Rabkin M, Blum J J
Biochem J. 1985 Feb 1;225(3):761-86. doi: 10.1042/bj2250761.
Hepatocytes were isolated from the livers of fed rats and incubated, in the presence and absence of 100 nM-glucagon, with a substrate mixture containing glucose (10 mM), fructose (4 mM), alanine (3.5 mM), acetate (1.25 mM), and ribose (1 mM). In any given incubation one substrate was labelled with 14C. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, glutamate, lipid glycerol and fatty acids was measured after 20 and 40 min of incubation under quasi-steady-state conditions [Borowitz, Stein & Blum (1977) J. Biol. Chem. 252, 1589-1605]. These data and the measured O2 consumption were analysed with the aid of a structural metabolic model incorporating all reactions of the glycolytic, gluconeogenic, and pentose phosphate pathways, and associated mitochondrial and cytosolic reactions. A considerable excess of experimental measurements over independent flux parameters and a number of independent measurements of changes in metabolite concentrations allowed for a stringent test of the model. A satisfactory fit to the data was obtained for each condition. Significant findings included: control cells were glycogenic and glucagon-treated cells glycogenolytic during the second interval; an ordered (last in, first out) model of glycogen degradation [Devos & Hers (1979) Eur. J. Biochem. 99, 161-167] was required in order to fit the experimental data; the pentose shunt contributed approx. 15% of the carbon for gluconeogenesis in both control and glucagon-treated cells; net flux through the lower Embden-Meyerhof pathway was in the glycolytic direction except during the 20-40 min interval in glucagon-treated cells; the increased gluconeogenesis in response to glucagon was correlated with a decreased pyruvate kinase flux and lactate output; fluxes through pyruvate kinase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase were not coordinately controlled; Krebs cycle activity did not change with glucagon treatment; flux through the malic enzyme was towards pyruvate formation except for control cells during interval II; and 'futile' cycling at each of the five substrate cycles examined (including a previously undescribed cycle at acetate/acetyl-CoA) consumed about 26% of cellular ATP production in control hepatocytes and 21% in glucagon-treated cells.
从喂食大鼠的肝脏中分离出肝细胞，在存在和不存在100 nM胰高血糖素的情况下，将其与含有葡萄糖（10 mM）、果糖（4 mM）、丙氨酸（3.5 mM）、乙酸盐（1.25 mM）和核糖（1 mM）的底物混合物一起孵育。在任何给定的孵育中，一种底物用14C标记。在准稳态条件下孵育20和40分钟后，测量14C掺入葡萄糖、糖原、CO2、乳酸、丙氨酸、谷氨酸、脂质甘油和脂肪酸中的量[Borowitz、Stein和Blum（1977年）《生物化学杂志》252，1589 - 1605]。借助一个包含糖酵解、糖异生和磷酸戊糖途径的所有反应以及相关线粒体和胞质反应的结构代谢模型，对这些数据和测量的氧气消耗进行了分析。实验测量值大大超过独立通量参数，并且对代谢物浓度变化进行了多次独立测量，从而能够对该模型进行严格测试。在每种条件下都获得了与数据的满意拟合。重要发现包括：在第二个时间段内，对照细胞具有糖原生成能力，而胰高血糖素处理的细胞具有糖原分解能力；为了拟合实验数据，需要一个糖原降解的有序（后进先出）模型[Devos和Hers（1979年）《欧洲生物化学杂志》99，161 - 167]；在对照细胞和胰高血糖素处理的细胞中，磷酸戊糖途径约为糖异生贡献15%的碳；除了在胰高血糖素处理的细胞的20 - 40分钟时间段内，通过较低的Embden - Meyerhof途径的净通量处于糖酵解方向；对胰高血糖素的反应中糖异生增加与丙酮酸激酶通量和乳酸输出减少相关；通过丙酮酸激酶、丙酮酸羧化酶和磷酸烯醇丙酮酸羧激酶的通量没有受到协同控制；用胰高血糖素处理后，三羧酸循环活性没有变化；除了在第二个时间段的对照细胞外，通过苹果酸酶的通量朝着丙酮酸形成的方向；在所研究的五个底物循环中的每一个（包括乙酸盐/乙酰辅酶A处一个先前未描述的循环）的“无效”循环消耗了对照肝细胞中约26%的细胞ATP产生量以及胰高血糖素处理的细胞中21%的细胞ATP产生量。
