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

Yuwei Wang,Roger S Mcleod,Zemin Yao

doi:10.1074/jbc.272.19.12272

Abstract

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 microg/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 microM 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.

Highlights

Two forms of apolipoprotein B1 are synthesized by the rat liver, the full-length apoB100 and apoB48, which represents the N-terminal 48% of apoB100 [1]
To demonstrate further that human apoB48 (hB48)-very low density lipoproteins (VLDL) secretion was comparable with endogenous rat B48 (rB48)-VLDL secretion, we tested the response of the transfected cell line to brefeldin A
We examined the effect of microsomal triglyceride transfer protein (MTP) inhibition on VLDL secretion by inactivating MTP prior to metabolic labeling of Normal MTP Activity Is Required for the Second Step of B48-VLDL Assembly—In the current work we have inquired whether or not MTP activity is required for B48-VLDL secretion using two experimental protocols: MTP was inactivated either before or after metabolic labeling to assess secretion of pre-existing or newly synthesized apoB and lipid as lipoproteins

Summary

EXPERIMENTAL PROCEDURES

Materials—Culture media and sera were obtained from Life Technologies, Inc. Reagents for polyacrylamide gel electrophoresis were obtained from Bio-Rad. VLDL and HDL were separated by ultracentrifugation, and lipoproteins containing rat B100 (rB100-VLDL, d Ͻ 1.02 g/ml) or hB48 (hB48-VLDL, d Ͻ 1.02 g/ml or hB48-HDL, d ϭ 1.08 –1.13 g/ml) were separated by immunoaffinity chromatography Lipids associated with these particles were extracted with chloroform/methanol. Twelve fractions obtained from sucrose gradient ultracentrifugation of the conditioned [35S]methionine labeling media were mixed with bovine serum albumin (final concentration 1%) and 100 ␮l of 1D1-affinity beads for 16 h at 4 °C to recover hB48-lipoprotein. Immunocomplexes with either hB48- or rB100-lipoproteins were washed five times with 1 ml of phosphate-buffered saline by centrifugation Since these lipoproteins were mostly confined to density fractions 1 and 2 (VLDL, d Ͻ 1.02 g/ml) and 8 –10 (HDL, d ϭ 1.08 –1.13 g/ml), only three combined fractions (i.e. hB48-VLDL, rB100-VLDL, and hB48-HDL) were subjected to lipid analysis. The recovery of hB48VLDL from the conditioned medium by immunoaffinity purification was greater than 90%, and the purified hB48-VLDL contained less than 15% endogenous rB100-VLDL as determined by Western blot analysis and by quantification of 35S-labeled apoBs (data not shown)

RESULTS

MTP and VLDL Secretion

DISCUSSION