms for Hcy across the microvillous plasma membrane (MVM) of the syncytiotrophoblast, the transporting epithelium of human placenta. Three Hcy transport systems have been identified, systems L, A and y(+)L. This was accomplished using a strategy of competitive inhibition to investigate the effects of Hcy on the uptake of well-characterised radiolabelled substrates for each transport system into isolated MVM vesicles. The reverse experiments were also performed, examining the effects of model substrates on [³⁵S]L-Hcy uptake. This article describes the evidence for systems L, A and y(+)L involvement in placental Hcy transport and discusses the physiological implications of these findings with respect to placental function and fetal development.

Both syncytiotrophoblast microvillous plasma membrane vesicles (MVM) and placental villous fragments are used to characterize the placental uptake of maternal substrate and to investigate changes in uptake associated with pathological conditions. However, the two techniques have not been directly compared. In this study uptake of (14)C-L-serine was compared in placental villous fragments and in MVM prepared from the same placentas. (14)C-L-serine uptake into MVM vesicles was mediated by System L and System A and smaller unidentified Na(+)-dependent and Na(+)-independent components. In villous fragments an unidentified Na(+)-dependent component mediated the majority of (14)C-L-serine uptake followed by System A and System L. The unidentified Na(+)-independent component of L-serine uptake was not detected in villous fragments. The ratio of System A activity to System L activity was similar in villous fragments and MVM vesicles. However, the unidentified Na(+)-dependent component in villous fragments was significantly higher than that in MVM vesicles. This indicates that the main differences in serine uptake mechanisms identified using the two techniques were not due to differences in System A and System L activity but to differences in the unidentified Na(+)-dependent component. This study suggests that uptake of L-serine into MVM vesicles and villous fragments via Systems A and L is comparable, but that this is not true for all components of L-serine uptake.

The pathophysiology of delirium remains elusive though neurotransmitters and their precursor large neutral amino acids (LNAAs) may play a role. This pilot study investigated whether alterations of tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr) plasma levels were associated with a higher risk of transitioning to delirium in critically ill patients.Plasma LNAA concentrations were determined on days 1 and 3 in mechanically ventilated (MV) patients from the MENDS randomized controlled trial (dexmedetomidine vs. lorazepam sedation). Three independent variables were calculated by dividing plasma concentrations of Trp, Phe, and Tyr by the sum of all other LNAA concentrations. Delirium was assessed daily using the confusion assessment method for the intensive care unit (CAM-ICU). Markov regression models were used to analyze independent associations between plasma LNAA ratios and transition to delirium after adjusting for covariates.The 97 patients included in the analysis had a high severity of illness (median APACHE II, 28; IQR, 24-32). After adjusting for confounders, only high or very low tryptophan/LNAA ratios (p = 0.0003), and tyrosine/LNAA ratios (p = 0.02) were associated with increased risk of transitioning to delirium, while phenylalanine levels were not (p = 0.27). Older age, higher APACHE II scores and increasing fentanyl exposure were also associated with higher probabilities of transitioning to delirium.In this pilot study, plasma tryptophan/LNAA and tyrosine/LNAA ratios were associated with transition to delirium in MV patients, suggesting that alterations of amino acids may be important in the pathogenesis of ICU delirium. Future studies evaluating the role of amino acid precursors of neurotransmitters are warranted in critically ill patients.

L-type amino acid transporter 1 (LAT1) is proposed to be a major nutrient transporter at the blood brain barrier. LAT1 requires the heavy chain of 4F2 cell surface antigen (4F2hc) for functional expression.We investigated the expression of this heterodimeric transporter after traumatic spinal cord injury in rat by using immunohistochemical and western blot analyses.LAT1 immunoreactivities were up-regulated in the capillary endothelia in close to the injury epicenter 24 hours after injury. It reached a peak at 48 hours after injury, and thereafter decreased. 4F2hc was abundant and unchanged all through the time course after SCI. Western blot analysis under reductive and non-reductive conditions showed that LAT1 and 4F2hc were conjugated as a heterodimeric transporter and the functional regulation was dependent on the light chain, LAT1.We suggest that LAT1 may be transiently upregulated as part of the tissue-repair process after traumatic contusion injury in the spinal cord.

Methylmercury (MeHg) is a neurotoxicant which enters the brain and may cause permanent change. Thus, the properties of MeHg transport across cell membranes are a key factor in designing an appropriate model for MeHg neurotoxicity. This study uses cell cultures to examine the uptake and efflux mechanisms of methylmercury in C6 glioma, B35 neuroblastoma and rat brain endothelial (RBE4) cells. The cellular uptake and efflux of MeHg was investigated using (14)C-labeled MeHg. The uptake of MeHg-chloride was temperature-independent while the uptake of MeHg-L-cysteine was temperature-dependent in all the three cell types. This indicates that uptake of MeHg-chloride is due to passive diffusion and uptake of MeHg-L-cysteine is due to a protein carrier. Substrates of the amino acid transport system L inhibited uptake of MeHg-L-cysteine in C6 and RBE4 cells, but not B35 cells, indicating a role for system L in MeHg-uptake in the former two. Probenecid, Na(+)-free medium, MeHg and several L-amino acids did not alter the efflux of MeHg from C6 and RBE4 cells. The amino acids L-cysteine and cystine however, increased the efflux. Both cysteine and cystine are important in the generation of glutathione (GSH), suggesting the involvement of GSH in MeHg efflux. HgCl(2) at low concentrations (0.5 and 1.0 microM) decreased the MeHg efflux and at high concentrations (5.0 and 10.0 microM) increased the efflux. This inhibiting effect of HgCl(2) at low concentrations is possibly due to binding to GSH while the effect of high HgCl(2) concentrations is attributed to disrupted membrane integrity, as measured by Trypan blue. This study demonstrates differing transport mechanisms of MeHg in the cell lines C6, B35 and RBE4.