Efectos de cortisol sobre la modulación temprana de atrogina-1 y muifl en músculo esquelético de trucha arcoíris (Onclzoíynclzus mykiss)
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Fecha
2019
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Profesor/a Guía
Facultad/escuela
Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
La acuicultura es una actividad económica relevante para la producción de proteína destinada a la alimentación humana, siendo Chile el segundo productor de salmónidos a nivel mundial. Sin embargo, el mantenimiento de peces en condiciones de cultivo intensivo y semi intensivo produce estrés en los ejemplares lo que finalmente impacta procesos biológicos como el crecimiento. La principal hormona encargada de regular las respuestas a estrés corresponde a cortisol. La interacción de cortisol con el receptor de glucocorticoides actúa modulando la expresión de diferentes genes blanco. Estudios recientes han relacionado el efecto del estrés sobre la regulación de los atrogenes muro y atrogina-1 en el músculo esquelético de vertebrados. Ambos atrogenes son considerados participantes claves que inducen la degradación de proteínas vía ubiquitin-proteasoma promoviendo el proceso de atrofia muscular. Sin embargo, en peces, la participación de cortisol sobre la regulación de ambos atrogenes es desconocida.
Por lo tanto, el objetivo de esta tesis fue evaluar la participación de cortisol sobre la regulación en la expresión de muro y atrogina-1 en el músculo esquelético de trucha arcoíris. Para ello, se utilizó un ensayo in vivo en donde truchas arcoíris fueron administradas intraperitonealmente con cortisol durante 1, 3 y 9 horas. Posteriormente, los niveles de transcrito de ambos atrogenes fueron evaluados en el músculo esquelético mediante PCR en tiempo real.
Los resultados muestran que los niveles de atrogina-1 disminuyen luego de 3 horas de tratamiento con cortisol, y luego aumentan significativamente a las 9 horas post-tratamiento. Por otro lado, los niveles de transcrito de murfl aumentan significativamente luego de 9 horas de administración con cortisol. El aumento significativo de ambos atrogenes fue validado en un modelo in vitro de miotubos administrados con dosis fisiológicas de cortisol. Finalmente, se logró identificar dos sitios half-GRE putativos en el promotor de muro, pero no en atrogina-1.
Considerando los resultados obtenidos en ambos ensayos in vivo e in vitro se logró determinar que cortisol modula la expresión temprana de muro y atrogina-1 en el músculo esquelético de trucha arcoíris. Este trabajo representa una importante contribución para entender los mecanismos básicos que regulan la respuesta al estrés y crecimiento en peces.
Aquaculture is a relevant economic activity to produce protein destined for human consumption, being Chile the second producer of salmonids worldwide. However, the maintenance of fish under intensive and semi-intensive conditions trigger stress in the specimens, which finally impacts biological processes such as growth. The main hormone responsible for the stress regulation corresponds to cortisol, which, by interacting with its glucocorticoid receptor, modulates the expression of different target genes. Recent studies have linked the effect of stress on the regulation of atrogenes murfl and atrogin-I in skeletal muscle of vertebrates. Both atrogenes are considered key participants that induce the degradation of proteins via ubiquitin proteasome pathway, promoting the process of muscular atrophy. However, in fish, the involvement of cortisol in the regulation of both atrogenes is unknown. Therefore, the goal of this thesis was to evaluate the direct role of cortisol in the early regulation of murfl and atrogin-1 in skeletal muscle of rainbow trout. For this propose, an in vivo protocol was performed in which rainbow trouts were intraperitoneally administered with vehicle and cortisol for 1, 3 and 9 hours. Subsequently, the transcript levels of both atrogenes were evaluated in the skeletal muscle by real time PCR. These results reveal that transcript levels of atrogin-1 decrease after 3 hours of treatment with cortisol, and then increase significantly at 9 hours post treatment. On the other hand, murfl transcript levels increase significantly after 9 hours of administration with cortisol. The significant increase of both atrogenes was validated using an in vitro model of myotubes administered with physiological doses of cortisol. Finally, two putative half-GRE sites were identified in the promoter of murfl but not in atrogin-1. The results obtained in both in vivo and in vitro experiments determined that cortisol modulates the early expression of murfl and atrogin-1 in skeletal muscle of rainbow trout. This work represents an important contribution to understand the basic mechanisms involve in the regulation of fish stress response.
Aquaculture is a relevant economic activity to produce protein destined for human consumption, being Chile the second producer of salmonids worldwide. However, the maintenance of fish under intensive and semi-intensive conditions trigger stress in the specimens, which finally impacts biological processes such as growth. The main hormone responsible for the stress regulation corresponds to cortisol, which, by interacting with its glucocorticoid receptor, modulates the expression of different target genes. Recent studies have linked the effect of stress on the regulation of atrogenes murfl and atrogin-I in skeletal muscle of vertebrates. Both atrogenes are considered key participants that induce the degradation of proteins via ubiquitin proteasome pathway, promoting the process of muscular atrophy. However, in fish, the involvement of cortisol in the regulation of both atrogenes is unknown. Therefore, the goal of this thesis was to evaluate the direct role of cortisol in the early regulation of murfl and atrogin-1 in skeletal muscle of rainbow trout. For this propose, an in vivo protocol was performed in which rainbow trouts were intraperitoneally administered with vehicle and cortisol for 1, 3 and 9 hours. Subsequently, the transcript levels of both atrogenes were evaluated in the skeletal muscle by real time PCR. These results reveal that transcript levels of atrogin-1 decrease after 3 hours of treatment with cortisol, and then increase significantly at 9 hours post treatment. On the other hand, murfl transcript levels increase significantly after 9 hours of administration with cortisol. The significant increase of both atrogenes was validated using an in vitro model of myotubes administered with physiological doses of cortisol. Finally, two putative half-GRE sites were identified in the promoter of murfl but not in atrogin-1. The results obtained in both in vivo and in vitro experiments determined that cortisol modulates the early expression of murfl and atrogin-1 in skeletal muscle of rainbow trout. This work represents an important contribution to understand the basic mechanisms involve in the regulation of fish stress response.
Notas
Tesis (Ingeniera en Biotecnología)
Palabras clave
Trucha Arcoiris, Genética, Estrés en Peces, Cortisol, Investigaciones.