Evaluating the growth axis of an ancient species such as the Shovelnose Sturgeon Scaphirhynchus platorynchus can aid in understanding the divergence of growth factors among a vast evolutionary span of vertebrates. It is hypothesized, that similar to teleosts and tetrapods, growth hormone (GH) mediates and stimulates the anabolic actions of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) in sturgeon. Using Shovelnose Sturgeon as a model, the objectives of this research were to identify the acute and chronic effects of exogenous recombinant bovine GH (rbGH) on IGF-I and IGF-II gene expression and investigate the roles IGF's have on whole animal growth and nutrient partitioning in the presence of chronic rbGH administration. In the first experiment, fish were injected with five different concentrations of rbGH (0, 30, 60, 120, 240 μg/g body weight (BW) rbGH)) and sampled at five time points (6, 12, 24, 36, 48-h post-injection). Hepatic and muscle tissue specimens were rapidly excised following euthanasia for mRNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) analysis of IGF expression. qRT-PCR analysis indicated hepatic IGF-I and IGF-II expression was significantly higher than muscle IGF-I expression, and IGF-II was not expressed in muscle. The highest rbGH concentration (240 μg/g BW) significantly increased hepatic IGF-I and IGF-II mRNA and muscle IGF-I mRNA expression levels at 48 hours post injection. After 6 weeks of bi-weekly rbGH administration in a second experiment, fish injected with rbGH at 240 μg/g BW gained significantly more length and weight than fish injected with the sham (0 μg rbGH; sesame oil), and whole body proximate analysis revealed rbGH treated fish had significantly higher amounts of protein. This research aids in understanding the regulatory and evolutionary principles of the vertebrate somatotropic axis through characterizing the endocrine regulation of growth in Scaphirhynchus sturgeon.

Le traumatisme cranio-cérébral (TCC) est l'une des plus grandes causes de mortalité et de morbidité de ce siècle. Les études menées ces dernières années sur ce sujet, ont montré que l'hypopituitarisme est une des conséquences les plus fréquentes et les plus durables consécutives à un TCC. L'hypopituitarisme se traduit par une ou des déficience(s) hormonale(s) et notamment une diminution de la sécrétion de l'hormone de croissance (GH). Mes travaux de thèse ont consisté à mettre en place un model murin pour étudier le déficit en GH après un TCC ; et ainsi rechercher à quel niveau l'axe somatotrope est-il touché. J'ai tout d'abord participé à des études qui m'ont permis de mettre en place des pré-requis expérimentaux à l'étude du TCC sur des souris (J. Endoc. 2009 ; PNAS, 2010). Dans ce modèle animal, 10% à 20% de ces souris traumatisées présentent un défaut de sécrétion de la GH trente jours après avoir subi un TCC au niveau du cortex pariétal droit, ont un défaut de sécrétion de la GH, alors que les animaux opérés mais non traumatisés (sham) ne présentent pas ce défaut. Les souris déficientes pour la sécrétion de GH ne présentent pas de dérégulation de l'architecture vasculaire hypophysaire ou hypothalamique. Au niveau moléculaire, la transcription des ARNm de l'hormone de croissance et de la neurohormone hypothalamique GHRH (qui stimule la sécrétion de GH) ne semble pas affectée par les effets du traumatisme. En revanche, très rapidement après un traumatisme localisé au niveau du cortex, on observe l'apparition d'une cicatrice gliale. Cette cicatrice astrocytaire apparait non seulement dans la région du traumatisme, mais également au niveau de l'éminence médiane. Cette réponse inflammatoire, associée à un défaut des tanycytes bordant le 3ème ventricule, pourrait expliquer, au moins en partie, ce déficit en GH post-TCC. En résumé, cette étude nous a permis de reproduire sur le modèle murin (avec un même fond génétique) les déficits en GH que l'on observe chez des patients traumatisés. / Traumatic Brain Injury (TBI) is a major cause of mortality and morbidity. Studies on this subject in recent years have revealed that hypopituitarism is one of the most common sequelae to TBI. Since the pituitary is an endocrine tissue, hypopituitarism results in marked hormonal deficiency, including decreased serum levels of growth hormone. As such, the work contained within this thesis has focused on developing a mouse model of TBI-induced GH deficiency, with the principal aim of discovering where the deficit lies in the somatotropic axis (i.e. hypothalamaic releasing hormone vs. pituitary cell activity). By subjecting mice to a controlled impact to the right parietal cortex, we observed a GH secretory defect in 10% to 20% of cases. These defects where apparent thirty days following trauma and were not detected in animals subjected to surgery, but not TBI (sham). Mice suffering hypopituitarism secondary to TBI did not show any obvious changes to pituitary or hypothalamic vascular architecture, and at the molecular level, GH and GHRH (the major GH-secretagogue) mRNA levels were normal. However, in the acute phase following TBI, a large glial scar developed which extended to the level of the median eminence (ME). This inflammatory response could explain, at least in part, GH deficiency post-TBI if associated with dysfunction of third ventricle tanycytes known to be involved in hypothalamic neuron output. In summary, by using an isogenic, age-matched mouse model of TBI, this study has provided insights into the causes of GH deficiency, a common consequence of cranial trauma in humans.

Durante a endotoxemia, observa-se alteração no eixo hormônio do crescimento(GH)/fator de crescimento semelhante à insulina (IGF)-1. Acredita-se que o aumento de citocinas pró-inflamatórias seja responsável por essa alteração, apesar do mecanismo para essa alteração ainda não estar completamente elucidado. A ghrelina é um hormônio peptídico que apresenta propriedades anti-inflamatórias, e, portanto, pode contribuir para a manutenção da integridade do eixo GH/IGF-1. O objetivo do presente estudo foi avaliar o efeito do tratamento sistêmico de ghrelina sobre o eixo GH/IGF-1 em ratos Wistar submetidos à endotoxemia. Para a indução da endotoxemia, foi administrado lipopolissacarídeo (LPS; 5mg/kg intraperitoneal) sistemicamente. Os animais foram tratados com ghrelina (15nmol/kg; endovenoso) concomitantemente à administração de LPS e tiveram o sangue e o fígado coletados após 2h,6h ou 12h. Foram quantificadas a concentração sanguínea do fator de necrose tumoral alfa (TNF-?), interleucina (IL)-1?, IL-6, nitrato, corticosterona, GH, IGF-1 e ghrelina endógena, assim como a concentração hepática de TNF-?, IL-1? e IL-6. O TNF-?, IL-1?, IL-6, GH, IGF-1 e ghrelina endógena foram quantificados pela técnica de ELISA. A corticosterona foi quantificada pela técnica de radioimunoensaio. O Nitrato foi quantificado pela técnica de quimioluminescência. Também foram quantificadas a expressão proteica hepática do receptor do hormônio secretador do GH (GHSR-1a) e do receptor do GH (GHR) pela técnica de Western Blott, bem como a expressão gênica hepática de IGF-1 e GHR pela técnica de PCR-RT. Os ratos submetidos à endotoxemia apresentaram redução sérica de IGF-1 e de GH, caracterizando a alteração do eixo GH/IGF-1. Os animais endotoxêmicos e tratados com ghrelina apresentaram menor redução dos níveis circulantes de IGF-1, além de apresentarem menores níveis de TNF-?, IL-1?, IL-6 e nitrato após administração de LPS. A menor redução de IGF-1 circulante após o tratamento com ghrelina não foi relacionada a alterações na expressão proteica de GHSR-1a ou GHR, nem relacionada a alterações na expressão gênica de IGF-1 ou GHR nos intervalos de tempo analisados. Portanto, a propriedade anti-inflamatória da ghrelina levou à redução do aumento dos mediadores pró-inflamatórios e contribuiu para a manutenção da integridade do eixo GH/IGF-1 ao atenuar a queda na concentração sanguínea de IGF-1. Endotoxemia. Inflamação. Eixo GH/IGF-1 / During the endotoxemia it is possible to observe a change on the growth hormone (GH) /insulin-like growth factor (IGF)-1 axis. It is believed that the pro inflammatory cytokines increase is responsible for this change even not having the mechanism for this change completely elucidated. The ghrelin is a peptidic hormone which has antiinflammatory properties and, because of that, can contribute to the GH/IGF-1 axis integrity maintenance. This research goal is to evaluate the ghrelin systemic treatment effect on the GH/IGF-1 axis on Wistar rats submitted to endotoxemia. To induct the endotoxemia it was given systemically to the rats a lipopolysaccharide (LPS; 5mg/kg intraperitoneal). The animals were treated with ghrelin (15nmol/kg; intravenous) while receiving the LPS and they had their blood and liver collected after 2h, 6h or 12h. Blood concentration of alfa tumoral necrose (TNF-?), interleukin (IL)-1?, IL-6, nitrate, corticosterone, GH, IGF-1 and endogenous ghrelin were quantified as well as their TNF-?, IL-1? e IL-6 hepatic concentration. The TNF-?, IL-1?, IL-6, GH, IGF-1 and the endogenous ghrelin were quantified through the ELISA technique. The corticosterone was quantified through the radioimmunoassay technique. The nitrate was quantified through the chemiluminescence technique. The hepatic protein expression from the growth hormone secretagogue receptor (GHSR)-1a and the receptor of the GH (GHR) were quantified through the Western Blott technique and the IGF-1 and the GHR hepatic gene expression through the PCR-RT technique. The rats submitted to the endotoxemia presented an IGF-1 and a GH serum decrease characterizing a change on the GH/IGF-1 axis. The endotoxemic animals treated with ghrelin showed a smaller reduction of the IGF-1 circulating levels besides presenting a smaller TNF-?, IL-1?, IL- 6 and nitrate levels after receiving the LPS. The smallest IGF-1 circulating decrease, after the treatment with ghrelin, was related neither to the changes on the GHSR-1a or GHR protein expressions nor to the IGF-1 or GHR gene expressions during the analyzed time intervals. Therefore, the ghrelin anti-inflammatory property inflected a reduction of the pro-inflammatory mediators increase and contributed for the GH/IGF- 1 axis integrity maintenance while mitigating the IGF-1 blood concentration fall.

Determining the mechanisms by which growth hormone (GH) enhances bone growth and development has proven difficult. GH can act either systemically via the stimulation of liver insulin like growth factor (IGF)-1, or locally via activation of the GH receptor (GHR). Furthermore, the local actions of GH may be IGF-1 dependent (indirect) or independent (direct). Suppressor of cytokine signalling 2 (SOCS2) has been identified as an important regulator of GH signalling via the JAK/STAT pathway. The SOCS2 knockout (Socs2-/-) mouse is characterised by its overgrowth phenotype despite no elevation in systemic GH and IGF-1 levels. It therefore offers a valid and novel model to investigate the local effects of enhanced GH signalling on the skeleton. The work presented in this thesis investigates the Socs2-/- mouse model to better understand the actions of local GH on longitudinal bone growth and bone accrual. Ex vivo metatarsal organ cultures, osteoblast cultures, and in vivo approaches are used to unravel the mechanisms of GH action on the skeleton. This thesis also explores the potential of SOCS2 as primary mediator of inflammatory induced bone loss through the utilisation of the dextran sulphate sodium (DSS) model of colitis. Embryonic and postnatal ex vivo metatarsal organ cultures are used to study the mechanism of GH action on longitudinal bone growth. Specifically, the present work highlights that enhanced linear growth in the absence of SOCS2 is associated with an increase in the GH regulated proteins, IGF-2 and IGF binding protein 3 (IGFBP3), but not IGF-1. This indicates that IGF-1 may not be essential for mediating GH action on bone growth. Completion of an in depth analysis of the bone phenotype of juvenile and adult, male and female Socs2-/- mice reveals an anabolic phenotype consistent with increased GH signalling. Male Socs2-/- mice are shown to have a greater enhancement of cortical parameters compared to females, resulting in increased bone strength. Investigation of the mechanisms behind the enhanced bone accrual in Socs2-/- mice identifies SOCS2 as the primary SOCS protein regulating GH signalling in primary osteoblasts. The JAK/STAT pathway is confirmed as the key signalling pathway targeted by SOCS2. Despite this enhanced signalling there is little evidence presented in this thesis to suggest that GH actions on osteoblasts and ultimately bone mass are mediated through increased Igf1 expression. GH treatment is shown to be anabolic to bone of young juvenile Socs2-/- mice, but not WT mice. This increase in bone mass is associated with increase bone p-STAT5 signalling, but no increase in Igf1 levels indicating that GH may have IGF-1 independent effects in the Socs2-/- mouse model. GH treatment of young mice also reveals an age and sex specific effect of GH action where GH does not stimulate growth until approximately 3 weeks of age. From 3 weeks of age, WT female mice show increased growth in response to GH, but males do not. The increased growth is associated with increased p-STAT5 signalling and increased bone area. This thesis also confirms SOCS2 a critical mediator of bone loss associated with inflammation. The present results show that deteriorated trabecular bone health in colitic mice is associated with elevated Socs2 expression in bone. Furthermore, despite similar levels of gut inflammation observed in Socs2-/- mice with DSS induced colitis these mice are partly protected from poor bone health. The work described herein has used the Socs2-/- mouse model to strengthen our understanding of the actions of local GH on skeletal growth and development. It also provides compelling evidence for the importance of SOCS2 as a mediator of bone loss in cases of inflammatory bowel disease.

Durante a endotoxemia, observa-se alteração no eixo hormônio do crescimento(GH)/fator de crescimento semelhante à insulina (IGF)-1. Acredita-se que o aumento de citocinas pró-inflamatórias seja responsável por essa alteração, apesar do mecanismo para essa alteração ainda não estar completamente elucidado. A ghrelina é um hormônio peptídico que apresenta propriedades anti-inflamatórias, e, portanto, pode contribuir para a manutenção da integridade do eixo GH/IGF-1. O objetivo do presente estudo foi avaliar o efeito do tratamento sistêmico de ghrelina sobre o eixo GH/IGF-1 em ratos Wistar submetidos à endotoxemia. Para a indução da endotoxemia, foi administrado lipopolissacarídeo (LPS; 5mg/kg intraperitoneal) sistemicamente. Os animais foram tratados com ghrelina (15nmol/kg; endovenoso) concomitantemente à administração de LPS e tiveram o sangue e o fígado coletados após 2h,6h ou 12h. Foram quantificadas a concentração sanguínea do fator de necrose tumoral alfa (TNF-?), interleucina (IL)-1?, IL-6, nitrato, corticosterona, GH, IGF-1 e ghrelina endógena, assim como a concentração hepática de TNF-?, IL-1? e IL-6. O TNF-?, IL-1?, IL-6, GH, IGF-1 e ghrelina endógena foram quantificados pela técnica de ELISA. A corticosterona foi quantificada pela técnica de radioimunoensaio. O Nitrato foi quantificado pela técnica de quimioluminescência. Também foram quantificadas a expressão proteica hepática do receptor do hormônio secretador do GH (GHSR-1a) e do receptor do GH (GHR) pela técnica de Western Blott, bem como a expressão gênica hepática de IGF-1 e GHR pela técnica de PCR-RT. Os ratos submetidos à endotoxemia apresentaram redução sérica de IGF-1 e de GH, caracterizando a alteração do eixo GH/IGF-1. Os animais endotoxêmicos e tratados com ghrelina apresentaram menor redução dos níveis circulantes de IGF-1, além de apresentarem menores níveis de TNF-?, IL-1?, IL-6 e nitrato após administração de LPS. A menor redução de IGF-1 circulante após o tratamento com ghrelina não foi relacionada a alterações na expressão proteica de GHSR-1a ou GHR, nem relacionada a alterações na expressão gênica de IGF-1 ou GHR nos intervalos de tempo analisados. Portanto, a propriedade anti-inflamatória da ghrelina levou à redução do aumento dos mediadores pró-inflamatórios e contribuiu para a manutenção da integridade do eixo GH/IGF-1 ao atenuar a queda na concentração sanguínea de IGF-1. Endotoxemia. Inflamação. Eixo GH/IGF-1 / During the endotoxemia it is possible to observe a change on the growth hormone (GH) /insulin-like growth factor (IGF)-1 axis. It is believed that the pro inflammatory cytokines increase is responsible for this change even not having the mechanism for this change completely elucidated. The ghrelin is a peptidic hormone which has antiinflammatory properties and, because of that, can contribute to the GH/IGF-1 axis integrity maintenance. This research goal is to evaluate the ghrelin systemic treatment effect on the GH/IGF-1 axis on Wistar rats submitted to endotoxemia. To induct the endotoxemia it was given systemically to the rats a lipopolysaccharide (LPS; 5mg/kg intraperitoneal). The animals were treated with ghrelin (15nmol/kg; intravenous) while receiving the LPS and they had their blood and liver collected after 2h, 6h or 12h. Blood concentration of alfa tumoral necrose (TNF-?), interleukin (IL)-1?, IL-6, nitrate, corticosterone, GH, IGF-1 and endogenous ghrelin were quantified as well as their TNF-?, IL-1? e IL-6 hepatic concentration. The TNF-?, IL-1?, IL-6, GH, IGF-1 and the endogenous ghrelin were quantified through the ELISA technique. The corticosterone was quantified through the radioimmunoassay technique. The nitrate was quantified through the chemiluminescence technique. The hepatic protein expression from the growth hormone secretagogue receptor (GHSR)-1a and the receptor of the GH (GHR) were quantified through the Western Blott technique and the IGF-1 and the GHR hepatic gene expression through the PCR-RT technique. The rats submitted to the endotoxemia presented an IGF-1 and a GH serum decrease characterizing a change on the GH/IGF-1 axis. The endotoxemic animals treated with ghrelin showed a smaller reduction of the IGF-1 circulating levels besides presenting a smaller TNF-?, IL-1?, IL- 6 and nitrate levels after receiving the LPS. The smallest IGF-1 circulating decrease, after the treatment with ghrelin, was related neither to the changes on the GHSR-1a or GHR protein expressions nor to the IGF-1 or GHR gene expressions during the analyzed time intervals. Therefore, the ghrelin anti-inflammatory property inflected a reduction of the pro-inflammatory mediators increase and contributed for the GH/IGF- 1 axis integrity maintenance while mitigating the IGF-1 blood concentration fall.

Context and objective: Experimental models demonstrate an important role of growth hormone (GH) in retinal development. However, the interactions between GH/IGF-I axis and the neuro-vascularization of the human retina are still not clear. A model of untreated congenital isolated GH deficiency (IGHD) may clarify the action of GH on the retina. The purpose of this work was to assess the retinal neuro-vascularization in adults with congenital IGHD. Methods: In a cross sectional study, we performed fundus photographs (to assess the number of retinal vascular branching points and the optic disc and cup size), and optical coherence tomography (to assess the thickness of macula) in 25 adults IGHD subjects (13 males, 50,9 yr. [12,0]) homozygous for a null mutation (c.57+1 G>A) in the GH releasing hormone receptor gene and 28 controls (14 males, 46,4 yr. [14,7]). Results: Fisher's exact test revealed that IGHD subjects presented more reduction of vascular branching points in comparison to controls (91% vs. 53% [p=0.049]). Conversely, the percentage of moderate reduction in IGHD was higher than in control (p=0,01). The rates of individuals with increased optic disc and cup size were increased in IGHD in comparison to controls (92,9% vs. 57,1 for optic disc and 92,9% vs. 66,7% for cup [p<0.0001 in both cases]). The percentage of increased optic disc and cup in IGHD was higher than in control (p=0,005 for optic disc and p=0,028 for cup). There was no difference in fovea thickness or in any of the macula areas. Conclusions: Most IGHD individuals present moderate reduction of vascular branching points, increase of optic disc and cup size, but equal thickness of the macula. / Contexto e Objetivo: O hormônio do crescimento (GH) tem importante papel no desenvolvimento da retina, como demonstram diversos modelos experimentais. Contudo, a relação entre o eixo GH/IGF-I e a neuro-vascularização da retina humana ainda não é completamente conhecida. O modelo da deficiência isolada, congênita e vitalícia do GH (DIGH) não tratada é ideal para estudar a ação do GH nos tecidos oculares. O objetivo deste trabalho é avaliar a neuro-vascularização retiniana em indivíduos adultos com DIGH. Casuística e Métodos: Neste estudo transversal, examinamos 25 indivíduos (13 homens, idade 50,9 anos [12,0]) com DIGH devido a mutação c.57+1G>A no gene do receptor do hormônio liberador do GH e 28 controles (14 homens, idade 46,4 anos [14,7]), pareados por idade e sexo. Ambos os grupos foram submetidos a retinografia (para avaliar o número de ramificações nos vasos retinianos, o tamanho do disco óptico e da escavação) e tomografia de coerência óptica (OCT) (para avaliar a espessura da mácula). Resultados: Os indivíduos com DIGH apresentaram maior redução no número de ramificações vasculares em relação aos controles (91% vs. 53% [p=0,049]), segundo teste exato de Fisher. A porcentagem de redução moderada foi maior na DIGH que nos controles (p=0,01). A taxa de indivíduos com aumento do disco óptico e da escavação foi maior na DIGH em relação aos controles (92,9% vs. 57,1% para o disco e 92,9% vs. 66,7% para a escavação [p<0,0001 em ambos os casos]). A porcentagem de aumento do disco óptico e da escavação foi maior na DIGH (p=0,005 e p=0,028, respectivamente). Não houve diferença na espessura da mácula. Conclusões: A DIGH congênita e vitalícia não tratada provoca diminuição no número de ramificações dos vasos da retina, aumento do disco óptico e da escavação, mas não altera a espessura da mácula.

Η αυξητική ορμόνη (GH), πολυλειτουργική ορμόνη που παράγεται από τα σωματοτρόπα κύτταρα του πρόσθιου λοβού της υπόφυσης, προάγει την μεταγεννητική ανάπτυξη σκελετικών και μαλακών ιστών. Επίσης, ασκεί ποικίλες άλλες βιολογικές δράσεις, όπως ρύθμιση του μεταβολισμού των υδατανθράκων, των πρωτεϊνών και του λίπους. Κατά συνέπεια, η ανεπάρκεια της εκτός από αναπτυξιακά μπορεί να προκαλέσει και σοβαρά μεταβολικά προβλήματα. Η GH δρα στους περιφερικούς ιστούς άμεσα αλλά και έμμεσα μέσω του ινσουλινόμορφου αυξητικού παράγοντα IGF-I. Μετά από πρόσδεση της GH στον υποδοχέα της (GHR), ο IGF-I παράγεται στο ήπαρ, όπου απελευθερώνεται στην γενική κυκλοφορία, αλλά παράγεται και τοπικά στους περιφερικούς ιστούς, όπου δρα με αυτοκρινή ή παρακρινή τρόπο. Η έκκριση της GH από την υπόφυση έχει παλμική μορφή και ρυθμίζεται κυρίως μέσω τριών υποφυσιοτρόπων παραγόντων: εκλυτική ορμόνη της GH (GHRH), σωματοστατίνη (SRIF) και γκρελίνη. Η απελευθέρωση της GHRH και της SRIH από τον υποθάλαμο επηρεάζεται και από μια ποικιλία άλλων νευροδιαβιβαστών, νευροορμονών και νευροπεπτιδίων. Έχει υπολογιστεί σε διάφορες μελέτες ότι κοντό ανάστημα συσχετιζόμενο με ανεπάρκεια της αυξητικής ορμόνης (GHD) παρατηρείται με συχνότητα 1 στις 4000 έως 1 στις 10000 γεννήσεις. Παρόλο που οι περισσότερες περιπτώσεις είναι σποραδικές και θεωρούνται αποτέλεσμα περιβαλλοντικών εγκεφαλικών προσβολών ή αναπτυξιακών ανωμαλιών, γενετική αιτιολογία προτείνεται περίπου στο 10% των GHD περιπτώσεων, λόγω του ότι έχει προσβληθεί ένας τουλάχιστον πρώτου βαθμού συγγενής. Η διάγνωση της GHD είναι μια πολύπλευρη διαδικασία που απαιτεί εκτενή κλινική εκτίμηση, αξιολόγηση σωματομετρικών παραμέτρων, βιοχημικές δοκιμασίες του GH-IGF άξονα, και ακτινολογική εκτίμηση. Η GHD μπορεί να παρουσιάζεται είτε ως μεμονωμένο πρόβλημα (IGHD) είτε σε συνδυασμό με πολλαπλές ορμονικές ανεπάρκειες (CPHD). Μοντέλα ζώων έχουν χρησιμοποιηθεί για μελέτη της φυσιολογικής λειτουργίας του υποθαλαμικού-GH άξονα και των πιθανών διαταραχών που οδηγούν σε IGHD/CPHD στους ανθρώπους. Σύμφωνα με τα κλινικά χαρακτηριστικά, τον τρόπο κληρονομικότητας και την ανταπόκριση στην εξωγενή θεραπεία, τέσσερις τύποι οικογενούς IGHD έχουν περιγραφεί στον άνθρωπο. Μεταλλαγές έχουν βρεθεί να συμβαίνουν στο GH γονίδιο (GH1) και στο γονίδιο του υποδοχέα της GHRH (GHRH-R). Πολυμορφισμοί στον υποκινητή του GH1 γονιδίου μειώνουν επίσης την έκφραση του. Πρόσφατα, μεταλλαγές στο γονίδιο του υποδοχέα της γκρελίνης (GHS-R) συσχετίστηκαν με IGHD. Μεταλλαγές σε διακριτά γονίδια μεταγραφικών παραγόντων, που είναι βασικά για την ανάπτυξη και διαφοροποίηση των κυττάρων του πρόσθιου λοβού της υπόφυσης, όπως Pit1/POU1F1, PROP1, HESX1, LHX3, LHX4, έχουν αναγνωρισθεί μέχρι σήμερα σε ανθρώπους με CPHD. Καθώς μεγάλο ποσοστό οικογενών περιπτώσεων IGHD/CPHD δεν οφείλεται σε μεταλλαγές σε κάποιο από τα ήδη γνωστά γονίδια, φαίνεται να εμπλέκονται μεταλλαγές σε επιπρόσθετα υποψήφια γονίδια. Περαιτέρω γενετικές μελέτες μπορούν να συμβάλλουν σε καλύτερη κατανόηση της GHD, σε πρώιμη διάγνωση και βελτίωση της θεραπευτικής αγωγής στα άτομα με GHD. / Growth hormone (GH), a multifunctional hormone which is synthesized in the somatotrope cells of the anterior pituitary gland, promotes postnatal development of skeletal and soft tissues. In addition, GH exerts multiple biological actions, such as regulating the metabolism of carbohydrates, proteins and fat. Consequently, GH deficiency (GHD) apart from causing developmental disorders can also have a deleterious effect on the body’s metabolism. GH acts on peripheral tissues both directly and indirectly, through the mediation of insulin-like growth factor-1 (IGF-1). Upon binding of GH to its receptor (GHR), IGF-1 is produced both in the liver, from where it is released into the general circulation, and locally in the peripheral tissues, such as bone, cartilage, and muscle, where it acts in an autocrine or paracrine fashion. GH is secreted from the pituitary gland in a pulsatile fashion. Major regulatory factors include three hypophysiotropic factors: GH releasing hormone (GHRH), somatostatin (SRIF), and ghrelin. Moreover, GH secretion can be affected by a variety of other neurotransmitters, neurohormones and neuropeptides. The diagnosis of GHD demands detailed clinical, auxological, radiological and biochemical evaluation of the GH-IGF axis. GHD may occur as isolated GHD (IGHD) or in combination with other pituitary hormone deficiencies (Combined Pituitary Hormone Deficiency, CPHD). The physiological actions of the hypothalamic-GH axis and the possible disorders leading to IGHD/CPHD in humans have been extensively studied in animal models. Short stature associated with GHD has been estimated to occur in about 1/4000-1/10000 in various studies. Whereas most cases are sporadic and believed to result from environmental cerebral insults or developmental anomalies, approximately 10% of the affected individuals have a first-degree relative with the same disorder, suggesting a hereditary trend and genetic factors affecting the disorder. Four types of familial IGHD have been described in humans according to clinical characteristics, the mode of inheritance and the response to exogenous therapy. Mutations reducing gene expression have been described in the GH1 gene and in the GHRH receptor (GHRH-R) gene. Polymorphisms found in the promoter of the GH1 gene can also reduce its expression. Recently, mutations in the ghrelin receptor (GHS-R) gene were associated with IGHD. Mutations in discrete genes of transcriptional factors necessary for the development and differentiation of anterior pituitary cells, such as Pit1/POU1F1, PROP1, HESX1, LHX3, LHX4 have been recognized in individuals with CPHD. Considering that a large proportion of familial cases of IGHD/CPHD are not caused by mutations in any of the known genes, mutations in additional candidate genes may be involved. Further genetic studies may contribute to a better understanding of GHD, earlier diagnosis and better therapeutic approaches for this disorder.

Ghréline et obestatine sont deux hormones isolées à partir du tractus gastro-intestinal et issues du clivage protéolytique du même précurseur : la préproghréline. La ghréline est un peptide de 28 acides aminés qui subit une acylation sur sa sérine en position 3 lui permettant de se lier au récepteur des GH sécrétagogues (GHS-R). Ainsi la ghréline stimule la sécrétion de l’hormone de croissance (GH), hormone ayant également un rôle dans la modulation du métabolisme énergétique. En plus de son rôle en faveur de la sécrétion de GH, la ghréline est le seul peptide orexigène du tractus gastro-intestinal et un puissant facteur adipogène. L’obestatine isolée plus récemment à partir du tractus gastro-intestinal a été initialement décrite comme ayant un rôle anorexigène mais les données physiologiques concernant le rôle de ce peptide sont rapidement devenues contradictoires. Parallèlement, des données issues du laboratoire ont montré que l’obestatine avait une action antagoniste des effets de la ghréline exogène sur la sécrétion de GH et la prise alimentaire chez le rongeur mais que cette interaction n’avait pas lieu au niveau hypophysaire. Ainsi, nous avons émis l’hypothèse selon laquelle une interaction au niveau central entre la ghréline et l’obestatine est nécessaire au maintien de l’homéostasie des systèmes neuroendocrines contrôlant la croissance, la composition corporelle et la balance énergétique chez l’adulte. Dans un premier temps, il était nécessaire de déterminer le lieu de l’interaction entre ces deux peptides. Nous nous sommes donc intéressés aux neurones à NPY et GHRH du noyau arqué de l’hypothalamus (ArcN) qui expriment le GHS-R et sont la cible de la ghréline pour ses actions sur la prise alimentaire et la sécrétion de GH. Nos résultats montrent que l’obestatine et un variant naturel, l’obestatine Q90L, retrouvé parmi les patientes anorexiques à indice de masse corporelle (IMC) bas, ont un effet antagoniste sur la sécrétion de GH, la prise alimentaire et l’activité neuronale induites par la ghréline au niveau des neurones à NPY et GHRH de l’ArcN chez la souris. Une grande variabilité interindividuelle en réponse à la ghréline est observée et les effets antagonistes de l’obestatine ne sont visibles que chez les souris qui répondent bien à la ghréline, ce qui pourrait expliquer pourquoi les effets de l’obestatine ont été difficiles à caractériser jusqu’à présent.Afin de déterminer le rôle de la balance ghréline/obestatine dans le contrôle de la sécrétion de GH et la prise alimentaire, nous avons tiré parti de souris déficientes pour le gène de la préproghréline (ghrl-/-) qui n’expriment ni ghréline ni obestatine et chez lesquelles les deux peptides peuvent être remplacés pour en étudier l’impact. En effet, les ratios ghréline/obestatine sont modifiés dans plusieurs pathologies associées à des déséquilibres de la balance énergétique et de l’axe GH/IGF-1 mais l’impact physiologique de ces ratios déséquilibrés n’est pas connu. Nous avons, tout d’abord, caractérisé l’axe GH/IGF-1 ainsi que le comportement alimentaire chez les souris ghrl-/- et montré qu’elles n’avaient pas de phénotype permettant de les différencier des souris sauvages en ce qui concerne la taille, le poids ou encore le comportement alimentaire. Ces données sont en accord avec d’autres modèles de souris ghrl-/- déjà décrits au moment de notre étude. Néanmoins, nous observons que l’amplitude des pics sécrétoires de GH des souris ghrl-/- jeunes adultes (7 semaines) est réduite comparée à celle des souris sauvages du même âge. Plus âgées (36 semaines), ces souris retrouvent des profils de sécrétion de GH identiques à ceux des souris sauvages, soulignant un rôle de la ghréline endogène en période de croissance. / Role of peptides derived from preproghrelin in the control of GH secretion and feeding behavior

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Wachstumshormon (GH) besteht aus 191 Aminosäuren, hat eine Molekülmasse von 22kD und ist essentiell für postnatales Wachstum. Es wird aus der Adenohypophyse freigesetzt. GH bindet an einen GH-Rezeptor (GHR) und aktiviert somit über intrazelluläre Signalvorgänge Zielgene, insbesondere das, welches für die Kodierung von insulin-like growth factor (IGF-1) zuständig ist. IGF-1 vermittelt den Großteil aller GH-Signale. Zusammen mit den bereits bekannten GH Mutanten R77C und D112G ist in dieser Studie der neue GH Mutant d188-190 charakterisiert worden. Alle drei Mutanten wurden in heterozygoter Form in kleinwüchsigen Patienten identifiziert. Diesen Patientendaten zu Folge schien es möglich, dass d188-190 eine GH-antagonistische Wirkung besitzt. Zusätzlich wurde die extrem konservierte C-terminale Disulfidbrücke des GH im Mutanten d188-190 unterbrochen vorgefunden. Die Auswirkung der Unterbrechung wurde durch Substitution einer oder beider involvierter Cysteine durch Alanine untersucht. Alle Mutanten und Wildtypen des GH wurden in menschlichen embryonalen Nierenzellen (HEK-293) angezüchtet und eine Reihe von in vitro Experimenten sind für deren Charakterisierung etabliert worden. Es zeigte sich, dass d188-190 keine GH-antagonistische Wirkung besitzt. Im Vergleich zum Wildtyp weist der Mutant eine verminderte Bindungsaffinität zu GH, schwächere biologische Aktivität und höhere Stabilität auf. R77C und D112G sind dem Wildtyp GH sehr ähnlich. Die Disulfidbrücke ist wichtig für die Rezeptorbindung und für die biologische Aktivität von GH. Wenn ein Cystein entfernt wird vermindert sich die Stabilität des Moleküls. Dieser Effekt kann durch Entfernen des zweiten Cysteins wieder rückgängig gemacht werden. Die in dieser Studie etablierten Experimente können Verwendung finden in der Charakterisierung bislang nicht bekannter GH Mutanten und können darüber hinaus zur Behandlung von Patienten eingesetzt werden. / Growth hormone (GH) is a 22 kD, 191-aa, pituitary-derived peptide hormone that is essential for postnatal growth. GH signals via binding to GH receptor (GHR), which initiates intracellular signal transduction pathways. This leads to activation of target genes, most importantly the one encoding insulin-like growth factor (IGF)-1, which mediates most GH action. In this study a novel GH mutant, d188-190, was characterized along with previously reported GH mutants R77C and D112G. All of these mutants had been identified in heterozygous form in patients with retarded growth. Based on patient data, d188-190 was thought to be a GHR antagonist. Moreover, the extremely conserved C-terminal disulfide bridge of GH was disrupted in mutant d188-190 and its role was studied by substituting one or both of the involved cysteines with alanines. All mutants and wild type (wt) GH were produced in human embryonic kidney (HEK)-293 cells and an array of in vitro experiments was established for their characterization. It turned out that the novel d188-190 mutant is not a GHR antagonist after all. It has a diminished binding affinity to GHR, low biological activity and high stability compared to wt GH. R77C and D112G are rather similar to wt GH. The disulfide bridge is important for receptor binding and biological activity of GH. If one of the cysteines is removed the stability of the molecule drops but this can be reversed by removing both cysteines. If further GH mutants are to be identified, the established array of experiments will be useful for their fast characterization and could even contribute to correct treatment of patients.