Scott D K, O'Doherty R M, Stafford J M, Newgard C B, Granner D K
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232, USA.
J Biol Chem. 1998 Sep 11;273(37):24145-51. doi: 10.1074/jbc.273.37.24145.
Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-controlling enzyme in hepatic gluconeogenesis, and it therefore plays a central role in glucose homeostasis. The rate of transcription of the PEPCK gene is increased by glucagon (via cAMP) and glucocorticoids and is inhibited by insulin. Under certain circumstances glucose also decreases PEPCK gene expression, but the mechanism of this effect is poorly understood. The glucose-mediated stimulation of a number of glycolytic and lipogenic genes requires the expression of glucokinase (GK) and increased glucose metabolism. HL1C rat hepatoma cells are a stably transfected line of H4IIE rat hepatoma cells that express a PEPCK promoter-chloramphenicol acetyltransferase fusion gene that is regulated in the same manner as the endogenous PEPCK gene. These cells do not express GK and do not normally exhibit a response of either the endogenous PEPCK gene, or of the trans-gene, to glucose. A recombinant adenovirus that directs the expression of glucokinase (AdCMV-GK) was used to increase glucose metabolism in HL1C cells to test whether increased glucose flux is also required for the repression of PEPCK gene expression. In AdCMV-GK-treated cells glucose strongly inhibits hormone-activated transcription of the endogenous PEPCK gene and of the expressed fusion gene. The glucose effect on PEPCK gene promoter activity is blocked by 5 mM mannoheptulose, a specific inhibitor of GK activity. The glucose analog, 2-deoxyglucose mimics the glucose response, but this effect does not require GK expression. 3-O-methylglucose is ineffective. Glucose exerts its effect on the PEPCK gene within 4 h, at physiologic concentrations, and with an EC50 of 6.5 mM, which approximates the Km of glucokinase. The effects of glucose and insulin on PEPCK gene expression are additive, but only at suboptimal concentrations of both agents. The results of these studies demonstrate that, by inhibiting PEPCK gene transcription, glucose participates in a feedback control loop that governs its production from gluconeogenesis.
磷酸烯醇式丙酮酸羧激酶（PEPCK）是肝脏糖异生过程中的一种限速酶，因此在葡萄糖稳态中起着核心作用。胰高血糖素（通过环磷酸腺苷）和糖皮质激素可提高PEPCK基因的转录速率，而胰岛素则起抑制作用。在某些情况下，葡萄糖也会降低PEPCK基因的表达，但其作用机制尚不清楚。葡萄糖对许多糖酵解和脂肪生成基因的刺激作用需要葡萄糖激酶（GK）的表达及葡萄糖代谢的增强。HL1C大鼠肝癌细胞是H4IIE大鼠肝癌细胞的稳定转染细胞系，它表达一种PEPCK启动子-氯霉素乙酰转移酶融合基因，该基因与内源性PEPCK基因的调控方式相同。这些细胞不表达GK，内源性PEPCK基因或转基因通常对葡萄糖均无反应。一种指导葡萄糖激酶表达的重组腺病毒（AdCMV-GK）被用于增强HL1C细胞中的葡萄糖代谢，以测试葡萄糖通量增加是否也是抑制PEPCK基因表达所必需的。在经AdCMV-GK处理的细胞中，葡萄糖强烈抑制内源性PEPCK基因和所表达融合基因的激素激活转录。葡萄糖对PEPCK基因启动子活性的影响被5 mM甘露庚酮糖阻断，甘露庚酮糖是GK活性的特异性抑制剂。葡萄糖类似物2-脱氧葡萄糖可模拟葡萄糖反应，但这种作用不需要GK表达。3-O-甲基葡萄糖则无效。葡萄糖在生理浓度下4小时内即可对PEPCK基因产生作用，其半数有效浓度（EC50）为6.5 mM，接近葡萄糖激酶的米氏常数（Km）。葡萄糖和胰岛素对PEPCK基因表达的影响具有相加性，但仅在两种药物的亚最佳浓度时如此。这些研究结果表明，通过抑制PEPCK基因转录，葡萄糖参与了一个反馈控制环，该环调控着糖异生过程中葡萄糖的生成。
