Microsomal Triglyceride Transfer Protein Promoter Research Articles

The atypical antipsychotic quetiapine induces hyperlipidemia by activating intestinal PXR signaling.

Quetiapine, one of the most prescribed atypical antipsychotics, has been associated with hyperlipidemia and an increased risk for cardiovascular disease in patients, but the underlying mechanisms remain unknown. Here, we identified quetiapine as a potent and selective agonist for pregnane X receptor (PXR), a key nuclear receptor that regulates xenobiotic metabolism in the liver and intestine. Recent studies have indicated that PXR also plays an important role in lipid homeostasis. We generated potentially novel tissue-specific PXR-KO mice and demonstrated that quetiapine induced hyperlipidemia by activating intestinal PXR signaling. Quetiapine-mediated PXR activation stimulated the intestinal expression of cholesterol transporter Niemann-Pick C1-Like 1 (NPC1L1) and microsomal triglyceride transfer protein (MTP), leading to increased intestinal lipid absorption. While NPC1L1 is a known PXR target gene, we identified a DR-1-type PXR-response element in the MTP promoter and established MTP as a potentially novel transcriptional target of PXR. Quetiapine's effects on PXR-mediated gene expression and cholesterol uptake were also confirmed in cultured murine enteroids and human intestinal cells. Our findings suggest a potential role of PXR in mediating adverse effects of quetiapine in humans and provide mechanistic insights for certain atypical antipsychotic-associated dyslipidemia.

The flavonoid derivative 4'-nitro-6-hydroxyflavone suppresses the activity of HNF4α and stimulates the degradation of HNF4α protein through the activation of AMPK.

Hepatocyte nuclear factor 4α (HNF4α) is a nuclear receptor that regulates the expression of genes involved in a number of critical metabolic pathways. The modulation of HNF4α activity is thought to be a promising drug target pathway for hyperlipidemia. To identify compounds that reduce the activity of HNF4α, we conducted luciferase reporter assays using the promoter region of microsomal triglyceride transfer protein (MTP) gene, which contains an HNF4α-responsive element. Using this system, we show here that the flavonoid derivative 4'-nitro-6-hydroxyflavone (NOHF) suppresses MTP promoter activity. Treatment with NOHF caused a decrease in the expression of the HNF4α target gene. We also identified that NOHF triggers the AMP-activated protein kinase (AMPK) and accelerates the degradation of HNF4α protein. Knock-down of AMPK diminishes the effect of NOHF. These results indicate that NOHF is an AMPK activator and attenuates the transcriptional activity of HNF4α, at least in part, by accelerating HNF4α protein degradation.

RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP

Multifunctional RNA helicase DDX3 participates in HCV infection, one of the major causes of hepatic steatosis. Here, we investigated the role of DDX3 in hepatic lipid metabolism. We found that HCV infection severely reduced DDX3 expression. Analysis of intracellular triglyceride and secreted ApoB indicated that lipid accumulations were increased while ApoB secretion were decreased in DDX3 knockdown HuH7 and HepG2 cell lines. Down-regulation of DDX3 significantly decreased protein and transcript expression of microsomal triglyceride transfer protein (MTP), a key regulator of liver lipid homeostasis. Moreover, DDX3 interacted with hepatocyte nuclear factor 4 (HNF4) and small heterodimer partner (SHP), and synergistically up-regulated HNF4-mediated transactivation of MTP promoter via its ATPase activity. Further investigation revealed that DDX3 interacted with CBP/p300 and increased the promoter binding affinity of HNF4 by enhancing HNF4 acetylation. Additionally, DDX3 partially relieved the SHP-mediated suppression on MTP promoter by competing with SHP for HNF4 binding which disrupted the inactive HNF4/SHP heterodimer while promoted the formation of the active HNF4 homodimer. Collectively, these results imply that DDX3 regulates MTP gene expression and lipid homeostasis through interplay with HNF4 and SHP, which may also reveal a novel mechanism of HCV-induced steatosis.

FoxO6 Integrates Insulin Signaling With MTP for Regulating VLDL Production in the Liver

Excessive production of triglyceride-rich very low-density lipoproteins (VLDL-TG) contributes to hypertriglyceridemia in obesity and type 2 diabetes. To understand the underlying mechanism, we studied hepatic regulation of VLDL-TG production by (forkhead box O6) FoxO6, a forkhead transcription factor that integrates insulin signaling to hepatic metabolism. We showed that transgenic mice expressing a constitutively active FoxO6 allele developed hypertriglyceridemia, culminating in elevated VLDL-TG levels and impaired postprandial TG clearance. This effect resulted in part from increased hepatic VLDL-TG production. We recapitulated these findings in cultured HepG2 cells and human primary hepatocytes, demonstrating that FoxO6 promoted hepatic VLDL-TG secretion. This action correlated with the ability of FoxO6 to stimulate hepatic production of microsomal triglyceride transfer protein (MTP), a molecular chaperone that catalyzes the rate-limiting step in VLDL-TG assembly and secretion. FoxO6 was shown to bind to the MTP promoter and stimulate MTP promoter activity in HepG2 cells. This effect was inhibited by insulin, consistent with the ability of insulin to promote FoxO6 phosphorylation and disable FoxO6 DNA-binding activity. Mutations of the FoxO6 target site within the MTP promoter abrogated FoxO6-mediated induction of MTP promoter activity. Hepatic FoxO6 expression became deregulated in insulin-resistant mice with obesity and type 2 diabetes. FoxO6 inhibition in insulin-resistant liver suppressed hepatic MTP expression and curbed VLDL-TG overproduction, contributing to the amelioration of hypertriglyceridemia in obese and diabetic db/db mice. These results characterize FoxO6 as an important signaling molecule upstream of MTP for regulating hepatic VLDL-TG production.

Circadian Regulation of Intestinal Lipid Absorption by Apolipoprotein AIV Involves Forkhead Transcription Factors A2 and O1 and Microsomal Triglyceride Transfer Protein

We have shown previously that Clock, microsomal triglyceride transfer protein (MTP), and nocturnin are involved in the circadian regulation of intestinal lipid absorption. Here, we clarified the role of apolipoprotein AIV (apoAIV) in the diurnal regulation of plasma lipids and intestinal lipid absorption in mice. Plasma triglyceride in apoAIV(-/-) mice showed diurnal variations similar to apoAIV(+/+) mice; however, the increases in plasma triglyceride at night were significantly lower in these mice. ApoAIV(-/-) mice absorbed fewer lipids at night and showed blunted response to daytime feeding. To explain reasons for these lower responses, we measured MTP expression; intestinal MTP was low at night, and its induction after food entrainment was less in apoAIV(-/-) mice. Conversely, apoAIV overexpression increased MTP mRNA in hepatoma cells, indicating transcriptional regulation. Mechanistic studies revealed that sequences between -204/-775 bp in the MTP promoter respond to apoAIV and that apoAIV enhances expression of FoxA2 and FoxO1 transcription factors and their binding to the identified cis elements in the MTP promoter at night. Knockdown of FoxA2 and FoxO1 abolished apoAIV-mediated MTP induction. Similarly, knockdown of apoAIV in differentiated Caco-2 cells reduced MTP, FoxA2, and FoxO1 mRNA levels, cellular MTP activity, and media apoB. Moreover, FoxA2 and FoxO1 expression showed diurnal variations, and their expression was significantly lower in apoAIV(-/-) mice. These data indicate that apoAIV modulates diurnal changes in lipid absorption by regulating forkhead transcription factors and MTP and that inhibition of apoAIV expression might reduce plasma lipids.

NR2F1 disrupts synergistic activation of the MTTP gene transcription by HNF-4α and HNF-1α

Regulation of microsomal triglyceride transfer protein (MTP) expression mainly occurs at the transcriptional level. We have previously shown that MTTP gene expression was repressed in nondifferentiated intestinal cells by nuclear receptor 2 family 1 (NR2F1). However, mechanisms involved in the repression of MTP by NR2F1 were not elucidated. Here, we show that MTP expression requires hepatic nuclear factor (HNF)-4α transcription factor. Different HNF-1 proteins synergistically enhance MTP promoter activity along with HNF-4α by binding to different cis elements. NR2F1 does not alter individual effects of HNF-4α and HNF-1 proteins on the MTTP gene promoter. However, NR2F1 suppresses synergistic activation of the MTP promoter by HNF-4α/HNF-1α by binding to a direct repeat 1 (DR1) element. This suppression is further enhanced in the presence of nuclear receptor corepressor 1. In short, these studies identified a novel mechanism of MTP repression that involves binding of NR2F1 to the DR1 element and recruitment of corepressors. In this mechanism, NR2F1 does not affect activities of individual transcription factors; instead, it abrogates synergistic activation by HNF-4α and HNF-1 proteins.

Genetic polymorphisms in non-alcoholic fatty liver disease in obese Egyptian children

Background/Aim:Polymorphisms in the promoter of microsomal triglyceride transfer protein (MTP) lead to decreased MTP transcription, less export of triglyceride from hepatocytes, and greater intracellular triglyceride accumulation. Therefore, functional polymorphisms in MTP may be involved in determining susceptibility to nonalcoholic steatohepatitis (NASH). The aim of this study is to examine the effect of some genetic influences among a group of obese Egyptian children.Patients and Methods:A cross-sectional study was conducted on 76 overweight and obese children presenting to the Pediatric Endocrinology Unit, Cairo University Children's Hospital, Egypt, as well as on 20 healthy controls. Anthropometric measurements were taken for all the patients and they underwent clinical examination, ultrasonographic examination of the liver, and liver biopsy when appropriate. Liver functions, blood glucose, serum insulin, C-peptide, and lipid profile were assessed and HOMA-IR calculated. Blood samples from biopsy-proven NASH patients and controls were analyzed by polymerase chain reaction (PCR) and restriction fragment length polymorphism for the –493 G/T polymorphism in the promoter of MTP and the 1183 T/C polymorphism in the mitochondrial targeting sequence of manganese superoxide dismutase (MnSOD).Results:Eight had biopsy-proven simple steatosis and 7 had NASH. NASH patients had a much higher incidence of the MTP G/G genotype (P = 0.002, CI: 2.9–392) compared with the controls. NASH patients also had a 100% prevalence of the MnSOD T/T genotype.Conclusion:Certain genotypes in MTP and MnSOD are significantly more prevalent among obese children with NASH and may be responsible for such a phenotype.

Diurnal Regulation of MTP and Plasma Triglyceride by CLOCK Is Mediated by SHP

We examined the role of clock genes in the diurnal regulation of plasma triglyceride-rich apolipoprotein B-lipoproteins and their biosynthetic chaperone, microsomal triglyceride transfer protein (MTP). Clock(mt/mt) mice showed sustained hypertriglyceridemia and high MTP expression. CLOCK knockdown activated MTP promoter and reduced small heterodimer partner (SHP, NROB2). CLOCK upregulated SHP by binding to its E box. SHP suppressed MTP expression by binding to the HNF4alpha/LRH-1 at the MTP promoter. Cyclic expression of MTP after serum shock was abrogated by siCLOCK and siSHP. Plasma triglyceride and MTP showed reduced diurnal variations in Shp(-/-) mice. Whereas peaks and nadirs in SHP expression were inversely correlated with those of MTP, these changes were reduced in Clock(mt/mt) mice. Expression of Shp abrogated hypertriglyceridemia in Clock(mt/mt) mice. Together, these studies describe a role of Clock/Shp in the diurnal regulation of MTP and plasma triglyceride and indicate that disruptions in circadian regulation might cause hyperlipidemia.

Hepatocyte nuclear factor 1 binding element within the promoter of microsomal triglyceride transfer protein (MTTP) gene is crucial for MTTP basal expression and insulin responsiveness

Insulin inhibits the transcription of the microsomal triglyceride transfer protein (MTTP), which plays a pivotal role in lipoprotein assembly and secretion. Here, we provide evidence that a hepatocyte nuclear factor 1 binding element (HNF1A element) within the MTTP promoter serves as a novel negative insulin-responsive element. Deletion/mutation mapping of the MTTP gene promoter identified a modified HNF1A element that is crucial to the negative insulin effect. Chimeric promoter containing this HNF1A element and minimal TEAD1 promoter also responded negatively toward insulin treatment. Gel shift assay demonstrated that HNF1A but not HNF1B binds to this element. Enforced expression of HNF1A was sufficient to reconstitute the negative insulin responsiveness of MTTP promoter in TM4SF1 myocytes that are HNF1A negative. Furthermore, replacing this element with consensus HNF1A element preserved the negative insulin response, suggesting that negative insulin responsiveness is a generic characteristic of HNF1A element. Given that many genes implicated in diabetes contain HNF1A element, the potential regulation of these genes by insulin via HNF1A element may provide important clues for the manifestation and treatment of diabetic metabolic syndromes.

FoxO1 mediates insulin-dependent regulation of hepatic VLDL production in mice

Excessive production of triglyceride-rich VLDL is attributable to hypertriglyceridemia. VLDL production is facilitated by microsomal triglyceride transfer protein (MTP) in a rate-limiting step that is regulated by insulin. To characterize the underlying mechanism, we studied hepatic MTP regulation by forkhead box O1 (FoxO1), a transcription factor that plays a key role in hepatic insulin signaling. In HepG2 cells, MTP expression was induced by FoxO1 and inhibited by exposure to insulin. This effect correlated with the ability of FoxO1 to bind and stimulate MTP promoter activity. Deletion or mutation of the FoxO1 target site within the MTP promoter disabled FoxO1 binding and resulted in abolition of insulin-dependent regulation of MTP expression. We generated mice that expressed a constitutively active FoxO1 transgene and found that increased FoxO1 activity was associated with enhanced MTP expression, augmented VLDL production, and elevated plasma triglyceride levels. In contrast, RNAi-mediated silencing of hepatic FoxO1 was associated with reduced MTP and VLDL production in adult mice. Furthermore, we found that hepatic FoxO1 abundance and MTP production were increased in mice with abnormal triglyceride metabolism. These data suggest that FoxO1 mediates insulin regulation of MTP production and that augmented MTP levels may be a causative factor for VLDL overproduction and hypertriglyceridemia in diabetes.

The MTP −493TT genotype is associated with peripheral arterial disease: Results from the Linz Peripheral Arterial Disease (LIPAD) Study

Objectives Microsomal triglyceride transfer protein (MTP) transfers lipids into apoprotein B-containing lipoproteins for secretion from liver, intestine, and heart. We hypothesized the −493T single nucleotide polymorphism in the MTP promoter region to be associated with altered lipoprotein levels and with presence of peripheral arterial disease (PAD). Design and methods 433 patients with symptomatic PAD and 433 controls matched for sex and age from the Linz Peripheral Arterial Disease (LIPAD) study were genotyped cross-sectionally for the −493T single nucleotide polymorphism in the promoter region of the MTP gene. Results The frequency of the −493T allele in patients with PAD was 0.320, whereas it was 0.255 in controls ( p < 0.001). The MTP −493TT genotype was independently associated with PAD, even after adjustment for LDL cholesterol. The odds ratio of the −493TT MTP genotype for PAD was 3.18 (95% CI, 1.76–5.71) when adjusted for current smoking, arterial hypertension, LDL cholesterol, triglycerides, glycohemoglobin, C-reactive protein, and homocysteine. Furthermore, we found an association between the MTP promoter polymorphism and the apolipoprotein B-containing lipoproteins total-cholesterol ( p = 0.011), LDL cholesterol ( p = 0.002) and apolipoprotein B ( p = 0.034). Conclusions Our results provide preliminary evidence for a potential role of the MTP −493TT genotype in the pathogenesis of PAD.

Secretion of atherogenic risk factor apolipoprotein B‐100 is increased by a potential mechanism of JNK/PKC‐mediated insulin resistance in liver cells

Apolipoprotein B-100 (ApoB) is the main protein of the atherogenic lipoproteins and plasma ApoB levels reflect the total numbers of atherogenic lipoproteins. Induction of insulin resistance was accompanied by a considerable rise in the production of hepatic very low density lipoprotein (VLDL) containing ApoB and triglyceride. Increased plasma levels of ApoB and triglyceride in VLDL are common characteristics of the dyslipidemia associated with insulin resistance and type 2 diabetes mellitus. Thus, we investigate whether phorbol 12-myristate-13-acetate (PMA)-induced insulin resistance affects the increase of ApoB secretion. PMA increased ApoB secretion and transcriptional level of microsomal triglyceride transfer protein (MTP). PMA treatment also resulted in increase of insulin receptor substrate 1 (IRS1) serine312 (Ser312) and serine1101 (Ser1101) phosphorylation and induction of IRS1 degradation. Additionally, PMA induced activation of c-jun N-terminal kinase (JNK) and protein kinase C (PKC) isoforms (alpha, betaI, delta, zeta, theta), and reduced AKT8 virus oncogene cellular homolog (AKT) activation in a time dependent manner. PMA-induced ApoB secretion, MTP promoter activities, and IRS1 degradation was significantly decreased by treatment of JNK and PKCs inhibitors. Orthovanadate, a potent tyrosine phosphatase inhibitor, increased tyrosine phosphorylation of IRS1 and decreased ApoB secretion of Chang liver cells although PMA was co-treated. From the results, it was concluded that PMA-induced insulin resistance, through induction of serine phosphorylation of IRS1 mediated by activated JNK and PKCs, increases ApoB secretion in Chang liver cells.

Molecular characterization of the role of orphan receptor small heterodimer partner in development of fatty liver

The orphan receptor Small Heterodimer Partner (SHP, NROB2) regulates metabolic pathways, including hepatic bile acid, lipid, and glucose homeostasis. We reported that SHP-deletion in leptin-deficient OB(-/-) mice increases insulin sensitivity, and prevents the development of fatty liver. The prevention of steatosis in OB(-/-)/SHP(-/-) double mutants is not due to decreased body weight but is associated with increased hepatic very-low-density lipoprotein (VLDL) secretion and elevated microsomal triglyceride transfer protein (MTP) mRNA and protein levels. SHP represses the transactivation of the MTP promoter and the induction of MTP mRNA by LRH-1 in hepatocytes. Adenoviral overexpression of SHP inhibits MTP activity as well as VLDL-apoB protein secretion, and RNAi knockdown of SHP exhibits opposite effects. The expression of SHP in induced in fatty livers of OB(-/-) mice and other genetic or dietary models of steatosis, and acute overexpression of SHP by adenovirus, result in rapid accumulation of neutral lipids in hepatocytes. In addition, the pathways for hepatic lipid uptake and lipogenic program are also downregulated in OB(-/-)/SHP(-/-) mice, which may contribute to the decreased hepatic lipid content. These studies demonstrate that SHP regulates the development of fatty liver by modulating hepatic lipid export, uptake, and synthesis, and that the improved peripheral insulin sensitivity in OB(-/-)/SHP(-/-) mice is associated with decreased hepatic steatosis.

Functional analysis of promoter variants in the microsomal triglyceride transfer protein (MTTP) gene

The microsomal triglyceride transfer protein (MTTP) is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins from the intestine and liver. According to this function, polymorphic sites in the MTTP gene showed associations to low-density lipoprotein (LDL) cholesterol and related traits of the metabolic syndrome. Here we studied the functional impact of common MTTP promoter polymorphisms rs1800804:T>C (-164T>C), rs1800803:A>T (-400A>T), and rs1800591:G>T (-493G>T) using gene-reporter assays in intestinal Caco-2 and liver Huh-7 cells. Significant results were obtained in Huh-7 cells. The common MTTP promoter haplotype -164T/-400A/-493G showed about two-fold lower activity than the rare haplotype -164C/-400T/-493T. MTTP promoter mutant constructs -164T/-400A/-493T and -164T/-400T/-493T exhibited similar activity than the common haplotype. Activities of mutants -164C/-400A/-493G and -164C/-400A/-493T resembled the rare MTTP promoter haplotype. Electrophoretic mobility shift assays (EMSAs) revealed higher binding capacity of the transcriptional factor Sterol regulatory element binding protein1a (SREBP1a) to the -164T probe in comparison to the -164C probe. In conclusion, our study indicates that the polymorphism -164T>C mediates different activities of common MTTP promoter haplotypes via SREBP1a. This suggested that the already described SREBP-dependent modulation of MTTP expression by diet is more effective in -164T than in -164C carriers.

The Ile128Thr polymorphism influences stability and ligand binding properties of the microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTTP) is essential for the assembly of VLDLs. We recently observed that a polymorphism in the MTTP promoter (-493G>T), which is in allelic association with an isoleucine-to-theronine substitution at position 128 (Ile128Thr) in the expressed protein, confers an increased risk of coronary heart disease. Two variant proteins comprising amino acids 16-297 of intact MTTP, MTTP(N)-Ile128 and MTTP(N)-Thr128, had similar native secondary structure content, as judged by circular dichroism. However, the thermal stability of MTTP(N)-Thr128 was greatly reduced, and this protein was also more extensively cleaved in limited proteolysis experiments compared with MTTP(N)-Ile128; both of these findings support a less compact fold. On adding LDL, which includes natively folded apolipoprotein B (apoB), decreased stability of the MTTP(N)-Thr128-LDL complex was observed compared with that of the MTTP(N)-Ile128-LDL complex. In a refined model of the N-terminal domain of MTTP, residue 128 is located in a surface-exposed position, in the same region as an identified MTTP binding site in the homologous apoB protein. Thus, the Ile128Thr polymorphism confers reduced structural stability, leading to decreased binding of MTTP to LDL particles. Because the major MTTP binding target on LDL is apoB, the Ile128Thr polymorphism could target the MTTP-apoB interaction.

Association of a microsomal triglyceride transfer protein gene polymorphism with blood pressure in Japanese women

Genetic variants of the microsomal triglyceride transfer protein (MTP) have been associated with the serum concentration of low density lipoprotein-cholesterol, predisposition to coronary heart disease, or longevity. The relation of a -493Gright curved arrow T polymorphism in the promoter of MTP to blood pressure was examined in a population-based study. The subjects (1124 men, 1108 women) were aged 40-79 years and were randomly recruited to a population-based prospective cohort study of aging and age-related diseases in Japan. Blood pressure was measured at least twice with subjects in the sitting position. The serum lipid profile was determined after the subjects had fasted overnight. The -493Gright curved arrow T genotype of MTP was determined with a fluorescence-based allele-specific DNA primer assay system. There was no difference in the serum concentrations of total cholesterol, high density lipoprotein-cholesterol, low density lipoprotein-cholesterol, or triglycerides among MTP genotypes for men or for women. Systolic or diastolic blood pressure was not related to the -493Gright curved arrow T polymorphism in men. For women, however, systolic and diastolic blood pressures were significantly related to MTP genotype, with the T allele of the polymorphism being associated with low blood pressure. The relation between MTP genotype and the prevalence of hypertension was almost significant (P=0.055) for all women. Although MTP genotype was not associated with the prevalence of hypertension in premenopausal women, the relation between these parameters was significant (P=0.040) in postmenopausal women, with the TT genotype protecting against this condition. These results suggest that MTP genotype is a determinant of blood pressure in Japanese women.

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.

Influence of microsomal triglyceride transfer protein promoter polymorphism −493 GT on fasting plasma triglyceride values and interaction with treatment response to atorvastatin in subjects with heterozygous familial hypercholesterolaemia

Familial hypercholesterolaemia (FH) is an autosomal dominant disease characterized by elevated levels of low-density lipoprotein-cholesterol (LDL-C). Phenotypic expression is highly variable, being influenced by diet, age, gender, body mass index, apolipoprotein E genotype and type of LDL-receptor gene mutation. Microsomal triglyceride (TG) transfer protein (MTP) is a protein involved in lipid metabolism. Polymorphism MTP -493 GT has been shown to modulate lipid levels in several populations. To analyse the effect of this polymorphism in the lipid phenotype expression of FH and treatment response, we studied a sample of 222 Spanish FH patients, of whom 147 were studied before and after treatment with 20 mg of atorvastatin daily during 6 weeks. The variant was analysed by polymerase chain reaction amplification and single-strand confirmation polymorphism. Treatment reduced LDL-C, total cholesterol and TGs. Baseline fasting TGs and very-low-density lipoprotein cholesterol levels were lower in female T allele carriers (TG: 111+/-51 mg/dl GG, 89+/-35 mg/dl GT, 83+/-26 mg/dl TT, P=0.022; very-low-density lipoprotein cholesterol: 24+/-13 mg/dl GG, 16+/-5 mg/dl GT, 17+/-5 mg/dl TT, P=0.018). Triglyceride response to atorvastatin was modulated by this polymorphism in men (P=0.009), but not in women, although differences between genotypes were maintained after treatment. In conclusion, the MTP -493 GT polymorphism modulates pre- and post-treatment plasma TG values of FH in Spanish subjects in a gender-specific way. Other environmental and genetic factors likely also modulate this response.

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.