Evidence for Prolonged Cell-Surface Contact of Acetyl-LDL Before Entry Into Macrophages

Abstract

Abstract Acetyl-LDL stimulates acyl-CoA:cholesterol acyltransferase (ACAT) much more effectively than LDL in mouse peritoneal macrophages. Previous work with another potent ACAT stimulator, β-VLDL, suggested that atherogenic lipoproteins may use internalization pathways distinct from that of LDL. Brief incubation of fluorescently labeled acetyl-LDL and LDL followed by a short chase period without lipoproteins was used to compare endocytic pathways. LDL was delivered rapidly to perinuclear vesicles, corresponding to late endosomes and lysosomes. A substantial fraction (>40%) of acetyl-LDL was initially retained in the cell periphery, while the rest was rapidly delivered to late endosomes that also contained LDL. Fluorescence of peripheral 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)–acetyl-LDL could be quenched by TNBS, indicating accessibility of the peripheral acetyl-LDL to the extracellular space. Quantification of fluorescence intensities demonstrated that >40% of the cell-associated DiI–acetyl-LDL but only about 10% of DiI-LDL fluorescence was quenchable by TNBS after a 3-minute chase. Fucoidin can efficiently displace DiI–acetyl-LDL bound to cells at 0°C. DiI–acetyl-LDL in the TNBS-quenchable peripheral compartments, however, was resistant to fucoidin. Electron microscopy of colloidal gold–acetyl-LDL showed that acetyl-LDL on the cell surface was often associated with microvilli or ruffles. After clearance from the surface, the peripheral acetyl-LDL was also delivered to the late endosomes and lysosomes. These results indicate that a substantial portion of acetyl-LDL enters macrophages through a pathway that initially differs from that of LDL. This pathway involves a prolonged retention of acetyl-LDL on the plasma membrane. This surface retention may affect ACAT activation in macrophages.