Owen M R, Halestrap A P
Department of Biochemistry, School of Medical Sciences, University of Bristol, UK.
Biochim Biophys Acta. 1993 Apr 5;1142(1-2):11-22. doi: 10.1016/0005-2728(93)90079-u.
(1) Liver cells from starved rats were incubated with 10 mM L-lactate, 1 mM pyruvate and 0.3 microM glucagon in the presence and absence of the mild respiratory inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) at 0.5 mM. (2) The whole cell concentrations of phosphoenolpyruvate, 2-phosphoglycerate and 3-phosphoglycerate increased about 2-fold, whilst the triose and hexose phosphate concentrations all decreased significantly. Similar results were obtained with 0.15 microM oligomycin and 10 microM atractyloside. (3) These data can be explained by a substantial decrease in the cytosolic free concentration ratio of ATP/ADP acting on the equilibrium of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. (4) The increase in cytosolic phosphoenolpyruvate concentration can account for the observed increase in pyruvate kinase flux that occurs under these conditions (Pryor et al. (1987) Biochem. J. 247, 449-457). (5) An inhibition of pyruvate carboxylase was also implied by a decrease in calculated tissue oxaloacetate concentrations, confirming a role for both enzymes in the inhibition of gluconeogenesis. (6) Whole cell concentrations of effectors of pyruvate carboxylase activity were measured; only the ATP/ADP ratio decreased significantly. (7) Subcellular fractionation studies showed a good correlation between the measured mitochondrial ATP/ADP ratio and rates of gluconeogenesis both in the presence and absence of oleate. (8) A similar correlation could be observed between rates of pyruvate carboxylation and the measured matrix ATP/ADP ratio in isolated liver mitochondria from starved rats. (9) Data are also presented suggesting an additional effect of DCMU on the rate pyruvate carboxylation in situ under some circumstances, mediated by decreases in mitochondrial acetyl-CoA and cytosolic pyruvate concentrations. (10) It is noted that the effects of phenylethylbiguanide (phenformin) on the rate of gluconeogenesis and metabolite profiles in the perfused liver (Cooke et al. (1973) J. Biol. Chem. 248, 5272-5277) are similar to those caused by DCMU, supporting a mitochondrial locus of action for this hypoglycaemic agent.
(1) 将饥饿大鼠的肝细胞与10 mM L-乳酸、1 mM丙酮酸和0.3 μM胰高血糖素一起孵育，分别在存在和不存在0.5 mM轻度呼吸抑制剂3-(3,4-二氯苯基)-1,1-二甲基脲（DCMU）的情况下进行。(2) 磷酸烯醇丙酮酸、2-磷酸甘油酸和3-磷酸甘油酸的全细胞浓度增加了约2倍，而磷酸丙糖和磷酸己糖的浓度均显著降低。用0.15 μM寡霉素和10 μM苍术苷也得到了类似的结果。(3) 这些数据可以通过作用于甘油醛-3-磷酸脱氢酶和磷酸甘油酸激酶平衡的胞质游离ATP/ADP浓度比大幅降低来解释。(4) 胞质磷酸烯醇丙酮酸浓度的增加可以解释在这些条件下观察到的丙酮酸激酶通量的增加（Pryor等人，(1987)《生物化学杂志》247, 449 - 457）。(5) 计算得到的组织草酰乙酸浓度降低也暗示了丙酮酸羧化酶的抑制作用，证实了这两种酶在糖异生抑制中的作用。(6) 测量了丙酮酸羧化酶活性效应物的全细胞浓度；只有ATP/ADP比值显著降低。(7) 亚细胞分级分离研究表明，在存在和不存在油酸的情况下，测量的线粒体ATP/ADP比值与糖异生速率之间具有良好的相关性。(8) 在饥饿大鼠分离的肝线粒体中，丙酮酸羧化速率与测量的线粒体基质ATP/ADP比值之间也能观察到类似的相关性。(9) 还提供了数据表明，在某些情况下，DCMU对原位丙酮酸羧化速率有额外影响，这是由线粒体乙酰辅酶A和胞质丙酮酸浓度降低介导的。(10) 值得注意的是，苯乙双胍（苯乙福明）对灌注肝脏中糖异生速率和代谢物谱的影响（Cooke等人，(1973)《生物化学杂志》248, 5272 - 5277）与DCMU引起的影响相似，支持了这种降血糖药物的线粒体作用位点。
