Use of reversed phase HP liquid chromatography to assay conversion of N-acylglycines to primary fatty acid amides by peptidylglycine-alpha-amidating monooxygenase

Primary fatty acid amides (R-CO-NH2) and N-acylglycines (R-CO-NH-CH2-COOH) are classes of compounds that have only recently been isolated and characterized from biological sources. Key questions remain regarding how these lipid amides are produced and degraded in biological systems. Relative to the fatty acids, little has been done to develop methods to separate and quantify the fatty acid amides and N-acylglycines. We describe reversed phase HPLC methods for the separation of C2-C12 primary fatty acid amides and N-acylglycines and also C12-C22 fatty acid amides. Separation within each class occurs primarily on the basis of simple interactions between the acyl chain and the chromatographic stationary phase, but the polar headgroups on these and related fatty acids and N-acylethanolamides modulate the absolute retention in reversed phase mode. We use these methods to measure the enzyme-mediated, two-step conversion of N-octanoylglycine to octanoamide.     

The endogenous cannabinoid system and its role in nociceptive behavior

The analgesic properties of exogenous cannabinoids have been recognized for many years and suggest a regulatory role for the endogenous cannabinoid ("endocannabinoid") system in mammalian nociceptive pathways. The endocannabinoid system includes: (1) at least two families of lipid signaling molecules, the N-acyl ethanolamines (e.g., anandamide) and the monoacylglycerols (e.g., 2-arachidonoyl glycerol); (2) multiple enzymes involved in the biosynthesis and degradation of these lipids, including the integral membrane enzyme fatty acid amide hydrolase; and (3) two G-protein coupled receptors, CB1 and CB2, which are primarily localized to the nervous system and immune system, respectively. Here, we review recent genetic, behavioral, and pharmacological studies that have tested the function of the endocannabinoid system in pain sensation. Collectively, these investigations support a role for endocannabinoids in modulating behavioral responses to acute, inflammatory, and neuropathic pain stimuli.     

Occurrence, metabolism, and prospective functions of N-acylethanolamines in plants

N-Acylethanolamines (NAEs) are fatty acid amides that are derived from an N-acylated phoshatidylethanolamine presursor, a minor membrane lipid constituent of plant and animal cells. Historically, the formation of N-acylethanolamines was associated with cellular stress and tissue damage in mammals, but more recently has been shown to be part of the endocannabinoid signaling system that regulates a variety of normal physiological functions, including neurotransmission, immune responses, vasodilation, embryo development and implantation, feeding behavior, cell proliferation, etc. The widespread regulation of vertebrate physiology by this class of lipid mediators and the conservation of the mechanisms for NAE formation, perception and degradation in higher plants raises the possibility that the metabolism of NAEs represents an evolutionarily conserved lipid signaling pathway that regulates an array of physiological processes in multicellular eukaryotes. Here the recent information on NAEs in plants is reviewed in the context of the occurrence, metabolism and functions of this bioactive class of lipid mediators.     

Mass spectral characterization of fatty acid amides from alfalfa trichomes and their deterrence against the potato leafhopper

A homologous series of N-(3-methylbutyl)amides of normal saturated C14, C15, C16, C17 and C18 fatty acids were identified as major components of glandular trichome extracts from Medicago sativa G98A, an alfalfa genotype resistant to the potato leafhopper, Empoasca fabae. A second homologous series of N-(2-methylpropyl)amides of C14 through C18 normal fatty acids were minor components. Saturated free fatty acids C12, C13, C14, C15, C16, C17 and C18 were present in trace amounts, as was the N-(3-methylbutyl)amide of linoleic acid (C18:2). N-(3-methylbutyl)amides and N-(2-methylpropyl)amides of C14 through C18 fatty acids, along with the N-(3-methylbutyl)amide of linoleic acid, were synthesized and bioassayed for leafhopper deterrence by applying the compounds to the surface of a sachet containing an artificial diet. Leafhoppers were then offered a two-way choice between diet surfaces treated with the synthetic amides or an untreated control. N-(3-methylbutyl)amides and N-(2-methylpropyl)amides of C14 through C18 fatty acids did not deter leafhopper settling in a dose-dependent fashion. In contrast, when tested singly, N-(3-methylbutyl)amide of linoleic acid exhibited dose-dependent deterrence against leafhopper settling. Fatty acid amides localized in alfalfa glandular trichomes likely contribute to leafhopper resistance.     

A FAAH-regulated class of N-acyl taurines that activates TRP ion channels

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that catabolizes several bioactive lipids in vivo. Most of the physiological substrates of FAAH characterized to date belong to the N-acyl ethanolamine (NAE) class of fatty acid amides, including the endocannabinoid anandamide, the anti-inflammatory lipid N-palmitoyl ethanolamine, and the satiating factor N-oleoyl ethanolamine. We recently identified a second structural class of fatty acid amides regulated by FAAH in vivo: the N-acyl taurines (NATs). Global metabolite profiling revealed high concentrations of long chain (> or = C20) saturated NATs in the central nervous system (CNS) of FAAH(-/-) mice. Here, we use metabolite profiling to characterize the FAAH-NAT system in peripheral mouse tissues. Livers and kidneys of FAAH(-/-) mice possessed dramatic elevations in NATs, which, in contrast to those detected in the CNS, were enriched in polyunsaturated acyl chains (e.g., C20:4, C22:6). Peripheral NATs rose more than 10-fold within 1 h following pharmacological inactivation of FAAH and reached levels up to approximately 5000 pmol/g tissue (C22:6 in kidney), implicating a constitutive and highly active pathway for NAT metabolism in which FAAH plays an integral part. Interestingly, NATs were found to activate multiple members of the transient receptor potential (TRP) family of calcium channels, including TRPV1 and TRPV4, which are both expressed in kidney. The dramatic elevation in endogenous levels of NATs following acute or chronic inactivation of FAAH, in conjunction with the pharmacological effects of these lipids on TRP channels, suggests the existence of a second major lipid signaling system regulated by FAAH in vivo.     

A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs

Fatty acids, which are essential nutritional components, are also involved in cardiovascular and metabolic diseases. Here we report a human cell surface receptor that we name free fatty acid receptor (FFAR), because it is specifically activated by medium to long-chain free fatty acids. The receptor belongs to the class of seven-transmembrane, G-protein coupled receptors (GPCRs) and also mediates responses to antidiabetic drugs of the thiazolidinedione type. It is expressed in skeletal muscle, heart, liver, and pancreatic beta-cells. Stimulation of FFAR increases the intracellular calcium concentration in cells expressing the receptor in a native (pancreatic beta-cell line) or in a recombinant form. In view of the nature of the activating substances, their physiological role in the body, and the tissue distribution of FFAR we suggest the term "nutrient sensing receptor" for receptors acting at the interface between dietary components and signalling molecules.     

New metabolically stable fatty acid amide ligands of cannabinoid receptors: Synthesis and receptor affinity studies

We investigated the structure-activity relationships for the interactions of fatty acid amide analogs of the endocannabinoid anandamide with human recombinant cannabinoid receptors. Thirty-five novel fatty acid amides were synthesized using five different types of acyl chains and 11 different aromatic amine 'heads.' Although none of the new compounds was a more potent ligand than anandamide, we identified three amine groups capable of improving the metabolic stability of arachidonoylamides and their CB(1)/CB(2) selectivity ratio to over 20-fold, and several aromatic amines capable of improving the affinity of short chain or monosaturated fatty acids for cannabinoid CB(1) receptors. For the first time a tertiary amide of arachidonic acid was found to possess moderate affinity (K(i)=300 nM) for cannabinoid CB(1), but not CB(2), receptors.     

On the importance of fatty acid composition of membranes for aging

The membrane pacemaker theory of aging is an extension of the oxidative stress theory of aging. It emphasises variation in the fatty acid composition of membranes as an important influence on lipid peroxidation and consequently on the rate of aging and determination of lifespan. The products of lipid peroxidation are reactive molecules and thus potent damagers of other cellular molecules. It is suggested that the feedback effects of these peroxidation products on the oxidative stress experienced by cells is an important part of the aging process. The large variation in the chemical susceptibility of individual fatty acids to peroxidation coupled with the known differences in membrane composition between species can explain the different lifespans of species, especially the difference between mammals and birds as well as the body-size-related variation in lifespan within mammals and birds. Lifespan extension by calorie-restriction can also be explained by changes in membrane fatty acid composition which result in membranes more resistant to peroxidation. It is suggested that lifespan extension by reduced insulin/IGF signalling may also be mediated by changes in membrane fatty acid composition.     

Hub and switches: endocannabinoid signalling in midbrain dopamine neurons

The last decade has provided a wealth of experimental data on the role played by lipids belonging to the endocannabinoid family in several facets of physiopathology of dopamine neurons. We currently suggest that these molecules, being intimately connected with diverse metabolic and signalling pathways, might differently affect various functions of dopamine neurons through activation not only of surface receptors, but also of nuclear receptors. It is now emerging how dopamine neurons can regulate their constituent biomolecules to compensate for changes in either internal functions or external conditions. Consequently, dopamine neurons use these lipid molecules as metabolic and homeostatic signal detectors, which can dynamically impact cell function and fitness. Because dysfunctions of the dopamine system underlie diverse neuropsychiatric disorders, including schizophrenia and drug addiction, the importance of better understanding the correlation between an unbalanced endocannabinoid signal and the dopamine system is even greater. Particularly, because dopamine neurons are critical in controlling incentive-motivated behaviours, the involvement of endocannabinoid molecules in fine-tuning dopamine cell activity opened new avenues in both understanding and treating drug addiction. Here, we review recent advances that have shed new light on the understanding of differential roles of endocannabinoids and their cognate molecules in the regulation of the reward circuit, and discuss their anti-addicting properties, particularly with a focus on their potential engagement in the prevention of relapse.     

A second fatty acid amide hydrolase with variable distribution among placental mammals

Fatty acid amides constitute a large and diverse class of lipid transmitters that includes the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The magnitude and duration of fatty acid amide signaling are controlled by enzymatic hydrolysis in vivo. Fatty acid amide hydrolase (FAAH) activity in mammals has been primarily attributed to a single integral membrane enzyme of the amidase signature (AS) family. Here, we report the functional proteomic discovery of a second membrane-associated AS enzyme in humans that displays FAAH activity. The gene that encodes this second FAAH enzyme was found in multiple primate genomes, marsupials, and more distantly related vertebrates, but, remarkably, not in a number of lower placental mammals, including mouse and rat. The two human FAAH enzymes, which share 20% sequence identity and are referred to hereafter as FAAH-1 and FAAH-2, hydrolyzed primary fatty acid amide substrates (e.g. oleamide) at equivalent rates, whereas FAAH-1 exhibited much greater activity with N-acyl ethanolamines (e.g. anandamide) and N-acyl taurines. Both enzymes were sensitive to the principal classes of FAAH inhibitors synthesized to date, including O-aryl carbamates and alpha-keto heterocycles. These data coupled with the overlapping, but distinct tissue distributions of FAAH-1 and FAAH-2 suggest that these proteins may collaborate to control fatty acid amide catabolism in primates. The apparent loss of the FAAH-2 gene in some lower mammals should be taken into consideration when extrapolating genetic or pharmacological findings on the fatty acid amide signaling system across species.     

The endocannabinoid system,anandamide and the regulation of mammalian cell apoptosis

Endocannabinoids are a new class of lipid mediators, which include amides, esters and ethers of long-chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the main endogenous agonists of cannabinoid receptors able to mimic several pharmacological effects of Delta-9-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. The pathways leading to the synthesis and release of AEA and 2-AG from neuronal and non-neuronal cells are still rather uncertain. Instead, it is known that the activity of AEA is limited by cellular uptake through a specific membrane transporter, followed by intracellular degradation by a fatty acid amide hydrolase. Together with AEA and congeners these proteins form the 'endocannabinoid system'. Here, the involvement of AEA in apoptosis and the underlying signal transduction pathways will be reviewed, along with the metabolic routes and the molecular targets of this endocannabinoid. Also, recent findings on the apoptotic potential of AEA for neuronal cell differentiation and brain development will be discussed.     

Altered hepatic lipid metabolism in C57BL/6 mice fed alcohol: a targeted lipidomic and gene expression study

Chronic alcohol consumption is associated with fatty liver disease in mammals. The object of this study was to gain an understanding of dysregulated lipid metabolism in alcohol-fed C57BL/6 mice using a targeted lipidomic approach. Liquid chromatography tandem mass spectrometry was used to analyze several lipid classes, including free fatty acids, fatty acyl-CoAs, fatty acid ethyl esters, sphingolipids, ceramides, and endocannabinoids, in plasma and liver samples from control and alcohol-fed mice. The interpretation of lipidomic data was augmented by gene expression analyses for important metabolic enzymes in the lipid pathways studied. Alcohol feeding was associated with i) increased hepatic free fatty acid levels and decreased fatty acyl-CoA levels associated with decreased mitochondrial fatty acid oxidation and decreased fatty acyl-CoA synthesis, respectively; ii) increased hepatic ceramide levels associated with higher levels of the precursor molecules sphingosine and sphinganine; and iii) increased hepatic levels of the endocannabinoid anandamide associated with decreased expression of its catabolic enzyme fatty acid amide hydrolase. The unique combination of lipidomic and gene expression analyses allows for a better mechanistic understanding of dysregulated lipid metabolism in the development of alcoholic fatty liver disease.     

Antigen chemically coupled to the surface of liposomes are cross-presented to CD8+ T cells and induce potent antitumor immunity

We have previously demonstrated that liposomes with differential lipid components display differential adjuvant effects when Ags are chemically coupled to their surfaces. In the present study, Ag presentation of liposome-coupled OVA was investigated in vitro, and it was found that OVA coupled to liposomes made using unsaturated fatty acid was presented to both CD4+ and CD8+ T cells, whereas OVA coupled to liposomes made using saturated fatty acid was presented only to CD4+ T cells. Confocal laser scanning microscopic analysis demonstrated that a portion of the OVA coupled to liposomes made using unsaturated, but not saturated fatty acid, received processing beyond the MHC class II compartment, suggesting that the degradation of OVA might occur in the cytosol, and that the peptides generated in this manner would be presented to CD8+ T cells via MHC class I. The ability to induce cross-presentation of an Ag coupled to liposomes consisting of unsaturated fatty acid was further confirmed by in vivo induction of CTL and by the induction of tumor eradication in mice; E.G7 tumors in mice that received combined inoculation with OVA(257-264)-liposome conjugates, CpG, and anti-IL-10 mAbs were completely eradicated. In those mice, the frequency of CD8+ T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased. These results suggested that, by choosing lipid components for liposomes, surface-coupled liposomal Ags might be applicable for the development of tumor vaccines to present tumor Ags to APCs and induce antitumor responses.     

Primary fatty acid amide metabolism: conversion of fatty acids and an ethanolamine in N18TG2 and SCP cells

Primary fatty acid amides (PFAM) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotion, angiogenesis, and many other processes. Oleamide is the best-studied of the primary fatty acid amides, whereas the other known PFAMs are significantly less studied. Herein, quantitative assays were used to examine the endogenous amounts of a panel of PFAMs, as well as the amounts produced after incubation of mouse neuroblastoma N(18)TG(2) and sheep choroid plexus (SCP) cells with the corresponding fatty acids or N-tridecanoylethanolamine. Although five endogenous primary amides were discovered in the N(18)TG(2) and SCP cells, a different pattern of relative amounts were found between the two cell lines. Higher amounts of primary amides were found in SCP cells, and the conversion of N-tridecanoylethanolamine to tridecanamide was observed in the two cell lines. The data reported here show that the N(18)TG(2) and SCP cells are excellent model systems for the study of PFAM metabolism. Furthermore, the data support a role for the N-acylethanolamines as precursors for the PFAMs and provide valuable new kinetic results useful in modeling the metabolic flux through the pathways for PFAM biosynthesis and degradation.     

Lipid signalling in plant responses to abiotic stress

Lipids are one of the major components of biological membranes including the plasma membrane, which is the interface between the cell and the environment. It has become clear that membrane lipids also serve as substrates for the generation of numerous signalling lipids such as phosphatidic acid, phosphoinositides, sphingolipids, lysophospholipids, oxylipins, N‐acylethanolamines, free fatty acids and others. The enzymatic production and metabolism of these signalling molecules are tightly regulated and can rapidly be activated upon abiotic stress signals. Abiotic stress like water deficit and temperature stress triggers lipid‐dependent signalling cascades, which control the expression of gene clusters and activate plant adaptation processes. Signalling lipids are able to recruit protein targets transiently to the membrane and thus affect conformation and activity of intracellular proteins and metabolites. In plants, knowledge is still scarce of lipid signalling targets and their physiological consequences. This review focuses on the generation of signalling lipids and their involvement in response to abiotic stress. We describe lipid‐binding proteins in the context of changing environmental conditions and compare different approaches to determine lipid–protein interactions, crucial for deciphering the signalling cascades.     

The endocannabinoid system in neurodegeneration

Endocannabinoids are bioactive lipids, that comprise amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the best studied endocannabinoids, and act as agonists of cannabinoid receptors. Thus, AEA and 2-AG mimic several pharmacological effects of the exogenous cannabinoid delta9-tetrahydrocannabinol, the psychoactive principle of hashish and marijuana. It is known that the activity of endocannabinoids at their receptors is limited by cellular uptake through specific membrane transporters, followed by intracellular degradation by a fatty acid amide hydrolase (for AEA and partly 2-AG) or by a monoacylglycerol lipase (for 2-AG). Together with AEA, 2-AG and congeners, the proteins that bind, transport and metabolize these lipids form the "endocannabinoid system". This new system will be briefly presented in this review, in order to put in a better perspective the role of the endocannabinoid pathway in neurodegenerative disorders, like Parkinson's disease, Huntington's disease, and multiple sclerosis. In addition, the potential exploitation of antagonists of endocannabinoid receptors, or of inhibitors of endocannabinoid metabolism, as next-generation therapeutics will be discussed.