Merrill A H, Lingrell S, Wang E, Nikolova-Karakashian M, Vales T R, Vance D E
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322-3050, USA.
J Biol Chem. 1995 Jun 9;270(23):13834-41. doi: 10.1074/jbc.270.23.13834.
Sphingolipids are constituents of liver and lipoproteins, but relatively little is known about their synthesis and secretion. Incubation of rat hepatocytes with [14C]- or [3H]serine labeled the long-chain base backbones of mainly ceramide and sphingomyelin. Most of the labeled sphingolipids were associated with the cells; however, 1-5% (the majority of which was ceramide) was released into the medium as part of very low density lipoproteins (VLDL). Since this is the first report that lipoproteins contain ceramide, lipoproteins were isolated from rat plasma, and the ceramide contents were (per mg of protein): 6.5 nmol for VLDL (d < 1.018), 0.6 nmol for low density lipoproteins (1.018 < d < 1.063), 0.2 nmol for high density lipoproteins (1.063 < d < 1.18), and 0.1 nmol for the albumin fraction; the lipoproteins also contained 0.1-0.4 nmol of free sphingosine/mg of protein. A number of factors affected the secretion of radiolabeled sphingolipids: 1) addition of palmitic acid, but not stearic or oleic acid, enhanced secretion due to an increase in long-chain base synthesis de novo. 2) Choline deficiency caused a 42% reduction in the secretion of radiolabeled sphingomyelin, but this was due to an effect on VLDL secretion rather than a decrease in sphingolipid synthesis. Removal of choline was examined because previous studies (Yao, Z. M., and Vance, D. E. (1988) J. Biol. Chem. 263, 2998-3004) have shown that choline deficiencies depress phosphatidylcholine synthesis and lipoprotein secretion. 3) Incubation of the cells with fumonisin B1, a mycotoxin inhibitor of sphinganine (sphingosine) N-acyltransferase, reduced overall sphingolipid synthesis and secretion by 90%, but had no effect on the secretion of apoB, phosphatidylcholine, or cholesterol. All together, these findings demonstrate that rat hepatocytes synthesize ceramide and sphingomyelin de novo and incorporate them into both cellular membranes and secreted VLDL, but normal sphingolipid synthesis is not required for lipoprotein secretion.
鞘脂是肝脏和脂蛋白的组成成分，但人们对其合成和分泌了解相对较少。用[14C] - 或[3H]丝氨酸孵育大鼠肝细胞，标记了主要为神经酰胺和鞘磷脂的长链碱基骨架。大多数标记的鞘脂与细胞相关；然而，1 - 5%（其中大部分是神经酰胺）作为极低密度脂蛋白（VLDL）的一部分释放到培养基中。由于这是关于脂蛋白含有神经酰胺的首次报道，从大鼠血浆中分离出脂蛋白，其神经酰胺含量（每毫克蛋白质）分别为：VLDL（d < 1.018）为6.5 nmol，低密度脂蛋白（1.018 < d < 1.063）为0.6 nmol，高密度脂蛋白（1.063 < d < 1.18）为0.2 nmol，白蛋白部分为0.1 nmol；这些脂蛋白还含有0.1 - 0.4 nmol游离鞘氨醇/毫克蛋白质。许多因素影响放射性标记鞘脂的分泌：1）添加棕榈酸而非硬脂酸或油酸，由于从头合成的长链碱基增加而增强了分泌。2）胆碱缺乏导致放射性标记鞘磷脂的分泌减少42%，但这是由于对VLDL分泌的影响而非鞘脂合成的减少。研究胆碱的去除是因为先前的研究（Yao, Z. M., and Vance, D. E. (1988) J. Biol. Chem. 263, 2998 - 3004）表明胆碱缺乏会抑制磷脂酰胆碱合成和脂蛋白分泌。3）用伏马菌素B1（一种鞘氨醇（鞘脂）N - 酰基转移酶的霉菌毒素抑制剂）孵育细胞，使总体鞘脂合成和分泌减少90%，但对载脂蛋白B、磷脂酰胆碱或胆固醇的分泌没有影响。总之，这些发现表明大鼠肝细胞从头合成神经酰胺和鞘磷脂，并将它们整合到细胞膜和分泌的VLDL中，但脂蛋白分泌不需要正常的鞘脂合成。
