Activation of LXR increases acyl-CoA synthetase activity through direct regulation of ACSL3 in human placental trophoblast cells

Placental fatty acid transport and metabolism are important for proper growth and development of the feto-placental unit. The nuclear receptors, liver X receptors α and β (LXRα and LXRβ), are key regulators of lipid metabolism in many tissues, but little is known about their role in fatty acid transport and metabolism in placenta. The current study investigates the LXR-mediated regulation of long-chain acyl-CoA synthetase 3 (ACSL3) and its functions in human placental trophoblast cells. We demonstrate that activation of LXR increases ACSL3 expression, acyl-CoA synthetase activity, and fatty acid uptake in human tropholast cells. Silencing of ACSL3 in these cells attenuates the LXR-mediated increase in acyl-CoA synthetase activity. Furthermore, we show that ACSL3 is directly regulated by LXR through a conserved LXR responsive element in the ACSL3 promoter. Our results suggest that LXR plays a regulatory role in fatty acid metabolism by direct regulation of ACSL3 in human placental trophoblast cells.     

茄科雷尔氏菌脂酰-CoA合成酶的功能鉴定

【目的】茄科雷尔氏菌是一种常见的农作物致病菌,引起植物青枯病。研究其脂肪酸代谢途径将有助于寻找新的抗菌药物靶点,为防治青枯病害提供新的思路。【方法】利用大肠杆菌FadD序列,进行同源比对发现茄科雷尔氏菌GMI1000中RSc2857(RsFadD)具有较高的相似性,推测其具有脂酰-CoA合成酶活性。采用PCR扩增方法获得RsfadD基因,连入表达载体pBAD24M后互补大肠杆菌fadD突变株,并检测转化子的生长情况。RsfadD与pET-28b连接后,在大肠杆菌BL(DE3)中表达,并利用Ni-NTA纯化获得带有组氨酸标签的RsFadD,体外测定RsFadD的活性。利用同源重组方法,获得RsfadD敲除突变株,分析突变株的生长性状。【结果】RsfadD异体互补大肠杆菌fadD突变株,恢复突变株在以脂肪酸为碳源的基础培养基上生长。体外活性测定RsFadD具有脂酰-CoA合成酶活性,对不同链长的脂肪酸都具有活性,但活性低于大肠杆菌FadD。RsfadD突变株在添加不同链长脂肪酸的基础培养上仅能微弱生长,而在丰富培养基上生长无差异。【结论】茄科雷尔氏菌中RsfadD编码脂酰-CoA合成酶,在脂肪酸利用过程中发挥重要作用。但RsfadD突变株在基础培养基上微弱生长,说明茄科雷尔氏菌基因组中还有其他的脂酰-CoA合成酶基因。以上研究结果为进一步研究茄科雷尔氏菌中脂酰-CoA合成酶以及脂肪酸利用机制奠定了基础。     

Long-chain acyl-CoA synthetase isoforms differ in preferences for eicosanoid species and long-chain fatty acids

Because the signaling eicosanoids, epoxyeicosatrienoic acids (EETs) and HETEs, are esterified to membrane phospholipids, we asked which long-chain acyl-CoA synthetase (ACSL) isoforms would activate these molecules and whether the apparent FA substrate preferences of each ACSL isoform might differ depending on whether it was assayed in mammalian cell membranes or as a purified bacterial recombinant protein. We found that all five ACSL isoforms were able to use EETs and HETEs as substrates and showed by LC-MS/MS that ACSLs produce EET-CoAs. We found differences in substrate preference between ACS assays performed in COS7 cell membranes and recombinant purified proteins. Similarly, preferences and Michaelis-Menten kinetics for long-chain FAs were distinctive. Substrate preferences identified for the purified ACSLs did not correspond to those observed in ACSL-deficient mouse models. Taken together, these data support the concept that each ACSL isoform exhibits a distinct substrate preference, but apparent substrate specificities depend upon multiple factors including membrane character, coactivators, inhibitors, protein interactions, and posttranslational modification.     

A revised nomenclature for mammalian acyl-CoA thioesterases/hydrolases

Acyl-CoA thioesterases, also known as acyl-CoA hydrolases, are a group of enzymes that hydrolyze CoA esters such as acyl-CoAs (saturated, unsaturated, branched-chain), bile acid-CoAs, CoA esters of prostaglandins, etc., to the corresponding free acid and CoA. However, there is significant confusion regarding the nomenclature of these genes. In agreement with the HUGO Gene Nomenclature Committee and the Mouse Genomic Nomenclature Committee, a revised nomenclature for mammalian acyl-CoA thioesterases/hydrolases has been suggested for the 12 member family. The family root symbol is ACOT, with human genes named ACOT1-ACOT12, and rat and mouse genes named Acot1-Acot12. Several of the ACOT genes are the result of splicing events, and these splice variants are cataloged.     

Effect of the non-steroidal anti-inflammatory drugs on the acyl-CoA synthetase activity toward medium-chain,long-chain and polyunsaturated fatty acids in mitochondria of mouse liver and brain

Effect of eleven non-steroidal anti-inflammatory drugs on the acyl-CoA synthetase activities toward octanoic, palmitic, arachidonic and docosahexaenoic acids was evaluated in mouse liver and brain mitochondria. The drugs tested were aspirin, salicylic acid, diflunisal, mefenamic acid, indomethacin, etodolac, ibuprofen, ketoprofen, naproxen, loxoprofen, flurbiprofen. In mouse liver mitochondria, diflunisal and mefenamic acid exhibited the inhibitory activities not only for octanoic acid (IC₅₀?=?78.7 and 64.7 µM) and but also for palmitic acid (IC₅₀?=?236.5 and 284.4 µM), respectively. Aspirin was an inhibitor for the activation of octanoic acid only (IC₅₀?=?411.0 µM). In the brain, mefenamic acid and diflunisal inhibited strongly palmitoyl-CoA formation (IC₅₀?=?57.3 and 114.0 µM), respectively. The activation of docosahexaenoic acid in brain was sensitive to inhibition by diflunisal and mefenamic acid compared with liver.     

A novel murine long-chain acyl-CoA synthetase expressed in brain participates in neuronal cell proliferation

Refsum disease (RfD) is an autosomal recessive neurologic disorder of the lipid metabolism. We have identified a novel murine long-chain acyl-CoA synthetase (mLACS) associated with the RfD gene using yeast two-hybrid assay. Northern blot analyses revealed that mLACS was expressed mainly in the brain and testis. mLACS was highly expressed in the brain at 2 weeks after birth and maintained through adult life. Expressions of the brain-specific LACS family increased in the PC12 cells undergoing neurite outgrowth by nerve growth factor. mLACS preferentially catalyzed the formation of arachidonoyl-CoA more than palmitoyl-CoA or oleoyl-CoA in PC12 cells. Triacsin C, an inhibitor of LACS, suppressed the cell proliferation and decreased mLACS expression in parent PC12 cells, but not in stably anti-sense mLACS cDNA-transfected cells. Our results indicate that mLACS participates in neuronal cell proliferation and differentiation, and interaction of the RfD gene with brain-selective mLACS may be involved in the pathogenesis of RfD.     

Rat liver acyl-CoA synthetase 4 is a peripheral-membrane protein located in two distinct subcellular organelles,peroxisomes, and mitochondrial-associated membrane

Obesity and non-insulin-dependent diabetes favor storage of fatty acids in triacylglycerol over oxidation. Recently, individual acyl-CoA synthetase (ACS) isoforms have been implicated in the channeling of fatty acids either toward lipid synthesis or toward oxidation. Although ACS1 had been localized to three different subcellular regions in rat liver, endoplasmic reticulum, mitochondria, and peroxisomes, the study had used an antibody raised against the full-length ACS1 protein which cross-reacts with other isoforms, probably because all ACS family members contain highly conserved amino acid sequences. Therefore, we examined the subcellular location of ACS1, ACS4, and ACS5 in rat liver to determine which isoform was present in peroxisomes, whether the ACSs were intrinsic membrane proteins, and which ACS isoforms were up-regulated by PPAR alpha ligands. Non-cross-reacting ACS1, ACS4, and ACS5 peptide antibodies showed that ACS4 was the only ACS isoform present in peroxisomes isolated from livers of gemfibrozil-treated rats. ACS4 was also present in fractions identified as mitochondria-associated membrane (MAM). ACS1 was present in endoplasmic reticulum fractions and ACS5 was present in mitochondrial fractions. Incubation with troglitazone, a specific inhibitor of ACS4, decreased ACS activity in the MAM fractions 30-45% and in the peroxisomal fractions about 30%. Because the signal for ACS4 protein in peroxisomes was so strong compared to the MAM fraction, we examined ACS4 mRNA abundance in livers of rats treated with the PPAR alpha agonist GW9578. Treatment with GW9578 increased ACS4 mRNA abundance 40% and ACS1 mRNA 25%. Although we had originally proposed that ACS4 is linked to triacylglycerol synthesis, it now appears that ACS4 may also be important in activating fatty acids destined for peroxisomal oxidation. We also determined that, unlike ACS1 and 5, ACS4 is not an intrinsic membrane protein. This suggests that ACS4 is probably targeted and linked to MAM and peroxisomes by interactions with other proteins.     

油茶长链脂肪酰基CoA合成酶基因1(CoLACS1)分子特征与表达分析

长链脂肪酰基Co A合成酶(LACS)在脂肪酸的合成与分解代谢中起着重要作用。本研究以油茶(Camellia oleifera Abel)国家审定品种华硕(Camellia oleifera Huashuo)种仁转录组数据为基础,根据LACS基因Unigene序列设计引物,分离克隆了油茶LACS1基因全长c DNA序列,命名为CoLACS1(Gene Bank登录号:KJ960228),全长2114 bp,开放阅读框2088 bp,编码695个氨基酸;生物信息学分析显示CoLACS1具有3个B1ock,从分子特征可判断CoLACS1属于LACS家族;氨基酸同源比对显示与其他物种的LACS氨基酸序列具有较高的相似性,其中与拟南芥LACS7相似性为78%,与麻风树、大豆、毛果杨等物种LACS6(peroxisomal)相似性可达80%以上;对CoLACS1进行原核表达分析,构建的p ET30a-CoLACS1载体成功转化至BL21(DE3)中经1 mmol/L IPTG诱导表达,菌液检测获得预测的目的蛋白(分子量约为76 k D);分析转录组数据中CoLACS1的Unigene序列RTKM值并对CoLACS1进行实时荧光定量PCR分析,结果表明CoLACS1在油茶华硕种子发育各时期平稳表达,表达丰度变化不大,荧光定量结果变化规律与转录组数据分析一致;同时分析华硕种仁不同时期含油率和脂肪酸成分变化,双变量统计分析发现CoLACS1表达模式与油茶油脂积累规律呈显著相关性。本研究为进一步研究油茶油脂积累与代谢的基因调控提供理论依据。     

Characterization of a novel plant acyl-CoA synthetase that is expressed in lipogenic tissues of Brassica napus L.

A cDNA encoding a novel isoform of acyl-CoA synthetase (ACS6) was isolated from embryos of oilseed rape. Homology searches show it is most closely related to ACS4 from rat and human brain rather than the other oilseed rape ACSs. The ACS6 is strongly expressed in embryos and flowers, tissues of Brassica napus that synthesize lipids at high rates. The activity of recombinantly expressed ACS6 was recovered in the insoluble fraction (214000×g, 1 h pellet). CHAPS-solubilized recombinant ACS6 protein preferred utilising long-chain fatty acids that contained a cis-9 double bond, i.e. palmitoleic, oleic, linoleic and linolenic acids. Western blot analysis showed that the ACS6 protein is membrane-bound.     

Carnitine palmitoyltransferase activities: Effects of serum albumin,acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low M concentrations of octanoyl-CoA. However, even a large excess (500 M) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low M concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above effect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolismin vivo.Abbreviations ACBP acyl-CoA binding protein - BSA bovine serum albumin - CPT carnitine palmitoyltransferase - CPT₀ malonyl-CoA sensitive CPT of the outer mitochondrial membrane - CPT malonyl-CoA insensitive CPT of the inner mitochondrial membrane - OG octylglucoside - OMV outer membrane vesicles - IMV inner membrane vesicles Affiliated to the Department of Experimental Medicine, University of Montreal     

Inhibition of long chain fatty acyl-CoA synthetase (ACSL) and ischemia reperfusion injury

Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [¹⁴C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion.     

Peroxisome proliferator-activated receptor gamma 2 and acyl-CoA synthetase 5 polymorphisms influence diet response

Peroxisome proliferator-activated receptor gamma (PPARgamma) and its response gene, Acyl CoA synthetase 5 (ACSL5), which has an important role in fatty acid metabolism, may affect weight loss in response to caloric restriction. Therefore, we aimed to determine whether these genes were involved in the interindividual response to dietary treatment. Genotypic/phenotypic comparisons were made between selected obese women from the quintiles losing the most (diet responsive, n = 74) and the quintiles losing the least (diet-resistant, n = 67) weight in the first 6 weeks of a 900-kcal formula diet. Two common PPARgamma single nucleotide polymorphisms, Pro(12)Ala and C1431T, and eight polymorphisms across the ACSL5 gene were selected for single locus and haplotypic association analyses. The PPARgamma Pro(12)Ala single nucleotide polymorphism was associated with diet resistance (odds ratio = 3.48, 95% confidence interval = 1.41 to 8.56, p = 0.03), and the rs2419621, located in the 5'untranslated region of the ACSL5 gene, displayed the strongest association with diet response (odds ratio = 3.45, 95% confidence interval = 1.61 to 7.69, p = 0.001). Skeletal muscle ACSL5 mRNA expression was significantly lower in carriers of the wildtype compared with the variant rs2419621 allele (p = 0.03). Our results suggest a link between PPARgamma2 and ACSL5 genotype and diet responsiveness.     

The metabolic capacity of lipid droplet localized acyl-CoA synthetase 3 is not sufficient to support local triglyceride synthesis independent of the endoplasmic reticulum in A431 cells

ACSL3 is the only long chain fatty acyl-CoA synthetase consistently found on growing and mature lipid droplets (LDs), suggesting that this specific localization has biological relevance. Current models for LD growth propose that triglycerides are synthesized by enzymes at the LD surface, with activated fatty acids provided by LD localized ACSL3, thus allowing growth independent of the ER. Here, we tested this hypothesis by quantifying ACSL3 on LDs from human A431 cells.RNAi of ACSL3 reduced the oleoyl-CoA synthetase activity by 83%, suggesting that ACSL3 is by far the dominant enzyme of A431 cells. Molar quantification revealed that there are 1.4 million ACSL3 molecules within a single cell. Metabolic labeling indicated that each ACSL3 molecule contributed a net gain of 3.1 oleoyl-CoA/s. 3D reconstruction of confocal images demonstrated that 530 individual lipid droplets were present in an average oleate fed A431 cell. A representative single lipid droplet with a diameter of 0.66?μm contained 680 ACSL3 molecules on the surface. Subcellular fractionation showed that at least 68% of ACSL3 remain at the ER even during extensive fatty acid supplementation. High resolution single molecule microscopy confirmed the abundance of cytoplasmic ACSL3 outside of LDs. Model calculations for triglyceride synthesis using only LD localized ACSL3 gave significant slower growth of LDs as observed experimentally.In conclusion, although ACSL3 is an abundant enzyme on A431 LDs, the metabolic capacity is not sufficient to account for LD growth solely by the local synthesis of triglycerides.     

A spectrophotometric method for the determination of free fatty acid in serum using acyl-coenzyme A synthetase and acyl-coenzyme A oxidase

A mixture of Ti(IV) and 4-(2-pyridylazo)resorcinol was found to be useful in the spectrophotometric determination of trace amounts of hydrogen peroxide. The absorbance at 508 nm was proportional to the concentration of hydrogen peroxide added. The reagent was successfully applied to the assay of free fatty acid in serum through the combined use of acyl-CoA synthetase and acyl-CoA oxidase. The latter enzyme produces H₂O₂. As a result, hydrogen peroxide was produced through the enzymatic oxidation of free fatty acid. It was possible to determine free fatty acid in 50 μl of serum at concentrations ranging from 0.02 to 1.5 mm. The coefficient of variation was less than 3% at concentrations ranging from 0.1 to 1.5 mm. In the present method, there is the advantage of minimal influence from reducible substances as well as greater simplicity and accuracy.     

Functional conversion of fatty acyl-CoA synthetase to firefly luciferase by site-directed mutagenesis: A key substitution responsible for luminescence activity

We demonstrated that firefly luciferase has a catalytic function of fatty acyl-CoA synthesis [Oba, Y., Ojika, M. and Inouye, S. (2003) Firefly luciferase is a bifunctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase. FEBS Lett. 540, 251-254] and proposed that the evolutionary origin of beetle luciferase is a fatty acyl-CoA synthetase (FACS) in insect. In this study, we performed the functional conversion of FACS to luciferase by replacing a single amino acid to serine. This serine residue is conserved in luciferases and possibly interacts with luciferin. The mutants of FACSs in non-luminous click beetle Agrypnus binodulus (AbLL) and Drosophilamelanogaster (CG6178) gave luminescence enhancement, suggesting that the serine residue is a key substitution responsible for luminescence activity.     

A new,simple assay for long-chain acyl-CoA dehydrogenase in cultured skin fibroblasts using stable isotopes and GC-MS

In this paper, we present a new method for measurement of long-chain acyl-CoA dehydrogenase (LCAD) activities in cultured skin fibroblasts. The method is based upon gas chromatographic/mass spectrometric determination of 3-OH-hexadecanoic acid formed during incubation of fibroblasts in a medium containing palmitoyl-CoA and crotonase, to convert the enoyl-CoA ester produced into the 3-hydroxyacyl-CoA ester. The validity of the method demonstrated by the finding of a full deficiency of LCAD in fibroblasts from three patients with an established deficiency of LCAD.     

Purification, characterization, and mass spectrometric sequencing of a medium chain acyl-CoA synthetase from mouse liver mitochondria and comparisons with the homologues of rat and bovine

Medium chain acyl-CoA synthetases catalyze the first reaction of amino acid conjugation of many xenobiotic carboxylic acids and fatty acid metabolism. This paper reports studies on purification, characterization, and the partial amino acid sequence of mouse liver enzyme. The medium chain acyl-CoA synthetase was isolated from mouse liver mitochondria. The purified enzyme catalyzes this reaction not only for straight medium chain fatty acids but also for aromatic and arylacetic acids. Maximal activity was found with hexanoic acid. High activities were obtained with benzoic acid having methyl, pentyl, and methoxy groups in the para- or meta-positions of the benzene ring. However, the enzyme was less active with valproic acid and ketoprofen. Salicylic acid exhibited no activity. The medium chain acyl-CoA synthetases from mouse and bovine liver mitochondria were subjected to in-gel tryptic digestion, followed by LC-MS/MS sequence analysis. The amino acid sequence of each tryptic peptide of mouse liver mitochondrial medium chain acyl-CoA synthetase differed from that from bovine liver mitochondria only in one or two amino acids. LC-MS/MS analysis provided the information about these differences in amino acid sequences. In addition, we compared the properties of this protein with the homologues from rat and bovine.     

Corresponding increase in long-chain acyl-CoA and acylcarnitine after exercise in muscle from VLCAD mice

Long-chain acylcarnitines accumulate in long-chain fatty acid oxidation defects, especially during periods of increased energy demand from fat. To test whether this increase in long-chain acylcarnitines in very long-chain acyl-CoA dehydrogenase (VLCAD(-/-)) knock-out mice correlates with acyl-CoA content, we subjected wild-type (WT) and VLCAD(-/-) mice to forced treadmill running and analyzed muscle long-chain acyl-CoA and acylcarnitine with tandem mass spectrometry (MS/MS) in the same tissues. After exercise, long-chain acyl-CoA displayed a significant increase in muscle from VLCAD(-/-) mice [C16:0-CoA, C18:2-CoA and C18:1-CoA in sedentary VLCAD(-/-): 5.95 +/- 0.33, 4.48 +/- 0.51, and 7.70 +/- 0.30 nmol x g(-1) wet weight, respectively; in exercised VLCAD(-/-): 8.71 +/- 0.42, 9.03 +/- 0.93, and 14.82 +/- 1.20 nmol x g(-1) wet weight, respectively (P < 0.05)]. Increase in acyl-CoA in VLCAD-deficient muscle was paralleled by a significant increase in the corresponding chain length acylcarnitine. Exercise resulted in significant lowering of the free carnitine pool in VLCAD(-/-) muscle. This is the first study demonstrating that acylcarnitines and acyl-CoA directly correlate and concomitantly increase after exercise in VLCAD-deficient muscle.