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Background The activation of the hypothalamic–pituitary–adrenal (HPA) axis is essential for metabolic adaptation in response to fasting. However, the neurocircuitry connecting changes in the peripheral energy stores to the activity of hypothalamic paraventricular corticotrophin-releasing factor (CRFPVN) neurons, the master controller of the HPA axis activity, is not completely understood. Our main goal was to determine if hypothalamic arcuate nucleus (ARC) POMC and AgRP neurons can communicate fasting-induced changes in peripheral energy stores, associated to a fall in plasma leptin levels, to CRFPVN neurons to modulate the HPA axis activity in mice. Results We observed increased plasma corticosterone levels associate with increased CRFPVN mRNA expression and increased CRFPVN neuronal activity in 36 h fasted mice. These responses were associated with a fall in plasma leptin levels and changes in the mRNA expression of Agrp and Pomc in the ARC. Fasting-induced decrease in plasma leptin partially modulated these responses through a change in the activity of ARC neurons. The chemogenetic activation of POMCARC by DREADDs did not affect fasting-induced activation of the HPA axis. DREADDs inhibition of AgRPARC neurons reduced the content of CRFPVN and increased its accumulation in the median eminence but had no effect on corticosterone secretion induced by fasting. Conclusion Our data indicate that AgRPARC neurons are part of the neurocircuitry involved in the coupling of PVNCRF activity to changes in peripheral energy stores induced by prolonged fasting.

The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor that regulates essential physiological functions. In particular, activation of GHSR in response to its endogenous agonist ghrelin promotes food intake and blood glucose increase. Therefore, compounds aimed at blocking GHSR signaling constitute potential options against obesity-related metabolic disorders. We have previously developed potent ligands of GHSR based on a triazole scaffold. Here, we report a new 3,4,5-trisubstituted 1,2,4-triazole compound, named JMV 6616, that potently blocks GHSR activity in vitro and in vivo. Specifically, in HEK293T cells JMV 6616 behaves as an inverse agonist since it binds to GHSR and inhibits its ghrelin-independent signaling. Accordingly, using purified labeled GHSR assembled into lipid nanodiscs we found that JMV 6616 decreases GHSR-catalyzed G protein activation and stabilizes an inactive receptor conformation. Importantly, JMV 6616 also acts on native GHSR since it blocks the insulinostatic effect of ghrelin in pancreatic islets. In mice, JMV 6616 inhibits blood glucose-raising effects of ghrelin treatment and the orexigenic actions of acute ghrelin administration. Together, our data suggest that this triazole-derived modulator of GHSR holds promise to mitigate several pathological features associated with eating and metabolic disorders.

Introduction: Ghrelin regulates a variety of functions by acting in the brain. The targets of ghrelin in the mouse brain have been mainly mapped using immunolabeling against c-Fos, a transcription factor used as a marker of cellular activation, but such analysis has several limitations. Here, we used positron emission tomography in mice to investigate the brain areas responsive to ghrelin. Methods: We analyzed in male mice the brain areas responsive to systemically injected ghrelin using positron emission tomography imaging of 18F-fluoro-2-deoxyglucose (18F-FDG) uptake, an indicator of metabolic rate. Additionally, we studied if systemic administration of fluorescent ghrelin or native ghrelin displays symmetric accessibility or induction of c-Fos, respectively, in the brain of male mice. Results: Ghrelin increased 18F-FDG uptake in few specific areas of the isocortex, striatum, pallidum, thalamus, and midbrain at 0–10-min posttreatment. At the 10–20 and 20–30 min posttreatment, ghrelin induced mixed changes in 18F-FDG uptake in specific areas of the isocortex, striatum, pallidum, thalamus, and midbrain, as well as in areas of the olfactory areas, hippocampal and retrohippocampal regions, hypothalamus, pons, medulla, and even the cerebellum. Ghrelin-induced changes in 18F-FDG uptake were transient and asymmetric. Systemically administrated fluorescent-ghrelin-labeled midline brain areas known to contain fenestrated capillaries and the hypothalamic arcuate nucleus, where a symmetric labeling was observed. Ghrelin treatment also induced a symmetric increased c-Fos labeling in the arcuate nucleus. Discussion/Conclusion: Systemically injected ghrelin transiently and asymmetrically affects the metabolic activity of the brain of male mice in a wide range of areas, in a food intake-independent manner. The neurobiological bases of such asymmetry seem to be independent of the accessibility of ghrelin into the brain.

Prolonged fasting is a major challenge for living organisms. An appropriate metabolic response to food deprivation requires the activation of the corticotropin-releasing factor-producing neurons of the hypothalamic paraventricular nucleus (PVHWe estimated the activation of the PVHWe found that food deprivation results in the activation of the PVHFood deprivation-induced activation of the PVH

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.

Objective Arcuate nucleus (ARC) neurons producing Agouti-related peptide (AgRP) and neuropeptide Y (NPY; ARCAgRP/NPY neurons) are activated under energy-deficit states. ARCAgRP/NPY neurons innervate the hypothalamic paraventricular nucleus (PVH), and ARC→PVH projections are recognized as key regulators of food intake. Plasma ghrelin levels increase under energy-deficit states and activate ARCAgRP/NPY neurons by acting on the growth hormone secretagogue receptor (GHSR). Here, we hypothesized that activation of ARCAgRP/NPY neurons in fasted mice would promote morphological remodeling of the ARCAgRP/NPY→PVH projections in a GHSR-dependent manner. Methods We performed 1) fluorescent immunohistochemistry, 2) imaging of green fluorescent protein (GFP) signal in NPY-GFP mice, and 3) DiI axonal labeling in brains of ad libitum fed or fasted mice with pharmacological or genetic blockage of the GHSR signaling and then estimated the density and strength of ARCAgRP/NPY→PVH fibers by assessing the mean fluorescence intensity, the absolute area with fluorescent signal, and the intensity of the fluorescent signal in the fluorescent area of the PVH. Results We found that 1) the density and strength of ARCAgRP/NPY fibers increase in the PVH of fasted mice, 2) the morphological remodeling of the ARCAgRP/NPY→PVH projections correlates with the activation of PVH neurons, and 3) PVH neurons are not activated in ARC-ablated mice. We also found that fasting-induced remodeling of ARCAgRP/NPY→PVH fibers and PVH activation are impaired in mice with pharmacological or genetic blockage of GHSR signaling. Conclusion This evidence shows that the connectivity between hypothalamic circuits controlling food intake can be remodeled in the adult brain, depending on the energy balance conditions, and that GHSR activity is a key regulator of this phenomenon., Graphical abstract Image 1, HIGHLIGHTS • The density and strength of ARCAgRP/NPY→PVH fibers increase in fasted mice. • Remodeling of ARCAgRP/NPY→PVH projections correlates with the activation of PVH neurons. • GHSR signaling is required for fasting-induced ARCAgRP/NPY→PVH projection remodeling.

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.

Ghrelin is an octanoylated peptide hormone that potently and rapidly increases food intake. The orexigenic action of ghrelin involves the hypothalamic arcuate nucleus (ARC), which is accessible to plasma ghrelin and expresses high levels of the ghrelin receptor. Local administration of ghrelin in a variety of other brain nuclei also increases food intake. It is currently unclear, however, whether these non-ARC ghrelin brain targets are impacted by physiological increases of plasma ghrelin. Thus, the present study aimed to clarify which ghrelin brain targets participate in the short-term orexigenic actions of ghrelin. First, c-Fos induction into mouse brains centrally or peripherally treated with ghrelin was analysed. It was confirmed that peripherally administered ghrelin dose-dependently increases food intake and mainly activates c-Fos in ARC neurones. By contrast, centrally administered ghrelin activates c-Fos in a larger number of brain nuclei. To determine which nuclei are directly accessible to ghrelin, mice were centrally or peripherally injected with a fluorescent ghrelin tracer. It was found that peripherally injected tracer mainly accesses the ARC, whereas centrally injected tracer reaches most brain areas known to express ghrelin receptors. Subsequently, the effects of ghrelin were tested in ARC-ablated mice and it was found that these mice failed to increase food intake in response to peripherally administered ghrelin but fully responded to centrally administered ghrelin. ARC-ablated mice showed patterns of ghrelin-induced c-Fos expression similar to those seen in control mice with the exception of the ARC, where no c-Fos was found. Thus, peripheral ghrelin mainly accesses the ARC, which is required for the orexigenic effects of the hormone. Central ghrelin accesses a variety of nuclei, which can mediate the orexigenic effects of the hormone, even in the absence of an intact ARC. Fil: Cabral, Agustina Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina Fil: Valdivia Torres, Lesly Spring. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina Fil: Fernandez, Gimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina Fil: Reynaldo, Mirta Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina Fil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina

Ghrelin is a stomach-derived peptide hormone that acts in the brain to regulate many important physiological functions. Ghrelin receptor, named the growth hormone secretagogue receptor (GHSR), is present in many brain areas with or without obvious direct access to ghrelin circulating in the bloodstream. Ghrelin is also present in the cerebrospinal fluid (CSF) but the brain targets of CSF ghrelin are unclear. Here, we studied which brain areas are accessible to ghrelin present in the CSF. For this purpose, we centrally injected mice with fluorescein-labeled ghrelin (F-ghrelin) peptide tracer and then systematically mapped the distribution of F-ghrelin signal through the brain. Our results indicated that centrally injected F-ghrelin labels neurons in most of the brain areas where GHSR is present. Also, we detected F-ghrelin uptake in the ependymal cells of both wild-type and GHSR-null mice. We conclude that CSF ghrelin is able to reach most of brain areas expressing GHSR. Also, we propose that the accessibility of CSF ghrelin to the brain parenchyma occurs through the ependymal cells in a GHSR-independent manner. Fil: Cabral, Agustina Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina Fil: Fernandez, Gimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina Fil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina

Ghrelin is an octanoylated peptide hormone that plays a key role in the regulation of the body weight and glucose homeostasis. In plasma, ghrelin circulates bound to larger proteins whose identities are partially established. Here, we used size exclusion chromatography, mass spectrometry and isothermal titration microcalorimetry to show that ghrelin interacts with serum albumin. Furthermore, we found that such interaction displays an estimated dissociation constant (KD) in the micromolar range and involves albumin fatty-acid binding sites as well as the octanoyl moiety of ghrelin. Notably, albumin-ghrelin interaction reduces the spontaneous deacylation of the hormone. Both in vitro experiments-assessing ghrelin ability to inhibit calcium channels-and in vivo studies-evaluating ghrelin orexigenic effects-indicate that the binding to albumin affects the bioactivity of the hormone. In conclusion, our results suggest that ghrelin binds to serum albumin and that this interaction impacts on the biological activity of the hormone. Fil: Lufrano, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Trejo, Sebastian Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Universitat Autònoma de Barcelona; España Fil: Llovera, Ramiro Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Salgueiro, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad Nacional de Quilmes; Argentina Fil: Fernandez, Gimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Martínez Damonte, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Raingo, Jesica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Ermacora, Mario Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Universidad Nacional de Quilmes; Argentina Fil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina