Mutagenesis of rat acyl-CoA synthetase 4 indicates amino acids that contribute to fatty acid binding

Although each of the five mammalian long-chain acyl-CoA synthetases (ACSL) can bind saturated and unsaturated fatty acids ranging from 12 to 22 carbons, ACSL4 prefers longer chain polyunsaturated fatty acids. In order to gain a better understanding of ACSL4 fatty acid binding, we based a mutagenesis approach on sequence alignments related to ttLC-FACS crystallized from Thermus thermophilus HB8. Four residues selected for mutagenesis corresponded to residues in ttLC-FACS that comprise the fatty acid binding pocket; the fifth residue aligned with a region thought to be involved in fatty acid selectivity of the Escherichia coli acyl-CoA synthetase, FadD. Changing an amino acid at the entry of the putative fatty acid binding pocket, G401L, resulted in an inactive enzyme. Mutating a residue near the pocket entry, L399M, did not significantly alter enzyme activity, but mutating a residue at the hydrophobic terminus of the pocket, S291Y, altered ACSL4's preference for 20:5 and 22:6 and increased its apparent K(m) for ATP. Mutating a site in a region previously identified as important for fatty acid binding also altered activation of 20:4 and 20:5. These studies suggested that the preference of ACSL4 for long-chain polyunsaturated fatty acids can be modified by altering specific amino acid residues.     

Fatty acid transport across the cell membrane: Regulation by fatty acid transporters

Transport of long-chain fatty acids across the cell membrane has long been thought to occur by passive diffusion. However, in recent years there has been a fundamental shift in understanding, and it is now generally recognized that fatty acids cross the cell membrane via a protein-mediated mechanism. Membrane-associated fatty acid-binding proteins (‘fatty acid transporters’) not only facilitate but also regulate cellular fatty acid uptake, for instance through their inducible rapid (and reversible) translocation from intracellular storage pools to the cell membrane. A number of fatty acid transporters have been identified, including CD36, plasma membrane-associated fatty acid-binding protein (FABP_pm), and a family of fatty acid transport proteins (FATP1–6). Fatty acid transporters are also implicated in metabolic disease, such as insulin resistance and type-2 diabetes. In this report we briefly review current understanding of the mechanism of transmembrane fatty acid transport, and the function of fatty acid transporters in healthy cardiac and skeletal muscle, and in insulin resistance/type-2 diabetes. Fatty acid transporters hold promise as a future target to rectify lipid fluxes in the body and regain metabolic homeostasis.     

Adipose tissue fatty acid metabolism and cardiovascular disease

PURPOSE OF REVIEW: Fatty acid and triacylglycerol metabolism in adipose tissue may be involved in the generation of risk factors for cardiovascular disease and type 2 diabetes. Pharmaceutical companies are targeting adipocyte metabolism in their search for drugs for treating, or reducing the risk of, these conditions. We review new developments in adipose tissue fatty acid metabolism and how that might relate to cardiovascular disease. RECENT FINDINGS: Fatty acid release from human adipose tissue is oscillatory, with a period of about 12 min. Remarkably, oscillatory fatty acid release is also seen in isolated adipocytes. Further evidence has emerged that not all adipose depots are equal, and that lower-body adipose tissue may exert protective effects against cardiovascular disease. There have been a number of developments in the area of fatty acid handling by adipocytes. Fatty acid binding proteins are clearly important in regulating fatty acid metabolism, with striking protection against atherosclerosis in mice deficient in both the binding proteins expressed in adipocytes. The demonstration that adipocytes lacking hormone-sensitive lipase still display lipolysis has led to the identification of novel lipases that may play crucial roles in adipose tissue fatty acid metabolism. Further evidence has accrued of the interaction between hormone-sensitive lipase and perilipin, the protein that coats the adipocyte lipid droplet. SUMMARY: Recent developments in our understanding of adipose tissue fatty acid metabolism open up the possibility of new pharmaceutical targets. However, interference with adipose tissue fatty acid metabolism is not to be undertaken lightly and needs a clear understanding of the normal role of adipocyte lipolysis.     

Regulation of fatty acid biosynthesis in Escherichia coli.

Our understanding of fatty acid biosynthesis in Escherichia coli has increased greatly in recent years. Since the discovery that the intermediates of fatty acid biosynthesis are bound to the heat-stable protein cofactor termed acyl carrier protein, the fatty acid synthesis pathway of E. coli has been studied in some detail. Interestingly, many advances in the field have aided in the discovery of analogous systems in other organisms. In fact, E. coli has provided a paradigm of predictive value for the synthesis of fatty acids in bacteria and plants and the synthesis of bacterial polyketide antibiotics. In this review, we concentrate on four major areas of research. First, the reactions in fatty acid biosynthesis and the proteins catalyzing these reactions are discussed in detail. The genes encoding many of these proteins have been cloned, and characterization of these genes has led to a better understanding of the pathway. Second, the function and role of the two essential cofactors in fatty acid synthesis, coenzyme A and acyl carrier protein, are addressed. Finally, the steps governing the spectrum of products produced in synthesis and alternative destinations, other than membrane phospholipids, for fatty acids in E. coli are described. Throughout the review, the contribution of each portion of the pathway to the global regulation of synthesis is examined. In no other organism is the bulk of knowledge regarding fatty acid metabolism so great; however, questions still remain to be answered. Pursuing such questions should reveal additional regulatory mechanisms of fatty acid synthesis and, hopefully, the role of fatty acid synthesis and other cellular processes in the global control of cellular growth.     

海洋真菌Thraustochytrium sp.FJN-10 Δ~4脱饱和酶基因的cDNA克隆及结构分析

海洋真菌Thraustochytriumsp.FJN-10是二十二碳六烯酸(Docosahexaenoic acid,DHA,C22:6n-3)的高产菌株。为阐明其DHA生物合成机制及采用基因工程技术提高DHA生物合成能力,通过RT-PCR扩增了DHA生物合成相关脱饱和酶基因的保守区,采用cDNA末端扩增技术(Rapid amplification of cDNA ends,RACE)获取全长基因,结果分析表明其克隆基因为Δ4脂肪酸脱饱和酶基因,开放阅读框由1560个核苷酸组成,共编码519个氨基酸,这是Thraus-tochytriumsp.FJN-10Δ4脂肪酸脱饱和酶基因研究的首次报道。     

Industrial oils from transgenic plants

Unusual fatty acids that have useful industrial properties occur widely in the seed oils of many non-agronomic plant species. Researchers are attempting to use biotechnology to produce high levels of these fatty acids in the seeds of existing crop plants. cDNAs for a wide variety of unusual fatty acid biosynthetic enzymes have been identified, particularly through the use of expressed sequence tags. However, it has not yet been possible to use these cDNAs to produce large amounts of unusual fatty acids in seeds of transgenic plants. This difficulty points to the need for a greater understanding of fatty acid metabolism in oilseeds.     

Carbon flux and fatty acid synthesis in plants.

The de novo synthesis of fatty acids in plants occurs in the plastids through the activity of fatty acid synthetase. The synthesis of the malonyl-coenzyme A that is required for acyl-chain elongation requires the import of metabolites from the cytosol and their subsequent metabolism. Early studies had implicated acetate as the carbon source for plastidial fatty acid synthesis but more recent experiments have provided data that argue against this. A range of cytosolic metabolites including glucose 6-phosphate, malate, phosphoenolpyruvate and pyruvate support high rates of fatty acid synthesis by isolated plastids, the relative utilisation of which depends upon the plant species and the organ from which the plastids are isolated. The import of these metabolites occurs via specific transporters on the plastid envelope and recent advances in the understanding of the role of these transporters are discussed. Chloroplasts are able to generate the reducing power and ATP required for fatty acid synthesis by capture of light energy in the reactions of photosynthetic electron transport. Regulation of chloroplast fatty acid synthesis is mediated by the response of acetyl-CoA carboxylase to the redox state of the plastid, which ensures that the carbon metabolism is linked to the energy status. The regulation of fatty acid synthesis in plastids of heterotrophic cells is much less well understood and is of particular interest in the tissues that accumulate large amounts of the storage oil, triacylglycerol. In these heterotrophic cells the plastids import ATP and oxidise imported carbon sources to produce the required reducing power. The sequencing of the genome of Arabidopsis thaliana has now enabled a number of aspects of plant fatty acid synthesis to be re-addressed, particularly those areas in which in vitro biochemical analysis had provided equivocal answers. Examples of such aspects and future opportunities for our understanding of plant fatty acid synthesis are presented and discussed.     

The memory of the fatty acid system

Mental memory system has sensory memory, short-term memory, working memory, and long-term memory. Working memory “keeps things in mind in parallel” when performing complex tasks. Similar aspects can be found for immunological memory. However, there exists another one, the memory of the fatty acid system.This article shows sensory memory of the fatty acid system, which is the perception apparatus of small intestine enterocytes (CD36, SR-B1, FATP4, FABP1, FABP2) and hepatocytes. In these cells, the fatty acid short-term memory is located, consisting of a cytoplasmic lipid droplet cycle. Similar like a working memory in the brain, the short-term memory of enterocytes and hepatocytes use parallel processing and recourse to long-term fatty acid memory. The fatty acid long-term memory is far away from these primary points of uptake. It is located in the adipocyte and in cellular membranes.The process of building a fatty acid memory is described with constructs like sensing environmental material, encoding, consolidation, long-term storage, retrieval, re-encoding, re-consolidation, and renewed long-term storage.The article illustrates the dynamics of building a fatty acid memory, the information content of fatty acids including the code, the roles of fatty acids in the body, and a new understanding of the expression “you are what you eat”. The memory of the fatty acid system, plays a decisive role in integrating environmental signals over time (diet and microbiome).     

Mouse GPR40 heterologously expressed in Xenopus oocytes is activated by short-, medium-, and long-chain fatty acids

Several orphan G protein-coupled receptors, including GPR40, have recently been shown to be responsive to fatty acids. Although previous reports have suggested GPR40 detects medium- and long-chain fatty acids, it has been reported to be unresponsive to short chain fatty acids. In this study, we have heterologously expressed mouse GPR40 in Xenopus laevis oocytes and measured fatty acid-induced increases in intracellular Ca²⁺, via two electrode voltage clamp recordings of the endogenous Ca²⁺-activated chloride conductance. Exposure to 500 µM linoleic acid (C18:2), a long-chain fatty acid, stimulated significant currents in mGPR40-injected oocytes (P < 0.01, ANOVA), but not in water-injected control oocytes (not significant, ANOVA). These currents were confirmed as Ca²⁺-activated chloride conductances because they were biphasic, sensitive to changes in external pH, and inhibited by DIDS. Similar currents were observed with medium-chain fatty acids, such as lauric acid (C12:0) (P < 0.01, ANOVA), and more importantly, with short-chain fatty acids, such as butyric acid (C4:0) (P < 0.01, ANOVA). In contrast, no responses were observed in mGPR40-injected oocytes exposed to either acetic acid (C2:0) or propionic acid (C3:0). Therefore, GPR40 has the capacity to respond to fatty acids with chain lengths of four or greater. This finding has important implications for understanding the structure:function relationship of fatty acid sensors, and potentially for short-chain fatty acid sensing in the gastrointestinal tract. fatty acid chain length; nutrient sensing     

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.     

FATP4 contributes as an enzyme to the basal and insulin-mediated fatty acid uptake of C₂C₁₂ muscle cells

The function of membrane proteins in long-chain fatty acid transport is controversial. The acyl-CoA synthetase fatty acid transport protein-4 (FATP4) has been suggested to facilitate fatty acid uptake indirectly by its enzymatic activity, or directly by transport across the plasma membrane. Here, we investigated the function of FATP4 in basal and insulin mediated fatty acid uptake in C(2)C(12) muscle cells, a model system relevant for fatty acid metabolism. Stable expression of exogenous FATP4 resulted in a twofold higher fatty acyl-CoA synthetase activity, and cellular uptake of oleate was enhanced similarly. Kinetic analysis demonstrated that FATP4 allowed the cells to reach apparent saturation of fatty acid uptake at a twofold higher level compared with control. Short-term treatment with insulin increased fatty acid uptake in line with previous reports. Surprisingly, insulin increased the acyl-CoA synthetase activity of C(2)C(12) cells within minutes. This effect was sensitive to inhibition of insulin signaling by wortmannin. Affinity purified FATP4 prepared from insulin-treated cells showed an enhanced enzyme activity, suggesting it constitutes a novel target of short-term metabolic regulation by insulin. This offers a new mechanistic explanation for the concomitantly observed enhanced fatty acid uptake. FATP4 was colocalized to the endoplasmic reticulum by double immunofluorescence and subcellular fractionation, clearly distinct from the plasma membrane. Importantly, neither differentiation into myotubes nor insulin treatment changed the localization of FATP4. We conclude that FATP4 functions by its intrinsic enzymatic activity. This is in line with the concept that intracellular metabolism plays a significant role in cellular fatty acid uptake.     

Strange organelles--Plasmodium mitochondria lack a pyruvate dehydrogenase complex

Our understanding of the Plasmodium mitochondrion and apicoplast has been greatly assisted by the genome sequence project. Sequence data have seeded recent research showing that the apicoplast is the site of several anabolic pathways including fatty acid synthesis. The discovery of an active apicoplast pyruvate dehydrogenase complex implies this enzyme generates the acetyl-CoA needed for fatty acid synthesis. However, the absence of a corresponding mitochondrial complex suggests that energy generation in Plasmodium is considerably different from pathways described in other eukaryotes.     

Conformational effects in enzyme catalysis: reaction via a high energy conformation in fatty acid amide hydrolase

Quantum mechanics/molecular mechanics and molecular dynamics simulations of fatty acid amide hydrolase show that reaction (amide hydrolysis) occurs via a distinct, high energy conformation. This unusual finding has important implications for fatty acid amide hydrolase, a key enzyme in the endocannabinoid system. These results demonstrate the importance of structural fluctuations and the need to include them in the modeling of enzyme reactions. They also show that approaches based simply on studying enzyme-substrate complexes can be misleading for understanding biochemical reactivity.     

Oxidation of hepatic carnitine palmitoyl transferase-I (CPT-I) impairs fatty acid beta-oxidation in rats fed a methionine-choline deficient diet

There is growing evidence that mitochondrial dysfunction, and more specifically fatty acid β-oxidation impairment, is involved in the pathophysiology of non-alcoholic steatohepatitis (NASH). The goal of the present study was to achieve more understanding on the modification/s of carnitinepalmitoyltransferase-I (CPT-I), the rate-limiting enzyme of the mitochondrial fatty acid β-oxidation, during steatohepatitis. A high fat/methionine-choline deficient (MCD) diet, administered for 4 weeks, was used to induce NASH in rats.We demonstrated that CPT-I activity decreased, to the same extent, both in isolated liver mitochondria and in digitonin-permeabilized hepatocytes from MCD-diet fed rats.At the same time, the rate of total fatty acid oxidation to CO(2) and ketone bodies, measured in isolated hepatocytes, was significantly lowered in treated animals when compared to controls. Finally, an increase in CPT-I mRNA abundance and protein content, together with a high level of CPT-I protein oxidation was observed in treated rats. A posttranslational modification of rat CPT-I during steatohepatitis has been here discussed.     

Energy response and fatty acid metabolism in Onychostoma macrolepis exposed to low-temperature stress

Temperature is a key environmental factor, and understanding how its fluctuations affect physiological and metabolic processes is critical for fish. The present study characterizes the energy response and fatty acid metabolism in Onychostoma macrolepis exposed to low temperature (10 °C). The results demonstrated that cold stress remarkably disrupted the energy homeostasis of O. macrolepis, then the AMP-activated protein kinase (AMPK) could strategically mobilize carbohydrates and lipids. In particular, when the O. macrolepis were faced with cold stress, the lipolysis was stimulated along with the enhanced fatty acid β-oxidation for energy, while the fatty acid synthesis was supressed in the early stage. Additionally, the fatty acid composition analysis suggested that saturated fatty acid (SFA) might accumulate while monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) in storage lipids (mainly containing non-polar lipid, NPL) could be utilized to supply energy during cold acclimation. Altogether, this study may provide some meritorious for understanding the cold-tolerant mechanism of fish in the viewpoint of energy balance combined with fatty acid metabolism, and thus to contribute to this species rearing in fish farms in the future.     

A global perspective on the crosstalk between saturated fatty acids and Toll-like receptor 4 in the etiology of inflammation and insulin resistance

Obesity is featured by chronic systemic low-grade inflammation that eventually contributes to the development of insulin resistance. Toll-like receptor 4 (TLR4) is an important mediator that triggers the innate immune response by activating inflammatory signaling cascades. Human, animal and cell culture studies identified saturated fatty acids (SFAs), the dominant non-esterified fatty acid (NEFA) in the circulation of obese subjects, as non-microbial agonists that trigger the inflammatory response via activating TLR4 signaling, which acts as an important causative link between fatty acid overload, chronic low-grade inflammation and the related metabolic aberrations. The interaction between SFAs and TLR4 may be modulated through the myeloid differentiation primary response gene 88-dependent and independent signaling pathway. Greater understanding of the crosstalk between dietary SFAs and TLR4 signaling in the pathogenesis of metabolic imbalance may facilitate the design of a more efficient pharmacological strategy to alleviate the risk of developing chronic diseases elicited in part by fatty acid overload. The current review discusses recent advances in the impact of crosstalk between SFAs and TLR4 on inflammation and insulin resistance in multiple cell types, tissues and organs in the context of metabolic dysregulation.     

Comparative biochemical studies of the murine fatty acid transport proteins (FATP) expressed in yeast

The fatty acid transport protein (FATP) family is a group of proteins that are predicted to be components of specific fatty acid trafficking pathways. In mammalian systems, six different isoforms have been identified, which function in the import of exogenous fatty acids or in the activation of very long-chain fatty acids. This has led to controversy as to whether these proteins function as membrane-bound fatty acid transporters or as acyl-CoA synthetases, which activate long-chain fatty acids concomitant with transport. The yeast FATP orthologue, Fat1p, is a dual functional protein and is required for both the import of long-chain fatty acids and the activation of very long-chain fatty acids; these activities intrinsic to Fat1p are separable functions. To more precisely define the roles of the different mammalian isoforms in fatty acid trafficking, the six murine proteins (mmFATP1-6) were expressed and characterized in a genetically defined yeast strain, which cannot transport long-chain fatty acids and has reduced long-chain acyl-CoA synthetase activity (fat1Delta faa1Delta). Each isoform was evaluated for fatty acid transport, fatty acid activation (using C18:1, C20:4, and C24:0 as substrates), and accumulation of very long-chain fatty acids. Murine FATP1, -2, and -4 complemented the defects in fatty acid transport and very long-chain fatty acid activation associated with a deletion of the yeast FAT1 gene; mmFATP3, -5, and -6 did not complement the transport function even though each was localized to the yeast plasma membrane. Both mmFATP3 and -6 activated C20:4 and C20:4, while the expression of mmFATP5 did not substantially increase acyl-CoA synthetases activities using the substrates tested. These data support the conclusion that the different mmFATP isoforms play unique roles in fatty acid trafficking, including the transport of exogenous long-chain fatty acids.     

Saturated and polyunsaturated fatty acids reciprocally modulate dendritic cell functions mediated through TLR4

TLRs provide critical signals to induce innate immune responses in APCs such as dendritic cells (DCs) that in turn link to adaptive immune responses. Results from our previous studies demonstrated that saturated fatty acids activate TLRs, whereas n-3 polyunsaturated fatty acids inhibit agonist-induced TLR activation. These results raise a significant question as to whether fatty acids differentially modulate immune responses mediated through TLR activation. The results presented in this study demonstrate that the saturated fatty acid, lauric acid, up-regulates the expression of costimulatory molecules (CD40, CD80, and CD86), MHC class II, and cytokines (IL-12p70 and IL-6) in bone marrow-derived DCs. The dominant negative mutant of TLR4 or its downstream signaling components inhibits lauric acid-induced expression of a CD86 promoter-reporter gene. In contrast, an n-3 polyunsaturated fatty acid, docosahexaenoic acid, inhibits TLR4 agonist (LPS)-induced up-regulation of the costimulatory molecules, MHC class II, and cytokine production. Similarly, DCs treated with lauric acid show increased T cell activation capacity, whereas docosahexaenoic acid inhibits T cell activation induced by LPS-treated DCs. Together, our results demonstrate that the reciprocal modulation of both innate and adaptive immune responses by saturated fatty acid and n-3 polyunsaturated fatty acid is mediated at least in part through TLRs. These results imply that TLRs are involved in sterile inflammation and immune responses induced by nonmicrobial endogenous molecules. These results shed new light in understanding how types of dietary fatty acids differentially modulate immune responses that could alter the risk of many chronic diseases.     

Resistance to Diet-Induced Obesity in Mice with Synthetic Glyoxylate Shunt

Given the success in engineering synthetic phenotypes in microbes and mammalian cells, constructing non-native pathways in mammals has become increasingly attractive for understanding and identifying potential targets for treating metabolic disorders. Here, we introduced the glyoxylate shunt into mouse liver to investigate mammalian fatty acid metabolism. Mice expressing the shunt showed resistance to diet-induced obesity on a high-fat diet despite similar food consumption. This was accompanied by a decrease in total fat mass, circulating leptin levels, plasma triglyceride concentration, and a signaling metabolite in liver, malonyl-CoA, that inhibits fatty acid degradation. Contrary to plants and bacteria, in which the glyoxylate shunt prevents the complete oxidation of fatty acids, this pathway when introduced in mice increases fatty acid oxidation such that resistance to diet-induced obesity develops. This work suggests that using non-native pathways in higher organisms to explore and modulate metabolism may be a useful approach.     

Identification of a new fatty acid synthesis-transport machinery at the peroxisomal membrane

The neurodegenerative disease X-linked adrenoleukodystrophy (X-ALD) is characterized by the abnormal accumulation of very long chain fatty acids. Mutations in the gene encoding the peroxisomal ATP-binding cassette half-transporter, adrenoleukodystrophy protein (ALDP), are the primary cause of X-ALD. To gain a better understanding of ALDP dysfunction, we searched for interaction partners of ALDP and identified binary interactions to proteins with functions in fatty acid synthesis (ACLY, FASN, and ACC) and activation (FATP4), constituting a thus far unknown fatty acid synthesis-transport machinery at the cytoplasmic side of the peroxisomal membrane. This machinery adds to the knowledge of the complex mechanisms of peroxisomal fatty acid metabolism at a molecular level and elucidates potential epigenetic mechanisms as regulatory processes in the pathogenesis and thus the clinical course of X-ALD.