Sodium-dependent Amino Acid Transporter Research Articles

New Insight on Vitality Differences for the Penaeid Shrimp, Fenneropenaeus chinensis, in Low Salinity Environment Through Transcriptomics

Excessive rainfall changes salinity in shrimp farming ponds in short period and exerts low salinity stress on the outdoor breeding shrimp under global warming. Fenneropenaeus chinensis can have different performance on vitality in low salinity environments. To reveal mechanisms of vitality difference in shrimp living in low saline environments. This study based on the normal and moribund F. chinensis in 10 ppt salinity environment using high-throughput sequencing identifies 1,429 differentially expressed genes (DEGs), 586 of which are upregulated, while 843 of which are downregulated in the normal group (FCN10) as compared to the moribund group (FCM10). Meanwhile, another transcriptomic analysis is conducted on the normal and moribund shrimp from 25 ppt (FCN25 vs. FCM25) salinity environment as the control, in which 1,311 DEGs (upregulated: 327 genes, downregulated: 984 genes) are identified. In this study, intersective pathways, GO (Gene Ontology) categories and DEGs from the two groups of comparative transcriptome are investigated. The two intersective pathways (Metabolism of xenobiotics by cytochrome P450, Pentose, and glucuronate interconversions) significantly enriched by DEGs are related to detoxification. In these two pathways, there is one vitality regulation-related gene (VRRG), the Dhdh (dihydrodiol dehydrogenase), which is upregulated in both the groups of FCN10 and FCN25 as compared to the groups of FCM10 and FCM25, respectively. Similarly, in the 25 top intersective GO categories, four VRRGs are revealed. Three of them are upregulated (Itgbl, kielin/chordin-like protein, Slc2a8, solute carrier family 2, facilitated glucose transporter member 8-like protein and Cyp3a30, cytochrome P450 3A30-like protein); one of them is downregulated (Slc6a9, sodium-dependent nutrient amino acid transporter 1-like protein isoform X2). These GO categories are related to transmembrane transporter activity of substance, enzyme inhibitor activity, monooxygenase activity. RT-qPCR analysis further verifies the VRRGs. The study gives new insight into understanding the vitality differences for F. chinensis, in low salinity environment. The pathways and DEGs in response to low salinity stress in modulating the vitality of F. chinensis that could serve as tools in future genetic studies and molecular breeding.

Analysis of L-leucine amino acid transporter species activity and gene expression by human blood brain barrier hCMEC/D3 model reveal potential LAT1, LAT4, B0AT2 and y+LAT1 functional cooperation

In the CNS, amino acid (AA) neurotransmitters and neurotransmitter precursors are subject to tight homeostatic control mediated by blood-brain barrier (BBB) solute carrier amino acid transporters (AATs). Since the BBB is composed of multiple closely apposed cell types and opportunities for human in vivo studies are limited, we used in vitro and computational approaches to investigate human BBB AAT activity and regulation. Quantitative real-time PCR (qPCR) of the human BBB endothelial cell model hCMEC/D3 (D3) was used to determine expression of selected AAT, tight junction (TJ), and signal transduction (ST) genes under various culture conditions. L-leucine uptake data were interrogated with a computational model developed by our group for calculating AAT activity in complex cell cultures. This approach is potentially applicable to in vitro cell culture drug studies where multiple “receptors” may mediate observed responses. Of 7 Leu AAT genes expressed by D3 only the activity of SLC7A5-SLC3A2/LAT1-4F2HC (LAT1), SLC43A2/LAT4 (LAT4) and sodium-dependent AATs, SLC6A15/B0AT2 (B0AT2), and SLC7A7/y+LAT1 (y+LAT1) were calculated to be required for Leu uptake. Therefore, D3 Leu transport may be mediated by a potentially physiologically relevant functional cooperation between the known BBB AAT, LAT1 and obligatory exchange (y+LAT1), facilitative diffusion (LAT4), and sodium symporter (B0AT2) transporters.

PSV-11 Efficacy of Quisqualic Acid as an Amino Acid Transporter Inhibitor in Pig Oocytes

Abstract Quisqualic acid is a known inhibitor of sodium-dependent amino acid transporters. However, it is unknown if quisqualic acid has similar effects in in vitro mature oocytes. Therefore, the objective of this study was to determine the optimal dose and effects of quisqualic acid supplemented during maturation. Oocytes (n=362) were supplemented during maturation with quisqualic acid (0, 0.5, 0.75, 1.0, 2.5 mM) to determine the minimum concentration of quisqualic acid that had no effect on oocyte maturation but significantly decreased the intracellular glutathione concentration. The addition of 1.0 mM quisqualic acid was the lowest concentration observed to cause intracellular glutathione levels to be significantly less (P < 0.05) without affecting maturation compared to no quisqualic acid. Based on those results, oocytes were supplemented with or without 1.0 mM quisqualic acid then evaluated for cumulus cell expansion (n=410) and stage of meiosis (n=380) at the end of maturation. Additional oocytes were fertilized and assessed for cortical granule exocytosis (n=400) and kinetics at 12 h after IVF (n=420). Supplementing quisqualic acid to the media did not have an effect on stage of meiosis, fertilization, polyspermic penetration, or cortical granule exocytosis. Supplementing 1.0 mM quisqualic acid significantly decreased (P < 0.05) cumulus cell expansion by the end of maturation and male pronuclear formation by 12 h after IVF. These results suggest that quisqualic acid supplementation during maturation in pigs inhibits sodium-dependent amino acid transporters.

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.

GPNA inhibits the sodium-independent transport system L for neutral amino acids.

L-γ-Glutamyl-p-nitroanilide (GPNA) is widely used to inhibit the glutamine transporter ASCT2, although it is known that it also inhibits other sodium-dependent amino acid transporters. In a panel of human cancer cell lines, which express the system L transporters LAT1 and LAT2, GPNA inhibits the sodium-independent influx of leucine and glutamine. The kinetics of the effect suggests that GPNA is a low affinity, competitive inhibitor of system L transporters. In Hs683 human oligodendroglioma cells, the incubation in the presence of GPNA, but not ASCT2 silencing, lowers the cell content of leucine. Under the same conditions the activity of mTORC1 is inhibited. Decreased cell content of branched chain amino acids and mTORC1 inhibition are observed in most of the other cell lines upon incubation with GPNA. It is concluded that GPNA hinders the uptake of essential amino acids through system L transporters and lowers their cell content.

Abstract 1007: The amino acid transporter SNAT4: Potential role as a tumor suppressor in melanoma

Abstract Cancer cells utilize amino acids to satisfy their need for nutrients and fuel their accelerated growth. The amino acid glutamine has been identified as one of the key building blocks in cancer cells, utilized for macromolecular synthesis and energy production. Due to the increased requirement for glutamine and other amino acids, cancer cells commonly increase expression of amino acid transporters, such as SLC1A5 (ASCT2). Amino acid transporters are membrane transport proteins that are used by cells to move important amino acids in and out of the cell. Interestingly, most amino acid transporters are upregulated in cancer to ensure continued access to nutrients. We have recently shown that ASCT2 plays a critical role in regulating glutamine uptake in melanoma, prostate and breast cancer. Many other glutamine transporters are upregulated in different cancers, including SLC38 family members SNAT1, SNAT2 and SNAT3. The role of SLC38A4 (SNAT4) in amino acid uptake and cancer growth, however, has not been examined. Like the other SNATs, SNAT4 is a sodium-dependent amino acid transporter that transports neutral amino acids, such as alanine, across the plasma membrane, although it has low affinity for glutamine. Interestingly, unlike the other SNAT family members, our analysis showed that SNAT4 expression is downregulated in 879/917 cancer cell lines in Oncomine. Analysis of SNAT4 mutations in patient samples using cBioPortal showed infrequent mutations in the majority of cancers. However, the TCGA melanoma data (cBioPortal) showed that 32 of 278 melanoma cases (11.5%) have a point mutation in SNAT4 suggesting an important role in melanoma. These mutations included 9 SNAT4 hotspot mutations, present in 2-4 patients each. Using a SNAT4 homology model to predict loss-of-function mutants, we selected five of these mutations to assess in a SNAT4 transport assay in Xenopus oocytes. Two of the five hotspot mutations (G31E, S76F, G78E, G150E and S371F) were able to completely inactivate SNAT4 alanine transport in oocytes. Further analysis of 52 melanoma patient samples in Oncomine showed SNAT4 downregulation in all patients, suggesting that, in contrast to most amino acid transporters, SNAT4 plays a tumor suppressor role in melanoma. We are currently examining these mutations in melanoma cell lines to determine their effects on cell growth. We are also examining how SNAT4 is involved in either the import or export of amino acids and the downstream metabolic consequences that may directly inhibit melanoma cell growth. Through this research we will gain a better understanding of the role of SNAT4- mediated amino acid metabolism in preventing melanoma cell growth. Citation Format: Nicholas J. Otte, Angelika Broer, Patrick O’Young, Michelle van Geldermalsen, Qian Wang, Charles G. Bailey, Stefan Broer, Jeff Holst. The amino acid transporter SNAT4: Potential role as a tumor suppressor in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1007.

Enhanced intracellular accumulation of a non-nucleoside anti-cancer agent via increased uptake of its valine ester prodrug through amino acid transporters

The phenomenon known as multiple-drug resistance, whereby anti-cancer agents are expelled from cancer cells, makes it necessary to develop methods that will reliably increase the accumulation of anti-cancer agents within cancer cells. To accomplish this goal, a new model compound, Val-SN-38, was synthesized by introducing valine to SN-38, an active ingredient of irinotecan.Val-SN-38 improved intracellular accumulation approximately 5-fold in MCF7 cells, compared with SN-38, and rather than changes in membrane permeability, the amino acid transporter ATB0,+ played a role, whereas the dipeptide transporter PEPT1 did not. Other sodium-dependent amino acid transporters, namely ATA1, ATA2, and ASCT2, were unexpectedly involved in the uptake of Val-SN-38 as well. The efflux of Val-SN-38 by major efflux transporters was variably changed, but not significantly.In summary, the enhanced accumulation of Val-SN-38 in cancer cells was due to augmented uptake via various amino acid transporters. The results of the present study make a compelling argument in favour of a prodrug concept that can improve intracellular accumulation and take advantage of amino acid transporters without significantly inducing multiple-drug resistance.

Quantitative PCR tissue expression profiling of the human SGLT2 gene and related family members

SGLT2 (for “Sodium GLucose coTransporter” protein 2) is the major protein responsible for glucose reabsorption in the kidney and its inhibition has been the focus of drug discovery efforts to treat type 2 diabetes. In order to better clarify the human tissue distribution of expression of SGLT2 and related members of this cotransporter class, we performed TaqMan™ (Applied Biosystems, Foster City, CA, USA) quantitative polymerase chain reaction (PCR) analysis of SGLT2 and other sodium/glucose transporter genes on RNAs from 72 normal tissues from three different individuals. We consistently observe that SGLT2 is highly kidney specific while SGLT5 is highly kidney abundant; SGLT1, sodium-dependent amino acid transporter (SAAT1), and SGLT4 are highly abundant in small intestine and skeletal muscle; SGLT6 is expressed in the central nervous system; and sodium myoinositol cotransporter is ubiquitously expressed across all human tissues.Electronic Supplementary MaterialSupplementary material is available for this article at 10.1007/s13300-010-0006-4 and is accessible for authorized users.

Structure-Function Relationships of Inhibitors for the EAAC1 and ASCT2 Amino Acid Transporters

The SLC1 family of sodium-dependent amino acid transporters includes the high-affinity neuronal glutamate transporter EAAC1 and the closely related neutral-amino-acid transporter ASCT2, which exchanges neutral amino acids, such as alanine, serine, cysteine and glutamine across the cell membrane. ASCT2 may be important for glutamate release from astrocytes, but no high-affinity inhibitors are known that would allow one to test this hypothesis.

Docking of Multiple Ligands into a New Homology Model of the Serotonin Transporter

The serotonin transporter (SERT) is a presynaptic membrane protein that terminates neuronal transmission of serotonin (5HT) by transporting its substrate into the emitting neuron. Recently new crystal structures of the Leucine Transporter (LeuT) with multiple bound ligands have been published [1]. LeuT is a bacterial sodium dependent amino acid transporter, which is a homologue of the mammalian neurotransmitter sodium symporter (NSS) family (tcdb-code 2.A.22, www.tcdb.org), such as SERT. In light of our studies on the molecular basis of inhibitor binding we constructed a new homology model of SERT and used this for docking studies of a series of pharmacologically active ligands.

Mutations in SLC6A19, encoding B0AT1, cause Hartnup disorder.

Hartnup disorder, an autosomal recessive defect named after an English family described in 1956 (ref. 1), results from impaired transport of neutral amino acids across epithelial cells in renal proximal tubules and intestinal mucosa. Symptoms include transient manifestations of pellagra (rashes), cerebellar ataxia and psychosis. Using homozygosity mapping in the original family in whom Hartnup disorder was discovered, we confirmed that the critical region for one causative gene was located on chromosome 5p15 (ref. 3). This region is homologous to the area of mouse chromosome 13 that encodes the sodium-dependent amino acid transporter B(0)AT1 (ref. 4). We isolated the human homolog of B(0)AT1, called SLC6A19, and determined its size and molecular organization. We then identified mutations in SLC6A19 in members of the original family in whom Hartnup disorder was discovered and of three Japanese families. The protein product of SLC6A19, the Hartnup transporter, is expressed primarily in intestine and renal proximal tubule and functions as a neutral amino acid transporter.

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.

The generalized aminoaciduria seen in patients with hepatocyte nuclear factor-1alpha mutations is a feature of all patients with diabetes and is associated with glucosuria.

Hepatocyte nuclear factor-1alpha (HNF-1alpha) mutations are the most common cause of maturity-onset diabetes of the young. HNF-1alpha homozygous knockout mice exhibit a renal Fanconi syndrome with glucosuria and generalized aminoaciduria in addition to diabetes. We investigated glucosuria and aminoaciduria in patients with HNF-1alpha mutations. Sixteen amino acids were measured in urine samples from patients with HNF-1alpha mutations, age-matched nondiabetic control subjects, and age-matched type 1 diabetic patients, type 2 diabetic patients, and patients with diabetes and chronic renal failure. The HNF-1alpha patients had glucosuria at lower glycemic control (as shown by HbA1c) than type 1 and type 2 diabetic patients, consistent with a lower renal glucose threshold. The HNF-1alpha patients had a generalized aminoaciduria with elevated levels of 14 of 16 amino acids and an increased mean Z score for all amino acids compared with control subjects (0.66 vs. 0.00; P < 0.0005). Generalized aminoaciduria was also present in type 1 diabetic (Z score, 0.80; P < 0.0001), type 2 diabetic (Z score, 0.71; P < 0.0002), and chronic renal failure (Z score, 0.65; P < 0.01) patients. Aminoaciduria was not associated with microalbuminuria or proteinuria but was associated with glucosuria (1.00 glucosuria vs. 0.19 no glucosuria; P = 0.002). In type 1 diabetic patients, urine samples taken on the same day showed significantly more aminoaciduria when glucosuria was present compared with when it was absent (P < 0.01). In conclusion, HNF-1alpha mutation carriers have a mutation-specific defect of proximal tubular glucose transport, resulting in increased glucosuria. In contrast, the generalized aminoaciduria seen in patients with HNF-1alpha mutations is a general feature of patients with diabetes and glucosuria. Glucose may depolarize and dissipate the electrical gradient of the sodium-dependent amino acid transporters in the proximal renal tubule, causing a reduction in amino acid resorption.

Neutral amino acid transport in bovine articular chondrocytes.

1. The sodium-dependent amino acid transport systems responsible for proline, glycine and glutamine transport, together with the sodium-independent systems for leucine and tryptophan, have been investigated in isolated bovine chondrocytes by inhibition studies and ion replacement. Each system was characterized kinetically. 2. Transport via system A was identified using the system-specific analogue alpha-methylaminoisobutyric acid (MeAIB) as an inhibitor of proline, glycine and glutamine transport. 3. Uptake of proline, glycine and glutamine via system ASC was identified by inhibition with alanine or serine. 4. System Gly was identified by the inhibition of glycine transport with excess sarcosine (a substrate for system Gly) whilst systems A and ASC were inhibited. This system, having a very limited substrate specificity and tissue distribution, was also shown to be Na+ and Cl- dependent. Evidence for expression of the system Gly component GLYT-1 was obtained using the reverse transcriptase-polymerase chain reaction (RT-PCR). 5. System N, also of narrow substrate specificity and tissue distribution, was shown to be present in chondrocytes. Na+-dependent glutamine uptake was inhibited by high concentrations of histidine (a substrate of system N) in the presence of excess MeAIB and serine. 6. System L was identified using the system specific analogue 2-aminobicyclo(2,2, 1)heptane-2-carboxylic acid (BCH) and D-leucine as inhibitors of leucine and tryptophan transport. 7. The presence of system T was tested by using leucine, tryptophan and tyrosine inhibition. It was concluded that this system was absent in the chondrocyte. 8. Kinetic analysis showed the Na+-independent chondrocyte L system to have apparent affinities for leucine and tryptophan of 125 +/- 27 and 36 +/- 11 microM, respectively. 9. Transport of the essential amino acids leucine and tryptophan into bovine chondrocytes occurs only by the Na+-independent system L, but with a higher affinity than the conventional L system.

Sodium-Dependent Amino Acid Transport Is Preserved in Lyophilized Reconstituted Apical Membranes from Intestinal Epithelium

We demonstrate for the first time that functional electrogenic Na+-dependent amino acid transport is preserved for extended periods when purified brush border membranes prepared in hypotonic media are lyophilized and then rehydrated in buffer containing mannitol, NaSCN, and/or KSCN/valinomycin. Reconstituted lyophilized apical membranes from small intestine formed morphologically, physiologically, and thermodynamically normal vesicles which transportedl-alanine via system B into an osmotically active space energized by secondary active transport, as measured under equilibrium and nonequilibrium conditions. The lyophilized membranes are readily prepared and stored, thereby providing a means to pool large quantities of formed vesicles that are useful in examining cloned and reconstituted native amino acid transporter polypeptides.

Emerging roles for sodium dependent amino acid transport in mesenchymal cells

The functional aspects of sodium dependent amino acid transport in mesenchymal cells are the subject of this contribution. In a survey of the cross-talk existing among the various transport mechanisms, particular attention is devoted to the role played by substrates shared by several transport systems, such as L-glutamine. Intracellular levels of glutamine are determined by the activity of System A, the main transducer of ion gradients built on by Na,K-ATPase into neutral amino acid gradients. Changes in the activity of the System are employed to regulate intracellular amino acid pool and, hence, cell volume. System A activity has been found increased in hypertonically shrunken cells and in proliferating cells. Under both these conditions cells have to increase their volume; therefore, System A can be employed as a convenient mechanism to increase cell volume both under hypertonic and isotonic conditions. Although less well characterized, the uptake of anionic amino acids performed by System X(-) AG may be involved in the maintenance of intracellular amino acid pool under conditions of limited availability of neutral amino acids substrates of System A.

Feasibility of labeled α-acetamido-aminoisobutyric acid as new tracer compound for kinetic labeling of neutral amino acid transport: Preparation of α-( N-[1- 11C]Acetyl)- and α-( N-[1- 14C]Acetyl)-aminoisobutyric acid

The nonphysiological, nonracemic, branched-chain α-acetamido-aminoisobutyric acid was labeled with the carbon isotope 11C with the intention to use it in conjunction with positron emission tomography (PET) to measure the kinetics of amino acid transport in vivo. It was produced by the reaction of the novel 11C-precursor N-[1- 11C]acetylpyridinium chloride with α-aminoisobutyric acid. Typically, 2 GBq of α-( N-[1- 11C]acetyl)-aminoisobutyric acid were isolated with a specific activity of 12 to 20 GBq · μmol −1 at the time of application, and with a radiochemical purity of >98%. The chemical identity of α-( N-[1- 11C]acetyl)-aminoisobutyric acid was confirmed by comparison with α-( N-[1- 14C]acetyl)-aminoisobutyric acid that was independently prepared by a standard acetylation procedure of α-aminoisobutyric acid using [1- 14C]acetic anhydride. In vivo, both labeled substrates were not metabolized. In cell-culture experiments, 84% of the substrate entered the cells by the sodium-dependent amino acid transport system A, whereas 16% was taken up by the sodium-independent system. The uptake of the radiotracer was measured 20 min and 40 min postinjection in tumor-bearing male Copenhagen rats for assessment of its in vivo biodistribution.

Expression of a Novel Insulin-Activated Amino Acid Transporter Gene During Differentiation of 3T3-L1 Preadipocytes into Adipocytes

A cDNA encoding a novel insulin-activated adipocyte amino acid transporter (designated AAAT) was cloned from a mouse 3T3-L1 adipocyte library. The deduced amino acid sequence of the cDNA corresponds to a protein of 553 amino acids that possesses 56% amino acid sequence identity to the human neutral amino acid transporter and 42% identity to the rat brain glutamate transporter. Transient transfection of 3T3-L1 preadipocytes with an AAAT expression vector led to insulin-dependent uptake of L-serine and to a lesser extent, uptake of L-alanine and L-glutamate. Expression of the AAAT message is tissue-specific, with the highest level occurring in mouse adipose tissue and a lower level in lung. Unlike other sodium-dependent amino acid transporter mRNAs, the AAAT message is not expressed in brain, kidney, liver or heart and only traces are detected in spleen, thymus and skeletal muscle. Consistent with its high level in adipose tissue, expression of the AAAT message is markedly increased when 3T3-L1 preadipocytes are induced to differentiate into adipocytes.

The effects of cocaine on neutral amino acid uptake by human placental basal membrane vesicles

Prior studies have demonstrated that cocaine binds to human placental microvillous membrane vesicles at a single high-affinity site and that both 10 and 500 nmol/L cocaine inhibit sodium-dependent alanine uptake. The purpose of this study was to characterize cocaine binding to human placental basal plasma membrane and to determine the effects of cocaine on basal vesicle uptake of alanine and leucine. Basal vesicles were isolated from the placentas of uncomplicated human pregnancies with no history of cocaine use. The binding of tritiated cocaine to basal vesicle membrane and the uptakes of tritiated cocaine, alanine, and leucine were determined with filtration assays. Alanine and leucine uptakes were measured in the presence and absence of sodium and 10 and 500 nmol/L cocaine. Cocaine binding was characterized with Scatchard analyses, and uptakes were compared by means of Student t tests. Tritiated cocaine bound to basal membrane at two separate high-affinity sites. Sodium-dependent alanine uptake was significantly inhibited only by 500 nmol/L cocaine. Sodium-independent amino acid uptake was unaffected by cocaine. Cocaine may interfere with fetal growth by impairing the activity of sodium-dependent amino acid transporters in both the microvillous and basal membrane. These membranes may be differentially sensitive to the effects of cocaine on such transporters.

Complex subcellular distribution of sodium-dependent amino acid transport systems in kidney cortex and LLC-PK1/Cl4 cells

To characterize the amino acid transport system in basolateral membranes and to test for possible intracellular loci of amino acid transport activity, we surveyed the distribution of L-alanine transport activity in rabbit proximal tubular cells and LLC-PK1/Cl4 cells. A three-dimensional separation procedure based on differential sedimentation, density gradient centrifugation, and counter-current distribution resolved 21 physically and biochemically distinct membrane populations from rabbit cortex. Inhibition of L-alanine transport by phenylalanine and N-(methylamino)isobutyric acid was used to delineate parallel amino acid transport pathways. Population n was identified as brush border membranes by virtue of its 16-fold maltase enrichment; 94% of its Na(+)-dependent alanine transport activity was mediated by systems previously shown to be characteristic of brush border membranes. Two populations, c' and c", which accounted for 25% of the total Na,K-ATPase activity, were identified as basalateral membranes on the basis of Na,K-ATPase cumulative enrichment factors of 15 and 21; 82% of the total alanine transport in these populations was mediated by a Na(+)-independent system similar to the classical system L. Na,K-ATPase, Na(+)-independent and Na(+)-dependent alanine transport activities were associated with intracellular membrane populations as well as with the plasma membranes. The major intracellular locus of Na,K-ATPase activity, population i accounted for roughly 31% of the Na,K-ATPase, maximally enriched ninefold; it contained 29% of the total system L transport activity. Population l, which was identified as endoplasmic reticulum because it was the major locus of membrane-bound NADPH cytochrome c reductase activity, contained 44% of the total system A transport. Three distinct Golgi-derived populations, m', m", and o, accounted for 39% of the total system A transport. A survey of the amino acid transport systems in LLC-PK1/Cl4 cells showed that the majority of system A-mediated amino acid transport was present in membranes of intracellular and possibly apical origin. The presence of large intracellular pools of amino acid transport activities might reflect newly synthesized transport proteins, ongoing membrane recycling or, perhaps, intracellular reserves available for rapid recruitment to the plasma membrane.