Roles Of Endoplasmic Reticulum Stress, Autophagy, And MiR-375 In Apoptosis Of Pancreatic β Cells Induced By Hyperglycemia And Hyperlipidemia

Objective:To investigate the roles of ERS, autophagy, and miR-375 in the apoptosis of RINm5 F cells under high-glucose and high-fat conditions. Methods:RINm5F cells were cultured in vitro. The concentrations of 5.5mM, 16.7mM, 33.3 mM of glucose and 0mM, 0.25 mM and 0.5mM of palmitate acid were used to prepare culture medium. The RINm5 F cells were cultured under these conditions for 24 h, and under one specific high glycolipid concentration,the cells was cultured with liraglutide(400 nM). Annexin-V/PI double staining was used to detect cell apoptosis. The Cell Counting Kit-8 was used to measure the proliferative activity of cells. Quantitative real-time PCR was performed to measure the expression levels of ERS-related genes and miR-375, and Western blot was used to measure the expression levels of ERS-related proteins and autophagy-related proteins. Results:1. The test results of AnnexinV/PI and CCK-8 indicated that palmitate acid could restrain the proliferation and promote the apoptosis of RINm5 F cells, and the degree of its impact was related to its concentration. The glucose of high concentration literally had no significant impact on proliferation.2. The expression of CHOP mRNA and XBP-1 mRNA was positively related to the concentration of palmitic acid(P<0.05)in 5.5mM、16.7mM and 33.3 mM GLU groups, and under the same palmitate acid condition, compared with 5.5mM GLU group, CHOP mRNA expression increased in 33.3m M GLU group(P<0.05).3. The expression of Caspase-3、CHOP、LC3-II and p62 protein was tested by Western Blot. The results of above tests indicated that at the same level of glucose concentration, the expression of Caspase-3、CHOP 、LC3-II in RINm5 F cells in palmitic acid of high concentration was higher than that of lower concentration, while the expression of p62 showed the opposite trend. The expression of all above indicators showed the dependency on concentration. In addition, glucose could amplify the effect of palmitate acid(P<0.05).4. The expression of miR-375 was also tested by QT-PCR. Under the 5.5mM glucose condition, miR-375 expressed more with the palmitate acid concentration raising. Moreover, the level of expression was much lower in 16.7mM and 33.3mM GLU groups than that in 5.5mM GLU group(P<0.05).5. Under the specific high glicolipid concentration, the cells cultured with liraglutide(400 nM) showed higher proliferation, less apoptosis. The expression of CHOP mRNA, XBP-1 mRNA,miR-375 and the protein Caspase-3,CHOP also decreased with the intervention; while protein LC3-II expressed more. There was no significant change of p62 between the groups with or without liraglutide intervention. Conclusion:1. Palmitic acid-mediated lipotoxicity can inhibit the proliferation of RINm5 F pancreatic β cells and promote their apoptosis.2. Under high-glucose and high-fat conditions, the endoplasmic reticulum stress and unfolded protein response in pancreatic β cells were promoted.3. The autophagy of RINm5 F cells was enhanced under high-glucose and high-fat conditions.4. Glucose could inhibit miR-375 expressing, while palmitate acid could promote miR-375 expression.5. Liraglutide protects pancreatic β cells under high-glucose and high-fat condition through reducing endoplasmic reticulum stress, controlling autophagy, and downregulating miR-375 expression.