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

Summary

The protocol uses an adipocyte-specific CD63-GFP reporter mouse line to visualize and quantify the secretion of adipocyte-derived extracellular vesicles (EVs) and demonstrate their uptake by progenitor cells, revealing a paracrine pathway within adipose tissue.

Abstract

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.

Introduction

Adipose tissue is increasingly recognized as a dynamic endocrine organ with key roles in whole-body energy homeostasis^1,2,3,4,5. Beyond its classical function as a lipid storage depot, adipocytes regulate systemic insulin sensitivity, and disturbances in adipose mass-whether excess or deficiency-are linked to insulin resistance and metabolic disease^6,7,8,9. One mechanism proposed ....

Protocol

In accordance with the animal care and use guidelines of Albert Einstein College of Medicine, male mice were group-housed at 22 °C with 30-70% relative humidity on a 12 h light/12 h dark cycle, provided ad libitum access to food and water, and maintained on a C57BL/6J background.

1. In vivo detection of secreted AdEVs through uptake by APCs

1. Buffer preparation
   1. Digestion buffer: Prepare HEPES-buffered DMEM by adding 0.5 mL of 1 M HEPES and 0.5 g fatty acid-poor bovine serum albumin (BSA) to 50 mL of DMEM. Mix until dissolved.
   2. Wash buffer: Prepare PBS with 2.5 mM ethyle....

Results

Using this protocol of AdEVs detection through APC uptake by confocal microscopy, we isolated APCs from the inguinal adipose tissues of AdipCD63-GFP and Stop^fl/fl/CD63-GFP mice and stained them with DAPI for visualization of the nuclei. When examined by confocal microscopy, APCs from Stop^fl/fl/CD63-GFP mice showed no detectable GFP puncta (Figure 1A). In contrast, APCs isolated from AdipCD63-GFP mice exhibited numerous distinct GFP puncta throughout the cytoplasm (

Discussion

The described protocol fills an important methodological gap by providing a robust and reproducible system to isolate and characterize AdEVs and their uptake by APCs. Compared to EVs from other sources, AdEVs are more lipid-rich and buoyant, making the classical ultracentrifugation-based EV isolation techniques less efficient³⁵. However, most published protocols for AdEV purification still depend on ultracentrifugation, which can affect vesicle morphology, lead to EV aggregates, and cause protein .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5 M EDTA, pH 8.0	Fisher	AM9260G	500x dilution
10x PBS	Fisher Scientific	70-013-032	1000x dilution with water
1 M HEPES	Gibco	15630080	1000x dilution
Adipose Tissue Progenitor Isolation Kit, mouse	Miltenyi biotec	30-106-639	Ready for use
Amicon Ultra Centrifugal Filter, 100 kDa MWCO	Sigma	UFC9100	15 mL sample volume
Amicon Ultra Centrifugal Filter, 100 kDa MWCO	Sigma	UFC9100	4 mL sample volume
BSA	Sigma	A6003	Ready for use
Calnexin antibody	calnexin	ab22595	1000x dilution
CD63 antibody	abcam	ab217345	500x dilution
CD81	cell signaling Technology	10037s	500x dilution
Corning BioCoat Collagen I Culture Slide	Sigma	CLS354630	Ready for use
Corning cell strainer	Sigma	CLS431750	40 μm pore size
Corning cell strainer	Sigma	CLS431752	100 μm pore size
COX IV antibody	Cell Signaling Technology	4844	1000x dilution
DMEM	Gibco	11965092	Ready for use
DMEM/F-12, GlutaMAX supplement	Thermofisher	10565018	Ready for use
Dnase 1	Sigma	D4513	Stock 5 KU/mL at -20 °C
GlutaMAX Supplement	Gibco	35050061	100x dilution
Halt™ Protease and Phosphatase Inhibitor Cocktail (100X)	Thermofisher	78444	Ready for use
HSP70 antibody	abcam	ab181606	1000x dilution
Invitrogen eBioscience 10X RBC Lysis Buffer (Multi-species)	Fisher Scientific	50-112-9743	10x dilution in water
Izon Science Usa Ltd QEVORIGINAL 70NMGEN2 5/PK SDP	Fisher Scientific	NC2042225	Ready for use
LEICA STELLARIS 8 confocal microscope	Leica Microscopes	N/A
Liberase TM Research Grade	Sigma	LIBTM-RO	Stock 5 mg/mL at -20 °C
Millex MCE syringe filter	Sigma	SLAA025	0.8 μm pore size
NanoSight Pro	Malvern Panalytical	N/A
Novex Tris-Glycine Mini Protein Gels, 4–12%,	Thermofisher	XP04120BOX	Ready for use
Novex Tris-Glycine SDS Sample Buffer (2x)	Thermofisher	LC2676	Ready for use
Pacific Blue anti-mouse Ly-6A/E (Sca-1) Antibody	BioLegend	108119	25x dilution
Paraformaldehyde solution (16%)	Fisher Scientific	50-980-487	dilute to 4% with PBS
pluriStrainer S / 30 µm (Cell Strainer)	Fisher Scientific	NC0922459	30 μm pore size
Ponceau S solution	Sigma	P7170-1L	Ready for use
Primocin	Invivogen	NC9141851	500x dilution
ProLong Gold Antifade Reagent with DAPI	Cell Signaling Technology	8961S	Ready for use
Purified anti-mouse CD16/32 Antibody	BioLegend	101301	100x dilution
RIPA buffer	Fisher Scientific	PI89900	10x dilution in water
Spectral flow cytometer (Aurora 3)	Cytek Biosciences	N/A
Syntaxin 4 antibody	Synaptic Systems	110042	5000x dilution
Tecnai 20 (Transmission Electron Microscope)	FEI	N/A
Tergeo-EM	PIE Scientific LLC	N/A
Vinculin antibody	abcam	ab18508	5000x dilution