Abstract
Atheroma macrophages accumulate large amounts of free cholesterol (FC) as well as cholesteryl ester (CE). An important adaptive response to FC loading might be increased cellular phospholipid to accommodate the excess FC. To explore this idea, J774 macrophages were incubated for 48 h without lipid, with acetyl-low density lipoprotein to induce mostly CE loading, or with acetyl-low density lipoprotein plus an acyl-CoA:cholesterol O-acyltransferase inhibitor (58035) to induce marked FC loading. The total phospholipid content approximately doubled in FC-loaded versus control or CE-loaded macrophages, with phosphatidylcholine showing the largest increase (approximately 2.5-fold versus control). Electron micrographs revealed the presence of multiple intracellular membrane whorls in the FC-loaded macrophages but not in the control or CE-loaded macrophages. [3H]Choline incorporation into phosphatidylcholine was also greater in FC-loaded macrophages versus control or CE-loaded macrophages, whereas [3H]phosphatidylcholine degradation was similar in all of the macrophages. In these experiments and in others that used non-lipoprotein cholesterol, there was a very close correlation between cellular FC content and phosphatidylcholine biosynthesis. To determine the mechanism of increased phosphatidylcholine synthesis, FC-loaded and CE-loaded macrophages were pulsed with [3H]choline, then chased and assayed for labeled phosphatidylcholine biosynthetic precursors. The only major differences were a 2-fold greater disappearance of label from [3H]choline phosphate and a 5-fold greater appearance of label in CDP-[3H]choline in the FC-loaded macrophages. These data suggest a stimulation of CTP:phosphocholine cytidylyltransferase (CT), which was confirmed by microsomal CT assays. Further studies revealed that the increase in phosphatidylcholine biosynthesis in FC-loaded macrophages was: (a) reversible under conditions of high density lipoprotein3-mediated cellular cholesterol efflux; (b) not blocked by cycloheximide-induced protein synthesis inhibition; and (c) not associated with increased CT mRNA levels. Thus, FC loading of macrophages leads to an increase in phosphatidylcholine mass which is caused by increased phosphatidylcholine biosynthesis. The mechanism appears to be FC-mediated post-translational activation of CT. This adaptive response may be important for atheroma macrophage survival, and disruption of the response may lead to macrophage necrosis and lesion progression.
Highlights
Atheroma macrophages accumulatlearge amounts of important for atheroma macrophagesurvival, and disfree cholesterol(FC)as well as cholesterylester (CE).An ruption of the responsemaylead to macrophage necrosis important adaptiveresponse to FC loading might be ina-nd lesion progression
( b )not blocked by cycloheximide-induced protein syn- we show that FC loading of cultured macrophages thesis inhibition;and (c)not associated with increased leads to a marked increase in the cellular content of phospho
Known to up-regulate cellular phosphatidylcholine biosynthesis [54,55,56] and which might be expected to be increased in aminedthe effect of FC loading on phosphatidylcholine macrophages incubated withacetyl-LDL plus 58035
Summary
Time courseof incorporation of [%]choline into [%]phosphatidylcholine(panel A), [SHlcholine uptake and phosphorylation (panelB ) ,and choline and choline phosphate pool siz(epsanel C)in control and cholesterol-loaded macrophagePsa. nel A, monolayers of 5774 macrophages were incubated fo2r4 h in DMEM,10% LPDS alone(opencircles) or medium containing pg of 58035/m1(closed circles)or 50 pg of acetyl-LDWml (open triangles)or acetyl-LDL plus 58035(closed triangles). Known to up-regulate cellular phosphatidylcholine biosynthesis [54,55,56] and which might be expected to be increased in aminedthe effect of FC loading on phosphatidylcholine macrophages incubated withacetyl-LDL plus 58035. That in these cells, as with 5774 macrophages, FC loading by Consistent with thperevious data, phosphatidylcholine biosynincubation with acetyl-LDL plus 58035 was associated with an thesis wasincreased -%fold in thecells incubated withacetylincrease inphosphatidylcholine biosynthesis.
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