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REV-ERBα ( nr1d1, nuclear receptor subfamily 1 group D member 1) is a transcriptional repressor that in mammals regulates nutrient metabolism, and has effects on energy homeostasis, although its role in teleosts is poorly understood. To determine REV-ERBα's involvement in fish energy balance and metabolism, we studied the effects of acute and 7-day administration of its agonist SR9009 on food intake, weight and length gain, locomotor activity, feeding regulators, plasma and hepatic metabolites, and liver enzymatic activity. SR9009 inhibited feeding, lowering body weight and length gain. In addition, the abundance of ghrelin mRNA decreased in the intestine, and abundance of leptin-aI mRNA increased in the liver. Hypocretin , neuropeptide y ( npy ), and proopiomelanocortin ( pomc ) mRNA abundance was not modified after acute or subchronic SR9009 administration, while hypothalamic cocaine- and amphetamine-regulated transcript ( cartpt-I ) was induced in the subchronic treatment, being a possible mediator of the anorectic effects. Moreover, SR9009 decreased plasma glucose, coinciding with increased glycolysis and a decreased gluconeogenesis in the liver. Decreased triglyceride levels and activity of lipogenic enzymes suggest a lipogenesis reduction by SR9009. Energy expenditure by locomotor activity was not significantly affected by SR9009. Overall, this study shows for the first time in fish the effects of REV-ERBα activation via SR9009, promoting a negative energy balance by reducing energetic inputs and regulating lipid and glucose metabolism.

The objective of this study was to investigate the role of palmitoylethanolamide (PEA) in the regulation of energy homeostasis in goldfish (Carassius auratus). We examined the effects of acute or chronic intraperitoneal treatment with PEA (20 μg·g -1 body weight) on parameters related to food intake and its regulatory mechanisms, locomotor activity, glucose and lipid metabolism, and the possible involvement of transcription factors and clock genes on metabolic changes in the liver. Acute PEA treatment induced a decrease in food intake at 6 and 8 h post-injection, comparable to that observed in mammals. This PEA anorectic effect in goldfish could be mediated through interactions with leptin and NPY, as PEA increased hepatic expression of leptin aI and reduced hypothalamic expression of npy. The PEA chronic treatment reduced weight gain, growth rate, and locomotor activity. The rise in glycolytic potential together with the increased potential of glucose to be transported into liver suggests an enhanced use of glucose in the liver after PEA treatment. In addition, part of glucose may be exported to be used in other tissues. The activity of fatty acid synthase (FAS) increased after chronic PEA treatment, suggesting an increase in the hepatic lipogenic capacity, in contrast with the mammalian model. Such lipogenic increment could be linked with the PEA-induction of REV-ERBα and BMAL1 found after the chronic treatment. As a whole, the present study shows the actions of PEA in several compartments related to energy homeostasis and feeding behavior, supporting a regulatory role for this N-acylethanolamine in fish., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Oleoylethanolamide (OEA) is an acylethanolamide synthesized mainly in the gastrointestinal tract with known effects in mammals on food intake and body mass through activation of peroxisome proliferator-activated receptor type α (PPARα). Since we previously demonstrated that acute treatment with OEA in goldfish resulted in decreased food intake and locomotor activity, as in mammals, we hypothesize that OEA would be involved in the control of energy metabolism in fish. Therefore, we assessed the effects of acute (for 6 h) and chronic (for 11 days) treatments with OEA (5 µg g -1 body mass) on metabolite concentrations and enzyme activities related to glucose and lipid metabolism in liver of goldfish (Carassius auratus). In the chronic treatment, OEA impairs the increase in body mass and reduces locomotor activity, without any signs of stress. The lipolytic capacity in liver decreased after both acute and chronic OEA treatments, whereas lipogenic capacity increased after acute and decreased after chronic treatment with OEA. These results are different from those observed to date in mammalian adipose tissue, but not so different from those known in liver, and might be attributed to the absence of changes in the expression of pparα, and/or to the increase in the expression of the clock gene bmal1a after chronic OEA treatment. As for glucose metabolism, a clear decrease in the capacity of hepatic tissue to use glucose was observed in OEA-treated fish. These results support an important role for OEA in the regulation of liver lipid and glucose metabolism, and could relate to the metabolic changes associated with circadian activity and the regulation of food intake in fish.

Ghrelin is a potent orexigenic signal mainly synthesized in the stomach and foregut of vertebrates. Recent studies in rodents point out that ghrelin could also act as an input for the circadian system and/or as an output of peripheral food-entrainable oscillators, being involved in the food anticipatory activity (FAA). In this study we pursue the possible interaction of ghrelin with the circadian system in a teleost, the goldfish (Carassius auratus). First, we analyzed if ghrelin is able to modulate the core clock functioning by regulating clock gene expression in fish under a light/dark cycle 12L:12D and fed at 10 am. As expected the acute intraperitoneal (IP) injection of goldfish ghrelin (gGRL[1-19], 44 pmol/g bw) induced the expression of hypothalamic orexin. Moreover, ghrelin also induced (∼ 2-fold) some Per clock genes in hypothalamus and liver. This effect was partially counteracted in liver by the ghrelin antagonist ([D-Lys(3)]-GHRP-6, 100 pmol/g bw). Second, we investigated if ghrelin is involved in daily FAA rhythms. With this aim locomotor activity was studied in response to IP injections (5-10 days) of gGRL[1-19] and [D-Lys(3)]-GHRP-6 at the doses above indicated. Ghrelin and saline injected fish showed similar 24h activity patterns. However, ghrelin antagonist treatment abolished the FAA in schedule fed fish under 24h light, suggesting the involvement of the endogenous ghrelin system in this pre-feeding activity. Altogether these results suggest that ghrelin could be acting as an input for the entrainment of the food-entrainable oscillators in the circadian organization of goldfish., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Daily rhythms of feeding regulators are currently arousing research interest due to the relevance of the temporal harmony of endocrine regulators for growth and welfare in vertebrates. However, it is unknown the leptin circadian pattern in fish. The aim of this study is to investigate if leptin (gLep-aI and gLep-aII) expression is rhythmic in goldfish (Carassius auratus) liver and brain, and if such rhythms are driven by feeding time through a food entrainable oscillator. Fish maintained under 12-h light:12-h dark photoperiod and a scheduled feeding time showed 24-h locomotor activity and glycaemia rhythms. Moreover, hepatic gLep-aI and brain gLep-aI and gLep-aII expression were rhythmic with different daily profiles, showing a postprandial increase of leptin expression in the liver but not in the brain. Under constant light and different feeding regimes (scheduled fed at 10:00, 22:00 or randomly fed), feeding time synchronized daily rhythms in locomotor activity, glycaemia and clock gene expression (gPer1a, gPer3 and gCry3), but the rhythmic expression of hepatic gLep-aI and brain gLep-aII only remained in fed fish at 10:00. In summary, daily rhythms of leptin expression in goldfish are differently regulated at central and peripheral level, and they are not directly driven by clock genes. The role of food entrained oscillators on leptin expression rhythms in fish remains to be demonstrated., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Orexins are neuropeptides mainly known for regulating feeding behavior and sleep-wakefulness cycle in vertebrates. Daily variations of orexin-A expression have been reported in fish, with the highest levels preceding feeding time. However, it is unknown if such variations could be related with daily rhythms of clock genes, which form the molecular core of circadian oscillators. The aim of the present study was to identify the possible role of orexin as an input element of the goldfish circadian system. It was investigated the effects of orexin-A (10ng/gbw) intracerebroventricular injections on the expression of clock genes, NPY and ghrelin, as well as on daily locomotor activity rhythms. Goldfish held under 12L:12D photoperiod and injected at midday with orexin or saline, were sacrificed at 1 and 3h post-injection. The analysis of genes expression by qReal Time PCR showed an increment of Per genes in hypothalamus and foregut at 3h post-injection, but not in hindgut and liver. The gBmal1a expression remained unaltered in all the studied tissues. Orexin induced NPY in the hypothalamus and ghrelin in the foregut. Locomotor activity was studied in fish daily injected with orexin for several consecutive days under different experimental conditions. Orexin synchronized locomotor activity in goldfish maintained in 24L and fasting conditions. Present results support a cross-talking between orexin-A and other feeding regulators at central and peripheral level, and suggest, for the first time, a role of this peptide as an input of the circadian system in fish., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Leptin is a hormone involved in feeding and body weight regulation in vertebrates, but the relationship between energy status and leptin has not been clearly established in fish. The aim of this study was to investigate in a teleost, the goldfish (Carassius auratus), the tissue expression pattern of two leptins (gLep-aI and gLep-aII) and leptin receptor (gLepR); and the effect of feeding on expression of these genes. Leptin system expression in goldfish was firstly analyzed in fish under overfeeding (2 weeks) or fasting (1 week), and secondly, at different postfeeding times (0, 3, 6, 9 and 12h). Goldfish has two Lep-a paralog genes, gLep-aI was widely expressed in central and peripheral tissues, whereas gLep-aII was preferentially expressed in brain. This different distribution pattern of leptins suggests that they can play different physiological roles in goldfish. The gLepR mRNA was ubiquitous expressed, with the highest expression in the telencephalon and hypothalamus. No significant differences in the leptin system expression were found among control, overfed and fasting groups, suggesting an apparent lack of correlation between nutritional status and leptin system in goldfish. Hepatic expression of gLep-aI significantly increased 9h after feeding time, while hypothalamic leptin system expression did not change after feeding. In summary, leptin in goldfish could signal short-term changes in food intake, as postprandial satiety, but seems to be independent of fasting/overfeeding conditions in this teleost. The widespread distribution of leptins and leptin receptor in goldfish strongly supports that this hormone may have pleitropic actions in fish., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Melatonin is a key neuroendocrine transducer in the circadian organization of vertebrates. However, its role in gastrointestinal physiology has not been explored in depth. In goldfish, a role for melatonin as a modulator of intestinal motility has been reported, whereby it attenuates the cholinergic contraction. The aim of the present work was to investigate this relaxation induced by melatonin in the gut smooth muscle of the goldfish, studying the possible involvement of nitric oxide. An in vitro model of isolated goldfish intestine was used to test the effects on intestinal motility. The addition of melatonin (10 pM-100 μM) to the organ bath relaxed acetylcholine- and serotonin-stimulated gut strips, but no effect was observed on KCl-contracted preparations. The addition of L-NAME (nitric oxide synthase inhibitor) increased the amplitude of the spontaneous slow waves, while sodium nitroprusside (SNP, nitric oxide donor) abolished them. All these results support a role for the nitrergic system in goldfish gut motility. However, neither L-NAME, nor SNP nor the nitric oxide precursor, l-arginine, modified the melatonin relaxing effect. These results highlight the existence of a basal nitrergic tone in the gut of goldfish, where melatonin would exert a calcium-dependent, nitric oxide-independent relaxing effect on serotonergic and cholinergic contraction., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The present study focused on the effects of a subchronic melatonin treatment on locomotor activity and cortisol plasma levels in goldfish. We compared two different administration routes: peripheral (10 microg/g body weight) versus central (1 microg/microl) injections of melatonin for 7 or 4 days, respectively. Daily locomotor activity, including both diurnal and nocturnal activities, food anticipatory activity and circulating cortisol at 11:00 (under 24 h of food deprivation and 17 h postinjection) were significantly reduced after repeated intraperitoneal injections with melatonin for 7 days, but not after intracerebroventricular treatment. Taking in mind the anoretic effect of melatonin in this species, we investigated if such feeding reduction is directly responsible for the reduction in motor activity induced by melatonin treatment. Food restriction (50%) for 10 days did not significantly modify either daily locomotor activity or plasma cortisol levels in goldfish, indicating that the peripheral action of melatonin diminishing locomotor activity in goldfish is not a direct consequence of its anoretic action. In summary, our results indicate that, as previously described in other vertebrate species, melatonin can regulate locomotor activity and cortisol levels in goldfish, suggesting a sedative effect of this hormone in this teleost., (Copyright 2010 Elsevier Inc. All rights reserved.)

The objective of the present study was to analyze the effects of chronic melatonin (10 microg/g body weight) on body weight and the main energetic reserves, particularly hepatic and muscle content of proteins, lipids and glycogen in goldfish. In addition, we studied plasma leptin and ghrelin, and hypothalamic content of neuropeptide Y (NPY) and monoamines after chronic melatonin treatment in order to elucidate a possible interplay between melatonin and these feeding regulators on the body weight regulation in this species. Body weight gain and specific growth rate were reduced (74% and 76%, respectively) after chronic (10 days) intraperitoneal (i.p.) treatment with melatonin. The carbohydrate and lipid metabolism was regulated by melatonin in goldfish, because this indoleamine reduced muscle glycogen stores and increased lipid mobilization. A suppressive trend, but not statistically significant, in circulating ghrelin was observed after chronic treatment with melatonin. Chronic melatonin administration significantly reduced noradrenergic metabolism and increased dihydroxiphenylacetic acid content in the hypothalamus, without significant modifications in the serotoninergic system. Thus, it could be suggested that melatonin may mediate its action on energy balance in fish, at least in part, via interactions with hypothalamic catecholaminergic system. Plasma leptin and hypothalamic NPY remained unaltered after melatonin treatment, suggesting that these feeding regulators may not be involved in the effects of melatonin on energy homeostasis in fish.

Simple Summary: This study investigated the response of goldfish (Carassius auratus) to different management conditions by assessing their oxygen consumption using an intermittent respirometry system, which serves as an indicator of metabolic rate. Goldfish consumed more oxygen during the day than at night, agreeing with the diurnal pattern of this teleost. Feeding to satiety increased their oxygen consumption by 35%, peaking 3 h after eating and returning to the normal routine values after 7 h. Handling the fish for five minutes caused a 140% increase in oxygen use, which returned to normal after 2.5 h. Increasing the water temperature gradually to 30 °C resulted in a 200% increase in oxygen consumption 2.5 h after the start of the temperature increase. Common aquaculture anesthetics also affected oxygen use for at least 4 h post-recovery. These findings suggest that metabolic rate is a valuable indicator of how goldfish respond to different practices that might increase their energy expenditure and stress. Therefore, intermittent respirometry, which measures oxygen consumption over time, is recommended to help understand and improve fish welfare in aquaculture. This study examined the metabolic rate (MO2, oxygen consumption) of goldfish (Carassius auratus) under normal management conditions in aquaculture. Using an intermittent respirometry system, we assessed daily variations and the effects of feeding, handling, temperature increase, and anesthetics. MO2 exhibited a daily rhythm, with higher values during day. Feeding to satiety produced a 35% increase in MO2 compared to fasted animals, with a maximum peak after 3 h and returning to baseline after 7 h. Handling stress (5 min) produced a 140% MO2 peak (from 180 to 252 mg O2 kg−1 h−1), returning to the routine MO2 after 2.5 h. An increase in water temperature (+0.1 °C min−1) up to 30 °C caused MO2 to peak at 200% after 2.5 h from the start of the temperature increase. The use of common anesthetics in aquaculture (MS-222, 2-phenoxyethanol and clove oil in deep anesthesia concentration) affects MO2 during the first few minutes after anesthetic recovery, but also during the following 4 h. It can be concluded that the metabolic rate is a good indicator of the goldfish's response to aquaculture practices involving energy expenditure and stress. Thus, intermittent respirometry is a valuable non-invasive tool for understanding and improving fish welfare in aquaculture. [ABSTRACT FROM AUTHOR]

Oleoylethanolamide (OEA) is a bioactive lipid mediator, produced in the intestine and other tissues, which is involved in energy balance regulation in mammals, modulating feeding and lipid metabolism. The purpose of the present study was to investigate the presence and possible role of OEA in feeding regulation in goldfish (Carassius auratus). We assessed whether goldfish peripheral tissues and brain contain OEA and their regulation by nutritional status. OEA was detected in all studied tissues (liver, intestinal bulb, proximal intestine, muscle, hypothalamus, telencephalon and brainstem). Food deprivation (48 h) reduced intestinal OEA levels and levels increased upon re-feeding, suggesting that this compound may be involved in the short-term regulation of food intake in goldfish, as a satiety factor. Next, the effects of acute intraperitoneal administration of OEA on feeding, swimming and plasma levels of glucose and triglycerides were analysed. Food intake, swimming activity and circulating triglyceride levels were reduced by OEA 2 h post-injection. Finally, the possible interplay among OEA and other feeding regulators (leptin, cholecystokinin, ghrelin, neuropeptide Y, orexin and monoamines) was investigated. OEA actions on energy homeostasis in goldfish could be mediated, at least in part, through interactions with ghrelin and the serotonergic system, as OEA treatment reduced ghrelin expression in the intestinal bulb, and increased serotonergic activity in the telencephalon. In summary, our results indicate for the first time in fish that OEA could be involved in the regulation of feeding, swimming and lipid metabolism, suggesting a high conservation of OEA actions in energy balance throughout vertebrate evolution.

Background: Melatonin is a key hormone in regulation of circadian rhythms, and involved in many rhythmic functions, such as feeding and locomotor activity. Melatonin reportedly counteracts stress responses in many vertebrates, including fish. However, targets for this action of melatonin and underlying mechanisms remain unknown. Results: This study reports potential anti-stress properties of melatonin in goldfish (Carassius auratus), with a focus on its effect on plasma cortisol, food intake, and locomotor activity, all of them involved in the responses to stress exposure. Indeed, acute injection of melatonin counteracted stress-induced hypercortisolinemia and reduced food intake. The reduced locomotor activity following melatonin treatment suggests a possible sedative role in fish. To assess whether this anti-stress effects of melatonin involve direct actions on interrenal tissue, in vitro cultures of head kidney (containing the interrenal cortisolproducing tissue) were carried out in presence of ACTH, melatonin, and luzindole, an antagonist of melatonin receptors. Melatonin in vitro reduced ACTH-stimulated cortisol release, an effect attenuated by luzindole; this suggests the presence of specific melatonin receptors in interrenal tissue. Conclusions: Our data support a role for melatonin as an anti-stress signal in goldfish, and suggest that the interrenal tissue of teleosts may be a plausible target for melatonin action decreasing cortisol production. [ABSTRACT FROM AUTHOR]

Simple Summary: The disruption of circadian rhythms is considered a potential source of distress, but the extent of its consequences is under study. This research investigated whether the disruption of the light/dark and feeding/fasting cycles can trigger stress and anxiety-like behavior in fish. For this purpose, we first optimized behavioral tests to measure anxiety-like states in goldfish. Second, we studied anxiety and stress responses in two groups of goldfish exposed to either continuous light or randomly scheduled meals for two months. Both conditions led to anxiety-like behavior, evidenced by increased thigmotaxis in the open field with object approach task. Fish exposed to constant light also showed higher locomotor activity, suggesting greater energy expenditure. Additionally, chronodisruption led to increased cortisol levels throughout the experiment, with evidence of a possible axis fatigue due to chronic stress in fish under continuous light. Altogether, these findings support that these chronodisruptive conditions cause distress in the animals, and they should be considered for welfare improvement. The correlation analyses suggested that cortisol, thigmotaxis, and scototaxis are not dependent on each other, while there is a high influence of individual behavioral traits. Thus, the need for combining multiple parameters when assessing discomfort in fish is emphasized. Chronodisruption caused by factors such as light at night and mistimed meals has been linked to numerous physiological alterations in vertebrates and may be an anxiogenic factor affecting welfare. This study aims to investigate whether chronodisruption causes measurable changes in the anxiety responses of goldfish under two conditions: randomly scheduled feeding (RF) and continuous light (LL). Anxiety-like behavior was assessed in the open field with object approach and black/white preference tests, which had been validated using diazepam. An increased thigmotaxis response and decreased object exploration under both chronodisruption protocols indicated anxiety states. Furthermore, locomotor activity was increased in LL fish. The black/white preference test discriminated anxiolysis induced by diazepam but was unable to detect anxiety caused by chronodisruption. Plasma cortisol increased in both RF and LL fish throughout the experiment, confirming that both conditions caused stress. The LL fish also showed an apparently desensitized hypothalamus–pituitary–interrenal HPI axis, with a decrease in pomc and crf expression. Individual analysis found no correlation between anxiety-like behavior and stress axis activation nor between scototaxis and thigmotaxis responses. However, individual differences in sensitivity to each test were detected. Altogether, these results highlight circadian disruption as a stressor for fish and endorse a multiple variable approach for reliably assessing animal discomfort. [ABSTRACT FROM AUTHOR]

The liver circadian clock plays a pivotal role in driving metabolic rhythms, being primarily entrained by the feeding schedule, although the underlying mechanisms remain elusive. This study aimed to investigate the potential role of insulin as an intake signal mediating liver entrainment in fish. To achieve this, the expression of clock genes, which form the molecular basis of endogenous oscillators, was analyzed in goldfish liver explants treated with insulin. The presence of insulin directly increased the abundance of per1a and per2 transcripts in the liver. The dependency of protein translation for such insulin effects was evaluated using cycloheximide, which revealed that intermediate protein translation is seemingly unnecessary for the observed insulin actions. Furthermore, the putative interaction between insulin and glucocorticoid signaling in the liver was examined, with the results suggesting that both hormones exert their effects by independent mechanisms. Finally, to investigate the specific pathways involved in the insulin effects, inhibitors targeting PI3K/AKT and MEK/ERK were employed. Notably, inhibition of PI3K/AKT pathway prevented the induction of per genes by insulin, supporting its involvement in this process. Together, these findings suggest a role of insulin in fish as a key element of the multifactorial system that entrains the liver clock to the feeding schedule. [ABSTRACT FROM AUTHOR]

N -acylethanolamines (NAEs) are a family of endogenous lipid signaling molecules that are involved in regulation of energy homeostasis in vertebrates with a putative role on circadian system. The aim of this work was to study the existence of daily fluctuations in components of NAEs system and their possible dependence on food intake. Specifically, we analyzed the content of oleoylethanolamide (OEA), palmitoylethanolamide (PEA), stearoylethanolamide (SEA), their precursors (NAPEs), as well as the expression of nape-pld (NAEs synthesis enzyme), faah (NAEs degradation enzyme), and pparα (NAEs receptor) in gastrointestinal and brain tissues of goldfish (Carassius auratus) throughout a 24-h cycle. Daily profiles of bmal1a and rev -erbα expression in gastrointestinal tissues were also quantified because these clock genes are also involved in lipid metabolism, are PPAR-targets in mammals, and could be a link between NAEs and circadian system in fish. Gastrointestinal levels of NAEs exhibited daily fluctuations, with a pronounced and rapid postprandial increase, the increment being likely caused by food intake as it is not present in fasted animals. Such periprandial differences were not found in brain, supporting that NAEs mobilization occurs in a tissue-specific manner and suggesting that these three NAEs could be acting as peripheral satiety signals. The abundance of pparα mRNA displayed a daily rhythm in the intestine and the liver, suggesting a possible rhythmicity in the NAEs functionality. The increment of pparα expression during the rest phase can be related with its role stimulating lipid catabolism to obtain energy during the fasting state of the animals. In addition, the clock genes bmal1a and rev -erbα also showed daily rhythms, with a bmal1a increment after feeding, supporting its role as a lipogenic factor. In summary, our data show the existence of all components of NAEs system in fish (OEA, PEA, SEA, precursors, synthesis and degradation enzymes, and the receptor PPARα), supporting the involvement of NAEs as peripheral satiety signals. [ABSTRACT FROM AUTHOR]

The circadian system controls temporal homeostasis in all vertebrates. The light-dark (LD) cycle is the most important zeitgeber ("time giver") of circadian system, but feeding time also acts as a potent synchronizer in the functional organization of the teleost circadian system. In mammals is well known that food intake during the rest phase promotes circadian desynchrony which has been associated with metabolic diseases. However, the impact of a misalignment of LD and feeding cycles in the entrainment of fish circadian oscillators is largely unknown. The objective of this work was to investigate how a time-lag feeding alters temporal homeostasis and if this could be considered a stressor. To this aim, goldfish maintained under a 12 h light-12 h darkness were fed at mid-photophase (SF6) or mid-scotophase (SF18). Daily rhythms of locomotor activity, clock genes expression in hypothalamus, liver, and head kidney, and circulating cortisol were studied. Results showed that SF6 fish showed daily rhythms of bmal1a and clock1a in all studied tissues, being in antiphase with rhythms of per1 genes, as expected for proper functioning clocks. The 12 h shift in scheduled feeding induced a short phase advance (4–5-h) of the clock genes daily rhythms in the hypothalamus, while in the liver the shift for clock genes expression rhythms was the same that the feeding time shift (∼12 h). In head kidney, acrophases of per genes underwent a 12-h shift in SF18 animals, but only 6 h shift for clock1a. Plasma cortisol levels showed a significant daily rhythm in animals fed at SF6, but not in SF18 fish fed, which displayed higher cortisol values throughout the 24-h. Altogether, results indicate that hypothalamus, liver, and head kidney oscillate in phase in SF6 fish, but these clocks are desynchronized in SF18 fish, which could explain cortisol alterations. These data reinforce the hypothesis that the misalignment of external cues (daily photocycle and feeding time) alters fish temporal homeostasis and it might be considered a stressor for the animals. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate how photocycle and feeding-time cues regulate the daily expression of Per1a, Per2a, Per3, and Cry3 in the goldfish hindgut. For this purpose, we studied the daily rhythmicity of these genes in fish maintained under different lighting conditions and under different feeding regimes (scheduled or not). We also studied whether the timing of just one meal is able to reset the hindgut molecular clock. In a first experiment, randomly fed fish were divided into four groups and kept under different light conditions for 30 d: 12 h light and 12 h dark (12L:12D), an inverted photoperiod (12D:12L), constant darkness (24D), and constant light (24L). In a second study, fish maintained under 24L were divided into four groups fed at different time points for 35 d: (1) fish scheduled-fed once a day (at 10:00 h); (2) fish fed with a 12-h shifted schedule (at 22:00 h), (3) fish fed at 10:00 h throughout the experiment, except the last day when fed at 22:00 h; and (4) a randomly fed group of fish. Fish were sacrificed every 6 h throughout a 24-h cycle. In both experiments, g Per1a, g Per2a, g Per3, and g Cry3 transcripts were quantified using Real Time-qPCR in the hindgut. Results show the clock genes gPer1a, gPer2a, and gCry3 are synchronized by both zeitgebers, the photocycle and feeding regime, in goldfish hindgut. Moreover, such clock genes anticipate light-on and food delivery, when these cues appear in a cyclic manner. In the absence of both zeitgebers, gCry3 and gPer2a rhythmicity disappeared. In contrast, the gPer1 rhythm was maintained under 24L and random feeding conditions, but not always, suggesting that food when randomly supplied is able to reset the clock depending on other factors, such as the energetic and metabolic conditions of the fish. The expression of gPer2a was not activated during the light phase of the cycle, suggesting the hindgut of goldfish is a non-direct photosensitive organ. In contrast to the other three genes, gPer3 expression in the goldfish hindgut seemed to be dependent on the timing of the last food delivery, even in the presence of a photocycle. This gene was the only one that maintained daily rhythms under both constant lighting conditions (24D and 24L), although with lower amplitude than when a photocycle was present. This indicates that, although the acrophase (peak time) of the gPer3 expression rhythm seems to be driven by feeding time, there is an interaction of both zeitgebers, food and light, to regulate its expression. In conclusion, present data indicate: (1) the hindgut of goldfish can be synchronized in vivo by both the photocycle and feeding time; (2) food is a potent signal that entrains this peripheral oscillator; and (3) both environmental cues seems to target different elements of the molecular clock. (Author correspondence: ) [ABSTRACT FROM AUTHOR]

Little is known about the feeding time dependence of clock gene expression in fish. The aim of the present study was to investigate whether a scheduled feeding time can entrain the rhythmic expression of several clock genes (period and cryptocrome) in the brain and liver of a teleost, the goldfish. Fish maintained under continuous light (LL) conditions were divided into 3 groups. Two groups were fed daily at 1000 h and 2200 h, respectively, and the third group was subjected to a random schedule regime. After 30 days, the fishes under 24-h food deprivation were sacrificed through a 24-h cycle, and clock gene expression in the optic tectum, hypothalamus, and liver was quantified by real-time PCR. The findings pointed to differences between the central and peripheral tissues studied. In the absence of a light-dark cycle (constant light), a scheduled feeding regime was necessary and sufficient to maintain both the rhythmic expression of several clock genes in the optic tectum and hypothalamus, as well as daily rhythms in locomotor activity. In contrast, neither locomotor activity nor clock gene expression in brain tissues was synchronized in randomly fed fish. However, in the liver, most of the clock genes studied presented significant daily rhythms in phase (related to the time of the last meal) in all 3 experimental groups, suggesting that the daily rhythm of clock genes in this organ only depends on the last meal time. The data suggest that, as in mammals, the smooth running of the food entrainable oscillator (FEO) in fish involves the rhythmic expression of several clock genes (Per1 and Cry3) in the central and peripheral structures. The results also indicate that the food anticipatory activity (FAA) in goldfish is not only the result of rhythmic clock gene expression in the liver because rhythmic clock gene expression was observed in randomly fed fishes, while FAA was not observed. [ABSTRACT FROM PUBLISHER]

Simple Summary: The circadian system synchronizes physiology and behavior to predictable environmental variations (daily cycles in light, temperature, or food availability). To this purpose, animals possess endogenous oscillators that provide internal temporal signals, thanks to rhythmic clock gene expression. In mammals, disruption of circadian oscillators correlates to overactivation of the stress axis, but this connection is less well known in teleosts. This work aims to learn how the absence of two main external signals (light-dark cycle and feeding schedule) impacts the oscillators in the hypothalamus-pituitary-interrenal) axis of goldfish, studying rhythms in locomotor activity, circulating cortisol, and clock gene expression. The removal of environmental synchronizators caused an increase in basal cortisol levels, indicating stress. Constant darkness also overrode clock gene rhythms in the whole axis but did not impede food anticipatory activity. Feeding at random times disrupted the interrenal clock, but cortisol and activity rhythms remained, concluding that clock gene rhythms in the interrenal tissue are unnecessary for rhythmic cortisol release or diurnality. This work evidences how environmental conditions impact the HPI axis, affecting endocrine and circadian functioning, which need to be considered when progressing towards optimizing fish welfare. Vertebrates possess circadian clocks, driven by transcriptional–translational loops of clock genes, to orchestrate anticipatory physiological adaptations to cyclic environmental changes. This work aims to investigate how the absence of a light-dark cycle and a feeding schedule impacts the oscillators in the hypothalamus-pituitary-interrenal axis of goldfish. Fish were maintained under 12L:12D feeding at ZT 2; 12L:12D feeding at random times; and constant darkness feeding at ZT 2. After 30 days, fish were sampled to measure daily variations in plasma cortisol and clock gene expression in the hypothalamus-pituitary-interrenal (HPI) axis. Clock gene rhythms in the HPI were synchronic in the presence of a light-dark cycle but were lost in its absence, while in randomly fed fish, only the interrenal clock was disrupted. The highest cortisol levels were found in the randomly fed group, suggesting that uncertainty of food availability could be as stressful as the absence of a light-dark cycle. Cortisol daily rhythms seem to depend on central clocks, as a disruption in the adrenal clock did not impede rhythmic cortisol release, although it could sensitize the tissue to stress. [ABSTRACT FROM AUTHOR]

The REV-ERBα nuclear receptor is a key component of the molecular machinery of circadian oscillators in mammals. While the rhythmic expression of this receptor has been described in teleosts, several critical aspects of its regulation remain unknown, such as which synchronizers entrain its rhythm, and whether it can modulate the expression of other clock genes. The objective of this study was to gain deeper understanding of the role of REV-ERBα in the fish circadian system. To this end, we first investigated the cues that entrain the rhythm of rev-erbα expression in the goldfish (Carassius auratus) liver and hypothalamus. A 12-h shift in feeding time induced a parallel shift in the hepatic rhythm of rev-erbα expression, confirming that this gene is food-entrainable in the goldfish liver. In contrast, light seems the main driver of rev-erbα rhythmic expression in the hypothalamus. Next, we examined the effects of REV-ERBα activation on locomotor activity and hepatic expression of clock genes. Subchronic treatment with the REV-ERBα agonist SR9009 slightly decreased locomotor activity anticipating light onset and food arrival, and downregulated hepatic bmal1a , clock1a , cry1a , per1a and pparα expression. This generalized repressing action of REV-ERBα on the expression of hepatic clock genes was confirmed in vitro by using agonists (SR9009 and GSK4112) and antagonist (SR8278) of this receptor. Overall, the present work reveals that REV-ERBα modulates the daily expression of the main genes of the teleostean liver clock, reinforcing its role in the liver temporal homeostasis, which seems highly conserved in both fish and mammals. [Display omitted] • rev-erbα rhythm is entrained by feeding time in liver but not in hypothalamus. • REV-ERBα agonist SR9009 slightly reduces FAA, not affecting activity daily rhythms. • REV-ERBα activation downregulates clock gene expression in the hepatic oscillator. • SR8278 antagonist counteracts the repression of liver clock genes by REV-ERBα. • REV-ERBα shows a conserved circadian role in mammals and fish. [ABSTRACT FROM AUTHOR]