Proteomics and metalloproteomics tools in the characterization of plasmatic and hepatic biomarkers from rats submitted to diabetes type 1

Author(s)

Camila Pereira Braga

• Document type: Doctoral Thesis
• Press: Botucatu. 2016-08-26.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2016-07-21
• Advisor: Pedro de Magalhães Padilha; Ana Angélica Henrique Fernandes

Abstract

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. (AU)

• FAPESP's process: 11/21672-1 - PROTEOMICS AND METALLOPROTEOMICS TOOLS IN THE CHARACTERIZATION OF PLASMATIC AND HEPATIC BIOMARKERS FROM RATS SUBMITTED TO DIABETES TYPE 1
• Grantee: Camila Pereira Braga
• Support Opportunities: Scholarships in Brazil - Doctorate