Regulation of enzyme activity and gene expression of pyruvate kinase by dietary factors

1.肝脏L型丙酮酸激酶(PK)基因的转录受胰岛素/葡萄糖通过增强子单位的刺激。HNF1和NF1家族蛋白分别与该单位的L-I和L-II结合。我们发现，仅NF1家族的四个成员并不能刺激LPK基因的启动子活性，但它们能增强HNF1的活性。此外，我们还鉴定了SHARP2是一个L-III结合蛋白，并且发现SHARP2在大鼠肝脏中是由饮食中的葡萄糖诱导的。与B盒结合的SP1/SP3和与C盒结合的NFY对于PKM基因的转录刺激是重要的。SP1/SP3通过与NF-Y.3的A亚基相互作用，协同反式激活PKM基因。胰岛素刺激3T3-L1脂肪细胞PKM基因的表达，即使在无糖的情况下也是如此。部分胰岛素反应元件可能存在于PKM基因的2.2k上游区域。提示PI-3信号通路和MAPKK信号通路共同参与了PKM基因的胰岛素调节。PKM_1是一种非变构同工酶，对1，6-二磷酸果糖缺乏异质性变构作用。我们发现，Glu-432通过排斥静电相互作用阻止果糖-1，6-二磷酸的结合，从而阻碍了异向性变构效应。氧化应激对肝癌细胞M_2PK活性无明显影响。

1. Transcription of hepatic L-type pyruvate kinase (PK) gene is stimulated by insulin/glucose through an enhancer unit. HNF1 and NF1 family proteins bind to L-I and L-II of this unit, respectively. We showed that four members of NF1 family alone did not stimulate the promoter activity of the LPK gene, but that they enhanced the activity of HNF1. In addition,we identified SHARP2 as a L-III binding protein and found that SHARP2 was induced by dietary glucose in rat liver.2. Sp1/Sp3 bound to box B and NFY bound to box C were important for transcriptional stimulation of the PKM gene. Sp1/Sp3 synergistically transactivated the PKM gene by interacting with A subnit of NF-Y.3. Insulin stimulated the PKM gene expression in 3T3-L1 adipocytes even in the absence of glucose. A part of insulin response element was suggested to be present in 2.2k upstream region of the PKM gene. It was suggested that both PI-3 kinase and MAPKK pathways were involved in the insulin regulation of the PKM gene.4. PKM_1 , a nonallosteric isozyme, lacks heterotropic allosteric effect involving fructose-1,6-bisphosphate. We found that Glu-432 hinders the heterotropic allosteric effect by preventing the binding of fructose-1,6-bisphosphate through a repulsive electrostatic interaction. The M_2PK activity was not altered under oxidative stress in hepatoma cells.