Abstract
The regulation of lipoprotein assembly and secretion at a molecular level is incompletely understood. To begin to identify the determinants of apoprotein synthesis and distribution among lipoprotein classes, we have examined the effects of chylomicron remnants which deliver triglyceride and cholesterol, and beta very low density lipoprotein (beta VLDL), which deliver primarily cholesterol, on apolipoprotein synthesis and secretion by the human hepatoma Hep G2. Hep G2 cells were incubated with remnants or beta VLDL for 24 h, the medium was changed and the cells then incubated with [35S]methionine. The secreted lipoproteins were separated by gradient ultracentrifugation and the radiolabeled apoproteins were isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and counted. Remnants caused a 14-fold, and beta VLDL a 7-fold, increase in VLDL apoprotein (apo) secretion; the apoB/apoE ratio in this class was unchanged. Preincubation with either of the lipoproteins also stimulated low density lipoprotein apoB secretion. Preincubation with beta VLDL, but not with remnants, significantly increased apoE and apoA-I secreted in high density lipoprotein (HDL). In addition, the apoE/apoA-I ratio precipitated from the HDL of beta VLDL-treated cells by anti-apoE was 2.2-fold higher than that precipitated by anti-apoA-I. There was no difference in the ratios precipitated from control HDL. This was due to the secretion of a lipoprotein, subsequently isolated by immunoaffinity chromatography, that contained predominantly apoE. When Hep G2 cells were preincubated with oleic acid alone, total apoprotein secretion was not altered. However, cholesterol-rich liposomes stimulated secretion of newly synthesized apoE, but not apoB, while apoA-I secretion was variably affected. Cholesterol-poor liposomes had no effect. Thus, lipid supply is a determinant of apoprotein synthesis and secretion, and cholesterol may be of particular importance in initiating apoprotein synthesis.
Highlights
From the Department of Medicine, Stanford University, Stanford,California 94305and the Research Institute, Palo Alto Medical Foundation, Palo Alto, Californiu94301
Hep G2 cells were incubated with remnants oBr VLDL for 24 h, the medium was changed and thecells incubatedwith [3SS]methionine.The secreted lipoproteins were separated by gradient ultracentrifugation and the radiolabeled apoproteins were isolated by immunoprecipitatissues for uptake by receptor-mediated endocytosis, while apoA-I [8] and apoC-I1 [9, 10] are important in regulating enzymes which control the breakdown and assimilation of lipoprotein lipid
One set of flasks contained only fresh medium, a second set contained medium in which Hep G2 cells had been grown for 24 h, and a third seot f flasks contained near-confluentHep G2 cells.The medium was spun a t 2,000 rpm for 5 min to remove cell debris, 0.5 ml of human serum was added as a source of carrier lipoprotein, and this mixture was brought to density 1.21with KBr
Summary
Foundation for Blood Research, P. 0.Box 190, Scarborough, ME 04074. § To whom correspondence should be addressed Research Institute, Palo Alto Medical Foundation, 860 Bryant St., Palo Alto, CA. One set of flasks contained only fresh medium, a second set contained medium in which Hep G2 cells had been grown for 24 h (no cells were present during the incubation with "'1-VLDL), and a third seot f flasks contained near-confluentHep G2 cells.The medium was spun a t 2,000 rpm for 5 min to remove cell debris, 0.5 ml of human serum was added as a source of carrier lipoprotein, and this mixture was brought to density 1.21with KBr. The lipoproteins were separated by the method of Redgrave and West [37]. Anti-apoA-I Affinity Column-Hep G2 cells were incubated for 24 h with complete media containing either 40 pg/ml OVLDL, 40 pg/ml chylomicron remnants, or an equivalent volume of PBS. Triglyceride and lipoprotein cholesterol were determined using an enzymatic kit (Sigma)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
