SLC38A2 and glutamine signalling in cDC1s dictate anti-tumour immunity.

Cancer cells evade T cell-mediated killing through tumour-immune interactions whose mechanisms are not well understood1,2. Dendritic cells (DCs), especially type-1 conventional DCs (cDC1s), mediate T cell priming and therapeutic efficacy against tumours3. DC functions are orchestrated by pattern recognition receptors3-5, although other signals involved remain incompletely defined. Nutrients are emerging mediators of adaptive immunity6-8, but whether nutrients affect DC function or communication between innate and adaptive immune cells is largely unresolved. Here we establish glutamine as an intercellular metabolic checkpoint that dictates tumour-cDC1 crosstalk and licenses cDC1 function in activating cytotoxic T cells. Intratumoral glutamine supplementation inhibits tumour growth by augmenting cDC1-mediated CD8+ T cell immunity, and overcomes therapeutic resistance to checkpoint blockade and T cell-mediated immunotherapies. Mechanistically, tumour cells and cDC1s compete for glutamine uptake via the transporter SLC38A2 to tune anti-tumour immunity. Nutrient screening and integrative analyses show that glutamine is the dominant amino acid in promoting cDC1 function. Further, glutamine signalling via FLCN impinges on TFEB function. Loss of FLCN in DCs selectively impairs cDC1 function in vivo in a TFEB-dependent manner and phenocopies SLC38A2 deficiency by eliminating the anti-tumour therapeutic effect of glutamine supplementation. Our findings establish glutamine-mediated intercellular metabolic crosstalk between tumour cells and cDC1s that underpins tumour immune evasion, and reveal glutamine acquisition and signalling in cDC1s as limiting events for DC activation and putative targets for cancer treatment.

Asparagine transport through SLC1A5/ASCT2 and SLC38A5/SNAT5 is essential for BCP-ALL cell survival and a potential therapeutic target.

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) blasts strictly depend on the transport of extra-cellular asparagine (Asn), yielding a rationale for L-asparaginase (ASNase) therapy. However, the carriers used by ALL blasts for Asn transport have not been identified yet. Exploiting RS4;11 cells as BCP-ALL model, we have found that cell Asn is lowered by either silencing or inhibition of the transporters ASCT2 or SNAT5. The inhibitors V-9302 (for ASCT2) and GluγHA (for SNAT5) markedly lower cell proliferation and, when used together, suppress mTOR activity, induce autophagy and cause a severe nutritional stress, leading to a proliferative arrest and a massive cell death in both the ASNase-sensitive RS4;11 cells and the relatively ASNase-insensitive NALM-6 cells. The cytotoxic effect is not prevented by coculturing leukaemic cells with primary mesenchymal stromal cells. Leukaemic blasts of paediatric ALL patients express ASCT2 and SNAT5 at diagnosis and undergo marked cytotoxicity when exposed to the inhibitors. ASCT2 expression is positively correlated with the minimal residual disease at the end of the induction therapy. In conclusion, ASCT2 and SNAT5 are the carriers exploited by ALL cells to transport Asn, and ASCT2 expression is associated with a lower therapeutic response. ASCT2 may thus represent a novel therapeutic target in BCP-ALL.

METTL3-mediated m6A methylation of SLC38A1 stimulates cervical cancer growth.

The objective of this study was to better characterize the role of the glutamine transporter SLC38A1 in cervical cancer and explore the underlying mechanisms. Data from public databases and clinical cervical cancer tissue samples were used to assess the expression of SLC38A1 and its prognostic significance. Immunohistochemical staining, qRT-PCR, and Western blotting were used to evaluate the expression of relevant genes and proteins. Cell viability, cell cycle, apoptosis, and intracellular glutamine content were measured using CCK-8, flow cytometry, and biochemical assays. Additionally, the RNA immunoprecipitation (RIP) assay was used to examine the impact of METTL3/IGF2BP3 on the m6A modification of the SLC38A1 3'UTR. Both cervical cancer specimens and cells showed significantly increased expression of SLC38A1 and its expression correlated with an unfavorable prognosis. Knockdown of SLC38A1 inhibited cell viability and cell cycle progression, induced apoptosis, and suppressed tumor growth in vivo. Glutaminase-1 inhibitor CB-839 reversed the effects of SLC38A1 overexpression. METTL3 promoted m6A modification of SLC38A1 and enhanced its mRNA stability through IGF2BP3 recruitment. Moreover, METTL3 silencing inhibited cell viability, cell cycle progression, intracellular glutamine content, and induced apoptosis, but these effects were reversed by SLC38A1 overexpression. In conclusion, METTL3-mediated m6A methylation of SLC38A1 stimulates cervical cancer progression. SLC38A1 inhibition is a potential therapeutic strategy for cervical cancer.

Structure-activity relationship of amino acid analogs to probe the binding pocket of sodium-coupled neutral amino acid transporter SNAT2.

The sodium-coupled neutral amino acid transporter SNAT2 (SLC38A2) has been shown to have important physiological functions and is implicated in various diseases like cancer. However, few compounds targeting this transporter have been identified and little is known about the structural requirements for SNAT2 binding. In this study, the aim was to establish the basic structure-activity relationship for SNAT2 using amino acid analogs. These analogs were first studied for their ability to inhibit SNAT2-mediated 3H-glycine uptake in hyperosmotically treated PC-3 cells. Then to identify substrates a FLIPR membrane potential assay and o-phthalaldehyde derivatization of intracellular amino with subsequent quantification using HPLC-Fl was used. The results showed that ester derivatives of the C-terminus maintained SNAT2 affinity, suggesting that the negative charge was less important. On the other hand, the positive charge at the N-terminus of the substrate and the ability to donate at least two hydrogen bonds to the binding site appeared important for SNAT2 recognition of the amine. Side chain charged amino acids generally had no affinity for SNAT2, but their non-charged derivatives were able to inhibit SNAT2-mediated 3H-glycine uptake, while also showing that amino acids of a notable length still had affinity for SNAT2. Several amino acid analogs appeared to be novel substrates of SNAT2, while γ-benzyl L-glutamate seemed to be inefficiently translocated by SNAT2. Elaborating on this structure could lead to the discovery of non-translocated inhibitors of SNAT2. Thus, the present study provides valuable insights into the basic structural binding requirements for SNAT2 and can aid the future discovery of compounds that target SNAT2.

Radiation-induced exosomes promote oral squamous cell carcinoma progression via enhancing SLC1A5-glutamine metabolism.

BACKGROUND: Radiotherapy (RT) can drive cancer cells to enter a state of cellular senescence in which cells can secrete senescence-associated secretory phenotype (SASP) and produce small extracellular vesicles (sEVs) to interact with cells in the tumor microenvironment (TME). Tumor-derived sEVs that are taken up by recipient cells contribute to cancer cell metabolic plasticity, resistance to anticancer therapy, and adaptation to the TME. However, how radiation-induced sEVs support oral squamous cell carcinoma (OSCC) progression remains unclear. METHODS: Beta-galactosidase staining and SASP mRNA expression analysis were used to evaluate the senescence-associated activity of OSCC cells after irradiation. Nanoparticle tracking analysis was performed to identify radiation-induced sEVs. Liquid chromatography-tandem mass spectrometry (LC-MS) was used to explore changes in the levels of proteins in radiation-induced sEVs. Cell Counting Kit-8 and colony formation assays were performed to investigate the function of radiation-induced SASP and sEVs in vitro. A xenograft tumor model was established to investigate the functions of radiation-induced sEVs and V-9302 in vivo as well as the underlying mechanisms. Bioinformatics analysis was performed to determine the relationship between glutamine metabolism and OSCC recurrence. RESULTS: We determined that the radiation-induced SASP triggered OSCC cell proliferation. Additionally, radiation-induced sEVs exacerbated OSCC cell malignancy. LC-MS/MS and bioinformatics analyses revealed that SLC1A5, which is a cellular receptor that participates in glutamine uptake, was significantly enriched in radiation-induced sEVs. In vitro and in vivo, inhibiting SLC1A5 could block the oncogenic effects of radiation-induced sEVs in OSCC. CONCLUSION: Radiation-induced sEVs might promote the proliferation of unirradiated cancer cells by enhancing glutamine metabolism; this might be a novel molecular mechanism underlying radiation resistance in OSCC patients.

Abnormal Weakening of DNA Methylation around the SLC6A1 Gene Promoter in Temporal Lobe Epilepsy.

BACKGROUND: The solute carrier (SLC) superfamily, which transports solutes across biological membranes, includes four members (SLC2A1, SLC6A1, SLC9A64, and SLC35A2) that have been linked to epilepsy. This study sought to examine the DNA methylation patterns near the promoters of these genes in temporal lobe epilepsy (TLE), as DNA methylation is a crucial epigenetic modification that can impact gene expression. METHODS: The study comprised 38 individuals with TLE and 38 healthy controls. Methylation experiments were performed using peripheral blood, while demethylation experiments were carried out using SH-SY5Y cells with the DNA methylation inhibitor decitabine. RESULTS: A significant difference was observed in the DNA methylation rate of SLC6A1 between TLE patients and controls, with TLE patients showing a lower rate (4.81% vs. 5.77%, p = 0.0000), which remained significant even after Bonferroni correction (p = 0.0000). Based on the hypomethylated SLC6A1 in TLE, a predictive model was established that showed promise in distinguishing and calibrating TLE. In the TLE group, there were differences in DNA methylation rates of SLC6A1 between the young patients and the older controls (4.42% vs. 5.22%, p = 0.0004). A similar trend (p = 0.0436) was noted after adjusting for sex, age at onset, and drug response. In addition, the study found that DNA methylation had a silencing impact on the expression of the SLC6A1 gene in SH-SY5Y cells, which were treated with decitabine at a set dose gradient. CONCLUSIONS: The evidence suggests that lower methylation of SLC6A1 may stimulate transcription in TLE, however, further investigation is necessary to confirm the exact mechanism.

Comprehensive analysis of the biological function and immune infiltration of SLC38A2 in gastric cancer.

BACKGROUND: Solute carrier family 38 member 2 (SLC38A2) has previously been reported to participate in carcinogenesis. However, its expression and function in gastric cancer (GC) remain unclear. The present study aimed to investigate the role of SLC38A2 in GC. METHODS: The prognostic value and expression of SLC38A2 in GC was analyzed by combining bioinformatics and experimental analyses. Colony formation, Cell Counting Kit-8, wound healing, Transwell and tumor formation assays were performed to assess the biological function of SLC38A2. The cBioPortal, GeneMANIA and LinkedOmics databases were mined to determine the underlying regulatory mechanisms of SLC38A2. The role of SLC38A2 in tumor immune infiltration was explored using the TIMER database. RESULTS: Our results demonstrated that SLC38A2 was upregulated and was correlated with a poor prognosis in GC patients. SLC38A2 downregulation significantly inhibited the proliferation, invasion and migration of GC cells. Abnormal genetic alteration and epigenetic regulation may contribute to the upregulation of SLC38A2 expression levels in GC. The results of enrichment analysis demonstrated that SLC38A2 was associated with 'hippo signaling' and 'ubiquitinyl hydrolase activity'. The results also indicated that SLC38A2 may be a key factor in GC immune infiltration and M2 macrophage polarization. CONCLUSION: Overall, these data identified that SLC38A2 may serve as a potential prognostic biomarker and therapeutic target in GC.

Long non-coding RNA OGFRP1 regulates cell proliferation and ferroptosis by miR-299-3p/SLC38A1 axis in lung cancer.

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.

SNAT2/SLC38A2 Confers the Stemness of Gastric Cancer Cells via Regulating Glutamine Level.

BACKGROUND: 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). AIMS: This work aims to explore the roles of Gln transporters in gastric cancer cell stemness. METHODS: 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. RESULTS: 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. CONCLUSIONS: SNAT2 can promote the stemness of GC cells in a Gln-dependent manner.

Paternal knockout of Slc38a4/SNAT4 causes placental hypoplasia associated with intrauterine growth restriction in mice.

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.

Hypoxia-induced switch in SNAT2/SLC38A2 regulation generates endocrine resistance in breast cancer.

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.

SLC38A4 Amino Acid Transporter Expression Is Significantly Lower in Early Preterm Intrauterine Growth Restriction Complicated Placentas.

Intrauterine growth restriction (IUGR), predominantly caused by placental insufficiency, affects partitioning of nutrients to the fetus. The system A sodium-coupled transporters (SNAT or SLC38), of types A1, A2, and A4, control non-essential amino acid uptake and supply. Here, we aimed to investigate the expression of these transporters across different placental disease cohorts and cells. To determine disease impact, transporter expressions at the gene (qPCR) and protein (western blots) level were assessed in gestationally matched placental tissues. Early (<34 weeks), and late (34−36 weeks) onset IUGR cases with/out preeclampsia were compared to preterm controls. We also investigated level of transporter expression in primary trophoblasts under glucose deprivation (n = 6) and hypoxia conditions (n = 7). SLC38A4 protein was significantly downregulated in early preterm pregnancies complicated with IUGR with/out preeclampsia. There were no differences in late preterm IUGR cohorts. Furthermore, we demonstrate for the first time in primary trophoblast cells, that gene expression of the transporters was sensitive to and induced by glucose starvation. SLC38A4 mRNA expression was also significantly upregulated in response to hypoxia. Thus, SLC38A4 expression was persistently low in early preterm IUGR pregnancies, regardless of disease aetiology. This suggests that gestational age at delivery, and consequently IUGR severity, may influence loss of its expression.

D-Serine Signaling and NMDAR-Mediated Synaptic Plasticity Are Regulated by System A-Type of Glutamine/D-Serine Dual Transporters.

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 serine racemase knock-out 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 synaptosomes 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-serine 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.SIGNIFICANCE STATEMENT 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 extracellular 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.

ChREBP downregulates SNAT2 amino acid transporter expression through interactions with SMRT in response to a high-carbohydrate diet.

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.NEW & NOTEWORTHY This study highlights the key role of carbohydrate responsive element-binding protein (ChREBP) in the fine-tuned regulation between glucose and amino acid metabolism in the liver via regulation of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) expression after the consumption of a high-carbohydrate diet. ChREBP binds to a carbohydrate response element (ChoRE) site in the SNAT2 promoter region and recruits silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor to reduce SNAT2 transcription. This study revealed that ChREBP prevents the uptake of glucogenic amino acids upon the consumption of a high-carbohydrate diet.

Increased expression of glutamine transporter SNAT2/SLC38A2 promotes glutamine dependence and oxidative stress resistance, and is associated with worse prognosis in triple-negative breast cancer.

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

SLC38A4 functions as a tumour suppressor in hepatocellular carcinoma through modulating Wnt/ß-catenin/MYC/HMGCS2 axis.

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/ß-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.

V-9302 inhibits proliferation and migration of VSMCs, and reduces neointima formation in mice after carotid artery ligation.

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.

Ceftriaxone regulates glutamate production and vesicular assembly in presynaptic terminals through GLT-1 in APP/PS1 mice.

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.

Maternal intermittent fasting during pregnancy induces fetal growth restriction and down-regulated placental system A amino acid transport in the rat.

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.

Prenatal Hypoxia Induces Premature Aging Accompanied by Impaired Function of the Glutamatergic System in Rat Hippocampus.

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.