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Title: Study of extracellular vesicles secreted by experimental models of metabolic syndrome, and their effects on adipocytes
Author: Mleczko, J. E.
ISNI:       0000 0004 7428 4309
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Extracellular vesicles (EVs) is a collective name given to nanometer-sized membrane vesicles (30-200 nm) secreted by all cells and found in most body fluids. They originate either from endosomal multivesicular bodies, which by fusion with the plasma membrane are released into the extracellular environment as exosomes, or directly through the budding off the plasma membrane as microvesicles. They were first described to take part in the removal of unwanted plasma membrane proteins during the maturation of reticulocytes to erythrocytes, however, following the decades of research they are now recognized as being important mediators of cell-to-cell communication and vehicles to transfer active biological material. They contain lipids, proteins and different species of nucleic acids. Remarkably, their composition has been shown to be altered in pathological conditions, and although this feature has been widely explored to find low-invasive biomarkers, the physiological effects of such content alteration is greatly unknown. Obesity is reaching epidemic proportions worldwide mostly due to the growing incidence of metabolic syndrome (MetS), which is a cluster of metabolic abnormalities such as obesity, insulin resistance, dyslipidemia and hypertension that when occurring together increase the risk of cardiovascular complications and type 2 diabetes mellitus. In the current study, we focused on investigating the nature and the effect of different metabolic alterations observed in MetS development, such as the adipose hypertrophy, hepatic steatosis, inflammation and adipose tissue hypoxia, on the secretion and content of EVs. Furthermore, the work also addressed the effects of these EVs on the biology and metabolism of adipocytes. To achieve our aim, we established in vitro cellular models, that resemble the mentioned metabolic conditions and that are suitable for subsequent production and isolation of EVs. We performed detailed biochemical and structural characterization of EVs isolated from conditioned medium of hypertrophic adipocytes, adipocytes cultured in hypoxia or macrophage- conditioned medium. In addition, we also analyse EVs from chemically-induced steatosis in hepatic cell lines and steatotic primary hepatocytes obtained from Zucker rat hepatocytes that represent a genetic model of obesity. We used cryo-EM technology to visualize the isolated EVs in their native state, and Nanoparticle Tracking Analysis to measure their concentration and size distribution. Furthermore, by Western blot analysis, we identified the abundance of EV-related and cell type-specific proteins, which could be affected by metabolic perturbation of the parental cell. In the case of Zucker hepatocytederived EVs, we performed further analysis of their protein, RNA and lipid content. Our study demonstrates that the profile of EVs secreted by all our in vitro models of metabolic syndrome is highly affected in terms of the concentration and protein abundance. We found that steatotic hepatocytes secrete EVs with significantly reduced exosomal-markers such as Aip1/Alix, CD63, CD81, Tsg101 and Flotillin1, and increased abundane in Hsp70 and 90. Furthermore, a proteomic analysis of EVs released by steatotic Zucker hepatocytes revealed a very specific protein signature, which reflected the metabolic state of the cell. The majority of proteins upregulated were related with the fat metabolism, fatty acid synthesis, glycolysis and pentose phosphate pathway, in turn, the downregulated ones were mainly mitochondrial. Interestingly, the abundance of diverse enzymes involved in xenobiotics metabolism was significantly downregulated in EVs from the steatotic model. Finally, we show that these EVs influence insulin sensitivity in recipient 3T3-L1 adipocytes. Moreover, we found the proinflammatory cytokine Tnf to be associated with EVs secreted by LPS-stimulated Raw264.7 macrophage-like cells. Interestingly, they were able to significantly reduce the mitochondrial functions in 3T3-L1 adipocytes. In conclusion, our research supports the hypothesis that EVs play an important role in the development of pathologies associated with MetS.
Supervisor: Mora, Silvia ; Varro, Andrea ; Falcon-Perez, Juan Manuel Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral