Establishment of pancreatic β cell iron overload model and β cell injury induced by iron overload

AIM: To establish rat insulinoma INS-1 cell iron overload model, and to study the effect of iron overload on the viability, insulin secretion, mitochondrial defect and oxidative stress change in the INS-1 cells. METHODS:INS-1 cells were cultured with ferric ammonium citrate (FAC) at different concentrations (0, 5, 10, 20, 40, 80, 160 and 320 μmol/L). Labile iron pool (LIP) were calculated by detecting calcein-AM fluorescence in 24 h, 48 h and 72 h. The cell viability was measured by CCK8 assay. Iron overload model was established by screening for the best combination to ensure both high LIP level and cell viability. Reactive oxygen species (ROS) level was further analyzed by flow cytometry after fluorescent probe staining. The function of insulin secretion was measured by ELISA. The mitochondrial membrane potential was detected by JC-1 kit, and the mitochondrial changes were observed by transmission electron microscopy. RESULTS: Intracellular LIP levels were significantly increased in FAC groups in a concentration-dependent manner (P<0.05). The viability of INS-1 cells was suppressed with increasing FAC concentration or culture time (P<0.05). The highest LIP level and cell viability over 50% were observed with the condition of exposure to FAC for 48 h, indicating that INS-1 cell iron overload model was established. With the increase in the FAC concentrations, the insulin secretion was also increased and then decreased, and that in 160 and 320 mol/L groups showed statistical difference compared with control group (P<0.05). The ROS level was significantly increased by FAC exposure as compared with control group (P<0.05). Mitochondrial membrane potential was decreased with the increase in the iron concentration (P<0.05). After iron overload, the mitochondria of INS-1 cells were swollen, the internal cristae were expanded, and the normal structure was lost. With the increase in the FAC concentration, the mitochondrial structure was destroyed more obviously. CONCLUSION: Co-culture of INS-1 cells with FAC for 48 h successfully establish the iron overload model. Iron overload significantly damages mitochondrial structure and increases intracellular ROS level. The viability of pancreatic β cells is sensitive to iron, even lower doses of iron can damage β cells. The insulin secretion is reduced when the number of β cells is decreased to a certain extent.