ACAT Activity Research Articles

Molecular structures of human ACAT2 disclose mechanism for selective inhibition

Endoplasmic reticulum-localized acyl-CoA:cholesterol acyltransferases (ACAT), including ACAT1 and ACAT2, convert cholesterol to cholesteryl esters that become incorporated into lipoproteins or stored in cytosolic lipid droplets. Selective inhibition of ACAT2 has been shown to considerably attenuate hypercholesterolemia and atherosclerosis in mice. Here, we report cryogenic electron microscopy structures of human ACAT2 bound to its specific inhibitor pyripyropene A or the general ACAT inhibitor nevanimibe. Structural analysis reveals that ACAT2 has a topology in membranes similar to that of ACAT1. A catalytic core with an entry site occupied by a cholesterol molecule and another site for allosteric activation of ACAT2 is observed in these structures. Enzymatic assays show that mutations within sites of cholesterol entry or allosteric activation attenuate ACAT2 activity invitro. Together, these results reveal mechanisms for ACAT2-mediated esterification of cholesterol, providing a blueprint to design new ACAT2 inhibitors for use in the prevention of cardiovascular disease.

Long-term outcome in patients with juvenile dermatomyositis: A case series

BackgroundFollow-up of juvenile dermatomyositis (JDM) patients has demonstrated the impact of the disease on several organs in the long term. ObjectiveAs there is little information on the long-term outcome of JDM, we aimed to assess long-term outcomes in a series of JDM patients. MethodsAfter selection of JDM patients, a consultation with a dermatologist and a rheumatologist was held for each patient. Cutaneous, muscle, and disease damage was assessed using different validated scores including the abbreviated Cutaneous Assessment Tool (aCAT), 8-muscle Manual Muscle Testing (MMT8), Childhood Myositis Assessment Scale (CMAS), Myositis Damage Index (MDI), Childhood Health Assessment Questionnaire (CHAQ), and Health Assessment Questionnaire (HAQ). Long-term disease outcomes were recorded including growth and pubertal development, educational and vocational achievement, and development of comorbidities. ResultsSeven patients were included in the study. After a mean follow-up of 14.9±8.8 years, the mean aCAT score was 0.57±1.4 and only one patient had a positive aCAT activity score. The mean aCAT damage score was 1.4±1.3 and five (71%) patients had a score of ≥1. Five (71.4%) patients had normal muscle strength with an MMT8 score of >72, and none had severe muscle weakness (MMT8 ≤32, and CMAS<35). The mean total extent of damage according to the MDI was ≥1 in five (71%) patients and mainly involved the skin. Two (29%) patients had mild disability according to the CHAQ/HAQ disability index. In terms of quality of life, no patient had a score of<40 (1 SD below the mean for healthy controls). ConclusionsBased on validated cutaneous and musculoskeletal scores, our study demonstrated the good functional outcomes of JDM at long-term follow-up.

Dietary caffeic acid, ferulic acid and coumaric acid supplements on cholesterol metabolism and antioxidant activity in rats

This study was designed to test the lipid-lowering and antioxidative activities of three phenolic compounds, caffeic acid, ferulic acid and coumaric acid. Four groups of rats were given a semisynthetic diet containing 3% of cholesterol for 6 weeks. The control group only received a high cholesterol diet, whereas the other three groups received a diet including 0.2% of caffeic acid, 0.2% of ferulic acid and 0.2% of coumaric acid. The caffeic acid, ferulic acid and coumaric acid significantly lowered the plasma lipid and hepatic cholesterol levels compared to those in the control (p < 0.05). The hepatic HMG-CoA reductase activity was significantly lower in the caffeic acid group than in the ferulic acid and coumaric acid groups (p < 0.05), while the hepatic ACAT activity was significantly lower in the caffeic acid, ferulic acid and coumaric acid groups compared to the control group (p < 0.05). The overall potential of the antioxidant system was significantly enhanced by the caffeic acid, ferulic acid and coumaric acid supplements as the plasma and hepatic TBARS levels were lowered while the hepatic SOD activities and GSH concentration were elevated in the high-cholesterol-fed rats (p < 0.05). Furthermore, these results indicated that the supplementation of caffeic acid, ferulic acid and coumaric acid boosted the antioxidant activity in rats and promoted the excretion of neutral sterol and acidic sterol (p < 0.05), thereby leading to a decreased absorption of dietary cholesterol as well as lower plasma, hepatic cholesterol and promoted excretion of fecal sterols.

Molecular insight into the mechanism of lipid regulating effect of Alisma orientalis based on ACAT

We studied the lipid-regulating effect and molecular mechanism of the medical components of Alisma orientalis: alisol A, alisol B, 23-acetyl alisol C (23C) and the 3 (alisol A): 1(alisol B):1(23C) and 2(alisol A):2(alisol B):1(23C) mixtures designed based on the ratio of them in Alisma orientalis from Fujian, Guangxi Province, China. The animal experiment and network pharmacology showed that ACAT was one of its lipid-regulating targets and alisols may reduce the level of TC by inhibiting ACAT activity. The molecular simulation and homologous modeling results suggested that the binding of alisol mixtures with ACAT was stronger than that of monomers because alisol monomers acted on different active regions of ACAT resulting in the superposition effect and caused the synergistic effect. The lipid-regulating effect of Fujian mixture was stronger than that of Guangxi mixture showing that 3:1:1 was a better ratio. The N-terminal lipid-regulating activity of ACAT was stronger than that of transmembrane domain 1.

The influence of different amounts of zinc citrate on blood biochemical indices and productivity of rabbit organism

Deficiency of micro- and macronutrients in the organism of young rabbits causes disorders of metabolism and decreased productivity. Inclusion of essential micronutrients made by nanotechnology to the rabbit diet can eliminate this problem. Therefore, the purpose of our research was to investigate the influence of different amounts of zinc citrate in combination with cobalt and chromium citrates on the biochemical processes in the organism and the productivity of rabbits from 62 to 86 days of age. Researches were performed on rabbits 40 days of age, weighing 1.2–1.4 kg. The animals of the experimental groups were fed with fodder ration of the control group and during the day were fed with cobalt citrate in the amount of 40 μg Co/kg of body weight, chromium citrate – 40 mcg Cr/kg of body weight and zinc citrate, calculated respectively in I, II and III of experimental groups 0.25; 0.50 and 0.75 mg Zn/kg of body weight. In the preparatory period – on the 10th day and in the experimental on the 12th, 24th and 36th days of drinking the supplements in 4 animals from the group blood samples were taken for biochemical researches. Body weight gain was also determined by weighing. It was found that in the blood of animals of the first experimental group on the 12th day of drinking mineral additives increased the cholesterol content on 28.8%, on 24th day – decrease by 39.1% and increase – creatinine by 11.6%, and on 36th day – increase in blood of total protein on 12.3%, increase of alkaline phosphatase activity by 23.0%, creatinine content by 15.5% and average daily growth by 9.4%. A similar additive containing zinc citrate of 0.50 mg Zn/kg of body weight in the blood of rabbits of the second experimental group increased the activity of ACAT by 12 and 24 days, respectively, by 37.3 and 22.1%, respectively, and reduced their cholesterol content by 33.9 and 63.1% compared to the control. Whereas in the same group the growth of ALT activity was noted by 23.7%, and the average daily increase was higher than the control indicator by 7.9% 36 days of the research in comparison with the control group. The high content of zinc citrate in the mineral supplement (0.75 mg Zn/kg) in the blood of rabbits of the third experimental group contributed to the increase of the total protein concentration in all periods of the research, AST activity increased by 26.1% on the 24th day, AAT by 34.9% and the content of triacylglycerols and cholesterol was decreased, while on the 36th day the AAT activity increased by 20.3%, under these conditions, the average daily weight gain of rabbits was the highest and amounted to 38.4 g, which was 12.9% higher than the control. Therefore, the additive in the content of citrates of Co and Cr in the amount of 40 μg/kg of body weight and Zn citrate in the amount of 0.75 mg Zn/kg of body weight contributed to the improvement of metabolic processes and increase of growth indices, which may indicate the dose-dependent influence of the additives used in the rabbit young body.

Enhanced acyl-CoA:cholesterol acyltransferase activity increases cholesterol levels on the lipid droplet surface and impairs adipocyte function

Cholesterol plays essential structural and signaling roles in mammalian cells, but too much cholesterol can cause cytotoxicity. Acyl-CoA:cholesterol acyltransferases 1 and 2 (ACAT1/2) convert cholesterol into its storage form, cholesteryl esters, regulating a key step in cellular cholesterol homeostasis. Adipose tissue can store >50% of whole-body cholesterol. Interestingly, however, almost no ACAT activity is present in adipose tissue, and most adipose cholesterol is stored in its free form. We therefore hypothesized that increased cholesterol esterification may have detrimental effects on adipose tissue function. Here, using several approaches, including protein overexpression, quantitative RT-PCR, immunofluorescence, and various biochemical assays, we found that ACAT1 expression is significantly increased in the adipose tissue of the ob/ob mice. We further demonstrated that ACAT1/2 overexpression partially inhibited the differentiation of 3T3-L1 preadipocytes. In mature adipocytes, increased ACAT activity reduced the size of lipid droplets (LDs) and inhibited lipolysis and insulin signaling. Paradoxically, the amount of free cholesterol increased on the surface of LDs in ACAT1/2-overexpressing adipocytes, accompanied by increased LD localization of caveolin-1. Moreover, cholesterol depletion in adipocytes by treating the cells with cholesterol-deficient media or β-cyclodextrins induced changes in cholesterol distribution that were similar to those caused by ACAT1/2 overexpression. Our results suggest that ACAT1/2 overexpression increases the level of free cholesterol on the LD surface, thereby impeding adipocyte function. These findings provide detailed insights into the role of free cholesterol in LD and adipocyte function and suggest that ACAT inhibitors have potential utility for managing disorders associated with extreme obesity.

Anti-ApoA-1 IgGs in Familial Hypercholesterolemia Display Paradoxical Associations with Lipid Profile and Promote Foam Cell Formation.

Aims: Anti-Apolipoprotein A-1 autoantibodies (anti-ApoA-1 IgG) promote atherogenesis via innate immune receptors, and may impair cellular cholesterol homeostasis (CH). We explored the presence of anti-ApoA-1 IgG in children (5–15 years old) with or without familial hypercholesterolemia (FH), analyzing their association with lipid profiles, and studied their in vitro effects on foam cell formation, gene regulation, and their functional impact on cholesterol passive diffusion (PD). Methods: Anti-ApoA-1 IgG and lipid profiles were measured on 29 FH and 25 healthy children. The impact of anti-ApoA-1 IgG on key CH regulators (SREBP2, HMGCR, LDL-R, ABCA1, and miR-33a) and foam cell formation detected by Oil Red O staining were assessed using human monocyte-derived macrophages. PD experiments were performed using a validated THP-1 macrophage model. Results: Prevalence of high anti-ApoA-1 IgG levels (seropositivity) was about 38% in both study groups. FH children seropositive for anti-ApoA-1 IgG had significant lower total cholesterol LDL and miR-33a levels than those who were seronegative. On macrophages, anti-ApoA-1 IgG induced foam cell formation in a toll-like receptor (TLR) 2/4-dependent manner, accompanied by NF-kB- and AP1-dependent increases of SREBP-2, LDL-R, and HMGCR. Despite increased ABCA1 and decreased mature miR-33a expression, the increased ACAT activity decreased membrane free cholesterol, functionally culminating to PD inhibition. Conclusions: Anti-ApoA-1 IgG seropositivity is frequent in children, unrelated to FH, and paradoxically associated with a favorable lipid profile. In vitro, anti-ApoA-1 IgG induced foam cell formation through a complex interplay between innate immune receptors and key cholesterol homeostasis regulators, functionally impairing the PD cholesterol efflux capacity of macrophages.

TOXICOLOGICAL EVALUATION OF INDOMETHACIN AS A RISK FACTOR FOR WORKERS’ HEALTH

Introduction. The toxicity of indomethacin was studied for its hygienic regulation. Material and methods. The toxic properties of indomethacin in the experiments on out-bred and linear mice, rats, Guinea pigs and rabbits contained in standard vivarium conditions and quarantined have been studied. In the experiments, various modes (single, repeated, chronic) and ways of exposure (intragastric, inhalation, epicutaneous) were modeled. The average lethal dose (LD50) of Indomethacin and the threshold of a single acute action (Limac) were determined; irritant effect on the skin and mucous membranes, cumulative and allergenic activity were revealed. In subacute and chronic intake to the body, the main target organs were determined on the based of the results of biochemical and hematological studies. Results. DL50 for male rats, females and male mice, when introduced into the stomach, were have been established to be 20, 15 and 25.6 mg/kg respectively. It refers to the substances of hazard class 2. DL50 in the intraperitoneal introduction for the rats accounted for 13.8 mg/kg, for Guinea pigs - 500 mg/kg. The clinical picture of acute poisoning in mice and rats was characterized by low mobility, decreasing breathing, ataxia, muscle relaxation, anorexia, diarrhea, ulceration with the perforation of the intestines, and the death on the 2-4th days after the poisoning. In the experiments on Guinea pigs, the ulcerogenic effect was not detected. Local irritant effect on the skin and mucous membranes of the eyes was not revealed. It has a marked skin-resorptive action causing ulcerogenic effect and the death of the animals after 6 applications. The introduction of verospiron to the rats in a dose of 25 mg/kg simultaneously with the application of indomethacin ointment on the skin prevented the ulcer development in the gastrointestinal tract and the death of the animals. No sensitizing effect was detected. It has an average cumulative ability: the cumulation coefficient amounted to 2.6. In a subacute experiment, there was a decrease in the body temperature and summation-threshold index, an increase in the vertical motor activity and a threshold of pain sensitivity. During the study of blood serum an increase in AcAt activity, a rise of chlorides in the blood serum and their decrease in the urine, and an increase in the number of erythrocytes and hemoglobin in peripheral blood were revealed. In the pathomorphological study, there was an increase in the coefficients of liver mass and ulceration of the stomach and intestines. The threshold of acute inhalation action accounted for 0.52 mg/m3 (by the reduction of the summation-threshold index and the content of sodium and chlorides in the urine). Conclusion. The maximum permissible concentration of indomethacin in the air of the working area was of 0.05 mg/m3 with the mark “special protection of the skin and eyes”, hazard class 1, aerosol.

The effect of bergamot polyphenolic fraction on lipid transfer protein system and vascular oxidative stress in a rat model of hyperlipemia

BackgroundExperimental and epidemiological studies show that bergamot polyphenolic fraction (BPF) ameliorates the serum lipemic profile, normalizes blood pressure and improves non alcoholic fatty liver disease in patients suffering from metabolic syndrome. Despite this evidence, the molecular mechanisms responsible for these beneficial effects remain unclear. The aim of our study is to clarify the effects of BPF on the lipoprotein assembly and to identify oxidative stress biomarkers correlating hyperlipidaemia and BPF-induced metabolic changes.MethodsMale Wistar rats (180–200 g) were randomly assigned to receive a standard diet, a hypercholesterolemic diet or a hypercholesterolemic diet+BPF (20 mg/Kg/rat daily, gavage), respectively, for 90 days. Total cholesterol (tChol), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglycerides (TG) and fasting plasma glucose were evaluated at the baseline as well as at the end of the treatment. To assess the effect of BPF on the Lipid Transfer Protein System, detection of ACAT, LCAT, CETP, PON1, Apo A1 and Apo B have also been carried out. Finally, the lipid peroxidation biomarker (TBARS) and oxyLDL were also measured.ResultsBPF prevented tChol, LDL-C, TG and fasting plasma glucose enhancement and improved HDL-C. Treatment of hyperlipæmic rats with BPF significantly restored altered the serum concentration of lipemic biomarkers and the activity of ACAT, LCAT, CETP and PON1, an effect accompanied by the concomitant normalization of Apo A1 and APO B levels. In addition, TBARS levels were reduced significantly by the treatment with BPF.ConclusionsBPF prevents diet-induced alteration of the lipid profile in rats, counteracting oxidative stress and improving the dysregulation of the Lipid Transfer Protein System. These data add new insights into the molecular mechanisms underlying the beneficial role of BPF in the therapy of hyperlipidaemia, thus suggesting a novel approach in the prevention of cardiovascular disease.

A simple method to disrupt and restore subunit interaction of acyl-CoA:cholesterol acyltransferase 1

Acyl-CoA:cholestereol acyltransferase 1 (ACAT1) is a two-fold dimer (homotetramer) and has two distinct dimerization domains. One domain is in an alpha-helical rich region near the cytoplasmic N-terminus. The other is proposed to be near the C-terminus where multiple transmembrane domains promote hydrophobic interactions between two ACAT1 subunits. The truncation of the ACAT1 N-terminal dimerization domain, Δ1-65, creates a dimer which is fully enzymatically active. It is currently not known how the C-terminal dimerization domain contributes to ACAT1 enzymatic activity. Here we describe a simple method that dissociates ACAT1 dimers through the addition of the non-ionic detergents Triton X-100 or octyl glucoside which disrupt the C-terminal dimerization domain. We also document the protocols for a method to exchange Triton X-100 with CHAPS to restore C-terminal dimerization of the ACAT1 protein, and an optimized liposomal assay to assess ACAT enzymatic activity.•This method can be applied to dissociate ACAT1 subunits by using Triton X-100 or octyl glucoside.•ACAT1 dimerization can be restored by exchanging Triton X-100 with CHAPS.•The liposomal ACAT activity assay conditions have been optimized.

A New Class of EutT ATP:Co(I)rrinoid Adenosyltransferases Found in Listeria monocytogenes and Other Firmicutes Does Not Require a Metal Ion for Activity.

ATP:Co(I)rrinoid adenosyltransferases (ACATs) are involved in de novo adenosylcobamide (AdoCba) biosynthesis and in salvaging complete and incomplete corrinoids from the environment. The ACAT enzyme family is comprised of three classes of structurally and evolutionarily distinct proteins (i.e., CobA, PduO, and EutT). The structure of EutT is unknown, and an understanding of its mechanism is incomplete. The Salmonella enterica EutT ( SeEutT) enzyme is the best-characterized member of its class and is known to be a ferroprotein. Here, we report the identification and initial biochemical characterization of an enzyme representative of a new class of EutTs that does not require a metal ion for activity. In vivo and in vitro evidence shows that the metal-free EutT homologue from Listeria monocytogenes ( LmEutT) has ACAT activity and that, unlike other ACATs, the biologically active form of LmEutT is a tetramer. In vitro studies revealed that LmEutT was more efficient than SeEutT and displayed positive cooperativity. LmEutT adenosylated cobalamin, but not cobinamide, showed specificity for ATP and 2'-deoxyATP and released a triphosphate byproduct. Bioinformatics analyses suggest that metal-free EutT ACATs are also present in other Firmicutes.

In vitro exploration of ACAT contributions to lipid droplet formation during adipogenesis

As adipose tissue is the major cholesterol storage organ and most of the intracellular cholesterol is distributed to lipid droplets (LDs), cholesterol homeostasis may have a role in the regulation of adipocyte size and function. ACATs catalyze the formation of cholesteryl ester (CE) from free cholesterol to modulate the cholesterol balance. Despite the well-documented role of ACATs in hypercholesterolemia, their role in LD development during adipogenesis remains elusive. Here, we identify ACATs as regulators of de novo lipogenesis and LD formation in murine 3T3-L1 adipocytes. Pharmacological inhibition of ACAT activity suppressed intracellular cholesterol and CE levels, and reduced expression of genes involved in cholesterol uptake and efflux. ACAT inhibition resulted in decreased de novo lipogenesis, as demonstrated by reduced maturation of SREBP1 and SREBP1-downstream lipogenic gene expression. Consistent with this observation, knockdown of either ACAT isoform reduced total adipocyte lipid content by approximately 40%. These results demonstrate that ACATs are required for storage ability of lipids and cholesterol in adipocytes.

ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis

Mammalian cells synthesize various sterol molecules, including the C30 sterol, lanosterol, as cholesterol precursors in the endoplasmic reticulum. The build-up of precursor sterols, including lanosterol, displays cellular toxicity. Precursor sterols are found in plasma HDL. How these structurally different sterols are released from cells is poorly understood. Here, we show that newly synthesized precursor sterols arriving at the plasma membrane (PM) are removed by extracellular apoA-I in a manner dependent on ABCA1, a key macromolecule for HDL biogenesis. Analysis of sterol molecules by GC-MS and tracing the fate of radiolabeled acetate-derived sterols in normal and mutant Niemann-Pick type C cells reveal that ABCA1 prefers newly synthesized sterols, especially lanosterol, as the substrates before they are internalized from the PM. We also show that ABCA1 resides in a cholesterol-rich membrane domain resistant to the mild detergent, Brij 98. Blocking ACAT activity increases the cholesterol contents of this domain. Newly synthesized C29/C30 sterols are transiently enriched within this domain, but rapidly disappear from this domain with a half-life of less than 1 h. Our work shows that substantial amounts of precursor sterols are transported to a certain PM domain and are removed by the ABCA1-dependent pathway.

Geranyl Favonoid from Breadfruit Regulate Dyslipidemia in Hypercholesterolemic Rat

Hpercholesterolemia is closely related to oxidative damages from high fat and cholesterol intakes. The geranyl flavonoid (GF) from Artocarpus altilis (Breadfruit) has potential antioxidant, anti-hyperlipidemia and anti-atherogenic properties. This work was to investigate the influence of GF on synthesis of cholesterol and lipogenesis in hypercholesterolemic rat fed with high-cholesterol diet. Treatment with GF significantly increased SOD, CAT, GPx and GR activities in organ tissues, as well as decreased TBARS activity compared to the untreated group. Total glutathione, NO and α-tocopherol contents in liver, kidney and adipose tissues in GF treated group were also higher than that in untreated group. In addition, the GF treatment increased ACAT and fatty acid synthase activities, and decreased HMG-CoA reductase activity. The results suggest that treatment of GF could inhibit the synthesis of cholesterol and the lipogenesis by regulating ACAT, HMG-CoA reductase and fatty acid synthase activities and improving the antioxidant defense status in hypercholesterolemic rat.

Barley sprout extract containing policosanols and polyphenols regulate AMPK, SREBP2 and ACAT2 activity and cholesterol and glucose metabolism in vitro and in vivo

Mechanism of barley sprout extract (BS), which contains policosanols and polyphenols, on cholesterol and glucose metabolism was investigated. BS reduced the intracellular cholesterol concentrations in the HepG2 cells and the plasma cholesterol concentrations in the mice by the activation of AMPK and the subsequent phosphorylation inhibition of HMGCR. BS suppressed the nuclear translocation of SREBP2, reducing the transcription of HMGCR. AMPK activation with BS reduced the fasting glucose and hepatic triglyceride concentrations in mice by repressing the hepatic gluconeogenic genes, including fructose-1, 6-bisphosphatase and pyruvate carboxylase and the plasma levels of the proinflammatory cytokines tumor necrosis factor-α and interleukin-6. The activation of hepatic autophagy by BS was confirmed by induced protein expressions of LC3-II and LAMP. In conclusion, BS activates AMPK and hepatic autophagy and inhibits SREBP2, resulting in hypocholesterolemic and hypoglycemic activities and improvements in the symptoms of hepatic steatosis.

Ameliorative effect of Pracparatum mungo extract on high cholesterol diets in hamsters

This study was designed to test the lipid-lowering and antioxidative activities of Pracparatum mungo extract (PME), in comparison to its components berberine and glycyrrhizin in cholesterol-fed hamsters. The PME and berberine significantly lowered the atherogenic index compared to glycyrrhizin and the control (P < 0.05). The hepatic HMG-CoA reductase activity was significantly lower in the PME and berberine groups than in the glycyrrhizin group (P < 0.05), while the hepatic ACAT activity was significantly decreased by all treatments with respect to the high cholesterol fed group (P < 0.05). The overall potential of the antioxidant system was significantly enhanced by the PME, berberine and glycyrrhizin supplements as the plasma and hepatic TBARS levels were lowered while the hepatic superoxide dismutase activity and glutathione levels, HMG-CoA reductase, LDL receptor, PPAR, SREBP-2 and CYP7A1 mRNA expressions were increased by the treatments of PME and berberine in comparison with the high cholesterol fed hamsters (P < 0.05). Collectively, these results suggest that the supplementation of PME, berberine and glycyrrhizin increased antioxidant activity in hamsters. Furthermore, we observed that PME and berberine groups promoted the excretion of neutral and acidic sterols (P < 0.05), that could contribute to explain the lower plasma and hepatic cholesterol levels found in the treated animals.

Acat1 Knockdown Gene Therapy Decreases Amyloid-β in a Mouse Model of Alzheimer's Disease

Both genetic inactivation and pharmacological inhibition of the cholesteryl ester synthetic enzyme acyl-CoA:cholesterol acyltransferase 1 (ACAT1) have shown benefit in mouse models of Alzheimer's disease (AD). In this study, we aimed to test the potential therapeutic applications of adeno-associated virus (AAV)-mediated Acat1 gene knockdown in AD mice. We constructed recombinant AAVs expressing artificial microRNA (miRNA) sequences, which targeted Acat1 for knockdown. We demonstrated that our AAVs could infect cultured mouse neurons and glia and effectively knockdown ACAT activity in vitro. We next delivered the AAVs to mouse brains neurosurgically, and demonstrated that Acat1-targeting AAVs could express viral proteins and effectively diminish ACAT activity in vivo, without inducing appreciable inflammation. We delivered the AAVs to the brains of 10-month-old AD mice and analyzed the effects on the AD phenotype at 12 months of age. Acat1-targeting AAV delivered to the brains of AD mice decreased the levels of brain amyloid-β and full-length human amyloid precursor protein (hAPP), to levels similar to complete genetic ablation of Acat1. This study provides support for the potential therapeutic use of Acat1 knockdown gene therapy in AD.

HPLC‐FLD determination of NBD‐cholesterol, its ester and other metabolites in cellular lipid extracts

22-[N(-7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol), a fluorescent cholesterol analog, was an extragenous cholesterol tracer used to study cholesterol absorption and metabolism in cultured cells. In order to measure free intracellular cholesterol and its esters, a precise and sensitive method employing high-performance liquid chromatography/fluorescence detection (HPLC-FLD) was developed for the first time. Method validation showed a limit of detection at 30 ng/mL. The calibration curve was linear within the range of 0.0625-10.0 µg/mL (r(2) = 0.999). Accuracy and precision were highlighted by good recovery and low variations. Apart from NBD-cholesteryl oleate, two additional cellular metabolites of NBD-cholesterol, probably an isomer and an oxidation product, were determined in the lipid extracts of Caco-2 human colon adenocarcinoma cells according to mass spectrometry. In AC29 mouse malignant mesothelioma cells overexpressing acyl-CoA:cholesterol acyltransferase-1 (ACAT1) or ACAT2, only the oxidized metabolite was detected. Using the newly developed method, YIC-C8-434, a known ACAT inhibitor, was shown to inhibit ACAT activity in Caco-2 cells, as well as in AC29/ACAT1 or AC29/ACAT2 cells. In conclusion, the sensitive and specific HPLC-FLD method is a powerful tool for simultaneous quantification of intracellular NBD-cholesterol and its oleoyl-ester.

ACAT Inhibitory Activity of Exudates from Calocedrus Macrolepis Var. Formosana

Cholesterol acyltransferase (ACAT) is an enzyme controlling cholesterol esterification in cells. Large amounts of cholesterol esters accumulate in macrophages and smooth muscle cells of blood vessel walls resulting in the initial stages of atherosclerosis. Thus, atherosclerosis might be inhibited through inhibition of the activity of ACAT. In the present study, we identified by spectral analysis and chromatographic quantification that ferruginol was the most abundant component of exudates secreted from the wounding site of Calocedrus macrolepis Kurz var. formosana. Results obtained from the cholesterol absorption assay revealed that ferruginol exhibited a significant inhibitory activity on cholesterol absorption in mice macrophages (RAW 264.7 cell). Based on the results from analyzing the ratio of cholesterol esterification, ferruginol dose-dependently suppressed cholesterol esterification and the IC50 value was 2.0 microg/mL. In conclusion, ferruginol revealed strong inhibitory activities that retarded the absorption and esterification of cholesterol in cells. Our finding indicates that ferruginol might possess a potential for development as a pharmaceutical product for preventing arteriosclerosis.

Effect of RYR, MFA and MFB on serum lipids and antioxidant activity in rats

summary Background & aims: This study was designed to test the lipid-lowering and antioxidative activities of red yeast rice (RYR), in comparison to its components monasfluore A (MFA) and monasfluore B (MFB) in cholesterol-fed rats. Methods: The control group only received cholesterol supplemented diet, whereas the other three groups received the same diet added with 1% RYR, or 1% MFA, or 1% MFB. Results: All treatments (RYR, MFA and MFB) reduced the atherogenic index. Simply, MFA and MFB are statistically but slightly more effective than RYR (55e58% reduction versus 46% reduction). The MFA and MFB significantly lowered the atherogenic index compared to the RYR and control (P < 0.05). The hepatic HMG-CoA reductase activity was significantly lower in the MFA group than in the RYR and MFB groups (P < 0.05), while the hepatic ACAT activity was significantly decreased by all treatments with respect to the high cholesterol fed group (P < 0.05). The overall potential of the antioxidant system was significantly enhanced by the RYR, MFA and MFB supplements as the plasma and hepatic TBARS levels were lowered while the hepatic superoxide dismutase activity and glutathione levels were increased by the treatments in comparison with the high cholesterol fed rats (P < 0.05). Conclusions: Collectively, these results suggest that the supplementation of RYR, MFA and MFB increased antioxidant activity in rats. Furthermore, we observed that all treatments promoted the excretion of neutral and acidic sterols (P < 0.05), that could contribute to explain lower plasma and, hepatic cholesterol levels found in treated animal.