ABCA1 mRNA Levels Research Articles

Dietary Phytosterols and Phytostanols Alter the Expression of Sterol-Regulatory Genes in SHRSP and WKY inbred Rats

Background/Aims: We elucidated the molecular mechanism(s) underlying sterol trafficking by investigating alterations in gene expression in response to increased retention of dietary phytosterols and phytostanols in stroke-prone spontaneously hypertensive (SHRSP) and normotensive Wistar Kyoto (WKY) inbred rats. Methods: SHRSP and WKY inbred rats were fed a control diet or a diet supplemented with phytosterols or phytostanols (2 g/kg diet). Results: Intake of phytosterols and phytostanols increased their incorporation in plasma, red blood cells, liver, aorta and kidney, but decreased cholesterol levels in liver and aorta in both rat strains. Phytosterol intake up-regulated mRNA expression of intestinal Npc1l1 and Abcg8, and hepatic Abcg5, Abca1, Cyp27a1 and Hmgcr. Phytostanol intake up-regulated Npc1l1 and Srebp2, but down-regulated Abcg5 mRNA expression in small intestine. Phytostanols also up-regulated Abca1 expression in SHRSP rats, but down-regulated Abca1 expression in WKY inbred rats. Compared to phytosterols, dietary phytostanols reduced phytosterol levels in plasma, red blood cells, and kidney, as well as altered mRNA levels of hepatic Abca1,Cyp27a1, and Hmgcr and intestinal Abcg5/8, Hmgcr and Srebp2. Conclusions: Altered expression of multiple sterol-regulatory genes may contribute to the incorporation and cholesterol-lowering actions of phytosterols and phytostanols. Phytosterols and phytostanols may act through different mechanism(s) on cholesterol and phytosterol/phytostanol trafficking.

Liver X receptor regulates expression of MRP2 but not that of MDR1 and BCRP in the liver

Liver X receptors (LXRs) belong to the nuclear hormone receptor superfamily. Multidrug resistance-associated protein 2 (MRP2), multidrug resistance 1 (MDR1) and breast cancer resistance protein (BCRP) play an important role in the efflux of a broad range of endogenous and xenobiotic compounds from hepatocytes. Since the effects of LXR activation on there transporters have been obscure, we investigated the effects of LXR agonists, TO901317 and 25-hydroxycholesterol, on MRP2, MDR1, BCRP expression in HepG2 cells and the rat liver. In an in vitro study, TO901317 increased ABCA1, an LXR target gene, and MRP2 mRNA and protein levels. On the other hand, TO901317 had little effect on MDR1 and BCRP mRNA levels. In an in vivo study, Abca1 and Mrp2 mRNA and protein levels were increased by TO901317, but TO901317 had no effect on Mdr1a and Bcrp mRNA levels in the rat liver. Moreover, TO901317-induced MRP2 mRNA expression was blocked by LXRα knockdown. In this study, we demonstrated that LXR activation induced expression of MRP2 but not that of MDR1 and BCRP in hepatocytes. The results suggest that agonists for LXR activate transcription of the MRP2 gene in order to promote excretion of endogenous and xenobiotic compounds from hepatocytes into bile.

Liver X Receptor–Mediated Induction of Cholesteryl Ester Transfer Protein Expression Is Selectively Impaired in Inflammatory Macrophages

Cholesteryl ester transfer protein (CETP) is a target gene for the liver X receptor (LXR). The aim of this study was to further explore this regulation in the monocyte-macrophage lineage and its modulation by lipid loading and inflammation, which are key steps in the process of atherogenesis. Exposure of bone marrow-derived macrophages from human CETP transgenic mice to the T0901317 LXR agonist increased CETP, PLTP, and ABCA1 mRNA levels. T0901317 also markedly increased CETP mRNA levels and CETP production in human differentiated macrophages, whereas it had no effect on CETP expression in human peripheral blood monocytes. In inflammatory mouse and human macrophages, LXR-mediated CETP gene upregulation was inhibited, even though ABCA1, ABCG1, and SREBP1c inductions were maintained. The inhibition of CETP gene response to LXR agonists in inflammatory cells was independent of lipid loading (ie, oxidized LDL increased CETP production in noninflammatory macrophages with a synergistic effect of synthetic LXR agonists). LXR-mediated induction of human CETP expression is switched on during monocyte-to-macrophage differentiation, is magnified by lipid loading, and is selectively lost in inflammatory macrophages, which suggests that inflammatory cells may not increase the circulating CETP pool on LXR agonist treatment.

Polyunsaturated fatty acids down-regulate in vitro expression of the key intestinal cholesterol absorption protein NPC1L1: no effect of monounsaturated nor saturated fatty acids

Several transporter proteins regulate intestinal cholesterol absorption. Of these proteins, NPC1L1 is a major contributor to this process. Fatty acids (FAs) modulate cholesterol absorption by a mechanism that remains unknown. We evaluate the effect of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) on the expression of NPC1L1 and others proteins associated with cholesterol absorption (SR-BI, ABCG5, ABCG8, ABCA1, CAV-1, ANX-2) in human enterocytes in vitro. The role of SREBPs, PPARs, LXR and RXR in this process was also investigated. Caco-2/TC-7 enterocytes were incubated for 24 h with a wide range of concentrations of FA–bovine serum albumin (50–300 μM). Gene expression was analyzed by quantitative real-time PCR. The NPC1L1 protein present in enterocyte membranes was analyzed using Western blot. NPC1L1 mRNA levels were reduced 35–58% by the n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ( P<.05). Linoleic acid (n-6), palmitic acid and oleic acid did not affect NPC1L1 mRNA expression. ABCA1 mRNA levels were reduced 44–70% by n-6 arachidonic acid and 43–55% by n-3 EPA ( P<.05). LXR and LXR+RXR agonists decreased NPC1L1 mRNA expression by 28% and 57%, respectively ( P<.05). A concentration of 200 μM of EPA and DHA decreased NPC1L1 protein expression in enterocyte membranes by 58% and 59%, respectively. We have demonstrated that the PUFAs n-3 EPA and DHA down-regulate NPC1L1 mRNA expression. In addition, PUFAs also down-regulate NPC1L1 protein expression in enterocyte membranes. LXR and RXR activation induced a similar repression effect. The lipid-lowering effect of n-3 PUFAs could be mediated in part by their action at the NPC1L1 gene level.

Cancer preventive phytochemicals uniquely activate liver x receptor responsive genes through receptor dependent and independent mechanisms in prostate cancer cells

Activation of liver X receptor (LXR) in models of prostate cancer (PCa) results in reduction of cell growth and hormone signaling. We hypothesized that cancer protective phytochemicals may interact with the LXR pathway in an in vitro model of PCa. In the present study we examined the effects of three diet‐derived cancer preventive phytochemicals: genistein, resveratrol, and diindolylmethane (DIM), on activation of two classic LXR responsive genes ATP binding cassette transporter A1 and G1, (ABCA1) and (ABCG1) mRNA in LNCaP human prostate cancer cells. LNCaP cells treated with either 5 μM genistein, resveratrol or DIM resulted in a 3‐7 fold increase in ABCG1 and ABCA1 mRNA levels. Using siRNA technology, we also examined which LXR isotype (LXRα or LXRβ) may be involved in any interactions between these phytochemicals and the LXR pathway. Transfection of LNCaP cells with LXRβ siRNA, but not LXRα siRNA, completely inhibited genistein's ability to induce ABCG1 mRNA but only had a partial effect on DIM. Use of siRNA against LXRα or LXRβ had no effect on resveratrol's induction of ABCG1 mRNA. Increased ABCA1 mRNA expression by all three phytochemicals could not be reversed by either LXRα or LXRβ siRNA. Our data revealed that in LNCaP cells: i) Dietary phytochemicals activate mRNA expression of LXR responsive genes ABCA1 and ABCG1, and ii) Genistein and DIM can modulation ABCG1 mRNA with a unique requirement for the LXRβ isoform.

Inhibition of cellular lipid biosynthesis by sulfated oxysterol is mediated via the LXR pathway

Nuclear oxysterol receptor, Liver X receptor (LXR) is a key transcriptional regulator of liver lipid metabolism. We recently identified the oxysterol 25‐diol 3‐sulfate (25HC3S) as an important regulatory molecule in liver. We now have studied the effects of 25HC3S and its precursor, 25‐hydroxycholesterol (25HC) on the LXR/SREBP‐1 signaling pathway in microphages. Addition of 25HC3S to human macrophages markedly decreased nuclear LXR protein levels. Real time PCR and western blot results showed 25HC3S inhibited SREBP‐1 mRNA and protein mature form.25HC3S decreased the expression of SREBP‐1‐responsive genes including acetyl CoA carboxylase‐1 and fatty acid synthase, as well as HMGR and LDLR, which are key proteins involved in lipids metabolism.Subsequently, Oil‐Red O staining and TLC found 25HC3S inhibited intracellular lipids accumulation.25HC as a LXR ligand had opposite effects, increasing ABCA1, ABCG1, SREBP‐1, and FAS mRNA levels in the macrophages. In the presence of 25HC3S, 25HC and LXR agonist T0901317 failed to increase LXR targeting gene expression. We conclude that 25HC3S acts in macrophage as cholesterol satiety signal, down‐regulating cholesterol and fatty acid biosynthetic pathways via inhibition of LXR/SREBP signaling. These studies suggest that 25HC3S is a potent regulator of lipid metabolism in macrophages.

Hibernation increases cholesterol and apolipoprotein A‐I levels in ground squirrels

Studies in two ground squirrel species suggest expression of ApoA‐I increases in gut and liver during hibernation. Since ApoA‐I is the major lipoprotein in HDL particles, we hypothesized that hibernation alters cholesterol metabolism. To test this idea we measured cholesterol and lipoprotein levels, and changes in expression of genes that regulate their metabolism in summer (SUM) and hibernating (HIB) 13‐lined ground squirrels. Plasma cholesterol was ~2‐fold greater in HIB squirrels during torpor (T) (474.6 ± 44.8 mg/dl) and interbout arousal (IBA) (436.1 ± 56.2 mg/dl) than in SUM (219.1 ± 12.8 mg/dl, P&lt;0.01); VLDL‐, LDL‐, and HDL‐cholesterol showed the same trend. ApoA‐I mRNA levels in liver and gut, and protein levels in plasma, liver and gut were greater in HIB (P&lt;0.05 for all). Liver mRNA levels of ApoB, LXR, SREBP2 and the rate limiting enzyme in cholesterol synthesis (HMGR) were similar in SUM and HIB, but SREBP1c expression was lower in T compared with SUM and IBA (P&lt;0.001). Protein expression of CYP7A1 (bile acid synthesis) was lower in HIB ((P&lt;0.05). ABCA1 mRNA levels rose 13‐fold in gut of HIB, suggesting altered cholesterol processing in gut during hibernation. These results confirm significant seasonal changes in cholesterol and lipoprotein dynamics in ground squirrels. Understanding their causes and functional significance will shed light on adaptations to the lipid‐centric metabolism of hibernation.

Overexpression of Human 15( S )-Lipoxygenase-1 in RAW Macrophages Leads to Increased Cholesterol Mobilization and Reverse Cholesterol Transport

The purpose of this study was to determine the effect of 15-lipoxygenase-1 (15-LO-1) on cholesterol mobilization from macrophages. Overexpression of human 15-LO-1 in RAW mouse macrophages led to enhanced cholesterol efflux, increased cholesteryl ester (CE) hydrolysis, and increased reverse cholesterol transport (RCT). Efflux studies comparing 15-LO-1 overexpressing cells to mock-transfected RAW macrophages resulted in a 3- to 7-fold increase in cholesterol efflux to apolipoprotein A-I and a modest increase in efflux to HDL. Additional experiments revealed an increase in mRNA and protein levels of ABCA1 and ABCG1 in the RAW expressing 15-LO-1 compared to controls. Efforts to examine whether the arachidonic acid metabolite of 15-LO-1, (15S)-hydroxyeicosatetraenoic acid (HETE), was responsible for the enhanced efflux revealed this eicosanoid metabolite did not play a role. Enhanced steryl ester hydrolysis was observed in 15-LO-1 overexpressing cells suggesting that the CE produced in the 15-LO-1 expressing cells was readily mobilized. To measure RCT, RAW macrophages overexpressing 15-LO-1 or mock-transfected cells were cholesterol enriched by exposure to acetylated low-density lipoprotein and [(3)H]-cholesterol. These macrophages were injected into wild-type animals and RCT was measured as a percent of injected dose of (3)H appearing in the feces at 48 hours. We found 7% of the injected (3)H in the feces of mice that received macrophages overexpressing 15-LO-1 and 4% in the feces of mice that received mock-transfected cells. These data are consistent with a model in which overexpression of human 15-LO-1 in RAW macrophages promotes RCT through increased CE hydrolysis and ABCA1-mediated cholesterol efflux.

25-Hydroxycholesterol-3-sulfate regulates macrophage lipid metabolism via the LXR/SREBP-1 signaling pathway

The oxysterol receptor LXR is a key transcriptional regulator of lipid metabolism. LXR increases expression of SREBP-1, which in turn regulates at least 32 genes involved in lipid synthesis and transport. We recently identified 25-hydroxycholesterol-3-sulfate (25HC3S) as an important regulatory molecule in the liver. We have now studied the effects of 25HC3S and its precursor, 25-hydroxycholesterol (25HC), on lipid metabolism as mediated by the LXR/SREBP-1 signaling in macrophages. Addition of 25HC3S to human THP-1-derived macrophages markedly decreased nuclear LXR protein levels. 25HC3S administration was followed by dose- and time-dependent decreases in SREBP-1 mature protein and mRNA levels. 25HC3S decreased the expression of SREBP-1-responsive genes, acetyl-CoA carboxylase-1, and fatty acid synthase (FAS) as well as HMGR and LDLR, which are key proteins involved in lipid metabolism. Subsequently, 25HC3S decreased intracellular lipids and increased cell proliferation. In contrast to 25HC3S, 25HC acted as an LXR ligand, increasing ABCA1, ABCG1, SREBP-1, and FAS mRNA levels. In the presence of 25HC3S, 25HC, and LXR agonist T0901317, stimulation of LXR targeting gene expression was repressed. We conclude that 25HC3S acts in macrophages as a cholesterol satiety signal, downregulating cholesterol and fatty acid synthetic pathways via inhibition of LXR/SREBP signaling. A possible role of oxysterol sulfation is proposed.

Altered cholesterol ester cycle in ex vivo skin fibroblasts from Alzheimer patients

AbstractRecent studies in both animal and cell models of Alzheimer's disease (AD) indicated that sub-cellular cholesterol distribution seems to regulate amyloid-beta (A[beta]) generation in the brain. In particular, cholesterol-esters (CE), rather than total cholesterol levels, appear directly correlated with A[beta] production. Here we observed that, similarly to brain cells, skin fibroblasts obtained from AD patients produce and accumulate more CE than skin fibroblasts from age-matched healthy controls do. AD fibroblasts also exhibited a 2 fold increase in the expression of ACAT1, in addition to lower levels of SREBP2, nCEH, Caveolin-1 and ABCA1 mRNA levels, all of which are involved in the CE cycle. HMGCoA-reductase and LDL-receptor mRNAs levels did not show statistically significant changes in AD, compared to non-AD, cells. Furthermore, although APP mRNA did not significantly vary, neprilysin (NEP), the most important enzyme in the proteolysis of A[beta], was expressed at very low levels in skin fibroblasts of sporadic AD patients. Our results contribute to the concept that AD may be the consequence of a basic and systemic defect in the CE cycle. Moreover, our results identify new possible targets for the diagnosis, prevention, and cure or, at least, amelioration of the symptoms of AD.

Niemann-Pick C1-Like 1 Is Required for an LXR Agonist to Raise Plasma HDL Cholesterol in Mice

Activation of liver x receptor (LXR) raises plasma HDL-cholesterol (HDL-C) in mice. Interestingly, the LXR agonist GW3965 fails to raise plasma HDL-C in mice lacking intestinal ABCA1, indicating that intestinal ABCA1 plays a predominant role in GW3965-mediated HDL production. How this is coupled to intestinal function remains elusive. Because cholesterol is essential for HDL assembly and directly regulates intestinal ABCA1 expression via activating LXR, we hypothesized that cholesterol absorption, a major function of intestine, modulates LXR-dependent HDL formation. Mice lacking Niemann-Pick C1-Like 1 (NPC1L1) (L1-KO mice), a gene that is essential for cholesterol absorption, were treated with LXR agonist T0901317 for 7 days. Intriguingly, this treatment failed to significantly raise plasma HDL-C but caused a much greater fecal cholesterol excretion in L1-KO mice. The intestinal ABCA1 mRNA level was about 4-fold lower in L1-KO versus wild-type mice, and increased 3.9-fold and 8.8-fold after T0901317 treatment in wild-type and L1-KO mice, respectively. Hepatic ABCA1 failed to respond to T0901317 in mice of both genotypes, although hepatic mRNAs for many LXR target genes were higher in the T0901317-treated versus untreated wild-type animals. NPC1L1 is required for an LXR agonist to increase plasma HDL-C in mice.

Interleukin‐10 inhibits the down‐regulation of ATP binding cassette transporter A1 by tumour necrosis factor‐alpha in THP‐1 macrophage‐derived foam cells

It is suggested that cholesterol efflux mediated by ATP binding cassette transporter A1 (ABCA1) plays an important role in anti-atherogenesis. However, the effects of inflammatory cytokines on ABCA1 expression and cholesterol accumulation in foam cells are little known. This study investigates the effects of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) on ABCA1 expression and cholesterol content in THP-1 macrophage-derived foam cells. ABCA1mRNA and protein levels were determined by RT-PCR and Western blot, respectively. The total cholesterol content in THP-1 macrophage-derived foam cells was detected by the zymochemistry method. Results revealed that TNF-alpha could increase cholesterol content by down-regulating ABCA1 expression in a time-dependent manner in THP-1 macrophage-derived foam cells, which may contribute to its pro-atherosclerotic effect. In addition IL-10 time-dependently decreased cholesterol accumulation by up-regulating ABCA1 expression and inhibited the down-regulation of ABCA1 by TNF-alpha in THP-1 macrophage-derived foam cells, which may be one of the mechanisms of IL-10 contributing to its anti-atherosclerotic action.

Human Aldehyde Oxidase 1 Interacts with ATP-binding Cassette Transporter-1 and Modulates its Activity in Hepatocytes

AOX1, a member of the cytosolic molybdenum hydroxylase family, has been identified by us earlier as an ABCA1-interacting protein. AOX1 is well-described as xenobiotic metabolizing enzyme, which upon oxidation of acetaldehyde and retinaldehyde to acetic acid and retinoic acid generates reactive oxygen species. Here we show that knock-down of AOX1 in HepG2 by small interfering RNA significantly reduced ABCA1-dependent lipid efflux and enhanced phagocytic uptake of microspheres similar to ABCA1 deficiency, without affecting ABCA1 mRNA and protein levels. ABCA1 and AOX1 are coexpressed in human hepatocytes, kidney proximal tubular epithelial cells, Leydig, and adrenocortical cells. Expression of ABCA1 and AOX1 was investigated by immunohistochemistry in liver tissue arrays. A strong AOX1 expression was found in normal liver, and in cirrhosis. In contrast, hepatocellular carcinomas showed either a complete loss or reduced expression of AOX1. Significant correlations were found between reduced AOX1 expression and tumor stage, or metastatic or regional lymph node states. Deregulation was also observed for ABCA1 expression but to a lesser extent. Our findings show that the interaction of ABCA1 with AOX1 modulates ABCA1-linked cellular functions such as lipid efflux and phagocytosis in hepatocytes, and the reduced expression of AOX1 in malignant transformed hepatocytes supports the differentiation dependent upregulation of AOX1.

Abstract 828: Adiponectin Enhanced ApoA-1- and HDL- mediated Cellular Cholesterol Efflux from Human Monocyte-Derived Macrophages

(Background) Plasma HDL-cholesterol levels are inversely correlated to the risk of CAD. HDL plays a crucial role as a shuttle carrying cholesterol derived from peripheral tissues and transporting it back to the liver. Recently, ATP-binding cassette transporters (ABCA1, ABCG1) have been identified as important membrane receptors to generate HDL by removing cholesterol and phospholipids from macrophage foam cells in the arterial wall. On the other hand, adiponectin (APN) abundantly secreted from adipocytes is one of the important molecules to inhibit the development of atherosclerosis. Epidemiological studies have revealed a positive correlation between plasma HDL-cholesterol and APN concentrations in humans, and APN levels are decreased in patients with CAD. In the current study, we investigated the role of APN on cellular cholesterol efflux from human macrophages. (Methods) Recombinant human APN was prepared. Human monocyte-derived macrophages were isolated from whole blood of healthy volunteers and incubated with RPMI-1640 medium containing 10% type AB serum. After 7 days incubation, the indicated concentrations of recombinant APN were added to the medium containing 0.1% BSA and incubated for 24 hours. The expression of ABCA1 and ABCG1 was measured by real-time quantitative PCR and western blot analyses. Cholesterol efflux from macrophages, previously incubated with [ 3 H]-labeled acetylated LDL, was measured with or without APN and expressed by a percentage of radioactivity of the medium to total radioactivity (cells plus medium). APN was added into the medium containing 0.1% BSA and apoA-1 (10 μg/ml) or HDL (50 μg/ml HDL protein) and incubated for 24 hours. (Results) APN significantly up-regulated the mRNA and protein levels of ABCA1 in human macrophages by 3.6- and 2-fold, respectively. ApoA-1-mediated cholesterol efflux from macrophages was 8.1-fold-increased by APN treatment. Furthermore, the expression of ABCG1 was also significantly increased by APN. HDL-mediated cholesterol efflux was slightly increasd by APN. (Conclusions) In conclusion, APN might act as a protective factor against atherosclerosis by increasing cholesterol efflux from human macrophages in both ABCA1- and ABCG1-dependent pathways.

Expression of nuclear receptor mRNA and liver X receptor-mediated regulation of ABC transporter A1 at rat blood–brain barrier

The aim of the present study was to investigate the expression of nuclear receptor mRNA and regulation of the expression of ATP-binding cassette (ABC) transporters by nuclear receptor agonists in rat brain capillary endothelial cells, which form the blood–brain barrier, by using rat brain capillary fraction from 8-week-old rats and a conditionally immortalized brain capillary endothelial cell line (TR-BBB13). RT-PCR analysis revealed that liver X receptor α and β, retinoid X receptor α and β and peroxisome proliferator-activating receptor α and β mRNAs were expressed in the rat brain capillary endothelial cells and TR-BBB cells. In contrast, pregnane X receptor, farnesoid X receptor and constitutive androstane receptor were not detected. Furthermore, treatment with a liver X receptor agonist increased the ABCA1 mRNA level in TR-BBB13 cells, while ABCG2 mRNA expression was not affected. Treatment with a rat pregnane X receptor agonist did not affect the ABCB1 mRNA level in TR-BBB13 cells. These results demonstrate that the rat blood–brain barrier has an expressional regulation mechanism via sterol-related nuclear receptor, and indicate that the blood–brain barrier in 8-week-old rats lacks ABCB1 regulation via pregnane X receptor.

Cholesterol and Plant Sterol Efflux from Cultured Intestinal Epithelial Cells Is Mediated by ATP-Binding Cassette Transporters

In this study we analyzed functions of ATP-binding cassette (ABC) transporters involved in sterol transport from Caco-2 cells. Treatment with a synthetic liver x receptor ligand elevated both mRNA and protein levels of ABCG5, G8, and ABCA1. The ligand stimulated cholesterol efflux, suggesting that ABC transporters are involved in it. To identify the acceptors of cholesterol, potential molecules such as apolipoprotein A-I, glycocholic acid, phosphatidylcholine, and bile acid micelles were added to the medium. Apo A-I, a known acceptor of cholesterol transported by ABCA1, elevated cholesterol efflux on the basal side, whereas the others raised cholesterol efflux on the apical side. Moreover, bile acid micelles preferentially augmented plant sterol efflux rather than cholesterol. Finally, in HEK293 cells stably expressing ABCG5/G8, bile acid micelle-mediated sterol efflux was significantly accelerated. These results indicate that ABCG5/G8, unlike ABCA1, together with bile acids should participate in sterol efflux on the apical surface of Caco-2 cells.

Adiponectin accelerates reverse cholesterol transport by increasing high density lipoprotein assembly in the liver

Plasma high density lipoprotein (HDL)–cholesterol levels are negatively correlated with the incidence of coronary artery disease. HDL plays an important role in protecting against atherosclerosis by removing cholesterol from atheroma and transporting it back to the liver. The ATP-binding cassette transporters (ABCA1 and ABCG1) and scavenger receptor BI (SR-BI) are thought to be one of the rate-limiting factors to generate HDL in the liver. Adiponectin (APN) secreted from adipocytes is also one of the important molecules to inhibit the development of atherosclerosis. Recently, it has been reported that plasma HDL–cholesterol levels are positively correlated with plasma APN concentrations in humans. Therefore, we investigated the association of APN with HDL assembly in the liver. Human hepatoma cell line, HepG2 cells, were incubated for 24 h in the culture medium with the indicated concentrations of recombinant APN. APN enhanced the mRNA level of apolipoprotein A-I (apoA-I) in HepG2 cells and increased the secretion of apoA-I from the cells to the medium. Furthermore, APN increased both mRNA and protein levels of ABCA1, but not ABCG1 and SR-BI, in HepG2 cells. Taken together, the current study demonstrates that APN might protect against atherosclerosis by increasing HDL assembly through enhancing ABCA1 pathway and apoA-1 synthesis in the liver.

Physiologically regulated transgenic ABCA1 does not reduce amyloid burden or amyloid-β peptide levels in vivo

ABCA1-deficient mice have low levels of poorly lipidated apolipoprotein E (apoE) and exhibit increased amyloid load. To test whether excess ABCA1 protects from amyloid deposition, we crossed APP/PS1 mice to ABCA1 bacterial artificial chromosome (BAC) transgenic mice. Compared with wild-type animals, the ABCA1 BAC led to a 50% increase in cortical ABCA1 protein and a 15% increase in apoE abundance, demonstrating that this BAC supports modest ABCA1 overexpression in brain. However, this was observed only in animals that do not deposit amyloid. Comparison of ABCA1/APP/PS1 mice with APP/PS1 controls revealed no differences in levels of brain ABCA1 protein, amyloid, Abeta, or apoE, despite clear retention of ABCA1 overexpression in the livers of these animals. To further investigate ABCA1 expression in the amyloid-containing brain, we then compared ABCA1 mRNA and protein levels in young and aged cortex and cerebellum of APP/PS1 and ABCA1/APP/PS1 animals. Compared with APP/PS1 controls, aged ABCA1/APP/PS1 mice exhibited increased ABCA1 mRNA, but not protein, selectively in cortex. Additionally, ABCA1 mRNA levels were not increased before amyloid deposition but were induced only in the presence of extensive Abeta and amyloid levels. These data suggest that an induction of ABCA1 expression may be associated with late-stage Alzheimer's neuropathology.

Telmisartan Enhances Cholesterol Efflux from THP-1 Macrophages by Activating PPAR&amp;gamma;

The ATP binding cassette transporters A1 and G1 (ABCA1/G1) and scavenger receptor class B type I (SR-BI) are key molecules in cholesterol efflux and atherogenesis. These genes are regulated by peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor (LXR). Telmisartan is an angiotensin type 1 receptor blocker which has been reported to act as a ligand for PPARgamma. We investigated whether PPARgamma-activating ARBs affect the expression of these genes and cholesterol efflux from macrophages. Telmisartan increased ABCA1, ABCG1 and SR-BI mRNA levels in THP-1 macrophages in a dose- and time-dependent fashion. It also increased their protein levels and enhanced apoA-I- and HDL-mediated cholesterol efflux from macrophages. The knockdown of PPARgamma by siRNA abolished the telmisartan-induced expression of these genes. The knockdown of LXRalpha resulted in the complete and partial abolishment of telmisartan-induced ABCA1 and ABCG1 expression, respectively. We also demonstrated that telmisartan-induced SR-BI expression was dependent on the PPARgamma pathway but not on the LXRalpha pathway. A luciferase assay using an ABCA1 promoter construct showed that telmisartan activated ABCA1 transcription, which was abolished if the LXR binding element was mutated, indicating that increased ABCA1 transcription by telmisartan is LXR-dependent. Our results showed that telmisartan enhanced both apoA-I- and HDL-mediated cholesterol efflux from macrophages by increasing ABCA1, ABCG1 and SR-BI expression via PPARgamma-dependent and LXR-dependent/independent pathways.

Regulation of ABCA1 expression in human keratinocytes and murine epidermis

Keratinocytes require abundant cholesterol for cutaneous permeability barrier function; hence, the regulation of cholesterol homeostasis is of great importance. ABCA1 is a membrane transporter responsible for cholesterol efflux and plays a pivotal role in regulating cellular cholesterol levels. We demonstrate that ABCA1 is expressed in cultured human keratinocytes (CHKs) and murine epidermis. Liver X receptor (LXR) activation markedly stimulates ABCA1 mRNA and protein levels in CHKs and mouse epidermis. In addition to LXR, activators of peroxisome proliferator-activated receptor (PPAR)alpha, PPARbeta/delta, and retinoid X receptor (RXR), but neither PPARgamma nor retinoic acid receptor, also increase ABCA1 expression in CHKs. Increases in cholesterol supply induced by LDL or mevalonate stimulate ABCA1 expression, whereas inhibiting cholesterol synthesis with statins or cholesterol sulfate decreases ABCA1 expression in CHKs. After acute permeability barrier disruption by either tape-stripping or acetone treatment, ABCA1 expression declines, and this attenuates cellular cholesterol efflux, making more cholesterol available for regeneration of the barrier. In addition, during fetal epidermal development, ABCA1 expression decreases at days 18-22 of gestation (term = 22 days), leaving more cholesterol available during the critical period of barrier formation. Together, our results show that ABCA1 is expressed in keratinocytes, where it is negatively regulated by a decrease in cellular cholesterol levels or altered permeability barrier requirements and positively regulated by activators of LXR, PPARs, and RXR or increases in cellular cholesterol levels.