Proteomic analysis of thermogenic adipocyte-derived extracellular vesicles in mice

Thermogenic brown adipocytes (BAs) are not only an energy sink but also regulate systemic metabolic health through secreted factors and extracellular vehicles (EVs). Here we investigate BA-EVs under both control and cold stimulation. We first established and validated transgenic mouse models expressing human CD63 (hCD63)-GFP for visualization and isolation of EVs exclusively from adipocytes and BAs. We then used super-resolution microscopy to compare the subcellular localization and maturation of EVs in adipocytes grown in culture or directly isolated from mice, revealing distinct features. We further used dynabeads to enrich BA-EVs and employed mass spectrometry to define the EV-proteomes. We discovered that cold-stimulation elevated contents of mitochondrial and lysosomal proteins in BA-EVs. We validated the presence and activity of creatine kinase B (CKB) in BA-EVs. We finally showed that BA-EVs promote thermogenic gene expression in white adipocytes, but cold-stimulation surprisingly blunted EV’s effect in up-regulating Prdm16 expression. These data establish a novel system for analyzing EV contents in vivo and provide insight into how EV proteomes dynamically adapt to non-shivering thermogenesis.