17 beta-estradiol protects INS-1 insulinoma cells from mitophagy via G protein-coupled estrogen receptors and the PI3K/Akt signaling pathway

摘要

17 beta-estradiol (17-E2) is a steroid hormone that is known to exert effects on blood glucose homeostasis. The G protein-coupled estrogen receptor (GPER) has been identified as a non-genomic estrogenic receptor, and is involved in numerous physiological processes, including cell survival, energy provision and metabolism. 17-E2 may decrease apoptosis by binding to the GPER. The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is involved in physiological and pathological functions such as autophagy. The purpose of the present study was to investigate the role of the PI3K/Akt signaling pathway in the mediation of the effects of GPERs, and the effects of 17-E2 on mitophagy in INS-1 cells, a rat insulin-secreting -cell line. In vitro, INS-1 cells were treated with different concentrations of 17-E2 with and without pretreatment with a GPER antagonist (G15) or PI3K antagonist (LY294002) and compared with a negative control. An immunofluorescence assay demonstrated that GPERs are expressed in INS-1 cells. Western blot assays demonstrated that 17-E2 increased GPER levels and the phosphorylation of Akt. Transmission electronic microscopy revealed that 17-E2 reduced the formation of mitophagosomes and autophagosomes in INS-1 cells. An immunofluorescence staining assay indicated that the co-localization of translocase of mitochondrial outer membrane complex 20 (TOM20) with lysosomal-associated membrane protein 2 (LAMP2) was decreased in INS-1 cells treated with 17-E2 alone. Western blotting demonstrated that 17-E2 reduced the protein levels of activated microtubule-associated protein-1 light chain 3, and increased those of TOM20 and mitochondrial heat-shock protein 60. Notably, the protective effects of 17-E2 were significantly diminished by G15 or LY294002. In conclusion, the present study suggests that 17-E2 activates the PI3K/Akt pathway via the GPER in INS-1 cells. Furthermore, 17-E2 may be involved in mitophagy by the regulating the GPER/PI3K/Akt pathway.