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Aim To investigate the effects of scutellarein on the macrophage foam cell formation and cholesterol efflux, and the underlying mechanism. Methods THP-1 cells were differentiated with PMA, and the cell viability was detected by MTT assays. The effects of scutellarein on the cholesterol efflux and macrophage foam cell formation were evaluated by using NBD-la-beled cholesterol and the cholesterol detection kit. The effects of scutellarein on the activation of PPARγ-LXRα-ABCA1 signaling pathway were determined by molecular docking, ELISA, dual-luciferase reporter and Western blot. The effects of PPARγ knowdown on scutellarein-induced cholesterol efflux and inhibiting macrophage foaming were analyzed by siRNA interference. Results Scutellarein dose-dependently inhibited oxLDL-induced cholesterol accumulation, accelerated cholesterol efflux and significantly increased the protein expression of LXRα and ABCA1. At the same time, scutellarein could bind PPARγ and initiate its downstream LXRa-ABCAl signaling pathway. In addition, gene silencing of PPARγ not only significantly inhibited scutellarein-induced LXRα-ABCA1 signaling pathway and cholesterol efflux, but also reversed the inhibitory effect of scutellarein on macrophage foaming. Conclusions Scutellarein could promote the cholesterol efflux by activating PPARγ and initiating the downstream LXR-ABCA1 signaling pathway, thereby prevent the macrophage foam cell formation.
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Hepatic cholesterol accumulation is an important contributor to hypercholesterolemia, which results in atherosclerosis and cardiovascular disease (CVD). ATP-citrate lyase (ACLY) is a key lipogenic enzyme that converts cytosolic citrate derived from tricarboxylic acid cycle (TCA cycle) to acetyl-CoA in the cytoplasm. Therefore, ACLY represents a link between mitochondria oxidative phosphorylation and cytosolic de novo lipogenesis. In this study, we developed the small molecule 326E with an enedioic acid structural moiety as a novel ACLY inhibitor, and its CoA-conjugated form 326E-CoA inhibited ACLY activity with an IC50 = 5.31 ± 1.2 μmol/L in vitro. 326E treatment reduced de novo lipogenesis, and increased cholesterol efflux in vitro and in vivo. 326E was rapidly absorbed after oral administration, exhibited a higher blood exposure than that of the approved ACLY inhibitor bempedoic acid (BA) used for hypercholesterolemia. Chronic 326E treatment in hamsters and rhesus monkeys resulted in remarkable improvement of hyperlipidemia. Once daily oral administration of 326E for 24 weeks prevented the occurrence of atherosclerosis in ApoE-/- mice to a greater extent than that of BA treatment. Taken together, our data suggest that inhibition of ACLY by 326E represents a promising strategy for the treatment of hypercholesterolemia.
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Lipid metabolism disorder is an important risk factor for obesity,type 2 diabetes mellitus,non-alcoholic fatty liver disease and other chronic metabolic diseases.Micro RNAs(miRNAs)are short stranded RNA molecules with the size of 19 to 25 nucleotides that regulate lipid metabolism,within a number of physiological functions.Studies have shown that miRNAs can participate in the regulation of lipid metabolism by polyphenolic phytochemicals.In this paper,the mechanism of plant polyphenols improving lipid metabolism disorders and maintaining lipid balance through miRNA is summarized from the synthesis of triglyceride,fatty acid β oxidation,cholesterol efflux and cholesterol esterification,aiming to provide a new thought for the application and development of plant polyphenols to improve the diseases related to lipid metabolism disorders,and offer theoretical basis for the research and development of miRNA as a potential target for the prevention and treatment of diseases related to lipid metabolism disorders.
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AIM: To observe the effect of Tangshen formula (TSF) treatment on TGF-β1/Smad3 pathway and cellular cholesterol efflux and explore its potential mechanism in HG-induced mouse tubular epithelial cells (mTECs). METHODS: After 25 mmol/L high glucose induced mTECs, TSF and Smad3 inhibitor SIS3 were given to intervene respectively. The lipid content in the cells was detected by ELISA kit; TGF-β1/Smad3 pathway and PGC-1α, LXR, ABCA1, ABCG1 were detected by Western blot and real-time PCR. RESULTS: TSF diminished the levels of TC, TG, LDL-C and increased the levels of HDL-C in HG-induced mTECs. Western blot and real-time PCR analysis showed that expression levels of TGF-β1, Smad3, Collagen and Fibronectin were significantly downregulated in the HG-induced mTECs with TSF and SIS3 treatment. And PGC-1α, LXR, ABCA1, ABCG1 expression levels were significantly upregulated in the HG-induced mTECs with TSF and SIS3 treatment. CONCLUSION: TSF can promote the cellular cholesterol efflux in HG-induced mTECs vis suppression of TGF-β1/Smad3 pathway.
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Objective@#To explore the impact of arsenic on cholesterol efflux and the expression of ATP-binding cassette, sub-family A, member 1 ( ABCA1 ), ATP-binding cassette transporter G1 ( ABCG1 ), and scavenger receptor class B member I ( SRBI ) in macrophages, so as to provide the evidence for the mechanism of arsenic induced atherosclerosis.@*Methods@#The human myeloid leukemia mononuclear cells ( THP-1 ), induced by phorbol myristate acetate, and mouse primary macrophages were treated with 0, 0.625, 1.25, 2.5 and 5 μmol/L NaAsO2 for 48 hours. Then the cells treated with 2.5 μmol/L NaAsO2 were changed to arsenic free mediums for 48 hours and collected every 12 hours to analyze the time effect of arsenic. The expression levels of ABCA1, ABCG1 and SRBI were determined by quantitative polymerase chain reaction and western blotting. Cholesterol efflux rates were measured by 3H isotope tracer. @*Results@#Arsenic significantly down-regulated the expression levels of ABCA1 and ABCG1, and cholesterol efflux in a dose-dependent manner. The levels of ABCA1 mRNA decreased by 69% and 72%, the levels of ABCG1 mRNA decreased by 42% and 34%, and the rate of cholesterol efflux decreased by 55% and 59% in THP-1 and mouse primary macrophages cells treated with 5 μmol/L NaAsO2 ( all P<0.05 ). Arsenic had no significant effect on SRBI expression ( all P>0.05 ). Arsenic inhibited ABCA1 expression and cholesterol efflux in THP-1 in a time-dependent manner. Compared with cells before the exposure of arsenic, the level of ABCA1 mRNA and the rate of cholesterol efflux in THP-1 bottomed at 48 hours by 43% and 42%, and gradually recovered when arsenic was removed. @*Conclusions@#Arsenic inhibits cholesterol efflux by down-regulating the expression of ABCA1 and ABCG1 in macrophages.
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Objective: To explore the effect of astragalus polysaccharide (APS) on oxidized low-density lipoprotein (ox-LDL)-induced lipid metabolism of macrophages and its underlying mechanism. Methods: The small interfering RNA (siRNA) targeting ATP binding cassette transporter A1 (ABCA1) was transfected into RAW 264.7 macrophages. Then the cells were stimulated with various concentrations of APS (20 mg/L, 60 mg/L and 150 mg/L), followed by the incubation with 50 mg/L ox-LDL for 24 h. qRT-PCR and Western blot were used to investigate the expression of ABCA1 mRNA and protein. Oil red O was used to analyze the level of foam cells. Lipid accumulation level was assessed by high performance liquid chromatography. [3H]-cholesterol was used to evaluate cholesterol efflux. Results: APS dose-dependently inhibited ox-LDL-induced formation of macrophage-derived foam cell compared with those in control group (P<0.05). HPLC analysis confirmed that APS attenuated lipid accumulation in a dose-dependent manner based on the decrease in ratio of cholesterol ester (CE)/total cholesterol (TC), concomitant with up-regulation of cholesterol efflux (P<0.05), indicating that APS might inhibit lipid deposition in macrophage by enhancing reverse cholesterol transport. Further more, APS dose-dependently increased ABCA1 mRNA and protein levels (P<0.05). When silencing ABCA1 expression with its specific siRNA, APS-inhibited lipid accumulation was significantly up-regulated, accompanied with the down-regulation of cholesterol efflux (P<0.05). Conclusion: APS may regulate lipid metabolism of macrophages by ABCA1-mediated progress of reverse cholesterol transport. Therefore, this study provides a potential target for the treatment of cardiovascular diseases triggered by vulnerable plaque.
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Objective: To investigate the inhibitory effect of macrophage foaming by Yinlan Tianzhi formula (YLTZ) and to explain its effects on lipid-induced inflammation and LXRα-ABCA1 signal pathway. Methods: The model of macrophage foaming was induced by incubating the RAW264.7 cells or BMMs with ox-LDL (50 mg·L-1). The serum containing YLTZ was prepared. The cells were divided into blank group, model group, and drug group. After drug intervention, MTT method was used to detect cell proliferation. The lipid accumulation in cells was observed by oil red O staining, and GPO-PAP method was used to determine the total cholesterol content in cells. Protein and mRNA levels were determined by Western blot and RT- qPCR. Results: Compared with control group, after YLTZ treatment, the lipid level was significantly decreased, and the level of mRNA and protein of LXRα and ABCA1 were significant increased. The expression of inflammatory factor COX2 and iNOS was significantly decreased. Conclusion: YLTZ inhibits macrophage foaming through enhancing LXRα-ABCA1 pathway and suppressing of inflammatory response.
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Macrophage cholesterol efflux is a central step in reverse cholesterol transport, which helps to maintain cholesterol homeostasis and to reduce atherosclerosis. Lipophagy has recently been identified as a new step in cholesterol ester hydrolysis that regulates cholesterol efflux, since it mobilizes cholesterol from lipid droplets of macrophages via autophagy and lysosomes. In this review, we briefly discuss recent advances regarding the mechanisms of the cholesterol efflux pathway in macrophage foam cells, and present lipophagy as a therapeutic target in the treatment of atherosclerosis.
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Atherosclerosis , Autophagy , Cholesterol , Foam Cells , Homeostasis , Hydrolysis , Lipid Droplets , Lysosomes , MacrophagesABSTRACT
Objective:To explore the effects of PPARγ on the cholesterol efflux of C57 mice peritoneal macrophage.Methods:Firstly constructing C57 mice model under different metabolic situation including high-fat diet and acute inflammation then isolate and culture its peritoneal macrophage,observing the expressions of PPARγ and IκB-α,identify the character of macrophage cholesterol efflux in every group.Then pretreat the normol C57 mice peritoneal macrophage with PPARγ ligand ciglitazone and PPARγ antisense oligonucleotide,observing the effect to cholesterol efflux after simulated with LPS in vitro.Results:The level of mice serum lipids of high fat diet group was significantly higher than that of normal diet group.The results of Western-blotting showed that the expression of PPARγ protein in groups of HFD and stimulated by LPS were significantly higher than that of control group.The expression in groups stimulated by LPS was all lower significantly than in control grouph.The determination of cholesterol efflux showed that this function of macrophage with HFD was more enhanced than that of control group but was inhibited in group stimulated by LPS.To normol peritoneal macrophage pretreat with PPARγ antisense oligonucleotide and stimulated by LPS,the expression of PPARγ protein was lower than that of control group but the expression of IκB-α was depressed obviously.Conclusion:The PPARγ ligand ciglitazone can increase the cholesterol efflux of C57 mice peritoneal macrophage and weaken the inhibition stimulated by LPS.The PPARγ antisense oligonucleotide can depress it and aggravate the inhibition.
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ATP-binding cassette protein A1 (ABCA1) is a cholesterol transporter that contributes to the active transport/removal of excess cellular cholesterol. ABCA1 expression is up-regulated when cells accumulate cholesterol. Aims: The purpose of this study was to determine any correlation between extracellular phospholipid levels and ABCA1 expression and function. Methodology: Human foreskin fibroblasts were incubated with cholesterol alone or cholesterol and phosphatidylcholine. Total RNA was isolated and subjected to end-point RT-PCR to compare ABCA1 transcript levels. Cell lysates were subjected to Western blot analysis to compare ABCA1 protein levels. Cells were loaded with radiolabeled cholesterol and cellular cholesterol efflux was measured in the presence and absence of apoE, a cholesterol acceptor. ApoE-dependent efflux was calculated as a measure of ABCA1-mediated efflux. Results: Here we show that incubation of cholesterol-loaded human skin fibroblasts with L-- phosphatidylcholine (PC) decreases ABCA1 mRNA and protein levels by 93% and 57%, respectively, compared to cells loaded with cholesterol alone. Similarly, PC treatment results in a 25% reduction in ABCG1 mRNA levels compared to cells treated with cholesterol alone, but there is no change in SR-BI transcript levels. Subsequent incubation of phospholipid-treated cells with a cholesterol acceptor such as apoE for 24 hours shows a 65% reduction in ABCA1-mediated cholesterol efflux compared to efflux in cells not treated with PC. During the lipid treatment itself, there is a 2.7-fold greater loss of cholesterol from PC treated cells compared to cells treated with cholesterol alone. Measurement of cholesterol in cellular lipid extracts reveals that cells incubated in the presence of phosphatidylcholine are significantly depleted of cholesterol having only 20% of the cholesterol compared to cells loaded with cholesterol alone. Conclusion: Thus, phosphatidylcholine facilitates removal of cellular cholesterol, thereby negating the cholesterol-dependent induction of ABCA1 message, protein and function.
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Objective To establish a high performance liquid chromatography (HPLC) method for determining the choles‐terol efflux from macrophage‐derived foam cells mediated by apolipoprotein A‐1(apoA‐1) .Methods Human THP‐1 monocytic cells ,pre‐treated with 160 nmol/L phorbol‐12‐myristate acetate (PMA) for 24 h to differentiate into adherence macrophages ,then incubated with 50 μg/mL acetylated low density lipoprotein (ac‐LDL) for 48 h to induce foam cells formation ,then added apoA‐1 for 24 h .THP‐1‐derived macrophage foam cells were identified by oil red O‐staining ,and the cellular cholesterol content by meas‐ured by HPLC method .Cholesterol efflux from macrophage foam cells was determined by HPLC analysis and liquid scintillation counting ,respectively .Results Oil red O‐stainable lipid droplet accumulation were observed in entire cytoplasm of THP‐1‐derived macrophage foam cells .Measuring cellular cholesterol content showed that free cholesterol ,total cholesterol and cholesterol ester content in macrophage foam cells were increased remarkably than PMA group macrophages (P<0 .01) .After treated with ac‐LDL for 48 h ,the macrophage foam cells accumulated 80 .25 μg/mg cell protein and 47 .65 μg/mg cell protein respectively ,and the cho‐lesterol ester accounted for 59 .38% of the cellular total cholesterol (P<0 .01) .The ratio of cholesterol efflux reached 5 .63% and 7 .08% respectively by HPLC analysis and liquid scintillation counting using apoA‐1 mediation (P<0 .01) .Conclusion Combina‐tion of an enzymatic catalysis and HPLC method for determining cholesterol efflux from foam cells is successfully established in this study , thus providing a technical foundation for the further study of cellular lipid homeostasis .
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O HDL-c é um fator de risco cardiovascular negativo e sua concentração plasmática apresenta relação inversa com a incidência de eventos cardiovasculares. Entretanto, as evidências relativas ao grupo de indivíduos com níveis de HDL-c acima do percentil 95 da população geral ainda são escassas e o impacto da hiperalfalipoproteinemia (HALP) sobre o risco cardiovascular continua representando motivo de controvérsia na literatura médica. Alguns estudos em populações específicas associam a HALP a aumento do risco cardiovascular. Ao mesmo tempo, outros estudos identificaram populações de indivíduos hipoalfalipoproteinêmicos com marcada longevidade. Assim, demonstrou-se aparente dissociação entre níveis de HDL-c e risco cardiovascular em determinadas populações, reconduzível a aspectos disfuncionais da HDL. O objetivo do presente estudo foi verificar o papel da HALP na determinação do risco cardiovascular; comparar a prevalência de doença cardiovascular subclínica, avaliada por meio da quantificação ultrassonográfica da Espessura Íntimo-Medial Carotídea (EIMC), entre portadores de HDL-c >= 90mg/dL (grupo HALP) e portadores de concentrações de HDL-c atualmente consideradas normais (entre 40 e 50mg/dL para os homens e entre 50 e 60mg/dL para as mulheres); e avaliar características e função da HDL em portadores de HALP por meio do estudo de sua composição, de sua capacidade de efluxo de colesterol, e de sua atividade anti-inflamatória e antioxidante, correlacionando estas características com a presença de doença cardiovascular subclínica avaliada por meio da determinação da EIMC, da Velocidade de Onda de Pulso (VOP) e da presença de Calcificação Arterial Coronariana (CAC) avaliada pela TCMD. Para responder estas perguntas, o presente estudo foi articulado em dois braços: Braço 1: Análise da coorte do estudo ELSA com o objetivo de determinar a prevalência de HALP em uma população geral; definir o perfil demográfico, antropométrico e metabólico dos portadores de HALP; e...
HDL-c is a negative cardiovascular risk factor and its plasma concentration is inversely related to the incidence of cardiovascular events. However, evidence of benefit among subjects with HDL-c levels above the 95th percentile of the general population is still scarce and the impact of hyperalphalipoproteinemia (HALP) on cardiovascular risk continues to represent matter of debate in the medical literature. Some studies with specific populations indicated an increased cardiovascular risk associated with HALP. In addition, other reports identified groups of patients with marked hypoalphalipoproteinemia and longevity. Hence, there could be a dissociation between HDL-c levels and cardiovascular risk in certain populations, possibly due to dysfunctional HDL particles. The aim of this study was to investigate the role of HALP phenotype in determining cardiovascular risk; to compare the prevalence of subclinical cardiovascular disease, assessed by ultrasound measurement of Carotid Intima-Media Thickness (CIMT) among patients with HDL-c >= 90mg/dL (HALP group) and patients with HDL-c currently considered normal (40-50mg/dL for men and 50-60mg/dL for women); and to evaluate HDL functionality in patients with HALP through the study of its composition, its cholesterol efflux capacity, and its anti-inflammatory and antioxidant activity; correlating those characteristics with the presence of subclinical cardiovascular disease assessed by CIMT, Pulse Wave Velocity (PWV) and Coronary Artery Calcification (CAC). To answer these questions, the present study was articulated into two arms: Arm 1: ELSA-Brasil study cohort analysis in order to assess HALP prevalence in a general population, defining demographic, anthropometric and metabolic profile of HALP individuals; and comparing the prevalence of subclinical vascular disease among HALP subjects with controls with normal HDL-c. Arm 2: Recruitment of 80 healthy volunteers with HALP to study the correlation...
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Humans , Male , Female , Middle Aged , Aged , Atherosclerosis , Carotid Intima-Media Thickness , Cholesterol, HDL , Tumor Necrosis Factor-alphaABSTRACT
Objective: To investigate the effects of quercetin on cholesterol accumulation and cholesterol flow in RAW264.7 macrophages and explore the potential mechanism underlying its anti-atherogenic activity. Methods: The inhibitory effect of quercetin on cholesterol accumulation induced by oxidized low-density lipoprotein (ox-LDL) was assessed by oil red O staining and total cholesterol (TC) specific kits in RAW264.7 macrophages. And the action of cholesterol efflux and influx was tested by fluorescent assays. Cholesterol flow-associated genes expression was detected by real-time quantitative PCR (RT-PCR). Results: Quercetin significantly inhibited the cholesterol accumulation. Treatment with quercetin (10 μmol/L) significantly enhanced cholesterol efflux and substantially inhibited cholesterol influx. RT-PCR showed that quercetin significantly increased the mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ), liver X receptor alpha (LXRα), ATP-binding cassette, subfamily A1 (ABCA1) and subfamily G1 (ABCG1), decreased scavenger receptor (SR)-A1 and SR-A2. Conclusion: Quercetin might be a new inhibitor on intracellular cholesterol accumulation. Upregulation of the classical PPARγ-LXRα-ABCA1/ ABCG1 pathway and down-regulation of SR-A1 and SR-A2 may participate in its suppressive effect on intracellular cholesterol accumulation.
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Objective: To clarify the potential inhibitive effect of epipinoresinol on macrophage foam and potential mechanisms. Methods: The inhibition of epipinoresinol on foam cell formation after stained with oil red O was assessed by Image-Pro Plus and Microplate Reader, and the effect on cholesterol efflux and influx was tested by fluorescence detection to obtain cholesterol inflows-time curve and outflow rate. Additionally the cholesterol flow-associated genes expression was checked by real-time PCR (RT-PCR). Results: Epipinoresinol dose-dependently inhibited the enhanced cholesterol accumulation elicited by oxidized low-density lipoprotein cholesterol (ox-LDL) in RAW264.7 cells. Treatment with epipinoresinol significantly enhanced the cholesterol efflux mediated by high-density lipoprotein (HDL) and substantially inhibited the cholesterol influx. RT-PCR showed that epipinoresinol significantly increased the mRNA levels of PPARγ, LXRα, ABCA1, and ABCG1, decreased those of SR-A1 and SR-A2. Conclusion: Epipinoresinol is a new inhibitor on foam cell formation that may stimulate the cholesterol efflux through up-regulating the PPARγ-LXRα-ABCA1/ABCG1 pathway and prevent cholesterol influx through down-regulating SR-A1 and SR-A2, which may be useful on atherosclerosis treatment.
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Objective To investigate the effect of hydrogen sulfide (H2S) on the cholesterol efflux and ATP-binding cassette transporter A1 (ABCA1) expression in foam cells .Methods RAW 264 .7 macrophages were incubated with oxidized low density lipoprotein to induce foam cells .Foam cells were incubated with H2S donor sodium hydrosulfide .Cholesterol efflux from macropha-ges was tested by labed cholesterol .The cellular levels of free cholesterol (FC) ,cholesterol ester (CE) and total cholesterol (TC) were measured by high performance liquid chromatography assays .The mRNA and protein expressions of ABCA1 were detected by Real-time PCR and Western blot .Results Compared with the foam cells ,the rates of cholesterol efflux were significantly in-creased ,the levels of TC ,FC ,CE and CE/TC ratio were significantly decreased(P<0 .05) and expression of ABCA1 was signifi-cantly increased by treatment with H2S in dose-and time-dependent manner(P<0 .05) .Conclusion H2S up-regulates of expres-sion ABCA1 and promotes cholesterol efflux in RAW 264 .7 macrophage-derived foam cells .
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AIM: To study the characteristic of liver X receptor alpha (LXRα), its target gene expression and cholesterol efflux in human macrophages treated with atorvastatin. METHODS: Human monocyte-derived macrophages were collected and cultured. Macrophages were treated with or without atorvastatin. Apolipoprotein A-I mediated human monocyte-derived macrophage cholesterol efflux was detected by liquid scintillation counting method. Reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate the mRNA expression of LXRα and some of its target genes ABCA1, SREBP2, CETP, PLTP, apoE, MMP-9 and MIP-1α. The protein expression of LXRα, ABCA1, MMP-9 and MIP-1α was determined by Western blotting. RESULTS: Pre-incubation of human monocyte-derived macrophages with atorvastatin dose dependently (1-2 μmol/L) stimulated cholesterol efflux mediated by apolipoprotein A-I. Atorvastatin also increased the mRNA expression of LXRα, ABCA1, SREBP2, CETP, PLTP, and protein expression of LXRα, ABCA1, but decreased the expression of MMP-9 and MIP-1α at both mRNA and protein levels. CONCLUSION: Atorvastatin enhances the cholesterol efflux, upregulates LXR and some genes associated with cholesterol metabolism and inhibits inflammatory responses in macrophages, indicating that statins may affect the formation of foam cells by activating LXR signaling pathway.
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Objective To identify potential linkage of cholesterol efflux with the expressions of ATP-binding cas-sette receptor A1 (ABCA1), ABCG1 and scavenger receptor B1 (SR-B1) in monocytes derived macrophages of patients with type 2 diabetes mellitus. Methods and Results Blood was collected from subjects with or without type 2 diabetes mellitus. Peripheral blood monocytes were differentiated for 72 hours into macrophages, and cholesterol efflux assays, Real-time quantitative PCR and western blot were performed. Macrophages from patients with type 2 diabetes mellitus showed a reduction in cholesterol efllux. The mRNA and protein expressions of ABCG1 in macrophages from patients with type 2 diabetes mellitus were significantly reduced. In contrast, the expression of ABCA1 and SR-B1 was not significantly different in both control subjects and diabetic patients. In addition, cellular cholesterol efflux from macrophages to autologous serum and pool serum was significantly correlated with the expression of ABCG1. Conclusion ABCG1 expression and cholesterol efflux are reduced in patients with type 2 diabetes mellitus. This impaired cholesterol efflux significantly correlates with decreased expression of ABCG1.
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Objective To investigate effects of atorvastatin on the development from macrophages (HMDM) to foam cells.Methods Monocytes were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation and plastic adsorptive process.The isolated cells were stimulated by phorbol 12-myristate 13-acetate (PMA) (50 nmol/L) for 48 h and transformed to macrophages.Macrophages were co-incubated with 80 mg/L ox- idized low density lipoprotein (ox-LDL) and atorvastatin (0-100 ?mol/L),respectively for 0,6,12 and 24 h. Total cholesterol (TC),free cholesterol (FC) and protein (Pro) in cultured cells were quantitatively analyzed by high performance chromatography (HPLC) analysis and modified Lowry protein assay.Results When macropha- ges were incubated with 80 mg/L ox-LDL,the ratio of TC/Pro was greater than 20,and large amount of lipid drop- lets were displayed indicating the formation of foam cells.Atorvastatin decreased TC/Pro ratio in foam cells in a concentration and time dependent manner (0-100 ?mol/L)(P
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Objective To identify potential linkage of cholesterol efflux with the expressions of ATP-binding cassette receptor A1(ABCA1),ABCG1 and scavenger receptor B1(SR-B1) in monocytes derived macrophages of patients with type 2 diabetes mellitus.Methods and Results Blood was collected from subjects with or without type 2 diabetes mellitus.Peripheral blood monocytes were differentiated for 72 hours into macrophages,and cholesterol efflux assays,Real-time quantitative PCR and western blot were performed.Macrophages from patients with type 2 diabetes mellitus showed a reduction in cholesterol efflux.The mRNA and protein expressions of ABCG1 in macrophages from patients with type 2 diabetes mellitus were significantly reduced.In contrast,the expression of ABCA1 and SR-B1 was not significantly different in both control subjects and diabetic patients.In addition,cellular cholesterol efflux from macrophages to autologous serum and pool serum was significantly correlated with the expression of ABCG1.Conclusion ABCG1 expression and cholesterol efflux are reduced in patients with type 2 diabetes mellitus.This impaired cholesterol efflux significantly correlates with decreased expression of ABCG1.
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Objective To examine the effects of high-density lipoprotein(HDL) and lipoprotein-deficient serum(LPDS) isolated from patients with type 2 diabetes mellitus on cholesterol efflux through human skin fibroblast(HSF) and human hepatoma cell line(HepG2).Methods and Results Blood was collected from 13 patients with type 2 diabetes mellitus and 17 healthy volunteers,HDL and LPDS were isolated.Cholesterol efflux assays,RT-PCR and Western blot were performed with HSF and HepG2 cells.The HepG2 cells showed a high expression of scavenger receptor B1(SR-B1) and lack of functional ATP-binding cassette receptor A1(ABCA1) and ATP-binding cassette receptor G1(ABCG1) while HSF cells express SR-B1 at very low level and have a high expression of ABCA1 pretreated with 22-OH cholesterol.The cholesterol efflux from HepG2 cells to HDL isolated from patients with diabetes decreased significantly as compared to controls.However,cholesterol efflux from HSF cells to LPDS was not different between groups.Conclusion The function of HDL involving cholesterol efflux in type 2 diabetes mellitus was impaired while cholesterol efflux induced by LPDS from HSF cells was maintained,suggesting that HDL plays a critical role in mediation of intracellular cholesterol accumulation and progression of atherosclerosis inpatients with type 2 diabetes.