肝脏17-α羟化酶在糖异生中的作用及机制研究

Role and mechanism of hepatic Cyp17A1 gene in regulation of hepatic gluconeogenesis

目的:探讨肝脏17-α羟化酶(Cyp17A1)在糖异生中的作用。方法:采用实时定量PCR和蛋白印迹法检测正常C57BL/6小鼠在进食-饥饿-再进食状态下的肝脏糖异生关键酶[磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase,PEPCK)、葡萄糖-6-磷酸酶(glucose-6-phosphatase,G6Pase)]基因及Cyp17A1的表达,比较正常小鼠与瘦素受体缺陷(db/db)小鼠肝脏中上述基因的表达差异;给予正常小鼠腹腔注射Cyp17A1下游产物17-羟孕酮(17-hydroxyprogesterone,17-OHP),并检测小鼠糖异生能力的改变和糖异生相关基因的表达;采用荧光素酶报告基因实验,检测17-OHP对糖皮质激素受体转录活性的调控作用。结果:在正常小鼠饥饿状态下及db/db小鼠中,肝脏糖异生关键酶基因(PEPCK及G6Pase)表达上调,肝脏Cyp17A1表达上调,其下游产物17-OHP含量升高;17-OHP处理组小鼠血糖升高,肝脏PEPCK及G6Pase mRNA水平显著增加;17-OHP通过激活糖皮质激素受体的转录活性,呈剂量依赖性上调糖异生关键酶基因(PEPCK、G6Pase)的mRNA水平。结论:肝脏Cyp17A1酶通过其下游产物17-OHP激活糖皮质激素受体转录活性,上调糖异生关键酶基因的m RNA水平,从而促进糖异生过程。

Objective： To investigate the role of hepatic Cyp17A1 in regulation of gluconeogenesis. Methods： Expres- sion levels of gluconeogenesis enzymes （phosphoenolpyruvate carboxykinase, PEPCK; glucose-6-phosphatase, G6Pase） and Cypl7A1 genes in both C57BL/6 and db/db mice were determined by real-time PCR and Western blotting. C57BL/6 mice were intraperitoneally administrated with 17-OHP, the downstream product of Cypl7A1 enzyme, and then the he- patic gluconeogenesis was measured by pyruvate tolerance test and real-time PCR. Luciferase reporter assay was per- formed to evaluate the role of 17-OHP in regulation of transcriptional activity of glucocorticoid receptor （GR）. Results： Expression levels of PEPCK, G6Pase and Cypl7A1 were increased in fasting C57BL/6 mice and db/db mice. Besides, the administration of 17-OHP elevated blood glucose level and upregulated transcriptional level of PEPCK and G6Pase. Luciferase reporter assay demonstrated that 17-OHP promoted the transcriptional activity of GR and upregulated the transcriptional level of PEPCK and G6Pase. Conclusions： Hepatic Cypl7A1 promotes hepatic gluconeogenesis by acti- vating the transcriptional activity of glucocorticoid receptor through its downstream product 17-OHP.