The Lipidation by Hepatocytes of Human Apolipoprotein A-I Occurs by Both ABCA1-dependent and -independent Pathways

Robert S Kiss,Dan C Mcmanus,Vivian Franklin,Wei Ling Tan,Andrea Mckenzie,Giovanna Chimini,Yves L Marcel

doi:10.1074/jbc.m300137200

Abstract

The pathways of hepatic intra- and peri-cellular lipidation of apolipoprotein A-I (apoA-I) were studied by infecting primary mouse hepatocytes from either apoA-I-deficient or ABCA1-deficient mice with a recombinant adenovirus expressing the human apoA-I (hapoA-I) cDNA (endo apoA-I) or incubating the hepatocytes with exogenously added hapoA-I (exo apoA-I) and examining the hapoA-I-containing lipoproteins formed. The cells, maintained in serum-free medium, were labeled with [(3)H]choline, and the cell medium was separated by fast protein liquid chromatography or immunoprecipitated to quantify labeled choline phospholipids specifically associated with hapoA-I. With the apoA-I-deficient hepatocytes, the high density lipoprotein fraction formed with endo apoA-I contained proportionally more phospholipids than that formed with exo apoA-I. However, the lipoprotein size and electrophoretic mobility and phospholipid profiles were similar for exo apoA-I and endo apoA-I. Taken together, these data demonstrate that a significant proportion of hapoA-I is secreted from hepatocytes in a phospholipidated state but that hapoA-I is also phospholipidated peri-cellularly. With primary hepatocytes from ABCA1-deficient mice, the expression and net secretion of adenoviral-generated endogenous apoA-I was unchanged compared with control mice, but (3)H-phospholipids associated with endo apoA-I and exo apoA-I decreased by 63 and 25%, respectively. The lipoprotein size and electrophoretic migration and their phospholipid profiles remained unchanged. In conclusion, we demonstrated that intracellular and peri-cellular lipidation of apoA-I represent distinct and additive pathways that may be regulated independently. Hepatocyte expression of ABCA1 is central to the lipidation of newly synthesized apoA-I but also contributes to the lipidation of exogenous apoA-I. However, a significant basal level of phospholipidation occurs in the absence of ABCA1.

Highlights

The pathways of hepatic intra- and peri-cellular lipidation of apolipoprotein A-I were studied by infecting primary mouse hepatocytes from either apoAI-deficient or ABCA1-deficient mice with a recombinant adenovirus expressing the human apoA-I cDNA or incubating the hepatocytes with exogenously added hapoA-I and examining the hapoA-I-containing lipoproteins formed
ABCA1 deficiency significantly reduced the proportion of apoA-I found in the high density lipoprotein (HDL) fraction, reduced the proportion of lipid associated with the HDL fraction, and significantly reduced the total 3H-phospholipids released from hepatocytes, but not the amount of apoA-I secreted, compared with control hepatocytes
We found that HDL formed by endogenously synthesized apoA-I was more profoundly reduced by ABCA1 deficiency than that formed by exogenously added apoA-I

Summary

Introduction

The pathways of hepatic intra- and peri-cellular lipidation of apolipoprotein A-I (apoA-I) were studied by infecting primary mouse hepatocytes from either apoAI-deficient or ABCA1-deficient mice with a recombinant adenovirus expressing the human apoA-I (hapoA-I) cDNA (endo apoA-I) or incubating the hepatocytes with exogenously added hapoA-I (exo apoA-I) and examining the hapoA-I-containing lipoproteins formed. With primary hepatocytes from ABCA1-deficient mice, the expression and net secretion of adenoviral-generated endogenous apoA-I was unchanged compared with control mice, but 3H-phospholipids associated with endo apoA-I and exo apoA-I decreased by 63 and 25%, respectively. ABCA1 is expressed in many tissues and at high levels in liver, brain, and small intestine, and testis, lung, spleen, and kidney [17,18,19] This suggests that in both liver and intestine, where apoA-I synthesis is high, ABCA1 may contribute to the lipidation of newly secreted or nascent lipoproteins. We characterized the nascent lipoproteins formed by primary hepatocytes cultured in lipoprotein-free medium compared with those formed by interaction of exogenous apoA-I with the same cells In both conditions hepatocytes generate a lipidated pool of apoA-I-containing lipoproteins via a pathway dependent on ABCA1. The lipidation of apoA-I is reduced but not abolished in experiments with ABCA1-deficient hepatocytes, suggesting the existence of alternate lipidation pathways

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