Des Rosiers C, Di Donato L, Comte B, Laplante A, Marcoux C, David F, Fernandez C A, Brunengraber H
Department of Nutrition, University of Montréal, Québec, Canada.
J Biol Chem. 1995 Apr 28;270(17):10027-36. doi: 10.1074/jbc.270.17.10027.
We conducted an extensive mass isotopomer analysis of citric acid cycle and gluconeogenic metabolites isolated from livers of overnight fasted rats perfused with 4 mM glucose, 0.2 mM octanoate, 1 mM [U-13C3]lactate, and 0.2 mM [U-13C3]pyruvate, in the anterograde or retrograde mode. In both perfusion modes, two distinct isotopomer patterns were observed: (i) those of phosphoenolpyruvate, glucose, malate, and aspartate and (ii) those of citrate, alpha-ketoglutarate, glutamate, and glutamine. Key citric acid cycle parameters and, hence, rates of gluconeogenesis, calculated (Lee, W.-N.P. (1989) J. Biol. Chem. 264, 13002-13004 and Lee, W.-N.P. (1993) J. Biol. Chem. 268, 25522-25526) from our mass isotopomer data did not only vary, but lead to conclusions inconsistent with Lee's citric acid cycle model. Compared to lactate and pyruvate uptake, which sets an upper limit to glucose production, rates of gluconeogenesis calculated (i) with the phosphoenolpyruvate and citrate data were similar, but those calculated (ii) with the glutamate data amounted to only 60%, which is unlikely. All these conclusions are independent of the perfusion modes. We provide evidence that the following processes contribute to the observed labeling discrepancy: (i) the reversibility of the isocitrate dehydrogenase reaction and (ii) an active citrate cleavage pathway for the transfer of the oxaloacetate carbon skeleton from mitochondria to the cytosol. Also, a good fit of our labeling data was obtained with a model of citric acid cycle and gluconeogenesis which we developed to incorporate the above reactions (Fernandez, C.A., and Des Rosiers, C. (1995) J. Biol. Chem. 270, 10037-10042). The following conclusions can be drawn from the calculated reaction rates: (i) about half of the lactate conversion to glucose occurs via the citrate cleavage pathway, (ii) the flux through the reversal of the isocitrate dehydrogenase reaction is almost as fast as that through the citrate synthase reaction, and (iii) the flux through citrate synthase and alpha-ketoglutarate dehydrogenase is 1.6- and 3.2-fold that through pyruvate carboxylase, respectively.
我们对从过夜禁食大鼠肝脏中分离出的柠檬酸循环和糖异生代谢物进行了广泛的质量同位素异构体分析，这些大鼠肝脏在顺行或逆行模式下灌注了4 mM葡萄糖、0.2 mM辛酸、1 mM [U-13C3]乳酸和0.2 mM [U-13C3]丙酮酸。在两种灌注模式下，均观察到两种不同的同位素异构体模式：（i）磷酸烯醇丙酮酸、葡萄糖、苹果酸和天冬氨酸的模式，以及（ii）柠檬酸、α-酮戊二酸、谷氨酸和谷氨酰胺的模式。根据我们的质量同位素异构体数据计算得出的关键柠檬酸循环参数以及糖异生速率（Lee, W.-N.P. (1989) J. Biol. Chem. 264, 13002 - 13004和Lee, W.-N.P. (1993) J. Biol. Chem. 268, 25522 - 25526）不仅有所不同，而且得出的结论与Lee的柠檬酸循环模型不一致。与为葡萄糖生成设定上限的乳酸和丙酮酸摄取相比，（i）用磷酸烯醇丙酮酸和柠檬酸数据计算出的糖异生速率相似，但（ii）用谷氨酸数据计算出的糖异生速率仅为60%，这是不太可能的。所有这些结论均与灌注模式无关。我们提供的证据表明，以下过程导致了观察到的标记差异：（i）异柠檬酸脱氢酶反应的可逆性，以及（ii）一条活跃的柠檬酸裂解途径，用于将草酰乙酸碳骨架从线粒体转移到细胞质中。此外，我们开发的一个包含上述反应的柠檬酸循环和糖异生模型很好地拟合了我们的标记数据（Fernandez, C.A., and Des Rosiers, C. (1995) J. Biol. Chem. 270, 10037 - 10042）。从计算出的反应速率可以得出以下结论：（i）约一半的乳酸转化为葡萄糖是通过柠檬酸裂解途径发生的，（ii）通过异柠檬酸脱氢酶反应逆向的通量几乎与通过柠檬酸合酶反应的通量一样快，以及（iii）通过柠檬酸合酶和α-酮戊二酸脱氢酶的通量分别是通过丙酮酸羧化酶通量的1.6倍和3.2倍。
