Early regulation of corticosteroid receptor expression in rainbow trout (Oncorhynchus mykiss) gills is mediated by membrane-initiated cortisol signaling.

Cortisol is a key stress-related hormone involved in the physiological adjustments of fish. In gills, cortisol contributes to acclimatization to changes in environmental salinity, promoting both ion uptake or salt excretion. Cortisol exerts its biological effects through its interaction with specific intracellular glucocorticoid (GR) and mineralocorticoid (MR) receptors. Additionally, the further identification of GR and MR on the surface of different tissues, together with the existence of cortisol-mediated effects observed using membrane-impermeable analogs (e.g., cortisol-BSA), supports the existence of membrane-initiated cortisol actions in fish. Nevertheless, the impact of this alternative cortisol mechanism in relevant tissues for fish salinity acclimation, such as gill, is unknown. In this work, we sought to explore the contribution of rapid membrane-initiated cortisol on GR and MR regulation in rainbow trout (Oncorhynchus mykiss) gills using in vivo and in vitro approaches. Juvenile rainbow trout intraperitoneally injected with cortisol or cortisol-BSA showed increased gr2 but no gr1 or mr mRNA levels in gills after one hour of treatment. This result was further confirmed using RT-gills-W1 cell lines stimulated with both versions of cortisol. Interestingly, after three and six hours of cortisol or cortisol-BSA treatment, there were no changes in the mRNA levels of any corticosteroid receptor in RT-gills-W1 cells. Finally, using immunofluorescence analysis, we identified GR and MR in rainbow trout gill cells localized on the cell surface. Considering the in vivo and in vitro results of this work, we suggest that membrane-initiated cortisol action contributes to the early expression of gr2 in rainbow trout gills during salinity acclimation.

Role of glucocorticoid and mineralocorticoid receptors in rainbow trout (Oncorhynchus mykiss) skeletal muscle: A transcriptomic perspective of cortisol action.

Cortisol is an essential regulator of neuroendocrine stress responses in teleost. Cortisol performs its effects through the modulation of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), activating gene expression. Until now the contribution of both receptors in the global transcriptional response in teleost skeletal muscle has not been explored. To understand in a comprehensive and global manner how GR and MR modulates the skeletal muscle transcriptomic response, we performed RNA-seq analysis. Juvenile rainbow trout (Oncorhynchus mykiss) pretreated with a suppressor of endogenous cortisol production were intraperitoneally injected with cortisol (10 mg/kg). We also included a treatment with mifepristone (GR antagonist) and eplerenone (MR antagonist) in the presence or absence of cortisol. cDNA libraries were constructed from the skeletal muscle of rainbow trout groups: vehicle, cortisol, mifepristone, eplerenone, mifepristone/cortisol and eplerenone/cortisol. RNA-seq analysis revealed that 135 transcripts were differentially expressed in cortisol vs. mifepristone/cortisol group, mainly associated to inflammatory response, ion transmembrane transport, and proteolysis. In the other hand, 68 transcripts were differentially expressed in cortisol vs. eplerenone/cortisol group, mainly associated to muscle contraction, and regulation of cell cycle. To validate these observations, we performed in vitro experiments using rainbow trout myotubes. In myotubes treated with cortisol, we found increased expression of cxcr2, c3, and clca3p mediated by GR, associated with inflammatory response, proteolysis, and ion transmembrane transport, respectively. Contrastingly, MR modulated the expression of myh2 and gadd45g mainly associated with muscle contraction and regulation of cell cycle, respectively. These results suggest that GR and MR have a differential participation in the physiological response to stress in teleost skeletal muscle.

Regulation of the early expression of MAFbx/atrogin-1 and MuRF1 through membrane-initiated cortisol action in the skeletal muscle of rainbow trout.

Glucocorticoids are key stress-related hormones in vertebrates, with cortisol being the main glucocorticoid in teleosts. Glucocorticoids exert their effects through two mechanisms of action: genomic/classic and membrane initiated. In mammals, cortisol-mediated stress has been found to be associated with increased expression of critical atrophy-related genes (atrogenes), such as MAFbx/atrogin-1 and murf1/trim63. However, the direct impact of cortisol on the early regulation of atrogene expression in teleost skeletal muscle and the contribution of membrane-initiated cortisol action to this process have not been identified. In this work, the mRNA levels of atrogin-1 and murf1 were assessed in isolated myotubes and skeletal muscle of rainbow trout administered with cortisol or cortisol-BSA. This latter compound is a membrane-impermeable cortisol analog that exclusively induces membrane-initiated effects. We found that cortisol (10 mg/kg) first decreased the expression of both atrogenes at 3 h of treatment and then increased their expression at 9 h of treatment in the skeletal muscle of rainbow trout. Additionally, the in vitro analysis suggested that membrane-initiated cortisol action regulates murf1 but not atrogin-1 in rainbow trout myotubes. Using RU486 to selectively block glucocorticoid receptor (GR), we found that early downregulation of murf1 is potentially mediated by membrane GR signaling in myotubes. Considering the results of both the in vivo and in vitro approaches, we suggest that membrane-initiated cortisol action regulates the early expression of atrophy-related processes in teleosts.

Contribution of Non-canonical Cortisol Actions in the Early Modulation of Glucose Metabolism of Gilthead Sea Bream (Sparus aurata).

Teleost fish are exposed to diverse stressors in farming and wildlife conditions during their lifespan. Cortisol is the main glucocorticoid hormone involved in the regulation of their metabolic acclimation under physiological stressful conditions. In this context, increased plasma cortisol is associated with energy substrate mobilization from metabolic tissues, such as liver and skeletal muscle, to rapidly obtain energy and cope with stress. The metabolic actions of cortisol have primarily been attributed to its genomic/classic action mechanism involving the interaction with intracellular receptors, and regulation of stress-responsive genes. However, cortisol can also interact with membrane components to activate rapid signaling pathways. In this work, using the teleost fish gilthead sea bream (Sparus aurata) as a model, we evaluated the effects of membrane-initiated cortisol actions on the early modulation of glucose metabolism. For this purpose, S. aurata juveniles were intraperitoneally administrated with cortisol and with its membrane impermeable analog, cortisol-BSA. After 1 and 6 h of each treatment, plasma cortisol levels were measured, together with glucose, glycogen and lactate in plasma, liver and skeletal muscle. Transcript levels of corticosteroids receptors (gr1, gr2, and mr) and key gluconeogenesis (g6pc and pepck)- and glycolysis (pgam1 and aldo) related genes in the liver were also measured. Cortisol and cortisol-BSA administration increased plasma cortisol levels in S. aurata 1 h after administration. Plasma glucose levels enhanced 6 h after each treatment. Hepatic glycogen content decreased in the liver at 1 h of both cortisol and cortisol-BSA administration, while increased at 6 h due to cortisol but not in response to cortisol-BSA. Expression of gr1, g6pc, pgam1, and aldo were preferentially increased by cortisol-BSA in the liver. Taking all these results in consideration, we suggest that non-canonical cortisol mechanisms contribute to the regulation of the early glucose metabolism responses to stress in S. aurata.

Early transcriptomic responses associated with the membrane-initiated action of cortisol in the skeletal muscle of rainbow trout (Oncorhynchus mykiss).

Cortisol is a critical neuroendocrine regulator of the stress response in fish. Cortisol practically affects all tissues by interacting with an intracellular receptor and modulating target gene expression. However, cortisol also interacts with components of the plasma membrane in a nongenomic process that activates rapid signaling. Until now, the implication of this novel cortisol signaling for the global transcriptional response has not been explored. In the present work, we evaluated the effects of the membrane-initiated actions of cortisol on the in vivo transcriptome of rainbow trout (Oncorhynchus mykiss) skeletal muscle. RNA-Seq analyses were performed to examine the transcriptomic changes in rainbow trout stimulated by physiological concentrations of cortisol and cortisol coupled with bovine serum albumin (cortisol-BSA), a membrane-impermeable analog of cortisol. A total of 660 million paired-ends reads were generated. Reads mapped onto the reference genome revealed that 1,737; 897; and 1,012 transcripts were differentially expressed after 1, 3, and 9 h of cortisol-BSA treatment, respectively. Gene Ontology analysis showed that this novel action of cortisol modulates several biological processes, such as mRNA processing, ubiquitin-dependent protein catabolic processes, and transcription regulation. In addition, a KEGG analysis revealed that focal adhesion was the main signaling pathway that was upregulated at all the times tested. Taking these results together, we propose that the membrane-initiated cortisol action contributes significantly in the regulation of stress-mediated gene expression.

Quantitative proteomics analysis of membrane glucocorticoid receptor activation in rainbow trout skeletal muscle.

Cortisol modulates energy metabolism promoting the mobilization of glucose and increasing proteolysis to overcome stressful situations in teleost. The cortisol metabolic effects are attributed to genomic mechanisms that involve the interaction of cortisol with its glucocorticoid intracellular receptor. Furthermore, cortisol can also interact with plasma membrane glucocorticoid receptors activating a rapid nongenomic signaling; however, its contribution during the early acute phase stress response in fish is unknown. In the present work, we evaluated the effects of membrane-initiated cortisol actions in vivo in the proteome of rainbow trout (Oncorhynchus mykiss) skeletal muscle. Quantitative iTRAQ analyses were performed to examine proteomic changes in rainbow trout stimulated with physiological concentrations of cortisol and cortisol-BSA, a membrane-impermeable cortisol conjugate. A total of 873 proteins were identified, among which 61 and 47 proteins were differentially expressed under cortisol and cortisol-BSA treatments, respectively. Functional clustering analysis revealed an upregulation of proteins associated with mitochondria and oxidative phosphorylation. These results were validated by Western blot analysis. Additionally, using rainbow trout myotubes, the participation of membrane glucocorticoid receptors in gene expression was evaluated. The results obtained suggest that cortisol acts through a membrane canonical glucocorticoid receptor and mediates the expression of proteins associated with mitochondrial oxidative phosphorylation.

Membrane-initiated cortisol action modulates early pyruvate dehydrogenase kinase 2 (pdk2) expression in fish skeletal muscle.

Cortisol is the main glucocorticoid hormone in teleosts involved in the regulation of metabolic adjustments under both normal and stressful physiological conditions. In the skeletal muscle, cortisol modulates the energetic metabolism promoting the mobilization of glucose and other energetic substrates to overcome the stress stimulus. The effects of cortisol-mediated stress response are attributed to canonical/genomic mechanisms which involve the interaction of the hormone with its intracellular glucocorticoid receptor and, consequently, modulation of target genes. However, cortisol also can interact with membrane components, activating rapid signaling pathways with unknown contribution during the early stress response. In the present work, we evaluated the impact of membrane-initiated cortisol action over the expression of the critical modulator of energetic metabolism, pyruvate dehydrogenase kinase 2 (pdk2), in fish skeletal muscle. Juvenile rainbow trout were intraperitoneally administered with stress-related doses of cortisol and cortisol-BSA, and the expression of pdk2 was assayed by using RT-qPCR. Our results reveal that pdk2 mRNA levels increased in the skeletal muscle at one hour in both cortisol- and cortisol-BSA-treated fish. Moreover, in vitro studies revealed a biphasic response over the pdk2 regulation in myotubes mediated first through membrane-cortisol signaling pathways followed by the classic cortisol action. Finally, pdk2 up-regulation owing to cortisol and cortisol-BSA is reverted in RU486 treated myotubes, suggesting that GR signaling participates in both cortisol signaling pathways. This work suggests that non-classical cortisol pathways contribute to regulate the early metabolic response to stress in fish skeletal muscle.

RNA-seq analysis of the head-kidney transcriptome response to handling-stress in the red cusk-eel (Genypterus chilensis).

Stress is a primary contributing factor of fish disease and mortality in aquaculture. We have previously reported that the red cusk-eel (Genypterus chilensis), an important farmed marine fish, demonstrates a handling-stress response that results in increased juvenile mortality, which is mainly associated with skeletal muscle atrophy and liver steatosis. To better understand the systemic effects of stress on red cusk-eel immune-related gene expression, the present study assessed the transcriptomic head-kidney response to handling-stress. The RNA sequencing generated a total of 61,655,525 paired-end reads from control and stressed conditions. De novo assembly using the CLC Genomic Workbench produced 86,840 transcripts and created a reference transcriptome with a N50 of 1426bp. Reads mapped onto the assembled reference transcriptome resulted in the identification of 569 up-regulated and 513 down-regulated transcripts. Gene ontology enrichment analysis revealed a significant up-regulation of the biological processes, like response to stress, response to biotic stimulus, and immune response. Conversely, a significant down-regulation of biological processes is associated with metabolic processes. These results were validated by RT-qPCR analysis for nine candidate genes involved in the immune response. The present data demonstrated that short term stress promotes the immune innate response in the marine teleost G. chilensis. This study is an important step towards understanding the immune adaptive response to stress in non-model teleost species.

Transcriptomic analysis of the hepatic response to stress in the red cusk-eel (Genypterus chilensis): Insights into lipid metabolism, oxidative stress and liver steatosis.

Teleosts exhibit a broad divergence in their adaptive response to stress, depending on the magnitude, duration, and frequency of stressors and the species receiving the stimulus. We have previously reported that the red cusk-eel (Genypterus chilensis), an important marine farmed fish, shows a physiological response to stress that results in increased skeletal muscle atrophy mediated by over-expression of components of the ubiquitin proteasome and autophagy-lysosomal systems. To better understand the systemic effects of stress on the red cusk-eel metabolism, the present study assessed the transcriptomic hepatic response to repetitive handling-stress. Using high-throughput RNA-seq, 259 up-regulated transcripts were found, mostly associated with angiogenesis, gluconeogenesis, and triacylglyceride catabolism. Conversely, 293 transcripts were down-regulated, associated to cholesterol biosynthesis, PPARα signaling, fatty acid biosynthesis, and glycolysis. This gene signature was concordant with hepatic metabolite levels and hepatic oxidative damage. Moreover, the increased plasmatic levels of AST (aspartate aminotransferase), ALT (alanine aminotransferase) and AP (alkaline phosphatase), as well as liver histology suggest stress-induced liver steatosis. This study offers an integrative molecular and biochemical analysis of the hepatic response to handling-stress, and reveals unknown aspects of lipid metabolism in a non-model teleost.

Cortisol Induces Reactive Oxygen Species Through a Membrane Glucocorticoid Receptor in Rainbow Trout Myotubes.

Cortisol is an essential regulator of neuroendocrine stress responses in teleosts. Cortisol predominantly affects target tissues through the genomic pathway, which involves interacting with cytoplasmic glucocorticoid receptors, and thereby, modulating stress-response gene expressions. Cortisol also produces rapid effects via non-genomic pathways, which do not involve gene transcription. Although cortisol-mediated genomic pathways are well documented in teleosts, non-genomic pathways are not fully understood. Moreover, no studies have focused on the contribution of non-genomic cortisol pathways in compensatory stress responses in fish. In this study, rainbow trout (Oncorhynchus mykiss) skeletal myotubes were stimulated with physiological concentrations of cortisol and cortisol-BSA, a membrane-impermeable agent, resulting in an early induction of reactive oxygen species (ROS). This production was not suppressed by transcription or translation inhibitors, suggesting non-genomic pathway involvement. Moreover, myotube preincubation with RU486 and NAC completely suppressed cortisol- and cortisol-BSA-induced ROS production. Subcellular fractionation analysis revealed the presence of cell membrane glucocorticoid receptors. Finally, cortisol-BSA induced a significant increase in ERK1/2 and CREB phosphorylation, as well as in CREB-dependent transcriptional activation of the pgc1a gene expression. The obtained results strongly suggest that cortisol acts through a non-genomic glucocorticoid receptor-mediated pathway to induce ROS production and contribute to ERK/CREB/PGC1-α signaling pathway activation as stress compensation mechanisms. J. Cell. Biochem. 118: 718-725, 2017. © 2016 Wiley Periodicals, Inc.

mRNA-seq reveals skeletal muscle atrophy in response to handling stress in a marine teleost, the red cusk-eel (Genypterus chilensis).

BACKGROUND: Fish reared under intensive conditions are repeatedly exposed to stress, which negatively impacts growth. Although most fish follow a conserved pattern of stress response, with increased concentrations of cortisol, each species presents specificities in the cell response and stress tolerance. Therefore, culturing new species requires a detailed knowledge of these specific responses. The red cusk-eel (Genypterus chilensis) is a new economically important marine species for the Chilean aquaculture industry. However, there is no information on the stress- and cortisol-induced mechanisms that decrease skeletal muscle growth in this teleost. RESULTS: Using Illumina RNA-seq technology, skeletal muscle sequence reads for G. chilensis were generated under control and handling stress conditions. Reads were mapped onto a reference transcriptome, resulting in the in silico identification of 785 up-regulated and 167 down-regulated transcripts. Gene ontology enrichment analysis revealed a significant up-regulation of catabolic genes associated with skeletal muscle atrophy. These results were validated by RT-qPCR analysis for ten candidates genes involved in ubiquitin-mediated proteolysis, autophagy and skeletal muscle growth. Additionally, using a primary culture of fish skeletal muscle cells, the effect of cortisol was evaluated in relation to red cusk-eel skeletal muscle atrophy. CONCLUSIONS: The present data demonstrated that handling stress promotes skeletal muscle atrophy in the marine teleost G. chilensis through the expression of components of the ubiquitin-proteasome and autophagy-lysosome systems. Furthermore, cortisol was a powerful inductor of skeletal muscle atrophy in fish myotubes. This study is an important step towards understanding the atrophy system in non-model teleost species and provides novel insights on the cellular and molecular mechanisms that control skeletal muscle growth in early vertebrates.