adipocyte ͉ Cide ͉ diabetes ͉ fat droplet ͉ fat metabolism

Macrophage-derived apoE has been shown to play an important role in the susceptibility of the vessel wall to atherosclerosis. Previous studies have shown that macrophage sterol content modulates apoE synthesis and secretion, associated with a large transcriptional response of the apoE gene. The current studies were undertaken to evaluate the existence of additional posttranscriptional regulatory loci for the effect of sterols on apoE synthesis and secretion. Using a macrophage cell line transfected to constitutively express an apoE cDNA to facilitate detection of a post-transcriptional regulatory locus, we demonstrated that preincubations in 25-hydroxycholesterol and cholesterol lead to increased apoE secretion in pulse/chase experiments. Examination of cell lysates in these experiments showed that apoE not secreted by control cells was degraded and not detectable, suggesting that the preincubation in sterols increased secretion by decreasing degradation of newly synthesized apoE. The measurement of total protein and apoE degradation in cell fractions revealed an intermediate density fraction that degraded significant amounts of newly synthesized total protein and newly synthesized apoE. In this fraction, degradation of total protein and apoE was unaffected by chloroquine but was substantially reduced by N-acetyl-Leu-Leu-norleucinal plus N-acetyl-Leu-Leu-methioninal or by lactacystin, suggesting the involvement of proteasomes. Preincubation in sterol/oxysterol or acetylated low density lipoprotein did not modify total protein degradation by this fraction but inhibited apoE degradation. Similar results were obtained using intermediate density fractions isolated from human monocyte-derived macrophages. The results of our studies indicate that newly synthesized apoE in the macrophage can be degraded in an intermediate density nonlysosomal cellular compartment, which is sensitive to proteasomal inhibitors. Alteration of cellular lipid homeostasis by preincubation in sterol/oxysterol or acetylated low density lipoprotein inhibits apoE, but not total protein, degradation in this fraction. Inhibition of the degradation of apoE in this fraction likely contributes to the increased apoE secretion observed in sterol-enriched cells.

Abstract-Sterol efflux importantly contributes to preservation of cellular cholesterol homeostasis, and multiple pathways may be involved for mediating such efflux. Recently, an important role has been ascribed to ABCA1 in facilitating lipid efflux from cells, including macrophages, to extracellular lipid-free apolipoproteins. Macrophages are relatively unique among cells because they express apoprotein E (apoE) as a major protein product, and this endogenous expression of apoE increases sterol and phospholipid efflux from macrophages. The studies in this article were designed to test whether the sterol efflux mediated by the endogenous expression of apoE in macrophages was dependent on ABCA1 expression. These studies were facilitated by comparing apoE-expressing J774 cells (J774E ϩ ) with nonexpressing parental cells (J774E Ϫ ). Sterol efflux was higher from J774E ϩ cells compared with J774E Ϫ cells, but the increment in efflux between these cell types was not increased by induction of ABCA1 expression with cAMP. Induction of ABCA1 with cAMP, however, did increase sterol efflux to exogenously added apoA1 from both cell types. Inhibitors of ABCA1 activity significantly reduced (by 40% to 50%) sterol efflux from both J774E ϩ and J774E Ϫ cells treated with cAMP and apoA1. This inhibitor did not, however, reduce the increment in sterol efflux due to the expression of endogenous apoE. The results of these studies indicate that the increment in sterol efflux mediated by the endogenous expression of apoE in macrophages does not depend on ABCA1 expression or activity. (Arterioscler Thromb Vasc

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