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

Regulación de la actividad alimentaria por los neuropéptidos CRF, (Beta)endorfina y galanina en peces ciprinidos ("Carassius auratus" y "Tinca tinca")

El conocimiento actual sobre la regulación de la alimentación por neuropeptidos esta restringido fundamentalmente a los mamíferos, planteándonos en esta tesis doctoral estudiar este aspecto en peces ciprinidos, concretamente en carassius auratus y tinca tinca. Inicialmente, realizamos unos estudios preliminares en tinca tinca para determinar su patrón alimentario y modificaciones en parametros metabólicos y hormonales. La administración intracerebroventricular, pero no la intraperitoneal, del crf reduce la ingesta en carassius auratus y tinca tinca; y la de b-endorfina y galanina la estimulan en carassius auratus, sugiriendo que dichos peptidos estan involucrados en la regulacion central de la alimentación en los peces. Además, se han establecido interacciones crf-eje tiroideo y eje tiroideo-estado nutricional en ambas especies de ciprinidos, asi como interacciones entre el sistema peptidergico y catecolaminergico en la regulación de la alimentación en carassius auratu

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

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

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 postinjection, 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

Two cholecystokinin receptor subtypes are identified in goldfish, being the CCKAR involved in the regulation of intestinal motility

Cholecystokinin (CCK) plays a key role in the digestive physiology of vertebrates. However, very little is known about the role of CCK on intestinal functions in fish. The present study identifies two CCK receptor subtypes in a stomachless teleost, the goldfish (Carassius auratus), and investigates by using an in vitro system their involvement mediating the effects of the sulfated octapeptide of CCK (CCK-8S) on the motility of isolated proximal intestine. Partial-length mRNAs encoding two CCK receptor isoforms (CCKAR and CCKBR.I) were sequenced and the structural analysis showed that both receptors belong to the G-protein coupled receptor superfamily. Both gold- fish CCK receptor sequences were more closely related to zebrafish sequences, sharing the lowest similarities with cavefish and tilapia. The highest expression of goldfish CCKAR was observed along the whole intestine whereas the CCKBR gen was predominantly expressed in the hypothalamus, vagal lobe and posterior intestine. Application of CCK-8S to the organ bath evoked a concentration-dependent contractile response in intestine strips. The contractions were not blocked by either tetrodotoxin or atropine, suggesting that CCK-8S acts on the gut smooth muscle directly. Preincubations of intestine strips with devazepide and L365,260 (CCKAR and CCKBR receptor selective antagonists) showed that the CCK-8S-induced contraction could be partially mediated by the CCKAR receptor subtype, which is also the most abundant CCK receptor found in gastrointestinal tissues. In conclusion, two CCK receptors with a differential distribution pattern has been identified in goldfish, and the CCKAR subtype is mainly involved in the regulation of intestinal motility by the CCK-8S

Adaptation to the European Space of Higher Education of the subject Projects and Studies in Biology

The subject projects and Studies in Biology is of a professional and applied nature, as the knowledge acquired can be applied in professional practice, because the students are initiated in the implementation of a Project (Planning, Alternative Solutions, Studies, Pre-projects and Projects. The general objective of this Project involved adapting the contents of the subject to the European Space of Higher Education (EEES) by installing it in the platform WebCT (Web Course Tools), adopted by Madrid's Complutense University for its Virtual Campus. The specific objectives to be met are the following: Conceptual objectives: To introduce students to the typology of Projects. This knowledge will enable people with a degree in Biology to work in Consultancy companies and different Organisms of the Administration and to adjust to the general work market. The students are also introduced to the Spanish System of Science and Technology and to the typology of Research Projects and Projects in collaboration with industries. Procedural objectives: handling procedures for searching for updated information, both offers and calls, and innovation in regulations or in science-technology and knowledge of software programs necessary for drafting a Budget for planning and implementing projects. Attitude-related projects: Considering that people with degrees in Biology traditionally work in Teaching and Research, this subject attempts to change the attitude of students towards what could involve practicing their profession in other areas tan these. In accordance wíth these objectives, the information on the development of the subject has been organized into a series of modules through which the basic knowledge the students should avail of is expounded. Each of these modules has línks to more elaborate documents in which the amount of material is increased and its implications and relationships with other contents of the subject are indicated. The modules are organízed into classes, practica! work aimed at problem- solving, common projects, presentation and debate, and the students must draft a Pre-project for implementation using the different documents it comprises. The subject requíres evaluation of the different conceptual, procedural and attitude-related objectives, and the general adoption of an ongoing evaluation process, in which the following aspects are evaluated: - Participation in classes requiring the presence of students, refresher courses and debates. - Evaluation of the seminars: presentation and defense of projects and of exercises completed. - Directed Academíc Work (creation and defense of a Pre-project with its corresponding documents). - Correct desígn of questionnaires. - Evaluation throughout the course with objective evaluation of the contents, by means of test-types exams