EVIDENCE THAT MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN IS LIMITING INTHE PRODUCTION OF APOLIPOPROTEIN B-CONTAINING LIPOPROTEINS IN HEPATICCELLS

The microsomal triglyceride transfer protein (MTP) is a heterodimeric Lipid transfer protein that is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins, A key unresolved question is whether the MTP-mediated step is rate limiting. To address this, a unique experimental strategy was used that allowed the in sit u modulation and measurement of MTP triglyceride transfer activity In order to accomplish this, an irreversible photoaffinity inhibitor, BMS -192951, tvas de signed and synthesized. When incubated with purified MTP and irradiated with UV light at 360 nm, BMS-192951 inhibits trigly ceride transfer by covalently binding to the protein. HepG2 cells were treated with either increasing concentrations of BMS-192951 (0-15 mu M) with 5 min of ultraviolet irradiation, or 3.0 mu M BMS-192951 with various lengths (0-15 min) of ultraviolet irradiation, Microsomal extr acts were prepared exhaustively dialyzed to remove unbound inhibitor, and assayed for MTP-mediated triglyceride transfer activity. BMS-19295 1 was shown to reduce MTP activity in both a dose- and UV exposure tim e-dependent fashion. Measurement of apoB concentration in the media sh owed that apoB secretion was reduced in proportion to the in situ inhi bition of MTP activity, while no change tvas observed in apoA-I secret ion. Experiments performed in McArdle RH-7777 rat hepatoma cells and p rimary rat hepatocytes gave nearly identical results; the decrease in apoB secretion was proportional to the decrease in NTP activity. These re suits indicate that MTP-mediated lipid transfer is limiting in the assembly and secretion of apoB-containing lipoproteins in hepatic cel ls under the conditions tested.