Dissertação de Mestrado Investigação Biomédica 2023 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

Diabetes entails numerous metabolic alterations across multiple organs, highlighting the critical role of inter-organ communication in its pathogenesis. Interestingly, extracellular vesicles (EVs) are agents of systemic communication, in both physiological and pathological contexts. Nevertheless, the intricate molecular mechanisms through which EVs operate in the context of diabetes remain unclear. Furthermore, plasma-derived extracellular vesicles (PDEVs) are increasingly recognized as pivotal mediators of inter-organ communication and potential sources of novel biomarkers for various medical conditions. To decipher the intricate messages encapsulated in EVs, under prediabetic conditions, we isolated PDEVs from diet induced prediabetic murine models. PDEVs presented mean size typical of small EVs, with no exclusive classification as exosomes due to the absence of intracellular origin analysis. Notably, prediabetic mice exhibited higher PDEV particle numbers and total protein concentrations, while average protein content per particle remained comparable between experimental groups. PDEVs proteomic profile unveiled a profound alteration in protein cargo under prediabetic conditions. Tandem mass spectrometry identified 5 proteins exclusively in HFD-PDEV, 112 exclusively in NCD-PDEV, and 223 shared across both groups. Prediabetes led to a decline in the diversity of PDEV protein cargo, suggesting a refined messaging system and more intense given that there are more circulating EVs in the prediabetic condition. Functional analysis of proteins exclusively identified in NCD-PDEV revealed significant enrichments in certain molecular functions, pointing to roles in protein folding, phosphatase activity, and hydrolase activity. The function of a protein is inherently linked to its conformation, and aberrations in protein folding give rise to a spectrum of cellular alterations associated with the etiology of various human diseases. These findings highlight the importance of proper folder in maintaining metabolic homeostasis. Noteworthy, the merely presence of a specific protein in the cell is insufficient for proper function, that is dependent on its post-translational modifications (PTMs). Next, we explored the PTMs within the identified proteins with a focus on acetylation and glycation. Intriguingly, prediabetic conditions correlated with an overall decreased acetylation and glycation events, indicating altered regulatory mechanisms in prediabetes. The gut plays a crucial role in regulating metabolism, and we have previously revealed that gut-derived extracellular vesicles (GDEVs) hold particular significance in the context of diabetes. Earlier we characterized the GDEV’ proteome. In this study, we revisit and expand upon our prior findings by analyzing the PTMs within GDEVs. Prediabetic conditions led to a decrease in both acetylation events, similar to the observations in PDEVs. Understanding the overlap between PDEVs and GDEVs is crucial in elucidating potential biomarkers and mechanisms underlying prediabetes. We identified four proteins exclusively present in the healthy state of both PDEVs and GDEVs, and the term "immune response" emerged as noteworthy. Hinting at immune regulation as pivotal in dysmetabolism. Importantly, their absence in prediabetes suggests their vital role in maintaining metabolic health. Complement Factor H, one of these proteins, has been associated with metabolic disorders. Additionally, overlapping iBAQ data between PDEVs and GDEVs identified four common proteins. Despite their shared presence, marked differences in fold change values between PDEVs and GDEVs precluded direct analogies or comparisons. This comprehensive exploration of proteomic alterations and PTMs in both PDEVs and GDEVs provides valuable insights into the intricate mechanisms underlying prediabetes, shedding light on potential biomarkers and pathways for future research