Quantitative analysis of flux along the gluconeogenic, glycolytic and pentose phosphate pathways under reducing conditions in hepatocytes isolated from fed rats.
Quantitative analysis of flux along the gluconeogenic, glycolytic and pentose phosphate pathways under reducing conditions in hepatocytes isolated from fed rats.
复制标题
在还原条件下对从进食大鼠中分离的肝细胞中沿糖异生、糖酵解和磷酸戊糖途径的通量进行定量分析。
DOI：
10.1042/bj2120585
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
Blum,JJ
中科院分区：
文献类型：
--
作者：
Crawford,JM;Blum,JJ
从喂食大鼠的肝脏中分离肝细胞，并与葡萄糖（10 mM）、核糖（1 mM）、甘露糖（4 mM）、甘油（3 mM）、乙酸盐（1.25 mM）和乙醇（5 mM）的混合物一起孵育，在任何给定孵育中均使用14 C标记一种底物。在孵育开始后20和40 min测量标记物掺入CO2、葡萄糖、糖原、脂质甘油和脂肪酸、乙酸盐和葡萄糖的C-1。这些数据（每个间隔约48次测量）与单室模型结合使用，单室模型是生血管、糖酵解和戊糖磷酸途径的反应，以及相关线粒体反应的简化模型。一种改进的计算机分析方法的方程描述的流量的标签通过每个碳原子的每个代谢物在稳态条件下，用于计算值的34个独立的通量参数在这个模型中。获得了与数据的良好拟合，从而可以对所考虑的途径中的大多数通量进行良好的估计。数据显示：高于磷酸丙糖水平的净流量是产酶的;磷酸己糖中的标记物在第二个20分钟间隔内完全平衡;磷酸丙糖异构酶步骤不平衡磷酸丙糖之间的标记物;底物循环在葡萄糖-葡萄糖6-磷酸、果糖6-磷酸-果糖1，6-二磷酸和磷酸烯醇化酸-丙酮酸-草酰乙酸循环下操作;并且虽然通过催化戊糖磷酸途径的非氧化步骤的酶的净通量小，但是双向通量大。
Hepatocytes were isolated from the livers of fed rats and incubated with a mixture of glucose (10 mM), ribose (1 mM), mannose (4 mM), glycerol (3 mM), acetate (1.25 mM), and ethanol (5 mM) with one substrate labelled with 14C in any given incubation. Incorporation of label into CO2, glucose, glycogen, lipid glycerol and fatty acids, acetate and C-1 of glucose was measured at 20 and 40 min after the start of the incubation. The data (about 48 measurements for each interval) were used in conjunction with a single-compartment model of the reactions of the gluconeogenic, glycolytic and pentose phosphate pathways and a simplified model of the relevant mitochondrial reactions. An improved method of computer analysis of the equations describing the flow of label through each carbon atom of each metabolite under steady-state conditions was used to compute values for the 34 independent flux parameters in this model. A good fit to the data was obtained, thereby permitting good estimates of most of the fluxes in the pathways under consideration. The data show that: net flux above the level of the triose phosphates is gluconeogenic; label in the hexose phosphates is fully equilibrated by the second 20 min interval; the triose phosphate isomerase step does not equilibrate label between the triose phosphates; substrate cycles are operating at the glucose-glucose 6-phosphate, fructose 6-phosphate-fructose 1,6-bisphosphate and phosphoenolpyruvate-pyruvate-oxaloacetate cycles; and, although net flux through the enzymes catalysing the non-oxidative steps of the pentose phosphate pathway is small, bidirectional fluxes are large.