Aplicação de ferramentas proteômicas e metaloproteômicas na caracterização de biomarcadores plasmáticos e hepáticos de ratos submetidos ao diabetes tipo 1
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Data
2016-07-21
Autores
Braga, Camila Pereira [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Diabetes mellitus tipo 1 é caracterizado pelo aumento significativo de glicose circulante no sangue, resultado da deficiência na secreção e/ou ação de insulina. O quadro hiperglicêmico, quando cronicamente instalado, leva a anormalidades no metabolismo de lipídios, proteínas e carboidratos, além de alterar o balanço entre pró-oxidantes e antioxidantes. É um importante problema de saúde pública, pois compromete a qualidade de vida e sobrevida dos indivíduos, além de envolver elevados custos no seu tratamento. Quando se consegue identificar os fatores determinantes para o desenvolvimento do diabetes, novas estratégias de prevenção e tratamento são necessárias. Nesse contexto, as proteínas e os minerais apresentam fundamental importância como componentes estruturais e funcionais dos seres vivos. Estudos proteômicos e metaloproteômicos, auxiliam na compreensão da variabilidade do diabetes, contribuindo assim na elucidação dos aspectos fisiológicos e funcionais das biomoléculas presentes em amostras biológicas, como plasma e tecido animal, além disso, a possível identificação de biomarcadores relacionados ao desenvolvimento de doenças crônicas degenerativas, como o diabetes. O objetivo geral do trabalho foi utilizar ferramentas proteômicas e metaloproteômicas na identificação de possíveis biomarcadores presentes no plasma e fígado de ratos diabéticos tipo 1 induzido experimentalmente com estreptozotocina. Foram utilizados 24 ratos Wistar, distribuídos em 3 grupos experimentais (n= 8): C (controle); DM1 (diabéticos tipo 1); DM1 + I (diabéticos tipo 1, tratadas com insulina). O diabetes mellitus tipo 1 foi induzido com estreptozotocina (60mg/kg, i.p., dose única) e os animais do grupo DM1 + I receberam reposição insulínica (Humulin N100UI Protamina Neutra de Hagedorn - NPH, marca Lilly), com dose inicial de 3U/animal (administrada diariamente, via subcutânea, período vespertino) e de acordo com os valores obtidos de glicemia a dose inicial foi reajustada/mantida para que se atingissem os níveis séricos de euglicemia. No plasma, foi realizado estudo metaloproteômico para as proteínas que apresentaram diferença de expressão entre os grupos experimentais, utilizando-se a 2D-PAGE no processo de fracionamento de proteínas das amostras de plasma, GFAAS e FAAS na determinação quantitativa de Cu, Mg, Se e Zn nos spots que apresentaram diferença de expressão e ESI/MS-MS na caracterização dessas proteínas. Trinta e cinco proteínas apresentaram diferença de expressão no plasma, indicando a alpha-1-macroglobulina e haptoglobulina como possíveis biomarcadoras do diabetes tipo 1 controlado (tratado com insulina); e as proteínas 2'-desoxinucleósido-5'-fosfato de N-hidrolase 1, proteína transmembranar 1, soro amilóide P-componente, vitamina D de ligação e biliverdina como possíveis candidatas a biomarcadoras do diabetes tipo 1 não controlado. Já a quantificação de Cu, Mg, Se e Zn nos spots revelaram como esses minerais poderiam estar ligados nessas proteínas e como estariam envolvidos na expressão proteica, progressão e complicações do diabetes tipo 1. No fígado, primeiramente foi realizado estudo proteômico, onde as proteínas foram caracterizadas pela técnica de gel-free e classificadas como dependentes ou independentes de insulina (tratamento), em relação aos dados obtidos para abundância e diferença de expressão. Esse estudo identificou 305 proteínas com diferença de expressão entre os grupos experimentais, entre elas 147 foram classificadas como dependentes de insulina e 152 como independentes de insulina. O controle dos níveis de glicose sanguínea pelo tratamento com insulina, restaurou os níveis da enzima frutose-1,6 bifosfatase indicando controle da gliconeogênese, e afetou a abundância das enzimas que funcionam na desintoxicação de EROs, restaurando o equilíbrio redox e a função mitocondrial. No fígado também foi feito o estudo das proteínas diferencialmente expressas pelo Oxi-proteoma, utilizando a 2D-DIGE com os fluoróforos Cy3 e Cy-5-hidrazida no processo de fracionamento e caracterização dos spots proteicos por ESI-MS/MS. A identificação das proteínas carboniladas indicou que o tratamento com insulina está associado com a diminuição do estresse oxidativo por meio da regulação positiva das enzimas antioxidantes e diminuição da produção de EROs, controle das vias metabólicas envolvidas com o metabolismo de carboidratos (glicólise e gliconeogênese) e metabolismo energético. Estudo metaloproteômico também foi realizado no fígado, utilizando a 2D-PAGE no processo de fracionamento de proteínas, GFAAS e FAAS na determinação quantitativa de Cu, Mg, Se e Zn nos spots obtidos no fracionamento das proteína e caracterização das proteínas por ESI/MS-MS. Os resultados obtidos nesse estudo sugeriram diferentes interações entre os minerais estudados e as proteínas entre os grupos experimentais. Essas interações podem estar associadas com alterações funcionais nessas proteínas e envolvimento na progressão e complicações do diabetes tipo 1. Assim, as informações obtidas no presente trabalho fornecem subsídios técnico-científicos na área de saúde de extrema importância, possibilitando o entendimento dos dados proteômicos e metaloproteômicos gerados relacionados com suas respectivas vias metabólicas, função molecular, processo biológico e componente celular. O caráter inédito da proposta proporcionou a elucidação de aspectos fisiológicos e funcionais das biomoléculas presentes em amostras biológicas, como plasma e tecido hepático.
Type 1 diabetes mellitus is characterized by the significant increase in circulating glucose in the blood as a result of deficiency in the secretion and/or insulin action. The hyperglycemic state, when chronically installed, leads to abnormalities in the metabolism of lipids, proteins and carbohydrates, in addition to changing the balance between pro-oxidants and antioxidants. This is an important public health problem because it affects the quality of life and the survival of individuals, often necessitating high-cost treatment. When one can identify the determining factors for the development of diabetes, new strategies for prevention and treatment are needed. In this context, our results reflect the fundamental importance of protein and minerals such as structural and functional components of living things, proteomic and metalloproteomic studies that can assist in the understanding of diabetes variability, thus helping to elucidate the physiological and functional aspects of biomolecules in biological samples such as plasma and animal tissue. In addition, the possible identification of biomarkers related to the development of chronic degenerative diseases like diabetes. The overall objective of the study was to use proteomics and metalloproteomic in identifying potential biomarkers present in the plasma and liver of diabetic rats type 1 experimentally induced with streptozotocin. The total of 24 rats, Wistar, were divided into 3 groups (n = 8): C (control); DM1 (type 1 diabetes); DM1 + I (type 1 diabetes treated with insulin). The type 1 diabetes mellitus was induced with streptozotocin (60 mg/kg, i.p., single dose) and animals of DM1 + I group received insulin replacement (Humulin N100UI Neutral Protamine Hagedorn - NPH, brand Lilly), with an initial dose of 3U/animal (daily administered subcutaneously evening period) and in accordance with the values obtained from the initial dose of glucose was readjusted/kept so that it reached serum euglycemia. In plasma was performed metalloproteomic study for proteins that showed differences in expression between the experimental groups, 2D-PAGE was used in protein fractionation process of plasma samples, GFAAS and FAAS for quantitative determination of Cu, Mg, Se and Zn in the spots that showed differences of expression and the proteins were characterized by ESI/MS-MS. Thirty-five proteins present in the plasma had difference of expression, indicating the alpha-1-macroglobulin and haptoglobulin as potential biomarkers of type 1 diabetes controlled (insulin treated); and 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, transmembrane protein 11, serum amyloid P-component, vitamin D-binding protein and biliverdin as possible candidates for biomarkers of type 1 diabetes not controlled. The quantification of Cu, Mg, Se and Zn in spots revealed how these minerals could be linked these proteins and how they would be involved in protein expression, progression and complications of type 1 diabetes. In the liver, was first conducted the proteomic study where proteins were characterized by gel-free technique and classified as insulin dependent or independent (treatment), compared to the data obtained for abundance and difference in expression. This study identified 305 proteins with differential expression between experimental groups, among which 147 were classified as insulin-dependent and 152 as insulin-independent. The restoration of glucose levels in the blood by insulin treatment restored levels of the enzyme fructose-1,6 bisphosphatase indicating control of gluconeogenesis, and affected the abundance of enzymes that function in the detoxification of ROS, restoring redox balance and mitochondrial function. In the liver it has also been a study of the proteins differentially expressed by Oxi-proteome, using the 2D-DIGE with the fluorophores Cy3 and Cy-5-hydrazide in the fractionation process and characterization of protein spots by ESI-MS/ MS. The identification of the carbonyl proteins indicated that treatment with insulin was associated with decreased oxidative stress by upregulating the antioxidant enzymes and decreased production of ROS, controlling the metabolic pathways involved in carbohydrate metabolism (glycolysis and gluconeogenesis) and energy metabolism. The metalloproteomic study was also performed on liver, using 2D-PAGE for the protein fractionation process, GFAAS and FAAS for quantitative determination of Cu, Mg, Se and Zn in the spots obtained in the fractionation of the protein and characterization of proteins by ESI/MS-MS. The results suggested different interactions between minerals and proteins between experimental groups, and may be associated with functional alterations in these proteins and involvement in progression and complications of diabetes type 1. However, the work provides technical and scientific support in extreme health importance, enabling the understanding of proteomic and metalloproteomic data generated in relation to their respective metabolic pathways, molecular function, biological process and cellular component. The unprecedented of the proposal provided the elucidation of physiological and functional aspects of biomolecules present in biological samples, such as plasma and hepatic tissue.
Type 1 diabetes mellitus is characterized by the significant increase in circulating glucose in the blood as a result of deficiency in the secretion and/or insulin action. The hyperglycemic state, when chronically installed, leads to abnormalities in the metabolism of lipids, proteins and carbohydrates, in addition to changing the balance between pro-oxidants and antioxidants. This is an important public health problem because it affects the quality of life and the survival of individuals, often necessitating high-cost treatment. When one can identify the determining factors for the development of diabetes, new strategies for prevention and treatment are needed. In this context, our results reflect the fundamental importance of protein and minerals such as structural and functional components of living things, proteomic and metalloproteomic studies that can assist in the understanding of diabetes variability, thus helping to elucidate the physiological and functional aspects of biomolecules in biological samples such as plasma and animal tissue. In addition, the possible identification of biomarkers related to the development of chronic degenerative diseases like diabetes. The overall objective of the study was to use proteomics and metalloproteomic in identifying potential biomarkers present in the plasma and liver of diabetic rats type 1 experimentally induced with streptozotocin. The total of 24 rats, Wistar, were divided into 3 groups (n = 8): C (control); DM1 (type 1 diabetes); DM1 + I (type 1 diabetes treated with insulin). The type 1 diabetes mellitus was induced with streptozotocin (60 mg/kg, i.p., single dose) and animals of DM1 + I group received insulin replacement (Humulin N100UI Neutral Protamine Hagedorn - NPH, brand Lilly), with an initial dose of 3U/animal (daily administered subcutaneously evening period) and in accordance with the values obtained from the initial dose of glucose was readjusted/kept so that it reached serum euglycemia. In plasma was performed metalloproteomic study for proteins that showed differences in expression between the experimental groups, 2D-PAGE was used in protein fractionation process of plasma samples, GFAAS and FAAS for quantitative determination of Cu, Mg, Se and Zn in the spots that showed differences of expression and the proteins were characterized by ESI/MS-MS. Thirty-five proteins present in the plasma had difference of expression, indicating the alpha-1-macroglobulin and haptoglobulin as potential biomarkers of type 1 diabetes controlled (insulin treated); and 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, transmembrane protein 11, serum amyloid P-component, vitamin D-binding protein and biliverdin as possible candidates for biomarkers of type 1 diabetes not controlled. The quantification of Cu, Mg, Se and Zn in spots revealed how these minerals could be linked these proteins and how they would be involved in protein expression, progression and complications of type 1 diabetes. In the liver, was first conducted the proteomic study where proteins were characterized by gel-free technique and classified as insulin dependent or independent (treatment), compared to the data obtained for abundance and difference in expression. This study identified 305 proteins with differential expression between experimental groups, among which 147 were classified as insulin-dependent and 152 as insulin-independent. The restoration of glucose levels in the blood by insulin treatment restored levels of the enzyme fructose-1,6 bisphosphatase indicating control of gluconeogenesis, and affected the abundance of enzymes that function in the detoxification of ROS, restoring redox balance and mitochondrial function. In the liver it has also been a study of the proteins differentially expressed by Oxi-proteome, using the 2D-DIGE with the fluorophores Cy3 and Cy-5-hydrazide in the fractionation process and characterization of protein spots by ESI-MS/ MS. The identification of the carbonyl proteins indicated that treatment with insulin was associated with decreased oxidative stress by upregulating the antioxidant enzymes and decreased production of ROS, controlling the metabolic pathways involved in carbohydrate metabolism (glycolysis and gluconeogenesis) and energy metabolism. The metalloproteomic study was also performed on liver, using 2D-PAGE for the protein fractionation process, GFAAS and FAAS for quantitative determination of Cu, Mg, Se and Zn in the spots obtained in the fractionation of the protein and characterization of proteins by ESI/MS-MS. The results suggested different interactions between minerals and proteins between experimental groups, and may be associated with functional alterations in these proteins and involvement in progression and complications of diabetes type 1. However, the work provides technical and scientific support in extreme health importance, enabling the understanding of proteomic and metalloproteomic data generated in relation to their respective metabolic pathways, molecular function, biological process and cellular component. The unprecedented of the proposal provided the elucidation of physiological and functional aspects of biomolecules present in biological samples, such as plasma and hepatic tissue.
Descrição
Palavras-chave
Diabetes, Biomarcadores, Eletroforese bidimensional, Metabolismo, Metaloproteínas, Proteínas, Biomarkers, Two-dimensional electrophoresis, Metabolism, Metalloproteins, Proteins