卫生研究

目的 探讨高硒条件下，小鼠正常肝细胞(AML12)中3-磷酸甘油酸(3-phosphoglycerate, 3-PG)的来源，重点阐明糖异生途径是否参与丝氨酸从头合成过程。方法 将AML12细胞分为4组：对照组、硒处理组(Se)、siPCK2组及Se+siPCK2组。通过siRNA干扰线粒体磷酸烯醇式丙酮酸羧激酶基因(PCK2),采用Western blot检测糖异生关键酶(PCK1、PCK2、G-6-Pase)、丝氨酸合成与代谢相关酶(PHGDH、PSAT1、SHMT1、MS)和硒蛋白(SELENOP和GPX1)及胰岛素信号通路相关蛋白(PI3K、AKT、mTOR、AMPK等)的表达变化。结果 与对照组相比，Se组糖异生关键酶PCK2和G-6-Pase的表达显著升高(P<0.05),丝氨酸合成与代谢相关酶PHGDH、PSAT1、SHMT1、MTHFR、MS以及硒蛋白SELENOP、GPX1的表达水平明显上调(P<0.05),同时，Se组中AMPK及AMPK S496的表达被激活(P<0.05),而mTOR、mTOR S2448、PI3K、AKT、AKT S473和AKT T308的蛋白表达水平均受到抑制(P<0.05)。在干扰PCK2后，高硒诱导的糖异生及丝氨酸合成与代谢相关酶的上调被显著逆转(P<0.05),PI3K和AKT蛋白表达的抑制得到部分恢复(P<0.05),但AMPK与mTOR的表达未完全恢复。PCK1的表达在各组之间差异无统计学意义。结论 高硒条件下，AML12细胞中糖异生途径(尤其是PCK2介导的线粒体糖异生)是丝氨酸从头合成途径所需3-PG的主要来源。

OBJECTIVE To investigate the source of 3-PG in mouse normal hepatocytes(AML12) under high-Se condition, and to clarify whether the gluconeogenic pathway contributes to SSP activation.METHODS AML12 cells were divided into four groups: control group, high-Se group(Se), siPCK2 group(siPCK2), and high-Se group with siPCK2 group(Se+siPCK2). Mitochondrial phosphoenolpyruvate carboxykinase(PCK2) expression was silenced by siRNA. Western blotting was employed to examine the expression of gluconeogenic enzymes(PCK1, PCK2, G-6-Pase), serine biosynthesis and metabolism-related enzymes(PHGDH, PSAT1, SHMT1, MTHFR, MS), selenoproteins(SELENOP, GPX1), and insulin signaling-related proteins(PI3K, AKT, mTOR, AMPK).RESULTS Compared with the control group, Se treatment significantly increased the expression of PCK2(P<0.05) and G-6-Pase(P<0.05). Key enzymes involved in serine biosynthesis and metabolism-related enzymes(PHGDH, PSAT1, SHMT1, MTHFR, MS) as well as selenoproteins(SELENOP, GPX1) were also upregulated(P<0.05). AMPK and its phosphorylated form(AMPK S496) were activated(P<0.05), while the PI3K-AKT-mTOR signaling components, including mTOR S2448, AKT S473, and AKT T308, were markedly suppressed(P<0.05). Interference with PCK2 significantly reversed the Se-induced upregulation of gluconeogenic and serine biosynthesis and metabolism-related enzymes(P<0.05) and partially restored PI3K and AKT expression(P<0.05), though AMPK and mTOR signaling remained incompletely recovered. PCK1 expression showed no significant difference among all groups.CONCLUSION Under high-Se conditions, gluconeogenesis—particularly PCK2-mediated mitochondrial gluconeogenesis—is the predominant source of 3-PG required for de novo serine synthesis in AML12 hepatocytes.