Clock genes are known to be the molecular core of biological clocks of vertebrates. They are expressed not only in those tissues considered central pacemakers, but also in peripheral tissues. In the present study, partial cDNAs for six of the principal clock genes (Period 1-3 and Cryptochrome 1-3) were cloned from a teleost fish, the goldfish (Carassius auratus). These genes showed high homology (approximately 90%) with the respective cDNAs of zebrafish (Danio rerio), the only other teleost from which clock genes have been cloned. The daily expression pattern of each gene in retina, gut and liver of goldfish was investigated using quantitative RT-PCR and cosinor analysis. All clock genes analyzed in the retina showed circadian rhythmicity; however, only Per 2-3 and Cry 2-3 were rhythmic in goldfish liver and gut. The amplitude and phase of the expression in liver and gut were different from those found in goldfish retina. Such differences suggest that other cues, such as feeding time, may contribute to the entrainment of oscillators in goldfish liver and gut. Our results support the use of goldfish as a teleost model to investigate the location and functioning of the circadian oscillators.

The environment which living organisms inhabit is not constant and many factors, such as light, temperature, and food availability, display cyclic and predictable variations. To adapt to these cyclic changes, animals present biological rhythms in many of their physiological variables, timing their functions to occur when the possibility of success is greatest. Among these variables, many endocrine factors have been described as displaying rhythms in vertebrates. The aim of the present review is to provide a thorough review of the existing knowledge on the rhythms of the endocrine system of fish by examining the hormones that show rhythmicity, how environmental factors control these rhythms and the variation in the responses of the endocrine system depending on the time of the day. We mainly focused on the hypothalamic-pituitary axis, which can be considered as the master axis of the endocrine system of vertebrates and regulates a great variety of functions, including reproduction, growth, metabolism, energy homeostasis, stress response, and osmoregulation. In addition, the rhythms of other hormones, such as melatonin and the factors, produced in the gastrointestinal system of fish are reviewed.

The effects of short-time fasting on appetite, growth, and nutrient were studied in Atlantic salmon (Salmo salar) smolts. Feed deprivation did change the energy metabolism with reduced plasma protein and muscle indispensible amino acid levels. Plasma levels of ghrelin were significantly higher in starved salmon compared with fed fish after 2 days, but no differences in circulating ghrelin were found between treatments after 14 days. Two mRNA sequences for ghrelin-1 and ghrelin-2, 430 and 533 bp long, respectively, were detected. In addition, the growth hormone secretagogues-receptor like receptor (GHSR-LR) 1a and 1b were identified. Ghrelin-1 but not ghrelin-2 mRNA levels were affected by starvation in the stomach. Lower ghrelin-1 mRNA levels were detected at day 2 in starved fish compared with fed fish. The mRNA levels of GHSR-LR1a were not affected by starvation. Fasting reduced the phenotypic growth and the transcription of insulin-like growth factor (IGF)-II together with IGF-IIR, but IGF-I mRNA were not regulated in fasted salmon after 14 days. Three IGF-binding proteins (IGFBP) at 23, 32, and 43 kDa were found in salmon, and circulating 23 kDa was significantly increased after 14 days of starvation compared with fed fish, indicating increased catabolism. The levels of IGFBP-1 mRNA were significantly higher in fed and starved fish after 14 days compared to those at the start of the experiment, but no significant difference was observed between the treatments. In conclusion, we have shown that circulating ghrelin and ghrelin-1 mRNA is related to changes in energy metabolism in Atlantic salmon.

Light at night is essential to a 24/7 society, but it has negative consequences on health. Basically, light at night induces an alteration of our biological clocks, known as chronodisruption, with effects even when this occurs during pregnancy. Indeed, an abnormal photoperiod during gestation alters fetal development, inducing long-term effects on the offspring. Accordingly, we carried out a longitudinal study in rats, exploring the impact of gestational chronodisruption on the adrenal biorhythms and function of the offspring. Adult rats (90 days old) gestated under chronic photoperiod shift (CPS) decrease the time spent in the open arm zone of an elevated plus maze to 62% and increase the rearing time to 170%. CPS adults maintained individual daily changes in corticosterone, but their acrophases were distributed from 12.00 h to 06.00 h. CPS offspring maintained clock gene expression and oscillation, nevertheless no daily rhythm was observed in genes involved in the regulation and synthesis of steroids. Consistent with adult adrenal gland being programmed during fetal life, blunted daily rhythms of corticosterone, core clock gene machinery, and steroidogenic genes were observed in CPS fetal adrenal glands. Comparisons of the global transcriptome of CPS versus control fetal adrenal gland revealed that 1078 genes were differentially expressed (641 down-regulated and 437 up-regulated). In silico analysis revealed significant changes in Lipid Metabolism, Small Molecule Biochemistry, Cellular Development and the Inflammatory Response pathway (z score: 48-20). Altogether, the present results demonstrate that gestational chronodisruption changed fetal and adult adrenal function. This could translate to long-term abnormal stress responses and metabolic adaptation, increasing the risk of developing chronic diseases.

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, gPer1a, gPer2a, gPer3, and gCry3 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.