Effects of chylomicron remnants and P-VLDL on

The regulation of lipoprotein secretion in the cell line HepG2 was studied. HepG2 cells were preincubated with chylomicron remnants (triglycerideand cholesterol-rich) or with beta very low density lipoproteins (P-VLDL) (cholesterolrich). The medium was removed and the cells were incubated for an additional 24 hr in a lipoprotein-free medium that contained either [2-3H]glycerol or DL-[2-’H]mevdondte. Cells and media were harvested, and lipoproteins were separated and fractionated. The mass and radioactivity of the lipids in cells and in the lipoproteins were measured. The activities of cellular acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl CaA (HMG-CoA) reductase were also determined. Preincubation with chylomicron remnants induced an increase in cellular triglyceride and stimulated both HMG-CoA reductase and ACAT. Preincubation with P-VLDL induced an increase in cellular free and esterified cholesterol, inhibited HMG-CoA reductase and stimulated ACAT. Although the absolute amount of VLDL is small, chylomicron remnants induced large relative increases in the amount of triglyceride and phospholipid secreted in VLDL and decreases in the amount of triglyceride secreted in low density (LDL) and high density (HDL) lipoproteins as well as a decrease in the amount of phospholipid secreted in HDL. IR contrast, preincubation with P-VLDL did not affect triglyceride secretion, but markedly stimulated the amount of phospholipid secreted in HDL. I Comparison of the mass of glycerolipid actually secreted with that calculated from the cellular specific activity suggested that glycerolipids are secreted from single, rapidly equilibrating pools. Cholesterol and cholesteryl ester secretion were affected differently. Preincubation with chylomicron remnants increased the amount of free cholesterol secreted in both VLDL and LDL, but did not alter cholesteryl ester secretion. Preincubation with P-VLDL increased free cholesterol secretion in all lipoprotein fractions and increased cholesteryl ester secretion in VLDL and LDL, but not HDL. Comparison of isotope and mass data suggested that the cholesteryl ester secreted came primarily from a preformed, rather than a newly synthesized, pool. In summary, these data provide insight to the mechanism whereby a liver cell regulates the deposition of exogenous lipid.-Craig, W. Y., and A. D. Cooper. Effects of chylomicron remnants and P-VLDL on the class and composition of newly secreted lipoproteins by HepG2 cells. J. Lipid Rcs. 1988. 29: 299-308. Supplementary key words liver triglyceride cholesterol phospholipid The factors influencing the relative amounts and composition of different lipoproteins secreted by the liver are largely unknown. Davis et al. (1) suggested that apoB synthesis limits the rate of lipoprotein secretion and that the cellular composition of neutral lipids determines the core composition of secreted hepatic very low density lipoprotein (VLDL). Miller and Lane (2) suggested that the lipid content of VLDL secreted by cultured chick hepatocytes determines the apoprotein content. Thus, understanding the determinants of the lipid content of lipoproteins is potentially of fundamental importance in understanding how serum lipoprotein and lipid levels are determined. It is difficult, if not impossible, to study the regulation of nascent lipoprotein secretion in vivo. Accordingly, a variety of model systems have been used. The isolated perfused liver has provided much information about the composition and characteristics of nascent lipoprotein, but this system is not readily manipulated. More recently, cultured animal hepatocytes have proved useful in elucidating some basic aspects of lipoprotein formation. Most recently, the human hepatoma-derived cell line HepG2 has been used to gain insights into the nature of the lipoproteins secreted by human liver. Although one must be cautious about extrapolating to normal liver, it has been shown that these cells synthesize apoA-I, apoE and apoB (3, 4). The synthesis of apoA-I (5) is under hormonal regulation. The accumulation of a high density lipoproteinlike particle in the media of HepG2 cells has been documented (6). In previous work from this laboratory, Abbreviations: VLDL, very low density lipoproteins; HDL, high density lipoproteins; LDL, low density lipoproteins; MEM, minimum essential medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; TLC, thin-layer chromatography; ACAT, acyl-CoA:cholesterol acyltransferase; HMG, 3-hydroxy-3-methylglutaryl; MVA, mevalonic acid; LCAT, lecithin: cholesterol acyltransferase. ‘Present address: Foundation for Blood Research, PO. Box 190, Scarborough, ME 04074. Journal of Lipid Research Volume 29, 1988 299 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 0, 2013 w w w .j.org D ow nladed fom the ability of the cells to secrete VLDL-like particles and a triglyceride-rich low density lipoprotein (LDL) particle was reported (7), while in other work, we found that the synthesis of apoB, apoE, and apoA-I could be induced by loading the cells with cholesterol-containing lipoproteins The purpose of the present work was to examine the determinants of lipoprotein lipid composition secreted by HepG2 and to assess whether specific pools of lipids were utilized preferentially. To study the effect of modulating cellular lipid levels on lipoprotein lipid secretion, we have investigated the effects of incubating HepG2 cells with lipoproteins of defined lipid composition. Chylomicron remnants are rich in triglyceride as well as cholesterol and cholesteryl ester while P-VLDL contain predominately cholesteryl ester, although both particles have similar apoprotein components. Our data demonstrate that lipid substrate supply is indeed important in determining both the composition and amount of hepatic lipoproteins secreted. Loading HepG2 cells with chylomicron remnants caused a redistribution of glycerolipid secretion to favor production of VLDL-like particles. When 0-VLDL were delivered to the cells, the secretion of free cholesterol and cholesteryl ester in lipoprotein was increased, and their distribution seemed to be independently regulated. Moreover, preformed, rather than newly synthesized cholesterol, was preferentially secreted. (8). MATERIALS AND METHODS