A sensitive method to analyze in vitro secretion of lipoproteins: distribution of apolipoproteins is modulated by oleic acid in HepG2 cells.
Lipoprotein metabolism can be studied by the analysis of lipoprotein production in cell culture. An inherent problem in such an analysis is the low concentration of lipoproteins in culture supernatants. The difficulty comes from the fact that the samples must be concentrated prior to any analysis. The concentrating methods (e.g., dialysis or ultrafiltration) induce a heterogeneous loss of components. In order to minimize these losses, we have developed a sensitive three-step method to analyze the distribution and the amount of apolipoproteins in the different classes of lipoproteins secreted by the human hepatoma cell line HepG2. Cells were labeled with [14C]acetate and [35S]methionine for 4 h in the presence of 0.08 mM BSA, complexed or not, with 0.75 mM oleic acid. The 14C-radiolabeled cellular lipids were extracted and analyzed by thin-layer chromatography and the secreted lipoproteins were analyzed by the following three-step method. First, the lipoproteins were isolated by flotation ultracentrifugation. Second, total lipoproteins were directly applied to native agarose-acrylamide gel electrophoresis in order to separate lipoproteins with respect to their diameter. After migration, the gel was sliced and each fragment was eluted in a buffer containing sodium dodecyl sulfate and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This allowed evaluation of the proportion of apolipoproteins in lipoproteins. Oleic acid (0.75 mM) increased the rate of triglyceride biosynthesis and apoB-100 secretion by 1.7-fold and 2.4-fold, respectively. Moreover, oleic acid treatment modified the profile of secreted lipoproteins. Oleic acid-treated cells secreted more apoB-100 within VLDL than control cells.
Benoist
F
F
Laboratoires Glaxo Wellcome, Centre de Recherche, Les Ulis, France.
Grand-Perret
T
T
D001053:Apolipoproteins / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008074:Lipoproteins / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D001053:Apolipoproteins; D008074:Lipoproteins; D009829:Oleic Acids; D019301:Oleic Acid