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目的:探讨环磷腺苷效应元件结合蛋白(CREB)辅激活物-活性调节2传感器(TORC2)在脂联素抑制肝糖异生过程中的作用及机制。方法:实验室前期已构建的人脂联素真核表达质粒,并成功转染人肝细胞系L-02细胞。在此基础上采用蛋白免疫印迹试验、免疫组织化学等方法,对比不同葡萄糖浓度处理后的脂联素转染组和转染空质粒对照组L-02细胞中TORC2细胞内定位,以及磷酸化腺苷酸活化蛋白激酶(p-AMPK)、CREB、p-CREB蛋白表达差异,同时监测各组葡萄糖-6-磷酸酶(G-6-Pase)活性。结果:与对照组相比,转染组p-AMPK蛋白表达量显著增高(P<0.05),CREB、p-CREB蛋白表达量差异无统计学意义(P>0.05)。定位分析显示转染组TORC2主要位于细胞质,而对照组TORC2主要位于细胞核内。葡萄糖关键酶检测发现转染组G-6-Pase活性显著低于对照组(P<0.05)。结论:脂联素抑制糖异生的机制可能与活化p-AMPK、抑制TORC2去磷酸化、阻止TORC2进入细胞核以及抑制糖异生关键酶基因表达有关。
AIM: To investigate the role and mechanism of adiponectin inhibitor glucagon-responsive element-binding protein (CREB) coactivator-activator-2 sensor (TORC2) in inhibiting hepatic gluconeogenesis. Methods: The human adiponectin eukaryotic expression plasmid was constructed in the laboratory and successfully transfected into human hepatocyte cell line L-02. On this basis, Western blotting and immunohistochemistry were used to compare the intracellular localization of TORC2 in adiponectin-transfected L-02 cells transfected with different concentrations of glucose and phosphorylated adenovirus The expressions of p-AMPK, CREB and p-CREB were detected by enzyme-linked immunosorbent assay (ELISA). The activity of G-6-Pase in each group was also monitored. Results: Compared with the control group, the expression of p-AMPK protein in the transfected group was significantly increased (P <0.05), and the expression of CREB and p-CREB protein was no significant difference (P> 0.05). Positioning analysis showed that transfection group TORC2 mainly in the cytoplasm, while the control group TORC2 mainly located in the nucleus. G-6-Pase activity in transfection group was significantly lower than that in control group (P <0.05). CONCLUSION: The mechanism of gluconeogenesis inhibiting gluconeogenesis may be related to activation of p-AMPK, inhibition of TORC2 dephosphorylation, prevention of TORC2 entry into the nucleus and inhibition of gluconeogenesis key enzyme gene expression.