曲古霉素调节肝细胞糖异生的机制研究

Mechanisms of trichostatin A in the regulation of hepatocyte gluconeogenesis

目的探讨组蛋白去乙酰化酶（HDAC）抑制剂曲古霉素参与调节肝细胞糖异生的机制。方法体外培养人肝细胞系HL7702，分为对照组（5txlDMSO）、胰岛素组（5ul DMSO＋100nmol／L胰岛素）、曲古霉素联合胰岛素组（2mol／L曲古霉素＋100nmol／L胰岛素）、地塞米松组（1mol／L地塞米松）、曲古霉素联合地塞米松组（2mol／L曲古霉素＋1mol／L地塞米松）。采用体外葡萄糖输出、Western印迹和实时荧光定量RT-PCR的方法检测肝细胞体外葡萄糖输出、胰岛素信号分子蛋白激酶B（Akt）、叉头转录因子01（Fox01）的磷酸化和磷酸烯醇式丙酮酸羧激酶（PEPCK）、葡萄糖-6-磷酸酶（G6Pase）基因的表达。结果与对照组相比，胰岛素组葡萄糖输出降低（t=5．35，P〈0．01），而与胰岛素组相比，曲古霉素联合胰岛素组葡萄糖输出增加（t=-14．049，P〈0．01）；与对照组相比，地塞米松组葡萄糖输出升高（t=-2．782，P〈0．01），与地塞米松组相比，曲古霉素联合地塞米松组葡萄糖输出升高（t=-2．955，P〈0．05）。与对照组相比，胰岛素组磷酸化Akt酪氨酸308、磷酸化Akt丝氨酸473、磷酸化Fox01丝氨酸256蛋白水平均升高（t=-5．356、-5．004、-3．073，P均〈0．05），PEPCK和G6PasemRNA水平均降低（t=5．215、4．777，P均〈0．01）；与胰岛素组相比，曲古霉素联合胰岛素组磷酸化Akt酪氨酸308、磷酸化Akt丝氨酸473、磷酸化Fox01丝氨酸256蛋白水平均降低（t=22．152、26．759、3．907，P均〈0．01），PEPCK和G6PasemRNA水平均升高（t=-3．144、-2．819，P均〈0．05）。结论曲古霉素能够通过抑制Akt和Fox01磷酸化活性，促进糖异生调节基因的表达，进而促进肝细胞葡萄糖输出。

Objective To investigate the mechanisms of histone deacetylase inhibitor （HDAC） in- hibitor trichostatin A （TSA） in the regulation of hepatocyte gluconeogenesis. Methods Human HL7702 cells were cultured and divided into control group（ 5 ~xl DMSO） , insulin group （5 ul DMSO ＋ 100 nmol/L insulin）, TSA combined with insulin group （2 moL/L TSA ＋ 100 nmol/L insulin）, dexamethasone group （ 1 mol/L dexamethasone） and TSA combined with dexamethasone group （2 mol/L TSA ＋ 1 mol/L dexam- ethasone）. In vitro glucose assay, Western blotting and real-time PCR were used to analyze the level of glu- cose production, phosphorylation of protein kinase B （Akt）, forkhead box transcription factor O1 （FoxO1）, gene expression of phosphoenolpyruvate carboxykinase （PEPCK） and glucose-6-phosphatase （G6Pase）. Results Compared with control group, the glucose production was decreased in insulin group （ t = 5.35, P 〈 0.01 ）. Compared with insulin group, the glucose production was increased in TSA combined with insulin group （t = -14. 049, P 〈0.01 ）. Compared with control group, the glucose production was increased in dexamethasone group （t = - 2. 782, P 〈 0. O1 ） , and compared with dexamethasone group, the glucose pro- duction was increased in TSA combined with dexamethasone group （ t = - 2. 955, P 〈 0.05 ）. Compared with control group, the phosphorylation of Akt at T308 and S473 as well as the phosphorylation of FoxO1 at S256 were increased （ t = - 5. 356, - 5. 004 - 3. 073, all P 〈 0.05 ） , but the level of PEPCK mRNA and G6Pase mRNA were decreased in insulin group （ t = 5. 215, 4.777, all P 〈 0.01 ）. Compared with insulin group, the phosphorylation of Akt at T308 and S473 as well as the phosphorylation of FoxO1 at S256 were decreased （t = 22. 152, 26. 759, 3. 907 ,all P 〈 0.01 ）, but the level of PEPCK mRNA and G6Pase mRNA were increased in TSA combined with insulin group （ t = - 3. 144, - 2. 819, all P 〈 0.05 ）. Conclusions TSA stimulates the expression of glueoneogentie related genes through its inhibition of the phosphorylation of Akt and FoxO1, and therefore promotes glucose output of hepatic cells.