Comprehensive analysis of ROS-induced protein phosphorylation and oxidation in physiology and pathophysiology of pancreatic beta cells
Anatomy and Physiology
Biochemistry
Endocrinology, Diabetology, Metabolism
Public Health, Healthcare Research, Social and Occupational Medicine
Final Report Abstract
Pancreatic β-cells represent the almost exclusive endogenous source of insulin that is essential for the maintenance of an appropriate glucose homeostasis. The regulation of insulin secretion is a complex process that involves protein phosphorylation as essential component. Additionally, oxidation events are also of central importance in physiological as well as pathophysiological insulin secretion process and accumulating evidence suggests a cross-talk of protein phosphorylation and oxidation in this context. Therefore, it was the aim of the research fellowship to characterize these processes and relations in more detail using cell culture as in vitro model. For this purpose, three projects were designed to (A) analyze protein phosphorylation and oxidation events during insulin secretion stimulated by glucose, GLP-1 and GLP-1 receptor agonist, (B) characterize the effects of increased H2O2 formation induced by MND during GSIS, and (C) determine changes due to H2O2 quenching based on catalase overexpression. Additionally, a fourth study was included based on previous work of Martin R. Larsen dealing with (D) the importance of FAK auto-phosphorylation for the insulin secretion pathway. However, because the position as a Junior Research Group leader was offered to me, I returned the fellowship ahead of schedule (12 months) and therefore it was not possible to finish all planned projects. Consequently, we decided to re-structure the research plan to finish at least acquisition of mass spectrometric data for project A (parts) and project D. Detailed data interpretation of these data is ongoing and part of a post-fellowship cooperation that also aims to promote finalization/ realization of projects B and C. Briefly, with regard to project A we focused on GSIS in INS-1E cells and plan to analyze the effects of GLP-1 and GLP-1 receptor agonists in a future collaboration. In this context, we were able to finish the cell stimulation experiments as well as mass spectrometric data acquisition. Thereby, the preliminary data evaluation revealed distinct time-dependent changes in protein phosphorylation and oxidation that can be clustered into different regulation patterns. Additionally, it was possible to demonstrate timedependent changes in the cellular H2O2 formation that will be linked to the proteomics data during ongoing data interpretation process. In line with this it was also possible to finish cell culture experiments as well as mass spectrometric analyses of project D. In this context, the preliminary data analysis confirmed the importance of FAK for the insulin secretion process and ongoing data interpretation as well as in-depth characterization of insulin secretion will highlight affected cellular pathways as well as their interaction with each other. In project B it was possible to finish the preliminary experiments concerning the optimal MDN dose for stimulation of cellular H2O2 formation that will be used for future experiments. Additionally, experiments concerning the effects of short- and medium-term glucolipotoxicity could be realized and preliminary data evaluation indicated specific changes that are subject of ongoing detailed data interpretation. Due to time reasons it was not possible to obtain any data concerning protein phosphorylation and oxidation in project C. However, the project was already initiated and the successful adenoviral overexpression of catalase in INS-1E cells could be characterized. Future efforts will evaluate the stability of the transfection as well as effects on cell viability and consider mass spectrometric evaluation. In summary, the preliminary results of the research projects are encouraging and detailed data analysis will highlight the importance of protein oxidation as well as the cross-talk of protein oxidation and phosphorylation in the insulin secretion process. The obtained insights will contribute to the deeper understanding of the importance of oxidation events in the cellular metabolism and potentially contribute to the development of new therapeutic approaches.
