Proteomic and Super‐Resolution Analyses of Murine Brown Adipocyte‐Derived Extracellular Vesicles

Thermogenic brown adipocytes (BAs) are not only an energy sink but also regulate systemic metabolic health through secreted factors and extracellular vesicles (EVs). Here, we investigate the baseline profiles BA‐EVs and how cold‐induced thermogenesis alters the proteome of BA‐EVs. We first established and validated transgenic mouse models expressing human CD63 (hCD63)‐GFP for visualisation and isolation of EVs exclusively from adipocytes and BAs. We then used super‐resolution microscopy to compare the subcellular localisation and maturation of EVs in adipocytes grown in culture or directly isolated from mice, revealing distinct features. We then used immunoprecipitation to enrich BA‐EVs and employed mass spectrometry to define the EV‐proteomes. We discovered that cold stimulation elevated secretion 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 analysing EV contents in vivo and provide insight into how EV proteomes dynamically adapt to non‐shivering thermogenesis.