Expression of glutamine metabolism-related proteins in Hürthle cell neoplasm of thyroid: Comparison with follicular neoplasm.

PURPOSE: We evaluated the expression of glutaminolysis-related proteins in Hurthle cell neoplasms (HCN) and follicular neoplasms (FN) of the thyroid, and investigated its clinical implication. METHODS: Tissue microarrays were constructed from 264 FNs (112 follicular carcinomas [FCs] and 152 follicular adenomas [FAs]) and 108 HCNs (27 Hurthle cell carcinomas [HCCs] and 81 Hurthle cell adenomas [HCAs]. The immunohistochemical staining result of 3 glutaminolysis-related proteins (Glutaminase 1 [GLS1], glutaminate dehydrogenase [GDH] and alanine- serine, cysteine-preferring transporter 2 [ASCT2]) was analyzed. RESULTS: GLS1 and GDH showed significantly higher expression rates in HCN compared to FN (P<0.001). More HCN cases showed co-positivity of multiple glutaminolysis-related proteins than those of FN cases (P<0.001). In silico analysis, both GLUD1 and GLUD2 showed higher expression rate in HCA compared to FA (P=0.027 and P=0.018, respectively). SLC1A5 expression was highest in HCA, followed by FC and FA (HCA vs FC, P=0.023; FC vs FA, P=0.002). CONCLUSION: FN and HCN exhibit a different expression pattern for glutaminolysis-related proteins, and GLS1 and GDH have higher expression rates in HCN and FN.

Overexpression of SLC1a5 in lymph node metastases outperforms assessment in the primary as a negative prognosticator in non-small cell lung cancer.

Despite recent advances in therapeutic options, lung cancer is the leading cause of death among malignant diseases worldwide. Glutamine-dependence is an established attribute in cancer tissue with emerging importance as a diagnostic and therapeutic target. We analysed the expression of SLC1a5, a major glutamine transporter, in the primary tumour and corresponding nodal metastasis of non-small cell lung cancer (NSCLC) to investigate its biological impact. Expression of SLC1a5 was analysed by immunohistochemistry in 259 NSCLC and in 142 nodal metastases and correlated with clinicopathological parameters including overall survival. SLC1a5 expression in the primary tumour and in the corresponding lymph node metastasis revealed a positive correlation (p = 0.005). Moreover, overexpression of SLC1a5 was found to be an independent prognostic factor (p = 0.027) if assessed in lymph node metastases only. SLC1A5 expression was studied for the first time in both primary NSCLC and its corresponding nodal metastasis. Our results indicate that overexpression of SLC1a5 is associated with shorter overall survival. This proved to be an independent prognosticator if assessed in the lymph node metastases. Thus, diagnostics in lymph node metastasis provide superior prognostic information for SLC1a5 overexpression and may open target prediction for future therapeutic options.

Expression of glutamine metabolism-related proteins in thyroid cancer.

PURPOSE: This study aimed to investigate the expression of glutamine metabolism-related protein in tumor and stromal compartments among the histologic subtypes of thyroid cancer. RESULTS: GLS1 and GDH expression in tumor and stromal compartments were the highest in AC than in other subtypes. Tumoral ASCT2 expression was higher in MC but lower in FC (p < 0.001). In PTC, tumoral GLS1 and tumoral GDH expression was higher in the conventional type than in the follicular variant (p = 0.043 and 0.001, respectively), and in PTC with BRAF V600E mutation than in PTC without BRAF V600E mutation (p<0.001). Stromal GDH positivity was the independent factor associated with short overall survival (hazard ratio: 21.48, 95% confidence interval: 2.178-211.8, p = 0.009). METHODS: We performed tissue microarrays with 557 thyroid cancer cases (papillary thyroid carcinoma [PTC]: 344, follicular carcinoma [FC]: 112, medullary carcinoma [MC]: 70, poorly differentiated carcinoma [PDC]: 23, and anaplastic carcinoma [AC]: 8) and 152 follicular adenoma (FA) cases. We performed immunohistochemical staining of glutaminolysis-related proteins (glutaminase 1 [GLS1], glutamate dehydrogenase [GDH], and amino acid transporter-2 [ASCT-2]). CONCLUSION: Glutamine metabolism-related protein expression differed among the histologic subtypes of thyroid cancer.

Clinicopathological significance of LAT1 and ASCT2 in patients with surgically resected esophageal squamous cell carcinoma.

BACKGROUND: Amino acid transporters are highly expressed in various human cancers. L-type amino acid transporter 1 (LAT1) and system alanine-serine-cysteine amino acid transporter-2 (ASCT2) play a crucial role in tumor progression and survival. However, the clinicopathological significance of these transporters in patients with esophageal squamous cell carcinoma (ESCC) remains unclear. METHODS: One hundred and fifty-seven patients with surgically resected ESCC were evaluated. Immunohistochemical analysis was performed for LAT1, ASCT2, CD98, Ki-67, and micro-vessel density (MVD), as determined by CD34 expression. RESULTS: LAT1 and ASCT2 were positively expressed in 59% (93/157) and 48% (76/157) of tumors respectively. LAT1 and ASCT2 expression significantly correlated with T factor, N factor, lymphatic permeation, vascular invasion, and CD98 expression. The 5-year survival rates of LAT1-high and -low and ASCT2-high and -low expressing patients were 62.0% and 69.6% (P < 0.05) and 59.6% and 70.1% (P = 0.068), respectively. The combined positive expression of LAT1 and ASCT2 was a significant prognostic factor in univariate analysis. CONCLUSION: High expression of LAT1 and ASCT2 correlates with metastasis and invasion. Accordingly, these proteins could serve as prognostic biomarkers and therapeutic targets for treating patients with surgically resectable ESCC. J. Surg. Oncol. 2016;113:381-389. © 2016 Wiley Periodicals, Inc.

Altered expression of human endogenous retroviruses syncytin-1, syncytin-2 and their receptors in human normal and gestational diabetic placenta.

INTRODUCTION: Syncytins belong to the Human Endogenous Retrovirus family. The syncytin-1 receptor, SLC1A5, and syncytin-2 receptor, MFSD2, interact with their respective syncytin proteins to induce syncytiotrophoblast formation. However, there is no information about syncytins in gestational diabetic placenta. Therefore, we studied the expression and localization of syncytins and their receptors during normal placental development and in gestational diabetic placenta. METHODS: Immunohistochemistry and Western-blot methods were performed with antibodies against syncytin-1, syncytin-2, SLC1A5 and MFSD2 in human first trimester placental tissues, normal term and gestational diabetic placentas. Syncytin-1, syncytin-2 and MFSD2 mRNA transcripts were determined by qRT-PCR in normal and diabetic term placentas. RESULTS: Cytoplasmic syncytin-1, syncytin-2, SLC1A5 and MFSD2 immunoreactions were observed in the trophoblastic layers in all placental samples. Some of the stromal cells showed strong cytoplasmic punctate staining. There were significantly weak syncytin-2 and MFSD2 immunoreaction intensities in diabetic placentas by ImageJ analysis, in parallel with decreased syncytin-2 and MFSD2 proteins in diabetic placentas by Western-blot. Protein expression of SLC1A5 increased dramatically in early pregnancy compared to term placenta. Syncytin-1, syncytin-2 and MFSD2 mRNA transcripts showed similar relative expression pattern by qRT-PCR. DISCUSSION: Syncytins were localized not only in cytotrophoblast cells and the basement membrane of the syncytiotrophoblast but also in the apical microvillous membrane and cytoplasm of syncytiotrophoblast, and some of the stromal cells and endothelium. Decreased syncytin-2 and MFSD2 proteins in gestational diabetic placentas might cause abnormal syncytiotrophoblast formation and possibly be involved in the pathology. Therefore, our study highlights an important potential relationship between syncytins and gestational diabetic placenta.

Glutaminolysis and carcinogenesis of oral squamous cell carcinoma.

Glutaminolysis is a crucial factor for tumor metabolism in the carcinogenesis of several tumors but has not been clarified for oral squamous cell carcinoma (OSCC) yet. Expression of glutaminolysis-related solute carrier family 1, member 5 (SLC1A5)/neutral amino acid transporter (ASCT2), glutaminase (GLS), and glutamate dehydrogenase (GLDH) was analyzed in normal oral mucosa (n = 5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 42) by immunohistochemistry. SLC1A5/ASCT2 and GLS were significantly overexpressed in the carcinogenesis of OSCC compared with normal tissue, while GLDH was weakly detected. Compared with SIN I-III SLC1A5/ASCT2 and GLS expression were significantly increased in OSCC. GLDH expression did not significantly differ from SIN I-III compared with OSCC. This study shows the first evidence of glutaminolysis-related SLC1A5/ASCT2, GLS, and GLDH expression in OSCC. The very weak GLDH expression indicates that glutamine metabolism is rather related to nucleotide or protein/hexosamine biosynthesis or to the function as an antioxidant (glutathione) than to energy production or generation of lactate through entering the tricarboxylic acid cycle. Overcoming glutaminolysis by targeting c-Myc oncogene (e.g. by natural compounds) and thereby cross-activation of mammalian target of rapamycin complex 1 or SLC1A5/ASCT2, GLS inhibitors may be a useful strategy to sensitize cancer cells to common OSCC cancer therapies.

Targeting SLC1a5-mediated glutamine dependence in non-small cell lung cancer.

We previously elucidated the pleotropic role of solute carrier family A1 member 5 (SLC1A5) as the primary transporter of glutamine (Gln), a modulator of cell growth and oxidative stress in non-small cell lung cancer (NSCLC). The aim of our study was to evaluate SLC1A5 as a potential new therapeutic target and candidate biomarker predictive of survival and response to therapy. SLC1A5 targeting was examined in a panel of NSCLC and human bronchial cell lines by RNA interference and by a small molecular inhibitor, gamma-l-glutamyl-p-nitroanilide (GPNA). The effects of targeting SLC1A5 on cell growth, Gln uptake, ATP level, autophagy and cell death were examined. Inactivation of SLC1A5 genetically or pharmacologically decreased Gln consumption, inhibited cell growth, induced autophagy and apoptosis in a subgroup of NSCLC cell lines that overexpress SLC1A5. Targeting SLC1A5 function decreased tumor growth in NSCLC xenografts. A multivariate Cox proportional hazards analysis indicates that patients with increased SLC1A5 mRNA expression have significantly shorter overall survival (p = 0.01, HR = 1.24, 95% CI: 1.05-1.46), adjusted for age, gender, smoking history and disease stage. In an immunohistochemistry study on 207 NSCLC patients, SLC1A5 protein expression remained highly significant prognostic value in both univariate (p < 0.0001, HR = 1.45, 95% CI: 1.15-1.50) and multivariate analyses (p = 0.04, HR = 1.22, 95% CI: 1.01-1.31). These results position SLC1A5 as a new candidate prognostic biomarker for selective targeting of Gln-dependent NSCLC.

Clinicopathological significance of ASC amino acid transporter-2 expression in pancreatic ductal carcinoma.

AIMS: ASC amino acid transporter-2 (ASCT2) is highly expressed in cancer cells. However, the clinicopathological significance of ASCT2 expression in pancreatic cancer remains unclear. The aim of this study was to investigate the clinical significance of ASCT2 expression in pancreatic cancer. METHODS AND RESULTS: Ninety-seven patients with surgically resected pancreatic ductal adenocarcinoma were evaluated. Tumour sections were stained by immunohistochemistry for ASCT2, Ki67, CD34 (to determine microvessel density), phospho-AKT (p-AKT) and phospho-mammalian target of rapamycin (p-mTOR) expression. ASCT2 was expressed in 54% (52/97) of tumours. Statistically significant differences in patient age, T stage, N stage, lymphatic permeation, vascular invasion, Ki67, and CD34 and p-mTOR expression were observed between tumours with and without ASCT2 expression. Multivariate analysis confirmed that vascular invasion, ASCT2 expression and Ki67 expression were independent predictive factors for a poorer prognosis. CONCLUSIONS: ASCT2 expression plays an important role in tumour cell growth, and is a promising pathological marker for predicting a worse outcome in pancreatic cancer.

Targeting glutamine transport to suppress melanoma cell growth.

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAF(WT) (C8161 and WM852) and BRAF(V600E) mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2-mediated glutamine transport is a potential therapeutic target for both BRAF(WT) and BRAF(V600E) melanoma.

Control of glutamine metabolism by the tumor suppressor Rb.

Retinoblastoma (Rb) protein is a tumor suppressor that is dysregulated in a majority of human cancers. Rb functions to inhibit cell cycle progression in part by directly disabling the E2F family of cell cycle-promoting transcription factors. Because the de novo synthesis of multiple glutamine-derived anabolic precursors is required for cell cycle progression, we hypothesized that Rb also may directly regulate proteins involved in glutamine metabolism. We examined glutamine metabolism in mouse embryonic fibroblasts (MEFs) isolated from mice that have triple knock-outs (TKO) of all three Rb family members (Rb-1, Rbl1 and Rbl2) and found that loss of global Rb function caused a marked increase in (13)C-glutamine uptake and incorporation into glutamate and tricarboxylic acid cycle (TCA) intermediates in part via upregulated expression of the glutamine transporter ASCT2 and the activity of glutaminase 1 (GLS1). The Rb-controlled transcription factor E2F-3 altered glutamine uptake by direct regulation of ASCT2 mRNA and protein expression, and E2F-3 was observed to associate with the ASCT2 promoter. We next examined the functional consequences of the observed increase in glutamine uptake and utilization and found that glutamine exposure potently increased oxygen consumption, whereas glutamine deprivation selectively decreased ATP concentration in the Rb TKO MEFs but not the wild-type (WT) MEFs. In addition, TKO MEFs exhibited elevated production of glutathione from exogenous glutamine and had increased expression of gamma-glutamylcysteine ligase relative to WT MEFs. Importantly, this metabolic shift towards glutamine utilization was required for the proliferation of Rb TKO MEFs but not for the proliferation of the WT MEFs. Last, addition of the TCA cycle intermediate α-ketoglutarate to the Rb TKO MEFs reversed the inhibitory effects of glutamine deprivation on ATP, GSH levels and viability. Taken together, these studies demonstrate that the Rb/E2F cascade directly regulates a major energetic and anabolic pathway that is required for neoplastic growth.

Inactivation of the glutamine/amino acid transporter ASCT2 by 1,2,3-dithiazoles: proteoliposomes as a tool to gain insights in the molecular mechanism of action and of antitumor activity.

The ASCT2 transport system catalyses a sodium-dependent antiport of glutamine and other neutral amino acids which is involved in amino acid metabolism. A library of 1,2,3-dithiazoles was designed, synthesized and evaluated as inhibitors of the glutamine/amino acid ASCT2 transporter in the model system of proteoliposomes reconstituted with the rat liver transporter. Fifteen of the tested compounds at concentration of 20µM or below, inhibited more than 50% the glutamine/glutamine antiport catalysed by the reconstituted transporter. These good inhibitors bear a phenyl ring with electron withdrawing substituents. The inhibition was reversed by 1,4-dithioerythritol indicating that the effect was likely owed to the formation of mixed sulfides with the protein's Cys residue(s). A dose-response analysis of the most active compounds gave IC(50) values in the range of 3-30µM. Kinetic inhibition studies indicated a non-competitive inhibition, presumably because of a potential covalent interaction of the dithiazoles with cysteine thiol groups that are not located at the substrate binding site. Indeed, computational studies using a homology structural model of ASCT2 transporter, suggested as possible binding targets, Cys-207 or Cys-210, that belong to the CXXC motif of the protein.

Francisella tularensis regulates the expression of the amino acid transporter SLC1A5 in infected THP-1 human monocytes.

Francisella tularensis, a Gram-negative bacterium that causes the disease tularemia in a large number of animal species, is thought to reside preferentially within macrophages in vivo. F. tularensis has developed mechanisms to rapidly escape from the phagosome into the cytoplasm of infected cells, a habitat with a rich supply of nutrients, ideal for multiplication. SLC1A5 is a neutral amino acid transporter expressed by human cells, which serves, along with SLC7A5 to equilibrate cytoplasmic amino acid pools. We herein analysed whether SLC1A5 was involved in F. tularensis intracellular multiplication. We demonstrate that expression of SLC1A5 is specifically upregulated by F. tularensis in infected THP-1 human monocytes. Furthermore, we show that SLC1A5 downregulation decreases intracellular bacterial multiplication, supporting the involvement of SLC1A5 in F. tularensis infection. Notably, after entry of F. tularensis into cells and during the whole infection, the highly glycosylated form of SLC1A5 was deglycosylated only by bacteria capable of cytosolic multiplication. These data suggest that intracellular replication of F. tularensis depends on the function of host cell SLC1A5. Our results are the first, which show that Francisella intracellular multiplication in human monocyte cytoplasm is associated with a post-translational modification of a eukaryotic amino acid transporter.

Disruption of astrocytic glutamine turnover by manganese is mediated by the protein kinase C pathway.

Manganese (Mn) is a trace element essential for normal human development and is required for the proper functioning of a variety of physiological processes. Chronic exposure to Mn can cause manganism, a neurodegenerative disorder resembling idiopathic Parkinson's disease (PD). Mn(II) neurotoxicity is characterized by astrocytic impairment both in the expression and activity of glutamine (Gln) transporters. Because protein kinase C (PKC) activation leads to the downregulation of a number of neurotransmitter transporters and Mn(II) increases PKC activity, we hypothesized that the PKC signaling pathway contributes to the Mn(II)-mediated disruption of Gln turnover. Our results have shown that Mn exposure increases the phosphorylation of both the PKCα and PKCδ isoforms. PKC activity was also shown to be increased in response to Mn(II) treatment. Corroborating our earlier observations, Mn(II) also caused a decrease in Gln uptake. This effect was blocked by PKC inhibitors. Notably, PKC activation caused a decrease in Gln uptake mediated by systems ASC and N, but had no effect on the activities of systems A and L. Exposure to α-phorbol 12-myristate 13-acetate significantly decreased SNAT3 (system N) and ASCT2 (system ASC) protein levels. Additionally, a co-immunoprecipitation study demonstrated the association of SNAT3 and ASCT2 with the PKCδ isoform, and Western blotting revealed the Mn(II)-mediated activation of PKCδ by proteolytic cleavage. PKC activation was also found to increase SNAT3 and ubiquitin ligase Nedd4-2 binding and to induce hyperubiquitination. Taken together, these findings demonstrate that the Mn(II)-induced dysregulation of Gln homeostasis in astrocytes involves PKCδ signaling accompanied by an increase in ubiquitin-mediated proteolysis.

Age-related changes in the renal dopaminergic system and expression of renal amino acid transporters in WKY and SHR rats.

This study examined age-related changes in renal dopaminergic activity and expression of amino acid transporters potentially involved in renal tubular uptake of l-DOPA in Wistar Kyoto (WKY) and spontaneously hypertensive rats. Aging (from 13 to 91 weeks) was accompanied by increases in systolic blood pressure (SBP) in both WKY and SHR. The sum of urinary dopamine and DOPAC and the urinary dopamine/l-DOPA ratio were increased in aged SHR but not in aged WKY. The urinary dopamine/renal delivery of l-DOPA ratio was increased in both rat strains with aging. LAT2 abundance was increased in aged WKY and SHR. The expression of 4F2hc was markedly elevated in aged SHR but not in aged WKY. ASCT2 was upregulated in both aged WKY and SHR. Plasma aldosterone levels and urinary noradrenaline levels were increased in aged WKY and SHR though levels of both entities were more elevated in aged SHR. Activation of the renal dopaminergic system is more pronounced in aged SHR than in aged WKY and is associated with an upregulation of renal cortical ASCT2 in WKY and of LAT2/4F2hc and ASCT2 in SHR. This activation may be the consequence of a counter-regulatory mechanism for stimuli leading to sodium reabsorption.

Manganese disrupts astrocyte glutamine transporter expression and function.

Glutamine (Gln) plays an important role in brain energy metabolism and as a precursor for the synthesis of neurotransmitter glutamate and GABA. Previous studies have shown that astrocytic Gln transport is impaired following manganese (Mn) exposure. The present studies were performed to identify the transport routes and the respective Gln transporters contributing to the impairment. Rat neonatal cortical primary astrocytes treated with Mn displayed a significant decrease in Gln uptake mediated by the principle Gln transporting systems, N and ASC. Moreover, systems N, ASC and L were less efficient in Gln export after Mn treatment. Mn treatment caused a significant reduction of both in mRNA expression and protein levels of SNAT3 (system N), SNAT2 (system A) and LAT2 (system L), and lowered the protein but not mRNA expression of ASCT2 (system ASC). Mn exposure did not affect the expression of the less abundant systems N transporter SNAT5 and the system L transporter LAT1, at either the mRNA or protein level. Hence, Mn-induced decrease of inward and outward Gln transport can be largely ascribed to the loss of the specific Gln transporters. Consequently, deregulation of glutamate homeostasis and its diminished availability to neurons may lead to impairment in glutamatergic neurotransmission, a phenomenon characteristic of Mn-induced neurotoxicity.

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.

Segmental and complementary expression of L-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase and neutral amino acid transporter ASCT1 in the mouse kidney.

3-Phosphoglycerate dehydrogenase (Phgdh) is the initial step enzyme in the phosphorylated pathway of L-serine biosynthesis. We have previously revealed in the brain that Phgdh is preferentially expressed in glial cells, but not in neurons, and that glia-borne L-serine exerts strong neurotrophic actions to neuronal survive, differentiation, and development. To investigate whether such an L-serine-meditated intercellular relationship is constructed in peripheral organs and tissues, we examined the kidney, which is one of the organs with the highest expression of Phgdh mRNA in the body. We found that Phgdh was distributed highly in the renal papilla and inner layer of the outer zone and moderately in the cortex, whereas it was almost negative in the outer layer of the outer zone. This heterogeneous distribution was due to selective expression in distinct tubular segments, i.e., the Bowman's capsule, proximal tubule, and thin limbs of the Henle's loop. Interestingly, neutral amino acid transporter ASCT1, which preferentially transports alanine, serine, cysteine, and threonine, was selectively expressed in Phgdh-negative tubular segments, i.e., the distal tubule and collecting duct. Therefore, either Phgdh or ASCT1 is provided to each segment of renal tubules, suggesting that metabolic interplay mediated by L-serine biosynthesis and supply may exist in the kidney too.

Underexpression of the Na+-dependent neutral amino acid transporter ASCT2 in the spontaneously hypertensive rat kidney.

This study examined the inward transport of l-[(14)C]alanine, an ASCT2 preferential substrate, in monolayers of immortalized renal proximal tubular epithelial (PTE) cells from Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. The expression of ASCT2 in WKY and SHR PTE cells and kidney cortices from WKY and SHR was also evaluated. l-[(14)C]alanine uptake was highly dependent on extracellular Na(+). Replacement of NaCl by LiCl or choline chloride abolished transport activity in SHR and WKY PTE cells. In the presence of the system L inhibitor BCH, Na(+)-dependent l-alanine uptake in WKY and SHR PTE cells was inhibited by alanine, serine, and cysteine, which is consistent with amino acid transport through ASCT2. The saturable component of Na(+)-dependent l-alanine transport under V(max) conditions in SHR PTE cells was one-half of that in WKY PTE cells, with similar K(m) values. Differences in magnitude of Na(+)-dependent l-alanine uptake through ASCT2 between WKY and SHR PTE cells correlated positively with differences in ASCT2 protein expression, this being more abundant in WKY PTE cells. Abundance of ASCT2 transcript and protein in kidney cortices of SHR rats was also lower than that in normotensive WKY rats. In conclusion, immortalized SHR and WKY PTE cells take up l-alanine mainly through a high-affinity Na(+)-dependent amino acid transporter, with functional features of ASCT2 transport. The activity and expression of the ASCT2 transporter were considerably lower in the SHR cells.

Manganese induces oxidative impairment in cultured rat astrocytes.

Excessive free radical formation has been implicated as a causative factor in neurotoxic damage associated with exposures to a variety of metals, including manganese (Mn). It is well established that Mn accumulates in astrocytes, affecting their ability to indirectly induce and/or exacerbate neuronal dysfunction. The present study examined the effects of Mn treatment on the following endpoints in primary astrocyte cultures: (1) oxidative injury, (2) alterations in high-energy phosphate (adenosine 5'-triphosphate, ATP) levels, (3) mitochondrial inner membrane potential, and (4) glutamine uptake and the expression of glutamine transporters. We quantified astrocyte cerebral oxidative damage by measuring F(2)-isoprostanes (F(2)-IsoPs) using stable isotope dilution methods followed by gas chromatography-mass spectrometry with selective ion monitoring. Our data showed a significant (p < 0.01) elevation in F(2)-IsoPs levels at 2 h following exposure to Mn (100 microM, 500 microM, or 1 mM). Consistent with this observation, Mn induced a concentration-dependent reduction in ATP and the inner mitochondrial membrane potential (DeltaPsi(m)), measured by the high pressure liquid chromatography method and the potentiometric dye, tetramethyl rhodamine ethyl ester, respectively. Moreover, 30 min of pretreatment with Mn (100 microM, 500 microM, or 1 mM) inhibited the net uptake of glutamine (GLN) ((3)H-glutamine) measured at 1 and 5 min. Expression of the messenger RNA coding the GLN transporters, SNAT3/SN1 and SNAT1, was inhibited after 100 and 500 microM Mn treatment for 24 h. Our results demonstrate that induction of oxidative stress, associated mitochondrial dysfunction, and alterations in GLN/glutamate cycling in astrocytes represent key mechanisms by which Mn exerts its neurotoxicity.

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.