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Journal of Lipid Research, Vol 36, 2513-2528, Copyright © 1995 by Lipid Research, Inc.
RK Avramoglu, K Cianflone and AD Sniderman
The rate of secretion of apoB-100-containing lipoprotein particles by HepG2
cells is determined to an important extent by post-translational
mechanisms, the mass of neutral lipids clearly playing a role in this
process. Our previous data indicated that cholesteryl ester might influence
the proportion of newly synthesized apoB-100 molecules that are
incorporated into nascent lipoproteins rather than being catabolized
intracellularly shortly after they have been synthesized. The present
studies, therefore, were designed: 1) to examine in more detail the
relationship between the mass of triglyceride and cholesteryl ester in
HepG2 cells and the rate of apoB-100 secretion, and 2) to determine whether
cholesteryl ester molecules that have been synthesized and stored within
these cells must undergo hydrolysis and re-esterification before being
secreted with newly synthesized apoB-100 molecules. Changes in apoB-100
secretion in HepG2 cells were assessed in response to changes in
intracellular triglyceride and/or cholesteryl ester pool size. This was
accomplished through lipid loading of the cells by incubating them
overnight with exogenously supplied very low density lipoprotein (VLDL)
(with lipoprotein lipase, LPL), low density lipoprotein (LDL), oleate, or
LDL + oleate. The medium was changed to fresh serum-free medium and
apoB-100 secretion was shown to increase over at least 8 h. After overnight
incubation with VLDL, intracellular triglyceride mass increased 6-fold,
while intracellular cholesteryl ester mass increased 2-fold. Medium
apoB-100 increased up to 3-fold, while apoB-100 mRNA increased by only 12%.
Both heparin (10 IU/ml) and lactoferrin (20 microM) independently blocked
the VLDL-mediated increases in intracellular cholesteryl ester mass (-56%
and -46%) without decreasing triglyceride mass. ApoB-100 secretion was also
reduced by 53% and 72%, respectively. Incubation of HepG2 cells with LDL
increased intracellular cholesteryl ester mass but triglyceride mass
remained unchanged. In this instance, apoB-100 secretion increased 2-fold
but there was no change in apoB-100 mRNA. Overall, there was little
relationship between the mass of intracellular triglyceride and the rate of
apoB-100 secretion (r2 = 0.034, NS) whereas there was a strong correlation
between the intracellular mass of cholesteryl ester and apoB-100 secretion
(r2 = 0.67, P < 0.0005). To examine the process of cholesteryl ester
secretion, intracellular triglyceride and cholesteryl ester mass were
increased after incubation with LDL + oleate. The medium was then changed
to fresh serum-free medium containing an acyl-CoA:cholesterol
acyltransferase (ACAT) inhibitor (Sandoz compound 58-035). Although de novo
cholesteryl ester synthesis was inhibited up to 89%, cholesteryl ester mass
secretion remained constant with up to 15% of total cholesteryl ester mass
secreted over the 8-h period. ApoB-100 secretion also remained elevated
above control, with 92% of the cholesteryl ester secreted associated with
apoB-100 particles (27% with d < 1.006 g/mL particles and 65% with d
1.006-1.063 g/mL particles). Therefore, not only newly synthesized
cholesteryl ester but also stored cholesteryl ester can associate with
newly synthesized apoB-100 molecules and can be secreted without the
necessity of an intracellular hydrolysis/re-esterification step.
ARTICLES
Role of the neutral lipid accessible pool in the regulation of secretion of apoB-100 lipoprotein particles by HepG2 cells
Unit for the Prevention of Cardiovascular Disease, McGill University, Montreal, Quebec, Canada.
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