Sodium-coupled Amino Acid Transporter Research Articles

Identification of SLC38A7 as a Prognostic Marker and Potential Therapeutic Target of Lung Squamous Cell Carcinoma.

No effective molecular targeted therapy has been established for SCC. We conducted a comprehensive study of SCC patients using RNA-sequencing and TCGA dataset to clarify the driver oncogene of SCC. Forty-six samples of 23 patients were totally analyzed with RNA-sequencing. We then searched for candidate-oncogenes of SCC using the TCGA database. To identify candidate oncogenes, we used the following 2 criteria: (1) the genes of interest were overexpressed in tumor tissues of SCC patients in comparison to normal tissues; and (2) using an integrated mRNA expression and DNA copy number profiling analysis using the TCGA dataset, the DNA copy number of the genes was positively correlated with the mRNA expression. We identified 188 candidate-oncogenes. Among those, the high expression of SLC38A7 was a strong prognostic marker that was significantly associated with a poor prognosis in terms of both overall survival (OS) and recurrence-free survival in the TCGA dataset (P < 0.05). Additionally, 202 resected SCC specimens were also subjected to an immunohistochemical analysis. Patients with the high expression of SLC38A7 (alternative name is sodium-coupled amino acid transporters 7) protein showed significantly shorter OS in comparison to those with the low expression of SLC38A7 protein [median OS 3.9 years (95% confidence interval, 2.4-6.4 years) vs 2.2 years (95% confidence interval, 1.9-4.1 years); log rank test: P = 0.0021]. SLC38A7, which is the primary lysosomal glutamine transporter required for the extracellular protein-dependent growth of cancer cells, was identified as a candidate therapeutic target of SCC.

Molecular networks of insulin signaling and amino acid metabolism in subcutaneous adipose tissue are altered by body condition in periparturient Holstein cows

Peripartal cows mobilize not only body fat but also body protein to satisfy their energy requirements. The objective of this study was to determine the effect of prepartum BCS on blood biomarkers related to energy and nitrogen metabolism, and mRNA and protein abundance associated with AA metabolism and insulin signaling in subcutaneous adipose tissue (SAT) in peripartal cows. Twenty-two multiparous Holstein cows were retrospectively classified into a high BCS (HBCS; n = 11, BCS ≥ 3.5) or normal BCS (NBCS; n = 11, BCS ≤ 3.17) group at d 28 before expected parturition. Cows were fed the same diet as a total mixed ration before parturition and were fed the same lactation diet postpartum. Blood samples collected at -10, 7, 15, and 30 d relative to parturition were used for analyses of biomarkers associated with energy and nitrogen metabolism. Biopsies of SAT harvested at -15, 7, and 30 d relative to parturition were used for mRNA (real time-PCR) and protein abundance (Western blotting) assays. Data were subjected to ANOVA using the MIXED procedure of SAS (v. 9.4; SAS Institute Inc., Cary, NC), with P ≤ 0.05 being the threshold for significance. Cows in HBCS had greater overall plasma nonesterified fatty acid concentrations, due to marked increases at 7 and 15 d postpartum. This response was similar (BCS × Day effect) to protein abundance of phosphorylated (p) protein kinase B (p-AKT), the insulin-induced glucose transporter (SLC2A4), and the sodium-coupled neutral AA transporter (SLC38A1). Abundance of these proteins was lower at -15 d compared with NBCS cows, and either increased (SLC2A4, SLC38A1) or did not change (p-AKT) at 7 d postpartum in HBCS. Unlike protein abundance, however, overall mRNA abundances of the high-affinity cationic (SLC7A1), proton-coupled (SLC36A1), and sodium-coupled amino acid transporters (SLC38A2) were greater in HBCS than NBCS cows, due to upregulation in the postpartum phase. Those responses were similar to protein abundance of p-mTOR, which increased (BCS × Day effect) at 7 d in HBCS compared with NBCS cows. mRNA abundance of argininosuccinate lyase (ASL) and arginase 1 (ARG1) also was greater overall in HBCS cows. Together, these responses suggested impaired insulin signaling, coupled with greater postpartum AA transport rate and urea cycle activity in SAT of HBCS cows. An in vitro study using adipocyte and macrophage cocultures stimulated with various concentrations of fatty acids could provide some insights into the role of immune cells in modulating adipose tissue immunometabolic status, including insulin resistance and AA metabolism.

Glutamine Cooperatively Upregulates Lipopolysaccharide-Induced Nitric Oxide Production in BV2 Microglial Cells through the ERK and Nrf-2/HO-1 Signaling Pathway

Glutamine (Gln) is a nonessential α-amino acid for protein biosynthesis. However, the mechanism through which Gln regulates NO production in microglial cells is still unclear. In this study, we investigated whether the presence or absence of Gln affects NO production in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Our data revealed that Gln depletion decreased cell viability accompanied by mild cytotoxicity, and blocked LPS-induced NO production concomitant with a significant decrease in inducible NO synthase (iNOS) expression. Additionally, Gln depletion for 24 h blocked the restoration of LPS-mediated NO production in the presence of Gln, suggesting that Gln depletion caused long-term immune deprivation. In particular, sodium-coupled amino acid transporter 1 and 2 (SNAT1 and SNAT2), which are the main Gln transporters, were highly upregulated in LPS-stimulated BV2 microglial cells, in the presence of Gln accompanied by NO production. Regardless of the presence of Gln, LPS positively stimulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression, and transient Nrf2 knockdown and HO-1 inhibition stimulated LPS-induced NO production and iNOS expression; however, transient Nrf2 knockdown did not affect SNAT1 and SNAT2 expression, indicating that Gln transporters, SNAT1 and SNAT2, were not regulated by Nrf2, which downregulated the HO-1-mediated NO production. Moreover, Gln depletion significantly reduced LPS-induced extracellular signal-regulated kinase (ERK) phosphorylation; furthermore, a specific ERK inhibitor, PD98059, and transient ERK knockdown attenuated LPS-stimulated NO production and iNOS expression, in the presence of Gln, accompanied by downregulation of SNAT1 and SNAT2, suggesting that the ERK signaling pathway was related to LPS-mediated NO production via SNAT1 and SNAT2. Altogether, our data indicated that extracellular Gln is vital for NO production from microglia in inflammatory conditions.

Bed rest impairs skeletal muscle amino acid transporter expression, mTORC1 signaling, and protein synthesis in response to essential amino acids in older adults

Skeletal muscle atrophy during bed rest is attributed, at least in part, to slower basal muscle protein synthesis (MPS). Essential amino acids (EAA) stimulate mammalian target of rapamycin (mTORC1) signaling, amino acid transporter expression, and MPS and are necessary for muscle mass maintenance, but there are no data on the effect of inactivity on this anabolic mechanism. We hypothesized that bed rest decreases muscle mass in older adults by blunting the EAA stimulation of MPS through reduced mTORC1 signaling and amino acid transporter expression in older adults. Six healthy older adults (67 ± 2 yr) participated in a 7-day bed rest study. We used stable isotope tracers, Western blotting, and real-time qPCR to determine the effect of bed rest on MPS, muscle mTORC1 signaling, and amino acid transporter expression and content in the postabsorptive state and after acute EAA ingestion. Bed rest decreased leg lean mass by ∼4% (P < 0.05) and increased postabsorptive mTOR protein (P < 0.05) levels while postabsorptive MPS was unchanged (P > 0.05). Before bed rest acute EAA ingestion increased MPS, mTOR (Ser(2448)), S6 kinase 1 (Thr(389), Thr(421)/Ser(424)), and ribosomal protein S6 (Ser(240/244)) phosphorylation, activating transcription factor 4, L-type amino acid transporter 1 and sodium-coupled amino acid transporter 2 protein content (P < 0.05). However, bed rest blunted the EAA-induced increase in MPS, mTORC1 signaling, and amino acid transporter protein content. We conclude that bed rest in older adults significantly attenuated the EAA-induced increase in MPS with a mechanism involving reduced mTORC1 signaling and amino acid transporter protein content. Together, our data suggest that a blunted EAA stimulation of MPS may contribute to muscle loss with inactivity in older persons.

Identification of SLC38A7 (SNAT7) Protein as a Glutamine Transporter Expressed in Neurons

The SLC38 family of transporters has in total 11 members in humans and they encode amino acid transporters called sodium-coupled amino acid transporters (SNAT). To date, five SNATs have been characterized and functionally subdivided into systems A (SLC38A1, SLC38A2, and SLC38A4) and N (SLC38A3 and SLC38A5) showing the highest transport for glutamine and alanine. Here we present identification of a novel glutamine transporter encoded by the Slc38a7 gene, which we propose should be named SNAT7. This transporter has L-glutamine as the preferred substrate but also transports other amino acids with polar side chains, as well as L-histidine and L-alanine. The expression pattern and substrate profile for SLC38A7 shows highest similarity to the known system N transporters. Therefore, we propose that SLC38A7 is a novel member of this system. We used in situ hybridization and immunohistochemistry with a custom-made antibody to show that SLC38A7 is expressed in all neurons, but not in astrocytes, in the mouse brain. SLC38A7 is unique in being the first system N transporter expressed in GABAergic and also other neurons. The preferred substrate and axonal localization of SLC38A7 close to the synaptic cleft indicates that SLC38A7 could have an important function for the reuptake and recycling of glutamate.

Cellular localization and protein concentration of the SNAT2 transporter in rat mammary gland during pregnancy and lactation

The sodium coupled amino acid transporter SNAT2 is expressed in mammary gland (MG). There is a special amino acid demand for cellular differentiation during pregnancy and for protein milk secretion during lactation in the MG. The purpose of the present work was to assess the amount and localization of SNAT along pregnancy and lactation in rat MG. Western blot analysis in MG of virgin rats showed the presence of a single band with a MW of 60 kD corresponding to SNAT2 protein. The intensity of this band was maintained during pregnancy and decreased dramatically one day before delivery. During lactation, SNAT2 increased rapidly after 5 days and remains constant along lactation and decreased after post-weaning. Immunohistochemical analysis revealed that MG SNAT2 of virgin rats was exclusively located in adipose cells. During gestation, there was an increase in the staining of SNAT2 in epithelial cells that will synthesize milk and decrease before delivery. During lactation there was a significant increase in the amount of SNAT2 in epithelial cells. Electron immunomicroscopy analysis of SNAT2 in MG during peak of lactation indicated that the SNAT2 protein was retained in vesicles originated from the endoplasmic reticulum and then translocated to the basolateral membrane of the epithelial cells. Our results suggested that presence of SNAT2 in the different cells and stages depends on the needs of the MG. CONACYT grant No 46134

A Conserved Na+ Binding Site of the Sodium-coupled Neutral Amino Acid Transporter 2 (SNAT2)

The SLC38 family of solute transporters mediates the coupled transport of amino acids and Na(+) into or out of cells. The structural basis for this coupled transport process is not known. Here, a profile-based sequence analysis approach was used, predicting a distant relationship with the SLC5/6 transporter families. Homology models using the LeuT(Aa) and Mhp1 transporters of known structure as templates were established, predicting the location of a conserved Na(+) binding site in the center of membrane helices 1 and 8. This homology model was tested experimentally in the SLC38 member SNAT2 by analyzing the effect of a mutation to Thr-384, which is predicted to be part of this Na(+) binding site. The results show that the T384A mutation not only inhibits the anion leak current, which requires Na(+) binding to SNAT2, but also dramatically lowers the Na(+) affinity of the transporter. This result is consistent with a previous analysis of the N82A mutant transporter, which has a similar effect on anion leak current and Na(+) binding and which is also expected to form part of the Na(+) binding site. In contrast, random mutations to other sites in the transporter had little or no effect on Na(+) affinity. Our results are consistent with a cation binding site formed by transmembrane helices 1 and 8 that is conserved among the SLC38 transporters as well as among many other bacterial and plant transporter families of unknown structure, which are homologous to SLC38.

Kinetics of glial glutamine efflux and the mechanism of neuronal uptake studied in vivo in mildly hyperammonemic rat brain

Kinetics of glial glutamine (GLN) transport to the extracellular fluid (ECF) and the mechanism of GLN(ECF) transport into the neuron--crucial pathways in the glutamine-glutamate cycle--were studied in vivo in mildly hyperammonemic rat brain, by NMR and microdialysis to monitor intra- and extracellular GLN. The minimum rate of glial GLN efflux, determined from the rate of GLN(ECF) increase during perfusion of alpha-(methylamino)isobutyrate (MeAIB), which inhibits neuronal GLN(ECF) uptake by sodium-coupled amino-acid transporter (SAT), was 2.88 +/- 0.22 micromol/g/h at steady-state brain [GLN] of 8.5 +/- 0.8 micromol/g. Our previous study showed that the rate of glutamine synthesis under identical experimental conditions was 3.3 +/- 0.3 micromol/g/h. At steady-state glial [GLN], this is equal to its efflux rate to the ECF. Comparison of the two rates suggests that SAT mediates at least 87 +/- 8% (= 2.88/3.3 x 100%) of neuronal GLN(ECF) uptake. While MeAIB induced > 2-fold elevation of GLN(ECF), no sustained elevation was observed during perfusion of the selective inhibitor of LAT, 2-amino-bicyclo[1,1,2]heptane-2-carboxylic acid (BCH), or of d-threonine, a putative selective inhibitor of ASCT2-mediated GLN uptake. The results strongly suggest that SAT is the predominant mediator of neuronal GLN(ECF) uptake in adult rat brain in vivo.

Suppression of glial glutamine release to the extracellular fluid studiedin vivoby NMR and microdialysis in hyperammonemic rat brain

Release of glial glutamine (GLN) to the extracellular fluid (ECF), mainly mediated by the bidirectional system N transporter SN1, was studied in vivo in hyperammonemic rat brain, using (15)N-nuclear magnetic resonance (NMR) to monitor intracellular [5-(15)N]GLN and microdialysis/gradient (1)H-(15)N heteronuclear single-quantum correlation NMR to analyse extracellular [5-(15)N]GLN. GLN(ECF) was elevated to 2.4 +/- 0.2 mm after 4.5 h of intravenous ammonium acetate infusion. The [GLN(i)]/[GLN(ECF)] ratio (i = intracellular) was 9.6 +/- 0.9, compared with 17-20 in normal brain. GLN(ECF) then decreased substantially at t = 4.9 +/- 0.1 h. Comparison of the time-courses of intra- and extra-cellular [5-(15)N]GLN strongly suggested that the observed decrease reflects partial suppression of glial GLN release to ECF. Suppression also followed elevation of GLN(ECF) to 1.9 mM, resulting in a [GLN](i)/[GLN(ECF)] ratio of 8.4, upon perfusion of alpha-(methylamino)isobutyrate which inhibits neuronal uptake of GLN(ECF) mediated by sodium-coupled amino acid transporter (SAT). The results provide first evidence for bidirectional operation of SN1 in vivo, and clarify the effect of transmembrane GLN gradient on glial GLN release at physiological Na(+) gradient. Implications of the results for SN1 as an additional regulatory site in the glutamine/glutamate cycle and utility of this approach for examining the role of GLN in an experimental model of fulminant hepatic failure are discussed.

Tyrosine phosphorylation-and epidermal growth factor-dependent regulation of the sodium-coupled amino acid transporter B 0 in the human placental choriocarcinoma cell line JAR

We have recently cloned an amino acid transporter from the human placental choriocarcinoma cell line JAR which, when functionally expressed in HeLa cells, induces an amino acid transport activity with characteristics known to be associated with the amino acid transport system B 0 (R. Kekuda, P.D. Prasad, Y.J. Fei, V. Torres-Zamorano, S. Sinha, T.L. Yang-Feng, F.H. Leibach, and V. Ganapathy, J. Biol. Chem. 271, 18657–18661, 1996). The presence of the amino acid transport system B 0 (ATB 0) has however not been previously described in these cells by functional studies. In the present investigation, we have obtained evidence for the existence of ATB 0 in JAR cells and delineated the functional characteristics of the transporter. The identifying characteristics include Na +-dependence and preference for neutral amino acids. In addition, we have used the JAR cells as a model system to investigate the regulatory aspects of ATB 0. Treatment of the cells with the neuroprotective agent aurintricarboxylic acid (ATA) for 16 h leads to a significant increase in ATB 0 activity. This increase is associated with enhanced maximal velocity of the transporter and with increased steady state levels of the transporter mRNA. The effect of ATA is blocked by the tyrosine kinase inhibitor genistein. ATA treatment results in increased tyrosine phosphorylation of two major proteins, 180 kDa and 140 kDa in size. The 180 kDa protein is likely to be the epidermal growth factor (EGF) receptor because exposure of the cells to EGF also leads to enhanced tyrosine phosphorylation of a protein of similar molecular size. Furthermore, the effects of ATA on ATB 0 activity and on ATB 0 mRNA levels can be reproduced by EGF. Treatment of the cells with EGF for 24 h results in a significant increase in ATB 0 activity and this effect is associated with an increase in the maximal velocity of the transporter and with an increase in the steady state levels of the transporter mRNA. These data suggest that ATA influences ATB 0 activity in JAR cells most likely by activating the EGF receptor through tyrosine phosphorylation. It is concluded that the human placental choriocarcinoma cells functionally express the amino acid transport system B 0 and that the expression of the system in these cells is stimulated by EGF.© 1997 Elsevier Science B.V. All rights reserved.

In vivo amino acid fluxes in regenerating liver after two-thirds hepatectomy in the rat

Background/Aims: Recent reports in the literaturesuggest that liver cell swelling following amino acid influx exerts anabolic and anti-catabolic effects. We have tested the possibility that rapid liver growth after partial hepatectomy is promoted by an increased amino acid-influx and that this is associated with an increased hepatic water content and a decreased rate of proteolysis. Methods: Two-thirds hepatectomy was performed in rats. Plasma liver flow and amino acid fluxes were measured after 24 or 48 h. Results: Plasma liver flow was increased 24 and 48 h after partial hepatectomy or sham-operation in pair-fed animals. At these time points, in both groups there was a specific two- to threefold increased net hepatic uptake of the amino acids alanine and glycine, both being transported by the sodium-coupled amino acid transport system A/ASC. No changes in uptake of system N transported amino acids were observed. Both in partially hepatectomized and sham-operated pair-fed animals, the hepatic uptake of alanine and glycine was accompanied by a minor increase in tissue water (from 68 to 70%). Proteolysis, measured by leucine efflux, was only reduced in regenerating livers. Conclusions: We conclude that cell swelling is not animportant factor in the stimulation of net protein synthesis during liver regeneration.

In vivo amino acid fluxes in regenerating liver after two-thirds hepatectomy in the rat

<h2>Abstract</h2><h3>Background/Aims:</h3> Recent reports in the literaturesuggest that liver cell swelling following amino acid influx exerts anabolic and anti-catabolic effects. We have tested the possibility that rapid liver growth after partial hepatectomy is promoted by an increased amino acid-influx and that this is associated with an increased hepatic water content and a decreased rate of proteolysis. <h3>Methods:</h3> Two-thirds hepatectomy was performed in rats. Plasma liver flow and amino acid fluxes were measured after 24 or 48 h. <h3>Results:</h3> Plasma liver flow was increased 24 and 48 h after partial hepatectomy or sham-operation in pair-fed animals. At these time points, in both groups there was a specific two- to threefold increased net hepatic uptake of the amino acids alanine and glycine, both being transported by the sodium-coupled amino acid transport system A/ASC. No changes in uptake of system N transported amino acids were observed. Both in partially hepatectomized and sham-operated pair-fed animals, the hepatic uptake of alanine and glycine was accompanied by a minor increase in tissue water (from 68 to 70%). Proteolysis, measured by leucine efflux, was only reduced in regenerating livers. <h3>Conclusions:</h3> We conclude that cell swelling is not animportant factor in the stimulation of net protein synthesis during liver regeneration.

Sodium-coupled amino acid transport in renal tubule

Amino acids are reabsorbed from the tubular lumen by a saturable, carrier-mediated, concentrative transport mechanism driven by a Na+ electrochemical gradient across the luminal membrane. This process is followed by efflux mainly via carrier-mediated, Na+-independent facilitated diffusion across the basolateral membrane. Individual amino acids may have two or more Na+-dependent transport systems with different kinetic characteristics along the luminal membrane of the proximal tubule, thereby enabling very efficient amino acid reabsorption. Dual Na+-coupled transport pathways for some amino acids located in both the luminal and the peritubular membranes may operate in concert to provide the tubular epithelial cell with essential nutrients. One or more Na+ ions, H+, Cl- and in the case of acidic amino acids, K+ ion, may be involved in the translocation of the carrier complex. For most amino acids this process is electrogenic positive, favored by a negative cell interior. At least seven distinct, but largely interacting, Na+-dependent amino acid transport systems have been identified in the brush border membrane. A diet-induced adaptation in Na+-coupled taurine transport and acidosis-induced adaptive response in Na+-dependent glutamine transport are expressed at the luminal and the basolateral membrane surfaces, respectively. The aminoaciduria of early life may be related to a rapid dissipation of the Na+ electrochemical gradient necessary for amino acid reabsorption.

Kinetic and Thermodynamic Aspects of Sodium-Coupled Amino Acid Transport by Marine Invertebrates

SYNOPSIS. Marine invertebrates absorb amino acids directly across their external body surfaces. This absorption process occurs via carrier-mediated transport systems which, recent evidence suggests, may be sodium-dependent. Steady-state amino acid gradients are maintained at levels exceeding 103–106 times that of the external environment. Examination of the standing gradients of total free amino acids, Na, and K in seven invertebrates suggests that an Na/amino acid cotransport model, or an Na/K/amino acid cotransport model can account for amino acid gradients of this magnitude. However, the Na : amino acid coupling coefficients must be 2 or 3 depending on factors such as membrane potential and the intracellular Na activities. Evidence from studies of L-alanine transport in the integument of the polychaete Glycera dibranchiata is presented showing that, for this case, the Na : alanine coupling coefficient is 3. It is concluded that the models presented are plausible and readily testable explanations for the observed amino acid gradients in marine invertebrates.

Alterations in membrane permeability with trypsin treatment.

Trypsin treatment of Ehrlich cells reduced sodium-coupled amino acid transport, lowered ATP levels, and abolished cation gradients. The data suggest that the decrease in active amino acid transport results from an alteration in membrane permeability caused by DNA released from a fraction of the cells upon trypsin treatment. The reduced amino acid transport appears to be the result of an abolition of ion gradients required for transport since vesicles prepared from these cells show near normal transport activity. The altered permeability of the membrane can be restored by incubation of the cells with serum. Protein synthesis does not appear to play a role in the restoration of Na+-dependent amino acid transport since restoration is unaffected by the presence of cycloheximide. The recovery of activity does depend partly on the presence of Ca2+ in the incubation medium since reversal of the trypsin inhibition of glycine transport can be obtained on incubation with Ca2+ and glucose.