Mineralocorticoid receptor activates postnatal adiposity in zebrafish lacking proopiomelanocortin.

The proopiomelanocortin (Pomc)-derived peptides, including adrenocorticotropic hormone and α-melanocyte stimulating hormone (α-Msh), play both a central and a peripheral role in modulating the stress response. The central role is predominantly associated with nutrient homeostasis, while peripherally they play an important role in the synthesis of glucocorticoids (GCs) in response to stress. Pomc mutations are a major risk factor in the development of early-onset childhood obesity in humans. This is attributed primarily to their central effects on melanocortin receptor dysfunction leading to hyperphagia and reduced energy expenditure, while the peripheral mechanism contributing to obesity has largely been unexplored. Here, we tested the hypothesis that Pomc mutation-mediated adrenal insufficiency and the associated changes in GC signaling contribute to postnatal adiposity using zebrafish as a model. We generated a ubiquitous Pomc knockout zebrafish that mimicked the mammalian mutant phenotype of adrenal insufficiency and enhanced adiposity. The loss of Pomc inhibited stress-induced cortisol production and reprogrammed GC signaling by reducing glucocorticoid receptor responsiveness, whereas the mineralocorticoid receptor (Mr) signaling was enhanced. Larval feeding led to enhanced growth and adipogenesis in the Pomc mutants, and this was inhibited by eplerenone, an Mr antagonist. Altogether, our results underscore a key role for Mr signaling in early developmental adipogenesis and a possible target for therapeutic intervention for early-onset childhood obesity due to Pomc dysfunction.

Chronic cortisol stimulation enhances hypothalamus-specific enrichment of metabolites in the rainbow trout brain.

The hypothalamus is a key integrating center that is involved in the initiation of the corticosteroid stress response, and in regulating nutrient homeostasis. Although cortisol, the principal glucocorticoid in humans and teleosts, plays a central role in feeding regulation, the mechanisms are far from clear. We tested the hypothesis that the metabolic changes to cortisol exposure signal an energy excess in the hypothalamus, leading to feeding suppression during stress in fish. Rainbow trout (Oncorhynchus mykiss) were administered a slow-release cortisol implant for 3 days, and the metabolite profiles in the plasma, hypothalamus, and the rest of the brain were assessed. Also, U-13C-glucose was injected into the hypothalamus by intracerebroventricular (ICV) route, and the metabolic fate of this energy substrate was followed in the brain regions by metabolomics. Chronic cortisol treatment reduced feed intake, and this corresponded with a downregulation of the orexigenic gene agrp, and an upregulation of the anorexigenic gene cart in the hypothalamus. The U-13C-glucose-mediated metabolite profiling indicated an enhancement of glycolytic flux and tricarboxylic acid intermediates in the rest of the brain compared with the hypothalamus. There was no effect of cortisol treatment on the phosphorylation status of AMPK or mechanistic target of rapamycin in the brain, whereas several endogenous metabolites, including leucine, citrate, and lactate were enriched in the hypothalamus, suggesting a tissue-specific metabolic shift in response to cortisol stimulation. Altogether, our results suggest that the hypothalamus-specific enrichment of leucine and the metabolic fate of this amino acid, including the generation of lipid intermediates, contribute to cortisol-mediated feeding suppression in fish.NEW & NOTEWORTHY Elevated cortisol levels during stress suppress feed intake in animals. We tested whether the feed suppression is associated with cortisol-mediated alteration in hypothalamus metabolism. The brain metabolome revealed a hypothalamus-specific metabolite profile suggesting nutrient excess. Specifically, we noted the enrichment of leucine and citrate in the hypothalamus, and the upregulation of pathways involved in leucine metabolism and fatty acid synthesis. This cortisol-mediated energy substrate repartitioning may modulate the feeding/satiety centers leading to the feeding suppression.

Valine administration in the hypothalamus alters the brain and plasma metabolome in rainbow trout.

Central administration of valine has been shown to cause hyperphagia in fish. Although mechanistic target of rapamycin (mTOR) is involved in this response, the contributions to feed intake of central and peripheral metabolite changes due to excess valine are unknown. Here, we investigated whether intracerebroventricular injection of valine modulates central and peripheral metabolite profiles and may provide insights into feeding response in fish. Juvenile rainbow trout (Oncorhynchus mykiss) were administered an intracerebroventricular injection of valine (10 µg·µL-1 at 1 µL·100·g-1 body wt), and the metabolite profile in plasma, hypothalamus, and rest of the brain (composing of telencephalon, optic tectum, cerebellum, and medulla oblongata) was carried out by liquid chromatography-mass spectrometry (LC/MS)-based metabolomics. Valine administration led to a spatially distinct metabolite profile at 1 h postinjection in the brain: enrichment of amino acid metabolism and energy production pathways in the rest of the brain but not in hypothalamus. This suggests a role for extrahypothalamic input in the regulation of feed intake. Also, there was enrichment of several amino acids, including tyrosine, proline, valine, phenylalanine, and methionine, in plasma in response to valine. Changes in liver transcript abundance and protein expression reflect an increased metabolic capacity, including energy production from glucose and fatty acids, and a lower protein kinase B (Akt) phosphorylation in the valine group. Altogether, valine intracerebroventricular administration affects central and peripheral metabolism in rainbow trout, and we propose a role for the altered metabolite profile in modulating the feeding response to this branched-chain amino acid.NEW & NOTEWORTHY Valine causes hyperphagia in fish when it is centrally administered; however, the exact mechanisms are far from clear. We tested how intracerebroventricular injection of valine in rainbow trout affected the brain and plasma metabolome. The metabolite changes in response to valine were more evident in the rest of the brain compared with the hypothalamus. Furthermore, we demonstrated for the first time that central valine administration affects peripheral metabolism in rainbow trout.

Zygotic Exposure to Venlafaxine Disrupts the Circadian Locomotor Activity Behaviour in Zebrafish Larvae.

The antidepressant venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is commonly prescribed to treat major depressive disorder and is found at high concentrations in the aquatic environment. Concerns have been raised related to the health of aquatic organisms in response to this nontargeted pharmaceutical exposure. For instance, we previously demonstrated that exposure to venlafaxine perturbs neurodevelopment, leading to behavioural alterations in zebrafish (Danio rerio). We also observed disruption in serotonin expression in the pineal and raphe, regions critical in regulating circadian rhythms, leading us to hypothesize that zygotic exposure to venlafaxine disrupts the circadian locomotor rhythm in larval zebrafish. To test this, we microinjected zebrafish embryos with venlafaxine (1 or 10 ng) and recorded the locomotor activity in 5-day-old larvae over a 24-h period. Venlafaxine deposition reduced larval locomotor activity during the light phase, but not during the dark phase of the diurnal cycle. The melatonin levels were higher in the dark compared to during the light photoperiod and this was not affected by embryonic venlafaxine deposition. Venlafaxine exposure also did not affect the transcript abundance of clock genes, including clock1a, bmal2, cry1a and per2, which showed a clear day/night rhythmicity. A notable finding was that exposure to luzindole, a melatonin receptor antagonist, decreased the locomotor activity in the control group in light, whereas the activity was higher in larvae raised from the venlafaxine-deposited embryos. Overall, zygotic exposure to venlafaxine disrupts the locomotor activity of larval zebrafish fish during the day, demonstrating the capacity of antidepressants to disrupt the circadian rhythms in behaviour. Our results suggest that disruption in melatonin signalling may be playing a role in the venlafaxine impact on circadian behaviour, but further investigation is required to elucidate the possible mechanisms in larval zebrafish.

Exposure to Total Suspended Solids (TSS) Impacts Gill Structure and Function in Adult Zebrafish.

Total suspended solids (TSS) are a major contributor of anthropogenic impacts to aquatic systems. TSS exposure have been shown to affect the function of gills, but the mode of action is unclear. Zebrafish (Danio rerio) is emerging as an excellent model for mechanistic toxicology, and as there are no baseline studies on TSS effects in zebrafish gills, we tested the hypothesis that environmental concentrations of TSS damages gill structure and function in this species. Adult zebrafish were exposed to either 0, 10, 100, 500, 1000, or 2000 mg/L TSS for 4 days to assess the gill morphology. The minimal concentration that affected the gill structure was further tested for the distribution of key ion transporters, including Na+/K+- ATPase (NKA) and vacuolar-type H+-ATPase (VHA), using confocal microscopy. Our results reveal that TSS concentration as low as 100 mg/L alters the morphology of gills, including greater filament thickness, lamellae thickness, and epithelial lifting. This was also associated with a reduction in NKA immunoreactive (IR) cell count and intensity in the 100 mg/L TSS group, while there was neither a change in the VHA-IR cell count or expression nor the transcript abundance of atp6v1a and atp1a1a4 in the gills. Markers of stress response in these animals, including levels of cortisol, glucose, lactate, and glycogen were not altered after 4 days of TSS exposure. Overall, environmentally relevant concentrations of TSS can damage the gill structure and function in zebrafish and has the potential to enhance the toxicity of contaminants acting via the gills.

Chronic cortisol elevation restricts glucose uptake but not insulin responsiveness in zebrafish skeletal muscle.

Although teleosts show an elevated insulin response to hyperglycemia, the circulating glucose levels are not normalized as rapidly as in mammals. While this may suggest a lack of target tissue insulin responsiveness, the underlying mechanisms are unclear. We investigated whether changes in skeletal muscle insulin sensitivity and glucose uptake underlie the cortisol-mediated elevated blood glucose levels. Adult zebrafish (Danio rerio) were exposed to water-borne cortisol for 3 days followed by an intraperitoneal injection of glucose with or without insulin. Cortisol treatment resulted in a temporal delay in the reduction in blood glucose levels, and this corresponded with a reduced glucose uptake capacity and lower glycogen content in the skeletal muscle. The transcript abundance of slc2a1b (which encodes for GLUT1b) and a suite of genes encoding enzymes involved in muscle glycogenesis and glycolysis were upregulated in the cortisol group. Both the control and cortisol groups showed higher whole body insulin expression in response to blood glucose elevation, which also resulted in enhanced insulin-stimulated phosphorylation of AKT in the skeletal muscle. The insulin-mediated phosphorylation of S6 kinase was lower in the cortisol group. Altogether, chronic cortisol stimulation restricts glucose uptake and enhances the glycolytic capacity without affecting insulin responsiveness in zebrafish skeletal muscle.

Negative feedback regulation in the hypothalamic-pituitary-interrenal axis of rainbow trout subjected to chronic social stress.

The formation of dominance hierarchies in pairs of juvenile rainbow trout (Oncorhynchus mykiss) results in subordinate individuals exhibiting chronically elevated plasma cortisol concentrations. Cortisol levels reflect a balance between cortisol production, which is coordinated by the hypothalamic-pituitary-interrenal (HPI) axis in teleost fish, and negative feedback regulation and hormone clearance, which act to lower cortisol levels. However, the mechanisms contributing to the longer-term elevation of cortisol levels during chronic stress are not well established in fishes. The current study aimed to determine how subordinate fish maintain elevated cortisol levels, by testing the prediction that negative feedback and clearance mechanisms are impaired by chronic social stress. Plasma cortisol clearance was unchanged by social stress based on a cortisol challenge trial, hepatic abundance of the cortisol-inactivating enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11ßHSD2), and tissue fate of labelled cortisol. The capacity for negative feedback regulation in terms of transcript and protein abundances of corticosteroid receptors in the preoptic area (POA) and pituitary appeared stable. However, changes in 11ßHSD2 and mineralocorticoid receptor (MR) expression suggest subtle regulatory changes in the pituitary that may alter negative feedback. The chronic cortisol elevation observed during social subordination likely is driven by HPI axis activation and compounded by dysregulated negative feedback.

Increased gut serotonin production in response to bisphenol A structural analogs may contribute to their obesogenic effects.

Obesogens are synthetic, environmental chemicals that can disrupt endocrine control of metabolism and contribute to the risk of obesity and metabolic disease. Bisphenol A (BPA) is one of the most studied obesogens. There is considerable evidence that BPA exposure is associated with weight gain, increased adiposity, poor blood glucose control, and nonalcoholic fatty liver disease in animal models and human populations. Increased usage of structural analogs of BPA has occurred in response to legislation banning their use in some commercial products. However, BPA analogs may also cause some of the same metabolic impairments because of common mechanisms of action. One key effector that is altered by BPA and its analogs is serotonin, however, it is unknown if BPA-induced changes in peripheral serotonin pathways underlie metabolic perturbations seen with BPA exposure. Upon ingestion, BPA and its analogs act as endocrine-disrupting chemicals in the gastrointestinal tract to influence serotonin production by the gut, where over 95% of serotonin is produced. The purpose of this review is to evaluate how BPA and its analogs alter gut serotonin regulation and then discuss how disruption of serotonergic networks influences host metabolism. We also provide evidence that BPA and its analogs enhance serotonin production in gut enterochromaffin cells. Taken together, we propose that BPA and many BPA analogs represent endocrine-disrupting chemicals that can influence host metabolism through the endogenous production of gut-derived factors, such as serotonin.

Coordinated Action of Corticotropin-Releasing Hormone and Cortisol Shapes the Acute Stress-Induced Behavioural Response in Zebrafish.

INTRODUCTION: The stress response mediated by the hypothalamus-pituitary-adrenal (HPA) axis activation is highly conserved in vertebrates. Hyperactivity is one such established acute stress response, and corticotropin-releasing hormone (CRH), the primary step in HPA activation, signalling has been implicated in this stressor-mediated behaviour. However, whether CRH mediates the acute behavioural effects either alone or in conjunction with glucocorticoids (GCs) are far from clear. We hypothesized that the CRH receptor 1 (CRHR1)-mediated rise in GCs post-stress is necessary for the initiation and maintenance of the acute stress-related behaviour. METHODS: We first generated zebrafish (Danio rerio) with a mutation in the CRHR1 gene (CRHR1-KO) to assess the function of CRH. The behavioural readout utilized for this study was the locomotor activity of larval zebrafish in response to an acute light exposure, a protocol that freezes the larvae in response to the light stimulus. To test whether cortisol signalling is involved in the stress-mediated hyperactivity, we treated wildtype fish with metyrapone (MET), an inhibitor of 11ß-hydroxylase, to suppress cortisol production. The temporal role for cortisol signalling in the stress-related hyperactivity was tested using the glucocorticoid receptor knockout (GRKO) and mineralocorticoid receptor knockout (MRKO) zebrafish mutants. RESULTS: CRHR1-KO larvae did not increase cortisol, the principal GC in teleosts, post-stress, confirming a functional knockout. An acute stress resulted in the hyperactivity of the larvae in light at 15, 60, and 240 min post-stress, and this was absent in CRHR1-KO larvae. Addition of MET effectively blocked the attendant rise in cortisol post-stress; however, the stress-mediated hyperactivity was inhibited only at 60 and 240 min but not at 15 min post-stress. Addition of human CRH peptide caused hyperactivity at 15 min, and this response was also abolished in the CRHR1-KO mutants. The stress-induced hyperactivity was absent in the MRKO fish, while GRKO mutants showed transient effects. CONCLUSIONS: The results suggest that the stress-induced hyperactivity is induced by the CRH/CRHR1 system, while the temporal activation of cortisol production and the associated GR/MR signalling is essential for prolonging the stressor-induced hyperactivity. This study underscores the importance of systems-level analysis to assess stress responsivity.

Venlafaxine deposition in the zygote disrupts the endocrine control of growth in juvenile zebrafish.

The antidepressant venlafaxine can be found at levels nearing µg/L in waterways receiving municipal wastewater effluent, exposing non-target organisms, such as fish, to this chemical. We showed previously that zygotic exposure to venlafaxine alters neurodevelopment and behaviour in zebrafish (Danio rerio) larvae. Here, we tested the hypothesis that the zygotic deposition of venlafaxine disrupts endocrine pathways related to growth in zebrafish. This was carried out by microinjecting embryos (1-4 cell stage) with either 0, 1, or 10 ng venlafaxine. Zygotic venlafaxine deposition reduced the growth of fish after 30 days post-fertilization. Specific growth rate was particularly impacted by 1 ng venlafaxine. This growth retardation corresponded with the disruption of endocrine pathways involved in growth and metabolism. Venlafaxine exposed embryos displayed reduced transcript abundance of key genes involved in anabolic hormone action. Early-life venlafaxine exposure also reduced whole-body insulin and glucose content in juveniles. Target-tissue glucose uptake measurements indicated that high venlafaxine deposition preferentially increased glucose uptake to the brain. Zygotic venlafaxine did not affect feed intake nor altered the transcript abundance of key feeding-related peptides. Taken together, zygotic venlafaxine deposition compromises zebrafish growth by disrupting multiple endocrine pathways, and this study has identified key markers for potential use in risk assessment.

Loss of the glucocorticoid receptor causes accelerated ovarian ageing in zebrafish.

Reproductive decline in mid-adult females is an established phenotype of the ageing process. Stress and the rise in glucocorticoids (GCs) accelerate reproductive ageing, but little is known about the mechanisms involved. During stress, GCs activate the glucocorticoid receptor (GR), a ubiquitously expressed, ligand-bound transcription factor, to elicit physiological changes for restoring homeostasis. Here, we tested the hypothesis that GC-GR signalling is essential for accelerating reproductive ageing. To test this, we used a ubiquitous GR knockout (GRKO) zebrafish, which is inherently hypercortisolemic, to delineate the role of high cortisol and GR signalling on reproductive ageing. The loss of GR led to premature ovarian ageing, including high frequency of typical and atypical follicular atresia in vitellogenic oocytes, yolk liquefaction and large inflammatory infiltrates. The reduction in oocyte quality was also associated with a decline in ovarian tert expression in the adult GRKO fish compared to the early adult GRKO and adult wild-type zebrafish. Accelerated ovarian ageing also impacted the progeny, including lower breeding success, fecundity, egg fertilization rate and delayed somitogenesis and embryo survival in the adult GRKO fish. We adduce that GR signalling is essential for prolonging the reproductive lifespan and improving the egg quality and embryo viability in zebrafish.

Vape flavourants dull sensory perception and cause hyperactivity in developing zebrafish embryos.

E-cigarette use (vaping) during pregnancy has been increasing, and the potential exists for the developing brain in utero to be exposed to chemical constituents in the vape. Vapes come in over 7000 unique flavours with and without nicotine, and while nicotine is a known neurotoxicant, the effects of vape flavouring alone, in the absence of nicotine, on brain function are not well understood. Here, we performed a screen of vape aerosol extracts (VAEs) to determine the potential for prenatal neurotoxicity using the zebrafish embryo photomotor response (PMR)-a translational biosensor of neurobehavioural effects. We screened three commonly used aerosolized vape liquids (flavoured and flavourless) either with or without nicotine. No neurobehavioural effects were detected in flavourless, nicotine-free VAEs, while the addition of nicotine to this VAE dulled sensory perception. Flavoured nicotine-free VAEs also dulled sensory perception and caused hyperactivity in zebrafish embryos. The combination of flavour and nicotine produced largely additive effects. Flavoured VAEs without nicotine had similar neuroactive potency to nicotine. Together, using zebrafish PMR as a high throughput translational behavioural model for prenatal exposure, our results demonstrate that e-cigarette flavourants that we screened elicit neurobehavioural effects worthy of further investigation for long-term neurotoxic potential and also have the potential to modulate nicotine impact on the developing brain.

Zygotic Venlafaxine Exposure Impacts Behavioral Programming by Disrupting Brain Serotonin in Zebrafish.

The antidepressant venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is present in surface waters downstream of wastewater treatment plants. We previously showed that zygotic venlafaxine deposition alters larval behavior in zebrafish (Danio rerio), but the mechanisms were unknown. Here we tested the hypothesis that venlafaxine disrupts central serotonergic development, leading to impaired behavioral responses in zebrafish larvae. This was tested by microinjecting embryos with venlafaxine immediately after fertilization and performing spatial distribution of serotonin immunoreactivity, as well as characterizing target genes involved in serotonin turnover in the zebrafish brain. We provide evidence that venlafaxine exposure reduces serotonin immunoreactivity and tyrosine hydroxylase-positive cell populations in specific larval brain regions, and this corresponded with reduced larval activity observed in the drug-exposed group. Lowered serotonin was not due to either reduced synthesis or increased breakdown capacity. However, co-injection of serotonin alongside venlafaxine in embryos recovered brain serotonin immunoreactivity, tyrosine hydroxylase-positive cell populations, and rescued venlafaxine-mediated behavioral changes. Overall, our results demonstrate for the first time that early life exposure to venlafaxine perturbs brain development, which may be due to reduced serotonin, leading to altered larval behavior in zebrafish.

Hypoxia affects the ontogeny of the hypothalamus-pituitary-interrenal axis functioning in the lake whitefish (Coregonus clupeaformis).

Early life stages are sensitive to environmental insults and changes during critical developmental periods; this can often result in altered adult behaviour and physiology. Examining the development of the hypothalamus-pituitary-interrenal (HPI) axis and its responsiveness, or lack thereof, during development are important for understanding the short- and long-term impacts of stressors on embryonic and larval fish. We examined the ontogeny of the HPI axis in embryonic (21, 38, 63, 83 and 103 days post-fertilisation (dpf)) and larval (1, 2, 3 and 4 weeks post-hatch (wph)) lake whitefish (Coregonus clupeaformis) by quantifying changes in mRNA levels of several genes associated with HPI axis functioning and whole animal cortisol levels throughout development and in response to a severe or mild hypoxic stress. Cortisol, and crh, crhbp1, pomc and star transcripts were detected from the earliest embryonic age studied. Cortisol levels in control embryos decreased between 21 and 63 dpf, suggesting the utilisation of maternal cortisol deposits. However, by 83 dpf (70% developed) endogenous de novo synthesis had generated a 4.5-fold increase in whole embryo cortisol. Importantly, we provide novel data showing that the HPI axis can be activated even earlier. Whole body cortisol increased in eyed lake whitefish embryos (38 dpf; ~32% developed) in response to hypoxia stress. Coincident with this hypoxia-induced increase in cortisol in 38 dpf embryos were corresponding increases in crh, crhbp1, pomc and star transcript levels. Beyond 38 dpf, the HPI axis in lake whitefish embryos was hyporesponsive to hypoxia stress at all embryonic ages examined (63, 83 and 103 dpf; 54, 72 and 85% developed, respectively). Post-hatch, larvae responded to hypoxia with an increase in cortisol levels and HPI axis genes at 1 wph, but this response was lost and larvae appeared hyporesponsive at subsequent ages (2, 3 and 4 wph). Collectively our work demonstrates that during fish embryogenesis and the larval stage there are windows where the HPI axis is responsive and windows where it is truly hyporesponsive; both could be beneficial in ensuring undisrupted development particularly in the face of increasing environmental changes.

Evaluating interactions between the melanocortin-5 receptor, MRAP1, and ACTH(1-24): A phylogenetic study.

The melanocortin-2 receptor (MC2R) and the melanocortin-5 receptor (MC5R) are found on the same chromosome in most vertebrate genomes, and for the species analyzed in this study, MC2R and MC5R are co-expressed in glucocorticoid-producing cells that also express the accessory protein MRAP1. Since MRAP1 affects the ligand sensitivity of MC2R orthologs, this study tested the hypothesis that co-expression of MC5R with MRAP1 would also affect the ligand sensitivity of MC5R. The hypothesis was confirmed for stingray, rainbow trout, and chicken, MC5R orthologs. However, elephant shark MC5R was not affected in the same way by co-expression of MRAP1. It appears that, for some MC5R orthologs (i.e., stingray, rainbow trout, and chicken), a docking site for the R/KKRRP motif of ACTH(1-24) may become exposed on the receptor following co-expression with MRAP1. However, for elephant shark MC5R co-expression with MRAP1 may not affect engagement ACTH(1-24). Hence during the radiation of the chordates, the interaction between MRAP1 and MC5R has diverged.

Postnatal triglyceride accumulation is regulated by mineralocorticoid receptor activation under basal and stress conditions.

KEY POINTS: Glucocorticoids (GCs) either enhance or reduce obesity in mammals, but limited information exists on the role of corticosteroid receptors in mediating the effect of GCs on lipid metabolism during postnatal development. Mineralocorticoid receptor (MR) activation leads to triglyceride (TG) accumulation post-feeding, whereas glucocorticoid receptor (GR) activation reduces TG levels. The TG profile was inversely related to the lipoprotein lipase (lpl) transcript abundance, and this gene was downregulated by MR activation. Cortisol plays an important role in adipogenesis during postnatal development in zebrafish, and this includes gene/pathway-specific signalling by GR, MR and GR/MR interactions. Ubiquitous MR and GR knockout in zebrafish provides an excellent model to study the mode of action of GCs in regulating lipid metabolism. ABSTRACT: Glucocorticoids (GCs) act through two receptors, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), which differ in both their affinity to bind GCs and their function. As MR has 10-fold higher affinity for GCs compared to GR, it has been postulated that MR activation occurs at basal levels, while stress levels of these steroid hormones activate GR signalling. There is a growing body of evidence that both these receptors are involved in GC-mediated lipid metabolism. However, the role of GCs in lipogenesis and lipolysis is controversial, as these steroids appear to both enhance and reduce obesity. As lipid synthesis is a critical part of early development, we hypothesized that both MR and GR contribute to lipid regulation by GCs during postnatal growth. Using MR and GR knockout zebrafish, we demonstrate that MR activation, but not GR activation, is involved in triglyceride (TG) accumulation during the larval development post feeding. Lack of MRs did not affect the gene expression of fatty acid synthase (fas), or acyl-CoA:diacylglycerol acyltransferase 2 (dgat2), but increased lipoprotein lipase (lpl) transcript abundance. Activation of GR with exogenous cortisol decreased TG levels and increased lpl mRNA levels, but these responses require the presence of MR. Larval transcriptome revealed that MR was the primary regulator of genes involved in lipid synthesis, while GR activation favoured lipid catabolism. Our results underscore a key role for MR activation in mediating postnatal lipid accumulation, as well as cooperatively regulating GR-mediated lipolysis during postnatal stress.

Loss of the glucocorticoid receptor in zebrafish improves muscle glucose availability and increases growth.

Chronic stress and the associated elevation in corticosteroid levels increase muscle protein catabolism. We hypothesized that the glucocorticoid receptor (GR)-regulated restriction of muscle glucose availability may play a role in the increased protein catabolism during chronic stress. To test this, we generated a ubiquitous GR knockout (GRKO) zebrafish to determine the physiological consequence of glucocorticoid stimulation on muscle metabolism and growth. Adult GRKO zebrafish had higher body mass, and this corresponded to an increased protein and lipid, but not carbohydrate, content. GRKO fish were hypercortisolemic, but they elicited a higher cortisol response to an acute stressor. However, the stressor-induced increase in plasma glucose level observed in the wild type was completely abolished in the GRKO fish. Also, the muscle, but not liver, capacity for glucose uptake was enhanced in the GRKO fish, and this corresponded with a higher hexokinase activity in the mutants. Zebrafish lacking GR also showed a higher capacity for protein synthesis, including increased phosphorylation of eukaryotic initiation factor 4B, higher expression of heat shock protein cognate 70, and total protein content. A chronic fasting stressor reduced body mass and muscle protein content in adult zebrafish, but this decrease was attenuated in the GRKO compared with the wild-type fish. Metabolomics analysis revealed that the free pool of amino acid substrates used for oxidation and gluconeogenesis were lower in the fasted GRKO fish muscle compared with the wild type. Altogether, chronic stressor-mediated GR signaling limits muscle glucose uptake, and this may play a role in protein catabolism, leading to the growth suppression in fish.

Effects of cortisol and lipopolysaccharide on expression of select growth-, stress- and immune-related genes in rainbow trout liver.

Many studies have shown that stress-induced cortisol levels negatively influence growth and immunity in finfish. Despite this knowledge, few studies have assessed the direct effects of cortisol on liver immune function. Using real-time PCR, the expression of three cortisol-responsive genes (GR: glucocorticoid receptor, IGF-1: insulin-like growth factor-I and SOCS-1: suppressor of cytokine signaling-I), genes involved with innate and adaptive immunity (IL-1ß: interleukin-1 beta, IgM: immunoglobin-M and Lyz: lysozyme), and liver-specific antimicrobial peptides (hepcidin and LEAP-2A: liver-expressed antimicrobial peptide-2A) was studied in vitro using rainbow trout liver slices. The abundances of GR, SOCS-1 and IGF-1 mRNAs were suppressed by cortisol treatment. Abundance of IL-1ß mRNA was upregulated by LPS and suppressed by cortisol treatment in a time-dependent manner. While abundance of IgM mRNA was suppressed by cortisol treatment and stimulated by LPS, there were no effects of cortisol or LPS on abundance of Lyz mRNA. Abundance of hepcidin and LEAP-2A mRNA levels were suppressed by cortisol treatment and stimulated by LPS. These results demonstrate that cortisol directly suppresses abundance of GR, IGF-1, IL-1ß, IgM, hepcidin, LEAP-2A and SOCS-1 mRNA transcripts in the rainbow trout liver. We report for the first time, a suppressive effect of cortisol (within 8 h of treatment) on hepcidin and LEAP-2A mRNAs in rainbow trout liver, which suggests that acute stress may negatively affect liver immune function in rainbow trout.

Bisphenol A in Eggs Impairs the Long-Term Stress Performance of Rainbow Trout in Two Generations.

Salmonids are ecologically, economically, and culturally important fish species in North America, but whether contaminants in the environment play a role in their population decline is unclear. We tested the hypothesis that bisphenol A (BPA) deposition in eggs, mimicking a maternal transfer scenario, compromises the stress axis functioning and target tissues stress response in two generations of a model salmonid species, rainbow trout ( Oncorhynchus mykiss). Eggs were enriched with 0, 4, or 40 ng of BPA, fertilized, and reared in clean water for two generations. The fish were subjected to an acute stressor after a year in both generations to test their stress performances. Trout raised from BPA-enriched eggs showed impaired stressor-mediated plasma cortisol and lactate response in the F1 and F2 generations, respectively. Key genes involved in cortisol biosynthesis in the head kidney, as well as stress- and growth-related transcripts in the liver and muscle, were impacted either in the F1 and/or F2 generations. Our results underscore the long-term impact associated with BPA in eggs, mimicking a maternal transfer scenario, on the stress performance of trout in two generations. The results highlight the need for developing novel biomarkers to predict long-term and generational toxicities in salmonids.

Lifelong Exposure to PCBs in the Remote Norwegian Arctic Disrupts the Plasma Stress Metabolome in Arctic Charr.

Lake Ellasjøen on the remote Norwegian island of Bjørnøya is populated by Arctic charr (Salvelinus alpinus) having 20-fold higher body burdens of polychlorinated biphenyls (PCB) compared to charr from the neighboring Lake Laksvatn. This provides a natural setting to test the hypothesis that lifelong exposure to PCBs compromises the energy metabolism in this northernmost living salmonid. To test this, blood was sampled from charr from both lakes immediately after capture and following a 1 h handling and confinement stressor to assess possible differences in their energy metabolism and energy substrate mobilization, respectively. The plasma metabolome of charr was assessed by metabolite detection/separation with LC-MS. Plasma metabolite profiles revealed differences in key pathways involved in amino acid metabolism between charr from each lake, underscoring an impact of PCBs on energy metabolism in Arctic charr residing in Lake Ellasjøen. Subjecting charr from either lake to an acute stressor altered the plasma metabolite profiles and revealed distinct stress metabolome in Lake Ellasjøen charr, suggesting a reduced metabolic capacity. Taken together, lifelong exposure to PCBs in Ellasjøen charr disrupts the plasma metabolome, and may impair the adaptive metabolic response to stressors, leading to a reduced fitness.