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Heritage agrochemicals like myclobutanil, oxyfluorfen, and pronamide, are extensively used in agriculture, with well-established studies on their animal toxicity. Yet, human toxicity assessment relies on conventional human risk assessment approaches including the utilization of animal-based ADME (Absorption, Distribution, Metabolism, and Excretion) data. In recent years, Physiologically Based Pharmacokinetic (PBPK) modelling approaches have played an increasing role in human risk assessment of many chemicals including agrochemicals. This study addresses the absence of PBPK-type data for myclobutanil, oxyfluorfen, and pronamide by generating in vitro data for key input PBPK parameters (Caco-2 permeability, rat plasma binding, rat blood to plasma ratio, and rat liver microsomal half-life), followed by generation of PBPK models for these three chemicals via the GastroPlusTM software. Incorporating these experimental input parameters into PBPK models, the prediction accuracy of plasma AUC (area under curve) was significantly improved. Validation against rat oral administration data demonstrated substantial enhancement. Steady-state plasma concentrations (Css) of pronamide aligned well with published data using measured PBPK parameters. Following validation, parent-based tissue concentrations for these agrochemicals were predicted in humans and rats after single or 30-day repeat exposure of 10 mg/kg/day. These predicted concentrations contribute valuable information for future human toxicity risk assessments of these agrochemicals. [ABSTRACT FROM AUTHOR]

Advanced compartmental absorption and transit (ACAT) based computational models have become increasingly popular in the industry for predicting oral drug product performance. However, due to its complexity, some compromises have been made in practice, and the stomach is often assigned as a single compartment. Although this assignment worked generally, it may not be sufficient to reflect the complexity of the gastric environment under certain conditions. For example, this setting was found to be less accurate in estimating stomach pH and solubilization of certain drugs under food intake, which leads to a misprediction of the food effect. To overcome the above, we explored the use of a kinetic pH calculation (KpH) for the single-compartment stomach setting. Several drugs have been tested with the KpH approach and compared with the default setting of Gastroplus. In general, the Gastroplus prediction of food effect is greatly improved, suggesting this approach is effective in improving the estimation of physicochemical properties related to food effect for several basic drugs by Gastroplus. [ABSTRACT FROM AUTHOR]

Cholesterol is an essential plasma membrane lipid for the maintenance of cellular homeostasis and cancer cell proliferation. Free cholesterol is harmful to cells; therefore, excessive free cholesterol must be quickly esterified by acetyl‐coenzyme A:cholesterol acetyltransferase (ACAT) and exported by scavenger receptor class B member I (SR‐BI) or ATP‐binding cassette protein A1 from specific cells such as macrophage foam cells, which contain cholesteryl ester‐derived vacuoles. Many vacuoles are present in the cytoplasm of Burkitt lymphoma cells. In this study, we observed that these vacuoles are often seen in high‐grade lymphomas. Cell culture study using lymphoma cell lines found that esterified cholesterol is the main component of these vacuoles and the expression of cholesterol metabolism‐related molecules was significantly upregulated in lymphoma cell lines, with SR‐BI and ACAT inhibitors (BLT‐1 and CI‐976, respectively) impeding lymphoma cell proliferation. Cytoplasmic free cholesterol was increased by ACAT and SR‐BI inhibitors, and the accumulation of free cholesterol induced lymphoma cell apoptosis by inducing endoplasmic reticulum stress. Furthermore, synergistic effects of SR‐BI and ACAT inhibitors were observed in a preclinical study. Treatment with SR‐BI inhibitor suppressed lymphoma progression in a tumor‐bearing mouse model, whereas ACAT inhibitor did not. Therefore, SR‐BI inhibitors are potential new antilymphoma therapeutics that target cholesterol metabolism. [ABSTRACT FROM AUTHOR]

An affordable, stable, reliable and sustainable power supply is the need of the hour, keeping in view the journey to a smart green grid. The present study and derivation of adaptive critical angle threshold (ACAT) contributes to this effect. The ACAT serves as a key index and an indicator to grid operator in proactive mitigation of angular instability in the grid, which is seen to be one of the major initiator of blackouts worldwide. The synchrophasor technology enables accurate calculation of ACAT on real time basis by time synchronised sampling of local voltage and current phasors. The real time calculation makes the index adaptive to variation in system topologies as well as system vulnerabilities. Furthermore, the local system model formed for deriving ACAT has less computational burden while capturing the dynamics of external system, without requirement of intricate power system details. The angular separation of the station bus under supervision can be monitored with respect to the critical angle and serve as a key indicator for initiation of preventive actions during contingencies. The devised methodology is tested using standard IEEE test systems as well as a practical 400/220 kV state grid system using ETAP/MATLAB. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Niemann‐Pick disease type C (NPC) is a genetic disorder in which patient cells have endosomal/lysosomal accumulation of cholesterol and sphingolipids. However, the relationship between sphingolipids and cholesterol accumulation in NPC cells has not been established. Here, we investigated the role of sphingomyelin (SM) on the accumulation of cholesterol in NPC cells. Reduction of SM by inhibition of the ceramide transfer protein CERT decreased the cholesterol accumulation in NPC cells. The accumulation of SM in NPC cells inhibited the transport of cholesterol to the endoplasmic reticulum. Overexpression of Rab9 in NPC cells reduced the cholesterol accumulation, which was recovered by treatment with SM. In NPC cells that overexpressed a Rab9 constitutively active mutant, SM treatment did not lead to the cholesterol accumulation. These results indicate that SM negatively regulates the Rab9‐dependent vesicular trafficking of cholesterol, and a reduction in SM levels in NPC cells recovers the Rab9‐dependent vesicular trafficking defect. [ABSTRACT FROM AUTHOR]

Introduction: Juvenile dermatomyositis (JDM) is the commonest childhood inflammatory myopathy. Outcome of children with JDM has improved significantly with current treatment protocols. We undertook this study to evaluate long‐term outcome of these children using validated outcome measures. Methods: All children diagnosed as JDM and on follow‐up for more than 2 years were eligible for enrolment. Cross‐sectional examination was performed to assess signs of disease activity, damage, physical function and complications by using multiple validated outcome measures/tools. Results: Thirty‐seven patients were enrolled, 19 were male. Median duration of disease at time of enrolment was 73 months (range 24‐219 months) and median duration of follow‐up was 60 months (range 24‐218 months). Disease course was monocyclic in two‐thirds of patients. Eight children were still on therapy at the time of enrolment. On Manual Muscle Testing 8, 3 and 7 children had severe weakness and mild to moderate weakness, respectively. Neck flexors were the most commonly affected muscle group. On abbreviated Cutaneous Assessment Tool, 14 children had evidence of cutaneous activity. More than 50% had at least 1 sign of cutaneous damage, most common signs being calcinosis and lipodystrophy. Nearly two‐thirds of patients had damage in at least 1 organ using Myositis Damage Index. Nine children had physical dysfunction when assessed by Child Health Assessment Questionnaire. Conclusions: Skin disease continued to be active in a significant proportion of patients. Features of damage, namely calcinosis and lipodystrophy, were seen in more than half. Muscle disease normalized in a large proportion of patients. [ABSTRACT FROM AUTHOR]

The purification and cloning of the acyl-coenzyme A: cholesterol acyltransferase (ACAT) enzymes and the sterol O-acyltransferase (SOAT) genes has opened new areas of interest in cholesterol metabolism given their profound effects on foam cell biology and intestinal lipid absorption. The generation of mouse models deficient in Soat1 or Soat2 confirmed the importance of their gene products on cholesterol esterification and lipoprotein physiology. Although these studies supported clinical trials which used non-selective ACAT inhibitors, these trials did not report benefits, and one showed an increased risk. Early genetic studies have implicated common variants in both genes with human traits, including lipoprotein levels, coronary artery disease, and Alzheimer’s disease; however, modern genome-wide association studies have not replicated these associations. In contrast, the common SOAT1 variants are most reproducibly associated with testosterone levels.

Cholesterol is an important lipid molecule and is needed for all mammalian cells. In various cell types, excess cholesterol is stored as cholesteryl esters; acyl-CoA:cholesterol acyltransferase 1 (ACAT1) plays an essential role in this storage process. ACAT1 is located at the endoplasmic reticulum and has nine transmembrane domains (TMDs). It is a member of the membrane-bound O-acyltransferase (MBOAT) family, in which members contain multiple TMDs and participate in a variety of biological functions. When solubilized in the zwitterionic detergent CHAPS, ACAT1 can be purified to homogeneity with full enzyme activity and behaves as a homotetrameric protein. ACAT1 contains two dimerization motifs. The first motif is located near the N-terminus and is not conserved in MBOATs. Deletion of the N-terminal dimerization domain converts ACAT1 to a dimer with full catalytic activity; therefore, ACAT1 is a two-fold dimer. The second dimerization domain, located near the C-terminus, is conserved in MBOATs; however, it was not known whether the C-terminal dimerization domain is required for enzyme activity. Here we show that treating ACAT1 with non-ionic detergent, Triton X-100 or octyl glucoside, causes the enzyme to become a two-fold monomer without any enzymatic activity. Detergent exchange of Triton X-100 with CHAPS restores ACAT1 to a two-fold dimer but fails to restore its enzymatic activity. These results implicate that ACAT1 requires hydrophobic subunit interactions near the C-terminus in order to remain active as a two-fold dimer. Our results also caution the use of Triton X-100 or octyl glucoside to purify other MBOATs. Image 1 • ACAT1 is a two-fold dimer and has two dimerization domains. • The N-terminal dimerization domain is not required for enzyme activity. • Triton X-100 disrupts C-terminal dimerization and inactivates the enzyme. • The C-terminal dimerization domain is required for enzyme activity. [ABSTRACT FROM AUTHOR]

Highlights • A proactive risk identification and analysis approach for operational processes was developed. • A new risk analysis framework was proposed based on FRAM and ACAT model. • The conventional FRAM was enriched with a detailed and rigorous description of functions. • More functional constraints were identified with the hybrid approach. Abstract Risks in the industrial operation processes involve complex system elements such as human, machine, organization, information, as well as nonlinear coupling relationships among them. Traditional risk analysis methods focus on the cause-effect relationships between the system elements and accidents, while ignoring what the correct and proper relationships should be. For a proactive risk identification and analysis, learning from success is suggested instead of learning from post hoc accidents, which requires that risk analysis identifies the normal functions and their couplings. Therefore, system functioning has been a subject of interest in the field of risk analysis. The Functional Resonance Analysis Method (FRAM) has been an effective tool to reveal the couplings and dependent relationships among different functions. However, the functions identification and interaction analysis in the FRAM is limited because there is no consistent or explicit stop rule. For a detailed and rigorous description of functions, the Accident Causation Analysis and Taxonomy (ACAT) model is used to enrich the FRAM by generating functions based on a closed-loop control system. Two operation processes in the hazardous industries are used as illustrations. The results show that more functional constraints and deep contributing factors to accidents can be identified with the hybrid approach. Graphical abstract Image, graphical abstract [ABSTRACT FROM AUTHOR]

The ability of Mycobacterium tuberculosis (Mtb) to persist in its human host relies on numerous immune evasion strategies, such as the deregulation of the lipid metabolism leading to the formation of foamy macrophages (FM). Yet, the specific host factors leading to the foamy phenotype of Mtb-infected macrophages remain unknown. Herein, we aimed to address whether host cytokines contribute to FM formation in the context of Mtb infection. Our approach is based on the use of an acellular fraction of tuberculous pleural effusions (TB-PE) as a physiological source of local factors released during Mtb infection. We found that TB-PE induced FM differentiation as observed by the increase in lipid bodies, intracellular cholesterol, and expression of the scavenger receptor CD36, as well as the enzyme acyl CoA:cholesterol acyl transferase (ACAT). Importantly, interleukin-10 (IL-10) depletion from TB-PE prevented the augmentation of all these parameters. Moreover, we observed a positive correlation between the levels of IL-10 and the number of lipid-laden CD14+ cells among the pleural cells in TB patients, demonstrating that FM differentiation occurs within the pleural environment. Downstream of IL-10 signaling, we noticed that the transcription factor signal transducer and activator of transcription 3 was activated by TB-PE, and its chemical inhibition prevented the accumulation of lipid bodies and ACAT expression in macrophages. In terms of the host immune response, TB-PE-treated macrophages displayed immunosuppressive properties and bore higher bacillary loads. Finally, we confirmed our results using bone marrow-derived macrophage from IL-10−/− mice demonstrating that IL-10 deficiency partially prevented foamy phenotype induction after Mtb lipids exposure. In conclusion, our results evidence a role of IL-10 in promoting the differentiation of FM in the context of Mtb infection, contributing to our understanding of how alterations of the host metabolic factors may favor pathogen persistence.

The ability of Mycobacterium tuberculosis (Mtb) to persist in its human host relies on numerous immune evasion strategies, such as the deregulation of the lipid metabolism leading to the formation of foamy macrophages (FM). Yet, the specific host factors leading to the foamy phenotype of Mtb-infected macrophages remain unknown. Herein, we aimed to address whether host cytokines contribute to FM formation in the context of Mtb infection. Our approach is based on the use of an acellular fraction of tuberculous pleural effusions (TB-PE) as a physiological source of local factors released during Mtb infection. We found that TB-PE induced FM differentiation as observed by the increase in lipid bodies, intracellular cholesterol, and expression of the scavenger receptor CD36, as well as the enzyme acyl CoA:cholesterol acyl transferase (ACAT). Importantly, interleukin-10 (IL-10) depletion from TB-PE prevented the augmentation of all these parameters. Moreover, we observed a positive correlation between the levels of IL-10 and the number of lipid-laden CD14+ cells among the pleural cells in TB patients, demonstrating that FM differentiation occurs within the pleural environment. Downstream of IL-10 signaling, we noticed that the transcription factor signal transducer and activator of transcription 3 was activated by TB-PE, and its chemical inhibition prevented the accumulation of lipid bodies and ACAT expression in macrophages. In terms of the host immune response, TB-PE-treated macrophages displayed immunosuppressive properties and bore higher bacillary loads. Finally, we confirmed our results using bone marrow-derived macrophage from IL-10-/- mice demonstrating that IL-10 deficiency partially prevented foamy phenotype induction after Mtb lipids exposure. In conclusion, our results evidence a role of IL-10 in promoting the differentiation of FM in the context of Mtb infection, contributing to our understanding of how alterations of the host metabolic factors may favor pathogen persistence. [ABSTRACT FROM AUTHOR]

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Direct-acting antivirals (DAAs), which target hepatitis C virus (HCV) proteins, have exhibited impressive efficacy in the management of chronic hepatitis C. However, the concerns regarding high costs, drug resistance mutations and subsequent unexpected side effects still call for the development of host-targeting agents (HTAs) that target host factors involved in the viral life cycle and exhibit pan-genotypic antiviral activity. Given the close relationship between lipid metabolism and the HCV life cycle, we investigated the anti-HCV activity of a series of lipid-lowering drugs that have been approved by government administrations or proven safety in clinical trials. Our results showed that avasimibe, an inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), exhibited marked pan-genotypic inhibitory activity and superior inhibition against HCV when combined with DAAs. Moreover, avasimibe significantly impaired the assembly of infectious HCV virions. Mechanistic studies demonstrated that avasimibe induced downregulation of microsomal triglyceride transfer protein expression, resulting in reduced apolipoprotein E and apolipoprotein B secretion. Therefore, the pan-genotypic antiviral activity and clinically proven safety endow avasimibe exceptional potential as a candidate for combination therapy with DAAs. In addition, the discovery of the antiviral properties of ACAT inhibitors also suggests that inhibiting the synthesis of cholesteryl esters might be an additional target for the therapeutic intervention for chronic HCV infection. [ABSTRACT FROM AUTHOR]

Physiologically based pharmacokinetic (PBPK) modeling has been extensively used to study the factors of effect drug absorption, distribution, metabolize and extraction progress in human. In this study, Compound A(CPD A) is a BCS Class II drug, which has been extensive applied in clinical as lipid-lowering drug, administered orally after food, they displayed positive food effects in human, A PBPK model was built to mechanistic investigate the food effect of CPD A tablet in our study. By using gastroplus™ software, the PBPK models accurately predicted the results of food effects and predicted data were within 2-fold error of the observed results. The PBPK model mechanistic illuminated the changes of pharmacokinetic values for the positive food effects of the compound in human. Here in, the PBPK modeling which were combined with ACAT absorption models in it, successfully simulated the food effect in human of the drug. The simulation results were proved that PBPK model can be able to serve as a potential tool to predict the food effect on certain oral drugs. [ABSTRACT FROM AUTHOR]

Cholesterol is always an issue because blood total cholesterol (TC) and low-density lipoprotein (LDL) correlate strongly with coronary heart disease. Cholesterol homeostasis is maintained by a complex mechanism of sterol absorption, anabolism, catabolism and excretion. Nutraceuticals and functional foods which lower TC must affect the genes which regulate cholesterol homeostasis. In general, cholesterol-lowering functional foods and nutraceuticals can be classified into seven types namely intestinal Niemann-Pick C1 like 1 (NPC1L1) competitors, intestinal acyl-CoA:cholesterol acyltransferase 2 (ACAT2) inhibitors, 3-hydroxy-3-methylglutaryl (HMG-CoA) reductase inhibitors, LDL receptor up-regulators, bile acid reabsorption inhibitors, cholesterol-7α-hydroxylase (CYP7A1) activators, and plasma cholesteryl ester transporting protein (CETP) inhibitors. This mini-review classifies the popular cholesterol-lowering nutraceuticals and functional foods, and explores their underlying mechanisms.

In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

Purpose Studies have shown altered pharmacokinetic patterns (PK) in patient suffering from acute pain. Thus, we aimed to simulate pharmacokinetics of meloxicam and ibuprofen in pain and pain-free states using a physiological based software program to identify the underlining mechanistic changes for the observed differences. Method Published in vivo data of meloxicam and ibuprofen were used for the simulations. Two drug formulations were studied: a fast dissolving (FD) and regular release (RR) tablet formulation. The oral bioavailability was compared between these formulations in vagally suppressed rats (gastric dysfunction) and a control group. For ibuprofen additional human data of a control and post dental surgery group were used. All simulations were performed using GastroPlus™. The in vivo drug release and PK of all formulations were estimated for both drugs using the software’s immediate release (IR) or gastric release (GR) models. Result For meloxicam, the IR model predicted the in vivo absorption in the control group after administration of the FD and RR formulations. When gastric dysfunction was induced, the IR model did not predict absorption while the GR model did for both formulations, FD and RR. For ibuprofen, the predictions were also very close for both formulations, using the IR model for the control group and the GR model for the vagally suppressed condition in rats and humans. Conclusions Gastric control of the drug release in pain/disease state was identified as the major factor causing the observed differences in the pharmacokinetics. Computer simulations of disease states can be employed to optimize drug release from dosage forms to overcome the reported shortfalls in the drug absorption. [ABSTRACT FROM AUTHOR]

The grapefruit flavonoid, naringenin, is hypocholesterolemic in vivo, and inhibits basal apolipoprotein B (apoB) secretion and the expression and activities of both ACAT and microsomal triglyceride transfer protein (MTP) in human hepatoma cells (HepG2). In this report, we examined the effects of naringenin on apoB kinetics in oleate-stimulated HepG2 cells and determined the contribution of microsomal lumen cholesteryl ester (CE) availability to apoB secretion. Pulse-chase studies of apoB secretion and intracellular degradation were analyzed by multicompartmental modeling. The model for apoB metabolism in HepG2 cells includes an intracellular compartment from which apoB can be either secreted or degraded by both rapid and slow pathways. In the presence of 0.1 mM oleic acid, naringenin (200 μM) reduced the secretion of newly synthesized apoB by 52%, due to a 56% reduction in the rate constant for secretion. Intracellular degradation was significantly increased due to a selective increase in rapid degradation, while slow degradation was unaffected. Incubation with either N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) or lactacystin showed that degradation via the rapid pathway was largely proteasomal. Although these changes in apoB metabolism were accompanied by significant reductions in CE synthesis and mass, subcellular fractionation experiments comparing naringenin to specific ACAT and HMG-CoA reductase inhibitors revealed that reduced accumulation of newly synthesized CE in the microsomal lumen is not consistently associated with reduced apoB secretion. However, naringenin, unlike the ACAT and HMG-CoA reductase inhibitors, significantly reduced lumenal TG accumulation.We conclude that naringenin inhibits apoB secretion in oleate-stimulated HepG2 cells and selectively increases intracellular degradation via a largely proteasomal, rapid kinetic pathway. Although naringenin inhibits ACAT, CE availability in the endoplasmic reticulum (ER) lumen does not appear to regulate apoB secretion in HepG2 cells. Rather, inhibition of TG accumulation in the ER lumen via inhibition of MTP is the primary mechanism blocking apoB secretion.

Previous studies have indicated that LCAT may play a role in the generation of cholesteryl esters (CE) in plasma apolipoprotein B (apoB) lipoproteins. The purpose of the present study was to examine the quantitative importance of LCAT on apoB lipoprotein CE fatty acid (CEFA) composition. LCAT−/− mice were crossed into the LDL receptor (LDLr)−/− and apoE−/− background to retard the clearance and increase the concentration of apoB lipoprotein in plasma. Plasma free cholesterol was significantly elevated but total and esterified cholesterol concentrations were not significantly affected by removal of functioning LCAT in either the LDLr−/− or apoE−/− mice consuming a chow diet. However, when functional LCAT was removed from LDLr−/− mice, the CEFA ratio (saturated + monounsaturated/polyunsaturated) of plasma LDL increased 7-fold because of a 2-fold increase in saturated and monounsaturated CE, a 40% reduction in cholesteryl linoleate, and a complete absence of long chain (>18 carbon) polyunsaturated CE (20:4, 20:5n-3, and 22:6n-3), from 29.3% to 0%. Removal of functional LCAT from apoE−/− mice resulted in only a 1.6-fold increase in the CEFA ratio, due primarily to a complete elimination of long chain CE (7.7% to 0%). Our results demonstrate that LCAT contributes significantly to the CEFA pool of apoB lipoprotein and is the only source of plasma long chain polyunsaturated CE in these mice. —Furbee, J. W., Jr., O. Francone, and J. S. Parks. In vivo contribution of LCAT to apolipoprotein B lipoprotein cholesteryl esters in LDL receptor and apolipoprotein E knockout mice.

The aim of this study was to determine the impact of diabetic macrosomia on cholesterol and lipoprotein metabolism. Age-related changes in the activities of serum LCAT, hepatic HMG-CoA reductase, cholesterol 7α-hydroxylase, and ACAT, the major enzymes involved in cholesterol metabolism, were determined in macrosomic offspring of streptozotocin-induced diabetic rats. Hepatic, serum, and lipoprotein cholesterol contents were also examined. Mild hyperglycemia in pregnant rats was induced by intraperitoneal injection of streptozotocin (40 mg/kg body weight) on day 5 of gestation. Control pregnant rats were injected with citrate buffer. At birth, macrosomic pups had higher serum, LDL-HDL1, and HDL2-3 cholesterol levels (P < 0.05) associated with increased LCAT activity (+57%) compared with control values. At 1 and 2 months of life, serum and lipoprotein cholesterol concentrations in macrosomic rats were similar to those of controls, whereas LCAT activity remained elevated about 1.5-fold. In addition, there was no change in hepatic cholesterol contents but hepatic HMG-CoA reductase, cholesterol 7α-hydroxylase, and ACAT activities were higher in both macrosomic males and females than in their respective controls (P < 0.01). By 3 months, macrosomic rats had developed hypercholesterolemia with a rise in all lipoproteins. Enzyme activities were still increased in these mature macrosomic rats, and hepatic cholesteryl esters were higher only in macrosomic females.These data demonstrate an overproduction, combined with overutilization, of cholesterol during the phase of rapid growth in macrosomic rats. However, cholesterol oversynthesis exceeded its removal and was a major contributor to hypercholesterolemia in adult macrosomic rats. In conclusion, macrosomia was associated with alterations in cholesterol metabolism through adulthood.—Merzouk, H., S. Madani, A. Boualga, J. Prost, M. Bouchenak, and J. Belleville. Age-related changes in cholesterol metabolism in macrosomic offspring of rats with streptozotocin-induced diabetes. J. Lipid Res. 2001. 42: 1152–1159.

We have previously shown that acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) protein content increases significantly during the human monocyte-macrophage differentiation process. To gain further insight, we used undifferentiated human monocytic THP-1 cells as a model system with which to examine whether ACAT-1 mRNA and protein content can be increased by treating cells with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or with 9-cis-retinoic acid (9-cis-RA), two agents known to upregulate the expression of various genes during the monocyte-macrophage differentiation process. Immunoblot analysis with anti-human ACAT-1 antibodies revealed that ACAT-1 protein was increased by 2.6-fold, using 1,25-(OH)2D3 at a physiological concentration (100 pM). ACAT-1 protein was also increased when using 9-cis-RA, but only at relatively high concentrations (0.1–1 μM). Northern blot analysis revealed that among the four ACAT-1 mRNA transcripts (2.8, 3.6, 4.2, and 7.0 kb) examined, only the 2.8- and 3.6-kb transcripts were selectively increased. On the basis of enzyme assays in vitro, ACAT activity was increased 3.0-fold by using 100 nM 1,25-(OH)2D3, and 1.8-fold by using 1 μM 9-cis-RA. Together, our results suggest that 1,25-(OH)3 participates in ACAT-1 gene expression during the monocyte-macrophage differentiation process. —Maung, K. K., A. Miyazaki, H. Nomiyama, C. C. Y. Chang, T-Y. Chang, and S. Horiuchi. Induction of acyl-coenzyme A:cholesterol acyltransferase-1 by 1,25-dihydroxyvitamin D3 or 9-cis-retinoic acid in undifferentiated THP-1 cells.

Class A scavenger receptors (SR-A) mediate the uptake of modified low density lipoprotein (LDL) by macrophages. Although not typically associated with the activation of intracellular signaling cascades, results with peritoneal macrophages indicate that the SR-A ligand acetylated LDL (AcLDL) promotes activation of cytosolic kinases and phospholipases. These signaling responses were blocked by the treatment of cells with pertussis toxin (PTX) indicating that SR-A activates Gi/o-linked signaling pathways. The functional significance of SR-A-mediated Gi/o activation is not clear. In this study, we investigated the potential role of Gi/o activation in regulating SR-A-mediated lipoprotein uptake. Treatment of mouse peritoneal macrophages with PTX decreased association of fluorescently labeled AcLDL with cells. This inhibition was dependent on the catalytic activity of the toxin confirming that the decrease in AcLDL uptake involved inhibiting Gi/o activation. In contrast to the inhibitory effect on AcLDL uptake, PTX treatment did not alter β-VLDL-induced cholesterol esterification or deposition of cholesterol. The ability of polyinosine to completely inhibit AcLDL uptake, and the lack of PTX effect on β-VLDL uptake, demonstrated that the inhibitory effect is specific for SR-A and not the result of non-specific effects on lipoprotein metabolism. Despite having an effect on an SR-A-mediated lipoprotein uptake, there was no change in the relative abundance of SR-A protein after PTX treatment. These results demonstrate that activation of a PTX-sensitive G protein is involved in a feedback process that positively regulates SR-A function.—Whitman, S. C., A. Daugherty, and S. R. Post. Regulation of acetylated low density lipoprotein uptake in macrophages by pertussis toxin-sensitive G proteins. J. Lipid Res. 2000. 41: 807–813.

The Th1-derived cytokine gamma interferon, IFN-γ, is present within the microenvironment of an atheromatous lesion and likely contributes to lesion progression through macrophage activation. While the inflammatory effects of IFN-γ are well known, the role of this cytokine in cholesterol metabolism in macrophage derived foam cells is unclear. In the present study, the incubation of foam cells with IFN-γ resulted in the reduction of HDL3-mediated cholesterol efflux. The decrease in cholesterol efflux was not observed with other macrophage-activating factors as colony-stimulating factors failed to demonstrate a similar effect. The reduction in cholesterol efflux was independent of apoE synthesis or SR-BI expression and was associated with a redistribution of intracellular cholesterol with an increase in cholesteryl ester accumulation. The increase in the esterified pool, primarily in cholesterol eicosapentadenoate, docosapentaenoate, arachidonate, and linoleate was associated with a 2-fold increase in acyl-CoA:cholesterol-O-acyltransferase, ACAT, activity and message without any change in neutral cholesteryl ester hydrolase activity. While CD36 message was reduced in IFN-γ-treated foam cells, the ability to reverse the decrease in efflux by the ACAT inhibitor A58035 in a dose-dependent manner suggests that the IFN-γ effect on efflux is primarily through the modulation of ACAT expression. Therefore, in addition to its inflammatory effects, IFN-γ can contribute to the progression of an atherosclerotic lesion by altering the pathway of intracellular cholesterol trafficking in macrophage derived foam cells. —Panousis, C. G., and S. H. Zuckerman. Regulation of cholesterol distribution in macrophage-derived foam cells by interferon-γ.

Three series of xanthone sulfonamides were synthesized, and their inhibitory activities against acyl-CoA: cholesterol acyltransferase (ACAT) were evaluated. Results showed that most of the title compounds exhibited strong inhibitory activity against ACAT, of which compounds 1c, 1e, 1f, 2d, 2e, and 3d were proved to be more active than the positive control Sandoz 58-035. Computational docking experiments indicated that the interaction between inhibitors and ACAT contained the H-bond interaction, the hydrophobic interaction, and the narrow hydrophobic cleft. [ABSTRACT FROM AUTHOR]

The fluorescent cholesterol analog 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (fluoresterol) was characterized as a tool for exploring the biochemistry and cell biology of intestinal cholesterol absorption. Hamsters absorbed fluoresterol in a concentration- and time-dependent manner, with an efficiency of about 15–30% that of cholesterol. Fluoresterol absorption was blocked by compounds known to inhibit cholesterol absorption, implying that fluoresterol interacts with those elements of the normal pathway for cholesterol absorption on which the inhibitors act. Confocal microscopy of small intestinal tissue demonstrated that fluoresterol was taken up by absorptive epithelial cells and packaged into lipoprotein particles, suggesting a normal route of intracellular trafficking. Uptake of fluoresterol was confirmed by biochemical analysis of intestinal tissue, and a comparison of [3H]cholesterol and fluoresterol content in the mucosa suggested that fluoresterol moved through the enterocytes more rapidly than did cholesterol. This interpretation was supported by measurements of fluoresterol esterification in the mucosa. Four hours after hamsters were given fluoresterol and [3H]cholesterol orally, 44% of the fluoresterol in the intestinal mucosa was esterified, compared to 8% of the [3H]cholesterol. Caco-2 cells took up 2- to 5-fold more [3H]cholesterol than fluoresterol from bile acid micelles, and esterified 21–24% of the fluoresterol but only 1–4% of the [3H]cholesterol. Thus fluoresterol apparently interacts with the proteins required for cholesterol uptake, trafficking, and processing in the small intestine.—Sparrow, C. P., S. Patel, J. Baffic, Y-S. Chao, M. Hernandez, M-H. Lam, J. Montenegro, S. D. Wright, and P. A. Detmers. A fluorescent cholesterol analog traces cholesterol absorption in hamsters and is esterified in vivo and in vitro. J. Lipid Res. 1999. 40: 1747–1757.

Hypertriglyceridemic (HTG) very low density lipoproteins (VLDL) from subjects with type IV hyperlipoproteinemia induce both cholesteryl ester (CE) and triglyceride (TG) accumulation in cultured J774 macrophages. We examined whether the cytokine interferon-γ (IFN-γ), which is expressed by lymphocytes in atherosclerotic lesions, would modulate macrophage uptake of HTG-VLDL. Incubation of cells with HTG-VLDL alone significantly increased cellular CE and TG mass 17- and 4.3-fold, respectively, while cellular free cholesterol (FC) was unaffected. Preincubation of cells with IFN-γ (50 U/ml) prior to incubation with HTG-VLDL caused a marked enhancement in cellular CE and TG 27- and 6-fold over no additions (controls), respectively, and a 1.5-fold increase in FC. IFN-γ increased low density lipoprotein (LDL)-induced cellular CE 2-fold compared to LDL alone. IFN-γ did not enhance the uptake of type III (apoE2/E2) HTG-VLDL or VLDL from apoE knock-out mice. Incubations in the presence of a lipoprotein lipase (LPL) inhibitor or an acylCoA:cholesterol acyltransferase (ACAT) inhibitor demonstrated that the IFN-γ-enhanced HTG-VLDL uptake was dependent on LPL and ACAT activities. IFN-γ significantly increased the binding and degradation of 125I-labeled LDL. Binding studies with 125I-labeled α2-macroglobulin, a known LDL receptor-related protein (LRP) ligand, and experiments with copper-oxidized LDL indicated that the IFN-γ-enhanced uptake was not due to increased expression of the LRP or scavenger receptors. Thus, IFN-γ may promote foam cell formation by accelerating macrophage uptake of native lipoproteins. IFN-γ-stimulated CE accumulation in the presence of HTG-VLDL occurs via a process that requires receptor binding-competent apoE and active LPL. IFN-γ-enhanced uptake of both HTG-VLDL and LDL is mediated by the LDL-receptor and requires ACAT-mediated cholesterol esterification.—Whitman, S. C., C. A. Argmann, C. G. Sawyez, D. B. Miller, R. A. Hegele, and M. W. Huff. Uptake of type IV hypertriglyceridemic VLDL by cultured macrophages is enhanced by interferon-γ. J. Lipid Res. 1999. 40: 1017–1028.

Origins of hyperlipidemia and cholestasis that occur during pregnancy were investigated by examining expression of key elements related to plasma and hepatic cholesterol metabolism during pregnancy, lactation, and post-lactation in the rat model. Among major findings were: during pregnancy, the activities of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, acyl coenzyme A:cholesterol acyltransferase, acyl coenzyme A:diacylglycerol acyltransferase, cholesterol 7α-hydroxylase, cholesterol ester hydrolases, low density lipoprotein receptors, LRP, and mdr2 were significantly lower or similar to non-pregnant controls while SR-B1 was elevated. Once lactation began, reductase, cholesterol acyltransferase, 7α-hydroxylase activities, low density lipoprotein receptors, and mdr2 increased while SR-B1 decreased. In later stages of lactation most hepatic elements returned to near control levels. Plasma cholesterol levels were higher than control at birth and during lactation with increase in LDL-size particles. By 24 h post-lactation, plasma triglycerides were 3.7-fold higher while cholesterol remained unchanged. Very large lipoproteins were present while LDL-size particles were now absent. Hepatic cholesterol acyltransferase had decreased to 27“% of control while diacylglycerol acyltransferase increased 3-fold and low density lipoprotein receptors doubled. Most elements were normalized 3 weeks after weaning except for LRP and low density lipoprotein receptors which were elevated. These studies provide an integrated picture of expression of key elements of hepatic and plasma cholesterol metabolism during pregnancy and lactation and advance understanding of hyperlipidemia and cholestasis during these states. —Smith, J. L., S. R. Lear, T. M. Forte, W. Ko, M. Massimi, and S. K. Erickson. Effect of pregnancy and lactation on lipoprotein and cholesterol metabolism in the rat. J. Lipid Res. 1998. 39: 2237–2249.

Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) is a resident endoplasmic reticulum enzyme that prevents the buildup of cholesterol in membranes by converting it to cholesterol esters. Blocking ACAT1 pharmacologically or by Acatl gene knock-out (KO) decreases amyloidopathy in mouse models for Alzheimer's disease. However, the beneficial actions of ACAT1 blockage to treat Alzheimer's disease remained not well understood. Microglia play essential roles in the proteolytic clearance of amyloid β (Aβ) peptides. Here we show that Acatl gene KO in mouse increases phagocytic uptake of oligomeric Aβl-42 and stimulates lysosomal Aβl-42 degradation in cultured microglia and in vivo. Additional results show that Acatl gene KO or a specific ACAT1 inhibitor K604 stimulates autophagosome formation and transcription factor EB-mediated lysosomal proteolysis. Surprisingly, the effect of ACAT1 blockage does not alter mTOR signaling or endoplasmic reticulum stress response but can be modulated by agents that disrupt cholesterol biosynthesis. To our knowledge, our current study provides the first example that a small molecule (K604) can promote autophagy in an mTOR-independent manner to activate the coordinated lysosomal expression and regulation network. Autophagy is needed to degrade misfolded proteins/ peptides. Our results implicate that blocking ACAT1 may provide a new way to benefit multiple neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

ABSTRACT Cholesteryl oleate present as a neutral lipid in low-density lipoprotein has been speculated to be a biomarker for atherosclerosis. Methods which are at hand for the quantification of cholesteryl oleate are either costly or entail the use of radioactive compounds. Charring of TLC plates has been used to identify cholesteryl esters for a long time but has never been applied to quantification of cholesteryl esters in biological matrices. Here, we report a novel method based on planar chromatography for the analysis of the products of the acyl CoA-cholesterol acyltransferase (ACAT) assay, viz. cholesteryl esters. Using silica gel 60 F254 as stationary phase, compounds were spotted on the plate and run using a solvent system comprising n-hexane-diethyl ether-glacial acetic acid (90:10:1, v/v/v). The plates were developed by dipping in anisaldehyde-sulfuric acid reagent and were scanned at 546 nm for quantification. The developed method shows good linear relationship in the concentration range of 100-500 ng/band with a correlation coefficient ( r) value of 0.9996. The method was validated for accuracy, precision and robustness. Percentage recovery of the method was found to be in the range 96.88-103.01% with intra- and inter-day precision analysis yielding <2% relative standard deviation at nominal concentrations for analysis. The limits of detection and quantification were found to be 6.45 and 19.54 ng, respectively. The method was validated for robustness by making deliberate changes in mobile phase composition, volume and temperature of analysis, and the standard deviations of peak areas for these intentional changes were found to be 1.07, 1.02 and 1.30 respectively. The method was applied to the estimation of cholesteryl esters in plasma samples from patients diagnosed with hypercholesterolemia. No interferences were found from the biological matrices used in the assay. The proposed method could be of immense potential for estimation of cholesteryl oleate as a marker of ACAT activity, for screening of ACAT inhibitors in drug discovery process and in the prognosis of atherosclerosis. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: To investigate the adrenal toxicity of a novel inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase, compound X (CX), histopathological examinations, fat staining, adrenal cholesterol measurement, blood biochemistry, plasma corticosterone and ACTH measurement, ACTH-stimulation assay, and adrenal gene-expression analyses were done in rats in repeated-dose studies (experiment 1: 0, 3, 10, 30 and 150mg/kg for 4, 8, 15 and 28 days; experiment 2: 0, 3, 10,30 and 150mg/kg for 28 days; experiment 3: 0, 10, 30, 100 and 300mg/kg for 28 days). CX induced morphologic changes such as vacuolation and hypertrophy in the zona fasciculata (ZF) at ≥10mg/kg, and eosinophilic changes in the ZF at 150mg/kg. Vacuolation decreased in a dose-dependent manner and was replaced by eosinophilic changes. Inflammatory and fibrous changes were observed at ≥30mg/kg. These changes were expressed at early stages of dosing and were not exacerbated by extension of the administration period. Oil-red-O/Filipin staining showed depletion of cholesterol ester in dose-dependent manner and enabled adrenal cholesterol measurement. Filipin staining also revealed vacuoles to be composed of cholesterol esters. No significant changes were observed during the dosing period of CX for plasma corticosterone and ACTH levels. Gene-expression analyses showed up-regulation of Star and Abca1 mRNA levels at 300mg/kg. In conclusion, CX induced adrenal toxicity, but CX did not influence adrenocortical functions, and exacerbation of adrenal toxicities by extension of the administration period was not observed. Up-regulation of genes related to the transport of FC, such as Star and Abca1, were observed in CX groups, and these genes may be involved in the maintenance of adrenal structure and function in rats given CX. [Copyright &y& Elsevier]

Sublingual administration of certain buffered propranolol may improve the rate and extent of absorption compared to oral administration. The main objectives of this study were to (1) compare the plasma propranolol concentrations (Cp-prop) following sublingual administration of a specially buffered formulation (Promptol™) to that following oral administration of Inderal and (2) evaluate the utility of a special pharmacokinetic model in describing the Cp-prop following sublingual administration. Eighteen healthy volunteers received 10 mg sublingual Promptol™ or oral Inderal. Multiple Cp-prop were determined and their pharmacokinetics compared. Additional data following sublingual 40 mg Promptol™ or Inderal were utilized for evaluation of a special advanced compartmental absorption and transit (ACAT) model. For model simulation, the physicochemical parameters were imported from AMET predictor, whereas the pharmacokinetic parameters were calculated and optimized by Gastroplus. Based on this model, the quantity of drug absorbed via buccal/sublingual mucosa was estimated. Cp-prop was higher at earlier times with 3-fold greater relative bioavailability following sublingual Promptol™ compared to that from oral Inderal. The special ACAT model provided excellent goodness of fit of Cp-prop-time curve and estimated a 56.6% increase in absorption rate from Promptol™ and higher initial Cp-prop compared to the regular formulation. The modified ACAT model provided a useful approach to describe sublingual absorption of propranolol and clearly demonstrated an improvement of absorption of Promptol™. The sublingual 10 mg Promptol™ achieved not only a similar systemic exposure as 30 mg oral Inderal but an earlier effective Cp-prop which may be advantageous for certain clinical conditions. [ABSTRACT FROM AUTHOR]

ABSTRACT Schnyder corneal dystrophy ( SCD) is an autosomal dominant disease characterized by germline variants in UBIAD1 introducing missense alterations leading to deposition of cholesterol in the cornea, progressive opacification, and loss of visual acuity. UBIAD1 was recently shown to synthesize menaquinone-4 ( MK-4, vitamin K2), but causal mechanisms of SCD are unknown. We report a novel c.864G>A UBIAD1 mutation altering glycine 177 to glutamic acid (p.G177E) in six SCD families, including four families from Finland who share a likely founder mutation. We observed reduced MK-4 synthesis by UBIAD1 altered by SCD mutations p.N102S, p.G177R/E, and p.D112N, and molecular models showed p.G177-mutant UBIAD1 disrupted transmembrane helices and active site residues. We show UBIAD1 interacts with HMGCR and SOAT1, enzymes catalyzing cholesterol synthesis and storage, respectively, using yeast two-hybrid screening and immunoprecipitation. Docking simulations indicate cholesterol binds to UBIAD1 in the substrate-binding cleft and substrate-binding overlaps with GGPP binding, an MK-4 substrate, suggesting potential competition between these metabolites. Impaired MK-4 synthesis is a biochemical defect identified in SCD suggesting UBIAD1 links vitamin K and cholesterol metabolism through physical contact between enzymes and metabolites. Our data suggest a role for endogenous MK-4 in maintaining cornea health and visual acuity. [ABSTRACT FROM AUTHOR]

Hyperlipidemia is an important factor to induce metabolic syndrome such as obesity, diabetes and cardiovascular diseases. Recently, some antihyperlipidemic agents from herbal medicines have been in the spotlight in the medical science field. Thus, the present study evaluated the antihyperlipidemic activities of the essential oil from the leaves of Pinus koraiensis SIEB (EOPK) that has been used as a folk remedy for heart disease. The reverse transcription polymerase chain reaction (RT-PCR) revealed that EOPK up-regulated low density lipoprotein receptor (LDLR) at the mRNA level as well as negatively suppressed the expression of sterol regulatory element-binding protein (SREBP)-1c, SREBP-2, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), fatty acid synthase (FAS) and glycerol-3-phosphate acyltransferase (GPAT) involved in lipid metabolism in HepG2 cells. Also, western blotting showed that EOPK activated LDLR and attenuated the expression of FAS at the protein level in the cells. Consistently, EOPK significantly inhibited the level of human acylcoenzyme A: cholesterol acyltransferase (hACAT)1 and 2 and reduced the low-density lipoprotein (LDL) oxidation activity. Furthermore, chromatography-mass spectrometry (GC-MS) analysis showed that EOPK, an essential oil mixture, contained camphene (21.11%), d-limonene (21.01%), α-pinene (16.74%) and borneol (11.52%). Overall, the findings suggest that EOPK can be a potent pharmaceutical agent for the prevention and treatment of hyperlipidemia. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Purpose: To introduce guidelines in selecting the particle size distribution ( n0, cm−1) that will guarantee optimal oral absorption for drugs with low solubility. Methods: Unlike other multi-compartmental models the gastrointestinal tract is modeled as a continuous tube with spatially varying properties. The transport through the intestinal lumen is described using the dispersion model. The model accounts for the dissolution of poly-dispersed powders. Results: The model was used to examine the sensitivity of the absorption on permeability (P) and water solubility (Cs) following administration in different log-normal powders. The absorption exhibits inverse sigmoidal dependence on the mean particle size (rm, µm) regardless of the administrated dose or drug properties. Thus, there is an optimal rm that maximizes the benefit-cost ratio of the formulation; finer particles do not improve the absorption while coarser particles decrease it. Using the model we find that the optimal rm depends mainly on the drug Cs and on the geometrical standard deviation (gSTD). Conclusions: The results of this work provide the formulator with guidelines to select both rm and gSTD that guarantee optimal absorption. [ABSTRACT FROM AUTHOR]

Abstract: Cholesterol is always an issue because blood total cholesterol (TC) and low-density lipoprotein (LDL) correlate strongly with coronary heart disease. Cholesterol homeostasis is maintained by a complex mechanism of sterol absorption, anabolism, catabolism and excretion. Nutraceuticals and functional foods which lower TC must affect the genes which regulate cholesterol homeostasis. In general, cholesterol-lowering functional foods and nutraceuticals can be classified into seven types namely intestinal Niemann-Pick C1 like 1 (NPC1L1) competitors, intestinal acyl-CoA:cholesterol acyltransferase 2 (ACAT2) inhibitors, 3-hydroxy-3-methylglutaryl (HMG-CoA) reductase inhibitors, LDL receptor up-regulators, bile acid reabsorption inhibitors, cholesterol-7α-hydroxylase (CYP7A1) activators, and plasma cholesteryl ester transporting protein (CETP) inhibitors. This mini-review classifies the popular cholesterol-lowering nutraceuticals and functional foods, and explores their underlying mechanisms. [Copyright &y& Elsevier]

Abstract: Alzheimer''s disease (AD) is a devastating neurodegenerative disorder. Accumulation and deposition of the beta-amyloid (Aβ) peptide generated from its larger amyloid precursor protein (APP) is one of the pathophysiological hallmarks of AD. Intracellular cholesterol was shown to regulate Aβ production. Recent genetic and biochemical studies indicate that not only the amount, but also the distribution of intracellular cholesterol is critical to regulate Aβ generation. Acyl-coenzyme A: cholesterol acyl-transferase (ACAT) is a family of enzymes that regulates the cellular distribution of cholesterol by converting membrane cholesterol into hydrophobic cholesteryl esters for cholesterol storage and transport. Using pharmacological inhibitors and transgenic animal models, we and others have identified ACAT1 as a potential therapeutic target to lower Aβ generation and accumulation. Here we discuss data focusing on ACAT inhibition as an effective strategy for the prevention and treatment of AD. [Copyright &y& Elsevier]

Abstract: Recent guidelines in North America and Europe recommend lowering low density lipoprotein associated cholesterol (LDLC) to achieve optimal coronary heart disease risk reduction. Statins have been the therapy of choice and proven successful and relatively safe. However, we are now facing new challenges and it appears that additional or alternative drugs are urgently needed. This boosts research in the field, reopening old cases like other inhibitors of cholesterol synthesis or making attractive tools from the latest technologies like gene silencing by anti-sense oligonucleotides. LDLs are cholesterol-enriched lipoproteins stabilized by the hepatic apolipoprotein B100, and derived from TG rich very low density lipoprotein. This review focuses on the molecular pathways involved in plasma LDLC production and elimination, in particular cholesterol absorption and the hepatobiliary route, apoB100 and VLDL production, and LDL clearance via the LDL receptor. We will identify important or rate-limiting proteins (including Niemann-Pick C1-like 1 (NPC1L1), microsomal TG transfer protein (MTP), acyl-coenzyme A/cholesterol acyltransferase (ACAT), Acyl-CoA:diacylglycerol acyltransferases 2 (DGAT2), proprotein convertase subtilisin kexin type 9 (PCSK9)), and nuclear receptors (farnesoid X receptor (FXR), thyroid hormone receptor (TR)) that constitute interesting therapeutic targets. Numerous compounds already in use modulate these pathways, such as phytosterols, ezetimibe, bile acids sequestrants, niacin, and fibrates. Many pathways can be considered to lower LDLC, but the road has been paved with disappointments and difficulties. With new targets identified and diversification of the drugs, a new era for better LDLC management is plausible. [Copyright &y& Elsevier]

Abstract: Cideb, a member of CIDE family proteins, has emerged as an important regulator in the development of obesity and diabetes by controlling fatty acid synthesis and VLDL secretion in hepatocytes. Here, we investigated the role of Cideb in cholesterol biosynthesis, uptake and storage in the liver by using Cideb-null mice as a model system. Cideb-null mice and wild-type mice were treated with normal diet (ND) or high cholesterol diet (HCD) for one month. The metabolic parameters of cholesterol metabolism and expression profiles of genes in cholesterol biosynthesis and storage were measured. Cideb-null mice had lower levels of plasma cholesterol and LDL when fed with both ND and HCD and increased rate of cholesterol absorption. Furthermore, the liver of Cideb-null mice has lower rates of cholesterol biosynthesis and reduced expression levels of sterol response element-binding protein (SREBP) cleavage-activation protein (SCAP), and lower levels of nuclear form of SREBP2 and its downstream target genes in cholesterol biosynthesis pathway under a normal diet treatment. On the contrary, hepatic cholesterol biosynthesis rate between wild-type and Cideb-null mice was similar after high cholesterol diet treatment. Interestingly, hepatic cholesterol storage in the liver of Cideb-null mice was significantly increased due to its increased LDL receptor (LDLR) and acyl-CoA cholesterol acyltransferase (ACAT) expression. Finally, we observed drastically reduced cholesterol levels in the heart of Cideb-null mice fed with a high cholesterol diet. Overall, our data suggest that Cideb is a novel regulator in controlling cholesterol homeostasis in the liver. Therefore, Cideb could serve as an important therapeutical target for the treatment of atherosclerosis and cardiovascular diseases. [Copyright &y& Elsevier]

The article examines the effect of Urotensin II (UII) receptor (UT) gene deletion in the atherosclerosis of a mouse. According to the article, UT gene deletion in an apolipoprotein E (ApoE)-deficient background boosts downregulation of acyl-coenzyme A cholesterol acyltransferase (ACAT1). In result, it weaken hepatic lipoprotein receptor-mediated uptake and lipid transporter expression.

Abstract: Oxysterol-induced macrophage apoptosis may have a role in atherosclerosis. Macrophages lacking the type 2 cannabinoid receptor (CB2) are partially resistant to apoptosis induced by 7-ketocholesterol (7KC). AM-251 and SR144528 are selective antagonists of CB1 and CB2 receptors, respectively. We observed that both compounds reduce 7KC-induced apoptosis in Raw 264.7 macrophages. As oxysterol-induced macrophage apoptosis requires acyl-coenzymeA:cholesterol acyltransferase (ACAT) activity, we tested their affects on ACAT activity. AM-251 and SR144528 both reduced cholesteryl ester synthesis in unstimulated and acetylated LDL-stimulated Raw 264.7 macrophages, CB2+/+ and CB2−/− peritoneal macrophages, as well as in vitro, in mouse liver microsomes. Consistent with inhibition of ACAT, the development of foam cell characteristics in macrophages by treatment with acetylated LDL was reduced by both compounds. This work is the first evidence that AM-251 and SR144528 are inhibitors of ACAT and as a result, might have anti-atherosclerotic activities independent of their affect on cannabinoid signaling. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Novel synthetic oxysterols (22S,23S)-3β-hydroxy-22,23-oxido-5α-ergost-8(14)-en-15-one ( I) and (22R,23R)-3β-hydroxy-22,23-oxido-5α-ergost-8(14)-en-15-one ( II) influenced biosynthesis of cholesteryl esters from [14C]acetate (85% and 180% of control at 5 μM concentration) in the human hepatoma Hep G2 cell line. Ketosterol ( I) increased the level of cholesteryl ester biosynthesis from [14C]oleate in Hep G2 cells in a dose dependent manner, whereas the level of cholesteryl esters biosynthesis in the presence of ketosterol ( II) reached the maximal value (269±20% of control) at 1 μM concentration of this compound. In a cell free system ketosterol ( I) increased the rate of ACAT-dependent cholesterol acylation similar to 25-hydroxycholesterol, however, ketosterol ( II)), efficiently stimulated an initial rate of ACAT-catalyzed cholesterol esterification, followed by rapid inactivation of this enzyme. [ABSTRACT FROM AUTHOR]

Growing evidence suggests that the small intestine may contribute to excessive postprandial lipemia, which is highly prevalent in insulin-resistantl Type 2 diabetic individuals and substantially increases the risk of cardiovascular disease. The aim of the present study was to determine the role of high glucose levels on intestinal cholesterol absorption, cholesterol transporter expression, enzymes controlling cholesterol homeostasis, and the status of transcription factors. To this end, we employed highly differentiated and polarized cells (20 days of culture), plated on permeable polycarbonate filters. In the presence of [[sup14]C]cholesterol, glucose at 25 mM stimulated cholesterol uptake compared with Caco-2/15 cells supplemented with 5 mM glucose (P < 0.04). Because combination of 5 mM glucose with 20mM of the structurally related mannitol or sorbitol did not change cholesterol uptake, we conclude that extracellular glucose concentration is uniquely involved in the regulation of intestinal cholesterol transport. The high concentration of glucose enhanced the protein expression of the critical cholesterol transporter NPC1L1 and that of CD36 (P < 0.02) and concomitantly decreased SR-BI protein mass (P < 0.02). No significant changes were observed in the protein expression of ABCA1 and ABCG8, which act as efflux pumps favoring cholesterol export out of absorptive cells. At the same time, 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity was decreased (P < 0.007), whereas ACAT activity remained unchanged. Finally, increases were noted in the transcription factors LXR-α, LXR-β, PPAR-β, and PPAR--γ along with a drop in the protein expression of SREBP-2. Collectively, our data indicate that glucose at high concentrations may regulate intestinal cholesterol transport and metabolism in Caco-2/15 cells, thus suggesting a potential influence on the cholesterol absorption process in Type 2 diabetes. [ABSTRACT FROM AUTHOR]

Abstract: Previously, using an oxysterol to induce cholesterol trafficking to the Endoplasmic Reticulum (ER), we reported a dissociation between cholesterol transport to two important cholesterol regulatory components in the ER: the cholesterol esterifying enzyme ACAT (Acyl CoA:Cholesterol Acyltransferase) and the membrane-bound transcription factor SREBP (Sterol Regulatory Element Binding Protein) (X. Du, Y.H. Pham and A.J. Brown, Effects of 25-hydroxycholesterol on cholesterol esterification and SREBP processing are dissociable: implications for cholesterol movement to the regulatory pool in the endoplasmic reticulum, J. Biol Chem. 279 (2004) 47010–47016). Here, we employed low-density lipoprotein (LDL) as a more physiologically-relevant mode of cholesterol delivery, and compared cholesterol transport to ACAT (determined by esterification) and SREBP (assessed by processing) in mutant Chinese Hamster Ovary cells that have cholesterol-trafficking defects (including Niemann–Pick type C). We showed clear differences in kinetics between the two, with impaired cholesterol trafficking to SREBP being resolved more rapidly than to ACAT. This is unlikely to be due to a reduced threshold of cholesterol sensed by the SREBP system relative to ACAT, since both responded to LDL-derived cholesterol within 2 h whereas the divergence observed between the two was prolonged (>20 h). Furthermore, ACAT inhibition did not expand the ER regulatory pool of cholesterol as judged by unaltered sensitivity of SREBP processing to LDL. Collectively, our data favor the contention that there are different cholesterol pools and/or transport pathways which feed ACAT and SREBP within the ER. [Copyright &y& Elsevier]

Abstract: Fungal beauveriolide III (1b), discovered as an inhibitor of lipid droplet accumulation in mouse macrophages and showing antiatherogenic activity in mouse model, consists of l-Phe, l-Ala, d-allo-Ile, and (3S, 4S)-3-hydroxy-4-methyloctanoic acid moieties. A combinatorial library of beauveriolide analogues focusing on l-Ala and d-allo-Ile of 1b was synthesized by combinatorial synthesis. Among them, d-Ala analogues consisting of A{2} improved their solubility, while those with 7{1, 3, 2},7{2, 3, 1}, and 7{2, 3, 2} were 20 times more potent than 1b. [Copyright &y& Elsevier]

Abstract: Accumulation of excess non-esterified free cholesterol (FC) in macrophages is a key factor in macrophage death during late stages of atheroslerosis. Raising FC content in macrophages has been shown to trigger Rac activation and actin polymerisation and to inhibit cell migration. Here, the plasma membrane distribution of the fluorescent cholesterol-mimicking sterol dehydroergosterol (DHE) was investigated in FC-loaded J774 macrophages. Wide field fluorescence and deconvolution microscopy were combined with quantitative assessment of sterol distribution in straightened plasma membrane image segments. DHE''s surface distribution matched exactly large ruffles and membrane protrusions which were pronounced in FC-loaded cells. Plasma membrane blebs, however, formed in FC-loaded J774 cells had a homogenous staining along the membrane bilayer at 20°C. The results show that even in FC-loaded cells with increased membrane cholesterol content, sterols do not form a separate phase in the plasma membrane. [Copyright &y& Elsevier]

Abstract: Diacylglycerol acyltransferase (DGAT) and acyl-CoA: cholesterol acyltransferase (ACAT) are the enzymes that catalyze the final reactions of triacylgycerol (TG) and cholesteryl ester (CE) synthesis, and accumulation of TG and CE in adipocytes and arteries causes obesity and atherosclerosis, respectively. Therefore, DGAT and ACAT have been viewed as potential therapeutic targets for these diseases. From the screening program for DGAT inhibitors, new compounds were discovered from fungal and plant extracts, and are expected to provide leads for drug development. From the screening programs for ACAT inhibitors and lipid droplet synthesis inhibitors, new compounds with chemical structures different from those of known synthetic inhibitors were discovered from the cultures of fungal and actinomycete strains. Among them, fungal beauveriolide III rather selectively inhibited ACAT1 isozyme, while fungal pyripyropene A was found to be a highly selective inhibitor of ACAT2 isozyme. Both inhibitors proved orally active in in vivo models. Furthermore, a library of beauveriolide and pyripyropene analogs was prepared by combinatorial and semisynthetic methods, respectively. The future prospects of these inhibitors are discussed. [Copyright &y& Elsevier]