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J. Biol. Chem., Vol. 280, Issue 22, 21612-21621, June 3, 2005

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ApoA-I Lipidation in Primary Mouse Hepatocytes

SEPARATE CONTROLS FOR PHOSPHOLIPID AND CHOLESTEROL TRANSFERS^*

Hui Zheng, Robert S. Kiss, Vivian Franklin, Ming-Dong Wang, Bassam Haidar, and Yves L. Marcel¶

From the Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Institute, Departments of Biochemistry, Microbiology, and Immunology, and of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario K1Y 4W7, Canada

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--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.

Received for publication, February 25, 2005 , and in revised form, March 25, 2005.

^* This work was supported in part by Canadian Institutes of Health Research Grant 44359 (to Y. L. M.) and Group Grant 64519 and by an academic grant in aid from Pfizer Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The recipient of a scholarship from the Ontario Graduate Student Science and Technology Award.

Supported by postdoctoral fellowships from the Heart and Stroke Foundation of Canada.

^¶ To whom correspondence should be addressed: University of Ottawa Heart Institute, 40 Ruskin St., Ottawa, Ontario K1Y 4W7, Canada. Tel.: 613-761-5254; Fax: 613-761-5281; E-mail: ylmarcel{at}ottawaheart.ca.

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