Abstract
This study aimed to examine whether expression of human hepatic lipase (hHL) exerted an intracellular effect on hepatic production of apolipoprotein (apo) A-I. The levels of secreted and cell-associated apoA-I were contrasted between primary hepatocytes isolated from Lipc-null and C57BL/6 mice, and between Lipc-null hepatocytes transfected with either hHL-encoding or control adenovirus. An HSPG-binding deficient hHL protein (hHLmt) was used to determine the impact of cell surface binding on HL action. Accumulation of apoA-I in conditioned media of primary hepatocytes isolated from Lipc-null mice was increased as compared to that from C57BL/6 mice. Metabolic labeling experiments showed that secretion of 35S-apoA-I from Lipc-null cells was significantly higher than that from C57BL/6 cells. Expression of hHL in Lipc-null hepatocytes, through adenovirus-mediated gene transfer, resulted in decreased synthesis and secretion of 35S-apoA-I, but not 35S-apoE, as compared with cells transfected with control adenovirus. Expression of HSPG-binding deficient hHLmt in Lipc-null cells also exerted an inhibitory effect on apoA-I production, even though hHLmt displayed impaired exit from the endoplasmic reticulum as compared with hHL. Subcellular fractionation revealed that expression of hHL or hHLmt led to increased microsome-association of apoA-I relative to non-transfected control. Expression of hHL negatively impacts hepatic production of apoA-I.
Highlights
A strong inverse correlation exists between the level of plasma high density lipoprotein (HDL)-associated cholesterol and the incidence of coronary heart disease[1]
A dual function has been ascribed to human hepatic lipase (hHL) - it acts as a triacylglycerol (TAG) hydrolase and phospholipase, and acts as a ligand for cell surface anchorage/uptake of various lipoproteins[5]
Structure-function analyses with HL and lipoprotein lipase (LPL) chimeric proteins suggested that the carboxyl-terminal domain of HL was important for HSPG-binding[15], and more recent systematic analysis of hHL showed that HSPG-binding domains span amino acids 301 through 320 and the carboxyl-terminal amino acids 465 through 476[16]
Summary
A strong inverse correlation exists between the level of plasma high density lipoprotein (HDL)-associated cholesterol and the incidence of coronary heart disease[1]. Deficiency of HL in humans is characterized by an elevation in plasma concentrations of cholesterol and TAG as well as large, buoyant HDL particles[ . Infection of Lipc-null mice with hHL-encoding adenovirus resulted in decreased cholesterol, TAG, phospholipids, HDL-cholesterol and apoA-I[11,12]. Mature hHL (476 amino acids) secreted from hepatocytes is mainly associated with HSPG on the cell surface[13] whereas secreted mouse HL is mostly blood borne[13,14]. A chimeric hHL protein (designated hHLmt), in which the carboxyl terminus of hHL (amino acid residues 406 through 476) was replaced with the corresponding mouse sequence, exhibited reduced HSPG binding activity and yet retained catalytic activity[13]. Expression of the HSPG-binding deficient hHLmt in C57BL/6 mice resulted in decreased apoA-I, HDL-cholesterol, and HDL-phospholipid in pre-heparin plasma relative to hHL-expressing mice[17]
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