Sensing Glucose in the Central Melanocortin Circuits of Rainbow Trout: A Morphological Study

In mammals, glucosensing markers reside in brain areas known to play an important role in the control of food intake. The best characterized glucosensing mechanism is that dependent on glucokinase (GK) whose activation by increased levels of glucose leads in specific hypothalamic neurons to decreased or increased activity, ultimately leading to decreased food intake. In fish, evidence obtained in recent years suggested the presence of GK-like immunoreactive cells in different brain areas related to food intake control. However, it has not been established yet whether or not those neuronal populations having glucosensing capacity are the same that express the neuropeptides involved in the metabolic control of food intake. Therefore, we assessed through dual fluorescent in situ hybridization the possible expression of GK in the melanocortinergic neurons expressing proopiomelanocortin (POMC) or agouti-related protein (AGRP). POMC and AGRP expression localized exclusively in the rostral hypothalamus, in the ventral pole of the lateral tuberal nucleus, the homolog of the mammalian arcuate nucleus. Hypothalamic GK expression confined to the ependymal cells coating the ventral pole of the third ventricle but some expression level occurred in the AGRP neurons. GK expression seems to be absent in the hypothalamic POMC neurons. These results suggest that AGRP neurons might sense glucose directly through a mechanism involving GK. In contrast, POMC neurons would not directly respond to glucose through GK and would require presynaptic inputs to sense glucose. Ependymal cells could play a critical role relying glucose metabolic information to the central circuitry regulating food intake in fish, especially in POMC neurons

Advances in the study of glucosensing systems in rainbow trout and their involvement in the control of food intake

En estudios previos de nuestro grupo de investigación tenemos demostrado en trucha arco iris como modelo de pez teleósteo, que los sistemas glucosensores centrales (hipotálamo y cerebro posterior) y periféricos (hígado y cuerpos de Brockmann) basados en la glucoquinasa (GK) se activan/inhiben con el incremento/descenso en los niveles de glucosa desencadenando en el hipotalámo cambios en la producción de factores orexigénicos y anorexigénicos (NPY, POMC e CART), lo que resulta en una inhibición/estimulación de la ingesta. Desconocemos sin embargo hasta la fecha la presencia y posible funcionalidad en peces de sistemas glucosensores alternativos, como los mediados por LXR, SGLT-1, actividad mitocondrial o alfa-gustducina, y su posible implicación en el control de la ingesta de alimento y en la anorexia que se produce en situaciones de estrés. Así mismo, desconocemos el impacto de los cambios en los niveles de glucosa sobre la actividad integradora de sistemas como AMPK o sirtuinas, y desconocemos también las vías de señalización intracelular implicadas en el proceso. Por todo esto, los objetivos del plan de investigación son los siguientes: 1. Caracterización de la presencia de sistemas glucosensores alternativos al mediado por GK en tejidos centrales (hipotálamo y cerebro posterior) y periféricos (hígado y cuerpos de Brockmann) de trucha arco iris. 2. Caracterización de la respuesta de dichos sistemas sensores frente a incrementos/descensos en los niveles circulantes de glucosa inducidos mediante tratamientos in vivo e in vitro. 3. Caracterización de la presencia en dichas áreas de sistemas integradores mediados por AMPK, mTOR, Akt, sirtuinas o Fox01, y de las vías intracelulares de señalización implicadas en su activación, así como de la activación/inhibición de dichas vías y sistemas en paralelo a la activación/inhibición de los distintos sistemas sensores de glucosa. 4. Caracterización de la relación entre la actividad de los distintos sistemas sensores de glucosa y el control de la ingesta de alimento. 5. Caracterización de la presencia de ANLS (lanzadera de lactato entre astrocitos y neuronas) en cerebro de trucha arco iris con la premisa de que el metabolismo del lactato sufre alteraciones frente a cambios en los niveles de glucosa. 6. Evaluar el impacto de distintos modelos de estrés en la respuesta de los diferentes sistemas sensores de glucosa. 7. Localización espacial de marcadores glucosensores en tejidos centrales como hipotálamo y área preóptica mediante técnicas inmunohistoquímicas. 8. Aplicación de técnicas de hibridación in situ para localizar la expresión de marcadores de glucosensibilidad, relacionados con el control metabólico de la ingesta en cerebro de trucha arco iris y su posible co-localización con neuropéptidos. 9. Caracterización de la presencia de diferentes sistemas sensores de glucosa en telencéfalo de trucha arco iris. 10. Evaluar la expresión a nivel central (hipotálamo, telencéfalo y cerebro posterior) de la proteína de unión al elemento de respuesta a carbohidratos (ChREBP).En estudos previos do grupo de investigación demostramos en troita arco da vella como modelo de peixe teleósteo, que os sistemas glucosensores centrais (hipotálamo e cerebro posterior) e periféricos (fígado e corpos de Brockmann) baseados na glucoquinasa (GK) actívanse/inhibense co incremento/descenso nos niveis de glicosa desencadeando no hipotalámo cambios na produción de factores orexixénicos e anorexixénicos (NPY, POMC e CART), o que resulta nunha inhibición/estimulación da inxesta. Descoñecemos sin embargo ata a data a presenza e posible funcionalidade en peixes de sistemas glucosensores alternativos, como os mediados por LXR, SGLT-1, actividade mitocondrial ou alfa-gustducina, e a súa posible implicación no control da inxesta de alimento e na anorexia que se produce en situacións de estrés. Así mesmo, descoñecemos o impacto dos cambios nos niveis de glicosa sobre a actividade integradora de sistemas como AMPK ou sirtuinas, e descoñecemos tamén as vías de sinalización intracelular implicadas no proceso. Por todo iso, os obxectivos do plan de investigación son os seguintes: 1. Caracterización da presenza de sistemas glucosensores alternativos ó mediado por GK en tecidos centrais (hipotálamo e cerebro posterior) e periféricos (fígado e corpos de Brockmann) da troita arco da vella. 2. Caracterización da resposta dos devanditos sistemas sensores fronte a incrementos/descensos nos niveis circulantes de glicosa inducidos mediante tratamentos in vivo e in vitro. 3. Caracterización da presenza nas devanditas áreas de sistemas integradores mediados por AMPK, mTOR, Akt, sirtuinas ou Fox01, e das vías intracelulares de sinalización implicadas na súa activación, así como da activación/inhibición das devanditas vías e sistemas en paralelo á activación/inhibición dos distintos sistemas sensores de glicosa 4. Caracterización da relación entre a actividade dos devanditos sistemas sensores de glicosa e o control da inxesta de alimento 5. Caracterización da presenza de ANLS (lanzadeira de lactato entre astrocitos e neuronas) en cerebro de troita arco da vella ca premisa de que o metabolismo do lactato sufre alteracións fronte a cambios nos niveis de glicosa. 6. Avaliar o impacto de distintos modelos de estrés na resposta dos devanditos sistemas sensores de glicosa. 7. Localización espacial de marcadores glucosensores en tecidos centrais como hipotálamo e área preóptica mediante técnicas inmunohistoquímicas. 8. Aplicación de técnicas de hibridación in situ para localizar a expresión de marcadores de glucosensibilidade relacionados co control metabólico da inxesta en cerebro de troita arco da vella e a súa posible co-localización con neuropéptidos. 9. Caracterización da presenza de diferentes sistemas sensores de glicosa en telencéfalo de troita arco da vella. 10. Avaliar a expresión a nivel central (hipotálamo, telencéfalo e cerebro posterior) da proteína de unión ó elemento de resposta a carbohidratos (ChREBP).Previous studies performed in our research group with rainbow trout have demonstrated the activation/inhibition of GK-based central (hypothalamus and hindbrain) and peripheral (liver and Brockmann bodies) glucosensor systems by the increase/decrease of glucose levels. This results in changes in the production of orexigenic (NPY) and anorexigenic factors( POMC and CART), with the subsequent inhibition/stimulation of food intake. To date little is known regarding the presence-absence of functional alternative glucosensor systems, such as those dependent on LXR, SGLT-1, mitochondrial activity and alpha-gustducin, and their involvement in the control of food intake and the stress-related anorectic effects. In adition, the impact of the changes in glucose levels on integratory systems, such as AMPK or sirtuins, is unknown, but also the intracellular signaling pathways involved as well. Accordingly, the main goals of the present research schedule are: 1. To characterize in rainbow trout the presence of alternative glucosensor systems based on GK in central (hypothalamus and hindbrain) and peripheral (liver and Brockmann bodies) tissues. 2. To evaluate in vivo and in vitro the response of such sensor systems to induced- increases/decreases of circulating glucose levels. 3. To test in such areas the presence of integratory systems based on AMPK, mTOR, Akt, sirtuins or Fox01, and the intracellular pathways involved in their activation. In parallel to the activation/inhibition of the different glucosensor systems, the activation/inhibition of such pathways will be also evaluated. 4. To characterize the interaction between the activity of glucosensor systems and food intake regulation. 5. To test the presence of ANLS (astrocyte-neuron lactate shuttle) in brain of rainbow trout, based on lactate metabolism being altered by changes in glucose levels. 6. To evaluate the response of glucosensor systems to different stress conditions. 7. To immunohistochemically localize glucosensing markers in central tissues such as hypothalamus and preoptic area. 8. To localize by in situ hybridization in trout brain the expression of the glucosensing markers related to metabolic control of food intake. Their co-localization with food intake-related neuropeptides will be also evaluated. 9. To characterize the presence of different glucosensor systems in telencephalon. 10. To evaluate the expression of the binding protein carbohydrate response element (ChREBP) in different brain regions (hypothalamus, telencephalon and hindbrain).Ministerio de Economía y Competitividad de España (BES-2014-068040)Ministerio de Economía y Competitividad de España (AGL2013-46448-C3-1-R)Xunta de Galicia (CN 2012/004)Ministerio de Economía y Competitividad de España (AGL2016-74857-C3-1-R

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 β-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.Agencia Estatal de Investigación (AEI) | Ref. AGL2016-74857-C3-1-RXunta de Galicia | Ref. ED413B 2019/37Ministerio de Educación, Cultura y Deporte | Ref. FPU16/00045Xunta de Galicia | Ref. ED481B2018/01

Localization of the co-expression of glucokinase and peptides related to food intake control in rainbow trout brain

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017Peer reviewe

Brain monoaminergic neurotransmitters during chronic stress in rainbow trout (Oncorhynchus mykiss)

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017Stress is negatively affecting animal welfare in such a way that most behavioral and physiological functions, such as food intake, are jeopardized. In fish, the stress response initiates with the activation of the hypothalamus-sympathetic-cromaffin (HPC), and the hypothalamus-pituitary-interrenal cells (HPI) axes leading to increased plasma catecholamines and cortisol levels. Our previous results point to the important role played by both dopaminergic and serotonergic systems in initiating the response to acute stress. However, little is known regarding the involvement of both systems under chronic stress and their influence on food intake. To address that question an experiment was performed consisting on stressing rainbow trout (Oncorhynchus mykiss) by high stocking density for 3 and 10 days. Food intake was evaluated all over the experiment in non-stressed and stressed fish. Plasma levels of cortisol, glucose and lactate, the content of DA and 5HT, and their main metabolites (DOPAC and 5HIAA) in different brain regions, and hypothalamic mRNA abundance of TH, TPH1 were assessed. Our results reveal that stressed animals showed reduced food intake, increased plasma cortisol levels, and enhanced dopaminergic and serotonergic activities in telencephalon, hypothalamus, optic tectum and hindbrain, independently of the stress duration. TH and TPH1 mRNA abundance also increased in stressed trout. Our present results support the hypothesis of a key role played by both dopaminergic and serotonergic systems in initiating and maintaining the neuroendocrine response to stress. In addition their involvement in mediating the stress-related food intake inhibition might be taken in consideration.Funded by Ministerio de Economía y Competitividad and European Fund for Regional Development (AGL2013-46448-3-1-R and FEDER) and AEO-ECIMAT. C. Otero-Rodiño was a recipient of FPI fellowship.Peer reviewe

Ghrelin modulates hypothalamic fatty acid-sensing and control of food intake in rainbow trout

There is no information available on fish as far as the possible effects of ghrelin on hypothalamic fatty acid metabolism and the response of fatty acid-sensing systems, which are involved in the control of food intake. Therefore, we assessed in rainbow trout the response of food intake, hypothalamic fatty acid-sensing mechanisms and expression of neuropeptides involved in the control of food intake to the central treatment of ghrelin in the presence or absence of a long-chain fatty acid such as oleate. We observed that the orexigenic actions of ghrelin in rainbow trout are associated with changes in fatty acid metabolism in the hypothalamus and an inhibition of fatty acid-sensing mechanisms, which ultimately lead to changes in the expression of anorexigenic and orexigenic peptides resulting in increased orexigenic potential and food intake. Moreover, the response to increased levels of oleate of hypothalamic fatty acid-sensing systems (activation), expression of neuropeptides (enhanced anorexigenic potential) and food intake (decrease) were counteracted by the simultaneous treatment with ghrelin. These changes provide evidence for the first time in fish of a possible modulatory role of ghrelin on the metabolic regulation by fatty acid of food intake occurring in the hypothalamusThis study was supported by research grants from Ministerio de Economía y Competitividad (Spain) and European Fund for Regional Development to J L S (AGL2013-46448-C3-1-R and FEDER) and J M C-R (AGL2013-46448-C3-3-R and FEDER). M L-P and C O-R were recipients of pre-doctoral fellowships from the Ministerio de Economía y Competitividad (BES-2011-043394 and BES-2014-068040 respectively).Peer reviewe

Glucosensing capacity of rainbow trout telencephalon

To assess the hypothesis of glucosensing systems present in fish telencephalon, we first demonstrated in rainbow trout, by in situ hybridisation, the presence of glucokinase (GK). Then, we assessed the response of glucosensing markers in rainbow trout telencephalon 6 hours after i.c.v. treatment with glucose or 2‐deoxyglucose (inducing glucoprivation). We evaluated the response of parameters related to the mechanisms dependent on GK, liver X receptor (LXR), mitochondrial activity, sweet taste receptor and sodium‐glucose linked transporter 1 (SGLT‐1). We also assessed mRNA abundance of neuropeptides involved in the metabolic control of food intake (agouti‐related protein, neuropeptide Y, pro‐opiomelanocortin, and cocaine‐ and amphetamine‐related transcript), as well as the abundance and phosphorylation status of proteins possibly involved in linking glucosensing with neuropeptide expression, such as protein kinase B (AkT), AMP‐activated protein kinase (AMPK), mechanistic target of rapamycin and cAMP response element‐binding protein (CREB). The responses obtained support the presence in the telencephalon of a glucosensing mechanism based on GK and maybe one based on LXR, although they do not support the presence of mechanisms dependent on mitochondrial activity and SGLT‐1. The mechanism based on sweet taste receptor responded to glucose but in a converse way to that characterised previously in the hypothalamus. In general, systems responded only to glucose but not to glucoprivation. Neuropeptides did not respond to glucose or glucoprivation. By contrast, the presence of glucose activates Akt and inhibits AMPK, CREB and forkhead box01. This is the first study in any vertebrate species in which the response to glucose of putative glucosensing mechanisms is demonstrated in the telencephalon. Their role might relate to processes other than homeostatic control of food intake, such as the hedonic and reward systemSpanish Agencia Estatal de Investigacion and FEDER, Grant/Award Number: AGL2016-74857-C3-1-R, AGL2016-74857-C3-3-R and BES -2014 -068040; the European Fund of Regional DevelopmentPeer reviewe

Evidence for the Presence of Glucosensor Mechanisms Not Dependent on Glucokinase in Hypothalamus and Hindbrain of Rainbow Trout (<i>Oncorhynchus mykiss</i>)

<div><p>We hypothesize that glucosensor mechanisms other than that mediated by glucokinase (GK) operate in hypothalamus and hindbrain of the carnivorous fish species rainbow trout and stress affected them. Therefore, we evaluated in these areas changes in parameters which could be related to putative glucosensor mechanisms based on liver X receptor (LXR), mitochondrial activity, sweet taste receptor, and sodium/glucose co-transporter 1 (SGLT-1) 6h after intraperitoneal injection of 5 mL.Kg^-1 of saline solution alone (normoglycaemic treatment) or containing insulin (hypoglycaemic treatment, 4 mg bovine insulin.Kg^-1 body mass), or D-glucose (hyperglycaemic treatment, 500 mg.Kg^-1 body mass). Half of tanks were kept at a 10 Kg fish mass.m^-3 and denoted as fish under normal stocking density (NSD) whereas the remaining tanks were kept at a stressful high stocking density (70 kg fish mass.m^-3) denoted as HSD. The results obtained in non-stressed rainbow trout provide evidence, for the first time in fish, that manipulation of glucose levels induce changes in parameters which could be related to putative glucosensor systems based on LXR, mitochondrial activity and sweet taste receptor in hypothalamus, and a system based on SGLT-1 in hindbrain. Stress altered the response of parameters related to these systems to changes in glycaemia.</p></div

Activities of CPT-1 (A) and HOAD (D), and mRNA abundance of CPT1c (B), CPT1d (C), HOAD (E), COX4 (F), and UCP2a (G) in hypothalamus of rainbow trout.

<p>Further details as in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128603#pone.0128603.g003" target="_blank">Fig 3</a>.</p