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.