Tubacin对β细胞葡萄糖刺激胰岛素分泌的影响

Effect of Tubacin on Glucose-stimulated Insulin Secretion in β Cell

目的:观察去乙酰化酶HDAC6特异性抑制剂Tubacin对MIN6细胞胰岛素分泌的影响,并探讨其可能机制。方法:用RT-PCR检测去乙酰化酶家族成员在MIN6细胞的表达,用免疫荧光技术观察HDAC6在小鼠胰岛和MIN6细胞内的定位;分别在5.6mmol/L和25mmol/L葡萄糖的DMEM中加和不加10μmol/L Tubacin,用其处理MIN6细胞24h,收集上清,ELISA检测胰岛素浓度。同时用MTT法检测Tubacin对MIN6细胞活力的影响,RT-PCR检测上述处理条件下Insulin基因的表达情况。以Western blot和免疫荧光技术检测Tubacin对MIN6细胞骨架蛋白α-tubulin乙酰化水平的影响。结果:MIN6细胞中各乙酰化酶家族成员mRNA的表达水平相差较大,其中HDAC6表达相对较高。HDAC6主要表达于小鼠胰岛β细胞及MIN6细胞胞浆中。在5.6mmol/L和25mmol/L葡萄糖条件下,Tubacin处理24h可以显著抑制胰岛素分泌,但不影响MIN6细胞活力。同时,在5.6mmol/L葡萄糖存在条件下Tubacin不影响胰岛素基因表达,在25mmol/L葡萄糖存在条件下Tubacin可轻度上调胰岛素基因表达。Western blot和免疫荧光结果发现,Tubacin抑制胰岛素分泌的同时伴有α-tubulin乙酰化水平增加。结论:HDAC6抑制剂Tubacin可能通过增加MIN6细胞α-tubulin乙酰化水平,改变细胞骨架的活动度而抑制胰岛素分泌。

Objective To investigate the effects of tubacin, a specific inhibitor of HDAC6,on insulin secretion of 13 cell, and to explore the possible mechanism. Methods The expression profile of HDACs in MIN6 was observe by RT-PCR and the expression of HDAC6 in MIN6 cell and mice islet was observed by immunocytochemistry simueltaneously. After MIN6 cells in 24-cells plates were incubated with tubacin for 24h in the presence of 5.6 and 25 mmol/L glucose, the culture medium was taken for insulin assay with RIA. MTY was used to observe the effect of tubacin on MIN6 cell viability. The expression of insulin gene in MIN6 cultured in the same condition as described above was observe by RT-PCR immunocytochemistry and Western blot technique were used to observe the effect of tubacin on the level of acetylation of α-tubulin. Results The expression levels of different member of HDACs were significantly dif- ferent in MIN6, and HDAC6 was expressed relatively high. Furthermore, HDAC6 was mainly located in cytoplasm of MIN6 cell and in 13 cell of mice islet. IRA showed tubacin inhibited basal and glucose-stimulated insulin secretion. There was no effect of tubacin on MIN6 cell viability showed by MTI＇. The increment of insulin gene＇s expression in MIN6 cell treated with tubacin and 25mmzl/L glucose was observed by RT-PCR. Meanwhile, immunocytochemistry and Western blot technique both presented that the level of acetylation of α-tubulin in MIN6 cell was increased by treatment of tubacin ,which inhibited insulin release as well. Conclusion Tubacin,the specific inhibitor of HDAC6, inhibited glucose-stimulated insulin secretion possibly by decreasing the level of acetylation of α-tubulin, which affected MIN6 cell cytoskeleton dynamics.