The Effect Of Naaso₂ On Insulin Synthesis And Secretion Of Pancreatic Islet β-cells

Object: To investigate the effect of NaAsO2on insulin synthesis and secretion of NIT-1 insulinoma cells (pancreatic isletβ-cells) and discuss the possible mechanism.Methods:NIT-1 cells were cultured with NaAsO2 (0,1,2,4,8,16and32μmol/L respectively) for 24 and 48h, and then the vability of cells were determined by MTT. NIT-1 cells were cultured with NaAsO2 (0,1,4and 8μmol/L respectively) for 24 and 48h, and then glucose stimulated insulin secretion(GSIS) was measured by ELISA, the levels of insulin mRNA expression were detected by RT-PCR method, AnnexinV/PI double labeling FCM was used to determine cell apoptotic rate, Laser sanning confocal microscope (LSCM) was used to examine the variation of reactive oxygen species. NIT-1 cells were cultured with NaAsO2 (0,4and 8μmol/L respectively) for 48h, Fluorescent Imunohistochemistry was used to investigated the distribution of PDX-1 protein. Meanwhile, the expression of p-JNK and PDX-1 protein was measured by Western-blot method.Results:(1) The proliferation of NIT-1 insulinoma cells was inhibited by NaAsO2 in time-and dose-dependent manner as the concentration of NaAsO2 was higher than 4μmol/L.(2) GSIS and insulin mRNA expression of NIT-1 insulinoma cells were evaluated after incubated with 0,1,4 and 8(μmol/L NaAsO2 for 24h and 48h:It showed a significant decrease in GSIS and insulin mRNA expression of cells exposed to 8μmol/L NaAsO2 during 24h and 4 or 8μmol/L NaAsO2 during 48h respectively. On the other hand, insulin secretion of NIT-1 cells were no longer able to distinguish between different glucose concentrations by 8μmol/L treatment for 48h. (3)Total apoptosis rate of cells was increased according to enhancement of concentration and action time of NaAsO2 was higher than 4μmol/L. (4)The level of ROS of each experiment group was increased by NaAsO2 in time-and dose-dependent manner. (5) NaAsO2 of each experiment group lead to an decrease in PDX-1 protein translocation to the nuclei of NIT-1 cells. (4) The expression of p-JNK of each experiment groups is higher than control and expression of PDX-1 protein was decreased.Conclusion:Sodium arsenite can exert inhibitory effects on insulin secretion and insulin mRNA expression ofβ-cells in time-and dose-dependent manner. Its possible mechanism:arsenic toxicity can lead to oxidative stress since it can stimulate production of reactive oxygen species (ROS). Accordingly, ROS activated the JNK signal pathway that can increase cell apoptosis. Meanwhile, the activated JNK signal pathway can lead to an decrease in PDX-1 expression and translocation of the protein to the nuclei ofβ-cells.Our data suggest that arsenic may contribute to the development of diabetes mellitus by impairing pancreaticβ-cell functions, particularly insulin synthesis and secretion.