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 [3H]glycerol for 12 h, washed, and supplemented with OA, TC, and [14C] 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.
Highlights
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)
And Secretion of CM—We have provided evidence for the synthesis and secretion of apoB-containing CM by differentiated Caco-2 cells based on four different criteria that are characteristic of CM assembly by enterocytes
We studied the effect of a physiologic inhibitor of CM secretion, Pluronic L81, to document that CM secretion was inhibited (Fig. 2B) in cell cultures as in animal studies [16, 47, 48]
Summary
Materials—Antibodies used for the determination of apoB have been described [18, 19]. Cells were incubated with 2 ml of medium containing 20% FBS, OA with either BSA or TC on the apical side of the Transwells for different times. Basolateral conditioned media were subjected to sequential density gradient ultracentrifugation. The recovery of apoB during ultracentrifugation ranged between 45 and 70% in earlier experiments when basolateral surfaces were exposed to serum-free media. Addition of 0.1% FBS to serum-free conditioned media increased the recovery to 60 –90% without affecting the distribution of apoB in different lipoprotein fractions. Basolateral conditioned media were subjected to density gradient ultracentrifugation as described above. The basolateral media from two Transwells were pooled and subjected to ultracentrifugation to obtain various lipoprotein fractions as described under “Experimental Procedures.”.
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