RESUMO
The hormone ghrelin displays several well-characterized functions, including some with pharmaceutical interest. The receptor for ghrelin, the growth hormone secretagogue receptor (GHSR), is expressed in the hypothalamic paraventricular nucleus (PVH), a critical hub for the integration of metabolic, neuroendocrine, autonomic, and behavioral functions. Here, we performed a neuroanatomical and functional characterization of the neuronal types mediating ghrelin actions in the PVH of male mice. We found that fluorescent ghrelin mainly labels PVH neurons immunoreactive for nitric oxide synthase 1 (NOS1), which catalyze the production of nitric oxide [NO]). Centrally injected ghrelin increases c-Fos in NOS1 PVH neurons and NOS1 phosphorylation in the PVH. We also found that a high dose of systemically injected ghrelin increases the ghrelin level in the cerebrospinal fluid and in the periventricular PVH, and induces c-Fos in NOS1 PVH neurons. Such a high dose of systemically injected ghrelin activates a subset of NOS1 PVH neurons, which do not express oxytocin, via an arcuate nucleus-independent mechanism. Finally, we found that pharmacological inhibition of NO production fully abrogates ghrelin-induced increase of calcium concentration in corticotropin-releasing hormone neurons of the PVH whereas it partially impairs ghrelin-induced increase of plasma glucocorticoid levels. Thus, plasma ghrelin can directly target a subset of NO-producing neurons of the PVH that is involved in ghrelin-induced activation of the hypothalamic-pituitary-adrenal neuroendocrine axis.
Assuntos
Hormônio Liberador da Corticotropina , Grelina , Camundongos , Masculino , Animais , Hormônio Liberador da Corticotropina/metabolismo , Grelina/farmacologia , Grelina/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Neurônios/metabolismoRESUMO
Ghrelin is a stomach-derived hormone that acts via the growth hormone secretagogue receptor (GHSR). Recent evidence suggests that some of ghrelin's actions may be mediated via the supramammillary nucleus (SuM). Not only does ghrelin bind to cells within the mouse SuM, but ghrelin also activates SuM cells and intra-SuM ghrelin administration induces feeding in rats. In the current study, we aimed to further characterize ghrelin action in the SuM. We first investigated a mouse model expressing enhanced green fluorescent protein (eGFP) under the promoter of GHSR (GHSR-eGFP mice). We found that the SuM of GHSR-eGFP mice contains a significant amount of eGFP cells, some of which express neuronal nitric oxide synthase. Centrally-, but not systemically-, injected ghrelin reached the SuM, where it induced c-Fos expression. Furthermore, a 5-day 40% calorie restriction protocol, but not a 2-day fast, increased c-Fos expression in non-eGFP+ cells of the SuM of GHSR-eGFP mice, whereas c-Fos induction by calorie restriction was not observed in GHSR-deficient mice. Exposure of satiated mice to a binge-like eating protocol also increased c-Fos expression in non-eGFP+ cells of the SuM of GHSR-eGFP mice in a GHSR-dependent manner. Finally, intra-SuM-injected ghrelin did not acutely affect food intake, locomotor activity, behavioral arousal or spatial memory but increased recognition memory. Thus, we provide a compelling neuroanatomical characterization of GHSR SuM neurons and its behavioral implications in mice.
Assuntos
Neurônios , Óxido Nítrico , Receptores de Grelina , Animais , Grelina/metabolismo , Hipotálamo Posterior , Camundongos , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Ratos , Receptores de Grelina/metabolismo , Transdução de SinaisRESUMO
Liver-expressed antimicrobial peptide 2 (LEAP2) was recently recognized as an endogenous ligand for the growth hormone secretagogue receptor (GHSR), which also is a receptor for the hormone ghrelin. LEAP2 blocks ghrelin-induced activation of GHSR and inhibits GHSR constitutive activity. Since fluorescence-based imaging and pharmacological analyses to investigate the biology of GHSR require reliable probes, we developed a novel fluorescent GHSR ligand based on the N-terminal LEAP2 sequence, hereafter named F-LEAP2. In vitro, F-LEAP2 displayed binding affinity and inverse agonism to GHSR similar to LEAP2. In a heterologous expression system, F-LEAP2 labeling was specifically observed in the surface of GHSR-expressing cells, in contrast to fluorescent ghrelin labeling that was mainly observed inside the GHSR-expressing cells. In mice, centrally-injected F-LEAP2 reduced ghrelin-induced food intake, in a similar fashion to LEAP2, and specifically labeled cells in GHSR-expressing brain areas. Thus, F-LEAP2 represents a valuable tool to study the biology of GHSR in vitro and in vivo.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Encéfalo/metabolismo , Corantes Fluorescentes/química , Grelina/metabolismo , Rim/metabolismo , Animais , Células Cultivadas , Ingestão de Alimentos , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Domínios Proteicos , Transdução de SinaisRESUMO
Ghrelin is a hormone produced in the gastrointestinal tract that acts via the growth hormone secretagogue receptor. In the central nervous system, ghrelin signalling is able to recruit different neuronal targets that regulate the behavioural, neuroendocrine, metabolic and autonomic effects of the hormone. Notably, several studies using radioactive or fluorescent variants of ghrelin have found that the accessibility of circulating ghrelin into the mouse brain is both strikingly low and restricted to some specific brain areas. A variety of studies addressing central effects of systemically injected ghrelin in mice have also provided indirect evidence that the accessibility of plasma ghrelin into the brain is limited. Here, we review these previous observations and discuss the putative pathways that would allow plasma ghrelin to gain access into the brain together with their physiological implications. Additionally, we discuss some potential features regarding the accessibility of plasma ghrelin into the human brain based on the observations reported by studies that investigate the consequences of ghrelin administration to humans.
Assuntos
Encéfalo/fisiologia , Grelina/fisiologia , Neurônios/fisiologia , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Grelina/sangue , Humanos , Neurônios/metabolismoRESUMO
Ghrelin is a potent orexigenic peptide hormone that acts through the growth hormone secretagogue receptor (GHSR), a G protein-coupled receptor highly expressed in the hypothalamus. In vitro studies have shown that GHSR displays a high constitutive activity, whose physiological relevance is uncertain. As GHSR gene expression in the hypothalamus is known to increase in fasting conditions, we tested the hypothesis that constitutive GHSR activity at the hypothalamic level drives the fasting-induced hyperphagia. We found that refed wild-type (WT) mice displayed a robust hyperphagia that continued for 5 days after refeeding and changed their food intake daily pattern. Fasted WT mice showed an increase in plasma ghrelin levels, as well as in GHSR expression levels and ghrelin binding sites in the hypothalamic arcuate nucleus. When fasting-refeeding responses were evaluated in ghrelin- or GHSR-deficient mice, only the latter displayed an â¼15% smaller hyperphagia, compared with WT mice. Finally, fasting-induced hyperphagia of WT mice was significantly smaller in mice centrally treated with the GHSR inverse agonist K-(D-1-Nal)-FwLL-NH2, compared with mice treated with vehicle, whereas it was unaffected in mice centrally treated with the GHSR antagonists D-Lys3-growth hormone-releasing peptide 6 or JMV2959. Taken together, genetic models and pharmacological results support the notion that constitutive GHSR activity modulates the magnitude of the compensatory hyperphagia triggered by fasting. Thus, the hypothalamic GHSR signaling system could affect the set point of daily food intake, independently of plasma ghrelin levels, in situations of negative energy balance.
Assuntos
Jejum/fisiologia , Grelina/fisiologia , Hiperfagia/etiologia , Receptores de Grelina/fisiologia , Animais , Ingestão de Alimentos/fisiologia , Grelina/metabolismo , Hiperfagia/genética , Hiperfagia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Grelina/genética , Receptores de Grelina/metabolismo , Transdução de Sinais/genéticaRESUMO
Ghrelin is an octanoylated peptide that acts via its specific receptor, the growth hormone secretagogue receptor type 1a (GHSR-1a), and regulates a vast variety of physiological functions. It is well established that ghrelin is predominantly synthesized by a distinct population of endocrine cells located within the gastric oxyntic mucosa. In addition, some studies have reported that ghrelin could also be synthesized in some brain regions, such as the hypothalamus. However, evidences of neuronal production of ghrelin have been inconsistent and, as a consequence, it is still as a matter of debate if ghrelin can be centrally produced. Here, we provide a comprehensive review and discussion of the data supporting, or not, the notion that the mammalian central nervous system can synthetize ghrelin. We conclude that no irrefutable and reproducible evidence exists supporting the notion that ghrelin is synthetized, at physiologically relevant levels, in the central nervous system of adult mammals.
Assuntos
Sistema Nervoso Central/metabolismo , Grelina/biossíntese , Animais , Expressão Gênica , Técnicas de Inativação de Genes , Grelina/genética , Humanos , Neurônios/metabolismo , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Ghrelin is known to act on the area postrema (AP), a sensory circumventricular organ located in the medulla oblongata that regulates a variety of important physiological functions. However, the neuronal targets of ghrelin in the AP and their potential role are currently unknown. In this study, we used wild-type and genetically modified mice to gain insights into the neurons of the AP expressing the ghrelin receptor [growth hormone secretagogue receptor (GHSR)] and their role. We show that circulating ghrelin mainly accesses the AP but not to the adjacent nucleus of the solitary tract. Also, we show that both peripheral administration of ghrelin and fasting induce an increase of c-Fos, a marker of neuronal activation, in GHSR-expressing neurons of the AP, and that GHSR expression is necessary for the fasting-induced activation of AP neurons. Additionally, we show that ghrelin-sensitive neurons of the AP are mainly γ-aminobutyric acid (GABA)ergic, and that an intact AP is required for ghrelin-induced gastric emptying. Overall, we show that the capacity of circulating ghrelin to acutely induce gastric emptying in mice requires the integrity of the AP, which contains a population of GABA neurons that are a target of plasma ghrelin.
Assuntos
Área Postrema/fisiologia , Neurônios GABAérgicos/fisiologia , Grelina/sangue , Animais , Área Postrema/efeitos dos fármacos , Jejum , Neurônios GABAérgicos/efeitos dos fármacos , Esvaziamento Gástrico/efeitos dos fármacos , Grelina/administração & dosagem , Grelina/metabolismo , Masculino , Camundongos , Proteínas Proto-Oncogênicas c-fos/genética , Receptores de Grelina/genética , Receptores de Grelina/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Previous work has established that the hormone ghrelin engages the hypothalamic-pituitary-adrenal neuroendocrine axis via activation of corticotropin-releasing factor (CRF) neurons of the hypothalamic paraventricular nucleus (PVN). The neuronal circuitry that mediates this effect of ghrelin is currently unknown. Here, we show that ghrelin-induced activation of PVN CRF neurons involved inhibition of γ-aminobutyric acid (GABA) inputs, likely via ghrelin binding sites that were localized at GABAergic terminals within the PVN. While ghrelin activated PVN CRF neurons in the presence of neuropeptide Y (NPY) receptor antagonists or in arcuate nucleus (ARC)-ablated mice, it failed to do it so in mice with ghrelin receptor expression limited to ARC agouti gene related protein (AgRP)/NPY neurons. These data support the notion that ghrelin activates PVN CRF neurons via inhibition of local GABAergic tone, in an ARC-independent manner. Furthermore, these data suggest that the neuronal circuits mediating ghrelin's orexigenic action vs. its role as a stress signal are anatomically dissociated.
Assuntos
Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Hormônio Liberador da Corticotropina/metabolismo , Grelina/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Corticosterona/sangue , Antagonistas GABAérgicos , Técnicas de Silenciamento de Genes , Grelina/administração & dosagem , Infusões Intraventriculares , Masculino , Camundongos , Muscimol/farmacologia , Neuropeptídeo Y/antagonistas & inibidores , Núcleo Hipotalâmico Paraventricular/metabolismo , Receptores de Grelina/efeitos dos fármacos , Receptores de Grelina/genética , Receptores de Grelina/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
The growth hormone secretagogue receptor type 1a (GHSR1a) has the highest known constitutive activity of any G protein-coupled receptor (GPCR). GHSR1a mediates the action of the hormone ghrelin, and its activation increases transcriptional and electrical activity in hypothalamic neurons. Although GHSR1a is present at GABAergic presynaptic terminals, its effect on neurotransmitter release remains unclear. The activities of the voltage-gated calcium channels, CaV2.1 and CaV2.2, which mediate neurotransmitter release at presynaptic terminals, are modulated by many GPCRs. Here, we show that both constitutive and agonist-dependent GHSR1a activity elicit a strong impairment of CaV2.1 and CaV2.2 currents in rat and mouse hypothalamic neurons and in a heterologous expression system. Constitutive GHSR1a activity reduces CaV2 currents by a Gi/o-dependent mechanism that involves persistent reduction in channel density at the plasma membrane, whereas ghrelin-dependent GHSR1a inhibition is reversible and involves altered CaV2 gating via a Gq-dependent pathway. Thus, GHSR1a differentially inhibits CaV2 channels by Gi/o or Gq protein pathways depending on its mode of activation. Moreover, we present evidence suggesting that GHSR1a-mediated inhibition of CaV2 attenuates GABA release in hypothalamic neurons, a mechanism that could contribute to neuronal activation through the disinhibition of postsynaptic neurons.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Grelina/metabolismo , Hipotálamo/fisiologia , Neurônios/fisiologia , Receptores de Grelina/metabolismo , Animais , Sequência de Bases , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Células Cultivadas , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Camundongos , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Receptores de Grelina/genéticaRESUMO
The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor involved in food intake and energy expenditure regulation. MC4R activation modifies neuronal activity but the molecular mechanisms by which this regulation occurs remain unclear. Here, we tested the hypothesis that MC4R activation regulates the activity of voltage-gated calcium channels and, as a consequence, synaptic activity. We also tested whether the proposed effect occurs in the amygdala, a brain area known to mediate the anorexigenic actions of MC4R signaling. Using the patch-clamp technique, we found that the activation of MC4R with its agonist melanotan II specifically inhibited 34.5 ± 1.5% of N-type calcium currents in transiently transfected HEK293 cells. This inhibition was concentration-dependent, voltage-independent and occluded by the Gαs pathway inhibitor cholera toxin. Moreover, we found that melanotan II specifically inhibited 25.9 ± 2.0% of native N-type calcium currents and 55.4 ± 14.4% of evoked inhibitory postsynaptic currents in mouse cultured amygdala neurons. In vivo, we found that the MC4R agonist RO27-3225 increased the marker of cellular activity c-Fos in several components of the amygdala, whereas the N-type channel blocker ω conotoxin GVIA increased c-Fos expression exclusively in the central subdivision of the amygdala. Thus, MC4R specifically inhibited the presynaptic N-type channel subtype, and this inhibition may be important for the effects of melanocortin in the central subdivision of the amygdala.
Assuntos
Tonsila do Cerebelo/fisiologia , Canais de Cálcio Tipo N/metabolismo , Terminações Pré-Sinápticas/fisiologia , Receptor Tipo 4 de Melanocortina/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Fármacos do Sistema Nervoso Central/farmacologia , Toxina da Cólera/farmacologia , Células HEK293 , Humanos , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Camundongos , Peptídeos/farmacologia , Peptídeos Cíclicos/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptor Tipo 4 de Melanocortina/agonistas , alfa-MSH/análogos & derivados , alfa-MSH/metabolismo , ômega-Conotoxina GVIA/farmacologiaRESUMO
The neuropeptide thyrotropin releasing hormone (TRH) is necessary for adequate cold-induced thermogenesis. TRH increases body temperature via both neuroendocrine and autonomic mechanisms. TRH neurons of the hypothalamic paraventricular nucleus (PVN) regulate thermogenesis through the activation of the hypothalamic-pituitary-thyroid axis during cold exposure. However, little is known about the role that TRH neurons play in mediating the sympathetic response to cold exposure. Here, we examined the response of TRH neurons of rats to cold exposure in hypothalamic regions including the PVN, the dorsomedial nucleus and the lateral hypothalamus along with areas of the ventral medulla including raphe obscurus, raphe pallidus (RPa) and parapyramidal regions. Our results using a double immunohistochemistry protocol to identify TRH and c-Fos (as a marker of cellular activity) followed by analysis of preproTRH gene expression demonstrate that only TRH neurons located in the PVN and the RPa are activated in animals exposed to short-term cold conditions.
Assuntos
Temperatura Baixa , Hipotálamo/fisiologia , Neurônios/metabolismo , Termogênese/fisiologia , Hormônio Liberador de Tireotropina/metabolismo , Animais , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Ghrelin is a stomach-derived hormone that regulates food intake and neuroendocrine function by acting on its receptor, GHSR (Growth Hormone Secretagogue Receptor). Recent evidence indicates that a key function of ghrelin is to signal stress to the brain. It has been suggested that one of the potential stress-related ghrelin targets is the CRF (Corticotropin-Releasing Factor)-producing neurons of the hypothalamic paraventricular nucleus, which secrete the CRF neuropeptide into the median eminence and activate the hypothalamic-pituitary-adrenal axis. However, the neural circuits that mediate the ghrelin-induced activation of this neuroendocrine axis are mostly uncharacterized. In the current study, we characterized in vivo the mechanism by which ghrelin activates the hypophysiotropic CRF neurons in mice. We found that peripheral or intra-cerebro-ventricular administration of ghrelin strongly activates c-fos--a marker of cellular activation--in CRF-producing neurons. Also, ghrelin activates CRF gene expression in the paraventricular nucleus of the hypothalamus and the hypothalamic-pituitary-adrenal axis at peripheral level. Ghrelin administration directly into the paraventricular nucleus of the hypothalamus also induces c-fos within the CRF-producing neurons and the hypothalamic-pituitary-adrenal axis, without any significant effect on the food intake. Interestingly, dual-label immunohistochemical analysis and ghrelin binding studies failed to show GHSR expression in CRF neurons. Thus, we conclude that ghrelin activates hypophysiotropic CRF neurons, albeit indirectly.