2-Aminopurine Inhibits Lipid Accumulation Induced by Apolipoprotein E-Deficient Lipoprotein in Macrophages: Potential Role of Eukaryotic Initiation Factor-2␣ Phosphorylation in Foam Cell Formation

ABSTRACT We previously reported that apolipoprotein (Apo) E-deficient, ApoB48-containing (E Ϫ /B48) lipoproteins inhibited expression of lysosomal hydrolase and transformed mouse peritoneal macrophages (MPMs) into foam cells. The present study examined the effect of 2-aminopurine (2-AP), an inhibitor of eukaryotic initiation factor (eIF)-2␣ phosphorylation, on E Ϫ /B48 lipoprotein-induced changes in gene expression and foam cell formation. Our data demonstrated that E Ϫ /B48 lipoproteins enhanced phosphorylation of eIF-2␣ in macrophages. Incubation of MPMs with E Ϫ /B48 lipoproteins inhibited the translation efficiency of mRNAs encoding lysosomal acid lipase, cathepsin B, and cation-dependent mannose 6 phosphate receptor, with a parallel reduction in the level of these proteins. Addition of 2-AP to the culture media alleviated the suppressive effect of E Ϫ /B48 lipoproteins on lysosomal hydrolase mRNA translation, increased macrophage degradation of E Ϫ /B48 lipoproteins, and inhibited foam cell formation. Transfection of MPMs with a nonphosphorylatable eIF-2␣ mutant also attenuated the suppressive effect of E Ϫ /B48 lipoproteins on expression of lysosomal acid lipase, associated with a reduced accumulation of cellular cholesterol esters. This is the first demonstration that ApoE-deficient lipoproteins inhibit lysosomal hydrolase synthesis and transform macrophages into foam cells through induction of eIF-2␣ phosphorylation

A Novel Function of Apolipoprotein E: Upregulation of ATP-Binding Cassette Transporter A1 Expression

Despite the well known importance of apolipoprotein (Apo) E in cholesterol efflux, the effect of ApoE on the expression of ATP-binding cassette transporter A1 (ABCA1) has never been investigated. The objective of this study was to determine the effect of ApoE on ApoB-carrying lipoprotein-induced expression of ABCA1, a protein that mediates cholesterol efflux. Our data demonstrate that ApoB-carrying lipoproteins obtained from both wild-type and ApoE knockout mice induced ApoAI-mediated cholesterol efflux in mouse macrophages, which was associated with an enhanced ABCA1 promoter activity, and an increased ABCA1 mRNA and protein expression. In addition, these lipoproteins increased the level of phosphorylated specificity protein 1 (Sp1) and the amount of Sp1 bound to the ABCA1 promoter. However, all these inductions were significantly diminished in cells treated with ApoE-free lipoproteins, when compared to those treated with wild-type lipoproteins. Enrichment with human ApoE3 reversed the reduced inducibility of ApoE-free lipoproteins. Moreover, we observed that inhibition of Sp1 DNA-binding by mithramycin A diminished ABCA1 expression and ApoAI-mediated cholesterol efflux induced by ApoB-carrying lipoproteins, and that mutation of the Sp1-binding motif in the ABCA1 promoter region diminished ApoB-carrying lipoprotein-induced ABCA1 promoter activity. Collectively, these data suggest that ApoE associated with ApoB-carrying lipoproteins has an upregulatory role on ABCA1 expression, and that induction of Sp1 phosphorylation is a mechanism by which ApoE upregulates ABCA1 expression

Transport of Apolipoprotein B-Containing Lipoproteins through Endothelial Cells Is Associated with Apolipoprotein E-Carrying HDL-Like Particle Formation

Passage of apolipoprotein B-containing lipoproteins (apoB-LPs), i.e., triglyceride-rich lipoproteins (TRLs), intermediate-density lipoproteins (IDLs), and low-density lipoproteins (LDLs), through the endothelial monolayer occurs in normal and atherosclerotic arteries. Among these lipoproteins, TRLs and IDLs are apoE-rich apoB-LPs (E/B-LPs). Recycling of TRL-associated apoE has been shown to form apoE-carrying high-density lipoprotein (HDL)-like (HDLE) particles in many types of cells. The current report studied the formation of HDLE particles by transcytosis of apoB-LPs through mouse aortic endothelial cells (MAECs). Our data indicated that passage of radiolabeled apoB-LPs, rich or poor in apoE, through the MAEC monolayer is inhibited by filipin and unlabeled competitor lipoproteins, suggesting that MAECs transport apoB-LPs via a caveolae-mediated pathway. The cholesterol and apoE in the cell-untreated E/B-LPs, TRLs, IDLs, and LDLs distributed primarily in the low-density (LD) fractions (d ≤ 1.063). A substantial portion of the cholesterol and apoE that passed through the MAEC monolayer was allotted into the high-density (HD) (d > 1.063) fractions. In contrast, apoB was detectable only in the LD fractions before or after apoB-LPs were incubated with the MAEC monolayer, suggesting that apoB-LPs pass through the MAEC monolayer in the forms of apoB-containing LD particles and apoE-containing HD particles

Cholecystokinin elevates mouse plasma lipids.

Cholecystokinin (CCK) is a peptide hormone that induces bile release into the intestinal lumen which in turn aids in fat digestion and absorption in the intestine. While excretion of bile acids and cholesterol into the feces eliminates cholesterol from the body, this report examined the effect of CCK on increasing plasma cholesterol and triglycerides in mice. Our data demonstrated that intravenous injection of [Thr28, Nle31]-CCK at a dose of 50 ng/kg significantly increased plasma triglyceride and cholesterol levels by 22 and 31%, respectively, in fasting low-density lipoprotein receptor knockout (LDLR(-/-)) mice. The same dose of [Thr28, Nle31]-CCK induced 6 and 13% increases in plasma triglyceride and cholesterol, respectively, in wild-type mice. However, these particular before and after CCK treatment values did not achieve statistical significance. Oral feeding of olive oil further elevated plasma triglycerides, but did not alter plasma cholesterol levels in CCK-treated mice. The increased plasma cholesterol in CCK-treated mice was distributed in very-low, low and high density lipoproteins (VLDL, LDL and HDL) with less of an increase in HDL. Correspondingly, the plasma apolipoprotein (apo) B48, B100, apoE and apoAI levels were significantly higher in the CCK-treated mice than in untreated control mice. Ligation of the bile duct, blocking CCK receptors with proglumide or inhibition of Niemann-Pick C1 Like 1 transporter with ezetimibe reduced the hypercholesterolemic effect of [Thr28, Nle31]-CCK in LDLR(-/-) mice. These findings suggest that CCK-increased plasma cholesterol and triglycerides as a result of the reabsorption of biliary lipids from the intestine

Overexpression of Catalase Enhances Benzo(a)pyrene Detoxification in Endothelial Microsomes.

UNLABELLED:We previously reported that overexpression of catalase upregulated xenobiotic- metabolizing enzyme (XME) expression and diminished benzo(a)pyrene (BaP) intermediate accumulation in mouse aortic endothelial cells (MAECs). Endoplasmic reticulum (ER) is the most active organelle involved in BaP metabolism. To examine the involvement of ER in catalase-induced BaP detoxification, we compared the level and distribution of XMEs, and the profile of BaP intermediates in the microsomes of wild-type and catalase transgenic endothelial cells. Our data showed that endothelial microsomes were enriched in cytochrome P450 (CYP) 1A1, CYP1B1 and epoxide hydrolase 1 (EH1), and contained considerable levels of NAD(P)H:quinone oxidoreductase-1 (NQO1) and glutathione S-transferase-pi (GSTP). Treatment of wild-type MAECs with 1μM BaP for 2 h increased the expression of microsomal CYP1A1, 1B1 and NQO1 by ~300, 64 and 116%, respectively. However, the same treatment did not significantly alter the expression of EH1 and GSTP. Overexpression of catalase did not significantly increase EH1, but upregulated BaP-induced expression of microsomal CYP1A1, 1B1, NQO1 and GSTP in the following order: 1A1>NQO1>GSTP>1B1. Overexpression of catalase did not alter the distribution of each of these enzymes in the microsomes. In contrast to our previous report showing lower level of BaP phenols versus BaP diols/diones in the whole-cell, this report demonstrated that the sum of microsomal BaP phenolic metabolites were ~60% greater than that of the BaP diols/diones after exposure of microsomes to BaP. Overexpression of catalase reduced the concentrations of microsomal BaP phenols and diols/diones by ~45 and 95%, respectively. This process enhanced the ratio of BaP phenol versus diol/dione metabolites in a potent manner. Taken together, upregulation of phase II XMEs and CYP1 proteins, but not EH1 in the ER might be the mechanism by which overexpression of catalase reduces the levels of all the BaP metabolites, and enhances the ratio of BaP phenolic metabolites versus diol/diones in endothelial microsomes

Effects of cholecystokinin on plasma cholesterol and triglycerides in oil-fed mice.

<p><b>A–B.</b> Wild-type and <i>LDLR^−/−</i> mice were gavage-fed with 0.15 ml olive oil and then intravenously injected 50 ng/kg of [Thr28, Nle31]-cholecystokinin (CCK). Blood samples were collected before (control) and at 2 h after the mice were treated with oil+PBS or oil and CCK. Plasma triglycerides, total cholesterol (TC) and free cholesterol (FC) were measured colorimetrically. Esterified cholesterol (EC) was calculated as the difference between TC and FC. <b>C.</b> Wild-type and <i>LDLR^−/−</i> mice were gavage-fed with 0.15 ml water. Blood samples were collected before (control) and at 2 h after water feeding for measuring plasma TC and triglycerides. Values represent the mean ± SEM of 4–6 independent experiments. Differences among samples obtained from mice before (control) and after treated with oil+PBS or oil plus CCK were analyzed by two-way ANOVA followed by Tukey post-hoc tests (A and B). The difference between controls and water feeding was analyzed by Student's paired <i>t</i>-test (C). * <i>P</i><0.05 compared to control.</p

Effect of cholecystokinin on mouse plasma apolipoproteins.

<p>Wild-type and <i>LDLR^−/−</i> mice were injected with 50 ng/kg of cholecystokinin (CCK) or an equal volume of PBS (control) via the tail vein. Blood samples were collected at 2 h after CCK or vehicle injection. <b>A–B.</b> The levels of plasma apolipoprotein (apo) AI, apo E, B100, and B48 were determined by western blot analysis. The levels of apolipoproteins were expressed as their immunoblot intensity relative to µl plasma. <b>C.</b> Mouse plasma was fractionated with a FPLC system. The apoAI, apoB48 and B100 in the VLDL, LDL and HDL fractions were determined with western blot analysis, and expressed relative to µl plasma or FPLC eluates. Values represent the mean ± SEM of four independent experiments. The difference between controlc and CCK treatment was analyzed by Student's unpaired <i>t</i>-test. * <i>P</i><0.05 compared to control.</p