Sakata N, Wu X, Dixon J L, Ginsberg H N
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
J Biol Chem. 1993 Nov 5;268(31):22967-70.
Under lipid-poor conditions, most newly synthesized apolipoprotein B100 (apoB) undergoes rapid degradation in Hep G2 cells such that only a small fraction of newly synthesized apoB is actually secreted. Addition of oleate to Hep G2 culture medium stimulates apoB secretion by a post-translational mechanism. In the current studies we have explored oleate-stimulation of apoB secretion by using calpain inhibitor I, N-acetyl-leucyl-leucyl-norleucinal (ALLN), a compound that inhibits the intracellular degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and the T cell receptor alpha subunit. Preincubation of Hep G2 cells with ALLN (40 micrograms/ml) for 1 h markedly inhibited degradation of newly synthesized apoB. Whereas only 32% of newly labeled apoB remained intact (cells+medium) in control cells after a 10-min pulse with [3H]leucine followed by a 60-min chase, 84% of labeled apoB was intact in ALLN-treated cells. However, most of the ALLN-protected apoB remained intracellular, as ALLN did not stimulate the rate of apoB secretion over the control rate (12 versus 9.2%). Although secretion of apoB was not accelerated, the protection afforded by ALLN continued for several hours, and labeled apoB continued to be secreted over 3 h of chase after which secretion ceased. The protection afforded by ALLN resulted in 37% of labeled apoB secreted by 3 h compared to 15% in control cells. In contrast, simultaneous treatment of cells with ALLN and oleate both accelerated and increased total apoB secretion, such that 36% of initially labeled apoB was recovered in the medium by 60 min and 71% of labeled apoB was secreted by 180 min of chase. These data show that ALLN and oleate affect apoB metabolism by different mechanisms. Although ALLN can protect nascent apoB from rapid early intracellular degradation, it does not accelerate apoB secretion. In contrast, although our results can not rule out the possibility that oleate may directly inhibit proteolysis of apoB, oleate appears to protect apoB mainly by facilitating transport of apoB out of a protease-containing compartment associated with the endoplasmic reticulum.
在低脂条件下，大多数新合成的载脂蛋白B100（apoB）在Hep G2细胞中会迅速降解，以至于只有一小部分新合成的apoB实际被分泌出来。向Hep G2培养基中添加油酸可通过翻译后机制刺激apoB分泌。在当前的研究中，我们使用钙蛋白酶抑制剂I，N - 乙酰 - 亮氨酰 - 亮氨酰 - 正亮氨酸（ALLN）来探究油酸对apoB分泌的刺激作用，ALLN是一种能抑制3 - 羟基 - 3 - 甲基戊二酰辅酶A还原酶和T细胞受体α亚基细胞内降解的化合物。用ALLN（40微克/毫升）预孵育Hep G2细胞1小时可显著抑制新合成的apoB的降解。在用[³H]亮氨酸脉冲标记10分钟后再追踪60分钟，对照细胞中只有32%的新标记apoB保持完整（细胞 + 培养基），而在ALLN处理的细胞中84%的标记apoB是完整的。然而，大多数ALLN保护的apoB仍留在细胞内，因为ALLN并没有使apoB的分泌速率超过对照速率（12%对9.2%）。尽管apoB的分泌没有加速，但ALLN提供的保护持续了几个小时，并且在追踪3小时后标记的apoB仍持续分泌，之后分泌停止。与对照细胞中15%相比，ALLN提供的保护使得3小时时37%的标记apoB被分泌出来。相反，同时用ALLN和油酸处理细胞既加速又增加了总的apoB分泌，以至于在60分钟时培养基中回收了36%最初标记的apoB，在追踪180分钟时71%的标记apoB被分泌出来。这些数据表明ALLN和油酸通过不同机制影响apoB代谢。尽管ALLN可以保护新生的apoB免于早期细胞内的快速降解，但它不会加速apoB的分泌。相反，尽管我们的结果不能排除油酸可能直接抑制apoB蛋白水解的可能性，但油酸似乎主要通过促进apoB从与内质网相关的含蛋白酶区室中转运出来来保护apoB。
