Binding properties of high-density lipoprotein subfractions and low-density lipoproteins to rabbit hepatocytes

Abstract

Primary cultures of rabbit hepatocytes which were preincubated for 20 h in a medium containing lipoproteindeficient serum subsequently bound, internalized and degraded 125I-labeled high-density lipoproteins 2 (HDL 2). The rate of degradation of HDL 2 was constant in incubations from 3 to 25 h. As the concentration of HDL 2 in the incubation medium was increased, binding reached saturation. At 37° C, half-maximal binding ( K m ) was achieved at a concentration of 7.3 μg of HDL 2 protein/ml (4.06·10 −8 M) and the maximum amount bound was 476 ng of HDL 2 protein/mg of cell protein. At 4°C, HDL 2 had a K m of 18.6 μg protein/ml (1.03·10 −7 M). Unlabeled low-density lipoproteins (LDL) inhibited only at low concentrations of 125I-labeled HDL 2. Quantification of 125I-labeled HDL 2 binding to a specific receptor (based on incubation of cells at 4°C with and without a 50-fold excess of unlabeled HDL) yielded a dissociation constant of 1.45·10 − M. Excess HDL 2 inhibited the binding of both 125I-labeled HDL 2 and 125I-labeled HDL 3, but excess HDL 3 did not affect the binding of 125I-labeled HDL 3. Preincubation of hepatocytes in the presence of HDL resulted in only a 40% reduction in specific HDL 2 receptors, whereas preincubation with LDL largely suppressed LDL receptors. HDL 2 and LDL from control and hypercholesterolemic rabbits inhibited the degrActation of 125I-labeled HDL 2, but HDL 3 did not. Treatment of HDL 2 and LDL with cyclohexanedione eliminated their capacity to inhibit 125I-labeled HDL 2 degradation, suggesting that apolipoprotein E plays a critical role in triggering the degradative process. The effect of incubation with HDL on subsequent 125I-labeled LDL binding was time-dependent: a 20 h preincubation with HDL reduced the amount of 125I-labeled LDL binding by 40%; there was a similar effect on LDL bound in 6 h but not on LDL bound in 3 h. The binding of 125I-labeled LDL to isolated liver cellular membranes demonstrated saturation kinetics at 4°C and was inhibited by EDTA or excess LDL. The binding of 125I-labeled HDL 2 was much lower than that of 125I-labeled LDL and was less inhibited by unlabeled lipoproteins. The binding of 125I-labeled HDL 3 was not inhibited by any unlabeled lipoproteins. EDTA did not affect the binding of either HDL 2 or HDL 3 to isolated liver membranes. Hepatocytes incubated with [2- 14C]acetate in the absence of lipoproteins incorporated more label into cellular cholesterol, nonsaponifiable lipids and total cellular lipid than hepatocytes incubated with [2- 14C]acetate in the presence of any lipoprotein fraction. However, the level of 14C-labeled lipids released into the medium was higher in the presence of medium lipoproteins, indicating that the effect of those lipoproteins was on the rate of release of cellular lipids rather than on the rate of synthesis.