High-Affinity Uptake and Degradation of Apolipoprotein E Free High-Density Lipoprotein and Low-Density Lipoprotein in Cultured Porcine Hepatocytes

Isolated pig liver membranes contain a specific “lipoprotein binding site” that recognizes low-density lipoprotein (LDL) and apolipoprotein E (apoE) free high-density lipoprotein (HDL) [Bachorik, P. S., Kwiterovich, P. O., & Cooke, J. (1978) Biochemistry 17, 5287–5299]. We report here that a similar site exists in cultured porcine hepatocytes and that it mediates the uptake and degradation of apoE-free HDL. The binding of ¹²⁵I-labeled HDL and ¹²⁵I-labeled LDL (¹²⁵I-HDL and ¹²⁵I-LDL, respectively) at 4 °C and the uptake and degradation of the lipoproteins at 37 °C were time dependent and saturable and were not inhibited by unrelated proteins. Chloroquine (6×10⁻⁵ M) inhibited the degradation of ¹²⁵I-HDL by 76% and of ¹²⁵I-LDL by >99%; leupeptin inhibited the degradation of both lipoproteins by about 25%. ^I25I-HDL binding (4 °C), uptake, and degradation (37 °C) were inhibited by LDL, methyl-LDL, and methyl-HDL about as well as by unlabeled HDL but were unaltered in Pronase-treated cells or in cells that were cultured for 24 h in either lipoprotein-free medium or medium containing HDL or LDL (200 µg/mL). In contrast, these conditions affected the uptake and degradation of ¹²⁵I-LDL disproportionately. HDL and methyl-LDL inhibited ¹²⁵I-LDL uptake by 50% or more but had little effect on degradation. ¹²⁵I-LDL binding was reduced by 12% and degradation by 57% in Pronase-treated cells. Preincubation of the cells with LDL (200 µg/mL) reduced uptake by 35% and degradation by 68%. Similar preincubation with HDL (200 µg/mL) increased ¹²⁵I-LDL degradation by 60% but did not affect ¹²⁵I-LDL uptake. The findings indicated the presence in porcine hepatocytes of at least two distinct sites for lipoproteins. One site resembled the LDL receptor and mediated ¹²⁵I-LDL degradation. A second, Pronase-insensitive site recognized both HDL and LDL. This site mediated almost all of the degradation of ¹²⁵I-HDL but little if any degradation of ¹²⁵I-LDL.