RESUMEN
Once considered as a "metabolic waste," lactate is now recognized as a major fuel for tricarboxylic acid (TCA) cycle. Our metabolic flux analysis reveals that skeletal muscle mainly uses lactate to fuel TCA cycle. Lactate is transported through the cell membrane via monocarboxylate transporters (MCTs) in which MCT1 is highly expressed in the muscle. We analyzed how MCT1 affects muscle functions using mice with specific deletion of MCT1 in skeletal muscle. MCT1 deletion enhances running performance, increases oxidative fibers while decreasing glycolytic fibers, and enhances flux of glucose to TCA cycle. MCT1 deficiency increases the expression of mitochondrial proteins, augments cell respiration rate, and elevates mitochondrial activity in the muscle. Mechanistically, the protein level of PGC-1α, a master regulator of mitochondrial biogenesis, is elevated upon loss of MCT1 via increases in cellular NAD+ level and SIRT1 activity. Collectively, these results demonstrate that MCT1-mediated lactate shuttle plays a key role in regulating muscle functions by modulating mitochondrial biogenesis and TCA flux.
Asunto(s)
Ciclo del Ácido Cítrico , Ácido Láctico , Transportadores de Ácidos Monocarboxílicos , Músculo Esquelético , Biogénesis de Organelos , Simportadores , Animales , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Músculo Esquelético/metabolismo , Simportadores/metabolismo , Simportadores/genética , Ácido Láctico/metabolismo , Ratones , Mitocondrias/metabolismo , Sirtuina 1/metabolismo , Sirtuina 1/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Ratones Noqueados , GlucólisisRESUMEN
A gene encoding 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) from the extremely thermophilic bacterium Rhodothermus obamensis STB05 was successfully cloned and expressed in Escherichia coli. Extracellular expression of the recombinant enzyme (R.o-GBE) was achieved with a yield of 1080â¯mg/L. Then it was purified and further characterized biochemically. R.o-GBE was optimally active at pH 7.0 and 65⯰C. It remained stable at temperatures up to 80⯰C and had a half-life at 85⯰C of approximately 31â¯min. Far-UV circular dichroism and intrinsic fluorescence analyses revealed that high temperatures reduced its activity by changing the secondary and tertiary structure of R.o-GBE. The enzyme had broad pH stability between pH 3.0 and 11.0â¯at 4⯰C, and preferred weakly acidic conditions at high temperatures. None of the metal ions enhanced the activity of R.o-GBE, but Ca2+ may be required for its activity. Its specific activity with amylopectin was 6651 U/mg, which is much higher than that reported for other GBEs. Its excellent thermostability, broad pH stability, and high specific activity make R.o-GBE highly suitable for industrial applications.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/genética , Proteínas Bacterianas/genética , Rhodothermus/genética , Enzima Ramificadora de 1,4-alfa-Glucano/química , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Calcio/metabolismo , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Expresión Génica , Concentración de Iones de Hidrógeno , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodothermus/química , Rhodothermus/metabolismoRESUMEN
1,4-α-Glucan branching enzyme (GBE, EC. 2.4.1.18), which plays a key role in the synthesis of starch and glycogen, has been overexpressed in E. coli as an intracellular enzyme by many researchers. In this study, it was found that the GBEs from Geobacillus thermoglucosidans and Rhodothermus obamensis were secreted into the culture medium when they were expressed separately, in E. coli. This occurred despite the absence of any signal peptide. In fact, although bioinformatics tools predicted that both of these proteins would localize to the cytoplasm, a high level of expression and non-classical secretion was found to achieve without addition of the inducer isopropyl ß-d-thiogalactopyranoside. Further experiments revealed that secretion was a two-step process that occurred via the periplasmic space. Results excluded the involvement of the Sec pathway or the TAT pathway. Instead, the findings indicated a relationship between cell membrane integrity and the secretion of the two GBEs, and suggested that their N-termini play an essential role in their expression and secretion.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Escherichia coli/enzimología , Enzima Ramificadora de 1,4-alfa-Glucano/química , Permeabilidad de la Membrana Celular , Estabilidad de Enzimas , Escherichia coli/citología , Geobacillus/enzimología , Rhodothermus/enzimologíaRESUMEN
1,4-α-Glucan branching enzyme (GBE) catalyzes the formation of α-1,6 branch points in starch or glycogen by hydrolyzing α-1,4-glucosidic linkages and then synthesizing α-1,6-glucosidic linkages. In the GBE from Geobacillus thermoglucosidans STB02, alanine 310 (Ala310) is located in conserved region II. An analysis of the amino acid sequence shows that Ala310 is highly conserved in the prokaryotic GBE subfamily. Site-directed mutagenesis was used to determine the function of Ala310 in GBE. Replacement of Ala310 with glycine, aspartate, asparagine, isoleucine, glutamate, or glutamine resulted in mutant enzymes with less than 10% to 25% of wild-type activity when amylopectin or amylose was used as substrate. Studies using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) showed that A310G mutant had no effect on the transfer pattern, but the branching activity had been repressed to a large extent. Kinetic analysis also showed that mutations of Ala310 had an effect on the KM value that changed the preferred substrate from amylopectin to amylose. These results show that Ala310 is important for the catalytic activity of the GBE from G. thermoglucosidans STB02.