Low- and high-density lipoprotein metabolism in HepG2 cells expressing various levels of apolipoprotein E.
To determine the importance of hepatic apolipoprotein (apo) E in lipoprotein metabolism, HepG2 cells were transfected with a constitutive expression vector (pRc/CMV) containing either the complete or the first 474 base pairs of the human apoE cDNA inserted in an antisense orientation, for apoE gene inactivation, or the full-length human apoE cDNA inserted in a sense orientation for overexpression of apoE. Stable transformants were obtained that expressed 15, 24, 226, and 287% the apoE level of control HepG2 cells. The metabolism of low-density lipoprotein (LDL) and high-density lipoprotein-3 (HDL(3)), two lipoprotein classes following both holoparticle and cholesteryl esters (CE)-selective uptake pathways, was compared between all these cells. LDL-protein degradation, an indicator of the holoparticle uptake, was greater in low apoE expressing cells than in control or high expressing cells, while HDL(3)-protein degradation paralleled the apoE levels of the cells (r(2) = 0.989). LDL- and HDL(3)-protein association was higher in low apoE expressing cells compared to control cells. In opposition, LDL- and HDL(3)-CE association was not different from control cells in low apoE expressing cells but rose in high apoE expressing cells. In consequence, the CE-selective uptake (CE/protein association ratio) was positively correlated with the level of apoE expression in all cells for both LDL (r(2) = 0.977) and HDL(3) (r(2) = 0.998). We also show that, although in normal and low apoE expressor cells, 92% of LDL- and 80% HDL(3)-CE hydrolysis is sensitive to chloroquine suggesting a pathway linked to lysosomes for both lipoproteins, cells overexpressing apoE lost 60% of chloroquine-sensitive HDL(3)-CE hydrolysis without affecting that of LDL-CE. Thus, the level of apoE expression in HepG2 cells determines the fate of LDL and HDL(3).
Charpentier
D
D
Département des Sciences Biologiques, Université du Québec à Montréal, C.P. 8888, Succ. Centre-ville, Montréal, Québec, Canada.
Tremblay
C
C
Rassart
E
E
Rhainds
D
D
Auger
A
A
Milne
R W
RW
Brissette
L
L
D001057:Apolipoproteins E / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D002788:Cholesterol Esters / Q000378:metabolism; D008076:Cholesterol, HDL / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007424:Intracellular Fluid / Q000378:metabolism; D008075:Lipoproteins, HDL / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D013696:Temperature; D014162:Transfection; D014316:Tritium; D014407:Tumor Cells, Cultured / Q000378:metabolism*
D001057:Apolipoproteins E; D002788:Cholesterol Esters; D008076:Cholesterol, HDL; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D014316:Tritium; D002738:Chloroquine