Analysis of low-density lipoprotein catabolism by primary cultures of hepatic cells from normal and low-density lipoprotein receptor knockout mice

Low-density lipoproteins (LDL) are taken up by LDL receptor (LDLr)-dependen t and -independent pathways; the role and importance of the latest being le ss well defined. We analyzed the importance of these pathways in the mouse by comparing LDL binding to primary cultures of hepatocytes from LDLr knock out (LDLr KO) and normal C57BL/6J mice. Saturation curve analysis shows tha t I-125-LDL bind specifically to normal and LDLr KO mouse hepatocytes with similar dissociation constants (K-d) (31.2 and 22.9 mu g LDL-protein/ml, re spectively). The maximal binding capacity (B-max) is, however, reduced by 4 8% in LDLr KO mouse hepatocytes in comparison to normal hepatocytes. Conduc ting the assay in the presence of a 200-fold excess of high-density lipopro tein-3 (HDL3) reduced by 39% the binding of I-125-LDL to normal hepatocytes and abolished the binding to the LDLr KO mouse hepatocytes. These data ind icate that in normal mouse hepatocytes, the LDLr is responsible for approxi mately half of the LDL binding while a lipoprotein binding site (LBS), inte racting with both LDL and HDL3, is responsible for the other half. It can a lso be deduced that both receptors/sites have a similar affinity for LDL. T he metabolism of LDL-protein and cholesteryl esters (CE) was analyzed in bo th types of cells. I-125-LDL-protein degradation was reduced by 95% in LDLr KO hepatocytes compared to normal hepatocytes. Comparing the association o f I-125-LDL and H-3-CE-LDL revealed a CE-selective uptake of 35.6- and 22-f old for normal and LDLr KO mouse hepatocytes, respectively. Adding a 200-fo ld excess of HDL3 in the assay reduced by 71% the CE-selective uptake in LD Lr KO hepatocytes and by 96% in normal hepatocytes. This indicates that mou se hepatocytes are able to selectively take up CE from LDL by the LBS, The comparison of LDL-CE association also showed that the LBS pathway provides 5-fold more LDL-CE to the cell than the LDLr. Overall, our results indicate that in mouse hepatocytes, LDLr is almost completely responsible for LDL-p rotein degradation while the LBS is responsible for the major part of LDL-C E entry by a CE-selective uptake pathway. (C) 2000 Elsevier Science B.V. Al l rights reserved.