Multi-layered proteomics identifies insulin-induced upregulation of the EphA2 receptor via the ERK pathway and dependent on low IGF1R level

Insulin resistance impairs the cellular insulin response and frequently precedes metabolic disorders, like type 2 diabetes, which are affecting an increasing number of people globally. Given the critical role of the liver in glucose and lipid metabolism, understanding the molecular mechanisms in hepatic insulin resistance is essential for early preventive treatments. To elucidate changes in insulin signal transduction associated with hepatocellular resistance, we employed a multi-layered mass spectrometry-based proteomics approach focusing on insulin receptor (IR) signaling at the interactome, phosphoproteome, and proteome levels.in a long-term hyperinsulinemia-induced insulin-resistant HepG2 cell line with a knockout of the insulin-like growth factor 1 receptor (IGF1R KO). Analysis of the dynamic insulin-induced IR interactome revealed recruitment of the PI3K complex in both insulin-sensitive and -resistant cells. From the phosphoproteomics dataset, a change in insulin-stimulated signaling responses in insulin resistance was observed and showed attenuated signaling via the metabolic PI3K-AKT pathway but sustained extracellular signal-regulated kinase (ERK) activity. At the proteome level, the ephrin type-A receptor 2 (EphA2) showed an insulin-induced increase in expression. This receptor belongs to the Eph receptor family and participates in various cellular processes, such as cell adhesion, migration, and tissue development. The protein abundance regulation of EphA2 occurred through the ERK signaling pathway and was concordantly independent of insulin resistance. Induction of EphA2 by insulin was confirmed in other cell lines and observed uniquely in cells with high levels of IR compared to IGF1R. The multi-layered proteomics dataset provided insights into insulin signaling in general and in the context of insulin resistance, and it can going forward serve as a resource to generate and test hypotheses, leading to an improved understanding of insulin resistance.