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Background Skeletal muscle mass and function are partly maintained by the supply of amino acids, altered amino acid transport is an important cause of frailty that can lead to decreased independence with increasing age and slow trauma recovery. The system‐A sodium coupled neutral amino acid transporter (SNAT)‐2 coded by gene family SLC38A2 generates a 506 amino acid 56 kDa protein that is an important transporter of amino acids in skeletal muscle. Ageing is associated with a decrease in expression of SNAT2 transporters. Methods In this study, we used the C2C12 cell line, using myoblast cells and cells differentiated into myotubes. We investigated if the expression of SNAT2 DNA would enhance intracellular amino acid levels and increase their availability for protein synthesis. Results In control myoblasts and myotubes, we found significantly decreased expression of SNAT2 (6.5× decrease, n = 4 per group, P

Studies have shown that melatonin (MLT) can delay ovarian aging, but the mechanism has not been fully elucidated. Here we show that granulosa cells isolated from mice follicles can synthesize MLT; the addition of MLT in ovary culture system inhibited follicle activation and growth; In vivo experiments indicated that injections of MLT to mice during the follicle activation phase can reduce the number of activated follicles by inhibiting the PI3K-AKT-FOXO3 pathway; during the early follicle growth phase, MLT administration suppressed follicle growth and atresia, and multiple pathways involved in folliculogenesis, including PI3K-AKT, were suppressed; MLT deficiency in mice increased follicle activation and atresia, and eventually accelerated age-related fertility decline; finally, we demonstrated that prolonged high-dose MLT intake had no obvious adverse effect. This study presents more insight into the roles of MLT in reproductive regulation that endogenous MLT delays ovarian aging by inhibiting follicle activation, growth and atresia., Chan Yang, Qinghua Liu, et al. use in vitro and SNAT-null mouse models to investigate the role of melatonin in ovarian aging. Their results suggest that melatonin can delay ovarian aging by inhibiting follicle activation, growth and atresia, providing more insight into how melatonin impacts reproduction.

Thyroid hormones, T3(triiodothyronine) and T4(thyroxine), induce a variety of long-term effects on important physiological functions, ranging from development and growth to metabolism regulation, by interacting with specific nuclear or cytosolic receptors. Extranuclear or nongenomic effects of thyroid hormones are mediated by plasma membrane or cytoplasmic receptors, mainly by αvβ3 integrin, and are independent of protein synthesis. A wide variety of nongenomic effects have now been recognized to be elicited through the binding of thyroid hormones to this receptor, which is mainly involved in angiogenesis, as well as in cell cancer proliferation. Several signal transduction pathways are modulated by thyroid hormone binding to αvβ3 integrin: protein kinase C, protein kinase A, Src, or mitogen-activated kinases. Thyroid hormone-activated nongenomic effects are also involved in the regulation of Na+-dependent transport systems, such as glucose uptake, Na+/K+-ATPase, Na+/H+exchanger, and amino acid transport System A. Of note, the modulation of these transport systems is cell-type and developmental stage-dependent. In particular, dysregulation of Na+/K+-ATPase activity is involved in several pathological situations, from viral infection to cancer. Therefore, this transport system represents a promising pharmacological tool in these pathologies.

Carbohydrate responsive element-binding protein (ChREBP) has been identified as a primary transcription factor that maintains energy homeostasis through transcriptional regulation of glycolytic, lipogenic, and gluconeogenic enzymes in response to a high-carbohydrate diet. Amino acids are important substrates for gluconeogenesis, but nevertheless, knowledge is lacking about whether this transcription factor regulates genes involved in the transport or use of these metabolites. Here, we demonstrate that ChREBP represses the expression of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) in response to a high-sucrose diet in rats by binding to a carbohydrate response element (ChoRE) site located -160 bp upstream of the transcriptional start site in the SNAT2 promoter region. Additionally, immunoprecipitation assays revealed that ChREBP and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) interact with each other, as part of the complex that repress SNAT2 expression. The interaction between these proteins was confirmed by an in vivo chromatin immunoprecipitation assay. These findings suggest that glucogenic amino acid uptake by the liver is controlled by ChREBP through the repression of SNAT2 expression in rats consuming a high-carbohydrate diet.

Background Glutamine (Gln) is an abundant nutrient used by cancer cells. Breast cancers cells and particularly triple-receptor negative breast cancer (TNBC) are reported to be dependent on Gln to produce the energy required for survival and proliferation. Despite intense research on the role of the intracellular Gln pathway, few reports have focussed on Gln transporters in breast cancer and TNBC. Methods The role and localisation of the Gln transporter SLC38A2/SNAT2 in response to Gln deprivation or pharmacological stresses was examined in a panel of breast cancer cell lines. Subsequently, the effect of SLC38A2 knockdown in Gln-sensitive cell lines was analysed. The prognostic value of SLC38A2 in a cohort of breast cancer was determined by immunohistochemistry. Results SLC38A2 was identified as a strongly expressed amino acid transporter in six breast cancer cell lines. We confirmed an autophagic route of degradation for SLC38A2. SLC38A2 knockdown decreased Gln consumption, inhibited cell growth, induced autophagy and led to ROS production in a subgroup of Gln-sensitive cell lines. High expression of SLC38A2 protein was associated with poor breast cancer specific survival in a large cohort of patients (p = 0.004), particularly in TNBC (p = 0.02). Conclusions These results position SLC38A2 as a selective target for inhibiting growth of Gln-dependent breast cancer cell lines.

The genetic pathogenesis of selective intrauterine growth restriction (sIUGR) remains elusive, with evidence suggesting an important role of epigenetic factors such as microRNAs. In this study, we explored the relevance of miR-373-3p to the occurrence of sIUGR. Hypoxia enhanced the levels of miR-373-3p and hypoxia-inducible factor (HIF)-1α, while HIF-1α knockdown not only boosted the migration and proliferation of HTR8 cells but also suppressed the hypoxia-induced upregulation of miR-373-3p and SLC38A1. By contrast, HIF-1α overexpression induced miR-373-3p downregulation and SLC38A1 upregulation, reducing cell growth and migration, which could be reversed by a miR-373-3p inhibitor. Importantly, the miR-373-3p inhibitor and mimic reproduced phenomena similar to those induced by HIF-1α downregulation and overexpression, respectively (including altered SLC38A1 expression, mTOR activation, cell growth, and migration). Mechanistically, the miRNA regulated cell behaviors and related mTOR signaling by targeting SLC38A1 expression through an interaction with the 3 ′ -untranslated region of SLC38A1. The placental tissues of smaller sIUGR fetuses exhibited miR-373-3p and HIF-1α upregulation, SLC38A1 downregulation, and activated mTOR. Overall, miR-373-3p appears to restrict the growth and migration of HTR8 trophoblast cells by targeting SLC38A1, as observed in the placental tissues associated with smaller sIUGR fetuses, and it could have utility in the diagnosis and treatment of this disorder.

Alcoholic steatohepatitis (ASH) is asymptomatic in the early stages and is typically advanced at the time of diagnosis. With the global rise in alcohol abuse, ASH is currently among the most detrimental diseases around the world. Hepatocellular carcinoma (HCC) is one of the final outcomes of numerous liver diseases. However, at present, HCC screening is mostly focused on liver cancer development. Moreover, there is no effective biomarker to predict the prognosis and recurrence of liver cancer. Meanwhile, there are limited studies on the prognosis and recurrence of HCC patients complicated with ASH. In this study, using bioinformatic analysis as well as cellular and animal models, we screened the differentially expressed (DE) miRNA-432 and SLC38A1 gene in ASH. Based on our analysis, miRNA-432 targeted SLC38A1, and the levels of miRNA-432 and SLC38A1 could accurately predict the overall survival (OS) and relapse free survival (RFS) in patients with liver cancer. Hence, these two genetic elements have the potential to synergistically predict the prognosis and recurrence of HCC complicated with ASH.

International audience; D-serine is a physiologic coagonist of NMDA receptors (NMDARs) required for synaptic plasticity, but mechanisms that terminate D-serine signaling are unclear. In particular, the identity of unidirectional plasma membrane transporters that mediate D-serine reuptake has remained elusive. We report that D-serine and glutamine share the same neuronal transport system, consisting of the classic system A transporters Slc38a1 and Slc38a2. We show that these transporters are not saturated with glutamine in vivo and regulate the extracellular levels of D-serine and NMDAR activity. Glutamine increased the NMDAR-dependent long-term potentiation and the isolated NMDAR potentials at the Schaffer collateral-CA1 synapses, but without affecting basal neurotransmission in male mice. Glutamine did not increase the NMDAR potentials in slices from ser-ine racemase knockout mice, which are devoid of D-serine, indicating that the effect of glutamine is caused by outcompeting D-serine for a dual glutamine-D-serine transport system. Inhibition of the system A reduced the uptake of D-serine in synap-tosomes and neuronal cultures of mice of either sex, while increasing the extracellular D-serine concentration in slices and in vivo by microdialysis. When compared with Slc38a2, the Slc38a1 transporter displayed more favorable kinetics toward the D-enantiomer. Biochemical experiments with synaptosomes from Slc38a1 knock-down mice of either sex further support its role as a D-serine reuptake system. Our study identifies the first concentrative and electrogenic transporters mediating D-ser-ine reuptake in vivo. In addition to their classical role in the glutamine-glutamate cycle, system A transporters regulate the synaptic turnover of D-serine and its effects on NMDAR synaptic plasticity. Despite the plethora of roles attributed to D-serine, the regulation of its synaptic turnover is poorly understood. We identified the system A transporters Slc38a1 and Slc38a2 as the main pathway for neuronal reuptake of D-serine. These transporters are not saturated with glutamine in vivo and provide an unexpected link between the serine shuttle pathway, responsible for regulating D-serine synaptic turnover, and the glutamine-glutamate cycle. Our observations suggest that Slc38a1 and Slc38a2 have a dual role in regulating neurotransmission. In addition to their classical role as the glutamine providers, the system A transporters regulate extracel-lular D-serine and therefore affect NMDAR-dependent synaptic plasticity. Higher glutamine export from astrocytes would increase extracellular D-serine, providing a feedforward mechanism to increase synaptic NMDAR activation.

Pulmonary fibrosis (PF) is a lethal fibrotic lung disease. The role of lncRNAs in multiple diseases has been confirmed, but the role and mechanism of lncRNA zinc finger antisense 1 (ZFAS1) in the progression of PF need to be elucidated further. Here, we found that lncRNA ZFAS1 was upregulated in bleomycin (BLM)-induced PF rats lung tissues and transforming growth factor-β1 (TGF-β1)-treated HFL1 cells, and positively correlated with the expression of solute carrier family 38 member 1 (SLC38A1), which is an important regulator of lipid peroxidation. Moreover, knockdown of lncRNA ZFAS1 significantly alleviated TGF-β1-induced fibroblast activation, inflammation and lipid peroxidation. In vivo experiments showed that inhibition of lncRNA ZFAS1 abolished BLM-induced lipid peroxidation and PF development. Mechanistically, silencing of lncRNA ZFAS1 attenuated ferroptosis and PF progression by lncRNA ZFAS1 acting as a competing endogenous RNA (ceRNA) and sponging miR-150-5p to downregulate SLC38A1 expression. Collectively, our studies demonstrated the role of the lncRNA ZFAS1/miR-150-5p/SLC38A1 axis in the progression of PF, and may provide a new biomarker for the treatment of PF patients.

The retromer complex is responsible for the endosomal sorting of numerous nutrient transporters. In this study, Curnock et al. show that in response to nutrient deprivation, retromer is transcriptionally regulated by TFEB and acts to support adaptive nutrient acquisition through endosomal recycling of the glutamine transporter, SNAT2 (SLC38A2)., Endosomal recycling maintains the cell surface abundance of nutrient transporters for nutrient uptake, but how the cell integrates nutrient availability with recycling is less well understood. Here, in studying the recycling of human glutamine transporters ASCT2 (SLC1A5), LAT1 (SLC7A5), SNAT1 (SLC38A1), and SNAT2 (SLC38A2), we establish that following amino acid restriction, the adaptive delivery of SNAT2 to the cell surface relies on retromer, a master conductor of endosomal recycling. Upon complete amino acid starvation or selective glutamine depletion, we establish that retromer expression is upregulated by transcription factor EB (TFEB) and other members of the MiTF/TFE family of transcription factors through association with CLEAR elements in the promoters of the retromer genes VPS35 and VPS26A. TFEB regulation of retromer expression therefore supports adaptive nutrient acquisition through endosomal recycling.

The integrated stress response (ISR) plays a pivotal role in adaptation of translation machinery to cellular stress. Here, we demonstrate an ISR-independent osmoadaptation mechanism involving reprogramming of translation via coordinated but independent actions of mTOR and plasma membrane amino acid transporter SNAT2. This biphasic response entails reduced global protein synthesis and mTOR signaling followed by translation of SNAT2. Induction of SNAT2 leads to accumulation of amino acids and reactivation of mTOR and global protein synthesis, paralleled by partial reversal of the early-phase, stress-induced translatome. We propose SNAT2 functions as a molecular switch between inhibition of protein synthesis and establishment of an osmoadaptive translation program involving the formation of cytoplasmic condensates of SNAT2-regulated RNA-binding proteins DDX3X and FUS. In summary, we define key roles of SNAT2 in osmotolerance.

Fetal growth restriction (FGR) is a complication of pregnancy that reduces birth weight, markedly increases infant mortality and morbidity and is associated with later-life cardiometabolic disease. No specific treatment is available for FGR. Placentas of human FGR infants have low abundance of sodium-coupled neutral amino acid transporter 2 (Slc38a2/SNAT2), which supplies the fetus with amino acids required for growth. We determined the mechanistic role of placental Slc38a2/SNAT2 deficiency in the development of restricted fetal growth, hypothesizing that placenta-specific Slc38a2 knockdown causes FGR in mice. Using lentiviral transduction of blastocysts with a small hairpin RNA (shRNA), we achieved 59% knockdown of placental Slc38a2, without altering fetal Slc38a2 expression. Placenta-specific Slc38a2 knockdown reduced near-term fetal and placental weight, fetal viability, trophoblast plasma membrane (TPM) SNAT2 protein abundance, and both absolute and weight-specific placental uptake of the amino acid transport System A tracer, 14C-methylaminoisobutyric acid (MeAIB). We also measured human placental SLC38A2 gene expression in a well-defined term clinical cohort and found that SLC38A2 expression was decreased in late-onset, but not early-onset FGR, compared with appropriate for gestational age (AGA) control placentas. The results demonstrate that low placental Slc38a2/SNAT2 causes FGR and could be a target for clinical therapies for late-onset FGR.

Assisted reproductive technologies (ART) may increase risk for abnormal placental development, preterm delivery and low birthweight. We investigated placental morphology, transporter expression and paired maternal/umbilical fasting blood nutrient levels in human term pregnancies conceived naturally (n = 10) or by intracytoplasmic sperm injection (ICSI, n = 11). Maternal and umbilical vein blood from singleton term (&gt, 37 weeks) C-section pregnancies were assessed for levels of free amino acids, glucose, free fatty acids (FFA), cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), very low-density lipoprotein (VLDL) and triglycerides. We quantified placental expression of GLUT1 (glucose), SNAT2 (amino acids), P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) (drug) transporters, and placental morphology and pathology. Following ICSI, placental SNAT2 protein expression was downregulated and umbilical cord blood levels of citrulline were increased, while FFA levels were decreased at term (p &lt, 0.05). Placental proliferation and apoptotic rates were increased in ICSI placentae (p &lt, 0.05). No changes in maternal blood nutrient levels, placental GLUT1, P-gp and BCRP expression, or placental histopathology were observed. In term pregnancies, ICSI impairs placental SNAT2 transporter expression and cell turnover, and alters umbilical vein levels of specific nutrients without changing placental morphology. These may represent mechanisms through which ICSI impacts pregnancy outcomes and programs disease risk trajectories in offspring across the life course.

BACKGROUND: Many molecular alterations are shared by embryonic liver development and hepatocellular carcinoma (HCC). Identifying the common molecular events would provide a novel prognostic biomarker and therapeutic target for HCC. METHODS: Expression levels and clinical relevancies of SLC38A4 and HMGCS2 were investigated by qRT-PCR, western blot, TCGA and GEO datasets. The biological roles of SLC38A4 were investigated by functional assays. The downstream signalling pathway of SLC38A4 was investigated by qRT-PCR, western blot, immunofluorescence, luciferase reporter assay, TCGA and GEO datasets. RESULTS: SLC38A4 silencing was identified as an oncofetal molecular event. DNA hypermethylation contributed to the downregulations of Slc38a4/SLC38A4 in the foetal liver and HCC. Low expression of SLC38A4 was associated with poor prognosis of HCC patients. Functional assays demonstrated that SLC38A4 depletion promoted HCC cellular proliferation, stemness and migration, and inhibited HCC cellular apoptosis in vitro, and further repressed HCC tumorigenesis in vivo. HMGCS2 was identified as a critical downstream target of SLC38A4. SLC38A4 increased HMGCS2 expression via upregulating AXIN1 and repressing Wnt/β-catenin/MYC axis. Functional rescue assays showed that HMGCS2 overexpression reversed the oncogenic roles of SLC38A4 depletion in HCC. CONCLUSIONS: SLC38A4 downregulation was identified as a novel oncofetal event, and SLC38A4 was identified as a novel tumour suppressor in HCC.

Meninges, or the membranous coverings of the brain and spinal cord, play host to dozens of morbid pathologies. In this study we provide a method to isolate the leptomeningeal cell layer, identify leptomeninges in histologic slides, and maintain leptomeningeal fibroblasts in in vitro culture. Using an array of transcriptomic, histological, and cytometric analyses, we identified ICAM1 and SLC38A2 as two novel markers of leptomeningeal cells in vivo and in vitro. Our results confirm the fibroblastoid nature of leptomeningeal cells and their ability to form a sheet-like layer that covers the brain and spine parenchyma. These findings will enable researchers in central nervous system barriers to describe leptomeningeal cell functions in health and disease.

The placenta is critical in mammalian embryonic development because the embryo’s supply of nutrients, including amino acids, depends solely on mother-to-embryo transport through it. However, the molecular mechanisms underlying this amino acid supply are poorly understood. In this study, we focused on system A amino acid transporters Slc38a1 /SNAT1, Slc38a2 /SNAT2, and Slc38a4 /SNAT4, which carry neutral, short-side-chain amino acids, to determine their involvement in placental or embryonic development. A triple-target CRISPR screen identified Slc38a4 /SNAT4 as the critical amino acid transporter for placental development in mice. We established mouse lines from the CRISPR founders with large deletions in Slc38a4 and found that, consistent with the imprinted paternal expression of Slc38a4 /SNAT4 in the placenta, paternal knockout (KO) but not maternal KO of Slc38a4 /SNAT4 caused placental hypoplasia associated with reduced fetal weight. Immunostaining revealed that SNAT4 was widely expressed in differentiating cytotrophoblasts and maturing trophoblasts at the maternal–fetal interface. A blood metabolome analysis revealed that amino acid concentrations were globally reduced in Slc38a4 /SNAT4 mutant embryos. These results indicated that SNAT4-mediated amino acid transport in mice plays a major role in placental and embryonic development. Given that expression of Slc38a4 in the placenta is conserved in other species, our Slc38a4 /SNAT4 mutant mice could be a promising model for the analysis of placental defects leading to intrauterine growth restriction in mammals.

The pathophysiological role of mammalian target of rapamycin complex 1 (mTORC1) in neurodegenerative diseases is established, but possible therapeutic targets responsible for its activation in neurons must be explored. Here we identified solute carrier family 38a member 1 (SNAT1, Slc38a1) as a positive regulator of mTORC1 in neurons. Slc38a1flox/flox and Synapsin I-Cre mice were crossed to generate mutant mice in which Slc38a1 was selectively deleted in neurons. Measurement of 2,3,5-triphenyltetrazolium chloride (TTC) or the MAP2-negative area in a mouse model of middle cerebral artery occlusion (MCAO) revealed that Slc38a1 deficiency decreased infarct size. We found a transient increase in the phosphorylation of p70S6k1 (pp70S6k1) and a suppressive effect of rapamycin on infarct size in MCAO mice. Autophagy inhibitors completely mitigated the suppressive effect of SNAT1 deficiency on neuronal cell death under in vitro stroke culture conditions. These results demonstrate that SNAT1 promoted ischemic brain damage via mTOR-autophagy system., Yamada et al. demonstrate that solute carrier family 38a member 1 (SNAT1, Slc38a1) is as a positive regulator of mTORC1 that promotes neuronal cell death, using neuron-specific Slc38a1 knockout mice. This study shows that SNAT1 exacerbates ischemic brain damage through mTOR-mediated suppression of autophagy.

Plasma amino acids and their transporters constitute an important part of the feedback loop between the liver and pancreatic α-cell function, and glucagon regulates hepatic amino acid turnover. Disruption of hepatic glucagon receptor action activates the loop and results in high plasma amino acids and hypersecretion of glucagon associated with α-cell hyperplasia. In the present study, we report a technique to rescue implanted human pancreatic islets from the mouse kidney capsule. Using this model, we have demonstrated that expression of the amino acid transporter SLC38A4 increases in α-cells after administration of a glucagon receptor blocking antibody. The increase in SLC38A4 expression and associated α-cell proliferation was dependent on mechanistic target of rapamycin pathway. We confirmed increased α-cell proliferation and expression of SLC38A4 in pancreas sections from patients with glucagon cell hyperplasia and neoplasia (GCHN) with loss-of-function mutations in the glucagon receptor. Collectively, using a technique to rescue implanted human islets from the kidney capsule in mice and pancreas sections from patients with GCHN, we found that expression of SLC38A4 was increased under conditions of disrupted glucagon receptor signaling. These data provide support for the existence of a liver-human α-cell endocrine feedback loop.

During Ramadan, many pregnant Muslim women fast between dawn and sunset. Although the impacts of prolonged maternal intermittent fasting (IF) on fetal growth and placental function are under-researched, reported effects include reduced placental weight and birth weight. In the present study, pregnant Wistar rats were used to model repeated cycles of IF on fetal development and placental function and to examine sex-specific effects. In the IF group, food was withdrawn daily from 17:00 to 09:00 over 21 days of gestation, while the control group received food ad libitum. Both groups had free water access. IF dams consumed less food, had significantly reduced weight compared with controls, with reduced plasma glucose and amino acids. Both fetal sexes were significantly lighter in the IF group with reduced fetal plasma amino acids. Placental weights and morphology were unchanged. The profile of placental metabolites was altered in the IF group with sex-specific responses evident. Transplacental flux of 14C-methylaminoisobutyric acid ( 14C-MeAIB), a system A amino acid transporter substrate, was significantly reduced in both fetal sexes in the IF group. Sodium-dependent 14C-MeAIB uptake into isolated placental plasma membrane vesicles was unchanged. The gene expression of system A transporter Slc38a1, Slc38a2 and Slc38a4 was up-regulated in IF male placentas only. No changes were observed in placental SNAT1 and SNAT2 protein expression. Maternal IF results in detrimental impacts on maternal physiology and fetal development with changes in the placental and fetal metabolite profiles. Reduced placental system A transporter activity may be responsible for fetal growth restriction in both sexes.

The constant transport of ions across the alveolar epithelial barrier regulates alveolar fluid homeostasis. Dysregulation or inhibition of Na+ transport causes fluid accumulation in the distal airspaces resulting in impaired gas exchange and respiratory failure. Previous studies have primarily focused on the critical role of amiloride-sensitive epithelial sodium channel (ENaC) in alveolar fluid clearance (AFC), yet activation of ENaC failed to attenuate pulmonary edema in clinical trials. Since 40% of AFC is amiloride-insensitive, Na+ channels/transporters other than ENaC such as Na+-coupled neutral amino acid transporters (SNATs) may provide novel therapeutic targets. Here, we identified a key role for SNAT2 ( SLC38A2) in AFC and pulmonary edema resolution. In isolated perfused mouse and rat lungs, pharmacological inhibition of SNATs by HgCl2 and α-methylaminoisobutyric acid (MeAIB) impaired AFC. Quantitative RT-PCR identified SNAT2 as the highest expressed System A transporter in pulmonary epithelial cells. Pharmacological inhibition or siRNA-mediated knockdown of SNAT2 reduced transport of l-alanine across pulmonary epithelial cells. Homozygous Slc38a2−/− mice were subviable and died shortly after birth with severe cyanosis. Isolated lungs of Slc38a2+/− mice developed higher wet-to-dry weight ratios (W/D) as compared to wild type (WT) in response to hydrostatic stress. Similarly, W/D ratios were increased in Slc38a2+/− mice as compared to controls in an acid-induced lung injury model. Our results identify SNAT2 as a functional transporter for Na+ and neutral amino acids in pulmonary epithelial cells with a relevant role in AFC and the resolution of lung edema. Activation of SNAT2 may provide a new therapeutic strategy to counteract and/or reverse pulmonary edema.

Background: Acute myeloid leukemia (AML) is one of the most common malignant and aggressive hematologic tumors, and its pathogenesis is associated with abnormal post-transcriptional regulation. Unbalanced competitive endogenous RNA (ceRNA) promotes tumorigenesis and progression, and greatly contributes to tumor risk classification and prognosis. However, the comprehensive analysis of the circular RNA (circRNA)-long non-coding RNA (lncRNA)-miRNA-mRNA ceRNA network in the prognosis of AML is still rarely reported. Method: We obtained transcriptome data of AML and normal samples from The Cancer Genome Atlas (TCGA), Genotype-tissue Expression (GTEx), and Gene Expression Omnibus (GEO) databases, and identified differentially expressed (DE) mRNAs, lncRNAs, and circRNAs. Then, the targeting relationships among lncRNA-miRNA, circRNA-miRNA, and miRNA-mRNA were predicted, and the survival related hub mRNAs were further screened by univariate and multivariate Cox proportional hazard regression. Finally, the AML prognostic circRNA-lncRNA-miRNA-mRNA ceRNA regulatory network was established. Results: We identified prognostic 6 hub mRNAs (TM6SF1, ZMAT1, MANSC1, PYCARD, SLC38A1, and LRRC4) through Cox regression model, and divided the AML samples into high and low risk groups according to the risk score obtained by multivariate Cox regression. Survival analysis verified that the survival rate of the high-risk group was significantly reduced (p &lt, 0.0001). The prognostic ceRNA network of 6 circRNAs, 32 lncRNAs, 8 miRNAs, and 6 mRNAs was established according to the targeting relationship between 6 hub mRNAs and other RNAs. Conclusion: In this study, ceRNA network jointly participated by circRNAs and lncRNAs was established for the first time. It comprehensively elucidated the post-transcriptional regulatory mechanism of AML, and identified novel AML prognostic biomarkers, which has important guiding significance for the clinical diagnosis, treatment, and further scientific research of AML.

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.

In cultured human fibroblasts, SNAT transporters (System A) account for the accumulation of non-essential neutral amino acids, are adaptively up-regulated upon amino acid deprivation and play a major role in cell volume recovery upon hypertonic stress. No information is instead available on the expression and activity of SNAT transporters in human bone marrow mesenchymal stromal cells (MSC), although they are increasingly investigated for their staminal and immunomodulatory properties and used for several therapeutic applications. The uptake of glutamine and proline, two substrates of SNAT1 and SNAT2 transporters, was measured in primary human MSC and an MSC line. The amino acid analogue MeAIB, a specific substrate of these carriers, has been used to selectively inhibit SNAT-dependent transport of glutamine and, through its sodium-dependent transport, as an indicator of SNAT1/2 activity. SNAT1/2 expression and localization were assessed with RT-PCR and confocal microscopy, respectively. Cell volume was assessed from urea distribution space. In all these experiments, primary human fibroblasts were used as the positive control for SNAT expression and activity. Compared with fibroblasts, MSC have a lower SNAT1 expression and hardly detectable membrane localization of both SNAT1 and SNAT2. Moreover, they exhibit no sodium-dependent MeAIB uptake or MeAIB-inhibitable glutamine transport, and exhibit a lower ability to accumulate glutamine and proline than fibroblasts. MSC exhibited an only marginal increase in MeAIB transport upon amino acid starvation and did not recover cell volume after hypertonic stress. In conclusion, the activity of SNAT transporters is low in human MSC. MSC adaptation to amino acid shortage is expected to rely on intracellular synthesis, given the absence of an effective up-regulation of the SNAT transporters.

Theanine is an amino acid abundant in green tea with an amide moiety analogous to glutamine (GLN) rather than glutamic acid (Glu) and GABA, which are both well-known as amino acid neurotransmitters in the brain. Theanine has no polyphenol and flavonoid structures required for an anti-oxidative property as seen with catechins and tannins, which are more enriched in green tea. We have shown marked inhibition by this exogenous amino acid theanine of the uptake of [3H]GLN, but not of [3H]Glu, in rat brain synaptosomes. Beside a ubiquitous role as an endogenous amino acid, GLN has been believed to be a main precursor for the neurotransmitter Glu sequestered in a neurotransmitter pool at glutamatergic neurons in the brain. The GLN transporter solute carrier 38a1 (Slc38a1) plays a crucial role in the incorporation of extracellular GLN for the intracellular conversion to Glu by glutaminase and subsequent sequestration at synaptic vesicles in neurons. However, Slc38a1 is also expressed by undifferentiated neural progenitor cells (NPCs) not featuring a neuronal phenotype. NPCs are derived from a primitive stem cell endowed to proliferate for self-renewal and to commit differentiation to several daughter cell lineages such as neurons, astrocytes, and oligodendrocytes. In vitro culture with theanine leads to the marked promotion of the generation of new neurons together with selective upregulation of Slc38a1 transcript expression in NPCs. In this review, we will refer to a possible novel neurogenic role of theanine for brain wellness through a molecular mechanism relevant to facilitated neurogenesis with a focus on Slc38a1 expressed by undifferentiated NPCs on the basis of our accumulating findings to date.

GABA signaling is involved in a wide range of neuronal functions, such as synchronization of action potential firing, synaptic plasticity and neuronal development. Sustained GABA signaling requires efficient mechanisms for the replenishment of the neurotransmitter pool of GABA. The prevailing theory is that exocytotically released GABA may be transported into perisynaptic astroglia and converted to glutamine, which is then shuttled back to the neurons for resynthesis of GABA&mdash, i.e., the glutamate/GABA-glutamine (GGG) cycle. However, an unequivocal demonstration of astroglia-to-nerve terminal transport of glutamine and the contribution of astroglia-derived glutamine to neurotransmitter GABA synthesis is lacking. By genetic inactivation of the amino acid transporter Solute carrier 38 member a1 (Slc38a1)&mdash, which is enriched on parvalbumin+ GABAergic neurons&mdash, and by intraperitoneal injection of radiolabeled acetate (which is metabolized to glutamine in astroglial cells), we show that Slc38a1 mediates import of astroglia-derived glutamine into GABAergic neurons for synthesis of GABA. In brain slices, we demonstrate the role of Slc38a1 for the uptake of glutamine specifically into GABAergic nerve terminals for the synthesis of GABA depending on demand and glutamine supply. Thus, while leaving room for other pathways, our study demonstrates a key role of Slc38a1 for newly formed GABA, in harmony with the existence of a GGG cycle.

Imprinted genes are characterized by monoallelic expression that is dependent on parental origin. Comparative analysis of imprinted genes between species is a powerful tool for understanding the biological significance of genomic imprinting. The slc38a4 gene encodes a neutral amino acid transporter and is identified as imprinted in mice. In this study, the imprinting status of SLC38A4 was assessed in bovine adult tissues and placenta using a polymorphism-based approach. Results indicate that SLC38A4 is not imprinted in eight adult bovine tissues including heart, liver, spleen, lung, kidney, muscle, fat, and brain. It was interesting to note that SLC38A4 showed polymorphic status in five heterogeneous placentas, with three exhibiting paternal monoallelic expression and two exhibiting biallelic expression. Monoallelic expression of imprinted genes is generally associated with allele-specific differentially methylation regions (DMRs) of CpG islands (CGIs)-encompassed promoter; therefore, the DNA methylation statuses of three CGIs in the SLC38A4 promoter and exon 1 region were tested in three placentas (two exhibiting paternal monoallelic and one showing biallelic expression of SLC38A4) and their corresponding paternal sperms. Unexpectedly, extreme hypomethylation (

Objective: To validate the gene expression profile obtained from the previous microarray analysis and to further study the biological functions of these genes in endometrial cancer. From our previous study, we identified 621 differentially expressed genes in laser-captured microdissected endometrioid endometrial cancer as compared to normal endometrial cells. Among these genes, 146 were significantly up-regulated in endometrial cancer. Materials and Methods: A total of 20 genes were selected from the list of up-regulated genes for the validation assay. The qPCR confirmed that 19 out of the 20 genes were up-regulated in endometrial cancer compared with normal endometrium. RNA interference (RNAi) was used to knockdown the expression of the upregulated genes in ECC-1 and HEC-1A endometrial cancer cell lines and its effect on proliferation, migration and invasion were examined. Results: Knockdown of MIF, SOD2, HIF1A and SLC7A5 by RNAi significantly decreased the proliferation of ECC-1 cells (p < 0.05). Our results also showed that the knockdown of MIF, SOD2 and SLC7A5 by RNAi significantly decreased the proliferation and migration abilities of HEC-1A cells (p < 0.05). Moreover, the knockdown of SLC38A1 and HIF1A by RNAi resulted in a significant decrease in the proliferation of HEC1A cells (p < 0.05). Conclusion: We have identified the biological roles of SLC38A1, MIF, SOD2, HIF1A and SLC7A5 in endometrial cancer, which opens up the possibility of using the RNAi silencing approach to design therapeutic strategies for treatment of endometrial cancer. Keywords: Endometrial cancer, Migration, Proliferation, RNA interference

Background/Aims: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner. Methods: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (Vmem) and swelling-dependent chloride (Cl-) currents (IClswell, VSOR, VRAC) were performed using the whole-cell patch clamp technique. Results: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by ∼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl- current under isotonic conditions resembling features of the swelling-activated Cl- current (IClswell). The cell membrane potential (Vmem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na+-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells. Conclusion: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling.

The influx of essential amino acids into skeletal muscle is primarily mediated by the large neutral amino acid transporter 1 (LAT1), which is dependent on the glutamine gradient generated by the sodium-dependent neutral amino acid transporter 2 (SNAT2). The protein expression and membrane localization of LAT1 may be influenced by amino acid ingestion and/or resistance exercise, although its acute influence on dietary amino acid incorporation into skeletal muscle protein has not been investigated. In a group design, healthy males consumed a mixed carbohydrate (0.75 g·kg−1) crystalline amino acid (0.25 g·kg−1) beverage enriched to 25% and 30% with LAT1 substrates L-[1-13C]leucine (LEU) and L-[ring-2H5]phenylalanine (PHE), respectively, at rest (FED: n = 7, 23 ± 5 y, 77 ± 4 kg) or after a bout of resistance exercise (EXFED: n = 7, 22 ± 2 y, 78 ± 11 kg). Postprandial muscle biopsies were collected at 0, 120, and 300 min to measure transporter protein expression (immunoblot), LAT1 membrane localization (immunofluorescence), and dietary amino acid incorporation into myofibrillar protein (ΔLEU and ΔPHE). Basal LAT1 and SNAT2 protein contents were correlated with each other (r = 0.55, p = 0.04) but their expression did not change across time in FED or EXFED (all, p > 0.05). Membrane localization of LAT1 did not change across time in FED or EXFED whether measured as outer 1.5 µm intensity or membrane-to-fiber ratio (all, p > 0.05). Basal SNAT2 protein expression was not correlated with ΔLEU or ΔPHE (all, p ≥ 0.05) whereas basal LAT1 expression was negatively correlated with ΔPHE in FED (r = −0.76, p = 0.04) and EXFED (r = −0.81, p = 0.03) but not ΔLEU (p > 0.05). Basal LAT1 membrane localization was not correlated with ΔLEU or ΔPHE (all, p > 0.05). Our results suggest that LAT1/SNAT2 protein expression and LAT1 membrane localization are not influenced by acute anabolic stimuli and do not positively influence the incorporation of dietary amino acids for de novo myofibrillar protein synthesis in healthy young males.

Summary: GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR) pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER)-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake. Here, we show that GADD34 promotes the processing of proteins synthesized on the ER during hyperosmotic stress independent of its action in the ISR. We show that GADD34/PP1 phosphatase activity reverses hyperosmotic-stress-induced Golgi fragmentation and is important for cis- to trans-Golgi trafficking of SNAT2, thereby promoting SNAT2 plasma membrane localization and function. These results suggest that GADD34 is a protective molecule for ocular diseases such as dry eye syndrome. : Here, Krokowski et al. show that GADD34/PP1 protects the microtubule network, prevents Golgi fragmentation, and preserves protein trafficking independent of its action in the integrated stress response (ISR). In osmoadaptation, GADD34 facilitates trans-Golgi-mediated processing of the endoplasmic reticulum (ER)-synthesized amino acid transporter SNAT2, which in turn increases amino acid uptake. Keywords: SNAT2, GADD34, hyperosmotic stress, amino acid transport, Golgi fragmentation, ISR

Nutrient ingestion is known to increase the exercise-induced stimulation of muscle protein synthesis following resistance exercise. Less is known about the effect of nutrients on muscle protein synthesis following sprint exercise. At two occasions separated by 1 mo, 12 healthy subjects performed three 30-s sprints with 20-min rest between bouts. In randomized order, they consumed a drink with essential amino acids and maltodextrin (nutrient) or flavored water (placebo). Muscle biopsies were obtained 80 and 200 min after the last sprint, and blood samples were taken repeatedly during the experiment. Fractional synthetic rate (FSR) was measured by continuous infusion of l-[2H5]phenylalanine up to 200 min postexercise. The mRNA expression and protein expression of SNAT2 were both 1.4-fold higher ( P < 0.05) after nutrient intake compared with placebo at 200 min postexercise. Phosphorylated Akt, mammalian target of rapamycin (mTOR), and p70S6k were 1.7- to 3.6-fold higher ( P < 0.01) 80 min after the last sprint with nutrient ingestion as compared with placebo. In addition, FSR was higher ( P < 0.05) with nutrients when plasma phenylalanine (FSRplasma) was used as a precursor but not when intracellular phenylalanine (FSRmuscle) was used. Significant correlations were also found between FSRplasma on the one hand and plasma leucine and serum insulin on the other hand in the nutrient condition. The results show that nutrient ingestion induces the expression of the amino acid transporter SNAT2 stimulates Akt/mTOR signaling and most likely the rate of muscle protein synthesis following sprint exercise. NEW & NOTEWORTHY There is limited knowledge regarding the effect of nutrients on muscle protein synthesis following sprint as compared with resistance exercise. The results demonstrate that nutrient ingestion during repeated 30-s bouts of sprint exercise induces expression of the amino acid transporter SNAT2 and stimulates Akt/mTOR signaling and most likely the rate of muscle protein synthesis. Future studies to explore the chronic effects of nutritional ingestion during sprint exercise sessions on muscle mass accretion are warranted.

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.

Background Resistance training is a potent stimulus to induce muscle hypertrophy. Supplemental protein intake is known to enhance gains in muscle mass through activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, which initiates protein translation. While the optimal dose of high quality protein to promote post exercise anabolism in young or older men has been investigated, little is known about the minimum doses of protein required to potentiate the resistance exercise activation of anabolic signalling in middle aged men. Methods Twenty healthy men (46.3 ± 5.7 years, BMI: 23.9 ± 6.6 kg/m2) completed a single bout of unilateral resistance exercise consisting of 4 sets of leg extension and press at 80% of 1 repetition maximum. Participants were randomised to consume either formulated milk product containing 9 g milk protein (FMP) or an isoenergetic carbohydrate placebo (CHO) immediately post exercise, in a double blind fashion. A single muscle biopsy was collected at pre-exercise baseline and then bilateral biopsies were collected 90 and 240 min after beverage consumption. Results P70S6KThr389 phosphorylation was increased with exercise irrespective of group, P70S6KThr421/Ser424 was increased with exercise only in the FMP group at 240 min. Likewise, rpS6 Ser235/236 phosphorylation was increased with exercise irrespective of group, rpS6 Ser240/244 increased to a greater extent following exercise in the FMP group. mRNA expression of the amino acid transporter, LAT1/ SLC7A5 increased with both exercise and beverage consumption irrespective of group. PAT1/ SLC36A1, CAT1/ SLC7A1 and SNAT2/ SLC38A2 mRNA increased only after exercise regardless of group. Conclusions Nine grams of milk protein is sufficient to augment some measures of downstream mTORC1 signalling after resistance exercise but does not potentiate exercise induced increases in amino acid transporter expression. Formulated products containing nine grams of milk protein would be expected stimulate muscle anabolism after resistance exercise. Trial registration New Zealand Clinical Trials Registry ACTRN12615001375549 . Registered: 17 December, 2015.

Vitamin D deficiency during pregnancy is linked to adverse perinatal outcomes such as small for gestational age infants. Recent evidence suggests that changes in placental amino acid transport contribute to altered fetal growth. We tested the hypothesis that 1,25-dihydroxy vitamin D3 increases the gene expression of System A and L amino acid transporter isoforms and stimulates placental amino acid transport activity in cultured primary human trophoblast cells mediated by mTOR signalling. Treatment with 1,25-dihydroxy vitamin D3 significantly increased mRNA expression of the System A isoform SNAT2 and System A activity, but had no effect on System L and did not affect mTOR signaling. siRNA silencing of the vitamin D receptor prevented 1,25-dihydroxy vitamin D3-stimulated System A transport. In conclusion, 1,25-dihydroxy vitamin D3 regulates System A activity through increased mRNA expression of SNAT2 transporters. Effects on placental amino acid transport may be the mechanism underlying the association between maternal vitamin D status and fetal growth.

Progressive myoclonus epilepsy is a heterogeneous group of disorders characterized by myoclonic and tonic-clonic seizures, ataxia and cognitive decline. We here present two affected brothers. At 9 months of age the elder brother developed ataxia and myoclonic jerks. In his second year he lost the ability to walk and talk, and he developed drug-resistant progressive myoclonus epilepsy. The cerebrospinal fluid level of glutamate was decreased while glutamine was increased. His younger brother manifested similar symptoms from 6 months of age. By exome sequencing of the proband we identified a novel homozygous frameshift variant in the potassium channel tetramerization domain 7 (KCTD7) gene (NM_153033.1:c.696delT: p.F232fs), which results in a truncated protein. The identified F232fs variant is inherited in an autosomal recessive manner, and the healthy consanguineous parents carry the variant in a heterozygous state. Bioinformatic analyses and structure modelling showed that KCTD7 is a highly conserved protein, structurally similar to KCTD5 and several voltage-gated potassium channels, and that it may form homo- or heteromultimers. By heterologous expression in Xenopus laevis oocytes, we demonstrate that wild-type KCTD7 hyperpolarizes cells in a K+ dependent manner and regulates activity of the neuronal glutamine transporter SAT2 (Slc38a2), while the F232fs variant impairs K+ fluxes and obliterates SAT2-dependent glutamine transport. Characterization of four additional disease-causing variants (R94W, R184C, N273I, Y276C) bolster these results and reveal the molecular mechanisms involved in the pathophysiology of KCTD7-related progressive myoclonus epilepsy. Thus, our data demonstrate that KCTD7 has an impact on K+ fluxes, neurotransmitter synthesis and neuronal function, and that malfunction of the encoded protein may lead to progressive myoclonus epilepsy.

Extracellular amino acid (AA) withdrawal/restriction invokes an integrated stress response (ISR) that induces global suppression of protein synthesis whilst allowing transcription and translation of a select group of genes, whose protein products facilitate cellular adaptation to AA insufficiency. Transcriptional induction of the System A/SNAT2 AA transporter represents a classic adaptation response and crucially depends upon activation of the General Control Nonderepressible-2 kinase/Activating transcription factor 4 (GCN2/ATF4) pathway. However, the ISR may also include additional signalling inputs operating in conjunction or independently of GCN2/ATF4 to upregulate SNAT2. Herein, we show that whilst pharmacological inhibition of MEK-ERK, mTORC1 and p38 MAP kinase signalling has no detectable effect on System A upregulation, inhibitors targeting GSK3 (e.g. SB415286) caused significant repression of the SNAT2 adaptation response. Strikingly, the effects of SB415286 persist in cells in which GSK3α/β have been stably silenced indicating an off-target effect. We show that SB415286 can also inhibit cyclin-dependent kinases (CDK) and that roscovitine and flavopiridol (two pan CDK inhibitors) are effective repressors of the SNAT2 adaptive response. In particular, our work reveals that CDK7 activity is upregulated in AA-deprived cells in a GCN-2-dependent manner and that a potent and selective CDK7 inhibitor, THZ-1, not only attenuates the increase in ATF4 expression but blocks System A adaptation. Importantly, the inhibitory effects of THZ-1 on System A adaptation are mitigated in cells expressing a doxycycline-inducible drug-resistant form of CDK7. Our data identify CDK7 as a novel component of the ISR regulating System A adaptation in response to AA insufficiency., Highlights • Roscovitine and flavopiridol (CDK inhibitors) block the System A adaptive response. • Extracellular amino acid (AA) withdrawal induces CDK7 activation. • Pharmacological inhibition of GCN2 represses CDK7 activation in AA-deprived cells. • Targeted suppression of CDK7 represses ATF4 expression and System A adaptation.

Significance The hypoxic microenvironment in solid tumors is known to reduce the efficacy of anticancer treatments in many cancer types, including breast cancer. This study shows hypoxia induces an amino acid transporter, SNAT2, which then causes resistance to antihormone therapy. We show major interplay between genes induced by estrogen receptor and hypoxia. Hypoxia-inducible factor 1α compensates for the loss of expression of estrogen receptor-α (ERα) for maintaining SNAT2 expression under hypoxia or endocrine therapies. SNAT2 overexpression produces complete resistance to antiestrogen therapy in vivo and is induced in tamoxifen resistance, and its expression is associated with poor survival in breast cancer and resistance to endocrine therapy in ERα+ luminal B patients. Our findings thus have revealed a previously unidentified mechanism for antiestrogen resistance driven by tumor metabolism., Tumor hypoxia is associated with poor patient outcomes in estrogen receptor-α–positive (ERα+) breast cancer. Hypoxia is known to affect tumor growth by reprogramming metabolism and regulating amino acid (AA) uptake. Here, we show that the glutamine transporter, SNAT2, is the AA transporter most frequently induced by hypoxia in breast cancer, and is regulated by hypoxia both in vitro and in vivo in xenografts. SNAT2 induction in MCF7 cells was also regulated by ERα, but it became predominantly a hypoxia-inducible factor 1α (HIF-1α)–dependent gene under hypoxia. Relevant to this, binding sites for both HIF-1α and ERα overlap in SNAT2’s cis-regulatory elements. In addition, the down-regulation of SNAT2 by the ER antagonist fulvestrant was reverted in hypoxia. Overexpression of SNAT2 in vitro to recapitulate the levels induced by hypoxia caused enhanced growth, particularly after ERα inhibition, in hypoxia, or when glutamine levels were low. SNAT2 up-regulation in vivo caused complete resistance to antiestrogen and, partially, anti-VEGF therapies. Finally, high SNAT2 expression levels correlated with hypoxia profiles and worse outcome in patients given antiestrogen therapies. Our findings show a switch in the regulation of SNAT2 between ERα and HIF-1α, leading to endocrine resistance in hypoxia. Development of drugs targeting SNAT2 may be of value for a subset of hormone-resistant breast cancer.

Amino acids are essential for cellular protein synthesis, growth, metabolism, signaling and in stress responses. Cell plasma membranes harbor specialized transporters accumulating amino acids to support a variety of cellular biochemical pathways. Several transporters for neutral amino acids have been characterized. However, Slc38a2 (also known as SA1, SAT2, ATA2, SNAT2) representing the classical transport system A activity stands in a unique position: Being a secondarily active transporter energized by the electrochemical gradient of Na+, it creates steep concentration gradients for amino acids such as glutamine: this may subsequently drive the accumulation of additional neutral amino acids through exchange via transport systems ASC and L. Slc38a2 is ubiquitously expressed, yet in a cell-specific manner. In this review, we show that Slc38a2 is regulated at the transcriptional and translational levels as well as by ions and proteins through direct interactions. We describe how Slc38a2 senses amino acid availability and passes this onto intracellular signaling pathways and how it regulates protein synthesis, cellular proliferation and apoptosis through the mechanistic (mammalian) target of rapamycin (mTOR) and general control nonderepressible 2 (GCN2) pathways. Furthermore, we review how this extensively regulated transporter contributes to cellular osmoadaptation and how it is regulated by endoplasmic reticulum stress and various hormonal stimuli to promote cellular metabolism, cellular signaling and cell survival. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

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.

We have previously shown that Na+-coupled neutral amino acid transporter 1 (SNAT1) modulates nitric oxide (NO) production in pulmonary arterial endothelial cells (PAECs) from newborn piglets. Specifically, the ability to increase NO production in response to the l-arginine-NO precursor l-citrulline is dependent on SNAT1 expression. Elucidating factors that regulate SNAT1 expression in PAECs could provide new insights and therapeutic targets relevant to NO production. Our major goals were to determine if reactive oxygen species (ROS) modulate SNAT1 expression in PAECs from newborn piglets and to evaluate the role of NADPH oxidase 1 (NOX1) and uncoupled endothelial NO synthase, enzymatic sources of ROS, in hypoxia-induced increases in SNAT1 expression. Treatment with either H2O2 or xanthine plus xanthine oxidase increased SNAT1 expression in PAECs from newborn piglets cultured under normoxic conditions. Hypoxia-induced increases in SNAT1 expression were inhibited by treatments with the ROS-removing agents catalase and superoxide dismutase, NOX1 siRNA, and the NO synthase inhibitor NG-nitro-l-arginine methyl ester. Both tetrahydropbiopterin (BH4) and l-citrulline, two therapies that decrease ROS by recoupling endothelial NO synthase, reduced the hypoxia-induced increase in SNAT1 expression. BH4 and l-citrulline treatment improved NO production in hypoxic PAECs despite a reduction in SNAT1 expression. In conclusion, SNAT1 expression is modulated by ROS in PAECs from newborn piglets. However, ROS-mediated decreases in SNAT1 expression per se do not implicate a reduction in NO production. Although SNAT1 may be critical to l-citrulline-induced increases in NO production, therapies designed to alter SNAT1 expression may not lead to a concordant change in NO production. NEW & NOTEWORTHY Na+-coupled neutral amino acid transporter 1 (SNAT1) modulates nitric oxide (NO) production in piglet pulmonary arterial endothelial cells. Factors that regulate SNAT1 expression in pulmonary arterial endothelial cells are unclear. Here, we show that ROS-reducing strategies inhibit hypoxia-induced increases in SNAT1 expression. l-Citrulline and tetrahydropbiopterin decrease SNAT1 expression but increase NO production. Although SNAT1 is modulated by ROS, changes in SNAT1 expression may not cause a concordant change in NO production.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. GABA signaling sustains fundamental brain functions, from nervous system development to the synchronization of population activity and synaptic plasticity. Despite these pivotal features, molecular determinants underscoring the rapid and cell-autonomous replenishment of the vesicular neurotransmitter GABA and its impact on synaptic plasticity remain elusive. Here, we show that genetic disruption of the glutamine transporter Slc38a1 in mice hampers GABA synthesis, modifies synaptic vesicle morphology in GABAergic presynapses and impairs critical period plasticity. We demonstrate that Slc38a1-mediated glutamine transport regulates vesicular GABA content, induces high-frequency membrane oscillations and shapes cortical processing and plasticity. Taken together, this work shows that Slc38a1 is not merely a transporter accumulating glutamine for metabolic purposes, but a key component regulating several neuronal functions.