Evidence that microsomal triglyceride transfer protein is limiting in the production of apolipoprotein B-containing lipoproteins in hepatic cells.
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 situ modulation and measurement of MTP triglyceride transfer activity. In order to accomplish this, an irreversible photoaffinity inhibitor, BMS-192951, was designed and synthesized. When incubated with purified MTP and irradiated with UV light at 360 nm, BMS-192951 inhibits triglyceride transfer by covalently binding to the protein. HepG2 cells were treated with either increasing concentrations of BMS-192951 (0-15 microM) with 5 min of ultraviolet irradiation, or 3.0 microM BMS-192951 with various lengths (0-15 min) of ultraviolet irradiation. Microsomal extracts were prepared exhaustively dialyzed to remove unbound inhibitor, and assayed for MTP-mediated triglyceride transfer activity. BMS-192951 was shown to reduce MTP activity in both a dose- and UV exposure time-dependent fashion. Measurement of apoB concentration in the media showed that apoB secretion was reduced in proportion to the in situ inhibition of MTP activity, while no change was observed in apoA-I secretion. Experiments performed in McArdle RH-7777 rat hepatoma cells and primary rat hepatocytes gave nearly identical results; the decrease in apoB secretion was proportional to the decrease in MTP activity. These results indicate that MTP-mediated lipid transfer is limiting in the assembly and secretion of apoB-containing lipoproteins in hepatic cells under the conditions tested.
Department of Metabolic Diseases, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA.
H
H
Jamil
C H
CH
Chu
J K
JK
Dickson
Y
Y
Chen
M
M
Yan
S A
SA
Biller
R E
RE
Gregg
J R
JR
Wetterau
D A
DA
Gordon
D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D001055:Apolipoproteins B / Q000096:biosynthesis*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology; D054833:Isoindoles; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D008113:Liver Neoplasms / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D010880:Piperidines / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D013462:Sulfur Radioisotopes; D014280:Triglycerides / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001055:Apolipoproteins B; C114701:BMS 192951; C101970:BMS 200150; D002352:Carrier Proteins; D007211:Indoles; D054833:Isoindoles; D010880:Piperidines; D013462:Sulfur Radioisotopes; D014280:Triglycerides; C106092:microsomal triglyceride transfer protein; D008715:Methionine; D003545:Cysteine