RESUMO
The risks of non-alcoholic fatty liver disease (NAFLD) include obese and non-obese stresses such as chronic hepatitis C virus (HCV) infection, but the regulatory determinants remain obscure. Apolipoprotein J (ApoJ) served as an ER-Golgi contact-site chaperone near lipid droplet (LD), facilitating HCV virion production. We hypothesized an interplay between hepatic ApoJ, cholesterol esterification and lipid deposit in response to NAFLD inducers. Exposures of HCV or free-fatty acids exhibited excess LDs along with increased ApoJ expression, whereas ApoJ silencing alleviated hepatic lipid accumulation. Both stresses could concomitantly disperse Golgi, induce closer ApoJ and sterol O-acyltransferase 2 (SOAT2) contacts via the N-terminal intrinsically disordered regions, and increase cholesteryl-ester. Furthermore, serum ApoJ correlated positively with cholesterol and low-density lipoprotein levels in normal glycaemic HCV patients, NAFLD patients and in mice with steatosis. Taken together, hepatic ApoJ might activate SOAT2 to supply cholesteryl-ester for lipid loads, thus providing a therapeutic target of stress-induced steatosis.
Assuntos
Clusterina/metabolismo , Metabolismo dos Lipídeos/fisiologia , Esterol O-Aciltransferase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Clusterina/fisiologia , Esterificação , Fígado Gorduroso/metabolismo , Feminino , Hepatite C Crônica/metabolismo , Humanos , Hipercolesterolemia/metabolismo , Gotículas Lipídicas/metabolismo , Lipídeos/fisiologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Esterol O-Aciltransferase/fisiologia , Esterol O-Aciltransferase 2RESUMO
Previous studies on ablation of several key genes of meibogenesis related to fatty acid elongation, omega oxidation, and esterification into wax esters have demonstrated that inactivation of any of them led to predicted changes in the meibum lipid profiles and caused severe abnormalities in the ocular surface and Meibomian gland (MG) physiology and morphology. In this study, we evaluated the effects of Soat1 ablation that were expected to cause depletion of the second largest class of Meibomian lipids (ML)-cholesteryl esters (CE)-in a mouse model. ML of the Soat1-null mice were examined using liquid chromatography high-resolution mass spectrometry and compared with those of Soat1+/- and wild-type mice. Complete suppression of CE biosynthesis and simultaneous accumulation of free cholesterol (Chl) were observed in Soat1-null mice, while Soat1+/- mutants had normal Chl and CE profiles. The total arrest of the CE biosynthesis in response to Soat1 ablation transformed Chl into the dominant lipid in meibum accounting for at least 30% of all ML. The Soat1-null mice had clear manifestations of dry eye and MG dysfunction. Enrichment of meibum with Chl and depletion of CE caused plugging of MG orifices, increased meibum rigidity and melting temperature, and led to a massive accumulation of lipid deposits around the eyes of Soat1-null mice. These findings illustrate the role of Soat1/SOAT1 in the lipid homeostasis and pathophysiology of MG.
Assuntos
Ésteres do Colesterol/metabolismo , Modelos Animais de Doenças , Disfunção da Glândula Tarsal/patologia , Glândulas Tarsais/patologia , Esterol O-Aciltransferase/fisiologia , Lágrimas/metabolismo , Animais , Homeostase , Masculino , Disfunção da Glândula Tarsal/etiologia , Disfunção da Glândula Tarsal/metabolismo , Glândulas Tarsais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Macrophages are phagocytes that play important roles in health and diseases. Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) converts cellular cholesterol to cholesteryl esters and is expressed in many cell types. Unlike global Acat1 knockout (KO), myeloid-specific Acat1 KO ( Acat1-) does not cause overt abnormalities in mice. Here, we performed analyses in age- and sex-matched Acat1-M/-M and wild-type mice on chow or Western diet and discovered that Acat1-M/-M mice exhibit resistance to Western diet-induced obesity. On both chow and Western diets, Acat1-M/-M mice display decreased adipocyte size and increased insulin sensitivity. When fed with Western diet, Acat1-M/-M mice contain fewer infiltrating macrophages in white adipose tissue (WAT), with significantly diminished inflammatory phenotype. Without Acat1, the Ly6Chi monocytes express reduced levels of integrin-ß1, which plays a key role in the interaction between monocytes and the inflamed endothelium. Adoptive transfer experiment showed that the appearance of leukocytes from Acat1-M/-M mice to the inflamed WAT of wild-type mice is significantly diminished. Under Western diet, Acat1-M/-M causes suppression of multiple proinflammatory genes in WAT. Cell culture experiments show that in RAW 264.7 macrophages, inhibiting ACAT1 with a small-molecule ACAT1-specific inhibitor reduces inflammatory responses to lipopolysaccharide. We conclude that under Western diet, blocking ACAT1 in macrophages attenuates inflammation in WAT. Other results show that Acat1-M/-M does not compromise antiviral immune response. Our work reveals that blocking ACAT1 suppresses diet-induced obesity in part by slowing down monocyte infiltration to WAT as well as by reducing the inflammatory responses of adipose tissue macrophages.
Assuntos
Acetil-CoA C-Acetiltransferase/genética , Acetil-CoA C-Acetiltransferase/fisiologia , Dieta , Inflamação/genética , Inflamação/patologia , Resistência à Insulina/genética , Macrófagos/patologia , Obesidade/genética , Esterol O-Aciltransferase/genética , Esterol O-Aciltransferase/fisiologia , Adipócitos/patologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Animais , Tamanho Celular , Feminino , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Inflamação/imunologia , Integrina beta1/metabolismo , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/fisiopatologia , Células RAW 264.7RESUMO
High levels of circulating TNF and its receptors, TNFR1 and TNFR2, predict the progression of diabetic kidney disease (DKD), but their contribution to organ damage in DKD remains largely unknown. Here, we investigated the function of local and systemic TNF in podocyte injury. We cultured human podocytes with sera collected from DKD patients, who displayed elevated TNF levels, and focal segmental glomerulosclerosis (FSGS) patients, whose TNF levels resembled those of healthy patients. Exogenous TNF administration or local TNF expression was equally sufficient to cause free cholesterol-dependent apoptosis in podocytes by acting through a dual mechanism that required a reduction in ATP-binding cassette transporter A1-mediated (ABCA1-mediated) cholesterol efflux and reduced cholesterol esterification by sterol-O-acyltransferase 1 (SOAT1). TNF-induced albuminuria was aggravated in mice with podocyte-specific ABCA1 deficiency and was partially prevented by cholesterol depletion with cyclodextrin. TNF-stimulated free cholesterol-dependent apoptosis in podocytes was mediated by nuclear factor of activated T cells 1 (NFATc1). ABCA1 overexpression or cholesterol depletion was sufficient to reduce albuminuria in mice with podocyte-specific NFATc1 activation. Our data implicate an NFATc1/ABCA1-dependent mechanism in which local TNF is sufficient to cause free cholesterol-dependent podocyte injury irrespective of TNF, TNFR1, or TNFR2 serum levels.
Assuntos
Colesterol/química , Nefropatias Diabéticas/sangue , Glomerulosclerose Segmentar e Focal/sangue , Fatores de Transcrição NFATC/fisiologia , Síndrome Nefrótica/sangue , Fator de Necrose Tumoral alfa/fisiologia , Transportador 1 de Cassete de Ligação de ATP/fisiologia , Adolescente , Albuminúria/sangue , Animais , Apoptose , Biópsia , Estudos de Casos e Controles , Criança , Pré-Escolar , Ciclodextrinas/metabolismo , Feminino , Regulação da Expressão Gênica , Taxa de Filtração Glomerular , Humanos , Inflamação , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Podócitos/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/sangue , Receptores Tipo II do Fator de Necrose Tumoral/sangue , Esterol O-Aciltransferase/fisiologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: DBA/2 apoE(-/-) mice have ≈10-fold larger lesions than AKR apoE(-/-) mice. The objective of this study was to determine whether macrophages from these 2 strains had altered cholesterol metabolism that might play a role in their divergent atherosclerosis susceptibility. APPROACH AND RESULTS: AKR and DBA/2 macrophages incubated with acetylated low-density lipoprotein resulted in higher cholesterol ester (CE) and lower free cholesterol accumulation in the DBA/2 cells. However, these strains had equivalent acetylated low-density lipoprotein uptake and cholesterol esterification activity. Cholesterol efflux from unloaded cells to apolipoprotein A-I or high-density lipoprotein was similar in the 2 strains. However, on acetylated low-density lipoprotein loading, cholesterol efflux was impaired in the DBA/2 cells, but this impairment was corrected by loading in the presence of an inhibitor of cholesterol esterification. Thus, the cholesterol efflux capabilities are similar in these strains, but there seemed to be a defect in lipid droplet-stored CE mobilization in DBA/2 cells. Lalistat 1, a specific inhibitor of lysosomal acid lipase, completely blocked the hydrolysis of lipid droplet-stored CE, implying that lipid droplet autophagy is responsible for CE turnover in these cells. CE turnover was 2-fold slower in DBA/2 versus AKR cells. Autophagic flux, estimated by a fluorescent light chain 3-II reporter and the increase in p62 levels after chloroquine treatment, was higher in AKR versus DBA/2 macrophages, which had an apparent decrease in autophagosome fusion with lysosomes. When autophagy was activated by amino acid starvation, CE levels decreased in DBA/2 cells. CONCLUSIONS: Physiological regulation of autophagy in macrophages controls CE accumulation and may modify atherosclerosis susceptibility.
Assuntos
Autofagia , Ésteres do Colesterol/metabolismo , Macrófagos/metabolismo , Animais , Colesterol/metabolismo , Hidrólise , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos DBA , Especificidade da Espécie , Esterol O-Aciltransferase/fisiologiaRESUMO
Modulation of the cholesterol-sensing liver X receptors (LXRs) and their downstream targets has emerged as promising therapeutic avenues in atherosclerosis. The intestine is important for its unique capabilities to act as a gatekeeper for cholesterol absorption and to participate in the process of cholesterol elimination in the feces and reverse cholesterol transport (RCT). Pharmacological and genetic intestine-specific LXR activation have been shown to protect against atherosclerosis. In this review we discuss the LXR-targeted molecular players in the enterocytes as well as the intestine-driven pathways contributing to cholesterol homeostasis with therapeutic potential as targets in the prevention and treatment of atherosclerosis..
Assuntos
Aterosclerose/tratamento farmacológico , Colesterol/metabolismo , Mucosa Intestinal/metabolismo , Receptores Nucleares Órfãos/fisiologia , Transportador 1 de Cassete de Ligação de ATP/fisiologia , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Aterosclerose/prevenção & controle , Homeostase , Humanos , Hipercolesterolemia/tratamento farmacológico , Absorção Intestinal/fisiologia , Lipoproteínas/genética , Receptores X do Fígado , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas de Membrana Transportadoras , Esterol O-Aciltransferase/fisiologia , Esterol O-Aciltransferase 2RESUMO
Acyl-CoA:cholesterol O-acyl transferase 2 (ACAT2) promotes cholesterol absorption by the intestine and the secretion of cholesteryl ester-enriched very low density lipoproteins by the liver. Paradoxically, mice lacking ACAT2 also exhibit mild hypertriglyceridemia. The present study addresses the unexpected role of ACAT2 in regulation of hepatic triglyceride (TG) metabolism. Mouse models of either complete genetic deficiency or pharmacological inhibition of ACAT2 were fed low fat diets containing various amounts of cholesterol to induce hepatic steatosis. Mice genetically lacking ACAT2 in both the intestine and the liver were dramatically protected against hepatic neutral lipid (TG and cholesteryl ester) accumulation, with the greatest differences occurring in situations where dietary cholesterol was elevated. Further studies demonstrated that liver-specific depletion of ACAT2 with antisense oligonucleotides prevents dietary cholesterol-associated hepatic steatosis both in an inbred mouse model of non-alcoholic fatty liver disease (SJL/J) and in a humanized hyperlipidemic mouse model (LDLr(-/-), apoB(100/100)). All mouse models of diminished ACAT2 function showed lowered hepatic triglyceride concentrations and higher plasma triglycerides secondary to increased hepatic secretion of TG into nascent very low density lipoproteins. This work demonstrates that inhibition of hepatic ACAT2 can prevent dietary cholesterol-driven hepatic steatosis in mice. These data provide the first evidence to suggest that ACAT2-specific inhibitors may hold unexpected therapeutic potential to treat both atherosclerosis and non-alcoholic fatty liver disease.
Assuntos
Fígado Gorduroso/prevenção & controle , Hiperlipidemias/prevenção & controle , Fígado/metabolismo , Esterol O-Aciltransferase/fisiologia , Triglicerídeos/metabolismo , Animais , Apolipoproteína B-100/fisiologia , Western Blotting , Ésteres do Colesterol/metabolismo , Colesterol na Dieta/administração & dosagem , Fígado Gorduroso/metabolismo , Feminino , Hiperlipidemias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de LDL/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esterol O-Aciltransferase/antagonistas & inibidores , Esterol O-Aciltransferase 2RESUMO
Cholesterol is esterified in mammals by two enzymes: LCAT (lecithin cholesterol acyltransferase) in plasma and ACAT(1) and ACAT(2) (acyl-CoA cholesterol acyltransferases) in the tissues. We hypothesized that the sterol structure may have significant effects on the outcome of esterification by these enzymes. To test this hypothesis, we analyzed sterol esters in plasma and tissues in patients having non-cholesterol sterols (sitosterolemia and Smith-Lemli-Opitz syndrome). The esterification of a given sterol was defined as the sterol ester percentage of total sterols. The esterification of cholesterol in plasma by LCAT was 67% and in tissues by ACAT was 64%. Esterification of nine sterols (cholesterol, cholestanol, campesterol, stigmasterol, sitosterol, campestanol, sitostanol, 7-dehydrocholesterol and 8-dehydrocholesterol) was examined. The relative esterification (cholesterol being 1.0) of these sterols by the plasma LCAT was 1.00, 0.95, 0.89, 0.40, 0.85, 0.82 and 0.80, 0.69 and 0.82, respectively. The esterification by the tissue ACAT was 1.00, 1.29, 0.75, 0.49, 0.45, 1.21 and 0.74, respectively. The predominant fatty acid of the sterol esters was linoleic acid for LCAT and oleic acid for ACAT. We compared the esterification of two sterols differing by only one functional group (a chemical group attached to sterol nucleus) and were able to quantify the effects of individual functional groups on sterol esterification. The saturation of the A ring of cholesterol increased ester formation by ACAT by 29% and decreased the esterification by LCAT by 5.9%. Esterification by ACAT and LCAT was reduced, respectively, by 25 and 11% by the presence of an additional methyl group on the side chain of cholesterol at the C-24 position. This data supports our hypothesis that the structure of the sterol substrate has a significant effect on its esterification by ACAT or LCAT.
Assuntos
Transtornos do Metabolismo dos Lipídeos/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/fisiologia , Sitosteroides/metabolismo , Síndrome de Smith-Lemli-Opitz/metabolismo , Esterol O-Aciltransferase/fisiologia , Esteróis/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Esterificação , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Adulto Jovem , Esterol O-Aciltransferase 2RESUMO
The enzymes acyl-coenzyme A (CoA):cholesterol acyltransferases (ACATs) are membrane-bound proteins that utilize long-chain fatty acyl-CoA and cholesterol as substrates to form cholesteryl esters. In mammals, two isoenzymes, ACAT1 and ACAT2, encoded by two different genes, exist. ACATs play important roles in cellular cholesterol homeostasis in various tissues. This chapter summarizes the current knowledge on ACAT-related research in two areas: 1) ACAT genes and proteins and 2) ACAT enzymes as drug targets for atherosclerosis and for Alzheimer's disease.
Assuntos
Esterol O-Aciltransferase/fisiologia , Sequência de Aminoácidos , Animais , Humanos , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Modelos Biológicos , Dados de Sequência Molecular , Conformação Proteica , Esterol O-Aciltransferase/química , Esterol O-Aciltransferase/genética , Esterol O-Aciltransferase/metabolismoRESUMO
We have previously reported that human ACAT1 mRNAs produce the 50 kDa protein using the AUG(11397-1399) initiation codon, and also a minor 56 kDa isoform using the upstream in-frame GGC(1274-1276) initiation codon. The GGC(1274-1276) codon is located at the optional long 5'-untranslated region (5'-UTR, nt 1-1396) of the mRNAs. The DNA sequences corresponding to this 5'-UTR are located in two different chromosomes, 7 and 1. In the current work, we report that the optional long 5'-UTR significantly impairs the production of human ACAT1 protein initiated from the AUG(1397-1399)codon, mainly by promoting its mRNA decay. The western blot analyses indicated that the optional long 5'-UTR potently impaired the production of different proteins initiated from the AUG(1397-1399) codon, meaning that this impairing effect was not influenced by the 3'-UTR or the coding sequence of ACAT1 mRNA. The results of reverse transcription-quantitative polymerase chain reaction demonstrated that this 5'- UTR dramatically reduced the contents of its linked mRNAs. Analyses of the protein to mRNA ratios showed that this 5'-UTR mainly decreased its mRNA stability rather than altering its translational efficiency. We next performed the plasmid transfection experiments and used actinomycin D to inhibit transcription. The results showed that this 5'-UTR promoted its mRNA decay. Additional transfection and nucleofection experiments using RNAs prepared in vitro illustrated that, in both the cytoplasm and the nucleus of cells, the optional long 5'-UTR-linked mRNAs decayed faster than those without the link. Overall, our study brings new insight to the regulation of the human ACAT1 gene expression at the post-transcription level.
Assuntos
Regiões 5' não Traduzidas , RNA Mensageiro/genética , Esterol O-Aciltransferase/fisiologia , Animais , Sequência de Bases , Western Blotting , Células CHO , Códon , Cricetinae , Cricetulus , Primers do DNA , Humanos , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esterol O-Aciltransferase/biossíntese , Esterol O-Aciltransferase/genéticaRESUMO
The conserved Rho-type GTPase Cdc42p is a key regulator of signal transduction and polarity in eukaryotic cells. In the yeast Saccharomyces cerevisiae, Cdc42p promotes polarized growth through the p21-activated kinases Ste20p and Cla4p. Previously, we demonstrated that Ste20p forms a complex with Erg4p, Cbr1p and Ncp1p, which all catalyze important steps in sterol biosynthesis. CLA4 interacts genetically with ERG4 and NCP1. Furthermore, Erg4p, Ncp1p and Cbr1p play important roles in cell polarization during vegetative growth, mating and filamentation. As Ste20p and Cla4p are involved in these processes it seems likely that sterol biosynthetic enzymes and p21-activated kinases act in related pathways. Here, we demonstrate that the deletion of either STE20 or CLA4 results in increased levels of sterols. In addition, higher concentrations of steryl esters, the storage form of sterols, were observed in cla4Delta cells. CLA4 expression from a multicopy plasmid reduces enzyme activity of Are2p, the major steryl ester synthase, under aerobic conditions. Altogether, our data suggest that Ste20p and Cla4p may function as negative modulators of sterol biosynthesis. Moreover, Cla4p has a negative effect on steryl ester formation. As sterol homeostasis is crucial for cell polarization, Ste20p and Cla4p may regulate cell polarity in part through the modulation of sterol homeostasis.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Esteróis/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/fisiologia , Polaridade Celular/efeitos dos fármacos , Homeostase , Peptídeos e Proteínas de Sinalização Intracelular/genética , MAP Quinase Quinase Quinases , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Esterol O-Aciltransferase/fisiologiaRESUMO
We investigated the mechanism of spontaneous cholesterol efflux induced by acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibition, and how an alteration of cholesterol metabolism in macrophages impacts on that in HepG2 cells. Oleic acid anilide (OAA), a known ACAT inhibitor reduced lipid storage substantially by promotion of cholesterol catabolism and repression of cholesteryl ester accumulation without further increase of cytotoxicity in acetylated low-density lipoprotein-loaded THP-1 macrophages. Analysis of expressed mRNA and protein revealed that cholesterol 7alpha-hydroxylase (CYP7A1), oxysterol 7alpha- hydroxylase (CYP7B1), and cholesterol 27-hydroxylase (CYP27) were highly induced by ACAT inhibition. The presence of a functional cytochrome P450 pathway was confirmed by quantification of the biliary cholesterol mass in cell monolayers and extracelluar medium. Notably, massively secreted biliary cholesterol from macrophages suppressed the expression of CYP7 proteins in a farnesoid X receptor (FXR)-dependent manner in HepG2 cells. The findings reported here provide new insight into mechanisms of spontaneous cholesterol efflux, and suggest that ACAT inhibition may stimulate cholesterol-catabolic (cytochrome P450) pathway in lesion-macrophages, in contrast, suppress it in hepatocyte via FXR induced by biliary cholesterol (BC).
Assuntos
Anilidas/farmacologia , Colesterol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hepatócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Ácidos Oleicos/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Esterol O-Aciltransferase/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Bile/metabolismo , Células Cultivadas , Ésteres do Colesterol/metabolismo , Proteínas de Ligação a DNA/agonistas , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Macrófagos/metabolismo , Modelos Biológicos , Receptores Citoplasmáticos e Nucleares/agonistas , Esterol O-Aciltransferase/fisiologia , Fatores de Transcrição/agonistasRESUMO
Targeted deletion of acyl-CoA:cholesterol acyltransferase 2 (ACAT2) (A2), especially in the liver, protects hyperlipidemic mice from diet-induced hypercholesterolemia and atherosclerosis, whereas the deletion of ACAT1 (A1) is not as effective, suggesting ACAT2 may be the more appropriate target for treatment of atherosclerosis. Among the numerous ACAT inhibitors known, pyripyropene A (PPPA) is the only compound that has high selectivity (>2000-fold) for inhibition of ACAT2 compared with ACAT1. In the present study we sought to determine the PPPA interaction site of ACAT2. To achieve this goal we made several chimeric proteins where parts of ACAT2 were replaced by the analogous region of ACAT1. Differences in the amino acid sequence and the membrane topology were utilized to design the chimeras. Among chimeras, A2:1-428/A1:444-550 had 50% reduced PPPA selectivity, whereas C-terminal-truncated ACAT2 mutant A2:1-504 (C-terminal last 22 amino acids were deleted) remained selectively inhibited, indicating the PPPA-sensitive site is located within a region between amino acids 440 and 504. Three additional chimeras within this region helped narrow down the PPPA-sensitive site to a region containing amino acids 480-504, representing the fifth putative transmembrane domain of ACAT2. Subsequently, for this region we made single amino acid mutants where each amino acid in ACAT2 was individually changed to its ACAT1 counterpart. Mutation of Q492L, V493L, S494A resulted in only 30, 50, and 70% inhibition of the activity by PPPA, respectively (as opposed to greater than 95% with the wild type enzyme), suggesting these three residues are responsible for the selective inhibition by PPPA of ACAT2. Additionally, we found that PPPA non-covalently interacts with ACAT2 apparently without altering the oligomeric structure of the protein. The present study provides the first evidence for a unique motif in ACAT2 that can be utilized for making an ACAT2-specific drug.
Assuntos
Piridinas/química , Sesquiterpenos/química , Esterol O-Aciltransferase/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Cinética , Modelos Biológicos , Dados de Sequência Molecular , Isoformas de Proteínas , Proteínas Recombinantes de Fusão/química , Esterol O-Aciltransferase 2RESUMO
Deletion of acyl-CoA:cholesterol O-acyltransferase 2 (ACAT2) in mice results in resistance to diet-induced hypercholesterolemia and protection against atherosclerosis. Recently, our group has shown that liver-specific inhibition of ACAT2 via antisense oligonucleotide (ASO)-mediated targeting likewise limits atherosclerosis. However, whether this atheroprotective effect was mediated by: 1) prevention of packaging of cholesterol into apoB-containing lipoproteins, 2) augmentation of nascent HDL cholesterol secretion, or 3) increased hepatobiliary sterol secretion was not examined. Therefore, the purpose of these studies was to determine whether hepatic ACAT2 is rate-limiting in all three of these important routes of cholesterol homeostasis. Liver-specific depletion of ACAT2 resulted in reduced packaging of cholesterol into apoB-containing lipoproteins (very low density lipoprotein, intermediate density lipoprotein, and low density lipoprotein), whereas high density lipoprotein cholesterol levels remained unchanged. In the liver of ACAT2 ASO-treated mice, cholesterol ester accumulation was dramatically reduced, yet there was no reciprocal accumulation of unesterified cholesterol. Paradoxically, ASO-mediated depletion of hepatic ACAT2 promoted fecal neutral sterol excretion without altering biliary sterol secretion. Interestingly, during isolated liver perfusion, ACAT2 ASO-treated livers had augmented secretion rates of unesterified cholesterol and phospholipid. Furthermore, we demonstrate that liver-derived cholesterol from ACAT2 ASO-treated mice is preferentially delivered to the proximal small intestine as a precursor to fecal excretion. Collectively, these studies provide the first insight into the hepatic itinerary of cholesterol when cholesterol esterification is inhibited only in the liver, and provide evidence for a novel non-biliary route of fecal sterol loss.
Assuntos
Colesterol/metabolismo , Fígado/metabolismo , Esterol O-Aciltransferase/genética , Esterol O-Aciltransferase/fisiologia , Esteróis/metabolismo , Animais , Apolipoproteínas B/metabolismo , Sistema Biliar/metabolismo , Ésteres do Colesterol/metabolismo , Fezes , Lipoproteínas LDL/metabolismo , Masculino , Camundongos , Camundongos Knockout , Oligonucleotídeos Antissenso/química , Fosfolipídeos/metabolismo , Esterol O-Aciltransferase 2RESUMO
Acyl-coenzyme A:cholesterol acyltransferase (ACAT) esterifies free cholesterol and stores cholesteryl esters in lipid droplets. Macrophage ACAT1 deficiency results in increased atherosclerotic lesion area in hyperlipidemic mice via disrupted cholesterol efflux, increased lipoprotein uptake, accumulation of intracellular vesicles, and accelerated apoptosis. The objective of this study was to determine whether lipid synthesis is affected by ACAT1. The synthesis, esterification, and efflux of new cholesterol were measured in peritoneal macrophages from ACAT1(-/-) mice. Cholesterol synthesis was increased by 134% (p=0.001) in ACAT1(-/-) macrophages compared to wildtype macrophages. Increased synthesis resulted in a proportional increase in the efflux of newly synthesized cholesterol. Although the esterification of new cholesterol was reduced by 93% (p<0.001) in ACAT1(-/-) macrophages, trace amounts of newly synthesized cholesteryl esters were detectable. Furthermore, the expression of SREBP1a mRNA was increased 6-fold in ACAT1(-/-) macrophages compared to wildtype macrophages, suggesting an up-regulation of cholesterol and fatty acid synthesis in ACAT1(-/-) macrophages. Increased cholesterol synthesis and up-regulation of SREBP in ACAT1(-/-) macrophages suggests that ACAT1 affects the regulation of lipid metabolism in macrophages. This change in cholesterol homeostasis may contribute to the atherogenic potential of ACAT1(-/-) macrophages.
Assuntos
Colesterol/biossíntese , Macrófagos Peritoneais/enzimologia , Esterol O-Aciltransferase/deficiência , Animais , Transporte Biológico , Células Cultivadas , Colesterol/sangue , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipídeos/biossíntese , Fosfolipídeos/sangue , Esterol O-Aciltransferase/fisiologiaRESUMO
Acyl-coenzyme A:cholesterol transferase (ACAT) is an integral membrane protein localized in the endoplasmic reticulum. ACAT catalyzes the formation of cholesteryl esters from cholesterol and fatty acyl coenzyme A. The cholesteryl esters are stored as cytoplasmic lipid droplets inside the cell. This process is very important to the organism as high cholesterol levels have been associated with cardiovascular disease. In mammals, two ACAT genes have been identified, ACAT1 and ACAT2. ACAT1 is ubiquitous and is responsible for cholesteryl ester formation in brain, adrenal glands, macrophages, and kidneys. ACAT2 is expressed in the liver and intestine. The inhibition of ACAT activity has been associated with decreased plasma cholesterol levels by suppressing cholesterol absorption and by diminishing the assembly and secretion of apolipoprotein B-containing lipoproteins such as very low density lipoprotein (VLDL). ACAT inhibition also prevents the conversion of macrophages into foam cells in the arterial walls, a critical event in the development of atherosclerosis. This review paper will focus on the role of ACAT in cholesterol metabolism, in particular as a target to develop novel therapeutic agents to control hypercholesterolemia, atherosclerosis, and Alzheimer's disease.
Assuntos
Aterosclerose/prevenção & controle , Inibidores Enzimáticos/farmacologia , Hipolipemiantes/farmacologia , Esterol O-Aciltransferase/antagonistas & inibidores , Animais , Aterosclerose/enzimologia , Colesterol/metabolismo , Humanos , Esterol O-Aciltransferase/fisiologia , Esterol O-Aciltransferase 2RESUMO
A gene encoding a sterol ester-synthesizing enzyme was identified in Arabidopsis. The cDNA of the Arabidopsis gene At1g04010 (AtPSAT) was overexpressed in Arabidopsis behind the cauliflower mosaic virus 35S promoter. Microsomal membranes from the leaves of overexpresser lines catalyzed the transacylation of acyl groups from phosphatidylethanolamine to sterols. This activity correlated with the expression level of the AtPSAT gene, thus demonstrating that this gene encodes a phospholipid:sterol acyltransferase (PSAT). Properties of the AtPSAT were examined in microsomal fractions from the tissues of an overexpresser. The enzyme did not utilize neutral lipids, had the highest activity with phosphatidylethanolamine, had a 5-fold preference for the sn-2 position, and utilized both saturated and unsaturated fatty acids. Various sterols and sterol intermediates, including triterpenic precursors, were acylated by the PSAT, whereas other triterpenes were not. Sterol selectivity studies showed that the enzyme is activated by end product sterols and that sterol intermediates are preferentially acylated by the activated enzyme. This indicates that PSAT both regulates the pool of free sterols as well as limits the amount of free sterol intermediates in the membranes. Two T-DNA insertion mutants in the AtPSAT gene, with strongly reduced (but still measurable) levels of sterol esters in their tissues, had no detectable PSAT activity in the microsomal fractions, suggesting that Arabidopsis possess other enzyme(s) capable of acylating sterols. The AtPSAT is the only intracellular enzyme found so far that catalyzes an acyl-CoA-independent sterol ester formation. Thus, PSAT has a similar physiological function in plant cells as the unrelated acyl-CoA:sterol acyltransferase has in animal cells.
Assuntos
Aciltransferases/química , Arabidopsis/genética , Arabidopsis/metabolismo , Ésteres/química , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/química , Esterol O-Aciltransferase/fisiologia , Esteróis/química , Aciltransferases/fisiologia , Sequência de Aminoácidos , Clonagem Molecular , DNA Bacteriano/química , DNA Complementar/metabolismo , Vetores Genéticos , Membranas Intracelulares/metabolismo , Lipídeos/química , Microssomos/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Plantas/genética , Plantas/metabolismo , Regiões Promotoras Genéticas , RNA/química , RNA/metabolismo , Esterol O-Aciltransferase/química , Especificidade por SubstratoRESUMO
7-Ketocholesterol (7KC) is a cytotoxic component of oxidized low density lipoproteins (OxLDLs) and induces apoptosis in macrophages by a mechanism involving the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we examined the role of ACAT in 7KC-induced and OxLDL-induced apoptosis in murine macrophages. An ACAT inhibitor, Sandoz 58-035, suppressed 7KC-induced apoptosis in P388D1 cells and both 7KC-induced and OxLDL-induced apoptosis in mouse peritoneal macrophages (MPMs). Furthermore, compared with wild-type MPMs, ACAT-1-deficient MPMs demonstrated significant resistance to both 7KC-induced and OxLDL-induced apoptosis. Macrophages treated with 7KC accumulated ACAT-derived [14C]cholesteryl and [3H]7-ketocholesteryl esters. Tandem LC-MS revealed that the 7KC esters contained primarily saturated and monounsaturated fatty acids. An inhibitor of cPLA2, arachidonyl trifluoromethyl ketone, prevented the accumulation of 7KC esters and inhibited 7KC-induced apoptosis in P388D1 cells. The decrease in 7KC ester accumulation produced by the inhibition of cPLA2 was reversed by supplementing with either oleic or arachidonic acid (AA); however, only AA supplementation restored the induction of apoptosis by 7KC. These results suggest that 7KC not only initiates the apoptosis pathway by activating cPLA2, as we have reported previously, but also participates in the downstream signaling pathway when esterified by ACAT to form 7KC-arachidonate.
Assuntos
Apoptose/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Macrófagos/fisiologia , Esterol O-Aciltransferase/fisiologia , Animais , Ácido Araquidônico/antagonistas & inibidores , Ácido Araquidônico/farmacologia , Radioisótopos de Carbono , Caspase 3 , Caspases/metabolismo , Linhagem Celular , Ésteres do Colesterol/metabolismo , Inibidores Enzimáticos/farmacologia , Esterificação/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , Cetocolesteróis/metabolismo , Cetocolesteróis/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/metabolismo , Fosfolipases A2 , Esterol O-Aciltransferase/antagonistas & inibidores , TrítioRESUMO
In contrast to some published studies of murine macrophages, we previously showed that ACAT inhibitors appeared to be anti-atherogenic in primary human macrophages in that they decreased foam cell formation without inducing cytotoxicity. Herein, we examined foam cell formation and cytotoxicity in murine ACAT1 knockout (KO) macrophages in an attempt to resolve the discrepancies. Elicited peritoneal macrophages from normal C57BL6 and ACAT1 KO mice were incubated with DMEM containing acetylated LDL (acLDL, 100 microg protein/ml) for 48h. Cells became cholesterol enriched and there were no differences in the total cholesterol mass. Esterified cholesterol mass was lower in ACAT1 KO foam cells compared to normal macrophages (p<0.04). Cytotoxicity, as measured by the cellular release of [(14)C]adenine from macrophages, was approximately 2-fold greater in ACAT1 KO macrophages as compared to normal macrophages (p<0.0001), and this was independent of cholesterol enrichment. cDNA microarray analysis showed that ACAT1 KO macrophages expressed substantially less collagen type 3A1 (26-fold), which was confirmed by RT-PCR. Total collagen content was also significantly reduced (57%) in lung homogenates isolated from ACAT1 KO mice (p<0.02). Thus, ACAT1 KO macrophages show biochemical changes consistent with increased cytotoxicity and also a novel association with decreased expression of collagen type 3A1.
Assuntos
Colágeno Tipo III/metabolismo , Células Espumosas/enzimologia , Macrófagos/enzimologia , Esterol O-Aciltransferase/fisiologia , Acetil-CoA C-Acetiltransferase , Animais , Sobrevivência Celular , Colesterol/metabolismo , Colágeno Tipo III/genética , Células Espumosas/metabolismo , Deleção de Genes , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismo , Esterol O-Aciltransferase/genéticaRESUMO
The relative contributions of ACAT2 and LCAT to the cholesteryl ester (CE) content of VLDL and LDL were measured. ACAT2 deficiency led to a significant decrease in the percentage of CE (37.2 +/- 2.1% vs. 3.9 +/- 0.8%) in plasma VLDL, with a concomitant increase in the percentage of triglyceride (33.0 +/- 3.2% vs. 66.7 +/- 2.5%). Interestingly, the absence of ACAT2 had no apparent effect on the percentage CE in LDL, whereas LCAT deficiency significantly decreased the CE percentage (38.6 +/- 4.0% vs. 54.6 +/- 1.9%) and significantly increased the phospholipid percentage (11.2 +/- 0.9% vs. 19.3 +/- 0.1%) of LDL. When both LCAT and ACAT2 were deficient, VLDL composition was similar to VLDL of the ACAT2-deficient mouse, whereas LDL was depleted in core lipids and enriched in surface lipids, appearing discoidal when observed by electron microscopy. We conclude that ACAT2 is important in the synthesis of VLDL CE, whereas LCAT is important in remodeling VLDL to LDL. Liver perfusions were performed, and perfusate apolipoprotein B accumulation rates in ACAT2-deficient mice were not significantly different from those of controls; perfusate VLDL CE decreased from 8.0 +/- 0.8% in controls to 0 +/- 0.7% in ACAT2-deficient mice. In conclusion, our data establish that ACAT2 provides core CE of newly secreted VLDL, whereas LCAT adds CE during LDL particle formation.