Suleiman S A, Vestling C S
J Biol Chem. 1979 Nov 10;254(21):10621-8.
Rat hepatocytes have been studied in suspension culture for 10-h periods. Levels of extractable lactate dehydrogenase (LDH) have been measured in these hepatocytes at hourly intervals in order to note the balance between biosynthesis and degradation of this enzyme. Newly synthesized LDH has been measured by following the rate of incorporation of [3H]leucine into radiochemically pure LDH of high specific catalytic activity as isolated by a rapid affinity chromatographic procedure. The effects of the addition of physiological concentrations of the following hormones at the beginning of 10-h culture periods immediately following preparation of the hepatocytes by the collagen perfusion procedure have been recorded. The hormones triiodothyronine (T3), insulin, glucagon, and dexamethasone have been added singly or in combination. The culture medium has supplied variable amounts of these hormones in the 10% of fetal calf (or other) serum added, and the hepatocytes themselves have provided intracellular amounts of hormones. In addition to the added hormones, N6,O2'-dibutyryl cyclic AMP (Bt2cAMP) has also been studied. Control suspensions of hepatocytes show reproducible initial levels of extractable LDH which are maintained or slightly increased during 10 h. Such control systems also incorporate [3H]leucine into total protein and into highly purified LDH at reproducible rates during 10 h of incubation. The effects of added hormones on LDH lavels are as follows: (a) T3 causes about a 2-fold increase in LDH at 7 to 8 h in hepatocytes from young adult animals, an effect which is lowered in either younger or older animals or in thyroidectomized animals. (b) Insulin leads to a similar increase in LDH at 5 to 6 h and a falling off at 8 to 10 h. (c) Glucagon also causes an approximate doubling of the amount of extractable LDH during a 10-doubling of the amount of extractable LDH during a 10-h period. (d) Dexamethasone does not produce an increase. (e) Bt2-cAMP produces an effect indistinguishable from that of glucagon. Paired combinations of these hormones fail to produce an additive response in any case. The combinations of T3 plus dexamethaseon and insulin plus dexamethasone lead to significant reductions in levels of extractable LDH when compared to the single hormone effects cited above. With respect to rates of synthesis of total protein as measured by [3H]leucine incorporation, only glucagon, glucagon plus Bt2-cAMP, glucagon plus insulin, T3 plus Bt2cAMP, and T3 plus insulin produce significant increases during a 10-h period. However, when [3H]leucine incorporation into highly purified LDH is measured as an index of LDH biosynthesis, T3, insulin, and glucagon consistently increase the biosynthetic rates during a 10-h period. Bt2cAMP produces a smaller increase. Dexamethasone fails to produce any significant change when compared to controls. Paired combinations of hormones again do not produce any additive effect on LDH biosynthesis when the hormone producing the higher level is taken as the reference...
已对大鼠肝细胞进行了10小时的悬浮培养研究。每隔一小时测量这些肝细胞中可提取的乳酸脱氢酶（LDH）水平，以观察该酶生物合成与降解之间的平衡。通过快速亲和色谱法分离出具有高比催化活性的放射化学纯LDH，跟踪[3H]亮氨酸掺入其中的速率来测定新合成的LDH。记录了在通过胶原灌注法制备肝细胞后，于10小时培养期开始时添加以下生理浓度激素的效果。已单独或组合添加了三碘甲状腺原氨酸（T3）、胰岛素、胰高血糖素和地塞米松。培养基在添加的10%胎牛（或其他）血清中提供了不同量的这些激素，肝细胞自身也含有细胞内水平的激素。除了添加的激素外，还研究了N6,O2'-二丁酰环磷酸腺苷（Bt2cAMP）。肝细胞的对照悬浮液显示出可提取LDH的可重复初始水平，在10小时内保持或略有增加。此类对照体系在10小时的孵育过程中也以可重复的速率将[3H]亮氨酸掺入总蛋白和高度纯化的LDH中。添加激素对LDH水平的影响如下：（a）在年轻成年动物的肝细胞中，T3在7至8小时使LDH增加约2倍，在较年轻或较年长动物或甲状腺切除动物中此效应降低。（b）胰岛素在5至6小时导致LDH有类似增加，在8至10小时下降。（c）胰高血糖素在10小时内也使可提取LDH的量增加约一倍。（d）地塞米松未导致增加。（e）Bt2-cAMP产生与胰高血糖素无法区分的效应。在任何情况下，这些激素的配对组合均未产生相加反应。与上述单一激素效应相比，T3加地塞米松以及胰岛素加地塞米松的组合导致可提取LDH水平显著降低。就通过[3H]亮氨酸掺入测量的总蛋白合成速率而言，在10小时内只有胰高血糖素、胰高血糖素加Bt2-cAMP、胰高血糖素加胰岛素、T3加Bt2cAMP和T3加胰岛素产生显著增加。然而，当将[3H]亮氨酸掺入高度纯化的LDH作为LDH生物合成的指标进行测量时，T3、胰岛素和胰高血糖素在10小时内持续增加生物合成速率。Bt2cAMP产生较小的增加。与对照相比，地塞米松未产生任何显著变化。当以产生较高水平的激素为参考时，激素的配对组合再次对LDH生物合成未产生任何相加效应……
