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Glutamine (Gln) is essential for cancer progression, however, few studies have been conducted to investigate the roles of Gln transporters in gastric cancer stem cells (CSCs). This work aims to explore the roles of Gln transporters in gastric cancer cell stemness. We collected spheres formed by gastric cancer (GC) cells through a 3-dimensional (3D) semisolid culture system which has been shown to hold CSC-like traits. Lentivirus package was used to construct GC cells with SNAT2 overexpression. Analysis of sphere-formation, stemness marker expression, ALDH activity were used to detect the effects of Gln transporters on GC cell stemness. Determination of reactive oxygen species (ROS) and Gln consumption combined with the methods analyzing cell stemness were performed to explore the underlying mechanisms. Gln consumption was upregulated in GC spheres compared to the parental GC cells. The Gln transporter SNAT2 was highly expressed in GC spheres compared to that in the parental GC cells. SNAT2 overexpression significantly increased the Gln consumption in GC cells and increased the expression of stemness markers, sphere-formation ability and ALDH activity. Notably, SNAT2-mediated promoting effects on GC cell stemness were rescued by Gln deprivation. What’s more, high expression of SNAT2 was associated with a poor GC patient survival through different online datasets. SNAT2 can promote the stemness of GC cells in a Gln-dependent manner.

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.

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

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.

Fetal growth and development are dependent on placental nutrient transport. The syncytiotrophoblast (ST) and its two polarized plasma membranes, the maternal-facing microvillous membrane (MVM) and fetal-facing basal membrane (BM), represent the primary barrier in the human placenta, controlling transplacental transfer of small solutes. MVM and BM nutrient transporter expression and activity are increased in obese mothers delivering large babies. However, placental nutrient transporter expression and activity in early gestation in normal and obese women are largely unknown. Placentas from normal BMI and obese women at 6–24 weeks of gestation, and term placentas from normal BMI women, were collected and ST plasma membranes isolated. The activity and protein expression of amino acid, glucose, and fatty acid transporters was assessed. No significant differences were observed in placental nutrient transporter protein expression between normal BMI and obese women in early pregnancy. In the MVM, system A amino acid activity (p = 0.02), SNAT2 (p

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.

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.

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.

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.

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.

Cancer cells have dramatically increased demands for energy as well as biosynthetic precursors to fuel their restless growth. Enhanced glutaminolysis is a hallmark of cancer metabolism which fulfills these needs. Two glutamine transporters, SLC1A5 and SLC38A2, have been previously reported to promote glutaminolysis in cancer with controversial perspectives. In this study, we harnessed the proximity labeling reaction to map the protein interactome using mass spectrometry-based proteomics and discovered a potential protein-protein interaction between SLC1A5 and SLC38A2. The SLC1A5/SLC38A2 interaction was further confirmed by bimolecular fluorescence complementation assay. We further investigated the metabolic influence of SLC1A5 and SLC38A2 overexpression in human cells, respectively, and found that only SLC38A2, but not SLC1A5, resulted in a cancer-like metabolic profile, where the intracellular concentrations of essential amino acids and lactate were significantly increased as quantified by nuclear magnetic resonance spectroscopy. Finally, we analyzed the 5-year survival rates in a large pan-cancer cohort and found that the SLC1A5hi /SLC38A2lo group did not relate to a poor survival rate, whereas the SLC1A5lo /SLC38A2hi group significantly aggravated the lethality. Intriguingly, the SLC1A5hi /SLC38A2hi group resulted in an even worse prognosis, suggesting a cooperative effect between SLC1A5 and SCL38A2. Our data suggest that SLC38A2 plays a dominant role in reprogramming the cancer-like metabolism and promoting the cancer progression, whereas SLC1A5 may augment this effect when co-overexpressed with SLC38A2. We propose a model to explain the relationship between SLC1A5, SLC38A2 and SCL7A5, and discuss their impact on glutaminolysis and mTOR signaling.

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.

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.

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/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.

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.

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.

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.

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.

Introduction Inflammation and underlying low-grade maternal infection can impair insulin signalling and upregulate nutrient transport in the placenta which contribute to fetal overgrowth associated with GDM and/or obese pregnancies. There are, however, no studies on the role of infection on placental nutrient transport in pregnancies complicated by GDM and/or obesity. Thus, the aims of this study were to determine the effect of the bacterial product lipopolysaccharide (LPS) or the viral dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) on the insulin signalling pathway and amino acid transport in primary human trophoblast cells. Methods Human primary villous trophoblast cells were treated with LPS or poly(I:C). Protein expression of insulin signalling pathway proteins, insulin receptor (IR)-β, insulin receptor substrate (IRS)-1 and protein kinase B (also known as Akt), and phosphatidylinositol-4,5-bisphosphate 3-kinase p85α subunit (PI3K-p85α) protein were assessed by Western blotting. Glucose and amino acid uptake were assessed by radiolabelled assay. Western blotting and qRT-PCR were used to determine amino acid transporter protein and mRNA expression, respectively. Results LPS and poly(I:C) significantly decreased phosphorylation of IR-β, IRS-1, Akt, total PI3K-p85α protein expression and glucose uptake. LPS and poly(I:C) also significantly increased expression of System A amino acid transporters SNAT1 and SNAT2, and System A-mediated uptake of amino acids. Discussion LPS and poly(I:C) induces insulin resistance and increases amino acid uptake in human primary trophoblast cells. This suggests that the presence of low-grade maternal infection can contribute to excess placental nutrient availability and promote fetal overgrowth in pregnancies complicated by GDM and/or obesity.

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.

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'.

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.

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.

Many cancer cells depend on glutamine as they use the glutaminolysis pathway to generate building blocks and energy for anabolic purposes. As a result, glutamine transporters are essential for cancer growth and are potential targets for cancer chemotherapy with ASCT2 (SLC1A5) being investigated most intensively. Here we show that HeLa epithelial cervical cancer cells and 143B osteosarcoma cells express a set of glutamine transporters including SNAT1 (SLC38A1), SNAT2 (SLC38A2), SNAT4 (SLC38A4), LAT1 (SLC7A5), and ASCT2 (SLC1A5). Net glutamine uptake did not depend on ASCT2 but required expression of SNAT1 and SNAT2. Deletion of ASCT2 did not reduce cell growth but caused an amino acid starvation response and up-regulation of SNAT1 to replace ASCT2 functionally. Silencing of GCN2 in the ASCT2(−/−) background reduced cell growth, showing that a combined targeted approach would inhibit growth of glutamine-dependent cancer cells.

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.

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.

Recent studies suggest that 2,4-DABA, a neurotoxic excitatory amino acid present in virtually all environments, but predominantly in aquatic ecosystems may be a risk factor for development of neurodegenerative diseases in animals and humans. Despite its neurotoxicity and potential environmental importance, mechanisms underlying the excitatory and putative excitotoxic action of 2,4-DABA in neurons are still unexplored. We previously reported on extensive two-stage membrane depolarization and functional disturbances in leech Retzius neurons induced by 2,4-DABA. Current study presents the first detailed look into the electrophysiological processes leading to this depolarization. Intracellular recordings were performed on Retzius neurons of the leech Haemopis sanguisuga using glass microelectrodes and input membrane resistance (IMR) was measured by injecting hyperpolarizing current pulses through these electrodes. Results show that the excitatory effect 2,4-DABA elicits on neurons’ membrane potential is dependent on sodium ions. Depolarizing effect of 5·10−3 mol/L 2,4-DABA in sodium-free solution was significantly diminished by 91% reducing it to 3.26 ± 0.62 mV and its two-stage nature was abrogated. In addition to being sodium-dependent, the depolarization of membrane potential induced by this amino acid is coupled with an increase of membrane permeability, as 2,4-DABA decreases IMR by 8.27 ± 1.47 MΩ (67.60%). Since present results highlight the role of sodium ions, we investigated the role of two putative sodium-dependent mechanisms in 2,4-DABA-induced excitatory effect – activation of ionotropic glutamate receptors and the electrogenic transporter for neutral amino acids. Excitatory effect of 5·10−3 mol/L 2,4-DABA was partially blocked by 10-5 mol/L 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) a non-NMDA receptor antagonist as the first stage of membrane depolarization was significantly reduced by 2.59 ± 0.98 mV (40%), whilst second stage remained unaltered. Moreover, involvement of the sodium-dependent transport system for neutral amino acids was investigated by equimolar co-application of 5·10−3 mol/L 2,4-DABA and L-alanine, a competitive inhibitor of this transporter. Although L-alanine exhibited no effect on the first stage of membrane depolarization elicited by 2,4-DABA, it substantially reduced the second stage (the overall membrane depolarization) from 39.63 ± 2.22 mV to 16.28 ± 2.58 mV, by 58.92%. We therefore propose that the electrophysiological effect of 2,4-DABA on Retzius neurons is mediated by two distinct mechanisms, i.e. by activation of ionotropic glutamate receptor that initiates the first stage of membrane depolarization followed by the stimulation of an electrogenic sodium-dependent neutral amino acid transporter, leading to additional influx of positive charge into the cell and the second stage of depolarization.

Amino acid transporters play an important role in cell growth and metabolism. MeAIB, a transporter-selective substrate, often represses the adaptive regulation of sodium-coupled neutral amino acid transporter 2 (SNAT2), which may act as a receptor and regulate cellular amino acid contents, therefore modulating cellular downstream signaling. The aim of this study was to investigate the effects of MeAIB to SNAT2 on cell proliferation, protein turnover, and the mammalian target of rapamycin (mTOR) signaling pathway in porcine enterocytes. Intestinal porcine epithelial cells (IPEC)-J2 cells were cultured in a high-glucose Dulbecco’s modified Eagle’s (DMEM-H) medium with 0 or 5 mmoL/L System A amino acid analogue (MeAIB) for 48 h. Cells were collected for analysis of proliferation, cell cycle, protein synthesis and degradation, intracellular free amino acids, and the expression of key genes involved in the mTOR signaling pathway. The results showed that SNAT2 inhibition by MeAIB depleted intracellular concentrations of not only SNAT2 amino acid substrates but also of indispensable amino acids (methionine and leucine), and suppressed cell proliferation and impaired protein synthesis. MeAIB inhibited mTOR phosphorylation, which might be involved in three translation regulators, EIF4EBP1, IGFBP3, and DDIT4 from PCR array analysis of the 84 genes related to the mTOR signaling pathway. These results suggest that SNAT2 inhibition treated with MeAIB plays an important role in regulating protein synthesis and mTOR signaling, and provide some information to further clarify its roles in the absorption of amino acids and signal transduction in the porcine small intestine.

Sodium-coupled neutral amino acid transporter 2 (SNAT2) is a subtype of the amino acid transport system A that is widely expressed in mammalian tissues. It plays critical roles in glutamic acid-glutamine circulation, liver gluconeogenesis and other biological pathway. However, the topology of the SNAT2 amino acid transporter is unknown. Here we identified the topological structure of SNAT2 using bioinformatics analysis, Methoxy-polyethylene glycol maleimide (mPEG-Mal) chemical modification, protease cleavage assays, immunofluorescence and examination of glycosylation. Our results show that SNAT2 contains 11 transmembrane domains (TMDs) with an intracellular N terminus and an extracellular C terminus. Three N-glycosylation sites were verified at the largest extracellular loop. This model is consistent with the previous model of SNAT2 with the exception of a difference in number of glycosylation sites. This is the first time to confirm the SNAT2 membrane topology using experimental methods. Our study on SNAT2 topology provides valuable structural information of one of the solute carrier family 38 (SLC38) members.

Inhibition of placental mechanistic target of rapamycin (mTOR) signalling, which activates NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2) ubiquitin ligase leading to increased sodium-coupled neutral amino acid transporter 2 (SNAT-2) ubiquitination and removal from the syncytiotrophoblast plasma membrane may constitute a key mechanism underlying decreased placental amino acid transport in human IUGR., Placental amino acid transport is decreased in intrauterine growth restriction (IUGR); however, the underlying mechanisms remain largely unknown. We have shown that mechanistic target of rapamycin (mTOR) signalling regulates system A amino acid transport by modulating the ubiquitination and plasma membrane trafficking of sodium-coupled neutral amino acid transporter 2 (SNAT-2) in cultured primary human trophoblast cells. We hypothesize that IUGR is associated with (1) inhibition of placental mTORC1 and mTORC2 signalling pathways, (2) increased amino acid transporter ubiquitination in placental homogenates and (3) decreased protein expression of SNAT-2 in the syncytiotrophoblast microvillous plasma membrane (MVM). To test this hypothesis, we collected placental tissue and isolated MVM from women with pregnancies complicated by IUGR (n=25) and gestational age-matched women with appropriately grown control infants (n=19, birth weights between the twenty-fifth to seventy-fifth percentiles). The activity of mTORC1 and mTORC2 was decreased whereas the protein expression of the ubiquitin ligase NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2; +72%, P

Hepatic encephalopathy (HE) is a neuropsychiatric disorder that occurs due to acute and chronic liver diseases, the hallmark of which is the increased levels of ammonia and subsequent alterations in glutamine synthesis, i.e. conditions associated with the pathophysiology of HE. Under physiological conditions, glutamine is fundamental for replenishment of the neurotransmitter pools of glutamate and GABA. The different isoforms of glutamine transporters play an important role in the transfer of this amino acid between astrocytes and neurons. A disturbance in the GABA biosynthetic pathways has been described in bile duct ligated (BDL) rats, a well characterized model of chronic HE. Considering that glutamine is important for GABA biosynthesis, altered glutamine transport and the subsequent glutamate/GABA-glutamine cycle efficacy might influence these pathways. Given this potential outcome, the aim of the present study was to investigate whether the expression of the glutamine transporters SAT1, SAT2, SN1 and SN2 would be affected in chronic HE. We verified that mRNA expression of the neuronal glutamine transporters SAT1 and SAT2 was found unaltered in the cerebral cortex of BDL rats. Similarly, no changes were found in the mRNA level for the astrocytic transporter SN1, whereas the gene expression of SN2 was increased by two-fold in animals with chronic HE. However, SN2 protein immuno-reactivity did not correspond with the increase in gene transcription since it remained unaltered. These data indicate that the expression of the glutamine transporter isoforms is unchanged during chronic HE, and thus likely not to participate in the pathological mechanisms related to the imbalance in the GABAergic neurotransmitter system observed in this neurologic condition.

The sodium coupled neutral amino acid transporter 2 (SNAT2/SAT2/ATA2) is expressed in the mammary gland (MG) and plays an important role in the uptake of alanine and glutamine which are the most abundant amino acids transported into this tissue during lactation. Thus, the aim of this study was to assess the amount and localization of SNAT2 before delivery and during lactation in rat MG, and to evaluate whether prolactin and the dietary protein/carbohydrate ratio might influence SNAT2 expression in the MG, liver and adipose tissue during lactation. Our results showed that SNAT2 protein abundance in the MG increased during lactation and this increase was maintained along this period, while 24h after weaning it tended to decrease. To study the effect of prolactin on SNAT2 expression, we incubated MG explants or T47D cells transfected with the SNAT2 promoter with prolactin, and we observed in both studies an increase in the SNAT2 expression or promoter activity. Consumption of a high-protein/low carbohydrate diet increased prolactin concentration, with a concomitant increase in SNAT2 expression not only in the MG during lactation, but also in the liver and adipose tissue. There was a correlation between SNAT2 expression and serum prolactin levels depending on the amount of dietary protein/carbohydrate ratio consumed. These findings suggest that prolactin actively supports lactation providing amino acids to the gland through SNAT2 for the synthesis of milk proteins.

Protein synthesis is one of the main cellular functions inhibited during hypertonic challenge. The subsequent accumulation of the compatible osmolyte betaine during the later adaptive response allows not only recovery of translation but also its stimulation. In this paper, we show that betaine modulates translation by enhancing the formation of cap-independent 48 S pre-initiation complexes, leaving cap-dependent 48 S pre-initiation complexes basically unchanged. In the presence of betaine, CrPV IRES- and sodium-dependent neutral amino acid transporter-2 (SNAT2) 5′-UTR-driven translation is 2- and 1.5-fold stimulated in MCF7 cells, respectively. Thus, betaine could provide an advantage in translation of messengers coding for proteins implicated in the response of cells to different stressors, which are often recognized by ribosomal 40 S subunit through simplified cap-independent mechanisms.

ATP-binding cassette (ABC) transporters are implicated in a diverse range of physiological and pathophysiological processes, such as cholesterol and lipid transportation and multidrug resistance. Despite the considerable efforts made in understanding of the cellular function of ABC proteins, the regulation mechanism of this type of protein is still poorly defined. Here we report the identification and functional characterization of a novel ATPase protein, protein associated with ABC transporters (PAAT), in humans. PAAT contains a nucleotide-binding domain (NBD)-like domain and a signal for intramitochondrial sorting. We showed that PAAT is localized in both the cytoplasm and the mitochondria and has an intrinsic ATPase activity. PAAT physically interacts with the 3 known mitochondrial inner membrane ABC proteins, ABCB7, ABCB8, and ABCB10, but not ABCB1, ABCB6, or ABCG2, and functionally regulates the transport of ferric nutrients and heme biosynthesis. Significantly, PAAT deficiency promotes cell death, reduces mitochondrial potential, and sensitizes mitochondria to oxidative stress-induced DNA damages. Our experiments revealed that PAAT is a novel ATPase and a trans-regulator of mitochondrial ABC transporters that plays an important role in the maintenance of mitochondrial homeostasis and cell survival.

We here investigated whether an acute bout of endurance exercise would induce the expression of amino acid transporters that regulate leucine transport across plasma and lysosomal membranes in rat skeletal muscle. Rats ran on a motor-driven treadmill at a speed of 28 m/min for 90 min. Immediately after the exercise, we observed that expression of mRNAs encoding L-type amino acid transporter 1 (LAT1) and CD98 was induced in the gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles. Sodium-coupled neutral amino acid transporter 2 (SNAT2) mRNA was also induced by the exercise in those three muscles. Expression of proton-assisted amino acid transporter 1 (PAT1) mRNA was slightly but not significantly induced by a single bout of exercise in soleus and EDL muscles. Exercise-induced mRNA expression of these amino acid transporters appeared to be attenuated by repeated bouts of the exercise. These results suggested that the expression of amino acid transporters for leucine may be induced in response to an increase in the requirement for this amino acid in the cells of working skeletal muscles.