ABCA1 Levels Research Articles

Hyperhomocysteinemia due to cystathionine beta synthase deficiency induces dysregulation of genes involved in hepatic lipid homeostasis in mice

Cystathionine beta synthase (CBS) deficiency leads to severe hyperhomocysteinemia, which confers diverse clinical manifestations, notably fatty liver. Recently, abnormal lipid metabolism has been demonstrated in CBS-deficient mice, a murine model of severe hyperhomocysteinemia. To gain further insights into effects of CBS deficiency on hepatic cholesterol metabolism, the expression of hepatic genes involved in biosynthesis, uptake and efflux was determined in CBS-deficient mice. Gene expression analysis was performed on liver of CBS-deficient mice using quantitative real-time PCR. We found that CBS-deficiency in liver mice significantly increases expression of genes induced by endoplasmic reticulum stress and genes that regulate the expression of enzymes required for cholesterol and fatty acid biosynthesis and uptake, notably the scavenger receptor class B type I (SR-BI), concomitant with overexpression of SR-BI at the protein level. Moreover, we also found increased mRNA levels of ABCG5, ABCG8, ABCG1 and ABCA1, which play important roles in reverse cholesterol transport, associated with an upregulation of liver X receptors and a downregulation of the peroxisome proliferators-activated receptor alpha. We found that several ATP-binding cassette transporters and nuclear hormone receptors involved in liver lipid homeostasis are differentially expressed in liver of CBS-deficient mice.

Both Hepatic and Extrahepatic ABCA1 Have Discrete and Essential Functions in the Maintenance of Plasma High-Density Lipoprotein Cholesterol Levels In Vivo

Extrahepatic tissues have long been considered critical contributors of cholesterol to nascent HDL particles in the reverse cholesterol transport pathway, in which ABCA1 plays the crucial role. Recent studies, however, including both overexpression and deletion of ABCA1 selectively in the liver, have highlighted the primary role of the liver in the maintenance of HDL levels in vivo. The availability of mice with complete deletion of ABCA1 (total knockout [TKO]) and with liver-specific deletion of ABCA1 (LSKO) has enabled us to dissect the discrete roles of hepatic relative to extrahepatic ABCA1 in HDL biogenesis. Delivery of adenoviral ABCA1 resulted in selective expression of physiological levels of ABCA1 in the livers of both LSKO and TKO mice, resulting in increased HDL cholesterol (HDL-C). Expression of ABCA1 in the liver of LSKO mice resulted in plasma HDL-C levels that were similar to those in wild-type mice and significantly above those seen in similarly treated TKO mice. HDL particles from ABCA1-expressing LSKO mice were larger and contained significantly increased cholesterol compared with TKO mice. Infusion of human apolipoprotein A-I/phospholipid reconstituted HDL particles normalized plasma HDL-C levels in LSKO mice but had no effect on HDL-C levels in TKO mice. Although hepatic ABCA1 appears crucial for phospholipid transport, extrahepatic tissues play an important role in cholesterol transfer to nascent HDL particles. These data highlight the discrete and specific roles of both liver and extrahepatic ABCA1 in HDL biogenesis in vivo and indicate that ABCA1 shows lipid cargo selectivity depending on its site of expression.

The effects of ABCA1 on cholesterol efflux and Aβ levels in vitro and in vivo

ABCA1 promotes cholesterol efflux from cells and is required for maintaining plasma cholesterol levels. Cholesterol homeostasis is important in the production of beta-amyloid (Abeta), a peptide that is overproduced in Alzheimer's disease (AD). Overexpression of ABCA1 can be achieved by stimulating Liver X Receptors (LXR), and changes in Abeta have been reported after LXR stimulation in vitro. To determine whether ABCA1 could alter endogenous Abeta levels, we used two different in vivo systems. We first examined the effects of an LXR agonist (TO-901317) on wild-type mice and found an increase in brain ABCA1 and apoE levels, which caused an increase in plasma cholesterol. This was accompanied by a decrease in brain Abeta levels. We then examined endogenous Abeta levels in ABCA1 knockout mice and found that, despite having no ABCA1, lowered brain apoE levels, and lowered plasma cholesterol, there was no change in Abeta levels. To assess these in vivo models in an in vitro system, we designed a model in which cholesterol transport via ABCA1 (or related transporters) was prevented. Switching off cholesterol efflux, even in the presence of TO-901317, caused no change in Abeta levels. However, when efflux capability was restored, TO-901317 reduced Abeta levels. These data show that promoting cholesterol efflux is a viable target for Abeta reducing strategies; however, knockout of cholesterol transporters is not sufficient to alter Abeta in vitro or in vivo.

Characterization of nascent HDL particles and microparticles formed by ABCA1-mediated efflux of cellular lipids to apoA-I

The nascent HDL created by ABCA1-mediated efflux of cellular phospholipid (PL) and free (unesterified) cholesterol (FC) to apolipoprotein A-I (apoA-I) has not been defined. To address this issue, we characterized the lipid particles released when J774 mouse macrophages and human skin fibroblasts in which ABCA1 is activated are incubated with human apoA-I. In both cases, three types of nascent HDL containing two, three, or four molecules of apoA-I per particle are formed. With J774 cells, the predominant species have hydrodynamic diameters of approximately 9 and 12 nm. These discoidal HDL particles have different FC contents and PL compositions, and the presence of acidic PL causes them to exhibit alpha-electrophoretic mobility. These results are consistent with ABCA1 located in more than one membrane microenvironment being responsible for the production of the heterogeneous HDL. Activation of ABCA1 also leads to the release of apoA-I-free plasma membrane vesicles (microparticles). These larger, spherical particles released from J774 cells have the same PL composition as the 12 nm HDL and contain CD14 and ganglioside, consistent with their origin being plasma membrane raft domains. The various HDL particles and microparticles are created concurrently, and there is no precursor-product relationship between them. Importantly, a large fraction of the cellular FC effluxed from these cells by ABCA1 is located in microparticles. Collectively, these results show that the products of the apoA-I/ABCA1 interaction include discoidal HDL particles containing different numbers of apoA-I molecules. The cellular PLs and cholesterol incorporated into these nascent HDL particles originate from different cell membrane domains.

Unsaturated Fatty Acids Phosphorylate and Destabilize ABCA1 through a Phospholipase D2 Pathway

Abnormal high density lipoprotein (HDL) metabolism among patients with diabetes and insulin resistance may contribute to their increased risk of atherosclerosis. ATP-binding cassette transporter ABCA1 mediates the transport of cholesterol and phospholipids from cells to HDL apolipoproteins and thus modulates HDL levels and atherogenesis. Unsaturated fatty acids, which are elevated in diabetes, impair the ABCA1 pathway in cultured cells by destabilizing ABCA1 protein. Here we examined the cellular pathway that mediates the ABCA1 destabilizing effects of fatty acids. The long-chain acyl-CoA synthetase inhibitor triacsin C completely reversed fatty acid-induced ABCA1 destabilization, indicating that fatty acids need to be activated to their CoA derivatives to enhance ABCA1 degradation. Unsaturated but not saturated fatty acids stimulated phospholipase D (PLD) activity, the PLD inhibitor 1-butanol prevented the unsaturated fatty acid-induced reduction in ABCA1 levels, and the PLD2 activator mastoparan markedly reduced ABCA1 protein levels, implicating a role for PLD2 in the ABCA1 destabilizing effects of fatty acids. Unsaturated fatty acids and mastoparan increased phosphorylation of ABCA1 serines. PLD2 small interfering RNA abolished the ability of unsaturated fatty acids to inhibit lipid transport activity, to reduce protein levels, and to increase serine phosphorylation of ABCA1. The diacylglycerol analog oleoylacetylglycerol also reduced ABCA1 protein levels and increased its serine phosphorylation, suggesting that PLD2-generated diacylglycerols promote the destabilizing phosphorylation of ABCA1. These data provide evidence that intracellular unsaturated acyl-CoA derivatives destabilize ABCA1 by activating a PLD2 signaling pathway.

Genes of Cholesterol Metabolism in Human Atheroma

Accumulation of cholesterol in foam cells of atheroma plaques depends on the balance between uptake and efflux of cholesterol. It may also depend on proteins surrounding lipid droplets, adipophilin, and perilipins. They favor triglyceride storage in adipocytes and could play a similar role for cholesterol in atheroma. We measured in human atheroma and nearby macroscopically intact tissue (MIT) the expression of perilipin, adipophilin, and regulatory factors of cholesterol metabolism. We identified perilipin A in human arterial wall. Its expression was largely increased in atheroma compared with MIT, and perilipin was present in macrophages and vascular smooth muscle cells. Adipophilin, ACAT1, and CD36 were also overexpressed in atheroma. mRNA levels of low-density lipoprotein receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and SREBP-2 were unchanged. With respect to efflux of cholesterol, the mRNA levels of NCEH and ABCA-1 were unchanged, whereas CLA-1 mRNA was slightly higher in atheroma. Importantly, immunoblotting of ABCA-1 showed a dramatic decrease of ABCA1 protein, the key molecule of cholesterol efflux, in atheroma compared with MIT. We show the presence and induction of perilipin in atheroma. This overexpression and the coordinated modifications of expression of key regulatory factors for cholesterol metabolism could favor cholesterol accumulation.

Abca7 Null Mice Retain Normal Macrophage Phosphatidylcholine and Cholesterol Efflux Activity despite Alterations in Adipose Mass and Serum Cholesterol Levels

Mutations in the A class of ATP-binding cassette transporters (ABCA) are causally implicated in three human diseases: Tangier disease (ABCA1), Stargadt's macular degeneration (ABCA4), and neonatal respiratory failure (ABCA3). Both ABCA1 and ABCA4 have been shown to transport lipids across cellular membranes, and ABCA3 may play a similar role in transporting pulmonary surfactant. Although the functions of the other 10 ABCA class transporters identified in the human genome remain obscure, ABCA7-transfected cells have been shown to efflux lipids in response to stimulation by apolipoprotein A-I. In an effort to elucidate the physiologic role of ABCA7, we generated mice lacking this transporter (Abca7-/- mice). Homozygous null mice were produced from intercrosses of heterozygous null mice at the expected Mendelian frequency and developed normally without any obvious phenotypic abnormalities. Cholesterol and phospholipid efflux stimulated by apolipoprotein A-I from macrophages isolated from wild type and Abca7-/- mice did not differ, suggesting that these activities may not be central to the physiological role of the transporter in vivo. Abca7-/- females, but not males, had significantly less visceral fat and lower total serum and high density lipoprotein cholesterol levels than wild type, gender-matched littermates. ABCA7 expression was detected in hippocampal and cortical neurons by in situ hybridization and in brain and white adipose tissue by Western blotting. Induction of adipocyte differentiation from 3T3 fibroblasts in culture led to a marked increase in ABCA7 expression. These studies suggest that ABCA7 plays a novel role in lipid and fat metabolism that Abca7-/- mice can be used to elucidate.

Disruption of cholesterol homeostasis by plant sterols.

The ABC transporters ABCG5 and ABCG8 limit absorption and promote excretion of dietary plant sterols. It is not known why plant sterols are so assiduously excluded from the body. Here we show that accumulation of plant sterols in mice lacking ABCG5 and ABCG8 (G5G8-/- mice) profoundly perturbs cholesterol homeostasis in the adrenal gland. The adrenal glands of the G5G8-/- mice were grossly abnormal in appearance (brown, not white) due to a 91% reduction in cholesterol content. Despite the very low cholesterol levels, there was no compensatory increase in cholesterol synthesis or in lipoprotein receptor expression. Moreover, levels of ABCA1, which mediates sterol efflux, were increased 10-fold in the G5G8-/- adrenals. Adrenal cholesterol levels returned to near-normal levels in mice treated with ezetimibe, which blocks phytosterol absorption. To determine which plant sterol(s) caused the metabolic changes, we examined the effects of individual plant sterols on cholesterol metabolism in cultured adrenal cells. Addition of stigmasterol, but not sitosterol, inhibited SREBP-2 processing and reduced cholesterol synthesis. Stigmasterol also activated the liver X receptor in a cell-based reporter assay. These data indicate that selected dietary plant sterols disrupt cholesterol homeostasis by affecting two critical regulatory pathways of lipid metabolism.

Cyclosporin A traps ABCA1 at the plasma membrane and inhibits ABCA1-mediated lipid efflux to apolipoprotein A-I.

ABCA1 mediates cellular cholesterol and phospholipid efflux to apolipoprotein A-I and other apolipoprotein acceptors. In this study, we analyzed the effect of the immunosuppressant cyclosporin A on the ABCA1-mediated lipid effluxes reactions. Cyclosporin A acted as a potent inhibitor of ABCA1 activity in several cell lines. Using the RAW264.7 mouse macrophage cell line, in which ABCA1 and its associated cholesterol efflux activity are inducible by cAMP analogues, cyclosporin A inhibition of cholesterol efflux to apolipoprotein A-I was rapidly reversible after its removal from the culture media, implying that ABCA1 levels were not drastically reduced by cyclosporin A. In fact, cyclosporin A treatment decreased ABCA1 turnover and yielded a 2-fold increase in cell-surface ABCA1. Despite the increase in cell-surface ABCA1, cyclosporin A decreased apolipoprotein A-I uptake, resecretion, and degradation in RAW cells. Finally, consistent with the inhibition of ABCA1 in vitro, cyclosporin A treatment induced a 33% reduction of high-density lipoprotein (HDL) levels in mice. ABCA1 inhibition by cyclosporin A supports a role for ABCA1 endocytic trafficking in ABCA1-mediated lipid efflux and could explain in part the low HDL levels observed in some patients with transplants.

Probucol Inactivates ABCA1 in the Plasma Membrane with Respect to Its Mediation of Apolipoprotein Binding and High Density Lipoprotein Assembly and to Its Proteolytic Degradation

Probucol has been shown to inhibit the release of cellular lipid by helical apolipoprotein and thereby to reduce plasma high density lipoprotein. We attempted to explore the underlying mechanism for this effect in human fibroblast WI-38. Probucol inhibited the apoA-I-mediated cellular lipid release and binding of apoA-I to the cells in a dose-dependent manner. It did not influence cellular uptake of low density lipoprotein, transport of cholesterol to the cell surface whether de novo synthesized or delivered as low density lipoprotein, and overall cellular content of cholesterol, although biosynthesis of lipids from acetate was somewhat increased. Probucol did not affect the mRNA level of ABCA1, and ABCA1 was recovered along with marker proteins for plasma membrane regardless of the presence of probucol. However, the protein level of ABCA1 increased, and the rate of its decay in the presence of cycloheximide was slower in the probucol-treated cells. ABCA1 in the probucol-treated cells was resistant to digestion by calpain but not by trypsin. We concluded that probucol inactivates ABCA1 in the plasma membrane with respect to its function in mediating binding of and lipid release by apolipoprotein and with respect to proteolytic degradation by calpain.

Dietary cholesterol stimulates CYP7A1 in rats because farnesoid X receptor is not activated.

Cholesterol feeding upregulates CYP7A1 in rats but downregulates CYP7A1 in rabbits. To clarify the mechanism responsible for the upregulation of CYP7A1 in cholesterol-fed rats, the effects of dietary cholesterol (Ch) and cholic acid (CA) on the activation of the nuclear receptors, liver X-receptor (LXR-alpha) and farsenoid X-receptor (FXR), which positively and negatively regulate CYP7A1, were investigated in rats. Studies were carried out in four groups (n = 12/group) of male Sprague-Dawley rats fed regular chow (control), 2% Ch, 2% Ch + 1% CA, and 1% CA alone for 1 wk. Changes in mRNA expression of short heterodimer partner (SHP) and bile salt export pump (BSEP), target genes for FXR, were determined to indicate FXR activation, whereas the expression of ABCA1 and lipoprotein lipase (LPL), target genes for LXR-alpha, reflected activation. CYP7A1 mRNA and activity increased twofold and 70%, respectively, in rats fed Ch alone when the bile acid pool size was stable but decreased 43 and 49%, respectively, after CA was added to the Ch diet, which expanded the bile acid pool 3.4-fold. SHP and BSEP mRNA levels did not change after feeding Ch but increased 88 and 37% in rats fed Ch + CA. This indicated that FXR was activated by the expanded bile acid pool. When Ch or Ch + CA were fed, hepatic concentrations of oxysterols, ligands for LXR-alpha increased to activate LXR-alpha, as evidenced by increased mRNA levels of ABCA1 and LPL. Feeding CA alone enlarged the bile acid pool threefold and increased the expression of both SHP and BSEP. These results suggest that LXR-alpha was activated in rats fed both Ch or Ch + CA, whereas CYP7A1 mRNA and activity were induced only in Ch-fed rats where the bile acid pool was not enlarged such that FXR was not activated. In rats fed Ch + CA, the bile acid pool expanded, which activated FXR to offset the stimulatory effects of LXR-alpha on CYP7A1.

Endotoxin down-regulates ABCG5 and ABCG8 in mouse liver and ABCA1 and ABCG1 in J774 murine macrophages: differential role of LXR

Several of the ATP binding cassette (ABC) transporters have recently been shown to play important roles in reverse cholesterol transport (RCT) and prevention of atherosclerosis. In the liver, ABCG5 and ABCG8 have been proposed to efflux sterols into the bile for excretion. ABCG5 and ABCG8 also limit absorption of dietary cholesterol and plant sterols in the intestine. In macrophages, ABCA1 and ABCG1 mediate cholesterol removal from these cells to HDL. Many of these ABC transporters are regulated by the liver X receptor (LXR). We have previously shown that endotoxin (lipopolysaccharide) down-regulates LXR in rodent liver. In the present study, we examined the in vivo and in vitro regulation of these ABC transporters by endotoxin. We found that endotoxin significantly decreased mRNA levels of ABCG5 and ABCG8 in the liver, but not in the small intestine. When endotoxin or cytokines (tumor necrosis factor and interleukin-1) were incubated with J774 murine macrophages, the mRNA levels of ABCA1 were decreased. This effect was rapid and sustained, and was associated with a reduction in ABCA1 protein levels. Endotoxin and cytokines also decreased ABCG1 mRNA levels in J774 cells. Although LXR is a positive regulator of ABCA1 and ABCG1, we did not observe a reduction in protein levels of LXR or in binding of nuclear proteins to an LXR response element in J774 cells. The decrease in ABCG5 and ABCG8 levels in the liver as well as a reduction in ABCA1 and ABCG1 in macrophages during the host response to infection and inflammation coupled with other previously described changes in the RCT pathway may aggravate atherosclerosis.

Experimental Glomerulopathy Alters Renal Cortical Cholesterol, SR-B1, ABCA1, and HMG CoA Reductase Expression

Previous studies indicate that acute tubular injury causes free cholesterol (FC) and cholesteryl ester (CE) accumulation within renal cortex/proximal tubules. This study assessed whether similar changes occur with glomerulopathy/nephrotic syndrome, in which high-circulating/filtered lipoprotein levels increase renal cholesterol supply. Potential adaptive changes in cholesterol synthetic/transport proteins were also assessed. Nephrotoxic serum (NTS) or passive Heymann nephritis (PHN) was induced in Sprague-Dawley rats. Renal injury (blood urea nitrogen, proteinuria) was assessed 2 and 7 days (NTS), or 10 and 30 days (PHN) later. FC and CE levels in renal cortex, isolated glomeruli, and proximal tubule segments were determined. SR-B1 (a CE influx protein), ABCA1 (a FC exporter), and HMG CoA reductase protein/mRNA levels were also assessed. FC was minimally elevated in renal cortex (0 to 15%), the majority apparently localizing to proximal tubules. More dramatic CE elevations were found ( approximately 5 to 15x), correlating with the severity of proteinuria at any single time point (r >/= 0.85). Cholesterol increments were associated with decreased SR-B1, increased ABCA1, and increased HMG CoA reductase (HMGCR) protein and its mRNA. Tubule (HK-2) cell culture data indicated that SR-B1 and ABCA1 levels are responsive to cholesterol supply. Experimental nephropathy can increase renal FC, and particularly CE, levels, most notably in proximal tubules. These changes are associated with adaptations in SR-B1 and ABCA1 expression, which are physiologically appropriate changes for a cholesterol overload state. However, HMGCR protein/mRNA increments can also result. These seem to reflect a maladaptive response, potentially contributing to a cell cholesterol overload state.

A PEST sequence in ABCA1 regulates degradation by calpain protease and stabilization of ABCA1 by apoA-I.

Cholesterol-loaded macrophage foam cells are a central component of atherosclerotic lesions. ABCA1, the defective molecule in Tangier disease, mediates the efflux of phospholipids and cholesterol from cells to apoA-I, reversing foam cell formation. In ABCA1, we identified a sequence rich in proline, glutamic acid, serine, and threonine (PEST sequence) that enhances the degradation of ABCA1 by calpain protease and thereby controls the cell surface concentration and cholesterol efflux activity of ABCA1. In an apparent positive feedback loop, apoA-I binds ABCA1, promotes lipid efflux, inhibits calpain degradation, and leads to increased levels of ABCA1. ApoA-I infusion also increases ABCA1 in vivo. These studies reveal a novel mode of regulation of ABCA1 by PEST sequence-mediated calpain proteolysis that appears to be reversed by apolipoprotein-mediated phospholipid efflux. Inhibition of ABCA1 degradation by calpain could represent a novel therapeutic approach to increasing macrophage cholesterol efflux and decreasing atherosclerosis.

Rapid quantification of murine ABC mRNAs by real time reverse transcriptase-polymerase chain reaction

Several ATP-binding cassette (ABC) transporters are critically involved in cholesterol and phospholipid efflux, reverse cholesterol transport, and play an important role in the development of atherosclerosis. Quantification of ABC mRNA is important in studying the regulation of cellular cholesterol homeostasis and mechanisms related to the pathogenesis of atherosclerosis. We have developed a one-step real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method for measuring mRNA levels of ABCA1, ABCG1, and ABCA2 in murine tissues using the TaqMan(TM) technology. It has significant methodological benefits when compared to classic Northern blotting or semi-quantitative RT-PCR analysis. Using this method, we found high expression levels of ABCA1 in liver and macrophages, and of ABCG1 in the brain and macrophages. The expression of ABCA1 and ABCG1 were further induced in macrophages loaded with acLDL. In contrast, ABCA2 was expressed exclusively in the brain with low expression levels in the macrophages. This method provides a rapid, highly sensitive, specific, and reproducible quantification of ABC mRNA, and can be performed with nanograms of total RNA sample, thus making it a superior method for studying the regulation of ABC transporters in cholesterol efflux and its role in the pathogenesis of atherosclerosis in murine models.