Objective: To establish INS-1 cell iron overload model, and study the effect on iron overload, proliferation, insulin secretion, mitochondria defect and oxidative stress change.
Methods: INS-1 cells were cultured with different concentrations (0 as control and 5, 10, 20, 40, 80,160,320µmol/L respectively) of ferric ammonium citrate (FAC). Labile iron pool (LIP) were calculated by detecting calcein-AM fluorescence in 24 h, 48 h and 72 h, when cell proliferation was accessed using CCK8. Iron overload model was established by screening for the best combination to ensure both high LIP level and cell proliferation. Reactive oxygen species (ROS) level was further detected by flow cytometer after fluorescent probe staining. The function of insulin secretion was detected by ELISA. The mitochondrial membrane potential was detected by jc-1 kit, and the mitochondrial changes were observed by transmission electron microscopy.
Results: 1. Intracellular LIP level significantly increased in the FAC groups, and was concentration dependent. 2. The INS-1 cell proliferation was suppressed (P<0.05) as FAC concentration or culture time increase in the FAC groups. INS-1 iron overload model was established with conditions of 48 h given its highest LIP level and a cell viability of >50%. 3. With the increase of FAC concentration, the insulin secretion increased and then decreased, and the 160 and 320mol/L groups showed statistical difference compared with the control group (P<0.05). 4. ROS level significantly increased when compared with control (P<0.05). 5. Mitochondrial membrane potential decreased with the increase of iron concentration. The difference was statistically significant (P<0.05). 6. After iron overload, the mitochondria of ins-1 cells were swollen, the internal cristae was expanded, and the normal structure was lost. With the increase of FAC concentration, the mitochondrial structure was destroyed more obviously.
Conclusion: Co-culture with FAC for 48h can successfully establish the iron overload model of ins-1 cells. Iron overload can significantly damage mitochondrial structure and increase intracellular ROS level. The survival of pancreatic beta cells is sensitive to iron, even lower doses of iron can damage beta cells. The insulin secretion can be reduced when the number of beta cells is decreased to a certain extent.
19 - 21 Sep 2019
European Society for Paediatric Endocrinology