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.

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.

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.

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.

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.