Air pollution exposure increases ABCB1 and ASCT1 transporter levels in mouse cortex.

Membrane transporters are important for maintaining brain homeostasis by regulating the passage of solutes into, out of, and within the brain. Growing evidence suggests neurotoxic effects of air pollution exposure and its contribution to neurodegenerative disorders, including Alzheimer's disease (AD), yet limited knowledge is available on the exact cellular impacts of exposure. This study investigates how exposure to ubiquitous solid components of air pollution, ultrafine particles (UFPs), influence brain homeostasis by affecting protein levels of membrane transporters. Membrane transporters were quantified and compared in brain cortical samples of wild-type and the 5xFAD mouse model of AD in response to subacute exposure to inhaled UFPs. The cortical ASCT1 and ABCB1 transporter levels were elevated in wild-type and 5xFAD mice subjected to a 2-week UFP exposure paradigm, suggesting impairment of brain homeostatic mechanisms. This study provides new insight on the molecular mechanisms underlying adverse effects of air pollution on the brain.

Functional analysis of OCTN2 and ATB0,+ in normal human airway epithelial cells.

In human, OCTN2 (SLC22A5) and ATB0,+ (SLC6A14) transporters mediate the uptake of L-carnitine, essential for the transport of fatty acids into mitochondria and the subsequent degradation by ß-oxidation. Aim of the present study was to characterize L-carnitine transport in EpiAirway™, a 3D organotypic in vitro model of primary human tracheal-bronchial epithelial cells that form a fully differentiated, pseudostratified columnar epithelium at air-liquid interface (ALI) condition. In parallel, Calu-3 monolayers grown at ALI for different times (8d or 21d of culture) were used as comparison. OCTN2 transporter was equally expressed in both models and functional at the basolateral side. ATB0,+ was, instead, highly expressed and active on the apical membrane of EpiAirway™ and only in early-cultures of Calu-3 (8d but not 21d ALI). In both cell models, L-carnitine uptake on the apical side was significantly inhibited by the bronchodilators glycopyrrolate and tiotropium, that hence can be considered substrates of ATB0,+; ipratropium was instead effective on the basolateral side, indicating its interaction with OCTN2. Inflammatory stimuli, such as LPS or TNFα, caused an induction of SLC6A14/ATB0,+ expression in Calu-3 cells, along with a 2-fold increase of L-carnitine uptake only at the apical side; on the contrary SLC22A5/OCTN2 was not affected. As both OCTN2 and ATB0,+, beyond transporting L-carnitine, have a significant potential as delivery systems for drugs, the identification of these transporters in EpiAirway™ can open new fields of investigation in the study of drug inhalation and pulmonary delivery.

Increased Glutaminolysis Marks Active Scarring in Nonalcoholic Steatohepatitis Progression.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) occurs in the context of aberrant metabolism. Glutaminolysis is required for metabolic reprograming of hepatic stellate cells (HSCs) and liver fibrogenesis in mice. However, it is unclear how changes in HSC glutamine metabolism contribute to net changes in hepatic glutaminolytic activity during fibrosis progression, or whether this could be used to track fibrogenic activity in NASH. We postulated that increased HSC glutaminolysis marks active scarring in NASH. METHODS: Glutaminolysis was assessed in mouse NASH fibrosis models and in NASH patients. Serum and liver levels of glutamine and glutamate and hepatic expression of glutamine transporter/metabolic enzymes were correlated with each other and with fibrosis severity. Glutaminolysis was disrupted in HSCs to examine if this directly influenced fibrogenesis. 18F-fluoroglutamine positron emission tomography was used to determine how liver glutamine assimilation tracked with hepatic fibrogenic activity in situ. RESULTS: The serum glutamate/glutamine ratio increased and correlated with its hepatic ratio, myofibroblast content, and fibrosis severity. Healthy livers almost exclusively expressed liver-type glutaminase (Gls2); Gls2 protein localized in zone 1 hepatocytes, whereas glutamine synthase was restricted to zone 3 hepatocytes. In fibrotic livers, Gls2 levels reduced and glutamine synthase zonality was lost, but both Slc1a5 (glutamine transporter) and kidney-type Gls1 were up-regulated; Gls1 protein was restricted to stromal cells and accumulated in fibrotic septa. Hepatocytes did not compensate for decreased Gls2 by inducing Gls1. Limiting glutamine or directly inhibiting GLS1 inhibited growth and fibrogenic activity in cultured human HSCs. Compared with healthy livers, fibrotic livers were 18F-fluoroglutamine-avid by positron emission tomography, suggesting that glutamine-addicted myofibroblasts drive increased hepatic utilization of glutamine as fibrosis progresses. CONCLUSIONS: Glutaminolysis is a potential diagnostic marker and therapeutic target during NASH fibrosis progression.

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.

Prognostic significance of amino-acid transporter expression (LAT1, ASCT2, and xCT) in surgically resected tongue cancer.

BACKGROUND: Amino-acid transporters are necessary for the tumour cell growth and survival, and have a crucial role in the development and invasiveness of cancer cells. But, it remains unclear about the prognostic significance of L-type amino-acid transporter 1 (LAT1), system ASC amino-acid transporter-2 (ASCT2), and xCT expression in patients with tongue cancer. We conducted the clinicopathological study to investigate the protein expression of these amino-acid transporters in tongue cancer. METHODS: Eighty-five patients with surgically resected tongue cancer were evaluated. Tumour sections were stained by immunohistochemistry for LAT1, ASCT2, xCT, 4F2hc/CD98hc (4F2hc), Ki-67, and microvessel density (MVD) determined by CD34, and p53. RESULTS: L-type amino-acid transporter 1 and 4F2hc were highly expressed in 61% (52 out of 85) and 45% (38 out of 47), respectively. ASC amino-acid transporter-2 and xCT were positively expressed in 59% (50 out of 85) and 21% (18 out of 85), respectively. The expression of both LAT1 and ASCT2 was significantly associated with disease staging, lymph-node metastasis, lymphatic permeation, 4F2hc expression and cell proliferation (Ki-67). xCT expression indicated a significant association with advanced stage and tumour factor. By univariate analysis, disease staging, lymphatic permeation, vascular invasion, LAT1, ASCT2, 4F2hc, and Ki-67 had a significant relationship with overall survival. Multivariate analysis confirmed that LAT1 was an independent prognostic factor for predicting poor prognosis. CONCLUSIONS: L-type amino-acid transporter 1 and ASCT2 can serve as a significant prognostic factor for predicting worse outcome after surgical treatment and may have an important role in the development and aggressiveness of tongue cancer.

Na+-dependent neutral amino acid transporter ASCT2 is downregulated in seriously traumatized human intestinal epithelial cells.

OBJECTIVE: Serious trauma to the body often is associated with changes in protein metabolism in multiple organs and tissues. Clinically, the catabolic response results in a generalized negative nitrogen balance. Nutrition support has been an important component of the care of seriously traumatized patients. However, during states of severe trauma, enterocyte transport function remains unclear. This study aims to quantitate the Na+-dependent neutral amino acid transport and expression of its transporter in traumatically injured Caco-2 cell lines. MATERIALS AND METHODS: Transport and transporter of Na+-dependent neutral amino acid in Caco-2 cell lines were characterized. Then the cell lines were cultured under hypoxic, nutrient-deprived, and ischemic conditions for 1, 2, 4, and 6 hours. After severe trauma was performed, we investigated the transport of Na+-dependent neutral amino acids and the expression of transporter protein and mRNA in apical membrane vesicles. RESULTS: Among the neutral amino acid transporters, only ASCT2 mRNA was amplified successfully. Under nutrient-deprived and ischemic conditions, transport of L-alanine and L-glutamine decreased significantly compared with control (P < 0.01), whereas hypoxia had no significant effect. The changes were associated with a decrease in maximum transport velocity without an influence on transport affinity. Expression of relative transporter proteins and mRNA decreased significantly compared with control (P < 0.01). CONCLUSIONS: Na+-dependent neutral amino acid transport and its key transporter are differently regulated during state of traumatic injury. It may be of use to provide some strategies targeting the special nutrient requirements and transport capabilities of seriously traumatized patients.

Molecular identification and characterisation of the glycine transporter (GLYT1) and the glutamine/glutamate transporter (ASCT2) in the rat lens.

Glutathione (GSH) is an essential antioxidant required for the maintenance of lens transparency. In the lens, GSH is maintained at unusually high concentrations as a result of direct GSH uptake and/or intracellular de novo synthesis from its precursor amino acids; cysteine, glycine and glutamine/glutamate. With increasing age, the levels of GSH, particularly in the core of the lens, are significantly reduced. It has been proposed that alterations in the transport of GSH and/or its precursor amino acids may contribute to the changes in GSH levels in older lenses. As considerable uncertainty exists about the molecular identity of GSH transporters in the lens, we have focused on identifying transporters involved in the uptake of the precursor amino acids required for GSH synthesis. Previously, we identified an uptake system for cyst(e)ine mediated by the Xc(-) exchanger and the Excitatory Amino Acid Transporters (EAATs) in the rat lens. In this current study, we have identified and localised additional uptake systems that contribute to GSH synthesis. Transcripts for GLYT1 (glycine transporter) and ASCT2 (glutamine/glutamate transporter) were detected in rat lens fiber cells using the reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis confirmed the expression of both GLYT1 and ASCT2 at the protein level. Immunocytochemistry revealed GLYT1 expression to be restricted to cortical regions of the lens. Labelling was predominantly cytoplasmic with some labelling of the membrane. In contrast, ASCT2 was expressed throughout the lens extending from the outer cortex through to the core. In the outer cortex, ASCT2 expression was predominantly cytoplasmic. However, with deeper distance into the lens, labelling became more membraneous indicating insertion of ASCT2 into the membranes of mature fiber cells of the lens core. The molecular identification and localisation of GLYT1 and ASCT2 in the lens suggests that these transporters may be responsible for the uptake of the precursor amino acids, glycine and glutamine, which are involved in GSH synthesis. Moreover, the presence of ASCT2 in the centre of the lens raises the possibility that ASCT2 may work with the Xc(-) exchanger to accumulate cysteine where it can potentially act as a low molecular mass antioxidant.

Characterization of rapid and high-affinity uptake of L-serine in neurons and astrocytes in primary culture.

The non-essential amino acid L-serine was shown to be required to support the survival of rat cerebellar Purkinje neurons because of lack of the expression of the L-serine biosynthesis enzyme 3-phosphoglycerate dehydrogenase in them. In the present study, we investigated L-[(3)H]serine uptake in primary cultures of neurons and astrocytes from the rat telencephalon. In both neurons and astrocytes, L-[(3)H]serine uptake was dependent on temperature and Na(+) ions, and exhibited a single component of high-affinity uptake sites (K(m)=15.0 and 17.2 micro M for neurons and astrocytes, respectively). Kinetic analysis of L-[(3)H]serine uptake also revealed that the uptake into neurons was faster than that into astrocytes. The selectivity of inhibition by amino acids of the L-[(3)H]serine uptake resembled that of the system ASC transporters ASCT1 and ASCT2. Neutral amino acids L-alanine, L-serine, L-cysteine, and L-threonine strongly inhibited the uptake by both cell types. Furthermore, in astrocytes, but not in neurons, L-valine and L-proline also inhibited L-[(3)H]serine uptake. Neither alpha-methyl aminoisobutyric acid (a system A-specific substrate) nor 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (a system L-specific substrate) inhibited the uptake of L-[(3)H]serine in both neurons and astrocytes. Expression of ASCT transporters in both neurons and astrocytes was examined by use of reverse transcriptase polymerase chain reaction and immunoblot analysis. Whereas transcripts (mRNAs) of both ASCT1 and ASCT2 transporters were detected in astrocytes, only the mRNA of the former subtype was detected in neurons. Immunoblot analysis confirmed the presence of ASCT1 in both neurons and astrocytes. These findings indicate that neurons accumulate a high level of L-serine by using a Na(+)-dependent, high-affinity transport system, operating predominantly through the ASCT1 transporter subtype.

Epidermal growth factor activation of intestinal glutamine transport is mediated by mitogen-activated protein kinases.

Glutamine is an essential nutrient for gut functions, but the regulation of its uptake by intestinal mucosal cells is poorly understood. Given the pivotal role of epidermal growth factor (EGF) in regulating gut metabolism, growth, and differentiation, this in vitro study was designed to investigate the intracellular signaling pathways involved in the regulation of EGF-mediated intestinal glutamine transport in intestinal epithelia. Continuous incubation with EGF (>30 hours, 100 ng/ml) stimulated glutamine transport activity across intestinal epithelial Caco-2 cell apical membrane. Exposure to EGF for 48 hours resulted in an increase in transport activity (50%) and glutamine transport system B gene ATB(0) mRNA levels (ninefold). EGF stimulated glutamine transport activity by increasing the glutamine transporter maximal velocity (V(max)) without altering the transporter apparent affinity (K(m)). Furthermore, EGF stimulated both intracellular protein kinase C and mitogen-activated protein kinase MEK1/2 activities. The EGF-stimulated glutamine transport activity was attenuated individually by the specific protein kinase C inhibitor chelerythrine chloride and the mitogen-activated protein kinase MEK1 inhibitor PD 98059. These data suggest that EGF activates glutamine transport activity across intestinal epithelial membrane via a signaling mechanism that involves activation of protein kinase C and the mitogen-activated protein kinase MEK1/2 cascade. EGF activates glutamine transport via alterations in transporter mRNA levels and the number of functional copies of transporter units.

Ontogeny and localization of the neutral amino acid transporter ASCT1 in rat brain.

ASCT1 is a protein that encodes System ASC, a sodium-dependent amino acid transport activity that transports primarily zwitterionic amino acids at physiological pH. ASCT1 has a 39-44% identity to the EAAT family of glutamate transporters. At extracellular pH values below 7.4, ASCT1 shifts substrate specificity to transport anionic amino acids. In this study we have examined the location of the ASCT1 transporter by immunohistochemistry in the developing rat brain. In addition, we have examined the cellular localization of ASCT1 in glial and neuronal cultures. The presence of ASCT1 immunoreactivity (ASCT1ir) in the developing brain was detectable as early as 14 days of gestation. At the cellular level, ASCT1ir was prominent in hippocampal pyramidal and dentate granule neurons. In the cerebellum, Purkinje cells and their dendrites were intensely labeled, whereas the granule and molecular layers were moderately labeled. In the cerebral cortex, neuronal cell bodies in all lamina and scattered astrocytes showed intense ASCT1ir. Double labeling experiments in vitro confirmed that ASCT1 was localized to both glia and neurons. These data illustrate that the rat ASCT1 transporter is expressed in the developing brain at levels equivalent to those observed in adult tissue. In addition, the expression and localization of ASCT1 are consistent with its possible role in pathophysiological processes that involve glutamate toxicity.