Assembly and Secretion of Chylomicrons by Differentiated Caco-2 Cells

Abstract

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 ul-tracentrifugations to obtain large CM, small CM, and very low density lipoproteins (VLDL), and the distribu-tion of apoB in these fractions was quantified. In the absence of OA, apoB was secreted as VLDL/LDL size particles. Addition of OA (>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 associ-ated 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 Plu-ronic 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 sim-ilar to enterocytes in vivo. Next, experiments were per-formed to identify the sources of lipids used for lipopro-tein assembly. Cells were labeled with [ 3 H]glycerol for 12 h, washed, and supplemented with OA, TC, and [ 14 C] 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 con-tained 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 lipopro-teins is dependent on the synthesis and delivery of nas-cent TG to these particles.