Fatty acid oxidation pathways
Eicosanoids
Eicosanoids are metabolic products of three degradation pathways of arachidonic acid. The name comes from Greek word “eikosiâ€, what means “twenty†for the 20- carbon fatty acid of arachidonate. They constitute a class of oxygenated hydrophobic compounds that mostly act as paracrine mediators.
Prostaglandins
Prostanoids, comprised of prostaglandins and thromboxanes, are oxygenated metabolites of C20 polyunsaturated fatty acids (Cha et al. 2006). The precursors are arachidonic acid (AA) and eicosapentaenoic acid (EPA), released from membrane phospholipids by phospholipase A2 (PLA2). The activation of PLA2 is the first step in the arachidonic acid cascade. The formation and accumulation of the products of this cascade leads to various physiological responses: stimulation of JNK and MAPK, changes in transmembrane signalling (Hernandez et al. 2002), formation of free radicals and lipid hydroperoxide products (Muralikrishna et al. 2006). Liberated from phospholipids arachidonic acid is converted by cyclooxygenase (COX) to prostaglandin H2, PGH2.Cyclooxygenase is a membrane-bound hemoprotein, present in two isoforms: COX1 and COX2. COX1 is a constitutive form, almost ubiquitously expressed, that regulates low prostaglandin synthesis required for cell homeostasis, while COX2 is an inducible form almost undetectable or present at low level in resting state but is synthesised de novo in response to intracellular and extracellular stimuli in inflammatory processes (Hetu et al.,
2005). However, COX2 can be also expressed constitutively in brain (Minghetti et al., 2004), kidney (Harris et al.1994) and lung (Ermert et al. 1998). It has been shown that COX-1 is located in the endoplasmic reticulum (ER) and perinuclear membranes, whereas COX-2 resides predominantly in the perinuclear envelope (Ueno et al., 2005).
There is a third type of cyclooxygenase,COX3, also known as COX-1b, which is an acetaminophen-sensitive splice variant of COX1 and identified in canine tissues of
still unknown function (Botting, 2000). Prostaglandins are ubiquitously produced and act locally in an autocrine and juxtacrine manner and modulate many physiological systems including the CNS, cardiovascular, gastrointestinal, endocrine, respiratory and immune system (Tab.4). As potent proinflammatory mediators they are synthesised in cancer, inflammation, cardiovascular disease, hypertension. Pharmacologically their synthesis is blocked by the use of the cyclooxygenase-inhibiting nonsteroidal anti-inflammatory drugs (NSAIDs).
Prostaglandin H2 is subsequently isomerised by tissue specific prostaglandin synthases to structurally similar prostaglandins: PGE2, PGD2, PGF2?, PGI2 (known as prostacyclin) and thromboxane A2, which represent active lipid molecules. There are at least 9 known prostaglandin receptors. Four bind PGE2 (EP1-EP4), two bind PGD2 (DP1, DP2), FP, IP and TP (TPa and TPb) receptors bind PGF2?, PGI2 and TXA2 respectively (Cha et al. 2006, Reid HM, 2003). With the exception of the DP2 receptor, which is a member of the chemoattractant receptor subgroup, the rest belongs to the G protein-coupled receptor superfamily and signal through different G proteins (Tab.5).
PGD2 is formed by the actions of two types of PGD2 synthase isoforms, one is present in the central nervous system, testis, and the human heart and is called lipocalin PGD2 synthase and the second is present in the spleen and hematopoietic system (hPGD2S), being widely distributed in antigen presenting cells, T helper Th2 lymphocytes, megakaryocytes and mast cells (Trivedi et al. 2006). Dendritic cells, helper Th2-type and T cells produce PGD2 suggesting a modulatory role of this prostaglandin in antigen-specific immune responses. PGD2 is released into the airways and skin following acute allergic response (Barr et al. 1988). Additionally, PGD2 can inhibit platelet aggregation, smooth muscle relaxation and contraction, vasodilation and vasoconstriction (Hata et al. 2004). PGD2 binds and activates two distinct GPCRs – DP and CRTH2 (DP2), which both mediate inflammatory properties oftheir ligand. CRTH2 is expressed in TH2 lymphocytes, eosinophils and basophils and mediates PGD2-stimulated chemotaxis and leukocyte mobilization (Hirai et al. 2001, Shichijo et al. 2003). The DP receptor is expressed on bronchial epithelium and mediates production of chemokines and cytokines that recruit inflammatory lymphocytes and eosinophils leading to sympthoms resembling asthma (Kabashima et al. 2003).
Prostaglandin E2 (PGE2) is synthesized mainly in kidney, platelets, blood vessels and macrophages. It is a proinflammatory mediator, which induces fever, increases vascular
permeability and vasodilation, enhances pain and oedema, caused by other factors as bradykinin and histamine (Calder, 2005). Studies in cultured fibroblasts revealed that PGE2
could stimulate its own generation through the induction of COX2 (Bagga et al. 2003). It has been reported however that PGE2 acts also anti-inflammatory as it can inhibit 5-LOX and decrease the production of inflammatory leukotrienes (Levy et al. 2001). Simultaneously 15- LOX is induced and anti-inflammatory lipoxins are formed. PGE2 signals through four receptors (EP1-EP4)
Prostacyclin I2 (PGI2) is synthesized in endothelial cells, macrophages, lung and kidney. It plays a role as a potent vasodilator and inhibitor of platelet aggregation and this activity is mediated through coupling of the PGI2 IP receptor to Gs-type G protein (Cook, 2005).
Thromboxane A2 is synthesized in platelets, monocytes, macrophages and lung. It promotes platelet aggregation. It signals through the TP receptor that exists in two alternatively splice variants, TP? (placenta, platelets) and TPß (endothelial cells) (Hata et al. 2004). It couples to Gs, Gi, Gh and G12-type of G proteins and coupling to Gq leads to activation of PLC ß, IP3/DAG generation and mobilization of intracellular calcium. This pathway is responsible for thromboxane receptor-mediated platelet aggregation. Thromboxane A2 and PGI2 are biological antagonists and the balance between them is crucial for maintaining a healthy state of the vasculature.
PGF2? is produced during menstrual cycle by secretory endometrium and plays a role in mammalian reproduction (Hata et al. 2004) and changes in PGF2? lead to reproductive
abnormalities. Increased expression of receptor for PGF2? has been documented in endometrial adenocarcinoma growth (Sales et al. 2004). Additionally, PGF2? is important in
renal function, cardiac hypertrophy and regulation of intraocular pressure. As well as the above mentioned PGJ2 andPGA2 can be also synthesized from PGD2 and PGE2, respectively. Bell-Parikh LC et al have shown that the dehydration process can occur not just in vitro but also in vivo. PGJ2 has further derivates (see figure 15.).
Delta-12-PGJ2 has antitumor and antiviral activity. It is also able to induce neuronal death and to impair 26 S proteasome assembly (Zhiyou Wang et al.: 2006). 15-deoxy-PGJ2 has an anti-inflammatory property as a ligand of PPAR-gamma meanwhile it also blocks IKK (Straus DS, Glass CK.: 2001). Interestingly, it has as well a positive effect on VEGF
generation which is a know factor of neoangiogenesis in different tumor cells. (Ginger L et al., 2005)
In the presence of omega-3 PUFAs (polyunsaturated fatty acid) 3-series of prostaglandins are synthesized by COX enzymes instead of 2-series of PGs. There are different types of PUFAs. Alpha-linolenic acid can be found in plants, especially in flaxseed. The best sources of EPA (eicosapentaenoic-acid) and DHA (docosahexaenoic acid) are oily, cold-water fish. There is clinical evidence of anti-inflammatory property of omega3s in different inflammatory diseases, such as rheumatoid arthritis, skin dermatitis and posterior blepharitis. In the Western diets the ratio of omega-3 and omega-6 PUFAs is about 1:10-20. The desired ratio would be 1: 1.4-2.0. (Simopoulos AP: Poult Sci. 2000 Jul;79(7):961-70.) As these two essential fatty acid groups have antagonistic effect their balance in the diet is highly recommended. Hence, it is advised to decrease omega-6 and increase omega-3 PUFA level. (Simopoulos AP: Lipids. 2001;36 Suppl:S83-9.)
Isoprostanes:
IsoPs and COX-derived PGs have similar structures. The difference is that in IsoP-s the side chains are predominantly in cis-formation in relation to the prostane ring whereas this
orientation is trans in PGs. These molecules are synthesized from the free-radical induced peroxidation of AA. Hence, they are a potential biomarker of oxidative stress. In a contrast of other IsoPs, cyclopentenone IsoPs (A2, J2) can readily conjugated with GSH. This can be one of the reasons while they can be detected just in a small content in humans. Cyclopentanone IsoPs are bioactive molecules. They have anti-inflammatory property: they can inhibit IKK, inhibit NF-kappaB DNA binding ability. They have also a positive effect on hsp70 and heme oxygenase-1 synthesis. As well as the above mentioned they are able to induce VEGF, too. (Ginger et al., 2005)
Epoxyeicosatrienoic acids (EETs)
The NADPH-dependent epoxidation of arachidonic acid by cytochrome P450 (CYP450) leads to the formation of epoxyeicosatrienoic acids (EETs) (Fig.15).Four regioisomeric cis-epoxyeicosatienoic acids have been reported: 5,6-, 8,9-, 11,12-, and 14,15-EET (Spiecker et al. 2005). All forms are present at similar concentration in heart, endothelium and human plasma and they act anti-inflammatory, antioxidative, antimigratory and profibrinolytic. Soluble epoxide hydrolase (sEH) converts EETs into less biologically active and more stable metabolites, dihydroxyeicosatrienoic acids (DHETs).Several mammalian CYP isoforms that generate EETs (CYP1A, CYP2B, CYP2C, CYP2G, and CYP2J, CYP2N, CYP4A) differ in their selectivity for regioisomers, catalytic efficiency and tissue distribution. The most predominant epoxygenase isoforms participating in EET production belong to the CYP2 gene family and whereas CYP2C isoforms regulate EET biosynthesis in human liver and kidney, CYP2J isoforms have been reported to be involved in epoxidation of endogenous arachidonic acid in human and rat heart (Wu et al. 1996,1997). EETs have numerous functions in the endothelial cell, including protection from hypoxiareoxygenation
injury, inhibition of cytokine-induced cellular adhesion molecule expression, and activation of tissue plasminogen activator (tPA) expression. In the vascular smooth muscle cell, 20-hydroxyeicosatetraenoic acid (HETE) is the major product of cytochrome P450-(CYP4A and CYP4F) catalyzed arachidonic acid metabolism. Membrane stretch and vasoactive agents activate phospholipase C (PLC) leading to release of IP3 and diacylglycerol (DAG). Increased intracellular Ca²+, resulting from inositol 1,4,5-triphosphate (IP3) release, signals the activation of Ca²+-sensitive PLA2 and DAG lipase to stimulate the formation of 20-HETE. Inhibition of CYP4A enzymes and therefore 20-HETE formation by
nitric oxide (NO) is an important mechanism for maintaining basal tone in the vascular smooth muscle cell. The pathway by which 20-HETE inhibits large-conductance Ca²+-
activated K+-channels (BKCa) involves protein kinase C (PKC) and Raf/mitogen-activated protein kinase (MAPK). Inhibition of BKCa elevates the membrane potential, which enhances Ca²+-entry via L-type Ca²+-channels, and vasoconstrictions. Endothelial-derived EETs can activate BKCa leading to membrane hyperpolarization and vasodilation. The multiple and often opposing effects of EETs and 20-HETE in vascular endothelial and smooth muscle cells illustrate the intricate regulation of these complex biological pathways by cytochrome P450-derived eicosanoids. EETs may represent the putative endothelial-derived hyperpolarizing factor (EDHF) that relaxes VSMC by opening large-conductance, Ca²+-activated K+ channels (BKCa) in the coronary vessels (Pratt 2001, Fisslthaler 1999) a process that envolves G?-dependent activation of BKCa channel alpha subunit and requires ADP-ribosylation of G?s (Li 1999, Fukao 2001). EETs possess potent inflammatory effects by inhibiting cytokine-induced endothelial cell adesion molecule expression and preventing leukocyte adhesion to the vascular wall (Node 1999,Campbell 2000, Zeldin 2000). The mechanism involves NF?B and ?B-kinase. EETs also increase tPA expression and enhance fibrinolityic activity via a mechanism that involves activation of G?s, increases intracellular cAMP and cAMP response element (CRE)-mediated tPA promoter activation (Node 2001). EETs were shown to protect EC against hypoxic injury (Yang 2001) and to effect L-type and P/Q type Ca²+ channels (Qu 2001,Chen 1999) and to suppress urinary Na+-excretion (Brand-Schieber 2000). 14,15-EET inhibits apoptosis induced by serum withdrawal, H2O2, etoposide and excess AA in renal proximal tubular epithelial cells (Chen 2001). The mechanism involves PI-3 kinase and PKB/AKT but not MAPK and mice overexpressing 14,15 epoxygenase (CYP102F87V mutant) are protected against induced apoptosis. In renal epithelial cells 14,15-EET is a potent mitogen that requires Src kinase, MAPK,ERK1/2 and PI-3 kinase (Chen 1998). 14,15-EET increases Src kinase activity and overexpression of C-terminal Src-kinase, which inhibits a family of kinases, and blocked EET-induced Tyr-phosphorylation and mitogenesis (Chen 2000). EETs may act via cell surface receptors (K1=226 nM) that are attenuated by cAMP and PKA activation (Wong 2000). Once formed, EETs can be further metabolized along a number of pathways (Zeldin 2001, Fang 2000, Fang 2001, Widstrom 2001) including hydratation by soluble epoxide hydrolase (SEH), conjugation by glutathione S-transferase, oxidation by cytochrome P450s and cyclooxygenases, esterification to glycerophospholipids, conversion to chain shortened epoxy-fatty acids via peroxisomal ß-oxydation, affinities of heart-, liver- and intestinal FABPs were 20-fold greater for EETs that for DHET (dihydroxyeicosatrienoic acid).
Hydroxyeicosatetraenoic acids (HETEs)
Arachidonic acid is also a precursor for hydroxyeicosatatraenoic acids (HETEs) and 9- and 20-hydroxyeicosatetraenoic acids are formed by cytochrome P450 enzymes in brain, lung, kidney and peripheral blood vessels and are known to play a role regulation in vascular tone in renal, cerebral and coronary circulation. 20-Hydroxyeicosatetraenoic acid (HETE) is a potent vasoconstrictor in vascular smooth muscle cells that is formed in reaction by ?-hydroxylation of arachidonic acid by enzymes of the CYP4A and CYP4F families and CYP4A11, CYP4F2 and CYP4F3B are considered to be major 20-HETE synthesis enzymes (Miyata et al 2005). 20-HETE is a vasoconstrictor that causes blockade of Ca2+-activated K+-channels, plays a role as a vascular oxygen sensor, in the thick ascending loop of Henle (TAHL), where it inhibits sodium transport via the sodiumpotassium- two chloride co-transporter and Na+/H+ exchanger (Sacerdoti et al., 2003; Escalante et al. 1991; Good et al. 1999). 20-HETE also inhibits sodium reabsorption in kidney proximal tubules by reducing Na+/K+ ATPase activity (Quigley et al. 2000). It has been documented that in experimental rat models of hypertension the expression of enzymes of the CYP4A family and subsequently the generation of 20-HETE was elevated (Sacerdoti et al. 1989; Alonso-Garcia et al. 2002).
Additionally, 20-HETE acts as a second messenger for parathyroid hormone, dopamine and angiotensin II, and also for serotonin and phenylephrine and in TAHL serves as a second messenger controlling potassium recycling and sodium uptake from the filtrate (Satarug et al. 2006, Sarkis et al. 2004).Besides 20-HETE other types can also synthesized through cytochrome P450 dependent pathway (see figure 21.)
Leukotrienes
Leukotrienes (LO) are eicosanoids produced in leukocytes and endothelial cells and have a triene structure (Samuelsson et al. 1979). Leukotrienes are final metabolites of 5-HPETE, derived from the conversion of AA by 5- lipoxygenase (5-LO), which is dehydrated to labile 5,6 epoxide intermediate LTA4. The LTA4 hydrolase converts LTA4 to biologically active LTB4, which in the presence of LTC4 synthase conjugates with reduced glutathione to LTC4. Removal of glutamic acid byglutamyl transpeptidase from LTC4 generates LTD4 and LTE4 results from subsequent cleavage of glycine by dipeptidase. LTC4 and its metabolites are known as cysteinyl leukotrienes (Khan et al. 2003). LTC4 synthase is a membrane protein, found in cells of hematopoietic origin (Lam, 2003).LTB4 and cysteinyl leukotrienes signal through receptors, which are member of the G
protein-coupled receptor superfamily. LTC4, LTD4 and LTE4 bind to CysLT1 and CysLT2 receptors to exert their biological effects. The production of LTB4 from endogenous but not from exogenous sources requires the presence of 5-LO activating protein (FLAP) (Dixon et al. 1990). FLAP and leukotriene C4 synthase and also prostaglandin E synthase belong to membrane-associated proteins in eicosanoid and glutathione metabolism superfamily (MAPEG) (Bresell et al. 2005). LTB4 increases vascular permeability, induces the release of lysosomal enzymes, is potent leukocyte chemoatractant and enhances generation of reactive oxygen species and production of proinflammatory cytokines like TNF?, IL-1 and IL-6 (Calder, 2005). LTC4, LTD4 and LTE4 are among the most potent bronchoconstrictors; additionally they exert mitogenic effects, modify vascular integrity and play a pathological role in asthma (Silverman et al. 1998).
Lipoxygenase interaction products - Lipoxins
Lipoxins (LXs), an anocrym for lipoxygenase interaction products, are specific lipid mediators, formed during cell-cell interaction and play an important role in anti-inflammation
and as a signal for a resolution phase of acute inflammatory responses in human and mammalian systems (McMahon et al., 2001). Three enzymes are required for formation of
leukotrienes: 5-lipoxygenase (5-LO), LTC4 synthase and LTA4 epoxide hydrolase and there are three major pathways leading to biosythesis of lipoxins. The first pathway involves platelet-leukocyte interaction and starts with the insertion of molecular oxygen at the C5 position in arachidonic acid (Fig.18).The production of leukotriene LTA4 involves 5-LO in neutrophils and is followed by the abstraction of hydrogen from C13 and insertion of oxygen at C15, is released from neutrophils as LTA4 and converted to LXA4 and LXB4 by 12-LO in platelets upon their adherence. The bioactive LXA4 and LXB4 are end products of these reactions. Platelets don’t produce lipoxins on their own, but become the major source of lipoxins when they adhere to PMN (Chiang et al. 2005). In the second type of LX generation oxygenation of arachidonic acid at the C15 position in the presence of 15-LO generates 15S-hydroperoxyeicosatetraenoic acid (15S-HpETE) or the reduced alcohol form 15S-hydroxyeicosatetraenoic acid (15S-HETE), which serve as substrates for 5-LO. The product of this reaction is 5S-hydroxyperoxy, 15S-hydro(peroxy)- DiH(p)ETE, which is rapidly converted to 5(6)-epoxytetraene. Once formed, 5(6)- epoxytetraene is subject to hydrolase activity to generate lipoxin A4 or lipoxin B4. 5-
lipoxygenase, the first enzyme of this biosythesis, is highly expressed in neutrophils and monocytes and is upregulated upon stimulation with IL-4 and IL-13 (Serhan et al. 1997). 5- lipoxygenase has been found in cytosol and in the nucleus. Upon activation the enzyme translocates to the perinuclear membrane, where it becomes catalytically active (Peters- Golden et al. 1993). The 5-LO action is regulated at the level of gene transcription,translation and enzyme translocation but also availability of free arachidonic acid as an
important factor stimulating 5-LO activity. Esterification of 15-HETE in phospholipids, especially inositol-containing phospholipds, within the membrane of neutrophiles can play important source of lipoxins in tissues during inflammation (Serhan, 2005). Cells rapidly take up and esterify 15-HETE into their inositolcontaining lipids, which upon agonist stimulation is released and converted into LXs. This suggests that precursors of lipoxins may be stored within the membranes and released upon inflammatory conditions (Brezinski et al. 1990). The third pathway to generate lipoxins is triggered by aspirin. Aspirin, in inflammatory state, acetylates COX-2 and shifts its activity from endothelial cell prostanoid production towards 15-HETE generation. COX-2 –derived HETE can be converted by 5-LO to carbon-15 positio R epimers, known as 15-epi-LX or aspirin-triggered lipoxin (ATL) (Claria et al. 1995). The anti-inflammatory effects of LXA4 are mediated via ALX receptor belonging to the G protein-coupled receptors superfamily. ALX has been identified in various cell types, including PMN, monocytes, activated T cells and enterocytes (Chiang et al. 2005). However, ALX is not a specific receptor for LXA4 as it has been shown to interact with other small lipids and to transmit different signals. Both lipoxins and ATL are rapidly inactivated by local metabolism, via dehydrogenation and ? oxidation mediated by prostaglandin dehydrogenase in monocytes (Kieran et al. 2004).
Resolvins, Docosatrienes and Neuroprotectins
Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are precursors of novel oxygenated bioactive products that were identified in resolving inflammatory exudates
(Serhan et al. 2000). They were named resolvins, docosatrienes and neuroprotectins and, unlike other omega-3 fatty acids products, are present in anti-inflammatory and
immunoregulatory processes. The classification of resolvins, the products of interaction between aspirin acetylated COX-2 and omega-3 polyunsaturated fatty acids, is based on
their origin: resolvins originated from EPA constitute resolvins of E series (Resolvin E1 or RvE1) and those from DHA constitute resolvins of D series (Resolvin D1 or RvD1). The antiinflammatory and neuroprotective products, which carry conjugated triene structures, are denoted as docosatrienes (DT) and docosatrienes that contain dihydroxy acid are termed neuroprotectins (NPD1) (Sehran et al. 2004). Both resolvins of D and E series are potent regulators of PMN infiltration in brain and their strong anti-inflammatory properties are mediated partially by modulation of cytokines expression (Serhan et al. 2002, Serhan et al. 2003). Additionally, RvE1 reduces dermal inflammation, peritonitis, dendritic cell migration and IL-12 production (Arita et al. 2005).The anti-inflammatory action of RvE1 is mediated via G-protein-coupled receptor, ChemR23 (Arita et al. 2005), which is upregulated by cytokines during resolution; a process that counter regulates proinflammatory gene expression and cell trafficking and stimulates inflammatory cell clearance (Nathan, 2002). ChemR23 is structurally similar to LXA4 receptor and similarly can interact with other lipid ligands.RvE1 may serve as a paracrine or autocrine signal during resolution to repress NF-kB activation and further cytokine synthesis.
NSAID and aspirin-triggered lipid mediators
The RvE1 is synthesized from EPA in vascular endothelial cells treated with aspirin. EPA is converted to 18R-HEPE that is released and rapidly turned by activated PMN to 5(6)-epoxide containing intermediate and subsequently to bioactive 5,12,18R-trixydroxy-EPE, termed resolvin RvE1 (Serhan 2005). 5S,18R-HEPE, termed RvE2 is generated via reduction of 5Shydro( peroxy), 18R-hydroxy EPE.Upon treatment with aspirin, DHA is converted by COX-2 to 17R-D series of resolvins and gives rise to RvD1 through RvD6 resolvins as well as docosatrienes. In the presence of 5-LO 17S series of resolvins (RvD1 through RvD6) and docosatrienes are produced.
Neuroprotectin D1 (10,17-docosatriene; NPD1, when generated by neuronal cells; otherwise Protectin D1), formed from DHA in cornea in a lipoxygenase-dependent pathway,
protects from thermal injury and promotes wound healing. It has been documented that in retinal pigment epithelium cells (RPE), NPD1 protects cells from apoptosis through
increasing expression of anti-apoptotic Bcl-2 and BclxL proteins and decreasing proapoptotic Bax and Bad (Bazan, 2005; 2006). It inhibits oxidative stress-induced caspase 3 activation and IL-1ß stimulated expression of COX-2. NPD1 possesses natural isomers, that all act anti-inflammatory (Serhan et al. 2006).
Categories: Fatty Acyls (FA) | Lipid signalling
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