INTRODUÇÃO Na puberdade, tanto fisiológica quanto precoce, um dos eventos marcantes é o estirão de crescimento. Análogos do GnRH tem sido utilizado como tratamento da puberdade precoce central (PPC) tendo como consequência o bloqueio do eixo gonadal e diminuição da velocidade de crescimento, porém mantém níveis séricos elevados de IGF-I e IGFBP-3. Não existem relatos sobre a expressão do IGF1R durante a puberdade. Considerando-se que este poderia ser um ponto de regulação da velocidade de crescimento, o presente estudo propõe avaliar a expressão do IGF1R em meninas com PPC antes e durante o tratamento. MÉTODOS: Avaliamos meninas com PPC que foram divididas em dois grupos: Grupo A foi constituído por 16 meninas avaliadas antes do início do tratamento com idade média de 8,0+-0,7anos e o Grupo B constituído por 16 pacientes em uso regular do análogo do GnRH com idade média de 9,4+-0,8 anos. O grupo controle foi composto por 18 crianças saudáveis, pré-púberes com idade média 7,1+-1,3 anos. RESULTADOS: A expressão do mRNA do gene do IGF1R foi maior no grupo B quando comparado ao grupo A (p= 0,04) e ao grupo controle (p=0,004). Não foi observado diferença estatística entre o Grupo A e o grupo controle (p=0,17). Os níveis séricos de IGF-I e IGFBP3 assim como a relação molar IGF-I/IGFBP3 foram significativamente maiores no grupo A em relação ao grupo controle. (p<0,0001). Não encontramos diferença nas concentrações de IGF-I, IGFBP3 ou na relação molar entre os grupos A e B. O grupo controle apresentou níveis mais elevados de IGFBP1 quando comparado com os grupos A e B (p<0,001). Ao compararmos somente os grupos A e B, o grupo B apresentou número estatisticamente maior de valores indetectáveis de IGFBP1 quando comparado com o grupo A (p=0,01) mostrando uma tendência a valores menores no grupo B. A dosagem de insulina foi significativamente menor no grupo controle quando comparado ao grupo A (p<0,001) e não apresentou diferença entre os grupos A e B. Ao analisarmos os 3 grupos (controle, A e B) não encontramos a correlação negativa entre insulina e IGFBP-1. Essa correlação aparece quando avaliamos somente o grupo controle e o grupo A (r= - 0,5; p=0,007) e desaparece quando acrescentamos o grupo B na análise. CONCLUSÃO: As variações nas concentrações séricas de IGF-I, IGFBP-3, IGFBP-1 e insulina não explicam a desaceleração da velocidade de crescimento durante o tratamento de meninas com puberdade precoce central com análogos do GnRH. O aumento da expressão do IGF1R parece refletir uma redução da sinalização intracelular do IGF1R com consequente diminuição da bioatividade do IGF-I em um mecanismo de feedback de alça ultra curta. O aumento da secreção do hormônio de crescimento devido a redução do feedback negativo na hipófise, explica as concentrações encontradas de IGF-I, IGFBP3 e IGFBP1.Estudos outros serão necessários para confirmar esta hipótese, avaliando diferentes pontos de sinalização na cascata pós-receptor / BACKGROUND: Growth spurt is a major event in central precocious puberty (CPP). GnRH analogues (GnRHa) treatment inhibit gonadal axis and decrease height velocity. However, serum IGF-I and IGFBP-3 remain high as before treatment. No reports regarding IGF type 1 receptor (IGF1R) in CPP is available. Considering that this could be a point of regulation of height velocity, the present study aims to study IGF1R mRNA expression in girls with CPP before and during GnRHa treatment. MÉTHODS: Sixteen girls with CPP (8.0±0.7yr) were evaluated before treatment (Group A) and sixteen (9.4±0.8yr) in use of GnRHa (Group B). Age-matched pre pubertal children were studied as controls (n=18). Fasting blood sample were collect for IGF1R mRNA expression analysis in peripheral lymphocytes (RT-PCR) and serum IGF-I, IGFBP-3, IGFBP-1 and insulin determination. RESULTS: The expression of IGF1R mRNA was higher in Group B than in Group A (p=0.04) and Controls (p=0.004). No difference was observed between Groups A and Controls. IGF-I, IGFBP-3 and IGF-I/IGFBP3 molar ratio were similar in Group A and B but higher than in Controls (p<0.0001). IGFBP-1 was higher (p<0.0001) in Controls than in Groups A and B. When we compare only Groups A and B, group B showed more IGFBP1 undetectable values than group A (p = 0.01) showing a tendency to lower values in group B. Insulin levels were lower in Controls than in Group A (p<0.001), but no difference were observed between Groups B and A. Negative correlation was found between insulin and IGFBP-1 when controls and Group A were put together (r= -0.5; p=0.007). This correlation disappear if Group B is included in the analysis. CONCLUSION: Serum concentrations of IGF-I, IGFBP-3, IGFBP-1 and insulin do not explain the decrease in height velocity during CPP treatment with GnRH analogue. The increase in IGF1R mRNA expression suggest impairment of IGF-I signaling and compensatory up regulation of the IGF1R. Increased GH concentrations due to reduction of IGF-I feedback could explain the IGF-I, IGFBP-3 and IGFBP-1 findings. Other studies are necessary to confirm this hypothesis by studying different signaling points in post-receptor cascade

A subnutrição é uma doença causada pela ingestão inadequada e/ou insuficiente de energia ou de outras biomoléculas, que pode comprometer o crescimento e o desenvolvimento dos tecidos. Em conjunto, a cartilagem do processo condilar da mandíbula e a sincondrose basioccipital representam os dois centros efetivos do crescimento craniofacial, que depende, dentre outros, de fatores locais como a produção do fator de crescimento semelhante à insulina I (IGF-I) e seu receptor (IGF-IR). Alterações tanto no peptídeo quanto no receptor estão associadas a graves atrasos no crescimento e desenvolvimento ósseo. Dentre os nutrientes alternativos com a capacidade de promoção da recuperação nutricional, destaca-se o resveratrol, um polifenol contido na uva e derivados que pode promover a melhora do crescimento através de propriedades osteogênicas, além de atuar como condroprotetor. Assim, a presente pesquisa teve por objetivo avaliar os efeitos da subnutrição proteica na cartilagem do processo condilar da mandíbula (PC) e na sincondrose basioccipital (SBo) de ratos, bem como os processos de renutrição e suplementação com resveratrol nos períodos pré-púbere (21 dias) e púbere (60 dias). Os grupos nutrido (N) e subnutrido (S) foram formados por filhotes cujas mães receberam dieta normoproteica e hipoproteica, respectivamente, desde o período de acasalamento; ao completarem 21 dias de vida extrauterina, época determinada para o desmame, os filhotes foram eutanasiados. A partir do 22&deg; dia de vida, filhotes dos grupos N e S foram mantidos com as respectivas dietas até o 60&deg; dia de vida quando constituíram, respectivamente, os grupos NN e SS. Dois grupos considerados renutridos foram formados por animais do grupo S, que a partir do 22&deg; dia passaram a receber, o primeiro, a dieta normoproteica com 20% de caseína (grupo RN), e o segundo, a dieta normoproteica acrescida de resveratrol (RRv). A cartilagem do PC e a SBo foram processadas através das técnicas histológicas rotineiras e posteriormente submetidas às colorações da HE, Picrossíruis e Safranina-O, para a evidenciação dos componentes celular e colágeno e imunohistoquímica para a marcação de células reativas ao IGF-I e IGF-IR. Os aspectos ultraestruturais também foram analisados através da microscopia eletrônica de transmissão. Os resultados mostraram que a subnutrição foi capaz de alterar os componentes da matriz extracelular (MEC), acarretando em modificações na secreção das fibrilas colágenas e acúmulo de proteoglicanas nos tecidos; diminuição do número de células imunorreativas ao IGF-I e IGF-IR na cartilagem do PC e atraso no crescimento ósseo endocondral, comprovado através das avaliações quantitativas e morfométricas, em ambos os tecidos. A renutrição com dieta normal foi parcialmente eficaz na recuperação dos parâmetros avaliados, e pode ter provocado uma diminuição da capacidade de resposta celular ao IGF-I. Os efeitos condroprotetores do resveratrol foram fundamentados através da recuperação dos componentes da MEC e organelas dos condrócitos e uma possível diminuição da insensibilidade ao IGF-I; o atraso no crescimento ósseo endocondral foi minimizado através do seu potencial osteogênico (mais acentuado na SBo). Muito embora a recuperação dos tecidos não tenha ocorrido totalmente, diante dos dados apresentados, pode-se afirmar que a suplementação de resveratrol na dieta auxiliou de forma mais efetiva no restabelecimento dos parâmetros afetados pela subnutrição do que somente a recuperação proteica com dieta normal / Undernutrition is a disease caused by inadequate and / or insufficient intake of energy or other biomolecules, which can compromise growth and development of tissues. Together, the mandibular condylar cartilage and basioccipital synchondrosis represent the two effective centers of craniofacial growth, which depends of the factors such as the local production of insulin like growth factor I (IGF-I) and receptor (IGF-IR). Changes in both the peptide and receptor are associated with serious delays in growth and bone development. Among the alternative nutrients to the promotion capacity of nutritional recovery, there is resveratrol, a polyphenol contained in grapes and derivatives that can promote improved growth through osteogenic properties, besides acting as chondroprotective. Thus, the present study aimed to evaluate in rats the effects of protein undernutrition in the mandibular condylar cartilage and basioccipital synchondrosis, as well renutrition and supplementation with resveratrol in pre pubertal periods (21 days) and pubertal (60 days). The nourished groups (N) and undernourished (S) were formed by young whose mothers received normal protein and hypoproteic diet respectively since the breeding season; to complete 21 days of extra-uterine life, given time to weaning, puppies were euthanized. From the 22nd day of life, puppies from N and S groups were kept with their diets until the 60th day of life when constituted, respectively, NN and SS groups. Two groups considered re-nourished were formed by the S group, which from day 22 began receiving, the first, the normal protein diet with 20% casein (RN group), and the second, the normal protein diet plus of resveratrol (RRv). The cartilage of PC and SBo were processed through routine histological techniques, and then subjected to HE, Sirius red and Safranin-O staining for the disclosure of cellular components and collagen and immunohistochemistry for marking cells responsive to IGF-I and IGF-IR. The ultrastructural aspects were also analyzed by transmission electron microscopy. The results showed that undernutrition was able to change the components of the extracellular matrix (ECM) leading to changes in the secretion of collagen fibrils and proteoglycans accumulation in the tissues; decrease in the number of immunoreactive cells to IGF-I and IGF-IR in PC cartilage and delayed endochondral bone growth, confirmed by quantitative and morphometric analysis in both tissues. Renutrition with normal diet was partially effective in recovering the parameters evaluated, and may have caused a decrease in cell responsiveness to IGF-I. The chondroprotective effects of resveratrol were founded by recovering the ECM components and organelles of chondrocytes and a possible decrease in the insensitivity to IGF-I; the delay in endochondral bone growth was minimized by osteogenic potential (more pronounced in SBo). Although the tissue recovery has not fully occurred, the resveratrol supplementation in the diet helped more effectively in restoring the parameters affected by undernutrition than just protein recovery with normal diet

A prática regular de exercícios físicos durante a infância e adolescência pode induzir o crescimento e desenvolvimento tanto da massa muscular assim como da massa óssea. Nesse contexto, o exercício físico está intimamente ligado à função anabólica provocada pela ação do eixo GH/IGF-I. Níveis basais de IGF-I estão correlacionados positivamente com massa muscular em crianças, adolescentes e adultos. A cinética do IGF-I e IGFBP-3 durante o treinamento crônico ainda não está totalmente esclarecida e um ótimo estimulo para maximizar uma resposta anabólica no treinamento com pesos ainda permanece obscuro. O objetivo do presente estudo foi analisar a cinética das concentrações de IGF-I e da proteína de ligação IGFBP-3 em adolescentes submetidos a dez semanas de treinamento de hipertrofia. As concentrações séricas de IGF-I e IGFBP-3 foram determinadas na 1ª, 5ª e 10ª semana de treinamento com pesos. A composição corporal avaliada mediante cálculo da massa magra, porcentagem de gordura e do índice de massa corporal foi também realizada na 1ª, 5ª e 10ª semana de treinamento e comparada à cinética de IGF-I e da IGFBP-3. O IGF-I apresentou aumento significativo no primeiro momento de avaliação entre o pré e pós-treino (p=0,03) e ao longo das 10 semanas de treinamento (p=0,003). Em relação à IGFBP-3 não foi possível identificar variação significativa entre o pré e o pós-treino em nenhuma das avaliações ou ao longo das 10 semanas de treinamento. A massa corporal, massa magra, a porcentagem de gordura e índice de massa corporal dos voluntários mantiveram-se inalterados ao longo das 10 semanas de treinamento. Correlação negativa foi observada entre a variação na massa muscular e a variação das concentrações séricas de IGF-I quando comparados os dados das avaliações da 1ª e 10ª semanas (r=-0,62; p= 0,002). Em resumo, o IGF-I, mas não a IGFBP-3, mostrou-se sensível aos efeitos agudos e crônicos do treinamento com pesos apresentando-se como um biomarcador de estado de treinamento em voluntários não atletas. A relação negativa entre a variação do IGF-I e a variação da massa magra pode sinalizar um aumento da sensibilidade ao IGF-I com o treinamento. / Regular physical exercise during childhood and adolescence can promote growth and development of muscle mass and bone mass. In this context, physical exercise is closely linked to the anabolic function of the GH / IGF-I axis. Baseline IGF-I levels are positively correlated with muscle mass in children, adolescents and adults. The kinetics of IGF-I and IGFBP-3 during chronic training is not fully understood yet; a good stimulus to maximize the anabolic response in resistance training remains unclear. The aim of the present study was to analyse the kinetics of IGF-I and IGFbinding protein 3 (IGFBP-3) in adolescents undergoing ten weeks of hypertrophy training. Serum IGF-I and IGFBP-3 concentrations were determined at the 1st, 5th and 10th week of resistance training. Body composition, lean mass, fat percentage and body mass index were also evaluated at the 1st, 5th and 10th week and compared to the changes in serum IGF-I and IGFBP-3. IGF-I levels increased during the training session at the first evaluation (p=0,03) and also increased during the 10 weeks of training (p=0,003). No changes in IGFBP-3 levels were observed during a training session or during the 10 weeks of training. Body mass, lean mass, fat percentage and body mass index of the volunteers remained unchanged throughout the 10 weeks of training. Negative correlation was observed between the variation in muscle mass and changes in serum IGF-I when data from the 1st and the 10th weeks were compared (r=-0,62;p=0,002). In summary, IGF-I but not IGFBP-3 was sensitive to the acute and chronic effects of resistance training and can be considered as a biomarker of training status in non-athlete volunteers. The negative correlation between the variations in lean mass and IGF-I could sign to a training induced increase in IGF-I sensitivity.

Objective: Previous population-based studies provided conflicting results regarding the association of total serum insulin-like growth factor I (IGF-I) and mortality. The aim of the present study was to assess the relation of IGF-I levels with all-cause mortality in a prospective study. Design: DETECT (Diabetes Cardiovascular Risk-Evaluation: Targets and Essential Data for Commitment of Treatment) is a large, multistage, and nationally representative study of primary care patients in Germany. The study population included 2463 men and 3603 women. Death rates were recorded by the respective primary care physician. Serum total IGF-I levels were determined by chemiluminescence immunoassays and categorized into three groups (low, moderate, and high) according to the sex- and age-specific 10th and 90th percentiles. Results: Adjusted analyses revealed that men with low [hazard ratio (HR) 1.70 (95% confidence interval [CI] 1.05–2.73), p=0.03] and high [HR 1.76 (95% CI 1.09–2.85), p=0.02] IGF-I levels had higher risk of all-cause mortality compared to men with moderate IGF-I levels. The specificity of low IGF-I and high IGF-I levels increased with lower and higher cut-offs, respectively. No such association became apparent in women. Conclusions: The present study revealed a U-shaped relation between IGF-I and all-cause mortality in male primary care patients.

Im Rahmen der vorliegenden Arbeit wurden zentrale Aspekte der Schilddrüsenphysiologie am Beispiel von Iod, TSHR und IGF-IR untersucht. Der Pathologie der Schilddrüsenautonomie liegen konstitutiv aktivierende Mutationen des TSHR zugrunde. Die Prävalenz der Schilddrüsenautonomie ist in Iod armen Regionen deutlich erhöht. Als Ursache für Mutationen im TSHR wird vermehrter oxidativer Stress unter Iodmangel angenommen (Krohn et al. 2007; Maier et al. 2007). Die genauen molekularen Mechanismen konnten bisher noch nicht hinreichend aufgeklärt werden. In diesem Zusammenhang interessierte uns inwiefern eine ausreichende Iodversorgung die Entwicklung bereits autonomer Zellen beeinflussen kann. Das verwendete in vitro Modell der Schilddrüsenautonomie mit konstitutiv aktivierenden Mutationen im TSHR wurde bereits in früheren Arbeiten charakterisiert (Führer et al. 2003). Mit Hilfe von Microarray Untersuchungen und Funktionsanalysen, konnten wir deutliche Genregulationen durch Iod an Hand von normalen und autonomen Thyreozyten erkennen. Besonders auffällig war die differentielle Regulation von Genen, die z.B. in der Proliferation, dem Zellzyklus und metabolischen Prozessen involviert sind. Wesentlich ist, dass trotz einer konstitutiven Aktivierung des TSHR Iod dennoch die Proliferation und Funktion einer frühzeitigen Schilddrüsenautonomie herabsetzt. Die physiologische Rolle des IGF-IR in der Schilddrüsenphysiologie in vivo wurde noch nicht systematisch erforscht. Um die Rolle des IGF-IR in der Schilddrüse im Hinblick auf deren Entwicklung und Metabolismus näher zu untersuchen, wurde ein Mausmodell generiert bei dem der IGF-IR schilddrüsenspezifisch über eine durch den TG Promoter regulierte Cre Rekombinase (Igf1rTgCre) ausgeschaltet wurde. Ziel war es nun zu untersuchen, welche Folgen ein thyreoidaler Igf1r Knockout auf die Funktion, Morphologie und Entwicklung der murinen Schilddrüse und metabolischer Parameter hat. Dieser Knockout zeigte in den Mäusen keine Veränderungen des Schilddrüsengewichtes und der Serum T3 Werte, wobei das Serum T4 nach 8 Wochen leicht absank, nach 4 Monaten aber wieder Normalwerte zeigte. Allerdings waren die Serum TSH Werte bis zu 9fach erhöht. Die Histologie der Igf1r-/- Mäuse zeigten mit einer Rate von 86% papilläre Schilddrüsenhyperplasien sowie eine starke Heterogenität der Follikelstruktur, die auch bei den Igf1r-/- Mäuse zu finden war. Die molekulare Kompensation des Igf1r Knockouts in der Schilddrüse besonders durch TSH konnte durch unsere Untersuchungen nicht hinreichend geklärt werden. Die Daten aus unseren Ergebnissen und eines reversen Mausmodells (Überexpression des IGF-IR und IGF-I) (Clement et al. 2001) weisen daraufhin, dass das IGF-IR Signal weniger essentiell für die Schilddrüsenhormonsynthese ist als für das Aufrechterhalten einer Homöostase und normaler Schilddrüsenmorphogenese.

Darbo tikslas: Ištirti insulino augimo faktoriaus (IGF – 1) geno poveikį galvijų produktyvumo savybėms. Darbo uždaviniai: 1. Surinkti ir išanalizuoti mokslinę literatūrą apie insulino augimo faktoriaus (IGF – 1) geną ir jo įtaką galvijų produktyvumo savybėms. 2. Nustatyti insulino augimo faktoriaus (IGF – 1) geno alelių ir genotipų dažnius. 3. Įvertinti insulino augimo faktoriaus (IGF – 1) geno įtaką galvijų priesvoriui. Pirmą kartą Lietuvoje buvo ištirtas insulino augimo faktoriaus (IGF – 1) genas bei nustatyta jo įtaka galvijų priesvoriui. Darbo praktinė reikšmė: Rastas insulino augimo faktoriaus (IGF – 1) geno polimorfizmas bei nustatyta jo įtaka galvijų priesvoriui leidžia atrinkti gyvulius su pageidaujamu genotipu ir tokiu būdu, vykdant selekciją genetinių žymenų pagalba, gerinti galvijų priesvorį. Išvados: 1. Tirtoje galvijų grupėje insulino augimo faktoriaus (IGF – 1) geno rasti du aleliai – A ir B. A alelis rastas 0,471 dažniu, o B alelis, didinantis galvijų priesvorį, rastas 0,529 dažniu. 2. Didžiausiu dažniu B alelis rastas Limuzinų galvijų veislėje (0,711). 3. Tirtoje galvijų grupėje insulino augimo faktoriaus (IGF – 1) geno rasti trys AA, AB ir BB genotipai. AA genotipą turėjo 24,0 proc. galvijų, AB genotipą turėjo 47,0 proc. galvijų, o BB genotipą, kuris didina galvijų priesvorį, turėjo 29,0 proc. galvijų. 4. Didžiausią kiekį, net 47 proc., BB genotipą turėjo Limuzinų veislės galvijai. 5. BB genotipo galvijai statistiškai reikšmingai turėjo didesnį priesvorį (86... [toliau žr. visą tekstą] / Object and tasks of work: Collect and analyze scientific literature on IGF – 1 gene and examine its effect on cattle production traits. Estimate genotype distribution and allelic frequencies at the IGF – 1 gene. Examine the effect of IGF – 1 gene on weight gain in cattle. Research methodology: Analysis of scientific literature, scientific articles, and statistical data. The practice part involved research of cattle IGF – 1 gene. Research findings and results were discussed using method of generalization. Theoretical analysis used scientific papers by different foreign and Lithuanian researchers. Different articles and research material offering information on the issue under consideration were used as the basis for the research. Results and conclusions. The IGF – 1 gene located on chromosome 5 was found to be a factor that regulates growth rate in cattle. Our research found that IGF – 1 gene has two alleles – A and B. Allele A frequency was 0.471, and allele B – 0.529. Allele A at its highest frequency of 0.708 was found in Lithuanian Black and White cattle breed, and allele B at its highest frequency of 0.711 – in Limousin cattle breed (see Table 2). The following three different genotypes were found for IGF – 1 gene: AA, AB, and BB. AA genotype of IGF – 1 gene showed the lowest frequency and varied from 0.053 in Limousin cattle breed to 0.500 in Lithuanian Black and White cattle breed, whereas AB genotype showed its highest frequency and varied from 0.400 in Lithuanian Red... [to full text]

Previous studies demonstrated that IGF-II binds directly to vitronectin (VN) while IGF-I binds poorly. However, binding of VN to integrins has been demonstrated to be essential for a range of IGF-I-stimulated biological effects including IGF binding protein-5 (IGFBP-5) production, IGF type-1 receptor autophosphorylation and cell migration. Thus, this study examined the hypothesis that a link between IGF-I and VN must occur and may be mediated through IGFBPs. Studies using competitive binding assays with VN and [125I]-labelled IGFs in the absence and presence of IGFBPs revealed IGFBP-4, IGFBP-5 and non-glycoyslated IGFBP-3 significantly enhance binding of IGF-I to VN, while IGFBP-2 and glycosylated IGFBP-3 had a smaller effect. Furthermore, binding studies with analogues indicate that glycosylation status of IGFBP-3 and the heparin-binding domains of IGFBP-3 and IGFBP-5 are important in this interaction. The functional significance of IGFs binding to VN on cell migration in MCF-7 breast carcinoma cells was examined and cell migration was found to be enhanced when VN was pre-bound to IGF-I in the presence of IGFBP-3, -4 and -5. The effect required IGF:IGFBP:VN complex formation; this was demonstrated by use of a non-IGFBP-binding analogue, des(1- 3)IGF-I. Additionally, higher doses of IGFs in the presence of VN also could stimulate cell migration. Together, these data indicated the importance of IGFBPs in modulating IGF-I binding to VN and that this binding has functional consequences in cells. Future directions for this work include investigations into the mechanisms underlying formation of the trimeric complex and the associated signalling pathways involved.

Members of the insulin-like growth factor (IGF) family are mitogenic growth factors which have been shown to play critical roles in both normal growth and development, and tumour biology. The IGF system is complex and the biological effects of the IGFs are determined by diverse interactions between many molecules, including interactions with the extracellular matrix (ECM). Recent observations have demonstrated that IGFs can associate with the ECM protein vitronectin (VN) and this interaction can modulate IGF-stimulated biological functions. It has been demonstrated previously that IGF-II can bind directly to VN, while IGF-I associates with VN indirectly via the involvement of IGF-binding proteins (IGFBPs) -2, -3, -4 and -5. As the IGF system plays important roles in both normal breast development and in the transformation and progression of breast cancer, this study aimed to describe the effects of substrate-bound IGF-I:IGFBP:VN complexes on breast cell functions and to dissect the mechanisms underlying these responses. The studies reported in this thesis demonstrate that substrate-bound IGF-I:IGFBP:VN complexes, containing IGFBP-3 and IGFBP-5, are potent stimulators of proliferation and migration in the "normal", non-tumourigenic MCF-10A breast epithelial and MCF-7 breast carcinoma cell lines. Interestingly, substrate-bound IGF-I:IGFBP:VN complexes were less effective in increasing the migration of the metastatic MDA-MB-231 breast cancer cell line. This, however, is due to these cells expressing the αvβ3 integrin which can support a highly migratory phenotype independent of IGF-I-stimulation. Taken together this suggests a particularly important role for these complexes in stimulating a highly migratory phenotype in pre-invasive or poorly metastatic breast cells. Studies using IGF-I analogues were also undertaken to establish if there was a requirement for ternary complex formation and the type-1-IGF receptor (IGF-1R) in the enhanced migration responses observed. These studies determined IGF-I:IGFBP:VN-stimulated migration to be dependent upon both heterotrimeric IGF-I:IGFBP:VN complex formation and activation of the IGF-1R. Furthermore, the enhanced cellular migration was abolished upon incubation of MCF-7 and MCF-10A cells with function blocking antibodies directed at VN-binding integrins and the IGF-IR. In addition, analysis of the signal transduction pathways underlying the enhanced cell migration revealed that the complexes stimulate a transient activation of the ERK/MAPK signaling pathway, while simultaneously producing a sustained activation of the PI3-K/AKT pathway. Optimal intracellular signaling required activation of both the IGF-1R and VN-binding integrins, as antibody mediated inhibition of either receptor led to substantial decreases in both ERK/MAPK and PI3-K/AKT pathway activation. Furthermore, experiments using pharmacological inhibitors of these pathways determined a pivotal role for PI3-K/AKT activation in substrate-bound IGF-I:IGFBP:VN-stimulated cell migration. In order to confirm an important role for the PI3-K/AKT pathway in these responses, wild-type and activated-AKT was transiently overexpressed in MCF-10A cells. Overexpression of both wild-type and activated-AKT further enhanced cellular migration in response to substrate-bound IGF-I:IGFBP:VN complexes. However, these responses still required co-activation of the IGF-1R and VN-binding integrins. In an attempt to obtain a global view of the possible molecular mechanisms underpinning IGF-I:IGFBP:VN-stimulated cell migration, oligonucleotide microarrays were used to screen for candidate genes important for the observed migratory responses. The microarray studies identified 165 genes which were differentially expressed in cells migrating in response to substrate-bound IGF-I:IGFBP:VN complexes. Gene ontology and functional analysis revealed many of these genes to be significantly associated with biological functions relevant to cancer transformation and progression, including cell growth and proliferation, cell death and cellular movement. In regard to cell migration, a number of the genes identified have previously reported roles in cellular movement, migration and metastasis, which may provide future targets to augment IGF-I:IGFBP:VN-stimulated cell migration. Taken together, the studies reported throughout this thesis have provided the first mechanistic insights into the action of IGF-I:IGFBP:VN complexes and add further evidence to support the involvement of VN-binding integrins and their co-operativity with the IGF-IR in the promotion of tumour cell migration. Importantly, identifying the molecular mechanisms by which IGF:VN complexes enhance breast cell function will lead to not only a better understanding of this critical interaction, but also aid in developing diagnostic tests and therapeutics directed at treating breast cancer.

Malnutrition, when it is chronic disease in childhood, causes a reduction in growth and endocrine adaptive changes to ensure the maintenance of life. In this regard, the IGF-1 and cortisol have been identified as markers of nutritional status of children with growth deficiency. These high levels of cortisol and low levels of IGF-1 favor gliconoegenese and release of fatty acids by adipose tissue and inhibit the action of GH-dependent somatomedin-C on the growth. For these reasons, it is also important to investigate levels of total cholesterol, and triglycerides. Associated with energy deficiency, observed in these malnourished children, there is a lack of vitamins and minerals. Anemia due to iron deficiency is currently the most common nutritional deficiency in the world, followed by vitamin A deficiency. The iron and vitamin A are essential for normal growth and development. In addition to inadequate food intake, intestinal parasites have been considered important factors in the etiology of the anemia and the malnutrition. In this context, this study found a population with a poor and homogeneous socio-economic status, where the number of children was associated with the degree of malnutrition presented by the children studied (p = 0.03). Most of the children had between 12 and 36 months, was poly-infected (79.7%) and anemic (44.3%), the latter also highly associated with the degree of malnutrition (p = 0.01). For the levels of cortisol, the majority of children were in normal limits. Moreover, most of the levels of IGF-1 was next to the lower limit, showing its fall during the chronic malnutrition and its association with the same (p = 0.02). The results of lipid profile revealed that the vast majority of children had at least one type of dyslipidemia (98.9%) and low levels of HDL was associated with the degree of malnutrition (p = 0.02). It is, therefore, that chronic malnutrition presented by the children studied generates endocrine adaptations that cause permanent changes in lipid profile. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A desnutrição, quando ocorre de forma crônica na infância, causa redução no crescimento e alterações endócrinas adaptativas para garantir a manutenção da vida. Neste sentido, o IGF-1 e o cortisol têm sido indicados como marcadores do estado nutricional dessas crianças com déficit de estatura. Os altos níveis de cortisol e os baixos níveis de IGF-1 favorecem a gliconoegênese e a liberação de ácidos graxos pelo tecido adiposo e inibem a ação do GH dependentes de IGF-1 no crescimento. Por estes motivos, é importante investigar também o perfil lipídico. Associada a deficiência energética, verificada nessas crianças desnutridas, observa-se a carência de vitaminas e minerais. A anemia por carência de ferro é, atualmente, a deficiência nutricional mais freqüente no mundo, seguida pela hipovitaminose A. O ferro e a vitamina A são essenciais para o crescimento e desenvolvimento normais. Além da inadequada ingestão de alimentos, as parasitoses intestinais têm sido consideradas importantes fatores na etiologia das anemias carênciais e da DEP. Dentro deste contexto, o presente estudo objetivou estudar o perfil sócio-econômico, nutricional e bioquímico de crianças atendidas no Centro de Recuperação e Educação Nutricional (CREN/AL). Para tal foram selecionadas 79 crianças com idades entre 12 e 71 meses. Os dados sócio-econômicos e a avaliação antropométrica foram realizados em consulta nutricional. As dosagens bioquímicas foram realizadas entre os meses de junho e julho de 2008. Nesta época, foi coletada uma amostra de 10 ml de sangue em jejum de 12 horas, para dosagens de IGF-1, vitamina A sérica, hemograma e lipidograma completos. A dosagem do cortisol salivar foi realizada em um segundo momento para que o estresse da retirada do sangue não interferisse no resultado do mesmo. A presente pesquisa encontrou uma população com uma condição sócio-econômica precária e homogênea, onde o número de filhos encontrou-se associado ao grau de desnutrição apresentado pelas crianças estudadas (p= 0,03). A maior parte das crianças tinha entre 12 e 36 meses, encontrava-se poliparasitada (79,7%) e apresentaram anemia (44,3%), esta última altamente associada também ao grau de desnutrição (p= 0,01). As dosagens de vitamina A demonstraram um resultado surpreendente, pois não foi encontrado nenhum caso de hipovitaminose A, revelando que a suplementação de vitamina A implementada pelo Governo Federal tem sido eficiente. Com relação aos níveis de cortisol, a maioria das crianças encontrava-se dentro da normalidade. Por outro lado, a maior parte dos níveis de IGF-1 permearam o limite inferior, revelando sua queda durante a desnutrição crônica e sua forte associação com a mesma (p= 0,02). Os resultados do lipidograma revelaram que quase a totalidade das crianças apresentou pelo menos um tipo de dislipidemia (98,9%), sendo os níveis baixos de HDL associados ao grau de desnutrição (p= 0,02). Conclui-se, portanto, que a desnutrição crônica apresentada pelas crianças estudadas gera adaptações endócrinas que provocam modificações permanentes no perfil lipídico.

HMb, metabólito da leucina, utilizado para aumentar a síntese protéica. Investigamos o efeito do HMb sobre o eixo somatotrófico, bem como o mRNA de IGF-I e miostatina muscular. Ratos tratados com HMb (320 mg/Kg de peso corporal /mL de salina-0,9%), ou salina (controle), gavagem, 4 semanas, decapitados, sangue para avaliação sérica: insulina (RIE), glicose (colorimetria) e IGF-I (RIE). Extração de RNA total, para avaliação do mRNA de IGF-I e miostatina (Fígado, músculo extensor digital longo, Sóleo), avaliação da expressão do mRNA do GH, por Northern Blot, e expressão do GH ,Western blotting (hipófise). Dados analisados pelo teste-T de Student (P<0,05). Tratamento aumentou o conteúdo de mRNA de GH (> 60%), da proteína GH (>20%), do mRNA do IGF-I (~24%), da concentração sérica de IGF-I (p<0,05), indicando uma ativação do eixo somatotrófico pelo HMb, sem alterações no mRNA de miostatina e IGF-I muscular, ainda um aumento da insulina (~2x), sem alterações na glicose sérica, resultado do efeito hiperglicemiante do GH, ou um efeito direto do HMb na secreção de insulina. / HMb, metabolite of leucine, used to increase protein synthesis. Evaluate the effect HMb on the somatotrophic axis activity, as well as muscle mRNA IGF-I and myostatin. Rats treated with HMb (320 mg / kg body weight / mL of saline-0, 9%) or saline (control), gavage, 4 weeks, decapitated, blood for evaluation of serum: insulin (RIA), glucose (colorimetric) and IGF-I (RIA). Extraction of RNA total, for evaluation the mRNA IGF-I and myostatin (liver, muscle extensor digitalis longus (EDL) and soleus), evaluation of the GH mRNA expression of by Northern blot, and GH content, western blotting (pituitaries). Data analyzed by Student t-test (P <0.05). HMb treatment increased the content of GH mRNA (> 60%), GH (> 20%), IGF-I mRNA (~ 24%), IGF-I (p <0.05), indicates that the somatotrophic axis activity is increased by the HMb, without changes in mRNA of myostatin and muscle IGF-I, insulin also increased (~ 2x), without changes in serum glucose, hyperglycemiant result of the effect of GH or a direct effect of HMb in the secretion of insulin.