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Ames dwarf (df/df) mice are deficient in anterior pituitary hormones: growth hormone (GH), thyroid stimulating hormone (TSH), and prolactin (PRL) due to a spontaneous, homozygous mutation of prop1[superscript df] gene. These dwarf mice exhibit characteristics such as delayed growth and development coupled with delayed aging, increased lifespan, overall increased insulin sensitivity, as well as resistance to certain diseases and cancers. The mutant mice possess low blood glucose, low serum insulin, and lower body temperature. Their enhanced longevity (about 40-60% longer lifespan than normal mice) is associated with their GH deficiency and disruption in the somatotropic axis (GH/IGF-1 hormonal pathway) as well as increased insulin sensitivity, which is supported by other mutant mouse models for longevity like Snell dwarfs and growth hormone receptor knock-out (GHRKO) mice. When young male Ames dwarf mice were treated with GH replacement therapy, they showed increased body growth to nearly match the normal mouse phenotype. In conjunction to an increase in physical growth, however, GH treatment also decreases the longevity and insulin sensitivity that are characteristic of these mice to levels seen in normal mice. Because of the lack of TSH, they also have undetectable levels of Thyroxine (T4). While T4 treatment didn't increase bodyweight of dwarfs to the same extent as GH treatment, the T4 treated mice retained their enhanced lifespan. Although df/df mice have enhanced whole-body insulin sensitivity, the male skeletal muscle was previously shown to be less responsive to insulin than their liver. In our study we analyzed the insulin signaling pathway in skeletal muscle from female mice after treatment with GH or GH combined with T4. Gene expression and protein expression were investigated in the skeletal muscle of female Ames dwarf mice that were treated with GH or GH and T4 therapy. Real Time Polymerase Chain Reaction (RT-PCR) was used to analyze the expression of mRNA involved with insulin and GH signaling, while western blots were used to analyze protein expression. This project found that female Ames skeletal muscle didn't respond to GH treatment to the same extent as males, and that GH and T4 treatment tends to neutralize the effects seen in GH-only treatment.

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This study represents the behavioural component of a larger project investigating the life history tactics, physiological resource allocation and behavioural time budgeting of a genetically engineered animal. The "supermouse" is a transgenic strain (mMT-1/rGH) that has one chromosome genetically engineered with extra copies of rat growth hormone genes, each fused to a metallothionein-1 promoter. The GH transgenes are permanently incorporated into the genome of the mouse and are inherited as a block, in a Mendelian manner. "Supermice" exhibit an accelerated growth rate and reach body weights twice that of their normal siblings: both transgenic mice and normal mice are obtained by crossing transgenic males to normal females. Although there must be increased costs associated with achieving their higher growth rate, these: mice show no increases in their specific feeding rates. Consequently there must be a reallocation of resources among various physiological and behavioural demands. The reality of such tradeoffs is known as the Principle of Allocation and predicts that reductions in behavioural activities might be one avenue for realizing extra growth. To test this, six components of the behavioural time budget (resting, locomotion, wheel running, feeding, drinking and grooming) were compared between transgenic and normal mice. Infra-red videocameras recorded the activities of individual male mice in artificial enclosures over 24 hours. The time spent in each bout of activity was recorded and compared. Transgenic mice out-slept their normal counterparts by 126% (an increase of 3.4 h) and were only 53.83% as active in terms of locomotion and wheel running as normal mice. Pooling the data revealed that on average, large mice spent more time at rest and less time engaged in locomotion. Slight but significant decreases in time spent drinking and grooming were also found. Transgenic mice spent only 77.01% as much time drinking, and 69.01% as much time grooming as normal mice. No difference in the amount of time spent feeding was found. / Thesis / Master of Science (MS)

The overall objective of this research was to understand the mechanims by which growth hormone (GH) regulates insulin-like growth factor-I (IGF-I) gene expression in liver. Previous studies have suggested that GH regulation of IGF-I gene expression in liver is mediated by binding of the transcription factor signal transducer and activator of transcription (STAT) 5 to four binding sites located distantly from the IGF-I promoter. The first specific objective of this research was to determine whether additional STAT5 binding sites were involved in GH stimulation of IGF-I gene expression in liver. Sequence analysis of 170 kb of mouse genomic DNA revealed nineteen consensus STAT5 binding sequences corresponding to fourteen ~200 bp chromosomal regions that were conserved in the corresponding human DNA sequence. Eight of these chromosomal regions were able to mediate STAT5 activation of reporter gene expression in cotransfection experiments. Two of these chromosomal regions corresponded to those previously identified. Gel-shift assays indicated that the eight new STAT5 binding sites and three of the four previously identified STAT5 binding sites could bind GH-activated STAT5 from mouse liver. Together, these results suggest that GH stimulation of IGF-I gene transcription in the mouse liver may be mediated by at least eleven STAT5 binding sites located distantly from the IGF-I promoter. In a previous study, I found that liver expression of liver-enriched transcription factor hepatocyte nuclear factor 3γ (HNF-3γ) was increased by GH in cattle. Therefore, the second specific objective of this research was to determine how GH stimulates HNF-3γ gene expression and whether the increased HNF-3γ mediates GH stimulation of IGF-I gene expression in bovine liver. Sequence analysis of the bovine HNF-3γ promoter revealed the presence of two putative binding sites for STAT5. The proximal putative STAT5 binding site appears to be conserved in other mammals. Chromatin immunoprecipitation (ChIP) assays demonstrated that GH increased the binding of STAT5 to the HNF-3γ promoter in bovine liver and that this binding was associated with increased HNF-3γ expression. Gel-shift assays demonstrated that the proximal STAT5 binding site in the HNF-3γ promoter could bind GH-activated STAT5 from bovine liver. Cotransfection analyses showed that the proximal STAT5 binding site was necessary for the HNF-3γ promoter to be activated by GH. The promoter of the bovine IGF-I gene contains three putative HNF-3 binding sites that seem to be evolutionarily conserved. ChIP assays indicated that GH stimulated the binding of HNF-3γ to the IGF-I promoter in bovine liver. Gel-shift assays showed that one of the putative HNF-3 binding sites could bind HNF-3γ protein from bovine liver. Co-transfection analyses demonstrated that this HNF-3 binding site was necessary for HNF-3γ activation of reporter gene expression from the IGF-I promoter. In summary, the results of this dissertation research suggest that GH-activated STAT5 directly stimulates IGF-I gene transcription in liver by binding to at least eleven distantly located STAT5 binding sites in the IGF-I locus and indirectly stimulates IGF-I gene transcription by enhancing HNF-3γ gene expression in the liver. / Ph. D.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal. / Il est maintenant bien établi que les GHRPs constituent une classe de peptides synthétiques stimulant la sécrétion de l'hormone de croissance par interaction avec leur récepteur spécifique au niveau hypophysaire. Ce récepteur constitué de 364 acides aminés appartient à la famille des récepteurs à sept passages transmembranaires couplé aux protéines G. Il est exprimé non seulement au niveau de l'hypophyse mais également au niveau de l'hypothalamus, des noyaux arqués et paraventriculaires. Récemment, il a été découvert par étude de photomarquage covalent que l'hexarelin, un hexapeptide de la famille des GHRPs se lie de façon spécifique à une glycoprotéine membranaire de poids moléculaire de 84 kDa exprimée dans les membranes de coeur de rat. La liaison de l'hexarelin à ce récepteur au niveau cardiaque induit une forte vasoconstriction coronarienne de façon dose­dépendante dans le modèle de coeur de rat perfusé. Les buts de la présente étude sont premièrement de déterminer si la liaison est spécifique seulement à l'hexarelin ou à d'autres analogues peptidiques de la famille des GHRPs, notamment le dérivé EP 70905. Dans un deuxième temps nous déterminerons si les analogues se liant au récepteur cardiaque des GHRPs exercent un effet vasoconstricteur sur les coronaires utilisant le modèle de cœur de rat perfusé. Troisièmement nous caractériserons les voies de signalisation impliquées dans la vasoconstriction induite par les GHRPs. Les études de compétition entre le radioligand photoactivable [125] I-Tyr-Ala-Bpa­hexarelin et le EP 70905 effectuées sur les membranes de cœur de rat démontrent une affinité de liaison comparable du EP 70905 et de l'hexarelin au récepteur cardiaque des GHRPs. Cependant, le EP 70905 induit une augmentation de la pression coronarienne significativement plus élevée que celle induite par l'hexarelin. Les études des voies de signalisation démontrent que les tyrosine kinases ne semblent pas être impliquées dans la vasoconstriction induite par le EP 70905, puisque la perfusion simultanée avec la génistéine n'affecte pas la vasoconstriction induite par ce peptide. Le rôle de l'endothéline endogène dont la libération est stimulée par le EP 70905 semble être mineur dans la vasoconstriction observée considérant les taux femtomolaires d'endothéline libérés dans le perfusat. De plus, la perfusion simultanée d'un antagoniste des récepteurs d'endothéline, le LU 302872 affecte que faiblement la vasoconstriction induite par le EP 70905. Par contre, la vasoconstriction coronarienne induite par le EP 70905 est inhibée de façon majeure suite à la perfusion de la nifédipine, un bloqueur des canaux calciques de type L. La perfusion simultanée d'un inhibiteur de la protéine kinase C, la chélérythrine affecte de façon significative la vasoconstriction induite par le EP 70905.

Introdução: Alterações em genes relacionados com a secreção de GH ou a organogênese hipofisária foram identificadas em pacientes com deficiência de hormônio do crescimento (DGH) congênita. Entretanto, poucos casos de DGH têm sua etiologia esclarecida. O GHRH é um candidato óbvio para explicar a deficiência isolada de GH (DIGH). Na literatura, os estudos de análise do GHRH não conseguiram identificar mutações, porém são antigos e utilizaram uma metodologia com limitações. A maioria dos pacientes com deficiência hipotálamo-hipofisária múltipla (DHHM) apresenta neuroipófise ectópica sugerindo a importância do estudo de genes que atuam no início do desenvolvimento hipofisário, com expressão inclusive no infundíbulo. O GLI2 é um fator de transcrição na sinalização Sonic Hedgehog, envolvido com o início da embriogênese hipofisária, expresso na bolsa de Rathke primordial e no diencéfalo ventral. Previamente, mutações no GLI2 foram encontradas em pacientes com holoprosencefalia, e também alterações hipofisárias. Objetivos: Analisar o GHRH em 151 pacientes com DIGH (42 brasileiros e 109 encaminhados de centros internacionais) e analisar o GLI2 em 180 pacientes brasileiros com DIGH ou DHHM por PCR e sequenciamento automático dos genes; e descrever o fenótipo dos pacientes com mutações identificadas. Resultados: No GHRH foram identificadas seis variantes em heterozigose com previsão benigna pelas análises in silico. A análise do GLI2 identificou três mutações novas em heterozigose com códon de parada prematuro (p.L788fsX794, p.L694fsX722 e p.E380X), e geração de proteínas truncadas, com perda do domínio responsável pela ativação transcricional. A mutação p.L788fsX794 foi identificada numa paciente com baixa estatura, polidactilia, epilepsia e hipoglicemias. Apresentava deficiência de GH, TSH, ACTH, prolactina, LH e FSH. Na investigação familiar foi diagnosticada DIGH em dois tios e DHHM numa prima. Estes familiares, além de sua mãe e outros parentes maternos também apresentaram a mutação e polidactilia. A mutação p.L694fsX722 foi identificada num menino com baixa estatura por deficiência de GH, além de lábio leporino e fenda palatina. Seu pai, embora saudável, também apresentou a mutação. A mutação p.E380X foi identificada numa lactente com retardo no desenvolvimento, hipoglicemias, poliúria e polidipsia. Apresentava deficiência de GH, ACTH, TSH e ADH. Sua mãe aparentemente normal também apresentou a mutação. Todos os pacientes com DGH e mutação no GLI2 apresentaram neuroipófise ectópica (não visualizada na paciente com p.E380X), adenoipófise hipoplásica e ausência de holoprosencefalia na ressonância magnética. Dezoito variantes não-sinônimas também foram identificadas no GLI2 em 24 pacientes. Dezesseis dessas variantes foram consideradas deletérias em pelo menos um programa de predição in silico e dez delas não foram encontradas em população controle. O fenótipo dos pacientes foi predominante de DHHM e com neuroipófise ectópica e sem holoprosencefalia. Variantes silenciosas, intrônicas e polimorfismos foram identificados no GLI2, mas aparentemente sem alteração funcional. Conclusão: Não identificamos mutação no GHRH e se realmente existe mutação neste gene como causa de DGH, deve ser muito rara. Variantes no GLI2 são frequentes (15%), indicando seu importante papel na etiologia da DGH congênita. Além disso, ampliamos o espectro fenotípico dos pacientes com mutações no GLI2, que foi caracterizado por DIGH ou DHHM, inclusive com diabetes insipidus, neuroipófise ectópica (maioria) e ausência de holoprosencefalia. Outras características observadas foram polidactilia, defeito de linha média facial e herança autossômica dominante com penetrância incompleta / Introduction: Alterations in genes related to GH secretion and pituitary organogenesis have been identified in patients with congenital GH deficiency (GHD). However, in only few cases of GHD the etiology has been established. GH-releasing hormone (GHRH) is an obvious candidate to explain isolated GH deficiency (IGHD). Previous reports in the literature did not identify mutations in GHRH, however, the methodology used was limited. Most patients with combined pituitary hormone deficiency (CPHD) have an ectopic posterior pituitary lobe (EPP) suggesting the study of genes involved in early pituitary development and also expressed in the infundibulum. GLI2 is a transcription factor in Sonic hedgehog signaling expressed in the primordial Rathkes pouch and ventral diencephalon during early pituitary development. Previously, GLI2 mutations were found in patients with holoprosencephaly and pituitary abnormalities. Aim: Analyse GHRH in 151 patients with IGHD (42 Brazilian and 101 from international centers) and GLI2 in 180 Brazilian patients with IGHD or CPHD by PCR and automatic sequencing, and describe the phenotype of patients with mutations. Results: In GHRH, six heterozygous variants that are benign according to in silico analysis were identified. GLI2 study revealed three novel heterozygous mutations leading to premature stop codons (p.L788fsX794, p.L694fsX722 e p.E380X) and truncated proteins, without the transcriptional activator domain. p.L788fsX794 was identified in a girl with short stature, polydactyly, epilepsy and hypoglycemia. She had GH, TSH, ACTH, prolactina, LH and FSH deficiencies. Two uncles had IGHD and one cousin CPHD. These relatives, the mother and other maternal relatives had polydactyly and carried the mutation. p.L694fsX722 was identified in a boy with short stature due to GHD who also had cleft lip and palate. His healthy father also carried the mutation. p.E380X was identified in an infant with delayed development, hypoglycemia, polyuria and polydipsia. She had GH, ACTH, TSH and ADH deficiencies. Her apparently normal mother also carried the mutation. All patients with GHD and GLI2 mutations had an EPP (not visualized in the patient with p.E380X), hypoplastic anterior pituitary lobe and absence of holoprosencephaly on MRI. Eighteen non-synonymous variants in GLI2 were identified in 24 patients. Sixteen of these were considered deleterious in at least one in silico prediction program and ten of these were not found in the control population. The phenotype was mainly of CPHD and EPP without holoprosencephaly. Several synonymous and intronic GLI2 variants and polymorphisms apparently without functional consequences were identified. Conclusions: No mutations in GHRH were identified and if mutations in this gene exist as a cause of IGHD, they must be very rare. Variants in GLI2 are frequent (15%) indicating its important role in the etiology of GHD. Furthermore, we expanded the clinical spectrum of patients with GLI2 mutations characterized by IGHD or CPHD including diabetes insipidus, ectopic posterior pituitary lobe (in most patients) and absence of holoprosencephaly. Additional features were polydactyly and midline facial defects and the inheritance was autosomal dominant with incomplete penetrance