ApoB in the medium and cell lysate was then immunoprecipitated by an antibody against apoB (Abcam Inc

ApoB in the medium and cell lysate was then immunoprecipitated by an antibody against apoB (Abcam Inc., Cambridge, MA) plus protein A/G-agarose, and precipitates were analyzed by 4% SDS-polyacrylamide gel electrophoresis. by reducing apoB secretion. Accumulating evidence has shown that phospholipid transfer protein (PLTP) plays an important role in the metabolism of lipoproteins (Tall and Lalanne, 2003). PLTP belongs to the family of lipid transfer/lipopolysaccharide-binding proteins, including cholesteryl ester transfer protein (CETP), lipopolysaccharide-binding protein, and bactericidal permeability increasing protein (Tollefson et al., 1988; Day et al., 1994). It has been shown that PLTP facilitates the transfer and exchange of phospholipids between VLDL and HDL (Tall et al., 1985). It also transfers phospholipids between HDL particles, resulting in the conversion of HDL3 into larger and smaller HDL particles (Jauhiainen et al., 1993; Huuskonen et al., 2000). PLTP can also bind several other amphipathic molecules, including -tocopherol, diacylglycerides, cerebrosides, and lipopolysaccharides (Lagrost et al., 1998). Several clinical studies suggest that high plasma PLTP activity is a risk factor for coronary artery disease and a determinant of carotid intima-media thickness in type 2 diabetes mellitus (Schlitt et al., 2003; de Vries et al., 2006). Studies using genetically modified mice strongly suggest that PLTP functions as a proatherogenic factor (Jiang et al., 2001; van Haperen et al., 2002; Yang et al., 2003). Zafirlukast PLTP has complex effects on lipoprotein metabolism and atherogenesis. PLTP deficiency causes a marked decrease in HDL lipids and apoA-I, due to higher catabolism, in both chow and 2-week Western diet-fed mice (Jiang et al., 1999; Kawano et al., 2002). Deletion of PLTP in hyperlipidemic apoE-deficient and human apoB transgenic mouse strains results in reduced LDL, attributable to decreased production of apoB, which was measured by Triton WR1339 injection (Jiang et al., 2001). Atherosclerotic lesion areas are also decreased in PLTP knockout mice in LDL receptor or apoE-deficient or apoB transgenic background, despite decreased HDL (Jiang et al., 2001). The proatherogenic role of PLTP is further supported by results from PLTP transgenic approaches. It has been reported that overexpression of PLTP in hyperlipidemic mouse models increased susceptibility to atherosclerosis, mainly due to reduced HDL (van Haperen et al., 2002, 2008; Yang et al., 2003; Samyn et al., 2008). Elevation of PLTP in adult mice increased VLDL and reduced HDL, leading to aggravation of preexisting atherosclerotic lesions (Moerland et al., 2008). In addition to its function in the circulation, intracellular PLTP F2R has been shown to regulate apoB-containing lipoprotein secretion in murine hepatocytes (Jiang et al., 2001). PLTP deficiency reduces apoB secretion from mouse primary hepatocytes; however, the mechanism remains unknown. Because the previous studies were carried out using hepatocytes isolated from PLTP-deficient mice, it is not known whether it is PLTP activity or PLTP protein per se that plays a role in apoB secretion. To address these questions, in this study we developed compounds that inhibit phospholipid transfer activity of PLTP. We found that inhibition of PLTP activity reduces the secretion of apoB from human hepatoma cells and mouse primary hepatocytes. Moreover, the PLTP inhibitor had no effect on apoB secretion from PLTP-deficient hepatocytes. These studies support the hypothesis that PLTP-mediated phospholipid transfer activity regulates apoB-containing lipoprotein secretion. Materials and Methods Materials 1-Palmitoyl-2-oleoyl-for 2 h at 7C. MTP activity was determined by adding 200 l of buffer containing 5% BSA with either DMSO alone or compounds to a mixture containing 50 l of donor liposomes, 100 l of acceptor liposomes and 150 l of MTP protein. After incubation at 37C for 45 min, triglyceride transfer was terminated by addition of Zafirlukast 300 l of Zafirlukast a 50% (wt/v) DEAE-cellulose suspension in assay buffer. After thorough mixing, the donor liposomes, bound to DEAE-cellulose, were selectively precipitated by centrifugation at 3000for 5 min. An aliquot of.