Quantitation of fluxes in the gluconeogenic, glycolytic, and pentose phosphate pathways in isolated rat hepatocytes: energetic considerations.
Quantitation of fluxes in the gluconeogenic, glycolytic, and pentose phosphate pathways in isolated rat hepatocytes: energetic considerations.
复制标题
离体大鼠肝细胞中糖异生、糖酵解和磷酸戊糖途径中通量的定量：能量考虑。
DOI：
10.1007/978-1-4684-5107-8_19
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发表时间：
1986
影响因子：
--
通讯作者：
Rabkin,MS
中科院分区：
文献类型：
--
作者：
Blum,JJ;Rabkin,MS
对于任何给定的底物混合物和激素状态，大量的因子起着调节代谢产物沿着肝细胞中碳水化合物代谢的主要途径流动的作用。除了酶的数量外，这些因素还包括各种效应物的细胞内水平，如果糖2，6-二磷酸、ADP、ATP和丙氨酸等，以及几种关键酶的磷酸化状态。酶水平受到饮食和荷尔蒙影响的长期调节，而调节酶的效应器水平和磷酸化状态则受制于荷尔蒙状态的时刻变化。虽然对纯化的酶或组织匀浆的研究有助于我们目前对许多有助于调节代谢通量的因素的了解，但要更全面地了解体内调节中间代谢的情况，需要对灌流的肝脏进行研究，或者在可以被认为代表完整肝脏的情况下，对分离的肝细胞进行研究。测量主要代谢途径上的通量模式问题的一种方法是，在任何给定的实验中，将细胞与一种用14C标记的底物的合适底物混合物孵育，并在准稳态条件下测量糖原、脂类等产品中标记积累的速度。这种方法背后的理论考虑已经被回顾(Blum和Stein，1982)，这里不再重复。该方法最初是在研究四膜虫的代谢时发展起来的，但已被证明适用于分离的肝细胞(Crawford和Blum，1983)。在后一项研究中，从饲养的大鼠分离的肝细胞与葡萄糖(10 MM)、核糖(1~~)、
For any given substrate mixture and hormonal state, a large number of factors serve to regulate the flow of metaboliles along the major pathways of carbohydrate metabolism in hepatocytes. In addition to the amounts of enzymes present, these factors include the intracellular levels of various effectors such as fructose 2, 6-bisphosphate, ADP, ATP, and alanine, among others, and the phosphorylation states of several key enzymes. Enzyme levels are subject to long term regulation by dietary and hormonal influences, while effector levels and the phosphorylation state of regulatory enzymes are subject to moment-to-moment changes in hormonal states. While studies on purified enzymes or on tissue homogenates have contributed enormously to our present understanding of the many factors that help regulate metabolic fluxes, a more completeu~ der standing of in vivo regulation of intermediary metabolism requires studies on perfused liver or, in so far as they can be considered as representative of the intact liver, on isolated hepatocytes. One approach to the problem of measuring flux patterns along major pathways of metabolism is to incubate the cells with a suitable substrate mixture with one substrate in any given experiment being labeled with l4C, and measure the rate of label accumulation in products such as glycogen, lipids, etc., under quasi-steady-state conditions. The theoretical considerations underlying this approach have been reviewed (Blum and Stein, 1982) and will not be repeated here. The method was originally developed in studies on the metabolism of the ciliate Tetrahymena pyriformis, but has been shown to be applicable to isolated hepatocytes (Crawford and Blum, 1983). In this latter study, hepatocytes isolated from fed rats were incubated with a mixture of glucose (10 mM), ribose (1~~),