Adaptation of the secretome of Echinostoma caproni may contribute to parasite survival in a Th1 milieu.

Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode, broadly employed to study the host-dependent mechanisms that govern the evolution of intestinal helminth infections. Resistance against E. caproni homologous secondary infections has been reported in mice and appears to be related to the generation of a local Th2 response, whereas Th1 responses promote the development of chronic primary infections. Herein, the ability of E. caproni to modulate its secretome according to the host environment is investigated. A two-dimensional differential in gel electrophoresis (2D-DIGE) analysis was performed to elucidate changes in the excretory/secretory products of E. caproni adults after primary and secondary infections in mice. A total of 16 protein spots showed significant differences between groups, and 7 of them were successfully identified by mass spectrometry. Adult worms exposed to a primary infection appear to upregulate proteins involved in detoxification (aldo-keto reductase), stress response (GroEL), and enhancement of parasite survival (acetyl-CoA A-acetyltransferase and UTP-glucose-1-phosphate urydyltransferase). In contrast, any protein was found to be significantly upregulated after secondary infection. Upregulation of such proteins may serve to withstand the hostile Th1 environment generated in primary infections in mice. These results provide new insights into the resistance mechanisms developed by the parasites to ensure their long-term survival.

Application of acetyl-CoA acetyltransferase (AtoAD) in Escherichia coli to increase 3-hydroxyvalerate fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

Polyhydroxyalkanoate (PHA) is a family of biodegradable polymers, and incorporation of different monomers can alter its physical properties. To produce the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) containing a high level of 3-hydroxyvalerate (3HV) by altering acetyl-CoA pool levels, we overexpressed an acetyl-CoA acetyltransferase (atoAD) in an engineered E. coli strain, YH090, carrying PHA synthetic genes bktB, phaB, and phaC. It was found that, with introduction of atoAD and with propionate as a co-substrate, 3HV fraction in PHA was increased up to 7.3-fold higher than a strain without atoAD expressed in trans (67.9 mol%). By the analysis of CoA pool concentrations in vivo and in vitro using HPLC and LC-MS, overexpression of AtoAD was shown to decrease the amount of acetyl-CoA and increase the propionyl-CoA/acetyl-CoA ratio, ultimately resulting in an increased 3HV fraction in PHA. Finally, synthesis of P(3HB-co-3HV) containing 57.9 mol% of 3HV was achieved by fed-batch fermentation of YJ101 with propionate.

Advanced glycation end products increase lipids accumulation in macrophages through upregulation of receptor of advanced glycation end products: increasing uptake, esterification and decreasing efflux of cholesterol.

BACKGROUND: Previous reports have suggested that advanced glycation end products (AGEs) participate in the pathogenesis of diabetic macroangiopathy. Our previous study have found that AGEs can increase the lipid droplets accumulation in aortas of diabetic rats, but the current understanding of the mechanisms remains incomplete by which AGEs affect lipids accumulation in macrophages and accelerate atherosclerosis. In this study, we investigated the role of AGEs on lipids accumulation in macrophages and the possible molecular mechanisms including cholesterol influx, esterification and efflux of macrophages. METHODS: THP-1 cells were incubated with PMA to differentiate to be macrophages which were treated with AGEs in the concentration of 300 µg/ml and 600 µg/ml with or without anti-RAGE (receptor for AGEs) antibody and then stimulated by oxidized-LDL (oxLDL) or Dil-oxLDL. Lipids accumulation was examined by oil red staining. The cholesterol uptake, esterification and efflux were detected respectively by fluorescence microscope, enzymatic assay kit and fluorescence microplate. Quantitative RT-PCR and Western blot were used to measure expression of the moleculars involved in cholesterol uptake, synthesis/esterification and efflux. RESULTS: AGEs increased lipids accumulation in macrophages in a concentration-dependent manner. 600 µg/ml AGEs obviously upregulated oxLDL uptake, increased levels of cholesterol ester in macrophages, and decreased the HDL-mediated cholesterol efflux by regulating the main molecular expression including CD36, Scavenger receptors (SR) A2, HMG-CoA reductase (HMGCR), ACAT1 and ATP-binding cassette transporter G1 (ABCG1). The changes above were inversed when the cells were pretreated with anti-RAGE antibody. CONCLUSIONS: The current study suggest that AGEs can increase lipids accumulation in macrophages by regulating cholesterol uptake, esterification and efflux mainly through binding with RAGE, which provide a deep understanding of mechanisms how AGEs accelerating diabetic atherogenesis.

Loss of ß-carotene 15,15'-oxygenase in developing mouse tissues alters esterification of retinol, cholesterol and diacylglycerols.

We provide novel insights into the function(s) of ß-carotene-15,15'-oxygenase (CMOI) during embryogenesis. By performing in vivo and in vitro experiments, we showed that CMOI influences not only lecithin:retinol acyltransferase but also acyl CoA:retinol acyltransferase reaction in the developing tissues at mid-gestation. In addition, LC/MS lipidomics analysis of the CMOI-/- embryos showed reduced levels of four phosphatidylcholine and three phosphatidylethanolamine acyl chain species, and of eight triacylglycerol species with four or more unsaturations and fifty-two or more carbons in the acyl chains. Cholesteryl esters of arachidonate, palmitate, linoleate, and DHA were also reduced to less than 30% of control. Analysis of the fatty acyl CoA species ruled out a loss in fatty acyl CoA synthetase capability. Comparison of acyl species suggested significantly decreased 18:2, 18:3, 20:1, 20:4, or 22:6 acyl chains within the above lipids in CMOI-null embryos. Furthermore, LCAT, ACAT1 and DGAT2 mRNA levels were also downregulated in CMOI-/- embryos. These data strongly support the notion that, in addition to cleaving ß-carotene to generate retinoids, CMOI serves an additional function(s) in retinoid and lipid metabolism and point to its role in the formation of specific lipids, possibly for use in nervous system tissue.

Intratumor cholesteryl ester accumulation is associated with human breast cancer proliferation and aggressive potential: a molecular and clinicopathological study.

BACKGROUND: The metabolic effect of intratumor cholesteryl ester (CE) in breast cancer remains poorly understood. The objective was to analyze the relationship between intratumor CE content and clinicopathological variables in human breast carcinomas. METHODS: We classified 30 breast carcinoma samples into three subgroups: 10 luminal-A tumors (ER+/PR+/Her2-), 10 Her-2 tumors (ER-/PR-/Her2+), and 10 triple negative (TN) tumors (ER-/PR-/Her2-). We analyzed intratumor neutral CE, free cholesterol (FC) and triglyceride (TG) content by thin layer chromatography after lipid extraction. RNA and protein levels of lipid metabolism and invasion mediators were analyzed by real time PCR and Western blot analysis. RESULTS: Group-wise comparisons, linear regression and logistic regression models showed a close association between CE-rich tumors and higher histologic grade, Ki-67 and tumor necrosis. CE-rich tumors displayed higher mRNA and protein levels of low-density lipoprotein receptor (LDLR) and scavenger receptor class B member 1 (SCARB1). An increased expression of acetyl-Coenzyme A acetyltransferase 1 (ACAT1) in CE-rich tumors was also reported. CONCLUSIONS: Intratumor CE accumulation is intimately linked to proliferation and aggressive potential of breast cancer tumors. Our data support the link between intratumor CE content and poor clinical outcome and open the door to new antitumor interventions.

[Microbial metabolites that inhibit sterol biosynthesis, their chemical diversity and characteristics of mode of action].

Inhibitors of sterol biosynthesis (ISB) are widespread in nature and characterized by appreciable diversity both in their chemical structure and mode of action. Many of these inhibitors express noticeable biological activity and approved themselves in development of various pharmaceuticals. In this review there is a detailed description of biologically active microbial metabolites with revealed chemical structure that have ability to inhibit sterol biosynthesis. Inhibitors of mevalonate pathway in fungous and mammalian cells, exhibiting hypolipidemic or antifungal activity, as well as inhibitors of alternative non-mevalonate (pyruvate gliceraldehyde phosphate) isoprenoid pathway, which are promising in the development of affective antimicrobial or antiparasitic drugs, are under consideration in this review. Chemical formulas of the main natural inhibitors and their semi-synthetic derivatives are represented. Mechanism of their action at cellular and biochemical level is discussed. Special attention is given to inhibitors of 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase (group of lovastatin) and inhibitors of acyl-CoA-cholesterol-acyl transferase (ACAT) that possess hypolipidemic activity and could be affective in the treatment of atherosclerosis. In case of inhibitors of late stages of sterol biosynthesis (after squalene formation) special attention is paid to compounds possessing evident antifungal and antitumoral activity. Explanation of mechanism of anticancer and antiviral action of microbial ISB, as well as the description of their ability to induce apoptosis is given.

Potent Anticancer Activities of Beauvericin Against KB Cells In Vitro by Inhibiting the Expression of ACAT1 and Exploring Binding Affinity.

BACKGROUND AND OBJECTIVE: Beauvericin (BEA), a cyclic hexadepsipeptide mycotoxin, is a potent inhibitor of the acyl-CoA: cholesterol acyltransferase enzyme 1 (ACAT1), involved in multiple tumor-correlated pathways. However, the binding mechanisms between BEA and ACAT1 were not elucidated. METHODS: BEA was purified from a mangrove entophytic Fusarium sp. KL11. Single-crystal X-ray diffraction was used to determine the structure of BEA. Wound healing assays of BEA against KB cell line and MDA-MB-231 cell line were evaluated. Inhibitory potency of BEA against ACAT1 was determined by ELISA assays. Molecular docking was carried out to illuminate the bonding mechanism between BEA and ACAT1. RESULTS: The structure of BEA was confirmed by X-ray diffraction, indicating a monoclinic crystal system with P21 space group (α = 90°, ß = 92.2216(9)°, γ= 90°). BEA displayed migration-inhibitory activities against KB cells and MDA-MB-231 cells In Vitro. ELISA assays revealed that the protein expression level of ACAT1 in KB cells was significantly decreased after BEA treatment (P ＜0.05). Molecular docking demonstrated that BEA formed hydrogen bond with His425 and pi-pi staking with Tyr429 in ACAT1. CONCLUSION: BEA sufficiently inhibited the proliferation and migration of KB cells and MDA-MB-231 cells by downregulating ACAT1 expression. In addition, BEA potentially possessed a strong binding affinity with ACAT1. BEA may serve as a potential lead compound for the development of a new ACAT1-targeted anticancer drug.

Thieno[2,3-c]isoquinolin-5-one, a potent poly(ADP-ribose) polymerase inhibitor, promotes atherosclerotic plaque regression in high-fat diet-fed apolipoprotein E-deficient mice: effects on inflammatory markers and lipid content.

We recently showed that poly(ADP-ribose) polymerase (PARP) is activated within atherosclerotic plaques in an animal model of atherosclerosis. Pharmacological inhibition of PARP or reduced expression in heterozygous animals interferes with atherogenesis and may promote factors of plaque stability, possibly reflecting changes in inflammatory and cellular factors consistent with plaque stability. The current study addresses the hypothesis that pharmacological inhibition of PARP promotes atherosclerotic plaque regression. Using a high-fat diet-induced atherosclerosis apolipoprotein E(-/-) mouse model, we demonstrate that administration of the potent PARP inhibitor, thieno[2,3-c]isoquinolin-5-one (TIQ-A), when combined with a regular diet regimen during treatment, induced regression of established plaques. Plaque regression was associated with a reduction in total cholesterol and low-density lipoproteins. Furthermore, plaques of TIQ-A-treated mice were highly enriched with collagen and smooth muscle cells, displayed thick fibrous caps, and exhibited a marked reduction in CD68-positive macrophage recruitment and associated foam cell presence. These changes correlated with a significant decrease in expression of monocyte chemoattractant protein-1 and intercellular cell adhesion molecule-1, potentially as a result of a robust reduction in tumor necrosis factor expression. The PARP inhibitor appeared to affect cholesterol metabolism by affecting acyl-coenzymeA/cholesterol acyltransferase-1 expression but exerted no effect on cholesterol influx or efflux as assessed by an examination of the ATP-binding cassette transporter-1 and the scavenger receptor-A expression levels in the different experimental groups. In accordance, PARP inhibition may prove beneficial not only in preventing atherogenesis but also in promoting regression of preexisting plaques.

Isolation and characterization of Chinese hamster ovary cell mutants defective in assembly of peroxisomes.

We made use of autoradiographic screening to isolate two Chinese hamster ovary (CHO) cell mutants deficient in peroxisomal dihydroxyacetonephosphate acyltransferase, a key enzyme for the biosynthesis of ether glycerolipids such as plasmalogens. Morphological analysis revealed no evidence of peroxisome in these mutants. Catalase was as active as in the normal cells but was not sedimentable. Pulse-chase radiolabeling experiments and cell-free translation of RNA demonstrated that acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation system, was synthesized as the 75-kD form but was not converted to 53- and 22-kD mature components that were present in the wild-type CHO cells; rather, degradation was apparent. Peroxisomal thiolase was synthesized as in normal cells but remained as a larger, 44-kD precursor, whereas maturation to the 41-kD enzyme was detected in the wild-type cells. The peroxisomal 70-kD integral membrane protein was also equally synthesized, as in the wild-type cells, and was not degraded. These results suggest that assembly of the peroxisomes is defective in the mutants, whereas the synthesis of peroxisomal proteins appears to be normal. Cell-fusion studies revealed that the two mutants are recessive to the wild-type CHO cells and belong to different complementation groups. Thus, these mutants presumably contain different lesions in gene(s) encoding factor(s) required for peroxisome assembly.

Peb1p (Pas7p) is an intraperoxisomal receptor for the NH2-terminal, type 2, peroxisomal targeting sequence of thiolase: Peb1p itself is targeted to peroxisomes by an NH2-terminal peptide.

Peb1 is a peroxisome biogenesis mutant isolated in Saccharomyces cerevisiae that is selectively defective in the import of thiolase into peroxisomes but has a normal ability to package catalase, luciferase and acyl-CoA oxidase (Zhang, J. W., C. Luckey, and P. B. Lazarow. 1993. Mol. Biol. Cell. 4:1351-1359). Thiolase differs from these other peroxisomal proteins in that it is targeted by an NH2-terminal, 16-amino acid peroxisomal targeting sequence type 2 (PTS 2). This phenotype suggests that the PEB1 protein might function as a receptor for the PTS2. The PEB1 gene has been cloned by functional complementation. It encodes a 42,320-D, hydrophilic protein with no predicted transmembrane segment. It contains six WD repeats that comprise the entire protein except for the first 55 amino acids. Peb1p was tagged with hemagglutinin epitopes and determined to be exclusively within peroxisomes by digitonin permeabilization, immunofluorescence, protease protection and immuno-electron microscopy (Zhang, J. W., and P. B. Lazarow. 1995. J. Cell Biol. 129:65-80). Peb1p is identical to Pas7p (Marzioch, M., R. Erdmann, M. Veenhuis, and W.-H. Kunau. 1994. EMBO J. 13: 4908-4917). We have now tested whether Peb1p interacts with the PTS2 of thiolase. With the two-hybrid assay, we observed a strong interaction between Peb1p and thiolase that was abolished by deleting the first 16 amino acids of thiolase. An oligopeptide consisting of the first 16 amino acids of thiolase was sufficient for the affinity binding of Peb1p. Binding was reduced by the replacement of leucine with arginine at residue five, a change that is known to reduce thiolase targeting in vivo. Finally, a thiolase-Peb1p complex was isolated by immunoprecipitation. To investigate the topogenesis of Peb1p, its first 56-amino acid residues were fused in front of truncated thiolase lacking the NH2-terminal 16-amino acid PTS2. The fusion protein was expressed in a thiolase knockout strain. Equilibrium density centrifugation and immunofluorescence indicated that the fusion protein was located in peroxisomes. Deletion of residues 6-55 from native Peb1p resulted in a cytosolic location and the loss of function. Thus the NH2-terminal 56-amino acid residues of Peb1p are necessary and sufficient for peroxisomal targeting. Peb1p is found in peroxisomes whether thiolase is expressed or not. These results suggest that Peb1p (Pas7p) is an intraperoxisomal receptor for the type 2 peroxisomal targeting signal.

Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations.

Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity.

Defect in biosynthesis of mitochondrial acetoacetyl-coenzyme A thiolase in cultured fibroblasts from a boy with 3-ketothiolase deficiency.

The etiology of 3-ketothiolase deficiency has been attributed to a defect of mitochondrial acetoacetyl-CoA thiolase because the acetoacetyl-CoA thiolase activity in related materials is not activated by K+, a property characteristic for this enzyme. We studied the enzyme protein and the biosynthesis of mitochondrial acetoacetyl-CoA thiolase, using cultured skin fibroblasts from a 5-yr-old boy with 3-ketothiolase deficiency. The following results were obtained. (a) Activation of acetoacetyl-CoA thiolase activity by K+ was nil; (b) The enzyme activity was not affected by treatment with the antibody against mitochondrial acetoacetyl-CoA thiolase; (c) A signal for mitochondrial acetoacetyl-CoA thiolase protein was not detected in the immunoblot analysis; and (d) Pulse-chase experiments of skin fibroblasts, using [35S]methionine, revealed no incorporation of radioactivity into this enzyme. Therefore, fibroblasts from this patient lacked mitochondrial acetoacetyl-CoA thiolase protein due to a defect in its biosynthesis.

Expression of genes encoding peroxisomal proteins in Saccharomyces cerevisiae is regulated by different circuits of transcriptional control.

In Saccharomyces cerevisiae induction of the FOX3 gene, encoding peroxisomal 3-oxoacyl-CoA thiolase, by growth on oleate as sole carbon source, is exerted via the cis-acting DNA element designated oleate response element (ORE) (Einerhand et al. (1991) Eur. J. Biochem. 200, 113-122). The transcription factor(s) binding to this upstream activation site (UAS) are still unknown, however. Induction of another peroxisomal enzyme, citrate synthase (CIT2) is dependent on the products of two genes called RTG1 and RTG2 (Liao and Butow (1993) Cell 72, 61-71). In the present study we have investigated whether RTG1 controls other genes coding for peroxisomal proteins, and whether such control takes place via the ORE. A number of genes coding for a variety of peroxisomal proteins such as: thiolase and catalase (peroxisomal matrix proteins), PAS3p (a peroxisomal membrane protein) and PAS10p (a protein involved in the import of peroxisomal proteins) were studied in their response to RTG1. Although the RTG1 and 2 products proved to be required for the increase in number and volume of peroxisomes upon induction by oleate, the single promoter output of the chosen set of genes remained practically unchanged in a rtg1 mutant strain. In addition gel retardation experiments indicated that RTG1 does not bind to the ORE. The behavior of genes coding for the various proteins also varied during repression, derepression and induction, indicating that probably a number of proteins are involved in tuning the output of each gene to cellular demand.

Up-regulation of the peroxisomal beta-oxidation system occurs in butyrate-grown Candida tropicalis following disruption of the gene encoding peroxisomal 3-ketoacyl-CoA thiolase.

In the yeast Candida tropicalis, two thiolase isozymes, peroxisomal acetoacetyl-CoA thiolase and peroxisomal 3-ketoacyl-CoA thiolase, participate in the peroxisomal fatty acid beta-oxidation system. Their individual contributions have been demonstrated in cells grown on butyrate, with C. tropicalis able to grow in the absence of either one. In the present study, a lack of peroxisomal 3-ketoacyl-CoA thiolase protein resulted in increased expression (up-regulation) of acetoacetyl-CoA thiolase and other peroxisomal proteins, whereas a lack of peroxisomal acetoacetyl-CoA thiolase produced no corresponding effect. Overexpression of the acetoacetyl-CoA thiolase gene did not suppress the up-regulation or the growth retardation on butyrate in cells without peroxisomal 3-ketoacyl-CoA thiolase, even though large amounts of the overexpressed acetoacetyl-CoA thiolase were detected in most of the peroxisomes of butyrate-grown cells. These results provide important evidence of the greater contribution of 3-ketoacyl-CoA thiolase to the peroxisomal beta-oxidation system than acetoacetyl-CoA thiolase in C. tropicalis and a novel insight into the regulation of the peroxisomal beta-oxidation system.

Single base substitutions at the initiator codon in the mitochondrial acetoacetyl-CoA thiolase (ACAT1/T2) gene result in production of varying amounts of wild-type T2 polypeptide.

Initiator codon mutations are relatively uncommon and less well characterized compared to other types of mutations. We identified a novel initiator codon mutation (c.2T>C) heterozygously in a Japanese patient (Patient GK30) with mitochondrial acetoacetyl-CoA thiolase (T2) gene deficiency (ACAT1 deficiency); c.149delC was on the other allele. We examined translation efficiencies of nine mutant T2 cDNAs harboring one-base substitutions at the initiator methionine codon using in vivo transient expression analysis. We found that all the mutants produced wild-type T2 polypeptide, to various degrees (wild type (100%) > c.1A>C (66%) > c.2T>C, c.3G>C, c.3G>T (22%) > c3G>A, c.1A>G (11%) > c.2T>A, c.2T>G, c.1A>T (7.4%)). T2 mRNA expression levels in Patient GK08 (a homozygote of c.2T>A) and Patient GK30 fibroblasts, respectively, were almost the same as in control fibroblasts, when examined using semiquantitative PCR. This means that initiator codon mutations did not affect T2 mRNA levels. We propose that all one-base substitutions at the initiator methionine codon in the T2 gene could be mutations, which retain some residual T2 activity.

Cholesterol homeostasis in the ovaries of neonatally diethylstilbestrol-treated mice.

In the ovary of neonatally DES-treated mice, lipid droplets accumulation was observed in the hypertrophied interstitial tissues. Our previous results demonstrated that the impaired steroidogenesis in the ovary of neonatally DES-treated mice was caused by altered gonadotropins levels, and resulted in the hypertrophy of ovarian interstitial cells. We speculated that lipid droplets in the ovary mainly consisted of cholesterol. This study was aimed to examine the effects of neonatal DES on cholesterol homeostasis in the ovary. The serum and ovarian total cholesterol concentrations in 3-month-old neonatally DES-treated mice were significantly higher than those in the neonatally oil-treated mice, but triglyceride concentrations were not altered. In the ovary of neonatally DES-treated mice, expression of Hmgcr, a rate-limiting enzyme in de novo cholesterol biosynthesis, was reduced but expression of Ldlr and Scarb1, involved in cholesterol uptake, was not changed. These results suggest that cholesterol uptake is not altered in the ovary of 3-month-old neonatally DES-treated mice. However, the expression of Acat1, the microsomal acyl coenzyme A cholesterol acyltransferase which is involved in cholesterol esterification and storing was increased compared with that in the ovary of neonatally oil-treated mice. Since ovarian steroidogenesis in neonatally DES-treated mice was impaired, synthesized and/or obtained cholesterol from the blood may not be used sufficiently. Thus, in the ovary of neonatally DES-treated mice, cholesterol is esterified by ACAT1 and stored in the interstitial cells.

Hydrogen sulfide inhibits macrophage-derived foam cell formation.

Recent evidence indicates that hydrogen sulfide (H(2)S) exerts an antiatherogenic effect, but the mechanism is unclear. Formation of macrophage-derived foam cells is a crucial event in the development of atherosclerosis. Thus, we explore the effect of H(2)S on the formation of macrophage-derived foam cells. Incubation of monocyte-derived macrophages with oxidized LDL (oxLDL) alone caused significant increases both in intracellular lipids revealed by Oil-red O staining and in intracellular total cholesterol (TC) and esterified cholesterol (EC) concentrations assessed by high-performance liquid chromatography. Sodium hydrosulfide (NaHS, an H(2)S donor) remarkably abrogated oxLDL-induced intracellular lipid accumulation, and attenuated TC and EC concentrations and EC/TC ratio, whereas dl-propargylglycine (PPG) (a H(2)S-generating enzyme cystathionine gamma lyase inhibitor) exacerbated lipid accumulation and augmented TC and EC concentrations and EC/TC ratio. Incubation of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-oxLDL led to lipoprotein binding and uptake of macrophages, which was blunted by NaHS, but enhanced by PPG. Furthermore, OxLDL markedly induced CD36, scavenger receptor A (SR-A) and acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) expressions in macrophages, which was suppressed by NaHS (50-200 µmol/L). Finally, the down-regulations of TC and EC concentrations as well as CD36 and ACAT-1 expressions by NaHS were suppressed by glibenclamide, a K(ATP) channel blocker, but facilitated by PD98059, an extracellular signal-regulated kinases 1 and 2 (ERK1/2) inhibitor. These results suggested that H(2)S inhibits foam cell formation by down-regulating CD36, SR-A and ACAT1 expressions via the K(ATP)/ERK1/2 pathway in human monocyte-derived macrophages.

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

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

Effect of dietary phosphatidylinositol on cholesterol metabolism in Zucker (fa/fa) rats.

Recent studies have shown that dietary phospholipids, especially phosphatidylcholine and phosphatidylserine, have various beneficial biological effects. However, there are not enough data concerning the physiological function of dietary phosphatidylinositol (PI). The metabolic syndrome, a cluster of metabolic abnormalities such as dyslipidemia, diabetes mellitus, and hypertension, is widespread and increasingly prevalent diseases in industrialized countries. In the present study, we evaluated that the effect of dietary PI on cholesterol metabolism in metabolic syndrome model Zucker (fa/fa) rats. For 4 weeks, rats were fed semisynthetic diets containing either 7% soybean oil or 5% soybean oil plus 2% PI. Dietary PI prevented the mild hypercholesterolemia and hepatic cholesterol accumulation in Zucker (fa/fa) rats. These effects were attributable to an increased fecal bile acid excretion and to the tendencies of decreased ACAT1 mRNA level and increased CYP7A1 mRNA level in the liver. Additionally, dietary PI markedly increased microsomal PI content in the liver of Zucker (fa/fa) rats. Our study suggests that dietary PI normalizes cholesterol metabolism through the enhancement of fecal bile acid excretion in the metabolic syndrome model rats.

Turnover and transformation of mitochondrial acetyl-CoA acetyltransferase into CoA-modified forms.

Rat liver mitochondrial acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase, EC 2.3.1.9) exists additionally in the CoA-modified forms A1 and A2. After a pulse of radioactivity using [35S]methionine in hepatocytes, the highest radioactivity was obtained in the unmodified enzyme. Over the chase time, the radioactivity in the unmodified enzyme decreased, but simultaneously increased in both CoA-modified forms, thus proving that the fully active unmodified enzyme exists before the partially active modified forms A1 and A2. Also, the specific radioactivity (ratio % radioactivity/% immunoreactive area) of A1 > A2 demonstrates a sequential CoA modification of form A1 to form A2. Acetyl-CoA acetyltransferase was degraded with an apparent half-life of 38.0 h: the modified forms A1 and A2 have half-lives of 24.5 and 7.2 h. The physiological meaning of the CoA modification of acetyl-CoA acetyltransferase is not yet understood.