Empirical Characterization of False Discovery Rates of Differentially Expressed Genes and Transcriptomic Benchmark Concentrations in Zebrafish Embryos.

High-throughput transcriptomics (HTTr) is increasingly applied to zebrafish embryos to survey the toxicological effects of environmental chemicals. Before the adoption of this approach in regulatory testing, it is essential to characterize background noise in order to guide experimental designs. We thus empirically quantified the HTTr false discovery rate (FDR) across different embryo pool sizes, sample sizes, and concentration groups for toxicology studies. We exposed zebrafish embryos to 0.1% dimethyl sulfoxide (DMSO) for 5 days. Pools of 1, 5, 10, and 20 embryos were created (n = 24 samples for each pool size). Samples were sequenced on the TempO-Seq platform and then randomly assigned to mock treatment groups before differentially expressed gene (DEG), pathway, and benchmark concentration (BMC) analyses. Given that all samples were treated with DMSO, any significant DEGs, pathways, or BMCs are false positives. As expected, we found decreasing FDRs for DEG and pathway analyses with increasing pool and sample sizes. Similarly, FDRs for BMC analyses decreased with increasing pool size and concentration groups, with more stringent BMC premodel filtering reducing BMC FDRs. Our study provides foundational data for determining appropriate experiment designs for regulatory toxicity testing with HTTr in zebrafish embryos.

Exploring transcriptional and post-transcriptional epigenetic regulation of crf and 11ßhsd2 in rainbow trout brain during chronic social stress.

Using dominance hierarchies in juvenile rainbow trout (Oncorhynchus mykiss) as a model of chronic social stress in fish, we explored whether epigenetic transcriptional and post-transcriptional mechanisms are involved in the gene expression of corticotropin-releasing factor (crf) and 11ß-hydroxysteroid dehydrogenase (11ßhsd2), key factors involved in the regulation of the endocrine stress axis response. In juvenile rainbow trout pairs, subordinate individuals display sustained elevation of circulating cortisol concentrations. Cortisol production is controlled by the hypothalamic-pituitary-interrenal (HPI) axis in fish and initiated by CRF release from the preoptic area (POA). Given that crf is modulated during chronic social stress, and that such stress has been implicated in the epigenetic regulation of crf in other taxa, we probed a role for epigenetic regulation of crf transcript abundance in chronically stressed rainbow trout. We also investigated the regulation of the cortisol-metabolising enzyme 11ßhsd2 in the POA, which is upregulated in subordinates. The potential involvement of DNA methylation and microRNAs (miRNAs) in the regulation of crf transcript abundance was investigated during social stress in the POA of fish, as was the potential involvement of miRNAs in 11ßhsd2 regulation. Although transcript abundances of crf were elevated in subordinate fish after 4 days, DNA methylation profiles within putative promoter sequences upstream of the crf gene were not significantly affected by chronic stress. An inverse relationship between crf and its predicted posttranscriptional regulator miR-103a-3p in the POA suggests that miRNAs may be involved in mediating the effects of chronic social stress on key components of the endocrine stress axis.

Lactate signaling and fuel selection in rainbow trout: mobilization of energy reserves.

Lactate is now recognized as a regulator of fuel selection in mammals because it inhibits lipolysis by binding to the hydroxycarboxylic acid receptor 1 (HCAR1). The goals of this study were to quantify the effects of exogenous lactate on: 1) lipolytic rate or rate of appearance of glycerol in the circulation (Ra glycerol) and hepatic glucose production (Ra glucose), and 2) key tissue proteins involved in lactate signaling, glucose transport, glycolysis, gluconeogenesis, lipolysis, and ß-oxidation in rainbow trout. Measurements of fuel mobilization kinetics show that lactate does not affect lipolysis as it does in mammals (Ra glycerol remains at 7.3 ± 0.5 µmol·kg-1·min-1), but strongly reduces hepatic glucose production (16.4 ± 2.0 to 8.9 ± 1.2 µmol·kg-1·min-1). This reduction is likely induced by decreasing gluconeogenic flux through the inhibition of cytosolic phosphoenolpyruvate carboxykinase (Pck1, alternatively called Pepck1; 60% and 24% declines in gene expression and protein level, respectively). It is also caused by lactate substituting for glucose as a fuel in all tissues except white muscle that increases glut4a expression and has limited capacity for monocarboxylate transporter (Mct)-mediated lactate import. We conclude that lipolysis is not affected by hyperlactatemia because trout show no activation of autocrine Hcar1 signaling (gene expression of the receptor is unchanged or even repressed in red muscle). Lactate regulates fuel mobilization via Pck1-mediated suppression of gluconeogenesis and by replacing glucose as a fuel. This study highlights important functional differences in the Hcar1 signaling system between fish and mammals for the regulation of fuel selection.

Variation in habitat use and its consequences for mercury exposure in two Eastern Ontario bat species, Myotis lucifugus and Eptesicus fuscus.

The St. Lawrence River in Eastern Ontario, Canada, has been a designated an area of concern due to past industrial contamination of sediment in some areas and transport of mercury from tributaries. Previous research using bats as sentinel species identified elevated concentrations of total mercury (THg) in fur of local bats and species-specific variation between little brown bats (Myotis lucifugus) and big brown bats (Eptesicus fuscus). Here, we investigated the mercury exposure pathways for these two species by testing the hypothesis that diet variation, particularly the reliance on aquatic over terrestrial insects, is a determinant of local bat mercury concentrations. We analyzed THg concentration and stable isotope ratios of δ15N and δ13C in fur of little and big brown bats, and in aquatic and terrestrial insects. Big brown bats, especially males, accumulated significantly higher THg concentrations in their fur compared to little brown bats. However, this difference was not related to diet because big brown bats consumed terrestrial insects, which were lower in mercury than aquatic insects, the primary prey for little brown bats. We also evaluated whether fur THg concentrations translate into molecular changes in tissues linked to (methyl)mercury toxicity by quantifying tissue changes in global DNA methylation and mitochondrial DNA abundance. No significant changes in DNA molecular markers were observed in relation to fur THg concentration, suggesting mercury exposure to local bats did not impact molecular level changes at the DNA level. Higher mercury in bats was not associated with local aquatic contamination or genotoxicity in this study area.

The mammalian insulin antagonist S961 does not exhibit insulin receptor antagonism in rainbow trout in vivo.

Due to their reported 'glucose-intolerant' phenotype, rainbow trout have been the focus of comparative studies probing underlying endocrine mechanisms at the organismal, tissue and molecular level. A particular focus has been placed on the investigation of the comparative role of insulin, an important glucoregulatory hormone, and its interaction with macronutrients. A limiting factor in the comparative investigation of insulin is the current lack of reliable assays to quantify circulating mature and thus bioactive insulin. To circumvent this limitation, tissue-specific responsiveness to postprandial or exogenous insulin has been quantified at the level of post-translational modifications of cell signalling proteins. These studies revealed that the insulin responsiveness of these proteins and their post-translational modifications are evolutionarily highly conserved and thus provide useful and quantifiable proxy indices to investigate insulin function in rainbow trout. While the involvement of specific branches of the intracellular insulin signalling pathway (e.g., mTor) in rainbow trout glucoregulation have been successfully probed through pharmacological approaches, it would be useful to have a functionally validated insulin receptor antagonist to characterize the glucoregulatory role of the insulin receptor pathway in its entirety for this species. Here, we report two separate in vivo experiments to test the ability of the mammalian insulin receptor antagonist, S961, to efficiently block insulin signalling in liver and muscle in response to endogenously released insulin and to exogenously infused bovine insulin. We found that, irrespective of the experimental treatment or dose, activation of the insulin pathway in liver and muscle was not inhibited by S961, showing that its antagonistic effect does not extend to rainbow trout.

A cross-species comparative approach to assessing multi- and transgenerational effects of endocrine disrupting chemicals.

A wide range of chemicals have been identified as endocrine disrupting chemicals (EDCs) in vertebrate species. Most studies of EDCs have focused on exposure of both male and female adults to these chemicals; however, there is clear evidence that EDCs have dramatic effects when mature or developing gametes are exposed, and consequently are associated with in multigenerational and transgenerational effects. Several publications have reviewed such actions of EDCs in subgroups of species, e.g., fish or rodents. In this review, we take a holistic approach synthesizing knowledge of the effects of EDCs across vertebrate species, including fish, anurans, birds, and mammals, and discuss the potential mechanism(s) mediating such multi- and transgenerational effects. We also propose a series of recommendations aimed at moving the field forward in a structured and coherent manner.

Transgenerational effects of polychlorinated biphenyls: 2. Hypothalamic gene expression in rats†.

Polychlorinated biphenyls (PCBs) are endocrine-disrupting chemicals (EDCs) with well-established effects on reproduction and behavior in developmentally-exposed (F1) individuals. Because of evidence for transgenerational effects of EDCs on the neuroendocrine control of reproductive physiology, we tested the hypothesis that prenatal PCB exposure leads to unique hypothalamic gene-expression profiles in three generations. Pregnant Sprague-Dawley rats were treated on gestational days 16 and 18 with the PCB mixture Aroclor 1221 (A1221), vehicle (3% DMSO in sesame oil), or estradiol benzoate (EB, 50 µg/kg), the latter a positive control for estrogenic effects of A1221. Maternal- and paternal-lineage F2 and F3 generations were bred using untreated partners. The anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC), involved in the hypothalamic control of reproduction, were dissected from F1 to F3 females and males, RNA extracted, and gene expression measured in a qPCR array. We detected unique gene-expression profiles in each generation, which were sex- and lineage-specific. In the AVPV, treatment significantly changed 10, 25, and 11 transcripts in F1, F2, and F3 generations, whereas 10, 1, and 12 transcripts were changed in these generations in the ARC. In the F1 AVPV and ARC, most affected transcripts were decreased by A1221. In the F2 AVPV, most effects of A1221 were observed in females of the maternal lineage, whereas only Pomc expression changed in the F2 ARC (by EB). The F3 AVPV and ARC were mainly affected by EB. It is notable that results in one generation do not predict results in another, and that lineage was a major determinant in results. Thus, transient prenatal exposure of F1 rats to A1221 or EB can alter hypothalamic gene expression across three generations in a sex- and lineage-dependent manner, leading to the conclusion that the legacy of PCBs continues for generations.

Alanine alters the carbohydrate metabolism of rainbow trout: glucose flux and cell signaling.

In rainbow trout, dietary carbohydrates are poorly metabolized compared with other macronutrients. One prevalent hypothesis suggests that high dietary amino acid levels could contribute to the poor utilization of carbohydrates in trout. In mammals, alanine is considered an important gluconeogenic precursor, but has recently been found to stimulate AMP-activated protein kinase (AMPK) to reduce glucose levels. In trout, the effect of alanine on glucose flux is unknown. The goal of this study was to determine the effects of 4 h exogenous alanine infusion on glucose metabolism in rainbow trout. Glucose flux, and the rate of glucose appearance (Ra) and disposal (Rd) were measured in vivo. Key glycolytic and gluconeogenic enzyme expression and activity, and cell signaling molecules relevant to glucose metabolism were assessed in the liver and muscle. The results show that alanine inhibits glucose Ra (from 13.2±2.5 to 7.3±1.6 µmol kg-1 min-1) and Rd (from 13.2±2.5 to 7.4±1.5 µmol kg-1 min-1) and the slight mismatch between Ra and Rd caused a reduction in glycemia, similar to the effects of insulin in trout. The reduction in glucose Rd can be partially explained by a reduction in glut4b expression in red muscle. In contrast to mammals, trout alanine-dependent glucose-lowering effects did not involve hepatic AMPK activation, suggesting a different mechanistic basis. Interestingly, protein kinase B (AKT) activation increased only in muscle, similar to effects observed in insulin-infused trout. We speculate that alanine-dependent effects were probably mediated through stimulation of insulin secretion, which could indirectly promote alanine oxidation to provide the needed energy.

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.

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.

Transgenerational effects of polychlorinated biphenyls: 1. Development and physiology across 3 generations of rats.

BACKGROUND: Polychlorinated biphenyls (PCBs) are persistent organic environmental contaminants and known endocrine-disrupting chemicals (EDCs). Previous studies demonstrated that developmental exposure to the weakly estrogenic PCB mixture Aroclor 1221 (A1221) in Sprague-Dawley rats altered sexual development, adult reproductive physiology and body weight. The current study tested the hypothesis that prenatal A1221 exposure not only disrupts these endpoints within an exposed individual's (F1 generation) lifespan, but may also affect subsequent generations (F2-F3). METHODS: We treated pregnant female rats on embryonic days (E) 16 and E18 with A1221 (1 mg/kg), estradiol benzoate (50 µg/kg, positive estrogenic control), or vehicle (3% DMSO in sesame oil, negative control). Endpoints related to sexually dimorphic developmental trajectories of reproductive and developmental physiology were measured, and as adults, reproductive endocrine status was assessed, in the F1, F2, and F3 generations. RESULTS: Significant effects of transgenerational EDCs were found for body weight and serum hormones. The A1221 descendants had significantly higher body weight in the F2-maternal lineage throughout postnatal development, and in F3-maternal lineage animals after weaning. In females, generation- and lineage-specific effects of exposure were found for serum progesterone and estradiol. Specifically, serum progesterone concentrations were lower in F2-A1221 females, and higher in F3-A1221 females, compared to their respective F2- and F3-vehicle counterparts. Serum estradiol concentrations were higher in F3-A1221 than F3-vehicle females. Reproductive and adrenal organ weights, birth outcomes, sex ratio, and estrous cycles, were unaffected. It is notable that effects of A1221 were only sometimes mirrored by the estrogenic control, EB, indicating that the mechanism of action of A1221 was likely via non-estrogenic pathways. CONCLUSIONS: PCBs caused body weight and hormonal effects in rats that were not observed in the directly exposed F1 offspring, but emerged in F2 and F3 generations. Furthermore, most effects were in the maternal lineage; this may relate to the timing of exposure of the F1 fetuses at E16 and 18, when germline (the future F2 generation) epigenetic changes diverge in the sexes. These results showing transgenerational effects of EDCs have implications for humans, as we are now in the 3rd generation since the Chemical Revolution of the mid-twentieth century, and even banned chemicals such as PCBs have a persistent imprint on the health of our descendants.

The nonapeptide isotocin in goldfish: Evidence for serotonergic regulation and functional roles in the control of food intake and pituitary hormone release.

Nonapeptides are a highly conserved family of peptides synthesized in the neuroendocrine brain and acting on central and peripheral receptors to regulate physiological functions in vertebrates. While the evolution of the two gene families of oxytocin-like and vasopressin-like nonapeptides and their receptors, as well as the neuroanatomy of their independent neuronal circuits have been well-characterized across vertebrate species, comparative studies on the physiological roles across vertebrates are lagging behind. In the current study, we focused on the comparative neuroendocrine functions and regulation of isotocin, the teleost homologue of mammalian oxytocin. Specifically, we address the hypothesis that isotocin exerts opposing effects on food intake and reproduction, which are well-established effects of its homologue oxytocin in mammalian species. Using goldfish, a well-characterized model of neuroendocrine regulation of both food intake and reproduction, we here showed that isotocin acts as an anorexigenic factor while exerting stimulatory effects on pituitary luteinizing hormone and growth hormone release. Given the dual inhibitory and stimulatory roles of serotonin on food intake and pituitary release of reproductive hormone in goldfish, we also investigated the potential crosstalk between both systems using immunohistochemistry and pharmacological approaches. Results provide neuroanatomical and pharmacological evidence for serotonergic regulation of magnocellular isotocinergic neurons in the preoptic area and pituitary. Together, these findings firstly provide the basis to investigate neuroendocrine cross-talk between serotonergic and nonapeptidergic systems in the regulation of both food intake and reproduction in goldfish, and secondly point to a conserved function of oxytocin-like peptides in the differential neuroendocrine control of both physiological processes in vertebrates.

Metabolic consequences of microRNA-122 inhibition in rainbow trout, Oncorhynchus mykiss.

BACKGROUND: MicroRNAs (miRNAs) are small regulatory molecules which post-transcriptionally regulate mRNA stability and translation. Several microRNAs have received attention due to their role as key metabolic regulators. In spite of the high evolutionary conservation of several miRNAs, the role of miRNAs in lower taxa of vertebrates has not been studied with regard to metabolism. The liver-specific and highly abundant miRNA-122 is one of the most widely studied miRNA in mammals, where it has been implicated in the control of hepatic lipid metabolism. Following our identification of acute postprandial, nutritional and endocrine regulation of hepatic miRNA-122 isomiRNA expression in rainbow trout, we used complementary in silico and in vivo approaches to study the role of miRNA-122 in rainbow trout metabolism. We hypothesized that the role of miRNA-122 in regulating lipid metabolism in rainbow trout is conserved to that in mammals and that modulation of miRNA-122 function would result in altered lipid homeostasis and secondarily altered glucose homeostasis, since lipogenesis has been suggested to act as glucose sink in trout. RESULTS: Our results show that miRNA-122 was functionally inhibited in vivo in the liver. Postprandial glucose concentrations increased significantly in rainbow trout injected with a miRNA-122 inhibitor, and this effect correlated with decreases in hepatic FAS protein abundance, indicative of altered lipogenic potential. Additionally, miRNA-122 inhibition resulted in a 20% decrease in plasma cholesterol concentration, an effect associated with increased expression of genes involved in cholesterol degradation and excretion. CONCLUSIONS: Overall evidence suggests that miRNA-122 may have evolved in early vertebrates to support liver-specific metabolic functions. Nevertheless, our data also indicate that metabolic consequences of miRNA-122 inhibition may differ quantitatively between vertebrate species and that distinct direct molecular targets of miRNA-122 may mediate metabolic effects between vertebrate species, indicating that miRNA-122 - mRNA target relationships may have undergone species-specific evolutionary changes.

A role for dopamine in control of the hypoxic ventilatory response via D2 receptors in the zebrafish gill.

Dopamine is a neurotransmitter involved in oxygen sensing and control of reflex hyperventilation. In aquatic vertebrates, oxygen sensing occurs in the gills via chemoreceptive neuroepithelial cells (NECs), but a mechanism for dopamine in autonomic control of ventilation has not been defined. We used immunohistochemistry and confocal microscopy to map the distribution of tyrosine hydroxylase (TH), an enzyme necessary for dopamine synthesis, in the gills of zebrafish. TH was found in nerve fibers of the gill filaments and respiratory lamellae. We further identified dopamine active transporter (dat) and vesicular monoamine transporter (vmat2) expression in neurons of the gill filaments using transgenic lines. Moreover, TH- and dat-positive nerve fibers innervated NECs. In chemical screening assays, domperidone, a D2 receptor antagonist, increased ventilation frequency in zebrafish larvae in a dose-dependent manner. When larvae were confronted with acute hypoxia, the D2 agonist, quinpirole, abolished the hyperventilatory response. Quantitative polymerase chain reaction confirmed expression of drd2a and drd2b (genes encoding D2 receptors) in the gills, and their relative abundance decreased following acclimation to hypoxia for 48 h. We localized D2 receptor immunoreactivity to NECs in the efferent gill filament epithelium, and a novel cell type in the afferent filament epithelium. We provide evidence for the synthesis and storage of dopamine by sensory nerve terminals that innervate NECs. We further suggest that D2 receptors on presynaptic NECs provide a feedback mechanism that attenuates the chemoreceptor response to hypoxia. Our studies suggest that a fundamental, modulatory role for dopamine in oxygen sensing arose early in vertebrate evolution.

Ontogenetic expression of metabolic genes and microRNAs in rainbow trout alevins during the transition from the endogenous to the exogenous feeding period.

As oviparous fish, rainbow trout change their nutritional strategy during ontogenesis. This change is divided into the exclusive utilization of yolk-sac reserves (endogenous feeding), the concurrent utilization of yolk reserves and exogenous feeds (mixed feeding) and the complete dependence on external feeds (exogenous feeding). The change in food source is accompanied by well-characterized morphological changes, including the development of adipose tissue as an energy storage site, and continuous muscle development to improve foraging. The aim of this study was to investigate underlying molecular mechanisms that contribute to these ontogenetic changes between the nutritional phenotypes in rainbow trout alevins. We therefore analyzed the expression of marker genes of metabolic pathways and microRNAs (miRNAs) important in the differentiation and/or maintenance of metabolic tissues. In exogenously feeding alevins, the last enzyme involved in glucose production (g6pca and g6pcb) and lipolytic gene expression (cpt1a and cpt1b) decreased, while that of gk, involved in hepatic glucose use, was induced. This pattern is consistent with a progressive switch from the utilization of stored (gluconeogenic) amino acids and lipids in endogenously feeding alevins to a utilization of exogenous feeds via the glycolytic pathway. A shift towards the utilization of external feeds is further evidenced by the increased expression of omy-miRNA-143, a homologue of the mammalian marker of adipogenesis. The expression of its predicted target gene abhd5, a factor in triglyceride hydrolysis, decreased concurrently, suggesting a potential mechanism in the onset of lipid deposition. Muscle-specific omy-miRNA-1/133 and myod1 expression decreased in exogenously feeding alevins, a molecular signature consistent with muscle hypertrophy, which may be linked to nutritional cues or increased foraging.

Post-prandial regulation of hepatic glucokinase and lipogenesis requires the activation of TORC1 signalling in rainbow trout (Oncorhynchus mykiss).

To assess the potential involvement of TORC1 (target of rapamycin complex 1) signalling in the regulation of post-prandial hepatic lipid and glucose metabolism-related gene expression in trout, we employed intraperitoneal administration of rapamycin to achieve an acute inhibition of the TOR pathway. Our results reveal that rapamycin inhibits the phosphorylation of TORC1 and its downstream effectors (S6K1, S6 and 4E-BP1), without affecting Akt and the Akt substrates Forkhead-box Class O1 (FoxO1) and glycogen synthase kinase 3α/ß (GSK 3α/ß). These results indicate that acute administration of rapamycin in trout leads to the inhibition of TORC1 activation. No effect is observed on the expression of genes involved in gluconeogenesis, glycolysis and fatty acid oxidation, but hepatic TORC1 inhibition results in decreased sterol regulatory element binding protein 1c (SREBP1c) gene expression and suppressed fatty acid synthase (FAS) and glucokinase (GK) at gene expression and activity levels, indicating that FAS and GK activity is controlled at a transcriptional level in a TORC1-dependent manner. This study demonstrates for the first time in fish that post-prandial regulation of hepatic lipogenesis and glucokinase in rainbow trout requires the activation of TORC1 signalling.

Rapid modulation of gene expression profiles in the telencephalon of male goldfish following exposure to waterborne sex pheromones.

Sex pheromones rapidly affect endocrine physiology and behaviour, but little is known about their effects on gene expression in the neural tissues that mediate olfactory processing. In this study, we exposed male goldfish for 6h to waterborne 17,20ßP (4.3 nM) and PGF2α (3 nM), the main pre-ovulatory and post-ovulatory pheromones, respectively. Both treatments elevated milt volume (P=0.001). Microarray analysis of male telencephalon following PGF2α treatment identified 71 unique transcripts that were differentially expressed (q<5%; 67 up, 4 down). Functional annotation of these regulated genes indicates that PGF2α pheromone exposure affects diverse biological processes including nervous system functions, energy metabolism, cholesterol/lipoprotein transport, translational regulation, transcription and chromatin remodelling, protein processing, cytoskeletal organization, and signalling. By using real-time RT-PCR, we further validated three candidate genes, ependymin-II, calmodulin-A and aldolase C, which exhibited 3-5-fold increase in expression following PGF2α exposure. Expression levels of some other genes that are thought to be important for reproduction were also determined using real-time RT-PCR. Expression of sGnRH was increased by PGF2α, but not 17,20ßP, whereas cGnRH expression was increased by 17,20ßP but not PGF2α. In contrast, both pheromones increase the expression of glutamate (GluR2a, NR2A) and γ-aminobutyric acid (GABAA γ2) receptor subunit mRNAs. Milt release and rapid modulation of neuronal transcription are part of the response of males to female sex pheromones.