Acyl-coenzyme A:cholesterol Acyltransferase-1 Expression Research Articles

A Dipeptidyl Peptidase-4 Inhibitor Inhibits Foam Cell Formation of Macrophages in Type 1 Diabetes via Suppression of CD36 and ACAT-1 Expression.

Dipeptidyl peptidase-4 (DPP-4) inhibitors have been reported to play a protective role against atherosclerosis in both animal models and patients with type 2 diabetes (T2D). However, since T2D is associated with dyslipidemia, hypertension and insulin resistance, part of which are ameliorated by DPP-4 inhibitors, it remains unclear whether DPP-4 inhibitors could have anti-atherosclerotic properties directly by attenuating the harmful effects of hyperglycemia. Therefore, we examined whether a DPP-4 inhibitor, teneligliptin, could suppress oxidized low-density lipoprotein (ox-LDL) uptake, foam cell formation, CD36 and acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) gene expression of macrophages isolated from streptozotocin-induced type 1 diabetes (T1D) mice and T1D patients as well as advanced glycation end product (AGE)-exposed mouse peritoneal macrophages and THP-1 cells. Foam cell formation, CD36 and ACAT-1 gene expression of macrophages derived from T1D mice or patients increased compared with those from non-diabetic controls, all of which were inhibited by 10 nmol/L teneligliptin. AGEs mimicked the effects of T1D; teneligliptin attenuated all the deleterious effects of AGEs in mouse macrophages and THP-1 cells. Our present findings suggest that teneligliptin may inhibit foam cell formation of macrophages in T1D via suppression of CD36 and ACAT-1 gene expression partly by attenuating the harmful effects of AGEs.

TLR4-mediated inflammation promotes foam cell formation of vascular smooth muscle cell by upregulating ACAT1 expression

Vascular smooth muscle cell (VSMC) foam cell formation is an important hallmark, especially in advanced atherosclerosis lesions. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) promotes foam cell formation by promoting intracellular cholesteryl ester synthesis. The present study tests the hypothesis that oxidized low-density lipoprotein (oxLDL) increases the ACAT1 expression by activating the Toll-like receptor 4 (TLR4)-mediated inflammation, and ultimately promotes VSMC foam cell formation. Wild-type, ApoE−/−, TLR4−/− and ACAT1−/− mice on a C57BL/6J background were used. Increased TLR4, proinflammatory cytokines and ACAT1 were observed in high-fat (HF) diet-induced atherosclerotic plaque formation and in oxLDL-stimulated VSMCs. ACAT1 deficiency impeded the HF diet-induced atherosclerotic plaque formation and impaired the TLR4-manipulated VSMC foam cell formation in response to oxLDL. TLR4 deficiency inhibited the upregulation of myeloid-differentiating factor 88 (MyD88), nuclear factor-κB (NF-κB), proinflammatory cytokines and ACAT1, and eventually attenuated the HF diet-induced atherosclerotic plaque formation and suppressed the oxLDL-induced VSMC foam cell formation. Knockdown of MyD88 and NF-κB, respectively, impaired the TLR4-manipulated VSMC foam cell formation in response to oxLDL. Rosiglitazone (RSG) attenuated HF diet-induced atherosclerotic plaque formation in ApoE−/− mice, accompanied by reduced expression of TLR4, proinflammatory cytokines and ACAT1 accordingly. Activation of peroxisome proliferator-activated receptor γ (PPARγ) suppressed oxLDL-induced VSMC foam cell formation and inhibited the expression of TLR4, MyD88, NF-κB, proinflammatory cytokines and ACAT1, whereas inhibition of PPARγ exerted the opposite effect. TLR4−/− mice and VSMCs showed impaired atherosclerotic plaque formation and foam cell formation, and displayed no response to PPARγ manipulation. In conclusion, our data showed that oxLDL stimulation can activate the TLR4/MyD88/NF-κB inflammatory signaling pathway in VSMCs, which in turn upregulates the ACAT1 expression and finally promotes VSMC foam cell formation.

Homocysteine-mediated cholesterol efflux via <italic>ABCA1</italic> and <italic>ACAT1</italic> DNA methylation in THP-1 monocyte-derived foam cells

Homocysteine (Hcy) has been recognized as a prevalent risk factor for cardiovascular events. Cholesterol-loaded foam cells are a central component of atherosclerotic lesions. ATP-binding cassette transporter A1 (ABCA1), which mediates the efflux of cellular cholesterol and phospholipids, is the rate-limiting step in lipid metabolism. Acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) promotes accumulation of cholesterol ester in macrophages, thereby resulting in the foam cell formation, a hallmark of early stage in atherosclerosis. In this study, cultured monocyte-derived foam cells were incubated with clinical relevant concentrations of Hcy for 24 h. Both increased number of foam cells and accumulation of cholesterol were found, and the mRNA and protein expression levels of ABCA1 were decreased, while ACAT1 expression was increased in the presence of Hcy. Furthermore, the DNA methylation level of ABCA1 gene was increased whereas ACAT1 DNA methylation was decreased by using different concentrations of Hcy. Moreover, our results showed that DNA methyltransferase (DNMT) activity and DNA methyltransferase 1 (DNMT1) mRNA expression were increased by Hcy. It is indicated that DNA methylation has the function to regulate the expression of ABCA1 and ACAT1 via DNMT. In conclusion, these results suggest that ABCA1 and ACAT1 DNA methylation induced by Hcy may play a potential role in ABCA1 and ACAT1 expression and the accumulation of cholesterol in monocyte-derived foam cells.

Ghrelin inhibits foam cell formation via simultaneously down-regulating the expression of acyl-coenzyme A:cholesterol acyltransferase 1 and up-regulating adenosine triphosphate-binding cassette transporter A1

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), revealed cardioprotective effects in both experimental models and human. There is far less information on the mechanisms that produce antiatherogenic effects. We assessed the expression of acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT-1) and adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1), which have been implicated in regulating cellular cholesterol homeostasis and therefore play critical roles in foam cell formation, in THP-1-derived foam cells in the presence of various concentration of ghrelin. After 48 h of culture in the presence of phorbol myristate acetate, THP-1 monocytes differentiated to macrophages. After another 24 h of culture with ox-LDL, the differentiated cells transformed to foam cells. Different concentrations of ghrelin and other intervention factors were added, respectively. The expression of ACAT-1 and ABCA1 was detected by a technique in molecular biology. The content of cellular cholesterol was measured by zymochemistry via a fluorospectrophotometer. Ghrelin could down-regulate the expression of ACAT-1 and up-regulate the expression of ABCA1 in a dose-dependent manner simultaneously. Ghrelin also decreased cellular cholesterol content and increased cholesterol efflux. These effects could be abolished by the specific antagonist of GHS-R and a peroxisome proliferator-activated receptor γ (PPARγ)-specific inhibitor, respectively. The results suggest that ghrelin inhibited foam cell formation via simultaneously down-regulating the expression of ACAT-1 and up-regulating ABCA1. Those effects may be achieved via pathways involving GHS-R and PPARγ.

Chlamydia pneumoniae induces macrophage-derived foam cell formation by up-regulating acyl-coenzyme A: cholesterol acyltransferase 1

In macrophages, the accumulation of cholesteryl esters synthesized by acyl-coenzyme A: cholesterol acyltransferase 1(ACAT1) plays a crucial role in foam cell formation, a hallmark of early atherosclerotic lesions. It is suggested that Chlamydia pneumoniae ( C. pneumoniae) induces foam cell formation. However, the mechanism of foam cell formation induced by C. pneumoniae has not been fully elucidated. In this study, we found that C. pneumoniae increased the expression of acyl-coenzyme A: cholesterol acyltransferase 1(ACAT1) mRNA and protein in a dose-dependent manner in THP-1-derived macrophages exposed to low density lipoprotein (LDL). In addition, C. pneumoniae dose-dependently suppressed the expression of peroxisome proliferator-activated receptor gamma (PPAR γ) mRNA and protein. Rosiglitazone, a specific PPAR γ agonist, not only dose-dependently alleviated the down-regulation of PPAR γ expression by C. pneumoniae infection, but also dose-dependently inhibited the C. pneumoniae-induced ACAT1 expression. Furthermore, higher doses of rosiglitazone (10 and 20 μM) suppressed the C. pneumoniae-induced foam cell formation from morphological (Oil red O staining) and biochemical (zymochemistry method) criteria. These results first demonstrate that C. pneumoniae induces macrophage-derived foam cell formation by up-regulating ACAT1 expression via PPAR γ-dependent pathway, which may contribute to its pro-atherogenic properties.

Rosiglitazone inhibits expression of acyl-coenzyme A: cholesterol acyltransferase-1 in THP-1 macrophages induced by advanced glycation end-products ⋆

Objective To investigate the effects of rosiglitazone, a synthetic ligand of peroxisome proliferators-activated receptor gamma (PPARγ), on the expression of acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) in phorbol myristate acetate (PMA)-pretreated THP-1 cells after the inducement of advanced glycation end products (AGEs). Methods After THP-1 cells were cultured in the presence of 0.1 μmol/L PMA for 72 h to induce phagocytic differentiation, the obtained THP-1 macrophages were treated with rosiglitazone for 4 h at different concentrations (1, 5 or 10 μmol/L) and then exposed to AGEs-modified bovine serum albumin (AGEs-BSA) for 24 h at a concentration of 200 mg/L. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis were performed to detect the mRNA and protein expressions of ACAT-1 respectively. Results Administration of AGEs-BSA (200 mg/L) into the THP-1 macrophages resulted in up-regulation of ACAT-1 at mRNA and protein levels when compared with the expressions in macrophages incubated with serum-free RPMI1640. Pretreatment of rosiglitazone inhibited significantly the increased expression of ACAT-1 induced by AGEs-BSA in a concentration-dependent manner. Conclusion PPARγ activation by rosiglitazone down-regulates ACAT-1 expression induced by AGEs in THP-1 macrophages, which might provide a new way for treating atherogenesis in diabetic patients.

Expression of two isozymes of acyl‐coenzyme A: Cholesterol acyltransferase‐1 and ‐2 in clear cell type renal cell carcinoma

The present study investigated the expression of acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) and -2 in clear cell type renal cell carcinoma (RCC). Clear cell type RCC and corresponding normal kidney tissue samples were obtained from 19 surgical cases (28-85 years of age). Tissue extracts were assayed for ACAT activity and protein expression by immunoblotting with anti-ACAT-1 and anti-ACAT-2 antibodies. Frozen sections were subjected to Oil red O staining for lipids, and were immunostained with ACAT-specific antibodies. Acyl-coenzyme A cholesterol acyltransferase activity was 5.7-fold higher (P < 0.01) in clear cell carcinoma (23.52 +/- 4.90 pmol/mg protein/min) than in normal kidney (4.12 +/- 0.36). Consistent with this, immunoblotting and immunohistochemical staining revealed strong expression of ACAT-1 in clear cell type RCC. Densitometric analysis showed that ACAT-1 expression was 2.9-fold higher in clear cell type RCC than in normal kidney. In contrast, ACAT-2 expression was negative in clear cell type RCC and normal kidney. Oil red O staining showed massive deposits of lipid in RCC cells. We identified strong expression of ACAT-1 in clear cell type RCC. Upregulation of ACAT-1 leads to high ACAT enzymatic activity, which accelerates the accumulation of cholesterol ester in clear cell type RCC.

Human Urotensin II Accelerates Foam Cell Formation in Human Monocyte-Derived Macrophages

Human urotensin II (U-II), the most potent vasoconstrictor peptide identified to date, and its receptor (UT) are involved in hypertension and atherosclerosis. Acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) converts intracellular free cholesterol into cholesterol ester (CE) for storage in lipid droplets and plays an important role in the formation of macrophage-derived foam cells in atherosclerotic lesions. We examined the effects of U-II on ACAT-1 expression and CE accumulation in human monocyte-derived macrophages. U-II increased ACAT activity in a concentration-dependent manner after 7 days in monocyte primary culture. Immunoblotting analysis showed that U-II at 25 nmol/L increased ACAT-1 protein expression level by 2.5-fold, which was completely abolished by anti-U-II antibody, selective UT receptor antagonists (urantide and 4-aminoquinoline), a G-protein inactivator (GDP-beta-S), a c-Src protein tyrosine kinase inhibitor (PP2), a protein kinase C (PKC) inhibitor (rottlerin), a mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059), or a Rho kinase (ROCK) inhibitor (Y27632). Northern blotting analysis indicated that among the 4 ACAT-1 mRNA transcripts (2.8-, 3.6-, 4.3-, and 7.0-kb), the 2.8- and 3.6-kb transcript levels were selectively upregulated by approximately 1.7-fold by U-II (25 nmol/L). Further, U-II (25 nmol/L) significantly increased acetylated LDL (acetyl-LDL)-induced CE accumulation in monocyte-derived macrophages but not scavenger receptor class A (SR-A) function as assessed by endocytic uptake of [(125)I]acetyl-LDL. Our results suggest that U-II may play a novel role in the formation of macrophage-derived foam cells by upregulating ACAT-1 expression via the UT receptor/G-protein/c-Src/PKC/MEK and ROCK pathways but not by SR-A, thus contributing to the relatively rapid development of atherosclerosis in hypertension.

Serotonin acts as an up-regulator of acyl-coenzyme A:cholesterol acyltransferase-1 in human monocyte–macrophages

Acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) converts intracellular free cholesterol into cholesterol ester for storage in lipid droplets and plays an important role in the formation of macrophage-derived foam cells in atherosclerotic lesions. Serotonin (5-HT), a potent vasoconstrictor that is released from activated platelets, increases uptake of oxidized low-density lipoprotein (LDL) by macrophages, leading to foam cell formation, and contributes to the development of atherosclerotic plaque. However, it is not yet known whether 5-HT affects ACAT-1 expression in human monocyte–macrophages as the molecular mechanism of enhanced foam cell formation by 5-HT remains unclear. We examined the effects of 5-HT on ACAT-1 expression during differentiation of cultured human monocytes into macrophages. Expression of ACAT-1 protein but not 5-HT 2A receptor increased in a time-dependent manner. 5-HT increased ACAT activity in a concentration-dependent manner after 7 days in primary monocyte culture. Immunoblotting analysis showed that 5-HT at 10 μM increased ACAT-1 protein expression level by two-fold, and this effect was abolished completely by a 5-HT 2A receptor antagonist (sarpogrelate), its major metabolite (M-1), a G protein inactivator (GDP-β-S), a protein kinase C (PKC) inhibitor (rottlerin), a Src family inhibitor (PP2), or a mitogen-activated protein kinase (MAPK) kinase inhibitor (PD98059). Northern blotting analysis indicated that among the four ACAT-1 mRNA transcripts (2.8-, 3.6-, 4.3-, and 7.0-kb), the levels of the 2.8- and 3.6-kb transcripts were selectively up-regulated by ∼1.7-fold by 5-HT (10 μM). The results of the present study suggested that 5-HT may play a crucial role in macrophage-derived foam cell formation by up-regulating ACAT-1 expression via the 5-HT 2A receptor/G protein/c-Src/PKC/MAPK pathway, contributing to the progression of atherosclerotic plaque.

Enhancement of human ACAT1 gene expression to promote the macrophage-derived foam cell formation by dexamethasone.

In macrophages, the accumulation of cholesteryl esters synthesized by the activated acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) results in the foam cell formation, a hallmark of early atherosclerotic lesions. In this study, with the treatment of a glucocorticoid hormone dexamethasone (Dex), lipid staining results clearly showed the large accumulation of lipid droplets containing cholesteryl esters in THP-1-derived macrophages exposed to lower concentration of the oxidized low-density lipoprotein (ox-LDL). More notably, when treated together with specific anti-ACAT inhibitors, the abundant cholesteryl ester accumulation was markedly diminished in THP-1-derived macrophages, confirming that ACAT is the key enzyme responsible for intracellular cholesteryl ester synthesis. RT-PCR and Western blot results indicated that Dex caused up-regulation of human ACAT1 expression at both the mRNA and protein levels in THP-1 and THP-1-derived macrophages. The luciferase activity assay demonstrated that Dex could enhance the activity of human ACAT1 gene P1 promoter, a major factor leading to the ACAT1 activation, in a cell-specific manner. Further experimental evidences showed that a glucocorticoid response element (GRE) located within human ACAT1 gene P1 promoter to response to the elevation of human ACAT1 gene expression by Dex could be functionally bound with glucocorticoid receptor (GR) proteins. These data supported the hypothesis that the clinical treatment with Dex, which increased the incidence of atherosclerosis, may in part due to enhancing the ACAT1 expression to promote the accumulation of cholesteryl esters during the macrophage-derived foam cell formation, an early stage of atherosclerosis.

Up-regulation of acyl-coenzyme A:cholesterol acyltransferase-1 by transforming growth factor-β1 during differentiation of human monocytes into macrophages

Expression of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) increases during differentiation of human monocytes into macrophages. To further elucidate the mechanism for ACAT-1 regulation in macrophages, we examined the effects of five cytokines including transforming growth factor-β1 (TGF-β1) on ACAT-1 expression in cultured human monocyte–macrophages. Immunoblot analyses showed that TGF-β1 increased ACAT-1 protein expression by two- to threefold when added during differentiation of human monocytes into macrophages. ACAT activity increased in parallel by 1.8-fold. Northern blot analyses revealed that among the three ACAT-1 mRNA transcripts detected (2.8-, 3.6-, and 4.3-kb), the 2.8- and 3.6-kb transcripts were selectively increased by TGF-β1. When TGF-β1 was added after differentiation, ACAT-1 expression was not altered. Since TGF-β1 is expressed in human atherosclerotic lesions, the current results suggest that ACAT-1 expression in monocytes infiltrating from the circulation to vascular walls may be enhanced by pre-existing TGF-β1.

Adiponectin down-regulates acyl-coenzyme A:cholesterol acyltransferase-1 in cultured human monocyte-derived macrophages

Acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) catalyzes the formation of cholesteryl esters (CE) and plays a significant role in formation of macrophage-derived foam cells in atherosclerotic lesions. Adiponectin was reported to play an anti-atherogenic role by inhibiting class A scavenger receptor (SR-A) expression in human macrophages. To further clarify its additional property, we examined its effect on ACAT-1 expression using human macrophages. Immunoblot analyses revealed a significant reduction of ACAT-1 protein by a low concentration (1 μg/ml) of adiponectin. The ACAT activity was also decreased in parallel by adiponectin. Northern blot analyses revealed that all four ACAT-1 mRNA transcripts (2.8, 3.6, 4.3, and 7.0 kb) were decreased almost equally by adiponectin. Furthermore, acetyl-LDL-induced CE-accumulation in these macrophages was reduced significantly by this adipocytokine. These results demonstrate the inhibitory effect of adiponectin on ACAT-1 expression, suggesting that adiponectin may play an anti-atherogenic role by down-regulating the expression of ACAT-1 as well as SR-A in human macrophages.