Endocrine Abstracts

Objective: Previously, we identified that knockdown of calcium/calmodulin-dependent serine protein kinase (Cask) in β cells in vitro resulted in decreased insulin secretion. We then used the Cre-loxP system to specifically delete the Cask gene in β cells of mice, and the mice exhibited impaired glucose tolerance and glucose-stimulated insulin secretion under a normal chow diet. However, under a high-fat diet (HFD), the insulin sensitivity and glucose tolerance of KO mice were significantly improved than wild-type. The insulin-stimulated phosphorylation levels of IRS1 and AKT in adipose tissue of HFD-KO mice increased significantly, but there were no significant changes in insulin sensitivity in liver and skeletal muscle. Study shows that β cells can regulate the function of target organs by secreting miRNAs via exosomes. Therefore, this study mainly discussed whether knockdown of Cask in β cells specifically affected the insulin sensitivity of adipocytes by regulating the release of exosomes and their contents.

Methods: Cask-siRNA was used to knockdown the Cask gene in MIN6 cells. The secretion of exosomes was detected by NTA and WB. Fluorescent labeling exosomes released by β cells were co-incubated with mature 3T3-L1 adipocytes for observing the uptake of exosomes. Adipocytes were treated with exosomes from the supernatant of MIN6 cells, and the expression of insulin signaling pathway related proteins was detected. The exosomes were sequenced, and differentially expressed miRNAs were screened and analyzed. 3T3-L1 adipocytes were transfected with miR-15b minics/inhibitors to detect the expression of its target gene INSR and the downstream proteins.

Results: We found that knockdown of Cask in β cells reduced the secretion of exosomes, and adipocytes could absorb exosomes derived from β cells. The number of exosomes absorbed by adipocytes also decreased after Cask deletion. The insulin sensitivity of adipocytes treated with exosomes from Cask-knockdown MIN6 cells was significantly higher than control. In addition, it was found that the content of miR-15b in the exosomes of MIN6 cells decreased after knockdown of Cask, and miR-15b can inhibit the insulin signaling pathway. Overexpression of miR-15b in 3T3-L1 adipocytes reduced the expression of INSR and the insulin signaling pathway was blocked. While inhibiting miR-15b in insulin-resistant adipocytes rescued the damaged insulin signaling.

Conclusion: Cask participates in the release of exosomes in β cells. Knockdown of Cask reduces the release of exosomes, thus reducing the content of miR-15b in them. Adipocytes can absorb the exosomes from β cells, which causes the changes of insulin sensitivity through miR-15b.