Assembly and secretion of chylomicrons by differentiated Caco-2 cells - Nascent triglycerides and preformed phospholipids are preferentially used for lipoprotein assembly
Assembly and secretion of chylomicrons by differentiated Caco-2 cells - Nascent triglycerides and preformed phospholipids are preferentially used for lipoprotein assembly
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DOI：
10.1074/jbc.274.28.19565
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发表时间：
1999-07-09
影响因子：
4.8
通讯作者：
Hussain, MM
中科院分区：
文献类型：
--
作者：
Luchoomun, J;Hussain, MM
为了开发乳糜微粒 (CM) 组装的细胞培养模型，分化的 Caco-2 细胞的顶端培养基添加了油酸 (OA) 以及白蛋白或牛磺胆酸盐 (TC)，对基底外侧培养基进行连续密度梯度超速离心以获得大 CM、小 CM 和极低密度脂蛋白 (VLDL)，并对这些组分中 apoB 的分布进行定量。在没有 OA 的情况下，apoB 被分泌为 VLDL/LDL 大小的颗粒。将 OA（大于或等于 0.8 mM）与 TC 一起添加，但不与白蛋白一起添加，导致三分之一的 apoB 分泌为 CM。脂质分析显示，一半的分泌磷脂 (PL) 和甘油三酯 (TG) 与 CM 相关。在CM中，TG比PL高7-11倍，表明CM是富含TG的颗粒。分泌的CM含有apoB100、apoB48和其他载脂蛋白。大CM的分泌被Pluronic L81特异性抑制，Pluronic L81是一种已知能抑制动物CM分泌的去污剂。这些研究表明，分化的 Caco-2 细胞以类似于体外肠细胞的方式组装和分泌 CM。接下来，进行实验以确定用于脂蛋白组装的脂质的来源。细胞用[H-3]甘油标记12小时，洗涤，并补充OA、TC和[C-14]甘油不同时间以诱导CM组装并放射性标记新生脂质，从细胞和培养基中提取TG和PL，并测定预先形成的和新生脂质与脂蛋白的关联。与新生 PL 相比，所有脂蛋白都含有更高含量的预形成 PL。 VLDL 含有等量的新生 TG 和预先形成的 TG，而 CM 含有更高含量的新生 TG，即使新生 TG 只占总细胞库的一小部分。这些研究表明，新生的TG和预先形成的PL优先用于CM组装，并为体内观察结果提供了分子解释，即分泌的CM的TG而非PL的脂肪酸组成反映了膳食脂肪的组成。据推测，在肠细胞中，内质网预先形成的 PL 与作为原始颗粒的 apoB 一起出芽，并且较大脂蛋白的组装依赖于新生 TG 的合成和向这些颗粒的递送。
To develop a cell culture model for chyclomicron (CM) assembly, the apical media of differentiated Caco-2 cells were supplemented with oleic acid (OA) together with either albumin or taurocholate (TC), The basolateral media were subjected to sequential density gradient ultracentrifugations to obtain large CM, small CM, and very low density lipoproteins (VLDL), and the distribution of apoB in these fractions was quantified. In the absence of OA, apoB was secreted as VLDL/LDL size particles. Addition of OA (greater than or equal to 0.8 mM) with TC, but not with albumin, resulted in the secretion of one-third of apoB as CM. Lipid analysis revealed that half of the secreted phospholipids (PL) and triglycerides (TG) were associated with CM. In CM, TG were 7-11-fold higher than PL indicating that CM were TG-rich particles. Secreted CM contained apoB100, apoB48, and other apolipoproteins, Secretion of large CM was specifically inhibited by Pluronic L81, a detergent known to inhibit CM secretion in animals. These studies demonstrate that differentiated Caco-2 cells assemble and secrete CM in a manner similar to enterocytes in vitro. Next, experiments were performed to identify the sources of lipids used for lipoprotein assembly. Cells were labeled with [H-3]glycerol for 12 h, washed, and supplemented with OA, TC, and [C-14] glycerol for various times to induce CM assembly and to radiolabel nascent lipids, TG and PL were extracted from cells and media and the association of preformed and nascent lipids with lipoproteins was determined. All the lipoproteins contained higher amounts of preformed PL compared with nascent PL. VLDL contained equal amounts of nascent and preformed TG, whereas CM contained higher amounts of nascent TG even when nascent TG constituted a small fraction of the total cellular pool. These studies indicate that nascent TG and preformed PL are preferentially used for CM assembly and provide a molecular explanation for the in vivo observations that the fatty acid composition of TG, but not PL, of secreted CM reflects the composition of dietary fat. It is proposed that in the intestinal cells the preformed PL from the endoplasmic reticulum bud off with apoB as primordial particles and the assembly of larger lipoproteins is dependent on the synthesis and delivery of nascent TG to these particles.