Inhibition of apolipoprotein A-I gene expression by obesity-associated endocannabinoids

Obesity is associated with increased serum endocannabinoid (EC) levels and decreased high-density lipoprotein cholesterol (HDLc). Apolipoprotein A-I (apo A-I), the primary protein component of HDL is expressed primarily in the liver and small intestine. To determine whether ECs regulate apo A-I gene expression directly, the effect of the obesity-associated ECs anandamide and 2-arachidonylglycerol on apo A-I gene expression was examined in the hepatocyte cell line HepG2 and the intestinal cell line Caco-2. Apo A-I protein secretion was suppressed nearly 50% by anandamide and 2-arachidonoylglycerol in a dose-dependent manner in both cell lines. Anandamide treatment suppressed both apo A-I mRNA and apo A-I gene promoter activity in both cell lines. Studies using apo A-I promoter deletion constructs indicated that repression of apo A-I promoter activity by anandamide requires a previously identified nuclear receptor binding site designated as site A. Furthermore, anandamide-treatment inhibited protein-DNA complex formation with the site A probe. Exogenous over expression of cannabinoid receptor 1 (CBR1) in HepG2 cells suppressed apo A-I promoter activity, while in Caco-2 cells, exogenous expression of both CBR1 and CBR2 could repress apo A-I promoter activity. The suppressive effect of anandamide on apo A-I promoter activity in Hep G2 cells could be inhibited by CBR1 antagonist AM251 but not by AM630, a selective and potent CBR2 inhibitor. These results indicate that ECs directly suppress apo A-I gene expression in both hepatocytes and intestinal cells, contributing to the decrease in serum HDLc in obese individuals.     

24, 25-dihydroxycholecalciferol but not 25-hydroxycholecalciferol suppresses apolipoprotein A-I gene expression

AimsLigands for the vitamin D receptor (VDR) regulate apolipoprotein A-I (apo A-I) gene expression in a tissue-specific manner. The vitamin D metabolite 24, 25-dihydroxycholecalciferol (24, 25-(OH)₂D₃) has been shown to possess unique biological effects. To determine if 24, 25-(OH)₂D₃ modulates apo A-I gene expression, HepG2 hepatocytes and Caco-2 intestinal cells were treated with 24, 25-(OH)₂D₃ or its precursor 25-OHD₃.Main methodsApo A-I protein levels and mRNA levels were measured by Western and Northern blotting, respectively. Changes in apo A-I promoter activity were measured using the chlorampenicol acetytransferase assay.Key findingsTreatment with 24, 25-(OH)₂D₃, but not 25-OHD₃, inhibited apo A-I secretion in HepG2 and Caco-2 cells and apo A-I mRNA levels and apo A-I promoter activity in HepG2 cells. To determine if 24, 25-(OH)₂D₃ represses apo A-I gene expression through site A, the nuclear receptor binding element that is essential for VDRs effects on apo A-I gene expression, HepG2 cells were transfected with plasmids containing or lacking site A. While the site A-containing plasmid was suppressed by 24, 25-(OH)₂D₃, the plasmid lacking site A was not. Likewise, treatment with 24, 25-(OH)₂D₃ suppressed reporter gene expression in cells transfected with a plasmid containing site A in front of a heterologous promoter. Finally, antisense-mediated VDR depletion failed to reverse the silencing effects of 24, 25-(OH)₂D₃ on apo A-I expression.SignificanceThese results suggest that the vitamin D metabolite 24, 25-(OH)₂D₃ is an endogenous regulator of apo A-I synthesis through a VDR-independent signaling mechanism.     

Apolipoprotein A-I gene expression is upregulated by polychlorinated biphenyls in rat liver

Xenobiotics such as polychlorinated biphenyls (PCB) increase serum cholesterol level (especially high density lipoprotein cholesterol) and apolipoprotein A-I (apo A-I) level in rats. The effect of PCB on serum apo A-I and hepatic apo A-I gene expression and the relationship between apo A-I and drug-metabolizing enzymes in rats were investigated. Serum levels of cholesterol and apo A-I were increased by dietary addition of PCB in a dose-dependent manner (0-500 mg/kg diet). Hepatic apo A-I mRNA level was also elevated by PCB in a similar fashion. Serum level of cholesterol gradually increased during feeding period of PCB (200 mg/kg diet, 105 days) and reached a two-fold higher level in PCB group than in controls. The levels of serum apo A-I and hepatic apo A-I mRNA linearly elevated during feeding period of PCB and were increased 3- or 4-fold, respectively, compared to controls. Although acute administration (16 hr) of PCB, 3-methylcholanthrene, and phenobarbital induced cytochrome P-450 gene expression in the liver, hepatic apo A-I gene expression was not increased by these xenobiotics. These results indicated that the serum levels of cholesterol and apo A-I had positive correlation with hepatic level of apo A-I mRNA in rats fed PCB, and that hepatic apo A-I gene expression was dependent upon intake of PCB but was not directly related to the induction of drug-metabolizing enzymes. This study demonstrated that xenobiotic-induced hyper-alpha-cholesterolemia would be caused by the increased apo A-I gene expression and cholesterol synthesis in the liver, coordinately.     

Ginsenoside Rf, a component of ginseng, regulates lipoprotein metabolism through peroxisome proliferator-activated receptor alpha

We investigated whether ginseng regulates lipoprotein metabolism by altering peroxisome proliferator-activated receptor alpha (PPARalpha)-mediated pathways, using a PPARalpha-null mouse model. Administration of ginseng extract, ginsenosides, and ginsenoside Rf (Rf) to wild-type mice not only significantly increased basal levels of hepatic apolipoprotein (apo) A-I and C-III mRNA compared with wild-type controls, but also substantially reversed the reductions in mRNA levels of apo A-I and C-III expected following treatment with the potent PPARalpha ligand Wy14,643. In contrast, no effect was detected in the PPARalpha-null mice. Testing of eight main ginsenosides on PPARalpha reporter gene expression indicated that Rf was responsible for the effects of ginseng on lipoprotein metabolism. Furthermore, the inhibition of PPARalpha-dependent transactivation by Rf seems to occur at the level of DNA binding. These results demonstrate that ginseng component Rf regulates apo A-I and C-III mRNA and the actions of Rf on lipoprotein metabolism are mediated via interactions with PPARalpha.     

Characterization of lipoprotein particles isolated by immunoaffinity chromatography. Particles containing A-I and A-II and particles containing A-I but no A-II

Two populations of A-I-containing lipoprotein particles: A-I-containing lipoprotein with A-II (Lp (A-I with A-II], and A-I-containing lipoprotein without A-II (Lp (A-I without A-II] have been isolated from plasma of 10 normolipidemic subjects by immunoaffinity chromatography and characterized. Both types of particles possess alpha-electrophoretic mobility and hydrated density in the range of plasma high-density lipoproteins (HDL). Lp (A-I without A-II) and Lp (A-I with A-II) are heterogeneous in size. Lp (A-I without A-II) comprised two distinct particle sizes with mean apparent molecular weight and Stokes diameter of 3.01 X 10(5), and 10.8 nm for Lp (A-I without A-II)1, and 1.64 X 10(5), and 8.5 nm for Lp (A-I without A-II)2. Lp (A-I with A-II) usually contained particles of at least three distinct molecular sizes with mean apparent molecular weight and Stokes diameter of 2.28 X 10(5) and 9.6 nm for Lp (A-I with A-II)1, 1.80 X 10(5) and 8.9 nm for Lp (A-I with A-II)2, and 1.25 X 10(5) and 8.0 nm for Lp (A-I with A-II)3. Apoproteins C, D, and E, and lecithin:cholesterol acyltransferase (LCAT) were detected in both Lp (A-I without A-II) and Lp (A-I with A-II) with most of the apoprotein D, and E, and LCAT (EC 2.3.1.43) in Lp (A-I with A-II) particles. Lp (A-I without A-II) had a slightly higher lipid/protein ratio than Lp (A-I with A-II). Lp (A-I with A-II) had an A-I/A-II molar ratio of approximately 2:1. The percentage of plasma A-I associated with Lp (A-I without A-II) was highly correlated with the A-I/A-II ratio of plasma (r = 0.96, n = 10). The variation in A-I/A-II ratio of HDL density subfractions therefore reflects different proportions of two discrete types of particles: particles containing A-I and A-II in a nearly constant ratio and particles containing A-II but no A-II. Each type of particle is heterogeneous in size and in apoprotein composition.     

The role of cadherin,beta-catenin,and AP-1 in retinoid-regulated carcinoma cell differentiation and proliferation

Vitamin A derivatives (retinoids) are potent regulators of cell proliferation and differentiation. Retinoids inhibit the function of the oncogenic AP-1 and beta-catenin/TCF pathways and also stabilize components of the adherens junction, a tumor suppressor complex. When treated with retinoic acid (RA), the breast cancer cell line, SKBR3, undergoes differentiation and reduction in cell proliferation. The present work demonstrates that in SKBR3 cells, which exhibit high AP-1 activity, RA-regulation of cadherin expression and function, but not changes in AP-1 (or beta-catenin/TCF) signaling, is responsible for the epithelial differentiation. However, cadherin function and recruitment of beta-catenin to the membrane is not required for RA to regulate DNA synthesis in these cells. RA also reduces the activity of an AP-1 and TCF-sensitive cyclin D1 reporter in SKBR3 cells in a manner that is independent of the TCF site. In contrast, in SW480 cells, which have high levels of beta-catenin/TCF signaling, the activity and retinoid responsiveness of the cyclin D1 promoter was markedly inhibited by mutation of the TCF site. These data indicate that the remarkably broad effects of RA on the growth and differentiation of many different epithelial cancers may well be explained by the ability of RA to differentially regulate the activity of RAR/RXR, AP-1, and beta-catenin/TCF pathways.     

Fluorescence quenching studies of apolipoprotein A-I in solution and in lipid-protein complexes: protein dynamics

Fluorescence lifetime and intensity quenching studies of human plasma apolipoprotein A-I (apo A-I) in aqueous solution and in recombinant lipoprotein complexes with dimyristoylphosphatidylcholine (DMPC) indicate differences in conformational dynamics. In aqueous solution, the bimolecular quenching constants (k*) for lipid-free apo A-I fluorescence quenching by oxygen and acrylamide are 2.4 X 10(9) and 0.38 X 10(9) M-1 s-1, respectively. These values are independent of the oligomeric form of the protein. There is no correlation between the relatively small k* for apo A-I, which reflects rapid, low-amplitude protein fluctuations, and the labile conformational changes of apo A-I folding reactions, like denaturation, which occur on a slower time scale. In recombinant DMPC/apo A-I complexes (100:1 molar ratio) the protein increases in amphiphilic alpha-helical structure as it blankets the lipid matrix. The apparent k* for oxygen quenching of apo A-I fluorescence in the complex is large and increases in a temperature-dependent manner. We have introduced a two-compartment model, which discriminates the source of quencher molecules as aqueous or lipid, to describe oxygen quenching of DMPC/apo A-I fluorescence. The magnitude and temperature dependence of the apparent k* predominantly reflect the partitioning of oxygen between the two phases rather than being a probe of the lipid physical state. Calculations of the helical hydrophobic moment in apo A-I indicate that tryptophan residues 8 and 72 occur at the lipid-protein interface of amphiphilic alpha-helices, whereas the other two tryptophan residues (50, 108) lie on the nonpolar faces of amphiphilic helices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of apolipoprotein A-I, C-II, C-III, and E mRNA in fetal human tissues. Time-dependent induction of apolipoprotein E mRNA by cultures of human monocyte-macrophages

Recently developed molecular probes for human apolipoprotein (apo) genes have been used to study the specificity of human tissue expression of the apo A-I, apo C-II, apo C-III, and apo E genes. We have found that apo E mRNA was present in all tissues examined. On the basis of total RNA concentration the relative abundance of apo E mRNA expressed as a percentage of the liver value is as follows: adrenal gland and macrophages, 74-100%; gonads and kidney, 12-15%; spleen, brain, thymus, ovaries, intestine, and pancreas, 3-9%; heart, 1.5%; stomach, striated muscle, and lung, less than 1%. The relative concentration of apo E mRNA in cultures of human peripheral blood monocyte-macrophages increases dramatically as a function of time in culture, and after 5 days, it compares to that of liver. The human tissues shown to synthesize apo E mRNA were also examined for their ability to synthesize apo A-I, apo C-II, and apo C-III mRNA. The relative abundance of apo A-I, apo C-III, and apo C-II mRNA expressed as a percentage of the liver value is as follows: apo A-I, intestine, 50%; apo A-I, pancreas and gonads, 12%; apo A-I, kidney, 4%; apo A-I, adrenal, 2.5%; apo A-I, ovaries and heart, 1%; apo A-I, stomach and thymus, less than 1%; apo C-III, intestine, 62%; apo C-III, pancreas, 7%; apo C-II, intestine, 3%; apo C-II, pancreas, less than 1%. The knowledge of tissue specificities in the synthesis of apolipoproteins is important for our understanding of the regulation of apolipoproteins and lipoprotein metabolism.     

Characterization of human high-density lipoprotein subclasses LP A-I and LP A-I/A-II and binding to HepG2 cells

Plasma HDL can be classified according to their apolipoprotein content into at least two types of lipoprotein particles: lipoproteins containing both apo A-I and apo A-II (LP A-I/A-II) and lipoproteins with apo A-I but without apo A-II (LP A-I). LP A-I and LP A-I/A-II were isolated by immuno-affinity chromatography. LP A-I has a higher cholesterol content and less protein compared to LP A-I/A-II. The average particle mass of LP A-I is higher (379 kDa) than the average particle weight of LP A-I/A-II (269 kDa). The binding of 125I-LP A-I to HepG2 cells at 4 degrees C, as well as the uptake of [3H]cholesteryl ether-labelled LP A-I by HepG2 cells at 37 degrees C, was significantly higher than the binding and uptake of LP A-I/A-II. It is likely that both binding and uptake are mediated by apo A-I. Our results do not provide evidence in favor of a specific role for apo A-II in the binding and uptake of HDL by HepG2 cells.