Stress Effects on the Mechanisms Regulating Appetite in Teleost Fish

The homeostatic regulation of food intake relies on a complex network involving peripheral and central signals that are integrated in the hypothalamus which in turn responds with the release of orexigenic or anorexigenic neuropeptides that eventually promote or inhibit appetite. Under stress conditions, the mechanisms that control food intake in fish are deregulated and the appetite signals in the brain do not operate as in control conditions resulting in changes in the expression of the appetite-related neuropeptides and usually a decreased food intake. The effect of stress on the mechanisms that regulate food intake in fish seems to be mediated in part by the corticotropin-releasing factor (CRF), an anorexigenic neuropeptide involved in the activation of the HPI axis during the physiological stress response. Furthermore, the melanocortin system is also involved in the connection between the HPI axis and the central control of appetite. The dopaminergic and serotonergic systems are activated during the stress response and they have also been related to the control of food intake. In addition, the central and peripheral mechanisms that mediate nutrient sensing capacity and hence implicated in the metabolic control of appetite are inhibited in fish under stress conditions. Finally, stress also affects peripheral endocrine signals such as leptin. In the present minireview, we summarize the knowledge achieved in recent years regarding the interaction of stress with the different mechanisms that regulate food intake in fish

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

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

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

Neuroendocrine and Immune Responses Undertake Different Fates following Tryptophan or Methionine Dietary Treatment: Tales from a Teleost Model

Methionine and tryptophan appear to be fundamental in specific cellular pathways involved in the immune response mechanisms, including stimulation of T-regulatory cells by tryptophan metabolites or pro-inflammatory effects upon methionine supplementation. Thus, the aim of this study was to evaluate the immunomodulatory effect of these amino acids on the inflammatory and neuroendocrine responses in juveniles of European seabass, Dicentrarchus labrax. To achieve this, goal fish were fed for 14 days methionine and tryptophan-supplemented diets (MET and TRP, respectively, 2× dietary requirement level) or a control diet meeting the amino acids requirement levels (CTRL). Fish were sampled for immune status assessment and the remaining fish were challenged with intraperitoneally injected inactivated Photobacterium damselae subsp. piscicida and sampled either 4 or 24 h post-injection. Respiratory burst activity, brain monoamines, plasma cortisol, and immune-related gene expression showed distinct and sometimes opposite patterns regarding the effects of dietary amino acids. While neuroendocrine intermediates were not affected by any dietary treatment at the end of the feeding trial, both supplemented diets led to increased levels of plasma cortisol after the inflammatory insult, while brain monoamine content was higher in TRP-fed fish. Peripheral blood respiratory burst was higher in TRP-fed fish injected with the bacteria inoculum but only compared to those fed MET. However, no changes were detected in total antioxidant capacity. Complement factor 3 was upregulated in MET-fed fish but methionine seemed to poorly affect other genes expression patterns. In contrast, fish fed MET showed increased immune cells numbers both before and after immune challenge, suggesting a strong enhancing effect of methionine on immune cells proliferation. Differently, tryptophan effects on inflammatory transcripts suggested an inhibitory mode of action. This, together with a high production of brain monoamine and cortisol levels, suggests that tryptophan might mediate regulatory mechanisms of neuroendocrine and immune systems cooperation. Overall, more studies are needed to ascertain the role of methionine and tryptophan in modulating (stimulate or regulate) fish immune and neuroendocrine responses

Efectos del estrés sobre la ingesta de alimento y su regulación por mecanismos neuroendocrinos y metabólicos en peces teleósteos

Una respuesta comportamental genérica al estrés en peces es la reducción de la ingesta. Se ha demostrado que varios tipos de agentes estresantes medioambientales, sociales, patológicos o físicos inhiben la ingesta y coartan el crecimiento en varias especies de peces. Actualmente se acepta que la ingesta de alimento está bajo el control de un sistema central que integra la acción de numerosos neuropéptidos y monoaminas con carácter orexigénico y anorexigénico. Por otro lado, se tiene conocimiento de que en la regulación de la actividad alimenticia en los peces participan factores relacionados con la homeostasis energética, entre los que destacan los niveles de nutrientes. En trabajos previos en la trucha arco iris se ha demostrado la existencia de mecanismos glucosensores a nivel central y periférico cuya actividad es capaz de influir de forma importante en la cantidad de alimento ingerido por el pez. No obstante, no existe información sobre como el estrés podría modificar la capacidad glucosensora y la expresión de neuropéptidos y con ello, modular los circuitos neuronales implicados en el control de la ingesta. Los sistemas cerebrales de regulación de la ingesta, fundamentalmente hipotalámicos, adquieren información ambiental suministrada por osciladores circadianos sincronizados por el ciclo de luz/oscuridad y por el propio horario de alimentación, lo que permite la integración temporal de la regulación homeostática de la ingesta. La melatonina, neurohormona sintetizada en la glándula pineal, la retina y el tracto gastrointestinal de los peces, es una molécula clave del sistema circadiano. Además, en vertebrados hay evidencias de que esta hormona podría ejercer una acción anti-estresante y con efectos secundarios sobre la actividad alimenticia, pero poco se conoce de esta capacidad de la melatonina en los peces. En base a ello, la presente tesis tiene como objetivo general evaluar el efecto del estrés sobre los mecanismos neuroendocrinos y metabólicos que regulan la ingesta en peces teleósteos, usando como modelo la trucha arco iris. Para ello se han desarrollado una serie de trabajos experimentales en los que se ha evaluado el efecto del estrés sobre los sistemas glucosensores centrales y periféricos descritos en trucha arco iris y sobre la expresión de neuropéptidos orexigénicos (NPY) y anorexigénicos (CART, POMC y CRF). Se ha evaluado también la interacción de la melatonina sobre estos sistemas y su implicación en el control de la alimentación, así como también su posible papel anti-estrés en peces. Los resultados de esta tesis nos muestran evidencias de que el estrés produce una desregulación de los sistemas glucosensores tanto centrales como periféricos en la trucha arco iris, lo que les impide responder a los cambios en los niveles de glucosa circulante. Además esto lleva a que bajo una situación de estrés, el animal no pueda regular adecuadamente la ingesta en base a los cambios producidos en los niveles de glucemia. Los cambios producidos en la ingesta debido a variaciones en los niveles de glucosa están relacionados con cambios en la expresión de neuropéptidos orexigénicos y anorexigénicos, sobre todo en condiciones de hiperglucemia. Además, el estrés altera esta respuesta de los neuropéptidos, lo que indica que la desregulación de la ingesta por cambios en la glucosa durante el estrés se podría deber a esta alteración. El CRF podría ser uno de los agentes a través de los cuales el estrés está desregulando la respuesta de los sistemas glucosensores centrales y su reflejo en la ingesta, ya que su tratamiento produce cambios sobre los parámetros glucosensores similares a los vistos en situaciones de estrés y también altera la respuesta a cambios en la glucemia de los neuropéptidos implicados en la regulación del apetito. También mostramos evidencias de que la síntesis y liberación de cortisol en presencia de ACTH por el riñón anterior de la trucha depende de los niveles circulantes de glucosa, de modo que en condiciones de hiperglucemia se sintetiza más cortisol. El efecto anti-estrés de la melatonina parece no estar relacionado con su interacción sobre el sistema glucosensor central, ya que los efectos de la hormona sobre los parámetros glucosensores van en la misma dirección que los que promueve el CRF. Más bien parece que el papel fisiológico de la Mel sobre los sistemas glucosensores estaría relacionado solo a nivel hepático con la modulación de los cambios diarios en la respuesta glucosensora, lo cual podría estar relacionado a su vez con los mecanismos involucrados en el control rítmico de la ingesta y la utilización de nutrientes. Los posibles efectos anti-estrés de la Mel parecen depender de la activación del eje HPI y de la interacción de esta hormona con sistemas de neurotransmisores como el serotoninérgico y dopaminérgico, suprimiendo el efecto que produce su activación durante situaciones de estrés. A modo de conclusión general, los resultados de la presente Tesis Doctoral ponen de manifiesto algunos de los mecanismos a través de los que el estrés interactúa con el control metabólico y neuroendocrino de la ingesta de alimento en la trucha arco iris como modelo de pez teleósteo.Ministerio de Educación y Ciencia-FEDER | Ref. AGL2007-65744-C03-01/ACUMinisterio de Ciencia e Innovación-FEDER | Ref. AGL2010-22247-C03-03/ACUUniversidade de VigoMinisterio de Innovación | Ref. BES-2008-00291

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 2h and 3h after oral administration, while glutamic acid showed a less pronounced satiating effect at 3h. The mRNA expression of taste receptor subunits tas1r1, tas1r2a, tas1r2b, and tas1r3 was measured in the epithelium overlying the bony basyhyal 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 mins. 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 since 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.Ministerio de Educación, Cultura y Deporte | Ref. FPU16/00045Xunta de Galicia | Ref. ED431B 2019/37Xunta de Galicia | Ref. ED481B2018/01

Central regulation of food intake is not affected by inclusion of defatted Tenebrio molitor larvae meal in diets for European sea bass (Dicentrarchus labrax)

The homeostatic regulation of food intake in fish has been thoroughly studied in the last years with dietary nutrient composition is one of the factors involved. Despite several studies addressed the impact of insect meal-based diets in food intake of fish, there is no knowledge about their impact in mechanisms of food intake regulation. This study aimed to explore underlying mechanisms through evaluation of dietary fishmeal (FM) replacement by defatted (d-) Tenebrio molitor (dTM) larvae meal in European sea bass. Fish (55 ± 2 g) fed a FM-based diet (CTRL) were compared with those fed two experimental diets containing increasing levels of dTM to replace 40 and 80% of FM (TM40 and TM80, respectively). After ten weeks of feeding, fish food intake was calculated; samples of plasma and different brain areas (hypothalamus and telencephalon) were collected at different post-prandial times (2, 6 and 24 h after feeding) to evaluate circulating metabolites and mRNA abundance in hypothalamus and telencephalon of neuropeptides involved in food intake regulation. No differences occurred in food intake and weight gain among diets. Plasma cholesterol levels decreased 24 h after feeding in fish fed TM40; fish fed dTM diets had higher cholesterol levels, 2 and 6 h after feeding, than those fed CTRL diet. Increased non-esterified fatty acids (NEFA) levels occurred in plasma of fish fed TM80, regardless of the sampling time. At central level, no changes occurred in the mRNA abundance of neuropeptide Y (npy), agouti-related protein 2 (agrp2), pro-opio melanocortin a (pomca) or cocaine- and amphetamine-related transcript 2 (cartpt2). The obtained results suggest that dietary FM replacement by dTM up to 80% in European sea bass does not affect food intake or its central homeostatic regulation, supporting the use of dTM as FM replacement in diets for European sea bass.Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGAgencia Estatal de Investigación | Ref. PID2019-103969RB-C31Xunta de Galicia | Ref. ED431B 2019/37Fundação para a Ciência e Tecnologia | Ref. SFRH/BD/138593/201