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

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 mu g/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 mu M 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.