The cholesterol oxidation product 7-ketocholesterol impairs pancreatic beta cell insulin secretion

Background: The impairment of pancreatic beta cell function caused by glucolipotoxicity plays an important role in the pathogenesis of type 2 diabetes. Previous studies have shown that cholesterol can induce beta cell dysfunction. However, the effect of the cholesterol oxidation product 7-ketocholesterol in beta-cell function remains unclear. Methods: Cell proliferation, Glucose-stimulated insulin secretion (GSIS), perifusion, calcium imaging, total internal reflection fluorescence microscopy (TIRFM), reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, qPCR, and Western blotting were used to evaluate the effect and mechanism of 7-ketocholesterol on INS1 cells and islets. N-Acetyl-L-cysteine was used to rescue insulin secretion of beta-cells. GSIS, perifusion, calcium levels and exocytosis events verified that early-phase insulin secretion was impaired after 7-ketocholesterol treatment. Results: The results of CCK 8 and GSIS demonstrated that 25 μmol/L 7-ketocholesterol significantly decreased insulin secretion in the INS1 cells ( P < 0.05), as did 50 μmol/L 7-ketocholesterol in the primary islets ( P < 0.05). The islet perifusion analysis verified that the insulin secretion function was impaired with 7-ketocholesterol( P < 0.001). Calcium imaging showed that the intracellular calcium levels were decreased following 7-ketocholesterol treatment( P <0.001). TIRFM imaging inferred that 7-ketocholesterol could reduced insulin-secretory-granule exocytosis by decreased fusion events and increased kiss-and-run events to the membrane to attenuate insulin secretion ( P < 0.01). Further data showed that the level of Snap25 gene and protein expression related to insulin exocytosis was substantially downregulated. Further study showed that the reactive oxygen species (ROS) in INS1 cells was upregulated, and both the mitochondrial membrane potential (MMP) and level of adenosine triphosphate (ATP) was downregulated ( P < 0.05). The regulation of nuclear factor erythroid 2-related factor (NRF2) is an important transcription factor for oxidative stress, for which its nuclear translocation results in the subsequent activation of gene transcription of Gpx4 , Sod1 , Txnip , Nqo1 , and Ho1 in INS1 cells. In addition, 7-ketocholesterol-induced pancreatic beta cell dysfunction and oxidative stress was ameliorated by pretreatment with the antioxidant, N-Acetyl-L-cysteine. Conclusions: These findings suggested that 7-ketocholesterol impacted insulin exocytosis to decrease the insulin secretion of pancreatic beta cells involved in the oxidative stress.