Dependent Neutral Amino Acid Transporter Research Articles

Effects of in ovo feeding of N-acetyl-L-glutamate on early intestinal development and growth performance in broiler chickens

The present study determined the effects of in ovo feeding (IOF) of N-acetyl-L-glutamate (NAG) on early intestinal development and growth performance of broilers. A total of 702 fertile broiler eggs were randomly divided into 3 treatments: 1) non-punctured control group, 2) saline-injected control group, and 3) NAG solution–injected group (1.5 mg/egg). At 17.5 D of incubation, 300 μL of each solution was injected into each egg of injected groups. Results indicated that the hatchability and healthy chicken rate were not affected by NAG injection (P > 0.05). Chicks from NAG solution–injected group had significantly decreased average daily feed intake and feed conversion ratio during 1–14 D than those in the non-punctured control group (P < 0.05). Compared with the non-punctured control group, IOF of NAG significantly increased the density of goblet cells in jejunum at hatch, duodenum at 7 D, and ileum at 14 D; decreased crypt depth in jejunum at hatch; and increased villus height in duodenum and jejunum and villus height:crypt depth ratio in duodenum at 7 D (P < 0.05). The intestinal mRNA expression of Na+-dependent neutral amino acid transporter, peptide transporter, and excitatory amino acid transporter 3 did not differ between groups at 7 or 14 D. However, the mRNA expression level of rBAT in jejunum significantly increased in the NAG solution–injected group than in the non-punctured control group at 7 D (P < 0.05). In conclusion, IOF of NAG (1.5 mg/egg) accelerated the early intestinal development by enhancing intestinal immune and absorption function, thereby positively affecting the feed efficiency for the first 2 wk post-hatch.

Effect of l-theanine on growth performance, intestinal development and health, and peptide and amino acid transporters expression of broilers.

l-Theanine has multiple beneficial biological activities. However, there is little information about the use of l-theanine in broiler production. Therefore, this study investigated the effect of l-theanine on growth performance, intestinal development and health, and the mRNA levels of intestinal peptide and amino acid (AA) transporters of broilers. Body weight and average daily gain were increased by l-theanine, whereas feed to gain ratio was decreased (quadratic, P < 0.05). Notably, the relative weight of duodenum, jejunum and ileum, villus height, villus height to crypt depth ratio, the jejunal activities of glutathione peroxidase, total antioxidant capacity, catalase and total superoxide dismutase were increased linearly and/or quadratically by l-theanine (P < 0.05), whereas crypt depth, serum d-lactic acid, and jejunal protein carbonyls and malondialdehyde content were decreased linearly and/or quadratically (P < 0.05). Moreover, l-theanine enhanced the jejunal mRNA levels of occludin, claudin-1, E-cadherin, zona occludens-1, di- and tripeptide transporter, excitatory AA transporter 3, Na+ -independent cationic AA transporter 1, Na+ -independent cationic and zwitterionic AA transporter, Na+ - and Cl- -dependent neutral and cationic AA transporter, Na+ -independent cationic and Na+ -dependent neutral AA transporter (y+LAT) 1, y+LAT2, Na+ -independent branched-chain and aromatic AA transporter, and heavy chain corresponding to the b°,+ transport system (linear and/or quadratic, P < 0.05). l-Theanine beneficially affected the growth performance of broilers by improving intestinal development and health, and the intestinal mRNA levels of AA and peptide transporters. Therefore, l-theanine has the potential to be a promising feed additive for broilers. © 2020 Society of Chemical Industry.

Accurate discrimination of Hartnup disorder from other aminoacidurias using a diagnostic ratio

IntroductionHartnup disorder is caused by a deficiency of the sodium dependent B0 AT1 neutral amino acid transporter in the proximal kidney tubules and jejunum. Biochemically, Hartnup disorder is diagnosed via amino acid excretion patterns. However, these patterns can closely resemble amino acid excretion patterns of generalized aminoaciduria, which may induce a risk for misdiagnosis and preclusion from treatment. Here we explore whether calculating a diagnostic ratio could facilitate correct discrimination of Hartnup disorder from other aminoacidurias. Methods27 amino acid excretion patterns from 11 patients with genetically confirmed Hartnup disorder were compared to 68 samples of 16 patients with other aminoacidurias. Amino acid fold changes were calculated by dividing the quantified excretion values over the upper limit of the age-adjusted reference value. ResultsIncreased excretion of amino acids is not restricted to amino acids classically related to Hartnup disorder (“Hartnup amino acids”, HAA), but also includes many other amino acids, not classically related to Hartnup disorder (“other amino acids”, OAA). The fold change ratio of HAA over OAA was 6.1 (range: 2.4–9.6) in the Hartnup cohort, versus 0.2 (range: 0.0–1.6) in the aminoaciduria cohort (p < .0001), without any overlap observed between the cohorts. DiscussionExcretion values of amino acids not classically related to Hartnup disorder are frequently elevated in patients with Hartnup disorder, which may cause misdiagnosis as generalized aminoaciduria and preclusion from vitamin B3 treatment. Calculation of the HAA/OAA ratio improves diagnostic differentiation of Hartnup disorder from other aminoacidurias.

Antioxidant response and bioavailability of methionine hydroxy analog relative to DL-methionine in broiler chickens.

This study was designed to compare the effect of methionine (Met) sources (DL-methionine [DLM] and DL-2-hydroxy-4-methylthio-butanoic acid [HMTBa]) and their supplementation levels on broiler growth performance and redox state. A 2 × 2 factorial arrangement was used with 2 sources (DLM and HMTBa) and 2 supplementation levels (0.05% and 0.25%) of Met. A total of 480 one-day-old broiler chicks were randomly divided into 4 treatments with 8 replicates per treatment (15 birds per replicate). The experiment lasted for 21 d. Broiler growth performance, redox capacity, redox-related genes expression, and Met transporters in different tissues were tested. Broilers fed high Met supplementation levels had improved (P < 0.05) body weight (BW), average daily gain (ADG) and feed conversion ratio (FCR). Similarly, broilers fed high Met levels had better (P < 0.05) antioxidant abilities in the serum, small intestine, and liver. Whereas, interactive effects (P < 0.05) were also observed between Met sources and levels. Compared with DLM, birds fed HMTBa diets had decreased (P < 0.05) total glutathione (T-GSH) and oxidized glutathione (GSSG) contents in duodenum, ileum, and liver. Similarly, broilers fed HMTBa supplemented diets had increased (P < 0.05) thioredoxin (Trx) gene expression in the duodenum and ileum, but decreased (P < 0.05) glutaredoxin (Grx), glutathione reductase (GSR), and glutathione synthetase (GSS) genes expression. Furthermore, lower gene expression of Na+ and Cl− dependent neutral and cationic amino acid transporter (ATB0, +), and Na+ dependent neutral amino acid transporter (B0AT) in the duodenum brush border, but higher gene expression of diamine acetyltransferase 1 (SAT1) and Na+-independent branched-chain and aromatic amino acid transporter (LAT1) in the jejunum and ileum basement membrane along with higher expression of the proton dependent monocarboxylate transporter 1 (MCT1) gene in the ileum were detected in birds fed HMTBa diets. In conclusion, DLM can be effectively used in glutathione synthesis to exert antioxidant functions, whereas HMTBa favors S-adenosylmethionine (SAM) synthesis and thus stimulates antioxidant-related genes expression.

Impact of mash and crumble diets on intestinal amino acids transporters, intestinal morphology and pancreatic enzyme activity of broilers.

The objective of this study was to evaluate effects of mash and crumble pre-starter diets on pancreatic enzyme activity, intestinal morphology, gene expression of intestinal peptide and amino acid (AA) transporters of broilers. Broilers in battery cages were assigned to different feed forms of pre-starter diet from 1 to 10days of age. Significantly increased body weight gain (BWG), feed intake (FI) and lowered FCR were observed in birds fed crumble pre-starter diet (CPD, p<0.05). Feed forms had no effect on whole and small intestine length, but relative intestinal length and relative small intestinal length significantly increased in the broilers fed a mash pre-starter diet (MPD, p<0.05). Feeding CPD increased the weight of pancreas (p<0.05), but relative weight of the pancreas was not influenced by treatments. Pancreatic protease and amylase activities significantly increased in the broilers fed CPD (p<0.05) but the activity of lipase was not influenced. Crypt depth (CD) and villus height (VH) were higher in broilers fed CPD (p<0.05) but villus width (VW), villus surface area (VSA) and villus height-to-crypt depth ratio (VCR) were not influenced by treatments. mRNA levels for peptide transporter 1 (PepT1), Na+ -independent cationic AA transporter1 (CAT1), Na+ -independent cationic and Na+ -dependent neutral AA transporter 1 (y+ LAT1) and Na+ -dependent neutral AA transporter (B0 AT) were lower in birds fed CPD (p<0.05). There were no differences in mRNA abundance of Na+ -independent cationic and zwitterionic AA transporter (b0,+ AT) among treatments. Overall, the present data showed that feeding crumble diet during first 10days of age, through higher FI, enhanced intestinal histomorphology, increased digestive enzyme activity is beneficial to growth performance of broilers. Indeed, dietary form can be an important factor in the expression of jejunal transporters.

Abstract LB-295: MEDI7247, a novel pyrrolobenzodiazepine ADC targeting ASCT2 with potent in vivo activity across a spectrum of hematological malignancies

Abstract MEDI7247 is a first in class ADC consisting of a human anti-ASCT2 monoclonal antibody site specifically conjugated to DNA cross-linking pyrrolobenzodiazepine (PBD) dimers. ASCT2 (SLC1A5) is a multi-pass, Na+-dependent neutral amino acid transporter that mediates the uptake of amino acids required for tumor growth and progression. ASCT2 is highly overexpressed in many hematologic cancers, most notably Multiple Myeloma (MM - 100% positive), Acute Myeloid Leukemia (AML - 100% positive) and Diffuse Large B cell lymphoma (DLBCL - 95% positive). ASCT2 expression is low in normal tissues. MEDI7247 (Q1Wx4) demonstrated a significant survival advantage in 3 disseminated AML cell line models, TF1α(ASCT2-High), MOLM-13(ASCT2-low) and M.V.411(ASCT2-High), when compared to the untreated control at the lowest dose levels examined: 0.05, 0.1 and 0.1 mg/kg, respectively. Further exemplifying the activity of MEDI7247, both the TF1α and MOLM-13 models did not reach 50% survival by the end of the study, with 80% survival at &amp;gt;200 days for TF1α and 70% survival at &amp;gt;180 days for MOLM-13. Similarly, a single dose of MEDI7247 in the TF1α model resulted in a 60% survival at &amp;gt;200 days at 0.05 mg/kg. MEDI7247 was also tested in a disseminated AML PDX(ASCT2-low) model at 0.05, 0.1 and 0.4 mg/kg. A significant improvement in survival was observed at both 0.1 and 0.4 mg/kg with the higher dose level extending survival by &amp;gt;80 days. MEDI7247 activity was further confirmed by monitoring peripheral blood CD33+ve cells, which initially receded, with the timing of reappearance preempting survival. Multiple Myeloma is another indication that exhibits a high level of ASCT2 expression. MEDI7247 (Q1Wx4) efficacy was examined in 3 disseminated MM cell line models, NCI-H929(ASCT2-High), MM.1S(ASCT2-medium) and OPM2(ASCT2-Medium), with a significant improvement in survival from control at the lowest dose levels examined: 0.1, 0.1 and 0.05 mg/kg, respectively. The activity of MEDI7247 (Q1Wx4) was also examined in the subcutaneous DLBCL model KARPAS 422(ASCT2-High). Tumor regressions were observed at all dose levels tested (0.1, 0.2, 0.3 and 0.4 mg/kg), with the higher two dose levels resulting in complete tumor regression without regrowth beyond 150 days. Additionally, MEDI7247 (Q1Wx4) is efficacious against the disseminated 697(ASCT2-Low) (Acute Lymphoblastic Leukemia - ALL) and RAJI(ASCT2-High) (Burkitt's lymphoma) models. A significant survival advantage was seen in both tumor models at the lowest dose examined of 0.05 mg/kg. In conclusion, MEDI7247 demonstrates antitumor efficacy across all tumor indications tested and varying levels of ASCT2 expression. These data support the use of MEDI7247 in ASCT2 positive hematological malignancies. MEDI7247 is currently in Phase 1 clinical trials. Citation Format: Noel R. Monks, Kevin P. Schifferli, Ravinder Tammali, M. Jack Borrok, Steven R. Coats, Ronald Herbst, David A. Tice, Nabendu Pore. MEDI7247, a novel pyrrolobenzodiazepine ADC targeting ASCT2 with potent in vivo activity across a spectrum of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-295.

Abstract LB-296: Discovery and development of MEDI7247, a novel Pyrrolobenzodiazepine (PBD)-based antibody drug conjugate targeting ASCT2, for treating hematological and solid cancers

Abstract The sodium dependent neutral amino acid transporter, ASCT2/SLC1A5 is frequently overexpressed in cancer to sustain “glutamine addiction” of cancer cells and to meet their increased demand for energy and nutrients. High expression of ASCT2 is often associated with poor disease prognosis. Immuno-histochemistry (IHC) data from 600+ tumor samples suggest ASCT2 expression in hematologic and solid cancers, including MM, AML, and DLBCL (≥ 95% positive in respective tumor samples), as well as squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), prostate, pancreatic, and colorectal cancer (66% to 95% positivity in respective tumors). Broad expression across various cancers and restricted expression in normal tissues warrant ASCT2 as an attractive candidate for an antibody drug conjugate. MEDI7247 is a novel investigational antibody-drug conjugate (ADC) comprising an anti-ASCT2 human monoclonal antibody site-specifically conjugated to pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker, with a drug to antibody ratio (DAR) of 2. MEDI7247 specifically binds to cell surface ASCT2 while exhibiting no affinity to the other members of the family, including ASCT1. Following binding, MEDI7247 is internalized and trafficked to the lysosomes to subsequently release the PBD warhead. PBD enters the nucleus where it induces DNA damage and results in tumor cell death. MEDI7247 shows potent in vitro cytotoxicity in several human cancer cell lines expressing variable levels of ASCT2. Dose dependent in vivo efficacy of MEDI7247 was determined in solid tumor xenografts and disseminated hematological malignancy models. MEDI7247 is cross-reactive with cynomolgous monkey ASCT2 and shows similar binding and killing with cyno ASCT2 expressing cells. MEDI7247 demonstrated acceptable safety profile in toxicity studies with non-human primates to support first in human trials. Based on its combined efficacy and safety, MEDI7247 is in clinical development for the treatment of ASCT2 positive cancers. Citation Format: Nabendu Pore, M Jack Borrok, Marlon Rebellato, MaryJane Hinrichs, Kevin P. Schifferli, Noel R. Monks, Ravinder Tammali, Ronald Herbst, Steven R. Coats, David A. Tice. Discovery and development of MEDI7247, a novel Pyrrolobenzodiazepine (PBD)-based antibody drug conjugate targeting ASCT2, for treating hematological and solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-296.

AP1G1 is involved in cetuximab-mediated downregulation of ASCT2-EGFR complex and sensitization of human head and neck squamous cell carcinoma cells to ROS-induced apoptosis

In this study, we expanded our recent work showing that ASCT2, a Na+-dependent neutral amino acid transporter that plays a major role in glutamine uptake in cancer cells, is physically associated with EGFR in human head and neck squamous cell carcinoma cells and in several other types of cancer cells. We found in our current study that ASCT2 can be downregulated by cetuximab, an approved anti-EGFR therapeutic antibody, via cetuximab-induced EGFR endocytosis independently of cetuximab-mediated inhibition of EGFR tyrosine kinase. We further found that ASCT2-EGFR association involves the adaptor-related protein complex 1 gamma 1 subunit (AP1G1), a subunit of clathrin-associated adaptor protein complex 1, which plays a role in membrane protein sorting in endosomes after receptor-mediated endocytosis. We found that AP1G1 is physically associated with both ASCT2 and EGFR and, together with those molecules, forms a heterotrimeric molecular complex. Knockdown of AP1G1 lowered the level of ASCT2-EGFR association, inhibited cetuximab-mediated internalization of ASCT2-EGFR complex, and decreased intracellular glutamine uptake and glutathione biosynthesis. These findings suggest a new therapeutic strategy to overcome cetuximab resistance in cancer cells through combination of cetuximab, which co-targets ASCT2 along with EGFR, with an ROS-inducing agent.

133. Use of 2As To Control Protein Subcellular Localization

A substantial proportion (~39%) of all human proteins are either secreted from the cell, located within the lumen/ membranes of cytoplasmic vesicular structures, or, are plasma membrane proteins. Given that such high proportion of proteins are initially translocated into the endoplasmic reticulum (ER), many therapeutic strategies rely on the ability to co-express multiple proteins – some, or all of which, might be targeted to such sites. This directly applies to in vivo gene therapy strategies, or, when therapeutic proteins may need to be co-expressed with selectable markers (e.g. ex vivo gene therapies).As was shown before, Picornavirus 2A (foot-and-mouth disease virus 2A; F2A) and ‘2A-like’ sequences are powerful tools that allow multiple proteins to be translated and co-expressed from a single transcript mRNA under the control of only one promoter. When 2A is positioned between sequences encoding two, or more, genes, it mediates a co-translational ‘cleavage’ at its own C-terminus. A major problem with co-expression of certain proteins targeted to, or transiting through, the ER is that the ‘cleavage’ activity of short F2As can be greatly inhibited by sequences immediately upstream leading to aberrant sub-cellular localisation of some proteins.We have also discovered a number of active cellular 2A-like sequences, associated with non-long terminal repeat (non-LTR) retrotransposons, but also with structural and metabolic proteins: ankyrin repeats, sodium dependent neutral amino acid transporters, and NOD-like receptor (NLR) proteins. Examination of the surrounding protein and gene structure revealed that in the majority of cases these 2A sequences occurred as N-terminal features. Interestingly, using SignalP, many of these novel 2As scored highly as N-terminal signal peptides.Here, we present our latest findings on 2A sequences. A series of test proteins (eGFP and mCherry) were expressed i) followed by ‘hybrid’ ‘self-cleaving’ F2A sequences (with different upstream contexts) or ii) downstream of a putative signal 2A. We demonstrate that inhibition of F2A-mediated cleavage in shorter sequences can be overcome by introduction of mutations upstream of 2A changing the context of the sequence between the C-terminus of the upstream protein and 2A sequence. In the case of N-terminal – cellular – (NLR) 2As, ‘uncleaved’ 2A indeed can act as a signal peptide. If 2A does not ‘cleave’, it directs a proportion of the newly synthesised reporter protein to the exocytic pathway: if 2A ‘cleaves’, the protein downstream is localised to the cytoplasm. This type of 2A mediates, therefore, a newly discovered form of dual protein targeting.

GLAST/EAAT1‐induced Glutamine release via SNAT3 in Bergmann glial cells: evidence of a functional and physical coupling

Glutamate, the major excitatory transmitter in the vertebrate brain, is removed from the synaptic cleft by a family of sodium-dependent glutamate transporters profusely expressed in glial cells. Once internalized, it is metabolized by glutamine synthetase to glutamine and released to the synaptic space through sodium-dependent neutral amino acid carriers of the N System (SNAT3/slc38a3/SN1, SNAT5/slc38a5/SN2). Glutamine is then taken up by neurons completing the so-called glutamate/glutamine shuttle. Despite of the fact that this coupling was described decades ago, it is only recently that the biochemical framework of this shuttle has begun to be elucidated. Using the established model of cultured cerebellar Bergmann glia cells, we sought to characterize the functional and physical coupling of glutamate uptake and glutamine release. A time-dependent Na⁺-dependent glutamate/aspartate transporter/EAAT1-induced System N-mediated glutamine release could be demonstrated. Furthermore, D-aspartate, a specific glutamate transporter ligand, was capable of enhancing the co-immunoprecipitation of Na⁺-dependent glutamate/aspartate transporter and Na⁺-dependent neutral amino acid transporter 3, whereas glutamine tended to reduce this association. Our results suggest that glial cells surrounding glutamatergic synapses may act as sensors of neuron-derived glutamate through their contribution to the neurotransmitter turnover.

Conserved Residues Serine 225 and Methionine 229 Attenuate the Sodium Transport Responses in the Sodium-Coupled Neutral Amino Acid Transporter, SNAT2

Sodium dependent neutral amino acid transporter (SNAT) 2, a member of the solute carrier (SLC) 38 family, system A, mediates the coupled transport of sodium and small neutral amino acids across a cell's membrane. There has been proposed structural homology between SNAT2 and the sodium/betaine symporter, BetP. We mutated two conserved amino acids that have been shown to be ligands involved in BetP sodium binding: serine-225 and methionine-229. Both residues are found in the predicted TMD 5. The functional consequences of the S225A mutation are a slight reduction in the apparent affinity for alanine, and a dramatic reduction of the apparent sodium affinity. M229A also shows a reduced apparent affinity for alanine. These results demonstrate there is involvement of these residues in the sodium coordination and/or amino acid interaction with SNAT2. It also shows the involvement of TMD 5, which previously had not been thought to be involved with sodium binding in SNAT2.

Leptin regulates sugar and amino acids transport in the human intestinal cell line Caco‐2

Studies in rodents have shown that leptin controls sugars and glutamine entry in the enterocytes by regulating membrane transporters. Here, we have examined the effect of leptin on sugar and amino acids absorption in the human model of intestinal cells Caco-2 and investigated the transporters involved. Substrate uptake experiments were performed in Caco-2 cells, grown on plates, in the presence and the absence of leptin, and the expression of the different transporters in brush border membrane vesicles was analysed by Western blot. Leptin inhibited 0.1 mm α-methyl-D-glucoside uptake after 5 or 30 min treatment and decreased SGLT1 protein abundance in the apical membrane. Uptake of 20 μm glutamine and 0.1 mm phenylalanine was also inhibited by leptin, indicating sensitivity to the hormone of the Na(+) -dependent neutral amino acid transporters ASCT2 and B(0) AT1. This inhibition was accompanied by a reduction in the transporters expression at the brush border membrane. Leptin also inhibited 1 mm proline and β-alanine uptake in Na(+) medium at pH 6, conditions for optimal activity of the H(+) -dependent neutral amino acid transporter PAT1. In this case, abundance of PAT1 in the brush border membrane after leptin treatment was not modified. Interestingly, leptin inhibitory effect on β-alanine uptake was reversed by the PKA inhibitor H-89 suggesting involvement of PKA pathway in leptin's regulation of PAT1 activity. These data show in human intestinal cells that leptin can rapidly control the activity of physiologically relevant transporters for rich-energy molecules, that is, D-glucose (SGLT1) and amino acids (ASCT2, B(0) AT1 and PAT1).

Dietary protein composition influences abundance of peptide and amino acid transporter messenger ribonucleic acid in the small intestine of 2 lines of broiler chicks

This study evaluated the effect of dietary protein composition on mRNA abundance of a peptide transporter (peptide transporter 1, PepT1), amino acid (AA) transporters [Na(+)-independent cationic and zwitterionic AA transporter (b(o,+)AT), excitatory AA transporter 3 (EAAT3), Na(+)-independent cationic and Na(+)-dependent neutral AA transporter 2 (y(+)LAT2), L-type AA transporter 1 (LAT1), and cationic AA transporter 1 (CAT1)], and a digestive enzyme (aminopeptidase N) in 2 lines (A and B) of broilers that differentially express PepT1 mRNA (line B > line A). From d 8 to 15 posthatch, birds were fed 1 of 3 diets. Protein sources included whey protein concentrate, a whey partial hydrolysate (WPH), or a mixture of free AA (AA) identical to the composition of whey. Quantities of mRNA were assayed by real-time PCR in the small intestine of males at d 8, 9, 11, 13, and 15. For all genes except LAT1, abundance of mRNA was greatest in line B birds that consumed the WPH diet (P < 0.006). When mRNA abundance was normalized to beta-actin quantities, this effect disappeared, demonstrating a generalized effect on gene expression in line B birds that consumed the hydrolysate. There was a greater villus height:crypt depth ratio (P < 0.05) in line B birds fed the WPH diet as compared with line A. In conclusion, line B birds, which express greater PepT1, displayed enhanced intestinal mucosal absorptive surface area and differential regulation of PepT1, AA transporters, and aminopeptidase N in response to dietary protein composition.

Competition between selenomethionine and methionine absorption in the intestinal tract of green sturgeon ( Acipenser medirostris)

l-Selenomethionine (SeMet) is a dominant form of selenium (Se) found in organisms at all levels of aquatic food chains and a key source of Se bioaccumulation and ecotoxicity. In mammals, intestinal absorption of SeMet is at least partly via the Na +-dependent neutral amino acid transporter. The mechanism of SeMet absorption and competitive effects of other dietary components on SeMet absorption in fish are unknown. Thus the in vitro uptake rates of l-methionine (Met) and the competitive effect of SeMet on Met absorption, an indicator that SeMet uses the same nutrient transporter(s) for absorption, in the various regions of the green sturgeon ( Acipenser medirostris) intestine were investigated using intact tissues (a modified everted sleeve method). Intestinal tissue was incubated in Ringer's solution containing 0–10 mmol L −1 Met or SeMet ( n = 5 for each substrate's concentration and intestinal region), respectively, as well as constant tracer levels of isotope-labeled Met. The data indicate that SeMet uptake was mediated by the same transporter(s) as Met and that the absorption kinetics were similar for both substrates. When there were differences in absorption they appeared to be mostly due to higher permeability (passive uptake) of the tissue for Met than for SeMet, particularly in the pyloric caeca (PC) and distal intestine (DI). Maximum rates of absorption, on the other hand, tended to be higher for SeMet than Met in the mid intestine and DI, whereas differences in affinity for the transporters varied between these tissues but were very similar in the PC. These differences may be due to differences in regional intestinal characteristics such as amount of mucus secreted and degree of tissue contraction, and/or substrate differences regarding solubility in and movement through the mucus, influence on tissue contraction, permeability through membranes or between cells, intracellular metabolism, as well as basolateral transport. Interestingly, an increasing proximal-to-distal gradient for rates of methionine and putative SeMet absorption was observed in green sturgeon which differs from the declining gradient usually observed for substrate absorption in other fish species and mammals.

Kidney amino acid transport

Near complete reabsorption of filtered amino acids is a main specialized transport function of the kidney proximal tubule. This evolutionary conserved task is carried out by a subset of luminal and basolateral transporters that together form the transcellular amino acid transport machinery similar to that of small intestine. A number of other amino acid transporters expressed in the basolateral membrane of proximal kidney tubule cells subserve either specialized metabolic functions, such as the production of ammonium, or are part of the cellular housekeeping equipment. A new finding is that the luminal Na(+)-dependent neutral amino acid transporters of the SLC6 family require an associated protein for their surface expression as shown for the Hartnup transporter B(0)AT1 (SLC6A19) and suggested for the L: -proline transporter SIT1 (IMINO(B), SLC6A20) and for B(0)AT3 (XT2, SLC6A18). This accessory subunit called collectrin (TMEM27) is homologous to the transmembrane anchor region of the renin-angiotensin system enzyme ACE2 that we have shown to function in small intestine as associated subunit of the luminal SLC6 transporters B(0)AT1 and SIT1. Some mutations of B(0)AT1 differentially interact with these accessory subunits, providing an explanation for differential intestinal phenotypes among Hartnup patients. The basolateral efflux of numerous amino acids from kidney tubular cells is mediated by heteromeric amino acid transporters that function as obligatory exchangers. Thus, other transporters within the same membrane need to mediate the net efflux of exchange substrates, controlling thereby the net basolateral amino transport and thus the intracellular amino acid concentration.

Na+‐dependent Neutral Amino Acid Transporter ASCT2 Is Downregulated in Seriously Traumatized Human Intestinal Epithelial Cells

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. 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. 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). 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.

Deletion of v7-3 (SLC6A15) transporter allows assessment of its roles in synaptosomal proline uptake, leucine uptake and behaviors.

V7-3 (SLC6A15) is the prototype for a gene subfamily whose members have sequence homologies to classical Na+- and Cl(-)-dependent neurotransmitter transporters but display unusual features that include characteristic large fourth extracellular loops. Interest in v7-3 has been increased by the elucidation of its expression in neurons located in cerebral cortex, hippocampus, cerebellum, midbrain and olfactory bulb. To help clarify the role of v7-3 in brain functions, we have created and characterized v7-3 knockout mice. These mice lack functional v7-3 protein but are viable and fertile. While our studies were in progress, v7-3 expression was reported to confer transport of proline and branched-chain amino acids in in vitro expression systems [Takanaga, H., Mackenzie, B., Peng, J.B., Hediger, M.A., 2005b. Characterization of a branched-chain amino-acid transporter SBAT1 (SLC6A15) that is expressed in human brain. Biochem. Biophys. Res. Commun. 337, 892-900; Broer, A., Tietze, N., Kowalczuk, S., Chubb, S., Munzinger, M., Bak, L.K., Broer, S., 2006. The orphan transporter v7-3 (slc6a15) is a Na+-dependent neutral amino acid transporter (B0AT2). Biochem. J. 393, 421-430]. Assessment of amino acid uptake into cortical synaptosomes of v7-3 knockouts identified 15% and 40% reductions in sodium-dependent proline and leucine transport, respectively, compared to wild type controls. Despite these biochemical changes, v7-3 knockout mice demonstrate only modest alterations in rotarod performance with aging and lack reproducible alterations in other motor, memory, anxiety or olfactory tests. Compensation for the lack of v7-3 via other amino acid carriers is likely to leave v7-3 knockouts without gross behavioral manifestations. The current results place v7-3 in the context of other brain transporters that accumulate proline and branched-chain amino acids.

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.

Glutamine Transporter ASCT2 Was Down‐Regulated in Ischemic Injured Human Intestinal Epithelial Cells and Reversed by Epidermal Growth Factor

Clinically, nutrition support has been an important component of the care of the hypoperfusion traumatized patient who is unable to accept complete volitional nutrition. However, enterocyte transport function during states of intestinal hypoperfusion remains unclear. Glutamine is essential for the viability and growth of intestine epithelial cells, and the Na(+)-dependent neutral amino acid transporter ASCT2 is thought to mainly mediate glutamine transport. This study aims to quantify the change of glutamine transporter ASCT2 expression in ischemic injured Caco-2 cell lines and the regulatory action of epidermal growth factor (EGF) on glutamine transport and its transporter. Cells were cultured under ischemic conditions for 2 hours. After ischemia was performed, Caco-2 cells were incubated with or without EGF (100 microg/mL) for 0-8 hours. Then we studied the cell membrane l-glutamine transport, the expression of ASCT2 protein, and mRNA. After ischemia was performed, Caco-2 cell membrane glutamine transport decreased significantly (p < .01), and the expression of ASCT2 proteins decreased significantly compared with control (p < .01). Under ischemic conditions, expression of ASCT2 mRNA was down-regulated by a real-time polymerase chain reaction (PCR) method. After EGF incubation for 1-2 hours, the proteins and mRNA of ASCT2 were reversed to normal levels (p > .05). In ischemic injured Caco-2 cells, ASCT2 protein expression and mRNA transcription were involved in the down-regulation of Na(+)-dependent glutamine transport. The decrease of glutamine transport and its transporter under ischemic conditions could be reversed by EGF action. These findings may help in the choice of the nutrition support manner and clinical therapy of ischemia-damaged intestinal epithelial cells.

Na + ‐dependent neutral amino acid transporter ASCT2 is under‐expressed in SHR kidney

Na ⁺ ‐dependent neutral amino acid transporter ASCT2 is under‐expressed in SHR kidney