Apolipoprotein E is resistant to intracellular degradation in vitro and invivo - Evidence for retroendocytosis

Apolipoprotein E (apoE) is an important determinant for the uptake of trigl yceride-rich lipoproteins and emulsions by the liver, but the intracellular pathway of apoE following particle internalization is poorly defined. In t he present study, we investigated whether retroendocytosis is a unique feat ure of apoE as compared with apoB by studying the intracellular fate of ver y low density lipoprotein-sized apoE-containing triglyceride-rich emulsion particles and LDL after LDLr-mediated uptake. Incubation of HepG2 cells wit h [B-3]cholesteryl oleate-labeled particles at 37 degrees C led to a rapid release of [H-3]cholesterol within 30 min for both LDL and emulsion particl es. In contrast, emulsion-derived I-125-apoE was more resistant to degradat ion (greater than or equal to 120 min) than LDL-derived I-125-apoB (30 min) . Incubation at 18 degrees C, which allows endosomal uptake but prevents ly sosomal degradation, with subsequent incubation at 37 degrees C resulted in a time-dependent release of intact apoE from the cells (up to 14% of the e ndocytosed apoE at 4 h). The release of apoE was accelerated by the presenc e of protein-free emulsion (20%) or high density lipoprotein (26%). Retroen docytosis of intact particles could be excluded since little intact [H-3]ch olesteryl oleate was released (<3%). In contrast, the degradation of LDL wa s complete with virtually no secretion of intact apoB into the medium. The intracellular stability of apoE was also demonstrated after hepatic uptake in C57Bl/6 mice. Intravenous injection of I-125-apoE and [H-3]cholesteryl o leate-labeled emulsions resulted in efficient LDLr-mediated uptake of both components by the liver (45-50% of the injected dose after 20 min). At 1 h after injection, only 15-20% of the hepatic I-125-apoE was degraded, wherea s 75% of the [H-3]cholesteryl oleate was hydrolyzed. From these data we con clude that following LDLr-mediated internalization by liver cells, apoE can escape degradation and can be resecreted. This sequence of events may allo w apoE to participate in its hypothesized intracellular functions such as m ediator of the post-lysosomal trafficking of Lipids and very low density li poprotein assembly.