No description available.

Pancreatic endocrine tumors and gastrointestinal stromal tumors are rare. Evidence regarding prognostic factors, and in the former also treatment, is scarce. We evaluated the survival and prognostic factors in a consecutive series of 324 patients with pancreatic endocrine tumors treated at a single institution. Radical surgery, WHO classification, TNM stage, age and Ki67 ≥2% emerged as independent prognostic factors. Having a non-functioning tumor was not an independent prognostic marker, and neither was hereditary tumor disease. We present the first evaluation of the newly proposed TNM staging system for these patients. A separate analysis of well-differentiated neuroendocrine carcinomas is reported, suggesting tumor size ≥5cm and Ki67 ≥2% as negative prognostic markers in this group. The first 36 patients with advanced neuroendocrine tumors treated with temozolomide at our clinic were evaluated. The median time to progression was seven months. Fourteen percent showed partial regression and 53% stabilization of disease. Side effects were generally mild. Investigation of O6-methylguanine DNA methyltransferase revealed a low expression in a subset of tumors. Four out of five patients responding to treatment had tumors with low expression. Concomitant expression of the orexigen ghrelin and its receptor in pancreatic endocrine tumors is demonstrated. No significant difference in mean plasma ghrelin between patients and controls were found, but elevated plasma ghrelin was seen in five patients. We provide the first report of expression of ghrelin and its receptor in gastrointestinal stromal tumors. Concomitant expression was frequent, indicating the presence of an autocrine loop. The tumors also expressed the neuroendocrine marker synaptic vesicle protein 2. Together, these findings are suggestive of neuroendocrine features.

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.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13362号 / 論医博第2204号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 稲垣 暢也, 教授 渡邉 大, 教授 影山 龍一郎 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

La ghréline acylée, une hormone produite par l’estomac, favorise la prise de poids corporel, majoritairement sous forme de masse grasse, par le biais de divers mécanismes centraux et périphériques via le récepteur sécrétagogue de l’hormone de croissance (GHSR). Le GHSR est un récepteur couplé aux protéines G qui, en plus de répondre à la ghréline acylée, possède une signalisation indépendante de la ghréline par le biais de son activité constitutive ou par une modulation de réponses dopaminergiques via oligomérisation du GHSR avec des récepteurs dopaminergiques. Malgré les puissantes réponses pharmacologiques à la ghréline acylée, des modèles animaux capables d’appréhender la complexité du système ghréline acylée-GHSR in vivo manquent, ce qui a considérablement ralenti l’élucidation des rôles physiologiques de cette hormone et de son récepteur. En effet, les modèles génétiques murins générés jusqu’à présent manquent de spécificité au niveau de l’hormone (incapacité à discriminer la ghréline acylée de la ghréline désacylée), et/ou au niveau du GHSR (incapacité à discriminer les différents modes de signalisation du GHSR). Dans ce contexte, de nouveaux modèles qui impacteraient de façon différentielle les voies de signalisation du GHSR seraient des outils pertinents pour contribuer au déchiffrage du système ghréline acylée-GHSR in vivo. Nous nous sommes ainsi attachés à caractériser un modèle de rats porteur d’une mutation ponctuelle dans le Ghsr qui prédit la délétion d’un domaine régulateur dans l’extrémité C-terminale du GHSR (GhsrQ343X). Dans des modèles cellulaires, nous avons montré que cette mutation découple le GHSR des processus d’internalisation du récepteur et de recrutement de la β-arrestine induits par la ghréline acylée, tout en augmentant la réponse aux agonistes du GHSR dans la voie des protéines G. Conformément à ce mécanisme, les rats mutants homozygotes GhsrM/M ont une réponse accrue à l’administration d’agonistes du GHSR sur le plan de la libération d’hormone de croissance, de la prise alimentaire ou de l’activité locomotrice. L’exploration physiologique et comportementale des rats GhsrM/M indique que la mutation GhsrQ343X est associée à une augmentation du poids et de l’adiposité indépendamment de la prise alimentaire, une diminution de l’oxydation globale des acides gras, de la flexibilité métabolique et de la tolérance au glucose, sans impact critique sur la prise alimentaire homéostatique. En outre, étant donné que la mutation GhsrQ343X n’augmente pas les niveaux circulants de ghréline, le phénotype métabolique général des rats GhsrM/M est en accord avec une sensibilité augmentée du GHSR en réponse au tonus endogène de ghréline acylée. Enfin, des résultats préliminaires suggèrent que la mutation GhsrQ343X pourrait être associée à des altérations relatives aux fonctions de récompense et de mémoire dont les mécanismes sous-jacents restent à décrypter. En résumé, nous proposons le modèle de rat mutant GhsrQ343X comme un nouvel outil, plus spécifique que les modèles murins d’invalidation génétique, pour explorer in vivo la signalisation du GHSR dans diverses fonctions biologiques, et à plus long terme aider au design de composés pharmacologiques ciblant le GHSR efficaces dans un cadre clinique. / The stomach-derived hormone acyl ghrelin promotes body weight gain, mostly in the form of fat mass, by means of several central and peripheral mechanisms mediated by the growth hormone secretagogue receptor (GHSR). The GHSR is a G protein-coupled receptor that, in addition to respond to acyl ghrelin, displays agonist-independent signaling through high constitutive activity and possibly heteromerization with dopamine receptors. Despite the potent biological properties of exogenous acyl ghrelin, the lack of animal models able to apprehend the complexity of the acyl ghrelin-GHSR system in vivo has been hampering the elucidation of its physiological roles. Indeed, genetic mouse models generated so far lack specificity either at the level of the hormone (not able to discriminate between acyl ghrelin versus desacyl ghrelin) and/or at the level of the GHSR (not able to discriminate between GHSR signaling modes). In this context, new models differentially affecting GHSR signaling pathways would represent valuable tools to decipher the acyl ghrelin-GHSR system in vivo. We therefore aimed at characterizing a new rat model carrying a point mutation in Ghsr that predicts truncation of a regulatory domain in the C-terminus, the GhsrQ343X mutation. In cellular models, this mutation was found to uncouple the GHSR from agonist-dependent receptor internalization and β-arrestin recruitment, while enhancing GHSR responsiveness in the G protein pathway. Accordingly, homozygous mutant GhsrM/M rats show enhanced responsiveness to exogenous GHSR agonists in terms of growth hormone release, food intake and locomotor activity. Physiological and behavioral exploration of GhsrM/M rats supports that the GhsrQ343X mutation is associated with increased body weight gain and adiposity independently of calorie intake, reduced whole-body fat oxidation, metabolic flexibility and glucose tolerance, without any critical impact on homeostatic feeding behavior. Moreover, given that circulating ghrelin levels are not increased by the GhsrQ343X mutation, the overall metabolic phenotype of GhsrM/M rats is consistent with enhanced GHSR sensitivity to the endogenous tone of acyl ghrelin. Furthermore, preliminary results suggest that the GhsrQ343X mutation could be associated with behavioral alterations related to reward and memory functions, through mechanisms that remain to be elucidated. Altogether, we propose the GhsrQ343X mutant rat model as a novel tool, more specific than knockout mouse models in its mechanism-of-action, to explore GHSR signaling across biological functions in vivo, and ultimately help in the design of efficient GHSR-targeting drugs.