Transcriptomic analysis of turbot (Scophthalmus maximus) treated with zymosan a reveals that lncRNAs and inflammation-related genes mediate the protection conferred against Aeromonas salmonicida.

Aeromonas salmonicida is one of the most harmful pathogens in finfish aquaculture worldwide. Immunostimulants such as ß-glucans are used to enhance the immunity of cultured fish. However, their effects on fish physiology are not completely understood. In the present work, we evaluated the effect of a single intraperitoneal (ip) injection of zymosan A on fish survival against A. salmonicida infection. A single administration of this compound protected fish against A. salmonicida challenge and reduce the bacterial load in the head kidney one week after its administration. Transcriptome analyses of head kidney samples revealed several molecular mechanisms involved in the protection conferred by zymosan A and their regulation by long noncoding RNAs. The transcriptome profile of turbot exposed only to zymosan A was practically unaltered one week after ip injection. However, the administration of this immunostimulant induced significant transcriptomic changes once the fish were in contact with the bacteria and increased the survival of the infected turbot. Our results suggest that the restraint of the infection-induced inflammatory response, the management of apoptotic cell death, cell plasticity and cellular processes involving cytoskeleton dynamics support the protective effects of zymosan A. All this information provides insights on the cellular and molecular mechanisms involved in the protective effects of this widely used immunostimulant.

Conserved function of zebrafish (Danio rerio) Gdf15 as a sepsis tolerance mediator.

GDF15 is frequently detected in patients suffering from various diseases, especially those associated with pro-inflammatory processes and/or metabolic disorders. Accordingly, sepsis, whose major complications are related to metabolic alterations and systemic inflammation, significantly increases the secretion of GDF15. Indeed, this cytokine could be considered a marker of sepsis severity. However, until the last several years, the involvement of GDF15 in these disorders had not been widely characterized. In mice, GDF15 was recently described as a pivotal inducer of sepsis tolerance by mediating metabolic alterations that reduce tissue damage. In this work we describe a zebrafish gdf15 gene. We found that gdf15 follows an expression pattern similar to that observed in mammals, being highly expressed in the liver and kidney and induced after pro-inflammatory stimuli. Moreover, larvae overexpressing gdf15 were more resistant to bacterial and viral challenges without affecting the pathogen load. Consequently, Gdf15 also protected zebrafish larvae against LPS-induced mortality. As in mice, zebrafish Gdf15 seems to induce sepsis tolerance by altering the metabolic parameters of the individuals.

Antiviral activity of palmitic acid via autophagic flux inhibition in zebrafish (Danio rerio).

Fatty acids (FAs) are key elements that affect not only growth but also different immune functions, and therefore, nutrition is important for growing healthy fish. Zebrafish (Danio rerio) is a good model for assessing the beneficial effects of immunostimulants, including FAs, before applying them in aquaculture. Accordingly, this study evaluated the effects of palmitic acid (PA) treatment on different immune parameters of zebrafish and on the mortality caused by the spring viremia of carp virus (SVCV). The results suggest that PA modulates the infection outcome in vivo, which benefits zebrafish and results in reduced mortality and viral titres. The antiviral protection elicited by this FA seems to be associated with the inhibition of autophagy and is independent of other immune processes, such as neutrophil proliferation or type I interferon (IFN) activity. The use of PA as an immunostimulant at low concentrations showed great potential in the prevention of SVCV infections; therefore, this FA could help to prevent the mortality and morbidity caused by viral agents in aquacultured fish. Nevertheless, the potentially detrimental effects of suppressing autophagy in the organism should be taken into account.

ß-glucan administration induces metabolic changes and differential survival rates after bacterial or viral infection in turbot (Scophthalmus maximus).

The innate immune response is able to ward off pathogens and remember previous infections using different mechanisms; this kind of immune reaction has been called "trained immunity". Changes in cellular metabolism (aerobic glycolysis) have been observed during training with some immunostimulants like ß-glucans or during viral and bacterial infections. We hypothesize that ß-glucans can induce metabolic changes used by the host to fight pathogens. Accordingly, we evaluated changes in metabolic parameters in turbot that could affect their survival after a previous intraperitoneal treatment with ß-glucans and subsequent administration of Viral Hemorrhagic Septicemia Virus (VHSV) or bacteria (Aeromonas salmonicida subsp. salmonicida). The results obtained support that ß-glucans, VHSV and A. salmonicida induce changes in lactate, glucose and ATP levels in plasma, head kidney and liver and in the mRNA expression of enzymes related to glucose and fatty acid metabolism in head kidney. Additionally, the metabolic changes induced by ß-glucans are beneficial for VHSV replication, but they are harmful to A. salmonicida, resulting in reduced mortality. ß-glucans appear to have great therapeutic potential and can induce trained immunity against bacterial disease but not against viral disease, which seems to take advantage of ß-glucan metabolic alterations.

Ceramides are involved in the regulation of food intake in rainbow trout (Oncorhynchus mykiss).

We hypothesize that ceramides are involved in the regulation of food intake in fish. Therefore, we assessed in rainbow trout (Oncorhynchus mykiss) the effects of intracerebroventricular treatment with C6:0 ceramide on food intake. In a second experiment, we assessed the effects in brain areas of ceramide treatment on neuropeptide expression, fatty acid-sensing systems, and cellular signaling pathways. Ceramide treatment induced a decrease in food intake, a response opposed to the orexigenic effect described in mammals, which can be related to enhanced mRNA abundance of cocaine and amphetamine-related transcript and proopiomelanocortin and decreased mRNA abundance of Agouti-related protein and neuropeptide Y. Fatty acid-sensing systems appear to be inactivated by ceramide treatment. The mRNA abundance of integrative sensors AMPK and sirtuin 1, and the phosphorylation status of cellular signaling pathways dependent on protein kinase B, AMPK, mammalian target of rapamycin (mTOR), and forkhead box protein O1 (FoxO1) are generally activated by ceramide treatment. However, there are differences between hypothalamus and hindbrain in the phosphorylation status of AMPK (decreased in hypothalamus and increased in hindbrain), mTOR (decreased in hypothalamus and increased in hindbrain), and FoxO1 (increased in hypothalamus and decreased in hindbrain) to ceramide treatment. The results suggest that ceramides are involved in the regulation of food intake in rainbow trout through mechanisms comparable to those characterized previously in mammals in some cases.

Intracerebroventricular ghrelin treatment affects lipid metabolism in liver of rainbow trout (Oncorhynchus mykiss).

We aimed to elucidate in rainbow trout (Oncorhynchus mykiss) the effects of central ghrelin (GHRL) treatment on the regulation of liver lipid metabolism, and the possible modulatory effect of central GHRL treatment on the simultaneous effects of raised levels of oleate. Thus, we injected intracerebroventricularly (ICV) rainbow trout GHRL in the presence or absence of oleate and evaluated in liver variables related to lipid metabolism. Oleate treatment elicited in liver of rainbow trout decreased lipogenesis and increased oxidative capacity in agreement with previous studies. Moreover, as demonstrated for the first time in fish in the present study, GHRL also acts centrally modulating lipid metabolism in liver, resulting in increased potential for lipogenesis and decreased potential for fatty acid oxidation, i.e. the converse effects to those elicited by central oleate treatment. The simultaneous treatment of GHRL and oleate confirmed these counteractive effects. Thus, the nutrient sensing mechanisms present in hypothalamus, particularly those involved in sensing of fatty acid, are involved in the control of liver energy metabolism in fish, and this control is modulated by the central action of GHRL. These results give support to the notion of hypothalamus as an integrative place for the regulation of peripheral energy metabolism in fish.

Glucosensing in liver and Brockmann bodies of rainbow trout through glucokinase-independent mechanisms.

We hypothesize that glucosensor mechanisms other than that mediated by glucokinase (GK) are present in the liver and Brockmann bodies (BB) of rainbow trout, and are affected by stress. We evaluated in these tissues changes in parameters related to putative glucosensor mechanisms based on liver X receptor (LXR), mitochondrial activity, sweet taste receptor, and SGLT-1 6h after intraperitoneal injection of saline solution alone (normoglycaemic treatment) or containing insulin (hypoglycaemic treatment), or d-glucose (hyperglycaemic treatment). Half of tanks were kept at normal stocking density (NSD; 10kgfishmass·m(-3)) whereas the remaining tanks were kept at high stocking density (HSD; 70kgfishmass·m(-3)). The results provide for the first time in fish evidence for the presence of putative glucosensor systems based on mitochondrial activity and sweet taste receptor in liver whereas in BB systems based on LXR, mitochondrial activity, sweet taste receptor, and SGLT-1 could be operative. We also obtained for the first time in fish evidence for the functioning of integrative metabolic sensors in response to changes in nutrient levels since changes in the mRNA abundance of sirtuin 1 (SIRT-1) were observed in response to increased glucose levels. The stress conditions elicited by HSD altered the response of the glucosensor systems based on mitochondrial activity, sweet taste receptor, and SGLT-1 in the liver, and LXR and SGLT-1 in the BB.

Ghrelin modulates hypothalamic fatty acid-sensing and control of food intake in rainbow trout.

There is no information available on fish as far as the possible effects of ghrelin on hypothalamic fatty acid metabolism and the response of fatty acid-sensing systems, which are involved in the control of food intake. Therefore, we assessed in rainbow trout the response of food intake, hypothalamic fatty acid-sensing mechanisms and expression of neuropeptides involved in the control of food intake to the central treatment of ghrelin in the presence or absence of a long-chain fatty acid such as oleate. We observed that the orexigenic actions of ghrelin in rainbow trout are associated with changes in fatty acid metabolism in the hypothalamus and an inhibition of fatty acid-sensing mechanisms, which ultimately lead to changes in the expression of anorexigenic and orexigenic peptides resulting in increased orexigenic potential and food intake. Moreover, the response to increased levels of oleate of hypothalamic fatty acid-sensing systems (activation), expression of neuropeptides (enhanced anorexigenic potential) and food intake (decrease) were counteracted by the simultaneous treatment with ghrelin. These changes provide evidence for the first time in fish of a possible modulatory role of ghrelin on the metabolic regulation by fatty acid of food intake occurring in the hypothalamus.

Daily rhythms in activity and mRNA abundance of enzymes involved in glucose and lipid metabolism in liver of rainbow trout, Oncorhynchus mykiss. Influence of light and food availability.

The present research aimed to investigate in a model of teleost fish (rainbow trout) the existence of daily changes in activity and mRNA abundance of several proteins involved in major pathways of carbohydrate and lipid metabolism in liver, and to test whether or not both the light-dark cycle and food availability might influence such rhythms. For this purpose, four cohorts of animals previously adapted to normal housing conditions (12L:12D; Lights on at ZT0; feeding time at ZT2) were subjected to: normal conditions (LD); 48-h constant darkness (DD); 96-h food deprivation (LD + Fasting); or constant darkness and food deprivation (DD + Fasting) respectively. After such time periods, fish were sacrificed and sampled every 4-h on the following 24-h period (ZT/CT0, 4, 8, 12, 16, 20 and 0'). Our results reveal that cortisol and all the analysed genes (gk, pepck, g6pase, pk, glut2, hoad and fas) exhibited well defined daily rhythms, which persisted even in the absence of light and/or food indicating the endogenous nature of such rhythms. Even when the variations of enzyme activities were not significant, their rhythms mostly paralleled those of the respective gene expression. The rhythms of mRNA abundance were apparently dependent on the presence of food, but the light/dark cycle also influenced such rhythms. Since cortisol does not appear to be mainly involved in generating such daily rhythms in liver, alternative mechanisms might be involved, such as a direct interaction between metabolism and the circadian system.

Response of lactate metabolism in brain glucosensing areas of rainbow trout (Oncorhynchus mykiss) to changes in glucose levels.

There is no evidence in fish brain demonstrating the existence of changes in lactate metabolism in response to alterations in glucose levels. We induced in rainbow trout through intraperitoneal (IP) treatments, hypoglycaemic or hyperglycaemic changes to assess the response of parameters involved in lactate metabolism in glucosensing areas like hypothalamus and hindbrain. To distinguish those effects from those induced by peripheral changes in the levels of metabolites or hormones, we also carried out intracerebroventricular (ICV) treatments with 2-deoxy-D-glucose (2-DG, a non-metabolizable glucose analogue thus inducing local glucopenia) or glucose. Finally, we also incubated hypothalamus and hindbrain in vitro in the presence of increased glucose concentrations. The changes in glucose availability were in general correlated to changes in the amount of lactate in both areas. However, when we assessed in these areas the response of parameters related to lactate metabolism, the results obtained were contradictory. The increase in glucose levels did not produce in general the expected changes in those pathways with only a minor increase in their capacity of lactate production. The decrease in glucose levels was, however, more clearly related to a decreased capacity of the pathways involved in the production and use of lactate, and this was especially evident after ICV treatment with 2-DG in both areas. In conclusion, the present results while addressing the existence of changes in lactate metabolism after inducing changes in glucose levels in brain glucosensing areas only partially support the possible existence of an astrocyte-neuron lactate shuttle in hypothalamus and hindbrain of rainbow trout relating glucose availability to lactate production and use.

Feeding rainbow trout with a lipid-enriched diet: effects on fatty acid sensing, regulation of food intake and cellular signaling pathways.

Using rainbow trout fed with low-fat or high-fat diets, we aimed to determine whether the response of food intake, mRNA abundance of hypothalamic neuropeptides involved in the metabolic regulation of food intake and fatty acid sensing systems in the hypothalamus and liver are similar to results previously observed when levels of specific fatty acids were raised by injection. Moreover, we also aimed to determine if the phosphorylation state of intracellular energy sensor 5'-AMP-activated protein kinase (AMPK), and proteins involved in cellular signaling such as protein kinase B (Akt) and target of rapamycin (mTOR) display changes that could be related to fatty acid sensing and the control of food intake. The increased levels of fatty acids in the hypothalamus and liver of rainbow trout fed with a high-fat diet only partially activated fatty acid sensing systems and did not elicit changes in food intake, suggesting that the fatty acid sensing response in fish is more dependent on the presence of specific fatty acids, such as oleate or octanoate, rather than to the global increase in fatty acids. We also obtained, for the first time in fish, evidence for the presence and function of energy sensors such as AMPK and proteins involved in cellular signaling, like mTOR and Akt, in the hypothalamus. These proteins in the hypothalamus and liver were generally activated in fish fed the high-fat versus low-fat diet, suggesting that cellular signaling pathways are activated in response to the increased availability of fatty acids.

Evidence for the Presence of Glucosensor Mechanisms Not Dependent on Glucokinase in Hypothalamus and Hindbrain of Rainbow Trout (Oncorhynchus mykiss).

We hypothesize that glucosensor mechanisms other than that mediated by glucokinase (GK) operate in hypothalamus and hindbrain of the carnivorous fish species rainbow trout and stress affected them. Therefore, we evaluated in these areas changes in parameters which could be related to putative glucosensor mechanisms based on liver X receptor (LXR), mitochondrial activity, sweet taste receptor, and sodium/glucose co-transporter 1 (SGLT-1) 6 h after intraperitoneal injection of 5 mL x Kg(-1) of saline solution alone (normoglycaemic treatment) or containing insulin (hypoglycaemic treatment, 4 mg bovine insulin x Kg(-1) body mass), or D-glucose (hyperglycaemic treatment, 500 mg x Kg(-1) body mass). Half of tanks were kept at a 10 Kg fish mass x m(-3) and denoted as fish under normal stocking density (NSD) whereas the remaining tanks were kept at a stressful high stocking density (70 kg fish mass x m(-3)) denoted as HSD. The results obtained in non-stressed rainbow trout provide evidence, for the first time in fish, that manipulation of glucose levels induce changes in parameters which could be related to putative glucosensor systems based on LXR, mitochondrial activity and sweet taste receptor in hypothalamus, and a system based on SGLT-1 in hindbrain. Stress altered the response of parameters related to these systems to changes in glycaemia.

Metabolic response in liver and Brockmann bodies of rainbow trout to inhibition of lipolysis; possible involvement of the hypothalamus-pituitary-interrenal (HPI) axis.

We previously demonstrated in rainbow trout that the decrease in circulating levels of fatty acid (FA) induced by treating fish with SDZ WAG 994 (SDZ) induced a counter-regulatory response in which the activation of the hypothalamus-pituitary-interrenal (HPI, equivalent to mammalian hypothalamus-pituitary-adrenal) axis was likely involved. This activation, probably not related to the control of food intake through FA sensor systems but to the modulation of lipolysis in peripheral tissues, liver and Brockmann bodies (BB, the main site of pancreatic endocrine cells in fish), would target the restoration of FA levels in plasma. To assess this hypothesis, we lowered circulating FA levels by treating fish with SDZ alone, or SDZ in the presence of metyrapone (an inhibitor of cortisol synthesis). In liver, the changes observed were not compatible with a direct FA-sensing response but with a stress response, which allows us to suggest that the detection of a FA decrease in the hypothalamus elicits a counter-regulatory response in liver, resulting in an activation of lipolysis to restore FA levels in plasma. The activation of these metabolic changes in liver could be attributable to the activation of the HPI axis and/or to the action of sympathetic pathways. In contrast, in BB, changes in circulating FA levels induce changes in several parameters compatible with the function of FA-sensing systems informing about the decrease in circulating FA levels.

Counter-regulatory response to a fall in circulating fatty acid levels in rainbow trout. Possible involvement of the hypothalamus-pituitary-interrenal axis.

We hypothesize that a decrease in circulating levels of fatty acid (FA) in rainbow trout Oncorhynchus mykiss would result in the inhibition of putative hypothalamic FA sensing systems with concomitant changes in the expression of orexigenic and anorexigenic factors ultimately leading to a stimulation of food intake. To assess this hypothesis, we lowered circulating FA levels treating fish with SDZ WAG 994 (SDZ), a selective A1 adenosine receptor agonist that inhibits lipolysis. In additional groups, we also evaluated if the presence of intralipid was able to counteract changes induced by SDZ treatment, and the possible involvement of the hypothalamus-pituitary-interrenal (HPI) axis by treating fish with SDZ in the presence of metyrapone, which decreases cortisol synthesis in fish. The decrease in circulating levels of FA in rainbow trout induced a clear increase in food intake that was associated with the decrease of the anorexigenic potential in hypothalamus (decreased POMC-A1 and CART mRNA abundance), and with changes in several parameters related to putative FA-sensing mechanisms in hypothalamus. Intralipid treatment counteracted these changes. SDZ treatment also induced increased cortisol levels and the activation of different components of the HPI axis whereas these changes disappeared in the presence of intralipid or metyrapone. These results suggest that the HPI axis is involved in a counter-regulatory response in rainbow trout to restore FA levels in plasma.

Central administration of oleate or octanoate activates hypothalamic fatty acid sensing and inhibits food intake in rainbow trout.

If levels of fatty acids like oleate and octanoate are directly sensed through different fatty acid (FA) sensing systems in hypothalamus of rainbow trout, intracerebroventricular (ICV) administration of FA should elicit effects similar to those previously observed after intraperitoneal (IP) treatment. Accordingly, we observed after ICV treatment with oleate or octanoate decreased food intake accompanied in hypothalamus by reduced potential of lipogenesis and FA oxidation, and decreased potential of ATP-dependent inward rectifier potassium channel (K(+)ATP). Those changes support direct FA sensing through mechanisms related to FA metabolism and mitochondrial activity. The FA sensing through binding to FAT/CD36 and subsequent expression of transcription factors appears to be also direct but an interaction with peripheral hormones cannot be rejected. Moreover, decreased expression of NPY and increased expression of POMC were observed in parallel with the activation of FA sensing systems and decreased food intake. These results allow us to suggest the involvement of at least these peptides in controlling the decreased food intake noted after oleate and octanoate treatment in rainbow trout.

Short-term time course of liver metabolic response to acute handling stress in rainbow trout, Oncorhynchus mykiss.

To elucidate the short-term time-course of liver metabolic response in rainbow trout to acute handling stress we subjected rainbow trout to 5min chasing and obtained samples 0 to 480min post-stress. Levels of cortisol, glucose and lactate were measured in plasma, whereas metabolite levels, enzyme activities, mRNA abundance of parameters related to energy metabolism, and glucocorticoid receptors were assessed in liver. Acute stress affected many parameters related to energy metabolism, with most of them turning back to normal levels after 480min. In general, the present results support the existence of two stages in the short-term time-course of metabolic response to handling stress. A first stage occurring few minutes post-stress (15-45min), was characterized by increased mobilization of liver glycogen resulting in increased production of endogenous glucose, reduced use of exogenous glucose and reduced lipogenic potential. A second stage, occurring 60-120min post-stress onwards was characterized by the recovery of liver glycogen levels, the increased capacity of liver for releasing glucose, and the recovery of lipogenic capacity whereas no changes were noted in gluconeogenic potential, which probably needs longer time periods to become enhanced.

In vitro response of putative fatty acid-sensing systems in rainbow trout liver to increased levels of oleate or octanoate.

In a previous study we provided evidence for the presence in liver of rainbow trout of fatty acid (FA) sensing systems responding to changes in levels of oleate (long-chain FA) or octanoate (medium-chain FA). Since those effects could be attributed to an indirect effect, we have evaluated in the present study in vitro (in the absence of extrahepatic regulatory mechanisms) whether or not liver responds to changes in FA concentration in a way similar to that previously observed in vivo. Accordingly, liver slices were exposed to increased oleate or octanoate concentrations to evaluate changes in parameters related to FA metabolism, FA transport, nuclear receptors and transcription factors, ROS effectors, and glucose metabolism. The responses observed in vitro in liver were in general not coincident with those previously observed in vivo allowing us to suggest that FA sensing capacity of liver in vivo is of indirect nature and could be related among other reasons to an interaction with other endocrine systems and/or to FA sensing in hypothalamus.

Oleic acid and octanoic acid sensing capacity in rainbow trout Oncorhynchus mykiss is direct in hypothalamus and Brockmann bodies.

In a previous study, we provided evidence for the presence in hypothalamus and Brockmann bodies (BB) of rainbow trout Oncorhynchus mykiss of sensing systems responding to changes in levels of oleic acid (long-chain fatty acid, LCFA) or octanoic acid (medium-chain fatty acid, MCFA). Since those effects could be attributed to an indirect effect, in the present study, we evaluated in vitro if hypothalamus and BB respond to changes in FA in a way similar to that observed in vivo. In a first set of experiments, we evaluated in hypothalamus and BB exposed to increased oleic acic or octanoic acid concentrations changes in parameters related to FA metabolism, FA transport, nuclear receptors and transcription factors, reactive oxygen species (ROS) effectors, components of the KATP channel, and (in hypothalamus) neuropeptides related to food intake. In a second set of experiments, we evaluated in hypothalamus the response of those parameters to oleic acid or octanoic acid in the presence of inhibitors of fatty acid sensing components. The responses observed in vitro in hypothalamus are comparable to those previously observed in vivo and specific inhibitors counteracted in many cases the effects of FA. These results support the capacity of rainbow trout hypothalamus to directly sense changes in MCFA or LCFA levels. In BB increased concentrations of oleic acid or octanoic acid induced changes that in general were comparable to those observed in hypothalamus supporting direct FA sensing in this tissue. However, those changes were not coincident with those observed in vivo allowing us to suggest that the FA sensing capacity of BB previously characterized in vivo is influenced by other neuroendocrine systems.

Response of hepatic lipid and glucose metabolism to a mixture or single fatty acids: Possible presence of fatty acid-sensing mechanisms.

To assess the hypothesis that an acute dietary fatty acid (FA) supply may improve glucose tolerance in rainbow trout, we orally administered fish with fish oil (FO; 10mL.kg(-1), one time), which were then subjected to a glucose tolerance test and sampled 6h after injection. Parameters related to glucose and lipid metabolism were then assessed. The results suggest that when both nutrients were administered at the same time, an increased potential for lipogenesis occurred concomitantly with a lower level of glycaemia. In a second experiment we administered intraperitoneally a single FA present in the FO mixture such as oleic acid (60 or 300µg.kg(-1)) whereas octanoic acid (60 or 300µg.kg(-1)) was used as negative control (absent from the FO). However, the effects of both FA were similar in reducing the potential of lipid synthesis and oxidation, and in enhancing the potential of glucose synthesis and glycogenesis. Differences found between FO and single FA administration show that response to FA was dependent on the treatment (mixture vs. single FA) but also comply with the idea that an interaction between FA and glucose rather than FA alone are in the origin of the results reported. The administration of individual FA such as oleic and octanoic acid failed in enhancing lipogenesis and reducing plasma glucose levels and thus in explaining results obtained with FO. However, results provide evidence that FA even provided at a low dose play a key role in the regulation of several putative components of a FA sensing system present in rainbow trout liver.

Melatonin treatment alters glucosensing capacity and mRNA expression levels of peptides related to food intake control in rainbow trout hypothalamus.

As demonstrated in previous studies, the functioning of brain glucosensing systems in rainbow trout is altered under stress conditions in a way that they are unable to respond properly to changes in glucose levels. Melatonin has been postulated as necessary for homeostatic control of energy metabolism in several vertebrate groups, and in fish it has been suggested as an anti-stress molecule. To evaluate the possible effects of melatonin on glucosensing, we have incubated hypothalamus and hindbrains of rainbow trout at different glucose concentrations in the presence of increased doses (0.01, 1, and 100nM) of melatonin assessing whether or not the responses to changes in glucose levels of parameters related to glucosensing (glucose, glycogen and glucose 6-phosphate levels, activities of GK, GSase and PK, and mRNA content of GK, GLUT2, Kir6.x-like, and SUR-like) are modified in the presence of melatonin. While no effects of melatonin were observed in hindbrain, in hypothalamus melatonin treatment up-regulated glucosensing parameters, especially under hypo- and normo-glycaemic conditions. The effects of melatonin in hypothalamus occurred apparently through MT(1) receptors since most effects were counteracted by the presence of luzindole but not by the presence of 4-P-PDOT. Moreover, melatonin treatment induced in hypothalamus increased mRNA expression levels of NPY and decreased mRNA levels of POMC, CART, and CRF. A role of the hormone in daily re-adjustment of hypothalamic glucosensor machinery is discussed.