ApoA-I lipidation in primary mouse hepatocytes. Separate controls for phospholipid and cholesterol transfers.
The liver is the major site of both apolipoprotein A-I (apoA-I) synthesis and ATP-binding cassette transporter A1 (ABCA1) expression. Here, we compare the lipidation with cholesterol and phospholipid of newly synthesized human apoA-I (hapoA-I) using adenoviral vector-mediated endogenous expression or exogenously added hapoA-I in wild type and ABCA1-null hepatocytes. Hepatocytes were labeled with [3H]cholesterol (delivered with LDL or methyl-beta-cyclodextrin), [3H]mevalonate, or [3H]choline. ABCA1 deficiency decreased apoA-I phospholipidation by 80%, but acquisition of de novo synthesized and exogenous cholesterol only decreased by 40-60%. The transfer of de novo synthesized cholesterol to apoA-I was decreased at all time points, but that of exogenously delivered cholesterol was independent of ABCA1 activity at the early time points. Progesterone does not affect apoA-I synthesis or its lipidation but inhibited the early phase of apoA-I cholesterol lipidation in both wild type and ABCA1-null hepatocytes. Fast protein liquid chromatography analysis of medium lipoproteins confirmed that with ABCA1 deficiency, the proportion of secreted high density lipoprotein-associated apoA-I and cholesterol decreased by about 50%. The very low density lipoprotein (VLDL)/LDL size fraction also contained a significant level of cholesterol in ABCA1 deficiency, consistent with the result of immunoprecipitations showing the presence of lipoproteins with both apoA-I and murine apoB. ApoA-I lipidation with newly synthesized cholesterol in ABCA1-null hepatocytes was significantly decreased by brefeldin A and monensin. In conclusion, we demonstrate that: (i) whereas most hepatic phospholipidation of apoA-I is mediated by ABCA1, acquisition of cholesterol depends on active transfer from intracellular compartments by ABCA1-dependent and -independent pathways, both sensitive to progesterone and (ii) there is separate regulation of phospholipid and cholesterol lipidation of apoA-I in hepatocytes.
Zheng
Hui
H
Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Institute, University of Ottawa, Ottawa, Ontario K1Y 4W7, Canada.
Kiss
Robert S
RS
Franklin
Vivian
V
Wang
Ming-Dong
MD
Haidar
Bassam
B
Marcel
Yves L
YL
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism*; D020126:Brefeldin A / Q000494:pharmacology; D002784:Cholesterol / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism*; D047468:Immunoprecipitation; D050356:Lipid Metabolism; D008074:Lipoproteins / Q000378:metabolism; D008075:Lipoproteins, HDL / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008985:Monensin / Q000494:pharmacology; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011374:Progesterone / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D019076:Transgenes
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D008074:Lipoproteins; D008075:Lipoproteins, HDL; D010743:Phospholipids; D020126:Brefeldin A; D011374:Progesterone; D008985:Monensin; D002784:Cholesterol