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.

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.

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.

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.

First evidence of nocturnin in fish: two isoforms in goldfish differentially regulated by feeding.

Nocturnin (NOC) is a unique deadenylase with robust rhythmic expression involved in the regulation of metabolic processes in mammals. Currently, the possible presence of NOC in fish is unknown. This report aimed to identify NOC in a fish model, the goldfish ( Carassius auratus), and to study the possible regulation of its expression by feeding. Two partial-length cDNAs of 293 and 223 bp, named nocturnin-a ( noc-a) and nocturnin-b ( noc-b), were identified and found to be highly conserved among vertebrates. Both mRNAs show a similar widespread distribution in central and peripheral tissues, with higher levels detected for noc-a compared with noc-b. The periprandial expression profile revealed that noc-a mRNAs rise sharply after a meal in hypothalamus, intestinal bulb, and liver, whereas almost no changes were observed for noc-b. Food deprivation was found to exert opposite effects on the expression of both NOCs (generally inhibitory for noc-a, and stimulatory for noc-b) in the three mentioned tissues. A single meal after a 48-h food deprivation period reversed (totally or partially) the fasting-induced decreases in noc-a transcripts in all studied tissues and the increases in noc-b expression in the intestinal bulb. Together, this study offers the first report of NOC in fish and shows a high dependence of its expression on feeding and nutritional status. The differential responses to feeding of the two NOCs raise the possibility that they might be underlying different physiological mechanisms (e.g., food intake, lipid mobilization, energy homeostasis) in fish.

Characterization of Ghrelin O-Acyltransferase (GOAT) in goldfish (Carassius auratus).

Ghrelin is the only known hormone posttranslationally modified with an acylation. This modification is crucial for most of ghrelin's physiological effects and is catalyzed by the polytopic enzyme ghrelin O-acyltransferase (GOAT). The aim of this study was to characterize GOAT in a teleost model, goldfish (Carassius auratus). First, the full-length cDNA sequence was obtained by RT-PCR and rapid amplification of cDNA ends methods. Two highly homologous cDNAs of 1491 and 1413 bp, respectively, named goat-V1 and goat-V2 were identified. Deduced protein sequences (393 and 367 amino acids, respectively) are predicted to present 11 and 9 transmembrane regions, respectively, and both contain two conserved key residues proposed to be involved in catalysis: asparagine 273 and histidine 304. RT-qPCR revealed that both forms of goat mRNAs show a similar widespread tissue distribution, with the highest expression in the gastrointestinal tract and gonads and less but considerable expression in brain, pituitary, liver and adipose tissue. Immunostaining of intestinal sections showed the presence of GOAT immunoreactive cells in the intestinal mucosa, some of which colocalize with ghrelin. Using an in vitro approach, we observed that acylated ghrelin downregulates GOAT gene and protein levels in cultured intestine in a time-dependent manner. Finally, we found a rhythmic oscillation of goat mRNA expression in the hypothalamus, pituitary and intestinal bulb of goldfish fed at midday, but not at midnight. Together, these findings report novel data characterizing GOAT, and offer new information about the ghrelinergic system in fish.

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.