Molecular mechanisms of the membrane-initiated cortisol actions on the teleost stress response
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Archivos
Fecha
2019
Autores
Profesor/a Guía
Facultad/escuela
Idioma
en
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Universidad Andrés Bello
Nombre de Curso
Licencia CC
Licencia CC
Resumen
La acuicultura es el sector productivo de alimentos de mayor crecimiento
abasteciendo cerca del 50% del pescado para consumo mundial. Sin embargo, los peces
sometidos a cultivos intensivos sufren de estrés lo que negativamente afecta su crecimiento,
reproducción y resistencia a enfermedades. Cortisol es la principal hormona involucrada en
la regulación de la respuesta fisiológica al estrés. El actual paradigma respecto a cómo
cortisol ejerce sus efectos se relaciona con su mecanismo genómico/clásico de acción que
involucra su interacción con receptores intracelulares y la modulación de genes blanco. Sin
embargo, cortisol también interacciona con componentes de la membrana plasmática en un
mecanismo no-genómico de acción con un impacto desconocido durante la respuesta a estrés.
En esta tesis nos propusimos evaluar la contribución de esta nueva acción de cortisol en la
respuesta compensatoria al estrés usando dos teleósteos importantes para la acuicultura como
modelo, trucha arcoíris (Oncorhynchus mykiss) y la dorada (Sparus aurata). Como primer
objetivo diseñamos un ensayo in vivo en el cual O. mykiss y S.aurata fueron administrados
intraperitonealmente con cortisol o con su análogo impermeable, cortisol-BSA, para emular
una condición de estrés agudo. Este protocolo fue replicado en S.aurata pero administrando
ambas versiones de cortisol disueltos en aceite de coco para evaluar efectos a largo plazo
iniciados en la superficie. Analizando parámetros de estrés en plasma y tejidos (cortisol,
glucosa, lactato, glicógeno), junto con aproximaciones transcriptómicas (RNA-seq, qPCR),
evaluamos la movilización de sustratos energéticos, y la respuesta transcripcional del
músculo esquelético mediada por esta nueva acción de cortisol en teleósteos. Encontramos
que la acción temprana de cortisol, potencialmente mediada por un GR de superficie, contribuye en el incremento de glucosa plasmática y regulación de genes asociados a
glicólisis y gluconeogénesis en O. mykiss y S.aurata. Además, esta nueva acción modula
diversos procesos biológicos en el músculo esquelético de O. mykiss como el procesamiento
de mRNA, procesos catabólicos dependientes de la ubiquitinación de proteínas, regulación
de la transcripción, entre otros; además de vías de señalización como fagosoma, adhesión
focal y espliceosoma. Como segundo objetivo, analizamos la contribución de la acción de
cortisol iniciada en la superficie sobre miotubos aislados de peces, determinando si la
señalización de GR participa en este proceso. Por lo tanto, miotubos fueron pre-tratados con
RU486 (antagonista de GR) y estimulados con cortisol o cortisol-BSA. Encontramos que la
mayoría de los genes relacionados al metabolismo de glucosa, tales como: eno2, pgaml y
pepck son potenciales blancos de la acción genómica de cortisol en los miotubos.
Sorprendentemente, nuestros resultados sugieren una respuesta bifásica en la regulación de
pdk2 en el músculo esquelético; en la que estaría participando primero la vía de cortisol
iniciada en la superficie, seguido de la acción genómica. Como tercer objetivo determinamos
el potencial receptor de corticoesteroide que mediaría la acción de cortisol iniciada en la
superficie mediante el análisis de los niveles de mRNA de grl, gr2 y mr en los ensayos in
vivo realizados en O. mykiss y S. aurata y también en los ensayos in vitro. Encontramos que
cortisol-BSA rápidamente disminuye la expresión de gr2 y luego incrementa su expresión
bajo este mismo tratamiento. Estos resultados sugieren que gr2 es potencialmente el receptor
que media la acción de cortisol iniciada en la superficie. Considerando todos los resultados
obtenidos en esta tesis, proponemos un nuevo paradigma de acción de cortisol que involucra
una participación fundamental de su vía iniciada en la superficie celular durante la respuesta
a estrés en teleósteos.
Aquaculture represents the fastest-growing food production sector, supplying about 50% of fish destined for world consumption. However, during intensive farming fish are exposed to severa( stressful conditions that negatively affects fish growth, reproduction and disease resistance. Cortisol is the main neuroendocrine stress hormone involved in the regulation of physiological response to stress. The actual paradigm of how cortisol regulates the stress response is associated with it genomic/classic mechanism involving the interaction of the hormone with intracellular receptors and the subsequently modulation of stress responsive genes. However, cortisol also could interact with membrane components in a rapid-non genomic signaling pathways with unknown impact during the stress response. In this thesis we set to evaluate the contribution of this novel cortisol action in the compensatory response to stress using two important aquaculture teleosts as a model, rainbow trout (Oncorhynchus mykiss) and gilthead seabream (Sparus aurata). As a first aim we design an in vivo assay in which O. mykiss and S. aurata were intraperitoneally administered with cortisol or with its impermeable analog, cortisol-BSA, to emulate a cortisol-mediated acute stress condition. This protocol was replicated in S.aurata but administrating both cortisol compounds dissolved in coconut oil to evaluate the long-term membrane- initiated cortisol action on fish. Determining the levels of severa! plasma and tissue stress-related parameters (e.g cortisol, glucose, glycogen, lactate) together with transcriptomic approaches (RNA-seq, RT-qPCR) we evaluated the mobilization of energetic substrates, as well as the transcriptional response of the skeletal muscle potentially mediated by this novel cortisol actions in teleosts. We found that early cortisol action, potentially mediated trough a membrane-GR, contributes on the increase of plasmatic glucose levels through the regulation of glycolysis and gluconeogenesis related genes in both O. mykiss and S.aurata. We also observed that the membrane-initiated cortisol actions impact diverse relevant biological process in the skeletal muscle of O. mykiss such mRNA processing, ubiquitin-dependent protein catabolic processes, transcription regulation, among others, as well as signaling pathways such phagosome, focal adhesion and spliceosome. As a second goal, we further analyzed the direct contribution of the membrane-initiated cortisol action on isolated fish myotubes determining whether membrane GR signaling pathways are participating in this process. Therefore, myotubes were pre-treated with RU486 (antagonist of GR) and stimulate with cortisol or cortisol-BSA. We found that mostly ofmetabolism glucose-related genes such eno2,pgaml and pepck are potentially target of genomic cortisol action in myotubes. Surprising, in vitro analysis suggests a biphasic response on the mRNA regulation of pdk2 in skeletal muscle; in which membrane-initiating cortisol pathway participate during the first phase, following by the classic cortisol participation in the second. Finally, as a third goal we determined the potential corticosteroid receptor that mediated the membrane-initiated cortisol action by analyzing the mRNA levels of grl, gr2 and mr in the in vivo assays performed in O. mykiss and S. aurata and also in the in vitro assay. We found that cortisol-BSA rapid decreased the gr2 expression and then increase their expression under cortisol-BSA treatment in myotubes. These results suggest that gr2 is potentially the receptor that mediate the rapid membraneinitiated cortisol action in fish skeletal muscle. Taken all the results obtained from this thesis, we pro pose a new paradigm of cortisol action involving the fundamental participation of the membrane-initiated cortisol signaling during the teleost stress response.
Aquaculture represents the fastest-growing food production sector, supplying about 50% of fish destined for world consumption. However, during intensive farming fish are exposed to severa( stressful conditions that negatively affects fish growth, reproduction and disease resistance. Cortisol is the main neuroendocrine stress hormone involved in the regulation of physiological response to stress. The actual paradigm of how cortisol regulates the stress response is associated with it genomic/classic mechanism involving the interaction of the hormone with intracellular receptors and the subsequently modulation of stress responsive genes. However, cortisol also could interact with membrane components in a rapid-non genomic signaling pathways with unknown impact during the stress response. In this thesis we set to evaluate the contribution of this novel cortisol action in the compensatory response to stress using two important aquaculture teleosts as a model, rainbow trout (Oncorhynchus mykiss) and gilthead seabream (Sparus aurata). As a first aim we design an in vivo assay in which O. mykiss and S. aurata were intraperitoneally administered with cortisol or with its impermeable analog, cortisol-BSA, to emulate a cortisol-mediated acute stress condition. This protocol was replicated in S.aurata but administrating both cortisol compounds dissolved in coconut oil to evaluate the long-term membrane- initiated cortisol action on fish. Determining the levels of severa! plasma and tissue stress-related parameters (e.g cortisol, glucose, glycogen, lactate) together with transcriptomic approaches (RNA-seq, RT-qPCR) we evaluated the mobilization of energetic substrates, as well as the transcriptional response of the skeletal muscle potentially mediated by this novel cortisol actions in teleosts. We found that early cortisol action, potentially mediated trough a membrane-GR, contributes on the increase of plasmatic glucose levels through the regulation of glycolysis and gluconeogenesis related genes in both O. mykiss and S.aurata. We also observed that the membrane-initiated cortisol actions impact diverse relevant biological process in the skeletal muscle of O. mykiss such mRNA processing, ubiquitin-dependent protein catabolic processes, transcription regulation, among others, as well as signaling pathways such phagosome, focal adhesion and spliceosome. As a second goal, we further analyzed the direct contribution of the membrane-initiated cortisol action on isolated fish myotubes determining whether membrane GR signaling pathways are participating in this process. Therefore, myotubes were pre-treated with RU486 (antagonist of GR) and stimulate with cortisol or cortisol-BSA. We found that mostly ofmetabolism glucose-related genes such eno2,pgaml and pepck are potentially target of genomic cortisol action in myotubes. Surprising, in vitro analysis suggests a biphasic response on the mRNA regulation of pdk2 in skeletal muscle; in which membrane-initiating cortisol pathway participate during the first phase, following by the classic cortisol participation in the second. Finally, as a third goal we determined the potential corticosteroid receptor that mediated the membrane-initiated cortisol action by analyzing the mRNA levels of grl, gr2 and mr in the in vivo assays performed in O. mykiss and S. aurata and also in the in vitro assay. We found that cortisol-BSA rapid decreased the gr2 expression and then increase their expression under cortisol-BSA treatment in myotubes. These results suggest that gr2 is potentially the receptor that mediate the rapid membraneinitiated cortisol action in fish skeletal muscle. Taken all the results obtained from this thesis, we pro pose a new paradigm of cortisol action involving the fundamental participation of the membrane-initiated cortisol signaling during the teleost stress response.
Notas
Tesis (Doctor en Biotecnología)
Tesis incluye paper publicado en Comparative Biochemistry and Physiology - Part A
Tesis incluye paper publicado en Comparative Biochemistry and Physiology - Part A
Palabras clave
Estrés en Peces, Trucha Arco Iris, Teleósteos, Investigaciones, Cortisol