Characterization of triacsin C inhibition of short-, medium-, and long-chain fatty acid: CoA ligases of human liver

Short-, medium-, and long-chain fatty acid:CoA ligases from human liver were tested for their sensitivity to inhibition by triacsin C. The short-chain fatty acid:CoA ligase was inhibited less than 10% by concentrations of triacsin C as high as 80 microM. The two mitochondrial xenobiotic/medium-chain fatty acid:CoA ligases (XM-ligases), HXM-A and HXM-B, were partially inhibited by triacsin C, and the inhibitions were characterized by low affinity for triacsin C (K(I) values > 100 microM). These inhibitions were found to be the result of triacsin C competing with medium-chain fatty acid for binding at the active site. The microsomal and mitochondrial forms of long-chain fatty acid:CoA ligase (also termed long-chain fatty acyl-CoA synthetase, or long-chain acyl-CoA synthetase LACS) were potently inhibited by triacsin C, and the inhibition had identical characteristics for both LACS forms. Dixon plots of this inhibition were biphasic. There is a high-affinity site with a K(I) of 0.1 microM that accounts for a maximum of 70% of the inhibition. There is also a low affinity site with a K(I) of 6 microM that accounts for a maximum of 30% inhibition. Kinetic analysis revealed that the high-affinity inhibition of the mitochondrial and microsomal LACS forms is the result of triacsin C binding at the palmitate substrate site.The high-affinity triacsin C inhibition of both the mitochondrial and microsomal LACS forms was found to require a high concentration of free Mg(2+), with the EC(50) for inhibition being 3 mM free Mg(2+). The low affinity triacsin C inhibition was also enhanced by Mg(2+). The data suggests that Mg(2+) promotes triacsin C inhibition of LACS by enhancing binding at the palmitate binding site. In contrast, the partial inhibition of the XM-ligases by triacsin C, which showed only a low-affinity component, did not require Mg(2+).     

Long-chain fatty acid transport in bacteria andyeast. Paradigms for defining the mechanism underlying this protein-mediated process

Protein-mediated transport of exogenous long-chain fatty acids across the membrane has been defined in a number of different systems. Central to understanding the mechanism underlying this process is the development of the appropriate experimental systems which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both [1] exhibit saturable long-chain fatty acid transport at low ligand concentration; [2] have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus; and [3] can be easily manipulated using the tools of molecular genetics. In E. coli, this process requires the outer membrane-bound fatty acid transport protein FadL and the inner membrane associated fatty acyl CoA synthetase (FACS). FadL appears to represent a substrate specific channel for long-chain fatty acids while FACS activates these compounds to CoA thioesters thereby rendering this process unidirectional. This process requires both ATP generated from either substrate-level or oxidative phosphorylation and the proton electrochemical gradient across the inner membrane. In S. cerevisiae, the process of long-chain fatty acid transport requires at least the membrane-bound protein Fat1p. Exogenously supplied fatty acids are activated by the fatty acyl CoA synthetases Faa1p and Faa4p but unlike the case in E. coli, there is not a tight linkage between transport and activation. Studies evaluating the growth parameters in the presence of long-chain fatty acids and long-chain fatty acid transport profiles of a fat1 strain support the hypothesis that Fat1p is required for optimal levels of long-chain fatty acid transport.     

Fatty acid transport in articular cartilage

Articular cartilage extracellular matrix imposes a significant transport barrier to albumin, the principal carrier of fatty acids. It has not been previously established whether it also influences the transport of fatty acids important for chondrocyte metabolism. Albumin was labelled with rhodamine-maleimide and bound to NBD-labelled lauric acid. Plugs of fresh equine metacarpal-phalangeal cartilage and subchondral bone were incubated with the complex at 4 degrees C for 2-160 h. The fluorophore distribution was quantified using quantitative microscopy in histological sections. The fluorescence intensity of both fluorophores fell steeply over 300 microm below the articular surface and remained relatively uniform through the mid zone but the ratio of lauric acid to albumin was higher than in the incubation medium. The effective diffusivity of lauric acid in the mid zone was (2.2+/-0.7) x 10(-12) m2 s(-1) (n = 33), higher than that of the carrier albumin, suggesting dissociation in the surface layer. Lauric acid accumulated reversibly at the tidemark.     

Role of fatty acid transport protein 4 in metabolic tissues: insights into obesity and fatty liver disease

Fatty acid (FA) metabolism is a series of processes that provide structural substances, signalling molecules and energy. Ample evidence has shown that FA uptake is mediated by plasma membrane transporters including FA transport proteins (FATPs), caveolin-1, fatty-acid translocase (FAT)/CD36, and fatty-acid binding proteins. Unlike other FA transporters, the functions of FATPs have been controversial because they contain both motifs of FA transport and fatty acyl-CoA synthetase (ACS). The widely distributed FATP4 is not a direct FA transporter but plays a predominant function as an ACS. FATP4 deficiency causes ichthyosis premature syndrome in mice and humans associated with suppression of polar lipids but an increase in neutral lipids including triglycerides (TGs). Such a shift has been extensively characterized in enterocyte-, hepatocyte-, and adipocyte-specific Fatp4-deficient mice. The mutants under obese and non-obese fatty livers induced by different diets persistently show an increase in blood non-esterified free fatty acids and glycerol indicating the lipolysis of TGs. This review also focuses on FATP4 role on regulatory networks and factors that modulate FATP4 expression in metabolic tissues including intestine, liver, muscle, and adipose tissues. Metabolic disorders especially regarding blood lipids by FATP4 deficiency in different cell types are herein discussed. Our results may be applicable to not only patients with FATP4 mutations but also represent a model of dysregulated lipid homeostasis, thus providing mechanistic insights into obesity and development of fatty liver disease.     

Fatty acid induced remodeling within the human liver fatty acid-binding protein

We crystallized human liver fatty acid-binding protein (LFABP) in apo, holo, and intermediate states of palmitic acid engagement. Structural snapshots of fatty acid recognition, entry, and docking within LFABP support a heads-in mechanism for ligand entry. Apo-LFABP undergoes structural remodeling, where the first palmitate ingress creates the atomic environment for placement of the second palmitate. These new mechanistic insights will facilitate development of pharmacological agents against LFABP.     

Amino acid sequence around the active serine in the acyl transferase domain of rabbit mammary fatty acid synthase

Rabbit mammary fatty acid synthase was labelled in the acyl transferase domain(s) by the formation of the O-ester intermediates after incubation with [¹⁴C]acetyl- or malonyl-CoA. Elastase peptides containing the labelled acyl groups were isolated using high performance liquid chromatography and sequenced by fast atom bombardment mass spectrometry. An identical peptide (acyl-Ser---Leu---Gly---Glu---Val---Ala) was obtained after labelling with acetyl- or malonyl-CoA. This confirms the hypothesis that, unlike Escherichia coli or yeast, a single transferase catalyses the transfer of both acetyl- and malonyl-groups in the mammalian complex. The sequence at this site is compared with that around the active serine in other acyl transferases and hydrolases.     

Fatty acid metabolism in adipocytes: functional analysis of fatty acid transport proteins 1 and 4

The role of fatty acid transport protein 1 (FATP1) and FATP4 in facilitating adipocyte fatty acid metabolism was investigated using stable FATP1 or FATP4 knockdown (kd) 3T3-L1 cell lines derived from retrovirus-delivered short hairpin RNA (shRNA). Decreased expression of FATP1 or FATP4 did not affect preadipocyte differentiation or the expression of FATP1 (in FATP4 kd), FATP4 (in FATP1 kd), fatty acid translocase, acyl-coenzyme A synthetase 1, and adipocyte fatty acid binding protein but did lead to increased levels of peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha. Both FATP1 and FATP4 kd adipocytes exhibited reduced triacylglycerol deposition and corresponding reductions in diacylglycerol and monoacylglycerol levels compared with control cells. FATP1 kd adipocytes displayed an approximately 25% reduction in basal (3)H-labeled fatty acid uptake and a complete loss of insulin-stimulated (3)H-labeled fatty acid uptake compared with control adipocytes. In contrast, FATP4 kd adipocytes as well as HEK-293 cells overexpressing FATP4 did not display any changes in fatty acid influx. FATP4 kd cells exhibited increased basal lipolysis, whereas FATP1 kd cells exhibited no change in lipolytic capacity. Consistent with reduced triacylglycerol accumulation, FATP1 and FATP4 kd adipocytes exhibited enhanced 2-deoxyglucose uptake compared with control adipocytes. These findings define unique and distinct roles for FATP1 and FATP4 in adipose fatty acid metabolism.     

A high yield optimized method for the production of acylated ACPs enabling the analysis of enzymes involved in P. falciparum fatty acid biosynthesis

The natural substrates of the enzymes involved in type-II fatty acid biosynthesis (FAS-II) are acylated acyl carrier proteins (acyl-ACPs). The state of the art method to produce acyl-ACPs involves the transfer of a phosphopantetheine moiety from CoA to apo-ACP by E. coli holo-ACP synthase (EcACPS), yielding holo-ACP which subsequently becomes thioesterified with free fatty acids by the E. coli acyl-ACP synthase (EcAAS). Alternatively, acyl-ACPs can be synthesized by direct transfer of acylated phosphopantetheine moieties from acyl-CoA to apo-ACP by means of EcACPS. The need for native substrates to characterize the FAS-II enzymes of P. falciparum prompted us to investigate the potential and limit of the two methods to efficiently acylate P. falciparum ACP (PfACP) with respect to chain length and β-modification and in preparative amounts. The EcAAS activity is found to be independent from the oxidation state at the β-position and accepts fatty acids as substrates with chain lengths starting from C8 to C20, whereas EcACPS accepts very efficiently acyl-CoAs with chain lengths up to C16, and with decreasing activity also longer chains (C18 to C20). Methods were developed to synthesize and purify preparative amounts of high quality natural substrates that are fully functional for the enzymes of the P. falciparum FAS-II system.     

Transacylase activity of lactating bovine mammary fatty acid synthase

An assay for the transacylation reaction catalyzed by fatty acid synthase was developed which does not require model substrates or labelled acyl-derivatives of CoA. It involves the transfer of the acyl group from unlabelled CoA to [³H]CoA. This assay shows the occurrence of transacylation at a relatively high rate with a variety of substrates that the enzyme is able to utilize. The activity is unaffected by dissociation of the enzyme or modification by iodoacetamide or 2-chloroacetyl-CoA.     

Fatty acid transporters in skin development,function and disease

Fatty acids in the epidermis can be incorporated into complex lipids or exist in a free form, and they are crucial to proper functions of the epidermis and its appendages, such as sebaceous glands. Epidermal fatty acids can be synthesized de novo by keratinocytes or taken up from extracutaneous sources in a process that likely involves protein transporters. Several proteins that are expressed in the epidermis have been proposed to facilitate the uptake of long-chain fatty acids (LCFA) in mammalian cells, including fatty acid translocase/CD36, fatty acid binding protein, and fatty acid transport protein (FATP)/very long-chain acyl-CoA synthetase. In this review, we will discuss the mechanisms by which these candidate transporters facilitate the uptake of fatty acids. We will then discuss the clinical implications of defects in these transporters and relevant animal models, including the FATP4 animal models and ichthyosis prematurity syndrome, a congenital ichthyosis caused by FATP4 deficiency. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Fatty acid binding protein 4 in human skeletal muscle

The mechanisms that regulate intramyocellular triglycerol (IMTG) storage and mobilization are largely unknown. However, during the last decades several intracellular fatty acid binding proteins (FABPs) have been identified. FABP3 is the dominating FABP in skeletal muscle. Expression of additional FABPs is suggested from findings in FABP3-null mutated mice. In the present study, our aims were to investigate if FABP4 is expressed within skeletal muscle fibers and if FABP3 and FABP4 are more abundant in skeletal muscle fibers in endurance-trained than in control subjects. We show that FABP4 protein is expressed within the skeletal muscle fibers and that FABP4 mRNA and protein are more abundant in the endurance trained subjects. Still, FABP4 is markedly less expressed than FABP3, which is the generally accepted dominating FABP in skeletal muscle tissue.     

Fatty acid analysis of triacylglycerols: Preparation of fatty acid methyl esters for gas chromatography

A method to prepare fatty acid methyl esters was developed for fatty acid analysis of triacylglycerols by gas chromatography (GC). Triacylglycerols were mixed with methanolic CH₃ONa in hexane containing a mid-polar solvent for 10 s at room temperature. Under these conditions, trioleoylglycerol was converted to methyl oleate with an average yield of 99.3%. This procedure gave reliable and reproducible data on fatty acid compositions determined by GC.     

Glutamate-41 of Vibrio harveyi acyl carrier protein is essential for fatty acid synthase but not acyl-ACP synthetase activity

Bacterial acyl carrier protein (ACP) is a small, acidic, and highly conserved protein that supplies acyl groups for biosynthesis of a variety of lipid products. Recent modelling studies predict that residues primarily in helix II of Escherichia coli ACP (Glu-41, Ala-45) are involved in its interaction with the condensing enzyme FabH of fatty acid synthase. Using recombinant Vibrio harveyi ACP as a template for site-directed mutagenesis, we have shown that an acidic residue at position 41 is essential for V. harveyi fatty acid synthase (but not acyl-ACP synthetase) activity. In contrast, various replacements of Ala-45 were tolerated by both enzymes. None of the mutations introduced dramatic structural changes based on circular dichroism and native gel electrophoresis. These results confirm that Glu-41 of ACP is a critical residue for fatty acid synthase, but not for all enzymes that utilize ACP as a substrate.     

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。     

The subcellular compartmentation of fatty acid transporters is regulated differently by insulin and by AICAR

Cellular fatty acid uptake is facilitated by a number of fatty acid transporters, FAT/CD36, FABPpm and FATP1. It had been presumed that FABPpm, was confined to the plasma membrane and was not regulated. Here, we demonstrate for the first time that FABPpm and FATP1 are also present in intracellular depots in cardiac myocytes. While we confirmed previous work that insulin and AICAR each induced the translocation of FAT/CD36 from an intracellular depot to the PM, only AICAR, but not insulin, induced the translocation of FABPpm. Moreover, neither insulin nor AICAR induced the translocation of FATP1. Importantly, the increased plasmalemmal content of these LCFA transporters was associated with a concomitant increase in the initial rate of palmitate uptake into cardiac myocytes. Specifically, the insulin-stimulated increase in the rate of palmitate uptake (+60%) paralleled the insulin-stimulated increase in plasmalemmal FAT/CD36 (+34%). Similarly, the greater AICAR-stimulated increase in the rate of palmitate uptake (+90%) paralleled the AICAR-induced increase in both plasmalemmal proteins (FAT/CD36 (+40%)+FABPpm (+36%)). Inhibition of palmitate uptake with the specific FAT/CD36 inhibitor SSO indicated that FABPpm interacts with FAT/CD36 at the plasma membrane to facilitate the uptake of palmitate. In conclusion, (1) there appears to be tissue-specific sensitivity to insulin-induced FATP1 translocation, as it has been shown elsewhere that insulin induces FATP1 translocation in 3T3-L1 adipocytes, and (2) clearly, the subcellular distribution of FABPpm, as well as FAT/CD36, is acutely regulated in cardiac myocytes, although FABPpm and FAT/CD36 do not necessarily respond identically to the same stimuli.     

Fatty acid transport by vectorial acylation in mammals: roles played by different isoforms of rat long-chain acyl-CoA synthetases

Mammals express multiple isoforms of acyl-CoA synthetase (ACSL1 and ACSL3-6) in various tissues. These enzymes are essential for fatty acid metabolism providing activated intermediates for complex lipid synthesis, protein modification, and beta-oxidation. Yeast in contrast express four major ACSLs, which have well-defined functions. Two, Faa1p and Faa4p, are specifically required for fatty acid transport by vectorial acylation. Four ACSLs from the rat were expressed in a yeast faa1delta faa4delta strain and their roles in fatty acid transport and trafficking characterized. All four restored ACS activity yet varied in substrate preference. ACSL1, 4, and 6 were able to rescue fatty acid transport activity and triglyceride synthesis. ACSL5, however, was unable to facilitate fatty acid transport despite conferring robust oleoyl-CoA synthetase activity. This is the first study evaluating the role of the mammalian ACSLs in fatty acid transport and supports a role for ACSL1, 4, and 6 in transport by vectorial acylation.     

Role of fatty acid-binding protein in cardiac fatty acid oxidation

Summary Although abundant in most biological tissues and chemically well characterized, the fatty acid-binding protein (FABP) was until recently in search of a function. Because of its strong affinity for long chain fatty acids and its cytoplasmic origin, this protein was repeatedly claimed in the literature to be the transcytoplasmic fatty acid carrier. However, techniques to visualize and quantify the movements of molecules in the cytoplasm are still in their infancy. Consequently the carrier function of FABP remains somewhat speculative. However, FABP binds not only fatty acids but also their CoA and carnitine derivatives, two typical molecules of mitochondrial origin. Moreover, it has been demonstrated and confirmed that FABP is not exclusively cytoplasmic, but also mitochondrial. A function for FABP in the mitochondrial metabolism of fatty acids plus CoA and carnitine derivatives would therefore be anticpated. Using spin-labelling techniques, we present here evidence that FABP is a powerful regulator of acylcarnitine flux entering the mitochondrial -oxidative system. In this perspective FABP appears to be an active link between the cytoplasm and the mitochondria, regulating the energy made available to the cell. This active participation of FABP is shown to be the consequence of its gradient-like distribution in the cardiac cell, and also of the coexistence of multispecies of this protein produced by self-aggregation.