Participação dos receptores de cininas no controle da homeostase glicêmica: análise do impacto sobre as catecolaminas e secreção de insulina
Data
2022-06-15
Autores
Gregnani, Marcos Antonio Fernandes da Silva [UNIFESP]
Orientadores
Araujo, Ronaldo Carvalho de [UNIFESP]
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: Os receptores de cininas são classicamente relacionados a eventos inflamatórios, seja através do recrutamento de células do sistema imunológico, modulando secreção de mediadores inflamatórios ou promovendo vasodilatação. Nosso grupo e outros pesquisadores vêm investigando uma outra perspectiva, que consiste em explorar efeitos metabólicos desses receptores. Após encontrarmos e descrevermos efeitos dos receptores sobre diversos órgãos relacionados ao metabolismo como fígado, músculo esquelético e tecido adiposo, buscamos nesse trabalho avaliar o papel desses receptores na modulação de 2 importantes mediadores endócrinos: catecolaminas e insulina. Métodos: Utilizamos como modelo para estudo das catecolaminas os animais B1KO, submetendo-os a avaliação das catecolaminas por meio de HPLC, diversos parâmetros glicêmicos e o comportamento dos animais quando submetidos a um teste máximo de esforço. Utilizamos também a linhagem PC12 para estudar diretamente o papel dos receptores de cininas sobre a secreção e síntese de catecolaminas. Para o estudo da secreção de insulina, usamos o animal ob/ob, que é um camundongo que apresenta um fenótipo de hiper-secreção desse hormônio. Nesse modelo, realizamos experimentos para avaliar a expressão gênica dos receptores de cininas, a secreção de insulina mediada por glicose (GSIS), modulação do influxo de cálcio mediante administração de peptídeos agonistas de B1R e B2R e realizamos também ensaios de expressão protéica para avaliar proteínas envolvidas com a ativação metabólica das células beta promovida pela glicose. Usamos também a linhagem Ins1E para avaliar respostas crônicas ao tratamento com DBK através de expressão gênica em condições de normoglicemia ou hiperglicemia, além de avaliar o impacto de DBK sobre o metabolismo por meio da quantificação de AMPc. Resultados: Os animais B1KO tiveram maior secreção de EPI plasmática em estado de repouso, o que provavelmente repercutiu nos resultados observados de menor conteúdo de glicogênio hepático e menor glicosúria. Quando esses animais foram confrontados com o desafio metabólico do exercício físico, foi observada ausência de resposta tanto de EPI quanto de NE, o que sugerimos ter relação com o déficit de desempenho que os animais demonstraram durante o exercício físico. Além das alterações em nível plasmático, foi observado padrão de resposta semelhante na glândula adrenal em relação a NE: ausência de resposta ao esforço físico nos animais B1KO. Ainda em relação à glândula adrenal, foram observadas diferenças na expressão gênica de enzimas-chave para a síntese e degradação de catecolaminas. Usando as células PC12, mostramos que o tratamento com agonistas dos receptores de cininas têm efeito positivo na secreção de catecolaminas e na expressão de enzimas relacionadas à sua síntese. Em relação ao estudo da secreção de insulina, observamos expressão dos receptores de cininas apenas nos animais ob/ob. Quando avaliamos os impactos sobre a GSIS, B1R mostrou-se um repressor por modular negativamente os processos metabólicos relacionados à GSIS, já que não observamos alterações relacionadas ao influxo de cálcio. Essas alterações também foram vistas na linhagem INS1E em experimentos que avaliaram parâmetros agudos, como a expressão de proteínas importantes para o metabolismo da glicose quanto em experimentos de expressão gênica onde realizamos tratamentos com glicose de longo prazo. Por outro lado, encontramos alguns resultados no sentido oposto quando avaliamos a ativação desses receptores em condição normal de glicose, como por exemplo o aumento de AMPc quando o agonista de B1R foi combinado a Forscolina. Conclusão: Nossos dados sugerem papel importante para os receptores de cininas na modulação de ambos hormônios, colocando esses receptores como potenciais moduladores endócrinos do metabolismo.
Introduction: Kinin receptors are classically related to inflammatory events, either by recruiting immune system cells, modulating secretion of inflammatory mediators or promoting vasodilation. Our group and other researchers have been investigating another issues, exploring metabolic effects of these receptors. After finding and describing the effects of the receptors on several organs related to metabolism, such as liver, skeletal muscle and adipose tissue, we sought to evaluate the role of these receptors in two important endocrine mediators: catecholamines and insulin. Methods: We used as a model for the study of catecholamines the B1KO animals, submitting them to evaluation of catecholamines by HPLC, glycemic parameters, and the animals performance when submited to a maximal exercise test. We also used the PC12 lineage to directly study the role of kinin receptors on catecholamine secretion and synthesis. For the study of insulin secretion, we used the ob/ob mice, which is a mouse that presents a phenotype of hyper secretion of this hormone. In this model, we performed experiments to evaluate the gene expression of kinin receptors, glucose-mediated insulin secretion (GSIS), modulation of calcium influx by acute administration of B1R and B2R agonist peptides, and also performed protein expression assays to evaluate proteins involved in glucose-promoted metabolic activation of beta cells. We also used the Ins1E strain to assess chronic responses to DBK treatment through gene expression under normoglycemic or hyperglycemic conditions, and to evaluate the impact of DBK on metabolism through cAMP quantification. Results: B1KO animals had higher plasma EPI secretion in the resting state, which likely impacted the results of lower liver glycogen content and lower glycosuria. When these animals were confronted with the metabolic challenge of exercise, no response of either EPI or NE was observed. In addition to changes at the plasma level, a similar response pattern was observed with respect to NE: no response to exercise in B1KO animals. Also in relation to the adrenal gland, differences in gene expression of key enzymes for catecholamine synthesis and degradation were observed. Using PC12 cells, we showed that treatment with kinin receptor agonists have a positive effect on catecholamine secretion and the expression of enzymes related to its synthesis. Regarding the study of insulin secretion, we observed expression of kininin receptors only in ob/ob animals. When we evaluated the impacts on GSIS, B1R proved to be a repressor by negatively modulating GSIS-related metabolic processes, since we did not observe alterations related to calcium influx. These changes were also seen in the INS1E strain in experiments that evaluated acute parameters, such as the expression of proteins important for glucose metabolism as well as in gene expression experiments where we performed long-term glucose treatments. On the other hand, we found some results in the opposite direction when we evaluated the activation of these receptors in normal glucose condition, as for example the increase of cAMP when the B1R agonist was combined with Forskolin. Conclusion: Our data suggest an important role for kinin receptors in the modulation of both hormones, placing these receptors as potential endocrine modulators of metabolism.
Introduction: Kinin receptors are classically related to inflammatory events, either by recruiting immune system cells, modulating secretion of inflammatory mediators or promoting vasodilation. Our group and other researchers have been investigating another issues, exploring metabolic effects of these receptors. After finding and describing the effects of the receptors on several organs related to metabolism, such as liver, skeletal muscle and adipose tissue, we sought to evaluate the role of these receptors in two important endocrine mediators: catecholamines and insulin. Methods: We used as a model for the study of catecholamines the B1KO animals, submitting them to evaluation of catecholamines by HPLC, glycemic parameters, and the animals performance when submited to a maximal exercise test. We also used the PC12 lineage to directly study the role of kinin receptors on catecholamine secretion and synthesis. For the study of insulin secretion, we used the ob/ob mice, which is a mouse that presents a phenotype of hyper secretion of this hormone. In this model, we performed experiments to evaluate the gene expression of kinin receptors, glucose-mediated insulin secretion (GSIS), modulation of calcium influx by acute administration of B1R and B2R agonist peptides, and also performed protein expression assays to evaluate proteins involved in glucose-promoted metabolic activation of beta cells. We also used the Ins1E strain to assess chronic responses to DBK treatment through gene expression under normoglycemic or hyperglycemic conditions, and to evaluate the impact of DBK on metabolism through cAMP quantification. Results: B1KO animals had higher plasma EPI secretion in the resting state, which likely impacted the results of lower liver glycogen content and lower glycosuria. When these animals were confronted with the metabolic challenge of exercise, no response of either EPI or NE was observed. In addition to changes at the plasma level, a similar response pattern was observed with respect to NE: no response to exercise in B1KO animals. Also in relation to the adrenal gland, differences in gene expression of key enzymes for catecholamine synthesis and degradation were observed. Using PC12 cells, we showed that treatment with kinin receptor agonists have a positive effect on catecholamine secretion and the expression of enzymes related to its synthesis. Regarding the study of insulin secretion, we observed expression of kininin receptors only in ob/ob animals. When we evaluated the impacts on GSIS, B1R proved to be a repressor by negatively modulating GSIS-related metabolic processes, since we did not observe alterations related to calcium influx. These changes were also seen in the INS1E strain in experiments that evaluated acute parameters, such as the expression of proteins important for glucose metabolism as well as in gene expression experiments where we performed long-term glucose treatments. On the other hand, we found some results in the opposite direction when we evaluated the activation of these receptors in normal glucose condition, as for example the increase of cAMP when the B1R agonist was combined with Forskolin. Conclusion: Our data suggest an important role for kinin receptors in the modulation of both hormones, placing these receptors as potential endocrine modulators of metabolism.
Descrição
Citação
Gregnani, M A F S. Participação dos receptores de cininas no controle da homeostase glicêmica: análise do impacto sobre as catecolaminas e secreção de insulina. São Paulo, 2022. 214 f. Tese (Doutorado em Biologia Molecular) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP). São Paulo, 2022.