RESUMO
The pharmacology of amino acid transporters in the SLC6 family is poorly developed compared to that of the neurotransmitter transporters. To identify new inhibitors of the proline transporter SIT1 (SLC6A20), its expression in Xenopus laevis oocytes was optimized. Trafficking of SIT1 was augmented by co-expression of angiotensin-converting enzyme 2 (ACE2) in oocytes but there was no strict requirement for co-expression of ACE2. A pharmacophore-guided screen identified tiagabine as a potent non-competitive inhibitor of SIT1. To understand its binding mode, we determined the cryo-electron microscopy (cryo-EM) structure of ACE2-SIT1 bound with tiagabine. The inhibitor binds close to the orthosteric proline binding site, but due to its size extends into the cytosolic vestibule. This causes the transporter to adopt an inward-open conformation, in which the intracellular gate is blocked. This study provides the first structural insight into inhibition of SIT1 and generates tools for a better understanding of the ACE2-SIT1 complex. These findings may have significance for SARS-CoV-2 binding to its receptor ACE2 in human lung alveolar cells where SIT1 and ACE2 are functionally expressed.
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The epithelial neutral amino acid transporter B0AT1 (SLC6A19) is the major transporter for the absorption of neutral amino acids in the intestine and their reabsorption in the kidney. Mouse models have demonstrated that lack of B0AT1 can normalize elevated plasma amino acids in rare disorders of amino acid metabolism such as phenylketonuria and urea-cycle disorders, implying a pharmacological approach for their treatment. Here we employ a medicinal chemistry approach to generate B0AT1 inhibitors with IC50-values of 31-90 nM. High-resolution cryo-EM structures of B0AT1 in the presence of two compounds from this series identified an allosteric binding site in the vestibule of the transporter. Mechanistically, binding of these inhibitors prevents a movement of TM1 and TM6 that is required for the transporter to make a conformational change from an outward open state to the occluded state.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Microscopia Crioeletrônica , Animais , Humanos , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/antagonistas & inibidores , Camundongos , Sítio Alostérico , Células HEK293 , Sítios de Ligação , Conformação ProteicaRESUMO
The Alanine-Serine-Cysteine transporter 1 (Asc-1 or SLC7A10) forms a crucial heterodimeric transporter complex with 4F2hc (SLC3A2) through a covalent disulfide bridge. This complex enables the sodium-independent transport of small neutral amino acids, including L-Alanine (L-Ala), Glycine (Gly), and D-Serine (D-Ser), within the central nervous system (CNS). D-Ser and Gly are two key endogenous glutamate co-agonists that activate N-methyl-d-aspartate (NMDA) receptors by binding to the allosteric site. Mice deficient in Asc-1 display severe symptoms such as tremors, ataxia, and seizures, leading to early postnatal death. Despite its physiological importance, the functional mechanism of the Asc-1-4F2hc complex has remained elusive. Here, we present cryo-electron microscopy (cryo-EM) structures of the human Asc-1-4F2hc complex in its apo state, D-Ser bound state, and L-Ala bound state, resolved at 3.6 Å, 3.5 Å, and 3.4 Å, respectively. Through detailed structural analysis and transport assays, we uncover a comprehensive alternating access mechanism that underlies conformational changes in the complex. In summary, our findings reveal the architecture of the Asc-1 and 4F2hc complex and provide valuable insights into substrate recognition and the functional cycle of this essential transporter complex.
Assuntos
Proteínas de Membrana Transportadoras , Serina , Camundongos , Humanos , Animais , Microscopia Crioeletrônica , Serina/metabolismo , Proteínas de Membrana Transportadoras/genética , Glicina , CisteínaRESUMO
The integrated stress response is a signaling network comprising four branches, each sensing different cellular stressors, converging on the phosphorylation of eIF2α to downregulate global translation and initiate recovery. One of these branches includes GCN2, which senses cellular amino acid insufficiency and participates in maintaining amino acid homeostasis. Previous studies have shown that GCN2 is a viable cancer target when amino acid stress is induced by inhibiting an additional target. In this light, we screened numerous drugs for their potential to synergize with the GCN2 inhibitor TAP20. The drug sensitivity of six cancer cell lines to a panel of 25 compounds was assessed. Each compound was then combined with TAP20 at concentrations below their IC50, and the impact on cell growth was evaluated. The strongly synergistic combinations were further characterized using synergy analyses and matrix-dependent invasion assays. Inhibitors of proteostasis and the MEK-ERK pathway, as well as the pan-CDK inhibitors, flavopiridol, and seliciclib, were potently synergistic with TAP20 in two cell lines. Among their common CDK targets was CDK7, which was more selectively targeted by THZ-1 and synergized with TAP20. Moreover, these combinations were partially synergistic when assessed using matrix-dependent invasion assays. However, TAP20 alone was sufficient to restrict invasion at concentrations well below its growth-inhibitory IC50. We conclude that GCN2 inhibition can be further explored in vivo as a cancer target.
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Amino acid antiporters mediate the 1:1 exchange of groups of amino acids. Whether substrate specificity can be different for the inward and outward facing conformation has not been investigated systematically, although examples of asymmetric transport have been reported. Here we used LC-MS to detect the movement of 12C- and 13C-labelled amino acid mixtures across the plasma membrane of Xenopus laevis oocytes expressing a variety of amino acid antiporters. Differences of substrate specificity between transporter paralogs were readily observed using this method. Our results suggest that antiporters are largely symmetric, equalizing the pools of their substrate amino acids. Exceptions are the antiporters y+LAT1 and y+LAT2 where neutral amino acids are co-transported with Na+ ions, favouring their import. For the antiporters ASCT1 and ASCT2 glycine acted as a selective influx substrate, while proline was a selective influx substrate of ASCT1. These data show that antiporters can display non-canonical modes of transport.
Assuntos
Aminoácidos , Antiporters , Aminoácidos/metabolismo , Especificidade por Substrato , Antiporters/metabolismo , Prolina/metabolismo , Transporte BiológicoRESUMO
SNAT2 (SLC38A2) is a sodium-dependent neutral amino acid transporter, which is important for the accumulation of amino acids as nutrients, the maintenance of cellular osmolarity, and the activation of mTORC1. It also provides net glutamine for glutaminolysis and consequently presents as a potential target to treat cancer. A high-throughput screening assay was developed to identify new inhibitors of SNAT2 making use of the inducible nature of SNAT2 and its electrogenic mechanism. Using an optimized FLIPR membrane potential (FMP) assay, a curated scaffold library of 33934 compounds was screened to identify 3-(N-methyl (4-methylphenyl)sulfonamido)-N-(2-trifluoromethylbenzyl)thiophene-2-carboxamide as a potent inhibitor of SNAT2. In two different assays an IC50 of 0.8-3 µM was determined. The compound discriminated against the close transporter homologue SNAT1. MDA-MB-231 breast cancer and HPAFII pancreatic cancer cell lines tolerated the SNAT2 inhibitor up to a concentration of 100 µM but in combination with tolerable doses of the glucose transport inhibitor Bay-876, proliferative growth of both cell lines was halted. This points to synergy between inhibition of glycolysis and glutaminolysis in cancer cells.
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Homeostasis is one of the fundamental concepts in physiology. Despite remarkable progress in our molecular understanding of amino acid transport, metabolism and signaling, it remains unclear by what mechanisms cytosolic amino acid concentrations are maintained. We propose that amino acid transporters are the primary determinants of intracellular amino acid levels. We show that a cell's endowment with amino acid transporters can be deconvoluted experimentally and used this data to computationally simulate amino acid translocation across the plasma membrane. Transport simulation generates cytosolic amino acid concentrations that are close to those observed in vitro. Perturbations of the system are replicated in silico and can be applied to systems where only transcriptomic data are available. This work explains amino acid homeostasis at the systems-level, through a combination of secondary active transporters, functionally acting as loaders, harmonizers and controller transporters to generate a stable equilibrium of all amino acid concentrations.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Homeostase/genética , Modelos Estatísticos , Neuroglia/metabolismo , Células A549 , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/classificação , Sistemas de Transporte de Aminoácidos/genética , Animais , Transporte Biológico , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Simulação por Computador , Expressão Gênica , Humanos , Cinética , Metabolômica/métodos , Neuroglia/citologia , Oócitos/citologia , Oócitos/metabolismo , Xenopus laevisRESUMO
Amino acid transporters play a vital role in metabolism and nutrient signaling pathways. Typically, transport activity is investigated using single substrates and competing amounts of other amino acids. We used GC-MS and LC-MS for metabolic screening of Xenopus laevis oocytes expressing various human amino acid transporters incubated in complex media to establish their comprehensive substrate profiles. For most transporters, amino acid selectivity matched reported substrate profiles. However, we could not detect substantial accumulation of cationic amino acids by SNAT4 and ATB0,+ in contrast to previous reports. In addition, comparative substrate profiles of two related sodium neutral amino acid transporters known as SNAT1 and SNAT2, revealed the latter as a significant leucine accumulator. As a consequence, SNAT2, but not SNAT1, was shown to be an effective activator of the eukaryotic cellular growth regulator mTORC1. We propose, that metabolomic profiling of membrane transporters in Xe nopus laevis oocytes can be used to test their substrate specificity and role in intracellular signaling pathways.
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The asymmetric distribution of phospholipids in the plasma/organellar membranes is generated and maintained through phospholipid flippases in resting cells, but becomes disrupted in apoptotic cells and activated platelets, resulting in phosphatidylserine (PS) exposure on the cell surface. Stable PS exposure during apoptosis requires inactivation of flippases to prevent PS from being reinternalized. Here we show that flippase ATP8A1 is highly expressed in both murine and human platelets, but is not present in the plasma membrane. ATP8A1 is cleaved by the cysteine protease calpain during apoptosis, and the cleavage is prevented indirectly by caspase inhibition, involving blockage of calcium influx into platelets and subsequent calpain activation. In contrast, in platelets activated with thrombin and collagen and exposing PS, ATP8A1 remains intact. These data reveal a novel mechanism of flippase cleavage and suggest that flippase activity in intracellular membranes differs between platelets undergoing apoptosis and activation.
Assuntos
Adenosina Trifosfatases/sangue , Plaquetas/metabolismo , Calpaína/sangue , Proteínas de Transferência de Fosfolipídeos/sangue , Fosfolipídeos/sangue , Animais , Apoptose/fisiologia , Plaquetas/enzimologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ativação PlaquetáriaRESUMO
The neutral amino acid transporter solute carrier family 1 member 5 (SLC1A5 or ASCT2) is overexpressed in many cancers. To identify its roles in tumors, we employed 143B osteosarcoma cells and HCC1806 triple-negative breast cancer cells with or without ASCT2 deletion. ASCT2ko 143B cells grew well in standard culture media, but ASCT2 was required for optimal growth at <0.5 mm glutamine, with tumor spheroid growth and monolayer migration of 143B ASCT2ko cells being strongly impaired at lower glutamine concentrations. However, the ASCT2 deletion did not affect matrix-dependent invasion. ASCT2ko 143B xenografts in nude mice exhibited a slower onset of growth and a higher number of small tumors than ASCT2wt 143B xenografts, but did not differ in average tumor size 25 days after xenotransplantation. ASCT2 deficiency was compensated by increased levels of sodium neutral amino acid transporter 1 (SNAT1 or SLC38A1) and SNAT2 (SLC38A2) in ASCT2ko 143B cells, mediated by a GCN2 EIF2α kinase (GCN2)-dependent pathway, but this compensation was not observed in ASCT2ko HCC1806 cells. Combined SNAT1 silencing and GCN2 inhibition significantly inhibited growth of ASCT2ko HCC1806 cells, but not of ASCT2ko 143B cells. Similarly, pharmacological inhibition of l-type amino acid transporter 1 (LAT1) and GCN2 significantly inhibited growth of ASCT2ko HCC1806 cells, but not of ASCT2ko 143B cells. We conclude that cancer cells with reduced transporter plasticity are more vulnerable to disruption of amino acid homeostasis than cells with a full capacity to up-regulate redundant transporters by an integrated stress response.
Assuntos
Sistema ASC de Transporte de Aminoácidos/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Antígenos de Histocompatibilidade Menor/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Sistema ASC de Transporte de Aminoácidos/deficiência , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Neoplasias Ósseas/metabolismo , Neoplasias da Mama/metabolismo , Feminino , Humanos , Camundongos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/metabolismo , Mutação , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Osteossarcoma/metabolismo , Células Tumorais CultivadasRESUMO
The glutamine transporter ASCT2 (SLC1A5) is actively investigated as an oncological target, but the field lacks efficient ASCT2 inhibitors. A new group of ASCT2 inhibitors, 2-amino-4-bis(aryloxybenzyl)aminobutanoic acids (AABA), were developed recently and shown to suppress tumor growth in preclinical in vivo models. To test its specificity, we deleted ASCT2 in two human cancer cell lines. Surprisingly, growth of parental and ASCT2-knockout cells was equally sensitive to AABA compounds. AABA compounds inhibited glutamine transport in cells lacking ASCT2, but not in parental cells. Deletion of ASCT2 and amino acid (AA) depletion induced expression of SNAT2 (SLC38A2), the activity of which was inhibited by AABA compounds. They also potently inhibited isoleucine uptake via LAT1 (SLC7A5), a transporter that is upregulated in cancer cells together with ASCT2. Inhibition of SNAT2 and LAT1 was confirmed by recombinant expression in Xenopus laevis oocytes. The reported reduction of tumor growth in pre-clinical models may be explained by a significant disruption of AA homeostasis.
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Astrocytes are glial cells that have an intimate physical and functional association with synapses in the brain. One of their main roles is to recycle the neurotransmitters glutamate and gamma-aminobutyric acid (GABA), as a component of the glutamate/GABA-glutamine cycle. They perform this function by sequestering neurotransmitters and releasing glutamine via the neutral amino acid transporter SNAT3. In this way, astrocytes regulate the availability of neurotransmitters and subsequently influence synaptic function. Since many plasma membrane transporters are regulated by protein kinase C (PKC), the aim of this study was to understand how PKC influences SNAT3 glutamine transport in astrocytes located immediately adjacent to synapses. We studied SNAT3 transport by whole-cell patch-clamping and fluorescence pH imaging of single astrocytes in acutely isolated brainstem slices, adjacent to the calyx of the Held synapse. Activation of SNAT3-mediated glutamine transport in these astrocytes was reduced to 77 ± 6% when PKC was activated with phorbol 12-myristate 13-acetate (PMA). This effect was very rapid (within ~20 min) and eliminated by application of bisindolylmaleimide I (Bis I) or 7-hydroxystaurosporine (UCN-01), suggesting that activation of conventional isoforms of PKC reduces SNAT3 function. In addition, cell surface biotinylation experiments in these brain slices show that the amount of SNAT3 in the plasma membrane is reduced by a comparable amount (to 68 ± 5%) upon activation of PKC. This indicates a role for PKC in dynamically controlling the trafficking of SNAT3 transporters in astrocytes in situ. These data demonstrate that PKC rapidly regulates the astrocytic glutamine release mechanism, which would influence the glutamine availability for adjacent synapses and control levels of neurotransmission.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Astrócitos/metabolismo , Proteína Quinase C/metabolismo , Sinapses/metabolismo , Animais , Encéfalo/metabolismo , Endocitose , Ativação Enzimática , Isoenzimas/metabolismo , Camundongos Endogâmicos C57BL , Ratos WistarRESUMO
Cells have a constant turnover of proteins that recycle most amino acids over time. Net loss is mainly due to amino acid oxidation. Homeostasis is achieved through exchange of essential amino acids with non-essential amino acids and the transfer of amino groups from oxidised amino acids to amino acid biosynthesis. This homeostatic condition is maintained through an active mTORC1 complex. Under amino acid depletion, mTORC1 is inactivated. This increases the breakdown of cellular proteins through autophagy and reduces protein biosynthesis. The general control non-derepressable 2/ATF4 pathway may be activated in addition, resulting in transcription of genes involved in amino acid transport and biosynthesis of non-essential amino acids. Metabolism is autoregulated to minimise oxidation of amino acids. Systemic amino acid levels are also tightly regulated. Food intake briefly increases plasma amino acid levels, which stimulates insulin release and mTOR-dependent protein synthesis in muscle. Excess amino acids are oxidised, resulting in increased urea production. Short-term fasting does not result in depletion of plasma amino acids due to reduced protein synthesis and the onset of autophagy. Owing to the fact that half of all amino acids are essential, reduction in protein synthesis and amino acid oxidation are the only two measures to reduce amino acid demand. Long-term malnutrition causes depletion of plasma amino acids. The CNS appears to generate a protein-specific response upon amino acid depletion, resulting in avoidance of an inadequate diet. High protein levels, in contrast, contribute together with other nutrients to a reduction in food intake.
Assuntos
Aminoácidos/metabolismo , Dieta , Homeostase , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Regulação do Apetite , Autofagia , Transporte Biológico , Sistema Nervoso Central/metabolismo , Dieta/efeitos adversos , Jejum/metabolismo , Regulação da Expressão Gênica , Humanos , Desnutrição/etiologia , Desnutrição/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Neurônios/metabolismo , Oxirredução , Período Pós-Prandial , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
SLC1A5 (solute carrier family 1, member 5) is a small neutral amino acid exchanger that is upregulated in rapidly proliferating lymphocytes but also in many primary human cancers. Furthermore, cancer cell lines have been shown to require SLC1A5 for their survival in vitro. One of SLC1A5's primary substrates is the immunomodulatory amino acid glutamine, which plays an important role in multiple key processes, such as energy supply, macromolecular synthesis, nucleotide biosynthesis, redox homeostasis, and resistance against oxidative stress. These processes are also essential to immune cells, including neutrophils, macrophages, B and T lymphocytes. We show here that mice with a stop codon in Slc1a5 have reduced glutamine uptake in activated lymphocytes and primary fibroblasts. B and T cell populations and maturation in resting mice were not affected by absence of SLC1A5. Antibody production in resting and immunized mice and the germinal center response to immunization were also found to be normal. SLC1A5 has been recently described as a novel target for the treatment of a variety of cancers, and our results indicate that inhibition of SLC1A5 in cancer therapy may be tolerated well by the immune system of cancer patients.
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BACKGROUND AND PURPOSE: The neutral amino acid transporter B0 AT1 (SLC6A19) has recently been identified as a possible target to treat type 2 diabetes and related disorders. B0 AT1 mediates the Na+ -dependent uptake of all neutral amino acids. For surface expression and catalytic activity, B0 AT1 requires coexpression of collectrin (TMEM27). In this study, we established tools to identify and evaluate novel inhibitors of B0 AT1. EXPERIMENTAL APPROACH: A CHO-based cell line was generated, stably expressing collectrin and B0 AT1. Using this cell line, a high-throughput screening assay was developed, which uses a fluorescent dye to detect depolarisation of the cell membrane during amino acid uptake via B0 AT1. In parallel to these functional assays, we ran a computational compound screen using AutoDock4 and a homology model of B0 AT1 based on the high-resolution structure of the highly homologous Drosophila dopamine transporter. KEY RESULTS: We characterized a series of novel inhibitors of the B0 AT1 transporter. Benztropine was identified as a competitive inhibitor of the transporter showing an IC50 of 44 ± 9 µM. The compound was selective with regard to related transporters and blocked neutral amino acid uptake in inverted sections of mouse intestine. CONCLUSION AND IMPLICATIONS: The tools established in this study can be widely used to identify new transport inhibitors. Using these tools, we were able to identify compounds that can be used to study epithelial transport, to induce protein restriction, or be developed further through medicinal chemistry.
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Sistemas de Transporte de Aminoácidos Neutros/antagonistas & inibidores , Benzotropina/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animais , Benzotropina/química , Células CHO , Cricetulus , Diabetes Mellitus Tipo 2/metabolismo , Relação Dose-Resposta a Droga , Drosophila , Feminino , Ensaios de Triagem em Larga Escala , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Relação Estrutura-AtividadeRESUMO
Many cancer cells depend on glutamine as they use the glutaminolysis pathway to generate building blocks and energy for anabolic purposes. As a result, glutamine transporters are essential for cancer growth and are potential targets for cancer chemotherapy with ASCT2 (SLC1A5) being investigated most intensively. Here we show that HeLa epithelial cervical cancer cells and 143B osteosarcoma cells express a set of glutamine transporters including SNAT1 (SLC38A1), SNAT2 (SLC38A2), SNAT4 (SLC38A4), LAT1 (SLC7A5), and ASCT2 (SLC1A5). Net glutamine uptake did not depend on ASCT2 but required expression of SNAT1 and SNAT2. Deletion of ASCT2 did not reduce cell growth but caused an amino acid starvation response and up-regulation of SNAT1 to replace ASCT2 functionally. Silencing of GCN2 in the ASCT2(-/-) background reduced cell growth, showing that a combined targeted approach would inhibit growth of glutamine-dependent cancer cells.
Assuntos
Sistema ASC de Transporte de Aminoácidos/genética , Sistema A de Transporte de Aminoácidos/fisiologia , Proliferação de Células , Deleção de Genes , Expressão Gênica , Glutamina/metabolismo , Células HeLa , Homeostase , Humanos , Transporte de Íons , Redes e Vias Metabólicas , Antígenos de Histocompatibilidade MenorRESUMO
Many solute carrier 6 (SLC6) family transporters require ancillary subunits to modify their expression and activity. The main apical membrane neutral amino acid transporters in mouse intestine and kidney, B(0)AT1 and B(0)AT3, require the ancillary protein collectrin or ACE2 for plasma membrane expression. Expression and activity of SLC6 neurotransmitter transporters are modulated by interaction with syntaxin 1A. Utilizing monocarboxylate-B(0)AT1/3 fusion constructs, we discovered that collectrin is also necessary for B(0)AT1 and B(0)AT3 catalytic function. Syntaxin 1A and syntaxin 3 inhibit the membrane expression of B(0)AT1 by competing with collectrin for access. A mutagenesis screening approach identified residues on trans-membrane domains 1α, 5, and 7 on one face of B(0)AT3 as a key region involved in interaction with collectrin. Mutant analysis established residues that were involved in collectrin-dependent functions as follows: plasma membrane expression of B(0)AT3, catalytic activation, or both. These results identify a potential binding site for collectrin and other SLC6 ancillary proteins.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animais , Sítios de Ligação , Biotinilação , Células CHO , Catálise , Cricetinae , Cricetulus , Drosophila melanogaster , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Mutagênese , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Qa-SNARE/metabolismo , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Sintaxina 1/metabolismo , Xenopus laevisRESUMO
OBJECTIVE: Type 2 diabetes arises from insulin resistance of peripheral tissues followed by dysfunction of ß-cells in the pancreas due to metabolic stress. Both depletion and supplementation of neutral amino acids have been discussed as strategies to improve insulin sensitivity. Here we characterise mice lacking the intestinal and renal neutral amino acid transporter B(0)AT1 (Slc6a19) as a model to study the consequences of selective depletion of neutral amino acids. METHODS: Metabolic tests, analysis of metabolite levels and signalling pathways were used to characterise mice lacking the intestinal and renal neutral amino acid transporter B(0)AT1 (Slc6a19). RESULTS: Reduced uptake of neutral amino acids in the intestine and loss of neutral amino acids in the urine causes an overload of amino acids in the lumen of the intestine and reduced systemic amino acid availability. As a result, higher levels of glucagon-like peptide 1 (GLP-1) are produced by the intestine after a meal, while the liver releases the starvation hormone fibroblast growth factor 21 (FGF21). The combination of these hormones generates a metabolic phenotype that is characterised by efficient removal of glucose, particularly by the heart, reduced adipose tissue mass, browning of subcutaneous white adipose tissue, enhanced production of ketone bodies and reduced hepatic glucose output. CONCLUSIONS: Reduced neutral amino acid availability improves glycaemic control. The epithelial neutral amino acid transporter B(0)AT1 could be a suitable target to treat type 2 diabetes.
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BACKGROUND: Despite homeostatic pH regulation, systemic and cellular pH changes take place and strongly influence metabolic processes. Transcription of the glutamine transporter SNAT3 (Slc38a3) for instance is highly up-regulated in the kidney during metabolic acidosis to provide glutamine for ammonia production. METHODS: Slc38a3 promoter activity and messenger RNA stability were measured in cultured cells in response to different extracellular pH values. RESULTS: Up-regulation of SNAT3 mRNA was mediated both by the stabilization of its mRNA and by the up-regulation of gene transcription. Stabilisation of the mRNA involved a pH-response element, while enhanced transcription made use of a second pH-sensitive Sp1 binding site in addition to a constitutive Sp1 binding site. Transcriptional regulation dominated the early response to acidosis, while mRNA stability was more important for chronic adaptation. Tissue-specific expression of SNAT3, by contrast, appeared to be controlled by promoter methylation and histone modifications. CONCLUSIONS: Regulation of SNAT3 gene expression by extracellular pH involves post-transcriptional and transcriptional mechanisms, the latter being distinct from the mechanisms that control the tissue-specific expression of the gene.
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Acidose/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/biossíntese , Sistemas de Transporte de Aminoácidos Neutros/genética , Acidose/genética , Animais , Células Cultivadas , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/genéticaRESUMO
Enterocytes are specialized to absorb nutrients from the lumen of the small intestine by expressing a select set of genes to maximize the uptake of nutrients. They develop from stem cells in the crypt and differentiate into mature enterocytes while moving along the crypt-villus axis. Using the Slc6a19 gene as an example, encoding the neutral amino acid transporter B(0)AT1, we studied regulation of the gene by transcription factors and epigenetic factors in the intestine. To investigate this question, we used a fractionation method to separate mature enterocytes from crypt cells and analyzed gene expression. Transcription factors HNF1a and HNF4a activate transcription of the Slc6a19 gene in villus enterocytes, whereas high levels of SOX9 repress expression in the crypts. CpG dinucleotides in the proximal promoter were highly methylated in the crypt and fully de-methylated in the villus. Furthermore, histone modification H3K27Ac, indicating an active promoter, was prevalent in villus cells but barely detectable in crypt cells. The results suggest that Slc6a19 expression in the intestine is regulated at three different levels involving promoter methylation, histone modification, and opposing transcription factors.