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J Biol Chem, Vol. 274, Issue 28, 19565-19572, July 9, 1999
From the Department of Biochemistry, School of Medicine, MCP
Hahnemann University, Philadelphia, Pennsylvania 19129
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 (
Assembly and Secretion of Chylomicrons by Differentiated
Caco-2 Cells
NASCENT TRIGLYCERIDES AND PREFORMED PHOSPHOLIPIDS ARE
PREFERENTIALLY USED FOR LIPOPROTEIN ASSEMBLY
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.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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