Cholesterol absorption is mainly regulated by the jejunal and ileal ATP-binding cassette sterol efflux transporters Abcg5 and Abcg8 in mice

In the present study, we investigated whether intestinal sterol efflux transporters Abcg5 and Abcg8 play a major role in determining variations in cholesterol (Ch) absorption efficiency, and we compared the physiological functions of the duodenal, jejunal, and ileal Abcg5 and Abcg8 on the absorption of Ch and sitostanol in inbred mice challenged with various amounts of Ch, sitostanol, hydrophilic, or hydrophobic bile acids. We found that Abcg5 and Abcg8 in the jejunum and ileum, but not in the duodenum, were main factors in determining, in part, variations in Ch absorption efficiency. The jejunal and ileal Abcg5 and Abcg8 played a major regulatory role in response to high dietary cholesterol and were more sensitive in the regulation of Ch absorption when compared with sitostanol absorption. These results, combined with different sterol uptake rates, suggest that the absorption efficiency of Ch and sitostanol is determined by the net results between influx and efflux of intraluminal Ch and sitostanol molecules crossing the apical membrane of the enterocyte. Hydrophilic and hydrophobic bile acids influenced Ch absorption through mediating Ch solubilization and its physical-chemical state within the small intestinal lumen. We conclude that Ch absorption is mainly regulated by the jejunal and ileal Abcg5 and Abcg8 in mice.     

Lipid efflux by the ATP-binding cassette transporters ABCA1 and ABCG1

Plasma levels of high-density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) are inversely correlated with the risk of cardiovascular disease. One major atheroprotective mechanism of HDL and apoA-I is their role in reverse cholesterol transport, i.e., the transport of excess cholesterol from foam cells to the liver for secretion. The ATP-binding cassette transporters ABCA1 and ABCG1 play a pivotal role in this process by effluxing lipids from foam cells to apoA-I and HDL, respectively. In the liver, ABCA1 activity is one rate-limiting step in the formation of HDL. In macrophages, ABCA1 and ABCG1 prevent the excessive accumulation of lipids and thereby protect the arteries from developing atherosclerotic lesions. However, the mechanisms by which ABCA1 and ABCG1 mediate lipid removal are still unclear. Particularly, three questions remain controversial and are discussed in this review: (1) Do apoA-I and HDL directly interact with ABCA1 and ABCG1, respectively? (2) Does cholesterol efflux involve retroendocytosis of apoA-I or HDL? (3) Which lipids are directly transported by ABCA1 and ABCG1?     

Impaired ATP-binding cassette transporter A1-mediated sterol efflux from oxidized LDL-loaded macrophages

We investigated the interaction of oxidized low density lipoprotein (OxLDL) with the ATP-binding cassette A1 (ABCA1) pathway in J774 macrophages. Cellular efflux to apolipoprotein AI (apo-AI) of OxLDL-derived cholesterol was lower than efflux of cholesterol derived from acetylated low density lipoprotein (AcLDL). ABCA1 upregulation by 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (cpt-cAMP) or 22 (R)-hydroxycholesterol (22-OH) and 9-cis retinoic acid (9cRA) increased the efflux to apo-AI of cellular sterols derived from AcLDL, but not of those from OxLDL. AcLDL, but not OxLDL, induced ABCA1 protein content and activity in J774. However, OxLDL did not influence J774 ABCA1 upregulation by cpt-cAMP or 22-OH/9cRA. We conclude that sterols released to cells by OxLDL are available neither as substrate nor as modulator of ABCA1.     

三磷酸腺苷结合盒转运体和细胞胆固醇外排

胆固醇是细胞质膜的重要组成成分。然而,过多的胆固醇累积可导致细胞中毒。异常的胆固醇胞内迁移与蓄积是造成许多心血管疾病如动脉粥样硬化的分子基础。细胞内胆固醇稳态由胆固醇的吸收、合成及外排等一系列过程调控。在哺乳动物细胞中,调节胆固醇合成、吸收和外排是维持体内胆固醇平衡的必要生理过程。本综述着重概述了三磷酸腺苷结合盒转运体(ABC)家族,如ABCA1、ABCG1、ABCG5和ABCG8的细胞功能及生理作用,以及这些转运体在调控胆固醇胞外转运中的分子机制。     

Cholesterol efflux from cultured adipose cells is mediated by LpAI particles but not by LpAI:AII particles

Cholesterol efflux was studied in cultured adipose cells after preloading with LDL cholesterol. Long-term exposure to LpAI and LpAI:AII particles isolated from the HDL fraction showed that LpAI particles only were able to promote cholesterol efflux. Liposomes containing different ApoAI/ApoAII molar ratios were tested: the larger the proportion of ApoAI, the faster the ability to remove cholesterol from Ob1771 cells. Dose-response curves showed that LpAI particles were active within a physiological range of concentrations, whereas LpAI:AII particles had no effect at all concentrations. The results are in favour of LpAI particles being the active components of the HDL fraction for the promotion of cholesterol efflux and suggest that LpAI particles and LpAI:AII particles represent distinct metabolic entities.     

Cholesterol and phospholipid efflux from cultured cells

The removal of phospholipids and cholesterol from tissues is the major mechanism mediating the initial assembly of high density lipoproteins (HDL), as well as being the main reason HDL are thought to protect against atherosclerosis. Investigations of the mechanisms of HDL assembly and testing of novel HDL-raising agents typically involve assays to determine phospholipid and/or cholesterol removal or "efflux" from cultured cells. The purpose of this chapter is to describe experimental protocols that can be used in the determination of cholesterol and phospholipid efflux from cultured cells by HDL apolipoproteins for the formation of new HDL particles, and the testing of novel HDL-raising therapies in vitro. A protocol is also provided for determining the size and nature of HDL particles formed in cell-conditioned medium using two-dimensional gel electrophoresis.     

节肢动物ABC转运蛋白及其介导的杀虫剂抗性

腺苷三磷酸结合盒转运蛋白（ATP-binding cassette transporter),简称ABC转运蛋白（ABC transporter）,是继细胞色素P450单加氧酶、羧酸酯酶、谷胱甘肽S-转移酶之后又一类参与解毒作用的重要蛋白家族,因其在杀虫剂解毒等方面起着非常重要的作用,近年来逐渐受到广泛关注。ABC转运蛋白是一大类跨膜蛋白,其核心结构通常由4个结构域组成,包括2个高度疏水的跨膜结构域（transmembrane domains , TMD）和2个核苷酸结合域（nucleotide binding domains, NBD）。根据序列相似性和保守结构域,可以把ABC转运蛋白家族分为8个亚家族,每个亚家族的成员数及功能不同。这类蛋白在各种生物体内均有分布,其主要功能包括转运物质、信号传导、细胞表面受体及参与细胞内DNA修复,转录及调节基因的表达过程等。此外,近年来的研究表明,ABC转运蛋白的突变或过表达不仅与节肢动物对化学农药的抗药性密切相关,而且在抗Bt毒素方面也起着非常重要的作用,对转Bt作物造成严重威胁。本文综述了节肢动物ABC转运蛋白的结构,ATP水解介导的作用机制,亚家族的分类、结构及生理功能,以及由ABC转运蛋白介导的抗药性研究进展,旨在深入了解ABC转运蛋白的研究现状及其在节肢动物抗药性方面的作用,为阐明节肢动物抗药性机制提供新的理论依据,对改进农业害虫的抗性监测和治理策略也具有一定的指导意义。     

Ethanol induces cholesterol efflux and up-regulates ATP-binding cassette cholesterol transporters in fetal astrocytes

Cholesterol plays an important role during brain development, since it is involved in glial cell proliferation, neuronal survival and differentiation, and synaptogenesis. Astrocytes produce large amounts of brain cholesterol and produce and release lipoproteins containing apoE that can extract cholesterol from CNS cells for elimination. We hypothesized that some of the deleterious effects of ethanol in the developing brain may be due to the disruption of cholesterol homeostasis in astrocytes. This study investigates the effect of ethanol on cholesterol efflux mediated by ATP-binding cassette (ABC) cholesterol transporters. In fetal rat astrocytes in culture, ethanol caused a concentration-dependent increase in cholesterol efflux and increased the levels of ABCA1 starting at 25 mm. Similar effects of ethanol on cholesterol efflux and ABCA1 were also observed in fetal human astrocytes. In addition, ABCA1 levels were increased in the brains of 7-day-old pups treated for 3 days with 2, 4, or 6 g/kg ethanol. Ethanol also increased apoE release from fetal rat astrocytes, and conditioned medium prepared from ethanol-treated astrocytes extracted more cholesterol than conditioned medium from untreated cells. In addition, ethanol increased the levels of another cholesterol transporter, ABCG1. Ethanol did not affect cholesterol synthesis and reduced the levels of intracellular cholesterol in rat astrocytes. Retinoic acid, which induces teratogenic effects similarly to ethanol, also caused up-regulation of ABCA1 and ABCG1.     

Induction of intestinal ATP-binding cassette transporters by a phytosterol-derived liver X receptor agonist

The nuclear receptors liver X receptor (LXR) alpha and LXRbeta serve as oxysterol receptors and regulate the expression of genes involved in lipid metabolism. LXR activation induces the expression of ATP-binding cassette (ABC) transporters, such as ABCG5 and ABCG8, which inhibit intestinal absorption of cholesterol and phytosterols. Although several synthetic LXR agonists have been generated, these compounds have limited clinical application, because they cause hypertriglycemia by inducing the expression of lipogenic genes in the liver. We synthesized derivatives of phytosterols and found some of them to act as LXR agonists. Among them, YT-32 [(22E)-ergost-22-ene-1alpha,3beta-diol], which is related to ergosterol and brassicasterol, is the most potent LXR agonist. YT-32 directly bound to LXRalpha and LXRbeta and induced the interaction of LXRalpha with cofactors, such as steroid receptor coactivator-1, as effectively as the natural ligands, 22(R)-hydroxycholesterol and 24(S),25-epoxycholesterol. Although the nonsteroidal synthetic LXR agonist T0901317 induced the expression of intestinal ABC transporters and liver lipogenic genes, oral administration of YT-32 selectively activated intestinal ABC transporters in mice. Unlike T0901317 treatment, YT-32 inhibited intestinal cholesterol absorption without increasing plasma triglyceride levels. The phytosterol-derived LXR agonist YT-32 might selectively modulate intestinal cholesterol metabolism.     

ATP-binding cassette transporters as pitfalls in selection of transgenic cells

Puromycin, hygromycin, and geneticin (G418) are antibiotics frequently used to select genetically engineered eukaryotic cells after transfection or transduction. Because intrinsic or acquired high expression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp/ABCB1) and multidrug resistance-associated proteins (MRP/ABCC1), can hamper efficient selection, it is important to know whether these antibiotics are substrates and/or inducers of efflux transporters. Therefore, we investigated the influence of these antibiotics on drug transporter expression by quantitative real-time polymerase chain reaction in the induction model cell line LS180. Moreover, we assessed whether ABC transporters influence the growth inhibitory effects of these antibiotics by proliferation assays using Madin-Darby canine kidney II (MDCKII) cells overexpressing the particular transporter. The results obtained indicate that puromycin and G418 are substrates of several ABC transporters, mainly Pgp/ABCB1. In contrast, hygromycin seems to be no good substrate for any of the ABC transporters investigated. Puromycin induced ABCC1/MRP1, whereas G418 suppressed ABCB1/Pgp, at the messenger RNA (mRNA) level. In contrast, hygromycin had no effect on ABC transporter mRNA expressions. In conclusion, this study emphasizes the significance of ABC transporters for the efficacy of selection processes. Consciousness of the results is supposed to guide the molecular biologist to the right choice of adequate experimental conditions for successful selection of genetically engineered eukaryotic cells.     

Scavenger receptor-BI inhibits ATP-binding cassette transporter 1- mediated cholesterol efflux in macrophages

Scavenger receptor BI (SR-BI) facilitates the efflux of cellular cholesterol to plasma high density lipoprotein (HDL). Recently, the ATP-binding cassette transporter 1 (ABC1) was identified as a key mediator of cholesterol efflux to apolipoproteins and HDL. The goal of the present study was to determine a possible interaction between the SR-BI and ABC1 cholesterol efflux pathways in macrophages. Free cholesterol efflux to HDL was increased ( approximately 2.2-fold) in SR-BI transfected RAW macrophages in association with increased SR-BI protein levels. Treatment of macrophages with 8-bromo-cAMP (cAMP) resulted in a 4.1-fold increase in ABC1 mRNA level and also increased cholesterol efflux to HDL (2.2-fold) and apoA-I (5.5-fold). However, in SR-BI transfected RAW cells, cAMP treatment produced a much smaller increment in cholesterol efflux to HDL (1.1-fold) or apoA-I (3.3-fold) compared with control cells. In macrophages loaded with cholesterol by acetyl-LDL treatment, SR-BI overexpression did not increase cholesterol efflux to HDL but did inhibit cAMP-mediated cholesterol efflux to apoA-I or HDL. SR-BI neutralizing antibody led to a dose- and time-dependent increase of cAMP-mediated cholesterol efflux in both SR-BI transfected and control cells, indicating that SR-BI inhibits ABC1-mediated cholesterol efflux even at low SR-BI expression level. Transfection of a murine ABC1 cDNA into 293 cells led to a 2.3-fold increase of cholesterol efflux to apoA-I, whereas co-transfection of SR-BI with ABC1 blocked this increase in cholesterol efflux. SR-BI and ABC1 appear to have distinct and competing roles in mediating cholesterol flux between HDL and macrophages. In nonpolarized cells, SR-BI promotes the reuptake of cholesterol actively effluxed by ABC1, creating a futile cycle.     

Cooperative transcriptional activation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 genes by nuclear receptors including Liver-X-Receptor

The ATP-binding cassette transporters ABCG5 and ABCG8 form heterodimers that limit absorption of dietary sterols in the intestine and promote cholesterol elimination from the body through hepatobiliary secretion. To identify cis-regulatory elements of the two genes, we have cloned and analyzed twenty-three evolutionary conserved region (ECR) fragments using the CMV-luciferase reporter system in HepG2 cells. Two ECRs were found to be responsive to the Liver-X-Receptor (LXR). Through elaborate deletion studies, regions containing putative LXREs were identified and the binding of LXRα was demonstrated by EMSA and ChIP assay. When the LXREs were inserted upstream of the intergenic promoter, synergistic activation by LXRα/RXRα in combination with GATA4, HNF4α, and LRH-1, which had been shown to bind to the intergenic region, was observed. In conclusion, we have identified two LXREs in ABCG5/ABCG8 genes for the first time and propose that these LXREs, especially in the ECR20, play major roles in regulating these genes. [BMB Reports 2013; 46(6): 322-327]     

The ATP-binding cassette transporter 1 mediates lipid efflux from Sertoli cells and influences male fertility

The liver X receptor/retinoid X receptor (LXR/RXR)-regulated gene ABCA1 effluxes cellular cholesterol and phospholipid to apolipoprotein A1 (apoA1), which is the rate-limiting step in high-density lipoprotein synthesis. The RXR pathway plays a critical role in testicular lipid trafficking, and RXRbeta-deficient male mice are sterile and accumulate lipids in Sertoli cells. Here, we demonstrate that ABCA1 mRNA and protein are abundant in Sertoli cells, whereas germ cells express little ABCA1. LXR/RXR agonists stimulate ABCA1 expression in cultured Sertoli MSC1 and Leydig TM3 cell lines. However, Sertoli TM4 cells lack ABCA1, and TM4 cells or primary Sertoli cells cultured from ABCA1(-/-) mice both fail to efflux cholesterol to apoA1. Expression of exogenous ABCA1 restores apoA1-dependent cholesterol efflux in Sertoli TM4 cells. In vivo, ABCA1-deficient mice exhibit lipid accumulation in Sertoli cells and depletion of normal lipid droplets from Leydig cells by 2 months of age. By 6 months of age, intratesticular testosterone levels and sperm counts are significantly reduced in ABCA1(-/-) mice compared with wild-type (WT) controls. Finally, a 21% decrease (P = 0.01) in fertility was observed between ABCA1(-/-) males compared with WT controls across their reproductive lifespans. These results show that ABCA1 plays an important role in lipid transport in Sertoli cells and influences male fertility.     

Review. Structure and mechanism of ATP-binding cassette transporters

ATP-binding cassette (ABC) transporters constitute a large superfamily of integral membrane proteins that includes both importers and exporters. In recent years, several structures of complete ABC transporters have been determined by X-ray crystallography. These structures suggest a mechanism by which binding and hydrolysis of ATP by the cytoplasmic, nucleotide-binding domains control the conformation of the transmembrane domains and therefore which side of the membrane the translocation pathway is exposed to. A basic, conserved two-state mechanism can explain active transport of both ABC importers and ABC exporters, but various questions remain unresolved. In this article, I will review some of the crystal structures and the mechanistic insight gained from them. Future challenges for a better understanding of the mechanism of ABC transporters will be outlined.     

ATP-binding cassette (ABC) transporters mediate nonvesicular, raft-modulated sterol movement from the plasma membrane to the endoplasmic reticulum

Little is known about the mechanisms of intracellular sterol transport or how cells maintain the high sterol concentration of the plasma membrane (PM). Here we demonstrate that two inducible ATP-binding cassette (ABC) transporters (Aus1p and Pdr11p) mediate nonvesicular movement of PM sterol to the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. This transport facilitates exogenous sterol uptake, which we find requires steryl ester synthesis in the ER. Surprisingly, while expression of Aus1p and Pdr11p significantly increases sterol movement from PM to ER, it does not alter intracellular sterol distribution. Thus, ER sterol is likely rapidly returned to the PM when it is not esterified in the ER. We show that the propensity of PM sterols to be moved to the ER is largely determined by their affinity for sterol sphingolipid-enriched microdomains (rafts). Our findings suggest that raft association is a primary determinant of sterol accumulation in the PM and that Aus1p and Pdr11p facilitate sterol uptake by increasing the cycling of sterol between the PM and ER.     

Fructose uptake in Bifidobacterium longum NCC2705 is mediated by an ATP-binding cassette transporter

Recently, a putative ATP-binding cassette (ABC) transport system was identified in Bifidobacterium longum NCC2705 that is highly up-regulated during growth on fructose as the sole carbon source. Cloning and expression of the corresponding ORFs (bl0033-0036) result in efficient fructose uptake by bacteria. Sequence analysis reveals high similarity to typical ABC transport systems and suggests that these genes are organized as an operon. Expression of FruE is induced by fructose, ribose, or xylose and is able to bind these sugars with fructose as the preferred substrate. Our data suggest that BL0033-0036 constitute a high affinity fructose-specific ABC transporter of B. longum NCC2705. We thus suggest to rename the coding genes to fruEKFG and the corresponding proteins to FruE (sugar-binding protein), FruK (ATPase subunit), FruF, and FruG (membrane permeases). Furthermore, protein-protein interactions between the components of the transporter complex were determined by GST pulldown and Western blot analysis. This revealed interactions between the membrane subunits FruF and FruG with FruE, which in vivo is located on the external side of the membrane, and with the cytoplasmatic ATPase FruK. This is in line with the proposed model for bacterial ABC sugar transporters.     

ATP-binding cassette transporters modulate both coelenterazine- and D-luciferin-based bioluminescence imaging

Bioluminescence imaging (BLI) of luciferase reporters provides a cost-effective and sensitive means to image biological processes. However, transport of luciferase substrates across the cell membrane does affect BLI readout intensity from intact living cells. To investigate the effect of ATP-binding cassette (ABC) transporters on BLI readout, we generated click beetle (cLuc), firefly (fLuc), Renilla (rLuc), and Gaussia (gLuc) luciferase HEK-293 reporter cells that overexpressed different ABC transporters (ABCB1, ABCC1, and ABCG2). In vitro studies showed a significant BLI intensity decrease in intact cells compared to cell lysates, when ABCG2 was overexpressed in HEK-293/cLuc, fLuc, and rLuc cells. Selective ABC transporter inhibitors were also applied. Inhibition of ABCG2 activity increased the BLI intensity more than two-fold in HEK-293/cLuc, fLuc, and rLuc cells; inhibition of ABCB1 elevated the BLI intensity two-fold only in HEK-293/rLuc cells. BLI of xenografts derived from HEK-293/ABC transporter/luciferase reporter cells confirmed the results of inhibitor treatment in vivo. These findings demonstrate that coelenterazine-based rLuc-BLI intensity can be modulated by ABCB1 and ABCG2. ABCG2 modulates d-luciferin-based BLI in a luciferase type-independent manner. Little ABC transporter effect on gLuc-BLI intensity is observed because a large fraction of gLuc is secreted. The expression level of ABC transporters is one key factor affecting BLI intensity, and this may be particularly important in luciferase-based applications in stem cell research.