Genetic ablation of bone marrow beta-adrenergic receptors in mice modulates miRNA-transcriptome networks of neuroinflammation in the paraventricular nucleus.

Elucidating molecular pathways regulating neuroimmune communication is critical for therapeutic interventions in conditions characterized by overactive immune responses and dysfunctional autonomic nervous system. We generated a bone marrow-specific adrenergic beta 1 and beta 2 knockout mouse chimera (AdrB1.B2 KO) to determine how sympathetic drive to the bone affects transcripts and miRNAs in the hypothalamic paraventricular nucleus (PVN). This model has previously exhibited a dampened systemic immune response and decreased blood pressure compared with control animals. Reduced sympathetic responsiveness of the bone marrow hematopoietic cells of AdrB1.B2 KO chimera led to suppression of transcriptional networks that included leukocyte cell adhesion and migration and T cell-activation and recruitment. Transcriptome responses related to IL-17a signaling and the renin-angiotensin system were also suppressed in the PVN. Based on the transcriptome response, we next computationally predicted miRNAs in the PVN that may underscore the reduced sympathetic responsiveness of the bone marrow cells. These included miR-27b-3p, miR-150, miR-223-3p, and miR-326. Using real-time PCR, we measured a downregulation in the expression of miR-150-5p, miR-205-5p, miR-223-3p, miR-375-5p, miR-499a-5p, miR-27b-3p, let-7a-5p, and miR-21a-5p in the PVN of AdrB1.B2 KO chimera, confirming computational predictions that these miRNAs are associated with reduced neuro-immune responses and the loss of sympathetic responsiveness in the bone marrow. Intriguingly, directional responses of the miRNA corresponded to mRNAs, suggesting complex temporal or circuit-dependent posttranscriptional control of gene expression in the PVN. This study identifies molecular pathways involved in neural-immune interactions that may act as targets of therapeutic intervention for a dysfunctional autonomic nervous system.

Profiling the rainbow trout hepatic miRNAome under diet-induced hyperglycemia.

Carnivorous rainbow trout exhibit prolonged postprandial hyperglycemia when fed a diet exceeding 20% carbohydrate content. This poor capacity to utilize carbohydrates has led to rainbow trout being classified as "glucose-intolerant" (GI). The metabolic phenotype has spurred research to identify the underlying cellular and molecular mechanisms of glucose intolerance, largely because carbohydrate-rich diets provide economic and ecological advantages over traditionally used fish meal, considered unsustainable for rainbow trout aquaculture operations. Evidence points to a contribution of hepatic intermediary carbohydrate and lipid metabolism, as well as upstream insulin signaling. Recently, microRNAs (miRNAs), small noncoding RNAs acting as negative posttranscriptional regulators affecting target mRNA stability and translation, have emerged as critical regulators of hepatic control of glucose-homeostasis in mammals, revealing that dysregulated hepatic miRNAs might play a role in organismal hyperglycemia in metabolic disease. To determine whether hepatic regulatory miRNA networks may contribute to GI in rainbow trout, we induced prolonged postprandial hyperglycemia in rainbow trout by using a carbohydrate-rich diet and profiled genome-wide hepatic miRNAs in hyperglycemic rainbow trout compared with fasted trout and trout fed a diet devoid of carbohydrates. Using small RNA next-generation sequencing and real-time RT-PCR validation, we identified differentially regulated hepatic miRNAs between these groups and used an in silico approach to predict bona fide mRNA targets and enriched pathways. Diet-induced hyperglycemia resulted in differential regulation of hepatic miRNAs compared with fasted fish. Some of the identified miRNAs, such as miRNA-27b-3p and miRNA-200a-3p, are known to be responsive to hyperglycemia in the liver of hyperglycemic glucose-tolerant fish and mammals, suggesting an evolutionary conserved regulation. Using Gene Ontology term-based enrichment analysis, we identify intermediate carbohydrate and lipid metabolism and insulin signaling as potential targets of posttranscriptional regulation by hyperglycemia-regulated miRNAs and provide correlative expression analysis of specific predicted miRNA-target pairs. This study identifies hepatic miRNAs in rainbow trout that exhibit differential postprandial expression in response to diets with different carbohydrate content and predicts posttranscriptionally regulated target mRNAs enriched for pathways involved in glucoregulation. Together, these results provide a framework for testable hypotheses of functional involvement of specific hepatic miRNAs in GI in rainbow trout.

Unexpected effect of insulin on glucose disposal explains glucose intolerance of rainbow trout.

The physiological reasons why salmonids show glucose intolerance are unclear. In mammals, rapid clearance of a glucose load is mainly achieved through insulin-mediated inhibition of hepatic glucose production ( Ra) and stimulation of glucose disposal ( Rd), but the effects of insulin on Ra and Rd glucose have never been measured in fish. The goal of this study was to characterize the impact of insulin on the glucose kinetics of rainbow trout in vivo. Glucose fluxes were measured by continuous infusion of [6-3H]glucose before and during 4 h of insulin administration. The phosphorylated form of the key signaling proteins Akt and S6 in the insulin cascade were also examined, confirming activation of this pathway in muscle but not liver. Results show that insulin inhibits trout Rd glucose from 8.6 ± 0.6 to 5.4 ± 0.5 µmol kg-1 min-1: the opposite effect than classically seen in mammals. Such a different response may be explained by the contrasting effects of insulin on gluco/hexokinases of trout versus mammals. Insulin also reduced trout Ra from 8.5 ± 0.7 to 4.8 ± 0.6 µmol·kg-1·min-1, whereas it can almost completely suppresses Ra in mammals. The partial inhibition of Ra glucose may be because insulin only affects gluconeogenesis but not glycogen breakdown in trout. The small mismatch between the responses to insulin for Rd (-37%) and Ra glucose (-43%) gives trout a very limited capacity to decrease glycemia. We conclude that the glucose intolerance of rainbow trout can be explained by the inhibiting effect of insulin on glucose disposal.

Glucagon regulation of carbohydrate metabolism in rainbow trout: in vivo glucose fluxes and gene expression.

Glucagon increases fish glycaemia, but how it affects glucose fluxes in vivo has never been characterized. The goal of this study was to test the hypothesis that glucagon stimulates hepatic glucose production (rate of appearance, Ra) and inhibits disposal (rate of disposal, Rd) in rainbow trout. Changes in the mRNA abundance of key proteins involved in glycolysis, gluconeogenesis and glycogen breakdown were also monitored. The results show that glucagon increases glycaemia (+38%) by causing a temporary mismatch between Ra and Rd before the two fluxes converge below baseline (-17%). A novel aspect of the regulation of trout gluconeogenesis is also demonstrated: the completely different effects of glucagon on the expression of three Pepck isoforms (stimulation of pck1, inhibition of pck2a and no response of pck2b). Glycogen phosphorylase was modulated differently among tissues, and muscle upregulated pygb and downregulated pygm Glucagon failed to activate the cAMP-dependent protein kinase or FoxO1 signalling cascades. We conclude that trout hyperglycaemia results from the combination of two responses: (i) an increase in Ra glucose induced by the stimulation of gluconeogenesis through transcriptional activation of pck1 (and possibly glycogen phosphorylase), and (ii) a decrease in Rd glucose via inhibition of glycogen synthase and glycolysis. The observed decrease in glucose fluxes after 4 h of glucagon administration may be caused by a counter-regulatory response of insulin, potentially linked to the decrease in pygm transcript abundance. Overall, however, these integrated effects of glucagon only lead to modest changes in glucose fluxes that partly explain why trout seem to be unable to control glycaemia very tightly.

Bioconcentration and Metabolic Effects of Emerging PFOS Alternatives in Developing Zebrafish.

The novel PFOS alternatives, 6:2 chlorinated polyfluorinated ether sulfonate (F-53B) and sodium p-perfluorous nonenoxybenzenesulfonate (OBS), are emerging in the Chinese market, but little is known about their ecological risks. In this study, zebrafish embryos were exposed to PFOS, F-53B, and OBS to evaluate their bioconcentration and acute metabolic consequences. Per- and polyfluoroalkyl substances (PFASs) accumulated in larvae in the order of F-53B > PFOS > OBS, with the bioconcentration factors ranging from 20 to 357. Exposure to F-53B and PFOS, but not OBS, increased energy expenditure, and reduced feed intake in a concentration-dependent manner and the expression of genes involved in metabolic pathways at the transcriptional and translational levels. Molecular docking revealed that the binding affinities of PFASs to glucokinase were decreased in the following order: F-53B > PFOS > OBS. Finally, the results of Point of Departure (PoD) indicate that metabolic end points at the molecular and organismal level are most sensitive to F-53B followed by PFOS and OBS. Collectively, F-53B has the highest bioconcentration potential and the strongest metabolism-disrupting effects, followed by PFOS and OBS. Our findings have important implications for the assessment of early developmental metabolic effects of PFOS alternatives F-53B and OBS in wildlife and humans.

Social status affects lipid metabolism in rainbow trout, Oncorhynchus mykiss.

Juvenile rainbow trout ( Oncorhynchus mykiss) confined in pairs form social hierarchies in which socially subordinate fish display characteristic traits, including reduced growth rates and altered glucose metabolism. These effects are, in part, mediated by chronically elevated cortisol levels and/or reduced feeding. To determine the effects of social status on lipid metabolism, trout were held in pairs for 4 days, following which organismal and liver-specific indexes of lipid metabolism were measured. At the organismal level, circulating triglycerides were elevated in dominant trout, whereas subordinate trout exhibited elevated concentrations of circulating free fatty acids (FFAs) and lowered plasma total cholesterol levels. At the molecular level, increased expression of lipogenic genes in dominant trout and cpt1a in subordinate trout was identified, suggesting a contribution of increased de novo lipogenesis to circulating triglycerides in dominant trout and reliance on circulating FFAs for ß-oxidation in the liver of subordinates. Given the emerging importance of microRNAs (miRNA) in the regulation of hepatic lipid metabolism, candidate miRNAs were profiled, revealing increased expression of the lipogenic miRNA-33 in dominant fish. Because the Akt-TOR-S6-signaling pathway is an important upstream regulator of hepatic lipid metabolism, its signaling activity was quantified. However, the only difference detected among groups was a strong increase in S6 phosphorylation in subordinate trout. In general, the changes observed in lipid metabolism of subordinates were not mimicked by either cortisol treatment or fasting alone, indicating the existence of specific, emergent effects of subordinate social status itself on this fuel.

Chronic social stress alters protein metabolism in juvenile rainbow trout, Oncorhynchus mykiss.

When confined in pairs, juvenile rainbow trout (Oncorhynchus mykiss) form dominance hierarchies in which subordinate fish exhibit characteristic physiological changes including reduced growth rates and chronically elevated plasma cortisol concentrations. We hypothesized that alterations in protein metabolism contribute to the reduced growth rate of socially stressed trout, and predicted that subordinate trout would exhibit reduced rates of protein synthesis coupled with increases in protein degradation. Protein metabolism was assessed in dominant and subordinate fish after 4 days of social interaction, and in fish that were separated after 4 days of interaction for a 4 days recovery period, to determine whether effects on protein metabolism recovered when social stress was alleviated. Protein metabolism was assessed in liver and white muscle by measuring the fractional rate of protein synthesis and markers of protein degradation. In the white muscle of subordinate fish, protein synthesis was inhibited and activities of the ubiquitin-proteasome pathway (UPP) and the autophagy lysosomal system (ALS) were elevated. By contrast, the liver of subordinate fish exhibited increased rates of protein synthesis and activation of the ALS. When allowed to recover from chronic social stress for 4 days, differences in protein metabolism observed in white muscle of subordinate fish during the interaction period disappeared. In liver, protein synthesis returned to baseline levels during recovery from social stress, but markers of protein degradation did not. Collectively, these data support the hypothesis that inhibition of muscle protein synthesis coupled with increases in muscle protein breakdown contribute to the reduced growth rates of subordinate rainbow trout.

Meta-analysis of differentially-regulated hepatic microRNAs identifies candidate post-transcriptional regulation networks of intermediary metabolism in rainbow trout.

MicroRNAs (miRNAs) are small non-coding RNAs which act as post-transcriptional regulators by decreasing targeted mRNA translation and stability. Principally targeting small 3' UTR elements of protein-coding mRNAs through complementary base-pairing, miRNAs are promiscuous regulators of the transcriptome. While potent roles for hepatic miRNAs in the regulation of energy metabolism have emerged in rodent models, comparative roles in other vertebrates remain largely unexplored. Indeed, while several miRNAs are deeply conserved among vertebrates, the acquisition of lineage- and species-specific miRNAs, as well as the rewiring between miRNA-mRNA target relationships beg the question of regulatory and functional conservation and innovation of miRNAs and their targets involved in energy metabolism. Here we provide a meta-analysis of differentially expressed hepatic miRNAs in rainbow trout, a scientifically and economically important teleost species with a 'glucose-intolerant' phenotype. Following exposure to nutritional and social context-dependent metabolic challenges, we analyzed differential miRNA expression from small-RNA-sequencing datasets generated with a consistent bioinformatics pipeline in conjunction with an in silico target prediction of metabolic transcripts and pathways. We provide evidence for evolutionary conserved (let-7, miRNA-27 family) and rewired (miRNA-30 family, miRNA-152, miRNA-722) miRNA-metabolic target gene networks in the context of the salmonid genome. These findings represent important first steps in our understanding of the comparative regulation and function of hepatic miRNAs in rainbow trout energy metabolism. We propose that the identified miRNA families should be prioritized for future comparative functional investigation in the context of hepatic energy- and glucose metabolism in rainbow trout.

Correction: Social status regulates the hepatic miRNAome in rainbow trout: Implications for posttranscriptional regulation of metabolic pathways.

[This corrects the article DOI: 10.1371/journal.pone.0217978.].

Social status regulates the hepatic miRNAome in rainbow trout: Implications for posttranscriptional regulation of metabolic pathways.

Juvenile rainbow trout develop social hierarchies when held in dyads, and the development of socially subordinate (SS) and social dominance (SD) phenotypes in this context has been linked to specific changes in the hepatic energy metabolism of all major macronutrients. Following our recently reported finding that transcript abundance of drosha, a key component of the microRNA (miRNA) biogenesis pathway, is increased in paired juvenile rainbow trout irrespective of social status compared to socially isolated (SI) controls, we here determined global changes of the hepatic miRNA pathway genes in detail at the transcript and protein level. Both SD and SS rainbow trout exhibited increased Ago2 protein abundance compared to SI rainbow trout, suggesting that hepatic miRNA function is increased in rainbow trout maintained in dyads. Given the well-described differences in hepatic intermediary metabolism between SD and SS rainbow trout, and the important role of miRNAs in the posttranscriptional regulation of metabolic pathways, we also identified changes in hepatic miRNA abundance between SS and SD rainbow trout using small RNA next generation sequencing. We identified a total of 24 differentially regulated miRNAs, with 15 miRNAs that exhibited increased expression, and 9 miRNAs that exhibited decreased expression in the liver of SS trout compared to SD trout. To identify potential miRNA-dependent posttranscriptional regulatory pathways important for social status-dependent regulation of hepatic metabolism in rainbow trout, we used an in silico miRNA target prediction and pathway enrichment approach. We identified enrichment for pathways related to metabolism of carbohydrates, lipids and proteins in addition to organelle-specific processes involved in energy metabolism, especially mitochondrial fusion and fission. Select predicted miRNA-mRNA target pairs within these categories were quantitatively analyzed by real-time RT-PCR to validate candidates for future studies that will probe the functional metabolic roles of specific hepatic miRNAs in the development of SD and SS metabolic phenotypes.

Epigenetics in teleost fish: From molecular mechanisms to physiological phenotypes.

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.