Chronic cortisol stimulation enhances hypothalamus-specific enrichment of metabolites in the rainbow trout brain.

The hypothalamus is a key integrating center that is involved in the initiation of the corticosteroid stress response, and in regulating nutrient homeostasis. Although cortisol, the principal glucocorticoid in humans and teleosts, plays a central role in feeding regulation, the mechanisms are far from clear. We tested the hypothesis that the metabolic changes to cortisol exposure signal an energy excess in the hypothalamus, leading to feeding suppression during stress in fish. Rainbow trout (Oncorhynchus mykiss) were administered a slow-release cortisol implant for 3 days, and the metabolite profiles in the plasma, hypothalamus, and the rest of the brain were assessed. Also, U-13C-glucose was injected into the hypothalamus by intracerebroventricular (ICV) route, and the metabolic fate of this energy substrate was followed in the brain regions by metabolomics. Chronic cortisol treatment reduced feed intake, and this corresponded with a downregulation of the orexigenic gene agrp, and an upregulation of the anorexigenic gene cart in the hypothalamus. The U-13C-glucose-mediated metabolite profiling indicated an enhancement of glycolytic flux and tricarboxylic acid intermediates in the rest of the brain compared with the hypothalamus. There was no effect of cortisol treatment on the phosphorylation status of AMPK or mechanistic target of rapamycin in the brain, whereas several endogenous metabolites, including leucine, citrate, and lactate were enriched in the hypothalamus, suggesting a tissue-specific metabolic shift in response to cortisol stimulation. Altogether, our results suggest that the hypothalamus-specific enrichment of leucine and the metabolic fate of this amino acid, including the generation of lipid intermediates, contribute to cortisol-mediated feeding suppression in fish.NEW & NOTEWORTHY Elevated cortisol levels during stress suppress feed intake in animals. We tested whether the feed suppression is associated with cortisol-mediated alteration in hypothalamus metabolism. The brain metabolome revealed a hypothalamus-specific metabolite profile suggesting nutrient excess. Specifically, we noted the enrichment of leucine and citrate in the hypothalamus, and the upregulation of pathways involved in leucine metabolism and fatty acid synthesis. This cortisol-mediated energy substrate repartitioning may modulate the feeding/satiety centers leading to the feeding suppression.

Endocannabinoid receptors are involved in enhancing food intake in rainbow trout.

The mechanisms involved in hedonic regulation of food intake, including endocannabinoid system (ECs) are scarcely known in fish. We recently demonstrate in rainbow trout the presence of a rewarding response mediated by ECs in hypothalamus and telencephalon when fish fed a lipid-enriched diet, and that central administration of main agonists of ECs namely AEA or 2-AG exert a bimodal effect on feed intake in fish with low doses inducing an increase that disappears with the high dose of both endocannabinoids (EC). To assess the precise involvement of the different receptors of the ECs (CNR1, TRPV1, and GPR55) in this response we injected intracerebroventricularly AEA or 2-AG in the absence/presence of specific receptor antagonists (AM251, capsazepine, and ML193; respectively). The presence of antagonists clearly counteracts the effect of EC supporting the specificity of EC action inducing changes not only in ECs but also in GABA and glutamate metabolism ultimately leading to the increase observed in food intake response.

Periprandial response of central cannabinoid system to different feeding conditions in rainbow trout Oncorhynchus mykiss.

BACKGROUND: The mechanisms that regulate food intake are very complex since they comprise several neuroendocrine and metabolic signals responding to energetic or reward requirements. Previous studies in mammals indicate that cannabinoid system is implicated in homeostatic and hedonic regulation of food intake. In fish, several studies describe the components of this system, but only a little information is available regarding their role in food intake and energy balance regulation. OBJECTIVES: The objective of this study was to evaluate the main components of cannabinoid system related to feeding conditions in fish. METHODS: Samples of blood and different brain areas (telencephalon and hypothalamus) were taken from rainbow trout under different nutritional status (fasted, fed and refed) at different periprandial times (-30, 0, +30 and +180 min). RESULTS: Changes in AEA and 2-AG levels were observed in plasma related to the nutritional status and the sampling times assessed. At central levels, changes in endocannabinoids levels were observed in hypothalamus and in mRNA abundance of cnr1 and tprv1 in telencephalon and faah, gpr55 and fos in both brain areas. DISCUSSION: The results obtained suggest a role of endocannabinoid system in the regulation of food intake in fish at central level but further studies are required to fully elucidate the mechanisms involved.

Central administration of endocannabinoids exerts bimodal effects in food intake of rainbow trout.

The endocannabinoid system (ECs) is known to participate in several processes in mammals related to synaptic signaling including regulation of food intake, appetite and energy balance. In fish, the relationship of ECs with food intake regulation is poorly understood. In the present study, we assessed in rainbow trout Oncorhynchus mykiss the effect of intracerebroventricular administration (ICV) of low and high doses of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) on food intake. We assessed endocannabinoid levels in hypothalamus, telencephalon and plasma as well as the effect of AEA and 2-AG administration at central level on gene expression of receptors involved in ECs (cnr1, gpr55 and trpv1) and markers of neural activity (fos, ntrk2 and GABA-related genes). The results obtained indicate that whereas high doses of endocannabinoids did not elicit changes in food intake levels, low doses of the endocannabinoids produce an orexigenic effect that could be due to a possible inhibition of gabaergic neurotransmission and the modulation of neural plasticity in brain areas related to appetite control, such as hypothalamus and telencephalon.

Role of the G protein-coupled receptors GPR84 and GPR119 in the central regulation of food intake in rainbow trout.

We evaluated the role of the G protein-coupled receptors GPR84 and GPR119 in food intake regulation in fish using rainbow trout (Oncorhynchus mykiss) as a model. In the first experiment, we assessed the effects on food intake of intracerebroventricular treatment with agonists of these receptors. In the second experiment, we assessed the impact of the same treatments on mRNA abundance in the hypothalamus and hindbrain of neuropeptides involved in the metabolic control of food intake (npy, agrp1, pomca1 and cartpt) as well as in changes in parameters related to signalling pathways and transcription factors involved in the integrative response leading to neuropeptide production. Treatment with both agonists elicited an anorectic response in rainbow trout attributable to changes observed in the mRNA abundance of the four neuropeptides. Changes in neuropeptides relate to changes observed in mRNA abundance and phosphorylation status of the transcription factor FOXO1. These changes occurred in parallel with changes in the phosphorylation status of AMPKα and Akt, the mRNA abundance of mTOR as well as signalling pathways related to PLCß and IP3. These results allow us to suggest that (1) at least part of the capacity of fish brain to sense medium-chain fatty acids such as octanoate depends on the function of GPR84, and (2) the capacity of fish brain to sense N-acylethanolamides or triglyceride-derived molecules occurs through the binding of these ligands to GPR119.

The endocannabinoid system is affected by a high-fat-diet in rainbow trout.

The endocannabinoid system (ECs) is a well known contributor to the hedonic regulation of food intake (FI) in mammals whereas in fish, the knowledge regarding hedonic mechanisms that control FI is limited. Previous studies reported the involvement of ECs in FI regulation in fish since anandamide (AEA) treatment induced enhanced FI and changes of mRNA abundance of appetite-related neuropeptides through cannabinoid receptor 1 (cnr1). However, no previous studies in fish evaluated the impact of palatable food like high-fat diets (HFD) on mechanisms involved in hedonic regulation of FI including the possible involvement of ECs. Therefore, we aimed to evaluate the effect of feeding a HFD on the response of ECs in rainbow trout (Oncorhynchus mykiss). First, we demonstrated a higher intake over 4 days of HFD compared with a control diet (CD). Then, we evaluated the postprandial response (1, 3 and 6 h) of components of the ECs in plasma, hypothalamus, and telencephalon after feeding fish with CD and HFD. The results obtained indicate that the increased FI of HFD occurred along with increased levels of 2-arachidonoylglycerol (2-AG) and AEA in plasma and in brain areas like hypothalamus and telencephalon putatively involved in hedonic regulation of FI in fish. Decreased mRNA abundance of EC receptors like cnr1, gpr55 and trpv1 suggest a feed-back counter-regulatory mechanism in response to the increased levels of EC. Furthermore, the results also suggest that neural activity players associated to FI regulation in mammals as cFOS, γ-Amino butyric acid (GABA) and brain derived neurotrophic factor (BDNF)/neurotrophic receptor tyrosine kinase (NTRK) systems could be involved in the hedonic eating response to a palatable diet in fish.

Oral and pre-absorptive sensing of amino acids relates to hypothalamic control of food intake in rainbow trout.

To assess the putative role of taste and pre-absorptive sensing of amino acids in food intake control in fish, we carried out an oral administration with l-leucine, l-valine, l-proline or l-glutamic acid in rainbow trout (Oncorhynchus mykiss). Treatment with proline significantly reduced voluntary food intake at 2 h and 3 h after oral administration, while glutamic acid showed a less pronounced satiating effect at 3 h. The mRNA expression of taste receptor subunits tas1r1, tas1r2a, tas1r2b and tas1r3 was measured in the epithelium overlying the bony basihyal of the fish (analogous to the tetrapod tongue) at 10, 20 or 30 min following treatment. No significant changes were observed, except for a tas1r down-regulation by valine at 30 min. Of the downstream taste signalling genes that were analysed in parallel, plcb2 and possibly trpm5 (non-significant trend) were down-regulated 20 min after proline and glutamic acid treatment. The signal originated in the oropharyngeal and/or gastric cavity presumably relays to the brain as changes in genes involved in the regulation of food intake occurred in hypothalamus 10-30 min after oral treatment with amino acids. In particular, proline induced changes consistent with an increased anorexigenic potential in the hypothalamus. We have therefore demonstrated, for the first time in fish, that the peripheral (pre-absorptive) detection of an amino acid (l-proline), presumably by taste-related mechanisms, elicits a satiety signal that in hypothalamus is translated into changes in cellular signalling and neuropeptides regulating food intake, ultimately resulting in decreased food intake.

In vitro insulin treatment reverses changes elicited by nutrients in cellular metabolic processes that regulate food intake in fish.

This research assessed the direct effects of insulin on nutrient-sensing mechanisms in the brain of rainbow trout (Oncorhynchus mykiss) using an in vitro approach. Cultured hypothalamus and hindbrain were exposed to 1 µmol l-1 insulin for 3 h, and signals involved in appetite regulation and nutrient-sensing mechanisms were measured. Additionally, the involvement of the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in the actions of insulin was studied by using the inhibitor wortmannin. Treatment with insulin alone did not elicit many changes in the appetite regulators and nutrient-sensing-related genes and enzymes tested in the hypothalamus and hindbrain. However, we found that, when insulin and nutrients were added together, insulin reversed most of the effects exerted by nutrients alone, suggesting that insulin changes responsiveness to nutrients at the central level. Effects reversed by insulin included expression levels of genes related to the sensing of both glucose (slc2a2, slc5a1, gck, pck1, pklr, g6pcb, gys1, tas1r3 and nr1h3 in the hindbrain, and slc2a2, pklr and pck1 in the hypothalamus) and fatty acid (cd36 in the hindbrain, and cd36 and acly in the hypothalamus). Nutrient-induced changes in the activity of Acly and Cpt-1 in the hindbrain and of Pepck, Acly, Fas and Hoad in the hypothalamus were also reversed by insulin. Most of the insulin effects disappeared in the presence of wortmannin, suggesting the PI3K/Akt pathway is a mediator of the effects of insulin reported here. This study adds new information to our knowledge of the mechanisms regulating nutrient sensing in fish.

Hypothalamic AMPKα2 regulates liver energy metabolism in rainbow trout through vagal innervation.

Hypothalamic AMPK plays a major role in the regulation of whole body metabolism and energy balance. Present evidence has demonstrated that this canonical mechanism is evolutionarily conserved. Thus, recent data demonstrated that inhibition of AMPKα2 in fish hypothalamus led to decreased food intake and liver capacity to use and synthesize glucose, lipids, and amino acids. We hypothesize that a signal of abundance of nutrients from the hypothalamus controls hepatic metabolism. The vagus nerve is the most important link between the brain and the liver. We therefore examined in the present study whether surgical transection of the vagus nerve in rainbow trout is sufficient to alter the effect in liver of central inhibition of AMPKα2. Thus, we vagotomized (VGX) or not (Sham) rainbow trout and then intracerebroventricularly administered adenoviral vectors tagged with green fluorescent protein alone or linked to a dominant negative isoform of AMPKα2. The inhibition of AMPKα2 led to reduced food intake in parallel with changes in the mRNA abundance of hypothalamic neuropeptides [neuropeptide Y (npy), agouti-related protein 1 (agrp1), and cocaine- and amphetamine-related transcript (cartpt)] involved in food intake regulation. Central inhibition of AMPKα2 resulted in the liver having decreased capacity to use and synthesize glucose, lipids, and amino acids. Notably, these effects mostly disappeared in VGX fish. These results support the idea that autonomic nervous system actions mediate the actions of hypothalamic AMPKα2 on liver metabolism. Importantly, this evidence indicates that the well-established role of hypothalamic AMPK in energy balance is a canonical evolutionarily preserved mechanism that is also present in the fish lineage.

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.

Na+/K+-ATPase is involved in the regulation of food intake in rainbow trout but apparently not through brain glucosensing mechanisms.

To assess the hypothesis that Na+/K+-ATPase (NKA) is involved in the central regulation of food intake in fish, we observed in a first experiment with rainbow trout (Oncorhynchus mykiss) that intracerebroventricular (ICV) treatment with ouabain decreased food intake. We hypothesized that this effect relates to modulation of glucosensing mechanisms in brain areas (hypothalamus, hindbrain, and telencephalon) involved in food intake control. Therefore, we evaluated in a second experiment, the effect of ICV administration of ouabain, in the absence or in the presence of glucose, on NKA activity, mRNA abundance of different NKA subunits, parameters related to glucosensing, transcription factors, and appetite-related neuropeptides in brain areas involved in the control of food intake. NKA activity and mRNA abundance of nkaα1a and nkaα1c in brain were inhibited by ouabain treatment and partially by glucose. The anorectic effect of ouabain is opposed to the orexigenic effect reported in mammals. The difference might relate to the activity of glucosensing as well as downstream mechanisms involved in food intake regulation. Ouabain inhibited glucosensing mechanisms, which were activated by glucose in hypothalamus and telencephalon. Transcription factors and neuropeptides displayed responses comparable to those elicited by glucose when ouabain was administered alone, but not when glucose and ouabain were administered simultaneously. Ouabain might therefore affect other processes, besides glucosensing mechanisms, generating changes in membrane potential and/or intracellular pathways finally modulating transcription factors and neuropeptide mRNA abundance leading to modified food intake.

SIRT1 mediates the effect of stress on hypothalamic clock genes and food intake regulators in rainbow trout, Oncorhynchus mykiss.

Stress negatively affects a wide range of physiological and behavioural functions (circadian physiology and food intake, among others), thus compromising animal welfare. Cortisol mediates the effect of stress on food intake, but other mediators (such as sirtuins) may participate in that related to circadian physiology. We evaluated 1) the effect of stress on the day-night variation of hypothalamic clock genes and food intake regulators, 2) changes of mRNA abundance in cortisol biosynthesis at the head kidney, and 3) changes of glucocorticoid receptors in both tissues of rainbow trout, together with the involvement of SIRT1 in such effect. Trout receiving or not SIRT1 inhibitor (EX527) and subjected or not to stress by high stocking density (72 h), were sampled at day- (ZT10) and night-time (ZT18). Our results indicate that SIRT1 mediates the effect of stress on mRNA abundance of clock genes in trout hypothalamus, but it also influences those changes occurring on food intake-related peptides. High stocking density inhibits clock genes expression, but enhances that of food intake-related peptides. EX527 treatment prevents stress-related changes observed in clock genes, thus evidencing a key role played by SIRT1 in mediating this effect on trout circadian oscillators. On the other hand, EX527 treatment partially prevents changes of food intake-related peptides, indicating that an interaction between SIRT1 and other mediators (such as cortisol) exists during response to stress. In support of that, our results reveal that SIRT1 influences cortisol biosynthesis during stress. Whatever the case is, further research will help understanding the underlying mechanisms involved.

Effects of CCK-8 and GLP-1 on fatty acid sensing and food intake regulation in trout.

We hypothesize that cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) are involved in the modulation of metabolic regulation of food intake by fatty acids in fish. Therefore, we assessed in rainbow trout (Oncorhynchus mykiss) the effects of intracerebroventricular treatment with 1 ng/g of CCK-8 and with 2 ng/g of GLP-1 on food intake, expression of neuropeptides involved in food intake control and the activity of fatty acid-sensing systems in hypothalamus and hindbrain. Food intake decreased up to 24 h post-treatment to 49.8-72.3% and 3.1-17.8% for CCK-8 and GLP-1, respectively. These anorectic responses are associated with changes in fatty acid metabolism and an activation of fatty acid-sensing mechanisms in the hypothalamus and hindbrain. These changes occurred in parallel with those in the expression of anorexigenic and orexigenic peptides. Moreover, we observed that the activation of fatty acid sensing and the enhanced anorectic potential elicited by CCK-8 and GLP-1 treatments occurred in parallel with the activation of mTOR and FoxO1 and the inhibition of AMPKα, BSX and CREB. The results are discussed in the context of metabolic regulation of food intake in fish.

Differential Role of Hypothalamic AMPKα Isoforms in Fish: an Evolutive Perspective.

In mammals, hypothalamic AMP-activated protein kinase (AMPK) α1 and α2 isoforms mainly relate to regulation of thermogenesis/liver metabolism and food intake, respectively. Since both isoforms are present in fish, which do not thermoregulate, we assessed their role(s) in hypothalamus regarding control of food intake and energy homeostasis. Since many fish species are carnivorous and mostly mammals are omnivorous, assessing if the role of hypothalamic AMPK is different is also an open question. Using the rainbow trout as a fish model, we first observed that food deprivation for 5 days did not significantly increase phosphorylation status of AMPKα in hypothalamus. Then, we administered adenoviral vectors that express dominant negative (DN) AMPKα1 or AMPKα2 isoforms. The inhibition of AMPKα2 (but not AMPKα1) led to decreased food intake. The central inhibition of AMPKα2 resulted in liver with decreased capacity of use and synthesis of glucose, lipids, and amino acids suggesting that a signal of nutrient abundance flows from hypothalamus to the liver, thus suggesting a role for central AMPKα2 in the regulation of peripheral metabolism in fishes. The central inhibition of AMPKα1 induced comparable changes in liver metabolism though at a lower extent. From an evolutionary point of view, it is of interest that the function of central AMPKα2 remained similar throughout the vertebrate lineage. In contrast, the function of central AMPKα1 in fish relates to modulation of liver metabolism whereas in mammals modulates not only liver metabolism but also brown adipose tissue and thermogenesis.

Response of rainbow trout's (Oncorhynchus mykiss) hypothalamus to glucose and oleate assessed through transcription factors BSX, ChREBP, CREB, and FoxO1.

We aimed to obtain information regarding mechanisms that link glucose- and fatty acid-sensing systems to expression of neuropeptides that regulate food intake in the fish brain. We assessed the relative expression and protein levels of the transcription factors BSX, ChREBP, FoxO1, and CREB in the hypothalamus of rainbow trout (Oncorhynchus mykiss) treated for 6 h with either glucose or oleate in vivo (intra-cerebroventricular treatment with 1 µl 100 g- 1 body weight of 40 µg glucose or 1 µmol oleate) or in vitro (incubation with 4-8 mM glucose or 100-500 µM oleate). BSX levels decreased after oleate treatment for mRNA (10% in vitro and 47% in vivo) and protein (25%), while minor changes occurred after glucose treatment. CREB values generally decreased after glucose or oleate treatment for mRNA (50% in vivo) as well as the phosphorylation status of protein (80%). Foxo1 mRNA levels increased in vivo with glucose (129%) and decreased in vivo with oleate (60%), and protein phosphorylation status increased with glucose (in vivo) and oleate. mRNA values of chrebpα decreased in response to glucose and oleate, while protein levels decreased with oleate and increased with glucose. The results support the association of several transcription factors with metabolic control of food intake in fish.

Involvement of cortisol and sirtuin1 during the response to stress of hypothalamic circadian system and food intake-related peptides in rainbow trout, Oncorhynchus mykiss.

Stress is conditioning animal welfare by negatively affecting a wide range of physiological and behavioral functions. This may be applied to circadian physiology and food intake. Cortisol, the stress-related hormone, may mediate such effect of stress, but other indirect mediators might be considered, such as sirtuin1. Then, either the independent modulatory effect or the existence of any interaction between mediators may be responsible. The circadian system is the main modulator of several integrative mechanisms at both central and peripheral levels that are rhythmically presented, thus influencing different processes such as food intake. In this way, food intake is controlled by the circadian system, as demonstrated by the persistence of such rhythms of food intake in the absence of environmental external cues. Our study aimed to evaluate the daily profile of hypothalamic mRNA abundance of circadian clock genes (clock1a, bmal1, per1 and rev-erbß-like), and food intake regulators (crf, pomc-a1, cart, and npy) in rainbow trout (Oncorhynchus mykiss), the impact of stress on such rhythms, and the involvement of cortisol and sirtuin1 as mediators. Four cohorts of trout were subjected to 1) normal stocking density (control group), 2) high stocking density for 72 hours (stress group), 3) normal stocking density and implanted with mifepristone, a glucocorticoid receptors antagonist, and 4) mifepristone administered and stressed for 72 hours. Fish from each group were sampled every 4-h along the 24-h LD cycle, and cortisol, glucose and lactate plasma levels were evaluated. Hypothalamic mRNA abundance of clock genes, food intake regulators, glucocorticoid receptors and sirtuin1 were qPCR assayed. Our results reveal the impact of stress on most of the genes assayed, but different mechanisms appear to be involved. The rhythm of clock genes displayed decreased amplitude and averaged levels in stressed trout, with no changes of the acrophase being observed. This effect was not prevented by mifepristone. On the contrary, the effect of stress on the daily profile of crf, pomc-a1, and npy was totally prevented by mifepristone administration. Accordingly, cortisol appears to mainly mediate the effect of stress on food intake regulators through binding to specific glucocorticoid receptors within trout hypothalamus, whereas sirtuin1 is apparently mediating such effects on the circadian system in the same brain region. Further research must be performed to clarify those mechanisms through which stress influences food intake and the circadian oscillator within the same brain region, hypothalamus, in rainbow trout, and the interaction among them all.

Influence of vegetable diets on physiological and immune responses to thermal stress in Senegalese sole (Solea senegalensis).

The substitution of fish resources as ingredients for aquafeeds by those based on vegetable sources is needed to ensure aquaculture sustainability in the future. It is known that Senegalese sole (Solea senegalensis) accepts high dietary content of plant ingredients without altering growth or flesh quality parameters. However, scarce information is available regarding the long-term impact of vegetable diets (combining the inclusion of both vegetable protein and oils) on the stress response and immunity of this fish species. This study aims to evaluate the concomitant effect of the extended use of vegetable protein-based diets with fish oil (FO) replacement (0, 50 or 100%) by vegetable oils (VO), on the response to acute (10 min) or prolonged (4 days) stress, induced by thermal shock. Plasma levels of cortisol, glucose and lactate as well as hepatic levels of glucose, glycogen and lactate were evaluated as primary and secondary responses to stress, 6 and 18 months after feeding the experimental diets (6 and 18 MAF). The brain monoaminergic activity in telencephalon and hypothalamus, and non-specific immune parameters were also evaluated. As expected, thermal shock induced an increase in values of plasma parameters related to stress, which was more evident in acute than in prolonged stress. Stress also affected lactate levels in the liver and the values of the alternative complement pathway-ACH50 in the plasma. Dietary substitution of FO induced an effect per se on some parameters such as decreased hepatic glucose and glycogen levels and peroxidase activity in plasma as well enhanced serotonergic activity in brain of non-stressed fish. The results obtained in some parameters indicate that there is an interaction between the use of vegetable diets with the physiological response to thermal stress, as is the case of the hepatic lactate, serotonergic neurotransmission in brain, and the activity of ACH50 in plasma. These results suggest that the inclusion of VO in plant protein based diets point to a slightly inhibited stress response, more evident for an acute than a prolonged stress.

Changes in the levels and phosphorylation status of Akt, AMPK, CREB and FoxO1 in hypothalamus of rainbow trout under conditions of enhanced glucosensing activity.

There is no available information about mechanisms linking glucosensing activation in fish and changes in the expression of brain neuropeptides controlling food intake. Therefore, we assessed in rainbow trout hypothalamus the effects of raised levels of glucose on the levels and phosphorylation status of two transcription factors, FoxO1 and CREB, possibly involved in linking these processes. We also aimed to assess the changes in the levels and phosphorylation status of two proteins possibly involved in the modulation of these transcription factors: Akt and AMPK. Therefore, in pooled preparations of hypothalamus incubated for 3 and 6 h in the presence of 2, 4 or 8 mmol l-1 d-glucose, we evaluated the response of parameters related to glucosensing mechanisms, neuropeptide expression and levels and phosphorylation status of the proteins of interest. The activation of hypothalamic glucosensing systems and the concomitant enhanced anorectic potential occurred in parallel with activation of Akt and inhibition of AMPK. The changes in these proteins relate to neuropeptide expression through changes in the level and phosphorylation status of transcription factors under their control, such as CREB and FoxO1, which displayed inhibitory (CREB) or activatory (FoxO1) responses to increased glucose.

Hypothalamic mechanisms linking fatty acid sensing and food intake regulation in rainbow trout.

We assessed in rainbow trout hypothalamus the effects of oleate and octanoate on levels and phosphorylation status of two transcription factors, FoxO1 and CREB, possibly involved in linking activation of fatty acid sensing with modulation of food intake through the expression of brain neuropeptides. Moreover, we assessed changes in the phosphorylation status of three proteins possibly involved in modulation of these transcription factors such as Akt, AMPK and mTOR. In a first experiment, we evaluated, in pools of hypothalamus incubated for 3 h and 6 h at 15°C in a modified Hanks' medium containing 100 or 500 µM oleate or octanoate, the response of fatty acid sensing, neuropeptide expression and phosphorylation status of proteins of interest. The activation of fatty acid sensing and enhanced anorectic potential occurred in parallel with the activation of Akt and mTOR, and the inhibition of AMPK. The changes in these proteins would relate to a neuropeptide expression through changes in the phosphorylation status of transcription factors under their control, such as CREB and FoxO1, which displayed inhibitory (CREB) or activatory (FoxO1) responses when tissues were incubated with oleate or octanoate. In a second experiment, we incubated hypothalamus for 6 h with 500 µM oleate or octanoate alone or in the presence of specific inhibitors of Akt, AMPK, mTOR, CREB or FoxO1. The presence of inhibitors counteracted the effects of oleate or octanoate on the phosphorylation status of the proteins of interest. The results support, for the first time in fish, the involvement of these proteins in the regulation of food intake by fatty acids.

Ceramide counteracts the effects of ghrelin on the metabolic control of food intake in rainbow trout.

In mammals, ceramides are involved in the modulation of the orexigenic effects of ghrelin (GHRL). We previously demonstrated in rainbow trout that intracerebroventricular (ICV) treatment with ceramide (2.5 µg/100 g fish) resulted in an anorexigenic response, i.e. a response opposed to that described in mammals, where ceramide treatment is orexigenic. Therefore, we hypothesized that the putative interaction between GHRL and ceramide must be different in fish. Accordingly, in a first experiment, we observed that ceramide levels in the hypothalamus of rainbow trout did not change after ICV treatment with GHRL. In a second experiment, we assessed whether the effects of GHRL treatment on the regulation of food intake in rainbow trout changed in the presence of ceramide. Thus, we injected ICV GHRL and ceramide alone or in combination to evaluate in hypothalamus and hindbrain changes in parameters related to the metabolic control of food intake. The presence of ceramide generally counteracted the effects elicited by GHRL on fatty acid-sensing systems, the capacity of integrative sensors (AMPK, mTOR and SIRT-1), proteins involved in cellular signalling pathways (Akt and FoxO1) and neuropeptides involved in the regulation of food intake (AgRP, NPY, POMC and CART). The results are discussed in the context of regulation of food intake by metabolic and endocrine inputs.