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Lung cancer is devastating cancer that ranks as the leading cause of cancer-related death. Long noncoding RNA (lncRNA) opioid growth factor receptor pseudogene 1 (OGFRP1) was recognized as an oncogene in many cancers. However, the molecular mechanism of OGFRP1 in lung cancer is still poorly understood. The expression of target RNAs and genes was detected by quantitative real-time PCR and western blot. The interaction between miR-299-3p and OGFRP1 or solute carrier family 38 member 1 (SLC38A1) was predicted by StarbaseV3.0 and verified by dual-luciferase reporter assay and Pearson's correlation coefficient. Besides, a transplantation model of human lung cancer in nude mice was established to evaluate the role of OGFRP1 in lung cancer. OGFRP1 and SLC38A1 were overexpressed, whereas miR-299-3p was lowly expressed in lung cancer tumors and cells. OGFRP1 knockdown suppressed cell proliferation and facilitated ferroptosis by promoting lipid peroxidation and iron accumulation in lung cancer. Besides, Furthermore, miR-299-3p inhibitor or SLC38A1 overexpression attenuated OGFRP1 depletion-induced suppression on cell proliferation and ferroptosis in lung cancer. Animal experiments indicated that OGFRP1 deficiency restrained tumor growth in vivo by regulating the miR-299-3p/SLC38A1 axis. OGFRP1 regulated cell proliferation and ferroptosis in lung cancer by inhibiting miR-299-3p to enhance SLC38A1 expression, providing a novel therapeutic strategy for lung cancer.

Rapidly proliferating cells such as vascular smooth muscle cells (VSMCs) require metabolic programs to support increased energy and biomass production. Thus, targeting glutamine metabolism by inhibiting glutamine transport could be a promising strategy for vascular disorders such as atherosclerosis, stenosis, and restenosis. V-9302, a competitive antagonist targeting the glutamine transporter, has been investigated in the context of cancer; however, its role in VSMCs is unclear. Here, we examined the effects of blocking glutamine transport in fetal bovine serum (FBS)- or platelet-derived growth factor (PDGF)-stimulated VSMCs using V-9302. We found that V-9302 inhibited mTORC1 activity and mitochondrial respiration, thereby suppressing FBS- or PDGF-stimulated proliferation and migration of VSMCs. Moreover, V-9302 attenuated carotid artery ligation-induced neointima in mice. Collectively, the data suggest that targeting glutamine transport using V-9302 is a promising therapeutic strategy to ameliorate occlusive vascular disease.

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.

Prenatal hypoxia is among leading causes of progressive brain pathologies in postnatal life. This study aimed to analyze the characteristics of the hippocampal glutamatergic system and behavior of rats in early (2 weeks), adult (3 months) and advanced (18 months) postnatal ontogenesis after exposure to prenatal severe hypoxia (PSH, 180 Torr, 5% O2, 3 h) during the critical period in the formation of the hippocampus (days 14-16 of gestation). We have shown an age-dependent progressive decrease in the hippocampal glutamate levels, a decrease of the neuronal cell number in the CA1 hippocampal region, as well as impairment of spatial long-term memory in the Morris water navigation task. The gradual decrease of glutamate was accompanied by decreased expression of the genes that mediate glutamate metabolism and recycling in the hippocampus. That deficiency apparently correlated with an increase of the metabotropic glutamate receptor type 1 (mGluR1) and synaptophysin expression. Generation of the lipid peroxidation products in the hippocampus of adult rats subjected to prenatal severe hypoxia (PSH rats) was not increased compared to the control animals when tested in a model of glutamate excitotoxicity induced by severe hypoxia. This demonstrates that excessive glutamate sensitivity in PSH rats does not compensate for glutamate deficiency. Our results show a significant contribution of the glutamate system dysfunction to age-associated decrease of this mediator, cognitive decline, and early neuronal loss in PSH rats.

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.

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.

Glioblastoma multiforme (GBM) is a primary brain tumor with devastating prognosis. Although the OThe function of Down syndrome critical region protein 3 (DSCR3) on MGMT-deficient GBM was investigated in vitro and in an orthotopic brain tumor model in mice. Purification of plasma membrane proteins by membrane-cytoplasmic separation and subsequent label free-based quantitative proteomics were used to identified potential protein partners for DSCR3. Immunofluorescence was performed to show the reverse transport of solute carrier family 38 member 1 (SLC38A1) mediated by DSCR3.DSCR3 is upregulated in MGMT-deficient GBM cells during TMZ treatment. Both DSCR3 and SLC38A1 were highly expressed in recurrent GBM patients. Silencing DSCR3 or SLC38A1 expression can increase TMZ sensitivity in MGMT-deficient GBM cells. Combination of proteomics and in vitro experiments show that DSCR3 directly binds internalized SLC38A1 to mediate its sorting into recycling pathway, which maintains the abundance on plasma membrane and enhances uptake of glutamine in MGMT-deficient GBM cells.DSCR3 is a crucial regulator of acquired TMZ resistance in MGMT-deficient GBM. The DSCR3-dependent recycling of SLC38A1 maintains its abundance on plasma membrane, leading to tumor progression and acquired TMZ resistance in MGMT-deficient GBM.

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.

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.

INTRODUCTION As an exogenous food contaminant, dietary oxidized lipid impairs growth and development, and triggers chronic diseases in humans or animals. This study explores the effects of soybean oil with different oxidative degree on the placental injury of gestational rats. METHODS AND RESULTS Thirty-two female adult rats are randomly assigned to four groups. The control group is fed the purified diet with fresh soybean oil (FSO), and the treatment groups are fed purified diets with lipid content replaced by oxidized soybean oil (OSO) at 200, 400, and 800 mEqO2 kg-1 from conception until delivery. On day 20 of gestation, OSO decreased placental and embryonic weights as the oxidative degree increased linearly and quadratically. The expression of Bax showed a linear increase, and Bcl-2 decreased as the oxidative degree increased. The expression of Fosl1 and Esx1 is linearly and quadratically decreased in OSO-treated groups than FSO group. OSO decreased the level of IL-10 but increased expression of IL-1β in placenta and plasma. OSO remarkably upregulates levels of Fatp1 and Glut1 and decreases expression of Snat2 and Glut3. CONCLUSION OSO aggravates placental injury by modulating nutrient transporters and apoptosis-related genes, impedes placental growth and development, and ultimately leads to the decrease of fetal weight.

Metastasis is the leading cause of colorectal cancer (CRC)-induced death. However, the underlying molecular mechanisms of CRC metastasis are poorly understood. Metabolic reprogramming is an intrinsic feature of cancer, which have complicated effects on cancer metastasis. Here, we find that a novel metastasis-related protein, cell migration-inducing and hyaluronan-binding protein (CEMIP), can act as a novel adaptor protein of O-GlcNAc transferase (OGT) to promote CRC metastasis through glutamine metabolic reprogramming. Mechanistically, CEMIP interacts with OGT and β-catenin, which leads to elevated O-GlcNAcylation of β-catenin and enhanced β-catenin nuclear translocation from cytomembrane. Furthermore, accumulated β-catenin in nucleus enhances the transcription of CEMIP to reciprocally regulate β-catenin and contributes to over-expression of glutaminase 1 and glutamine transporters (SLC1A5 and SLC38A2). Combinational inhibition of CEMIP and glutamine metabolism could dramatically attenuate the metastasis of CRC in vivo. Collectively, this study reveals the importance of glutamine metabolic reprogramming in CEMIP-induced CRC metastasis, indicating the great potential of CEMIP and glutamine metabolism for CRC metastasis prevention.

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.

We propose an original method for controlling BP by administration of Si~ODN nanocomposites containing antisense oligonucleotides fixed on silicon-organic nanoparticles. ODN in nanocomposites are targeted to mRNA of the genes encoding angiotensin-converting enzyme (ACE1) and type 1 angiotensin-II receptor (AT1A). The experiments were performed on hypertensive ISIAH rats, a genetic model of hypertension. Single inhalation or intraperitoneal administration of the nanocomposites targeted to ACE1 mRNA or ATA1 mRNA, respectively, led to a pronounced decrease (by ~30 mm Hg) in systolic BP in ISIAH rats over a week. The use of scrambled ODN in the nanocomposites had no effect. A decrease in the expression of ACE1 and AT1A genes under the effect of the corresponding antisense ODN was demonstrated, which attested to directed effect of the test preparations.

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.

Substance use disorders (SUD) often co-occur with other mental disorders such as major depression (MD). Our previous findings revealed sex-dependent changes in extracellular levels of glutamate (Glu) and glutamine (Gln) in the nucleus accumbens (NAc) in Long–Evans rats that were exposed to 21 days of chronic social defeat stress (CSDS), which models MD. The current study investigated the role of a Gln transporter called sodium-coupled neutral amino acid transporter subtype 1/2 (SNAT 1/2), phosphate-activated glutaminase (PAG), and astrocytic glutamate transporter-1 (GLT-1) on CSDS animals exposed to cocaine. Before cocaine exposure, CSDS males already showed decreased levels of SNAT 1/2 in the NAc and prefrontal cortex (PFC) compared to non-CSDS controls. The reduction in SNAT 1/2 levels was associated with an increase in Gln localization in the mitochondrial outer membrane in accumbal glutamatergic nerve terminals projecting from the PFC. CSDS females showed increased GLT-1 levels in the NAc and PFC compared to non-CSDS controls. Both acute and repeated cocaine exposure attenuated locomotor responses in CSDS males but increased those in CSDS females. Cocaine reduced SNAT 1/2 levels in the NAc but increased them in the PFC in CSDS males. Additionally, both PAG and GLT-1 levels were increased in the PFC in CSDS males. On the other hand, cocaine reduced SNAT 1/2 and GLT-1 levels in the NAc and PFC in CSDS females. Our results show that CSDS altered locomotor responses upon cocaine exposure in a sex-dependent manner that may be mediated by molecules associated with the Glu-Gln transfer.

Methionine (Met) plays a critical regulatory role in milk production, however, the molecular mechanism of action of Met is largely unknown. This study therefore aimed to investigate the influence of Met on milk synthesis in and proliferation of bovine mammary epithelial cells (BMECs) and explore the underlying mechanism. BMECs cultured in fetal bovine serum (FBS) free Dulbecco's modified eagle's medium (DMEM)/F-12 medium were treated with Met (0, 0.3, 0.6, 0.9, and 1.2 mM). Results showed that Met (0.6 mM) significantly increased milk protein and fat synthesis and cell proliferation. Met stimulation also increased mTOR phosphorylation and protein expression of SREBP-1c and Cyclin D1. Gene function study approaches further revealed that SNAT2 is a key regulator of these signaling pathways. PI3K inhibition experiments demonstrated that SNAT2 stimulates these pathways through regulating PI3K activity, and SNAT2 inhibition experiments further revealed that SNAT2 is required for Met to activate PI3K. Furthermore, immunofluorescence observation detected that Met stimulates SNAT2 cytoplasmic expression. Collectively, these findings demonstrate that Met positively regulates milk protein and fat synthesis and cell proliferation via the SNAT2-PI3K signaling pathway in BMECs.

Background Circular RNAs (circRNAs) have emerged as vital regulators in human cancers, including colorectal cancer (CRC). In this study, we aimed to explore the roles of circRUNX1 in CRC. Methods The levels of circRUNX1, RUNX1 mRNA, solute carrier family 38 member 1 (SLC38A1) mRNA and microRNA-485-5p (miR-485-5p) were determined by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The protein level of SLC38A1 was measured by Western blot assay. Cell colony formation, migration, invasion and apoptosis were assessed by colony formation assay, wound-healing assay, Transwell assay and flow cytometry analysis, respectively. The interaction between miR-485-5p and circRUNX1 or SLC38A1 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The levels of extracellular glutamine, intracellular glutamate and α-ketoglutarate (α-KG) were measured with specific kits. The functional role of circRUNX1 in CRC development in vivo was explored by murine xenograft model assay. Results CircRUNX1 was upregulated in CRC tissues and cells compared with normal tissues and cells. CircRUNX1 deficiency restrained CRC cell colony formation, migration, invasion and glutaminolysis and induced apoptosis in vitro as well as blocked tumour growth in vivo. CircRUNX1 directly sponged miR-485-5p, which negatively modulated SLC38A1 expression in CRC cells. The effects of circRUNX1 knockdown on CRC cell colony formation, migration, invasion, apoptosis and glutaminolysis were reversed by miR-485-5p inhibition. Moreover, miR-485-5p overexpression repressed the malignant behaviours of CRC cells, with SLC38A1 elevation overturned the impacts. Conclusion CircRUNX1 promoted CRC cell growth, metastasis and glutamine metabolism and repressed apoptosis by elevating SLC38A1 through sponging miR-485-5p, which might provide a novel target for CRC treatment.

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.

Perturbations in the glutamate-glutamine cycle and glutamate release from presynaptic terminals have been involved in the development of cognitive deficits in Alzheimer's disease (AD) patients and mouse models. Glutamate transporter-1 (GLT-1) removes glutamate from the synaptic cleft and transports it into astrocytes, where it is used as substrate for the glutamate-glutamine cycle. Ceftriaxone has been reported to improve cognitive deficits in AD mice by increasing GLT-1 expression, glutamate transformation to glutamine, and glutamine efflux from astrocytes. However, the impact of ceftriaxone on glutamine metabolism in neurons is unknown. The present study aimed to investigate whether ceftriaxone regulated the production and vesicular assembly of glutamate in the presynaptic terminals of neurons and to determine GLT-1 involvement in this process. We used the amyloid precursor protein (APP)/presenilin-1 (PS1) AD mouse model and GLT-1 knockdown APP/PS1 (GLT-1+/-/APP/PS1) mice. The expression levels of sodium-coupled neutral amino-acid transporter 1 (SNAT1) and vesicular glutamate transporters 1 and 2 (VGLUT1/2) were analyzed by immunofluorescence and immunohistochemistry staining as well as by Western blotting. Glutaminase activity was assayed by fluorometry. Ceftriaxone treatment significantly increased SNAT1 expression and glutaminase activity in neurons in APP/PS1 mice. Similarly, VGLUT1/2 levels were increased in the presynaptic terminals of APP/PS1 mice treated with ceftriaxone. The deletion of one GLT-1 allele in APP/PS1 mice prevented the ceftriaxone-induced upregulation of SNAT1 and VGLUT1/2 expression, indicating that GLT-1 played an important role in ceftriaxone effect. Based on the role of SNAT1, glutaminase, and VGLUT1/2 in the glutamate-glutamine cycle in neurons, the present results suggested that ceftriaxone improved the production and vesicular assembly of glutamate as a neurotransmitter in presynaptic terminals by acting on GLT-1 in APP/PS1 mice.

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.

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 (

Background Glutamine aminoacid regulates insulin exocytosis from pancreatic β-cells. Liraglutide is a glucagon-like peptide-1 (GLP-1) analogue that has fascinated function in inhibiting β-cell apoptosis and preserving pancreatic β-cell mass. The present study investigated the benefit of adding glutamine to a regimen of liraglutide in diabetic rats focusing on their role in increasing insulin production and upregulation of the expression of sodium-dependent neutral aminoacid transporter-2 (SNAT2). Methods In the present study, diabetes mellitus was induced in rats using streptozotocin (STZ, 50 mg/kg, ip). Male rats were allocated into 5 groups, (i) vehicle group, (ii) STZ-diabetic rats, (iii) STZ-diabetic rats treated with liraglutide (150 μg/kg, sc), (iv) STZ-diabetic rats treated with glutamine (po) and (v) STZ-diabetic rats treated with a combination of liraglutide and glutamine for four weeks. After finishing the therapeutic courses, the fasting blood glucose value was determined and rats were sacrificed. Pancreases were used for quantification of mRNA expression for SNAT2. Paraffin fixed samples were used for histologic staining and immunohistochemistry for insulin and apoptosis markers (activated caspase-3, BCL2 and BAX). Results Treatment with liraglutide and/or glutamine enhanced insulin production and hence glycemic control in diabetic male rats with favorable effects on apoptosis markers. Treatment with glutamine and its combination with liraglutide significantly increased pancreatic expression of SNAT2 by approximately 30–35 folds. Conclusion Addition of glutamine to liraglutide regimen enhances the glycemic control and may have utility in clinical settings.

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.

Branched chain amino acids (BCAAs) have protective effects against muscle atrophy. Although plasma BCAA concentrations are higher in patients with diabetes than in healthy subjects, diabetes is related to sarcopenia. We hypothesized that high glucose concentration reduces the quantity of BCAA transporters, and consequently, the effects of BCAAs are diminished despite their high levels. We examined whether glucose reduces the expression of L-type amino acid transporter 1 (LAT1), which transports neutral amino acids, including BCAA, in C2C12 myocytes. Glucose reduced LAT1 mRNA level by 80% in the C2C12 cells, compared with that in the glucose-free control cells. Regarding LAT1-related transporters, glucose also reduced the level of sodium-dependent neutral amino acid transporter 2 mRNA, but not that of 4F2 heavy chain. Although fructose reduced LAT1 mRNA levels, 2-deoxyglucose exhibited low effectiveness in reducing LAT1 mRNA level; galactose and mannitol had no effect. These results suggest a relationship between ATP produced during glycolysis and LAT1 mRNA levels. In fact, the AMP-activated protein kinase (AMPK) inhibitor dorsomorphin reduced LAT1 mRNA levels in the absence of glucose, whereas the AMPK activator 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside increased LAT1 mRNA levels even in the presence of glucose. Consistent with these findings, glucose reduced the levels of phospho-AMPKα (Thr172) compared with that in the glucose-free control. These findings indicate that glucose inactivates AMPK, leading to a reduction in LAT1 mRNA levels in the C2C12 cells. This glucose-induced reduction in LAT1 expression may explain the unresponsiveness to BCAA in the patients with diabetes.

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.

Foot-and-mouth disease (FMD) vaccines with improved stability and less reliant on a cold-chain are needed to improve the longevity of immune responses elicited in animals. This is especially so for serotypes O and SAT2 which are unstable in mildly acidic pH conditions or at elevated temperatures leading to dissociation of the capsid (146S particle) and loss of immunogenicity. Previously, stabilised SAT2 viruses were generated by reverse genetic approaches and assessed in vitro and in vivo with a guinea pig trial. Here we investigated the efficacy and comparative immunological responses of two thermostable and wild-type SAT2 vaccines over 5. months followed by challenge. We assessed humoral immune responses elicited in cattle in terms of total and neutralizing antibodies and IgG1/2 isotyping; and cell-mediated responses of IFN-γ as in vitro markers of protection. Whilst there were significant differences in total and neutralizing antibodies for the vSAT2-93H group compared to other vaccinated groups after the first vaccination, there were no significant differences after the second immunization. Following intra-dermolingual challenge all vaccinated groups were fully protected as determined by the absence of generalized lesions. These results provide proof that two vaccine doses, consisting of SAT2 antigen combined with ISA206B adjuvant, administered 4-6 weeks apart were able to protect animals up to 5. months pv. Additionally, vSAT2-93Y had significantly higher levels of IFN-γ after challenge and had a lower clinical score indicative of better protection compared to other vaccinated groups and the importance of cell mediated responses and antigen stability in protection. Fil: Scott, Katherine A.. Agricultural Research Council; Sudáfrica. University of Pretoria; Sudáfrica Fil: Rathogwa, N. M.. University of Pretoria; Sudáfrica. Agricultural Research Council; Sudáfrica Fil: Capozzo, Alejandra Victoria. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Maree, Francois F,. Agricultural Research Council; Sudáfrica. University of Pretoria; Sudáfrica

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.

The green tea amino acid theanine is abundant in green tea rather than black and oolong teas, which are all made of the identical tea plant “Chanoki” (Camellia sinensis). Theanine has a molecular structure close to glutamine (GLN) compared to glutamic acid (Glu), in terms of the absence of a free carboxylic acid moiety from the gamma carbon position. Theanine efficiently inhibits [3H]GLN uptake without affecting [3H]Glu uptake in rat brain synaptosomes. In contrast to GLN, however, theanine markedly stimulates the abilities to replicate and to commit to a neuronal lineage following prolonged exposure in cultured neural progenitor cells (NPCs) prepared from embryonic and adult rodent brains. Upregulation of transcript expression is found for one of the GLN transporter isoforms, Slc38a1, besides the promotion of both proliferation and neuronal commitment along with acceleration of the phosphorylation of mechanistic target of rapamycin (mTOR) and relevant downstream proteins, in murine NPCs cultured with theanine. Stable overexpression of Slc38a1 similarly facilitates both cellular replication and neuronal commitment in pluripotent embryonic carcinoma P19 cells. In P19 cells with stable overexpression of Slc38a1, marked phosphorylation is seen for mTOR and downstream proteins in a manner insensitive to further additional phosphorylation by theanine. Taken together, theanine would exhibit a novel pharmacological property to up-regulate Slc38a1 expression for activation of the intracellular mTOR signaling pathway required for neurogenesis after sustained exposure in undifferentiated NPCs in the brain. In this review, a novel neurogenic property of the green tea amino acid theanine is summarized for embryonic and adult neurogenesis with a focus on the endogenous amino acid GLN on the basis of our accumulating evidence to date.

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'.

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.

Zearalenone is an estrogenic mycotoxin produced by a variety of Fusarium fungi. There is evidence that exposure to zearalenone can cause intrauterine growth restriction, but little is known about the mechanism in the rat placenta caused by zearalenone. From gestational day 14–21, female Sprague Dawley rats (60 days old) were gavaged with zearalenone (0, 2.5, 5, 10, and 20 mg/kg/day body weight). Zearalenone dose-dependently reduced serum LH and FSH levels of dams at ≥ 5 mg/kg. RNA-seq and qPCR showed that zearalenone significantly down-regulated Slc38a1 expression at 2.5 mg/kg, Echs1 and Pc at 10 mg/kg, as well as Slc1a5, Cd36, Ldlr, Hadhb, and Cyp17a1 expression at a dose of 20 mg/kg, while it up-regulated the expression of Notch signal (Dvl1 and Jag 1). After zearalenone treatment, their proteins showed a similar trend. Zearalenone reduced the phosphorylation of AKT1, ERK1/2, and mTOR at 5 mg/kg or higher and 4EBP1 at 5 mg/kg. Zearalenone also increased BECLIN1, LC3B, and p62 levels and elevated BAX/BCL2 and CASP3/PROCASP3 ratios. In conclusion, zearalenone disrupts placental function such as reduction of nutrient transport and lipid metabolism possibly via AKT1/ERK1/2/mTOR-mediated autophagy and apoptosis.

The developmental outcomes of preimplantation mammalian embryos are regulated directly by the surrounding microenvironment, and inappropriate concentrations of amino acids, or the loss of amino acid-sensing mechanisms, can be detrimental and impact further development. A specific role for l-proline in the differentiation of embryonic stem (ES) cells, a cell population derived from the blastocyst, has been shown in culture. l-proline acts as a signalling molecule, exerting its effects through cell uptake and subsequent metabolism. Uptake in ES cells occurs predominantly through the sodium-coupled neutral amino acid transporter 2, Slc38a2 (SNAT2). Dynamic expression of amino acid transporters has been shown in the early mammalian embryo, reflecting functional roles for amino acids in embryogenesis. The expression of SNAT2 and family member Slc38a1 (SNAT1) was determined in mouse embryos from the 2-cell stage through to the early post-implantation pre-gastrulation embryo. Key changes in expression were validated in cell culture models of development. Both transporters showed temporal dynamic expression patterns and changes in intracellular localisation as differentiation progressed. Changes in transporter expression likely reflect different amino acid requirements during development. Findings include the differential expression of SNAT1 in the inner and outer cells of the compacted morula and nuclear localisation of SNAT2 in the trophectoderm and placental lineages. Furthermore, SNAT2 expression was up-regulated in the epiblast prior to primitive ectoderm formation, an expression pattern consistent with a role for the transporter in later developmental decisions within the pluripotent lineage. We propose that the differential expression of SNAT2 in the epiblast provides evidence for an l-proline-mediated mechanism contributing to the regulation of embryonic development.

L-glutamine (Gln) is the most abundant amino acid in plasma and cerebrospinal fluid and a precursor for the main central nervous system excitatory (L-glutamate) and inhibitory (γ-aminobutyric acid (GABA)) neurotransmitters. Concentrations of Gln and 13 other brain interstitial fluid amino acids were measured in awake, freely moving mice by hippocampal microdialysis using an extrapolation to zero flow rate method. Interstitial fluid levels for all amino acids including Gln were ∼5–10 times lower than in cerebrospinal fluid. Although the large increase in plasma Gln by intraperitoneal (IP) injection of 15N2-labeled Gln (hGln) did not increase total interstitial fluid Gln, low levels of hGln were detected in microdialysis samples. Competitive inhibition of system A (SLC38A1&2; SNAT1&2) or system L (SLC7A5&8; LAT1&2) transporters in brain by perfusion with α-(methylamino)-isobutyric acid (MeAIB) or 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) respectively, was tested. The data showed a significantly greater increase in interstitial fluid Gln upon BCH than MeAIB treatment. Furthermore, brain BCH perfusion also strongly increased the influx of hGln into interstitial fluid following IP injection consistent with transstimulation of LAT1-mediated transendothelial transport. Taken together, the data support the independent homeostatic regulation of amino acids in interstitial fluid vs. cerebrospinal fluid and the role of the blood–brain barrier expressed SLC7A5/LAT1 as a key interstitial fluid gatekeeper.

Neonatal pigs are used as a model to study and optimize the clinical treatment of infants who are unable to maintain oral feeding. Using this model, we have shown previously that pulsatile administration of leucine during continuous feeding over 24 h via orogastric tube enhanced protein synthesis in skeletal muscle compared with continuous feeding alone. To determine the long-term effects of leucine pulses, neonatal piglets ( n = 11–12/group) were continuously fed formula via orogastric tube for 21 days, with an additional parenteral infusion of either leucine (CON + LEU; 800 μmol·kg−1·h−1) or alanine (CON + ALA) for 1 h every 4 h. The results show that body and muscle weights and lean gain were ∼25% greater, and fat gain was 48% lower in CON + LEU than CON + ALA; weights of other tissues were unaffected by treatment. Fractional protein synthesis rates in longissimus dorsi, gastrocnemius, and soleus muscles were ∼30% higher in CON + LEU compared with CON + ALA and were associated with decreased Deptor abundance and increased mTORC1, mTORC2, 4E-BP1, and S6K1 phosphorylation, SNAT2 abundance, and association of eIF4E with eIF4G and RagC with mTOR. There were no treatment effects on PKB, eIF2α, eEF2, or PRAS40 phosphorylation, Rheb, SLC38A9, v-ATPase, LAMTOR1, LAMTOR2, RagA, RagC, and LAT1 abundance, the proportion of polysomes to nonpolysomes, or the proportion of mRNAs encoding rpS4 or rpS8 associated with polysomes. Our results demonstrate that pulsatile delivery of a leucine supplement during 21 days of continuous enteral feeding enhances lean growth by stimulating the mTORC1-dependent translation initiation pathway, leading to protein synthesis in skeletal muscle of neonates.

The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2−/− embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development.