Human Microsomal Triglyceride Transfer Protein Research Articles

17β-Estradiol-Induced Conformational Changes of Human Microsomal Triglyceride Transfer Protein: A Computational Molecular Modelling Study.

Human microsomal triglyceride transfer protein (hMTP) plays an essential role in the assembly of apoB-containing lipoproteins, and has become an important drug target for the treatment of several disease states, such as abetalipoproteinemia, fat malabsorption and familial hypercholesterolemia. hMTP is a heterodimer composed of a larger hMTPα subunit and a smaller hMTPβ subunit (namely, protein disulfide isomerase, hPDI). hPDI can interact with 17β-estradiol (E2), an endogenous female sex hormone. It has been reported that E2 can significantly reduce the blood levels of low-density lipoprotein, cholesterol and triglyceride, and modulate liver lipid metabolism in vivo. However, some of the estrogen’s actions on lipid metabolism are not associated with estrogen receptors (ER), and the exact mechanism underlying estrogen’s ER-independent lipid-modulating action is still not clear at present. In this study, the potential influence of E2 on the stability of the hMTP complex is investigated by jointly using multiple molecular dynamics analyses based on available experimental structures. The molecular dynamics analyses indicate that the hMTP complex in the presence of E2 has reduced interface contacts and surface areas. A steered molecular dynamics analysis shows that the forces required to separate the two subunits (namely, hPDI and hMTPα subunit) of the hMTP complex in the absence of E2 are significantly higher than the forces required to separate the complex in which its hPDI is already bound with E2. E2 makes the interface between hMTPα and hPDI subunits more flexible and less stable. The results of this study suggest that E2-induced conformational changes of the hMTP complex might be a novel mechanism partly accounting for the ER-independent lipid-modulating effect of E2.

Abstract 540: Very Low Density Lipoprotein Assembly is Required for cAMP Responsive Element-binding Protein H Processing and Hepatic Apolipoprotein A-IV Expression in Mouse Models of Acute Steatosis

Background: Our previous studies have demonstrated that hepatic expression of apolipoprotein A-IV (apoA-IV) is increased in mouse models of chronic steatosis and is closely correlated with hepatic triglyceride (TG) content. We have also shown that steatosis-induced hepatic apoA-IV gene expression is regulated by processing of the nuclear transcription factor cAMP responsive element-binding protein H (CREBH). Herein, we explored the mechanisms that mediate steatosis-induced CREBH processing. Methods: We measured hepatic CREBH processing, apoA-IV gene expression, and lipid content in several mouse models of attenuated or enhanced VLDL assembly that were subjected to acute steatosis induced by a 16 hour overnight fast or by feeding a ketogenic diet for 6 days. Results: Both fasting and the ketogenic diet induced acute hepatic TG accumulation associated with increased CREBH processing and apoA-IV gene expression, which were associated with hepatic TG content in C57BL/6 mice. All mouse models of attenuated VLDL secretion (shRNA-induced apoB knock down, liver specific microsomal triglyceride transfer protein (MTP) knockout, treatment with the MTP inhibitor BMS212122, and comparative gene identification-58 (CGI-58) deficiency) had increased hepatic TG accumulation, but displayed repressed CREBH processing and reduced apoA-IV gene expression compared to controls. When deficient VLDL assembly in liver-specific MTP knockout mice was reconstituted by adenoviral infection with a human MTP transgene, steatosis-induced CREBH processing and apoA-IV expression were restored. In a mouse model of enhanced VLDL assembly (transgenic overexpression of human MTP), apoA-IV gene expression correlated with bulk hepatic TG accumulation, but not with VLDL secretion rate, indicating that other steatosis-related factors participate in apoA-IV gene regulation. Conclusions: Taken together, these data provide compelling evidence that VLDL assembly and secretion is required for hepatic CREBH processing and enhanced apoA-IV gene expression in the setting of acute steatosis. These data further suggest that CREBH and apoA-IV play central roles in VLDL-mediated hepatic lipid efflux.

Identification of the Flavonoid Luteolin as a Repressor of the Transcription Factor Hepatocyte Nuclear Factor 4α

Hepatocyte nuclear factor 4α (HNF4α) is a nuclear receptor that regulates the expression of genes involved in the secretion of apolipoprotein B (apoB)-containing lipoproteins and in glucose metabolism. In the present study, we identified a naturally occurring flavonoid, luteolin, as a repressor of HNF4α by screening for effectors of the human microsomal triglyceride transfer protein (MTP) promoter. Luciferase reporter gene assays revealed that the activity of the MTP gene promoter was suppressed by luteolin and that the mutation of HNF4α-binding element abolished luteolin responsiveness. Luteolin treatment caused a significant decrease in the mRNA levels of HNF4α target genes in HepG2 cells and inhibited apoB-containing lipoprotein secretion in HepG2 and differentiated Caco2 cells. The interaction between luteolin and HNF4α was demonstrated using absorption spectrum analysis and luteolin-immobilized beads. Luteolin did not affect the DNA binding of HNF4α to the promoter region of its target genes but suppressed the acetylation level of histone H3 in the promoter region of certain HNF4α target genes. Short term treatment of mice with luteolin significantly suppressed the expression of HNF4α target genes in the liver. In addition, long term treatment of mice with luteolin significantly suppressed their diet-induced obesity and improved their serum glucose and lipid parameters. Importantly, long term luteolin treatment lowered serum VLDL and LDL cholesterol and serum apoB protein levels, which was not accompanied by fat accumulation in the liver. These results suggest that the flavonoid luteolin ameliorates an atherogenic lipid profile in vivo that is likely to be mediated through the inactivation of HNF4α.

Construction of New Ligation-Independent Cloning Vectors for the Expression and Purification of Recombinant Proteins in Silkworms Using BmNPV Bacmid System

A ligation independent cloning (LIC) system has been developed to facilitate the rapid and high-efficiency cloning of genes in a Bombyx mori expression system. This system was confirmed by the expression of human microsomal triglyceride transfer protein (hMTP) fused with EGFP in silkworm larvae and pupae. Moreover, hMTP and human protein disulfide isomerase (hPDI) genes were inserted into two LIC vectors harboring gcLINK sequences and were combined by using the LIC through gcLINK sequences. The constructed vector was incorporated into the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid, and injected into silkworm larvae. The expressed hMTP-hPDI complex was purified from the fat bodies of silkworm larvae. This LIC vector system was applied to express the E1, E2, and E3 subunits of human α-ketoglutarate dehydrogenase (KGDH) in silkworm larvae. The expressed proteins were purified easily from fat bodies using three different affinity chromatography steps. The LIC vectors constructed as described in this report allow for the rapid expression and purification of recombinant proteins or their complexes by using the BmNPV bacmid system.

Abstract 431: Impact of Liver-Specific Transgenic Overexpression of Drosophila and Human MTP on Diet-Induced Hepatic Steatosis and Lipoprotein Secretion

Microsomal triglyceride transfer protein (MTP) is essential for the formation of apolipoprotein B (apoB)-containing lipoproteins. Previous results established that invertebrate forms of MTP (e.g., Drosophila ) are capable of phospholipid (PL) transfer, whereas vertebrate forms of MTP (e.g., human) engage in both PL and triglyceride (TG) transfer. To determine, in vivo, whether the PL and TG transfer activities of MTP play different roles in lipoprotein formation and protection from hepatic steatosis, we created B6D2F1 mice that express human MTP (hMTP) or Drosophila MTP (dMTP) under the control of the apoE 5’ proximal promoter and 3’ hepatic control region in the plasmid pLIV11. Immunoblot analysis confirmed that transgenic expression was limited to the liver and that expression of the transgenes did not impact the abundance of endogenous mouse MTP. To assess the ability of MTP to protect mice from hepatic steatosis, dMTP transgenic mice, hMTP transgenic, and wild type (WT) littermates were placed on a high fat (45% from lard) and cholesterol (0.2%) diet for eight weeks. Mice displayed no significant differences in plasma TG or total plasma cholesterol; however, hMTP mice displayed a trend toward reduced liver TG content, indicating that chronic MTP overexpression may protect the liver from hepatic lipid accumulation. Surprisingly, however, a 2-fold increase in liver TG was observed in two separate founder lines of dMTP transgenic mice. H&E staining confirmed an increase in neutral lipid accumulation in mice expressing the dMTP transgene, relative to both WT and hMTP mice. Quantitative PCR analysis did not reveal changes in expression of genes responsible for hepatic lipogenesis or lipid oxidation. These data complement earlier studies using adenovirus-mediated expression, indicating that acute hMTP and dMTP expression in liver-specific MTP knockout mice can reverse hepatic steatosis caused by MTP deficiency. In contrast, chronic hepatic overexpression of dMTP in a wild type background appears to interfere with endogenous lipoprotein formation and secretion. Understanding the basis for this phenotype may provide insights into how the specific lipid transfer and other activities of MTP contribute to apoB lipoprotein formation.

Phospholipid transfer activity of microsomal triglyceride transfer protein produces apolipoprotein B and reduces hepatosteatosis while maintaining low plasma lipids in mice

Microsomal triglyceride transfer protein (MTP), essential for apolipoprotein B (apoB) biosynthesis, evolved as a phospholipid transfer protein and acquired triglyceride transfer activity during a transition from invertebrates to vertebrates. But it is unknown whether MTP directly transfers lipids onto apoB in vivo and, if it does, whether both neutral and polar lipid transfer activities of MTP are critical for lipoprotein assembly. The molecular bases for differences in lipid transfer activities with respect to distinct domains in Drosophila MTP (dMTP) and human MTP (hMTP) are not obvious because both proteins have very similar primary, secondary, and tertiary structures. We used an in vivo approach to delineate physiological significance of these distinct lipid transfer activities by expressing dMTP (transfers phospholipids) and hMTP (transfers phospholipids and triglycerides) orthologs using adenoviruses in liver-specific MTP-deficient (L-MTP(-/-)) mice that have low plasma and high hepatic lipids. Both orthologs improved plasma lipids but plasma triglycerides were lower in dMTP mice due to lower hepatic triglyceride and apoB production. Hepatosteatosis in L-MTP(-/-) mice was ameliorated to similar levels by both. Attenuation of hepatosteatosis upon dMTP expression pertained to enhanced β-oxidation with no changes in lipogenesis. Phospholipid transfer activity of MTP promoted biogenesis of both apoB48 and apoB100-containing very low density lipoprotein in addition to a phospholipid-rich apoB48-containing high-density lipoprotein particle. Triglyceride transfer activity augmented the biosynthesis of triglyceride-rich lipoproteins by increasing the formation of these particles in the lumen of the endoplasmic reticulum. Based on these findings, we posit that the selective inhibition of MTP triglyceride transfer activity might reduce hyperlipidemia while protecting liver from excess lipid accumulation.

Acute suppression of apo B secretion by insulin occurs independently of MTP

Secretion of apolipoprotein (apo) B-containing lipoproteins by the liver depends mainly upon apo B availability and microsomal triglyceride transfer protein (MTP) activity and is subject to insulin regulation. Hepatic MTP mRNA expression is negatively regulated by insulin which correlates with inhibition of apo B secretion suggesting that insulin might suppress apo B secretion through an MTP-dependent mechanism. To investigate this possibility, we examined the acute effect of insulin on hepatic MTP expression and activity levels in vivo utilizing apobec-1−/− mice. Insulin did not significantly alter hepatic MTP mRNA levels or lipid transfer activity 2h following injection, but suppressed expression of genes important in gluconeogenesis. To study the specific role of MTP, we expressed human MTP (hMTP) in primary rat hepatocytes using adenoviral gene transfer. Increased expression of hMTP resulted in a 47.6±17.9% increase in total apo B secreted. Incubation of hepatocytes with insulin suppressed apo B secretion by 50.1±10.8% in cells over-expressing hMTP and by 53.0±12.4% in control transfected hepatocytes. Results indicate that even under conditions of increased hepatic apo B secretion mediated by MTP, responsiveness of hepatocytes to insulin to suppress apo B secretion is maintained.

Pharmacological Characterization of Diethyl-2-({3-dimethylcarbamoyl-4-[(4′-trifluoromethylbiphenyl-2-carbonyl)amino]phenyl}acetyloxymethyl)-2-phenylmalonate (JTT-130), an Intestine-Specific Inhibitor of Microsomal Triglyceride Transfer Protein

Inhibitors of microsomal triglyceride transfer protein (MTP) expressed in the liver and small intestine are potential candidates for lipid-lowering agents. However, inhibition of hepatic MTP could lead to significant safety issues such as fatty liver disease. To develop a specific inhibitor of intestinal MTP, JTT-130 [diethyl-2-({3-dimethylcarbamoyl-4-[(4'-trifluoromethylbiphenyl-2-carbonyl)amino]phenyl}acetyloxymethyl)-2-phenylmalonate], was designed to be rapidly hydrolyzed in the absorption process. Here, we describe JTT-130, an intestine-specific MTP inhibitor, and evaluate its pharmacological properties. In in vitro metabolic stability tests, JTT-130 was readily hydrolyzed during incubation with liver S9 from humans, hamsters, and rats. In an in vitro triglyceride (TG) transfer assay with human intestinal MTP, JTT-130 potently inhibited TG transfer activity with an IC(50) value of 0.83 nM. When orally administered to hamsters, JTT-130 significantly suppressed an increase in chylomicron-TG after olive oil loading at 0.3 mg/kg and above but did not inhibit TG secretion from the liver at doses of up to 1000 mg/kg, indicating an inhibitory action highly specific for the small intestine. In rats orally administered [(14)C]triolein, JTT-130 potently suppressed an increase in blood (14)C radioactivity and increased (14)C radioactivity in the upper small intestine and the intestinal lumen. In hyperlipidemic hamsters fed a high-fat and high-cholesterol diet, repeated dosing with JTT-130 for 2 weeks reduced TG and cholesterol levels in the plasma and TG content in the liver. These results indicated that JTT-130 is a potent inhibitor specific to intestinal MTP and suggested that JTT-130 would be a useful compound for the treatment of dyslipidemia without inducing hepatotoxicity.

Microsomal Triglyceride Transfer Protein Enhances Cellular Cholesteryl Esterification by Relieving Product Inhibition

Cholesteryl ester synthesis by the acyl-CoA:cholesterol acyltransferase enzymes ACAT1 and ACAT2 is, in part, a cellular homeostatic mechanism to avoid toxicity associated with high free cholesterol levels. In hepatocytes and enterocytes, cholesteryl esters are secreted as part of apoB lipoproteins, the assembly of which is critically dependent on microsomal triglyceride transfer protein (MTP). Conditional genetic ablation of MTP reduces cholesteryl esters and enhances free cholesterol in the liver and intestine without diminishing ACAT1 and ACAT2 mRNA levels. As expected, increases in hepatic free cholesterol are associated with decreases in 3-hydroxy-3-methylglutaryl-CoA reductase and increases in ATP-binding cassette transporter 1 mRNA levels. Chemical inhibition of MTP also decreases esterification of cholesterol in Caco-2 and HepG2 cells. Conversely, coexpression of MTP and apoB in AC29 cells stably transfected with ACAT1 and ACAT2 increases cholesteryl ester synthesis. Liver and enterocyte microsomes from MTP-deficient animals synthesize lesser amounts of cholesteryl esters in vitro, but addition of purified MTP and low density lipoprotein corrects this deficiency. Enrichment of microsomes with cholesteryl esters also inhibits cholesterol ester synthesis. Thus, MTP enhances cellular cholesterol esterification by removing cholesteryl esters from their site of synthesis and depositing them into nascent apoB lipoproteins. Therefore, MTP plays a novel role in regulating cholesteryl ester biosynthesis in cells that produce lipoproteins. We speculate that non-lipoprotein-producing cells may use different mechanisms to alleviate product inhibition and modulate cholesteryl ester biosynthesis.

Acquisition of Triacylglycerol Transfer Activity by Microsomal Triglyceride Transfer Protein during Evolution

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of neutral-lipid-rich apolipoprotein B (apoB) lipoproteins. Previously we reported that the Drosophila MTP transfers phospholipids but does not transfer triglycerides. In contrast, human MTP transfers both lipids. To explore the acquisition of triglyceride transfer activity by MTP, we evaluated amino acid sequences, protein structures, and the biochemical and cellular properties of various MTP orthologues obtained from species that diverged during evolution. All MTP orthologues shared similar secondary and tertiary structures, associated with protein disulfide isomerase, localized to the endoplasmic reticulum, and supported apoB secretion. While vertebrate MTPs transferred triglyceride, invertebrate MTPs lacked this activity. Thus, triglyceride transfer activity was acquired during the transition from invertebrates to vertebrates. Within vertebrates, fish, amphibians, and birds displayed 27%, 40%, and 100% triglyceride transfer activity compared to mammals. We conclude that MTP triglyceride transfer activity first appeared in fish and speculate that the acquisition of triglyceride transfer activity by MTP provided for a significant advantage in the evolution of larger and more complex organisms.

Phospholipid Transfer Activity of Microsomal Triacylglycerol Transfer Protein Is Sufficient for the Assembly and Secretion of Apolipoprotein B Lipoproteins

Human microsomal triacylglycerol transfer protein (hMTP) is essential for apolipoprotein B (apoB)-lipoprotein assembly and secretion and is known to transfer triacylglycerols, cholesterol esters, and phospholipids. To understand the relative importance of each lipid transfer activity, we compared the ability of hMTP and its Drosophila ortholog (dMTP) to assemble apoB lipoproteins and to transfer various lipids. apoB48 secretion was induced when co-expressed with either hMTP or dMTP in COS cells, and oleic acid supplementation further augmented secretion without altering particle density. C-terminal epitope-tagged dMTP (dMTP-FLAG) facilitated the secretion of apoB polypeptides in the range of apoB48 to apoB72 but was approximately 50% as efficient as hMTP-FLAG. Comparison of lipid transfer activities revealed that although phospholipid transfer was similar in both orthologs, dMTP was unable to transfer neutral lipids. We conclude that the phospholipid transfer activity of MTP is sufficient for the assembly and secretion of primordial apoB lipoproteins and may represent its earliest function evolved for the mobilization of lipid in invertebrates. Identification of MTP inhibitors, which selectively affect transfer of a specific lipid class, may have therapeutic potential.

Transcriptional suppression of human microsomal triglyceride transfer protein by hypolipidemic insulin sensitizers

Microsomal triglyceride transfer protein (MTP) catalyzes the assembly and secretion of liver triglyceride-rich lipoproteins. The human MTP (hMTP) promoter activity is reported here to be suppressed by HNF-4α ligand antagonists (e.g., Medica analogs) or by PPARγ ligand agonists (e.g., thiazolidinediones), thus accounting for their hypolipidemic activity in humans. Suppression of liver hMTP by Medica analogs or by thiazolidinediones was mediated by the TAAA sequence that serves as non-canonical TATA box of the hMTP core promoter. MTP suppression was evident in the specific context of the wild type hMTP core promoter, but not in the context of the mutated rodent-conforming hMTP core promoter governed by a canonical TATA box conjoined with its proximal (−50/−38)DR-1 element. hMTP suppression by Medica analogs or thiazolidinediones mediated by hMTP TAAA was independent of HNF-4α or PPARγ. hMTP suppression by Medica analogs, but not by thiazolidinediones, was further complemented by inhibition of HNF-4α transcriptional activity transduced by the distal (−83/−70)DR-1 element of hMTP promoter. hMTP promoter activity was unaffected by PPARα activation. Furthermore, in contrast to hMTP, the promoter activity of the rodent-conforming hMTP was robustly activated by Wy-14,643-activated PPARα or by thiazolidinedione-activated PPARγ. Transcriptional activation by PPARα or PPARγ of the rodent-conforming, but not the wild type hMTP gene promoter, resulted from the species-specific context of the respective proximal DR-1 elements. Hence, suppression of hMTP transcription by hypolipidemic insulin sensitizers requires the specific context of hMTP core promoter. In light of the species-specific context of MTP core promoters, the rodent MTP promoter may not substitute for the human promoter when searching for hypolipidemic MTP suppressors.

Intestinal microsomal triglyceride transfer protein in type 2 diabetic and non-diabetic subjects: The relationship to triglyceride-rich postprandial lipoprotein composition

Background Microsomal triglyceride transfer protein (MTP) is responsible for the assembly of the triglyceride-rich lipoproteins (TRLs) and is increased in diabetic animal models. Human intestinal MTP expression has not been previously reported. This study examined the relationship between intestinal MTP gene expression and postprandial TRL composition in diabetic and non-diabetic subjects. Since the MTP promoter region has a sterol response element the effect of statins on intestinal MTP mRNA was analysed. Methods Twenty-seven diabetic and 24 non-diabetic subjects were examined. Duodenal biopsies were taken during gastroscopy and MTP mRNA was measured by RNase protection assay. Postprandial lipoprotein composition was determined. Results Diabetic subjects had significantly higher MTP mRNA than non-diabetic subjects. Statin therapy was associated with lower MTP mRNA in both groups. In the untreated diabetic patients compared to the untreated non-diabetic patients MTP mRNA was 25.0 ± 25.1 amol/μg versus 13.1 ± 5.6 amol/μg total RNA ( p < 0.05). In the statin-treated diabetic group compared to statin-treated non-diabetic group MTP mRNA was 17.7 ± 8.6 amol/μg versus 5.8 ± 4.1 amol/μg total RNA ( p < 0.05). In the whole group there was a positive correlation between the MTP mRNA and postprandial chylomicron cholesterol/B48 ( r = 0.36, p < 0.01). Conclusions This is the first study to demonstrate increased MTP expression in diabetic subjects. MTP mRNA expression was lower in statin-treated patients confirming the suggestion that the insulin and sterol response elements of the MTP gene are important regulators of MTP transcription in diabetes. Our results show that MTP plays a central role in regulating the cholesterol content of the chylomicron particle.

Microsomal Triglyceride Transfer Protein Promotes the Secretion of Xenopus laevis Vitellogenin A1

Vitellogenins (Vtg) are ancient lipid transport and storage proteins and members of the large lipid transfer protein (LLTP) gene family, which includes insect apolipophorin II/I, apolipoprotein B (apoB), and the microsomal triglyceride transfer protein (MTP). Lipidation of Vtg occurs at its site of synthesis in vertebrate liver, insect fat body, and nematode intestine; however, the mechanism of Vtg lipid acquisition is unknown. To explore whether Vtg biogenesis requires the apoB cofactor and LLTP family member, MTP, Vtg was expressed in COS cells with and without coexpression of the 97-kDa subunit of human MTP. Expression of Vtg alone gave rise to a approximately 220-kDa apoprotein, which was predominantly confined to an intracellular location. Coexpression of Vtg with human MTP enhanced Vtg secretion by 5-fold, without dramatically affecting its intracellular stability. A comparison of wild type and a triglyceride transfer-defective form of MTP revealed that both were capable of promoting Vtg secretion, whereas only wild type MTP could promote the secretion of apoB41 (amino-terminal 41% of apoB). These studies demonstrate that the biogenesis of Vtg is MTP-dependent and that MTP is the likely ancestral member of the LLTP gene family.

Oleate-Mediated Stimulation of Microsomal Triglyceride Transfer Protein (MTP) Gene Promoter: Implications for Hepatic MTP Overexpression in Insulin Resistance

Hepatic lipoprotein overproduction in a fructose-fed hamster model of insulin resistance was previously shown to be associated with a significant elevation of intracellular mass of microsomal triglyceride transfer protein (MTP) and elevated plasma levels of free fatty acids (FFA). Here, we further establish that fructose feeding and development of an insulin resistant state result in higher levels of MTP mRNA, protein mass, and lipid transfer activity. MTP protein mass was increased in fructose-fed hamster hepatocytes to 161 +/- 35.8% of control (p < 0.05), while MTP mRNA levels and MTP lipid transfer activity were increased to 147.5 +/- 30.8% (p < 0.05) and 177.5 +/- 14.5% (p < 0.05) of control levels, respectively. To identify underlying mechanisms, we also investigated the potential link between enhanced FFA flux and hepatic MTP gene expression. Direct modulation of MTP gene transcription by fatty acids was investigated by transfecting HepG2 cells with a reporter (luciferase) construct containing various base pair regions of the human MTP promoter including pMTP124 (with the sterol response element (SRE)), pMTP116, pMTP109 and pMTP100 (no SRE), and pMTP124SREKO (SRE sequences mutated). Treatment of HepG2 cells with oleic acid (360 muM) significantly increased luciferase activities in cells transfected with pMTP124 (136.6 +/- 11.0%, p < 0.05) and pMTP124SREKO (153.9 +/- 11.1%, p < 0.01) compared with control. Luciferase activity was also increased in a time and dose-dependent manner in the presence of oleic acid when transfected with pMTP124SREKO but not pMTP109 and pMTP100. Furthermore, long-term oleic acid treatment of HepG2 cells (10 days) induced higher levels of MTP mRNA (p < 0.05) confirming transcriptional stimulation of the MTP gene by oleic acid. In contrast, palmitate, arachidonic acid or linoleic acid did not significantly stimulate pMTP124 or pMTP124SREKO luciferase activity (p > 0.05). These data demonstrate that (1) MTP gene transcription may be directly up-regulated by oleic acid; (2) up-regulation of MTP gene transcription by oleic acid is SRE sequence independent; and (3) the sequence -116 to -109 in the MTP promoter region is essential for oleic acid-mediated stimulation. Stimulation of MTP gene expression may be a novel mechanism by which certain FFAs can induce hepatic lipoprotein secretion in insulin resistant states.

Transcriptional regulation of human microsomal triglyceride transfer protein by hepatocyte nuclear factor-4α

Microsomal triglyceride transfer protein (MTP) catalyzes the assembly of triglyceride (TG)-rich apolipoprotein B-containing liver (e.g., VLDL) and intestinal (e.g., chylomicron) lipoproteins. The human MTP gene promoter is reported here to associate in vivo with endogenous hepatocyte nuclear factor-4alpha (HNF-4alpha) and to be transactivated or transsuppressed by overexpressed or by dominant negative HNF-4alpha, respectively. Human MTP (hMTP) transactivation by HNF-4alpha is accounted for by the concerted activity of distal (-83/-70) and proximal (-50/-38) direct repeat 1 elements of the hMTP promoter that bind HNF-4alpha. Transactivation by HNF-4alpha is specifically antagonized by chicken ovalbumin upstream promoter. Transcriptional activation of hMTP by HNF-4alpha is mediated by HNF-4alpha domains engaged in ligand binding and ligand-driven transactivation and is further complemented by HNF-4alpha/HNF-1alpha synergism that involves the HNF-4alpha activation function 1 (AF-1) domain. hMTP transactivation by HNF-4alpha is specifically inhibited by beta,beta-tetramethyl-hexadecanedioic acid acting as an HNF-4alpha antagonist ligand. hMTP transactivation by HNF-4alpha may account for the activation or inhibition of MTP expression and the production of TG-rich lipoproteins by agonist (e.g., saturated fatty acids) or antagonist [e.g., (n-3) PUFA, hypolipidemic fibrates, or Methyl-substituted dicarboxylic acid (Medica) compounds] HNF-4alpha ligands.

Developmental expression and nutritional regulation of a zebrafish gene homologous to mammalian microsomal triglyceride transfer protein large subunit

The microsomal triglyceride transfer protein (MTP) large subunit is required for the assembly and secretion of apolipoprotein B-containing lipoproteins. We have found a zebrafish mtp homologous gene coding a protein with 54% identity with human MTP large subunit with the most conserved regions distributed in the corresponding predicted alpha-helical and C- and A-sheet domains. In situ hybridizations showed that zebrafish mtp transcripts were distributed in the yolk syncytial layer during early embryogenesis and in anterior intestine and liver from 48 hr postfertilization onward. Real-time quantitative RT-PCR confirmed the developmental regulation and tissue-specificity of mtp expression. A significant pretranslational up-regulation of mtp expression was observed in the anterior intestine after feeding. The nutritional regulation of zebrafish mtp expression observed in the anterior intestine supports the notion that this protein, similar to mammalian MTP large subunit, could be a factor implicated directly or indirectly in large lipid droplets accumulation observed in the fish enterocyte after feeding.

Bile Acid Reduces the Secretion of Very Low Density Lipoprotein by Repressing Microsomal Triglyceride Transfer Protein Gene Expression Mediated by Hepatocyte Nuclear Factor-4

Microsomal triglyceride transfer protein (MTP) is involved in the transfer of triglycerides, cholesterol esters, and phospholipids to newly synthesized apolipoprotein (apo) B. It is therefore essential for lipoprotein synthesis and secretion in the liver and the small intestine. Although several recent experiments have revealed the transcriptional regulation of the MTP gene, little has been revealed to date about hepatocyte nuclear factor-4 (HNF-4)-dependent regulation. We here report that the human MTP gene promoter contains a pair of functional responsive elements for HNF-4 and HNF-1, the latter of which is another target gene of HNF-4. Chromatin immunoprecipitation assays provide evidence that endogenous HNF-4 and HNF-1 can bind these elements in chromatin. In Hep G2 cells overexpression of either a dominant negative form of HNF-4 or small interfering RNAs (siRNAs) against HNF-4 dramatically reduces the activities of both the wild type and the HNF-4 site mutant MTP promoter. This suggests that HNF-4 regulates MTP gene expression either directly or indirectly through elevated HNF-1 levels. When Hep G2 cells were cultured with chenodeoxycholic acid (CDCA), a ligand for the farnesoid X receptor (FXR), mRNA levels for MTP and apo B were reduced because of increased expression of the factor small heterodimer partner (SHP), which factor suppresses HNF-4 activities. Chenodeoxycholic acid, but not a synthetic FXR ligand, attenuated expression of HNF-4, bringing about a further suppression of MTP gene expression. Over time the intracellular MTP protein levels and apo B secretion in the culture medium significantly declined. These results indicate that two nuclear receptors, HNF-4 and FXR, are closely involved in MTP gene expression, and the results provide evidence for a novel interaction between bile acids and lipoprotein metabolism.

CP-346086: an MTP inhibitor that lowers plasma cholesterol and triglycerides in experimental animals and in humans

A microsomal triglyceride transfer protein (MTP) inhibitor, CP-346086, was identified that inhibited both human and rodent MTP activity [concentration giving half-maximal inhibition (IC50) 2.0 nM]. In Hep-G2 cells, CP-346086 inhibited apolipoprotein B (apoB) and triglyceride secretion (IC50 2.6 nM) without affecting apoA-I secretion or lipid synthesis. When administered orally to rats or mice, CP-346086 lowered plasma triglycerides [dose giving 30% triglyceride lowering (ED30) 1.3 mg/kg] 2 h after a single dose. Coadministration with Tyloxapol demonstrated that triglyceride lowering was due to inhibition of hepatic and intestinal triglyceride secretion. A 2 week treatment with CP-346086 lowered total, VLDL, and LDL cholesterol and triglycerides dose dependently with 23%, 33%, 75%, and 62% reductions at 10 mg/kg/day. In these animals, MTP inhibition resulted in increased liver and intestinal triglycerides when CP-346086 was administered with food. When dosed away from meals, however, only hepatic triglycerides were increased. When administered as a single oral dose to healthy human volunteers, CP-346086 reduced plasma triglycerides and VLDL cholesterol dose dependently with ED50s of 10 mg and 3 mg, and maximal inhibition (100 mg) of 66% and 87% when measured 4 h after treatment. After a 2 week treatment (30 mg/day), CP-346086 reduced total and LDL cholesterol and triglycerides by 47%, 72%, and 75%, relative to either individual baselines or placebo, with little change in HDL cholesterol. Together, these data support further evaluation of CP-346086 in hyperlipidemia.

A Drosophila Microsomal Triglyceride Transfer Protein Homolog Promotes the Assembly and Secretion of Human Apolipoprotein B: IMPLICATIONS FOR HUMAN AND INSECT LIPID TRANSPORT AND METABOLISM

The assembly and secretion of triglyceride-rich lipoproteins in vertebrates requires apolipoprotein B (apoB) and the endoplasmic reticulum-localized cofactor, microsomal triglyceride transfer protein (MTP). Invertebrates, particularly insects, transport the majority of their neutral and polar lipids in lipophorins; however, the assembly of lipophorin precursor particles was presumed to be MTP-independent. A Drosophila melanogaster expressed gene sequence (CG9342), displaying 23% identity with human MTP, was recently identified. When coexpressed in COS cells, CG9342 promoted the assembly and secretion of apoB34 and apoB41 (N-terminal 34 and 41% of human apoB). The apoB34-containing particles assembled by human MTP and CG9342 displayed similar peak densities of approximately 1.169 g/ml and similar lipid compositions. However, CG9342 displayed differential sensitivities to two inhibitors of human MTP and low vesicle-based lipid transfer activity, in vitro. In addition, important predicted structural distinctions exist between the human and Drosophila proteins suggesting overlapping but not identical functional roles. We conclude that CG9342 and human MTP are orthologs that share only a subset of functions, consistent with known differences in intracellular and extracellular aspects of vertebrate and invertebrate lipid transport and metabolism.