Human monocyte-derived macrophages can efflux accumulated cholesterol without exogenously added cholesterol acceptors (Kruth, H. S., Skarlatos, S. I., Gaynor, P. M., and Gamble, W. (1994) J. Biol. Chem. 269, 24511-24518). Most of the effluxed cholesterol accumulates in the medium as apolipoprotein E-discoidal lipid particles. In the current study, we determined whether and to what degree cholesterol efflux from human monocytemacrophages depended on apolipoprotein E secretion. Unexpectedly, 2-week-old differentiated monocyte-macrophages secreted similar amounts of apolipoprotein E without or with cholesterol enrichment. Apolipoprotein E mRNA levels in these macrophages were not increased by cholesterol enrichment and were comparable with levels in HepG2 cells. Without cholesterol enrichment, monocyte-macrophages secreted lipid-poor apolipoprotein E with a density >1.21 g/ml. By contrast, cholesterol enrichment of monocyte-macrophages induced the association of apoE with phospholipid and cholesterol to form discoidal particles that floated at densities of 1.08 -1.10 g/ml. An anti-apolipoprotein E monoclonal antibody added to the culture medium significantly inhibited cholesterol and phospholipid efflux from the monocytemacrophages. This showed that apolipoprotein E was required for most of the cholesterol efflux, and that apolipoprotein E did not leave macrophages with lipid but rather associated with lipid after it was secreted. Thus, 1) apolipoprotein E was constitutively secreted by differentiated human monocyte-macrophages, 2) apolipoprotein E only formed discoidal particles following macrophage cholesterol enrichment, 3) apolipoprotein E was necessary for cholesterol efflux to occur in the absence of added cholesterol acceptors and, in addition 4) the level of macrophage unesterified cholesterol was not rate-limiting for this cholesterol efflux, and 5) net phospholipid synthesis occurred in macrophages secondary to apoE-mediated loss of macrophage phospholipid. In conclusion, apolipoprotein E functions in an autocrine pathway that mediates cholesterol efflux from human monocyte-derived macrophages. Apolipoprotein (apo)1 E shows an anti-atherogenic effect by facilitating clearance of atherogenic remnant lipoproteins from the plasma (1, 2). Recent studies suggest that apoE may be anti-atherogenic by additional mechanisms. Transgenic expression of apoE restricted to the blood vessel wall (3) or to macrophages (including those in the vessel wall) (4) inhibits development of atherosclerotic lesions in mice, even without altering plasma cholesterol levels. This suggests that expression of apoE locally within the vessel wall is sufficient to produce an anti-atherogenic effect. One mechanism by which locally produced apoE could be anti-atherogenic is by promoting reverse cholesterol transport from the vessel wall. This could occur through the association of apoE with plasma-derived high density lipoprotein (HDL) which increases HDL's capacity to carry cholesterol (5, 6). On the other hand, it is possible that apoE co...

The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDPdiacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined. Maximum activities were found in the exponential phase of cells grown in complete synthetic medium. As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5-to 5-fold. The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase. When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline. Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline. The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were derepressed. Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture. The phospholipid composition of cells in the exponential and stationary phase of growth was also examined. The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells. The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.

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