Previous studies have shown that, in rats, dietary cholesterol increases hepatic synthesis and secretion of triglyceride from preformed fatty acid and from fatty acid synthesized de novo. Clarification of the locus and mechanism of these effects will benefit from identifying a cellular model in which they can be further studied. The human hepatoma cell line, HepG2, retains essential hepatocyte functions relating to secretion of apoB containing lipoproteins. The applicant will determine the effect of cholesterol on fatty acid metabolism in this model. Cells will be deprived of cholesterol by treatment with lovastatin or made cholesterol replete by culture in the presence of human LDL, cholesterol-BSA complex, or mevalonate. The effect of these maneuvers on lipid content and rates of secretion of apoB containing lipoproteins will be determined. The applicant will also determine the effect of cholesterol depletion or repletion on synthesis of glycerolipids and cholesteryl ester from exogenous [1-14C] oleic acid as well as their effects on overall fatty acid uptake and oxidation. The applicant will determine the effects of cholesterol depletion or repletion on the rate of de novo fatty acid synthesis (incorporation of 3H2O into esterified and free fatty acids) and the activity and phosphorylation state of acetyl CoA carboxylase, the rate limiting enzyme for de novo synthesis of fatty acids. The effect of cholesterol content on secretion of newly synthesized lipids will also be determined. The potential clinical significance of these studies is to uncover the locus and mechanism for the development of hypertriglyceridemia concomitant with hypercholesterolemia with inclusion of cholesterol in a diet containing a constant amount of triglycerides.
