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.

miR-450a-5p within rat adipose tissue exosome-like vesicles promotes adipogenic differentiation by targeting WISP2

“…Connolly et al have reported that EVs from adipocytes are enriched in markers of adipogenesis (such as PREF-1, also known as DLK1, and PPARγ). The amount of PREF-1, a negative regulator of adipogenesis, decreased in EVs derived from cells during the later stages of differentiation (Connolly et al, 2015). Other adipogenesis-related proteins were also detected in adipocyte-derived exosomes including adenylate kinase 2 (AK2), and matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) (Lee et al, 2015;Aoki et al, 2007).…”

“…For example, BMP4, secreted by adipocytes, can induce ADSCs to undergo adipogenic commitment (Gustafson et al, 2015a). In addition to secreting soluble proteins, adipose tissue also releases extracellular vesicles (EVs) [including exosomes, small membrane vesicles (30-100 nm) that are released by multivesicular bodies fusing with the cell membrane and microvesicles, 100-1000 nm vesicles originated by shedding of the plasma membrane] (Deng et al, 2009;Connolly et al, 2015;Muller et al, 2011;Fleury et al, 2016). However, the size range of exosomes overlaps partially with that of microvesicles, which hinders a complete size-discrimination between these two populations of EVs .…”

Nitrite-derived nitric oxide reduces hypoxia-inducible factor 1α-mediated extracellular vesicle production by endothelial cells

“…Time-resolved fluorescence was used to assess the surface protein and content of the isolated EVs 223 derived from both normoxia and hypoxia, as described previously [44]. 1x10 9 EVs were loaded onto a 224 high protein binding 96-well plate (Greiner Bio-One, Germany) overnight at 4°C, before non-specific 225 sites were blocked with 1% BSA (R&D Systems) for two hours.…”

Extracellular Vesicles from Hypoxic Adipocytes and Obese Subjects Reduce Insulin‐Stimulated Glucose Uptake

“…Adipocytes have been shown to produce EVs of heterogeneous sizes which contribute to regulate lipid deposition and angiogenesis, reviewed in . Deng et al .…”