Microsomal Triglyceride Transfer Protein Gene Expression Research Articles

Regulating microsomal triglyceride transfer protein with natural products for the treatment of hyperlipidaemia

Hyperlipidaemia, characterised by elevated levels of lipids, particularly LDL-C, is a significant risk factor for atherosclerotic cardiovascular disease. While synthetic inhibitors of microsomal triglyceride transfer protein (MTP) have shown potential in lowering LDL-C, they are associated with adverse effects. This study explores a novel approach by screening natural products to identify plant extracts that down-regulate MTP gene expression, aiming to reduce hyperlipidaemia with fewer side effects. Modulating MTP expression, rather than direct inhibition, offers a promising avenue for lowering plasma lipids and mitigating cardiovascular risk. Various plant extracts were examined for their potential as MTP down-regulators, with Liquorice root and Pomegranate rind extracts demonstrating the highest efficacy. Additionally, the study assessed the total phenolic content of these extracts, revealing their ­antioxidant capacity. This research provides a foundation for further investigation into bioactive molecules as potential anti-hyperlipidemic agents with improved safety profiles, addressing a critical need in cardiovascular disease prevention.

Abstract 033: Differential Effects of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Production and Function on Microsomal Triglyceride Transfer Protein

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein that plays a key role in the regulation of plasma low-density lipoprotein (LDL) cholesterol levels. PCSK9 binding to the LDL receptor (LDLR) leads to receptor-mediated endocytosis and lysosomal degradation of LDLR. Prior studies have shown that PCSK9 increases production of triglyceride-rich apoB-lipoproteins via up-regulation of lipogenic genes, and that PCSK9 inhibition reduces plasma triglyceride levels. However, the effect of PCSK9 inhibition on hepatic lipogenic genes expression remain unclear. Using a human hepatocellular carcinoma cell line (HepG2) we show that overexpression of PCSK9 upregulated the expression of several lipogenic genes, including a 1.5-fold increase in ATP citrate lyase (ACLY), a 1.2-fold increase in Fatty Acid Synthase (FAS), a 1.2-fold increase in HMG-CoA-reductase, and a 1.8-fold increase in microsomal triglyceride transfer protein (MTTP), when compared with untransfected HepG2 cells. Overexpression of a gain-of-function mutant of PCSK9 (PCSK9-D374Y) had stronger effects in the same direction: a 2.3-fold increase in ACLY, a 1.8-fold increase in FAS, a 1.6-fold increase in HMG-CoA-reductase, and a 2.1-fold increase in MTTP. In HepG2 cells overexpressing either normal PCSK9 or PCSK9-D374Y, inhibition of PCSK9 function using a monoclonal antibody blocked PCSK9-mediated degradation of LDLR, and reduced expression of SREBP-dependent genes (ACLY, FAS and HMG-CoA-reductase) to levels of untransfected HepG2 cells. Interestingly, while inhibition of PCSK9 function via monoclonal antibody did not affect MTTP gene expression in these cells, inhibition of PCSK9 production via RNA interference reduced MTTP gene expression by 66%. We conclude that inhibition of PCSK9 production decreases MTTP gene expression, whereas blockade of PCSK9 function (and cellular re-entry) only decreases SREBP-dependent genes expression, but does not affect MTTP gene expression. This study shows that while PCSK9 exerts an influence on genes related to both lipogenesis and lipoprotein assembly, any inhibition of PCSK9 controls SREPB-dependent genes but only inhibiting PCSK9 production controls MTTP gene expression.

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.

Hepatic VLDL-TG Production and MTP Gene Expression are Decreased in Ovariectomized Rats: Effects of Exercise Training

The present study was designed to investigate the effects of estrogen withdrawal and exercise training on hepatic very low density lipoprotein-triglyceride (VLDL-TG) production and on expression of genes involved in hepatic VLDL synthesis in response to lipid infusion. Female Sprague-Dawley rats underwent ovariectomy (Ovx), sham surgery (Sham), and Ovx with 17β-estradiol supplementation (OvxE2) before being subdivided into sedentary (Sed) and trained (Tr) groups for 8 weeks. Exercise training consisted of continuous running on a rodent treadmill 5 times/wk. At the end of the 8-week period, all rats in the fasted state were intravenously infused with a 20% solution of Intralipid for 3-h followed by an injection of Triton WR-1339 to block lipoprotein lipase activity. Plasma TG accumulation was subsequently measured during 90 min to estimate VLDL-TG production. An additional control group consisting of Sham-Sed rats was infused with saline (0.9% NaCl). Estrogen withdrawal resulted in higher (p<0.01) liver fat accumulation concomitantly with lower (p<0.01) VLDL-TG production and lower mRNA and protein content of hepatic microsomal triglyceride transfer protein (MTP). All of these effects in Ovx rats were corrected with estrogen supplementation. Training in Ovx rats reduced (p<0.01) liver fat accumulation and further reduced (p<0.01) hepatic VLDL-TG production along with gene expression of MTP and diacylglycerol acyltransferase-2 (DGAT-2). It is concluded that VLDL-TG synthesis and/or secretion is decreased in Ovx rats probably via MTP regulation and that this decrease may constitute one of the factors involved in hepatic fat accumulation. The training effect on reducing VLDL production was independent of the estrogenic status.

Microsomal Triglyceride Transfer Protein Activity Is Not Required for the Initiation of Apolipoprotein B-containing Lipoprotein Assembly in McA-RH7777 Cells

We previously demonstrated that the N-terminal 1000 amino acid residues of human apolipoprotein (apo) B (designated apoB:1000) are competent to fold into a three-sided lipovitellin-like lipid binding cavity to form the apoB "lipid pocket" without a structural requirement for microsomal triglyceride transfer protein (MTP). Our results established that this primordial apoB-containing particle is phospholipid-rich (Manchekar, M., Richardson, P. E., Forte, T. M., Datta, G., Segrest, J. P., and Dashti, N. (2004) J. Biol. Chem. 279, 39757-39766). In this study we have investigated the putative functional role of MTP in the initial lipidation of apoB:1000 in stable transformants of McA-RH7777 cells. Inhibition of MTP lipid transfer activity by 0.1 microm BMS-197636 and 5, 10, and 20 microm of BMS-200150 had no detectable effect on the synthesis, lipidation, and secretion of apoB:1000-containing particles. Under identical experimental conditions, the synthesis, lipidation, and secretion of endogenous apoB100-containing particles in HepG2 and parental untransfected McA-RH7777 cells were inhibited by 86-94%. BMS-200150 at 40 microm nearly abolished the secretion of endogenous apoB100-containing particles in HepG2 and parental McA-RH cells but caused only 15-20% inhibition in the secretion of apoB: 1000-containing particles. This modest decrease was attributable to the nonspecific effect of a high concentration of this compound on hepatic protein synthesis, as reflected in a similar (20-25%) reduction in albumin secretion. Suppression of MTP gene expression in stable transformants of McA-RH7777 cells by micro-interfering RNA led to 60-70% decrease in MTP mRNA and protein levels, but it had no detectable effect on the secretion of apoB:1000. Our results provide a compelling argument that the initial addition of phospholipids to apoB:1000 and initiation of apoB-containing lipoprotein assembly occur independently of MTP lipid transfer activity.

Liver Microsomal Triglyceride Transfer Protein Is Involved in Hepatitis C Liver Steatosis

Hepatic steatosis is frequent in chronic hepatitis C. Several mechanisms might be implicated, including metabolic cofactors and direct viral effects on intracellular lipid pathways. In a transgenic mouse model, hepatitis C virus (HCV) was shown to inhibit microsomal triglyceride transfer protein (MTP) activity, which is essential for hepatic lipoprotein assembly and secretion. No data are available on liver MTP activity in HCV-infected patients. We therefore investigated liver MTP gene expression and its lipid transfer activity in untreated cases infected with the major HCV genotypes showing variable degrees of hepatic steatosis. MTP messenger RNA (mRNA) levels were measured by real-time polymerase chain reaction, and MTP activity was assessed by fluorescent assay in liver biopsy specimens of 58 HCV-positive patients. A set of metabolic and serum lipid markers was also measured at the time of liver biopsies. MTP mRNA levels showed a statistically significant (P = .001) inverse correlation with the degree of steatosis, independently of the HCV genotype. MTP mRNA levels also had an inverse correlation with serum insulin (P = .0002), homeostasis model assessment-insulin resistance (HOMA-IR) (P = .005), and body mass index (P = .02) in patients with HCV-1 and HCV-2 and with serum HCV-RNA (P = .02) in HCV-3 patients. Liver MTP-specific activity was significantly reduced in HCV-3 patients compared with those with other HCV genotypes (P = .004) and correlated with reduced serum cholesterol, apo B, and low-density lipoproteins. MTP may play a central role in HCV-related steatosis, being modulated by different genotype-specific mechanisms, mainly hyperinsulinemia in non-HCV-3 patients, and more profound and direct virus-related effects in HCV-3-infected individuals.

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.

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.

Possible involvement of enhanced intestinal microsomal triglyceride transfer protein (MTP) gene expression in acceleration of lipid absorption by a western-type diet in apolipoprotein E knockout mice

Evidence has been accumulating that triglyceride (TG)-rich lipoproteins are atherogenic. Microsomal TG transfer protein (MTP) is essential for the synthesis of both chylomicron in the intestine and very low density lipoprotein in the liver. To investigate whether a western-type diet, a so-called atherogenic diet, alters intestinal lipid absorption via change in intestinal MTP expression, the effects of two different diet regimes in apolipoprotein-E knockout (apoE KO) mice were examined. Male apoE KO mice aged 6 weeks were fed a western-type diet or a chow diet for 5 weeks. Then, measurement of plasma TG levels after oral fat-loading and analysis of jejunal MTP gene expression were performed. Both the maximum level and the 0–8 h area under the curve (AUC) of the increase in TG levels in the western-type diet-fed mice were almost three times greater than those in the chow diet-fed mice. MTP gene expression, determined by reverse transcriptase-polymerase chain reaction (RT-PCR), was obviously enhanced in the western-type diet-fed mice compared to the chow diet-fed mice. These results suggest that the enhancement of intestinal MTP gene expression is involved in the accelerated lipid absorption in the western-type diet-fed mice.

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.

Garlic Inhibits Microsomal Triglyceride Transfer Protein Gene Expression in Human Liver and Intestinal Cell Lines and in Rat Intestine

Epidemiologic studies have suggested that fresh garlic has lipid-lowering activity. Because the microsomal triglyceride transfer protein (MTP) plays a pivotal role in the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins, we evaluated the effect of garlic on the expression of the MTP gene in vitro in cell lines and in vivo in rats. Fresh garlic extract (FGE) reduced MTP mRNA levels in both the human hepatoma HepG2 and intestinal carcinoma Caco-2 cells in dose-dependent fashion; significant reductions were detected with 3 g/L FGE. Maximal 72 and 59% reductions, respectively, were observed with 6 g/L FGE. To evaluate the in vivo effect of garlic on MTP gene expression, rats were given a single oral dose of fresh garlic homogenate (FGH), with hepatic and intestinal MTP mRNA measured 3 h after dosing. Rats fed FGH had significantly (46% of the control) lower intestinal MTP mRNA levels compared with the control rats, whereas hepatic MTP mRNA levels were not affected. These results suggest a new mechanism for the hypolipidemic effect of fresh garlic. Long-term dietary supplementation of fresh garlic may exert a lipid-lowering effect partly through reducing intestinal MTP gene expression, thus suppressing the assembly and secretion of chylomicrons from intestine to the blood circulation.

Decreased microsomal triglyceride transfer protein activity contributes to initiation of alcoholic liver steatosis in rats

Background/Aims : To elucidate the role of microsomal triglyceride transfer protein (MTP) in the pathogenesis of alcoholic fatty liver, the effects of ethanol on MTP activity and gene expression were investigated. Methods and results : Male Sprague–Dawley rats fed an ethanol-containing liquid diet for 37 days, respectively, showed 2.9- and 4.9-fold increases in hepatic cholesterol and triglyceride content in comparison with rats fed an isocaloric ethanol-free diet ( P <0.01). Furthermore, a significant decrease in MTP activity and mRNA expression (by 27 and 58%, respectively) was observed after ethanol administration. Intravenous injection of human recombinant hepatocyte growth factor (hrHGF) on each of the last 7 days markedly suppressed ethanol-induced lipid accumulation in the liver. This inhibition of fatty change by hrHGF was accompanied by recovery of MTP activity and gene expression. No inhibitory effect of hrHGF on ethanol-induced acyl-CoA synthetase activation was observed. Experiments using human hepatoma-derived HepG2 cells indicated a direct positive effect of hrHGF on MTP gene expression as well as apolipoprotein B secretion. Conclusions : These results suggest that reduced MTP activity is crucial to development of alcoholic fatty liver, while promotion of MTP activity by HGF might serve as a therapeutic measure against alcoholic liver steatosis.

Sterol Regulatory Element-binding Protein Negatively Regulates Microsomal Triglyceride Transfer Protein Gene Transcription

We herein report that mRNA expression of microsomal triglyceride transfer protein (MTP) and its protein synthesis decline in response to sterol depletion in HepG2 cells, and we functionally characterized the MTP gene promoter in an effort to investigate the molecular mechanisms by which MTP gene transcription is regulated. Luciferase assays using truncated versions of the reporter gene revealed that the region at -124 to +33 base pairs of the human promoter contains the elements required for the suppression of transcription by sterol depletion. Enforced expression of an active form of sterol regulatory element-binding protein (SREBP)-1 (amino acids 1-487) or -2 (amino acids 1-481), both of which are activated under sterol-depleted conditions, is able to mimic sterol-mediated down-regulation. Either further truncation of the promoter region or mutation of the putative SREBP-binding sequence (5'-GCAGCCCAC-3', -124 to -116 base pairs) abolishes the sterol- and SREBP-dependent transcriptional regulation. Gel mobility shift assay showed that recombinant SREBP-2-(1-481) is able to bind the sequence. Enforced expression of a truncated form of SREBP-2 (amino acids 31-481), which acts as an inhibitor of transcription of the low density lipoprotein receptor gene because it lacks the transcriptional activation domain, also diminishes the luciferase activity, suggesting that direct binding to the promoter region might be sufficient and that the mechanism by which SREBPs inhibit MTP gene expression is distinct from that for the transcriptional stimulation of sterol-regulated genes. Although the SREBP-binding site overlaps a negative insulin-responsive element, insulin negatively regulates MTP gene expression even when the amount of the active form of SREBPs is quite low under the sterol-loaded conditions, indicating that SREBPs only slightly mediate, if at all, the insulin effects. Overall, we conclude that SREBPs are responsible for regulation of lipoprotein secretion via their control of MTP gene expression. Moreover, our results describe for the first time a novel mechanism by which SREBPs negatively regulate expression of the gene encoding the protein involved in lipid metabolism.

Hepatic microsomal triglyceride transfer protein gene expression in carbohydrate-induced hypertriglyceridemia

The microsomal triglyceride transfer protein (MTP) facilitates the assembly of very low density lipoproteins (VLDL). To investigate the effects of carbohydrate (CHO)-induced hypertriglyceridemia (HTG) on the hepatic expression of the MTP gene, Golden Syrian hamsters were fed fructose (FR) and sucrose (SUC) enriched diets. Changes in fasting plasma glucose (FPG), insulin, free fatty acids (FFA), TG, VLDL-TG and VLDL-protein were determined. Abundance of hepatic MTP-mRNA was compared to those of albumin (ALB)-mRNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-mRNA. Hepatic secretion rates (SR) of VLDL-TG and VLDL-PR were assessed after intravenous Triton WR-1339 injection. Lipoprotein lipase (LpL) activity of the epididymal fat pads was determined. As compared to the control and SUC, FR increased plasma insulin (FR = 238+24, SUC = 115±17, control = 82±16 μU/ml, p < 0.0001), FFA (FR = 1.93±0.24, SUC = 1.35±0.1, control = 1.47±0.13 mEq/L, p < 0.03) and TG (FR = 215±47, SUC = 93±7, control = 71±16 mg/dl, p < 0.01), without affecting FPG. There was no change in LpL activity. Fructose-feeding increased VLDL-TG-SR (FR = 2.95±0.26, SUC = 1.28±0.34, control = 2.18±0.26 mg/kg.wt/h −1, p < 0.004), but not VLDL-protein-SR. MTP-mRNA abundance did not change when compared to albumin mRNA, however FR-fed hamsters had lower MTP mRNA abundance when compared to GAPDH mRNA (FR = 0.96±0.13, SUC = 1.15±0.14, control = 1.49±0.14 MTP GAPDH-mRNA, p < 0.05). The finding that FR-feeding increased VLDL-TG secretion without any increase in MTP mRNA proves that CHO-induced HTG can occur without an increase in hepatic MTP gene expression.

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.

Transcriptional regulation of human and hamster microsomal triglyceride transfer protein genes. Cell type-specific expression and response to metabolic regulators.

In order to characterize the molecular mechanisms that dictate microsomal triglyceride transfer protein (MTP) gene transcription in human and hamster, two species with similar plasma lipoprotein profiles, the MTP gene promoters were cloned, sequenced, and functionally characterized by transient transfection analysis. The results presented in this report indicate that the 5' ends of human and hamster MTP genes share similar structural features. The promoter sequences are well conserved and consist of similar functional elements. Transient transfection analysis of MTP promoter-driven luciferase gene expression showed that the promoter is active in liver and intestinal cells but not in epithelial cells, consistent with endogenous MTP gene expression. The -123 to -85 bp region of the human promoter is critical for the expression and contains the consensus recognition sequences for liver cell-specific factors HNF-1 and HNF-4 and activator protein AP-1. The promoter contains a modified sterol response element and a negative insulin response element. The human promoter activity is positively regulated by cholesterol and negatively regulated by insulin. From the functional analysis of MTP promoters, it is concluded that the elements that regulate the cell type-specific expression in human and hamster are well conserved and that insulin and cholesterol can regulate the activity of the MTP promoter in opposite directions.