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Circulating plasma microRNAs (miRNAs) are well established as biomarkers of several diseases in humans and have recently been used as indicators of environmental exposures in fish. However, the role of plasma miRNAs in regulating acute stress responses in fish is largely unknown. Tissue and plasma miRNAs have recently been associated with excreted miRNAs; however, external miRNAs have never been measured in fish. The objective of this study was to identify the altered plasma miRNAs in response to acute stress in rainbow trout (Oncorhynchus mykiss), as well as altered miRNAs in fish epidermal mucus and the surrounding ambient water. Small RNA was extracted and sequenced from plasma, mucus, and water collected from rainbow trout pre- and 1 h-post a 3-min air stressor. Following small RNA-Seq and pathway analysis, we identified differentially expressed plasma miRNAs that targeted biosynthetic, degradation, and metabolic pathways. We successfully isolated miRNA from trout mucus and the surrounding water and detected differences in miRNA expression 1-h post air stress. The expressed miRNA profiles in mucus and water were different from the altered plasma miRNA profile, which indicated that the plasma miRNA response was not associated with or immediately reflected in external samples, which was further validated through qPCR. This research expands understanding of the role of plasma miRNA in the acute stress response of fish and is the first report of successful isolation and profiling of miRNA from fish mucus or samples of ambient water. Measurements of miRNA from plasma, mucus, or water can be further studied and have potential to be applied as non-lethal indicators of acute stress in fish. [ABSTRACT FROM AUTHOR]

Warm acclimation of rainbow trout can cause a decrease in the collagen content of the heart. This ability to remove cardiac collagen is particularly interesting considering that collagen deposition in the mammalian heart, following an injury, is permanent. We hypothesized that collagen removal can be facilitated by microRNA-29b (miR-29b), a highly conserved, small, non-coding RNA, as a reduction in this microRNA has been reported during the development of fibrosis in the mammalian heart. We also used a bioinformatics approach to investigate the binding potential of miR-29b to the seed sequences of vertebrate collagen isoforms. Cultured trout cardiac fibroblasts were transfected with zebrafish mature miR-29b mimic for 7 days with re-transfection occurring after 3 days. Transfection induced a 17.8-fold increase in miR-29b transcript abundance (P<0.05) as well as a 54% decrease in the transcript levels of the col1a3 collagen isoform, compared with non-transfected controls (P<0.05). Western blotting demonstrated that the level of collagen type I protein was 85% lower in cells transfected with miR-29b than in control cells (P<0.05). Finally, bioinformatic analysis suggested that the predicted 3'-UTR of rainbow trout col1a3 has a comparatively higher binding affinity for miR-29b than the 3'-UTR of col1a1. Together, these results suggest that miR-29b is a highly conserved regulator of collagen type I protein in vertebrates and that this microRNA decreases collagen in the trout heart by targeting col1a3. [ABSTRACT FROM AUTHOR]

Abstract: The role of methionine supplementation in fish metabolism remains largely unexplored. This study investigates the effects of methionine deprivation (MD) on glucose uptake in rainbow trout (Oncorhynchus mykiss) hepatocytes. To this end, primary hepatocytes were incubated in the presence (+M) or the absence (−M) of methionine for 48h and glucose uptake was assessed using a novel non-radioactive, fluorescent-linked enzymatic assay. Evidence indicated that glucose uptake increased under methionine deprivation, primarily due to the increased abundance of membrane bound sodium-glucose transporter 2 (SGLT2), which was likely facilitated by the cellular reduction in [ATP] resulting from increased mitochondrial uncoupling, as supported by elevated transcript levels of uncoupling protein 2a (UCP2a). This study is the first to suggest that the mechanisms responsible for the rapid glucose uptake associated with MR are facilitated by the greater abundance of SGLT2 glucose transporter and mitochondrial uncoupling. [Copyright &y& Elsevier]

AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, is proposed to function as a ''fuel gauge'' to monitor cellular energy status in response to nutritional environmental variations. However, in fish, few studies have addressed the metabolic consequences related to the activation of this kinase. This study demonstrates that the rainbow trout (Oncorhynchus mykiss) possesses paralogs of the three known AMPK subunits that co-diversified, that the AMPK protein is present in the liver and in isolated hepatocytes, and it does change in response to physiological (fasting-refeeding cycle) and pharmacological (AICAR and metformin administration and incubations) manipulations. Moreover, the phosphorylation of AMPK results in the phosphorylation of acetyl-CoA carboxylase, a main downstream target of AMPK in mammals. Other findings include changes in hepatic glycogen levels and several molecular actors involved in hepatic glucose and lipid metabolism, including mRNA transcript levels for glucokinase, glucose-6-phosphatase and fatty acid synthase both in vivo and in vitro. The fact that most results presented in this study are consistent with the recognized role of AMPK as a master regulator of energy homeostasis in living organisms supports the idea that these functions are conserved in this piscine model. [ABSTRACT FROM AUTHOR]

Hypoxia stress suppresses appetite in a variety of fish species, but the mechanisms mediating this response are not known. Therefore, given their anorexigenic and hypophysiotropic properties, we investigated the contribution of forebrain corticotropin-releasing factor (CRF) and urotensin I (UI) to the regulation of food intake and the hypothalamic-pituitary interrenal (HPI) stress axis in hypoxic rainbow trout. Exposure to 50 and 35% O2 saturation for 24 h decreased food intake by 28 and 48%, respectively. The 35% O2 treatment also increased forebrain CRF and UI mRNA levels, plasma cortisol, and lactate. Exposure for 72 h to the same conditions resulted in similar reductions in food intake, increases in plasma cortisol proportional to the hypoxia severity, and increases in forebrain CRF and UI mRNA levels in the 50% O2 treatment. Relative to saline-infused fish, chronic intracranial infusion of the CRF receptor antagonist a-helical CRF(9-41) reduced the appetite-suppressing effects of 24-h exposure to 35% O2 and blocked the hypoxia-induced increase in plasma cortisol. Finally, forebrain microdissection revealed that 50 and 35% O2 exposure for 24 h specifically increases preoptic area CRF and UI mRNA levels in proportion to the severity of the hypoxic challenge and either has no effect or elicits small decreases in other forebrain regions. These results show that CRF related peptides play a physiological role in regulating the HPI axis and in mediating at least a portion of the reduction in food intake under hypoxic conditions in rainbow trout and demonstrate that the response of forebrain CRF and UI neurons to this stressor is region specific. [ABSTRACT FROM AUTHOR]

The hypothalamic-pituitary-interrenal axis is an important regulator of stress and metabolism in teleosts. Cortisol is secreted by the head kidney where it increases gluconeogenesis in the liver to increase circulating glucose levels. MicroRNAs (miRNAs) are small, non-coding RNA molecules that bind to the 3′ untranslated region of specific mRNA to regulate their expression. MicroRNAs can also be secreted into circulation by association with extracellular vesicles (EVs) where they can influence the phenotype of other tissues. In this study, adult rainbow trout were exposed to a 3-minute acute air stress and allowed to recover for 1-, 3-, or 24-h to determine how miRNAs were altered. MicroRNAs measured in this study were chosen based on their high relative abundance in tissues that drive the stress response (miR-21a-3p, let-7a-5p, miR-143-3p) or their role in regulating DNA methylation (miR-29a-3p). In general, miRNAs increased in circulating EVs during the recovery period while decreasing in head kidney and liver at the same timepoints. Predicted targets for these miRNAs were analyzed using KEGG and DAVID functional enrichment analysis. Pathways involved in metabolism and cell signaling were predicted to be upregulated. Future studies can use these results to investigate how pathways are regulated after stress. Overall, our results indicate that miRNAs are regulated during teleost stress responses and could be supporting the cortisol-mediated changes that occur. Unlabelled Image • Rainbow trout were exposed to a 3 min air stressor and recovered for 1, 3 or 24 h. • Specific microRNAs were increased in extracellular vesicles. • Specific microRNAs were decreased in head kidney and liver. • These microRNAs are predicted to target pathways associated with stress. [ABSTRACT FROM AUTHOR]

While the field of epigenetics is increasingly recognized to contribute to the emergence of phenotypes in mammalian research models across different developmental and generational timescales, the comparative biology of epigenetics in the large and physiologically diverse vertebrate infraclass of teleost fish remains comparatively understudied. The cypriniform zebrafish and the salmoniform rainbow trout and Atlantic salmon represent two especially important teleost orders, because they offer the unique possibility to comparatively investigate the role of epigenetic regulation in 3R and 4R duplicated genomes. In addition to their sequenced genomes, these teleost species are well-characterized model species for development and physiology, and therefore allow for an investigation of the role of epigenetic modifications in the emergence of physiological phenotypes during an organism's lifespan and in subsequent generations. This review aims firstly to describe the evolution of the repertoire of genes involved in key molecular epigenetic pathways including histone modifications, DNA methylation and microRNAs in zebrafish, rainbow trout, and Atlantic salmon, and secondly, to discuss recent advances in research highlighting a role for molecular epigenetics in shaping physiological phenotypes in these and other teleost models. Finally, by discussing themes and current limitations of the emerging field of teleost epigenetics from both theoretical and technical points of view, we will highlight future research needs and discuss how epigenetics will not only help address basic research questions in comparative teleost physiology, but also inform translational research including aquaculture, aquatic toxicology, and human disease. [ABSTRACT FROM AUTHOR]

The acute stress response is well-characterized, with rainbow trout as a teleost model for physiological and molecular responses. Air exposure, which stimulates an acute stress response, modulates liver microRNAs in rainbow trout; however, these highly conserved non-coding RNAs that bind to mRNA and repress translation, have never been measured in brook trout and it is unknown how miRNA expression responds following air exposure in this less studied salmonid. Our objective was to characterize the effects of air exposure on rainbow and brook trout liver miRNA expression, as well as the mRNA expression and enzyme activity that the miRNAs are predicted to target. Brook and rainbow trout were sampled pre- and 1-, 3-, and 24-h post- a three-minute air exposure. Plasma cortisol, glucose, and lactate were measured. Relative expression of miR-21a-5p, miR-143-3p, let-7a-5p and relative expression and enzyme activities of five predicted targets (pyruvate kinase, glucokinase, citrate synthase, cytochrome c oxidase, and catalase) were measured in liver. Rainbow and brook trout both had increases in plasma cortisol and lactate, while only rainbow trout had significant post-stress increases in plasma glucose. Furthermore, both trout species had increased miR-143-3p and miR-21a-5p relative expression 24-h post-stress. Four of the five enzymes measured had altered activity following stress. Brook trout miRNAs had inverse relative expression with relative catalase mRNA expression and cytochrome c oxidase enzyme activity, but no relationship was found in rainbow trout. Therefore, we have further characterized the transcriptional and enzymatic response to air exposure in two salmonids. [Display omitted] • Characterized rainbow and brook trout response to 3-min air exposure. • Plasma metabolites increased post-air exposure with some variability. • Inverse relationships between miRNAs and predicted targets in brook trout. • Altered liver miRNAs and enzyme activities are linked to oxidative stress. [ABSTRACT FROM AUTHOR]