睾酮对胰岛素诱导的肝细胞糖原合成和Akt/GSK3β磷酸化水平的影响

Effect of testosterone on the rate of glycogen synthesis and the activity of Akt and GSK3β proteins stimulated by insulin in hepatocytes

目的:探讨睾酮(T)在肝脏胰岛素抵抗(IR)形成过程中的作用及其分子途径。方法:将成年C57BL/6雌鼠随机分为T组(n=11)及对照组(n=10),T组小鼠每日腹腔注射T(10μg/g体质量,溶剂为蓖麻油),对照组每日腹腔注射相同体积的蓖麻油,连续给药24周后处死,分离出原代小鼠肝细胞进行体外培养,用胰岛素(Ins)处理细胞后,通过液闪法检测原代肝细胞中的糖原合成率。分别用10^(-5) mol/L和10^(-7) mol/L浓度的T溶液短时间(1 h)或长时间(36 h)处理体外培养的人肝癌细胞系BEL-7404后,再用Ins处理BEL-7404细胞,然后通过液闪法检测细胞中的糖原合成率;并通过免疫印迹法检测细胞中Akt、GSK3β蛋白的表达水平和磷酸化水平。结果:Ins对T组小鼠原代肝细胞中糖原合成的诱导作用显著低于对照组(P<0.05),提示T组小鼠原代肝细胞对Ins的敏感性降低。BEL-7404细胞经T短时间(1 h)处理后,Ins对细胞中糖原合成率以及Akt和GSK3β蛋白活性的诱导作用显著提高(P<0.05);但当BEL-7404细胞经高浓度T(10^(-5) mol/L)长时间(36 h)处理后,Ins对细胞中糖原合成率以及Akt和GSK3β蛋白活性的诱导作用显著降低(P<0.05),提示高浓度T在短时间内能增强BEL-7404细胞对Ins的敏感性,但长时间暴露后会降低细胞对Ins的敏感性。结论:长时间的T暴露可能会降低肝细胞中Ins信号转导活性,从而干扰肝细胞对Ins的敏感性,导致IR的产生。

Objective： To investigate the role of testosterone （T） in the formation of insulin resistance （IR） in liver and its molecular pathway. Methods： Adult C57BL/6 female mice were randomly divided into T group （n= 11） and control group （n=10）. Mice in T group were daily intraperitoneal injected with T （1 mg T/100 g body weight, dissolved in castor oil）, mice in control group were treated with intraperitoneal injection of the same volume of castor oil. After 24 weeks, the mice were killed, and primary mice hepatocytes were isolated and cultured in vitro. After treatment of cells with insulin （Ins）, the rate of glycogen synthesis in primary hepatocytes was detected by a liquid scintillation method. The in vitro culture of human hepatocellular carcinoma cell line BEL-7404 were pretreated with 10.5 mol/L and 10.7 mol/L concentration ofT solution in short time （1 h） or long time （36 h） respectively, then treated by Ins, the rate of glycogen synthesis of the BEL-7404 cells were detected by a liquid scintillation method. The expression and phosphorylation leves of GSK3β and Akt proteins were detected by Western blotting. Results： The inducing effect of Ins on glycogen synthesis in primary hepatocytes of mice in T group was significantly lower than that in control group （P〈0.05）, suggesting that the sensitivity of primary hepatic cells to Ins decreased in T group. BEL-7404 cells were pretreated by T in short time （1 h） then the treated by Ins, the glycogen synthesis rate, as well as Akrt and GSK3β proteins activity were significantly increased （P〈0.05）. But when BEL-7404 cells were pretreated with high concentration （10.5 mol/L） T for a long time （36 h）, the rate of glycogen synthesis induced by Ins and the activity of Akt and GSK3β proteins were decreased significantly （P〈0.05）, which suggested that high concentration T can increase the sensitivity of BEL-7404 cells to Ins in a short time, but decrease the sensitivity to Ins in a prolonged exposure. Conclusion： T in a long time exposure may reduce insulin signaling activity in liver cells, which leads to IR.