ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL

• Published in: Journal of lipid research 2002-01, Vol.43 (1), p.36-44
• Main Authors: Chisholm, Jeffrey W, Burleson, Ellen R, Shelness, Gregory S, Parks, John S
• Format: Article
• Language: English
• Subjects: apolipoprotein A-I; Apolipoprotein A-I - metabolism; carbonate extraction; Carcinoma, Hepatocellular; Centrifugation, Density Gradient - methods; HDL biosynthesis; HDL metabolism; HDL secretion; human; Humans; Isotope Labeling - methods; Lipid Metabolism; Lipoproteins, HDL - metabolism; Particle Size; Tumor Cells, Cultured
• ISSN: 0022-2275
• DOI: 10.1016/s0022-2275(20)30184-x

The goal of this study was to determine whether apolipoprotein A-I (apoA-I) is lipidated before secretion by HepG2 cells. ApoA-I was extracted from microsomes after radiolabeling with [(35)S]Met/Cys. After ultracentrifugal flotation, d < 1.25 g/ml and d > 1.25 g/ml fractions were immunoprecipitated and analyzed by SDS-PAGE. Under steady state radiolabeling conditions, 20% of extracted microsomal apoA-I floated at d < 1.25 g/ml. Pulse-chase experiments demonstrated that the percentage of microsomal apoA-I associated with lipid peaked between 2 and 8 min postsynthesis. Density gradient ultracentrifugation, and nondenaturing gradient gel electrophoresis of HepG2 cell medium, indicated that 50% of secretory apoA-I existed as small HDL particles with a diameter of approximately 7.5 nm. These and additional data suggested that approximately 20% of newly secreted apoA-I is lipidated intracellularly and another 30% is secreted in lipid-free or lipid-poor form but acquires sufficient lipid to become small HDL within 1 h of secretion, with little further maturation over the time course of the incubation (2 h).These results indicate that a process exists for the presecretory intracellular assembly of apoA-I with lipid in HepG2 cells and that apoA-I is secreted in both lipid-poor and lipidated forms.