Insulin controls clock gene expression in the liver of goldfish probably via Pi3k/Akt pathway

The liver circadian clock plays a pivotal role in driving metabolic rhythms, being primarily entrained by the feeding schedule, although the underlying mechanisms remain elusive. This study aimed to investigate the potential role of insulin as an intake signal mediating liver entrainment in fish. To achieve this, the expression of clock genes, which form the molecular basis of endogenous oscillators, was analyzed in goldfish liver explants treated with insulin. The presence of insulin directly increased the abundance of per1a and per2 transcripts in the liver. The dependency of protein translation for such insulin effects was evaluated using cycloheximide, which revealed that intermediate protein translation is seemingly unnecessary for the observed insulin actions. Furthermore, the putative interaction between insulin and glucocorticoid signaling in the liver was examined, with the results suggesting that both hormones exert their effects by independent mechanisms. Finally, to investigate the specific pathways involved in the insulin effects, inhibitors targeting PI3K/AKT and MEK/ERK were employed. Notably, inhibition of PI3K/AKT pathway prevented the induction of per genes by insulin, supporting its involvement in this process. Together, these findings suggest a role of insulin in fish as a key element of the multifactorial system that entrains the liver clock to the feeding schedule.Agencia Estatal de Investigación | Ref. PID2019-103969RB-C32Agencia Estatal de Investigación | Ref. PID2019-103969RB-C31Xunta de Galicia | Ref. ED431B 2022/01Universidad Complutense de Madrid | Ref. CT42/18-CT43-1

Fatty acids of different nature differentially modulate feed intake in rainbow trout

Feed intake is subjected to a complex regulation involving a plethora of signals, among which nutrients stand as one of the most important. In mammals, the gastrointestinal tract is able to sense nutrients in the lumen, and respond with the release of signaling molecules that ultimately modulate brain circuits governing appetite, resulting in decreased/increased feeding. Whether equivalent mechanisms operate in fish remains unknown. In a recent study, we described that the gastrointestinal tract of rainbow trout contains several sensors for free fatty acids (FAs), and that the luminal presence of FAs of different length and degree of unsaturation modulates the levels of key gastrointestinal hormones involved in feed intake regulation. In this study, our aim was to characterize the impact of such a luminal presence of FAs on brain appetite-regulatory centers, as well as its effects on rainbow trout feed intake. Major results from this study demonstrated that: (i) FAs of different length and degree of unsaturation [medium-chain (MCFAs, octanoate), long-chain (LCFAs, oleate), long-chain polyunsaturated (PUFA, α-linolenate), and short-chain (SCFA, butyrate) FAs] differentially modulate feed intake levels when administered intragastrically, (ii) intragastrically-administered FAs modulate the phosphorylation status of appetite-related transcription factors, as well as mRNA levels of key appetite-regulating neuropeptides, in the hypothalamus and/or telencephalon, (iii) luminal presence of FAs results in changes in the central abundance of mRNAs encoding gastrointestinal hormone receptors, and (vi) luminal FA-derived central changes in neuropeptide mRNAs are not observed (or are lessened) in vagotomized fish. Together, these results provide comprehensive evidence in favor of a gut-brain axis in fish. In addition, we observed different responses in terms of feed intake regulation depending on the type of fatty acid administered into the lumen, which is very relevant for aquaculture considering differences in fatty acid composition in aquafeedsAgencia Estatal de Investigación | Ref. PID2019-103969RB-C31Agencia Estatal de Investigación | Ref. IJC2019-039166-IMinisterio de Universidades | Ref. FPU19/00122Ministerio de Educación, Cultura y Deporte | Ref. FPU16/0004

Impact of feeding diets with enhanced vegetable protein content and presence of umami taste-stimulating additive on gastrointestinal amino acid sensing and feed intake regulation in rainbow trout

The regulation of feed intake in fish is dependent upon different neuroendocrine and metabolic mechanisms including amino acid sensing in the gastrointestinal tract (GIT). However, there is little information regarding the impact of diets on such mechanisms. Therefore, in this study, we fed rainbow trout (Oncorhynchus mykiss) with 3 diets: a diet with high content of fishmeal and low content of soybean protein concentrate (SPC) (HF), a diet with a reduced content of fishmeal and high content of SPC (LF), and the LF diet supplemented with an umami tastestimulating additive (LFU). Fish were fed ad libitum once a day for 4 weeks, with no significant differences being registered in feed intake among groups. At the end of the feeding trial, we collected samples of different areas of the GIT (stomach, proximal and distal intestine) and hypothalamus at different times: after 48 h of fasting (time 0), and 1 h, 4 h, and 24 h after feeding. We evaluated the activity of pepsin in the stomach and trypsin and chymotrypsin in the proximal intestine, as well as mRNA abundance of transcripts encoding amino acid transporters and taste receptors, intracellular signalling molecules, and hormones. Moreover, we assessed the hypothalamic mRNA abundance of neuropeptides involved in feed intake regulation. Feeding rainbow trout with LF did not result in marked alterations in parameters related to digestive function and amino acid sensing in the rainbow trout GIT, nor in the expression of gastrointestinal hormones (except cck) and hypothalamic neuropeptides. In contrast, supplementation of the LF diet with an umami taste-stimulating additive resulted in a general improvement of digestive or absorptive function (increased protein, dry matter and energy digestibility, and earlier peak in plasma amino acid levels) and activation of gut-brain axis mechanisms involved in feed intake regulation through the transcriptional activation of amino acid transporters, taste receptors, signalling molecules, and hormones. These results demonstrate that the dietary inclusion of umami receptor stimulants has the potential to improve fish physiological responses to the rise in levels of vegetable protein in the diet.Xunta de Galicia | Ref. GPC-ED431B 2022/01Ministerio de Universidades | Ref. FPU19/00122Agencia Estatal de Investigación | Ref. IJC2019-039166-IUniversidade de Vigo/CISU

Fatty acid sensing in the gastrointestinal tract of rainbow trout: different to mammalian model?

It is well established in mammals that the gastrointestinal tract (GIT) senses the luminal presence of nutrients and responds to such information by releasing signaling molecules that ultimately regulate feeding. However, gut nutrient sensing mechanisms are poorly known in fish. This research characterized fatty acid (FA) sensing mechanisms in the GIT of a fish species with great interest in aquaculture: the rainbow trout (Oncorhynchus mykiss). Main results showed that: (i) the trout GIT has mRNAs encoding numerous key FA transporters characterized in mammals (FA transporter CD36 -FAT/CD36-, FA transport protein 4 -FATP4-, and monocarboxylate transporter isoform-1 -MCT-1-) and receptors (several free FA receptor -Ffar- isoforms, and G protein-coupled receptors 84 and 119 -Gpr84 and Gpr119-), and (ii) intragastrically-administered FAs differing in their length and degree of unsaturation (i.e., medium-chain (octanoate), long-chain (oleate), long-chain polyunsaturated (α-linolenate), and short-chain (butyrate) FAs) exert a differential modulation of the gastrointestinal abundance of mRNAs encoding the identified transporters and receptors and intracellular signaling elements, as well as gastrointestinal appetite-regulatory hormone mRNAs and proteins. Together, results from this study offer the first set of evidence supporting the existence of FA sensing mechanisms n the fish GIT. Additionally, we detected several differences in FA sensing mechanisms of rainbow trout vs. mammals, which may suggest evolutionary divergence between fish and mammals.Xunta de Galicia | Ref. ED431B 2022/01Agencia Estatal de Investigación española | Ref. PID2019-103969RB-C31Ministerio de Educación, Cultura y Deporte | Ref. FPU19/00122Ministerio de Educación, Cultura y Deporte | Ref. FPU16/00045Ministerio de Ciencia e Innovación | Ref. IJC2019-039166-

Evidence of gastrointestinal sensing and gut-brain communication in rainbow trout (Oncorhynchus mykiss) in response to the aqueous extract of fishmeal and its free amino acid fraction

Using rainbow trout (Oncorhynchus mykiss) as a model, we aimed to obtain information about the gastrointestinal tract (GIT) amino acid sensing capacity and hormone production along regions of the GIT, in response to proline (Pro), to a solution of free amino acids (FAA) mimicking the composition of a fishmeal (FM) aqueous extract (FM-FAA), or to the whole FM aqueous extract (FM-AQE). In addition, we evaluated central responses (in hypothalamus) in mechanisms regulating food intake, 2 h following intragastric administration of these treatments. The presence of Pro in the GIT elicited changes in amino acid sensing systems and in the production of GIT hormones, especially in the more proximal regions in parallel with an anorectic response in hypothalamus. The intragastric administration of FM-AQE induced increased production of the anorectic hormones peptide tyrosine-tyrosine (PYY) and cholecystokinin (CCK) that occurred 20 min post-treatment in the proximal and middle intestine of this treatment. These changes occurred in parallel with an anorectic response in the hypothalamus 2 h post-treatment. The treatment with FM-FAA elicited a comparable anorectic response in the hypothalamus at 2 h post-treatment, which was associated however with a more complex response in the GIT. This included a comparable increased production of the anorectic hormones PYY and CCK in the proximal and middle intestine, but also a decreased production of the orexigenic hormone ghrelin (GHRL) in the stomach, 20 min after FM-FAA administration. These effects were also accompanied by some changes in parameters related to amino acid sensing systems mediated by receptors, which were not observed in the FM-AQE treatment. Overall, results indicate that all treatments elicited a response in elements of gut sensing mechanisms and gut-brain axis, despite important differences in the specific genes (likely having different substrate specificities), GIT areas and times in which responses were observedXunta de Galicia | Ref. ED431B 2022/01Ministerio de Universidades | Ref. FPU19/00122Agencia Estatal de Investigación | Ref. IJC2019-039166-IFinanciado para publicación en acceso aberto: Universidade de Vigo/CISU

Unraveling the periprandial changes in brain serotonergic activity and its correlation with food intake-related neuropeptides in rainbow trout Oncorhynchus mykiss

This study explored changes in brain serotonin content and activity together with hypothalamic neuropeptide mRNA abundance around feeding time in rainbow trout, as well as the effect of one-day fasting. Groups of trout fed at two (ZT2) and six (ZT6) hours after lights on were sampled from 90 minutes before to 240 minutes after feeding, while additional groups of non-fed trout were also included in the study. Changes in brain amine and metabolite contents were measured in hindbrain, diencephalon and telencephalon, while in the diencephalon the mRNA abundance of tryptophan hydroxylase ( tph1 , tph2 ), serotonin receptors (5htr1a , 5htr1b and 5htr2c ) and several neuropeptides ( npy , agrp1 , cartpt , pomca1 , crfb ) involved in the control of food intake were also assessed. The results showed changes in the hypothalamic neuropeptides that were consistent with the expected role for each in the regulation of food intake in rainbow trout. Serotonergic activity increased rapidly at the time of food intake in the diencephalon and hindbrain and remained high for much of the postprandial period. This increase in serotonin abundance was concomitant with elevated levels of pomca1 mRNA in the diencephalon, suggesting that serotonin might act on brain neuropeptides to promote a satiety profile. Furthermore, serotonin synthesis and neuronal activity appear to increase already before the time of feeding, suggesting additional functions for this amine before and during food intake. Exploration of serotonin receptors in the diencephalon revealed only small changes for gene expression of 5htr1b and 5htr2c receptors during the postprandial phase. Therefore, the results suggest that serotonin may play a relevant role in the regulation of feeding behavior in rainbow trout during periprandial time, but a better understanding of its interaction with brain centers involved in receiving and processing food-related signals is still needed.Agencia Estatal de Investigación | Ref. PID2022-136288OB-C31Xunta de Galicia | Ref. ED431B 2019/37Agencia Estatal de Investigación | Ref. BES-2017-079708Xunta de Galicia | Ref. ED481B-2022-08

Oral administration of melatonin counteracts several of the effects of chronic stress in rainbow trout

To assess a possible antistress role of melatonin in fish, we orally administered melatonin to rainbow trout for 10 d and then kept the fish under normal or high stocking density conditions during the last 4 d. Food intake; biochemical parameters in plasma (cortisol, glucose, and lactate concentrations); liver (glucose and glycogen concentrations, and glycogen synthase activity); enzyme activities of amylase, lipase, and protease in foregut and midgut; and content of the hypothalamic neurotransmitters dopamine and serotonin, as well as their oxidized metabolites, 3,4-dihydroxyphenylacetic acid and 5-hydroxy-3-indoleacetic acid, were evaluated under those conditions. High stocking density conditions alone induced changes indicative of stress conditions in plasma cortisol concentrations, liver glycogenolytic potential, the activities of some digestive enzymes, and the 3,4-dihydroxyphenylacetic acid-to-dopamine and 5-hydroxy-3-indoleacetic acid-to-serotonin ratios in the hypothalamus. Melatonin treatment in nonstressed fish induced an increase in liver glycogenolytic potential, increased the activity of some digestive enzymes, and enhanced serotoninergic and dopaminergic metabolism in hypothalamus. The presence of melatonin in stressed fish resulted in a significant interaction with cortisol concentrations in plasma, glycogen content, and glycogen synthase activity in liver and dopaminergic and serotoninergic metabolism in the hypothalamus. In general, the presence of melatonin mitigated several of the effects induced by stress, supporting an antistress role for melatonin in rainbow trout.Ministerio de Ciencia e Innovación | Ref. AGL2010-22247-C03-03Xunta de Galicia | Ref. CN2012/ 00

Integration of nutrient sensing in fish hypothalamus

The knowledge regarding hypothalamic integration of metabolic and endocrine signaling resulting in regulation of food intake is scarce in fish. Available studies pointed to a network in which the activation of the nutrient-sensing (glucose, fatty acid, and amino acid) systems would result in AMP-activated protein kinase (AMPK) inhibition and activation of protein kinase B (Akt) and mechanistic target of rapamycin (mTOR). Changes in these signaling pathways would control phosphorylation of transcription factors cAMP response-element binding protein (CREB), forkhead box01 (FoxO1), and brain homeobox transcription factor (BSX) leading to food intake inhibition through changes in the expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), pro-opio melanocortin (POMC), and cocaine and amphetamine-related transcript (CART). The present mini-review summarizes information on the topic and identifies gaps for future research.Agencia Estatal de Investigación | Ref. PID2019-103969RB-C31Xunta de Galicia | Ref. ED431B 2019/3

Leucine sensing in rainbow trout hypothalamus is direct but separate from mTOR signalling in the regulation of food intake

In prior studies, we evidenced the presence in fish hypothalamus of different systems sensing changes in leucine levels to relate them to food intake control. To assess whether or not those effects are due to a direct action of leucine, we evaluated in the present in vitro study if rainbow trout (Oncorhynchus mykiss) hypothalamus respond to changes in leucine levels in a way comparable to that observed in vivo. Thus, we incubated hypothalamus in vitro with increased leucine concentrations (1, 5 and 25 mM). Then, we evaluated parameters related to mechanisms of amino acid sensing as well as transcription factors, and neuropeptides. Amino acid sensing systems related to metabolism of BCAA and glutamine and GCN2 kinase are activated by the presence of leucine, in a way comparable to that observed in vivo thus supporting a direct action of leucine on them. However, the activation of these systems did not trigger mTOR signalling pathway including downstream protein targets of mTOR, transcription factors, and mRNA abundance of neuropeptides involved in food intake the regulation of food intake. Therefore, the activation of hypothalamic mTOR observed after leucine administration in vivo is not a direct response to leucine but indirect. The absence in vitro of connections between hypothalamus and other brain areas and/or the lack of changes in levels of hormones, particularly insulin, could be responsible of this differential response.Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGXunta de Galicia | Ref. ED431B 2019/37Agencia Estatal de Investigación | Ref. PID2019-103969RB-C31Ministerio de Educación, Cultura y Deporte (España) | Ref. FPU16/0004

Leptin signalling in teleost fish with emphasis in food intake regulation

Leptin, the product of the obese (ob or Lep) gene, was first cloned in teleost fish in 2005, more than a decade after its identification in mammals. This was because bony fish and mammalian leptins share a very low amino acid sequence identity, which suggests different functionality of the leptin system in fish compared to that of mammals. Indeed, major differences are evident between the mammalian and fish leptin system. Thus, for instance, mammalian leptin is synthesized and released by the adipose tissue in response to the amount of fat depots, while several tissues (mainly the liver) are the main sources of leptin in fish, whose determining factors of production are still unclear. In mammals, the main physiological role for leptin is its involvement in the maintenance of energy balance by decreasing food intake and increasing energy expenditure, although a wide variety of actions have been attributed to this hormone (e.g., regulation of lipid and carbohydrate metabolism, reproduction and immune functions). In fish, available literature also points towards a multifunctional nature for leptin, although knowledge on its functions is limited. In this review, we offer an overview of teleostean leptin structure and mechanism of action, and discuss the available knowledge on the role of this hormone in food intake regulation in teleost fish, aiming to provide a comparative overview between the functioning of the teleostean and mammalian leptin systemsFinanciado para publicación en acceso aberto: Universidade de Vigo/CISUGAgencia Estatal de Investigación | Ref. PID2019-103969RB-C31Xunta de Galicia | Ref. ED431B 2019/37Xunta de Galicia | Ref. ED481B 2017/11