Deleterious impact of elaidic fatty acid on ABCA1-mediated cholesterol efflux from mouse and human macrophages

Consumption of trans fatty acids (TFA) increase cardiovascular risk more than do saturated FA, but the mechanisms explaining their atherogenicity are still unclear. We investigated the impact of membrane incorporation of TFA on cholesterol efflux by exposing J774 mouse macrophages or human monocyte-derived macrophages (HMDM) to media enriched or not (standard medium) with industrially produced elaidic (trans-9 18:1) acid, naturally produced vaccenic (trans-11 18:1) acid (34 h, 70 μM) or palmitic acid. In J774 macrophages, elaidic and palmitic acid, but not vaccenic acid, reduced ABCA1-mediated efflux by ~ 23% without affecting aqueous diffusion, SR-BI or ABCG1-mediated pathways, and this effect was maintained in cholesterol-loaded cells. The impact of elaidic acid on the ABCA1 pathway was weaker in cholesterol-normal HMDM, but elaidic acid induced a strong reduction of ABCA1-mediated efflux in cholesterol-loaded cells (− 36%). In J774 cells, the FA supplies had no impact on cellular free cholesterol or cholesteryl ester masses, the abundance of ABCA1 mRNA or the total and plasma membrane ABCA1 protein content. Conversely, TFA or palmitic acid incorporation induced strong modifications of the membrane FA composition with a decrease in the ratio of (cis-monounsaturated FA + polyunsaturated FA):(saturated FA + TFA), with elaidic and vaccenic acids representing each 20% and 13% of the total FA composition, respectively. Moreover, we demonstrated that cellular ATP was required for the effect of elaidic acid, suggesting that it contributes to atherogenesis by impairing ABCA1-mediated cholesterol efflux in macrophages, likely by decreasing the membrane fluidity, which could thereby reduce ATPase activity and the function of the transporter.     

ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann-Pick C2 but not Niemann-Pick C1 protein

Niemann-Pick disease type C (NPC) is caused by mutations leading to loss of function of NPC1 or NPC2 proteins, resulting in accumulation of unesterified cholesterol in late endosomes and lysosomes. We previously reported that expression of the ATP-binding cassette transporter A1 (ABCA1) is impaired in human NPC1^−/− fibroblasts, resulting in reduced HDL particle formation and providing a mechanism for the reduced plasma HDL cholesterol seen in the majority of NPC1 patients. We also found that treatment of NPC1^−/− fibroblasts with an agonist of liver X-receptor corrects ABCA1 expression and HDL formation and reduces lysosomal cholesterol accumulation. We have confirmed that ABCA1 expression is also reduced in NPC2^−/− cells, and found that α-HDL particle formation is impaired in these cells. To determine whether selective up-regulation of ABCA1 can correct lysosomal cholesterol accumulation in NPC disease cells and HDL particle formation, we produced and infected NPC1^−/− and NPC2^−/− fibroblasts with an adenovirus expressing full-length ABCA1 and enhanced green fluorescent protein (AdABCA1-EGFP). ABCA1-EGFP expression in NPC1^−/− fibroblasts resulted in normalization of cholesterol efflux to apolipoprotein A-I (apoA-I) and α-HDL particle formation, plus a marked reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes. In contrast, AdABCA1-EGFP treatment of NPC2^−/− fibroblasts to normalize ABCA1 expression had no effect on cholesterol efflux to apoA-I or accumulation of excess cholesterol in lysosomes, and only partially corrected α-HDL formation by these cells. These results suggest that correction of ABCA1 expression can bypass the mutation of NPC1 but not NPC2 to mobilize excess cholesterol from late endosomes and lysosomes in NPC disease cells. Expression of ABCA1-EGFP in NPC1^−/− cells increased cholesterol available for esterification and reduced levels of HMG-CoA reductase protein, effects that were abrogated by co-incubation with apoA-I. A model can be generated in which ABCA1 is able to mobilize cholesterol, to join the intracellular regulatory pool or to be effluxed for HDL particle formation, either directly or indirectly from the lysosomal membrane, but not from the lysosomal lumen. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Overexpression of stearoyl-coenzyme A desaturase 1 in macrophages promotes reverse cholesterol transport

Stearoyl-coenzyme A desaturase 1 (SCD1) is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids. However, the impact of SCD1 on atherosclerosis remains unclear. The aim of this study was to determine whether SCD1 affects macrophage reverse cholesterol transport (RCT) in mice. Compared to the control, adenoviral-mediated SCD1 overexpression in RAW264.7 macrophages increased cholesterol efflux to HDL, but not to apoA-I, without clear changes in ABCA1, ABCG1 and SR-BI expressions. While knockdown of ABCG1 and SR-BI did not affect the SCD1-induced cholesterol efflux to HDL, SCD1-overexpressing macrophages promoted the formation of both normal- and large-sized HDL in media, accompanying increased apolipoprotein A-I levels in HDL fractions. Transformation to larger particles of HDL was independently confirmed by nuclear magnetic resonance-based lipoprotein analysis. Interestingly, media transfer assays revealed that HDL generated by SCD1 had enhanced cholesterol efflux potential, indicating that SCD1 transformed HDL to a more anti-atherogenic phenotype. To study macrophage RCT in vivo, ³H-cholesterol-labeled RAW264.7 cells overexpressing SCD1 or the control were intraperitoneally injected into mice. Supporting the in vitro data, injection of SCD1-macrophages resulted in significant increases in ³H-tracer in plasma, liver, and feces compared to the control. Moreover, there was a shift towards larger particles in the ³H-tracer distribution of HDL fractions obtained from the mice.     

An ABCA1 truncation shows no dominant negative effect in a familial hypoalphalipoproteinemia pedigree with three ABCA1 mutations

The ATP binding cassette transporter (ABCA1) A1 is a key determinant of circulating high density lipoprotein cholesterol (HDL-C) levels. Mutations in ABCA1 are a major genetic contributor to low HDL-C levels within the general population. Following the finding of three different ABCA1 mutations, p.C978fsX988, p.T1512M and p.N1800H in a subject with hypoalphalipoproteinemia, we aimed to establish whether the p.C978fsX988 truncation exerted a dominant negative effect on the full-length ABCA1 alleles within family members as has been reported for other ABCA1 truncations. Characterisation of the p.C978fsX988 mutant in transfected HEK 293 cells showed it to be expressed as a GFP fusion protein but lacking in cholesterol efflux function. This was in keeping with results from cholesterol efflux assays in the fibroblasts of p.C978fsX988 carriers which also showed impaired efflux. Allele- specific quantification of p.C978fsX988 mRNA and analysis of ABCA1 protein levels in the fibroblasts of p.C978fsX988 heterozygotes showed negligible levels of mRNA and protein expression. There was no evidence of a dominant negative effect on wildtype or p.N1800H protein levels. We conclude that in the case of the p.C978fsX988 truncated mutant a lack of expression precludes it from having a dominant negative effect.     

Elevated COX2 expression and PGE2 production by downregulation of RXRα in senescent macrophages

Increased systemic level of inflammatory cytokines leads to numerous age-related diseases. In senescent macrophages, elevated prostaglandin E2 (PGE2) production contributes to the suppression of T cell function with aging, which increases the susceptibility to infections. However, the regulation of these inflammatory cytokines and PGE2 with aging still remains unclear. We have verified that cyclooxygenase (COX)-2 expression and PGE2 production are higher in LPS-stimulated macrophages from old mice than that from young mice. Downregulation of RXRα, a nuclear receptor that can suppress NF-κB activity, mediates the elevation of COX2 expression and PGE2 production in senescent macrophages. We also have found less induction of ABCA1 and ABCG1 by RXRα agonist in senescent macrophages, which partially accounts for high risk of atherosclerosis in aged population. Systemic treatment with RXRα antagonist HX531 in young mice increases COX2, TNF-α, and IL-6 expression in splenocytes. Our study not only has outlined a mechanism of elevated NF-κB activity and PGE2 production in senescent macrophages, but also provides RXRα as a potential therapeutic target for treating the age-related diseases.     

Adiponectin deficiency suppresses ABCA1 expression and ApoA-I synthesis in the liver

Plasma high density lipoprotein (HDL)-cholesterol levels are inversely correlated with the incidence of cardiovascular diseases. HDL is mainly assembled in the liver through the ATP-binding cassette transporter (ABCA1) pathway. In humans, plasma HDL-cholesterol levels are positively correlated with plasma adiponectin (APN) concentrations. Recently, we reported that APN enhanced apolipoprotein A-I (apoA-I) secretion and ABCA1 expression in HepG2 cells. In the present study, we investigated HDL assembly in APN-knockout (KO) mice. The apoA-I protein levels in plasma and liver were reduced in APN-KO mice compared with wild-type-mice. The ABCA1 expression in liver was also decreased in APN-KO mice. APN deficiency might cause the impaired HDL assembly by decreasing ABCA1 expression and apoA-I synthesis in the liver.     

Cholesterol efflux is differentially regulated in neurons and astrocytes: Implications for brain cholesterol homeostasis

Disruption of cholesterol homeostasis in the central nervous system (CNS) has been associated with neurological, neurodegenerative, and neurodevelopmental disorders. The CNS is a closed system with regard to cholesterol homeostasis, as cholesterol-delivering lipoproteins from the periphery cannot pass the blood–brain-barrier and enter the brain. Different cell types in the brain have different functions in the regulation of cholesterol homeostasis, with astrocytes producing and releasing apolipoprotein E and lipoproteins, and neurons metabolizing cholesterol to 24(S)-hydroxycholesterol. We present evidence that astrocytes and neurons adopt different mechanisms also in regulating cholesterol efflux. We found that in astrocytes cholesterol efflux is induced by both lipid-free apolipoproteins and lipoproteins, while cholesterol removal from neurons is triggered only by lipoproteins. The main pathway by which apolipoproteins induce cholesterol efflux is through ABCA1. By upregulating ABCA1 levels and by inhibiting its activity and silencing its expression, we show that ABCA1 is involved in cholesterol efflux from astrocytes but not from neurons. Furthermore, our results suggest that ABCG1 is involved in cholesterol efflux to apolipoproteins and lipoproteins from astrocytes but not from neurons, while ABCG4, whose expression is much higher in neurons than astrocytes, is involved in cholesterol efflux from neurons but not astrocytes. These results indicate that different mechanisms regulate cholesterol efflux from neurons and astrocytes, reflecting the different roles that these cell types play in brain cholesterol homeostasis. These results are important in understanding cellular targets of therapeutic drugs under development for the treatments of conditions associated with altered cholesterol homeostasis in the CNS.     

Contrasting effects of arachidonic acid and docosahexaenoic acid membrane incorporation into cardiomyocytes on free cholesterol turnover

The preservation of a constant pool of free cholesterol (FC) is critical to ensure several functions of cardiomyocytes. We investigated the impact of the membrane incorporation of arachidonic acid (C20:4 ω6, AA) or docosahexaenoic acid (C22:6 ω3, DHA) as ω6 or ω3 polyunsaturated fatty acids (PUFAs) on cholesterol homeostasis in primary cultures of neonatal rat cardiac myocytes. We measured significant alterations to the phospholipid FA profiles, which had markedly different ω6/ω3 ratios between the AA and DHA cells (13 vs. 1). The AA cells showed a 2.7-fold lower cholesterol biosynthesis than the DHA cells. Overall, the AA cells showed 2-fold lower FC masses and 2-fold higher cholesteryl ester masses than the DHA cells. The AA cells had a lower FC to phospholipid ratio and higher triglyceride levels than the DHA cells. Moreover, the AA cells showed a 40% decrease in ATP binding cassette transporter A1 (ABCA1)-mediated and a 19% decrease in ABCG1-mediated cholesterol efflux than the DHA cells. The differences in cholesterol efflux pathways induced by AA or DHA incorporation were not caused by variations in ABCs transporter expression and were reduced when ABC transporters were overexpressed by exposure to LXR/RXR agonists. These results show that AA incorporation into cardiomyocyte membranes decreased the FC turnover by markedly decreasing the endogenous cholesterol synthesis and by decreasing the ABCA1- and ABCG1-cholesterol efflux pathways, whereas DHA had the opposite effects. We propose that these observations may partially contribute to the beneficial effects on the heart of a diet containing a high ω3/ω6 PUFA ratio.     

Adiponectin prevents atherosclerosis by increasing cholesterol efflux from macrophages

Plasma high density lipoprotein (HDL)-cholesterol levels are inversely correlated to the risk of atherosclerotic cardiovascular diseases. Reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which HDL particles play a crucial role to carry cholesterol derived from peripheral tissues to the liver. Recently, ATP-binding cassette transporters (ABCA1, ABCG1) and scavenger receptor (SR-BI) have been identified as important membrane receptors to generate HDL by removing cholesterol from foam cells. Adiponectin (APN) 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, although its mechanism has not been clarified. Therefore, in the present study, we investigated the role of APN on RCT, in particular, cellular cholesterol efflux from human monocyte-derived and APN-knockout (APN-KO) mice macrophages. APN up-regulated the expression of ABCA1 in human macrophages, respectively. ApoA-1-mediated cholesterol efflux from macrophages was also increased by APN treatment. Furthermore, the mRNA expression of LXRα and PPARγ was increased by APN. In APN-KO mice, the expression of ABCA1, LXRα, PPARγ, and apoA-I-mediated cholesterol efflux was decreased compared with wild-type mice. In summary, APN might protect against atherosclerosis by increasing apoA-I-mediated cholesterol efflux from macrophages through ABCA1-dependent pathway by the activation of LXRα and PPARγ.     

Riccardin C: a natural product that functions as a liver X receptor (LXR)alpha agonist and an LXRbeta antagonist

Liver X receptors (LXRs) alpha and beta share considerable sequence homology and several functions, respond to the same endogenous and synthetic ligands, and play critical roles in maintaining lipid homeostasis. In this study, liverwort-derived riccardin C (RC) and F (RF) were identified as an LXRalpha agonist/LXRbeta antagonist and an LXRalpha antagonist, respectively. RC and RF bound to LXRs, but had different abilities to recruit a coactivator and thereby induce transactivation. Despite its unique subtype-selective activity, RC enhanced ABCA1 and ABCG1 expression and cellular cholesterol efflux in THP-1 cells. RC may provide a novel tool for identifying subtype-function and drug development.     

ABCG1 mediated oxidized LDL-derived oxysterol efflux from macrophages

Objectives

The uptake of oxidized LDL (oxLDL) by macrophages is a key initial event in atherogenesis, and the removal of oxidized lipids from artery wall via reverse cholesterol transport is considered antiatherogenic. The aims of this study were to investigate the pathways mediating the removal of oxysterols from oxLDL-loaded macrophages, and the subsequent uptake of the oxysterols by hepatocytes.

Methods

LDL was labeled with [3H]cholesterol, and LDL-[3H]cholesterol was oxidized by copper using a standard method. [3H]oxysterol formation in oxLDL was analyzed by thin layer chromatography. oxLDL-[3H]sterol was incubated with macrophages, allowing the uptake of [3H]sterol by macrophages. [3H]sterol efflux from macrophages mediated by ATP binding cassette transporters (ABCA1, ABCG1), or scavenger receptor class B type I (SR-BI) was measured. The subsequent uptake of the [3H]sterol by hepatocytes was also determined.

Results

7-Ketocholesterol was the major oxysterol formed in oxLDL, and it was significantly higher in oxLDL compared with that in native LDL (naLDL). oxLDL-derived sterol efflux to HDL from macrophages was significantly increased compared with naLDL-derived sterol, and it was mainly mediated by ABCG1, but not by ABCA1 or SR-BI. Moreover, although HDL dose-dependently induced sterol efflux from macrophages, only the exported sterol by ABCG1 pathway was efficiently taken up by hepatocytes.

Conclusions

ABCG1 mediates oxysterol efflux from oxLDL-loaded macrophages, and the exported oxysterol by ABCG1 pathway can be selectively taken up by hepatocytes.     

Placental ABCA1 and ABCG1 transporters efflux cholesterol and protect trophoblasts from oxysterol induced toxicity

ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 mediate the efflux of cholesterol and other sterols. Both transporters are expressed on the fetal capillaries of the placenta and are involved in maternal-to-fetal cholesterol delivery. In this study, we report that ABCA1 and ABCG1 are also present on the syncytiotrophoblast, the maternal facing placental membrane. Syncytial ABCA1 expression is apical, suggesting a role in cholesterol efflux to the mother, while ABCG1 is expressed basolaterally indicating transport to the fetus. Silencing of ABCA1 expression in primary trophoblasts in culture, or pharmacological antagonism by glyburide, decreased cholesterol efflux to apolipoprotein A-I (apoA-I) compared to controls, while ABCG1-silencing decreased cholesterol efflux to high density lipoproteins (HDL). In contrast, treatment with endogenous or synthetic LXR α/β ligands such as T0901317 increased ABCA1 and ABCG1 expression and enhanced cholesterol efflux to apoA-I and HDL, respectively, while treatment with pharmacological PPAR-α or -γ ligands was without effect. Trophoblasts transfected with ABCA1 or ABCG1 siRNA were more sensitive to toxic oxysterols substrates (25-hydroxycholesterol and 7-ketocholesterol) compared to mock-transfected cells, while prior treatment with T0901317 reduced oxysterol-mediated toxicity. These results identify syncytial ABCA1 and ABCG1 as important, inducible cholesterol transporters which also prevent placental accumulation of cytotoxic oxysterols.     

Retinoids synergized with insulin to induce Srebp-1c expression and activated its promoter via the two liver X receptor binding sites that mediate insulin action

We have reported that the rat liver lipophilic extract (LE) synergized with insulin to induce Gck and Srebp-1c in primary rat hepatocytes. After identification of retinol and retinal in LE, only their effects in the absence or presence of insulin on Gck, but not that on Srebp-1c, were investigated subsequently. The retinoid effects on the Srebp-1c expression and the activation of its promoter were examined with real-time PCR and reporter gene assays, respectively. In primary hepatocytes, retinal and retinoic acid (RA) synergized with insulin to induce Srebp-1c expression. This induction was followed by the elevation of its target gene, fatty acid synthase. Activation of retinoid X receptor, but not retinoic acid receptor, was responsible for the induction of Srebp-1c expression. RA, but not retinal, also induced Srebp-1c expression in a dose dependent manner in INS-1 cells. The RA responsive elements in Srebp-1c promoter were determined as previously identified two liver X receptor elements responsible for mediating insulin action. We conclude that retinoids regulate hepatic Srebp-1c expression through activation of retinoid X receptor. The RA- and insulin-induced Srebp-1c expression converged at the same sites in its promoter, indicating the roles of vitamin A in regulation of hepatic gene expression.     

Site-specific oxidation of apolipoprotein A-I impairs cholesterol export by ABCA1, a key cardioprotective function of HDL

The mechanisms that deprive HDL of its cardioprotective properties are poorly understood. One potential pathway involves oxidative damage of HDL proteins by myeloperoxidase (MPO) a heme enzyme secreted by human artery wall macrophages. Mass spectrometric analysis demonstrated that levels of 3-chlorotyrosine and 3-nitrotyrosine - two characteristic products of MPO - are elevated in HDL isolated from patients with established cardiovascular disease. When apolipoprotein A-I (apoA-I), the major HDL protein, is oxidized by MPO, its ability to promote cellular cholesterol efflux by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway is diminished. Biochemical studies revealed that oxidation of specific tyrosine and methionine residues in apoA-I contributes to this loss of ABCA1 activity. Another potential mechanism for generating dysfunctional HDL involves covalent modification of apoA-I by reactive carbonyls, which have been implicated in atherogenesis and diabetic vascular disease. Indeed, modification of apoA-I by malondialdehyde (MDA) or acrolein also markedly impaired the lipoprotein's ability to promote cellular cholesterol efflux by the ABCA1 pathway. Tandem mass spectrometric analyses revealed that these reactive carbonyls target specific Lys residues in the C-terminus of apoA-I. Importantly, immunochemical analyses showed that levels of MDA-protein adducts are elevated in HDL isolated from human atherosclerotic lesions. Also, apoA-I co-localized with acrolein adducts in such lesions. Thus, lipid peroxidation products might specifically modify HDL in vivo. Our observations support the hypotheses that MPO and reactive carbonyls might generate dysfunctional HDL in humans. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Cholesterol accumulation in prostate cancer: A classic observation from a modern perspective

Prostate cancer (PCa) is the most common cancer in men in developed countries. Epidemiological studies have associated high blood-cholesterol levels with an increased risk of PCa, whilst cholesterol-lowering drugs (statins) reduce the risk of advanced PCa. Furthermore, normal prostate epithelial cells have an abnormally high cholesterol content, with cholesterol levels increasing further during progression to PCa. In this review, we explore why and how this occurs.