Ghrelin is a gut-brain peptide hormone that induces appetite, stimulates the release of growth hormone, and has recently been shown to ameliorate inflammation. Recent studies have suggested that ghrelin may play a potential role in inflammation-related diseases such as inflammatory bowel diseases (IBD). A previous study with ghrelin in the TNBS mouse model of colitis demonstrated that ghrelin treatment decreased the clinical severity of colitis and inflammation and prevented the recurrence of disease. Ghrelin may be acting at the immunological and epithelial level as the ghrelin receptor (GHSR) is expressed by immune cells and intestinal epithelial cells. The current project investigated the effect of ghrelin in a different mouse model of colitis using dextran sodium sulphate (DSS) – a luminal toxin. Two molecular weight forms of DSS were used as they give differing effects (5kDa and 40kDa). Ghrelin treatment significantly improved clinical colitis scores (p=0.012) in the C57BL/6 mouse strain with colitis induced by 2% DSS (5kDa). Treatment with ghrelin suppressed colitis in the proximal colon as indicated by reduced accumulative histopathology scores (p=0.03). Whilst there was a trend toward reduced scores in the mid and distal colon in these mice this did not reach significance. Ghrelin did not affect histopathology scores in the 40kDa model. There was no significant effect on the number of regulatory T cells or TNF-α secretion from cultured lymph node cells from these mice. The discovery of C-terminal ghrelin peptides, for example, obestatin and the peptide derived from exon 4 deleted proghrelin (Δ4 preproghrelin peptide) have raised questions regarding their potential role in biological functions. The current project investigated the effect of Δ4 peptide in the DSS model of colitis however no significant suppression of colitis was observed. In vitro epithelial wound healing assays were also undertaken to determine the effect of ghrelin on intestinal epithelial cell migration. Ghrelin did not significantly improve wound healing in these assays. In conclusion, ghrelin treatment displays a mild anti-inflammatory effect in the 5kDa DSS model. The potential mechanisms behind this effect and the disparity between these results and those published previously will be discussed.

En production animale, notamment dans les filières bovines, il est dun intérêt économique daugmenter la quantité dhormone de croissance dans la circulation sanguine. La ghréline est un peptide principalement produit au niveau de la paroi stomacale. Ce ligand endogène au GHSR stimule la sécrétion hypophysaire de lhormone de croissance. Peptide orexigène, la ghréline est impliquée dans les mécanismes relatifs au maintien de lhoméostasie énergétique. Dans loptique daméliorer les performances animales, deux approches de la ghréline ont été effectuées. La première approche consiste en létude des gènes bovins codant pour la ghréline (bGHRL) et son récepteur (bGHSR). Ces deux gènes ont été respectivement localisés sur BTA 22 et BTA 1. Quatorze polymorphismes ont été détectés sur ces deux gènes et trois dentre eux affectent la structure primaire du GHSR bovin. Des associations, à différents niveaux de signification, entre certains de ces 14 sites polymorphiques et des traits de production et de conformation ont été mis en évidence au sein dun groupe de 127 taureaux Holstein sur base de leurs descendances directes présentes en Région Wallonne. La seconde approche aborde les effets dune immunisation passive contre la ghréline sur des rats mâles en croissance en comparaison à celles contre la leptine et la cholécystokinine. Lors dune alimentation équilibrée, le traitement envers la ghréline sur ces rats na pas influencé la croissance et lingestion par rapport aux animaux témoins. Des effets ont été observés entre les différentes immunomodulations au niveau des paramètres de croissance, dingestion et endocrinologiques. Les présents résultats invitent à de nouvelles investigations des gènes bGHRL et bGHSR sur des données relatives à dautres populations/races bovines et de limmunomodulation de la ghréline lors de conditions dexpérimentation différentes (alimentation déséquilibrée, stade physiologique, espèce, etc.). Ces investigations pourraient être valorisées en sélection et production animale mais également en médecine tant humaine que vétérinaire.

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

Die Regulation der Nahrungsaufnahme erfolgt zentral im Hypothalamus wo eine Vielzahl von G-Protein-gekoppelten Rezeptoren exprimiert werden die an der Gewichtsregulation beteiligt sind. Periphere hormonelle Signale aktivieren ihre korrespondierenden Rezeptoren im Nucleus arcuatus (ARC) oder im Nucleus paraventricularis (PVN) und modifizieren dadurch sowohl das anorexigene System, z.B. über die Stimulation des Melanocortin-4-Rezeptors (MC4R) im PVN, als auch das orexigene System mit dem Neuropeptide Y (NPY) sowie dem Agouti-related Protein (AgRP). Im Zuge einer systematischen Interaktionsstudie wurden verschiedene GPCRs, die entweder mit dem MC3R oder dem MC4R auf dem gleichen Neuron koexprimiert werden und nachweißlich die Appetit- und Gewichtregulation beeinflussen, untersucht. Basierend auf den Ergebnissen von Sandwich-ELISA und FRET- (Fluoreszenz-Energie-Transfer)Studien konnte eine Interaktion des MC3R mit dem Growth hormone secretagogues Rezeptor (GHSR) bestimmt werden, die beide auf den NPY/AgRP-Neuronen des ARC lokalisiert sind. Der MC3R gehört zu den Gαs bindenden Rezeptoren wohingegen GHSR über den Gαq vermittelten Signaltransduktionsweg signalisiert. Es konnte eine Erhöhung der induzierten cAMP-Spiegel infolge der Stimulation des MC3R sowohl mit α-, als auch β- und γ-MSH für die Koexpression von MC3R mit GHSR im Vergleich zum MC3R Homodimer ermittelt werden. Die Charakterisierung des neuen Signalisierungsverhaltens des Heterodimers unter der Verwendung verschiedener Inhibitoren zeigte eine Aktivierung von Gαi in Gegenwart der endogenen Agonisten beider Rezeptoren. Die Beobachtung unterschiedlicher Regulationsmuster nach der Kostimulation des Heterodimers in Abhängigkeit von α- oder γ-MSH jeweils in Anwesenheit von Ghrelin verweist auf komplexe Interaktionsmechanismen zwischen dem Melanocortin- und dem Ghrelin-Rezeptor innerhalb der hypothalamischen Gewichtsregulation. / Food intake is centrally regulated in hypothalamic nuclei where many GPCRs are expressed which are known to be involved in weight regulation.Peripheral hormonal signals activate their corresponding receptors in the arcuate nucleus (ARC) or paraventricular nucleus (PVN) and modulate the orexigenic (appetite-supressing) pathway mediated by stimulation of the melanocortin-4-receptor (MC4R) as well as the anorexigenic (appetite-stimulating) pathway including neuropeptide Y (NPY) and agouti-related protein (AgRP). In a systematic approach we investigated the interaction of a selective number of GPCRs which are co-expressed on the same neurons like MC3R or MC4R and know to play an essential role in hypothalamic weight regulation. Based on the results of a sandwich ELISA and fluorescence resonance energy transfer (FRET) approach we report the interaction of the MC3R and the growth hormone secretagogue receptor (GHSR) which are co-expressed on arcuate NPY/AgRP neurons. It is known that MC3R couple to the Gαs whereas GHSR couple to the Gαq signaling pathway. However, here the co-expression of MC3R and GHSR reveal a profoundly increase cAMP-accumulation after melanocortin (α-, β- and γ-MSH) challenge, that is higher compared to MC3R activation alone. In-depth characterization of the new signaling properties of the MC3R/GHSR heterodimer by different inhibitors revealed the activation of Gαi in the presents of both endogene agonists. The observation of different regulatory pattern after co-stimulation of the heterodimer depending on the endogenouse ligands (α- or γ-MSH) of MC3R reflect complex functional interaction mechanisms between melanocortin and ghrelin receptors within the hypothalamic signaling pathways of weight regulation.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / A grelina é um ligante endógeno do receptor secretagogo do hormônio do crescimento (GHSR), potente estimulador da liberação do hormônio de crescimento (GH), ingestão alimentar, e adiposidade. Além disso, sua ação hormonal inclui regulação do metabolismo energético cardíaco. Entretanto, a hipernutrição no início da vida leva ao desenvolvimento da obesidade, induz hipertrofia cardíaca, compromete a função cardíaca, e gera insuficiência cardíaca na vida adulta. Avaliar proteínas chaves no processo de sinalização da grelina no remodelamento cardíaco no coração de camundongos obesos após a hipernutrição na lactação. A obesidade foi induzida por redução de ninhada e camundongos adultos (180 dias) foram divididos em: grupo hiperalimentado, GH com obesidade decorrente de hipernutrição na lactação e controle, GC. Cardiomiócitos (cmi) do ventrículo esquerdo foram analisados por microscopia de luz e estereologia, o conteúdo e fosforilação de proteínas cardíacas: receptor de grelina (hormônio do crescimento secretagogo receptor 1a, GHSR-1a), proteína quinase-B (AKT e pAKT), phosphatidil inositol 3-quinase (PI3K), proteína quinase ativada por AMP (AMPK e pAMPK), m-TOR, pmTOR, Bax, Bcl2 e actina foram analizados por western blotting. A expressão gênica do GHSR-1a foi analisada por PCR em tempo real. A respirometria de alta resolução dos cardiomiócitos foi analisada por oxígrafo OROBOROS. Significância estatística (P< 0,05) determinada por teste t-Student não-pareado. Nossos dados demonstram que a hipernutrição na lactação induz aumento no peso corporal, iniciado aos 10 dias de idade, persistindo até os 180 dias de idade. A glicemia, peso do fígado, e da gordura visceral foram maiores no grupo GH. Além disso, o grupo GH também apresentou aumento no peso do coração e razão peso do coração/CT (comprimento da tíbia), indicando hipertrofia e remodelamento cardíaco, aumento na expressão e conteúdo de GHSR-1a no coração, associado ao maior conteúdo de PI3K e maior conteúdo e fosforilação de AKT, diminuição no conteúdo de Bcl2. Em contraste, o conteúdo e fosforilação da AMPK e mTOR no coração não foram diferentes entre os grupos. Os níveis de grelina plasmático no GH foram menores. A respiração do GH com grelina foi menor que no GC com grelina. A incubação das fibras cardíacas com grelina resultou em aumento do fluxo respiratório após adição de citocromo c nos grupos com grelina, indicando dano à membrana mitocondrial e extravazamento de citocromo c. Os grupos GC com grelina e GH sem grelina apresentaram RCR menor comparado ao GC sem grelina, indicando desacoplamento mitocondrial. Nossos resultados mostram que a hipernutrição na lactação induz diminuição do nível de grelina plasmática e aumento da expressão do GHS-R1a no cardiomiócito do animal quando adulto. Tal processo determina aumento da sensibilidade a grelina no coração, processo que ocorre independentemente de variações do AMPK e mTOR. Sugerimos uma redução no efeito protetor da ação da grelina na AMPK. Também, demonstramos que o remodelamento do miocárdio nestes animais adultos associa-se a GHSR-1a, PI3K, e fosforilação da AKT, mas não com AMPK e mTOR na vida adulta. / Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), has been suggested to be associated to obesity, insulin secretion, cardiovascular growth and homeostasis. GHS-R has been found in most of the tissues, and among the hormone action it is included the regulation of heart energy metabolism. Therefore, hypernutrition during early life leads to obesity, induces cardiac hypertrophy, compromises myocardial function, inducing heart failure in adulthood. We examined ghrelin signaling process in cardiac remodeling in these obese adult mice. We examined key proteins of cardiomyocyte metabolism in heart left ventricle from overfed (OG) and control (CG) groups from adult mice (180 days) overfed during lactation. Obesity was induced by litter reduction. Therefore, the study was done in adult mice 180 days old (OG, obese group (n=10) and CG, control group (n=10). The cardiomyocytes (cmy) of left ventricle were analyzed by light microscopy and stereology. The content and phosphorylation of cardiac proteins: growth hormone secretagogue receptor 1a (GHSR-1a), protein kinase B (AKT and pAKT), phosphatidil inositol 3 kinase (PI3K), AMP-activated protein kinase (AMPK and pAMPK), mTOR and pmTOR, BAX, Bcl2 and actin was achieved by western blotting. GHSR-1a gene expression was analyzed to RT-PCR. We performed high-resolution respirometry of cardiomyocytes with OROBOROS Oxygraph-2k. Statistical significance was determined by Student t-test for unpaired. P< 0.05 was considered statistical significant. Body weight, blood glucose, liver weight, and visceral fat weight were higher in OG than CG group. Obese mice had increased heart weight and heart weight/TL (tibia length) indicating cardiac remodeling and hypertrophy, increased GHSR-1a content and expression in the heart, associated to PI3K content, increased AKT content and phosphorylation (P< 0.05), decreased Bcl2 content. In contrast, AMPK and mTOR content and phosphorylation in heart were not different between the experimental groups. Ghrelin plasma levels in obese group were decreased when compared to control group. The O2 consumption of OG with ghrelin was lower than in the CG with ghrelin. Incubation of cardiac fibers with ghrelin resulted in increased respiration after addition of cytochrome c in groups with ghrelin, indicating mitochondrial membrane damage and leakage of cytochrome c. CG with ghrelin and OG without ghrelin showed RCR lower compared to GC without ghrelin, indicating mitochondrial uncoupling. In this study, our results showing a decrease of ghrelin level and an increase of GHS-R1a expression for a down regulation that leads a higher sensibility of ghrelin in heart, occurring independently of any association with AMPK and mTOR in hearts of obese mice. That suggests a reduction in protective effects through ghrelin action on AMPK. In addition, our data demonstrated that remodeled myocardial in obese mice exclusively overnourished in early life are associated with GHSR-1a, PI3K and AKT phosphorylation but not with AMPK and mTOR in adulthood.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / A grelina é um ligante endógeno do receptor secretagogo do hormônio do crescimento (GHSR), potente estimulador da liberação do hormônio de crescimento (GH), ingestão alimentar, e adiposidade. Além disso, sua ação hormonal inclui regulação do metabolismo energético cardíaco. Entretanto, a hipernutrição no início da vida leva ao desenvolvimento da obesidade, induz hipertrofia cardíaca, compromete a função cardíaca, e gera insuficiência cardíaca na vida adulta. Avaliar proteínas chaves no processo de sinalização da grelina no remodelamento cardíaco no coração de camundongos obesos após a hipernutrição na lactação. A obesidade foi induzida por redução de ninhada e camundongos adultos (180 dias) foram divididos em: grupo hiperalimentado, GH com obesidade decorrente de hipernutrição na lactação e controle, GC. Cardiomiócitos (cmi) do ventrículo esquerdo foram analisados por microscopia de luz e estereologia, o conteúdo e fosforilação de proteínas cardíacas: receptor de grelina (hormônio do crescimento secretagogo receptor 1a, GHSR-1a), proteína quinase-B (AKT e pAKT), phosphatidil inositol 3-quinase (PI3K), proteína quinase ativada por AMP (AMPK e pAMPK), m-TOR, pmTOR, Bax, Bcl2 e actina foram analizados por western blotting. A expressão gênica do GHSR-1a foi analisada por PCR em tempo real. A respirometria de alta resolução dos cardiomiócitos foi analisada por oxígrafo OROBOROS. Significância estatística (P< 0,05) determinada por teste t-Student não-pareado. Nossos dados demonstram que a hipernutrição na lactação induz aumento no peso corporal, iniciado aos 10 dias de idade, persistindo até os 180 dias de idade. A glicemia, peso do fígado, e da gordura visceral foram maiores no grupo GH. Além disso, o grupo GH também apresentou aumento no peso do coração e razão peso do coração/CT (comprimento da tíbia), indicando hipertrofia e remodelamento cardíaco, aumento na expressão e conteúdo de GHSR-1a no coração, associado ao maior conteúdo de PI3K e maior conteúdo e fosforilação de AKT, diminuição no conteúdo de Bcl2. Em contraste, o conteúdo e fosforilação da AMPK e mTOR no coração não foram diferentes entre os grupos. Os níveis de grelina plasmático no GH foram menores. A respiração do GH com grelina foi menor que no GC com grelina. A incubação das fibras cardíacas com grelina resultou em aumento do fluxo respiratório após adição de citocromo c nos grupos com grelina, indicando dano à membrana mitocondrial e extravazamento de citocromo c. Os grupos GC com grelina e GH sem grelina apresentaram RCR menor comparado ao GC sem grelina, indicando desacoplamento mitocondrial. Nossos resultados mostram que a hipernutrição na lactação induz diminuição do nível de grelina plasmática e aumento da expressão do GHS-R1a no cardiomiócito do animal quando adulto. Tal processo determina aumento da sensibilidade a grelina no coração, processo que ocorre independentemente de variações do AMPK e mTOR. Sugerimos uma redução no efeito protetor da ação da grelina na AMPK. Também, demonstramos que o remodelamento do miocárdio nestes animais adultos associa-se a GHSR-1a, PI3K, e fosforilação da AKT, mas não com AMPK e mTOR na vida adulta. / Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), has been suggested to be associated to obesity, insulin secretion, cardiovascular growth and homeostasis. GHS-R has been found in most of the tissues, and among the hormone action it is included the regulation of heart energy metabolism. Therefore, hypernutrition during early life leads to obesity, induces cardiac hypertrophy, compromises myocardial function, inducing heart failure in adulthood. We examined ghrelin signaling process in cardiac remodeling in these obese adult mice. We examined key proteins of cardiomyocyte metabolism in heart left ventricle from overfed (OG) and control (CG) groups from adult mice (180 days) overfed during lactation. Obesity was induced by litter reduction. Therefore, the study was done in adult mice 180 days old (OG, obese group (n=10) and CG, control group (n=10). The cardiomyocytes (cmy) of left ventricle were analyzed by light microscopy and stereology. The content and phosphorylation of cardiac proteins: growth hormone secretagogue receptor 1a (GHSR-1a), protein kinase B (AKT and pAKT), phosphatidil inositol 3 kinase (PI3K), AMP-activated protein kinase (AMPK and pAMPK), mTOR and pmTOR, BAX, Bcl2 and actin was achieved by western blotting. GHSR-1a gene expression was analyzed to RT-PCR. We performed high-resolution respirometry of cardiomyocytes with OROBOROS Oxygraph-2k. Statistical significance was determined by Student t-test for unpaired. P< 0.05 was considered statistical significant. Body weight, blood glucose, liver weight, and visceral fat weight were higher in OG than CG group. Obese mice had increased heart weight and heart weight/TL (tibia length) indicating cardiac remodeling and hypertrophy, increased GHSR-1a content and expression in the heart, associated to PI3K content, increased AKT content and phosphorylation (P< 0.05), decreased Bcl2 content. In contrast, AMPK and mTOR content and phosphorylation in heart were not different between the experimental groups. Ghrelin plasma levels in obese group were decreased when compared to control group. The O2 consumption of OG with ghrelin was lower than in the CG with ghrelin. Incubation of cardiac fibers with ghrelin resulted in increased respiration after addition of cytochrome c in groups with ghrelin, indicating mitochondrial membrane damage and leakage of cytochrome c. CG with ghrelin and OG without ghrelin showed RCR lower compared to GC without ghrelin, indicating mitochondrial uncoupling. In this study, our results showing a decrease of ghrelin level and an increase of GHS-R1a expression for a down regulation that leads a higher sensibility of ghrelin in heart, occurring independently of any association with AMPK and mTOR in hearts of obese mice. That suggests a reduction in protective effects through ghrelin action on AMPK. In addition, our data demonstrated that remodeled myocardial in obese mice exclusively overnourished in early life are associated with GHSR-1a, PI3K and AKT phosphorylation but not with AMPK and mTOR in adulthood.