Donald Schouten

Stimulation of the LDL receptor activity in the human hepatoma cell line Hep G2 by high-density serum fractions

The regulation of the LDL receptor activity in the human hepatoma cell line Hep G2 was studied. In Hep G2 cells, in contrast with fibroblasts, the LDL receptor activity was increased 2.5-fold upon increasing the concentration of normal whole serum in the culture medium from 20 to 100% by volume. Incubation of the Hep G2 cells with physiological concentrations of LDL (up to 700 micrograms/ml) instead of incubation under serum-free conditions resulted in a maximum 2-fold decrease in LDL receptor activity (10-fold decrease in fibroblasts). Incubation with physiological concentrations of HDL with a density of between 1.16 and 1.20 g/ml (heavy HDL) resulted in an approximately 7-fold increase in LDL receptor activity (1.5-fold increase in fibroblasts). This increased LDL receptor activity is due to an increase in the number of LDL receptors. Furthermore, simultaneous incubation of Hep G2 cells with LDL and heavy HDL (both 200 micrograms/ml) resulted in a 3-fold stimulation of the LDL receptor activity as compared with incubation in serum-free medium. 3-Hydroxy-3-methylglutaryl-CoA reductase activity was also stimulated after incubation of Hep G2 with heavy HDL (up to 3-fold). The increased LDL receptor activity in Hep G2 cells after incubation with heavy HDL was independent of the action of lecithin:cholesterol acyltransferase during that incubation. However, previous modification of heavy HDL by lecithin:cholesterol acyltransferase resulted in an enhanced ability of heavy HDL to stimulate the LDL receptor activity. Our results indicate that in Hep G2 cells the heavy HDL-mediated stimulation of the LDL receptor activity overrules the LDL-mediated down-regulation and raises the suggestion that in man the presence of heavy HDL and the action of lecithin:cholesterol acyltransferase in plasma may be of importance in receptor-mediated catabolism of LDL by the liver.

Characterization in vitro of interaction of human apolipoprotein E-free high density lipoprotein with human hepatocytes.

Characterization of the interaction of iodinated apolipoprotein (apo) E-free high density lipoprotein (HDL) with cultured human hepatocytes provides evidence for a saturable, Ca2(+)-independent, high affinity binding site with an apparent km value of 20 micrograms/ml of apolipoprotein. Nitrated HDL and low density lipoprotein (LDL) did not compete for the binding of HDL, in contrast to very low density lipoprotein (VLDL). It is suggested that VLDL competition is exerted by the presence of apo Cs. Degradation of HDL was relatively low and in some cases not detectable. In cases where degradation was found, inhibitors of the lysosomal pathway of protein degradation had no effect, while LDL degradation was inhibited more than 80%. In the presence of 10 microM of monensin, the cell-association of HDL was unaffected, but the degradation was inhibited by 30%. Under similar conditions, LDL association was inhibited by 40% and LDL degradation, by 90%. Incubation of human hepatocytes with fluorescently labeled HDL (Dil-HDL) revealed (in contrast to Dil-LDL) mainly strong membrane-bound fluorescence and hardly any labeling of small intracellular vesicles. It is concluded that human hepatocytes possess a specific high affinity site for human HDL with recognition properties similar to those described earlier on rat hepatocytes. No evidence that the binding of HDL is actively coupled to uptake and lysosomal degradation could be obtained, indicating that binding of LDL and HDL to human hepatocytes is coupled differently to intracellular pathways.

High-affinity uptake and degradation of acetylated low density lipoprotein by confluent human vascular endothelial cells.

Confluent human vascular endothelial cells take up and degrade acetylated low density lipoproteins (Ac-LDL) via a high-affinity binding process, comparable to that for native LDL. The degradation of 125I-labelled Ac-LDL by endothelial cells was inhibited by the addition of unlabelled Ac-LDL but not by the addition of unlabelled LDL. Similarly, the degradation of 125I-labelled LDL could be inhibited by unlabelled LDL but not by unlabelled Ac-LDL. Unlabelled apolipoprotein E-free HDL did not compete with the degradation of either 125I-labelled LDL or Ac-LDL. Electron microscopical studies, using gold-labelled LDL and gold-labelled Ac-LDL, showed that at 4 degrees C both LDL and Ac-LDL bind to indented regions of the endothelial cell plasma membrane. At 37 degrees C both LDL and Ac-LDL were taken up and associated with lysosomes. Although morphologically identical, we conclude that the binding of LDL and Ac-LDL to human endothelial cells proceeds via 2 different high-affinity receptors. The uptake and degradation of Ac-LDL by human endothelial cells was about 25 and 15%, respectively, of that of native LDL. The uptake and degradation of either LDL or Ac-LDL did not lead to a massive increase in cellular cholesterol content.