Effect of insulin on miRNA abundance in 3T3-L1 adipocytes and their secreted sEVs.

Adipocytes are a major source of secreted small extracellular vesicles (sEVs), which carry various cargo including small non-coding RNAs (smRNAs) and miRNAs to distal cells. Several miRNAs have been implicated in obesity and insulin resistance, and since they function like adipokines, we sought to assess whether insulin can regulate their secretion into sEVs from adipocytes. To this end, 3T3-L1 cells were differentiated using standard procedures, and following a washout period with no insulin, were stimulated with insulin (or PBS for control) for 48 hours to allow for accumulation of sEVs in the media. sEVs were isolated by ultracentrifugation and sEV and cellular RNA was isolated by trizol chloroform and prepared for sequencing by smRNAseq.