Expression Of ASCT1 Research Articles

Dynamic Evolution of Retroviral Envelope Genes in Egg-Laying Mammalian Genomes.

Independently acquired envelope (env) genes from endogenous retroviruses have contributed to the placental trophoblast cell-cell fusion in therian mammals. Egg-laying mammals (monotremes) are an important sister clade for understanding mammalian placental evolution, but the env genes in their genomes have yet to be investigated. Here, env-derived open reading frames (env-ORFs) encoding more than 400 amino acid lengths were searched in the genomes of two monotremes: platypus and echidna. Only two env-ORFs were present in the platypus genome, whereas 121 env-ORFs were found in the echidna genome. The echidna env-ORFs were phylogenetically classified into seven groups named env-Tac1 to -Tac7. Among them, the env-Tac1 group contained only a single gene, and its amino acid sequence showed high similarity to those of the RD114/simian type D retroviruses. Using the pseudotyped virus assay, we demonstrated that the Env-Tac1 protein utilizes echidna sodium-dependent neutral amino acid transporter type 1 and 2 (ASCT1 and ASCT2) as entry receptors. Moreover, the Env-Tac1 protein caused cell-cell fusion in human 293T cells depending on the expression of ASCT1 and ASCT2. These results illustrate that fusogenic env genes are not restricted to placental mammals, providing insights into the evolution of retroviral genes and the placenta.

Suppressed Expression of ASCT1 Contributes to the Maintenance of Leukemia Stemness Via the Enhancement of Antioxidant Capacity

Introduction: Acute myeloid leukemia (AML) is one of the most common hematological malignancies derived from self-renewing leukemic stem cells (LSCs). LSCs represent a small cell fraction harboring the self-renewal capacity and potential to give rise to mature leukemic cells and play a central role in the relapse of AML. In general, LSCs can be enriched within reactive oxygen species (ROS) low fraction. Glutathione (GSH), a major antioxidant small molecule, plays a central role in the maintenance of lower ROS levels; however, the molecular mechanisms of GSH maintenance in LSCs remain unclear. Result: First, we evaluated the intracellular GSH levels in the CD34+ immature fraction, in which LSCs can be enriched and CD34- mature AML cells from the identical patients (n=#). CD34+AML cells contained significantly higher levels of GSH. To clarify the molecular mechanisms how CD34+ primitive AML cells could maintain high GSH level, we analyzed the expression of cysteine transporters, because cysteine uptake is the rate-limiting step of GSH synthesis. In human, three amino acid transporters including ASCT1, ASC1 and xCT are known as cysteine/cystine transporters. The expression levels of ASC1 and xCT were not significantly different among the three cellular fractions of AML: the most immature CD34+CD38-, CD34+CD38+, and more differentiated CD34- cells. However, we found that CD34+CD38- LSCs completely lacked the expression of ASCT1 in all the cases examined (n=#). The expression of ASCT1 increased along with cellular differentiation; more differentiated CD34- AML cells expressed the higher ASCT1 expression than the most immature CD34+CD38- cells. We, therefore, hypothesized that the different expression pattern of ASCT1 might be associated with higher GSH levels in LSCs through the regulation of cellular cysteine levels. ASCT1 is known to transport neutral amino acids (alanine, serine, cysteine, threonine), no role for ASCT1 in leukemia has ever been investigated. We found that ASCT1 was highly expressed in M4/M5 monocytic leukemias according to the FAB classification. Analysis of beatAML RNA-seq data showed that patients with low ASCT1 expression had a worse prognosis. We found that primary AML cells with the higher ASCT1 expression had reduced intracellular GSH as compared to those with lower ASCT1 expression (n=#), suggesting the association of ASCT1 expression and GSH levels in primary AML cells. To test this hypothesis, we knocked down (KD) the expression of ASCT1 using shRNAs in cell lines. KD of ASCT1 induced an increase in intracellular GSH compared to control cells, suggesting that ASCT1 decreases intracellular GSH by transporting cysteine extracellularly. To clarify the function of ASCT1 in human AML-LSCs in vivo, we performed xenotransplantation experiments. The identical number of FACS-purified ASCT1+ and ASCT- AML cells from the identical patients were transplanted into the irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. We found that ASCT1- AML cells efficiently reconstituted human AML in vivo, whereas ASCT1+ AML cells completely failed in the engraftment in vivo in all the cases tested, suggesting that LSCs exclusively reside within ASCT1- AML cellular fraction. These results collectively suggest that LSCs maintain low ASCT1 expression, which is one of the molecular machineries to keep intracellular GSH high. Furthermore, only the ASCT1- fraction reconstitute AML in immunodeficient mice, indicating that suppression of ASCT1 is important for the maintenance of leukemia stemness.

In ovo threonine supplementation affects ileal gene expression of nutrient transporters in broilers inoculated post-hatch with Salmonella Enteritidis.

The effect of in ovo threonine (Thr) supplementation on the ileal expression of glucose, peptide and amino acid transporters was assessed in Salmonella Enteritidis-challenged broiler chicks. At 17.5days of incubation, fertile eggs were supplemented in the amniotic fluid with sterile saline or 3.5% threonine. Hatchlings were individually weighed, and Salmonella Enteritidis negative status was confirmed. At 2days of age, half of the birds of each group were inoculated with sterile nutrient broth or Salmonella Enteritidis inoculum. Relative expression of sodium-dependent glucose transporter 1 (SGLT1), glucose transporter 2 (GLUT2), di- and tri-peptide transporter 1 (PepT1) and alanine, serine, cysteine, threonine transporter (ASCT1) was assessed at hatch, 2 and 9days of age, i.e., before inoculation and 7days post-inoculation (dpi). At 9days of age (7dpi), threonine increased SGLT1 and GLUT2 expression, whereas GLUT2 expression decreased in Salmonella-challenged birds. There was a significant interaction between threonine and Salmonella for PepT1 and ASCT1. Threonine increased PepT1 expression only in non-challenged birds. In addition, in ovo supplementation increased expression of ASCT1 regardless of post-hatch inoculation; Salmonella inoculation resulted in decreased expression of ASCT1 only in supplemented birds. The results suggest that while intra-amniotic threonine administration in broiler embryos increases the expression of genes related to the absorption of monosaccharides and amino acids, Salmonella challenge may negatively affect the expression of protein related transporters in the ileum of broilers.

Maternal Immune Activation Alters Placental and Fetal Brain Amino Acid Transport in Rats

ProblemMaternal immune activation (MIA) during pregnancy is an environmental risk factor for neurodevelopmental disorders such as autism and schizophrenia. However, the exact mechanism through which this occurs is unknown. Due to the important roles that amino acids play in neurodevelopment, altered transport of amino acids across the placenta and throughout the developing fetal brain could play a role in neurodevelopmental disorders. Our objectives were to determine whether virally mediated MIA alters amino acid transporter expression in the placenta and fetal brain, and to explore the underlying mechanism(s) associated with these changes.MethodsPregnant rats received poly(I:C) on gestational day 14 and placental and fetal tissues were collected 6, 24, and 48 hours later. Amino acid transporter expression in the placenta and fetal brain was measured using qPCR and immunoblotting. Free amino acid concentrations in the fetal brain were quantified using HPLC. To explore a possible mechanism, the expression and phosphorylation of components of the placental mTOR and AMPK signaling pathways were investigated using immunoblotting.ResultsPoly(I:C) induced the mRNA expression of multiple amino acid transporters in the placenta and fetal brain over all three timepoints. Conversely, poly(I:C) significantly decreased protein expression of ASCT1 and EAAT2 in the placenta, and SNAT5, EAAT1, and GLYT1 in the fetal brain. Changes in transporter expression were accompanied by functional changes in the concentrations of numerous amino acids in the fetal brain. We observed activation of AMPK and inhibition of mTOR signaling in the placentas of poly(I:C)‐treated dams.ConclusionsMIA induced by poly(I:C) significantly alters placental and fetal brain amino acid transporter expression as well as amino acid concentrations in the fetal brain. Due to the importance of amino acids in neurodevelopment, changes in amino acid transporters could contribute to the development of a neurodevelopmental disorder. AMPK and mTOR have been previously shown to regulate amino acid transporter expression. Since poly(I:C) altered the activation of placental and AMPK and mTOR, these could provide novel treatment targets to alleviate MIA‐induced changes in amino acid transport and investigate the implications of these changes in neurodevelopment.Support or Funding InformationThis work was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) awarded to Dr. Piquette‐Miller [MOP 13346]. Eliza McColl was a recipient of a CIHR Canadian Graduate Scholarship (Master’s) and an Ontario Graduate Scholarship, and is currently funded by a CIHR Banting and Best Canadian Graduate Scholarship (Doctoral) [GSD‐164238].

Poly(I:C) alters placental and fetal brain amino acid transport in a rat model of maternal immune activation.

Maternal immune activation (MIA) during pregnancy is associated with increased chances of neurodevelopmental disorders including schizophrenia and autism spectrum disorder (ASD). However, the exact mechanism through which MIA contributes to altered neurodevelopment is unknown. Due to the important role that amino acids play in neurodevelopment, alteredamino acid transport could play a role in neurodevelopmental disorders. Indeed, altered plasma concentrations of multiple amino acids have been reported in individuals with ASD or schizophrenia. Therefore,our objective was to determine whether virally mediated MIA induces changes in amino acid transporters in the placenta and fetal brain. Pregnant rats were administered poly(I:C) on gestational day 14, and placental and fetal tissues were collected 6, 24, and 48hours later. Amino acid transporter expression was measured in the placenta and fetal brain using qPCR, Western blotting, and Simple Western. Free amino acid concentrations in the fetal brain were quantified using HPLC. Poly(I:C) increased mRNA expression of several amino acid transporters in the placenta and fetal brain over these timepoints. Conversely, poly(I:C) imposed significant decreases in the protein expression of ASCT1 and EAAT2 in placenta and expression of SNAT5, EAAT1, and GLYT1 in fetal brain. Functional consequences of altered transporter expression were demonstrated through widespread changes in the concentrations of free amino acids in the fetal brains. Together, these results represent novel findings with the poly(I:C) MIA model and contribute to the understanding of how MIA during pregnancy potentially leads to neurodevelopmental disorders.

Identification of ASCT1 As a Candidate Molecule Enhancing Antioxidant Activity in Primary Human AML Cells

Introduction:Recent studies have shown that the specific alteration of metabolic pathways are involved in the regulation of function of normal hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, little is known about the features of metabolic activity in human HSCs and LSCs. To reveal the metabolic pathway alterations in primary AML cells, we performed the comprehensive metabolome analysis by comparing normal human CD34+ hematopoietic stem/progenitor cells (HSPCs)(n=5) and CD34+ primitive AML cells containing LSCs (n=16) using highly sensitive CE-tandem mass spectrometry.Method:Metabolome analysisMetabolites were extracted from primitive CD34+ AML cells (n=16) and normal CD34+ bone marrow cells (n=4) and cord blood cells (n=1). Metabolome analysis was conducted by the C-SCOPE package of HMT using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) for cation analysis, and capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) for anion analysis. 116 metabolites were targeted for analysis in this study.Oxygen consumption rates and extracellular acidification rateO2 consumption rates (OCR) and extracellular acidification rate (ECAR) were measured by the Seahorse XF96 extracellular flux analyzer. Three replicate wells of 400,000 leukemic or normal cells per well were seeded in 96-well XF96 well plates coated with BD Cell-Tak (BD Biosciences) in serum-free unbuffered DMEM. Analyses were performed both at basal conditions and after injection of OLI (1 mg/ml), FCCP (1 mM), Antimycin A (5 mM).Result:We detected 101 metabolites involved in central carbon and energy metabolism. In glucose metabolism, the level of lactate, an end-product of aerobic glycolysis, were lower in CD34+ AML cells than normal HSPCs, whereas the level of pyruvate, a precursor of lactate, was not different. Thus, CD34+ AML cells had a significantly high pyruvate/lactate ratio as compared to normal HSPCs, suggesting that aerobic respiration is preferentially utilized in CD34+ AML cells. To confirm this observation, we directly measured the O2 consumption rate (OCR) and extracellular acidification rate (ECAR) of CD34+ AML cells (n=4) and normal HSPCs (n=5) by XF96 extracellular flux analyzer. OCR reflects the activity of aerobic respiration, whereas ECAR reflects lactate generation and correlates with anaerobic respiration activity. Therefore, OCR/ECAR ratio is a good index for the discrimination of aerobic and anaerobic respiration pattern. We found that the OCR/ECAR ratio of CD34+ primitive AML cells was significantly high as compared to that of HSPCs, suggesting that CD34+ AML cells predominantly utilized aerobic respiration.Although the aerobic respiration resulted in the production of reactive oxygen species (ROS), the intracellular ROS level was not different between CD34+ AML (n=7) cells and normal HSPCs (n=3), suggesting that the antioxidant activity should be strongly enhanced in CD34+ AML cells. Consistent with the observation, we found that CD34+ AML cells had a much higher level of glutathione (GSH), a primary intracellular antioxidant, than normal HSPCs.To clarify the molecular mechanisms how CD34+ primitive AML cells could maintain high GSH level, we analyzed the expression of cysteine transporters, because cysteine uptake is the rate-limiting step of GSH synthesis. In human, three amino acid transporters including ASCT1, ASC1 and xCT are known as cystine/cysteine transporters. Interestingly, ASCT1 was significantly highly expressed in CD34+ AML cells (n=10) as compared to normal CD34+ HSPCs (n=3). Of note, normal CD34+CD38- HSCs completely lacked ASCT1 expression, whereas CD34+CD38-LSCs expressed at a high level, indicating the possibility that the high expression of ASCT1 should be a LSC specific machinery for enhanced GSH synthesis.Thus, human AML cells predominantly utilize aerobic respiration that is supported by a high level of GSH, and AML specific ASCT1 expression presumably contributes to the high level of GSH. These data suggest that ASCT1 should be a promising molecule to specifically target AML stem/progenitor cells. DisclosuresAkashi:Celgene: Research Funding; Astellas Pharma: Research Funding; Shionogi & Co., Ltd: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol Meyers Squibb: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Sunitomo Dainippon Pharma: Consultancy.

Arginine vasopressin regulated ASCT1 expression in astrocytes from stroke-prone spontaneously hypertensive rats and congenic SHRpch1_18 rats

In stroke-prone spontaneously hypertensive rats (SHRSP/Izm), ischemia induces swelling of astrocytes, a process that subsequently leads to neuronal death. Following ischemic insult, arginine vasopressin (AVP) can induce edema and l-serine released by astrocytes supports the survival of neuronal cells. The purpose of this study was to examine whether AVP contributed to the regulation of l-serine production following ischemic stroke. Here, we used cultured astrocytes from SHRSP/Izm rats and Wistar Kyoto rats (WKY/Izm) to examine whether AVP changed the production of l-serine and/or altered gene expression levels of the neural amino acid transporter (Slc1a4), 3-phosphoglycerate dehydrogenase (Phgdh) and serine racemase (SRR). Furthermore, using astrocytes from the congenic rat SHRpch1_18 strain having quantitative trait loci (QTL) of stroke, we examined expression of those genes under conditions of hypoxia and reoxygenation (H/R). The expression levels of ASCT1 protein, the genes described above and l-serine levels were determined by Western blotting (WB), RT-PCR, real-time quantitative RT-PCR and HPLC. AVP increased the production of l-serine and the expression of Slc1a4 in WKY/Izm and SHRSP/Izm astrocytes. The production of l-serine and the expression of Slc1a4 were lower in SHRSP/Izm than in WKY/Izm cells. This difference was not seen with Phgdh. In the SHRpch1_18 strain, the expression of Slc1a4 and Phgdh significantly decreased after H/R. AVP-mediated enhanced expression of ASCT1 was blocked by the addition of bumetanide. These results suggest that the AVP-mediated attenuated expression of ASCT1 in astrocytes is associated with reduced l-serine production in SHRSP/Izm astrocytes. We hypothesize that reduction of gene expression by AVP might be related to the induction of stroke in the SHRpch1_18 rat strain.

Expression of l-Serine Biosynthetic Enzyme 3-Phosphoglycerate Dehydrogenase (Phgdh) and Neutral Amino Acid Transporter ASCT1 Following an Excitotoxic Lesion in the Mouse Hippocampus

The nonessential amino acid L-serine functions as a glia-derived trophic factor and strongly promotes the survival and differentiation of cultured neurons. The L-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase (Phgdh) and the small neutral amino acid transporter ASCT1 are preferentially expressed in specific glial cells in the brain. However, their roles in pathological progression remain unclear. We examined the expression of Phgdh and ASCT1 in kainic acid (KA)-induced neurodegeneration of the mouse hippocampus using immunohistochemistry and Western blots. Our quantitative analysis revealed that Phgdh and ASCT1 were constitutively expressed in the normal brain and transiently upregulated by KA-treatment. At the cellular level, Phgdh was expressed in astrocytes in control and in KA-treated mice while ASCT1 that was expressed primarily in the neurons of the normal brain appeared also in activated astrocytes in KA treated mouse brain. The preferential glial expression of ASCT1 was consistent with that of Phgdh. These results demonstrate injury-induced changes in Phgdh and ASCT1 expression. It is hypothesized that the secretion of L-serine is regulated by astrocytes in response to toxic molecules such as glutamate and free radicals that promote neurodegeneration, and may correspond to the level of L-serine needed for neuronal survival and glial activation during brain insults.

Changes in region- and cell type-specific expression patterns of neutral amino acid transporter 1 (ASCT-1) in the anterior cingulate cortex and hippocampus in schizophrenia, bipolar disorder and major depression

Although, the pathogenetic mechanisms of schizophrenia, bipolar disorder, and major depression are not clearly understood, various neurotransmitter systems are reported to have altered expression patterns of their receptor and transporter proteins. Changes in the expression of the neutral amino acid transporter 1 (ASCT-1) protein in the anterior cingulate gyrus and the hippocampus were investigated using immunohistochemistry and western blotting. A significant decrease in ASCT-1 immunoreactivity in neurons in the cingulate cortex as well as astrocytes of the white matter was seen in schizophrenia. In bipolar disorder and major depression, similar results were seen for neurons. In the hippocampus, there was a striking loss of immunoreactivity on astrocytes, neurons and interneurons in multiple regions in schizophrenia and bipolar disorder, while only minor changes were seen in major depression. The altered expression of ASCT-1 in neurons and astrocytes reflects profound changes in glutamatergic neurotransmission and highlights a significant role of astrocytes in the pathophysiology of neurotransmission in these major psychiatric disorders.

Increased Expression of Specific Intestinal Amino Acid and Peptide Transporter mRNA in Rats Fed by TPN Is Reversed by GLP-2

Intestinal function depends on the presence of luminal nutrients and is altered during starvation and refeeding. Amino acids are essential for enterocytes, but the luminal supply is compromised with changes in dietary intake. To test the hypothesis that during periods of restricted luminal nutrient availability mucosal cells undergo adaptations aimed toward preserving amino acid supply, the expression of amino acid and peptide transporter mRNAs was quantified in rats with no oral intake, whose nutritional status was maintained with total parenteral nutrition (TPN). The role of the intestinotrophic hormone glucagon-like peptide-2 (GLP-2) was investigated in the adaptive responses. Rats were administered TPN with or without exogenous GLP-2. Amino acid and peptide transporter mRNAs in small intestine mucosa were measured by semiquantitative RT-PCR. Compared with orally fed rats, removal of luminal nutrition increased the expression of ASCT1, SAT2, and GLYT1 mRNAs in the duodenum and of ASCT2, EAAC1, NBAT, and PepT1 mRNAs in the ileum. CAT1, PAT1, and SN2 mRNA abundances were unaffected. GLP-2 reversed these effects. Three subgroups of transporters were identified by regional differences in response to TPN. This may reflect differing roles for substrates of transporters located apically and basally and along the proximal-distal axis of the intestine. The importance of maintaining amino acid supply for intestinal mucosal cells is illustrated.

Neutral Amino Acid Transporter ASCT1 Is Preferentially Expressed in l-Ser-Synthetic/Storing Glial Cells in the Mouse Brain with Transient Expression in Developing Capillaries

Nonessential amino acid L-Ser plays an essential role in neuronal survival and differentiation, through preferential expression of the L-Ser biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH), in particular in glial cells but not in neurons. To seek the molecular candidates responsible for glia-borne L-Ser transport, we performed histochemical analyses on amino acid transporter ASCT1, which prefers small neutral amino acids, such as Ala, Ser, Cys, and Thr, and mediates their obligatory exchange. At early developmental stages, neuroepithelial cells constituting the ventricular zone expressed ASCT1 mRNA and protein ubiquitously. Thereafter, ASCT1 expression was gradually downregulated in neuronal populations during the late embryonic and neonatal periods, whereas its high expression was transmitted to radial glial cells and then to astrocytes. High levels of ASCT1 were also detected in the olfactory ensheathing glia. The preferential glial expression of ASCT1 was consistent with that of 3PGDH, and their extensive colocalization was demonstrated at the cellular level. Moreover, high cellular contents of L-Ser were revealed in these glial cells by using a specific antibody to L-Ser. These results strongly suggest that a large amount of L-Ser is synthesized and stored in these glial cells and is released through ASCT1 in exchange for other extracellular substrates. In addition, we observed prominent expression of ASCT1 in capillary endothelial cells of embryonic and neonatal brains. Therefore, ASCT1 appears to be regulated to meet metabolic demands by differentiating and mature neurons through the transport of glia- and blood-borne small neutral amino acids.

Novel neuroglial and glioglial relationships mediated by L-serine metabolism.

L-Serine is a non-essential amino acid that can be synthesized in the body. It derives from an intermediate of the glycolytic pathway, 3-phosphoglycerate, and utilized for the syntheses of proteins, other amino acids, membrane lipids, heme, and nucleotides. Emerging evidence indicates that L-serine functions as a glia-derived trophic factor, which strongly promotes the survival and differentiation of cultured neurons. L-Serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH) and small neutral amino acid transporter ASCT1 have been revealed to be expressed preferentially in the radial glia-astrocyte lineage and olfactory ensheathing glia of both adult and developing rodent brains. In contrast, these biosynthetic and transporter molecules for L-serine are faint or undetectable in neurons and phagocytic cells. In this review, we summarize recent progress to propose that L-serine synthesis in these glial cells and its supply to nearby neurons and other glia constitute a novel metabolic unit in the brain. Based on these neuroglial and glioglial relationships, glucose in neurons and phogocytes can be strategically used for energy production, while a variety of L-serine-derived biomolecules required for their proliferaton, survival, differentiation, and function are synthesized in and supplied from the radial glia-astrocyte lineage and olfactory ensheathing glia. A transient capillary expression of ASCT1 in fetal and neonatal brains further suggests that, in addition to the glia-borne L-serine, an active transport of blood-borne L-serine would play an essential role in neural development.