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Role of insulin and insulin-like peptides in bone formation: identification of bone-specific target genes and regulatory mechanisms, and characterization of the insulin-mimetic effect of vanadium
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Abstract(s)
Insulina e IGF-1 são pequenos péptidos circulantes conhecidos por regular a densidade
mineral e formação óssea em vertebrados através da activação vias de sinalização
celular PI-3K e MAPK. Por sua vez, demonstrou-se recentemente que o
vanádio/vanadato regula a proliferação e diferenciação de linhas celulares derivadas do
osso de mamíferos através mesmas vias. Neste estudo, os efeitos proliferativos e
mineralogénicos da insulina, IGF-1 e vanadato foram investigados em linhas derivadas
do osso de peixe: VSa13 (tipo condrócito) e VSa16 (tipo osteoblasto). Tratamentos
agudos com metavanadato (espécies n-méricas 1 a 5) nas células VSa13 revelaram uma
acumulação mais rápida e mais elevada de vanádio, bem como uma toxicidade mais
baixa do que decavanadato (n = 1 ou 10). Tratamentos crónicos com ambas as soluções
estimularam a proliferação das VSa13, tal como a IGF-1, e preveniram a mineralização,
tal como a insulina. Tratamentos com wortmanina e PD98959 demonstraram o
envolvimento das vias MAPK e PI-3K\Ras\ERK nos efeitos proliferativos e
mineralogénicos, respectivamente. Os dados obtidos são consistentes com observações
recentes nas células de mamífero ATDC5 (condrócitos) sugerindo uma conservação de
mecanismos ao longo da evolução. Aparentemente o vanadato não só terá interferido
com as vias de sinalização mas também com a actividade da fosfatase alcalina, uma
enzima muito importante na formação óssea. Nas células VSa16 foram observados
efeitos semelhantes na proliferação através de tratamentos com IGF-1 (estimulação), e
na mineralização através de tratamentos com vanadato e IGF-1 (inibição), sugerindo
uma conservação de mecanismos através dos tipos celulares de osso. Em paralelo, neste
trabalho foram identificadas em seabream 3 variantes da proIGF-1 (IGF-1a, 1b e 1c) e
associadas a diferentes modos de acção (local e sistémico) em tecidos de adultos e
durante o desenvolvimento, tal como previamente demonstrado em mamíferos.
vi
Propôs-se também que em peixes as variantes 1a e 1b estariam relacionadas com uma
adaptação a diferentes salinidades. Em geral, os resultados aqui apresentados indicam
uma conservação de mecanismos em vertebrados, demonstrando a importância dos
sistemas derivados de peixes no estudo dos efeitos semelhantes à insulina no osso.
Insulin and IGF-1 are small circulating peptides known to regulate bone mineral density and formation in vertebrates through the activation of intracellular PI-3K and MAPK signalling pathways. Vanadium/vanadate has recently been shown to regulate proliferation and differentiation of mammalian bone-derived cell lines also through the activation of both pathways. In this study, proliferative and mineralogenic effects of insulin, IGF-1 and vanadate have been investigated using fish bone-derived cell lines VSa13 (chondrocyte-like) and VSa16 (osteoblast-like). Short-term treatments of VSa13 cells with two different forms of vanadate in solution (meta- and decavanadate) reavealed that metavanadate (i) results in faster and higher accumulation of vanadate in the cell, and (ii) has a lower toxicity than decavanadate when administered in vitro to the cells. Chronic treatments with both solutions were found to partially mimic the effects of both factors, e.g. stimulated VSa13 cell proliferation, as IGF-1, and prevented ECM mineralization, as insulin. Treatments with wortmannin and PD98959 demonstrated the involvement of MAPK and PI-3K\Ras\ERK pathways in proliferative and mineralogenic effects, respectively. These data are consistent with recent observations in mammalian ATDC5 cells (chondrocytes) suggesting the conservation of mechanisms of action throughout evolution. Vanadate was also shown not only to interfere with regulatory pathways but also to inhibit alkaline phosphatase activity, a key enzyme in bone formation. In Vsa16 cells, similar effects have been observed on proliferation, using IGF-1 (i.e. stimulation), and ECM mineralization, using both IGF-1 and vanadate (i.e. inhibition), suggesting conservation of mechanisms across bone cell types. In parallel to this work, 3 spliced variants of seabream proIGF-1 (IGF-1a, 1b and 1c) were identified and associated to a different mode of action (local and systemic) in adult tissues and during development, as previously observed in mammals. Variants 1a iv and 1b were also proposed to play a role in fish osmoregulation. Altogether, results presented here indicate a conservation of mechanisms between fish and mammals, further demonstrating the suitability of fish systems to study vertebrate insulin-like effects in bone.
Insulin and IGF-1 are small circulating peptides known to regulate bone mineral density and formation in vertebrates through the activation of intracellular PI-3K and MAPK signalling pathways. Vanadium/vanadate has recently been shown to regulate proliferation and differentiation of mammalian bone-derived cell lines also through the activation of both pathways. In this study, proliferative and mineralogenic effects of insulin, IGF-1 and vanadate have been investigated using fish bone-derived cell lines VSa13 (chondrocyte-like) and VSa16 (osteoblast-like). Short-term treatments of VSa13 cells with two different forms of vanadate in solution (meta- and decavanadate) reavealed that metavanadate (i) results in faster and higher accumulation of vanadate in the cell, and (ii) has a lower toxicity than decavanadate when administered in vitro to the cells. Chronic treatments with both solutions were found to partially mimic the effects of both factors, e.g. stimulated VSa13 cell proliferation, as IGF-1, and prevented ECM mineralization, as insulin. Treatments with wortmannin and PD98959 demonstrated the involvement of MAPK and PI-3K\Ras\ERK pathways in proliferative and mineralogenic effects, respectively. These data are consistent with recent observations in mammalian ATDC5 cells (chondrocytes) suggesting the conservation of mechanisms of action throughout evolution. Vanadate was also shown not only to interfere with regulatory pathways but also to inhibit alkaline phosphatase activity, a key enzyme in bone formation. In Vsa16 cells, similar effects have been observed on proliferation, using IGF-1 (i.e. stimulation), and ECM mineralization, using both IGF-1 and vanadate (i.e. inhibition), suggesting conservation of mechanisms across bone cell types. In parallel to this work, 3 spliced variants of seabream proIGF-1 (IGF-1a, 1b and 1c) were identified and associated to a different mode of action (local and systemic) in adult tissues and during development, as previously observed in mammals. Variants 1a iv and 1b were also proposed to play a role in fish osmoregulation. Altogether, results presented here indicate a conservation of mechanisms between fish and mammals, further demonstrating the suitability of fish systems to study vertebrate insulin-like effects in bone.
Description
Tese de Doutoramento em Biologia, Especialidade em Biologia Molecular, Universidade do Algarve, 2008
Keywords
Biologia molecular Sparus aurata