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.

The microsomal triglyceride (TG) transfer protein (MTP) is a heterodimeric lipid transfer protein that catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between membranes. Previous studies showing that the proximal cause of abetalipoproteinemia is an absence of MTP indicate that MTP function is required for the assembly of the apolipoprotein B (apoB) containing plasma lipoproteins, i.e., very low density lipoproteins and chylomicrons. However, the precise role of MTP in lipoprotein assembly is not known. In this study, the role of MTP in lipoprotein assembly is investigated using an inhibitor of MTP-mediated lipid transport, 2-[1-(3, 3-diphenylpropyl)-4-piperidinyl]-2,3-dihydro-lH-isoindol-1-one (BMS-200150). The similarity of the IC50 for inhibition of bovine MTPmediated TG transfer (0.6 ,iM) Genetic studies (1)(2)(3)(4) have demonstrated that an absence of microsomal triglyceride (TG) transfer protein (MTP) causes abetalipoproteinemia, a disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing plasma lipoproteins. These studies indicate that MTP is required for the production of the apoB containing lipoproteins, very low density lipoproteins and chylomicrons by the liver and intestine. The requirement for MTP for lipoprotein production is further supported by studies using cell lines that are not of hepatic or intestinal origin. When a truncated form of apoB representing 53% of the full-length apoB-100 is expressed in HeLa cells, virtually no apoB is secreted (5). However, when MTP is coexpressed with apoB, apoB is secreted as a lipoprotein particle. Similar findings have been observed in COS cells (6).MTP is found in the lumen of microsomes isolated from liver and intestine (7). The protein purified from bovine liver is a heterodimer consisting of the multifunctional enzyme, protein disulfide isomerase, and a unique, large 97-kDa subunit (8-10). In vitro, MTP A mixture of compound A (62.5 mmol) and Zn dust (438 mmol) in AcOH (250 ml) was heated at reflux for 18 h, cooled to room temperature, filtered through Celite (Aldrich), and concentrated. The residue was dissolved in CH2Cl2 (500 ml), washed with saturated NaHCO3 (2 x 100 ml) and brine (100 ml), dried over MgSO4, and concentrated. The crude product was recrystallized from i-PrOH to yield 2,3-dihydro-2-[1-

In the current paper, we have conducted a detailed characterization of this reaction in test tube, intact cell culture, and animal models. Enzymatically, we found that triacylglycerol (TAG) synthesis from MAG by DGAT1 does not behave according to classic Michaelis-Menten kinetics. At low concentrations of 2-MAG (<50 M), the major acylation product by DGAT1 was TAG; however, increased concentrations of 2-MAG (50 -200 M) resulted in decreased TAG formation. This unique product/substrate relationship is similar to MGAT3 but distinct from DGAT2 and MGAT2. We have also found that XP620 is an inhibitor that selectively inhibits the acylation of MAG by DGAT1 (IC 50 of human DGAT1: 16.6 ؎ 4.0 nM (MAG as substrate) and 1499 ؎ 318 nM (DAG as substrate); IC 50 values of human DGAT2, MGAT2, and MGAT3 are >30,000 nM). Using this pharmacological tool, we have shown that ϳ76 and ϳ89% of the in vitro TAG synthesis initiated from MAG is mediated by DGAT1 in Caco-2 cell and rat intestinal mucosal membranes, respectively. When applied to intact cultured cells, XP620 substantially decreased but did not abolish apoB secretion in differentiated Caco-2 cells. It also decreased TAG and DAG syntheses in primary enterocytes. Last, when delivered orally to rats, XP620 decreased absorption of orally administered lipids by ϳ50%. Based on these data, we conclude that the acylation of acylglycerols by DGAT1 is important for dietary fat absorption in the intestine.Dietary fat absorption in mammals is critical for growth and development. In the small intestine, dietary triacylglycerol (TAG) 2 is first hydrolyzed by pancreatic lipases into free fatty acid and 2-monoacylglycerol (MAG) that are readily taken up by the enterocytes. Upon appearance in the enterocytes, 2-MAG is first acylated by acyl coenzyme A:monoacylglycerol (MGAT) to form diacylglycerol (DAG); DAG is further acylated by acyl coenzyme A:diacylglycerol acyltransferase (DGAT) to re-synthesize TAG. TAG molecules are then packaged with other lipids, such as cholesteryl ester, retinyl ester, and phospholipids to form chylomicron lipoprotein particles, which are ultimately secreted into the lymph to serve as an energy source for the whole body (1-3).Recently, multiple membrane-bound acyltransferases have been identified, which are implicated in the sequential MAG and DAG acylation reactions in the gut. Chronologically, DGAT1 was the first enzyme identified (4). It belongs to the acyl coenzyme A:cholesterol acyltransferase gene family (5), which possesses up to nine transmembrane domains (6). A detailed analysis of DGAT1 knock-out mice, which have been shown to be resistant to a high fat diet-induced obesity (7), revealed that DGAT1 deletion caused a substantial decrease, but not a total ablation, of chylomicron formation following an acute challenge of orally administered fat (8). Subsequently, DGAT2 and its homologues were identified as a seven-member gene family (9). Although DGAT1 and DGAT2 are capable of catalyzing the same DGAT enzyme reaction, they bear little sequence resemblan...

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Microsomal triglyceride transfer protein (MTP) is a lipid transfer protein that is required for the assembly and secretion of very low density lipoproteins by the liver and chylomicrons by the intestine. To further elucidate the nature of the lipid molecule binding and transport site on MTP, we have studied the relative rates at which MTP transports different lipid species. Assay conditions were chosen in which there were minimal changes in the physical properties of the substrate membranes so that transfer rates would reflect MTP-lipid interactions at a membrane surface. Lipid transport rates decreased in order of triglyceride > cholesteryl ester > diglyceride > cholesterol > phosphatidylcholine. Changes in the hydrophobic nature of a lipid molecule by the addition of a fatty acid, modulated the ability of MTP to transport it. Addition of one acyl chain from diglyceride to triglyceride, lysophosphatidylcholine to phosphatidylcholine, or cholesterol to cholesteryl ester increased the rate of MTP-mediated transport 10-fold. In contrast, the lipid transport rate was insensitive to the changes in the structure or charge of the polar head group on phospholipid substrates. Zwitterionic, net negative, or net positive charged phospholipid molecules were all transported at a comparable rate. The ability of MTP to transport lipids is strongly correlated to the binding of these lipids to MTP. Thus, MTP has a specific preference for binding and transporting nonpolar lipid compared with phospholipids, and within a class of lipid molecules, a decrease in polarity increases its tendency to be transported.

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The microsomal triglyceride transfer protein (MTP) is a heterodimeric lipid transfer protein that is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins. A key unresolved question is whether the MTP-mediated step is rate limiting. To address this, a unique experimental strategy was used that allowed the in situ modulation and measurement of MTP triglyceride transfer activity. In order to accomplish this, an irreversible photoaffinity inhibitor, BMS-192951, was designed and synthesized. When incubated with purified MTP and irradiated with UV light at 360 nm, BMS-192951 inhibits triglyceride transfer by covalently binding to the protein. HepG2 cells were treated with either increasing concentrations of BMS-192951 (0-15 m ) with 5 min of ultraviolet irradiation, or 3.0 m BMS-192951 with various lengths (0-15 min) of ultraviolet irradiation. Microsomal extracts were prepared exhaustively dialyzed to remove unbound inhibitor, and assayed for MTP-mediated triglyceride transfer activity. BMS-192951 was shown to reduce MTP activity in both a dose-and UV exposure time-dependent fashion. Measurement of apoB concentration in the media showed that apoB secretion was reduced in proportion to the in situ inhibition of MTP activity, while no change was observed in apoA-I secretion. Experiments performed in McArdle RH-7777 rat hepatoma cells and primary rat hepatocytes gave nearly identical results; the decrease in apoB secretion was proportional to the decrease in MTP activity. These results indicate that MTP-mediated lipid transfer is limiting in the assembly and secretion of apoB-containing lipoproteins in hepatic cells under the conditions tested.