β-Hydroxy-β-Methylbutyrate (HMB) Normalizes Dexamethasone-Induced Autophagy-Lysosomal Pathway in Skeletal Muscle

Dexamethasone-induced muscle atrophy is due to an increase in protein breakdown and a decrease in protein synthesis, associated with an over-stimulation of the autophagy-lysosomal pathway. These effects are mediated by alterations in IGF-1 and PI3K/Akt signaling. In this study, we have investigated the effects of β-Hydroxy-β-methylbutyrate (HMB) on the regulation of autophagy and proteosomal systems. Rats were treated during 21 days with dexamethasone as a model of muscle atrophy. Co-administration of HMB attenuated the effects promoted by dexamethasone. HMB ameliorated the loss in body weight, lean mass and the reduction of the muscle fiber cross-sectional area (shrinkage) in gastrocnemius muscle. Consequently, HMB produced an improvement in muscle strength in the dexamethasone-treated rats. To elucidate the molecular mechanisms responsible for these effects, rat L6 myotubes were used. In these cells, HMB significantly attenuated lysosomal proteolysis induced by dexamethasone by normalizing the changes observed in autophagosome formation, LC3 II, p62 and Bnip3 expression after dexamethasone treatment. HMB effects were mediated by an increase in FoxO3a phosphorylation and concomitant decrease in FoxO transcriptional activity. The HMB effect was due to the restoration of Akt signaling diminished by dexamethasone treatment. Moreover, HMB was also involved in the regulation of the activity of ubiquitin and expression of MurF1 and Atrogin-1, components of the proteasome system that are activated or up-regulated by dexamethasone. In conclusion, in vivo and in vitro studies suggest that HMB exerts protective effects against dexamethasone-induced muscle atrophy by normalizing the Akt/FoxO axis that controls autophagy and ubiquitin proteolysis.This project has been funded by Abbott Nutrition R&D

β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells

β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth.This project has been funded by Abbott Nutrition R&D

Comercio de organismos vivos modifcados(OVM) y su panorama para la agircultura

Trbajo final de graduación de de 168 págians en formato pdfLa manipulación genética es utilizada en diversos campos comerciales, sin embargo, es la agricultura el campo en el cual se considera que el comercio genético tendrá mayor impacto. Anuue hay mcuhas aplicaciones de la genética en la agricultura, el enfoque actual delas investigaciones está concentrado en desarrollar cultivos con características comerciales favorables, tales como una vida comercial más larga, resistencia a condiciciones ambientales agresivass, resistencia a herbicidas, plagas de insectos, enfermedades, mejores cualidades nutritivas, entre otras. La manipulación genética de cultivos se ha hecho desde hacientos años, mediante la denominadas cruzas; sin embargo, con las técnicas de ingenierí9a genética, el ser humano introduce características a los cultivos que incluso provienen de genes de organismos de diferentes reinos. La anterior situación ha puesto en la mesa de discursión consideraciones sobre los riesgos y beneficios que estas técncas de la biotecnología moderna pueden generar, de tal manera, que parece que el mundo se ha divididoentre los que apoyan el desarrollo de estas técnicas y aquellos que se muestran más celosos al respecto. Derivados de esta división de criterios surgen, los conflictos de indole comercial entre los países que producen y comercializan cultivos modificasos genéticamanete y aquellos que prefieren no comprar. Dentro de marco comercio internacional, la Organización Mundial del Comercio (OMC) es el entre rector, por excelencia, de la regulación comercial para evitar el surgimiento de conflictos entres sus miembros y garantizar igualdad de oportunidades. Sin embargo, tras el surgimeinto del Protocolo de Bioseguridad, cuyo objetivo primordial es la protección de la biodiversidad, surgen muchas disputas referentes a que dicho Protocol, dentro de sus enunciados, tiene un aire comercial, lo cual provoca que entre en disonancia con lo establecido por la OMC. Por otro lado, más del 50% de los países productores de cultivos transgénicos son países en vías de desarrollo, razón por la cual se considera que en el incremento de estas técnicas de agrobiotecnología puede encontrarse la clave para apoyar el crecimiento de los menos desarrollados. Finalmente, una consideración importante, dentro del dilema delas negociaciones comerciales y relaciones multilaterales entre países se encuentra el contenido social, donde el mejoramiento de la agricultura es evidentemente necsario para apoyar la seguridad alimentaria.Universidad Estatal a Distancia de Costa Ric

Aislamiento y caracterización del gen de la transglutaminasa de arroz (Oryza sativa)

[spa] En esta tesis se describen los resultados obtenidos en la clonación y caracterización de una nueva transglutaminasa (TGasa) vegetal aislada en arroz (Oryza sativa), la segunda transglutaminasa clonada en plantas y la primera en plantas C3. Las TGasas (E.C.2.3.2.13) son una familia de enzimas que promueven modificaciones postraduccionales de proteínas. Tienen especial importancia debido a sus implicaciones en enfermedades humanas y a sus aplicaciones biotecnológicas de interés industrial. Su investigación en plantas es aún incipiente y hasta la fecha, solo se ha clonado la TGasa cloroplástica de maíz (TGZ), a partir de una librería de ADN copia (ADNc) de plántulas de maíz. El aislamiento de la nueva transglutaminasa de arroz, descrita en esta tesis, se ha realizado a partir de ADNc y genómico. Utilizando cebadores diferenciales se realizó la clonación del gen (tgo), su inserción en un vector de expresión y su caracterización. Los análisis de la secuencia de tgo han permitido deducir que este nuevo gen está constituido por una secuencia de 1605 pb y que codifica para una proteína (TGO) de 352 aminoácidos. El análisis de la secuencia de TGO ha permitido detectar diferentes dominios relacionados con la actividad TGasa, como son la triada catalítica (Cys-His-Asp), secuencias repetidas en tándem y varios sitios de N-miristoilación. Además, otros dominios detectados, como los leucine-zipper, no eran conocidos en TGasas vegetales. La expresión de la proteína TGO en E. coli ha permitido realizar la caracterización de la enzima con respecto a diferentes parámetros. Así, mediante ensayos de actividad se ha podido confirmar la preferencia de la enzima frente a diferentes sustratos, así como su dependencia de calcio y la inhibición de su actividad frente a conocidos inhibidores de la actividad TGasa. Igualmente, la enzima fue capaz de polimerizar un sustrato in vitro y se comprobó su dependencia de la luz utilizando proteínas tilacoidales como sustrato. Éstas y otras características relevantes han permitido identificar a esta enzima como una verdadera TGasa. Finalmente, ensayos realizados con plantas de arroz para determinar su localización subcelular, así como su relación con proteínas implicadas en la fotosíntesis y su dependencia de la luz, llevan a sugerir que las funciones de TGO están asociadas a procesos fotosintéticos, probablemente relacionados con fotoprotección, al igual que ya se había descrito para la TGasa de maíz, aunque no se descartan otras funciones relacionadas.[eng] This thesis presents the results of cloning and characterization of a novel transglutaminase (TGase) from rice (Oryza sativa). This is the second transglutaminase cloned in plants and the first in C3 plants . TGases (EC2.3.2.13) are a family of enzymes that promote protein post-translational modifications. These enzymes are of particular importance because of their implication in human diseases and their biotechnological applications of industrial interest. Research on plant TGases is still incipient. To date, maize chloroplast TGase (TGZ) cloned from a DNA library is the only cloned TGase. The isolation of a new rice transglutaminase (TGO), described in this thesis, was performed using differential primers designed from both genomic and copy DNA, which has allowed the cloning of the gene (tgo), its insertion into an expression vector, and further characterization. Analysis of both the gene and protein sequences shows that this new gene is comprised of 1605 bp and encodes for a protein (TGO) of 352 amino acids. During sequence analysis different domains associated with TGase activity, such as a catalytic triad (Cys-His-Asp), tandem repeat sequences, and several N-myristoylation sites, were identified. Additionally, other domains not previously reported in plant TGases, such as a leucine-zipper domain, were observed. TGO protein expression in E. coli, allows enzyme characterization with respect to different parameters. Thus, the enzyme preference for different substrates, as well as calcium dependence and activity inhibition in the presence of known TGase inhibitors, was confirmed by these activity assays. The enzyme was able to polymerize in vitro a substrate and was also tested for light dependence using thylakoid proteins as substrate. All of these and other relevant TGO characteristics allowed the identification of this enzyme as a true TGase. Finally, studies with rice plants to determine TGO subcellular localization, as well as its relation to photosynthesis-involved proteins and light dependence, suggested that TGO functionality is associated with photosynthetic processes, probably related to photoprotection, which has already been described for maize TGase. In addition to photoprotection there are possibly additional functions that cannot be excluded

Rice transglutaminase gene: Identification, protein expression, functionality, light dependence and specific cell location

Transglutaminases (TGases), that catalyze post-translational modification of proteins, are scarcely known in plants. As part of a project to characterize transglutaminase genes in new plant species, the identification and characterization of a TGase in rice is presented. Using differential primers, a cDNA (tgo) of 1767 bp from genomic rice DNA amplification was obtained. The primers were designed from the rice DNA sequence relatively homologous to the gene encoding active maize chloroplast TGase. Amino acid sequence of the deduced rice TGase protein (TGO) indicated that it contains the enzyme catalytic triad (Cys–His–Asp), three repeats, myristoylation domains and a leucine zipper motif. The TGO recombinant protein was characterized, showing specific activity regulation, and indicating that tgo encoded for an authentic TGase. Substrate preference and Ca2+ dependent activity were also detected. In the rice plant TGO protein was immunolocalized in the grana chloroplasts, in protein vesicles near them, and in the bulliform cells. Immunoblot analyses, tgo mRNA expression, and TGase activity indicated that TGO expression in rice was light dependent and regulated by the illumination period. This work increases significantly our plant TGase understanding. Its functional role in rice, which is a good model system for C3 plants, is discussed.This study was supported by the Spanish projects MEC BFU2006-15115-01/BMC and BFU2009-08575. N. Campos has a pre-doctoral fellowship from the Agencia Española de Cooperación Internacional para el Desarrollo (AECID). The authors would especially like to thank Marc Boix (CRAG Microscopy Services), Oriol Casagran (CRAG Genomic Services), N. Cortadellas (Serveis Cientifico-Tècnics, UB), and the CRAG Greenhouse Services for their technical assistance and S. Burgess for the English correction. The authors would also like to thank the Spanish Ministry of Science and Innovation, Consolider-Ingenio 2010 Programme. CSD2007-00036 “CRAG” and Xarxa de Referencia en Biotecnologia of the Generalitat de Catalunya.Peer reviewe

Skeletal Muscle Regulates Metabolism via Interorgan Crosstalk: Roles in Health and Disease

Skeletal muscle is recognized as vital to physical movement, posture, and breathing. In a less known but critically important role, muscle influences energy and protein metabolism throughout the body. Muscle is a primary site for glucose uptake and storage, and it is also a reservoir of amino acids stored as protein. Amino acids are released when supplies are needed elsewhere in the body. These conditions occur with acute and chronic diseases, which decrease dietary intake while increasing metabolic needs. Such metabolic shifts lead to the muscle loss associated with sarcopenia and cachexia, resulting in a variety of adverse health and economic consequences. With loss of skeletal muscle, protein and energy availability is lowered throughout the body. Muscle loss is associated with delayed recovery from illness, slowed wound healing, reduced resting metabolic rate, physical disability, poorer quality of life, and higher health care costs. These adverse effects can be combatted with exercise and nutrition. Studies suggest dietary protein and leucine or its metabolite β-hydroxy β-methylbutyrate (HMB) can improve muscle function, in turn improving functional performance. Considerable evidence shows that use of high-protein oral nutritional supplements (ONS) can help maintain and rebuild muscle mass and strength. We review muscle structure, function, and role in energy and protein balance. We discuss how disease- and age-related malnutrition hamper muscle accretion, ultimately causing whole-body deterioration. Finally, we describe how specialized nutrition and exercise can restore muscle mass, strength, and function, and ultimately reverse the negative health and economic outcomes associated with muscle loss.Sin financiación5.775 JCR (2016) Q1, 4/49 Geriatrics & GerontologyUE

Activity of maize transglutaminase overexpressed in escherichia coli inclusion bodies: An alternative to protein refolding

Transglutaminases (TGases) catalyze protein post-translational modification by ε-(γ-glutamyl) links and covalent polyamine conjugation. In plants, this enzyme is poorly characterized and only the maize plastidial TGase gene (tgz) has been cloned. The tgz gene (Patent WWO03102128) had been subcloned and overexpressed in Escherichia coli cells, and the recombinant protein (TGZp) was present mainly in inclusion bodies (IB) fraction. In this work, after overexpression of TGZ15p and SDS-PAGE IB fraction analysis, bands about 65 and 56 kDa were obtained. Western blot, alkylation and MALDI-TOF/TOF analyses indicated that the 56 kDa band corresponded to a truncated sequence from the native TGZ15p (expected MW 65 kDa), by elimination of a chloroplast signal peptide fragment during expression processing. So that large-scale protein production and protein crystallization can be applied, we characterized the TGZ15p enzyme activity in the IB protein fraction, with and without refolding. Results indicate that it presented the biochemical characteristics of other described TGases, showing a certain plant-substrate preference. Solubilization of the IB fraction with Triton X-100 as nondenaturing detergent yielded active TGZ without the need for refolding, giving activity values comparable to those of the refolded protein, indicating that this is a valuable, faster way to obtain TGZ active protein.This study was supported by the Spanish projects MEC BFU2006-15115-01/BMC and BFU2009-08575. J. Gibert is the recipient of a pre-doctoral fellowship from the AGAUR (Generalitat de Catalunya) and N. Campos has a pre-doctoral fellowship from the Agencia Española de Cooperación Internacional (AECI). Authors especially thanks to Ana Coelho (ITQB, Univ. Nova de Lisboa, Portugal) for its valuable suggestions, E. Oliveira (Proteomic Services. Parc Cientific UB. Spain), N. Cortadellas (Serveis Cientifico-Tècnics, UB) for his technical assistance and Shirley Burgess for English corrections. Authors also thank the Spanish Ministry of Science and Innovation, Consolider-Ingenio 2010 Programme. CSD2007-00036 “CRAG” and to Xarxa de Referencia en Biotecnologia de la Generalitat de Catalunya.Peer reviewe

HMB activates mTOR and promotes protein synthesis in Neuro2a cells.

<p><b>(A)</b> Neuro2a cells were pre-treated with rapamycin 20nM or Torin1 10 nM and then were treated with 25 μM HMB for 30 min. Western blot analysis was performed using specific antibodies against phospho- and total-antibodies mTOR. <b>(B)</b> Protein synthesis was measured in Neuro2a cells incubated with 25 μM HMB for 2 hours in the absence (n = 10) or presence of inhibitors of PI3K/Akt (LY294002 20μM), ERK1/2 (PD98059 10μM) or mTOR (rapamycin 20nM). Pretreatment of inhibitors occurred as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135614#pone.0135614.g002" target="_blank">Fig 2</a>. Results represent means ± SEM (n = 4). * p<0.05 versus control cells. # p<0.05 versus HMB treated cells.</p

HMB induced neurite outgrowth is mediated by PI3K/Akt and ERK1/2 signaling pathways in Neuro2a cells.

<p>Neuro2a cells were treated with 25 μM HMB for 30 min. Western blot analysis was performed using specific antibodies against phospho- and total-antibodies against Akt <b>(A)</b> and ERK1/2 <b>(B)</b>. <b>(C)</b> Neuro2a cells were pre-treated with LY294002 20μM or PD98059 10μM and then treated with 25 μM HMB for 48 h. Inhibitors were maintained during the experiment. Cell morphology was measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135614#pone.0135614.g001" target="_blank">Fig 1</a>. Results represent means ± SEM (n = 4). * p<0.05 versus control cells. # p<0.05 versus HMB treated cells.</p