title: Characterisation of adipocyte-derived extracellular vesicles released pre- and post-adipogenesis.
abstract: Extracellular vesicles (EVs) are submicron vesicles released from many cell types, including adipocytes. EVs are implicated in the pathogenesis of obesity-driven cardiovascular disease, although the characteristics of adipocyte-derived EVs are not well described. We sought to define the characteristics of adipocyte-derived EVs before and after adipogenesis, hypothesising that adipogenesis would affect EV structure, molecular composition and function. Using 3T3-L1 cells, EVs were harvested at day 0 and day 15 of differentiation. EV and cell preparations were visualised by electron microscopy and EVs quantified by nanoparticle tracking analysis (NTA). EVs were then assessed for annexin V positivity using flow cytometry; lipid and phospholipid composition using 2D thin layer chromatography and gas chromatography; and vesicular protein content by an immuno-phenotyping assay. Pre-adipogenic cells are connected via a network of protrusions and EVs at both time points display classic EV morphology. EV concentration is elevated prior to adipogenesis, particularly in exosomes and small microvesicles. Parent cells contain higher proportions of phosphatidylserine (PS) and show higher annexin V binding. Both cells and EVs contain an increased proportion of arachidonic acid at day 0. PREF-1 was increased at day 0 whilst adiponectin was higher at day 15 indicating EV protein content reflects the stage of adipogenesis of the cell. Our data suggest that EV production is higher in cells before adipogenesis, particularly in vesicles <300 nm. Cells at this time point possess a greater proportion of PS (required for EV generation) whilst corresponding EVs are enriched in signalling fatty acids, such as arachidonic acid, and markers of adipogenesis, such as PREF-1 and PPARγ.
authors.affiliation: Institute of Molecular and Experimental Medicine, School of Medicine, Cardiff University, Cardiff, United Kingdom.
authors.forename: Katherine D
authors.initials: KD
authors.lastname: Connolly
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, United Kingdom.
authors.forename: Irina A
authors.initials: IA
authors.lastname: Guschina
authors.affiliation: Institute of Cancer Genetics, School of Medicine, Velindre Cancer Centre, Cardiff University, Cardiff, United Kingdom.
authors.forename: Vincent
authors.initials: V
authors.lastname: Yeung
authors.affiliation: Institute of Cancer Genetics, School of Medicine, Velindre Cancer Centre, Cardiff University, Cardiff, United Kingdom.
authors.forename: Aled
authors.initials: A
authors.lastname: Clayton
authors.affiliation: Institute of Molecular and Experimental Medicine, School of Medicine, Cardiff University, Cardiff, United Kingdom.
authors.forename: Mohd Shazli
authors.initials: MS
authors.lastname: Draman
authors.affiliation: Central Biotechnology Services, School of Medicine, Cardiff University, Cardiff, United Kingdom.
authors.forename: Christopher
authors.initials: C
authors.lastname: Von Ruhland
authors.affiliation: Institute of Molecular and Experimental Medicine, School of Medicine, Cardiff University, Cardiff, United Kingdom.
authors.forename: Marian
authors.initials: M
authors.lastname: Ludgate
authors.affiliation: School of Health Sciences, Cardiff Metropolitan University, Cardiff, United Kingdom.
authors.forename: Philip E
authors.initials: PE
authors.lastname: James
authors.affiliation: Institute of Molecular and Experimental Medicine, School of Medicine, Cardiff University, Cardiff, United Kingdom; reesda@cardiff.ac.uk.
authors.forename: D Aled
authors.initials: DA
authors.lastname: Rees
keywords: 3T3-L1; adipocytes; adipogenesis; microparticles; vesicles