THE DISTAL PATHWAY OF LIPOPROTEIN-INDUCED CHOLESTEROL ESTERIFICATION,BUT NOT SPHINGOMYELINASE-INDUCED CHOLESTEROL ESTERIFICATION, IS ENERGY-DEPENDENT

The stimulation of the intracellular cholesterol esterification pathwa y by atherogenic lipoproteins in macrophages is a key step in the deve lopment of atheroma foam cells. The esterification pathway can also be stimulated by hydrolysis of cell-surface sphingomyelin by the enzyme sphingomyelinase (SMase). In both cases, intracellular cholesterol tra nsport to the cholesterol esterifying enzyme, acyl-CoA:cholesterol O-a cyltransferase (ACAT), is thought to be critical, although the mechani sm of cholesterol transport is not known. In this report, we explore t wo fundamental properties of the cholesterol esterification pathway, n amely its dependence on energy and the effect of other treatments that block membrane vesicle trafficking. After the atherogenic lipoprotein , beta-very low density lipoprotein (beta-VLDL), was internalized by m acrophages and hydrolyzed in lysosomes, the cells were depleted of ene rgy by treatment with sodium azide and 2-deoxyglucose or by permeabili zation. Under these conditions, which allowed equal beta-VLDL-choleste ryl ester hydrolysis, cholesterol esterification was markedly decrease d in the energy-depleted cells. This effect was not due to blockage of lysosomal cholesterol export. IN the permeabilized cell system, energ y repletion restored beta-VLDL-induced, but not SMase-induced, cholest erol esterification in Chinese hamster ovary cells. Similar experiment s were carried out using N-ethylmaleimide, low potassium medium, or in hibitors of phosphatidylinositol 3-kinase, each of which blocks intrac ellular membrane vesicle trafficking. These treatments also inhibited beta-VLDL-induced, but not SMase-induced, cholesterol esterification. Finally, we show here that SMase treatment of cells leads to an increa se in plasma membrane vesiculation that is relatively resistant to ene rgy depletion. In summary, the stimulation of cholesterol esterificati on by lipoproteins, but not by SMase, is energy-dependent, N-ethylmale imide-sensitive, and blocked by both low potassium and phosphatidylino sitol 3-kinase inhibitors. The affected step or steps are distal to ch olesterol export form lysosomes and not due to direct inhibition of th e ACAT enzyme. Thus, the mechanisms, involved in lipoprotein-induced v ersus SMase-induced cholesterol esterification are different, perhaps due to the involvement of energy-dependent vesicular cholesterol trans port in the lipoprotein pathway and a novel, energy-independent vesicu lar transport mechanism in the SMase pathway.