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.

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The periprandial profile and effects of short- (7 days) and long-term (30 days) fasting on the ghrelinergic system were studied in goldfish (Carassius auratus). Plasma levels of acyl-ghrelin, desacyl-ghrelin, and ghrelin O-acyl transferase (GOAT) were analyzed by enzymoimmunoassays, and expression of preproghrelin, goat and growth hormone secretagogue receptors (ghs-r) was quantified by real-time PCR. Circulating levels of acyl-ghrelin and GOAT rise preprandially, supporting the role of acyl-ghrelin as a meal initiator in this teleost. Consistently, preproghrelin and ghs-r1a1 expression increases 1 h before feeding time in intestinal bulb, suggesting that this receptor subtype might be involved in the preprandial action of ghrelin in this tissue. Significant postfeeding variations are detected for preproghrelin in telencephalon, goat in telencephalon and hypothalamus, ghs-r1a1 in vagal lobe, ghs-r1a2 and ghs-r2a1 in hypothalamus and ghs-r2a2 in telencephalon and vagal lobe, especially in unfed fish. Short- and long-term fasting significantly increase preproghrelin expression in telencephalon and gut. Goat expression is upregulated by short-term fasting in telencephalon and hypothalamus, and by both short- and long-term fasting in gut. Expression of ghs-r increases by fasting in telencephalon, while an upregulation of type 2, but not type 1, receptors is observed in vagal lobe. In intestinal bulb, ghs-r1a2 transcripts increase after both short- and long-term fasting. These results show a high dependence of the ghrelinergic system on feeding and nutritional status in fish, and demonstrate for the first time a differential implication of the various components of this system suggesting different roles for the four ghrelinergic receptor subtypes.

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.

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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 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.

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.

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