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Defining the Proteomic and Phosphoproteomic Landscape of Circulating Extracellular Vesicles in the Diabetes Spectrum

The purpose of this study was to characterize the proteomic and phosphoproteomic profiles of circulating extracellular vesicles (EVs) from people with normal glucose tolerance (NGT), prediabetes (PDM), and diabetes (T2DM). Archived serum samples from 30 human subjects (N=10 per group, ORIGINS study, ClinicalTrials.gov NCT02226640) were used. EVs were isolated using EVTRAP (Tymora). Mass spectrometry (LC-MS)-based methods were used to detect the global EV proteome and phosphoproteome. Differentially expressed features, correlation networks, enriched pathways, and enriched tissue-specific protein sets were identified using custom R scripts. A total of 2372 unique EV proteins and 716 unique EV phosphoproteins were identified. Unsupervised clustering of the differentially expressed (fold change>2, P<0.05, FDR<0.05) proteins and, particularly, phosphoproteins, showed excellent discrimination among the three groups. Among characteristic changes in the PDM and T2DM EVs, "integrins switching" appeared to be a central feature. Proteins involved in oxidative phosphorylation (OXPHOS), known to be reduced in various tissues in diabetes, were significantly increased in EVs from PDM and T2DM, which suggests that an abnormally elevated EV-mediated secretion of OXPHOS components may underlie development of diabetes. We also detected a highly enriched signature of liver-specific markers among the downregulated EV proteins and phosphoproteins in both PDM and T2DM groups. This suggests that an alteration in liver EV composition and/or secretion may occur early in prediabetes. Levels of signaling molecules involved in cell death pathways were significantly altered in the circulating EVs. Consistent with the fact that patients with T2DM have abnormalities in platelet function, we detected a significant enrichment (FDR<<0.01) for upregulated EV proteins and phosphoproteins that play a role in platelet activation, coagulation, and chemokine signaling pathways in PDM and T2DM. Overall, this pilot study demonstrates the potential of EV proteomic and phosphoproteomic signatures to provide insight into the pathobiology of diabetes and its complications. These insights could lead to the development of new biomarkers of disease risk, classification, progression, and response to interventions that could allow personalization of interventions to improve outcomes.