Oleate-mediated stimulation of apolipoprotein B secretion from rat hepatoma cells. A function of the ability of apolipoprotein B to direct lipoprotein assembly and escape presecretory degradation.
Physiological concentrations of oleate stimulate apolipoprotein (apo) B-containing lipoprotein secretion from HepG2 cells without increasing apoB mRNA levels. The purpose of this study was to determine whether oleate acts by increasing translation of apoB mRNA or through posttranslational effects on the apoB protein. To address the mechanism of oleate-stimulated secretion of apoB, a series of carboxyl terminally truncated apoB constructs was made. Each contained the SV40 early promoter, the apoB 5'-untranslated region, and SV40 polyadenylation signals. Any difference in the response to oleate between endogenous apoB and the proteins encoded by the constructs or between the constructs themselves should thus depend on the protein sequence. Stable transformants were established for each of the constructs in the rat hepatoma cell line McArdle-RH7777. The effect of oleate on secretion of the apoB protein products was determined by labeling with [35S]methionine, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carboxyl-terminal truncation of apoB41 resulted in a loss of the ability of apoB secretion to respond to oleate. Ultracentrifugation of secreted proteins on continuous CsCl gradients from 1.0-1.4 g/ml revealed that this correlated with a decrease in the ability of apoB to be recovered as a buoyant lipoprotein particle. Addition of oleate decreased the densities at which the short forms of apoB secreted as lipoproteins were recovered. Pulse-chase analysis of the secretion of apoB100 and of the truncated proteins revealed that they all underwent rapid posttranslational intracellular degradation. We conclude that oleate has no effect on the translation of apoB mRNA but promotes the secretion of apoB-containing lipoproteins by reducing presecretory degradation of those forms of apoB that can produce buoyant lipoproteins.
White
A L
AL
Division of Molecular Medicine, Medical Research Council, Harrow, Middlesex, United Kingdom.
Graham
D L
DL
LeGros
J
J
Pease
R J
RJ
Scott
J
J
D000818:Animals; D001055:Apolipoproteins B / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011865:Radioisotope Dilution Technique; D051381:Rats; D013462:Sulfur Radioisotopes; D013997:Time Factors; D014162:Transfection
D001055:Apolipoproteins B; D009829:Oleic Acids; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D019301:Oleic Acid; D008715:Methionine