Stimulation by insulin of glycolysis in cultured hepatocytes is attenuated by extracellular ATP and puromycin through purine-dependent inhibition of phosphofructokinase 2 activation.
Stimulation by insulin of glycolysis in cultured hepatocytes is attenuated by extracellular ATP and puromycin through purine-dependent inhibition of phosphofructokinase 2 activation.
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DOI：
10.1111/j.1432-1033.1989.tb14843.x
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发表时间：
1989-06
期刊：
影响因子：
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通讯作者：
Irmelin Probst;Armin Quentmeier;Christa Schweickhardt;K. Unthan-Fechner
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文献类型：
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作者：
Irmelin Probst;Armin Quentmeier;Christa Schweickhardt;K. Unthan-Fechner
在培养的大鼠肝细胞中，胰岛素激活糖酵解之前是磷酸果糖激酶2（PFK 2）的激活和随后的果糖2，6-二磷酸[Fru（2，6）P2]水平的升高。细胞外添加ATP或嘌呤霉素阻止激素对糖酵解的影响。研究嘌呤类化合物抑制糖酵解刺激的机制。1. 50 μ M ATP完全阻止了糖酵解的3-5倍胰岛素依赖性增加，无论细胞最初是否具有低或高Fru（2，6）P2含量。50 μ M嘌呤霉素仅在初始Fru（2，6）P2水平较低且在糖酵解增加之前必须通过胰岛素增加的细胞中阻止胰岛素对糖酵解的刺激。它不拮抗初始高Fru（2，6）P2含量的胰岛素细胞的作用。2. ATP自身发挥作用;它在10分钟内将最初的高Fru（2，6）P2水平降低95%，并将基础糖酵解速率降低60%。使用约25 μ M ATP或15 μ M腺苷5 '[β，γ-亚甲基]三磷酸获得对Fru（2，6）P2水平的半数最大效应。ADP和腺苷-5-[γ-硫代]三磷酸与ATP一样有效，而100 μ M腺苷5 '[α，β-亚甲基]三磷酸没有引起任何影响。嘌呤霉素既不降低高Fru（2，6）P2水平，也不抑制基础糖酵解。3.细胞外ATP（100 μ M）导致PFK 2活性形式的抑制。细胞内Glc 6P、柠檬酸盐、ATP、ADP和AMP水平被细胞外ATP增加，磷酸烯醇式丙酮酸含量降低，Fru 6P和甘油3-磷酸水平保持恒定。嘌呤霉素不抑制PFK 2。4.嘌呤霉素和ATP均能抑制胰岛素依赖性的Fru（2，6）P2水平的升高，并能消除激素对PFK 2的激活作用。嘌呤霉素不能阻断葡萄糖引起的Fru（2，6）P2的积累; ATP也能拮抗葡萄糖依赖性的增加。5. 100 μ M ATP使cAMP依赖性蛋白激酶活性比从0.1升高到0.38，并在5分钟内使三磷酸肌醇水平增加16倍，而嘌呤霉素对这两个水平都没有影响。结论：这两种嘌呤通过阻止激素增加Fru（2，6）P2水平来阻断胰岛素对糖酵解的作用。然而，作用方式似乎不同：ATP拮抗胰岛素的作用，因为它导致PFK 2的抑制增加，而嘌呤霉素阻止胰岛素激活PFK 2。
Activation of glycolysis by insulin in cultured rat hepatocytes is preceded by an activation of phosphofructokinase 2 (PFK 2) and subsequent rise of the fructose 2,6-bisphosphate [Fru(2,6)P2] level. Extracellular addition of ATP or puromycin prevented the hormonal effect on glycolysis. The mechanism through which the purines abolished glycolytic stimulation was investigated. 1. 50 microM ATP completely prevented the 3-5-fold insulin-dependent increase of glycolysis, irrespective of whether the cells initially possessed a low or a high Fru(2,6)P2 content. 50 microM puromycin prevented the stimulation of glycolysis by insulin only in cells whose initial Fru(2,6)P2 levels were low and had to be increased by insulin prior to the increase in glycolysis. It did not antagonize the action of insulin cells with initial high Fru(2,6)P2 content. 2. ATP exerted effects on its own; it decreased initially high Fru(2,6)P2 levels by 95% within 10 min and decreased the basal glycolytic rate by 60%. Half-maximal effects on the Fru(2,6)P2 level were obtained with about 25 microM ATP or 15 microM adenosine 5'[beta, gamma-methylene]triphosphate. ADP and adenosine-5-[gamma-thio]triphosphate were as effective as ATP, whereas 100 microM adenosine 5'[alpha, beta-methylene]triphosphate elicited no effect. Puromycin neither decreased high Fru(2,6)P2 levels nor inhibited basal glycolysis. 3. Extracellular ATP (100 microM) led to inhibition of the active form of PFK 2. Intracellular levels of Glc6P, citrate, ATP, ADP and AMP were increased by extracellular ATP, the phosphoenolpyruvate content was decreased, Fru6P and glycerol 3-phosphate levels stayed constant. Puromycin did not inhibit PFK 2. 4. Both puromycin and ATP prevented the insulin-dependent rise of the Fru(2,6)P2 level, they abolished the activation of PFK 2 by the hormone. Puromycin did not block the accumulation of Fru(2,6)P2 provoked by glucose addition; ATP also antagonized the glucose-dependent increase. 5. 100 microM ATP elevated the cAMP-dependent protein kinase activity ratio from 0.1 to 0.38 and increased the level of inositol trisphosphate by 16-fold within 5 min, whereas puromycin was without effect on either level. It is concluded that the two purines block the insulin effect on glycolysis by preventing the hormone increasing the Fru(2,6)P2 level. The mode of action, however, seems to be different: ATP antagonizes insulin action in that it leads to increased inhibition of PFK 2 whereas puromycin prevents the activation of PFK 2 by insulin.