ABCA1 Protein Expression Research Articles

Butyrate Lowers Cellular Cholesterol through HDAC Inhibition and Impaired SREBP-2 Signalling

In animal studies, HDAC inhibitors such as butyrate have been reported to reduce plasma cholesterol, while conferring protection from diabetes, but studies on the underlying mechanisms are lacking. This study compares the influence of butyrate and other HDAC inhibitors to that of statins on cholesterol metabolism in multiple cell lines, but primarily in HepG2 hepatic cells due to the importance of the liver in cholesterol metabolism. Sodium butyrate reduced HepG2 cholesterol content, as did sodium valproate and the potent HDAC inhibitor trichostatin A, suggesting HDAC inhibition as the exacting mechanism. In contrast to statins, which increase SREBP-2 regulated processes, HDAC inhibition downregulated SREBP-2 targets such as HMGCR and the LDL receptor. Moreover, in contrast to statin treatment, butyrate did not increase cholesterol uptake by HepG2 cells, consistent with its failure to increase LDL receptor expression. Sodium butyrate also reduced ABCA1 and SRB1 protein expression in HepG2 cells, but these effects were not consistent across all cell types. Overall, the underlying mechanism of cell cholesterol lowering by sodium butyrate and HDAC inhibition is consistent with impaired SREBP-2 signalling, and calls into question the possible use of butyrate for lowering of serum LDL cholesterol in humans.

Low-molecular-weight fucoidan bidirectionally regulates lipid uptake and cholesterol efflux through the p38 MAPK phosphorylation

Atherosclerosis (AS) is the pathological basis of many cardiovascular and cerebrovascular diseases, in which macrophage-derived foam cells are the critical step and a typical pathological feature of early atherosclerosis. We previously confirmed that low-molecular-weight fucoidan (LMWF) had a good anti-AS effect, but the mechanism is still unclear. Here with aim to investigate the inhibitory effect of LMWF on foam cells and its molecular mechanism. Oil red O staining showed that LMWF effectively alleviated lipid accumulation and the formation of foam cells. Flow cytometry detection showed that LMWF promoted foam cells apoptosis. In addition, immunofluorescence showed that LMWF inhibited macrophage scavenger receptor A1 (SR-A1)-mediated lipid uptake and promoted ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol outflow. Western blot showed that LMWF downregulated SR-A1 protein expression and upregulated ABCA1 protein expression by inhibiting p38 mitogen activated protein kinase (p38MAPK) phosphorylation. Moreover, the mRNA transcriptions of Stat1, Elk-1, and Myc were downregulated when treated with LMWF. It concluded that, LMWF achieved bidirectional regulation of SR-A1 and ABCA1, then prevented the formation of foam cells, finally ameliorated the development of AS.

Utilizing the LoxP-Stop-LoxP System to Control Transgenic ABC-Transporter Expression In Vitro.

ABCA1 and ABCG1 are two ABC-transporters well-recognized to promote the efflux of cholesterol to apoAI and HDL, respectively. As these two ABC-transporters are critical to cholesterol metabolism, several studies have assessed the impact of ABCA1 and ABCG1 expression on cellular cholesterol homeostasis through ABC-transporter ablation or overexpressing ABCA1/ABCG1. However, for the latter, there are currently no well-established in vitro models to effectively induce long-term ABC-transporter expression in a variety of cultured cells. Therefore, we performed proof-of-principle in vitro studies to determine whether a LoxP-Stop-LoxP (LSL) system would provide Cre-inducible ABC-transporter expression. In our studies, we transfected HEK293 cells and the HEK293-derived cell line 293-Cre cells with ABCA1-LSL and ABCG1-LSL-based plasmids. Our results showed that while the ABCA1/ABCG1 protein expression was absent in the transfected HEK293 cells, the ABCA1 and ABCG1 protein expression was detected in the 293-Cre cells transfected with ABCA1-LSL and ABCG1-LSL, respectively. When we measured cholesterol efflux in transfected 293-Cre cells, we observed an enhanced apoAI-mediated cholesterol efflux in 293-Cre cells overexpressing ABCA1, and an HDL2-mediated cholesterol efflux in 293-Cre cells constitutively expressing ABCG1. We also observed an appreciable increase in HDL3-mediated cholesterol efflux in ABCA1-overexpressing 293-Cre cells, which suggests that ABCA1 is capable of effluxing cholesterol to small HDL particles. Our proof-of-concept experiments demonstrate that the LSL-system can be used to effectively regulate ABC-transporter expression in vitro, which, in turn, allows ABCA1/ABCG1-overexpression to be extensively studied at the cellular level.

Expression patterns of genes in steroidogenic, cholesterol uptake, and liver x receptor-mediated cholesterol efflux pathway regulating cholesterol homeostasis in natural and PGF2α induced luteolysis as well as early pregnancy in ovine corpus luteum

The aim was to evaluate the expression of genes of steroidogenic, cholesterol uptake, and liver X receptor (LXR) mediated cholesterol efflux pathway in ovine corpus luteum (CL) during natural and prostaglandin F2α (PGF2α) induced luteolysis and early pregnancy. For this study, two experiments were carried out 1); ewes were grouped into two sub-groups as cyclic 12 (C12, n = 4) and 16 (C16, n = 4) and pregnant 12 (P12, n = 4), 16 (P16, n = 4), and 22 (P22, n = 4). Additionally, 2) ewes were grouped into four groups following treatment of PGF2α, the duration of PGF2α challenge at 1 (PG1, n = 4), 4 (PG4, n = 4), and 16 (PG16, n = 4) hours on day 12 of the cycle was compared with 0 h. The corpus luteum tissue samples were collected on the corresponding estrus cycle and pregnancy days, and RNA was extracted using Trizol. mRNA expression levels of the steroidogenic (StAR, CYP11A1, and HSD3B1) and cholesterol uptake receptors (SCARB1 and LDLR) and LXR pathway (NR1H3, NR1H2, ABCA1, and ABCG1) were assessed using quantitative PCR (qPCR), and protein of LXR pathway was investigated using western blot. In-situ hybridization was used to detect mRNA localization. Steroidogenic and cholesterol uptake mRNAs were decreased in C16, while NR1H2 and ABCG1 were increased in C16, compared to C12. Steroidogenic and cholesterol uptake mRNA was greater in P16 than in C16. NR1H2 and ABCA1 protein expression were higher in P16 than in C16. LDLR mRNA was higher in P22 than in P12, while SCARB1 was higher in P16 than in P12. NR1H2 mRNA was greater in P22 than in P12. Steroidogenic and cholesterol uptake mRNA were decreased in PGF2α-induced luteolysis groups against C12. ABCG1 mRNA was higher in PG16 than in PG4 and PG1. The reduction of lipoprotein receptors rather than LXR-mediated reverse transport may contribute to the decline in progesterone (P4) in natural and functional luteolysis.

Caprylic Acid Improves Lipid Metabolism, Suppresses the Inflammatory Response and Activates the ABCA1/p-JAK2/p-STAT3 Signaling Pathway in C57BL/6J Mice and RAW264.7 Cells

ObjectiveThis study aimed to investigate the effects of caprylic acid (C8:0) on lipid metabolism and inflammation, and examine the mechanisms underlying these effects in mice and cells. MethodsFifty-six 6-week-old male C57BL/6J mice were randomly allocated to four groups fed a high-fat diet (HFD) without or with 2% C8:0, palmitic acid (C16:0) or eicosapentaenoic acid (EPA). RAW246.7 cells were randomly divided into five groups: normal, lipopolysaccharide (LPS), LPS+C8:0, LPS+EPA and LPS+cAMP. The serum lipid profiles, inflammatory biomolecules, and ABCA1 and JAK2/STAT3 mRNA and protein expression were measured. ResultsC8:0 decreased TC and LDL-C, and increased the HDL-C/LDL-C ratio after injection of LPS. Without LPS, it decreased TC in mice (P < 0.05). Moreover, C8:0 decreased the inflammatory response after LPS treatment in both mice and cells (P < 0.05). Mechanistic investigations in C57BL/6J mouse aortas after injection of LPS indicated that C8:0 resulted in higher ABCA1 and JAK2/STAT3 expression than that with HFD, C16:0 and EPA, and resulted in lower TNF-α, NF-κB mRNA expression than that with HFD (P < 0.05). In RAW 264.7 cells, C8:0 resulted in lower expression of pNF-κBP65 than that in the LPS group, and higher protein expression of ABCA1, p-JAK2 and p-STAT3 than that in the LPS and LPS+cAMP groups (P < 0.05). ConclusionOur studies demonstrated that C8:0 may play an important role in lipid metabolism and the inflammatory response, and the mechanism may be associated with ABCA1 and the p-JAK2/p-STAT3 signaling pathway.

Acid-Sensing Ion Channel 1/Calpain1 Activation Impedes Macrophage ATP-Binding Cassette Protein A1-Mediated Cholesterol Efflux Induced by Extracellular Acidification.

BackgroundExtracellular acidification is a common feature of atherosclerotic lesions, and such an acidic microenvironment impedes ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux and promotes atherogenesis. However, the underlying mechanism is still unclear. Acid-sensing ion channel 1 (ASIC1) is a critical H+ receptor, which is responsible for the perception and transduction of extracellular acidification signals.AimIn this study, we explored whether or how ASIC1 influences extracellular acidification-induced ABCA1-mediated cholesterol efflux from macrophage-derived foam cells.MethodsRAW 264.7 macrophages were cultured in an acidic medium (pH 6.5) to generate foam cells. Then the intracellular lipid deposition, cholesterol efflux, and ASIC1/calpain1/ABCA1 expressions were evaluated.ResultsWe showed that extracellular acidification enhanced ASIC1 expression and translocation, promoted calpain1 expression and lipid accumulation, and decreased ABCA1 protein expression as well as ABCA1-mediated cholesterol efflux. Of note, inhibiting ASIC1 activation with amiloride or Psalmotoxin 1 (PcTx-1) not only lowered calpain1 protein level and lipid accumulation but also enhanced ABCA1 protein levels and ABCA1-mediated cholesterol efflux of macrophages under extracellular acidification conditions. Furthermore, similar results were observed in macrophages treated with calpain1 inhibitor PD150606.ConclusionExtracellular acidification declines cholesterol efflux via activating ASIC1 to promote calpain1-mediated ABCA1 degradation. Thus, ASIC1 may be a novel therapeutic target for atherosclerosis.

AICAR upregulates ABCA1/ABCG1 expression in the retinal pigment epithelium and reduces Bruch's membrane lipid deposit in ApoE deficient mice

The etiology of age-related macular degeneration (AMD) is diverse; however, recent evidence suggests that the lipid metabolism-cholesterol pathway might be associated with the pathophysiology of AMD. The ATP-binding cassette (ABC) transporters, ABCA1 and ABCG1, are essential for the formation of high-density lipoprotein (HDL) and the regulation of macrophage cholesterol efflux. The failure of retinal or retinal pigment epithelium (RPE) cholesterol efflux to remove excess intracellular lipids causes morphological and functional damage to the retina. In this study, we investigated whether treatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMP-activated protein kinase (AMPK) activator, improves RPE cholesterol efflux and Bruch's membrane (BM) lipid deposits. The protein and mRNA levels of ABCA1 and ABCG1 in ARPE-19 cells and retinal and RPE/choroid tissue from apolipoprotein E-deficient (ApoE-/-) mice were evaluated after 24 weeks of AICAR treatment. The cholesterol efflux capacity of ARPE-19 cells and the cholesterol-accepting capacity of apoB-depleted serum from mice were measured. The thickness of the BM and the degree of lipid deposition were evaluated using electron microscopy. AICAR treatment increased the phosphorylation of AMPK and the protein and mRNA expression of ABCA1 and ABCG1 in vitro. It promoted cholesterol efflux from ARPE-19 cells and upregulated the protein and mRNA levels of ABCA1 and ABCG1 in the retina and RPE in vivo. ApoB-depleted serum from the AICAR-treated group showed enhanced cholesterol-accepting capacity. Long-term treatment with AICAR reduced BM thickening and lipid deposition in ApoE-/- mice. In conclusion, AICAR treatment increased the expression of lipid transporters in the retina and RPE in vivo, facilitated intracellular cholesterol efflux from the RPE in vitro, and improved the functionality of HDL to accept cholesterol effluxed from the cell, possibly via AMPK activation. Collectively, these effects might contribute to the improvement of early age-related pathologic changes in the BM. Pharmacological improvement of RPE cholesterol efflux via AMPK activation may be a potential treatment strategy for AMD.

Amyloid beta increases ABCA1 and HMGCR protein expression, and cholesterol synthesis and accumulation in mice neurons and astrocytes

IntroductionImbalanced cholesterol metabolism in the brain is one of the main pathophysiological mechanisms involved in Alzheimer's disease. We investigated the effect of amyloid-beta (Aβ) on the main proteins involved in regulation of cholesterol metabolism along with cholesterol content in astrocytes and neurons. MethodsAstrocytes and neurons were cultured and treated with Aβ. Apolipoprotein E (apoE) level in the cells and conditioned media, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), ATP-binding cassette transporter A1 (ABCA1), and cytochrome P450 46A1 (CYP46A1) in cell lysates were determined using immunoblotting. Astrocyte media was added to the Aβ-pretreated neurons then, HMGCR was assessed. Cholesterol was measured in both cells and media. ResultsAβ caused a significant increase in HMGCR and ABCA1 protein levels and cholesterol content in both cells without increasing cholesterol efflux. A similar increase was seen for cellular apoE level in astrocytes with no changes in media with a significant reduction of cholesterol efflux. HMGCR level was restored to near control level when Aβ-pretreated neurons were exposed to media from culture astrocytes. ConclusionAlmost all events related to cholesterol homeostasis in neurons and astrocytes, are somehow affected by Aβ. However, because ABCA1 has the most important role(s) in brain cholesterol homeostasis, all subsequent events associated with astrocytes-cholesterol synthesis and its shuttling to neurons are influenced by the effects of Aβ on ABCA1 which could likely be responsible for altered brain cholesterol metabolism in Alzheimer's disease.

Combined LXR and RXR Agonist Therapy Increases ABCA1 Protein Expression and Enhances ApoAI-Mediated Cholesterol Efflux in Cultured Endothelial Cells.

Endothelial ABCA1 expression protects against atherosclerosis and this atheroprotective effect is partially attributed to enhancing apoAI-mediated cholesterol efflux. ABCA1 is a target gene for LXR and RXR; therefore, treating endothelial cells with LXR and/or RXR agonists may increase ABCA1 expression. We tested whether treating cultured immortalized mouse aortic endothelial cells (iMAEC) with the endogenous LXR agonist 22(R)-hydroxycholesterol, synthetic LXR agonist GW3965, endogenous RXR agonist 9-cis-retinoic acid, or synthetic RXR agonist SR11237 increases ABCA1 protein expression. We observed a significant increase in ABCA1 protein expression in iMAEC treated with either GW3965 or SR11237 alone, but no significant increase in ABCA1 protein was observed in iMAEC treated with either 22(R)-hydroxycholesterol or 9-cis-retionic acid alone. However, we observed significant increases in both ABCA1 protein expression and apoAI-mediated cholesterol efflux when iMAEC were treated with a combination of either 22(R)-hydroxycholesterol and 9-cis-retinoic acid or GW3965 and SR11237. Furthermore, treating iMAEC with either 22(R)-hydroxycholesterol and 9-cis-retinoic acid or GW3965 and SR11237 did not trigger an inflammatory response, based on VCAM-1, ICAM-1, CCL2, and IL-6 mRNA expression. Based on our findings, delivering LXR and RXR agonists precisely to endothelial cells may be a promising atheroprotective approach.

The Susceptibility to Diet-Induced Atherosclerosis Is Exacerbated with Aging in C57B1/6 Mice.

The anti-atherogenic activity of HDL is mainly due to their capacity to mediate reverse cholesterol transport (RCT). However, it is not clear to what extent this activity is affected by aging or pro-atherogenic conditions. Three and 24-month-old C57Bl/6 mice were fed an atherogenic diet (high fat, high cholesterol) for 12 weeks. The aged mice displayed a significant reduction in the capacity of HDL to mediate RCT (29.03%, p < 0.0006). Interestingly, the atherogenic diet significantly stimulated the RCT process in both young and aged mice (241% and 201%, respectively, p < 0.01). However, despite this, significant amounts of cholesterol accumulated in the aortas of mice fed an atherogenic diet as compared to regular chow. The accumulation of cholesterol was more marked in the aortas of aged mice (110% increase, p < 0.002). ABCA1 and ABCG1 protein expression on macrophages decreased significantly (52 to 37% reduction, p < 0.002), whereas their expression on hepatic cells increased significantly (up to 590% for ABCA1 and 116% for ABCG1, p < 0.002). On the other hand, SR-BI protein expression on hepatic cells decreased significantly (42.85%, p < 0.0001). ABCG5, ABCG8, and CYP7a protein expression on hepatic cells was also higher in mice fed an atherogenic diet. The increase was age-dependent for both ABCG5 and ABCG8. Our results suggest that the susceptibility to diet-induced atherosclerosis is exacerbated with aging and is a consequence of the dysregulation of the expression levels of membrane cholesterol transporters.

Oleacein may intensify the efflux of oxLDL from human macrophages by increasing the expression of the SRB1 receptor, as well as ABCA1 and ABCG1 transporters

Abstract Oleacein is a polyphenol and can be found in unfiltered extra virgin olive oil and leaves Ligustrum vulgare L. (Oleaceae). The aim of the present study was to investigate whether oleacein could increase efflux cholesterol from macrophages. Oleacein in dose-dependent manner (OLEA20, OLEA50) significantly reduced lipid deposits in macrophages. The increase of SRB1 expression was found for OLEA50 and OLEA20 compared to oxLDL-induced macrophages. Oleacein significantly up-regulated protein expression of ABCA1 and ABCG1 transporter. Moreover, oleacein increased of HO-1 intracellular secretion, as well as increased activation of Nrf2. Whereas, the level of NF-κB protein was almost completely inhibited by oleacein at the same concentrations. For OLEA10, no significant changes were observed for the parameters tested. Our study demonstrated that oleacein may reduce foam cell formation. Therefore, we propose a novel explanation for the oleacein, that can be used to develop new therapeutic strategies for the treatment of atherosclerosis.

Testosterone stimulates cholesterol clearance from human macrophages by activating LXRα

AimsLow testosterone in men is associated with increased cardiovascular events and mortality. Testosterone has beneficial effects on several cardiovascular risk factors including cholesterol, endothelial dysfunction and inflammation as key mediators of atherosclerosis. Although evidence suggests testosterone is anti-atherogenic, its mechanism of action is unknown. The present study investigates whether testosterone exerts anti-atherogenic effects by stimulating cholesterol clearance from macrophages via activation of liver X receptor (LXRα), a nuclear master regulator of cellular cholesterol homeostasis, lipid regulation, and inflammation. Main methodsUsing human monocyte THP-1 cells differentiated into macrophages, the effect of testosterone (1–10 nM) treatment (24–72 h) on the expression of LXRα and LXR- targets apolipoprotein E (APOE), ATP-binding cassette transporter A1 (ABCA1), sterol regulatory element-binding transcription factor 1 (SREBF1) and fatty acid synthase (FAS), was investigated via qPCR and western blotting, with or without androgen receptor blockade with flutamide or LXR antagonism with CPPSS-50. Cholesterol clearance was measured by monitoring fluorescent dehydroergosterol (DHE) cellular clearance and ABCA1 cellular translocation was observed via immunocytochemistry in testosterone treated macrophages. Key findingsTestosterone increased mRNA and protein expression of LXRα, APOE, ABCA1, SREBF1 and FAS. These effects were blocked by flutamide and independently by LXR antagonism with CPPSS-50. Furthermore testosterone stimulated cholesterol clearance from the macrophages and promoted the translocation of ABCA1 toward the cell membrane. SignificanceTestosterone acts via androgen receptor-dependent pathways to stimulate LXRα and downstream targets to induce cholesterol clearance in human macrophages. This may, in part, explain the anti-atherogenic effects of testosterone frequently seen clinically.

ABCA1 is associated with the development of acquired chemotherapy resistance and predicts poor ovarian cancer outcome.

Aim: This study investigated the ATP binding cassette (ABC) transporter (ABCA1, ABCB1, ABCB3, ABCC2 and ABCG2) expression in high grade serous ovarian cancer (HGSOC) tissues, cell lines and primary cells to determine their potential relationship with acquired chemotherapy resistance and patient outcome.Methods: ABC transporter mRNA and protein expression (ABCA1, ABCB1, ABCB3, ABCC2 and ABCG2) was assessed in publicly available datasets and in a tissue microarray (TMA) cohort of HGSOC at diagnosis, respectively. ABC transporter mRNA expression was also assessed in chemosensitive ovarian cancer cell lines (OVCAR-5 and CaOV3) versus matching cell lines with acquired carboplatin resistance and in primary HGSOC cells from patients with chemosensitive disease at diagnosis (n = 10) as well as patients with acquired chemotherapy resistance at relapse (n = 6). The effects of the ABCA1 inhibitor apabetalone in carboplatin-sensitive and -resistant cell lines were also investigated.Results: High ABCA1 mRNA and protein expression was found to be significantly associated with poor patient outcome. ABCA1 mRNA and protein levels were significantly increased in ovarian cancer cell lines (OVCAR-5 CBPR and CaOV3 CBPR) with acquired carboplatin resistance. ABCA1 mRNA was significantly increased in primary HGSOC cells obtained from patients with acquired chemotherapy resistance. Apabetalone treatment reduced ABCA1 protein expression and increased the sensitivity of both parental and carboplatin-resistant ovarian cancer cells to carboplatin.Conclusion: These results suggest that inhibiting ABCA1 transporter may be useful in overcoming acquired chemotherapy resistance and improving outcome for patients with HGSOC.

Increased protein expression of ABCA1, HMG-CoA reductase, and CYP46A1 induced by garlic and allicin in the brain mouse and astrocytes-isolated from C57BL/6J.

Objective:Regulation of cholesterol level is essential for the brain optimal function. The beneficial effect of garlic consumption on cholesterol homeostasis is well known; however, the molecular mechanism to support its properties is unclear. Here, we investigated the beneficial effect of aqueous extract of garlic and allicin on lipid profile and the main players involved in brain cholesterol homeostasis including ABCA1, HMG-CoA reductase, and CYP46A1 in both C57BL/6J mice brain and astrocytes. Materials and Methods:Thirty mice were divided into control and garlic groups. Garlic group was fed with the aqueous extract of garlic. Serum lipids were measured and brain protein levels of ABCA1, HMGCR, and CYP46A1 were determined by western blotting. Changes in these proteins expression were also studied in the presence of allicin in cultured astrocytes. Results:A moderate decrease in serum total cholesterol and a significant increase in plasma HDL-C levels (p<0.05) were detected. A significant increase in ABCA1, HMGCR, and CYP46A1 protein levels was observed in the garlic group and in the cultured astrocytes treated with allicin by western blotting (p<0.05). Conclusion:Our findings indicated that the main players involved in cholesterol turnover including HMGCR that is involved in cholesterol synthesis, ABCA1 that is important in cholesterol efflux, and CYP46A1 that is necessary in cholesterol degradation, were up regulated by garlic/allicin in both animal and cell culture model. We concluded that increasing cholesterol turnover is a possible mechanism for the beneficial effects of garlic in cholesterol homeostasis.

Celastrol induces lipophagy via the LXRα/ABCA1 pathway in clear cell renal cell carcinoma.

Celastrol is a triterpene derived from the traditional Chinese medicine Tripterygium wilfordii Hook f, which displays potential anticancer activity. In the present study, we investigated the anticancer effects of celastrol against clear cell renal cell carcinoma (ccRCC) and the underlying mechanisms. Using Cancer Genome Atlas (TCGA) database and genotype-tissue expression (GTEx) database we conducted a bioinformatics analysis, which showed that the mRNA levels of liver-X receptors α (LXRα) and ATP-binding cassette transporter A1 (ABCA1) in ccRCC tissues were significantly lower than those in adjacent normal tissues. This result was confirmed by immunoblotting analysis of 4 ccRCC clinical specimens, which showed that the protein expression of LXRα and ABCA1 was downregulated. Similar results were obtained in a panel of ccRCC cell lines (786-O, A498, SN12C, and OS-RC-2). In 786-O and SN12C cells, treatment with celastrol (0.25-2.0 μM) concentration-dependently inhibited the cell proliferation, migration, and invasion as well as the epithelial-mesenchymal transition (EMT) process. Furthermore, we demonstrated that celastrol inhibited the invasion of 786-O cells through reducing lipid accumulation; celastrol concentration-dependently promoted autophagy to reduce lipid storage. Moreover, we revealed that celastrol dramatically activated LXRα signaling, and degraded lipid droplets by inducing lipophagy in 786-O cells. Finally, celastrol promoted cholesterol efflux from 786-O cells via ABCA1. In high-fat diet-promoted ccRCC cell line 786-O xenograft model, administration of celastrol (0.25, 0.5, 1.0 mg·kg-1·d-1, for 4 weeks, i.p.) dose-dependently inhibited the tumor growth with upregulated LXRα and ABCA1 protein in tumor tissue. In conclusion, this study reveals that celastrol triggers lipophagy in ccRCC by activating LXRα, promotes ABCA1-mediated cholesterol efflux, suppresses EMT progress, and ultimately inhibits cell proliferation, migration, and invasion as well as tumor growth. Thus, our study provides evidence that celastrol can be used as a lipid metabolism-based anticancer therapeutic approach.

Adiponectin's mechanisms in high-density lipoprotein biogenesis and cholesterol efflux

AimAmong adiponectin's beneficial properties is its ability to promote cellular cholesterol efflux, thereby generating high-density lipoprotein (HDL) particles. However, adiponectin's role in the regulation of macrophage lipid metabolism, a crucial process in atherogenesis, remains poorly investigated. The aim of this study was to characterize the adiponectin's role in HDL biogenesis. Methods and resultsWe perform kinetics studies in baby hamster kidney (BHK) and Tamm-Horsfall protein 1 (THP-1) cell lines to elucidate adiponectin's role in HDL biogenesis. In cholesterol-enriched cells, specific molar doses of adiponectin stimulated cholesterol efflux with high efficiency to apoA-I. In the presence of adiponectin, BHK cells expressing ATP binding cassette transporter A1 (ABCA1) or ABCG1 generated lipidated particles having α electrophoretic mobility (α-HDL) and a molecular size of 7.5–20 nm. Interestingly, in THP-1 macrophages, cholesterol efflux was associated with more lipidated preβ1-HDL particles. Direct molecular interaction of adiponectin with apoA-I enhanced the affinity of apoA-I to free cholesterol and resulted in an increase in preβ1-HDL particles from plasma ex vivo. Adiponectin increased ABCA1 and ABCG1 protein expression and activated the formation of ABCA1-linked cholesterol oxidase sensitive plasma membrane domains. ConclusionAdiponectin upregulated ABCA1 and ABCG1 protein expression, reduced lipid accumulation, and efficiently promoted nascent HDL formation. These results highlight that these cellular processes are interconnected through adiponectin and ABCA1- and ABCG1-dependent. In this pathway, adiponectin increased the affinity of apoA-I to cholesterol and effectively accelerated cholesterol removal from the plasma membrane to HDL particles. Thus, by accelerating HDL biogenesis, adiponectin may have therapeutic potential for atherosclerotic cardiovascular disease prevention and management.

VD3 and LXR agonist (T0901317) combination demonstrated greater potency in inhibiting cholesterol accumulation and inducing apoptosis via ABCA1-CHOP-BCL-2 cascade in MCF-7 breast cancer cells.

Obesity is associated with hypercholesterolemia and is a global epidemic. Epidemiological and animal studies revealed cholesterol is an essential regulator of estrogen receptor positive (ER+) breast cancer progression while inhibition of cholesterol accumulation was found to prevent breast tumor growth. Individually, vitamin D and LXR agonist T0901317 showed anticancer properties. The present study investigated the effects of vitamin D3 (VD3, calcitriol), LXR agonist (T0901317) and a combination of VD3 + T0901317 on cholesterol metabolism and cancer progression in ER+ breast cancer (MCF-7) cells. VD3 or T0901317 alone reduced cholesterol accumulation significantly in MCF-7 cells concomitant with an induction of ABCA1 protein and gene expression compared to the control treatment. Most importantly, VD3 + T0901317 combination showed higher effects in reducing cholesterol levels and increasing ABCA1 protein and gene expression compared to individual treatments. Importantly, VD3 + T0901317 combination showed higher effects in increasing apoptosis as measured by annexin apoptosis assay, cell viability and was associated with induction of CHOP protein and gene expression. Additionally, the VD3 + T0901317 exerted higher effects in reducing antiapoptotic BCL-2 while increased pro-apoptotic BAX gene expression compared to the individual treatments. The present results suggest that VD3 and T0901317 combination may have an important therapeutic application to prevent obesity and hyperlipidemia mediated ER+ breast cancer progression.

Isolation and purification of acidic polysaccharides from Agaricus blazei Murill and evaluation of their lipid-lowering mechanism

Polysaccharides are important active constituents of Agaricus blazei Morrill. In the present study, WABM-A was isolated from WABM using DEAE-cellulose, and subsequently purified using sepharose CL-6B to obtain the acidic polysaccharide WABM-A-b. WABM-A-b is mainly composed of Glc dextran, with a molecular weight of 10 KDa and β-1,6-D-Glcp as its main chain. The results of in vivo experiments show that in comparison with the MG, WABM-A significantly reduced the serum levels of TC, TG, and LDL-C, increased the serum levels of HDL-C (P < 0.01), and upregulated the liver expression of PPARγ, LXRα, ABCA1, and ABCG1 in rats with hyperlipidemia (P < 0.05). The results of in vitro experiments show that in comparison with the MG group, WABM-A-b-H significantly reduced the levels of TC and TG in HepG2 cells induced by oleic acid (P < 0.01), and significantly upregulated the protein expression of PPARγ, LXRα, ABCA1, and ABCG1 (P < 0.05). The present study demonstrates that WABM-A-b is an acidic glucan with lipid-lowering activity. The lipid-lowering mechanism of WABM-A-b is via the activation of the PPARγ/LXRα/ABCA1/ABCG1 cholesterol metabolism pathway. This is the first time that the hypolipidemic effect of Agaricus blazei Morrill acidic polysaccharides has been reported.

Role of pyruvate kinase M2 in oxidized LDL-induced macrophage foam cell formation and inflammation

Pyruvate kinase M2 (PKM2) links metabolic and inflammatory dysfunction in atherosclerotic coronary artery disease; however, its role in oxidized LDL (Ox-LDL)-induced macrophage foam cell formation and inflammation is unknown and therefore was studied. In recombinant mouse granulocyte-macrophage colony-stimulating factor-differentiated murine bone marrow-derived macrophages, early (1-6 h) Ox-LDL treatment induced PKM2 tyrosine 105 phosphorylation and promotes its nuclear localization. PKM2 regulates aerobic glycolysis and inflammation because PKM2 shRNA or Shikonin abrogated Ox-LDL-induced hypoxia-inducible factor-1α target genes lactate dehydrogenase, glucose transporter member 1, interleukin 1β (IL-1β) mRNA expression, lactate, and secretory IL-1β production. PKM2 inhibition significantly increased Ox-LDL-induced ABCA1 and ABCG1 protein expression and NBD-cholesterol efflux to apoA1 and HDL. PKM2 shRNA significantly inhibited Ox-LDL-induced CD36, FASN protein expression, DiI-Ox-LDL binding and uptake, and cellular total cholesterol, free cholesterol, and cholesteryl ester content. Therefore, PKM2 regulates lipid uptake and efflux. DASA-58, a PKM2 activator, downregulated LXR-α, ABCA1, and ABCG1, and augmented FASN and CD36 protein expression. Peritoneal macrophages showed similar results. Ox-LDL induced PKM2- SREBP-1 interaction and FASN expression in a PKM2-dependent manner. Therefore, this study suggests a role for PKM2 in Ox-LDL-induced aerobic glycolysis, inflammation, and macrophage foam cell formation.

Urotensin II, urotensin-related peptide, and their receptor in aortic valve stenosis

ObjectivesAortic valve stenosis (AVS) is the most common cause of surgical valve replacement worldwide. The vasoactive peptide urotensin II (UII) is upregulated in atherosclerosis and several other cardiovascular diseases; however, its role in the pathogenesis of AVS remains to be determined. Here, we investigated the expression of UII, urotensin-related peptide (URP), and the urotensin receptor (UT) and the role this system plays in AVS. MethodsImmunohistochemistry and reverse-transcriptase polymerase chain reaction were used to examine the cellular localization and mRNA expression, of UII, URP, and UT in calcified and noncalcified aortic valves. Human aortic valve interstitial cells were isolated from normal valves and treated with UII or URP, and changes in cell proliferation, cholesterol efflux, calcium deposition, and β-catenin translocation were assessed. ResultsThe mRNA expression of UII, URP, and UT was significantly greater in patients with AVS. There was abundant presence of UII, URP, and UT immunostaining in diseased compared with nondiseased valves and correlated significantly with presence of calcification (P < .0001) and fibrosis (P < .0001). Treating human aortic valve interstitial cells with UII or URP significantly increased cell proliferation (P < .0001) and decreased cholesterol efflux (P = .0011 and P = .0002, respectively). UII also significantly reduced ABCA1 protein expression (P = .0457) and increased β-catenin nuclear translocation (P < .0001) and mineral deposition (P < .0001). ConclusionsTogether, these data suggest that the urotensin system plays a role in the pathogenesis of AVS and warrants further investigation.