A novel series of highly potent benzimidazole-based microsomal triglyceride transfer protein inhibitors.

A series of benzimidazole-based analogues of the potent MTP inhibitor BMS-201038 were discovered. Incorporation of an unsubstituted benzimidazole moiety in place of a piperidine group afforded potent inhibitors of MTP in vitro which were weakly active in vivo. Appropriate substitution on the benzimidazole ring, especially with small alkyl groups, led to dramatic increases in potency, both in a cellular assay of apoB secretion and especially in animal models of cholesterol lowering. The most potent in this series, 3g (BMS-212122), was significantly more potent than BMS-201038 in reducing plasma lipids (cholesterol, VLDL/LDL, TG) in both hamsters and cynomolgus monkeys.

The role of the microsomal triglygeride transfer protein in abetalipoproteinemia.

The microsomal triglyceride transfer protein (MTP) is a dimeric lipid transfer protein consisting of protein disulfide isomerase and a unique 97-kDa subunit. In vitro, MTP accelerates the transport of triglyceride, cholesteryl ester, and phospholipid between membranes. It was recently demonstrated that abetalipoproteinemia, a hereditary disease characterized as an inability to produce chylomicrons and very low-density lipoproteins in the intestine and liver, respectively, results from mutations in the gene encoding the 97-kDa subunit of the microsomal triglyceride transfer protein. Downstream effects resulting from this defect include malnutrition, very low plasma cholesterol and triglyceride levels, altered lipid and protein compositions of membranes and lipoprotein particles, and vitamin deficiencies. Unless treated, abetalipoproteinemic subjects develop gastrointestinal, neurological, ophthalmological, and hematological abnormalities.

Anderson's disease: exclusion of apolipoprotein and intracellular lipid transport genes.

Anderson's disease is a rare, hereditary hypocholesterolemic syndrome characterized by chronic diarrhea, steatorrhea, and failure to thrive associated with the absence of apo B48-containing lipoproteins. To further define the molecular basis of the disease, we studied 8 affected subjects in 7 unrelated families of North African origin after treatment with a low-fat diet. Lipid loading of intestinal biopsies persisted, but the pattern and extent of loading was variable among the patients. Electron microscopy showed lipoprotein-like particles in membrane-bound compartments, the densities (0.65 to 7.5 particles/mu(2)) and the mean diameters (169 to 580 nm) of which were, in general, significantly larger than in a normal fed subject (0.66 particles/mu(2), 209 nm mean diameter). There were also large lipid particles having diameters up to 7043 nm (average diameters from 368 to 2127 nm) that were not surrounded by a membrane. Rarely, lipoprotein-like particles 50 to 150 nm in diameter were observed in the intercellular spaces. Intestinal organ culture showed that apo B and apo AIV were synthesized with apparently normal molecular weights and that small amounts were secreted in lipid-bound forms (density <1.006 g/mL). Normal microsomal triglyceride transfer protein (MTP) and activity were also detected in intestinal biopsies. Segregation analyses of 4 families excluded, as a cause of the disease, significant regions of the genome surrounding the genes for apo AI, AIV, B, CI, CII, CIII, and E, as were the genes encoding 3 proteins involved in intracellular lipid transport, MTP, and fatty acid binding proteins 1 and 2. The results suggest that a factor other than apoproteins and MTP are important for human intestinal chylomicron assembly and secretion.

An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.

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.

Evidence that microsomal triglyceride transfer protein is limiting in the production of apolipoprotein B-containing lipoproteins in hepatic cells.

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 microM) with 5 min of ultraviolet irradiation, or 3.0 microM 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.

Enhanced expression of hepatic acyl-coenzyme A synthetase and microsomal triglyceride transfer protein messenger RNAs in the obese and hypertriglyceridemic rat with visceral fat accumulation.

The liver plays a central role in lipoprotein metabolism. In particular, very-low density lipoprotein (VLDL) is assembled in the hepatocytes and secreted into the blood circulation. The VLDL is then catabolized to low-density lipoprotein by lipoprotein lipase and hepatic triglyceride lipase. Obese subjects, especially those with visceral fat accumulation, are frequently associated with hyperlipidemia, non-insulin-dependent diabetes mellitus (NIDDM), and hypertension. The mechanism of hyperlipidemia in visceral fat obesity has not yet been elucidated. Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model of NIDDM, characterized by obesity with visceral fat accumulation, hyperlipidemia, and late-onset insulin resistance. To elucidate the mechanism of hyperlipidemia observed in OLETF rats, we focused on the production of VLDL by the liver and investigated hepatic messenger RNA (mRNA) levels of microsomal triglyceride transfer protein (MTP), acyl-coenzyme A synthetase (ACS), and apolipoprotein B (apo B), which play important roles in VLDL synthesis and secretion. In 6-week-old OLETF rats, in which insulin resistance had not been manifested, visceral fat weight was already higher and portal free fatty acid (FFA) and VLDL-triglyceride levels were elevated compared with the control rats. Hepatic ACS activity and mRNA levels, and MTP mRNA levels were also increased in OLETF rats, whereas apo B mRNA levels were similar; these results suggest that the enhanced expression of both ACS and MTP genes associated with visceral fat accumulation before developing insulin resistance may be involved in the pathogenesis of hyperlipidemia in obese animal models with NIDDM.

A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase.

The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the ubiquitous multifunctional protein, protein disulfide isomerase, and a unique 97-kDa subunit. Mutations that lead to the absence of a functional 97-kDa subunit cause abetalipoproteinemia, an autosomal recessive disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing lipoproteins. Previous studies of abetalipoproteinemic patient, C.L., showed that the 97-kDa subunit was undetectable. In this report, [35S]methionine labeling showed that this tissue was capable of synthesizing the 97-kDa MTP subunit. Electrophoretic analysis showed two bands, one with a molecular mass of the wild type 97-kDa subunit and the other with a slightly lower molecular weight. Sequence analysis of cDNAs from additional intestinal biopsies showed this patient to be a compound heterozygote. One allele contained a perfect in-frame deletion of exon 10, explaining the lower molecular weight band. cDNAs of the second allele were found to contain 3 missense mutations: His297 --> Gln, Asp384 --> Ala, and Arg540 --> His. Transient expression of each mutant showed that only the Arg540 --> His mutant was non-functional based upon its inability to reconstitute apoB secretion in a cell culture system. The other amino acid changes are silent polymorphisms. High level coexpression in a baculovirus system of the wild type 97-kDa subunit or the Arg540 --> His mutant along with human protein disulfide isomerase showed that the wild type was capable of forming an active MTP complex while the mutant was not. Biochemical analysis of lysates from these cells showed that the Arg to His conversion interrupted the interaction between the 97-kDa subunit and protein disulfide isomerase. Replacement of Arg540 with a lysine residue maintained the ability of the 97-kDa subunit to complex with protein disulfide isomerase and form the active MTP holoprotein. These results indicate that a positively charged amino acid at position 540 in the 97-kDa subunit is critical for the productive association with protein disulfide isomerase. Of the 13 mutant MTP 97-kDa subunit alleles described to date, this is the first encoding a missense mutation.

An inhibitor of the microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells.

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-1H-isoindol-1-o ne (BMS-200150). The similarity of the IC50 for inhibition of bovine MTP-mediated TG transfer (0.6 microM) to the Kd for binding of BMS-200150 to bovine MTP (1.3 microM) strongly supports that the inhibition of TG transfer is the result of a direct effect of the compound on MTP. BMS-200150 also inhibits the transfer of phosphatidylcholine, however to a lesser extent (30% at a concentration that almost completely inhibits TG and cholesteryl ester transfer). When BMS-200150 is added to cultured HepG2 cells, a human liver-derived cell line that secretes apoB containing lipoproteins, it inhibits apoB secretion in a concentration dependent manner. These results support the hypothesis that transport of lipid, and in particular, the transport of neutral lipid by MTP, plays a critical role in the assembly of apoB containing lipoproteins.

The amino terminus of apolipoprotein B is necessary but not sufficient for microsomal triglyceride transfer protein responsiveness.

Human apolipoprotein (apo) B mediates the formation of neutral lipid-containing lipoproteins in the liver and intestine. The association of apoB with lipid is thought to be promoted by the microsomal triglyceride transfer protein complex. We have reconstituted lipoprotein assembly in an insect cell line that normally does not support this process. Expression of human microsomal triglyceride transfer protein (MTP) and apolipoprotein B48 (apoB48) together enabled Sf-21 insect cells to secrete approximately 60-fold more lipoprotein-associated triacylglycerol than control cells. This dramatic effect demonstrates that effective partitioning of triacylglycerol into the secretory pathway requires an endoplasmic reticulum-associated neutral lipid transporter (provided by MTP) and an apolipoprotein to shuttle the lipid through the pathway. Expression of the human apoB48 gene in insect cells resulted in secretion of the protein product. Including both MTP subunits with apoB48 and oleic acid specifically increased apoB48 secretion 8-fold over individual subunits alone. To assess whether specific regions of apoB are necessary for MTP responsiveness, nine apoB segments were expressed. These included NH2-terminal segments as well as internal and COOH-terminal regions of apoB fused with a heterologous signal sequence. ApoB segments containing the NH2-terminal 17% of the protein were secreted and responded to MTP activity; however, a segment containing only the NH2-terminal 17% of the protein was not significantly responsive to MTP. Segments lacking the NH2 terminus were not MTP-responsive, and five of six of these proteins were trapped intracellularly but, in certain cases, could be rescued by fusion to apoB17. These results suggest that the NH2 terminus of apoB is necessary but not sufficient for MTP responsiveness.

Crystallization of microsomal triglyceride transfer protein from bovine liver.

The microsomal triglyceride transfer protein (MTP) is a heterodimeric lipid transfer protein required for the assembly of plasma very low density lipoproteins in the liver and chylomicrons in the intestine. Bovine MTP was purified by a modification of a previously published procedure and crystals of MTP were grown reproducibly with polyethylene glycol as a precipitant at pH 7.0. MTP crystals, which diffract to Bragg spacings of better than 3.2 A, have the symmetry of space group P2(1)2(1)2(1) with refined lattice constants of a = 88.7, b = 100.9 and c = 201.1 A, with one heterodimer per asymmetric unit.

Microsomal triglyceride transfer protein: a protein complex required for the assembly of lipoprotein particles.

The mechanism of assembly of lipoprotein particles in the lumen of the endoplasmic reticulum is an important but poorly understood biological problem. A knowledge of this process is of great practical importance because possession of elevated levels of lipoproteins is one of the major risk factors for the development of atherosclerosis. This review describes a major advance in the delineation of the mechanisms involved in the assembly and secretion of apolipoprotein-B-containing lipoproteins: the demonstration of a requirement for microsomal triglyceride transfer protein.

Microsomal triglyceride transfer protein (MTP) regulation in HepG2 cells: insulin negatively regulates MTP gene expression.

The microsomal triglyceride transfer protein (MTP) is a heterodimeric lipid transfer protein that is required for the assembly and secretion of apoB-containing lipoproteins. In this study, four factors that modulate lipid and lipoprotein metabolism were tested for their ability to regulate MTP levels in HepG2 cells. Of the factors tested, only insulin (> or = 10(-9) M), and high concentrations of glucose (> 30 mM) were found to decrease MTP large subunit mRNA levels. Oleate and glucagon had no effect on MTP mRNA levels. The insulin effect was dose- and time-dependent and was mediated through the insulin receptor. In addition, insulin also decreased protein disulfide isomerase (the small subunit of MTP) mRNA levels, although to a lesser extent. Due to the slow turnover rate of MTP (t1/2 = 4.4 days), short-term insulin treatment (24 h) did not change MTP activity levels, indicating that the regulation of MTP mRNA levels by insulin is unrelated to insulin's acute inhibition of apoB secretion in HepG2 cells. In summary, MTP mRNA levels are acutely regulated by insulin in HepG2 cells; however, sustained changes in MTP mRNA levels would be required to affect MTP protein levels.

Microsomal triglyceride transfer protein. Specificity of lipid binding and transport.

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.

Identification of two classes of lipid molecule binding sites on the microsomal triglyceride transfer protein.

The gene for the microsomal triglyceride transfer protein (MTP) is defective in subjects with the genetic disease abetalipoproteinemia, indicating that MTP is essential for the assembly of apolipoprotein B containing lipoproteins. In vitro, MTP is a lipid molecule binding protein that catalyzes lipid transport between membranes by a shuttle mechanism. In this study, the lipid binding properties of MTP were examined. MTP was incubated with donor phosphatidylcholine vesicles of varying neutral lipid composition. MTP was subsequently reisolated by ultracentrifugation, and MTP-bound lipid was quantitated. When the triolein content of the vesicles was increased up to 4 mol %, neutral lipid binding to MTP increased proportionately, while phosphatidylcholine binding appeared to remain constant around two molecules per MTP. Using phosphatidylcholine emulsions containing 60 mol % triolein as the donor particles resulted in only a slight increase in triolein binding to MTP. The highest triolein:MTP ratio observed was (0.20-0.25):1. Differences in the neutral and phospholipid binding properties of MTP were observed by measuring the transport of lipid from MTP to acceptor vesicles. Transport of triolein was rapid and complete, while phosphatidylcholine transport was biphasic, containing rapid and slow phases. These results indicated that MTP contains more than one class of lipid molecule binding site. Measurements of fluorescent lipid transport from donor vesicles to MTP supported this hypothesis. The transport of pyrene-labeled triglyceride from donor particles to MTP was rapid, while phosphatidylcholine transfer had fast and slow phases. From these data, we propose that MTP contains at least two distinct classes of lipid molecule binding sites that differ in function. The fast site or sites are responsible for lipid transport.

Cloning and regulation of hamster microsomal triglyceride transfer protein. The regulation is independent from that of other hepatic and intestinal proteins which participate in the transport of fatty acids and triglycerides.

Microsomal triglyceride transfer protein (MTP) is a heterodimer consisting of protein disulfide isomerase and a unique large subunit. Recent studies showing that an absence of MTP is a cause of abetalipoproteinemia indicate that MTP is required for the assembly of very low density lipoproteins in the liver and chylomicrons in the intestine. In this study, complementary DNA encoding the large subunit of hamster MTP was cloned. The cDNA sequence was used to design a 50-base pair oligonucleotide probe for a solution hybridization assay to quantitate MTP large subunit mRNA levels in a study of MTP regulation in male Syrian Golden hamsters. In animals fed a low fat diet, MTP exhibited a proximal to distal gradient of expression in the intestine. MTP activity and large subunit mRNA levels in the liver were about 25 and 10% that found in the proximal intestine, respectively. To investigate the effect of diet on MTP, hamsters were maintained for 31 days on one of four diets: 1) control low fat, 2) high fat, 3) low fat, high sucrose, or 4) diet 1 followed by a 48-h fast. The high fat diet increased MTP large subunit mRNA levels in the liver and throughout the small and large intestine. A 55 and 126% increase was observed in the liver and intestine (duodenum and jejunum), respectively. A 40% increase of intestinal MTP protein mass was also observed. The high sucrose diet caused a significant 55% increase in hepatic MTP mRNA levels but did not significantly affect the intestinal mRNA levels. MTP mRNA levels were unchanged in response to fasting. A short term dietary study showed that intestinal MTP mRNA was up-regulated within 24 h after initiating a high fat diet. An acute hepatic response was not observed. The regulation of MTP mRNA levels by high fat diets was compared to that of the liver fatty acid binding protein (L-FABP) and apolipoprotein B (apoB). ApoB mRNA levels were not significantly affected by a high fat diet. Although L-FABP mRNA levels were increased in the liver and intestine, the onset of the changes did not parallel that of MTP. These results suggest that L-FABP, apoB, and MTP, three proteins which play important roles in the transport of fatty acids and triglyceride in the liver and intestine, are not coordinately regulated by diet in hamsters.

Human microsomal triglyceride transfer protein large subunit gene structure.

Microsomal triglyceride transfer protein (MTP) is a heterodimer consisting of the multifunctional enzyme protein disulfide isomerase and a unique, large 97-kDa subunit. MTP is required for the assembly and secretion of very low density lipoproteins and chylomicrons by the liver and intestine, respectively. In vitro, MTP catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between phospholipid surfaces. We have characterized the gene encoding the large subunit of human MTP. It contains 18 exons and spans approximately 55-60 kb. Fluorescent in situ hybridization localized this gene to band 4q24 of chromosome 4. A (CA)n repeat polymorphic marker, which may be useful for investigating a link between the MTP gene and genetic defects in lipid metabolism, was identified in intron 10. Sequence analysis of the 5' flanking region of the gene revealed potential sites which may bind transcriptional factors and control MTP expression.

The molecular basis of abetalipoproteinemia.

Abetalipoproteinemia is a recessive genetic disease in humans characterized by the virtual absence of apolipoprotein (apo)B and apoB-containing lipoproteins in plasma. Microsomal triglyceride transfer protein (MTP), a resident lipid transfer protein within the endoplasmic reticulum of hepatocytes and enterocytes, has been shown to be absent in enterocytes from subjects with this disease. MTP is a heterodimer of a unique large subunit and protein disulfide isomerase. It has been demonstrated that the absence of MTP in abetalipoproteinemia is secondary to mutations in the gene for the large subunit of MTP. Thus, mutations in the gene for the large subunit of MTP are a cause of abetalipoproteinemia, which indicates that the MTP is a necessary component for the assembly and secretion of apoB-containing lipoproteins from the liver and intestine.

Mechanism of microsomal triglyceride transfer protein catalyzed lipid transport.

The microsomal triglyceride transfer protein (MTP) is found in the lumen of microsomes isolated from liver and intestine. This protein, which catalyzes the transport of neutral lipids between membranes, appears to play an important role in the biogenesis of plasma very low density lipoproteins and chylomicrons. Enzyme kinetic studies were used to investigate the mechanism of MTP-catalyzed lipid transport. Initial rates of [14C]triolein and [14C]cholesteryl oleate transport from donor to acceptor small unilamellar vesicles were determined at varying donor and acceptor membrane concentrations. Results using two different kinetic analyses demonstrated lipid transport was best described by ping-pong bi-bi kinetics, indicating that MTP is a lipid binding protein which shuttles triglyceride and cholesteryl ester molecules between membranes. This model for lipid transport was supported by a fluorescent lipid binding assay in which MTP was able to extract pyrene-labeled cholesteryl ester from a vesicle. MTP-membrane interactions and lipid transport were regulated by membrane surface charge. Equilibrium gel filtration chromatography demonstrated MTP has a higher affinity for neutrally charged membranes than negatively charged membranes. In agreement with the membrane binding studies, MTP-mediated lipid transfer was inhibited by increasing the concentration of negatively charged phospholipid molecules in donor membranes.

Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia.

Abetalipoproteinemia is a human genetic disease that is characterized by a defect in the assembly or secretion of plasma very low density lipoproteins and chylomicrons. The microsomal triglyceride transfer protein (MTP), which is located in the lumen of microsomes isolated from the liver and intestine, has been proposed to function in lipoprotein assembly. MTP activity and the 88-kilodalton component of MTP were present in intestinal biopsy samples from eight control individuals but were absent in four abetalipoproteinemic subjects. This finding suggests that a defect in MTP is the basis for abetalipoproteinemia and that MTP is indeed required for lipoprotein assembly.