RESUMEN
Skeletal muscle (SM) is the most abundant tissue and the largest reservoir of protein in the body. It transports glucose in an insulin dependent manner by the glucose transporter type 4 (GLUT4) and contributes in the maintenance of serum amino acids concentration. By its mass and energetic requirements, it is fundamental for the systemic metabolic balance. In the present work, we present the effect of gestational undernourishment (GU) on the mechanical and metabolic properties of SM at birth and in old age in an animal model. Mechanical studies were performed on isolated muscles, while the GLUT4, amino acid transporters LAT2, SNAT2 and insulin receptors (IR) determination were performed on isolated transverse-tubule membranes (TT). The GU in offspring at birth, results in low muscle mass with increased contraction force and resistance to fatigue. However, in two-years old rats, there was muscle hypotrophy and sarcopenia, the force decreased between 50 and 70% in control rats and rats with GU respectively, accompanied by a lower expression of LAT2, SNAT2 and IR in TT. In conclusion, GU irreversibly affects the SM, an effect that could be similar in humans, which help us to understand the events that associate the GU with the metabolic debacle of SM and the metabolic diseases of human adulthood.
Asunto(s)
Desnutrición/complicaciones , Músculo Esquelético/fisiopatología , Atrofia Muscular/etiología , Efectos Tardíos de la Exposición Prenatal/etiología , Sarcopenia/etiología , Factores de Edad , Sistema de Transporte de Aminoácidos A , Sistema de Transporte de Aminoácidos y+/análisis , Sistemas de Transporte de Aminoácidos/análisis , Aminoácidos/sangre , Animales , Femenino , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/análisis , Glucosa , Transportador de Glucosa de Tipo 4/análisis , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Modelos Animales , Contracción Muscular/fisiología , Fuerza Muscular/fisiología , Músculo Esquelético/química , Músculo Esquelético/patología , Embarazo , Ratas , Receptor de Insulina/análisisRESUMEN
Wolff-Chaikoff effect is characterized by the blockade of thyroid hormone synthesis and secretion due to iodine overload. However, the regulation of monocarboxylate transporter 8 during Wolff-Chaikoff effect and its possible role in the rapid reduction of T4 secretion by the thyroid gland remains unclear. Patients with monocarboxylate transporter 8 gene loss-of-function mutations and monocarboxylate transporter 8 knockout mice were shown to have decreased serum T4 levels, indicating that monocarboxylate transporter 8 could be involved in the secretion of thyroid hormones from the thyroid gland. Herein, we aimed to evaluate the regulation of monocarboxylate transporter 8 during the Wolff-Chaikoff effect and the escape from iodine overload, besides the importance of iodine organification for this regulation. Monocarboxylate transporter 8 mRNA and protein levels significantly decreased after 1 day of NaI administration to rats, together with decreased serum T4; while no alteration was observed in LAT2 expression. Moreover, both monocarboxylate transporter 8 expression and serum T4 was restored after 6 days of NaI. The inhibition of thyroperoxidase activity by methimazole prevented the inhibitory effect of NaI on thyroid monocarboxylate transporter 8 expression, suggesting that an active thyroperoxidase is necessary for MCT8 downregulation by iodine overload, similarly to other thyroid markers, such as sodium iodide symporter. Therefore, we conclude that thyroid monocarboxylate transporter 8 expression is downregulated during iodine overload and that the normalization of its expression parallels the escape phenomenon. These data suggest a possible role for monocarboxylate transporter 8 in the changes of thyroid hormones secretion during the Wolff-Chaikoff effect and escape.
Asunto(s)
Yodo/metabolismo , Transportadores de Ácidos Monocarboxílicos/fisiología , Glándula Tiroides/metabolismo , Sistema de Transporte de Aminoácidos y+/análisis , Animales , Regulación hacia Abajo , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/análisis , Masculino , Transportadores de Ácidos Monocarboxílicos/análisis , Transportadores de Ácidos Monocarboxílicos/genética , Ratas , Ratas Wistar , Hormonas Tiroideas/metabolismoRESUMEN
Most tumor cell membranes overexpress L-type amino acid transporter 1, while normal cell membranes contain l-type amino acid transporter 2; both are Na(+)-independent amino acid transporters. Therefore, compounds that selectively inhibit L-type amino acid transporter 1 offer researchers with a novel cancer molecular target. Synthetic chemistry efforts and in vitro screening have produced a variety of novel compounds possessing high in vitrol-type amino acid transporter 1 selectivity; KYT-0353 was one such compound. The present studies illustrate that KYT-0353 inhibited (14)C-leucine uptake and cell growth in human colon cancer-derived HT-29 cells; IC(50)s were 0.06 microm and 4.1 microm, respectively. KYT-0353 also inhibited (14)C-leucine uptake in mouse renal proximal tubule cells expressing l-type amino acid transporter 1, and inhibited cell growth; IC(50)s were 0.14 microm and 16.4 microm, respectively. Compared to control animals, intravenously administered KYT-0353 (12.5 mg/kg and 25.0 mg/kg) showed statistically significant growth inhibition against HT-29 tumors transplanted to nude mice with maximal inhibition ratios of 65.9% and 77.2%, respectively. Body weight increase with time--a safety indicator--was slightly depressed at 12.5 mg/kg and 25.0 mg/kg with maximal ratios of 3.7% (day 2) and 6.3% (day 11), respectively. Thus, KYT-0353 showed significant growth inhibitory effects on HT-29 cells both in vitro and in vivo, whereas it only caused a slight body weight depression. Therefore, KYT-0353 appears to have potential as a novel anti-tumor agent, presumably via selective in vivol-type amino acid transporter 1 inhibition.
Asunto(s)
Antineoplásicos/farmacología , Benzoxazoles/farmacología , Transportador de Aminoácidos Neutros Grandes 1/fisiología , Tirosina/análogos & derivados , Sistema de Transporte de Aminoácidos y+/análisis , Animales , Proliferación Celular/efectos de los fármacos , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/análisis , Células HT29 , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Leucina/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Serina-Treonina Quinasas TOR , Tirosina/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have examined the expression and function of system l amino acid transporter in KB human oral epidermoid carcinoma cells. The KB cells express l-type amino acid transporter 1 (LAT1) in plasma membrane, but not l-type amino acid transporter 2 (LAT2). The [14C]l-leucine uptake by KB cells is inhibited by system l selective inhibitor BCH. The majority of [14C]l-leucine uptake is, therefore, mediated by LAT1. These results suggest that the transport of neutral amino acids including several essential amino acids into the KB cells mediated by LAT1 and the specific inhibition of LAT1 in oral cancer cells will be a new rationale for anti-cancer therapy.
Asunto(s)
Sistema de Transporte de Aminoácidos y+ , Células KB/química , Transportador de Aminoácidos Neutros Grandes 1/análisis , Aminoácidos Cíclicos/farmacología , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/análisis , Humanos , Inmunohistoquímica , Transportador de Aminoácidos Neutros Grandes 1/fisiología , Leucina/metabolismoRESUMEN
Infection with the nematode Nippostrongylus brasiliensis induces various types of cytological alterations in the intestinal villus epithelium. The aim of this study was to analyse the expression of hexose, peptide and amino acid transporters in the small intestinal epithelium after infection. Brown-Norway rats were infected with 2000 N. brasiliensis L3 larvae and villus epithelial cells were isolated at various time points after infection. Expression of hexose transporters Na(+)/glucose cotransporter SGLT1 and glucose transporter GLUT-1, -2 and -5, a peptide transporter (PepT1) and an amino acid transporter (LAT2) was examined by reverse transcription-PCR, Western blotting or immunohistochemistry. Semi-quantitative reverse transcription-PCR studies of separated jejunal epithelial cells showed that expression levels of GLUT5, PepT1 and LAT2 were significantly decreased 7 and 14 days after infection, while these changes were not observed in the ileal epithelium. Although the apical surface glucose transporter SGLT1 showed no significant alteration in mRNA expression, Western blotting analyses of jejunal epithelial cell lysate showed a marked decrease. Contrary to SGLT1, GLUT5, PepT1 and LAT2, expression of GLUT1, which is essential in maintaining high rates of glucose influx, was significantly up-regulated in the jejunal epithelium 7 and 14 days after infection in reverse transcription-PCR as in Western blotting analyses. Immunohistochemical studies showed that GLUT1 immunoreactivity was localised to the basolateral membrane of intestinal epithelial cells 7 days after infection. These results show that N. brasiliensis infection results in an increase in GLUT1 and a decrease in various hexose, amino acid and peptide transporter expression in jejunal epithelial cells. Up-regulation of GLUT1 might be a compensatory response in injured epithelial cells.
Asunto(s)
Sistema de Transporte de Aminoácidos y+ , Proteínas Portadoras/análisis , Células Epiteliales/metabolismo , Células Epiteliales/parasitología , Intestino Delgado/parasitología , Nippostrongylus , Infecciones por Strongylida/metabolismo , Simportadores , Actinas/análisis , Actinas/genética , Animales , Western Blotting/métodos , Proteínas Portadoras/genética , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/análisis , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/genética , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 2 , Transportador de Glucosa de Tipo 5 , Íleon/metabolismo , Íleon/parasitología , Inmunohistoquímica/métodos , Intestino Delgado/metabolismo , Yeyuno/metabolismo , Yeyuno/parasitología , Masculino , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Monosacáridos/análisis , Proteínas de Transporte de Monosacáridos/genética , Transportador de Péptidos 1 , ARN Mensajero/análisis , Ratas , Ratas Endogámicas BN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transportador 1 de Sodio-Glucosa , ATPasa Intercambiadora de Sodio-Potasio/análisis , ATPasa Intercambiadora de Sodio-Potasio/genética , Timidina Quinasa/análisis , Timidina Quinasa/genéticaRESUMEN
The plasma membrane transport system L is in many cells the only (efficient) pathway for the import of large branched and aromatic neutral amino acids. The corresponding transporters are hetero(di)mers composed of a catalytic subunit (LAT1 or LAT2=light chain=glycoprotein-associated amino acid transporter) associated covalently with the glycoprotein 4F2hc/CD98 (heavy chain). The tissue distribution of LAT1 suggests that it is involved mainly in transporting amino acids into growing cells and across some endothelial/epithelial secretory barriers, whereas the localization of LAT2 indicates that it is mainly involved in the basolateral efflux step of transepithelial (re)absorptive amino acid transport. However, system L transporters are obligatory amino acid exchangers with 1:1 stoichiometry, with similar (but not identical) intra- and extracellular substrate selectivities and with highly asymmetrical apparent affinities (low affinity inside). Therefore, net directional transport of large, neutral amino acids by system L depends on the parallel expression of a unidirectional transporter with overlapping selectivity (for instance systems A or N) that provides/recycles amino acids that drive system L exchange function. By mediating the regulated flux of these exchange substrates, unidirectional transporters control the activity of system L.
Asunto(s)
Sistema de Transporte de Aminoácidos L/fisiología , Sistema de Transporte de Aminoácidos y+ , Aminoácidos Neutros/metabolismo , Sistema de Transporte de Aminoácidos L/química , Animales , Transporte Biológico , Epitelio/metabolismo , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/análisis , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/metabolismo , Proteína-1 Reguladora de Fusión/análisis , Humanos , Membranas Intracelulares/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/análisis , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Concentración Osmolar , Distribución TisularRESUMEN
The heteromeric amino acid transporters b(0,+)AT-rBAT (apical), y(+)LAT1-4F2hc, and possibly LAT2-4F2hc (basolateral) participate to the (re)absorption of cationic and neutral amino acids in the small intestine and kidney proximal tubule. We show now by immunofluorescence that their expression levels follow the same axial gradient along the kidney proximal tubule (S1>S2S3). We reconstituted their co-expression in MDCK cell epithelia and verified their polarized localization by immunofluorescence. Expression of b(0,+)AT-rBAT alone led to a net reabsorption of l-Arg (given together with l-Leu). Coexpression of basolateral y(+)LAT1-4F2hc increased l-Arg reabsorption and reversed l-Leu transport from (re)absorption to secretion. Similarly, l-cystine was (re)absorbed when b(0,+)AT-rBAT was expressed alone. This net transport was further increased by the coexpression of 4F2hc, due to the mobilization of LAT2 (exogenous and/or endogenous) to the basolateral membrane. In summary, apical b(0,+)AT-rBAT cooperates with y(+)LAT1-4F2hc or LAT2-4F2hc for the transepithelial reabsorption of cationic amino acids and cystine, respectively. The fact that the reabsorption of l-Arg led to the secretion of l-Leu demonstrates that the implicated heteromeric amino acid transporters function in epithelia as exchangers coupled in series and supports the notion that the parallel activity of unidirectional neutral amino acid transporters is required to drive net amino acid reabsorption.