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 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 (>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 vivo. Next, experiments were performed to identify the sources of lipids used for lipoprotein 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 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.

The use of stable-isotopically labeled oleic acid to interrogate lipid assembly in vivo: assessing pharmacological effects in preclinical species

“…This suggests that the label may initially be incorporated into TG and CE in the enterocyte for secretion via chylomicrons and that accrual of the isotope by PC may be the result of synthesis/exchange mechanisms taking place in the periphery. These data support the conclusions of others that the enterocyte may draw from a pool of preformed phospholipid for chylomicron secretion ( 34 ). Analysis of the lipids retained in the intestinal tissue yields additional insight into disposition of the tracer.…”

A Drosophila Microsomal Triglyceride Transfer Protein Homolog Promotes the Assembly and Secretion of Human Apolipoprotein B

“…COS cells were transfected with human MTP or CG9342 and microsomal extracts were assayed for lipid transfer activity as described (33,44). Human MTP-transfected COS cells displayed transfer activity comparable to that observed with an equal mass of HepG2 cell microsomal extract.…”

Inhibiting Proteasomal Degradation of Microsomal Triglyceride Transfer Protein Prevents CCl4-induced Steatosis

“…Primary enterocytes were isolated and cultured as described (24). HepG2 and Caco-2 cell were cultured as described previously (25,26). Human apoB and apoAI secreted from cells were measured by enzyme-linked immunosorbent assay (23,27).…”

“…Radiolabeled triglycerides and phospholipids were separated using hexanes/ethyl ether/acetic acid (80:20:2) as a developing solvent. Lipids were visualized by exposure to iodine vapors; bands corresponding to the marker lipids were scraped, and radioactivity was measured by liquid scintillation counting (26).…”