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)-dependent and -independent pathways; the role and importance of the latest being less well defined. We analyzed the importance of these pathways in the mouse by comparing LDL binding to primary cultures of hepatocytes from LDLr knockout (LDLr KO) and normal C57BL/6J mice. Saturation curve analysis shows that (125)I-LDL bind specifically to normal and LDLr KO mouse hepatocytes with similar dissociation constants (K(d)) (31.2 and 22.9 microg LDL-protein/ml, respectively). The maximal binding capacity (B(max)) is, however, reduced by 48% in LDLr KO mouse hepatocytes in comparison to normal hepatocytes. Conducting the assay in the presence of a 200-fold excess of high-density lipoprotein-3 (HDL3) reduced by 39% the binding of (125)I-LDL to normal hepatocytes and abolished the binding to the LDLr KO mouse hepatocytes. These data indicate that in normal mouse hepatocytes, the LDLr is responsible for approximately half of the LDL binding while a lipoprotein binding site (LBS), interacting with both LDL and HDL3, is responsible for the other half. It can also be deduced that both receptors/sites have a similar affinity for LDL. The metabolism of LDL-protein and cholesteryl esters (CE) was analyzed in both types of cells. (125)I-LDL-protein degradation was reduced by 95% in LDLr KO hepatocytes compared to normal hepatocytes. Comparing the association of (125)I-LDL and (3)H-CE-LDL revealed a CE-selective uptake of 35.6- and 22-fold for normal and LDLr KO mouse hepatocytes, respectively. Adding a 200-fold excess of HDL3 in the assay reduced by 71% the CE-selective uptake in LDLr KO hepatocytes and by 96% in normal hepatocytes. This indicates that mouse 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-protein degradation while the LBS is responsible for the major part of LDL-CE entry by a CE-selective uptake pathway.
Truong
T Q
TQ
Département des Sciences Biologiques, Université du Québec à Montréal, C.P. 8888, Succ. Centre-ville, Montréal, Que., Canada.
Auger
A
A
Denizeau
F
F
Brissette
L
L
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D002788:Cholesterol Esters / Q000378:metabolism; D008078:Cholesterol, LDL / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D005260:Female; D006801:Humans; D007457:Iodine Radioisotopes; D008075:Lipoproteins, HDL / Q000494:pharmacology; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011973:Receptors, LDL / Q000172:deficiency* / Q000235:genetics; D014316:Tritium
D002788:Cholesterol Esters; D008078:Cholesterol, LDL; D007457:Iodine Radioisotopes; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D014316:Tritium