Up-regulation of the ATP-binding cassette transporter A1 inhibits hepatitis C virus infection.

International audienceHepatitis C virus (HCV) establishes infection using host lipid metabolism pathways that are thus considered potential targets for indirect anti-HCV strategies. HCV enters the cell via clathrin-dependent endocytosis, interacting with several receptors, and virus-cell fusion, which depends on acidic pH and the integrity of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1) mediates cholesterol efflux from hepatocytes to extracellular Apolipoprotein A1 and moves cholesterol within cell membranes. Furthermore, it generates high-density lipoprotein (HDL) particles. HDL protects against arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its cholesterol efflux function in Huh7.5 hepatoma cells, using the liver X receptor (LXR) agonist GW3965, impairs HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry, acting on virus-host cell fusion, but had no impact on virus attachment, replication, or assembly/secretion. It did not affect infectivity or properties of virus particles produced. Silencing of the ABCA1 gene and reduction of the specific cholesterol efflux function counteracted the inhibitory effect of the GW3965 on HCV infection, providing evidence for a key role of ABCA1 in this process. Impaired virus-cell entry correlated with the reorganisation of cholesterol-rich membrane microdomains (lipid rafts). The inhibitory effect could be reversed by an exogenous cholesterol supply, indicating that restriction of HCV infection was induced by changes of cholesterol content/distribution in membrane regions essential for virus-cell fusion. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent cholesterol efflux pathway disrupts membrane cholesterol homeostasis, leading to the inhibition of virus-cell fusion and thus HCV cell entry. Therefore besides other beneficial roles, ABCA1 might represent a potential target for HCV therapy

CETP deficiency due to a novel mutation in the CETP gene promoter and its effect on cholesterol efflux and selective uptake into hepatocytes

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ABCG1 is involved in vitamin E efflux

International audienceVitamin E membrane transport has been shown to involve the cholesterol transporters SR-BI, ABCA1 and NPC1L1. Our aim was to investigate the possible participation of another cholesterol transporter in cellular vitamin E efflux: ABCG1. In Abcg1-deficient mice, vitamin E concentration was reduced in plasma lipoproteins whereas most tissues displayed a higher vitamin E content compared to wild-type mice. alpha- and gamma-tocopherol efflux was increased in CHO cells overexpressing human ABCG1 compared to control cells. Conversely, alpha- and gamma-tocopherol efflux was decreased in ABCG1-knockdown human cells (Hep3B hepatocytes and THP-1 macrophages). Interestingly, alpha- and gamma-tocopherol significantly downregulated ABCG1 and ABCA1 expression levels in Hep3B and THP-1, an effect confirmed in vivo in rats given vitamin E for 5 days. This was likely due to reduced LXR activation by oxysterols, as Hep3B cells and rat liver treated with vitamin E displayed a significantly reduced content in oxysterols compared to their respective controls. Overall, the present study reveals for the first time that ABCG1 is involved in cellular vitamin E efflux. (C) 2014 Elsevier B.V. All rights reserved

Multimerization of GPIHBP1 and Familial Chylomicronemia from a Serine-to-Cysteine Substitution in GPIHBP1's Ly6 Domain

GPIHBP1, a glycosylphosphatidylinositol-anchored glycoprotein of microvascular endothelial cells, binds lipoprotein lipase (LPL) within the interstitial spaces and transports it across endothelial cells to the capillary lumen. The ability of GPIHBP1 to bind LPL depends on the Ly6 domain, a three-fingered structure containing 10 cysteines and a conserved pattern of disulfide bond formation. Here, we report a patient with severe hypertriglyceridemia who was homozygous for a GPIHBP1 point mutation that converted a serine in the GPIHBP1 Ly6 domain (Ser-107) to a cysteine. Two hypertriglyceridemic siblings were homozygous for the same mutation. All three homozygotes had very low levels of LPL in the preheparin plasma. We suspected that the extra cysteine in GPIHBP1-S107C might prevent the trafficking of the protein to the cell surface, but this was not the case. However, nearly all of the GPIHBP1-S107C on the cell surface was in the form of disulfide-linked dimers and multimers, whereas wild-type GPIHBP1 was predominantly monomeric. An insect cell GPIHBP1 expression system confirmed the propensity of GPIHBP1-S107C to form disulfide-linked dimers and to form multimers. Functional studies showed that only GPIHBP1 monomers bind LPL. In keeping with that finding, there was no binding of LPL to GPIHBP1-S107C in either cell-based or cell-free binding assays. We conclude that an extra cysteine in the GPIHBP1 Ly6 motif results in multimerization of GPIHBP1, defective LPL binding, and severe hypertriglyceridemia

Analysis of HCV particles secreted from cells that over-express ABCA1.

<p>Physical properties of the nascent virus particles produced in cells stimulated or not with GW3965 were analysed by centrifugation in iodixanol gradient. Huh7.5 cells were pre-incubated with solvent (panel A) or 1 µM GW3965 (panel B) and the drug was maintained until 72 h post-infection when cell supernatants were collected, concentrated and subjected to gradient centrifugation. HCV RNA in gradient fractions was quantified by qRT-PCR and core antigen, ApoB and ApoE by ELISA assays.</p

GW3965 treatment up-regulates ABCA1 expression and its cholesterol efflux function.

<p>(A) Cell toxicity of GW3965. Huh7.5 cells were cultured in the presence of indicated concentrations of the drug for 24 h. The luminescent signal is expressed in luminescence units (RLU). (B) Up-regulation of ABCA1 mRNA expression by GW3695 treatment. Huh7.5 cells were treated for 24 h with 1 µM GW3695 or drug solvent (DMSO). Then ABCA1 mRNA was determined by qRT-PCR. (C) ABCA1 protein production in drug-stimulated Huh7.5 cells. Cells were treated for 24 h with 1 µM GW3965 and analysed by Western blot (shown in the insert). Protein content in the ABCA1 band (220 kDA) in GW3965-(GW), and DMSO-(solv) treated cells was quantified relative to the calnexin band using the Odyssey Infrared Imaging System. (D) GW3965 stimulation promotes ABCA1-mediated cholesterol efflux to ApoA1. Huh7.5 cells were labelled with [³H] cholesterol then incubated with GW3965 or drug solvent. ABCA1-dependent [³H] cholesterol efflux was assayed by comparing cell-associated and free radioactivity. (E) Kinetics of ABCA1 gene expression following stimulation of cells with GW3965. Huh7.5 cells were treated with 1 µM GW3965 for the indicated time and ABCA1 mRNA was determined by qRT-PCR. Results were expressed as relative values compared to ABCA1 expression in cells treated with drug solvent. (F) Kinetics of cholesterol efflux in cells stimulated with GW3965. Huh7.5 cells were labelled with [³H] cholesterol for 24 h, and incubated for an additional 16 h with 1 μM GW3965 or drug solvent. ABCA1-dependent [³H] cholesterol efflux was assayed in the presence of ApoA1 and either GW3965 or solvent for the indicated period of time.</p

Adipocyte ATP-Binding Cassette G1 Promotes Triglyceride Storage, Fat Mass Growth, and Human Obesity

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Over-expression of ABCA1 inhibits HCV infection of primary human hepatocytes and human liver slices.

<p>(A–B) Inhibition of HCV infection of primary human hepatocytes. (A) Primary human hepatocytes were treated with 2–10 μM GW3965 (non-toxic concentrations for cells) or with drug solvent, prior to HCV infection. Twenty-four hours post-infection, ABCA1 mRNA was determined by qRT-PCR and expressed in arbitrary units, taking into account ABCA1 levels in liver cells pre-treated with the drug. (B) GW3965-treated and solvent-treated primary human hepatocytes were inoculated with HCV. After 24 h, intracellular HCV RNA was quantified by qRT-qPCR. The efficiency of infection in drug pre-treated cells was expressed as the percentage of infection compared to solvent-treated cells. (C–D) ABCA1 over-expression inhibits HCV infection of human liver slices. Human liver slices were cultured for 24 h, treated with 5 or 10 μM GW3965 or with DMSO before infection with HCVcc. At 24 h post-infection, total RNA was extracted and ABCA1 mRNA (C) and HCV RNA (D) were quantified by corresponding qRT-PCR assays and expressed as the percentage of RNA compared to the values obtained for solvent-treated cells.</p