INHIBITION OF THE MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN BLOCKS THEFIRST STEP OF APOLIPOPROTEIN-B LIPOPROTEIN ASSEMBLY BUT NOT THE ADDITION OF BULK CORE LIPIDS IN THE 2ND STEP

The microsomal triglyceride transfer protein (MTP) is required for ass embly and secretion of the lipoproteins containing apolipoprotein B (a poB): very low density lipoproteins and chylomicrons. Evidence indicat es that the subclasses of these lipoproteins that contain apoB-48 are assembled in a distinct two-step process; first a relatively lipid-poo r primordial lipoprotein precursor is produced, and then bulk neutral lipids are added to form the core of these spherical particles, To det ermine if either step is mediated by MTP, a series of clonal cell line s stably expressing apoB-53 and MTP was established in non-lipoprotein producing HeLa cells, MTP activity in these cells was similar to 30%, and apoB secretion was 7-33% of that in HepG2 cells on a molar basis, Despite having robust levels of triglyceride and phospholipid synthes is, these cell lines, as exemplified by HLMB53-59, secreted >90% of th e apoB-53 on relatively lipid-poor particles in the density range of 1 .063-1.21 g/ml, These results suggested that coexpression of MTP and a poB only reconstituted the first but not the second step in lipoprotei n assembly, To extend this observation, additional studies were carrie d out in McArdle RH-7777 rat hepatoma cells, in which the second step of apoB-48 lipoprotein assembly is well defined, Treatment of these ce lls with the MTP photoaffinity inhibitor BMS-192951 before pulse label ing with [S-35]methionine/cysteine led to an 85% block of both apoB-48 and apoB-100 but not apoAI secretion, demonstrating inhibition of the first step of lipoprotein assembly, After a 30-min [S-35]methioneine/ cysteine pulse labeling and 120 min of chase, all of the nascent apoB- 48 was observed to have a density of high density lipoproteins (1.063- 1.21 g/ml), indicating that only the first step of lipoprotein assembl y had occurred, The addition of oleic acid to the cell culture media a ctivated the second step as evidenced by the conversion of the apoB-48 high density lipoproteins to very low density lipoproteins (d < 1.006 g/ml) during an extended chase period, Inactivation of MTP after comp letion of the first step, but before stimulation of the second step by the addition of oleic acid, did not block this conversion, Thus, inhi bition of MTP did not hinder the addition of bulk core lipid to the pr imordial lipoprotein precursor particles, indicating that MTP is not r equired for the second step of apoB-48 lipoprotein assembly.