Role of the neutral lipid accessible pool in the regulation of secretion of apoB-100 lipoprotein particles by HepG2 cells.

The rate of secretion of apoB-100-containing lipoprotein particles by HepG2 cells is determined to an important extent by post-translational mechanisms, the mass of neutral lipids clearly playing a role in this process. Our previous data indicated that cholesteryl ester might influence the proportion of newly synthesized apoB-100 molecules that are incorporated into nascent lipoproteins rather than being catabolized intracellularly shortly after they have been synthesized. The present studies, therefore, were designed: 1) to examine in more detail the relationship between the mass of triglyceride and cholesteryl ester in HepG2 cells and the rate of apoB-100 secretion, and 2) to determine whether cholesteryl ester molecules that have been synthesized and stored within these cells must undergo hydrolysis and re-esterification before being secreted with newly synthesized apoB-100 molecules. Changes in apoB-100 secretion in HepG2 cells were assessed in response to changes in intracellular triglyceride and/or cholesteryl ester pool size. This was accomplished through lipid loading of the cells by incubating them overnight with exogenously supplied very low density lipoprotein (VLDL) (with lipoprotein lipase, LPL), low density lipoprotein (LDL), oleate, or LDL + oleate. The medium was changed to fresh serum-free medium and apoB-100 secretion was shown to increase over at least 8 h. After overnight incubation with VLDL, intracellular triglyceride mass increased 6-fold, while intracellular cholesteryl ester mass increased 2-fold. Medium apoB-100 increased up to 3-fold, while apoB-100 mRNA increased by only 12%. Both heparin (10 IU/ml) and lactoferrin (20 microM) independently blocked the VLDL-mediated increases in intracellular cholesteryl ester mass (-56% and -46%) without decreasing triglyceride mass. ApoB-100 secretion was also reduced by 53% and 72%, respectively. Incubation of HepG2 cells with LDL increased intracellular cholesteryl ester mass but triglyceride mass remained unchanged. In this instance, apoB-100 secretion increased 2-fold but there was no change in apoB-100 mRNA. Overall, there was little relationship between the mass of intracellular triglyceride and the rate of apoB-100 secretion (r2 = 0.034, NS) whereas there was a strong correlation between the intracellular mass of cholesteryl ester and apoB-100 secretion (r2 = 0.67, P < 0.0005). To examine the process of cholesteryl ester secretion, intracellular triglyceride and cholesteryl ester mass were increased after incubation with LDL + oleate. The medium was then changed to fresh serum-free medium containing an acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor (Sandoz compound 58-035). Although de novo cholesteryl ester synthesis was inhibited up to 89%, cholesteryl ester mass secretion remained constant with up to 15% of total cholesteryl ester mass secreted over the 8-h period. ApoB-100 secretion also remained elevated above control, with 92% of the cholesteryl ester secreted associated with apoB-100 particles (27% with d < 1.006 g/mL particles and 65% with d 1.006-1.063 g/mL particles). Therefore, not only newly synthesized cholesteryl ester but also stored cholesteryl ester can associate with newly synthesized apoB-100 molecules and can be secreted without the necessity of an intracellular hydrolysis/re-esterification step.