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.     

Calmodulin binding to human spectrin

Human hepatocellular carcinoma cells (Hep G2) were shown to secrete apo A-I as a proprotein . No apo A-I synthesis could be detected with endothelial cells from human umbilical cord veins. Conversion of proapo A-I into apo A-I is a slow (of the order of hours) process, mediated by a Ca2+/Mg2+-dependent enzyme which is present on the surface of plasma lipoprotein particles, endothelial cells and Hep G2 cells, and is probably synthesized by Hep G2 cells.     

Effect of contact inhibition on the regulation of cholesterol metabolism in cultured vascular endothelial cells.

Cholesterol synthesis in actively growing bovine vascular endothelial cells is regulated by low density lipoprotein (LDL) at a step prior to mevalonate formation, in a manner comparable to that found in aortic smooth muscle cells. LDL uptake by these cells is associated with induction of cholesterol esterification, an increase in total cell cholesterol, and an inhibition of endogenous sterol synthesis. In contrast, cholesterol metabolism in confluent contact-inhibited endothelial cultures was not significantly affected by LDL even though the cells bind the lipoprotein at high affinity receptor sites. Lysosomal degradation and subsequent regulatory effects on cellular cholesterol metabolism, however, were observed in contact-inhibited endothelial cells incubated with cationized rather than native LDL. Cationized LDL enter the cells independently of the high affinity sites. Therefore, the primary regulation of cholesterol metabolism in these cells is neither through the appropriate intracellular enzymes nor through the high affinity surface receptors, but via an inhibition of LDL internalization. It is suggested that this inhibition is due to a strict contact-inhibited morphology which enables the endothelium of the larger arteries to function as a selective barrier to the high circulating levels of plasma LDL.     

Mechanism of ATP-binding cassette transporter A1-mediated cellular lipid efflux to apolipoprotein A-I and formation of high density lipoprotein particles

The ATP-binding cassette transporter A1 (ABCA1) plays a critical role in the biogenesis of high density lipoprotein (HDL) particles and in mediating cellular cholesterol efflux. The mechanism by which ABCA1 achieves these effects is not established, despite extensive investigation. Here, we present a model that explains the essential features, especially the effects of ABCA1 activity in inducing apolipoprotein (apo) A-I binding to cells and the compositions of the discoidal HDL particles that are produced. The apo A-I/ABCA1 reaction scheme involves three steps. First, there is binding of a small regulatory pool of apo A-I to ABCA1, thereby enhancing net phospholipid translocation to the plasma membrane exofacial leaflet; this leads to unequal lateral packing densities in the two leaflets of the phospholipid bilayer. Second, the resultant membrane strain is relieved by bending and by creation of exovesiculated lipid domains. The formation of highly curved membrane surface promotes high affinity binding of apo A-I to these domains. Third, this pool of bound apo A-I spontaneously solubilizes the exovesiculated domain to create discoidal nascent HDL particles. These particles contain two, three, or four molecules of apo A-I and a complement of membrane phospholipid classes together with some cholesterol. A key feature of this mechanism is that membrane bending induced by ABCA1 lipid translocase activity creates the conditions required for nascent HDL assembly by apo A-I. Overall, this mechanism is consistent with the known properties of ABCA1 and apo A-I and reconciles many of the apparently discrepant findings in the literature.     

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.     

Lipid and lipoprotein metabolism in Hep G2 cells

Lipid composition, lipid synthesis and lipoprotein secretion by the Hep G2 cell line have been studied with substrate and insulin supplied under different conditions. The lipid composition of Hep G2 cells was close to that of normal human liver, except for a higher content in sphingomyelin (P less than 0.005) and a lower phosphatidylcholine/sphingomyelin ratio. Most of the [14C]triacylglycerols secreted into the medium were recovered by ultracentrifugation at densities of 1.006 to 1.020 g/ml. The main apolipoproteins secreted were apo B-100 and apo A-I. Hep G2 mRNA synthesized in vitro the pro-apolipoproteins A-I and E. Triacylglycerol secretion was 7.38 +/- 1.04 micrograms/mg cell protein per 20 h with 5.5 mM glucose in the medium and increased linearly with glucose concentration. Oleic acid (1 mM) increased the incorporation of [3H]glycerol into the medium and cell triacylglycerols by 251 and 899%, with a concomitant increment in cell triacylglycerols and cholesterol ester. Insulin (1 mU or 7 pmol/ml) inhibited triacylglycerol secretion and [35S]methionine incorporation into secreted protein by 47 and 28%, respectively, with a corresponding increase in the cells. Preincubation of cells with 2.5-10 mM mevalonolactone decreased the incorporation of [14C]acetate into cholesterol 6.2-fold, indicating an inhibitory effect on HMG-CoA reductase. It is concluded that in spite of some differences between Hep G2 and normal human hepatocytes, this line offers an alternative and reliable model for studies on liver lipid metabolism.     

Cholesterol transport via ABCA1: New insights from solid-phase binding assay

It is now well established that the ATP-binding cassette transporter A1 (ABCA1) plays a pivotal role in HDL metabolism, reverse cholesterol transport and net efflux of cellular cholesterol and phospholipids. We aimed to resolve some uncertainties related to the putative function of ABCA1 as a mediator of lipid transport by using a methodology developed in the laboratory to isolate a protein and study its interactions with other compounds. ABCA1 was tagged with the 1D4 peptide at the C terminus and expressed in human HEK 293 cells. Preliminary experiments showed that the tag modified neither the protein expression/localization within the cells nor the ability of ABCA1 to promote cholesterol cellular efflux to apolipoprotein A-I. ABCA1-1D4 was then purified and reconstituted in liposomes. ABCA1 displayed an ATPase activity in phospholipid liposomes that was significantly decreased by cholesterol. Finally, interactions with either cholesterol or apolipoprotein A-I were assessed by binding experiments with protein immobilized on an immunoaffinity matrix. Solid-phase binding assays showed no direct binding of cholesterol or apolipoprotein A-I to ABCA1. Overall, our data support the hypothesis that ABCA1 is able to mediate the transport of cholesterol from cells without direct interaction and that apo A-I primarily binds to membrane surface or accessory protein(s).     

Cholesterol-induced alteration of apolipoprotein A-I conformation in reassembled high density lipoprotein.

Recent reports have shown that apolipoprotein A-I (apo A-I), the major protein of high density lipoprotein (HDL) may exist in different conformational states. We studied the effects of apolipoprotein A-II and/or cholesterol on the conformation of apo A-I in reassembled HDL. Analysis of tryptophan fluorescence quenching in the presence of iodine suggested that cholesterol increased the number of apo A-I tryptophan residues accessible to the aqueous phase, but decreased their mean degree of hydration. These observations cannot be totally explained on the basis of the effect of cholesterol on phospholipid viscosity as determined by fluorescence anisotropy of diphenyl hexatriene. We did not observe any effect of apo A-II on the conformation of apo A-I.     

Specificity of ABCA7-mediated cell lipid efflux

Adenosine triphosphate-binding cassette transporter subfamily A member 7 (ABCA7) performs incompletely understood biochemical functions that affect pathogenesis of Alzheimer's disease. ABCA7 is most similar in primary structure to ABCA1, the protein that mediates cell lipid efflux and formation of high-density lipoprotein (HDL). Lipid metabolic labeling/tracer efflux assays were employed to investigate lipid efflux in BHK-ABCA7(low expression), BHK-ABCA7(high expression) and BHK-ABCA1 cells. Shotgun lipid mass spectrometry was used to determine lipid composition of HDL synthesized by BHK-ABCA7 and BHK-ABCA1 cells. BHK-ABCA7(low) cells exhibited significant efflux only of choline-phospholipid and phosphatidylinositol. BHK-ABCA7(high) cells had significant cholesterol and choline-phospholipid efflux to apolipoprotein (apo) A-I, apo E, the 18A peptide, HDL, plasma and cerebrospinal fluid and significant efflux of sphingosine-lipid, serine-lipid (which is composed of phosphatidylserine and phosphatidylethanolamine in BHK cells) and phosphatidylinositol to apo A-I. In efflux assays to apo A-I, after adjustment to choline-phospholipid, ABCA7-mediated efflux removed ~4 times more serine-lipid and phosphatidylinositol than ABCA1-mediated efflux, while ABCA1-mediated efflux removed ~3 times more cholesterol than ABCA7-mediated efflux. Shotgun lipidomic analysis revealed that ABCA7-HDL had ~20 mol% less phosphatidylcholine and 3–5 times more serine-lipid and phosphatidylinositol than ABCA1-HDL, while ABCA1-HDL contained only ~6 mol% (or ~1.1 times) more cholesterol than ABCA7-HDL. The discrepancy between the tracer efflux assays and shotgun lipidomics with respect to cholesterol may be explained by an underestimate of ABCA7-mediated cholesterol efflux in the former approach. Overall, these results suggest that ABCA7 lacks specificity for phosphatidylcholine and releases significantly but not dramatically less cholesterol in comparison with ABCA1.     

Apolipoprotein A-I induces translocation of cholesterol, phospholipid, and caveolin-1 to cytosol in rat astrocytes.

Intercellular cholesterol transport in the brain is carried by high density lipoprotein (HDL) generated in situ by cellular interaction with the apolipoprotein apoE, which is mainly synthesized by astrocytes, and with apoA-I secreted by cells such as endothelial cells. Rat astrocytes in fact generate HDL with extracellular apoA-I in addition to releasing HDL with endogenously synthesized apoE, seemingly by the same mechanism as the HDL assembly for systemic circulation. Relating to this reaction, apoA-I induced translocation of newly synthesized cholesterol and phospholipid to the cytosol prior to extracellular assembly of HDL, accompanied by an increase of caveolin-1 in the cytosol, activation of sterol regulatory element-binding protein, and enhancement of cholesterol synthesis. The lipid translocated into the cytosol was recovered in the fraction with a density of 1.09-1.16 g/ml as well as caveolin-1 and cyclophilin A. Cyclosporin A inhibited these apoA-I-mediated reactions and suppressed apoA-I-mediated cholesterol release. The findings suggest that such translocation of cholesterol and phospholipid into the cytosol is related to the apo A-I-mediated HDL assembly in astrocytes through functional association with caveolin-1 and a cyclosporin A-sensitive cyclophilin protein(s).     

Putative mechanisms of action of probucol on high-density lipoprotein apolipoprotein A-I and its isoproteins kinetics in rabbits

Probucol is a widely prescribed lipid-lowering agent, the major effects of which are to lower cholesterol in both low- and high-density lipoproteins (LDL and HDL, respectively). The mechanism of action of probucol on HDL apolipoprotein (apo) A-I kinetics was investigated in rabbits, with or without cholesterol feeding. 125I-labeled HDL was injected intravenously, and blood samples were taken periodically for 6 days. Kinetic parameters were calculated from the apo A-I-specific radioactivity decay curves. Fractional catabolic rate (FCR) and synthetic rate (SR) of apo A-I in rabbits fed a normal chow and normal chow with 1% probucol were similar. Apo A-I FCR of the rabbits fed 0.5% cholesterol was significantly increased but there were no changes in SR, compared to findings in the normal chow-fed group. Apo A-I FCR of the rabbits fed 1% probucol with 0.5% cholesterol (both 1 month and 2 months) was significantly increased compared to findings in rabbits fed the normal chow as well as 0.5% cholesterol diet group, while SR of apo A-I was significantly reduced in the former groups. Kinetics at 1 month after discontinuation of 1% probucol (under cholesterol feeding) showed a similar FCR of HDL-apo A-I to that of the rabbits fed 0.5% cholesterol, but the SR of apo A-I remained lower. Apo A-I isoproteins kinetics assessed by autoradiography of isoelectric focusing slab gels showed that the synthesis of proapo A-I was significantly reduced in the 1% probucol with 0.5% cholesterol administered, compared to the 0.5% cholesterol group. Thus, the action of probucol on HDL apo A-I kinetics was only prominent in case of higher serum cholesterol levels. The decreased HDL or apo A-I seen with probucol was apparently the result of an increase in FCR and a decrease in SR of HDL-apo A-I. A decreased synthesis of apo A-I remained evident even 1 month after discontinuing probucol. The action of probucol on the intracellular synthetic processes of apo A-I was revealed by the reduced synthesis of proapo A-I.     

Iron-ascorbate alters the efficiency of Caco-2 cells to assemble and secrete lipoproteins

Although oxidative stress has been implicated in development of gut pathologies, its role in intestinal fat transport has not been investigated. We assessed the effect of Fe(2+)-ascorbate-mediated lipid peroxidation on lipid synthesis, apolipoprotein biogenesis, and lipoprotein assembly and secretion. Incubation of postconfluent Caco-2 cells with iron(II)-ascorbate (0.2 mM/2 mM) in the apical compartment significantly promoted malondialdehyde formation without affecting sucrase activity, transepithelial resistance, DNA and protein content, and cell viability. However, addition of the oxygen radical-generating system reduced 1) [(14)C]oleic acid incorporation into cellular triglycerides (15%, P < 0.0002) and phospholipids (16%, P < 0.0005); 2) de novo synthesis of cellular apolipoprotein A-I (apo A-I) (18%, P < 0.05), apo A-IV (38%, P < 0.05), and apo B-48 (45%, P < 0.003) after [(35)S]methionine addition; and 3) production of chylomicrons (50%), VLDL (40%), LDL (37%), and HDL (30%) (all P < 0.0001). In contrast, increased total cellular cholesterol formation (96%, P < 0.0001), assayed by [(14)C]acetate incorporation, was noted, attributable to marked elevation (70%, P < 0.04) in activity of DL-3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. The ratio of Acyl-CoA to cholesterol acyltransferase, the esterifying cholesterol enzyme, remained unchanged. Fe(2+)-ascorbate-mediated lipid peroxidation modifies intracellular fat absorption and may decrease enterocyte efficiency in assembling and transporting lipids during gut inflammation.     

Structural models of human apolipoprotein A-I: a critical analysis and review

Human apolipoprotein (apo) A-I has been the subject of intense investigation because of its well-documented anti-atherogenic properties. About 70% of the protein found in high density lipoprotein complexes is apo A-I, a molecule that contains a series of highly homologous amphipathic alpha-helices. A number of significant experimental observations have allowed increasing sophisticated structural models for both the lipid-bound and the lipid-free forms of the apo A-I molecule to be tested critically. It seems clear, for example, that interactions between amphipathic domains in apo A-I may be crucial to understanding the dynamic nature of the molecule and the pathways by which the lipid-free molecule binds to lipid, both in a discoidal and a spherical particle. The state of the art of these structural studies is discussed and placed in context with current models and concepts of the physiological role of apo A-I and high-density lipoprotein in atherosclerosis and lipid metabolism.     

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.