Use of cyclodextrin to deliver lipids and to modulate apolipoprotein B-100 production in HepG2 cells.

Abstract

2-Hydroxypropyl-beta-cyclodextrin (cyclodextrin), cyclodextrin-solubilized oleate, and cyclodextrin-solubilized cholesterol were used to modulate proteolysis and secretion of newly-synthesized apolipoprotein B-100 (apoB) in HepG2 cells. Following cyclodextrin and lipid treatments, cells were pulse-labeled with [3H] leucine, and quantitative immunoprecipitation was used to measure apoB synthesis, apoB secreted into the medium, and the cellular content of undegraded apoB that was not secreted. Three-hour treatment with cyclodextrin-solubilized oleate (0.2 mM) increased secreted apoB from 4% (control cells) to 32% and cellular undegraded apoB from 15% (control cells to 64% of apoB synthesized, which is consistent with earlier studies using bovine serum albumin to complex exogenous oleate. Prolonged daily (4 d or more) administration of 0.5% (3.5 mM) cyclodextrin with medium containing 10% fetal bovine serum increased the secretion of nascent apoB from 5-10% (control) to 17-28% and cellular undegraded apoB from 15-20% (control) to 25-31% of apoB synthesized, respectively. Subsequent administration of cyclodextrin solubilized cholesterol (10-40 micrograms) for only 3 h reversed the cyclodextrin-mediated increase in apoB secretion. The application of 0.5% cyclodextrin to HepG2 cells can rapidly (within minutes) stimulate cholesterol efflux, and transiently (over a 1-2 d period) increase cholesterol synthesis. In the current studies, the cyclodextrin-mediated increase in cholesterol synthesis was not concurrent with the increase in apoB secretion. However, prolonged (15 d) administration of cyclodextrin was shown to increase the cellular free cholesterol concentration by 25-41%, reduce the cellular triglyceride concentration by 59%, and increase apoB secretion 3- to 4-fold, without affecting the cellular cholesteryl ester concentration. In comparison, 14-d treatment with cyclodextrin-solubilized cholesterol (20 micrograms/mL) followed by 1-d equilibration without cholesterol was shown to increase the cellular free cholesterol and cholesteryl ester concentrations by 76% and 10-fold, respectively, although apoB secretion was not affected. It is hypothesized that chronic daily administration of 0.5% cyclodextrin increased the cellular cholesterol concentration and flux in discrete putative regulatory compartments, which "shielded" nascent apoB from rapid proteolysis and facilitated apoB secretion. In conclusion, cyclodextrin was used independently and in combination with cholesterol or oleate to modulate apoB proteolysis and secretion. We speculate that subcellular changes in cholesterol concentration and flux may modulate apoB production in HepG2 cells.