Apolipoprotein E gene expression is reduced in apolipoprotein A-I transgenic mice

The levels of plasma apolipoprotein (apo) E, an anti-atherogenic protein involved in mammalian cholesterol transport, were found to be 2-3 fold lower in mice over-expressing human apoA-I gene. ApoE is mainly associated with VLDL and HDL-size particles, but in mice the majority of the apoE is associated with the HDL particles. Over-expression of the human apoA-I in mice increases the levels of human apoA-I-rich HDL particles by displacing mouse apoA-I from HDL. This results in lowering of plasma levels of mouse apoA-I. Since plasma levels of apoE also decreased in the apoA-I transgenic mice, the mechanism of apoE lowering was investigated. Although plasma levels of apoE decreased by 2-3 fold, apoB levels remained unchanged. As expected, the plasma levels of human apoA-I were almost 5-fold higher in the apoAI-Tg mice compared to mouse apoA-I in WT mice. If the over-expression of human apoA-I caused displacement of apoE from the HDL, the levels of hepatic apoE mRNA should remain the same in WT and the apoAI-Tg mice. However, the measurements of apoE mRNA in the liver showed 3-fold decreases of apoE mRNA in apoAI-Tg mice as compared to WT mice, suggesting that the decreased apoE mRNA expression, but not the displacement of the apoE from HDL, resulted in the lowering of plasma apoE in apoAI-Tg mice. As expected, the levels of hepatic apoA-I mRNA (transgene) were 5-fold higher in the apoAI-Tg mice. ApoE synthesis measured in hepatocytes also showed lower synthesis of apoE in the apoAI-Tg mice. These studies suggest that the integration of human apoA-I transgene in mouse genome occurred at a site that affected apoE gene expression. Identification of this locus may provide further understanding of the apoE gene expression.     

Limited effects of combined dietary copper deficiency/iron overload on oxidative stress parameters in rat liver and plasma

Copper (Cu) deficiency decreases the activity of Cu-dependent antioxidant enzymes such as Cu,zinc-superoxide dismutase (Cu,Zn-SOD) and may be associated with increased susceptibility to oxidative stress. Iron (Fe) overload represents a dietary oxidative stress relevant to overuse of Fe-containing supplements and to hereditary hemochromatosis. In a study to investigate oxidative stress interactions of dietary Cu deficiency with Fe overload, weanling male Long–Evans rats were fed one of four sucrose-based modified AIN-93G diets formulated to differ in Cu (adequate 6 mg/kg diet vs. deficient 0.5 mg/kg) and Fe (adequate 35 mg/kg vs. overloaded 1500 mg/kg) in a 2×2 factorial design for 4 weeks prior to necropsy. Care was taken to minimize oxidation of the diets prior to feeding to the rats. Liver and plasma Cu content and liver Cu,Zn-SOD activity declined with Cu deficiency and liver Fe increased with Fe overload, confirming the experimental dietary model. Liver thiobarbituric acid reactive substances were significantly elevated with Fe overload (pooled across Cu treatments, 0.80±0.14 vs. 0.54±0.08 nmol/mg protein; P<.0001) and not affected by Cu deficiency. Liver cytosolic protein carbonyl content and the concentrations of several oxidized cholesterol species in liver tissue did not change with these dietary treatments. Plasma protein carbonyl content decreased in Cu-deficient rats and was not influenced by dietary Fe overload. The various substrates (lipid, protein and cholesterol) appeared to differ in their susceptibility to the in vivo oxidative stress induced by dietary Fe overload, but these differences were not exacerbated by Cu deficiency.     

Apolipoprotein B gene expression in rat intestine. The effect of dietary fiber.

The effect of the dietary fiber on apo B mRNA level was studied in the intestine of rats that were fed either fiber-free or high-fiber (30% sugar-beet fiber) low-fat diets for 3 weeks. The fiber diet studied does not affect jejunal apo B mRNA levels but decreases the level of ileal apo B mRNA. In the rat cecum, in both fiber-free and fiber groups, we failed to detect the apo B mRNA. The test fiber diet feeding markedly increased fecal bile salt and cholesterol excretions. We suggest that dietary fiber can modify apo B expression in the intestine. The increased fecal bile salt excretion might be involved in such a modification.     

The effect of apolipoprotein E polymorphism on plasma cholesteryl ester transfer protein activity in type 2 diabetic patients

We studied the relationship between apo E polymorphism and cholesteryl ester transfer protein (CETP) activity in 127 type 2 diabetic patients who did not take lipid lowering drugs. Furthermore, we studied the relationship between apo E and cholesteryl ester transfer protein (CETP) in modulating plasma triglyceride and HDLcholesterol. Apo E genotypes were determined by PCR-RFLP, and CETP activity was measured using an exogenous way. Our results showed that the CETP activity increased significantly in the E2 carrier group compared to E4 carriers and E3/E3 homozygous (84.7 ± 43.9 vs. 62.5 ± 35.9 vs. 52.6 ± 23.6 nmol CE/ml/2h, respectively; p = 0.015). However, there was no association between apo E polymorphism and lipid parameter variations. Even after adjustment for CETP activity, the results remained unchanged, showing that CETP did not step in the relationship between apo E and lipid parameter variations. In conclusion there is an association between apo E polymorphism and CETP activity, and this association did not affect the relationship between apo E polymorphism and triglyceride and HDLcholesterol concentrations. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 6, pp. 931–935. The text was submitted by the authors in English.     

转基因鼠载脂蛋白E基因异常表达与血脂代谢水平的相关性

探讨载脂蛋白E(apoE)基因在转基因鼠体内的表达及其在血脂代谢中的作用.以单克隆抗体酶联免疫吸附法分别测定apoE4、apoE7转基因鼠(tg4、tg7)的血清apoE含量.用甘油磷酸化酶(GPO)法和胆固醇氧化酶(CHOD-PAP)法对转基因鼠及对照组鼠(control)的血清甘油三酯(TG)和胆固醇(TC)进行测定.tg4血清apoE含量为20.3±7.2ug/dl,tg7血清apoE含量为1.8±5.4ug/dl.血清TG水平转基因鼠[tg4(19.16±0.31)mmol/L,tg7(18.15±0.46)mmol/L]与对照组鼠[(4.95±2.25)mmol/L]的差异均有显著性(p＜0.05).血清TC水平tg4[(4.44±0.04)mmol/L]与对照组鼠[(1.49±0.01)mmol/L]也表现出显著性的差异(p＜0.05).提示apoE基因异常表达影响了转基因鼠的血脂代谢.     

重组人载脂蛋白AI米兰突变体在大肠杆菌中的高表达

用RT-PCR法从人肝总RNA库中克隆出人载脂蛋白Al的cDNA序列,再通过重叠PCR将载脂蛋白AI的第179位精氨酸密码子突变成半胱氨酸密码子,即载胎蛋白AI米兰突变体基因。将此目的基因克隆至表达载体pQE30,重组质粒转化JMl09宿主菌,经表达试验筛选出高表达克隆;工程菌经诱导后表达出含6个氨基酸前肽的载脂蛋白AI米兰突变体。表达产物主要以可溶形式存在,但也有部分为包涵体。     

Investigations into combined dietary deficiencies of copper,selenium, and vitamin E in the rat

Interactions between dietary Cu, Se, and vitamin E in ascorbate-induced hemolysis of erythrocytes obtained from rats fed diets deficient or adequate in these elements were investigated. Hemolysis was affected by all three dietary factors, through closely interrelated but distinct mechanisms. In vitamin E-deficient cells, hemolysis was increased and the amount of hemolysis was directly related to the amount of hemoglobin breakdown. Deficiency of Cu or Se decreased hemolysis, but only in vitamin E-deficient cells. Vitamin E did not affect the breakdown of hemoglobin, but Cu and Se did. Hemolysis and hemoglobin breakdown were decreased by the addition of glucose, through mechanisms independent of that involving reduced glutathione metabolism. These results suggest that vitamin E acts within erythrocyte membranes to prevent products of hemoglobin breakdown from initiating peroxidation and subsequent hemolysis. Effects of Cu and Se are linked with that of vitamin E by the involvement of glutathione peroxidase and Cu superoxide dismutase in the cytoplasmic breakdown of hemoglobin, rather than by a direct effect of these enzymes on lipid peroxidation. It is concluded that the erythrocyte, because of its high heme content, probably represents a special system in terms of peroxidative pathways, and these findings may not be directly applicable to other tissues.     

Effect of apolipoprotein A-V on plasma triglyceride, lipoprotein size, and composition in genetically engineered mice

Transgenic (Tg) mice that overexpress the human apolipoprotein A-V gene (APOA5) yet lack an endogenous mouse apoa5 gene (APOA5 Tg mice) were generated. Subsequently, the effect of human apoA-V expression on plasma triglyceride (TG) concentration and lipoprotein and apolipoprotein distribution was determined and compared with that in mice deficient in apoA-V (apoa5(-/-) mice). NMR analysis of plasma lipoproteins revealed that APOA5 Tg mice had a very low VLDL concentration (26.4 +/- 7.7 nmol/dl), whereas VLDL in apoa5(-/-) mice was 18- fold higher (467 +/- 152 nmol/dl). SDS-PAGE analysis of the d < 1.063 g/ml plasma fraction revealed that the apoB-100/apoB-48 ratio was 14-fold higher in APOA5 Tg versus apoa5(-/-) mice and that the apoE/total apoB ratio was 7-fold greater in APOA5 Tg versus apoa5(-/-) mice. It is anticipated that a reduction in apoB-100/apoB-48 ratio as well as that for apoE/apoB would impair the uptake of VLDL and remnants in apoa5(-/-) mice, thereby contributing to increased plasma TG levels. The concentration of apoA-V in APOA5 Tg mice was 12.5 +/- 2.9 microg/ml, which is approximately 50- to 100-fold higher than that reported for normolipidemic humans. ApoA-V was predominantly associated with HDL but was rapidly and efficiently redistributed to apoA- V-deficient VLDL upon incubation. Consistent with findings reported for human subjects, apoA-V concentration was positively correlated with TG levels in normolipidemic APOA5 Tg mice. It is conceivable that, in a situation in which apoA-V is chronically overexpressed, complex interactions among factors regulating TG homeostasis may result in a positive correlation of apoA-V with TG concentrations.     

A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes

Apolipoprotein E (apoE) is an important protein involved in lipoprotein clearance and cholesterol redistribution. ApoE is abundantly expressed in astrocytes in the brain and is closely linked to the pathogenesis of Alzheimer's disease (AD). We report here that small molecule ligands that activate either liver X receptors (LXR) or retinoid X receptor (RXR) lead to a dramatic increase in apoE mRNA and protein expression as well as secretion of apoE in a human astrocytoma cell line (CCF-STTG1 cells). Examination of primary mouse astrocytes also revealed significant induction of apoE mRNA, and protein expression and secretion following incubation with LXR/RXR agonists. Moreover, treatment of mice with a specific synthetic LXR agonist T0901317 resulted in up-regulation of apoE mRNA and protein in both hippocampus and cerebral cortex, indicating that apoE expression in brain can be up-regulated by LXR agonists in vivo. Along with a dramatic induction of ABCA1 cholesterol transporter expression, these ligands effectively mediate cholesterol efflux in both CCF-STTG1 cells and mouse astrocytes in the presence or absence of apolipoprotein AI (apoAI). Our studies provide strong evidence that small molecule LXR/RXR agonists can effectively mediate apoE synthesis and secretion as well as cholesterol homeostasis in astrocytes. LXR/RXR agonists may have significant impact on the pathogenesis of multiple neurological diseases, including AD.     

Regulation of the apolipoprotein B in heterozygous hypobeta-lipoproteinemic knock-out mice expressing truncated apoB,B81. Low production and enhanced clearance of apoB cause low levels of apoB

Meat quality of pigs is dependent on biochemical and biophysical processes in the time course post mortem (p.m.) and is associated with the intracellular Ca2+ homeostasis. However, there is little known about changes in the Ca2+ transporting proteins controlling the Ca2+ uptake of sarcoplasmic reticulum (SR) in the time course p.m. In this study changes in the Ca2+ transporting proteins were investigated in homogenates of longissimus muscles of 4 malignant hyperthermia susceptible (MHS) and 6 malignant hyperthermia resistant (MHR) Pietrain pigs. Muscle samples were obtained at different time intervals: biopsy 2 h prior slaughtering and from the carcass immediately after exsanguination (0 h), 45 min, 4 h, and 22 h p.m. The SR Ca2+ uptake rate was measured immediately after homogenization with closed calcium release channel (CRC), with opened CRC and without manipulation of CRC. Additionally the SR Ca2+ ATPase activity was determined.The results show: (i) The ability of SR to sequester Ca2+ declined to about 60% in the first 45 min p.m. in MHS samples irrespective of CRC state, whereas in MHR samples this decline was about 5%; (ii) Ca2+ uptake and Ca2+ ATPase activity were not different between the biopsy and 0 h samples, i.e. the stress of slaughter was of no immediate influence; (iii) The Ca2+ ATPase activity of the SR declined at about the same rate as the Ca2+ uptake in both MHS and MHR pig samples in the course of time p.m.; (iv) In samples, taken immediately after exsanguination, the Ca2+ ATPase activity of MHS pigs was higher than that of MHR pigs. However, in samples taken 4 h p.m. Ca2+ ATPase activity of MHS pigs has declined to about 30% of the value at 0 h; (v) The CRC can be closed and opened in all samples up to 22 h p.m. and seems to be fully functional at all sampling times; (vi) The CRC of MHS pigs is almost fully open, whereas the CRC of MHR pigs is only partially open at all sampling times; (vii) The permeability of the SR membrane to Ca2+ (determined as the ratio of SR Ca2+ ATPase with and without ionophore A23187) is the same in both MHS and MHR and did not change with ongoing time; (viii) No uncoupling of uptake from ATP hydrolysis occurred up to 4 h p.m., but the coupling differed between MHS and MHR for all time intervals with lower values for MHS pigs. The results suggest that the decreasing Ca2+ uptake rate of homogenates, sampled at different times p.m., is essentially caused by changes in the Ca2+ pump and not by changes in the CRC or an increased phospholipid membrane permeability to Ca2+.     

The recycling of apolipoprotein E in macrophages: influence of HDL and apolipoprotein A-I

The ability of apolipoprotein E (apoE) to be spared degradation in lysosomes and to recycle to the cell surface has been demonstrated by our group and others, but its physiologic relevance is unknown. In this study, we characterized apoE recycling in primary murine macrophages and probed the effects of HDL and apoA-I on this process. In cells pulsed with (125)I.apoE bound to VLDL, intact apoE was found in the chase medium for up to 24 h after the pulse. Approximately 27 +/- 5% of the apoE internalized during the pulse was recycled after 4 h of chase. Addition of apoA-I and HDL increased apoE recycling to 45 +/- 3% and 46 +/- 3%, respectively, similar to the amount of apoE recycled after pulsing the cells with (125)I.apoE.HDL. In addition, apoA-I-producing macrophages from transgenic mice showed increased apoE recycling at 4 h (38 +/- 3%). Increased ABCA1 expression potentiated apoE recycling, suggesting that recycling occurs via ABCA1. Finally, in the presence of apoA-I, recycled apoE exited the cells on HDL-like particles. These results suggest that apoE recycling in macrophages may be part of a larger signaling loop activated by HDL and directed at maximizing cholesterol losses from the cell.     

Effect of apolipoprotein E genotype on apolipoprotein B-100 metabolism in normolipidemic and hyperlipidemic subjects

The effect of apolipoprotein (apo) E genotype on apoB-100 metabolism was examined in three normolipidemic apoE2/E2, five type III hyperlipidemic apoE2/E2, and five hyperlipidemic apoE3/E2 subjects using simultaneous administration of ¹³¹I-VLDL and ¹²⁵I-LDL, and multi-compartmental modeling. Compared with normolipidemic apoE2/E2 subjects, type III hyperlipidemic E2/E2 subjects had increased plasma and VLDL cholesterol, plasma and VLDL triglycerides, and VLDL and intermediate density lipoprotein (IDL) apoB concentrations (P < 0.05). These abnormalities were chiefly a consequence of decreased VLDL and IDL apoB fractional catabolic rate (FCR). Compared with hyperlipidemic E3/E2 subjects, type III hyperlipidemic E2/E2 subjects had increased IDL apoB concentration and decreased conversion of IDL to LDL particles (P < 0.05). In a pooled analysis, VLDL cholesterol was positively associated with VLDL and IDL apoB concentrations and the proportion of VLDL apoB in the slowly turning over VLDL pool, and was negatively associated with VLDL apoB FCR after adjusting for subject group. VLDL triglyceride was positively associated with VLDL apoB concentration and VLDL and IDL apoB production rates after adjusting for subject group. A defective apoE contributes to altered lipoprotein metabolism but is not sufficient to cause overt hyperlipidemia. Additional genetic mutations and environmental factors, including insulin resistance and obesity, may contribute to the development of type III hyperlipidemia.     

Bacterial expression and characterization of rat apolipoprotein E

Apolipoprotein (apo) E is a protein involved in both lipid metabolism and neuroprotection. Recently, it has been suggested that apoE may play a role in the regulation of food intake and body weight in rodents. However, rodent plasma apoE is difficult to purify in reasonable amounts due to numerous time-consuming steps. To circumvent this, we created a bacterial expression system for the efficient production of large amounts of rat apoE. We inserted rat apoE DNA into the pET30 expression vector and overexpressed the proteins in Escherichia coli strain BL21 (DE3). A histidine tag present at the N-terminus allowed for easy purification of the recombinant protein. The tag was removed with an IgA protease (Igase) from Neisseria gonorrhoeae leaving the mature form of the protein. The use of Igase was important as several more common proteases routinely cleave apolipoproteins at undesired sites. The recombinant protein was then compared both structurally and functionally to rat plasma apoE. This expression system will be highly useful for probing the ability of rat apoE to mediate food intake in rats.     

Combined effect of high-fat diet and copper deficiency during gestation on fetal copper status in the rat

We have previously shown that a low-copper (Cu) diet produced alterations in placental Cu transport and fetal Cu stores. Because Cu deficiency has been associated with lipid deposition in rat dam liver, we hypothesized that a high fat intake, a prevalent dietary habit in many populations, may worsen fetal Cu status and its closely linked iron (Fe) deposits. Pregnant rats were fed one of four diets during the second half of gestation: NFNCu: normal fat (7%), normal Cu (6 mg/kg); HFNCu: high fat (21%), normal Cu; NFLCu: normal fat, low Cu (0.6 mg/kg), and HFLCu: high fat, low Cu. One day before delivery, dams were anesthetized, and maternal as well as fetal plasma and tissues were obtained. Maternal, fetal, and placental weights were indistinguishable regardless of the group. Dam plasma Cu and placental Cu were lower in both LCu groups than in the NFNCu or the HFNCu groups. However, fetal plasma Cu was similar in all treatment groups. Dam and fetal liver Cu stores were reduced in the LCu groups compared to the NCu groups. This resulted in lower fetal/maternal liver Cu ratios in the NFLCu (1.79 ± 0.14,p < 0.05) and HFLCu (1.59 ± 0.21,p < 0.05) as compared to the NFNCu (4.12 ± 0.44) and the HFNCu (4.15 ± 0.27). Dam liver Fe was higher in the NFNCu than in HFNCu group (1.10 ± 0.8 vs. 0.89 ± 0.06 μmol/g,p < 0.05); fetal liver Fe from HFNCu and NFLCu dams was lower than that from NFNCu fetuses (NFNCu: 2.42 ± 0.14; HFNCu: 1.92 ± 0.15,p < 0.05; NFLCu: 1.81 ± 0.10,p < 0.01). Fetuses of the HFLCu group had a lower heart Fe than the NFNCu group (0.56 ± 0.03 vs. 44.0 ± 3.0 μg/g,p < 0.01). These data indicate that a maternal high-fat diet can potentially aggravate the effects of Cu deficiency by further altering fetal Cu and Fe tissue stores.     

Role of apolipoprotein E and B gene variation in determining response of lipid, lipoprotein, and apolipoprotein levels to increased dietary cholesterol.

A large segment of the population is modifying its dietary cholesterol intake to achieve a healthier life-style. However, all individuals do not respond equally. We have investigated the effects that that two physiologically important polymorphisms in the apolipoprotein (apo) E and B genes have on the responses of plasma lipid, lipoprotein, and apolipoprotein levels to a high-cholesterol diet. Over a 6-wk period, individuals were prescribed two diets, one consisting of 300 mg dietary cholesterol/d for 3 wk and one consisting of 1,700 mg dietary cholesterol/d for 3 wk. Total cholesterol, low-density-lipoprotein cholesterol (LDL-C), and apo B levels were significantly increased on the high-cholesterol diet. Average total cholesterol (numbers in parentheses are SDs) went from 167.6 (23.4) mg/dl on the low-cholesterol diet to 190.8 (36.2) mg/dl on the high-cholesterol diet; LDL-C went from 99.9 (24.8) mg/dl to 119.2 (33.4) mg/dl, and apo B went from 74.9 (24.5) mg/dl to 86.8 (29.5) mg/dl. In 71 individuals, the frequencies of the apo epsilon 2, epsilon 3, and epsilon 4 alleles were .09, .84, and .07, respectively. The frequency of the longer, apo B signal peptide allele (5'beta SP27) was .68. Apo epsilon 2/3 individuals had significantly lower LDL-C levels than did epsilon 3/3 homozygotes, on both the low-cholesterol diet (LDL-C lower by 21 mg/dl) and the high-cholesterol diet (LDL-C lower by 27 mg/dl). Average triglyceride levels were significantly different among apo B signal peptide genotypes, with the 5'beta SP27/37 homozygotes having the lowest levels (70 mg/dl). When individuals were switched from the low-cholesterol diet to the high-cholesterol diet, in no case were the average responses in lipid levels significantly different among apo E or B genotypes. Therefore, these gene loci do not have a major effect on the response of lipid levels to increased dietary cholesterol.     

The role of apolipoprotein E in neurodegeneration and cardiovascular disease

Apolipoprotein E (ApoE) is an abundant plasma protein that interacts with low density lipoprotein receptors and other proteins, participating in the transport of cholesterol and lipids. Research has revealed many other roles for this multifunctional protein. ApoE is polymorphic and exists in three major isoforms: ApoE2, ApoE3 (the most common isoform) and ApoE4, which differ by only one amino acid, at positions 112 and 158. The altered binding to lipids and receptors by ApoE isoforms E2 and E4 results in an elevated risk for neurological, cerebrovascular and cardiovascular pathologies. Most notably, ApoE4 is associated with an elevated risk (relative to E3) for Alzheimer’s disease. The application of mass spectrometry for genotyping and also direct measurement of ApoE protein isoforms is a recent development and is well suited to high-throughput applications. The precise quantification of protein isoforms will allow better characterization of effects resulting from heterozygous APOE genotypes.