Variation at the apolipoprotein (apo) AI-CIII-AIV gene cluster and apo B gene loci is associated with lipoprotein and apolipoprotein levels in Italian children.

We have used RFLPs of the apolipoprotein (apo) B gene and apo AI-CIII-AIV gene cluster to estimate the genetic contribution of variation at these loci to the variability of plasmid lipid, lipoprotein, and apolipoprotein levels in 209 children from Sezze in central Italy. The sample was randomly divided into group I (107 children) and group II (102 children). Four site polymorphisms (PvuII, XbaI, MspI, and EcoRI) of the apo B gene and five site polymorphisms (XmnI, PstI, SstI, PvuII-CIII, and PvuII-AIV) of the apo AI-CIII-AIV gene cluster were examined in group I children. After adjustment for gender, age, and body-mass index, polymorphisms at both gene loci (PvuII-B, PvuII-CIII, and PvuII-AIV) were associated with significant effects on the levels of plasma apo AI, apo B, or high-density lipoprotein-cholesterol. RFLPs that showed significant effects in group I were genotyped in group II. All three polymorphisms were associated with similar effects on apolipoprotein levels, though for all RFLPs the magnitude of the effects was smaller in the group II children and only statistically significant for the effect of the PvuII-B genotype on apo AI levels. In the total sample of 209 children 7.4% of the sample variance in apo AI levels was explained by variation associated with the apo B PvuII-B RFLP. In addition, the PvuII-B RFLP was associated with significant effects on plasma apo B levels and explained 5.7% of the sample variance. The PvuII-CIII and PvuII-AIV polymorphisms were both associated with differences in apo AI levels, explaining 3.7%-5.7% of the sample variance. Taken together, the three PvuII polymorphisms explained 17.7% of the phenotypic variance in apo AI levels. There was significant evidence for an effect of nonlinearity of the PvuII-CIII genotypes on apo AI levels, with the individuals heterozygous for the polymorphism having the highest apo AI levels. No evidence of interaction between genotype and gender, age, and body-mass index was shown by covariance analysis. The molecular explanation of this effect is unclear. Our data show that variation at both the apo AI-CIII-AIV and apo B loci are associated with lipoprotein and apolipoprotein levels in this sample of Italian children.     

Helper-dependent adenoviral vector-mediated long-term expression of human apolipoprotein A-I reduces atherosclerosis in apo E-deficient mice

Apolipoprotein A-I (APOA-I) is the major protein component of high-density lipoproteins (HDL). It has been shown that over-expression of human APOA-I increases HDL cholesterol and decreases atherosclerosis. We constructed a helper-dependent adenoviral (HD-Ad) vector that contains the entire human APOA-I gene (hgAI). Intravenous delivery of 1x10(13) viral particles/kg of this vector was followed by high levels of human APOA-I expression (up to 200 mg/dl) in the absence of detectable hepatic toxicity. We treated apo E-deficient mice with the hgAI vector and fed them either with a high-fat diet or with regular chow. As a control, two groups of mice were treated with PBS. The apo E-deficient mice treated with the hgAI vector showed supraphysiological levels of expression of human APOA-I at week 4 and high levels of HDL cholesterol compared to the control groups. Analysis of aortic atherosclerotic lesions 20 weeks after treatment, showed a significant reduction of lesion size in the treated mice with both diets. In conclusion, liver-directed gene transfer of human APOA-I using a HD-Ad vector resulted in a reduction of the development of atherosclerosis with the absence of significant toxicity.     

Polymorphisms in the apolipoprotein (apo) AI-CIII-AIV gene cluster: detection of genetic variation determining plasma apo AI,apo CIII and apo AIV concentrations

We have examined the associations between levels of plasma apolipoprotein (apo) AI, apo CIII and apo AIV and genetic variation in the apo AI-CIII-AIV gene cluster in 162 boys and young men from Belgium aged from 7 to 23 years. Genotypes were determined for six restriction enzymes XmnI, PstI, SstI, PvuIIA-CIII, PvuIIB-AIV and XbaI, and for the G to A substitution at -75 bp in the 5' region of the apo AI gene. The polymorphism most strongly associated with apo AI levels was the G to A substitution (P = 0.025, R2 x 100 = 3.6%) confirming previous observations. The polymorphism most strongly associated with apo CIII levels was that of PvuIIA-CIII (P = 0.023, R2 x 100 = 2.9%) in the apo CIII gene. This novel association must be interpreted with caution until it has been confirmed in an independent sample. The polymorphism associated with the largest effect on apo AIV levels was that detected with XbaI in the apo AIV gene, but this association was not statistically significant. Previously reported associations between the SstI polymorphism and triglyceride levels, and between the PstI polymorphism and apo AI levels, were weakly detected in the present sample. Our results show that variation associated with some of the polymorphisms in the apo AI-CIII-AIV cluster makes a small, but statistically significant, contribution to the determination of apo AI and apo CIII levels in this sample of young men and boys. These observations may, in part, explain reported associations between polymorphisms in this gene cluster, differences in plasma lipid and lipoprotein levels, and prevalence of coronary artery disease.     

Nonsynonymous polymorphic sites in the apolipoprotein (apo) A-IV gene are associated with changes in the concentration of apo B- and apo A-I-containing lipoproteins in a normal population.

The aims of this study were to detect polymorphic sites in the apolipoprotein (apo) A-IV gene, to establish their frequencies, to determine potential haplotypes, and to investigate the role of these polymorphisms in lipid metabolism. A sequencing study of four individuals led to the identification of two synonymous mutations (codons 9 and 54) and three nonsynonymous mutations (Val-8----Met, Gln360----His, and Thr347----Ser) and of a VNTR polymorphism within a series of three or four CTGT repeats in the noncoding region of exon 3. Frequencies of these polymorphisms were determined in 291 students by using naturally occurring (BstEII for the synonymous mutation in codon 54, HinfI for Thr347----Ser, and Fnu4HI for Gln360----His) or artificially introduced restriction-enzyme cutting sites (BstEII for the synonymous mutation in codon 9 and MamI for Val-8----Met), subsequent to PCR amplification. The four-base deletion/insertion polymorphism and its localization cis or trans to the mutations in codons 347 and 360 were studied by direct sequencing of PCR-amplified DNA from 87 students. Frequencies of the rarer alleles were .007 for apo A-IV-8:Met, .04 for the synonymous mutation in codon 9, .14 for the synonymous mutation in codon 54, .16 for apo A-IV347:Ser, .07 for apo A-IV360:His, and .39 for the four-base of insertion. Apo A-IV360:His in all cases was cis-localized to the (CTGT)3 repeat and apo A-IV347:Thr; and apo A-IV347:Ser was cis-localized to the (CTGT)4 repeat and apo A-IV360:Gln. Four haplotypes formed from these three polymorphic sites were thus found. The apo A-IV347:Ser allele was associated both with significantly lower plasma apo B concentrations in both sexes and with significantly lower LDL-cholesterol concentrations in men. Heterozygous carriers of apo A-IV360:His exhibited significantly higher concentrations of LDL-cholesterol and lower Lp(a) concentrations, compared with apo A-IV360:Gln homozygotes. We could not confirm the previously reported association of apo A-IV360:His with elevated HDL-cholesterol concentrations. In the population, the Val-8----Met polymorphism was not associated with significantly different lipid concentrations, but in a family study the Met-8 allele was associated with lower HDL-cholesterol and higher LDL-cholesterol concentrations. In conclusion, our results indicate an important role of the apo A-IV gene locus in the metabolism of apo B and, to a lesser extent, apo A-I containing lipoproteins.     

High molecular weight adiponectin reduces apolipoprotein B and E release in human hepatocytes

Low circulating levels of high molecular weight adiponectin (HMW-Apm) have been linked to dyslipidaemia and systemic HMW-Apm negatively correlates with very low density lipoprotein (VLDL), apolipoprotein B (ApoB), and ApoE and is positively associated with ApoA-I. Therefore, it was investigated whether HMW-Apm alters the hepatic synthesis of ApoB, ApoE, and ApoA-I or the activity of the hepatic ATP-binding cassette transporter A1 (ABCA1), as the main determinant of plasma HDL. HMW-Apm reduces hepatic ApoB and ApoE release whereas ABCA1 protein, activity and ApoA-I were not altered. Global gene expression analysis revealed that hepatic nuclear factor 4-alpha (HNF4-alpha) and HNF4-alpha regulated genes like ApoB are downregulated by HMW-Apm and this was confirmed at the mRNA and protein level. Therefore it is concluded that HMW-adiponectin may ameliorate dyslipidaemia by reducing the hepatic release of ApoB and ApoE, whereas ABCA1 function and ApoA-I secretion are not influenced.     

There are two gene sequences for human apolipoprotein CI (apo CI) on chromosome 19, one of which is 4 kb from the gene for apo E

A cDNA probe corresponding to the mRNA sequence for apolipoprotein E (apo E) was used to screen two independently-constructed human genomic libraries. Two recombinants (lambda E-2, and lambda E2-1), isolated using the apo E cDNA probe, also contain part or all of the apo CI gene. Hybridisation studies using both apo E and apo CI cDNA probes show that these two genes are in the same orientation and separated by 4 kb.     

Aspirin reduces hypertriglyceridemia by lowering VLDL-triglyceride production in mice fed a high-fat diet

Systemic inflammation is strongly involved in the pathophysiology of the metabolic syndrome, a cluster of metabolic risk factors that includes hypertriglyceridemia. Aspirin treatment lowers inflammation via inhibition of NF-κB activity but also reduces hypertriglyceridemia in humans. The aim of this study was to investigate the mechanism by which aspirin improves hypertriglyceridemia. Human apolipoprotein CI (apoCI)-expressing mice (APOC1 mice), an animal model with elevated plasma triglyceride (TG) levels, as well as normolipidemic wild-type (WT) mice were fed a high-fat diet (HFD) and treated with aspirin. Aspirin treatment reduced hepatic NF-κB activity in HFD-fed APOC1 and WT mice, and in addition, aspirin decreased plasma TG levels (-32%, P < 0.05) in hypertriglyceridemic APOC1 mice. This TG-lowering effect could not be explained by enhanced VLDL-TG clearance, but aspirin selectively reduced hepatic production of VLDL-TG in both APOC1 (-28%, P < 0.05) and WT mice (-33%, P < 0.05) without affecting VLDL-apoB production. Aspirin did not alter hepatic expression of genes involved in FA oxidation, lipogenesis, and VLDL production but decreased the incorporation of plasma-derived FA by the liver into VLDL-TG (-24%, P < 0.05), which was independent of hepatic expression of genes involved in FA uptake and transport. We conclude that aspirin improves hypertriglyceridemia by decreasing VLDL-TG production without affecting VLDL particle production. Therefore, the inhibition of inflammatory pathways by aspirin could be an interesting target for the treatment of hypertriglyceridemia.     

Metformin reduces cellular lysophosphatidylcholine and thereby may lower apolipoprotein B secretion in primary human hepatocytes

The biguanide metformin is an oral antihyperglycemic drug for the treatment of type 2 diabetes mellitus. Further, a moderate improvement of dyslipidemia by metformin was reported, and therefore, the effect of metformin on the release of apolipoprotein B (ApoB) and ApoE in primary human hepatocytes was determined. Metformin at 0.5 and 1 mM reduced hepatic ApoB secretion but ApoE was not altered. Metformin is well known to stimulate the AMP kinase that subsequently reduces hepatic nuclear factor 4-alpha (HNF4-alpha) and HNF4-alpha regulated genes like ApoB. However, HNF4-alpha was only diminished by 1 mM metformin and ApoB mRNA was not suppressed indicating that this pathway may not explain reduced ApoB release. Lower abundance of lysophosphatidylcholine (lysoPC) may also diminish ApoB secretion. Therefore, electrospray ionization tandem mass spectrometry was applied to measure cellular lipids. PC, lysoPC (produced by hydrolysis of PC), phosphatidylserine and sphingomyelin (derived from PC) were lower in metformin-treated hepatocytes whereas phosphatidylethanolamine, an alternative precursor of PC, was not affected. In addition, ABCB4, the canalicular membrane flippase essential for biliary PC secretion, was diminished. Supplementation with lysoPC led to a selective elevation of endogenous lysoPC and rescued ApoB secretion in metformin-treated cells. Therefore, it is concluded that metformin reduces lysoPC in human hepatocytes and this may secondarily lead to a therapeutically beneficial lower release of ApoB.     

Limited suppression of the interferon-beta production by hepatitis C virus serine protease in cultured human hepatocytes

Toll-like receptors and RNA helicase family members [retinoic acid-inducible gene I (RIG-I) and melanoma differentiation associated gene-5 (MDA5)] play important roles in the induction of interferon-beta as a major event in innate immune responses after virus infection. TRIF (adaptor protein of Toll-like receptor 3)-mediated and Cardif (adaptor protein of RIG-I or MDA5)-mediated signaling pathways contribute rapid induction of interferon-beta through the activation of interferon regulatory factor-3 (IRF-3). Previously, it has been reported that the hepatitis C virus NS3-4A serine protease blocks virus-induced activation of IRF-3 in the human hepatoma cell line HuH-7, and that NS3-4A cleaves TRIF and Cardif molecules, resulting in the interruption of antiviral signaling pathways. On the other hand, it has recently been reported that non-neoplastic human hepatocyte PH5CH8 cells retain robust TRIF- and Cardif-mediated pathways, unlike HuH-7 cells, which lack a TRIF-mediated pathway. In the present study, we further investigated the effect of NS3-4A on antiviral signaling pathways. Although we confirmed that PH5CH8 cells were much more effective than HuH-7 cells for the induction of interferon-beta, we obtained the unexpected result that NS3-4A could not suppress the interferon-beta production induced by the TRIF-mediated pathway, although it suppressed the Cardif-mediated pathway by cleaving Cardif at the Cys508 residue. Using PH5CH8, HeLa, and HuH-7-derived cells, we further showed that NS3-4A could not cleave TRIF, in disagreement with a previous report describing the cleavage of TRIF by NS3-4A. Taken together, our findings suggest that the blocking of the interferon production by NS3-4A is not sufficient in HCV-infected hepatocyte cells.     

Cholesterol can stimulate secretion of apolipoprotein B by cultured human hepatocytes

During a 5 day cultivation of human hepatocytes in a primary culture the secretion of apolipoprotein B was measured by enzyme-linked immunosorbent assay. Density-gradient ultracentrifugation demonstrated that the majority of the secreted apolipoprotein B was associated with the very-low-density lipoprotein fraction. Exposure of the cells to cholesterol (5-100 micrograms/ml) resulted in a dose-dependent increase in apolipoprotein B secretion rate.     

Inhibition of plasminogen activation by apo(a): role of carboxyl-terminal lysines and identification of inhibitory domains in apo(a)

Apo(a), the distinguishing protein component of lipoprotein(a) [Lp(a)], exhibits sequence similarity to plasminogen and can inhibit binding of plasminogen to cell surfaces. Plasmin generated on the surface of vascular cells plays a role in cell migration and proliferation, two of the fibroproliferative inflammatory events that underlie atherosclerosis. The ability of apo(a) to inhibit pericellular plasminogen activation on vascular cells was therefore evaluated. Two isoforms of apo(a), 12K and 17K, were found to significantly decrease tissue-type plasminogen activator-mediated plasminogen activation on human umbilical vein endothelial cells (HUVECs) and THP-1 monocytes and macrophages. Lp(a) purified from human plasma decreased plasminogen activation on THP-1 monocytes and HUVECs but not on THP-1 macrophages. Removal of kringle V or the strong lysine binding site in kringle IV₁₀ completely abolished the inhibitory effect of apo(a). Treatment with carboxypeptidase B to assess the roles of carboxyl-terminal lysines in cellular receptors leads in most cases to decreases in plasminogen activation as well as plasminogen and apo(a) binding; however, inhibition of plasminogen activation by apo(a) was unaffected. Our findings directly demonstrate that apo(a) inhibits pericellular plasminogen activation in all three cell types, although binding of apo(a) to cell-surface receptors containing carboxyl-terminal lysines does not appear to play a major role in the inhibition mechanism.     

Identification of human apolipoprotein E variant gene: apolipoprotein E7 (Glu244,245----Lys244,245)

Apolipoprotein E (apoE) is one of the protein moieties of the human serum lipoproteins. Three major isoforms of apoE (apoE2, apoE3, and apoE4) and minor variant isoforms (apoE1, apoE5, and apoE7) have been detected by isoelectric focusing. In this study we have cloned the apoE7 gene from a patient with the apoE3/E7 phenotype associated with hypertriglyceridemia and diabetes mellitus. DNA sequencing revealed that the apoE7 gene has two base substitutions (G----A) changing Glu244,245----Lys244,245, compared with the apoE3 gene. The replacement of the two amino acids is consistent with the result of isoelectric focusing of the apoE7 isoprotein, which shifts to four positively charged units compared with the apoE3 isoprotein.