High-affinity uptake and degradation of ApoE-free high-density lipoprotein and low-density lipoprotein in cultured porcine hepatocytes

Abstract

Isolated pig liver membranes contain a specific "lipoprotein binding site" that recognizes low-density lipoproteins (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 degrees C and the uptake and degradation of the lipoproteins at 37 degrees C were time dependent and saturable and were not inhibited by unrelated proteins. Chloroquine (6 x 10(-5)M) inhibited the degradation of 125I-HDL by 76% and of 125I-LDL by greater than 99%; leupeptin inhibited the degradation of both lipoproteins by about 25%. 125I-HDL binding (4 degrees C), uptake, and degradation (37 degrees 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 containing HDL or LDL (200 micrograms/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 micrograms/mL) reduced uptake by 35% and degradation by 68%. Similar preincubation with HDL (200 micrograms/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.

Reversible high affinity uptake of apo E-free high density lipoproteins in cultured pig hepatocytes.

“…20 The LDL contained no detectable apo A-1. The HDL was treated by heparin-Sepharose affinity chromatography 27 to remove possible traces of apo B-and apo E-containing lipoproteins.…”

“…Heparin-Sepharose-treated HDL was examined by SDS-polyacrylamide gel electrophoresis and contained no apo B or apo E. The major apoprotein was apo A-1, M r 25,500; the minor apoproteins were a rapidly migrating component, possibly apo C, and traces of two unidentified proteins, M r 66,500 and 58,000, noted previously. 20 The lipoproteins were sterilized by ultrafiltration (0.45 /x/filters) and were stored at 4° C. Porcine lipoprotein-deficient serum (LPDS, d > 1.25 g/ml) was dialyzed, sterilized, and stored at -70° C until used.…”

“…125 I-HDL was cleared at least 54-fold faster, and 125 I-LDL at least 309-fold faster than ^-sucrose. 20 This observation suggests that although the high affinity catabolism of both lipoproteins occurred readily, 125 I-HDL was degraded more slowly than 125 I-LDL. This might result from differences in the ease with which the two lipoproteins reached the lysosomes or to differences in their rates of proteolysis once they had entered the lysosomes.…”

“…We called the site the "lipoprotein binding site" because it recognized both lipoproteins. 20 During those studies, we compared clearance rates for 12S I-HDL and 125 I-LDL with that of 1 4C-sucrose, which was used to measure the rate of bulk-phase pinocytosis. 125 I-HDL was cleared at least 54-fold faster, and 125 I-LDL at least 309-fold faster than ^-sucrose.…”

Novel Effects of the Acyl-Coenzyme A:Cholesterol Acyltransferase Inhibitor 58-035 on Foam Cell Development in Primary Human Monocyte–Derived Macrophages

“…23 Unbound 125 I was removed by dialysis against 7 changes (4 L each) of 0.15 mol/L NaCl containing 0.05% (wt/vol) Na 2 EDTA, pH 7.5. The labeled lipoprotein was then acetylated as described above and dialyzed against 4 changes of 0.15 mol/L NaCl-Na 2 EDTA buffer, pH 7.5.…”