Animals ; Atherosclerosis ; etiology ; metabolism ; Gene Expression Regulation ; Humans ; Lipase ; analysis ; genetics ; physiology ; Lipoproteins ; metabolism ; Lipoproteins, HDL ; metabolism ; Lipoproteins, LDL ; metabolism ; Polymorphism, Genetic ; Tissue Distribution

BACKGROUND: Plasma cholesteryl ester transfer protein (CETP) functions to transfer cholesteryl ester from HDL to triglyceride-rich lipoproteins and regulates plasma HDL cholesterol level. A common mutation, the exon 15 A to G substitution at codon 442 (D442G) results in reduced plasma CETP activity and increased plasma HDL cholesterol level. Meanwhile, hormone replacement therapy (HRT) in postmenopausal women increases plasma HDL cholesterol level. METHODS: We investigated the frequency of D442G mutation and its effect on plasma HDL cholesterol level in Korean women. We also examined if the mutation has any effect on an increase in plasma HDL cholesterol level during HRT. RESULTS: Two hundred and twenty eight women aged over 40 years were recruited in this study. Of 228 women, 22 (9.6%) were identified as having the D442G mutation; 21 heterozygotes and 1 homozygote. The subjects with the mutation had higher plasma HDL cholesterol levels than those without the mutation (61.6 +/- 17.3 vs. 55.1 +/- 14.0 mg/dL, p < 0.05). After 12 month HRT, HDL cholesterol increased by 6.4% (3.6 +/- 13.2 mg/dL, p < 0.05) and D442G mutation did not have any significant effect on the change of plasma HDL cholesterol level. CONCLUSION: D442G mutation is common in Korean postmenopausal women and it is associated with increased plasma HDL cholesterol level. HRT for postmenopausal women increased plasma HDL cholesterol level in similar amounts regardless of the presence or absence of D442G mutation.
Carrier Proteins/*genetics ; Estrogen Replacement Therapy ; Female ; Gene Frequency ; Human ; Korea ; Lipoproteins, HDL/*blood ; Menopause/*blood/*genetics ; Middle Age ; *Point Mutation

The work was aimed to investigate the differential expressions of lipid metabolism related genes in the early stage of atherosclerosis in the young apolipoprotein E deficient (apoE(-/-)) mice at different ages with normal chow diet. The genotypes of mice were identified by using multiplex polymerase chain reaction (multi-PCR) analysis. The semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR were used to analyze the expressions of lipid metabolism related genes in the liver of apoE(-/-) and age-matched wild type (WT) mice of 14-day old, 1-month old, 2-month old, 3-month old. The serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) contents were assayed using COD-PAP and GPO-PAP methods. The serum apolipoprotein B100 (apoB100) content was quantitated by immune turbidimetry. The hearts were perfusion-fixed in 4% formaldehyde, infiltrated with 30% gum sucrose for 24 h at 4 °C, and embedded in OCT compound. The aortic sinus tissues were serially sectioned at -15 °C, stained with Sudan IV, and counterstained with light green. The results were shown as follows. Compared with that in WT mice, the mRNA levels of apoA I and apoA IV in apoE(-/-) mice aged from 14-day old to 3-month old changed prominently (P<0.05), with apoA I up-regulated and apoA IV down-regulated. At the age of 1 month, the expression of apoB100 in apoE(-/-) mice was higher than that in WT mice (P<0.05). The expression of apoA V was up-regulated (P<0.05) and there was obvious lipid deposition in the aortic intima in apoE(-/-) mice at the age of 2 months. The expressions of fatty acid translocase (Fat/CD36) and angiopoietin-like protein 3 (Angptl 3) in apoE(-/-) mice were higher than those in WT mice at the age of 3 months (P<0.05), while the expressions of peroxisome proliferator-activated receptor α (PPARα), liver X receptor α (LXRα), carnitine palmitoyl transferase I (CPT I) and acyl coenzyme A oxidase 1 (ACOX1) showed no significant changes. The serum TC, TG, LDL-C and HDL-C contents in apoE(-/-) mice aged from 14-day old to 3-month old were higher than those in age-matched WT mice. apoE(-/-) mice showed a marked increase in serum apoB100 content, consistent with the trend of serum LDL-C content and apoB100 mRNA content in the liver. The results suggest that the mRNA expressions of apoA I, apoA IV, apoA V, apoB100 and Angptl 3 in apoE(-/-) mice change significantly compared with those in WT mice, and these genes might be relevant to the complicated lipid metabolism network, and involved in the early stage of atherogenesis.
Animals ; Apolipoprotein A-I ; metabolism ; Apolipoprotein B-100 ; blood ; Apolipoproteins A ; metabolism ; Apolipoproteins E ; genetics ; Atherosclerosis ; genetics ; Gene Expression ; Lipid Metabolism ; genetics ; Lipoproteins, HDL ; blood ; Lipoproteins, LDL ; blood ; Liver ; metabolism ; Mice ; Mice, Knockout ; Triglycerides ; blood

OBJECTIVETo develop a new high-throughput screening model for human high-density lipoprotein (HDL) receptor (CD36 and LIMPII analogous-1, CLA-1) agonists using CLA-1-expressing insect cells.

METHODSWith the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA (cDNA) of CLA-1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) into donor vector pFastBac1 and recombinant pFastBac1-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacmid DNA. Recombinant bacmid-CLA-1 was transfected into Spodoptera frugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA-1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized.

RESULTSWestern blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries.

CONCLUSIONHuman HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.

Animals ; Baculoviridae ; genetics ; metabolism ; Biological Assay ; Carbocyanines ; metabolism ; Cell Line, Tumor ; Cholesterol, HDL ; metabolism ; DNA, Complementary ; genetics ; metabolism ; Fluorescent Dyes ; metabolism ; Gene Expression ; Humans ; Lipoproteins, HDL ; agonists ; genetics ; metabolism ; Lipoproteins, LDL ; metabolism ; Receptors, Lipoprotein ; agonists ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Scavenger Receptors, Class B ; agonists ; genetics ; metabolism ; Spodoptera ; genetics ; metabolism

OBJECTIVETo investigate the effects of simvastatin on atherosclerosis and central aortic pressure in ApoE-knockout (ApoE-/-) mice.

METHODSTen 5-week-old male ApoE-/- mice and 5 C57 mice were fed with high-lipid diet for 3 weeks, and then C57 mice (WT group) and 5 ApoE-/- mice (ApoE-/- group) were given 1% carboxymethyl cellulose solution (8 ml·kg-1·d-1), and another 5 ApoE-/- mice (ApoE-/-/S group) were given simvastatin solution (50 mg·kg-1·d-1) by gavege for 3 weeks. The areas of atherosclerotic lesion in aortic root, central aortic pressure and serum lipid levels were examined.

RESULTSNo atherosclerotic plaques were observed in WT group. Compared with ApoE-/- group, simvastatin significantly decreased atherosclerotic lesion area in aortic root (89 818.05±16 980.93 μm2 vs 34 937.01±13 280.65 μm2, P<0.05). The systolic pressure (SP), mean arterial pressure (MAP), pulse pressure (PP) and diastolic pressure (DP) of central aortic pressure were significantly increased in ApoE-/- group compared with those in WT group (P<0.05). Compared to ApoE-/- group, the SP, MAP and PP of central aortic pressure were significantly reduced in ApoE-/-/S group (P<0.05). SP and MAP of central aortic pressure were positively correlated with atherosclerotic lesion area (SP: r=0.7152, P=0.0461; PP: r=0.7594， P=0.0288). Compared with WT group, serum triglyceride, total cholesterol and low-density lipoprotein levels were markedly increased in ApoE-/- group (P<0.05). Serum high-density lipoprotein level was decreased in ApoE-/- group compared with WT group. No differences in serum triglyceride, total cholesterol, low-density lipoprotein and high-density lipoprotein levels were found between ApoE-/- group and ApoE-/-/S group.

CONCLUSIONSimvastatin can attenuate atherosclerosis of aorta in ApoE-/- mice, which is associated with the reduced central aortic systolic pressure but not with the serum lipids levels.

Animals ; Apolipoproteins E ; genetics ; Arterial Pressure ; drug effects ; Atherosclerosis ; drug therapy ; genetics ; physiopathology ; Cholesterol ; blood ; Disease Models, Animal ; Lipoproteins, HDL ; blood ; Lipoproteins, LDL ; blood ; Male ; Mice ; Mice, Knockout ; Simvastatin ; pharmacology ; Triglycerides ; blood

OBJECTIVETo study the effects of high-density lipoprotein (HDL) and oxidized high-density lipoprotein (ox-HDL) on the expression of ATP-binding cassette transporter A1 (ABCAl) and cholesterol efflux in human umbilical vein endothelial cells (HUVECs).

METHODSIn vitro cultured HUVECs were incubated in the presence of 100 microg/ml HDL or 100 microg/ml ox-HDL for 24 h, using PBS as the negative control. ABCA1 mRNA level and cholesterol efflux rate were determined using RT-PCR and a liquid scintillator, respectively.

RESULTSHDL and ox-HDL significantly elevated the level of ABCA1 mRNA by 58% and 23% relative to the control level, respectively (P<0.05). The cholesterol efflux rate in ox-HDL group was significantly lower than that in HDL group (P<0.01).

CONCLUSIONHDL increases ABCAl expression and cholesterol efflux in HUVECs. Oxidative modification of HDL decrease cholesterol efflux by inhibiting the expression of ABCAl, suggesting a possible mechanism of ox-HDL in the pathogenesis of atherosclerosis.

ATP Binding Cassette Transporter 1 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; Cells, Cultured ; Cholesterol ; metabolism ; Endothelial Cells ; metabolism ; Humans ; Lipoproteins, HDL ; metabolism ; physiology ; Umbilical Veins ; cytology

The aim of this study was to determine the effects of polymorphisms in the apolipoprotein E gene (APOE) on lipid levels in Korean adults and to investigate the interactions between these polymorphisms and environmental factors in determining lipid levels. We performed a cross-sectional study of 1,900 subjects (668 men and 1,232 women; 45-74 yr old) in Namwon, Korea, in 2004. APOE polymorphisms were determined by polymerase chain reaction and restriction enzyme analysis. Carriers of the APOE*E2 (E2) allele had significantly lower total cholesterol and low-density lipoprotein cholesterol (LDL-C) concentrations than did carriers of the APOE*E3 (E3) or APOE*E4 (E4) alleles, regardless of gender. The APOE allele type had significant effect on high-density lipoprotein cholesterol (HDL-C) and triglyceride levels in women, but not in men. The effect of APOE allele type on HDL-C levels was modified by age in women. In addition, in men, the effect of APOE allele type on triglyceride levels was modified by smoking. These findings highlight the important effect of gene-environment interactions on lipid levels.
Aged ; Analysis of Variance ; Apolipoproteins E/*genetics ; Comparative Study ; Cross-Sectional Studies ; Female ; Gene Frequency ; Genotype ; Humans ; Korea ; Lipids/*blood ; Lipoproteins, HDL Cholesterol/blood ; Lipoproteins, LDL Cholesterol/blood ; Male ; Middle Aged ; *Polymorphism, Genetic ; Triglycerides/blood

OBJECTIVESTo study the contribution of the genes and environment to variation of serum levels of lipids and lipoprotein.

METHODSOne hundred and forty-three healthy monozygotic (MZ) twin pairs and 93 dizygotic (DZ) ones aged 5 to 19 [with a mean of (11.2 +/- 3.4) years]. Microsatellite polymorphism (STR) was used to diagnose zygosity of twins, and intraclass correlation coefficient method and Falconer formula were performed to investigate heritability of serum lipids and lipoproteins unadjusted or adjusted for age and sex. Logarithmic transformation was used for data with skewed distribution. Influence of relevant physical and biochemical indicators on serum lipids and other components was analyzed with partial coefficients of correlation adjusted for age and sex.

RESULTSIn the twin samples, difference in serum level of triglycerides (TG) between MZ and DZ was not statistically significant with intraclass variation and intraclass correlation. There was significant difference in serum levels of total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), and lipoprotein [Lp(a)] between MZ and DZ, with their heritability estimates of 0.56, 0.55, 0.49 and 0.58 unadjusted, respectively, and 0.63, 0.63, 0.55 and 0.64 adjusted for age and sex, respectively. Serum levels of TC, HDL-C, LDL-C and Lp(a) correlated reversely with age. Serum levels of TC, HDL-C and LDL-C in girls were slightly higher than those in boys. Most indicators for serum levels of lipids and lipoprotein, except for serum level of Lp(a) correlated with body mass index (BMI), body fat ratio, Pelidisi index, and other indexes such as blood pressure, blood sugar, serum level of calcium, adjusted for age and sex.

CONCLUSIONSSerum levels of TC, HDL-C and Lp(a) were influenced more greatly by genetic factors, and serum level of TG was mainly influenced by environmental ones. Levels of blood lipids in children were influenced by age and sex, and correlated with indicators that reflect their body fat and nutritional status.

Adolescent ; Adult ; Age Factors ; Child ; Cholesterol, HDL ; blood ; genetics ; Cholesterol, LDL ; blood ; genetics ; Female ; Humans ; Lipids ; blood ; genetics ; Lipoproteins ; blood ; genetics ; Male ; Sex Factors ; Triglycerides ; blood ; genetics ; Twin Studies as Topic ; Twins, Dizygotic ; Twins, Monozygotic

OBJECTIVETo study the relationship between polymorphisms of interleukin 10 (IL10QX) promoter and serum levels of lipoprotein in the healthy Chinese Han population.

METHODSPCR restriction fragment length polymorphism assay was used to detect the distribution of genotypes of IL10 -592,-819,-1082 in 200 healthy Chinese Han subjects. Serum levels of total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and very low-density lipoprotein (VLDL) in all subjects were measured to analyze the relationship with the polymorphisms of IL10 promoter.

RESULTSComparing with AA genotype, the group with GA genotype at IL10 promoter -1082 position had a significant elevation of serum HDL-C level [(1.514+/-0.501) mmol/L vs. (1.261+/-0.346) mmol/L, t=-2.225, P=0.028] and a lower serum TG level[(1.701+/-1.836) mmol/L vs. (0.981+/-0.314) mmol/L,Z=-2.096,P=0.036]. The TG, TC, HDL-C, LDL-C and VLDL levels did not show any statistically significant differences among different genotypes (CC, AA, CA) of the IL10 -592, as well as the genotypes (TT, TA, AA) ofIL10 -819 (P>0.05).

CONCLUSIONThe results suggest that in the Chinese Han population, the polymorphism at position -1082 in the promoter region of IL10 gene may be associated with the serum HDL-C level and TG level.

Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; China ; Cholesterol ; blood ; Cholesterol, HDL ; blood ; Cholesterol, LDL ; blood ; Genotype ; Humans ; Interleukin-10 ; genetics ; Lipoproteins ; blood ; Male ; Middle Aged ; Polymorphism, Genetic ; genetics ; Promoter Regions, Genetic ; genetics ; Triglycerides ; blood ; Young Adult

OBJECTIVETo investigate polymorphisms in the gene for lipoprotein lipase (LPL) in Chinese populations with coronary heart disease (CHD) and to inquire into the relationship between these polymorphisms in LPL gene and CHD.

METHODSGenomic DNA was extracted from patients with CHD and normal control subjects using a salting out method. The entire coding region and flanking sequences of all coding exons of the LPL gene were amplified by PCR technique and PCR products were detected by denaturing high-performance liquid chromatography (DHPLC) and sequenced with a dideoxy terminal termination method.

RESULTSA novel polymorphic site, G830A, that is within the fifth exon of the LPL gene was found. The 192 codon CGA was changed into CAA and resulted in the substitution of glutamine for arginine. Between the control and CHD groups, chi-square test showed no significant difference in the frequencies of the A/A genotype and A allele (P > 0.05). However, the frequencies of A/A genotype and A allele (0.653 and 0.786) in CHD patients with high plasma triglyceride/lowed plasma high density lipoprotein cholesterol were higher than those (0.415 and 0.642) in CHD patients without hyperlipidemia (P < 0.05).

CONCLUSIONNo direct association was found between the LPL Arg192-->Gln substitution polymorphism and CHD, but there is a significant positive correlation between the A/A genotype of the LPL gene and CHD associated with high triglyceride/lowed high density lipoprotein cholesterol. This study may provide new data for exploring the molecular mechanism of CHD.

Alleles ; Apolipoproteins ; blood ; Cholesterol, HDL ; blood ; Chromatography, High Pressure Liquid ; methods ; Coronary Disease ; blood ; enzymology ; genetics ; DNA ; chemistry ; genetics ; DNA Mutational Analysis ; Gene Frequency ; Humans ; Hypertriglyceridemia ; blood ; genetics ; Lipoprotein Lipase ; genetics ; Lipoproteins ; blood ; Polymorphism, Genetic