Volume 272, Number 19,
Issue of May 9, 1997
pp. 12272-12278
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Normal Activity of Microsomal Triglyceride Transfer Protein Is
Required for the Oleate-induced Secretion of Very Low Density
Lipoproteins Containing Apolipoprotein B from McA-RH7777 Cells
(Received for publication, January 15, 1997, and in revised form, February 11, 1997)
Yuwei
Wang
,
Roger S.
McLeod
and
Zemin
Yao
From the Lipoprotein and Atherosclerosis Group and the Departments
of Pathology & Laboratory Medicine and Biochemistry, University of
Ottawa Heart Institute, Ottawa, Ontario K1Y 4E9, Canada
The requirement of the activity of
microsomal triglyceride transfer protein (MTP) for very low density
lipoprotein (VLDL) secretion was determined using McA-RH7777 cells
stably transfected with human apoB48 (hB48). Secretion of VLDL
containing hB48 (hB48-VLDL) by the transfected cells was induced by
exogenous oleate (0.4 mM), and
oleate-dependent VLDL secretion was selectively inhibited by brefeldin A (0.2 µg/ml). Two protocols were used to determine the
effect of MTP inhibition on VLDL secretion. In the first protocol, cell
protein and lipid were labeled with radioactive amino acids and oleate
prior to MTP inhibition (using 5 µM of the photoaffinity inhibitor BMS-192951 to reduce MTP activity by 65-70%), and secretion of prelabeled apoB and triacylglycerol (TG) associated with
lipoproteins was monitored during oleate-supplemented chase. In control
cells, a 6-fold increase in incorporation of prelabeled TG into
hB48-VLDL was observed after oleate supplement, while incorporation of
prelabeled TG into VLDL containing endogenous rat apoB100 (rB100-VLDL)
was unaffected. Inhibition of MTP activity abolished the oleate-induced utilization of prelabeled TG (by 80%) and hB48 (by 70%) for hB48-VLDL secretion but decreased utilization of pre-existing TG (by <25%) and
B100 (by 45%) for rB100-VLDL secretion to a lesser extent. Inhibition
of MTP did not affect incorporation of prelabeled TG or hB48 into high
density lipoproteins containing hB48 (hB48-HDL). In the second
protocol, MTP was inactivated prior to metabolic labeling of protein
and lipid, and secretion of newly labeled apoB and TG as lipoproteins
was monitored after oleate supplement. Under this condition, MTP
inhibition decreased incorporation of newly labeled TG (by 80%) and
hB48 (80%) into hB48-VLDL but did not affect their incorporation into
hB48-HDL. Additionally, MTP inhibition decreased incorporation of newly
labeled TG (by 50%) and rB100 (by 90%) into rB100-VLDL. Thus, normal
activity of MTP is required for the oleate-induced secretion of
hB48-VLDL from McA-RH7777 cells.
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