Publicação
Metabolic biomarkers of chick embryonic lung development: impact of retinoic acid
| Resumo: | Pulmonary development is a complex process that depends on the activation of conserved signaling pathways that regulate cellular processes such as proliferation, differentiation and migration. Retinoic acid (RA) signaling is one of the active pathways during lung organogenesis, since it modulates branching morphogenesis in a dose-dependent manner. It is widely accepted that the cellular events underlying embryonic development require high amounts of energy and nutrients to form new biomass. However, the metabolic changes that occur during lung branching morphogenesis have not been described so far. In this work, we have characterized, for the first time, the metabolic profile of chick lung branching in early stages of development: b1, b2 and b3 (1, 2 or 3 secondary bronchi, respectively). Furthermore, the impact of RA on lung branching metabolism was evaluated. Ex vivo lung explant culture was performed in the presence or absence of RA, and the medium collected to analyze the production/consumption of metabolites associated with glucose catabolism (glucose, lactate, acetate, alanine), by 1 H-NMR. qRT-PCR and in situ hybridization were performed to assess the expression levels and patterns of key enzymes and transporters from glucose catabolic pathway. In normal conditions, the major metabolite variations occur from stage b1 to stage b3. In b3, there is an increase in lactate and acetate production. Still, glucose consumption is maintained from b1 to b3 stage with a concurrent decrease of glucose transporter 3 (glut3) transcript levels, and a decrease in hexokinase 1 (hk1) levels in b3 stage. This phenomenon suggests an increase in the glycolytic efficiency and a shift to lactate production. In fact, we observed a decrease on pyruvate dehydrogenase B (pdhb) and an increase in lactate dehydrogenase A (ldha) expression levels in b3 stage, while lactate dehydrogenase B (ldhb) levels decrease. Embryonic lung expression pattern of glut3, hk1, ldha, ldhb and pdha were characterized suggesting hypothetical interactions between the mesenchymal and epithelial compartments. When b2 lung explants were exposed to RA, glucose consumption, alanine and lactate production decreased. On the other hand, glutamate, glutamine consumption, and acetate production remain unaltered whereas pyruvate production increased. This phenomenon is accompanied by an increase in ldha expression levels. This study describes, for the first time, the temporal metabolic changes associated with chick pulmonary branching. It seems that glycolytic efficiency is increased, with a concurrent metabolic shift from Krebs cycle to lactate production. Furthermore, acetate and lactate are potentially seen as metabolic biomarkers of lung development. On the other hand, RA triggers a metabolic shift where lipids may be potentially seen as source of carbons and energy during RA-induced branching morphogenesis. |
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| Autores principais: | Silva, Hugo Miguel Fernandes |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Pulmonary development is a complex process that depends on the activation of conserved signaling pathways that regulate cellular processes such as proliferation, differentiation and migration. Retinoic acid (RA) signaling is one of the active pathways during lung organogenesis, since it modulates branching morphogenesis in a dose-dependent manner. It is widely accepted that the cellular events underlying embryonic development require high amounts of energy and nutrients to form new biomass. However, the metabolic changes that occur during lung branching morphogenesis have not been described so far. In this work, we have characterized, for the first time, the metabolic profile of chick lung branching in early stages of development: b1, b2 and b3 (1, 2 or 3 secondary bronchi, respectively). Furthermore, the impact of RA on lung branching metabolism was evaluated. Ex vivo lung explant culture was performed in the presence or absence of RA, and the medium collected to analyze the production/consumption of metabolites associated with glucose catabolism (glucose, lactate, acetate, alanine), by 1 H-NMR. qRT-PCR and in situ hybridization were performed to assess the expression levels and patterns of key enzymes and transporters from glucose catabolic pathway. In normal conditions, the major metabolite variations occur from stage b1 to stage b3. In b3, there is an increase in lactate and acetate production. Still, glucose consumption is maintained from b1 to b3 stage with a concurrent decrease of glucose transporter 3 (glut3) transcript levels, and a decrease in hexokinase 1 (hk1) levels in b3 stage. This phenomenon suggests an increase in the glycolytic efficiency and a shift to lactate production. In fact, we observed a decrease on pyruvate dehydrogenase B (pdhb) and an increase in lactate dehydrogenase A (ldha) expression levels in b3 stage, while lactate dehydrogenase B (ldhb) levels decrease. Embryonic lung expression pattern of glut3, hk1, ldha, ldhb and pdha were characterized suggesting hypothetical interactions between the mesenchymal and epithelial compartments. When b2 lung explants were exposed to RA, glucose consumption, alanine and lactate production decreased. On the other hand, glutamate, glutamine consumption, and acetate production remain unaltered whereas pyruvate production increased. This phenomenon is accompanied by an increase in ldha expression levels. This study describes, for the first time, the temporal metabolic changes associated with chick pulmonary branching. It seems that glycolytic efficiency is increased, with a concurrent metabolic shift from Krebs cycle to lactate production. Furthermore, acetate and lactate are potentially seen as metabolic biomarkers of lung development. On the other hand, RA triggers a metabolic shift where lipids may be potentially seen as source of carbons and energy during RA-induced branching morphogenesis. |
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