Suppression of cytosolic triacylglycerol recruitment for very low density lipoprotein assembly by inactivation of microsomal triglyceride transfer protein results in a delayed removal of apoB-48 and apoB-100 from microsomal and Golgi membranes of primary rat hepatocytes

Cellular apoB in primary rat hepatocyte cultures was pulse-labeled with [S- 35]methionine for 1 h, Cells were then chased with excess unlabeled methion ine for periods of up to 16 h in the presence or absence of BMS-200150, an inhibitor of microsomal triglyceride transfer protein (MTP). The secretion of apoB-48-VLDL was more sensitive to MTP inhibition than was apoB-100-VLDL , Inhibition of MTP had no inhibitory effect on the secretion of denser par ticles (apoB-48 HDL and apoB-100 HDL), BMS-200150 delayed the net removal o f newly synthesized apoB-48 and apoB-100 from the microsomal and Golgi memb ranes, but not from the corresponding lumenal compartments, Only minor prop ortions of the microsomal lumen apoB-48 and apoB-100 (12-16% and 17-19%, re spectively) were present as VLDL irrespective of whether MTP uas inactivate d or not. The HDL fraction contained most of the lumenal apoB-48 (67-73%) a nd a somewhat smaller proportion of apoB-100 (44-47%), The remainder of the lumenal apoB was associated with the IDL/LDL fraction. These proportions w ere unaffected by MTP inactivation, Excess labeled apoB which accumulated i n the membranes in the presence of BMS-200150 was degraded. Inhibition of M TP prevented the removal of pre-synthesized triacylglycerol (TAG) from the hepatocytes as apoB-VLDL. Under these conditions intracellular TAG accumula ted mainly in the cell cytosol, but also, to a lesser extent, in the micros omal membranes. The results suggest that inactivation of MTP inhibits a pat hway of VLDL assembly which does not involve the bulk lumenal compartments of the microsomes, Suppression of this pathway ultimately prevents the net transfer of cytosolic TAG into mature apoB-VLDL.