The pattern of growth hormone action in the mouse brain.

Growth hormone (GH) acts in numerous organs expressing the GH receptor (GHR), including the brain. However, the mechanisms behind the brain's permeability to GH and how this hormone accesses different brain regions remain unclear. It is well-known that an acute GH administration induces phosphorylation of the signal transducer and activator of transcription 5 (pSTAT5) in the mouse brain. Thus, the pattern of pSTAT5 immunoreactive cells was analyzed at different time points after intraperitoneal or intracerebroventricular GH injections. After a systemic GH injection, the first cells expressing pSTAT5 were those near circumventricular organs, such as arcuate nucleus neurons adjacent to the median eminence. Both systemic and central GH injections induced a medial-to-lateral pattern of pSTAT5 immunoreactivity over time, as GH-responsive cells were initially observed in periventricular areas and were progressively detected in lateral brain structures. Very few choroid plexus cells exhibited GH-induced pSTAT5. Additionally, Ghr mRNA was poorly expressed in the mouse choroid plexus. In contrast, some tanycytes lining the floor of the third ventricle expressed Ghr mRNA and exhibited GH-induced pSTAT5. The transport of radiolabeled GH into the hypothalamus did not differ between wild-type and dwarf Ghr knockout mice, indicating that GH transport into the mouse brain is GHR-independent. Also, single-photon emission computed tomography confirmed that radiolabeled GH rapidly reaches the ventral part of the tuberal hypothalamus. In conclusion, our study provides novel and valuable information about the pattern and mechanisms behind GH transport into the mouse brain.

Ghrelin Action in the PVH of Male Mice: Accessibility, Neuronal Targets, and CRH Neurons Activation.

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.

Ghrelin's orexigenic action in the lateral hypothalamic area involves indirect recruitment of orexin neurons and arcuate nucleus activation.

OBJECTIVE: Ghrelin is a potent orexigenic hormone, and the lateral hypothalamic area (LHA) has been suggested as a putative target mediating ghrelin's effects on food intake. Here, we aimed to investigate the presence of neurons expressing ghrelin receptor (a.k.a. growth hormone secretagogue receptor, GHSR) in the mouse LHA (LHAGHSR neurons), its physiological implications and the neuronal circuit recruited by local ghrelin action. METHODS: We investigated the distribution of LHAGHSR neurons using different histologic strategies, including the use of a reporter mice expressing enhanced green fluorescent protein under the control of the GHSR promoter. Also, we investigated the physiological implications of local injections of ghrelin within the LHA, and the extent to which the orexigenic effect of intra-LHA-injected ghrelin involves the arcuate nucleus (ARH) and orexin neurons of the LHA (LHAorexin neurons) RESULTS: We found that: 1) LHAGHSR neurons are homogeneously distributed throughout the entire LHA; 2) intra-LHA injections of ghrelin transiently increase food intake and locomotor activity; 3) ghrelin's orexigenic effect in the LHA involves the indirect recruitment of LHAorexin neurons and the activation of ARH neurons; and 4) LHAGHSR neurons are not targeted by plasma ghrelin. CONCLUSIONS: We provide a compelling neuroanatomical and functional characterization of LHAGHSR neurons in male mice that indicates that LHAGHSR cells are part of a hypothalamic neuronal circuit that potently induces food intake.

Systemic Ghrelin Treatment Induces Rapid, Transient, and Asymmetric Changes in the Metabolic Activity of the Mouse Brain.

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.

Rol de la Comisión Honoraria para la Lucha Antituberculosa y Enfermedades Prevalentes en la Estrategia Nacional de Vacunación contra la COVID-19 en Uruguay: período noviembre 2020 - julio 2021 / Role of the Honorary Commission for the Fight against Tuberculosis and Prevalent Diseases (CHLA-EP) in the National Vaccination Strategy against COVID-19 in Uruguay: november 2020 - July 2021 / Papel da Comissão Honorária de Luta contra a Tuberculose e Doenças Prevalentes (CHLA-EP) na Estratégia Nacional de Vacinação contra a COVID-19 no Uruguai: período de novembro de 2020 a julho de 2021

La pandemia por la infección por el nuevo coronavirus SARS-CoV-2 determinó que en Uruguay a inicio de marzo de 2020 el gobierno diseñara el Plan Nacional de Contingencia para la enfermedad COVID-19 causada por este virus. Este plan incluyó medidas preventivas no farmacológicas como: higiene de manos, distanciamiento físico, uso de mascarilla de diferentes tipos de acuerdo al riesgo de quien debía utilizarlos, en algunos períodos restricciones varias sobre circulación de personas, actividades educativas o espectáculos públicos, y tránsitos por tierra, aire o mar. Se apeló fuertemente a la libertad responsable. A partir de diciembre 2020 se decidió incorporar a la prevención primaria una Estrategia de Vacunación. El 1/3/2021 se inició la Campaña Vacunación COVID-19 2021, que fue no obligatoria, poblacional, inicialmente para personas mayores de 18 años y a partir de junio incluyó adolescentes entre 12 y 17 años y mujeres embarazadas. El Grupo Asesor Científico Honorario (GACH) del Gobierno Nacional junto a la Comisión Nacional Asesora de Vacunas (CNAV) del Ministerio de Salud Pública (MSP) que cuenta con largos años de aconsejar a las autoridades sanitarias en decisiones científicas sobre la introducción de vacunas, y una Comisión ad Hoc creada para vacunas COVID-19 reunieron a expertos del país para asesorar sobre la estrategia de vacunación. La Comisión Honoraria para la Lucha Antituberculosa y Enfermedades Prevalentes (CHLA-EP) como brazo operativo del Departamento de Inmunizaciones del MSP estuvo desde el mes de diciembre involucrada con todos los actores para implementar la Campaña de Vacunación poblacional COVID-19 sin descuidar las tareas que le competen en la vacunación para prevenir otras enfermedades infecciosas. Metodología: se realizó un estudio descriptivo, retrospectivo de las estrategias implementadas por el Dpto. Operativo de Inmunizaciones (brazo operativo del Departamento de Inmunizaciones del MSP), Laboratorio A. Calmette, Comisión Honoraria, Coordinación General y los departamentos administrativos: entre los meses de noviembre de 2020 a julio de 2021. Fuente de datos: informes difundidos o aportados especialmente para este trabajo por el Dpto. Operativo de Inmunizaciones, Laboratorio A. Calmette, Dpto. de Inmunizaciones del Ministerio de Salud Pública, Recursos Humanos, Recursos Materiales, Servicio de Apoyo, Comunicación y Diseño, Centros Periféricos, Dirección Ejecutiva y Secretaría General. Resultados: se muestran los resultados de 4 períodos: 1) planificación (noviembre-diciembre 2020); 2) capacitación, contratación de recursos humanos y asesoramiento para la adquisición de recursos materiales (enero-febrero 2021); 3) apertura de centros (hubs), puestos de vacunación COVID-19 y puesta en marcha de la Campaña COVID-19 (marzo 2021); 4) superposición campaña antigripal 2021 (abril-junio 2021) y mantenimiento coberturas de las vacunas del (PNI) 1. Planificación: se tomaron en cuenta las características de las vacunas que se recibirían vacunas a virus muertos, vacunas vectoriales y en particular la RNAm con requerimiento de almacenamiento a -70ºC, traslado y distribución a -25ºC, y reconstitución y uso a +28ºC en los diferentes escenarios. Se realizó análisis de riesgo y análisis de costo. Se intercambió con diferentes empresas que brindan dicho servicio, participando de actividades de capacitación con alguna de ellas, fundamentales a la hora de diseñar una cadena de frío sólida y sin fallas (por ejemplo actividad de entrenamiento con Vac-Q-tec). Se diseñó el traslado de las vacunas hacia los hubs, vacunatorios terciarios o centros periféricos de vacunación. Teniendo en cuenta el sitio de llegada de las vacunas, se plantearon los diferentes escenarios posibles, no solo a nivel central (Montevideo-Aeropuerto Internacional de Carrasco), sino también a nivel nacional utilizando las diferentes terminales aéreas. Así también se planificaron estrategias de distribución de las vacunas por vía terrestre involucrando diferentes actores (públicos o privados), basados en la experiencia de la institución debido al cotidiano trabajo que realizan, tanto en campaña de vacunación como en el mantenimiento del PNI. Se realizó la previsión de compras de insumos para el almacenamiento y distribución de la vacuna, como para la vacunación en sí. Se trabajó en conjunto con Departamento de Compras de Ministerio de Economía y Dirección de Secretaria del MSP. Se participó en la conformación de equipos de vacunación con la consigna de vacunación rápida, universal, gratuita y segura. Se estableciendo roles por equipo de vacunación constituidos por 6 personas. 2. Capacitación, contratación de recursos humanos y asesoramiento para la adquisición de recursos materiales. Se confeccionaron cursos y materiales en calidad y cantidad para que el personal adquiriera conocimientos actualizado sobre SARS-CoV-2 y COVID-19, mecanismos de transmisión, utilización y planificación de uso de equipos de protección personal (EPP) y seguimiento detallado de las vacunas en desarrollo. Análisis minucioso de los posibles candidatos a llegar a Uruguay, focalizándose en tres de ellos (Pfizer BioNtech, CoronaVac, Oxford AZ). Se realizó capacitación por roles (vacunadores, preparadores, administradores, referente de puesto, backup). Especial énfasis tuvo la capacitación en el cuidado de la persona a vacunar, registro del acto vacunal, cadena de frío en todos sus puntos, descarte de materiales utilizados y llenado de planillas de fiscalización. Todos los contenidos y materiales quedaron disponibles en la página web de la institución. Recursos humanos: se destinaron funcionarios para la confección, firma y papeleo de altas de 1.463 nuevos contratos de los equipos de todo el país, RRHH dedicados no sólo a la vacunación en sí, sino a participar activamente en cada uno de los puestos de vacunación. Esto significó que el trabajo se multiplicó por seis para las tareas requeridas durante este período. El Dpto. Recursos Materiales pasó de empaquetar y distribuir de 7 a 15 paquetes a 80 paquetes mensuales para cada centro periférico/vacunatorio. El envío habitual de materiales se cuadruplicó y en los comienzos de la campaña se llegó a quintuplicar y sextuplicar. Algunas de las cifras a destacar son: 3.300.000 jeringas con aguja, 8.800 Litros de alcohol eucaliptado, 852.000 guantes de látex y nitrilo y 141.000 sobreáticos. 3. Apertura de centros (hubs), puestos de vacunación COVID-19 y puesta en marcha de la Campaña COVID-19. Esto generó en forma progresiva la habilitación de centros (hubs) de máxima respuesta (vacunación masiva): ocho en Montevideo, uno en Canelones y uno en Maldonado. Dado que se decidió vacunar de 3 a 5 personas cada 15 minutos, en el hub del Hospital de Clínicas se administaron 211.153 dosis en sus 32 puestos de vacunación y en el hub Antel Arena se administraron 615.637 dosis en sus 15 puestos de vacunación. Al 31 de julio habían 144 puestos de vacunación COVID 19 habilitados en todo el territorio Nacional. La distribución se realizó a nivel nacional: 3.229. 320 dosis de vacuna Sinovac, 97.790 dosis de vacunas AstraZeneca y 1.585452 de vacuna Pfizer. Se participó en la conformación de 60 equipos con un referente (identificación, registro, control), preparador, 2 vacunadores y chofer para la vacunación en los Establecimientos de Larga Estadía de Personas Mayores (ELEPEM) y personas con situaciones especiales. Se participó activamente en la vacunación en ELEPEM, refugios y escuelas especiales, visitándose un total de 1679 Instituciones y se vacunaron 35.006 personas. 4. Superposición campaña antigripal 2021 (abril-junio 2021) y mantenimiento coberturas de las vacunas del (PNI). A partir del 24/04/2021 comenzó la Campaña Antigripal 2021, superponiéndose a la campaña descripta de COVID-19, lo que implicó reforzar las estrategias y ampliar las acciones para poder cumplir con ambas campaña paralelamente, siempre con el objetivo de no desatender el Programa Nacional de Inmunizaciones. Dentro de ésta se llevó a cabo la vacunación en los ELEPEM, como anualmente realiza la comisión y vacunación en territorio de vacunas COVID-19, antigripal y del PNI. Según los datos recibidos al 17 de Julio en el informe de la Unidad de Inmunizaciones del MSP (semana 12), fueron 605.802 dosis registradas, de las cuales al menos 87.802 dosis fueron administradas por la institución. Esta diferencia en relación a las campañas anteriores podría ser atribuida al intenso trabajo realizado en la estrategia de vacunación COVID-19. En el marco de la actividad interinstitucional barrio a barrio las personas vacunadas con vacuna antigripal en el móvil de la CHLA-EP en Montevideo fueron 7.225. La CHLA-EP ha contribuido desde la logística y la vacunación con esquema de dos dosis para las tres plataformas y una dosis de refuerzo para los que recibieron vacunas a virus muertos. La CHLA-EP ha contribuido para alcanzar las cifras de la Campaña de Vacunación COVID-19 entre el 1/3 y el 29/8/21: 5.487.181 dosis aplicadas, 2.65.269 personas que recibieron una dosis de vacuna y de ellas 2.496.290 tienen dos dosis y han recibido dosis de refuerzo 333.662. Conclusiones: la planificación, capacitación, provisión de recursos humanos y materiales lograron llevar a cabo dos campañas de vacunación coexistentes en hubs, vacunatorios, ELEPEM y en territorio que se han considerado exitosas e históricas.

The new Coronavirus SARS CoV-2 pandemic was declared at the beginning of March 2020 when the Uruguayan government appointed the National Contingency Plan for the COVID-19 disease caused by this virus. This plan included non-pharmacological preventive measures such as: hand hygiene, physical distancing, use of different types of masks according to the risk of who should wear them, in some periods, restrictions to mobility, educational activities or public shows, and movement by land, air or sea. Responsible freedom was strongly called for. In December 2020, the Vaccination Strategy was included as a primary prevention mechanism. On 3/1/2021, the COVID-19 2021 Vaccination Campaign began, it was non-mandatory, population-based, initially for people over 19 years of age, and for adolescents between 12 and 17 years of age and pregnant women since June. The National Honorary Scientific Advisory Group (GACH) together with the National Vaccine Advisory Committee (CNAV) of the Ministry of Public Health (MSP) that has vast experience in advising health authorities in scientific decisions on the introduction of vaccines, and an ad-Hoc Commission created for COVID-19 vaccines, brought together experts to advise on the vaccination strategy. The Honorary Commission for the Fight Against Tuberculosis and Prevalent Diseases (CHLA-EP), as the operational branch of the MSP's Department of Immunizations, has participated with all stakeholders since December to implement the COVID-19 Population Vaccination Campaign without neglecting their tasks regarding the vaccination to prevent other infectious diseases. Methodology: a descriptive, retrospective study was carried out regarding the strategies implemented by the Immunization Operational Department (operational branch of the Ministry of Health's Immunization Department), A. Calmette Laboratory, Honorary Commission, General Coordination and Administrative Departments between November, 2020 and July 2021. Data source: reports were issued or provided specially for this work by the Immunization Operational Department, A. Calmette Laboratory, by the Ministry of Public Health's immunization Department, Human Resources, Material Resources, Support, Communication and Design Service, Peripheral Centers, Executive Board and General secretariat. Results: the results of the 4 periods are as follows: 1- Planning (Period November - December 2020), 2- Training, hiring of human resources and advice for the acquisition of qualitative and quantitative material resources (January - February 2021), 3- Opening of COVID-19 vaccination centers (Hubs), posts and launch of the COVID-19 Campaign (March 2021), 4- Overlap of the 2021 flu campaign (April-June) and maintenance of vaccine coverage (National Immunization Programme - PNI). 1- Planning: The characteristics of the different vaccines (dead virus vaccines, vector vaccines and in particular mRNA vaccines, with storage requirements of -70ºC, transfer and distribution requirements of -25ºC, and reconstitution and use of +2 + 8ºC) were taken into account in the different scenarios. Risk and costs analysis were performed. We interchanged data with different companies that provided this service, we participated in training activities with some of them, mainly in the design of a solid cold chain without failures (e.g. a Vac-Q-tec training activity). The transfer of vaccines to hubs, tertiary vaccination centers or peripheral vaccination centers was designed. The different destination site of the vaccines, the different possible scenarios were taken into account, both at local central level (Montevideo - Carrasco International Airport), but also at National level using the different air terminals. Similarly, strategies for the distribution of vaccines by land were planned involving different public or private actors based on the institutions' experience, both regarding the vaccination campaign and in the PNI maintenance. We made a supply acquisition plan for the vaccine's storage and distribution, for the vaccination itself, and for the purchase of supplies jointly with the Procurement Department of the Ministry of Economy and Executive Secretariat of the MSP. Six-people vaccination teams were created with clear roles and with the slogan of rapid, universal, free and safe vaccination for all. 2- Training: Courses and materials were organized for the staff to acquire updated knowledge about SARS CoV 2 and COVID-19, transmission mechanisms, use and planning of use of personal protective equipment (PPE) and of course, a detailed monitoring of the vaccines being developed. We carried out a thorough analysis of the vaccine companies to arrive in Uruguay, we focused on 3 of them (Pfizer BioNtech, CoronaVac, Oxford AZ). Training was carried out by roles (vaccinators, preppers, administrators, position referents, backups). Special emphasis was placed on the training of the care of the person to be vaccinated, registration of the vaccination act, end-to-end cold chain, disposal of used materials and filing out inspection forms. All the contents and materials were available on the Institution's webpage. Human resources: human resources were allocated for the creation, signature and registration of the paperwork for 1,463 new contracts for teams across the country. These resources were allocated not only to vaccination itself, but also to actively participate in each of the vaccination posts. This meant that the work was multiplied by 6 for the tasks required during this period. The Department of Material Resources, went from packaging and distributing from 7 to 15 packages a month to 80 packages a month for each Peripheral/Vaccination Center. Material shipment quadrupled at the beginning of the campaign and it eventually grew by five and sixfold. Some of the key figures to highlight are: 3, 300,000 needle syringes, 8,800 liters of Eucalyptus Alcohol, 852,000 latex and nitrile gloves and 141,000 isolation gowns. 3. COVID-19 Vaccination Hubs, Posts and Campaigns. We gradually opened maximum response mass vaccination Hubs in Montevideo (8), Canelones (1) and Maldonado (1). Since it was had been decided to vaccinate 3 to 5 people every 15 minutes, the primary results at the Hospital de Clínicas Hub were 211,153 doses administered in 32 vaccination posts, and at Antel Arena Hub 615,637 doses were administered in its 15 vaccination posts. As of July 31, there were 144 COVID 19 vaccination posts throughout the national territory. The distribution was made nationwide: 3,229,320 Sinovac vaccine doses, 97,790 Oxford-Astra Zeneca vaccine doses, and 1,585,452 Pfizer-BioNtech vaccine doses. We devised 60 teams including a referent (identification, registration, control), a trainer, 2 vaccinators and vaccination driver for Homes for the Elderly at ELEPEM and people needing special care. They actively participated in the vaccination at ELEPEM, Shelters and Schools for Children with Special Needs and visited a total of 1679 Institutions and overall, 35,006 people were vaccinated. 4- Overlap of the 2021 flu campaign (April-June) and the maintenance of the COVID vaccine coverage (National Immunization Programme - PNI). Since 04/24/2021, the Anti-Flu Campaign began, and it overlapped the COVID-19 campaign described above, which meant reinforcing strategies and expanding actions to be able to carry out both campaigns simultaneously, always with the objective of maintaining the National Immunization Program. Vaccination was carried out at the ELEPEM, as it is performed annually by the commission and vaccination of COVID-19, influenza and PNI was performed on the territory. According to the data received on July 17 in the Ministry of Public Health Immunization Unit Report (week 12), there were 605,802 registered doses, of which at least 87,802 had been administered by the Institution. This difference regarding previous campaigns could have been caused by the intense work carried out during the COVID19 Vaccination Campaign. Within the framework of the inter-institutional activity "Barrio a Barrio" (Vaccination in all Neighborhoods), 7,225 people were vaccinated with the influenza vaccine at the CHLA-EP mobile vaccination bus in Montevideo. The CHLA-EP has contributed with logistics and vaccination with a 2-dose schedule for the 3 platforms and a booster dose for those who received virus vaccines. The CHLA-EP has contributed to achieving the target of the COVID-19 Vaccination Campaign between 1/3 and 8/29/21: 5,487,181 doses were applied, 2,065,269 people received 1 dose of vaccine and 2,496,290 of them received 2 doses and 333,662 have received booster doses. Conclusion: the planning, training, provision of human and material resources has enabled us to successfully carry out two historical simultaneous vaccination campaigns in hubs, vaccination centers, ELEPEM and all along the territory.

A pandemia causada pela infecção pelo novo Coronavírus SARS CoV-2 determinou que no Uruguai no início de março de 2020 o governo elaborasse o Plano Nacional de Contingência para a doença COVID-19 causada por este vírus. Este plano incluía medidas preventivas não farmacológicas como: higiene das mãos, distanciamento físico, uso de diferentes tipos de máscaras dependendo do risco de quem as deve usar, em alguns períodos várias restrições à circulação de pessoas, atividades educativas ou espetáculos públicos e a trânsito por terra, ar ou mar). Foi feito um forte apelo à liberdade responsável. A partir de dezembro de 2020, decidiu-se incorporar uma Estratégia de Vacinação à prevenção primária. Em 01/03/2021 teve início a Campanha de Vacinação COVID-19 2021, não obrigatória, de base populacional, inicialmente para maiores de 18 anos e a partir de junho contempla adolescentes entre 12 e 17 anos e gestantes. O Grupo Honorário de Assessoramento Científico do Governo Nacional juntamente com a Comissão Nacional de Assessoramento de Vacinas (CNAV) do Ministério da Saúde Pública (MSP), que assessora autoridades de saúde em decisões científicas sobre a introdução de vacinas, e uma Comissão ad hoc criada para vacinas contra a COVID-19 reuniu especialistas do país para aconselhar sobre a estratégia de vacinação. A Comissão Honorária de Luta contra a Tuberculose e Doenças Prevalentes (CHLA-EP) como braço operacional do Departamento de Vacinação do MSP envolveu-se desde dezembro com todos os intervenientes na implementação da Campanha de Vacinação da População COVID-19, aliás de realizar as tarefas de vacinação para prevenir outras doenças infecciosas. Metodologia: foi realizado um estudo descritivo e retrospectivo das estratégias implementadas pelo Departamento Operacional de Imunizações (braço operacional do Departamento de Imunizações do MSP), Laboratório A. Calmette, Comissão Honorária, Coordenação Geral e departamentos administrativos: entre os meses de novembro de 2020 a julho de 2021. Fonte de dados: relatórios divulgados ou oferecidos especialmente para este trabalho pelo Departamento Operacional de Imunizações, Laboratório A. Calmette, Departamento de Imunizações do Ministério da Saúde Pública, Recursos Humanos, Recursos Materiais, Serviço de Apoio, Comunicação e Design, Centros Periféricos, Direção Executiva e Secretaria Geral. Resultados: são apresentados os resultados de 4 períodos: 1- Planejamento (novembro - dezembro de 2020), 2- Capacitação, contratação de recursos humanos e assessoria para aquisição de recursos materiais em qualidade e quantidade (janeiro - fevereiro de 2021), 3- Abertura de centros (Hubs), postos de vacinação COVID-19 e implementação da Campanha COVID-19 (março 2021), 4- Sobreposição da campanha da gripe 2021 (abril-junho 2021) e manutenção da cobertura vacinal (PNI) 1- Planejamento: As características das vacinas a receber eram vacinas de vírus mortos, vacinas vetoriais e em particular mRNA com requisitos de armazenamento de temperaturas de -70ºC, transferência e distribuição de -25ºC, e reconstituição e uso de +2 +8ºC nos diferentes cenários. Análise de risco e análise de custo foram realizadas. Realizaram-se intercâmbios com diferentes empresas que prestam este serviço, participando em ações de formação com algumas delas, essenciais na criação de uma cadeia de frio sólida e sem falhas (ex. ação de formação com Vac-Q-tec). Foi projetada a transferência de vacinas para os hubs, centros de vacinação terciários ou periféricos. Tendo em conta o local de chegada das vacinas, foram considerados os diferentes cenários possíveis, não só a nível central (Montevidéu - Aeroporto Internacional de Carrasco), mas também a nível nacional utilizando os diferentes terminais aéreas. Da mesma forma, foram planejadas estratégias de distribuição de vacinas por via terrestre envolvendo diferentes atores (públicos ou privados), com base na experiência da instituição, tanto na campanha de vacinação quanto na manutenção do PNI. A previsão de compra de insumos foi feita tanto para o armazenamento e distribuição da vacina, quanto para a própria vacinação, como também na tomada de decisão na compra de insumos, atuando em conjunto com o Departamento de Compras do Ministério da Economia e Gestão Secretário do MSP. Eles participaram da formação de equipes de 6 pessoas com funções especificas de vacinação com o slogan de vacinação rápida, universal, gratuita e segura. 2- Capacitação, contratação de recursos humanos e assessoria para aquisição de recursos materiais em qualidade e quantidade. Foram organizados cursos e materiais para que os funcionários adquirissem conhecimento atualizado sobre SARS CoV2 e COVID-19, mecanismos de transmissão, uso e planejamento de uso de equipamentos de proteção individual (EPI) e também para o acompanhamento minucioso das vacinas em desenvolvimento. Fizemos a análise minuciosa das possíveis vacinas candidatas para chegar ao Uruguai, e focamos em 3 delas (Pfizer BioNtech, CoronaVac, Oxford AZ). O treinamento foi realizado por funções (vacinadores, treinadores, administradores, posição de referência, backup). Foi dado especial destaque à formação no cuidado da pessoa a vacinar, registo do ato vacinal, rede de frio em todos os seus pontos, eliminação de materiais utilizados e preenchimento de fichas de controlo. Todos os conteúdos e materiais foram disponibilizados na página web da Instituição. Recursos humanos: funcionários foram alocados para a preparação, assinatura e registro de 1.463 novos contratos para complementar as equipes em todo o país, estes recursos humanos estiveram dedicados não apenas à vacinação em si, mas a participar ativamente de cada um dos postos de vacinação. Isso significou que o trabalho se multiplicou por 6 para as tarefas exigidas durante esse período. O Departamento de Recursos Materiais, passou de embalar e distribuir de 7 para 15 embalagens por mês, passando para 80 embalagens para cada Periférico/Centro de Vacinação. A remessa habitual de materiais se quadruplicou e no início da campanha aumentou cinco e seis vezes. Alguns dos números a destacar são: 3.300.000 seringas com agulha, 8.800 litros de álcool de eucalipto, 852.000 luvas de látex e nitrílica e 141.000 túnicas. 3. Abertura de centros e postos de vacinação COVID-19 e lançamento da Campanha COVID-19. Isso gerou progressivamente o estabelecimento de centros de resposta máxima (Hubs) (vacinação em massa) 8 em Montevidéu, um em Canelones e um em Maldonado. Já que decidiu-se vacinar de 3 a 5 pessoas a cada 15 minutos, no Hub. O Hospital de Clínicas administrou 211.153 doses em seus 32 postos de vacinação e na Arena Hub Antel foram administradas 615.637 doses em seus 15 postos de vacinação. Até 31 de julho, havia 144 postos de vacinação contra a COVID 19 habilitados em todo o território nacional. A distribuição foi realizada, 3.229 foram distribuídos em todo o país, 320 doses de vacina Sinovac, 97.790 doses de vacinas AstraZeneca e 1.585452 de vacina Pfizer. Participaram no treinamento de 60 equipes de referência (identificação, registo, controle), treinador, 2 vacinadores e motorista para vacinação em estabelecimentos de longa permanência para idosos (ELEPEM) e pessoas com situações especiais em albergues e escolas especiais, visitando um total de 1.679 Instituições e vacinando a 35.006 pessoas. 4- Sobreposição da campanha de gripe de 2021 (abril a junho de 2021) e manutenção da cobertura vacinal (PNI). A partir de 24/04/2021 teve início a Campanha da Gripe 2021, sobrepondo-se à campanha COVID-19 descrita, o que implicou o reforço de estratégias e ampliação das ações para poder cumprir ambas campanhas em paralelo, sempre com o objetivo de não descurar o Programa Nacional de Imunizações. Foi realizada a vacinação no ELEPEM, conforme anualmente realizada pela comissão e a vacinação no território das vacinas COVID-19, influenza e PNI. De acordo com os dados recebidos até 17 de julho no relatório da Unidade de Imunizações dos Ministérios da Saúde Pública (semana 12), foram registradas 605.802 doses, das quais pelo menos 87.802 doses foram administradas pela Instituição. Essa diferença em relação às campanhas anteriores pode ser atribuída ao intenso trabalho realizado na estratégia de Vacinação COVID19. No âmbito da atividade interinstitucional Bairro a Bairro, 7.225 pessoas foram vacinadas com a vacina contra gripe no celular do CHLAEP de Montevidéu. O CHLA-EP tem contribuído desde a logística e vacinação com um esquema de 2 doses para as 3 plataformas e uma dose de reforço para quem recebeu vacinas de vírus mortos. O CHLA-EP contribuiu para atingir os números da Campanha de Vacinação COVID-19 entre 1/3 e 29/8/21: 5.487.181 doses aplicadas, 2.065.269 pessoas que receberam 1 dose de vacina e delas 2.496.290 têm 2 doses e 333.662 receberam doses de reforço. Conclusões: O planejamento, capacitação, disponibilização de recursos humanos e materiais permitiram a realização de duas campanhas de vacinação coexistentes nos hubs, vacinatórios, ELEPEM e no território, o que pode ser considerado um processo bem sucedido e histórico.

Arcuate AgRP, but not POMC neurons, modulate paraventricular CRF synthesis and release in response to fasting.

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.

Design and characterization of a triazole-based growth hormone secretagogue receptor modulator inhibiting the glucoregulatory and feeding actions of ghrelin.

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.

The controversial role of the vagus nerve in mediating ghrelin's actions: gut feelings and beyond.

Ghrelin is a stomach-derived peptide hormone that acts via the growth hormone secretagogue receptor (GHSR) and displays a plethora of neuroendocrine, metabolic, autonomic and behavioral actions. It has been proposed that some actions of ghrelin are exerted via the vagus nerve, which provides a bidirectional communication between the central nervous system and peripheral systems. The vagus nerve comprises sensory fibers, which originate from neurons of the nodose and jugular ganglia, and motor fibers, which originate from neurons of the medulla. Many anatomical studies have mapped GHSR expression in vagal sensory or motor neurons. Also, numerous functional studies investigated the role of the vagus nerve mediating specific actions of ghrelin. Here, we critically review the topic and discuss the available evidence supporting, or not, a role for the vagus nerve mediating some specific actions of ghrelin. We conclude that studies using rats have provided the most congruent evidence indicating that the vagus nerve mediates some actions of ghrelin on the digestive and cardiovascular systems, whereas studies in mice resulted in conflicting observations. Even considering exclusively studies performed in rats, the putative role of the vagus nerve in mediating the orexigenic and growth hormone (GH) secretagogue properties of ghrelin remains debated. In humans, studies are still insufficient to draw definitive conclusions regarding the role of the vagus nerve mediating most of the actions of ghrelin. Thus, the extent to which the vagus nerve mediates ghrelin actions, particularly in humans, is still uncertain and likely one of the most intriguing unsolved aspects of the field.

GHSR controls food deprivation-induced activation of CRF neurons of the hypothalamic paraventricular nucleus in a LEAP2-dependent manner.

OBJECTIVE: 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 (PVHCRF neurons), which are a part of the hypothalamic-pituitary-adrenal axis (HPA), as well as the growth hormone secretagogue receptor (GHSR) signaling, whose activity is up- or down-regulated, respectively, by the hormones ghrelin and the liver-expressed antimicrobial peptide 2 (LEAP2). Since ghrelin treatment potently up-regulates the HPA axis, we studied the role of GHSR in mediating food deprivation-induced activation of the PVHCRF neurons in mice. METHODS: We estimated the activation of the PVHCRF neurons, using immuno-staining against CRF and the marker of neuronal activation c-Fos in brain sections, and assessed plasma levels of corticosterone and glucose in different pharmacologically or genetically manipulated mouse models exposed, or not, to a 2-day food deprivation protocol. In particular, we investigated ad libitum fed or food-deprived male mice that: (1) lacked GHSR gene expression, (2) had genetic deletion of the ghrelin gene, (3) displayed neurotoxic ablation of the hypothalamic arcuate nucleus, (4) were centrally treated with an anti-ghrelin antibody to block central ghrelin action, (5) were centrally treated with a GHSR ligand that blocks ghrelin-evoked and constitutive GHSR activities, or (6) received a continuous systemic infusion of LEAP2(1-12). RESULTS: We found that food deprivation results in the activation of the PVHCRF neurons and in a rise of the ghrelin/LEAP2 molar ratio. Food deprivation-induced activation of PVHCRF neurons required the presence and the signaling of GHSR at hypothalamic level, but not of ghrelin. Finally, we found that preventing the food deprivation-induced fall of LEAP2 reverses the activation of the PVHCRF neurons in food-deprived mice, although it has no effect on body weight or blood glucose. CONCLUSION: Food deprivation-induced activation of the PVHCRF neurons involves ghrelin-independent actions of GHSR at hypothalamic level and requires a decrease of plasma LEAP2 levels. We propose that the up-regulation of the actions of GHSR associated to the fall of plasma LEAP2 level are physiologically relevant neuroendocrine signals during a prolonged fasting.

Growth hormone secretagogue receptor signaling in the supramammillary nucleus targets nitric oxide-producing neurons and controls recognition memory in mice.

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.

Ghrelin Gene Deletion Alters Pulsatile Growth Hormone Secretion in Adult Female Mice.

Using preproghrelin-deficient mice (Ghrl-/-), we previously observed that preproghrelin modulates pulsatile growth hormone (GH) secretion in post-pubertal male mice. However, the role of ghrelin and its derived peptides in the regulation of growth parameters or feeding in females is unknown. We measured pulsatile GH secretion, growth, metabolic parameters and feeding behavior in adult Ghrl-/- and Ghrl+/+ male and female mice. We also assessed GH release from pituitary explants and hypothalamic growth hormone-releasing hormone (GHRH) expression and immunoreactivity. Body weight and body fat mass, linear growth, spontaneous food intake and food intake following a 48-h fast, GH pituitary contents and GH release from pituitary explants ex vivo, fasting glucose and glucose tolerance were not different among adult Ghrl-/- and Ghrl+/+ male or female mice. In vivo, pulsatile GH secretion was decreased, while approximate entropy, that quantified orderliness of secretion, was increased in adult Ghrl-/- females only, defining more irregular GH pattern. The number of neurons immunoreactive for GHRH visualized in the hypothalamic arcuate nucleus was increased in adult Ghrl-/- females, as compared to Ghrl+/+ females, whereas the expression of GHRH was not different amongst groups. Thus, these results point to sex-specific effects of preproghrelin gene deletion on pulsatile GH secretion, but not feeding, growth or metabolic parameters, in adult mice.

Circulating ghrelin crosses the blood-cerebrospinal fluid barrier via growth hormone secretagogue receptor dependent and independent mechanisms.

Ghrelin is a peptide hormone mainly secreted from gastrointestinal tract that acts via the growth hormone secretagogue receptor (GHSR), which is highly expressed in the brain. Strikingly, the accessibility of ghrelin to the brain seems to be limited and restricted to few brain areas. Previous studies in mice have shown that ghrelin can access the brain via the blood-cerebrospinal fluid (CSF) barrier, an interface constituted by the choroid plexus and the hypothalamic tanycytes. Here, we performed a variety of in vivo and in vitro studies to test the hypothesis that the transport of ghrelin across the blood-CSF barrier occurs in a GHSR-dependent manner. In vivo, we found that the uptake of systemically administered fluorescent ghrelin in the choroid plexus epithelial (CPE) cells and in hypothalamic tanycytes depends on the presence of GHSR. Also, we detected lower levels of CSF ghrelin after a systemic ghrelin injection in GHSR-deficient mice, as compared to WT mice. In vitro, the internalization of fluorescent ghrelin was reduced in explants of choroid plexus from GHSR-deficient mice, and unaffected in primary cultures of hypothalamic tanycytes derived from GHSR-deficient mice. Finally, we found that the GHSR mRNA is detected in a pool of CPE cells, but is nearly undetectable in hypothalamic tanycytes with current approaches. Thus, our results suggest that circulating ghrelin crosses the blood-CSF barrier mainly by a mechanism that involves the GHSR, and also possibly via a GHSR-independent mechanism.

Ghrelin treatment induces rapid and delayed increments of food intake: a heuristic model to explain ghrelin's orexigenic effects.

Ghrelin is a stomach-derived peptide hormone with salient roles in the regulation of energy balance and metabolism. Notably, ghrelin is recognized as the most powerful known circulating orexigenic hormone. Here, we systematically investigated the effects of ghrelin on energy homeostasis and found that ghrelin primarily induces a biphasic effect on food intake that has indirect consequences on energy expenditure and nutrient partitioning. We also found that ghrelin-induced biphasic effect on food intake requires the integrity of Agouti-related peptide/neuropeptide Y-producing neurons of the hypothalamic arcuate nucleus, which seem to display a long-lasting activation after a single systemic injection of ghrelin. Finally, we found that different autonomic, hormonal and metabolic satiation signals transiently counteract ghrelin-induced food intake. Based on our observations, we propose a heuristic model to describe how the orexigenic effect of ghrelin and the anorectic food intake-induced rebound sculpt a timely constrain feeding response to ghrelin.

Development of Nonpeptidic Inverse Agonists of the Ghrelin Receptor (GHSR) Based on the 1,2,4-Triazole Scaffold.

GHSR controls, among others, growth hormone and insulin secretion, adiposity, feeding, and glucose metabolism. Therefore, an inverse agonist ligand capable of selectively targeting GHSR and reducing its high constitutive activity appears to be a good candidate for the treatment of obesity-related metabolic diseases. In this context, we present a study that led to the development of several highly potent and selective inverse agonists of GHSR based on the 1,2,4-triazole scaffold. We demonstrate that, depending on the nature of the substituents on positions 3, 4, and 5, this scaffold leads to ligands that exert an intrinsic inverse agonist activity on GHSR-catalyzed G protein activation through the stabilization of a specific inactive receptor conformation. Thanks to an in vivo evaluation, we also show that one of the most promising ligands not only exerts an effect on insulin secretion in rat pancreatic islets but also affects the orexigenic effects of ghrelin in mice.

Fasting induces remodeling of the orexigenic projections from the arcuate nucleus to the hypothalamic paraventricular nucleus, in a growth hormone secretagogue receptor-dependent manner.

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.

Evidence Supporting a Role for the Blood-Cerebrospinal Fluid Barrier Transporting Circulating Ghrelin into the Brain.

The stomach-derived hormone ghrelin mainly acts in the brain. Studies in mice have shown that the accessibility of ghrelin into the brain is limited and that it mainly takes place in some circumventricular organs, such as the median eminence. Notably, some known brain targets of ghrelin are distantly located from the circumventricular organs. Thus, we hypothesized that ghrelin could also access the brain via the blood-cerebrospinal fluid (CSF) barrier, which consists of the choroid plexus and the hypothalamic tanycytes. Using systemic injection of ghrelin or fluorescent-ghrelin in mice, we found that cells of the blood-CSF barrier internalize these molecules. In time-response studies, we found that peripherally injected fluorescent-ghrelin quickly reaches hypothalamic regions located in apposition to the median eminence and more slowly reaches the periventricular hypothalamic parenchyma, adjacent to the dorsal part of the third ventricle. Additionally, we found that CSF ghrelin levels increase after the systemic administration of ghrelin, and that central infusions of either an anti-ghrelin antibody, which immuno-neutralizes CSF ghrelin, or a scrambled version of ghrelin, which is also internalized by cells of the blood-CSF barrier, partially impair the orexigenic effect of peripherally injected ghrelin. Thus, current evidence suggests that the blood-CSF barrier can transport circulating ghrelin into the brain, and that the access of ghrelin into the CSF is required for its full orexigenic effect.

Brain accessibility delineates the central effects of circulating ghrelin.

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.

Evidence Supporting a Role for Constitutive Ghrelin Receptor Signaling in Fasting-Induced Hyperphagia in Male Mice.

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 ∼15% 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.

Circulating Ghrelin Acts on GABA Neurons of the Area Postrema and Mediates Gastric Emptying in Male Mice.

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.