Apolipoprotein Gene Expression Research Articles

Creb-binding protein (CBP) is a transcriptional coactivator for hepatocyte nuclear factor-4 HNF-4) and enhances apolipoprotein gene expression

Creb-binding protein (CBP) is a transcriptional coactivator for hepatocyte nuclear factor-4 HNF-4) and enhances apolipoprotein gene expression

CREB-binding Protein Is a Transcriptional Coactivator for Hepatocyte Nuclear Factor-4 and Enhances Apolipoprotein Gene Expression

Hepatocyte nuclear factor-4 (HNF-4) is a liver-enriched transcription factor that is crucial in the regulation of a large number of genes involved in glucose, cholesterol, and fatty acid metabolism and in determining the hepatic phenotype. We have previously shown that HNF-4 contains transcription activation functions at the N terminus (AF-1) and the C terminus (AF-2) which work synergistically to confer full HNF-4 activity. Here, we show that HNF-4 recruits the CREB-binding protein (CBP) coactivator on promoters of genes that contain functional HNF-4 sites. HNF-4 interacts with the N-terminal region of CBP (amino acids 1-771) and the C-terminal region of CBP (amino acids 1812-2441). The two activating functions of HNF-4, AF-1 and AF-2, interact with the N terminus and the N and C terminus of CBP, respectively. In addition, we show that in contrast to the other nuclear hormone receptors the interaction between HNF-4 and CBP is ligand-independent. Recruitment of CBP by HNF-4 results in an enhancement of the transcriptional activity of the latter. CBP does not activate gene expression in the absence of HNF-4, and dominant negative forms of HNF-4 prevent transcriptional activation by CBP, suggesting that the mere recruitment of CBP by HNF-4 is not sufficient for enhancement of gene expression. These findings demonstrate that CBP acts as a transcriptional coactivator for HNF-4 and provide new insights into the regulatory function of HNF-4.

Effects of Pinus pinaster and Pinus koraiensis seed oil supplementation on lipoprotein metabolism in the rat.

The aim of the present study was to assess the effect of vegetal oils obtained from Pinus pinaster and P. koraiensis seeds on plasma lipoprotein levels and apolipoprotein (apo) gene expression in rats. These oils contain two particular fatty acids of the delta5-unsaturated polymethylene-interrupted fatty acid (delta5-UPIFA) family: all-cis-5,9,12-1 8:3 (pinolenic) and/or all-cis-5,11,14-20:3 (sciadonic) acids. Rats were fed for 28 d a diet containing 5% (w/w) oil supplement. Two control diets were prepared to match the fatty acid composition of P. pinaster or P. koraiensis oils with the exception of delta5-UPIFA, which were replaced by oleic acid. Pinus pinaster seed oil decreased serum triglycerides by 30% (P < 0.02), very low density lipoprotein (VLDL)-triglycerides by 40% (P < 0.01), and VLDL-cholesterol by 33% (P < 0.03). Pinus koraiensis seed oil decreased serum triglycerides by 16% [not statistically significant (ns)] and VLDL-triglycerides by 21% (ns). Gel permeation chromatography and nondenaturating polyacrylamide gel electrophoresis showed a tendency of high density lipoprotein to shift toward larger particles in pine seed oil-supplemented rats. Finally, P. pinaster seed oil treatment was associated with a small decrease of liver apoC-III (P < 0.02) but not in apoE, apoA-I, or apoA-II mRNA levels. The levels of circulating apo were not affected by pine seed oil supplementation. In conclusion, P. pinaster seed oil has a triglyceride-lowering effect in rats, an effect that is due to a reduction in circulating VLDL.

Zinc deficiency decreases plasma level and hepatic mRNA abundance of apolipoprotein A-I in rats and hamsters.

The influence of Zn deficiency on the plasma level as well as the hepatic and intestinal gene expression of apolipoprotein (apo) A-I was examined in rats and hamsters. Male Sprague-Dawley rats (8 wk old) and Golden Syrian hamsters (7 wk old) were assigned to three dietary treatments: Zn adequate (ZA, 30 mg Zn/kg diet), Zn deficient (ZD, <0.5 mg Zn/kg diet), and Zn replete (ZDA, ZD animals fed the ZA diet for the last 2 days). The dietary treatments lasted for 18 days for rats or 6 wk for hamsters. For the measurement of apoA-I mRNA abundance, hamster apoA-I cDNA was cloned from the small intestine. The full-length 905-base pair cDNA shared approximately 80% similarity with the human, rat, and mouse apoA-I cDNAs. Hepatic and plasma Zn levels were reduced in ZD animals but normalized in ZDA rats and increased in ZDA hamsters compared with ZA animals. Zn deficiency reduced plasma apoA-I and hepatic apoA-I mRNA levels 13 and 38%, respectively, in ZD rats. The 2 days of Zn replenishment raised plasma apoA-I and hepatic apoA-I mRNA levels in ZDA rats by 34 and 28%, respectively, higher than ZA rats. Similarly, these levels were decreased by 18 and 25%, respectively, in ZD hamsters but normalized in ZDA hamsters compared with ZA hamsters. In contrast to the alterations of hepatic apoA-I mRNA levels, neither Zn deficiency nor subsequent Zn repletion produced alterations in the intestinal apoA-I mRNA abundance. Data from this study demonstrated that Zn deficiency specifically decreases hepatic apoA-I gene expression, which may at least be partly responsible for the reduction of plasma apoA-I levels.

Developmental Changes in Apolipoprotein B Gene Expression in the Liver of Fetal Calves

Changes in apolipoprotein (apo B) gene expression in the liver were determined in fetal calves from 90 to 260 days of intrauterine life. Results were compared with those obtained from the livers of 1-month-old calves and adult cows. By Western blot analysis using a rabbit antiserum to bovine apo B, apo B100 was detected from 90 days of intrauterine life. Apo B100 was shown to be the only form of apo B detected in the liver of fetal calves whatever the gestational age indicating the lack of editing process during the prenatal period. Hepatic apo B contents were stable during intrauterine life and comparable to those of calves and lower than those of cows. Hepatic contents of apo B mRNA increased with the gestational age similarly to total RNA. Values of apo B mRNA at 260 days of intrauterine life were comparable to those in the liver of 1-month-old calves and adult cows. These results suggested that hepatic synthesis of calf apo B during fetal development is specifically regulated at a posttranscriptional level, either by a decrease in the rate of translation and/or by an increase in the rate of intracellular apo B degradation.

Two Hepatic Enhancers, HCR.1 and HCR.2, Coordinate the Liver Expression of the Entire Human Apolipoprotein E/C-I/C-IV/C-II Gene Cluster

We show that the liver-specific expression of all four genes in the human apolipoprotein (apo) E/C-I/C-IV/C-II gene cluster in transgenic mice is determined by the coordinate action of two distinct hepatic control regions (HCR). These enhancers are positioned 15 kilobases (kb) (HCR.1) and 26 kb (HCR.2) downstream of the apoE gene. To investigate the action of each HCR, transgenic mice were generated with a 70-kb human genomic fragment that contained the complete apoE gene cluster or with this fragment modified by the specific deletion of HCR.1, HCR.2, or both HCR domains. Hepatic expression of all four apolipoprotein genes was observed in transgenic mice in which either HCR.1 or HCR.2 was deleted, but no transgene expression was found in the liver in the absence of both HCR domains. The overall patterns of transgene expression suggested that HCR.2 was the dominant element for apoC-IV and apoC-II expression and that HCR.1 was dominant for the apoE/C-I expression. No liver-specific transcriptional activity was identified for the proximal promoter of any gene in the cluster; all liver-specific activity was associated with HCR.1 and HCR.2. Thus, the HCRs of the apoE gene cluster constitute unique regulatory domains for determining the requirements for apolipoprotein gene expression in the liver.

A Complete Epithelial Organization of Caco-2 Cells Induces I-FABP and Potentializes Apolipoprotein Gene Expression

The culture of Caco-2 cells on plastic support impairs the expression of several genes involved in lipid metabolism. We describe culture conditions that permit the expression of the I-FABP gene and better expression of the apolipoprotein A-I, C-III, and A-IV genes. Basal lamina deposited on filters as well as the nature of nutrients on the apical side differentially modulated mRNA expression of I-FABP, APOBEC-1, and apolipoprotein genes. Growing cells on a filter led to functional polarization, illustrated by a secretion of apo B at the basal side, which induced the expression of the I-FABP, APOBEC-1, and apo A-IV genes and highly increased the expression of the apo C-III gene. Moreover, basal lamina deposited on the filter enhances the mRNA expression of apo A-I. Apo C-III and A-IV mRNA levels were decreased when cells were grown on a filter covered with basal lamina in the presence of a medium deprived of protein and lipid on the apical side, whereas these conditions had no effect on I-FABP, apo A-I, and APOBEC-1 mRNA levels. The addition of lipid micelles on the apical side had various effects, according to the genes. Caco-2 cells cultured under the conditions described here closely resembled enterocytes and represent a useful tool for studying the regulation of genes involved in lipid metabolism.

1.P.4 Regulation of apolipoprotein gene expression by the orphan intracellular receptor HNF-4

1.P.4 Regulation of apolipoprotein gene expression by the orphan intracellular receptor HNF-4

Comparison of human apoA-I expression in mouse models of atherosclerosis after gene transfer using a second generation adenovirus

Gene transfer and expression of apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins (HDL), is a potentially attractive method for investigating the effects of apoA-I on atherosclerosis. We constructed a second generation recombinant adenovirus encoding the human apoA-I cDNA. This adenoviral vector or a control vector was injected intravenously into apoE-deficient mice fed a chow diet and low density lipoprotein (LDL) receptor (LDLR)-deficient mice fed Western diet, as well as control wild-type C57BL/6 mice. The mean peak plasma human apoA-I concentrations were 235, 324, and 276 mg/dL in apoE-deficient, LDLR-deficient, and wild-type mice, respectively. Human apoA-I concentrations decreased rapidly in apoE-deficient mice and were barely detectable 6 weeks after injection. In contrast, substantially higher levels of human apoA-I were sustained in LDLR-deficient mice. In wild-type mice, human apoA-I levels decreased more rapidly than in LDLR-deficient mice, but could still be detected in plasma for up to 8 months after virus injection. In apoE-deficient mice a substantial fraction of human apoA-I was found associated with triglyceride (TG)-rich lipoproteins; in contrast, in LDLR-deficient and wild-type mice the majority of human apoA-I was found in the HDL fraction. Finally, expression of human apoA-I caused a transient but significant increase in triglyceride levels in all three mouse models. In summary: 1) a second generation recombinant adenovirus resulted in high-level expression of human apoA-I in mice; 2) significantly higher levels of human apoA-I persisted for a longer time in LDLR-deficient mice compared with apoE-deficient mice; and 3) substantial human apoA-I was found associated with TG-rich lipoproteins in apoE-deficient but not LDLR-deficient mice.

Effects of estrus cycle, ovariectomy, and treatment with estrogen, tamoxifen, and progesterone on apolipoprotein(a) gene expression in transgenic mice.

Plasma levels of lipoprotein(a) (Lp(a)), are regulated by the synthetic rate of apolipoprotein(a) (apo(a)), a major protein component of this atherogenic lipoprotein. Exogenously administered sex steroid hormones are potent regulators of plasma Lp(a) concentrations. We utilized a recently developed apo(a) yeast artificial chromosome (YAC) transgenic mouse model to study the effects of ovariectomy, estrus cycle, and exogenous administration of ethinyl-estradiol, the partial estrogen receptor agonist, tamoxifen, and progesterone on circulating apo(a) plasma levels. Analysis of liver RNA revealed that estrogen and tamoxifen exerts their plasma apo(a) lowering effect at the level of apo(a) mRNA. This action of estrogen and tamoxifen may contribute to their antiatherosclerotic and cardiovascular protective effect.

138 The effect of the obesity mutation “fatty” on hepatic apolipoprotein gene expression in rats is modified by the genetic background

138 The effect of the obesity mutation “fatty” on hepatic apolipoprotein gene expression in rats is modified by the genetic background

Hepatic Gene Expression of Apolipoprotein B100 During Early Lactation in Underfed, High Producing Dairy Cows

The hepatic gene expression of apolipoprotein B, the major protein of very low density lipoproteins in plasma, was studied using 8 Holstein × Friesian cows during the first 12 wk of lactation. Cows were fattened during gestation and were underfed just after parturition to increase fat mobilization and subsequent hepatic steatosis. Intracellular concentrations of apolipoprotein B and apolipoprotein B mRNA and control parameters (albumin, total lipids, RNA, and proteins) were determined in liver samples obtained by biopsy from each cow on four occasions at 1, 2, 4, and 12 wk after calving. Results were compared with those obtained from 5 dry cows in late pregnancy and 4 dry nonpregnant cows. The hepatic concentration of apolipoprotein B was lower (approximately 25%) during wk 1, 2, and 4 after calving, a period of intense liver steatosis (44.2 to 95.7mg of triglycerides/g of fresh tissue), than for nonsteatotic dry cows (pregnant or nonpregnant); hepatic concentrations were also lower than those during wk 12. In contrast, hepatic concentrations of mRNA coding for apolipoprotein B, total proteins, RNA, and albumin did not vary significantly during early lactation. These results suggested that synthesis of apolipoprotein B during early lactation is specifically regulated at a posttranscriptional level by a decrease in the rate of translation, or by a higher rate of intracellular degradation of apolipoprotein B, or both.

Modulation of apolipoprotein gene expression in fatty liver of obese rats: enhanced APOA-IV, but no APOB expression by a high sucrose diet.

Hepatic lipoprotein production is important to the understanding of mechanisms involved in the development of fatty liver and hyperlipidemia. Previously, we have reported that hepatic fatty acid synthesis and apolipoproteinB transcription are increased in obese rats. Here, we describe the effects of a high sucrose diet on hepatic fatty acid synthesis and apolipoprotein gene expression in obese rats. Obese rats with ventromedial hypothalamic lesions were fed on a high sucrose diet (30.3% of cal) or lab chow for 11 weeks. Serum triglycerides and plasma immuno-reactive insulin concentrations were further increased in the obese rats fed a high sucrose diet. The experimental diet increased the activity of hepatic acetyl-CoA carboxylase, the rate limiting enzyme for hepatic fatty acid synthesis, and triglycerides content, concurrent with an increase abundance of apolipoproteinA-IC mRNA in the obese rats. Despite further accumulation of hepatic triglycerides there was no further increase in hepatic apolipoproteinB mRNA abundance in the obese rats fed the high sucrose diet. These data suggest that the synthesis of hepatic fatty acids but not of apolipoproteinB is further increased in obese rats fed the high sucrose diet, and that apolipoproteinA-IV gene expression may be modulated in response to alterations in hepatic triglycerides flux.

Studies on the role of PPAR in the fibrate-modulated gene expression of apolipoprotein A-I, plasminogen activator inhibitor 1, and fibrinogen in primary hepatocyte cultures from cynomolgus monkey.

Chemicals/CAS: 5,8,11,14-Eicosatetraynoic Acid, 1191-85-1; Antilipemic Agents; Apolipoprotein A-I; Fibrinogen, 9001-32-5; pirinixic acid, 50892-23-4; Plasminogen Activator Inhibitor 1; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Transcription Factors

Hepatic apolipoprotein A-I, C-III and E mRNA expressions in human parenchymal liver diseases

In order to understand the mechanism underlying in dyslipoproteinemia accompanied with liver diseases, levels in apolipoproteins A-I, C-III and E mRNA expressions in various human parenchymal liver diseases were assessed by Northen blot analysis. The mRNA expressions were significantly decreased in liver cirrhosis and acute hepatitis as compared with controls and were correlated with serum albumin levels. This finding indicates that liver biosynthesis of these apolipoproteins is suppressed in parenchymal human liver disease. This could be one of the mechanisms causing the dyslipoproteinemia in the liver diseases. This is the first study to show decreased apolipoprotein gene expression in human parenchymal liver diseases.

1] Apolipoproteins: Pathophysiology and clinical implications

The term apolipoprotein was initially coined by John Oncley to refer to the protein constituents of plasma lipoproteins. Plasma apolipoproteins are of interest principally because of their central role in plasma lipid transport and, hence their role in atherogenesis. Over the past several decades, a great deal has been learned about apolipoprotein structure and function. The regulation of apolipoprotein gene expression and the molecular mechanisms of apolipoprotein function are beginning to be revealed. Given the rate of progress in this research, therapeutic applications that target the expression or metabolism of specific apolipoproteins can be expected in the near future. Central to the functions of all apolipoproteins (apo) is the ability to bind phospholipids that reside in specialized regions termed “amphiphathic helices.” The characteristic feature of the amphipathic helix is the spatial arrangement of hydrophobic and hydrophilic amino acids. The hydrophobic face of the helix is intercalated between the fatty acyl chains of phospholipids, whereas the hydrophilic face is located close to the polar head groups of phospholipids. Such an orientation allows the interaction of protein domains with lipoprotein-modifying enzymes and cellular receptors that control the catabolism of lipoproteins (Lp) and their removal from the circulation. The apoB gene differs from other apolipoprotein genes in genomic organization, in that it contains 29 exons, 2 of which are extremely long. In addition to the amphipathic helix motifs, located mainly in the carboxyterminal half, apoB contains unique proline-rich sequences predicted to form amphipathic β sheets. The evolutionary relationship between apoB and the other apolipoproteins is, therefore, not clear. ApoD is not a member of the apolipoprotein gene family, as it differs from all other apolipoproteins in genomic organization, primary structure, and tissue sites of expression. Similar arguments can be used to qualify apo[a] and minor apolipoproteins, such as aport and apoJ, as atypical apolipoproteins.

9- cis-retinoic acid increases apolipoprotein AI secretion and mRNA expression in HepG2 cells

HepG2 cells were studied as a model for regulation of hepatic apolipoprotein AI (apo AI) secretion and gene expression by 9- cis-retinoic acid. HepG2 cells cultured on plastic dishes were exposed to 9- cis-retinoic acid (9- cis-RA) for 48 h with a complete media change at 24 h. Apo AI mass in culture media was determined by ELISA, by quantitative immunoblotting and by steady-state 35S-methionine labeling. Messenger RNA levels were determined by RNase protection using probes for apo AI and the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH). 9- cis-RA increased secretion of apo AI by 52% at doses of 10 and 1 μM (6.3 ± 0.6 vs. 4.2 ± 0.3; P < 0.005; 6.1 ± 0.3 vs. 4.0 ± 0.7 ng of apo AI/mg cell protein, P < 0.05) and by 35% at 0.1 μM (5.5 ± 0.6 vs. 4.1 ± 0.4 ng apo AI/mg protein, P < 0.05, n = 4). Immunoblotting results were consistent with results from ELISA (70% increase at 10 μM 9- cis-RA, P < 0.001; 34% increase at 1 μM, P < 0.005, n = 3). Metabolically labeled apoAI in the medium was increased by 39% following steady-state labeling in the presence of 10 μM 9- cis-RA (597 ± 7 vs. 430 ± 13 DPM/μl media; P < 0.001; n = 4). 9- cis-RA (10 μM) also increased HepG2 cell apo AI mRNA expression by 76% (68 700 ± 400 vs. 38 900 ± 2700 DPM, P < 0.01, n = 4), whereas expression of G3PDH mRNA was slightly decreased (14%, P < 0.05). Thus, 9- cis-RA stimulates apo AI expression in HepG2 cells, suggesting a role for retinoids in activating endogenous apo AI gene expression.

Hepatic apolipoprotein and LDL receptor gene expression in the genetically hypercholesterolemic (RICO) rat

The present study was designed to examine apolipoprotein and LDL receptor gene expression in genetically hypercholesterolemic RICO rats. In the plasma of RICO rats as compared to SW (control) rats, the hypercholesterolemia (+ 41%) was associated with a significant increase in plasma apo B (+ 23%) and apo E (+ 68%) concentrations. Study of apolipoprotein synthesis in the liver has shown that this increase in plasma apo B and apo E concentrations was not associated with modification in their synthesis and mRNA levels. Study of apo E mRNA level in various tissues has shown only the modification in adrenals in RICO as compared to SW rats (2.7-fold increase). Study of LDL binding, LDL receptor mass and LDL receptor mRNA level in the liver of RICO and SW rats has shown no significant differences between these two strains. EDTA-resistant binding of rat LDL was lower in RICO than in SW rats suggesting that binding sites others than the LDL receptor are present in lesser amount in this hypercholesterolemic strain.

Magnesium deficiency modulates hepatic lipogenesis and apolipoprotein gene expression in the rat

The present study was performed to determine the effects of magnesium (Mg) deficiency upon plasma lipoproteins and hepatic apolipoprotein gene expression in the rat. The most obvious effect of Mg-deficiency on plasma lipids is a marked increase in post-prandial triacylglycerol concentration. This increased triglyceridemia persists in fasted rats. Density gradient ultracentrifugation analysis revealed marked alterations in the distribution of plasma lipoproteins in Mg-deficient rats. An increase in triacylglycerol-rich lipoproteins (TGRLP) was associated with a significant increase in plasma apolipoprotein B (apo B) concentration and was accompanied by selective accumulation of apo B-48. A decrease in high-density lipoproteins (HDL) was accompanied by a corresponding decrease in plasma apo E concentration and a concordant decrease in hepatic apo E mRNA abundance and biosynthesis. Hepatic apo B-100 synthesis was reduced by over 75% in Mg-deficient animals despite an increase in hepatic apo B mRNA abundance. However, this change in hepatic apo B gene expression was not associated with alterations in posttranscriptional apo B mRNA editing. These changes in apolipoprotein gene expression were associated with increased hepatic lipogenesis, despite the observation that net triacylglycerol secretion rates were not different between Mg-deficient and control animals. Taken together, the data demonstrate a complex pattern of alterations in hepatic lipid metabolism and apolipoprotein gene expression in the Mg-deficient rat and suggest a defect in the catabolism rather than secretion of TGRRP as the major factor underlying the altered plasma lipoprotein profile.

P-204 Effect of high sucrose diet on apolipoprotein gene expression in fatty liver with obesity

P-204 Effect of high sucrose diet on apolipoprotein gene expression in fatty liver with obesity