Insulin Controls Clock Gene Expression in the Liver of Goldfish Probably via Pi3k/Akt Pathway.

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.

Assessing Chronodisruption Distress in Goldfish: The Importance of Multimodal Approaches.

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.

Daily rhythms of REV-ERBα and its role as transcriptional repressor of clock genes in fish hepatic oscillator.

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.

Economic Improvement of Artisanal Fishing by Studying the Survival of Discarded Plectorhinchus mediterraneus.

Europe calls for the end to fisheries discards, which means bringing all caught fish (subject to minimum sizes or quotas) to land. This decision is beneficial to the ecosystem, since it forces the selectivity of the fishing gears to improve. However, artisanal fishermen find themselves in a vulnerable situation where their subsistence depends on catches with small profit margins. An exemption to this landing obligation exists, as it is also ruled that those animals whose survival is scientifically guaranteed may be returned to the sea. Here we study the survival of Plectorhinchus mediterraneus captured by hookline and gillnet, as well as their physiological recovery. Survival exceeds 93% in both cases. The physiological assessment of primary (cortisol) and secondary (energy mobilization, acid-base and hydromineral balance, and immune system) stress responses indicates that surviving animals are able to recover after fishing. Thus, we propose the optimal size of capture of this species to achieve greater economic benefit. For this, we rely on the prices according to size in recent years, as well as on the growth curves of the species. In this way, by releasing fish of less than 1 kg, the current benefits could be multiplied between 2.3 and 9.6 times. This pilot study lays the groundwork for regulating artisanal fisheries through scientific data related to survival of discards along with information on the sale prices.

Nuclear Receptors (PPARs, REV-ERBs, RORs) and Clock Gene Rhythms in Goldfish (Carassius auratus) Are Differently Regulated in Hypothalamus and Liver.

The circadian system is formed by a network of oscillators located in central and peripheral tissues that are tightly linked to generate rhythms in vertebrates to adapt the organism to the cyclic environmental changes. The nuclear receptors PPARs, REV-ERBs and RORs are transcription factors controlled by the circadian system that regulate, among others, a large number of genes that control metabolic processes for which they have been proposed as key genes that link metabolism and temporal homeostasis. To date it is unclear whether these nuclear receptors show circadian expression and which zeitgebers are important for their synchronization in fish. Therefore, the objective of this study was to investigate whether the two main zeitgebers (light-dark cycle and feeding time) could affect the synchronization of central (hypothalamus) and peripheral (liver) core clocks and nuclear receptors in goldfish. To this aim, three experimental groups were established: fish under a 12 h light-12 h darkness and fed at Zeitgeber Time 2; fish with the same photoperiod but randomly fed; and fish under constant darkness and fed at Circadian Time 2. After one month, clock genes and nuclear receptors expression in hypothalamus and liver and circulating glucose were studied. Clock genes displayed daily rhythms in both tissues of goldfish if the light-dark cycle was present, with shifted-acrophases of negative and positive elements, as expected for proper functioning clocks. In darkness-maintained fish hypothalamic clock genes were fully arrhythmic while the hepatic ones were still rhythmic. Among studied nuclear receptors, in the hypothalamus only nr1d1 was rhythmic and only when the light-dark cycle was present. In the liver all nuclear receptors were rhythmic when both zeitgebers were present, but only nr1d1 when one of them was removed. Plasma glucose levels showed significant rhythms in fish maintained under random fed regimen or constant darkness, with the highest levels at 1-h postprandially in all groups. Altogether these results support that hypothalamus is mainly a light-entrained-oscillator, while the liver is a food-entrained-oscillator. Moreover, nuclear receptors are revealed as clear outputs of the circadian system acting as key elements in the timekeeping of temporal homeostasis, particularly in the liver.

REV-ERBα Agonist SR9009 Promotes a Negative Energy Balance in Goldfish.

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 Lack of Light-Dark and Feeding-Fasting Cycles Alters Temporal Events in the Goldfish (Carassius auratus) Stress Axis.

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.

Pituitary Hormones mRNA Abundance in the Mediterranean Sea Bass Dicentrarchus labrax: Seasonal Rhythms, Effects of Melatonin and Water Salinity.

In fish, most hormonal productions of the pituitary gland display daily and/or seasonal rhythmic patterns under control by upstream regulators, including internal biological clocks. The pineal hormone melatonin, one main output of the clocks, acts at different levels of the neuroendocrine axis. Melatonin rhythmic production is synchronized mainly by photoperiod and temperature. Here we aimed at better understanding the role melatonin plays in regulating the pituitary hormonal productions in a species of scientific and economical interest, the euryhaline European sea bass Dicentrarchus labrax. We investigated the seasonal variations in mRNA abundance of pituitary hormones in two groups of fish raised one in sea water (SW fish), and one in brackish water (BW fish). The mRNA abundance of three melatonin receptors was also studied in the SW fish. Finally, we investigated the in vitro effects of melatonin or analogs on the mRNA abundance of pituitary hormones at two times of the year and after adaptation to different salinities. We found that (1) the reproductive hormones displayed similar mRNA seasonal profiles regardless of the fish origin, while (2) the other hormones exhibited different patterns in the SW vs. the BW fish. (3) The melatonin receptors mRNA abundance displayed seasonal variations in the SW fish. (4) Melatonin affected mRNA abundance of most of the pituitary hormones in vitro; (5) the responses to melatonin depended on its concentration, the month investigated and the salinity at which the fish were previously adapted. Our results suggest that the productions of the pituitary are a response to multiple factors from internal and external origin including melatonin. The variety of the responses described might reflect a high plasticity of the pituitary in a fish that faces multiple external conditions along its life characterized by marked daily and seasonal changes in photoperiod, temperature and salinity.

First evidence on the role of palmitoylethanolamide in energy homeostasis in fish.

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.

Diurnal Profiles of N-Acylethanolamines in Goldfish Brain and Gastrointestinal Tract: Possible Role of Feeding.

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.

Time-Lag in Feeding Schedule Acts as a Stressor That Alters Circadian Oscillators in Goldfish.

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.

Ghrelin induces clock gene expression in the liver of goldfish in vitro via protein kinase C and protein kinase A pathways.

The liver is the most important link between the circadian system and metabolism. As a food-entrainable oscillator, the hepatic clock needs to be entrained by food-related signals. The objective of the present study was to investigate the possible role of ghrelin (an orexigenic peptide mainly synthesized in the gastrointestinal tract) as an endogenous synchronizer of the liver oscillator in teleosts. To achieve this aim, we first examined the presence of ghrelin receptors in the liver of goldfish. Then, the ghrelin regulation of clock gene expression in the goldfish liver was studied. Finally, the possible involvement of the phospholipase C/protein kinase C (PLC/PKC) and adenylate cyclase/protein kinase A (AC/PKA) intracellular signalling pathways was investigated. Ghrelin receptor transcripts, ghs-r1a, are present in the majority of goldfish hepatic cells. Ghrelin induced the mRNA expression of the positive (gbmal1a, gclock1a) and negative (gper genes) elements of the main loop of the molecular clock machinery, as well as grev-erbα (auxiliary loop) in cultured liver. These effects were blocked, at least in part, by a ghrelin antagonist. Incubation of liver with a PLC inhibitor (U73122), a PKC activator (phorbol 12-myristate 13-acetate) and a PKC inhibitor (chelerythrine chloride) demonstrated that the PLC/PKC pathway mediates such ghrelin actions. Experiments with an AC activator (forskolin) and a PKA inhibitor (H89) showed that grev-erbα regulation could be due to activation of PKA. Taken together, the present results show for the first time in vertebrates a direct action of ghrelin on hepatic clock genes and support a role for this hormone as a temporal messenger in the entrainment of liver circadian functions.

Interplay between the endocrine and circadian systems in fishes.

The circadian system is responsible for the temporal organisation of physiological functions which, in part, involves daily cycles of hormonal activity. In this review, we analyse the interplay between the circadian and endocrine systems in fishes. We first describe the current model of fish circadian system organisation and the basis of the molecular clockwork that enables different tissues to act as internal pacemakers. This system consists of a net of central and peripherally located oscillators and can be synchronised by the light-darkness and feeding-fasting cycles. We then focus on two central neuroendocrine transducers (melatonin and orexin) and three peripheral hormones (leptin, ghrelin and cortisol), which are involved in the synchronisation of the circadian system in mammals and/or energy status signalling. We review the role of each of these as overt rhythms (i.e. outputs of the circadian system) and, for the first time, as key internal temporal messengers that act as inputs for other endogenous oscillators. Based on acute changes in clock gene expression, we describe the currently accepted model of endogenous oscillator entrainment by the light-darkness cycle and propose a new model for non-photic (endocrine) entrainment, highlighting the importance of the bidirectional cross-talking between the endocrine and circadian systems in fishes. The flexibility of the fish circadian system combined with the absence of a master clock makes these vertebrates a very attractive model for studying communication among oscillators to drive functionally coordinated outputs.

The satiety factor oleoylethanolamide impacts hepatic lipid and glucose metabolism in goldfish.

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.

Performing a hepatic timing signal: glucocorticoids induce gper1a and gper1b expression and repress gclock1a and gbmal1a in the liver of goldfish.

Glucocorticoids have been recently proposed as input signals of circadian system, although the underlying molecular mechanism remains unclear. This work investigates the role of glucocorticoids as modulators of clock genes expression in the liver of goldfish. In fish maintained under a 12L:12D photoperiod, an intraperitoneal injection at Zeitgeber Time 2 of a glucocorticoid analog, dexamethasone (1 µg/g body weight) induced per1 genes while decreased gbmal1a and gclock1a expression in the liver at 8 h post-injection. A 4-h in vitro exposure of goldfish liver to cortisol (0.1-10 µM) also induced gper1 genes in a concentration-dependent manner. Similarly, the exposure of the goldfish cultured liver to dexamethasone produced a concentration-dependent induction of gper1 genes. Moreover, this glucocorticoid analog led to a decrease in gbmal1a and gclock1a transcripts, while the other clock genes analyzed were unaffected. The induction of gper1a and gper1b by dexamethasone in vitro was observed at short times (2 h), whereas the reductions of gbmal1a and gclock1a transcripts needed longer exposure times (8 h) to the glucocorticoid to be significant. Additionally, a 2-h exposure to dexamethasone in the liver culture was enough to extend the induction of per genes for more than 12 h. Present results indicate that gper1 genes are targets for glucocorticoids in the regulation of goldfish hepatic oscillator, as previously reported in mammals, suggesting a conserved role of glucocorticoids in the functional organization of the peripheral circadian system in vertebrates. The repression of clock1a and bmal1a is not so well established, and suggests that other clock genes could be glucocorticoid targets in the goldfish liver.

In Situ Localization and Rhythmic Expression of Ghrelin and ghs-r1 Ghrelin Receptor in the Brain and Gastrointestinal Tract of Goldfish (Carassius auratus).

Ghrelin is a gut-brain peptide hormone, which binds to the growth hormone secretagogue receptor (GHS-R) to regulate a wide variety of biological processes in fish. Despite these prominent physiological roles, no studies have reported the anatomical distribution of preproghrelin transcripts using in situ hybridization in a non-mammalian vertebrate, and its mapping within the different encephalic areas remains unknown. Similarly, no information is available on the possible 24-h variations in the expression of preproghrelin and its receptor in any vertebrate species. The first aim of this study was to investigate the anatomical distribution of ghrelin and GHS-R1a ghrelin receptor subtype in brain and gastrointestinal tract of goldfish (Carassius auratus) using immunohistochemistry and in situ hybridization. Our second aim was to characterize possible daily variations of preproghrelin and ghs-r1 mRNA expression in central and peripheral tissues using real-time reverse transcription-quantitative PCR. Results show ghrelin expression and immunoreactivity in the gastrointestinal tract, with the most abundant signal observed in the mucosal epithelium. These are in agreement with previous findings on mucosal cells as the primary synthesizing site of ghrelin in goldfish. Ghrelin receptor was observed mainly in the hypothalamus with low expression in telencephalon, pineal and cerebellum, and in the same gastrointestinal areas as ghrelin. Daily rhythms in mRNA expression were found for preproghrelin and ghs-r1 in hypothalamus and pituitary with the acrophase occurring at nighttime. Preproghrelin, but not ghs-r1a, displayed a similar daily expression rhythm in the gastrointestinal tract with an amplitude 3-fold higher than the rest of tissues. Together, these results described for the first time in fish the mapping of preproghrelin and ghrelin receptor ghs-r1a in brain and gastrointestinal tract of goldfish, and provide the first evidence for a daily regulation of both genes expression in such locations, suggesting a possible connection between the ghrelinergic and circadian systems in teleosts.

Anatomical distribution and daily profile of gper1b gene expression in brain and peripheral structures of goldfish (Carassius auratus).

The functional organization of the circadian system and the location of the main circadian oscillators vary through phylogeny. Present study investigates by in situ hybridization the anatomical location of the clock gene gPer1b in forebrain and midbrain, pituitary, and in two peripheral locations, the anterior intestine and liver, in a teleost fish, the goldfish (Carassius auratus). Moreover, the daily expression profiles of this gene were also studied by quantitative Real Time-PCR. Goldfish were maintained under a 12L-12D photoperiod and fed daily at 2 h after lights were switched on. A wide distribution of gPer1b mRNA in goldfish brain and pituitary was found in telencephalon, some hypothalamic nuclei (including the homologous to mammalian SCN), habenular nucleus, optic tectum, cerebellum and torus longitudinalis. Moreover, gPer1b expression was observed, for the first time in teleosts, in the pituitary, liver and anterior intestine. Day/night differences in gper1b mRNA abundance were found by in situ hybridization, with higher signal at nighttime that correlates with the results obtained by RT-PCR, where a rhythmic gPer1b expression was found in all tissues with acrophases at the end of the night. Amplitudes of gper1b rhythms vary among tissues, being higher in liver and intestine than in the brain, maybe because different cues entrain clocks in these locations. These results support the existence of functional clocks in many central and peripheral locations in goldfish coordinated, ticking at the same time.

The liver of goldfish as a component of the circadian system: Integrating a network of signals.

The circadian system drives daily physiological and behavioral rhythms that allow animals to anticipate cyclic environmental changes. The discovery of the known as "clock genes", which are very well conserved through vertebrate phylogeny, highlighted the molecular mechanism of circadian oscillators functioning, based on transcription and translation cycles (∼ 24 h) of such clock genes. Studies in goldfish have shown that the circadian system in this species is formed by a net of oscillators distributed at central and peripheral locations, as the retina, brain, gut and liver, among others. In this work we review the existing information about the hepatic oscillator in goldfish due to its relevance in metabolism, and its key role as target of a variety of humoral signals. Different input signals modify the molecular clockwork in the liver of goldfish. Among them, there are environmental cues (photocycle and feeding regime) and different encephalic and peripheral endogenous signals (orexin, ghrelin and glucocorticoids). Per clock genes seem to be a common target for different signals. Thus, this genes family might be important for shifting the hepatic oscillator. The physiological relevance of the crosstalking between metabolic and feeding-related hormones and the hepatic clock sets the stage for the hypothesis that these hormones could act as "internal zeitgebers" communicating oscillators in the goldfish circadian system.

Distinct expression profiles of three melatonin receptors during early development and metamorphosis in the flatfish Solea senegalensis.

Melatonin actions are mediated through G protein-coupled transmembrane receptors. Recently, mt1, mt2, and mel1c melatonin receptors were cloned in the Senegalese sole. Here, their day-night and developmental expressions were analyzed by quantitative PCR. These results revealed distinct expression patterns of each receptor through development. mel1c transcripts were more abundant in unfertilized ovulated oocytes and declined during embryonic development. mt1 and mt2 expression was higher at the earliest stages (2-6 days post-fertilization), decreasing before (mt2) or during (mt1) metamorphosis. Only mt1 and mel1c expression exhibited day-night variations, with higher nocturnal mRNA levels. These results suggest different roles and transcriptional regulation of these melatonin receptors during flatfish development and metamorphosis.

Leptin expression is rhythmic in brain and liver of goldfish (Carassius auratus). Role of feeding time.

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.