Microsomal triglyceride transfer protein (MTP) is critical for the assembly and secretion of apolipoprotein B (apoB) lipoproteins. Its activity is classically measured by incubating purified MTP or cellular homogenates with donor vesicles containing radiolabeled lipids, precipitating the donor vesicles, and measuring the radioactivity transferred to acceptor vesicles. Here, we describe a simple, rapid, and sensitive fluorescence assay for MTP. In this assay, purified MTP or cellular homogenates are incubated with small unilamellar donor vesicles containing quenched fluorescent lipids (triacylglycerols, cholesteryl esters, and phospholipids) and different types of acceptor vesicles made up of phosphatidylcholine or phosphatidylcholine and triacylglycerols. Increases in fluorescence attributable to MTP-mediated lipid transfer are measured after 30 min. MTP's lipid transfer activity could be assayed using apoB lipoproteins but not with high density lipoproteins as acceptors. The assay was used to measure MTP activity in cell and tissue homogenates. Furthermore, the assay was useful in studying the inhibition of the cellular as well as purified MTP by its antagonists.This new method is amenable to automation and can be easily adopted for large-scale, high-throughput screening. -Athar, H., J. Iqbal, X-C. Jiang, and M. M. Hussain. A simple, rapid, and sensitive fluorescence assay for microsomal triglyceride transfer protein.

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Patients with abetalipoproteinemia, a disease caused by defects in the microsomal triglyceride transfer protein (MTP), do not produce apolipoprotein B-containing lipoproteins. It was hypothesized that small molecule inhibitors of MTP would prevent the assembly and secretion of these atherogenic lipoproteins. To test this hypothesis, two compounds identified in a high-throughput screen for MTP inhibitors were used to direct the synthesis of a highly potent MTP inhibitor. This molecule (compound 9) inhibited the production of lipoprotein particles in rodent models and normalized plasma lipoprotein levels in Watanabe-heritable hyperlipidemic (WHHL) rabbits, which are a model for human homozygous familial hypercholesterolemia. These results suggest that compound 9, or derivatives thereof, has potential applications for the therapeutic lowering of atherogenic lipoprotein levels in humans.

To Recent studies showing that a defect in the MTP is the proximal cause of abetalipoproteinemia indicate that this protein is required for assembly and secretion of apoBcontaining lipoproteins (10, 11). Abetalipoproteinemic patients have only trace amounts of plasma apoB-containing lipoproteins, resulting in extremely low TG and cholesterol levels (12). The cause of this phenotype is a defect in the pathway responsible for assembly and secretion of apoBcontaining lipoproteins since the apoB gene (13, 14) and lipid synthesis (12) have been shown to be normal. Thus, it is clear that MTP is required for the efficient assembly and secretion of apoB-containing lipoprotein particles.Although studies of abetalipoproteinemic patients indicate that MTP is required for the production of plasma lipoproteins containing apoB, the role of MTP in this process remains unclear. Also, it is not known whether MTP is the only tissue-specific factor needed by hepatocytes and enterocytes to synthesize and secrete these particles. To address these issues, apoB-53 was expressed in either a nonlipoprotein-producing cell line (HeLa) or a derivative cell line stably expressing the large subunit ofMTP and MTP activity. These cells were evaluated for their ability to secrete apoBcontaining lipoproteins into the tissue culture medium. tTo whom reprint requests should be addressed. MATERIALS AND METHODS 7628The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Highly active anti-retroviral therapies, which incorporate HIV protease inhibitors, resolve many AIDS-defining illnesses. However, patients receiving protease inhibitors develop a marked lipodystrophy and hyperlipidemia. Using cultured human and rat hepatoma cells and primary hepatocytes from transgenic mice, we demonstrate that protease inhibitor treatment inhibits proteasomal degradation of nascent apolipoprotein B, the principal protein component of triglyceride and cholesterol-rich plasma lipoproteins. Unexpectedly, protease inhibitors also inhibited the secretion of apolipoprotein B. This was associated with inhibition of cholesteryl-ester synthesis and microsomal triglyceride transfer-protein activity. However, in the presence of oleic acid, which stimulates neutral-lipid biosynthesis, protease-inhibitor treatment increased secretion of apolipoprotein B-lipoproteins above controls. These findings suggest a molecular basis for protease-inhibitor-associated hyperlipidemia, a serious adverse effect of an otherwise efficacious treatment for HIV infection.