Cholesteryl Ester Formation Research Articles

Bryonolic Acid Blocks Cancer Cell Clonogenicity and Invasiveness through the Inhibition of Fatty Acid: Cholesteryl Ester Formation

Bryonolic acid (BrA) is a pentacyclic triterpene present in several plants used in African traditional medicine such as Anisophyllea dichostyla R. Br. Here we investigated the in vitro anticancer properties of BrA. We report that BrA inhibits acyl-coA: cholesterol acyl transferase (ACAT) activity in rat liver microsomes in a concentration-dependent manner, blocking the biosynthesis of the cholesterol fatty acid ester tumour promoter. We next demonstrated that BrA inhibits ACAT in intact cancer cells with an IC50 of 12.6 ± 2.4 µM. BrA inhibited both clonogenicity and invasiveness of several cancer cell lines, establishing that BrA displays specific anticancer properties. BrA appears to be more potent than the other pentacyclic triterpenes, betulinic acid and ursolic acid studied under similar conditions. The inhibitory effect of BrA was reversed by exogenous addition of cholesteryl oleate, showing that ACAT inhibition is responsible for the anticancer effect of BrA. This report reveals new anticancer properties for BrA.

Arginine 123 of apolipoprotein A-I is essential for lecithin:cholesterol acyltransferase activity

ApoA-I activates LCAT that converts lipoprotein cholesterol to cholesteryl ester (CE). Molecular dynamic simulations suggested earlier that helices 5 of two antiparallel apoA-I molecules on discoidal HDL form an amphipathic tunnel for migration of acyl chains and unesterified cholesterol to the active sites of LCAT. Our recent crystal structure of Δ(185-243)apoA-I showed the tunnel formed by helices 5/5, with two positively charged residues arginine 123 positioned at the edge of the hydrophobic tunnel. We hypothesized that these uniquely positioned residues Arg123 are poised for interaction with fatty acids produced by LCAT hydrolysis of the sn-2 chains of phosphatidylcholine, thus positioning the fatty acids for esterification to cholesterol. To test the importance of Arg123 for LCAT phospholipid hydrolysis and CE formation, we generated apoA-I[R123A] and apoA-I[R123E] mutants and made discoidal HDL with the mutants and WT apoA-I. Neither mutation of Arg123 changed the particle composition or size, or the protein conformation or stability. However, both mutations of Arg123 significantly reduced LCAT catalytic efficiency and the apparent Vmax for CE formation without affecting LCAT phospholipid hydrolysis. A control mutation, apoA-I[R131A], did not affect LCAT phospholipid hydrolysis or CE formation. These data suggest that Arg123 of apoA-I on discoidal HDL participates in LCAT-mediated cholesterol esterification.

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

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

Lecithin:Cholesterol Acyltransferase Activation by Sulfhydryl-Reactive Small Molecules: Role of Cysteine-31.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in familial lecithin:cholesterol acyltransferase deficiency (FLD), an autosomal recessive disorder characterized by low high-density lipoprotein, anemia, and renal disease. This study aimed to investigate the mechanism by which compound A [3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile], a small heterocyclic amine, activates LCAT. The effect of compound A on LCAT was tested in human plasma and with recombinant LCAT. Mass spectrometry and nuclear magnetic resonance were used to determine compound A adduct formation with LCAT. Molecular modeling was performed to gain insight into the effects of compound A on LCAT structure and activity. Compound A increased LCAT activity in a subset (three of nine) of LCAT mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-Cys31Gly prevented activation by compound A. Substitution of Cys31 with charged residues (Glu, Arg, and Lys) decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe, and Met) increased activity up to 3-fold (P < 0.005). Mass spectrometry of a tryptic digestion of LCAT incubated with compound A revealed a +103.017 m/z adduct on Cys31, consistent with the addition of a single hydrophobic cyanopyrazine ring. Molecular modeling identified potential interactions of compound A near Cys31 and structural changes correlating with enhanced activity. Functional groups important for LCAT activation by compound A were identified by testing compound A derivatives. Finally, sulfhydryl-reactive β-lactams were developed as a new class of LCAT activators. In conclusion, compound A activates LCAT, including some FLD mutations, by forming a hydrophobic adduct with Cys31, thus providing a mechanistic rationale for the design of future LCAT activators.

The Spectrum of Niemann-Pick Type C Disease in Greece.

Niemann-Pick type C disease (NPC) is a neurovisceral lysosomal storage disease caused by mutations in either the NPC1 or the NPC2 gene. It is a cellular lipid trafficking disorder characterized by the accumulation of unesterified cholesterol and various sphingolipids in the lysosomes and late endosomes, and it exhibits a broad clinical spectrum. Today, over 420 disease-causing mutations have been identified in the NPC1 and the NPC2 genes. We present the clinical, biochemical, and molecular findings in 14 cases diagnosed in Greece during the last 28 years. Age at diagnosis ranged from 2.5 months to 48 years. Systemic manifestations were present in 7/14 patients. All developed neurological manifestations (age of onset 5 months to 42 years). Six patients are still alive (age: 5-50 years). Classical filipin staining pattern was observed in all but four patients (3 NPC1, 1 NPC2). The rate of LDL-induced cholesteryl ester formation was severely reduced in 4/7 and significantly reduced in 3/7 patients studied. Increased chitotriosidase activity was observed in 9/12 patients. Mutation analysis in 11 unrelated patients identified 12 different mutations in the NPC1 gene: eight previously described p.E1089K (c.3265G>A), p.F284Lfs*26 (c.852delT), p.A1132P(c.3394G>C), del promoter region and exons 1-10, p.R1186H (c.3557G>A), p.P1007A (c.3019C>G), p.Q92R(c.275A>G),p.S940L (c.2819C>T), and four novel ones: (p.N701K fs*13 (c.2102-2103insA), p.K1057R (c.3170A>G), IVS23+3insT(c.3591+3insT), p.C1119*(c.3357T>C); and the previously described IVS2+5G>A(c.190+5G>A) mutation in the NPC2 gene. All patients were of Greek origin. Assuming a birth rate of 100,000/year, a rough incidence estimate for NPC disease in Greece would be 0.5/100,000 births.

MOLECULAR EVALUATION OF -75G/A AND +83C/T APOLIPOPROTEIN A-1 GENE POLYMORPHISMS IN NON ALCOHOLIC FATTY LIVER DISEASE

Apolipoprotein A-1 (APOA1) is the major protein component of High Density Lipoprotein in plasma, promotes cholesterol efflux from tissues to the liver for excretion, it is a cofactor for Lecithin Cholesterolacyl Transferase (LCAT) which is responsible for the formation of most plasma cholesteryl esters. Therefore, the main objective of the present study was carried out to investigate the association of APOA1 gene polymorphisms among patients with Non Alcoholic Fatty Liver Disease (NAFLD) by using PCR-RFLP analysis. Samples were collected from 100 cases affected with NAFLD as well as 100 healthy samples as a negative control. APOA1 +83C/T gene polymorphism was showed that CC genotype was significantly higher frequency (p=0.0001) in healthy control than cases (70% vs. 10%), CT genotype was significantly higher frequency (p=0.001) in cases than healthy control (65% vs. 25%) and TT genotype was significantly higher frequency (p=0.001) in cases than healthy control (25% vs. 5%). Regarding all types of APOA1 -75G/A gene polymorphism, they were showed that all cases were non-significantly (p>0.05) when compared to healthy control. So, NAFLD among Egyptian was strongly associated with mutations related to APOA1 gene polymorphism especially APOA1 +83C/T gene polymorphism.

ATR-101, a Selective and Potent Inhibitor of Acyl-CoA Acyltransferase 1, Induces Apoptosis in H295R Adrenocortical Cells and in the Adrenal Cortex of Dogs.

ATR-101 is a novel, oral drug candidate currently in development for the treatment of adrenocortical cancer. ATR-101 is a selective and potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase 1 (ACAT1), an enzyme located in the endoplasmic reticulum (ER) membrane that catalyzes esterification of intracellular free cholesterol (FC). We aimed to identify mechanisms by which ATR-101 induces adrenocortical cell death. In H295R human adrenocortical carcinoma cells, ATR-101 decreases the formation of cholesteryl esters and increases FC levels, demonstrating potent inhibition of ACAT1 activity. Caspase-3/7 levels and terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeled-positive cells are increased by ATR-101 treatment, indicating activation of apoptosis. Exogenous cholesterol markedly potentiates the activity of ATR-101, suggesting that excess FC that cannot be adequately esterified increases caspase-3/7 activation and subsequent cell death. Inhibition of calcium release from the ER or the subsequent uptake of calcium by mitochondria reverses apoptosis induced by ATR-101. ATR-101 also activates multiple components of the unfolded protein response, an indicator of ER stress. Targeted knockdown of ACAT1 in an adrenocortical cell line mimicked the effects of ATR-101, suggesting that ACAT1 mediates the cytotoxic effects of ATR-101. Finally, in vivo treatment of dogs with ATR-101 decreased adrenocortical steroid production and induced cellular apoptosis that was restricted to the adrenal cortex. Together, these studies demonstrate that inhibition of ACAT1 by ATR-101 increases FC, resulting in dysregulation of ER calcium stores that result in ER stress, the unfolded protein response, and ultimately apoptosis.

A Systematic Investigation of Structure/Function Requirements for the Apolipoprotein A-I/Lecithin Cholesterol Acyltransferase Interaction Loop of High-density Lipoprotein

The interaction of lecithin-cholesterol acyltransferase (LCAT) with apolipoprotein A-I (apoA-I) plays a critical role in high-density lipoprotein (HDL) maturation. We previously identified a highly solvent-exposed apoA-I loop domain (Leu(159)-Leu(170)) in nascent HDL, the so-called "solar flare" (SF) region, and proposed that it serves as an LCAT docking site (Wu, Z., Wagner, M. A., Zheng, L., Parks, J. S., Shy, J. M., 3rd, Smith, J. D., Gogonea, V., and Hazen, S. L. (2007) Nat. Struct. Mol. Biol. 14, 861-868). The stability and role of the SF domain of apoA-I in supporting HDL binding and activation of LCAT are debated. Here we show by site-directed mutagenesis that multiple residues within the SF region (Pro(165), Tyr(166), Ser(167), and Asp(168)) of apoA-I are critical for both LCAT binding to HDL and LCAT catalytic efficiency. The critical role for possible hydrogen bond interaction at apoA-I Tyr(166) was further supported using reconstituted HDL generated from apoA-I mutants (Tyr(166) → Glu or Asn), which showed preservation in both LCAT binding affinity and catalytic efficiency. Moreover, the in vivo functional significance of NO2-Tyr(166)-apoA-I, a specific post-translational modification on apoA-I that is abundant within human atherosclerotic plaque, was further investigated by using the recombinant protein generated from E. coli containing a mutated orthogonal tRNA synthetase/tRNACUA pair enabling site-specific insertion of the unnatural amino acid into apoA-I. NO2-Tyr(166)-apoA-I, after subcutaneous injection into hLCAT(Tg/Tg), apoA-I(-/-) mice, showed impaired LCAT activation in vivo, with significant reduction in HDL cholesteryl ester formation. The present results thus identify multiple structural features within the solvent-exposed SF region of apoA-I of nascent HDL essential for optimal LCAT binding and catalytic efficiency.

Functional characterization of two single nucleotide polymorphisms of acyl-coenzyme A:cholesterol acyltransferase 2

BackgroundAcyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) plays a critical role in the formation of cholesteryl esters from cholesterol and fatty acids, and is a potential target for treating hypercholesterolemia. We recently reported the significant effects of two human ACAT2 gene polymorphisms, 41A>G (Glu14Gly, rs9658625) and 734C>T (Thr254Ile, rs2272296), on plasma lipid levels and coronary artery disease susceptibility in a case–control association study. In the present study, we evaluated the possible biological influence of the two polymorphism using two approaches. MethodsIn the first approach, the functional impact of the two polymorphisms was predicted in-silico using available web-based software, and in the second approach, the varying functions of the two polymorphisms were characterized in in vitro experiments, using ACAT2-deficient AC-29 cells. ResultsOur results show that the enzymatic activity of mutant Glu14Gly is approximately two times higher than wildtype, and that this increase is primarily due to the increased expression and/or stability of the mutant ACAT2 protein. ConclusionsThese results suggest that the genetic variation at Glu14Gly is functionally important and may contribute to ACAT2 protein expression and stability.

Role of Acyl‐Coenzyme A:Cholesterol Acyltransferase 1 in Lipogenesis in Adipocytes

Dysregulated lipogenesis in adipose tissue contributes to obesity and its metabolic disorders. However, the molecular mechanisms regulating lipogenesis in adipocytes are not fully understood. Acyl‐coenzyme A: cholesterol acyltransferase (ACAT), an enzyme that catalyzes the formation of cholesteryl ester from free cholesterol (FC), has been identified as a therapeutic target to treat atherosclerosis. However, the role of ACAT in adipogenesis and lipogenesis in adipocytes is unknown. Here, we employed both pharmaceutical inhibitors and a lentivirus‐mediated shRNA‐based gene knockdown technique to investigate the role of ACAT in adipogenesis using murine 3T3‐L1 preadipocytes as an in vitro model of adipogenesis. Avasimibe, an ACAT inhibitor, was added at non‐toxic concentrations and replaced every other day during the course of adipogenesis. Avasimibe treatment inhibited lipid accumulation in a dose‐dependent manner, as judged by Oil red O staining and thin layer chromatography. Consistently, ACAT1 knockdown in 3T3‐L1 cells reduced lipid droplet accumulation by 40%. Real‐time PCR revealed that ACAT inhibition down‐regulated expressions of sterol regulatory element‐binding protein‐1 and genes involved in lipogenesis, with little effect on adipogenic transcription factors. These results suggest that ACAT is required for lipid droplet biogenesis in adipocytes. In addition, ACAT inhibition reduced 37% of intracellular FC and impaired cholesterol uptake in adipocytes, indicating a role of ACAT1 in the biochemical link between lipogenesis and cholesterol homeostasis. Taken together, our results reveal a novel function of ACAT1 in lipogenesis and highlight an important role of ACAT1 in integrating cholesterol metabolism and neutral lipid synthesis in adipocytes.

Box C/D Small Nucleolar RNA (snoRNA) U60 Regulates Intracellular Cholesterol Trafficking

Mobilization of plasma membrane (PM) cholesterol to the endoplasmic reticulum is essential for cellular cholesterol homeostasis. The mechanisms regulating this retrograde, intermembrane cholesterol transfer are not well understood. Because mutant cells with defects in PM to endoplasmic reticulum cholesterol trafficking can be isolated on the basis of resistance to amphotericin B, we conducted an amphotericin B loss-of-function screen in Chinese hamster ovary (CHO) cells using insertional mutagenesis to identify genes that regulate this trafficking mechanism. Mutant line A1 displayed reduced cholesteryl ester formation from PM-derived cholesterol and increased de novo cholesterol synthesis, indicating a deficiency in retrograde cholesterol transport. Genotypic analysis revealed that the A1 cell line contained one disrupted allele of the U60 small nucleolar RNA (snoRNA) host gene, resulting in haploinsufficiency of the box C/D snoRNA U60. Complementation and mutational studies revealed the U60 snoRNA to be the essential feature from this locus that affects cholesterol trafficking. Lack of alteration in predicted U60-mediated site-directed methylation of 28 S rRNA in the A1 mutant suggests that the U60 snoRNA modulates cholesterol trafficking by a mechanism that is independent of this canonical function. Our study adds to a growing body of evidence for participation of small noncoding RNAs in cholesterol homeostasis and is the first to implicate a snoRNA in this cellular function.

Abstract 18673: Human Enzyme Replacement Therapy in a Patient With Familial Lecithin Cholesterol Acyltransferase Deficiency: Rapid Appearance of Normal Appearing HDL

Introduction: Lecithin cholesterol acyltransferase (LCAT) is a liver-derived enzyme in plasma that catalyzes the formation of cholesteryl esters (CE). Mutations in the human LCAT gene result in familial LCAT deficiency (FLD), characterized by absent high-density lipoprotein cholesterol (HDL-C), corneal opacities, anemia, and severe renal disease. Replacing the defective enzyme should restore the normal level of plasma CE (LCAT product) and lead to the appearance of alpha HDL (α-HDL) lipoproteins. Aim: To assess the formation of CE and the appearance of HDL over time in a FLD patient with recombinant human LCAT (ACP-501). Methods: A FLD subject with very low HDL-C (&lt;5 mg/dL), corneal opacities, stage 4/5 renal disease, anemia, and splenomegaly received a 1 hour infusion of recombinant human LCAT (rhLCAT) and serial plasma samples were collected over 7 days. Results: HDL-C increased from &lt;5 mg/dL to 17 mg/dL at 8 hours and remained measurable for 7 days. CE increased rapidly, doubling in concentration within 2 hours, and sustained a higher concentration than HDL over 7 days. An increase in LDL-C was delayed about 6 hours suggesting CETP transfer of CE from HDL to LDL. Small sized particles were rapidly converted to HDL2 and HDL3 sized particles on FPLC. Large α-HDL appeared on agarose electrophoresis within 30 minutes. ApoAI Western blot analysis confirmed the rapid generation of apoAI staining α-HDL. α-1, 2 and 3 particles rapidly appeared by 2-D gel electrophoresis. Filipin stained 1-D gels revealed large α particles that persisted for 7 days. α-4 sized particles decreased as α-HDL formed. Sudan stained 1-D gels showed the generation of CE-rich α-HDL. Conclusions: The infusion of rhLCAT (ACP-501) in a lecithin cholesterol acyltransferase deficient subject with no HDL rapidly generated CE and α-HDL with evidence of sustained activity over 7 days. CE appeared in LDL suggesting the transfer of CE from HDL to LDL by CETP. The lipid and lipoprotein changes are consistent with normal HDL maturation.

Role of renin-angiotensin system in activation of macrophages by modified lipoproteins

Angiotensin II favors the development of atherosclerosis. Our goal was to determine if foam cell formation increases angiotensin II generation by the endogenous renin-angiotensin system (RAS) and if endogenously produced angiotensin II promotes lipid accumulation in macrophages. Differentiated THP-1 cells were treated with acetylated low-density lipoproteins (ac-LDL), native LDL (n-LDL), or no LDL. Expression of RAS genes was assessed and angiotensin I/II levels were quantified in media and cell lysate. Ac-LDL increased angiotensin I/II levels and the angiotensin II/I ratio in cells and media after foam cell formation. Renin mRNA or activity did not change, but renin blockade completely inhibited the increase in angiotensin II. Angiotensinogen mRNA but not protein level was increased. Angiotensin-converting enzyme (ACE) and cathepsin G mRNA and activities were enhanced by ac-LDL. Inhibition of renin, ACE, or the angiotensin II receptor 1 (AT1-receptor) largely abolished cholesteryl ester formation in cells exposed to ac-LDL and decreased scavenger receptor A (SR-A) and acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT-1) protein levels. Inhibition of renin or the AT1-receptor in cells treated with oxidized LDL also decreased SR-A and ACAT-1 protein and foam cell formation. ac-LDL also increased angiotensin II by human peripheral blood monocyte-derived macrophages, whereas blockade of renin decreased cholesterol ester formation in these macrophages. These findings indicate that, during foam cell formation, angiotensin II generation by the endogenous RAS is stimulated and that endogenously generated angiotensin II is crucial for cholesterol ester accumulation in macrophages exposed to modified LDL.

Macrophage lipoprotein lipase modulates the development of atherosclerosis but not adiposity

The role of macrophage lipoprotein lipase (LpL) in the development of atherosclerosis and adiposity was examined in macrophage LpL knockout (MLpLKO) mice. MLpLKO mice were generated using cre-loxP gene targeting. Loss of LpL in macrophages did not alter plasma LpL activity or lipoprotein levels. Incubation of apolipoprotein E (ApoE)-deficient β-VLDL with peritoneal macrophages from ApoE knockout mice lacking macrophage LpL (MLpLKO/ApoEKO) led to less cholesteryl ester formation than that found with ApoEKO macrophages. MLpLKO/ApoEKO macrophages had reduced intracellular triglyceride levels, with decreased CD36 and carnitine palmitoyltransferase-1 mRNA levels compared with ApoEKO macrophages, when incubated with VLDL. Although both MLpLKO/ApoEKO and ApoEKO mice developed comparable hypercholesterolemia in response to feeding with a Western-type diet for 12 weeks, atherosclerosis was less in MLpLKO/ApoEKO mice. Epididymal fat mass and gene expression levels associated with inflammation did not differ between the two groups. In conclusion, macrophage LpL plays an important role in the development of atherosclerosis but not adiposity.

Sequence-specific apolipoprotein A-I effects on lecithin:cholesterol acyltransferase activity

Existing kinetic data of cholesteryl ester formation by lecithin:cholesterol acyltransferase in discoidal high-density lipoproteins with 34 mutations of apoA-I that involved all putative helices were grouped by cluster analysis into four noncoincident regions with mutations both without any functional impairment and with profound isolated (V- and K-mutations) or common (VK-mutations) effect on V(max)(app) and K(m)(app). Data were analyzed with a new kinetic model of LCAT activity at interface that exploits the efficiency of LCAT binding to the particle, particle dimensions, and surface concentrations of phosphatidylcholine and cholesterol. V-mutations with major location in the central part and C-domain affected the second-order rate constant of cholesteryl ester formation at the solvolysis of acyl-enzyme intermediate by cholesterol as nucleophile. The central region in apoA-I sequence is suggested to influence the proper positioning of cholesterol molecule toward LCAT active center with major contribution of arginine residue(s). K-mutations with major location in N-domain may affect binding and stability of enzyme-phosphatidylcholine complex. VK-mutations may possess mixed effects; the independent binding measurement may segregate individual steps.

Characterization of a second sterol‐esterifying enzyme in Toxoplasma highlights the importance of cholesterol storage pathways for the parasite

Lipid bodies are eukaryotic structures for temporary storage of neutral lipids such as acylglycerols and steryl esters. Fatty acyl-CoA and cholesterol are two substrates for cholesteryl ester (CE) synthesis via the ACAT reaction. The intracellular parasite Toxoplasma gondii is incapable of sterol synthesis and unremittingly scavenges cholesterol from mammalian host cells. We previously demonstrated that the parasite expresses a cholesteryl ester-synthesizing enzyme, TgACAT1. In this article, we identified and characterized a second ACAT-like enzyme, TgACAT2, which shares 56% identity with TgACAT1. Both enzymes are endoplasmic reticulum-associated and contribute to CE formation for storage in lipid bodies. While TgACAT1 preferentially utilizes palmitoyl-CoA, TgACAT2 has broader fatty acid specificity and produces more CE. Genetic ablation of each individual ACAT results in parasite growth impairment whereas dual ablation of ACAT1 and ACAT2 is not tolerated by Toxoplasma. ΔACAT1 and ΔACAT2 parasites have reduced CE levels, fewer lipid bodies, and accumulate free cholesterol, which causes injurious membrane effects. Mutant parasites are particularly vulnerable to ACAT inhibitors. This study underlines the important physiological role of ACAT enzymes to store cholesterol in a sterol-auxotrophic organism such as Toxoplasma, and furthermore opens up possibilities of exploiting TgACAT as targets for the development of antitoxoplasmosis drugs.

Simultaneous activation of p38 and JNK by arachidonic acid stimulates the cytosolic phospholipase A2-dependent synthesis of lipid droplets in human monocytes

Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. This effect appears specific for AA in that it is not mimicked by other fatty acids, whether saturated or unsaturated. LDs are formed by two different routes: (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling, leading to increased triacylglycerol and cholesteryl ester formation utilizing fatty acids coming from the de novo biosynthetic route. Both routes can be dissociated by the arachidonyl-CoA synthetase inhibitor triacsin C, which prevents the former but not the latter. LD formation by AA-induced signaling predominates, accounting for 60-70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A(2)α (cPLA(2)α), pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family members p38 and JNK, which correlates with inhibition of cPLA(2)α activation by phosphorylation. Collectively, these results suggest that concomitant activation of p38 and JNK by AA cooperate to activate cPLA(2)α, which is in turn required for LD formation possibly by facilitating biogenesis of this organelle, not by regulating neutral lipid synthesis.

Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation

The hypothesis tested in this study was that cholesterol esterification by ACAT2 would increase cholesterol absorption efficiency by providing cholesteryl ester (CE) for incorporation into chylomicrons. The assumption was that absorption would be proportional to Acat2 gene dosage. Male ACAT2⁺/⁺, ACAT2⁺/⁻, and ACAT2⁻/⁻ mice were fed a diet containing 20% of energy as palm oil with 0.2% (w/w) cholesterol. Cholesterol absorption efficiency was measured by fecal dual-isotope and thoracic lymph duct cannulation (TLDC) methods using [³H]sitosterol and [¹⁴C]cholesterol tracers. Excellent agreement among individual mice was found for cholesterol absorption measured by both techniques. Cholesterol absorption efficiency in ACAT2⁻/⁻ mice was 16% compared with 46-47% in ACAT2⁺/⁺ and ACAT2⁺/⁻ mice. Chylomicrons from ACAT2⁺/⁺ and ACAT2⁺/⁻ mice carried ∼80% of total sterol mass as CE, whereas ACAT2⁻/⁻ chylomicrons carried >90% of sterol mass in the unesterified form. The total percentage of chylomicron mass as CE was reduced from 12% in the presence of ACAT2 to ∼1% in ACAT2⁻/⁻ mice. Altogether, the data demonstrate that ACAT2 increases cholesterol absorption efficiency by providing CE for chylomicron transport, but one copy of the Acat2 gene, providing ∼50% of ACAT2 mRNA and enzyme activity, was as effective as two copies in promoting cholesterol absorption.

2,3,22,23-Tetrahydroxyl-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene, an Acyclic Triterpenoid Isolated from the Seeds of &lt;i&gt;Alpinia katsumadai&lt;/i&gt;, Inhibits Acyl-CoA : Cholesterol Acyltransferase Activity

In order to isolate a cholesterol-lowering compound from Alpinia katsumadai, an inhibitor for acyl-CoA : cholesterol acyltransferase (ACAT), an enzyme responsible for the cholesterol ester formation in liver, was purified, its chemical structure was determined, and in vivo and in vitro inhibition activities were performed. In a high fat diet mouse model, we discovered that the ethanol extract of Alpinia katsumadai reduced plasma cholesterol, triglyceride, and low density lipoprotein (LDL) levels. An acyclic triterpenoid showing ACAT inhibitory activity was isolated from the extract of seeds of A. katsumadai. By NMR spectroscopic analysis of its (1)H-NMR, (13)C-NMR, (1)H-(1)H correlation spectroscopy, heteronuclear multiple bond connectivity (HMBC), hetero multiquantum coherence (HMQC) and nuclear Overhauser effect, chemical structure of 2,3,22,23-tetrahydroxyl-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene (1), were elucidated. The acyclic triterpenoid was found to be responsible for the ACAT inhibition activities of rat liver microsomes with IC(50) values of 47.9 µM. It also decreased cholesteryl ester formation with IC(50) values of 26 µM in human hepatocyte HepG2 cell. The experimental study revealed that the ethanol extract of A. katsumadai has a hypolipemic effect in high fat diet mice, and the isolated acyclic triterpenoid has ACAT inhibition activity, showing a potential novel therapeutic approach for the treatment of hyperlipidemia and atherosclerosis.

STARD4 abundance regulates sterol transport and sensing

Nonvesicular transport of cholesterol plays an essential role in the distribution and regulation of cholesterol within cells, but it has been difficult to identify the key intracellular cholesterol transporters. The steroidogenic acute regulatory-related lipid-transfer (START) family of proteins is involved in several pathways of nonvesicular trafficking of sterols. Among them, STARD4 has been shown to increase intracellular cholesteryl ester formation and is controlled at the transcriptional level by sterol levels in cells. We found that STARD4 is very efficient in transporting sterol between membranes in vitro. Cholesterol levels are increased in STARD4-silenced cells, while sterol transport to the endocytic recycling compartment (ERC) and to the endoplasmic reticulum (ER) are enhanced upon STARD4 overexpression. STARD4 silencing attenuates cholesterol-mediated regulation of SREBP-2 activation, while its overexpression amplifies sterol sensing by SCAP/SREBP-2. To analyze STARD4's mode of action, we compared sterol transport mediated by STARD4 with that of a simple sterol carrier, methyl-β-cyclodextrin (MCD), when STARD4 and MCD were overexpressed or injected into cells. Interestingly, STARD4 and cytosolic MCD act similarly by increasing the rate of transfer of sterol to the ERC and to the ER. Our results suggest that cholesterol transport mediated by STARD4 is an important component of the cholesterol homeostasis regulatory machinery.