Cholesterol Transporter ATP-binding Cassette Research Articles

Heat-killed Lactiplantibacillus plantarum Shinshu N-07 exerts antiobesity effects in western diet-induced obese mice.

The aim of this study was to evaluate the antiobesity effects of heat-killed Lactiplantibacillus plantarum Shinshu N-07 (N-07) isolated from fermented Brassica rapa L. Male mice were divided into three groups (n=10/group); normal diet, western diet (WD), or WD+N-07 (N-07) group and administered each diet for 56 days. The N-07 group showed significant suppression of body weight gain and epididymal fat, perirenal fat, and liver weights compared with the WD group. Higher levels of fecal total cholesterol, triglyceride (TG), and free fatty acid (FFA) were observed in the N-07 group than in the WD group. The mRNA expression of the cholesterol transporter ATP-binding cassette transporter G5 (ABCG5) was significantly increased in the small intestine of N-07-fed mice compared with WD-fed mice. Moreover, N-07 supplementation significantly increased the mRNA expression of ABCG5 and ABCG8 in Caco-2 cells. Furthermore, the TG- and FFA-removal ability of N-07 was confirmed to evaluate its soybean oil- and oleic acid-binding capacities in in vitro experiments. The antiobesity effects of N-07 might be due to its ability to promote lipid excretion by regulating cholesterol transporter expression and lipid-binding ability.

Cholesterol accumulation in macrophages drives NETosis in atherosclerotic plaques via IL-1β secretion.

Neutrophil extracellular trap formation (NETosis) increases atherosclerotic plaque vulnerability and athero-thrombosis. However, mechanisms promoting NETosis during atherogenesis are poorly understood. We have shown that cholesterol accumulation due to myeloid cell deficiency of the cholesterol transporters ATP Binding Cassette A1 and G1 (ABCA1/G1) promotes NLRP3 inflammasome activation in macrophages and neutrophils and induces prominent NETosis in atherosclerotic plaques. We investigated whether NETosis is a cell-intrinsic effect in neutrophils or is mediated indirectly by cellular crosstalk from macrophages to neutrophils involving IL-1β. We generated mice with neutrophil or macrophage-specific Abca1/g1 deficiency (S100A8CreAbca1fl/flAbcg1fl/fl or CX3CR1CreAbca1fl/flAbcg1fl/fl mice, respectively), and transplanted their bone marrow into low-density lipoprotein receptor knockout mice. We then fed the mice a cholesterol-rich diet. Macrophage, but not neutrophil Abca1/g1 deficiency activated inflammasomes in macrophages and neutrophils, reflected by caspase-1 cleavage, and induced NETosis in plaques. NETosis was suppressed by administering an interleukin (IL)-1β neutralizing antibody. The extent of NETosis in plaques correlated strongly with the degree of neutrophil accumulation, irrespective of blood neutrophil counts, and neutrophil accumulation was decreased by IL-1β antagonism. In vitro, IL-1β or media transferred from Abca1/g1-deficient macrophages increased NETosis in both control and Abca1/Abcg1 deficient neutrophils. This cell-extrinsic effect of IL-1β on NETosis was blocked by an NLRP3 inhibitor. These studies establish a new link between inflammasome-mediated IL-1β production in macrophages and NETosis in atherosclerotic plaques. Macrophage-derived IL-1β appears to increase NETosis both by increasing neutrophil recruitment to plaques and by promoting neutrophil NLRP3 inflammasome activation.

Sphingosine-1-Phosphate (S1P) Lyase Inhibition Aggravates Atherosclerosis and Induces Plaque Rupture in ApoE-/-Mice.

Altered plasma sphingosine-1-phosphate (S1P) concentrations are associated with clinical manifestations of atherosclerosis. However, whether long-term elevation of endogenous S1P is pro- or anti-atherogenic remains unclear. Here, we addressed the impact of permanently high S1P levels on atherosclerosis in cholesterol-fed apolipoprotein E-deficient (ApoE−/−) mice over 12 weeks. This was achieved by pharmacological inhibition of the S1P-degrading enzyme S1P lyase with 4-deoxypyridoxine (DOP). DOP treatment dramatically accelerated atherosclerosis development, propagated predominantly unstable plaque phenotypes, and resulted in frequent plaque rupture with atherothrombosis. Macrophages from S1P lyase-inhibited or genetically deficient mice had a defect in cholesterol efflux to apolipoprotein A-I that was accompanied by profoundly downregulated cholesterol transporters ATP-binding cassette transporters ABCA1 and ABCG1. This was dependent on S1P signaling through S1PR3 and resulted in dramatically enhanced atherosclerosis in ApoE−/−/S1PR3−/− mice, where DOP treatment had no additional effect. Thus, high endogenous S1P levels promote atherosclerosis, compromise cholesterol efflux, and cause genuine plaque rupture.

Soraphen A enhances macrophage cholesterol efflux via indirect LXR activation and ABCA1 upregulation

Increased cholesterol efflux from macrophage foam cells in the subendothelial space confers protection against atherosclerosis. Soraphen A, a myxobacterial macrolactone, is an inhibitor of acetyl coenzyme A carboxylases (ACC), which control fatty acid synthesis and oxidation. To assess a potential direct link between macrophage cholesterol efflux and ACC inhibition, we examined [3H]-cholesterol efflux from human THP-1-derived foam cells in the presence of soraphen A. We dissected underlying molecular events by western blot analyses, RT-qPCR, reporter gene and coactivator recruitment assays as well as relative quantification of free and total cholesterol. Soraphen A increased cholesterol efflux from macrophage foam cells via upregulation of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1). Soraphen A enhanced transcription of ABCA1 in an LXR-dependent manner, however, without direct binding to the ligand-binding domain of this nuclear receptor. Soraphen A elevated the cellular level of free cholesterol, and failed to activate LXR upon exogenous supplementation with fatty acids or inhibition of cholesterol synthesis. Thus, impeded conversion from acetyl- to malonyl-CoA by soraphen A may lead to more unesterified cholesterol and thus potential LXR agonists. The present study reveals ACC inhibition as a previously unrecognized mechanism to regulate macrophage cholesterol efflux via indirect LXR activation and ABCA1 upregulation.

260Identification of differential roles of microRNA-33a and -33b during atherosclerosis progression with genetically modified mice

Abstract Background MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports, including ours, indicated that miR-33a located within the intron of sterol regulatory element-binding factor (SREBF) 2 targets cholesterol transporter ATP-binding cassette protein A1 (ABCA1) or other anti-atherogenic targets and contributes to atherogenesis. Its inhibition or deletion is known to result in the amelioration of atherosclerosis. However, mice lack the other member of miR-33 family, miR-33b, which exists in humans. Precise evaluation and comparison of the responsibilities of these two miRs during the progression of atherosclerosis are essential and need to be investigated. Methods and results The difference between miR-33a and miR-33b in vitro and in vivo were analyzed from multiple directions using genetically modified miR-33a knock-out (KO) and miR-33b knock-in (KI) humanized mice. At first, we performed transcriptomic analysis of primary cultured hepatocytes transfected with synthetic miR-33a, miR-33b, and a control miR and found similar potential target repression and targeting motif of miR-33a and miR-33b in vitro. However, we noticed distinct expression patterns of miR-33a and miR-33b in several organs. By crossing miR-33a KO and miR-33b KI mice, we established four strains with or without miR-33a and miR-33b. Comparison of these strains showed distinct distribution and regulation of miR-33 family. In particular, comparison between mice with only miR-33a (wild-type mice) and mice with only miR-33b (miR-33a−/− miR-33b+/+) revealed 4-fold predominant expression of miR-33b in the liver. Such differential expression resulted in a reduced expression of target genes such as ABCA1 and worsened serum cholesterol profile in mice with only miR-33b. On the contrary, in macrophages the expression levels of miR-33 family genes were similar and their effects on target genes and cholesterol efflux capacity to ApoA-I or HDL cholesterol (HDL-C) were almost comparable. To evaluate the whole body atherogenic potency, we developed ApoE−/− miR-33a+/+ miR-33b−/− mice and ApoE−/− miR-33a−/− miR-33b+/+ mice. ApoE−/− miR-33a−/− miR-33b+/+ mice developed increased atherosclerotic plaque compared with ApoE−/− miR-33a+/+ miR-33b−/− mice, in line with the predominant expression of miR-33b in the liver and decreased serum HDL-C levels whose lower cholesterol efflux capacity were confirmed in 3H-labeled macrophages. On the contrary, a bone marrow transplantation study showed no significant difference in atherosclerosis and serum cholesterol profile, and this was consistent with the relevant expression levels of miR-33a and miR-33b in bone marrow cells. Conclusions miR-33 family exhibited differences in distribution and regulation, and particularly in the progression of atherosclerosis, miR-33b would be more potent than miR-33a.

Abstract 605: Identification of Differential Roles of Microrna-33a and -33b During Atherosclerosis Progression with Genetically Modified Mice

Backgrounds: MicroRNA (miR)-33a targets cholesterol transporter ATP-binding cassette protein A1 or other anti-atherogenic targets and contributes to atherogenesis. Its inhibition or deletion is known to result in the amelioration of atherosclerosis. However, mice lack the other member of miR-33 family, miR-33b, which exists in humans. Thus, precise evaluation and comparison of the responsibilities of these two miRs during the progression of atherosclerosis has not been investigated. Methods and Results: The difference between miR-33a and miR-33b were analyzed from multiple directions using genetically modified mice. We generated four strains with or without miR-33a and miR-33b. Comparison of these strains showed distinct distribution and regulation of miR-33 family. In particular, comparison between mice with only miR-33a (wild-type mice) and mice with only miR-33b (miR-33a -/- /miR-33b +/+ ) revealed the prevalence of miR-33b in the liver. Such differential expression resulted in a worsened serum cholesterol profile in mice with only miR-33b. On the contrary, in macrophages the expression level of miR-33 family genes was similar and their effects on cholesterol efflux capacity were almost comparable. To evaluate the whole body atherogenic potency, we developed ApoE -/- /miR-33a +/+ /miR-33b -/- mice and ApoE -/- /miR-33a -/- /miR-33b +/+ mice. ApoE -/- /miR-33a -/- /miR-33b +/+ mice developed increased atherosclerotic plaque compared with ApoE -/- /miR-33a +/+ /miR-33b -/- mice, in line with the predominant expression of miR-33b in the liver and worsened serum cholesterol profile. On the contrary, a bone marrow transplantation study showed no significant difference, and this was consistent with the relevant expression levels of miR-33a and miR-33b in bone marrow cells. Conclusions: miR-33 family exhibited differences in distribution and regulation, and particularly in the progression of atherosclerosis, miR-33b would be more potent than miR-33a.

FMO3 and its metabolite TMAO contribute to the formation of gallstones

Trimethylamine-N-oxide (TMAO) is a metabolite derived from trimethylamine (TMA), which is first produced by gut microbiota and then oxidized by flavin-containing monooxygenase 3 (FMO3) in the liver. TMAO may contribute to the development of diseases such as atherosclerosis because of its role in regulating lipid metabolism. In this study, we found that high plasma TMAO levels were positively associated with the presence of gallstone disease in humans. We further found increased hepatic FMO3 expression and elevated plasma TMAO level in a gallstone-susceptible strain of mice C57BL/6J fed a lithogenic diet (LD), but not in a gallstone-resistant strain of mice AKR/J. Dietary supplementation of TMAO or its precursor choline increased hepatic FMO3 expression and plasma TMAO levels and induced hepatic canalicular cholesterol transporters ATP binding cassette (Abc) g5 and g8 expression in mice. Up-regulation of ABCG5 and ABCG8 expression was observed in hepatocytes incubated with TMAO in vitro. Additionally, in AKR/J mice fed a LD supplemented with 0.3% TMAO, the incidence of gallstones rose up to 70% compared with 0% in AKR/J mice fed only a LD. This was associated with increased hepatic Abcg5 and g8 expression induced by TMAO. Our study demonstrated TMAO could be associated with increased hepatic Abcg5/g8 expression, biliary cholesterol hypersecretion and gallstone formation.

Modelling interaction between HIV-1 Nef and calnexin.

HIV-associated atherosclerosis is a major comorbidity due, in part, to systemic effects of the virus on cholesterol metabolism. HIV protein Nef plays an important role in this pathology by impairing maturation of the main cellular cholesterol transporter ATP-Binding Cassette (ABCA) 1. ABCA1 maturation critically depends on calnexin, an integral endoplasmic reticulum membrane chaperone, and Nef binds to the cytoplasmic domain of calnexin and impairs interaction of calnexin with ABCA1. Overarching goal of the present study was to model Nef-calnexin interaction interface, and identify small molecule compounds potentially inhibiting this interaction. Molecular dynamics was utilized to build structure model of calnexin cytoplasmic domain, followed by global docking combined with application of QASDOM software developed by us for efficient analysis of receptor-ligand complexes. Structure-based virtual screening was performed for all sites identified by docking. A soluble analogue of a compound from the screening results list was tested for ability to down-regulate ABCA1. We identified major interaction sites in calnexin and reciprocal sites in Nef. Virtual screening yielded a number of small-molecule compounds potentially blocking a calnexin site. Interestingly, one of the compounds, NSC13987, was previously identified by us as an inhibitor targeting a Nef site. An analogue of NSC13987, AMS-55, potently reversed the negative effect of Nef on ABCA1 abundance. We have modelled Nef-calnexin interaction, predicted small molecule compounds that can potentially inhibit this interaction, and experimentally tested one of these compounds, confirming its effectiveness. These findings provide a platform for searching for new therapeutic agents to treat HIV-associated comorbidities.

Cholesterol Efflux Pathways Suppress Inflammasome Activation, NETosis, and Atherogenesis.

The CANTOS trial (Canakinumab Antiinflammatory Thrombosis Outcome Study) showed that antagonism of interleukin (IL)-1β reduces coronary heart disease in patients with a previous myocardial infarction and evidence of systemic inflammation, indicating that pathways required for IL-1β secretion increase cardiovascular risk. IL-1β and IL-18 are produced via the NLRP3 inflammasome in myeloid cells in response to cholesterol accumulation, but mechanisms linking NLRP3 inflammasome activation to atherogenesis are unclear. The cholesterol transporters ATP binding cassette A1 and G1 (ABCA1/G1) mediate cholesterol efflux to high-density lipoprotein, and Abca1/g1 deficiency in myeloid cells leads to cholesterol accumulation. To interrogate mechanisms connecting inflammasome activation with atherogenesis, we used mice with myeloid Abca1/g1 deficiency and concomitant deficiency of the inflammasome components Nlrp3 or Caspase-1/11. Bone marrow from these mice was transplanted into Ldlr-/- recipients, which were fed a Western-type diet. Myeloid Abca1/g1 deficiency increased plasma IL-18 levels in Ldlr-/- mice and induced IL-1β and IL-18 secretion in splenocytes, which was reversed by Nlrp3 or Caspase-1/11 deficiency, indicating activation of the NLRP3 inflammasome. Nlrp3 or Caspase-1/11 deficiency decreased atherosclerotic lesion size in myeloid Abca1/g1-deficient Ldlr-/- mice. Myeloid Abca1/g1 deficiency enhanced caspase-1 cleavage not only in splenic monocytes and macrophages, but also in neutrophils, and dramatically enhanced neutrophil accumulation and neutrophil extracellular trap formation in atherosclerotic plaques, with reversal by Nlrp3 or Caspase-1/11 deficiency, suggesting that inflammasome activation promotes neutrophil recruitment and neutrophil extracellular trap formation in atherosclerotic plaques. These effects appeared to be indirectly mediated by systemic inflammation leading to activation and accumulation of neutrophils in plaques. Myeloid Abca1/g1 deficiency also activated the noncanonical inflammasome, causing increased susceptibility to lipopolysaccharide-induced mortality. Patients with Tangier disease, who carry loss-of-function mutations in ABCA1 and have increased myeloid cholesterol content, showed a marked increase in plasma IL-1β and IL-18 levels. Cholesterol accumulation in myeloid cells activates the NLRP3 inflammasome, which enhances neutrophil accumulation and neutrophil extracellular trap formation in atherosclerotic plaques. Patients with Tangier disease, who have increased myeloid cholesterol content, showed markers of inflammasome activation, suggesting human relevance.

TRAK2, a novel regulator of ABCA1 expression, cholesterol efflux and HDL biogenesis.

The recent failures of HDL-raising therapies have underscored our incomplete understanding of HDL biology. Therefore there is an urgent need to comprehensively investigate HDL metabolism to enable the development of effective HDL-centric therapies. To identify novel regulators of HDL metabolism, we performed a joint analysis of human genetic, transcriptomic, and plasma HDL-cholesterol (HDL-C) concentration data and identified a novel association between trafficking protein, kinesin binding 2 (TRAK2) and HDL-C concentration. Here we characterize the molecular basis of the novel association between TRAK2 and HDL-cholesterol concentration. Analysis of lymphocyte transcriptomic data together with plasma HDL from the San Antonio Family Heart Study (n = 1240) revealed a significant negative correlation between TRAK2 mRNA levels and HDL-C concentration, HDL particle diameter and HDL subspecies heterogeneity. TRAK2 siRNA-mediated knockdown significantly increased cholesterol efflux to apolipoprotein A-I and isolated HDL from human macrophage (THP-1) and liver (HepG2) cells by increasing the mRNA and protein expression of the cholesterol transporter ATP-binding cassette, sub-family A member 1 (ABCA1). The effect of TRAK2 knockdown on cholesterol efflux was abolished in the absence of ABCA1, indicating that TRAK2 functions in an ABCA1-dependent efflux pathway. TRAK2 knockdown significantly increased liver X receptor (LXR) binding at the ABCA1 promoter, establishing TRAK2 as a regulator of LXR-mediated transcription of ABCA1. We show, for the first time, that TRAK2 is a novel regulator of LXR-mediated ABCA1 expression, cholesterol efflux, and HDL biogenesis. TRAK2 may therefore be an important target in the development of anti-atherosclerotic therapies.

RAGE Suppresses ABCG1-Mediated Macrophage Cholesterol Efflux in Diabetes.

Diabetes exacerbates cardiovascular disease, at least in part through suppression of macrophage cholesterol efflux and levels of the cholesterol transporters ATP binding cassette transporter A1 (ABCA1) and ABCG1. The receptor for advanced glycation end products (RAGE) is highly expressed in human and murine diabetic atherosclerotic plaques, particularly in macrophages. We tested the hypothesis that RAGE suppresses macrophage cholesterol efflux and probed the mechanisms by which RAGE downregulates ABCA1 and ABCG1. Macrophage cholesterol efflux to apolipoprotein A1 and HDL and reverse cholesterol transport to plasma, liver, and feces were reduced in diabetic macrophages through RAGE. In vitro, RAGE ligands suppressed ABCG1 and ABCA1 promoter luciferase activity and transcription of ABCG1 and ABCA1 through peroxisome proliferator–activated receptor-γ (PPARG)–responsive promoter elements but not through liver X receptor elements. Plasma levels of HDL were reduced in diabetic mice in a RAGE-dependent manner. Laser capture microdissected CD68+ macrophages from atherosclerotic plaques of Ldlr−/− mice devoid of Ager (RAGE) displayed higher levels of Abca1, Abcg1, and Pparg mRNA transcripts versus Ager-expressing Ldlr−/− mice independently of glycemia or plasma levels of total cholesterol and triglycerides. Antagonism of RAGE may fill an important therapeutic gap in the treatment of diabetic macrovascular complications.

Stimulation of Liver X Receptor Has Potent Anti-HIV Effects in a Humanized Mouse Model of HIV Infection.

Previous studies demonstrated that liver X receptor (LXR) agonists inhibit human immunodeficiency virus (HIV) replication by upregulating cholesterol transporter ATP-binding cassette A1 (ABCA1), suppressing HIV production, and reducing infectivity of produced virions. In this study, we extended these observations by analyzing the effect of the LXR agonist T0901317 [N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide] on the ongoing HIV infection and investigating the possibility of using LXR agonist for pre-exposure prophylaxis of HIV infection in a humanized mouse model. Pre-exposure of monocyte-derived macrophages to T0901317 reduced susceptibility of these cells to HIV infection in vitro. This protective effect lasted for up to 4 days after treatment termination and correlated with upregulated expression of ABCA1, reduced abundance of lipid rafts, and reduced fusion of the cells with HIV. Pre-exposure of peripheral blood leukocytes to T0901317 provided only a short-term protection against HIV infection. Treatment of HIV-exposed humanized mice with LXR agonist starting 2 weeks postinfection substantially reduced viral load. When eight humanized mice were pretreated with LXR agonist prior to HIV infection, five animals were protected from infection, two had viral load at the limit of detection, and one had viral load significantly reduced relative to mock-treated controls. T0901317 pretreatment also reduced HIV-induced dyslipidemia in infected mice. In conclusion, these results reveal a novel link between LXR stimulation and cell resistance to HIV infection and suggest that LXR agonists may be good candidates for development as anti-HIV agents, in particular for pre-exposure prophylaxis of HIV infection.

RNA-Seq Quantification of Hepatic Drug Processing Genes in Germ-Free Mice.

Intestinal bacteria have been shown to be important in regulating host intermediary metabolism and contributing to obesity. However, relatively less is known about the effect of intestinal bacteria on the expression of hepatic drug-processing genes in the host. This study characterizes the expression of hepatic drug-processing genes in germ-free (GF) mice using RNA-Seq. Total RNA were isolated from the livers of adult male conventional and GF C57BL/6J mice (n = 3 per group). In the livers of GF mice, the mRNA of the aryl hydrocarbon receptor target gene Cyp1a2 was increased 51%, and the mRNA of the peroxisome proliferator-activated receptor α (PPARα) target gene Cyp4a14 was increased 202%. Conversely, the mRNA of the constitutive androstane receptor (CAR) target gene Cyp2b10 was decreased 57%, and the mRNA of the pregnane X receptor target gene Cyp3a11 was decreased 87% in GF mice. Although other non-Cyp phase-1 enzymes in the livers of GF mice were only moderately affected, there was a marked down-regulation in the phase-2 enzymes glutathione S-transferases p1 and p2, as well as a marked up-regulation in the major bile acid transporters Na(+)-taurocholate cotransporting polypeptide and organic anion-transporting polypeptide 1b2, and the cholesterol transporter ATP-binding cassette transporter Abcg5/Abcg8. This study demonstrates that intestinal bacteria regulate the expression of a large number of drug-processing genes, which suggests that intestinal bacteria are responsible for some individual differences in drug responses.

HIV-1 infection of macrophages induces retention of cholesterol transporter ABCA1 in the endoplasmic reticulum.

HIV-1 infection is associated with a high risk of developing atherosclerosis,1 and studies in animal models and in vitro demonstrated that impairment of activity of the cholesterol transporter ATP-Binding Cassette A1 (ABCA1) is a significant contributor to this side effect of HIV infection.2,3 Our unpublished observations demonstrated that Nef binds to endoplasmic reticulum (ER) chaperone calnexin. While this binding does not affect the interaction between calnexin and gp160, it disrupts the interaction of calnexin and ABCA1. Given that calnexin is essential for the folding and maturation of ABCA1, this effect of Nef may explain Nef-mediated impairment of ABCA1 activity. Here, we provide images that demonstrate the effects of HIV infection on ABCA1 localization in macrophages. Macrophages are the key cells in the development of atherosclerosis, and HIV-mediated disruption of ABCA1 activity in these cells contributes to cholesterol accumulation and their transformation into “foam” cells, the signature cells in atherosclerosis.2 To investigate the mechanisms of ABCA1 inactivation, we infected monocyte-derived macrophages with VSV-G-pseudotyped HIV-1 and stained for HIV-1 Env (using anti-HIV gp120 sheep serum as a primary antibody and Alexa Fluor 488-conjugated donkey antisheep IgG as a secondary antibody), ABCA1 (using anti-ABCA1 rabbit polyclonal antibody as a primary antibody and Alexa Fluor 647-conjugated goat antirabbit IgG as a secondary antibody), and ER chaperone calnexin (using mouse monoclonal anticalnexin-ER membrane marker antibody as a primary antibody and goat antimouse DyLight 550-conjugated antibody as a secondary antibody), which regulates the folding and maturation of glycosylated proteins, including ABCA1 and gp160, in the ER. Nuclei were counterstained with DAPI. Images were captured with a Carl Zeiss LSM 710 confocal microscope, using a Plan Apochromat 100×/1.46 oil immersion lens. As shown in Fig. 1, ABCA1 in an infected (gp120-positive) cell localized predominantly to intracellular regions that stain with calnexin-ER antibody, whereas in uninfected cells the majority of ABCA1 molecules resided at the plasma membrane, the site of active cholesterol exchange between ABCA1 and cholesterol acceptors in the blood.4 These representative images were selected upon evaluation of several large tiled immunofluorescence images each containing over 100 cells. This relocalization of ABCA1 reflects its retention in the intracellular regions positive for calnexin, a marker for the ER. Such retention is consistent with Nef-mediated disruption of the calnexin–ABCA1 interaction, and may be responsible for subsequent degradation of ABCA1 and impairment of ABCA1-dependent cholesterol efflux. FIG. 1. ABCA1 localization in one gp120-positive and two gp120-negative cells of HIV-infected macrophage culture. The upper panel shows a composite image of four channels representing gp120 (green), calnexin (red), ABCA1 (white), and nuclei (blue), whereas panels ...

Cholesterol efflux from THP-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase

Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL.

Loss-of-function mutations in ABCA1 and enhanced β-cell secretory capacity in young adults.

Loss-of-function mutations affecting the cholesterol transporter ATP-binding cassette transporter subfamily A member 1 (ABCA1) impair cellular cholesterol efflux and are associated with reduced HDL-cholesterol (HDL-C) levels. ABCA1 may also be important in regulating β-cell cholesterol homeostasis and insulin secretion. We sought to determine whether loss-of-function ABCA1 mutations affect β-cell secretory capacity in humans by performing glucose-potentiated arginine tests in three subjects homozygous for ABCA1 mutations (age 25 ± 11 years), eight heterozygous subjects (28 ± 7 years), and eight normal control subjects pair-matched to the heterozygous carriers. To account for any effect of low HDL-C on insulin secretion, we studied nine subjects with isolated low HDL-C with no ABCA1 mutations (age 26 ± 6 years) and nine pair-matched control subjects. Homozygotes for ABCA1 mutations exhibited enhanced oral glucose tolerance and dramatically increased β-cell secretory capacity that was also greater in ABCA1 heterozygous subjects than in control subjects, with no differences in insulin sensitivity. Isolated low HDL-C subjects also demonstrated an increase in β-cell secretory capacity but in contrast to those with ABCA1 mutations, exhibited impaired insulin sensitivity, supporting β-cell compensation for increased insulin demand. These data indicate that loss-of-function mutations in ABCA1 in young adults may be associated with enhanced β-cell secretory capacity and normal insulin sensitivity and support the importance of cellular cholesterol homeostasis in regulating β-cell insulin secretion.

Prenatal Ethanol Exposure Up-Regulates the Cholesterol Transporters ATP-Binding Cassette A1 and G1 and Reduces Cholesterol Levels in the Developing Rat Brain.

Cholesterol plays a pivotal role in many aspects of brain development; reduced cholesterol levels during brain development, as a consequence of genetic defects in cholesterol biosynthesis, leads to severe brain damage, including microcephaly and mental retardation, both of which are also hallmarks of the fetal alcohol syndrome. We had previously shown that ethanol up-regulates the levels of two cholesterol transporters, ABCA1 (ATP binding cassette-A1) and ABCG1, leading to increased cholesterol efflux and decreased cholesterol content in astrocytes in vitro. In the present study we investigated whether similar effects could be seen in vivo. Pregnant Sprague-Dawley rats were fed liquid diets containing 36% of the calories from ethanol from gestational day (GD) 6 to GD 21. A pair-fed control groups and an ad libitum control group were included in the study. ABCA1 and ABCG1 protein expression and cholesterol and phospholipid levels were measured in the neocortex of female and male fetuses at GD 21. Body weights were decreased in female fetuses as a consequence of ethanol treatments. ABCA1 and ABCG1 protein levels were increased, and cholesterol levels were decreased, in the neocortex of ethanol-exposed female, but not male, fetuses. Levels of phospholipids were unchanged. Control female fetuses fed ad libitum displayed an up-regulation of ABCA1 and a decrease in cholesterol content compared with pair-fed controls, suggesting that a compensatory up-regulation of cholesterol levels may occur during food restriction. Maternal ethanol consumption may affect fetal brain development by increasing cholesterol transporters' expression and reducing brain cholesterol levels.

Peroxisome proliferator-activated receptor ligands regulate lipid content, metabolism, and composition in fetal lungs of diabetic rats

Maternal diabetes impairs fetal lung development. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors relevant in lipid homeostasis and lung development. This study aims to evaluate the effect of in vivo activation of PPARs on lipid homeostasis in fetal lungs of diabetic rats. To this end, we studied lipid concentrations, expression of lipid metabolizing enzymes and fatty acid composition in fetal lungs of control and diabetic rats i) after injections of the fetuses with Leukotriene B4 (LTB4, PPARα ligand) or 15deoxyΔ(12,14)prostaglandin J2 (15dPGJ2, PPARγ ligand) and ii) fed during pregnancy with 6% olive oil- or 6% safflower oil-supplemented diets, enriched with PPAR ligands were studied. Maternal diabetes increased triglyceride concentrations and decreased expression of lipid-oxidizing enzymes in fetal lungs of diabetic rats, an expression further decreased by LTB4 and partially restored by 15dPGJ2 in lungs of male fetuses in the diabetic group. In lungs of female fetuses in the diabetic group, maternal diets enriched with olive oil increased triglyceride concentrations and fatty acid synthase expression, while those enriched with safflower oil increased triglyceride concentrations and fatty acid transporter expression. Both olive oil- and safflower oil-supplemented diets decreased cholesterol and cholesteryl ester concentrations and increased the expression of the reverse cholesterol transporter ATP-binding cassette A1 in fetal lungs of female fetuses of diabetic rats. In fetal lungs of control and diabetic rats, the proportion of polyunsaturated fatty acids increased with the maternal diets enriched with olive and safflower oils. Our results revealed important changes in lipid metabolism in fetal lungs of diabetic rats, and in the ability of PPAR ligands to modulate the composition of lipid species relevant in the lung during the perinatal period.

ATP Binding Cassette Transporter G1 Deletion Induces IL-17–Dependent Dysregulation of Pulmonary Adaptive Immunity

Mice with genetic deletion of the cholesterol transporter ATP binding cassette G1 (ABCG1) have pulmonary lipidosis and enhanced innate immune responses in the airway. Whether ABCG1 regulates adaptive immune responses to the environment is unknown. To this end, Abcg1(+/+) and Abcg1(-/-) mice were sensitized to OVA via the airway using low-dose LPS as an adjuvant, and then challenged with OVA aerosol. Naive Abcg1(-/-) mice displayed increased B cells, CD4(+) T cells, CD8(+) T cells, and dendritic cells (DCs) in lung and lung-draining mediastinal lymph nodes, with lung CD11b(+) DCs displaying increased CD80 and CD86. Upon allergen sensitization and challenge, the Abcg1(-/-) airway, compared with Abcg1(+/+), displayed reduced Th2 responses (IL-4, IL-5, eosinophils), increased neutrophils and IL-17, but equivalent airway hyperresponsiveness. Reduced Th2 responses were also found using standard i.p. OVA sensitization with aluminum hydroxide adjuvant. Mediastinal lymph nodes from airway-sensitized Abcg1(-/-) mice produced reduced IL-5 upon ex vivo OVA challenge. Abcg1(-/-) CD4(+) T cells displayed normal ex vivo differentiation, whereas Abcg1(-/-) DCs were found paradoxically to promote Th2 polarization. Th17 cells, IL-17(+) γδT cells, and IL-17(+) neutrophils were all increased in Abcg1(-/-) lungs, suggesting Th17 and non-Th17 sources of IL-17 excess. Neutralization of IL-17 prior to challenge normalized eosinophils and reduced neutrophilia in the Abcg1(-/-) airway. We conclude that Abcg1(-/-) mice display IL-17-mediated suppression of eosinophilia and enhancement of neutrophilia in the airway following allergen sensitization and challenge. These findings identify ABCG1 as a novel integrator of cholesterol homeostasis and adaptive immune programs.

Membrane Transport Protein ABCA1 and Type 2 Diabetes Mellitus*

Beta cell dysfunction is a critical step in the pathogenesis of type 2 diabetes mellitus,while cellular cholesterol accumulation is an emerging mechanism for beta cell dysfunction in type 2 diabetes.Absence of the cholesterol transporter ATP-binding cassette transporter A1(ABCA1) results in increased islet cholesterol and impaired insulin secretion,indicating that impaired cholesterol efflux leads to beta cell dysfunction.