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1.
J Lipid Res ; 61(5): 734-745, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32217606

RESUMEN

Adipocytes take up long chain FAs through diffusion and protein-mediated transport, whereas FA efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating FA flux in adipocytes, the expression levels of 55 membrane transporters without known function were screened in subcutaneous adipose samples from obese patients before and after bariatric surgery using branched DNA methodology. Among the 33 solute carrier (SLC) transporter family members screened, the expression of 14 members showed significant changes before and after bariatric surgery. One of them, Slc43a3, increased about 2.5-fold after bariatric surgery. Further investigation demonstrated that Slc43a3 is highly expressed in murine adipose tissue and induced during adipocyte differentiation in primary preadipocytes and in OP9 cells. Knockdown of Slc43a3 with siRNA in differentiated OP9 adipocytes reduced both basal and forskolin-stimulated FA efflux, while also increasing FA uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased FA uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake.


Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Sistemas de Transporte de Aminoácidos/deficiencia , Sistemas de Transporte de Aminoácidos/genética , Animales , Transporte Biológico , Línea Celular , AMP Cíclico/metabolismo , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Proteínas de Transporte de Membrana/genética , Ratones , ARN Mensajero/genética , Adulto Joven
2.
mBio ; 8(6)2017 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-29259090

RESUMEN

Cystic fibrosis (CF) is caused by mutations in the CFTR gene and is associated with progressive and ultimately fatal infectious lung disease. There can be considerable variability in disease severity among individuals with the same CFTR mutations, and recent genome-wide association studies have identified secondary genetic factors that contribute to this. One of these modifier genes is SLC6A14, which encodes an amino acid transporter. Importantly, variants of this gene have been associated with age at first acquisition of Pseudomonas aeruginosa In this study, we aimed to determine the function of SLC6A14 in airway epithelia and how it might affect colonization by P. aeruginosa We show that SLC6A14 is expressed in respiratory epithelial cells and transports l-arginine out of the airway surface liquid (ASL). Exposure of airway epithelia to flagellin from P. aeruginosa led to upregulation of SLC6A14 expression and increased SLC6A14-dependent uptake of l-arginine from the ASL. In support of the hypothesis that l-arginine affects P. aeruginosa attachment, we showed that l-arginine supplementation promoted P. aeruginosa attachment to an abiotic surface in a dose-dependent manner. In a coculture model, we found that inhibition of SLC6A14-dependent l-arginine transport enhanced P. aeruginosa attachment. In Slc6a14-/y (knockout) mice, P. aeruginosa attachment to lung tissue was also significantly enhanced. Together, these findings suggest that SLC6A14 activity plays a role in the modification of the initial stages of airway infection by altering the level of l-arginine in the ASL, which in turn affects the attachment of P. aeruginosaIMPORTANCE CF patients with shared CFTR gene mutations show significant variability in their clinical presentation of infectious lung disease. Genome-wide association studies have been used to identify secondary genetic factors that may explain the variable susceptibility to infection by opportunistic pathogens, including P. aeruginosa, the leading cause of pathogen-induced lung damage in nonpediatric CF patients. Once identified and characterized, these secondary genetic modifiers may allow for the development of personalized medicine for patients and ultimately the extension of life. In this study, we interrogated the biological role of one of these modifiers, SLC6A14, and showed that it contributes to host defense by depleting extracellular arginine (an attachment-promoting metabolite for P. aeruginosa) from the airway surface liquid.


Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Adhesión Bacteriana , Células Epiteliales/microbiología , Pseudomonas aeruginosa/fisiología , Sistemas de Transporte de Aminoácidos/deficiencia , Animales , Arginina/metabolismo , Fibrosis Quística/complicaciones , Humanos , Ratones Noqueados , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/deficiencia , Infecciones por Pseudomonas/fisiopatología , Pseudomonas aeruginosa/metabolismo
3.
Biosci Biotechnol Biochem ; 80(6): 1125-30, 2016 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-26928127

RESUMEN

The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.


Asunto(s)
Sistemas de Transporte de Aminoácidos/genética , Antiportadores/genética , Vesículas Citoplasmáticas/metabolismo , Regulación Fúngica de la Expresión Génica , Histidina/metabolismo , Proteínas de la Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfato/metabolismo , Sistemas de Transporte de Aminoácidos/deficiencia , Antiportadores/deficiencia , Arginina/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Eliminación de Gen , Lisina/metabolismo , Proteínas de la Membrana/deficiencia , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo
4.
Sci Rep ; 6: 20910, 2016 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-26865023

RESUMEN

Arginine plays an important role in cellular function and metabolism. Arginine uptake mainly occurs through three amino acid permeases, Alp1p, Gap1p and Can1p, which act as both transporters and receptors for amino acid utilization. In this study, seven mutants were constructed with different combinations of permease deficiencies that inhibit arginine utilization. Their effects on arginine metabolism were measured. The three amino acid permeases were also individually overexpressed in wild-type (WT), Δalp1Δgap1Δcan1 and Δnpr1 strains. The growth and arginine utilization of Δcan1, Δgap1Δcan1 and Δalp1Δgap1Δcan1 mutants were suppressed in YNB medium when arginine was the sole nitrogen source. Meanwhile, overexpression of Alp1p and Can1p enhanced growth and arginine utilization in WT, Δalp1Δgap1Δcan1 and Δnpr1. Besides, overexpression of Can1p caused a 26.7% increase in OD600 and 29.3% increase in arginine utilization compared to that of Alp1p in Δalp1Δgap1Δcan1. Transcription analysis showed that the effects of three amino acid permeases on the arginine utilization and the regulation of related genes, were tightly related to their individual characteristics. However, their overall effects were different for different combinations of mutants. The results presented here suggest some possible synergistic effects of different amino acid permeases on regulation of amino acid utilization and metabolism.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Sistemas de Transporte de Aminoácidos/genética , Arginina/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Sistemas de Transporte de Aminoácidos/deficiencia , Transporte Biológico , Recuento de Colonia Microbiana , Mutación , Nitrógeno/metabolismo , Fosforilación , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Transcripción Genética
5.
PLoS One ; 8(6): e65717, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23755272

RESUMEN

Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3Δ nor sam3Δ single mutant is defective in polyamine transport, while the dur3Δ sam3Δ double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2Δ mutant. Studies of Agp2 localization indicate that in the double mutant dur3Δ sam3Δ, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport.


Asunto(s)
Sistemas de Transporte de Aminoácidos/genética , Carnitina/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Espermidina/metabolismo , Simportadores/genética , Transcripción Genética , Sistemas de Transporte de Aminoácidos/deficiencia , Sistemas de Transporte de Aminoácidos/metabolismo , Transporte Biológico , Eliminación de Gen , Proteínas de Transporte de Membrana/deficiencia , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Anotación de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Simportadores/metabolismo
6.
Eur J Neurosci ; 25(6): 1757-66, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17432963

RESUMEN

In the mammalian central nervous system, transporter-mediated reuptake may be critical for terminating the neurotransmitter action of D-serine at the strychnine insensitive glycine site of the NMDA receptor. The Na(+) independent amino acid transporter alanine-serine-cysteine transporter 1 (Asc-1) has been proposed to account for synaptosomal d-serine uptake by virtue of its high affinity for D-serine and widespread neuronal expression throughout the brain. Here, we sought to validate the contribution of Asc-1 to D-serine uptake in mouse brain synaptosomes using Asc-1 gene knockout (KO) mice. Total [(3)H]D-serine uptake in forebrain and cerebellar synaptosomes from Asc-1 knockout mice was reduced to 34 +/- 5% and 22 +/- 3% of that observed in wildtype (WT) mice, respectively. When the Na(+) dependent transport components were removed by omission of Na(+) ions in the assay buffer, D-serine uptake in knockout mice was reduced to 8 +/- 1% and 3 +/- 1% of that measured in wildtype mice in forebrain and cerebellum, respectively, suggesting Asc-1 plays a major role in the Na(+) independent transport of D-serine. Potency determination of D-serine uptake showed that Asc-1 mediated rapid high affinity Na(+) independent uptake with an IC(50) of 19 +/- 1 microm. The remaining uptake was mediated predominantly via a low affinity Na(+) dependent transporter with an IC(50) of 670 +/- 300 microm that we propose is the glial alanine-serine-cysteine transporter 2 (ASCT2) transporter. The results presented reveal that Asc-1 is the only high affinity D-serine transporter in the mouse CNS and is the predominant mechanism for D-serine reuptake.


Asunto(s)
Sistema de Transporte de Aminoácidos y+/deficiencia , Sistema de Transporte de Aminoácidos y+/fisiología , Sistema Nervioso Central/metabolismo , Serina/metabolismo , Sistemas de Transporte de Aminoácidos/deficiencia , Sistemas de Transporte de Aminoácidos/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Células Cultivadas , Sistema Nervioso Central/citología , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Serina/farmacocinética , Sodio/metabolismo , Sinaptosomas/metabolismo , Sinaptosomas/ultraestructura
8.
Cell ; 114(6): 739-49, 2003 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-14505573

RESUMEN

Organisms modulate their growth according to nutrient availability. Although individual cells in a multicellular animal may respond directly to nutrient levels, growth of the entire organism needs to be coordinated. Here, we provide evidence that in Drosophila, coordination of organismal growth originates from the fat body, an insect organ that retains endocrine and storage functions of the vertebrate liver. In a genetic screen for growth modifiers, we identified slimfast, a gene that encodes an amino acid transporter. Remarkably, downregulation of slimfast specifically within the fat body causes a global growth defect similar to that seen in Drosophila raised under poor nutritional conditions. This involves TSC/TOR signaling in the fat body, and a remote inhibition of organismal growth via local repression of PI3-kinase signaling in peripheral tissues. Our results demonstrate that the fat body functions as a nutrient sensor that restricts global growth through a humoral mechanism.


Asunto(s)
Sistemas de Transporte de Aminoácidos/aislamiento & purificación , Proteínas de Drosophila/aislamiento & purificación , Drosophila melanogaster/crecimiento & desarrollo , Privación de Alimentos/fisiología , Hormonas Juveniles/aislamiento & purificación , Fenómenos Fisiológicos de la Nutrición/fisiología , Sistemas de Transporte de Aminoácidos/deficiencia , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/deficiencia , Animales , Regulación hacia Abajo/fisiología , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Cuerpo Adiposo/metabolismo , Retroalimentación Fisiológica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Hormonas Juveniles/deficiencia , Hormonas Juveniles/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/fisiología
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