Dissecting The Effect Of Kdm2a On Pancreatic β Cells And The Underlying Mechanisms

Objective: To investigate the effect of Kdm2 a on pancreatic β cells and the underlying mechanism.Methods: 1.An NOD mouse-derived pancreatic beta cell line(NIT-1 cells)was cultured and stimulated by STZ or cytokines(TNF-α,IL-1β,IFN-γ)or PA.Western blot analysis was performed to detect the expression level of Kdm2 a.2.Establishment of beta cell-specific KDM2A-deficient model: The Kdm2af/f mice were crossed with Cre mice to generate the Kdm2af/f-Ins1-Cre ERT2 mice,which were intraperitoneally injected with tamoxifen for 5 consecutive days to generate beta cell-specific Kdm2 a KO mice.Kdm2af/f mice treated with tamoxifen were served as controls.Lysates from islets,liver,hypothalamus and skeletal muscle of KO and CON mice were used for western blot analysis to detect the expression level of Kdm2 a.3.Blood glucose levels were monitored in KO and CON mice every other day.Intraperitoneal glucose and insulin tolerance tests were undertaken at day 28 after tamoxifen treatment.At day 49,insulin concentrations of mice blood serum and GSIS experiment were analyzed using a Mouse Insulin ELISA kit.H&E and IF staining of pancreatic sections were performed for the quantifications of islet area and insulin content.4.Another group of mice was bred and treated with tamoxifen.At day 7 after treatment,the mice were stressed by multiple low doses of STZ for 5 consecutive days.Blood glucose levels were monitored and the mice were considered to have developed diabetes once non-fasting blood glucose levels exceeded 13.9 mmol/L for 2 consecutive days.At day 28 after the STZ treatment,incidence of diabetes was analyzed.Then the mice were sacrificed and blood insulin concentration was analyzed using an ELISA kit.H&E and IF staining of pancreatic sections were performed for the quantifications of islet area and insulin content.5.NIT-1 cells were cultured in DMEM low glucose medium and were transfected with Kdm2 a siRNA or Scramble siRNA using Lipofectamine 3000.Two days after transfection,the cells were stimulated with cytokines(TNF-α/IL-1β/IFN-γ)for 24 hours.Then the cells were stained with AnnexinⅤand PI.The proportions of apoptotic cells were analyzed by flow cytometry.Results: 1.The expression level of Kdm2 a in NIT-1 cells displayed an obvious increase after stimulation with STZ or cytokines,whereas the expression level of Kdm2 a showed a decrease after PA stimulation.2.Western blot analysis of islet lysates indicated a significant reduction in Kdm2 a protein expression in the KO mice compared with CON mice.Additionally,western blot analysis of other tissue lysates failed to detect obvious differences in terms of Kdm2 a level between KO and CON mice.3.At day 21 after tamoxifen treatment,there was a progressive increase of blood glucose levels in KO mice compared with CON group.Moreover,the KO mice displayed an abnormal glucose tolerance and a normal insulin sensitivity.At day 49,the KO mice showed a decrease in serum insulin level.There was also a decrease in GSIS in islets isolated from KO mice.Additionally,there was no obvious change in islet area,but KO mice displayed a significant decline of insulin content in islets.4.After 5 days of STZ treatment,there was a progressive increase of blood glucose level in both 2 groups of mice,whereas the change in blood glucose level was much higher in KO mice.By day 28 after STZ treatment,11 out of 12 KO mice became diabetic,but only 7 out of 13 CON mice became diabetic.There was also a decrease of serum insulin level in KO mice.Furthermore,KO mice displayed a significant decline both in islet area and insulin content of islets.5.Compared to the control group,the apoptosis rate was increased in Kdm2a-depleted NIT-1 cells stimulated with cytokines.Conclusion: The tamoxifen-treated Kdm2 a KO mice showed a deficient insulin secretion in beta cells and were more susceptible to the diabetes induced by STZ.