41-OR: Insulin Regulates Exosomal MicroRNA Secretion from Adipocytes Completing a Novel Feedback Loop

MicroRNAs (miRNAs) are critical regulators of gene expression. MiRNAs are also secreted by tissues in small extracellular vesicles known as exosomes and can act at a distance. We have shown that adipose tissue is a major source of circulating exosomal miRNAs and that some of these are taken up by distal organs, like the liver, where they regulate key metabolic genes. We have also shown that miRNA loading into exosomes is a tightly regulated process that involves the recognition of 4-8 nucleotide motifs by RNA binding proteins. Thus, exosomal miRNAs have the potential to act as endocrine factors, leading us to ask whether insulin regulates their secretion by adipocytes. To this end, we performed small RNA-seq on exosomes from differentiated 3T3-L1 adipocytes with and without insulin stimulation. Insulin induced the secretion of 52 miRNAs, including multiple members of the let-7 family, the miR-182/183 cluster, as well as miR-103-3p, miR-146b-5p, and miR-148a-3p, all of which have effects to regulate glucose homeostasis and insulin signaling. At the same time, insulin caused 36 miRNAs to be retained. These effects were largely independent of change in the cellular level of these miRNAs but depended on A/U-rich or poly-G motifs. Analysis of phosphoproteomic data of insulin-stimulated 3T3-L1 cells, revealed 26 RNA binding proteins known to bind A/U-rich or poly-G motifs and whose phosphorylation was altered by insulin, including hnRNPA1, FUS and KHSRP, some of which were phosphorylated at residues within their RNA-binding domains. By direct co-precipitation, we showed that hnRNPA1 binds miR-103-3p in an insulin- and motif-dependent manner, such that a 2-nucleotide mutation in the A/U motif of miR-103-3p abolished insulin-induced binding to hnRNPA1 and reduced insulin-induced secretion of miR-103-3p. Thus, insulin can regulate the secretion of exosomal miRNAs from adipocytes, which in turn can regulate metabolism in other tissues, adding a new layer to the endocrine regulation of metabolism.