The thesis is focused on the effect of ghrelin receptor (GHS-R1a) agonists and antagonist on food intake regulation. Ghrelin is the only known periferally produced orexigenic hormone and the only known acylated hormone. GHS-R1a agonists and antagonists could be useful in the treatment of cachexia and obesity, respectively. In the first part of the thesis, newly designed peptidic GHS-R1a agonists were characterized. The agonists were stabilized by replacing octanoylated Ser3 with a fatty acid coupled to diaminopropionic acid by a stable amide bond. Other noncoded amino acids were also incorporated. Ghrelin analogs were modified by replacing the octanoyl group with another fatty acid, incorporation of the second fatty acid or shortening the peptide chain. Most of the tested GHS-R1a agonists were found to possess high affinities for GHS-R1a (Ki = 10-9 - 10-10 nM) and to activate signaling pathways of ghrelin. After subcutaneous (SC) administration to mice, agonists showed significant and prolonged orexigenic effect. In the second part of the thesis, acute and long-term effects of pseudopeptide GHS-R1a agonist JMV1843 were tested in lean C57BL/6 mice. Acute SC administration of JMV1843 to fed mice increased food intake in a dose-dependent manner (ED50 = 1.94 mg/kg). JMV1843 was stable in blood serum in...

The thesis is focused on the effect of ghrelin receptor (GHS-R1a) agonists and antagonist on food intake regulation. Ghrelin is the only known periferally produced orexigenic hormone and the only known acylated hormone. GHS-R1a agonists and antagonists could be useful in the treatment of cachexia and obesity, respectively. In the first part of the thesis, newly designed peptidic GHS-R1a agonists were characterized. The agonists were stabilized by replacing octanoylated Ser3 with a fatty acid coupled to diaminopropionic acid by a stable amide bond. Other noncoded amino acids were also incorporated. Ghrelin analogs were modified by replacing the octanoyl group with another fatty acid, incorporation of the second fatty acid or shortening the peptide chain. Most of the tested GHS-R1a agonists were found to possess high affinities for GHS-R1a (Ki = 10-9 - 10-10 nM) and to activate signaling pathways of ghrelin. After subcutaneous (SC) administration to mice, agonists showed significant and prolonged orexigenic effect. In the second part of the thesis, acute and long-term effects of pseudopeptide GHS-R1a agonist JMV1843 were tested in lean C57BL/6 mice. Acute SC administration of JMV1843 to fed mice increased food intake in a dose-dependent manner (ED50 = 1.94 mg/kg). JMV1843 was stable in blood serum in...

A ghrelina, hormônio secretado principalmente por células gástricas, liga-se ao seu receptor, o receptor de secretagogo de GH (GHSR - Growth hormone secretagogue receptor), localizado no hipotálamo e na hipófise, estimulando a síntese e secreção do GH. Recentemente foram identificadas mutações no gene GHSR em crianças com baixa estatura idiopática (BEI) e com deficiência isolada de GH (DGH). No presente estudo investigamos a presença de mutações no gene GHSR em crianças com DGH isolada de causa não identificada e crianças com BEI, incluindo um subgrupo de crianças com atraso constitucional de crescimento e desenvolvimento (ACCD). Foram selecionados 14 pacientes com deficiência isolada de GH sem alterações anatômicas da região hipotálamo-hipofisária e 96 pacientes com BEI, destes 31 (32%) apresentavam ACCD. Também foram estudados 150 controles adultos e 197 crianças controle com crescimento e puberdade normais. A região codificadora do GHSR foi amplificada utilizando-se oligonucleotídeos iniciadores específicos, seguida de purificação enzimática e seqüenciamento automático. Encontramos 6 variantes alélicas em heterozigose no GHSR: nenhuma delas presente nos controles estudados, e quatro destas variantes estão localizadas em regiões conservadas do gene. Uma variante foi encontrada em uma paciente do grupo DGH (p.Val249Leu) e as outras cinco (c.-6 G>C, p.Ser84Ile, p.Val182Ala, p.Ala169Thr e p.Ala358Thr) foram encontradas em pacientes do subgrupo ACCD do grupo BEI. As variantes missense foram submetidas a estudo funcional que evidenciou que as mutações p.Ser84Ile e p.Val182Ala possuem diminuição na atividade basal associadas à diminuição da expressão do receptor na superfície celular. Adicionalmente, a mutação p.Ser84Ile também apresenta redução na atividade do GHSR induzida pelo ligante. A variante p.Val249Leu foi encontrada em uma paciente do sexo feminino com diagnóstico de DGH isolado. A falta de segregação familiar associada à ausência de déficit funcional da variante nos estudos in vitro sugere que, neste caso, a variante p.Val249Leu não é a causa do fenótipo de DGH nesta família e trata-se de uma variante alélica rara. As 5 variantes alélicas no GHSR (c.-6 G>C, p.Ser84Ile, p.Val182Ala, p.Ala169Thr e p.Ala358Thr) encontradas nos pacientes com BEI foram identificadas apenas naqueles com puberdade atrasada, ou seja, pertencentes ao subgrupo ACCD (3 do sexo masculino e 2 do sexo feminino). A freqüência de variantes neste grupo de pacientes foi de 16%, significativamente maior que nos outros grupos, e a ausência de variantes gênicas novas no grupo de crianças obesas com altura normal e mesmo no grupo de crianças com BEI sem ACCD sugere que nosso achado não foi casual e que as alterações descritas podem estar associadas ao fenótipo de ACCD. Os estudos in vitro mostraram prejuízos funcionais em 2 destas variantes (p.Ser84Ile e p.Val182Ala) porém, devido à limitação dos estudos funcionais (celulas heterólogas) não podemos afastar que as demais não tenham algum impacto funcional in vivo. Em conclusão, nossos resultados sugerem um envolvimento dos defeitos no GHSR na etiologia do atraso constitucional do crescimento e desenvolvimento em uma parcela de pacientes com esta condição / Ghrelin, hormone secreted by gastric cells, stimulates growth hormone secretion by acting on its receptor GHSR, located in the hypothalamus and pituitary. Recently, mutations in the GHSR gene were described in patients with growth hormone deficiency (GHD) and idiopathic short stature (ISS). In the present study we analyzed the GHSR gene in patients with isolated GHD and patients with ISS, including a subgroup of patients with constitutional delay of growth and puberty (CDGP). We studied 14 GHD patients with normal pituitary magnetic resonance imaging and 96 patients with ISS, 31 of them with CDGP. We also studied 150 adults and in 197 children with normal stature. The entire coding region as well as the exon-intron boundaries of GHSR were PCR amplified in all patients and control group and PCR products were bidirectionally sequenced. Six different heterozygous variants in GHSR were identified: none of them were found in the control group and four of these amino acid substitutions occurred at a conserved position within the GHSR. One variant (p.Val249Leu) was found in a GHD patient and the other five (c.-6 G>C, p.Ser84Ile, p.Val182Ala, p.Ala169Thr e p.Ala358Thr) were found in patients with CDGP. The missense variants were submitted to functional studies. Two of these variants (p.Ser84Ile and p.Val182Ala) result in a decrease in basal activity that was in part explained by a reduction in cell surface expression. The p.Ser84Ile mutation was also associated with a defect in ghrelin potency. The p.Val249Leu variant, found in a female patient with isolated GHD, did not segregate with the phenotype in the family and had no functional impairment in vitro. This suggests that p.Val249Leu is not the cause of the GHD in the family and may be a rare allelic variant. The other variants (c.-6 G>C, p.Ser84Ile, p.Val182Ala, p.Ala169Thr e p.Ala358Thr) were identified only in patients with CDGP (3 male and 2 female). The frequency of allelic variants observed in this group (16%) was higher than expected by chance in contrast with ISS and GHD children, and the absence of other GHSR mutations in the large group of control children suggests that the association between GHSR mutations and CDGP phenotype is unlikely to be fortuitous. Functional studies revealed that two of the identified missense variants (p.Ser84Ile and p.Val182Ala) are functionally significant. These functional studies were performed in heterologous cell expression systems; therefore it is not possible to completely rule out that the other identified variants might cause some unrevealed impairment on GHSR function or expression in vivo. In conclusion, our data raise the possibility that abnormalities in ghrelin receptor function may be implicated in the ethiology of CDGP in some patients

A ghrelina, hormônio secretado principalmente por células gástricas, liga-se ao seu receptor, o receptor de secretagogo de GH (GHSR - Growth hormone secretagogue receptor), localizado no hipotálamo e na hipófise, estimulando a síntese e secreção do GH. Recentemente foram identificadas mutações no gene GHSR em crianças com baixa estatura idiopática (BEI) e com deficiência isolada de GH (DGH). No presente estudo investigamos a presença de mutações no gene GHSR em crianças com DGH isolada de causa não identificada e crianças com BEI, incluindo um subgrupo de crianças com atraso constitucional de crescimento e desenvolvimento (ACCD). Foram selecionados 14 pacientes com deficiência isolada de GH sem alterações anatômicas da região hipotálamo-hipofisária e 96 pacientes com BEI, destes 31 (32%) apresentavam ACCD. Também foram estudados 150 controles adultos e 197 crianças controle com crescimento e puberdade normais. A região codificadora do GHSR foi amplificada utilizando-se oligonucleotídeos iniciadores específicos, seguida de purificação enzimática e seqüenciamento automático. Encontramos 6 variantes alélicas em heterozigose no GHSR: nenhuma delas presente nos controles estudados, e quatro destas variantes estão localizadas em regiões conservadas do gene. Uma variante foi encontrada em uma paciente do grupo DGH (p.Val249Leu) e as outras cinco (c.-6 G>C, p.Ser84Ile, p.Val182Ala, p.Ala169Thr e p.Ala358Thr) foram encontradas em pacientes do subgrupo ACCD do grupo BEI. As variantes missense foram submetidas a estudo funcional que evidenciou que as mutações p.Ser84Ile e p.Val182Ala possuem diminuição na atividade basal associadas à diminuição da expressão do receptor na superfície celular. Adicionalmente, a mutação p.Ser84Ile também apresenta redução na atividade do GHSR induzida pelo ligante. A variante p.Val249Leu foi encontrada em uma paciente do sexo feminino com diagnóstico de DGH isolado. A falta de segregação familiar associada à ausência de déficit funcional da variante nos estudos in vitro sugere que, neste caso, a variante p.Val249Leu não é a causa do fenótipo de DGH nesta família e trata-se de uma variante alélica rara. As 5 variantes alélicas no GHSR (c.-6 G>C, p.Ser84Ile, p.Val182Ala, p.Ala169Thr e p.Ala358Thr) encontradas nos pacientes com BEI foram identificadas apenas naqueles com puberdade atrasada, ou seja, pertencentes ao subgrupo ACCD (3 do sexo masculino e 2 do sexo feminino). A freqüência de variantes neste grupo de pacientes foi de 16%, significativamente maior que nos outros grupos, e a ausência de variantes gênicas novas no grupo de crianças obesas com altura normal e mesmo no grupo de crianças com BEI sem ACCD sugere que nosso achado não foi casual e que as alterações descritas podem estar associadas ao fenótipo de ACCD. Os estudos in vitro mostraram prejuízos funcionais em 2 destas variantes (p.Ser84Ile e p.Val182Ala) porém, devido à limitação dos estudos funcionais (celulas heterólogas) não podemos afastar que as demais não tenham algum impacto funcional in vivo. Em conclusão, nossos resultados sugerem um envolvimento dos defeitos no GHSR na etiologia do atraso constitucional do crescimento e desenvolvimento em uma parcela de pacientes com esta condição / Ghrelin, hormone secreted by gastric cells, stimulates growth hormone secretion by acting on its receptor GHSR, located in the hypothalamus and pituitary. Recently, mutations in the GHSR gene were described in patients with growth hormone deficiency (GHD) and idiopathic short stature (ISS). In the present study we analyzed the GHSR gene in patients with isolated GHD and patients with ISS, including a subgroup of patients with constitutional delay of growth and puberty (CDGP). We studied 14 GHD patients with normal pituitary magnetic resonance imaging and 96 patients with ISS, 31 of them with CDGP. We also studied 150 adults and in 197 children with normal stature. The entire coding region as well as the exon-intron boundaries of GHSR were PCR amplified in all patients and control group and PCR products were bidirectionally sequenced. Six different heterozygous variants in GHSR were identified: none of them were found in the control group and four of these amino acid substitutions occurred at a conserved position within the GHSR. One variant (p.Val249Leu) was found in a GHD patient and the other five (c.-6 G>C, p.Ser84Ile, p.Val182Ala, p.Ala169Thr e p.Ala358Thr) were found in patients with CDGP. The missense variants were submitted to functional studies. Two of these variants (p.Ser84Ile and p.Val182Ala) result in a decrease in basal activity that was in part explained by a reduction in cell surface expression. The p.Ser84Ile mutation was also associated with a defect in ghrelin potency. The p.Val249Leu variant, found in a female patient with isolated GHD, did not segregate with the phenotype in the family and had no functional impairment in vitro. This suggests that p.Val249Leu is not the cause of the GHD in the family and may be a rare allelic variant. The other variants (c.-6 G>C, p.Ser84Ile, p.Val182Ala, p.Ala169Thr e p.Ala358Thr) were identified only in patients with CDGP (3 male and 2 female). The frequency of allelic variants observed in this group (16%) was higher than expected by chance in contrast with ISS and GHD children, and the absence of other GHSR mutations in the large group of control children suggests that the association between GHSR mutations and CDGP phenotype is unlikely to be fortuitous. Functional studies revealed that two of the identified missense variants (p.Ser84Ile and p.Val182Ala) are functionally significant. These functional studies were performed in heterologous cell expression systems; therefore it is not possible to completely rule out that the other identified variants might cause some unrevealed impairment on GHSR function or expression in vivo. In conclusion, our data raise the possibility that abnormalities in ghrelin receptor function may be implicated in the ethiology of CDGP in some patients

Les sécrétines peptidiques de l’hormone de croissance (GHRPs) constituent une classe de peptides synthétiques capables de stimuler la sécrétion de l’hormone de croissance (GH). Cette activité est médiée par leur liaison à un récepteur couplé aux protéines G : le récepteur des sécrétines de l’hormone de croissance (GHS-R1a), identifié subséquemment comme le récepteur de la ghréline. La ghréline est un peptide de 28 acides aminés sécrété principalement par les cellules de la muqueuse de l’estomac, qui exerce de nombreux effets périphériques indépendamment de la sécrétion de l’hormone de croissance. Les effets indépendants de la sécrétion de GH incluent, entre autres, des actions sur le contrôle de la prise de nourriture, le métabolisme énergétique, la fonction cardiaque, le système immunitaire et la prolifération cellulaire. L’étude de la distribution périphérique des sites de liaison des GHRPs nous a permis d’identifier un second site, le CD36, un récepteur scavenger exprimé dans plusieurs tissus dont le myocarde, l’endothélium de la microvasculature et les monocytes/macrophages. Le CD36 exprimé à la surface du macrophage joue un rôle clé dans l’initiation du développement de l’athérosclérose par la liaison et l’internalisation des lipoprotéines de faible densité oxydées (LDLox) dans l’espace sous-endothélial de l’artère. L’hexaréline, un analogue GHRP, a été développé comme agent thérapeutique pour stimuler la sécrétion de l’hormone de croissance par l’hypophyse. Sa propriété de liaison aux récepteurs GHS-R1a et CD36 situés en périphérie et particulièrement sa capacité d’interférer avec la liaison des LDLox par le CD36 nous ont incité à évaluer la capacité de l’hexaréline à moduler le métabolisme lipidique du macrophage. L’objectif principal de ce projet a été de déterminer les effets de l’activation des récepteurs CD36 et GHS-R1a, par l’hexaréline et la ghréline, le ligand endogène du GHS-R1a, sur la physiologie du macrophage et de déterminer son potentiel anti-athérosclérotique. Les résultats montrent premièrement que l’hexaréline et la ghréline augmentent l’expression des transporteurs ABCA1 et ABCG1, impliqués dans le transport inverse du cholestérol, via un mécanisme contrôlé par le récepteur nucléaire PPARγ. La régulation de l’activité transcriptionnelle de PPARγ par l’activation des récepteurs CD36 et GHS-R1a se fait indépendamment de la présence du domaine de liaison du ligand (LBD) de PPARγ et est conséquente de changements dans l’état de phosphorylation de PPARγ. Une étude plus approfondie de la signalisation résultant de la liaison de la ghréline sur le GHS-R1a révèle que PPARγ est activé par un mécanisme de concertation entre les voies de signalisation Gαq/PI3-K/Akt et Fyn/Dok-1/ERK au niveau du macrophage. Le rôle de PPARγ dans la régulation du métabolisme lipidique par l’hexaréline a été démontré par l’utilisation de macrophages de souris hétérozygotes pour le gène de Ppar gamma, qui présentent une forte diminution de l’activation des gènes de la cascade métabolique PPARγ-LXRα-transporteurs ABC en réponse à l’hexaréline. L’injection quotidienne d’hexaréline à un modèle de souris prédisposées au développement de l’athérosclérose, les souris déficientes en apoE sous une diète riche en cholestérol et en lipides, se traduit également en une diminution significative de la présence de lésions athérosclérotiques correspondant à une augmentation de l’expression des gènes cibles de PPARγ et LXRα dans les macrophages péritonéaux provenant des animaux traités à l’hexaréline. L’ensemble des résultats obtenus dans cette thèse identifie certains nouveaux mécanismes impliqués dans la régulation de PPARγ et du métabolisme du cholestérol dans le macrophage via les récepteurs CD36 et GHS-R1a. Ils pourraient servir de cibles thérapeutiques dans une perspective de traitement des maladies cardiovasculaires. / Growth hormone-releasing peptides (GHRPs) are a class of small synthetic peptides known to stimulate GH release through their binding to a G protein-coupled receptor identified as GHS-R1a, later recognized as the ghrelin receptor. Ghrelin is an acetylated 28 amino acid hormone initially identified from the stomach, which induces the release of growth hormone (GH) from the pituitary but also regulates food intake, energy homeostasis, cardiovascular function, immune system and cell proliferation. In documenting the peripheral distribution of GHRPs binding sites, we uncovered the presence of another binding site for GHRPs, identified as CD36, a class B scavenger receptor. CD36 is expressed among several tissues, including myocytes, endothelial cells of the microvasculature and monocytes/macrophages. The macrophage CD36 contributes to excessive lipid loading and atherogenic formation of foam cells through uptake of oxidized low-density lipoprotein (oxLDL) in the subendothelial space of the artery. The properties of hexarelin, a ligand for GHS-R1a and CD36, which features overlapping binding sites with that of oxLDL binding domain on CD36, and thus interfering with the binding of oxLDL on CD36, have prompted us to evaluate the potential of hexarelin, as well as that of the endogenous ligand ghrelin in the modulation of macrophage cholesterol metabolism. We demonstrate here the ability of hexarelin and ghrelin to enhance the expression of ATP-binding cassette A1 and G1 transporters through a PPARγ-dependent mechanism. The hormone binding domain of PPARγ is not required to mediate PPARγ transcriptional activation by CD36 and GHS-R1a. Both hexarelin and ghrelin promotes phosphorylation of PPARγ in THP-1 macrophages. A more detailed study of GHS-R1a-initiated signaling revealed an intricate and complex signalling interplay triggered by ghrelin that involves modulation of Src-dependent Dok-1/ERK1/2 and Src-independent Gαq/PI3-K/Akt pathways, leading to PPARγ-dependent transcriptional competence in the macrophages. The central role of PPARγ on cholesterol metabolism in the macrophages has been demonstrated using peritoneal macrophages from PPARγ heterozygote mice whose response to hexarelin on PPARγ-LXRα-ABC transporters pathway was strongly impaired. Treatment of apolipoprotein E-null mice fed on a lipid-rich diet with hexarelin resulted in a significant reduction in atherosclerotic lesions, concomitant with an enhanced expression of PPARγ and LXRα target genes in peritoneal macrophages. The results presented in this thesis feature novel mechanisms by which the beneficial regulation of PPARγ and cholesterol metabolism in macrophages could be regulated by both CD36 and ghrelin receptor. The downstream effects following the activation of these receptors might be potential targets in the treatment of human coronary artery disease.

L’adaptation de l’organisme à son environnement est essentielle à sa survie. L’homéostasie énergétique permet l’équilibre entre les apports, les dépenses et le stockage d’énergie. Un surplus calorique important dérègle ce processus et mène au développement du syndrome métabolique caractérisé, entre autres, par une obésité, un diabète de type II, des maladies cardiovasculaires et des dyslipidémies. La ghréline participe au maintien de l’équilibre énergétique durant le jeûne en stimulant la production de glucose par le foie et le stockage lipidique dans le tissu adipeux. Le coactivateur transcriptionnel PGC-1alpha, surexprimé en situation de jeûne, est impliqué dans l’induction de la production de glucose par le foie et l’oxydation des acides gras. Notre hypothèse est que ces deux acteurs clés du métabolisme énergétique constituent un axe de régulation commun. Dans cette étude, nous montrons que la ghréline participe à la régulation de PGC-1alpha. Son récepteur GHS-R1a, possédant une forte activité constitutive, est également impliqué de façon indépendante au ligand. GHS-R1a réduit l’activité transcriptionnelle de PGC-1alpha tandis que l’ajout du ligand inverse modérément cette action. L’effet de GHS-R1a corrèle avec l’acétylation de PGC-1alpha qui est fortement augmentée de façon dose-dépendante. La stabilité de PGC-1alpha est également augmentée par le GHS-R1a indépendamment de l’ubiquitine. La ghréline diminue la capacité de PGC-1alpha à lier PPARbeta, un récepteur nucléaire partenaire de PGC-1alpha. De plus, la ghréline réduit, de façon ligand-dépendante, la capacité de coactivation de PGC-1alpha sur PPARbeta dans les hépatocytes. L’ensemble de ces résultats identifie PGC-1alpha comme cible du signal de la ghréline et suggère un axe de régulation ghréline/PGC-1alpha/PPARbeta.Une meilleure compréhension de cet axe de régulation va permettre la mise en évidence de nouvelles cibles thérapeutiques pour faire face aux pathologies associées au syndrome métabolique. / The adaptation of an organism to its environment is essential to its survival. Energy homeostasis is defined as the balance between intakes, expenses and storage of energy. An excess of calories disrupts this process and leads to the development of the metabolic syndrome that is characterized by obesity, type II diabetes, cardiovascular diseases and dyslipidemia. During fasting, ghrelin participates in the maintenance of energy balance by stimulating hepatic production of glucose and lipid storage in adipose tissue. The transcriptional coactivator PGC-1alpha is overexpressed in the liver during fasting and is involves in the induction of the hepatic glucose production and fatty acid oxidation. Our hypothesis is that these two key performers in the energy metabolism constitute a common axis control. In this study, we show that ghrelin plays a role in the regulation of PGC-1alpha. The ghrelin receptor GHS-R1a is also involved because of its strong constitutive activity in absence of ligand. We found that GHS-R1a inhibited PGC-1alpha transcriptional activity whereas adding ghrelin to cells moderated this effect. PGC-1alpha activation by GHS-R1a correlated with a dose-dependent increase of PGC-1alpha acetylation. The stability of PGC-1alpha was also increased by ghrelin receptor in a manner involving the ubiquitin-independent proteasome pathway. Ghrelin decreased the ability of PGC-1alpha to bind to PPARbeta, one of its nuclear receptor partners. Furthermore, ghrelin decreased the ability of PGC-1alpha to coactivate PPARbeta in a ligand-dependent manner in hepatocytes. Together, these results identify PGC-1alpha as a metabolic target of GHSR-1a signaling and defines a new regulatory axis involving ghrelin/PGC-1alpha/PPARbeta in hepatocytes. A better understanding of this regulation axis will provide novel aspects in therapeutic targeting of diseases associated with the metabolic syndrome.

A obesidade é causada pelo desequilíbrio entre a ingestão alimentar e o gasto energético corporal, com o armazenamento de energia na forma de gordura, no tecido adiposo. A obesidade causa alterações metabólicas, como resistência à insulina e dislipidemia, além de aumento de adipocinas e citocinas pró-inflamatórias. Este trabalho investigou a influência de polimorfismos nos genes da perilipina 1 (PLINl), visfatina (NAMPT) , resistina (RETN) e grelina (GHRL) na adiposidade e no perfil metabólico e inflamatório, antes e após um programa de orientação nutricional. Foram selecionados indivíduos obesos (OB, n=214), sobrepeso (SOB, n=71) e não obesos (NOB, n=69), com idade de 30 a 70 anos. Foram obtidos dados clínicos, antropométricos e de composição corporal. O recordatório de 24h foi aplicado a 87 indivíduos obesos para avaliação de consumo alimentar, antes e após o programa de orientação nutricional. Foi obtido sangue para extração de DNA e para analisar parâmetros laboratoriais (perfil lipídico e glicêmico, marcadores inflamatórios e adipocinas). Polimorfismos dos genes PLINl, NAMPT, RETN e GHRL foram analisados por PCR em tempo real. O grupo OB teve perfil antropométrico alterado e risco aumentado de hipertensão, diabetes tipo 2 e dislipidemia em comparação com os grupos SOB e NOB (p<0,05). As concentrações de glicose, colesterol total, LDL colesterol, VLDL colesterol, triglicérides, apolipoproteína B (ApoB), interleucina 1&#946; (IL-l &#946;) e fator de necrose tumoral alfa (TNF&#945;) foram maiores e as concentrações de HDL colesterol e apolipoproteina AI (apoAI) foram menores, no grupo OB que nos outros grupos (p<0,05). As frequências dos polimorfismos genéticos do grupo total foram similares as de outras populações. Os polimorfismos NAMPT rs1319501 C>T e rs10763861 C>T foram associados com obesidade (p<0,05). Os polimorfismos genéticos não influenciaram o perfil antropométrico do grupo total (p>0,05), mas no grupo de obesos, o polimorfismo GHRL rs4684677 T>A foi relacionado com maior porcentagem de gordura corporal (p=0,043). Após a orientação nutricional, observou-se diminuição da ingestão de calorias e do consumo de carboidratos, gorduras totais, sódio, magnésio e betacaroteno (p<0,05). Os polimorfismos genéticos não influenciaram o perfil antropométrico e o consumo alimentar de obesos, após a orientação nutricional. Em conclusão, polimorfismos dos genes NAMPT e GHRL contribuem para a adiposidade, mas não influenciam o comportamento alimentar e o perfil antropométrico, após orientação nutricional. / Obesity is caused by the imbalance between food intake and body energy expenditure, with the storage of energy in the form of fat, in adipose tissue. Obesity causes metabolic changes, such as insulin resistance and dyslipidemia, and an increase in adipokines and pro-inflammatory cytokines. This work investigated the influence of polymorphisms in the perilipine 1 (PLINI) , visfatin (NAMPT) , resistin (RETN) and ghrelin (GHRL) genes on adiposity and metabolic and inflammatory profile, before and after a nutritional orientation programo Obese (OB, n=214), overweight (SOB, n=71) and nonobese subjects (NOB, n=69), aged 30 to 70 years, were selected. ClinicaI, anthropometric and body composition data were obtained. The 24-hour dietary recall was applied to 87 obese subjects to eva1uate food intake before and after the nutritiona1 orientation programo Blood was obtained for DNA extraction and to analyze 1aboratory parameters (lipid and glycemic profile, inflammatory markers and adipokines). Po1ymorphisms of the PLINI, NAMPT, RETN and GHRL genes were analyzed by real-time PCR. The OB group had altered anthropometric profile and increased risk for hypertension, type 2 diabetes and dyslipidemia in comparison with SOB and NOB groups (p <0.05). Concentrations of glucose, total cholesterol, LDL cholesterol, VLDL cholesterol, triglycerides, apolipoprotein B (ApoB), interleukin 1&#946; (IL-1&#946;) and tumor necrosis factor alpha (TNF&#945;) were higher and the concentrations ofHDL cholesterol and apolipoprotein AI (apoAI) were lower in the OB than in the other groups (p <0.05). The frequencies of the genetic polymorphisms of the total group were similar to those of other populations. NAMPT rs1319501 C> T and rs10763861 C> T polymorphisms were associated with obesity (p <0.05). Genetic polymorphisms did not influence the anthropometric profile ofthe total group (p> 0.05), but in the obese group, the GHRL rs4684677 T> A polymorphism was related to a higher body fat percentage (p = 0.043). After nutritional orientation, a decrease in calorie intake and in the consumption of carbohydrates, total fats, sodium, magnesium and beta-carotene CP <0.05) were observed. Genetic polymorphisms did not influence the anthropometric profile and the dietary intake of obese individuaIs after nutritional orientation. In conclusion, NAMPT and GHRL gene polymorphisms contribute to adiposity but do not influence dietary behavior and anthropometric profile after nutritional orientation.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A obesidade é um dos maiores problemas de saúde pública que cresce em todo o mundo, resultante de um desequilíbrio entre ingestão alimentar e gasto energético. Pode-se dizer que a obesidade é o principal fator de risco para o desenvolvimento de doenças crônicas de maior prevalência como dislipidemias, doenças cardiovasculares, diabetes do tipo 2 e esteatose hepática não alcóolica, acarretando na redução da qualidade e expectativa de vida. A Grelina é um hormônio sintetizado pelo estômago, que atua em diferentes tecidos através de um receptor específico (GHS-R1a), incluindo hipotálamo e tecidos periféricos, como o fígado. Esse hormônio está envolvido no comportamento alimentar e adiposidade, modulando o armazenamento ou utilização dos substratos energéticos no coração, músculo esquelético, adipócitos e fígado, além disso, revela-se de grande importância na manutenção do metabolismo energético hepático. Estes dados suportam a hipótese de que as vias de sinalização responsivas à grelina são um importante componente da regulação do metabolismo energético hepático e da homeostase glicêmica. O objetivo deste trabalho, foi estudar o metabolismo energético hepático e a sinalização da grelina em camundongos Swiss adultos obesos submetidos a dieta ocidental rica em gordura saturada e carboidratos simples. Avaliamos o efeito desta dieta a partir do 21 dia de idade (desmame) até o 133 dia destes animais, através de parâmetros biométricos e bioquímicos, avaliação histomorfológica, respirometria de alta resolução, conteúdo de glicogênio hepático e conteúdo de algumas proteínas envolvidas na sinalização de insulina e grelina, além do metabolismo energético hepático. Baseado em nossos resultados observamos que o consumo de dieta ocidental rica em gordura saturada e carboidrato simples durante 16 semanas causa hiperfagia, levando ao quadro de obesidade na idade adulta e prejuízo nas vias de sinalização dos hormônios insulina e grelina, que são importantes moduladores do metabolismo energético hepático, favorecendo o desenvolvimento de esteatose hepática não alcoólica. / Obesity is a major public health problem growing around the world, resulting from an imbalance between food intake and energy expenditure. Obesity is one of the main risk factor for developing the most prevalent chronic diseases as dyslipidemia, cardiovascular disease, type 2 diabetes and non-alcoholic fat liver disease, resulting in lower life expectancy and quality of life. Ghrelin is a hormone synthesized into the stomach, which has an important role in different tissues by a specific receptor (GHS-R1a), including the hypothalamus and peripheral tissues such as the liver. This hormone is involved in feeding behavior and adiposity by modulating storage or use of energy substrates in heart, skeletal muscle, adipocytes and liver. Moreover, Ghrelin is important in maintaining liver energy metabolism. These data support the hypothesis that ghrelin signaling pathways is a key component in the regulation of energy metabolism and hepatic glucose homeostasis. The aim of this study was to investigate the hepatic energy metabolism and signaling of ghrelin in obese adults Swiss mice fed the Western diet, rich in saturated fat and simple carboidrate. We analyzed the effect of this diet starting from 21 days of age (weaning) up to 133 days, using biometric and biochemical parameters, histomorphological assessment, high resolution respirometry, hepatic glycogen content and proteins content involved in insulin and ghrelin signaling besides the hepatic energy metabolism. Based on our results we found that the consumption of rich Western diet for 16 weeks promoves overeating leading to obesity in adulthood, metabolic desorders and impairment in signaling pathways of hormones insulin and ghrelin, which are important metabolic modulators of liver energy, contributing to the development of NAFLD.

Ο εν τω βάθει ηλεκτρικός εγκεφαλικός ερεθισμός (DBS) αποτελεί μία ευρέως αποδεκτή και πολύ αποτελεσματική θεραπευτική μέθοδο για ασθενείς με φαρμακοανθεκτική ν. Πάρκινσον. Διάφορες μελέτες έδειξαν ότι το DBS στον υποθαλάμιο πυρήνα (STN) οδηγεί σε αύξηση του σωματικού βάρους, ο μηχανισμός της οποίας παραμένει άγνωστος. Τα τελευταία χρόνια διατυπώθηκαν διάφορες θεωρίες για τον πιθανό μηχανισμό αυτής της αύξησης βάρους. Σύμφωνα με μία από τις πιο ενδιαφέρουσες θεωρίες, η αύξηση βάρους οφείλεται σε διαταραχή του μηχανισμού ρύθμισης της λήψης τροφής σε υποθαλαμικό επίπεδο. Είναι γνωστό ότι ο υποθάλαμος κατέχει κεντρικό ρόλο στη ρύθμιση της ομοιόστασης της ενέργειας: δέχεται, επεξεργάζεται και ερμηνεύει ορεξιογόνα και ανορεξιογόνα σήματα όπως η γκρελίνη, το ΝΡΥ και η λεπτίνη. Σκοπός της παρούσας μελέτης ήταν η διερεύνηση της πιθανής συμμετοχής του ηλεκτρικού ερεθισμού του υποθαλάμιου πυρήνα στη ρύθμιση της ομοιόστασης της ενέργειας, μέσω της διαταραχής των ορεξιογόνων και ανορεξιογόνων πεπτιδίων γκρελίνη, ΝΡΥ και λεπτίνη. Για το σκοπό αυτό μελετήθηκαν 23 από τους ασθενείς με ν.Πάρκινσον που υποβλήθηκαν σε STN DBS στην κλινική μας (15 άντρες-8 γυναίκες, ηλικία: 65,2 ± 8,9χρόνια, διάρκεια νόσου:12,7 ± 6χρόνια). Κάθε ασθενής εξετάστηκε σε 3 διαδοχικές χρονικές στιγμές: 3 μέρες πριν το χειρουργείο, 3 και 6 μήνες μετά το χειρουργείο και υπεβλήθη σε μέτρηση του σωματικού βάρους και του BMI, λιπομέτρηση και μέτρηση των επιπέδων γκρελίνης, λεπτίνης, NPY και κορτιζόλης ορού. Τρεις μέρες πριν και 6 μήνες μετά το χειρουργείο, πραγματοποιήθηκε κλινική εκτίμηση των ασθενών με τη χρήση των: Unified Parkinson’s Desease Rating Scale (UPDRS), Schwab and England Scale και Hoehn Yahr scale, καθώς και υπολογισμός της ημερήσιας δόσης ντοπαμίνης (LEDD). Τα αποτελέσματα της μελέτης μας συνοψίζονται ως εξής: 3 μήνες μετά τη χειρουργική επέμβαση διαπιστώθηκε σημαντική αύξηση βάρους των ασθενών μας: (3.09±5.00kg, P=0.007), χωρίς περαιτέρω αύξηση στους 6 μήνες. Τα επίπεδα του ΝΡΥ στο περιφερικό αίμα αυξήθηκαν σημαντικά 3 μήνες μετά το χειρουργείο (p=0.05), ενώ τα επίπεδα της γκρελίνης αυξήθηκαν σημαντικά στους 6 μήνες (p=0.001). Η αύξηση του σωματικού βάρους συσχετίστηκε σημαντικά με τη μεταβολή των επιπέδων της γκρελίνης και της λεπτίνης στους 3 και 6 μήνες αντίστοιχα. Συμπερασματικά μπορούμε να πούμε ότι το STN DBS φαίνεται να προκαλεί μία προσωρινή δυσλειτουργία της υποθαλάμιας έκκρισης ΝΡΥ και γκρελίνης. Η μεταβολή του σωματικού βάρους μπορεί να αποδοθεί στην αυξημένη έκκριση γκρελίνης και λεπτίνης. Περαιτέρω μελέτες με μεγαλύτερο αριθμό ασθενών απαιτούνται για να επιβεβαιωθεί ο ρόλος της πεπτιδιακής δυσλειτουργίας στην αύξηση βάρους μετά τη νευροδιέγερση και για να διερευνηθεί η πιθανή νευροπροστατευτική δράση που το DBS μπορεί να ασκήσει μέσω της αύξησης των επιπέδων της γκρελίνης. / Deep brain stimulation (DBS) is a widely accepted and highly effective treatment method for patients with medically refractory idiopathic Parkinson's desease. Various studies have shown that DBS of the subthalamic nucleus (STN) results in increased body weight, the mechanism of which is still unknown. In recent years there were various theories as to the possible mechanism of this weight gain. According to the most interesting theory, weight gain is due to a disruption of the central mechanism that regulates food intake. It is known that the hypothalamus plays a central role in the regulation of energy homeostasis: it receives, processes and interprets orexigenic and anorexigenic signals such as ghrelin, NPY and leptin. The aim of this study was to investigate the possible involvement of STN DBS in the regulation of energy homeostasis, through the disruption of orexigenic and anorexigenic peptides ghrelin, leptin and NPY. Twenty three patients with idiopathic Parkinson’s desease who underwent STN DBS in our clinic were included in our study (15 males - 8 females, age : 65,2 ± 8,9 years, disease duration : 12,7 ± 6chronia ). Each patient was examined at three consecutive time points: 3 days before surgery, 3 and 6 months after surgery. At each clinical appointment all patients underwent body composition measurements including body weight, body mass index (BMI) and fat mass, as well as blood sampling for the measurement of the circulating levels of ghrelin, leptin, NPY and cortisol. Three days before and 6 months after surgery patients were clinically evaluated with the use of the Unified Parkinson's Desease Rating Scale (UPDRS), Schwab and England Scale and Hoehn Yahr scale and the L-dopa daily dose (LEDD) was recorded. The results of our study are summarized as follows : 3 months after surgery there was a significant increase of body weight: (3.09 ± 5.00kg, P = 0.007), with no further increase at 6 months. NPY levels increased significantly 3 months after surgery (p = 0.05), while ghrelin levels increased significantly at 6 months (p = 0.001). Weight gain was significantly correlated with the change of ghrelin and leptin levels at 3 and 6 months respectively. In conclusion, STN DBS seems to temporarily dysregulate the hypothalamic secretion of NPY and ghrelin and weight gain can be attributed to the increased secretion of leptin and ghrelin. Further studies with a larger number of patients are required to confirm the role of peptide dysfunction on weight gain after neurostimulation and to investigate the possible neuroprotective role of DBS, exerted through the increase of ghrelin levels.

Les sécrétines peptidiques de l’hormone de croissance (GHRPs) constituent une classe de peptides synthétiques capables de stimuler la sécrétion de l’hormone de croissance (GH). Cette activité est médiée par leur liaison à un récepteur couplé aux protéines G : le récepteur des sécrétines de l’hormone de croissance (GHS-R1a), identifié subséquemment comme le récepteur de la ghréline. La ghréline est un peptide de 28 acides aminés sécrété principalement par les cellules de la muqueuse de l’estomac, qui exerce de nombreux effets périphériques indépendamment de la sécrétion de l’hormone de croissance. Les effets indépendants de la sécrétion de GH incluent, entre autres, des actions sur le contrôle de la prise de nourriture, le métabolisme énergétique, la fonction cardiaque, le système immunitaire et la prolifération cellulaire. L’étude de la distribution périphérique des sites de liaison des GHRPs nous a permis d’identifier un second site, le CD36, un récepteur scavenger exprimé dans plusieurs tissus dont le myocarde, l’endothélium de la microvasculature et les monocytes/macrophages. Le CD36 exprimé à la surface du macrophage joue un rôle clé dans l’initiation du développement de l’athérosclérose par la liaison et l’internalisation des lipoprotéines de faible densité oxydées (LDLox) dans l’espace sous-endothélial de l’artère. L’hexaréline, un analogue GHRP, a été développé comme agent thérapeutique pour stimuler la sécrétion de l’hormone de croissance par l’hypophyse. Sa propriété de liaison aux récepteurs GHS-R1a et CD36 situés en périphérie et particulièrement sa capacité d’interférer avec la liaison des LDLox par le CD36 nous ont incité à évaluer la capacité de l’hexaréline à moduler le métabolisme lipidique du macrophage. L’objectif principal de ce projet a été de déterminer les effets de l’activation des récepteurs CD36 et GHS-R1a, par l’hexaréline et la ghréline, le ligand endogène du GHS-R1a, sur la physiologie du macrophage et de déterminer son potentiel anti-athérosclérotique. Les résultats montrent premièrement que l’hexaréline et la ghréline augmentent l’expression des transporteurs ABCA1 et ABCG1, impliqués dans le transport inverse du cholestérol, via un mécanisme contrôlé par le récepteur nucléaire PPARγ. La régulation de l’activité transcriptionnelle de PPARγ par l’activation des récepteurs CD36 et GHS-R1a se fait indépendamment de la présence du domaine de liaison du ligand (LBD) de PPARγ et est conséquente de changements dans l’état de phosphorylation de PPARγ. Une étude plus approfondie de la signalisation résultant de la liaison de la ghréline sur le GHS-R1a révèle que PPARγ est activé par un mécanisme de concertation entre les voies de signalisation Gαq/PI3-K/Akt et Fyn/Dok-1/ERK au niveau du macrophage. Le rôle de PPARγ dans la régulation du métabolisme lipidique par l’hexaréline a été démontré par l’utilisation de macrophages de souris hétérozygotes pour le gène de Ppar gamma, qui présentent une forte diminution de l’activation des gènes de la cascade métabolique PPARγ-LXRα-transporteurs ABC en réponse à l’hexaréline. L’injection quotidienne d’hexaréline à un modèle de souris prédisposées au développement de l’athérosclérose, les souris déficientes en apoE sous une diète riche en cholestérol et en lipides, se traduit également en une diminution significative de la présence de lésions athérosclérotiques correspondant à une augmentation de l’expression des gènes cibles de PPARγ et LXRα dans les macrophages péritonéaux provenant des animaux traités à l’hexaréline. L’ensemble des résultats obtenus dans cette thèse identifie certains nouveaux mécanismes impliqués dans la régulation de PPARγ et du métabolisme du cholestérol dans le macrophage via les récepteurs CD36 et GHS-R1a. Ils pourraient servir de cibles thérapeutiques dans une perspective de traitement des maladies cardiovasculaires. / Growth hormone-releasing peptides (GHRPs) are a class of small synthetic peptides known to stimulate GH release through their binding to a G protein-coupled receptor identified as GHS-R1a, later recognized as the ghrelin receptor. Ghrelin is an acetylated 28 amino acid hormone initially identified from the stomach, which induces the release of growth hormone (GH) from the pituitary but also regulates food intake, energy homeostasis, cardiovascular function, immune system and cell proliferation. In documenting the peripheral distribution of GHRPs binding sites, we uncovered the presence of another binding site for GHRPs, identified as CD36, a class B scavenger receptor. CD36 is expressed among several tissues, including myocytes, endothelial cells of the microvasculature and monocytes/macrophages. The macrophage CD36 contributes to excessive lipid loading and atherogenic formation of foam cells through uptake of oxidized low-density lipoprotein (oxLDL) in the subendothelial space of the artery. The properties of hexarelin, a ligand for GHS-R1a and CD36, which features overlapping binding sites with that of oxLDL binding domain on CD36, and thus interfering with the binding of oxLDL on CD36, have prompted us to evaluate the potential of hexarelin, as well as that of the endogenous ligand ghrelin in the modulation of macrophage cholesterol metabolism. We demonstrate here the ability of hexarelin and ghrelin to enhance the expression of ATP-binding cassette A1 and G1 transporters through a PPARγ-dependent mechanism. The hormone binding domain of PPARγ is not required to mediate PPARγ transcriptional activation by CD36 and GHS-R1a. Both hexarelin and ghrelin promotes phosphorylation of PPARγ in THP-1 macrophages. A more detailed study of GHS-R1a-initiated signaling revealed an intricate and complex signalling interplay triggered by ghrelin that involves modulation of Src-dependent Dok-1/ERK1/2 and Src-independent Gαq/PI3-K/Akt pathways, leading to PPARγ-dependent transcriptional competence in the macrophages. The central role of PPARγ on cholesterol metabolism in the macrophages has been demonstrated using peritoneal macrophages from PPARγ heterozygote mice whose response to hexarelin on PPARγ-LXRα-ABC transporters pathway was strongly impaired. Treatment of apolipoprotein E-null mice fed on a lipid-rich diet with hexarelin resulted in a significant reduction in atherosclerotic lesions, concomitant with an enhanced expression of PPARγ and LXRα target genes in peritoneal macrophages. The results presented in this thesis feature novel mechanisms by which the beneficial regulation of PPARγ and cholesterol metabolism in macrophages could be regulated by both CD36 and ghrelin receptor. The downstream effects following the activation of these receptors might be potential targets in the treatment of human coronary artery disease.