We studied the effect of inhibition of microsomal triglyceride transfer protein (MTP) on apolipoprotein (apo) B100 translation and secretion using HepG2 cells. The MTP-mediated lipid transfer activity was reduced using a specific MTP inhibitor. ApoB100 translation was synchronized by treatment with puromycin prior to L-[ 35 S]methionine pulse-chase labeling. During the first 4 min of chase, synthesis of apoB polypeptides the size of 100 -200 kDa was insensitive to the inhibitor, suggesting that inhibition of MTP did not affect the initiation of apoB100 translation. After 15 min of chase, the 100 -200-kDa species were chased into polypeptides larger than 320 kDa (i.e. apoB65 or 65% of full-length apoB100) in both control and inhibitor-treated cells. However, the amount of these polypeptides decreased (by 36% for apoB65-75, by 64% for apoB75-85, by 76% for apoB85-95, and by 77% for apoB100) upon MTP inhibition. No accumulation of smaller polypeptides was observed, but total immunoprecipitable apoB radioactivity was decreased suggesting that apoB could undergo cotranslational degradation when MTP activity was reduced. Inhibitors of the multicatalytic proteinase complex (proteasome) such as lactacystin or MG-115 could prevent apoB co-translational degradation. Nevertheless, MG-115 could not avoid the MTP inhibitor decreasing apoB100 secretion but rather induced the accumulation of secretion-incompetent apoB100 in the cell. These results indicate that MTP activity is required during the elongation of apoB100 polypeptides, particularly at the sequences downstream of carboxyl terminus of apoB65. Co-translational degradation might constitute a more general mechanism of early quality control for large or complex proteins.

The concept that selective transfer of high density lipoprotein (HDL)-derived cholesteryl esters (CE) does not require lipoprotein internalization has been challenged recently by evidence that implicates HDL recycling during the selective uptake process. This has prompted us to examine the role of the low density lipoprotein receptor-related protein (LRP) in selective uptake. LRP is an endocytic receptor for lipoprotein lipase (LpL) and apolipoprotein E (apoE) ligands that are able to mediate selective uptake. We report that molecules that interfere with ligand binding to LRP, such as the receptor-associated protein (RAP), suramin, ␣ 2 -macroglobulin, or lactoferrin, inhibit HDL-CE selective uptake by human primary adipocytes and SW872 liposarcoma cells by 35-50%. This partial inhibition of selective uptake from total HDL was not due to preferential inhibition of the HDL 2 or HDL 3 subfractions. Selective uptake by the scavenger receptor BI was not inhibited by RAP, excluding its involvement. Furthermore, in SW872 cells in which LRP was reduced to 14% of control levels by stable antisense expression, selective uptake was attenuated by at least 33%, confirming a role for LRP in this process. RAP, ␣ 2 -macroglobulin, lactoferrin, and suramin (individually or in paired combinations) also attenuated selective uptake of HDL-CE by primary human adipocytes by about 40%. On the other hand, human skin fibroblasts express LRP abundantly but lack the capacity for selective uptake, demonstrating that other molecules are required. In SW872 cells, exogenous apoE or LpL can facilitate selective uptake but only the apoE-enhanced uptake can be inhibited by RAP, implicating apoE as a likely co-mediator. We discuss the possible mechanisms by which the endocytic receptor, LRP, can mediate selective uptake.

The ␣ 2 -macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) is a large multifunctional receptor that interacts with a variety of molecules. It is implicated in biologically important processes such as lipoprotein metabolism, neurological function, tissue remodeling, protease complex clearance, and cell signal transduction. However, the regulation of LRP gene expression remains largely unknown. In this study, we have analyzed 2 kb of the 5-flanking region of the LRP gene and identified a predicted peroxisome proliferator response element (PPRE) from ؊1185 to ؊1173. Peroxisome proliferator-activated receptor ␥ (PPAR␥) ligands such as fatty acids and rosiglitazone increased functional cell surface LRP by 1.5-2.0-fold in primary human adipocytes and in the SW872 human liposarcoma cell line as assessed by activated ␣ 2 -macroglobulin binding and degradation. These agents were found to increase LRP transcription. Gel shift analysis of the putative PPRE demonstrated direct binding of PPAR␥/retinoid X receptor ␣ heterodimers to the PPRE in the LRP gene. Furthermore, these heterodimers could no longer interact with a mutated PPRE probe. The isolated promoter was functional in SW872 cells, and its activity was increased by 1.5-fold with the addition of rosiglitazone. Furthermore, the isolated response element was similarly responsive to rosiglitazone when placed upstream of an ideal promoter. Mutagenesis of the predicted PPRE abolished the ability of this construct to respond to rosiglitazone. These data demonstrate that fatty acids and rosiglitazone directly stimulate transcription of the LRP gene through activation of PPAR␥ and increase functional LRP expression.The ␣ 2 -macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) 1 is a 600-kDa multifunctional endocytic receptor that belongs to the low density lipoprotein receptor gene family (1). LRP binds and internalizes a broad range of biologically diverse ligands. These include proteases of the fibrinolytic pathway (2) and serpin-enzyme complexes (3) as well as proteins important in lipoprotein metabolism such as lipoprotein lipase, hepatic lipase, lipoprotein(a), and apoE-rich lipoproteins (4 -9). Targeted deletion of LRP in the mouse results in early embryonic death, demonstrating a critical function for LRP in prenatal development (10). LRP has also been shown to have a dual role in ␤-amyloid metabolism by enhancing ␤-amyloid precursor protein conversion to ␤-amyloid (11) and mediating the clearance of ␤-amyloid (12, 13). These data support a potentially complex role for LRP in the pathogenesis of Alzheimer's disease (14). In addition, LRP mediates signal transduction by interacting with cytosolic adaptor and scaffold proteins including DAB-1, JIP-2, and PSD-95 (15). A 39-kDa receptor-associated protein (RAP) is an endoplasmic reticulumresident protein that functions intracellularly as a molecular chaperone for LRP and regulates its ligand binding activity (16 -18). RAP is required for the proper folding and export...

Triglycerides (TGs), cholesteryl esters (CEs), cholesterol, and phosphatidylcholine have been independently proposed as playing regulatory roles in apoB-100 secretion; the results depended on the cellular model used. In this study, we reinvestigate the role of lipids in apoB-100 production in HepG2 cells and in particular, we clarify the respective roles of intracellular mass and the biosynthesis of lipids in the regulation of apoB-100 production. In a first set of experiments, the pool size of cholesterol, CEs, and TGs was modulated by a 3-day treatment with either lipid precursors or inhibitors of enzymes involved in lipid synthesis. We used simvastatin (a hydroxymethylglutaryl coenzyme A reductase inhibitor), 58-035 (an acyl coenzyme A cholesterol acyltransferase inhibitor), 5-tetradecyloxy-2-furancarboxylic acid (TOFA, an inhibitor of fatty acid synthesis), and oleic acid. The secretion rate of apoB-100 was not affected by the large modulation of lipid mass induced by these various pre-treatments. In a second set of experiments, the same lipid modulators were added during a 4-hour labeling period. Simvastatin and 58-035 inhibited cholesterol and CE synthesis without affecting apoB-100 secretion. By contrast, treatment of HepG2 cells with TOFA resulted in the inhibition of TG synthesis and apoB-100 secretion. This effect was highly specific for apoB-100 and was reversed by adding oleic acid, which stimulated both TG synthesis and apoB-100 secretion. Moreover, a combination of oleic acid and 58-035 inhibited CE biosynthesis and increased both TG synthesis and apoB-100 secretion. These results show that in HepG2 cells TG biosynthesis regulates apoB-100 secretion, whereas the rate of cholesterol or CE biosynthesis has no effect.

The role of microsomal triacylglycerol transfer protein (MTP) in the secretion of apolipoprotein B-1 00 (apoB-100) has been studied using an inhibitor of MTP: 4'-bromo-3'-methylmetaqualone. In vitro, this compound inhibits trioleoylglycerol transfer between lipid vesicles mediated by MTP with an IC,, of 0.9 pM whereas it does not inhibit the lipid transfer mediated by the cholesteryl ester transfer protein.In HepG2 cells, 4'-bromo-3'-methylmetaqualone inhibits the secretion of apoB-100 with an IC,, of 0.3 pM, without affecting the secretion of several other proteins like apoA-I or albumin. Moreover, there is no accumulation of apoB-100 in treated cells. Oleic acid, which increases apoB-100 secretion, only slightly modifies the IC,, of 4'-bromo-3'-methylmetaqualone (0.5 pM). The latter has no effect on the synthesis of major lipids within the cell, but decreases the secretion of triacylglycerol into apoB-100-containing lipoproteins. Pulsekhase experiments reveal that 4'-bromo-3'-methylmetaqualone acts on apoB-I00 production either at the co-translational or post-translational level. The cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal does not protect apoB-100 from the 4'-bromo-3'-methylmetaqualone effect but seems to be involved in a later step of apoB-100 intracellular degradation. By contrast, dithiothreitol can totally reverse the effect of the MTP inhibitor on apoB-100 production. The mechanism of MTP-mediated lipid assembly with apoB-100 is discussed.Keywords: lipoprotein assembly ; lipid transfer; microsomal triacylglycerol-transfer protein ; N-acetylleucinylleucinyl-2-aminohexanol; hepatocyte.Microsomal triacylglycerol transfer protein (MTP) mediates the transfer of triacylglycerol and cholesteryl ester between membranes 111. MTP displays a clear preference for transporting neutral lipids compared to polar lipids [2]. The transfer activity has only been reported to be present in liver and intestinal mucosa. MTP is a heterodimer containing two polypeptides of molecular masses 58 and 97 kDa. The 58-kDa subunit has been identified as a multifunctional protein, protein disulfide isomerase, whereas the 97-kDa subunit confers lipid transfer activity to the complex 131. In patients with abetalipoproteinemia, a rare human genetic disease characterized by a defect in very low-density lipoprotein (VLDL) and chylomicron production, mutations in the gene encoding the 97-kDa subunit prevent the expression of active MTP [4-71.Apolipoprotein B-100 (apoB-100), the structural protein of VLDL and low-density lipoprotein (LDL) cholesteryl esters are synthesized and assembled in the endoplasmic reticulum to form lipoprotein particles. The particles are subsequently transported to the Golgi apparatus and secreted. However, the sequence of events leading to the formation of mature lipoprotein particles in the lumen of the endoplasmic reticulum is still under investigation (for a review see 1191). Increasing evidence suggests that, at least in some cellular models, several steps of lipidation are required for l...