Characterization of Adipocyte-Derived Extracellular Vesicle Secretion Using a CD63-GFP Reporter Mouse Model <em>In Vivo</em> and <em>In Vitro</em>

Adipocytes are increasingly recognized as active endocrine cells that secrete substantial quantities of EVs enriched in proteins, lipids, and nucleic acids. However, the mechanisms governing EV secretion and the dynamics of EV release in different biological contexts are not fully understood. To address this gap, we generated a novel adipocyte-specific CD63-GFP reporter mouse line, in which expression of GFP-tagged CD63 is driven by the Adiponectin promoter, enabling selective labeling and tracking of AdEVs in vivo. Using this reporter model, we examined secretion of AdEVs and their delivery to APCs within adipose tissues. Confocal imaging and flow cytometry revealed that AdEVs are actively taken up by APCs, highlighting a previously underappreciated mechanism of paracrine communication within the adipose microenvironment. To complement the in vivo studies, an in vitro system was established, allowing for the direct monitoring of EV secretion from differentiated adipocytes. APCs were isolated from subcutaneous white adipose tissues of the reporter mice and induced to differentiate into mature adipocytes. In these cells, CD63-GFP signals were observed as discrete puncta and became enriched at the plasma membrane when EV secretion was inhibited, consistent with vesicle accumulation upon blocked release. Under normal growth conditions, EVs secreted into the culture medium were concentrated and subsequently purified using size exclusion chromatography, enabling downstream functional and biochemical analyses. This platform can be applied to APCs derived from other adipose depots, such as epididymal white and brown adipose tissue, or by differentiating them into depot-specific adipocytes, thereby enabling comparisons of EV secretion among different adipocyte types. Together, these approaches introduce a robust genetic tool and a complementary in vitro system to dissect the dynamics of AdEV secretion and targeting. These advances shed light on intra-adipose cellular communication and lay the groundwork for understanding the broader metabolic influence of AdEVs in health and disease.