Linoleic Acid-Enriched Phospholipids Act through Peroxisome Proliferator-Activated Receptors R To Stimulate Hepatic Apolipoprotein A-I Secretion ² Nihar R. Pandey, ‡ Joanna Renwick, § Ayesha Misquith, ‡ Ken Sokoll, ‡ and Daniel L. Sparks* ,‡,§ Liponex, Inc., 1740 Woodroffe AVenue, Ottawa, Ontario K2G 3R8, Canada, and Lipoprotein and Atherosclerosis Research Group, UniVersity of Ottawa Heart Institute, Ottawa, Ontario K1Y 4W7, Canada ReceiVed August 21, 2007; ReVised Manuscript ReceiVed NoVember 29, 2007 ABSTRACT: A uniquely formulated soy phospholipid, phosphatidylinositol (PI), is under development as a therapeutic agent for increasing plasma high-density lipoprotein (HDL) levels. Soy PI has been shown to increase plasma HDL and apolipoprotein A-I (apoA-I) levels in phase I human trials. Low micromolar concentrations of PI increase the secretion of apoA-I in model human hepatoma cell lines, through activation of G-protein and mitogen-activated protein (MAP) kinase pathways. Experiments were undertaken to determine the importance of the PI head group and acyl chain composition on hepatic apoA-I secretion. Phospholipids with choline and inositol head groups and one or more linoleic acid (LA) acyl chains were shown to stimulate apoA-I secretion by HepG2 cells and primary human hepatocytes. Phospholipids containing two LA groups (dilinoleoylphosphatidylcholine, DLPC) were twice as active as those with only one LA group and promoted a 4-fold stimulation in apoA-I secretion. Inhibition of cytosolic phospholipase A2 with pyrrolidine 1 (10 µM) resulted in complete attenuation of PI- and DLPC-induced apoA-I secretion. Pretreatment with the peroxisome proliferator-activated receptor R (PPARR) inhibitor MK886 (10 µM) also completely blocked PI- and DLPC-induced apoA-I secretion. Hepatic PPARR expression was significantly increased by both PI and DLPC. However, in contrast to that seen with the fibrate drugs, PI caused minimal inhibition of catalytic activities of cytochrome P450 and UGT1A1 enzymes. These data suggest that LA-enriched phospholipids stimulate hepatic apoA-I secretion through a MAP kinase stimulation of PPARR. LA-enriched phospholipids have a greater apoA-I secretory activity than the fibrate drugs and a reduced likelihood to interfere with concomitant drug therapies. Low plasma high-density lipoprotein (HDL) 1 levels and elevated low-density lipoprotein (LDL), cholesterol, and triglycerides (TG) are associated with increased risk of cardiovascular disease (1). Even with aggressive therapy to reduce plasma levels of LDL-C (i.e., statin therapy), sig- nificant residual cardiovascular risk remains (2, 3). Recent focus has shifted to targeting HDL elevation as an adjunctive therapy and have focused on the peroxisome proliferator- activated receptor (PPAR) activation profile of drugs includ- ing fibrates, statins, and niacin drugs (4) by increasing or modifying levels of HDL components, cholesterol, and apoA-I (5). Niacin (nicotinic acid) and fibrates (PPARR agonists) increase HDL cholesterol levels by 20-30% and 10-15%, respectively (reviewed in ref 5), as well as reducing plasma triglyceride levels by ∼30%. The statin drugs effectively reduce plasma triglyceride (TG) and LDL cho- lesterol levels (2, 6), while niacin and fibrates have been used to reduce plasma TG levels and raise HDL levels (7, 8). We previously have shown that soy PI increases apoA-I and HDL-cholesterol levels and decreases plasma triglycer- ides in healthy human subjects (9). The therapeutic effects of PI appeared similar to that of niacin and the fibrate drugs. PI uniquely affects hepatic lipid metabolism in rabbits (10) and in human hepatoma cell systems (11). PPARs comprise a three-member subgroup (R, γ, and /δ) within the nuclear hormone receptor family of ligand- activated transcription factors. Fibrates are considered to be PPAR agonists due to their ligand-specific activation of PPARR and heterodimerization of the 9-cis-retinoic acid receptor RXR and are able to uniquely regulate apolipopro- tein C-III and lipoprotein lipase gene expression, key players ² Studies were supported by a grant from the Heart and Stroke Foundation of Canada and from Liponex Inc. J.R. is supported by a Ph.D. studentship from the National Sciences and Engineering Research Council of Canada. * Corresponding author: University of Ottawa Heart Institute, 40 Ruskin Road, Ottawa, Ontario K1Y 4E9, Canada; tel (613) 761-4822; fax (613) 761-5102; e-mail dsparks@ottawaheart.ca. ‡ Liponex, Inc. § University of Ottawa Heart Institute. 1 Abbreviations: apoA-I, apolipoprotein A-I; cPLA2, cytosolic Ca 2+ - dependent phospholipase A2; CYP, cytochrome P450; DLPC, dilino- leoylphosphatidylcholine; DMSO, dimethyl sulfoxide; EDTA, ethyl- enediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; ERK1/2, extracellular-regulated protein kinases 1 and 2; HDL, high-density lipoprotein; HEPES, N-(2-hydroxyethyl)piperazine-N′- ethanesulfonic acid; LA, linoleic acid; LDL, low-density lipoprotein; MAPK, mitogen-activated protein kinase; PI, phosphatidylinositol; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B (Akt); PKC, protein kinase C; PLC, phospholipase C. PLPC, 1-palmitoyl-2- linoleoylphosphatidylcholine; PMSF, phenylmethanesulfonyl fluoride; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; PPARs, peroxisome proliferator-activated receptors; PPRE, peroxisome proliferator response element; TG, triglyceride; UDPGA, uridine 5′-diphosphoglucuronic acid; UGT, UDP-glucuronosyltransferase. 1579 Biochemistry 2008, 47, 1579-1587 10.1021/bi702148f CCC: $40.75 © 2008 American Chemical Society Published on Web 01/12/2008