Sex dimorphism of glucosensing parameters and appetite-regulating peptides in the hypothalamus of rainbow trout broodstocks.

Rainbow trout (Oncorhynchus mykiss) is traditionally considered as a poor user of digestible carbohydrates harbouring persistent postprandial hyperglycaemia and decreased growth performances when fed a diet containing more than 20% of digestible carbohydrates. While this glucose-intolerant phenotype is well-described in juveniles, evidence points to a particular regulation of glucose metabolism in rainbow trout broodstrocks. By detecting changes in glucose levels and triggering a specific metabolic response, the hypothalamus plays a key role in the regulation of peripheral glucose metabolism. Therefore, our objective was to assess, for the first time in fish, the short-term consequences in hypothalamus, the glucose sensing and feed intake regulation of feeding mature female and male, and neomale rainbow trout with a diet containing either no or a 33% carbohydrate. The hypothalamic glucosensing capacity was assessed through mRNA levels of glucosensing related-genes and feed intake regulation through appetite-regulating peptides. Our data indicate that a brief period of carbohydrate intake (5 meals at 8 °C) did not induce specific changes in glucosensing capacity and appetite-regulating peptides in the hypothalamus of rainbow trout broodstock. Our results did however demonstrate, for the first time in fish, the existence of sex dimorphism of glucosensing-related genes and appetite-regulating peptides.

Total fishmeal replacement by defatted Tenebrio molitor larvae meal induces alterations in intermediary metabolism of European sea bass (Dicentrarchus labrax).

The replacement of fishmeal (FM) by insect meal (IM) in aquafeed formulation has been thoroughly studied lately, but little is known about their impact on nutrient metabolism of fish. This study evaluated the impact not only of partial but also total FM replacement by IM on intermediary metabolism of European sea bass (Dicentrarchus labrax). A fishmeal-based diet was used as a control (CTRL) and two other diets were formulated to include 20% and 40% of defatted Tenebrio molitor larvae meal (dTM), replacing 50% (TM50) and 100% (TM100) of fishmeal (FM), respectively. After a 16-week feeding trial, a multidisciplinary approach including assessment of histological, biochemical, molecular, and enzymatic parameters was adopted to investigate hepatic and plasmatic responses to the different dietary formulations. The results obtained demonstrated that dTM can be successfully used to replace 50% of FM in diets for European sea bass, without adversely affecting liver health or intermediary metabolism of nutrients. As for TM100, although no signs of steatosis were observed in the liver, the activity of glycolytic and lipogenic genes and enzymes increased when compared to CTRL diet (P < 0.05), resulting in higher levels of plasmatic non-esterified fatty acids and triacylglycerides (P < 0.05), which in the long-term may compromise fish health, thus precluding such a high degree of substitution for use in practical diets for European sea bass.

Insect meal has been increasingly considered as novel protein source in diets for different animal models, including fish. European sea bass is one of the most important fish species in Europe, particularly in the Mediterranean. Fishmeal is considered a premium protein source in diets for aquaculture fish. Thus, this study evaluated the impact of partial and total fishmeal replacement by an insect meal (defatted Tenebrio molitor larvae meal) on intermediary metabolism of European sea bass. After 16 weeks of feeding, the results obtained demonstrated that selected insect meal can be successfully used to replace 50% of fish in diets for European sea bass, without adversely affecting liver health or intermediary metabolism of nutrients. On the other hand, total fishmeal replacement by insect meal may compromise fish health in the long-term.

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.

REV-ERBα Agonist SR9009 Promotes a Negative Energy Balance in Goldfish.

REV-ERBα (nr1d1, nuclear receptor subfamily 1 group D member 1) is a transcriptional repressor that in mammals regulates nutrient metabolism, and has effects on energy homeostasis, although its role in teleosts is poorly understood. To determine REV-ERBα's involvement in fish energy balance and metabolism, we studied the effects of acute and 7-day administration of its agonist SR9009 on food intake, weight and length gain, locomotor activity, feeding regulators, plasma and hepatic metabolites, and liver enzymatic activity. SR9009 inhibited feeding, lowering body weight and length gain. In addition, the abundance of ghrelin mRNA decreased in the intestine, and abundance of leptin-aI mRNA increased in the liver. Hypocretin, neuropeptide y (npy), and proopiomelanocortin (pomc) mRNA abundance was not modified after acute or subchronic SR9009 administration, while hypothalamic cocaine- and amphetamine-regulated transcript (cartpt-I) was induced in the subchronic treatment, being a possible mediator of the anorectic effects. Moreover, SR9009 decreased plasma glucose, coinciding with increased glycolysis and a decreased gluconeogenesis in the liver. Decreased triglyceride levels and activity of lipogenic enzymes suggest a lipogenesis reduction by SR9009. Energy expenditure by locomotor activity was not significantly affected by SR9009. Overall, this study shows for the first time in fish the effects of REV-ERBα activation via SR9009, promoting a negative energy balance by reducing energetic inputs and regulating lipid and glucose metabolism.

The Opioid System in Rainbow Trout Telencephalon Is Probably Involved in the Hedonic Regulation of Food Intake.

We hypothesize that opioids are involved in the regulation of food intake in fish through homeostatic and hedonic mechanisms. Therefore, we evaluated in rainbow trout (Oncorhynchus mykiss) hypothalamus and telencephalon changes in precursors, endogenous ligands and receptors of the opioid system under different situations aimed to induce changes in the homeostatic (through fasted/fed/refed fish) and hedonic (through feeding fish a control or a palatable high-fat diet) regulation of food intake. No major changes occurred in parameters assessed related with the nutritional condition of fish (fasted/fed/refed), allowing us to suggest that the opioid system seems not to have an important role in the homeostatic regulation of food intake in rainbow trout. The responses observed in telencephalon of rainbow trout fed the palatable high-fat diet included a decrease in mRNA abundance of the opioid precursor penka, in a way similar to that known in mammals, and increased mRNA abundance of the opioid receptors oprd1 and oprk1 supporting a role for telencephalic opioid system in the hedonic regulation of food intake in fish.

Involvement of Mechanistic Target of Rapamycin (mTOR) in Valine Orexigenic Effects in Rainbow Trout.

This study was aimed at clarifying the importance of a mechanistic target of rapamycin (mTOR) in the central orexigenic effect of valine in fish. For this, rainbow trout (Oncorhynchus mykiss) were intracerebroventricularly (ICV) injected with valine alone or in the presence of rapamycin as the mTOR inhibitor, and two experiments were performed. In the first experiment, we evaluated feed intake levels. In the second experiment, we evaluated in the hypothalamus and telencephalon the following: (1) the phosphorylation status of mTOR and its downstream effectors ribosomal protein S6 and p70 S6 kinase 1 (S6K1), (2) the abundance and phosphorylation status of transcription factors involved in appetite regulation, and (3) the mRNA levels of key neuropeptides associated with homeostatic regulation of feed intake in fish. Rising central levels of valine clearly resulted in an orexigenic response in rainbow trout. This response occurred in parallel with mTOR activation in both the hypothalamus and telencephalon, as supported by depressant changes in proteins involved in mTOR signalling (S6 and S6K1). Also, these changes disappeared in the presence of rapamycin. However, it is not clear which precise mechanisms link the activation of mTOR and the alteration in feed intake levels since we did not observe changes in mRNA levels of appetite-regulatory neuropeptides as well as in the phosphorylation status and levels of integrative proteins.

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.

First evidence for the presence of amino acid sensing mechanisms in the fish gastrointestinal tract.

This study aimed to characterize amino acid sensing systems in the gastrointestinal tract (GIT) of the carnivorous fish model species rainbow trout. We observed that the trout GIT expresses mRNAs encoding some amino acid receptors described in mammals [calcium-sensing receptor (CaSR), G protein-coupled receptor family C group 6 member A (GPRC6A), and taste receptors type 1 members 1 and 2 (T1r1, T1r2)], while others [taste receptor type 1 member 3 (T1r3) and metabotropic glutamate receptors 1 and 4 (mGlur1, mGlur4)] could not be found. Then, we characterized the response of such receptors, as well as that of intracellular signaling mechanisms, to the intragastric administration of L-leucine, L-valine, L-proline or L-glutamate. Results demonstrated that casr, gprc6a, tas1r1 and tas1r2 mRNAs are modulated by amino acids in the stomach and proximal intestine, with important differences with respect to mammals. Likewise, gut amino acid receptors triggered signaling pathways likely mediated, at least partly, by phospholipase C ß3 and ß4. Finally, the luminal presence of amino acids led to important changes in ghrelin, cholecystokinin, peptide YY and proglucagon mRNAs and/or protein levels. Present results offer the first set of evidence in favor of the existence of amino acid sensing mechanisms within the fish GIT.

Central serotonin participates in the anorexigenic effect of GLP-1 in rainbow trout (Oncorhynchus mykiss).

The incretin, glucagon-like peptide-1 (GLP-1) is a major player in the gut-brain axis regulation of energy balance and in fish it seems to exert a negative influence on food intake. In this study, we investigated the role of the brain serotonergic system in the effects promoted by a peripheral GLP-1 injection on food intake in rainbow trout (Oncorhynchus mykiss). For this, in a first experiment the incretin was intraperitoneally injected (100 ng/g body weight) alone or in combination with a 5HT2C receptor antagonist (SB 242084, 1 µg/g body weight) and food intake was measured 30, 90, and 180 min later. In a second experiment, we studied the effect of these treatments on mRNA abundance of hypothalamic neuropeptides that control food intake. In addition, the effect of GLP-1 on serotonin metabolism was assessed in hindbrain and hypothalamus. Our results show that GLP-1 induced a significant food intake inhibition, which agreed with the increased expression of anorexigenic neuropeptides pomc and cart in the hypothalamus. Furthermore, GLP-1 stimulated the synthesis of serotonin in the hypothalamus, which might be indicative of a higher use of the neurotransmitter. The effects of GLP-1 on food intake were partially reversed when a serotonin receptor antagonist, SB 242084, was previously administered to trout. This antagonist also reversed the stimulatory effect of the hormone in hypothalamic pomca1 mRNA abundance. We conclude that hypothalamic serotonergic pathways are essential for mediating the effects of GLP-1 on food intake in rainbow trout. In addition, the 5HT2C receptor subtype seems to have a prominent role in the inhibition of food intake induced by GLP-1 in this species.

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.

The long-chain fatty acid receptors FFA1 and FFA4 are involved in food intake regulation in fish brain.

We hypothesized that the free fatty acid receptors FFA1 and FFA4 might be involved in the anorectic response observed in fish after rising levels of long-chain fatty acids (LCFAs) such as oleate. In one experiment we demonstrated that intracerebroventricular (i.c.v.) treatment of rainbow trout with FFA1 and FFA4 agonists elicited an anorectic response 2, 6 and 24 h after treatment. In a second experiment, the same i.c.v. treatment resulted after 2 h in an enhancement in the mRNA abundance of anorexigenic neuropeptides pomca1 and cartpt and a decrease in the values of orexigenic peptides npy and agrp1 These changes occurred in parallel with those observed in the mRNA abundance and/or protein levels of the transcription factors Creb, Bsx and FoxO1, protein levels and phosphorylation status of Ampkα and Akt, and mRNA abundance of plcb1 and itrp3 Finally, we assessed in a third experiment the response of all these parameters after 2 h of i.c.v. treatment with oleate (the endogenous ligand of both free fatty acid receptors) alone or in the presence of FFA1 and FFA4 antagonists. Most effects of oleate disappeared in the presence of FFA1 and FFA4 antagonists. The evidence obtained supports the involvement of FFA1 and FFA4 in fatty acid sensing in fish brain, and thus involvement in food intake regulation through mechanisms not exactly comparable (differential response of neuropeptides and cellular signalling) to those known in mammals.

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.

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.

Central Treatment of Ketone Body in Rainbow Trout Alters Liver Metabolism Without Apparently Altering the Regulation of Food Intake.

We hypothesize that the presence in fish brain of a ketone body (KB) like ß-hydroxybutyrate (BHB) alters energy homeostasis through effects on food intake and peripheral energy metabolism. Using rainbow trout (Oncorhynchus mykiss) as a model, we intracerebroventricularly (ICV) administered 1 µl 100 g-1 body mass of saline solution alone (control) or containing 0.5 µmol of BHB. In a fist set of experiments, BHB did not affect food intake 6 and 24 h after treatment. In a second set of experiments, we evaluated 6 h after ICV BHB treatment changes in parameters putatively related to food intake control in brain areas (hypothalamus and hindbrain) involved in nutrient sensing and changes in energy metabolism in liver. The absence of changes in food intake might relate to the absence of major changes in the cascade of events from the detection of KB through ketone-sensing mechanisms, changes in transcription factors, and changes in the mRNA abundance of neuropeptides regulating food intake. This response is different than that of mammals. In contrast, central administration of BHB induced changes in liver energy metabolism suggesting a decreased use of glucose and probably an enhanced use of amino acid and lipid. These responses in liver are different to those of mammals under similar treatments but comparable to those occurring in fish under food deprivation conditions.

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.