Unravelling the role of reactive oxygen species in glucose-stimulated insulin secretion: insights from oxidative and phosphoproteomic analysis

Insulin-secreting beta cells are essential in regulating blood glucose levels and carbohydrate metabolism. However, β-cell dysfunction is a primary factor in the pathogenesis of diabetes. The process of insulin secretion is tightly regulated and involves shifts in cellular redox status, with hydrogen peroxide (H2O2) identified as a key component in glucose-stimulated insulin secretion. But, excessive production of reactive oxygen species (ROS) leads to β-cell exhaustion and loss. Therefore, understanding the redox proteome is crucial for understanding the precise interplay between ROS and insulin secretion. As changes in cysteine redox status can reflect alterations in redox signalling pathways and kinases and phosphatases are key regulators of insulin secretion and frequently modulated by redox signalling, in this study we analysed chances within the redox- and phosphor-proteome of beta-cells after glucose stimulation. Therefore, INS-1E cells were incubated with varying glucose concentrations following a period of starvation, lysed and non-oxidized cysteines were labelled using N-ethylmaleimide (NEM). Sequential alkylation enabled labeling of reversibly oxidized cysteines with cysteine-specific phosphonate adaptable tag (CysPAT), allowing simultaneous titanium dioxide-based enrichment of cysteine-oxPTM and phospho sites after tryptic digestion and labelling with TMT. Finally, samples were analysed using high-resolution LC-MS/MS (Orbitrap). This protocol was complemented by characterizing the oxidative status of β-cells during insulin secretion using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). INS-1E cells are demonstrating a typical biphasic insulin secretion pattern and a strong responsiveness to glucose and other secretagogues such as amino acids or ketone bodies. Furthermore, our findings reveal that major reversible oxidation events occur in proteins associated with localization, cellular, and metabolic processes. Notably, there were time-dependent alterations in glucose-stimulated insulin secretion and corresponding changes in cellular oxidative status. This highlights dynamic protein oxidation and increased ROS production upon glucose stimulation of beta cells, which also impacts the oxidative status of regulatory proteins. Through this comprehensive analysis, we aim to understand the complex signalling pathways governing insulin secretion and provide insights into the role of oxidative stress in beta-cell function and dysfunction.