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1.
Sci Rep ; 11(1): 21638, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34737339

RESUMO

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.


Assuntos
Transportador 1 de Aminoácidos Neutros Grandes/genética , Linfoma não Hodgkin/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Sistema L de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Linfoma não Hodgkin/fisiopatologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Transcriptoma/genética
2.
Nutrients ; 13(8)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445051

RESUMO

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.


Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo , Sistema L de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Dieta com Restrição de Proteínas , Retardo do Crescimento Fetal/etiologia , Troca Materno-Fetal , Placenta/metabolismo , Animais , Transporte Biológico , Modelos Animais de Doenças , Feminino , Desenvolvimento Fetal , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/fisiopatologia , Idade Gestacional , Fenômenos Fisiológicos da Nutrição Materna , Papio , Gravidez
3.
Placenta ; 103: 188-198, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33160252

RESUMO

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.


Assuntos
Sistema L de Transporte de Aminoácidos/genética , Placenta/metabolismo , Saco Vitelino/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sistema L de Transporte de Aminoácidos/classificação , Sistema L de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Feminino , Cadeias Leves da Proteína-1 Reguladora de Fusão/genética , Cadeias Leves da Proteína-1 Reguladora de Fusão/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Masculino , Gravidez , Ratos , Ratos Wistar
4.
Nature ; 585(7824): 277-282, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879489

RESUMO

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.


Assuntos
Sistema L de Transporte de Aminoácidos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Histonas/metabolismo , Metionina/metabolismo , Metilação , Neoplasias/metabolismo , Sistema L de Transporte de Aminoácidos/deficiência , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Epigênese Genética , Feminino , Histonas/química , Humanos , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Receptores de Antígenos de Linfócitos T/metabolismo , Fator de Transcrição STAT5/metabolismo
5.
Andrology ; 8(6): 1844-1858, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32741077

RESUMO

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.


Assuntos
Sistema L de Transporte de Aminoácidos/biossíntese , Sistema L de Transporte de Aminoácidos/metabolismo , Carcinoma Embrionário/patologia , Células-Tronco de Carcinoma Embrionário/metabolismo , Neoplasias Testiculares/patologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Antineoplásicos/farmacologia , Carcinogênese/patologia , Carcinoma Embrionário/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Cadeia Pesada da Proteína-1 Reguladora de Fusão/biossíntese , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/biossíntese , Masculino , Neoplasias Testiculares/tratamento farmacológico
6.
PLoS One ; 15(5): e0233863, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470053

RESUMO

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.


Assuntos
Sistema L de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Ritmo Circadiano , Proteínas na Dieta/farmacologia , Alimentos , Intestinos/fisiologia , Aminoácidos/metabolismo , Animais , Antiporters/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Grelina/administração & dosagem , Grelina/farmacologia , Insulina/metabolismo , Intestino Delgado/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Simportadores/metabolismo , Serina-Treonina Quinases TOR/metabolismo
7.
Int J Mol Sci ; 21(5)2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-32156054

RESUMO

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.


Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo , Sistema L de Transporte de Aminoácidos/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Retardo do Crescimento Fetal/fisiopatologia , Alvo Mecanístico do Complexo 1 de Rapamicina/biossíntese , Placenta/fisiopatologia , Animais , Linhagem Celular , Feminino , Retardo do Crescimento Fetal/genética , Humanos , Gravidez , Ratos , Ratos Sprague-Dawley , Estreptozocina
8.
Am J Physiol Endocrinol Metab ; 316(5): E810-E816, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30835509

RESUMO

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.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Apelina/metabolismo , Obesidade Materna/metabolismo , Trofoblastos/metabolismo , Proteínas Quinases Ativadas por AMP , Adulto , Sistema A de Transporte de Aminoácidos/metabolismo , Sistema L de Transporte de Aminoácidos/metabolismo , Receptores de Apelina/metabolismo , Peso ao Nascer , Estudos de Casos e Controles , Feminino , Macrossomia Fetal/metabolismo , Humanos , Recém-Nascido , Insulina/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Microvilosidades/metabolismo , Placenta/metabolismo , Gravidez , Cultura Primária de Células , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
9.
Growth Horm IGF Res ; 42-43: 66-73, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30273774

RESUMO

OBJECTIVE: Branched-chain amino acids (BCAAs) have been reported to inhibit several types of muscle atrophy via the activation of the mechanistic target of rapamycin complex 1 (mTORC1). However, we previously found that BCAA did not activate mTORC1 in growth hormone (GH)-deficient spontaneous dwarf rats (SDRs), and that GH restored the stimulatory effect of BCAAs toward the mTORC1. The objective of this study was to determine whether GH or Insulin-like growth factor-I (IGF-I) stimulated the expression of L-type amino acid transporters (LATs) that delivered BCAAs, and whether LATs were involved in the mTORC1 activation. DESIGN: After the continuous administration of GH, cross-sectional areas (CSAs) of muscle fibers and LAT mRNA levels in the skeletal muscles of SDRs were compared to those from the SDRs that received normal saline. The effect of GH and IGF-I on LAT mRNA levels were determined in L6 and C2C12 myocytes. The effects of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), a blocker for LATs, and LAT1 siRNA on mTORC1 activation and cell functions were examined in C2C12 cells. RESULTS: GH increased LAT1 and LAT4 mRNA levels in accordance with the increase in CSAs of muscle fibers in SDRs. IGF-I, and not GH, increased LAT1 mRNA levels in cultured L6 myocytes. IGF-I also increased LAT1 mRNA level in another myocyte line, C2C12. Furthermore, IGF-I reduced LAT3 and LAT4 mRNA levels in both cell lines. GH reduced LAT3 and LAT4 mRNA levels in L6 cells. BCH decreased basal C2C12 cell proliferation and reduced IGF-I-induced phosphorylation of 4E-BP1 and S6K, both of which are mTORC1 targets, but LAT1 siRNA did not affect the phosphorylation. This suggests that BCH may exert its effect via other pathway than LAT1. CONCLUSIONS: IGF-I increased LAT1 mRNA level in myocytes. However, the role of LAT1 in IGF-I-induced mTORC1 activation and cell functions remains unclear.


Assuntos
Sistema L de Transporte de Aminoácidos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Animais , Células Cultivadas , Masculino , Camundongos , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
10.
Int J Mol Sci ; 18(8)2017 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-28786956

RESUMO

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.


Assuntos
Sistema L de Transporte de Aminoácidos/metabolismo , Compostos de Metilmercúrio/metabolismo , Sistema L de Transporte de Aminoácidos/genética , Linhagem Celular Tumoral , Colforsina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/genética , Leucina/metabolismo , Metionina/metabolismo
11.
Thromb Haemost ; 117(7): 1402-1411, 2017 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-28382373

RESUMO

The 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.


Assuntos
Sistema L de Transporte de Aminoácidos/antagonistas & inibidores , Sistema L de Transporte de Aminoácidos/sangue , Células Endoteliais/fisiologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/fisiologia , Sistema L de Transporte de Aminoácidos/genética , Aminoácidos Cíclicos/farmacologia , Animais , Benzoxazóis/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Cisteína/análogos & derivados , Cisteína/farmacologia , Células Endoteliais/efeitos dos fármacos , Deformação Eritrocítica/efeitos dos fármacos , Deformação Eritrocítica/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Leucina/farmacologia , Camundongos , Camundongos Nus , RNA Mensageiro/sangue , RNA Mensageiro/genética , Reticulócitos/fisiologia , S-Nitrosotióis/sangue , S-Nitrosotióis/farmacologia , Tirosina/análogos & derivados , Tirosina/farmacologia
12.
Eur J Nucl Med Mol Imaging ; 44(5): 812-821, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27900521

RESUMO

PURPOSE: F-FDOPA is a highly sensitive and specific radiopharmaceutical for pheochromocytoma and paraganglioma (PPGL) imaging. However, 18F-FDOPA might be falsely negative in these tumors, especially those related to mutations in succinate dehydrogenase genes (SDHx). The aim of the present study was to evaluate the relationship between expression of L-DOPA transporters and 18F-FDOPA PET imaging results in PPGL. METHODS: From 2007 to 2015, 175 patients with non-metastatic PPGL were evaluated by 18F-FDOPA PET/CT for initial diagnosis/staging and follow-up. 18F-FDOPA PET/CT was considered as falsely negative for at least one lesion in 10/126 (8%) patients (two sporadic, six SDHD, two SDHB PPGLs). The mRNA and protein expression levels of CD98hc and LATs were evaluated in samples with different genetic backgrounds and imaging phenotypes. The qRT-PCR and immunohistochemical analyses were performed in 14 and 16 tumor samples, respectively. RESULTS: The SDHx mutated samples exhibited a significant decrease in mRNA expression of LAT3 when compared to sporadic PPGLs (P = 0.042). There was also a statistical trend toward decreased CD98hc (P = 0.147) and LAT4 (P = 0.012) levels in SDHx vs sporadic PPGLs. No difference was observed for LAT1/LAT2 mRNA levels. LAT1 protein was expressed in 15 out of 16 (93.75%) SDHx tumors, regardless of the 18F-FDOPA positivity. LAT1 and CD98hc were co-expressed in 6/8 18F-FDOPA-negative PPGLs. In contrast, in one case with absence of LAT1/CD98hc, 18F-FDOPA uptake was positive and attributed to LAT4 expression. CONCLUSIONS: We conclude that down-regulation of LAT1/CD98hc cannot explain the imaging phenotype of SDHx-related PPGLs. A reduced activity of LAT1 remains the primary hypothesis possibly due to a modification of intracellular amino acid content which may reduce 18F-FDOPA uptake.


Assuntos
Neoplasias das Glândulas Suprarrenais/diagnóstico por imagem , Sistema L de Transporte de Aminoácidos/genética , Di-Hidroxifenilalanina/análogos & derivados , Genótipo , Paraganglioma/diagnóstico por imagem , Feocromocitoma/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Neoplasias das Glândulas Suprarrenais/genética , Idoso , Reações Falso-Negativas , Feminino , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Paraganglioma/genética , Feocromocitoma/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Succinato Desidrogenase/genética
13.
Cancer Sci ; 107(10): 1499-1505, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27465934

RESUMO

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.


Assuntos
Sistema L de Transporte de Aminoácidos/antagonistas & inibidores , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Sistema L de Transporte de Aminoácidos/genética , Sistema L de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Animais , Antineoplásicos/administração & dosagem , Transporte Biológico/efeitos dos fármacos , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Proteína-1 Reguladora de Fusão/genética , Proteína-1 Reguladora de Fusão/metabolismo , Perfilação da Expressão Gênica , Humanos , Lactato Desidrogenases/metabolismo , Masculino , Camundongos , Terapia de Alvo Molecular , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma , Ensaios Antitumorais Modelo de Xenoenxerto
14.
J Mol Endocrinol ; 56(3): 175-87, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26647387

RESUMO

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 ß-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 ß-cell signalling and function. In this report, we show that the System-L transporters are required for BCAA uptake into clonal ß-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 ß-cell signalling and function in islets and thus may be a novel pharmacological/nutritional target for the treatment and prevention of type 2 diabetes.


Assuntos
Sistema L de Transporte de Aminoácidos/metabolismo , Células Secretoras de Insulina/metabolismo , Transdução de Sinais , Sistema L de Transporte de Aminoácidos/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Expressão Gênica , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Leucina/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/metabolismo , Ratos , Serina-Treonina Quinases TOR/metabolismo
15.
Endocrinology ; 156(11): 4345-55, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26305885

RESUMO

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 (125)I-labeled T4, T3, rT3, 3,3'-T2, and 10 µM [(125)I]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.


Assuntos
Sistema L de Transporte de Aminoácidos/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Transporte Biológico , Células COS , Chlorocebus aethiops , Humanos
16.
Biosci Biotechnol Biochem ; 79(12): 2057-62, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26125295

RESUMO

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.


Assuntos
Sistema L de Transporte de Aminoácidos/metabolismo , Aminoácidos/sangue , Leucina/farmacologia , Sistema L de Transporte de Aminoácidos/antagonistas & inibidores , Aminoácidos Cíclicos/farmacologia , Animais , Relação Dose-Resposta a Droga , Leucina/administração & dosagem , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo
17.
Reprod Biol Endocrinol ; 13: 57, 2015 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-26050671

RESUMO

BACKGROUND: System L transporters LAT1 (SLC7A5) and LAT2 (SLC7A8) mediate the uptake of large, neutral amino acids in the human placenta. Many System L substrates are essential amino acids, thus representing crucial nutrients for the growing fetus. Both LAT isoforms are expressed in the human placenta, but the relative contribution of LAT1 and LAT2 to placental System L transport and their subcellular localisation are not well established. Moreover, the influence of maternal body mass index (BMI) on placental System L amino acid transport is poorly understood. Therefore the aims of this study were to determine: i) the relative contribution of the LAT isoforms to System L transport activity in primary human trophoblast (PHT) cells isolated from term placenta; ii) the subcellular localisation of LAT transporters in human placenta; and iii) placental expression and activity of System L transporters in response to maternal overweight/obesity. METHODS: System L mediated leucine uptake was measured in PHT cells after treatment with si-RNA targeting LAT1 and/or LAT2. The localisation of LAT isoforms was studied in isolated microvillous plasma membranes (MVM) and basal membranes (BM) by Western blot analysis. Results were confirmed by immunohistochemistry in sections of human term placenta. Expression and activity System L transporters was measured in isolated MVM from women with varying pre-pregnancy BMI. RESULTS: Both LAT1 and LAT2 isoforms contribute to System L transport activity in primary trophoblast cells from human term placenta. LAT1 and LAT2 transporters are highly expressed in the MVM of the syncytiotrophoblast layer at term. LAT2 is also localised in the basal membrane and in endothelial cells lining the fetal capillaries. Measurements in isolated MVM vesicles indicate that System L transporter expression and activity is not influenced by maternal BMI. CONCLUSIONS: LAT1 and LAT2 are present and functional in the syncytiotrophoblast MVM, whereas LAT2 is also expressed in the BM and in the fetal capillary endothelium. In contrast to placental System A and beta amino acid transporters, MVM System L activity is unaffected by maternal overweight/obesity.


Assuntos
Sistema L de Transporte de Aminoácidos/metabolismo , Sobrepeso/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Adulto , Sistema L de Transporte de Aminoácidos/genética , Feminino , Humanos , Obesidade/genética , Obesidade/metabolismo , Sobrepeso/genética , Gravidez , Nascimento a Termo/metabolismo
18.
BMC Pregnancy Childbirth ; 14: 181, 2014 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-24886642

RESUMO

BACKGROUND: Sufficient amino acid transport activity (AAT) is indispensable for appropriate fetal growth. Studies suggest that placental nutrient uptake activity is responsive to both maternal and fetal nutrient demands. We hypothesize that under conditions of limited nutrient availability to the fetus, as often present in preeclampsia, intrauterine growth restriction (IUGR), and insufficient weight-gain during pregnancy, a general adaptive response aimed to increase amino acid transport activity may be observed in the placenta. METHOD: A total of 40 placentas from full-term (n = 10) and pre-term (average gestational period = 34.8 weeks, n = 10) normal pregnancies, IUGR (n = 10), and preeclampsia (n = 10) associated pregnancies were looked at by immunohistochemistry followed by relative qualitative scoring to compare expression levels and localization of System L, ASCT2, and mTOR proteins. RESULT: Microvillous syncytiotrophoblast (ST) in placenta of pregnancies complicated by IUGR or preeclampsia (PE) showed significant increases in the levels of System L amino acid transport proteins 4F2hc and LAT1 compared to both full-term control and pre-term (early gestation control) pregnancies seperately (p < 0.05). Elevated mTOR protein was uniquely higher in IUGR placentas compared to full-term controls (P = 0.0026). Total cellular ASCT2 transporter protein levels were similar in all groups, however, levels of ASCT2 protein localized to the ST microvillous membrane (MVM) were significantly lower in IUGR compared to both full-term and pre-term pregnancies (P = 0.0006, 0.03, respectively). Additionally, ASCT2 and mTOR protein levels were positively associated with maternal pre-pregnancy BMI (P = 0.046, 0.048, respectively). CONCLUSION: There are three important findings based upon the present study. First, in conditions of limited nutrient availability, such as PE or IUGR, there is an overall increase in the level of System L and mTOR protein expression in the ST, suggestive of an adaptive response. Second, a decrease in ASCT2 protein at the ST MVM suggests a post-translational event that may decrease AAT activity in IUGR placentas. Third, a physiological link between transporter expression and pre-pregnancy BMI is suggested based upon a positive association observed with ASCT2 and mTOR expression values.


Assuntos
Adaptação Fisiológica , Retardo do Crescimento Fetal/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Nascimento Prematuro/metabolismo , Nascimento a Termo/metabolismo , Adulto , Sistema ASC de Transporte de Aminoácidos/metabolismo , Sistema L de Transporte de Aminoácidos/metabolismo , Índice de Massa Corporal , Membrana Celular/metabolismo , Feminino , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Antígenos de Histocompatibilidade Menor , Gravidez , Serina-Treonina Quinases TOR/metabolismo , Trofoblastos/metabolismo , Aumento de Peso , Adulto Jovem
19.
PLoS One ; 9(6): e99217, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24901243

RESUMO

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.


Assuntos
Anticoagulantes/farmacologia , Aspirina/farmacologia , Dalteparina/farmacologia , Hipóxia , Placenta/efeitos dos fármacos , Placenta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sistema A de Transporte de Aminoácidos/metabolismo , Sistema L de Transporte de Aminoácidos/metabolismo , Transporte Biológico/efeitos dos fármacos , Radioisótopos de Carbono/metabolismo , Vilosidades Coriônicas/metabolismo , Feminino , Humanos , Técnicas In Vitro , Fosforilação/efeitos dos fármacos , Gravidez , Proteína Regulatória Associada a mTOR , Fator de Transcrição STAT3/metabolismo , Trítio/metabolismo
20.
Plant Cell Physiol ; 55(5): 855-61, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24590488

RESUMO

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.


Assuntos
Sistema L de Transporte de Aminoácidos/metabolismo , Paraquat/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Poliaminas/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Modelos Biológicos
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