Eicosanoid formation by a cytochrome P450 isoform expressed in the pharynx of Caenorhabditis elegans

Caenorhabditis elegans harbours several CYP (cytochrome P450) genes that are homologous with mammalian CYP isoforms important to the production of physiologically active AA (arachidonic acid) metabolites. We tested the hypothesis that mammals and C. elegans may share similar basic mechanisms of CYP-dependent eicosanoid formation and action. We focused on CYP33E2, an isoform related to the human AA-epoxygenases CYP2C8 and CYP2J2. Co-expression of CYP33E2 with the human NADPH-CYP reductase in insect cells resulted in the reconstitution of an active microsomal mono-oxygenase system that metabolized EPA (eicosapentaenoic acid) and, with lower activity, also AA to specific sets of regioisomeric epoxy- and hydroxy-derivatives. The main products included 17,18-epoxyeicosatetraenoic acid from EPA and 19-hydroxyeicosatetraenoic acid from AA. Using nematode worms carrying a pCYP33E2::GFP reporter construct, we found that CYP33E2 is exclusively expressed in the pharynx, where it is predominantly localized in the marginal cells. RNAi (RNA interference)-mediated CYP33E2 expression silencing as well as treatments with inhibitors of mammalian AA-metabolizing CYP enzymes, significantly reduced the pharyngeal pumping frequency of adult C. elegans. These results demonstrate that EPA and AA are efficient CYP33E2 substrates and suggest that CYP-eicosanoids, influencing in mammals the contractility of cardiomyocytes and vascular smooth muscle cells, may function in C. elegans as regulators of the pharyngeal pumping activity.     

Cytochrome P450-dependent metabolism of eicosapentaenoic acid in the nematode Caenorhabditis elegans

The genome of Caenorhabditis elegans contains 75 full length cytochrome P450 (CYP) genes whose individual functions are largely unknown yet. We tested the hypothesis that some of them may be involved in the metabolism of eicosapentaenoic acid (EPA), the predominant polyunsaturated fatty acid of this nematode. Microsomes isolated from adult worms contained spectrally active CYP proteins and showed NADPH-CYP reductase (CPR) activities. They metabolized EPA and with lower activity also arachidonic acid (AA) to specific sets of regioisomeric epoxy- and ω-/(ω-1)-hydroxy-derivatives. 17(R),18(S)-epoxyeicosatetraenoic acid was produced as the main EPA metabolite with an enantiomeric purity of 72%. The epoxygenase and hydroxylase reactions were NADPH-dependent, required the functional expression of the CPR-encoding emb-8 gene, and were inhibited by 17-ODYA and PPOH, two compounds known to inactivate mammalian AA-metabolizing CYP isoforms. Multiple followed by single RNAi gene silencing experiments identified CYP-29A3 and CYP-33E2 as the major isoforms contributing to EPA metabolism in C. elegans. Liquid chromatography/mass spectrometry revealed that regioisomeric epoxy- and hydroxy-derivatives of EPA and AA are endogenous constituents of C. elegans. The endogenous EPA metabolite levels were increased by treating the worms with fenofibrate, which also induced the microsomal epoxygenase and hydroxylase activities. These results demonstrate for the first time that C. elegans shares with mammals the capacity to produce CYP-dependent eicosanoids and may thus facilitate future studies on the mechanisms of action of this important class of signaling molecules.     

Enalapril reverses high-fat diet-induced alterations in cytochrome P450-mediated eicosanoid metabolism

Metabolism of arachidonic acid by cytochrome P450 (CYP) to biologically active eicosanoids has been recognized increasingly as an integral mediator in the pathogenesis of cardiovascular and metabolic disease. CYP epoxygenase-derived epoxyeicosatrienoic and dihydroxyeicosatrienoic acids (EET + DHET) and CYP ω-hydroxylase-derived 20-hydroxyeicosatetraenoic acid (20-HETE) exhibit divergent effects in the regulation of vascular tone and inflammation; thus, alterations in the functional balance between these parallel pathways in liver and kidney may contribute to the pathogenesis and progression of metabolic syndrome. However, the impact of metabolic dysfunction on CYP-mediated formation of endogenous eicosanoids has not been well characterized. Therefore, we evaluated CYP epoxygenase (EET + DHET) and ω-hydroxylase (20-HETE) metabolic activity in liver and kidney in apoE(-/-) and wild-type mice fed a high-fat diet, which promoted weight gain and increased plasma insulin levels significantly. Hepatic CYP epoxygenase metabolic activity was significantly suppressed, whereas renal CYP ω-hydroxylase metabolic activity was induced significantly in high-fat diet-fed mice regardless of genotype, resulting in a significantly higher 20-HETE/EET + DHET formation rate ratio in both tissues. Treatment with enalapril, but not metformin or losartan, reversed the suppression of hepatic CYP epoxygenase metabolic activity and induction of renal CYP ω-hydroxylase metabolic activity, thereby restoring the functional balance between the pathways. Collectively, these findings suggest that the kinin-kallikrein system and angiotensin II type 2 receptor are key regulators of hepatic and renal CYP-mediated eicosanoid metabolism in the presence of metabolic syndrome. Future studies delineating the underlying mechanisms and evaluating the therapeutic potential of modulating CYP-derived EETs and 20-HETE in metabolic diseases are warranted.     

Effects of starvation and neuroactive drugs on feeding in Caenorhabditis elegans

Caenorhabditis elegans concentrates its food, bacteria, by pharyngeal pumping. The rate of pumping is affected by the presence of bacteria. Using a new assay that allows measurement of pumping rate in a population of worms suspended in liquid by measuring their uptake of microscopic iron particles, we have confirmed and quantitated this effect. Furthermore, we demonstrated that starvation stimulates pumping. Worms that had been deprived of bacteria for more than 4 hours pumped in the absence of bacteria under conditions in which well-fed worms did not. Furthermore, starved worms responded to lower amounts of bacteria than did fed worms. The assay was also useful for measuring effects of drugs on pumping. Of about 30 chemicals screened, 5 had clear effects. The neurotransmitter serotonin and the serotonin uptake inhibitor imipramine stimulated pumping, while the serotonin antagonist gramine inhibits. Imipramine stimulation is greatly decreased in cat-1 and cat-4 mutants, which have low levels of serotonin. Muscimol, an agonist for the neurotransmitter GABA, and ivermectin, whose site of action may also be the GABA receptor, both inhibit pumping. Qualitative observations suggested a role for acetylcholine in the regulation of pumping.     

Mechanotransduction by Membrane Proteins: Worms find PEZO-1’s function easy to swallow

JGP study finds that the C. elegans orthologue of the PIEZO family is a mechanosensitive ion channel that regulates pharyngeal pumping and food sensation.

The PIEZO family of mechanosensitive cation channels has been implicated in a wide variety of physiological processes in mammals and is also associated with human disease. Mammalian genomes encode two family members, known as Piezo1 and Piezo2, but invertebrates such as the nematode Caenorhabditis elegans only possess a single Piezo-related gene (1). The function of the C. elegans orthologue, known as pezo-1, has largely remained obscure, but, in this issue of JGP, Millet et al. reveal that it encodes a bona fide mechanosensitive ion channel that regulates pharyngeal activity (2).Jonathan Millet (left), Valeria Vásquez (center), and colleagues reveal that pezo-1, the sole PIEZO family member in C. elegans, is a mechanosensitive ion channel that regulates pharyngeal pumping and food sensation, particularly when worms are fed with large and stiff bacterial filaments that are difficult to swallow (graphic created with BioRender.com).In 2020, an elegant study demonstrated that pezo-1 controls C. elegans ovulation and fertilization (3). However, explains Valeria Vásquez from the University of Tennessee Health Science Center, whether pezo-1 encodes for a mechanosensitive ion channel was unknown. “PEZO-1 is expressed in many tissues, including the pharynx, which is the organ we decided to concentrate on in our study,” Vásquez says.Muscle cells in the C. elegans pharynx rhythmically contract and relax to pump food into the worm’s intestine. Vásquez and colleagues, including first author Jonathan Millet, found that PEZO-1 is expressed in several different pharyngeal cell types (2), including the gland cells whose secretions lubricate the pharynx, and the proprioceptive NSM neurons that are thought to sense the presence of food within the pharynx lumen and release serotonin to increase the rate of pharyngeal pumping.Millet et al. analyzed pharyngeal pumping in worms lacking pezo-1, as well as in animals expressing a pezo-1 point mutant that, in human Piezo1, increases channel function by slowing channel deactivation and inactivation. Loss or gain of pezo-1 function had surprisingly little effect on pharyngeal activity, causing only mild alterations in the duration and frequency of pumping induced by serotonin, and more obvious effects when challenged with high osmolarity solutions.Worms cultured in the laboratory are usually fed a diet of small, easily ingested Escherichia coli cells and, both loss and gain of pezo-1 function increased the pharynx’s response to this type of food. In their natural habitat, however, C. elegans encounter bacteria of various shapes and sizes, some of which might be harder to swallow. “It occurred to me that it might make a difference if we fed the worms with bacteria that were stiffer and longer,” Vásquez says.The researchers therefore provided their pezo-1 mutants with E. coli treated with cephalexin, an antibiotic that inhibits cell separation and causes the bacteria to form long, spaghetti-like filaments. Compared with wild-type worms fed with this diet, pharyngeal activity was markedly enhanced by the gain-of-function pezo-1 mutant, but substantially reduced in the absence of pezo-1, almost as if the worms were “choking” on the bacterial filaments.Crucially, by performing patch-clamp experiments on both cultured C. elegans cells and insect cells expressing recombinant pezo-1, Millet et al. confirmed that PEZO-1 is, indeed, a mechanosensitive ion channel. However, it remains to be seen exactly how PEZO-1 helps the pharynx sense the physical parameters of food and adjust its pumping activity accordingly. One possibility is that the channel acts within the proprioceptive neurons to regulate the release of serotonin.Intriguingly, the Drosophila PIEZO orthologue controls feeding behavior in flies (4). “However, it’s not known which mechanosensitive channels are important in the pharyngeal system of mammals,” Vásquez says. “Our studies in C. elegans could therefore open an opportunity to understand food sensation in humans.”     

Formation of 20-hydroxyeicosatetraenoic acid, a vasoactive and natriuretic eicosanoid, in human kidney. Role of Cyp4F2 and Cyp4A11

20-hydroxyeicosatetraenoic acid (20-HETE), an omega-hydroxylated arachidonic acid (AA) metabolite, elicits specific effects on kidney vascular and tubular function that, in turn, influence blood pressure control. The human kidney's capacity to convert AA to 20-HETE is unclear, however, as is the underlying P450 catalyst. Microsomes from human kidney cortex were found to convert AA to a single major product, namely 20-HETE, but failed to catalyze AA epoxygenation and midchain hydroxylation. Despite the monophasic nature of renal AA omega-hydroxylation kinetics, immunochemical studies revealed participation of two P450s, CYP4F2 and CYP4A11, since antibodies to these enzymes inhibited 20-HETE formation by 65. 9 +/- 17 and 32.5 +/- 14%, respectively. Western blotting confirmed abundant expression of these CYP4 proteins in human kidney and revealed that other AA-oxidizing P450s, including CYP2C8, CYP2C9, and CYP2E1, were not expressed. Immunocytochemistry showed CYP4F2 and CYP4A11 expression in only the S2 and S3 segments of proximal tubules in cortex and outer medulla. Our results demonstrate that CYP4F2 and CYP4A11 underlie conversion of AA to 20-HETE, a natriuretic and vasoactive eicosanoid, in human kidney. Considering their proximal tubular localization, these P450 enzymes may partake in pivotal renal functions, including the regulation of salt and water balance, and arterial blood pressure itself.     

Octopamine and 5‐hydroxytryptamine mediate hemocytic phagocytosis and nodule formation via eicosanoids in the beet armyworm,Spodoptera exigua

Octopamine and 5‐hydroxytryptamine (5‐HT) have been known to mediate cellular immune responses, such as hemocytic phagocytosis and nodule formation, during bacterial invasion in some insects. In addition, eicosanoids also mediate these cellular immune reactions in various insects, resulting in clearing the bacteria circulating in the hemolymph. This study investigated a hypothesis on signal cross‐talk between both types of immune mediators in the beet armyworm, Spodoptera exigua, which had been observed in the effect of eicosanoids on mediating the cellular immune responses. In response to bacterial infection, octopamine or 5‐HT markedly enhanced both hemocytic phagocytosis and nodule formation in S. exigua larvae. Their specific antagonists, phentolamine (an octopamine antagonist) or ketanserin (a 5‐HT antagonist) suppressed both cellular immune responses of S. exigua. These effects of biogenic monoamines on the immune mediation were expressed through eicosanoids because the inhibitory effects of both antagonists were rescued by the addition of arachidonic acid (a precursor of eicosanoid biosynthesis). Furthermore, the stimulatory effects of both monoamines on the cellular immune responses were significantly suppressed by different inhibitors acting at their specific levels of eicosanoid biosynthesis. Taken together, this study suggests that octopamine and 5‐HT can mediate hemocytic phagocytosis and nodule formation through a downstream signal pathway relayed by eicosanoids in S. exigua. © 2009 Wiley Periodicals, Inc.     

Arachidonic Acid-metabolizing Cytochrome P450 Enzymes Are Targets of ω-3 Fatty Acids

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against cardiovascular disease by largely unknown mechanisms. We tested the hypothesis that EPA and DHA may compete with arachidonic acid (AA) for the conversion by cytochrome P450 (CYP) enzymes, resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J isoforms converting AA to epoxyeicosatrienoic acids (EETs) preferentially epoxidized the ω-3 double bond and thereby produced 17,18-epoxyeicosatetraenoic (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) from EPA and DHA. We found that these ω-3 epoxides are highly active as antiarrhythmic agents, suppressing the Ca²⁺-induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations. CYP4A/4F isoforms ω-hydroxylating AA were less regioselective toward EPA and DHA, catalyzing predominantly ω- and ω minus 1 hydroxylation. Rats given dietary EPA/DHA supplementation exhibited substantial replacement of AA by EPA and DHA in membrane phospholipids in plasma, heart, kidney, liver, lung, and pancreas, with less pronounced changes in the brain. The changes in fatty acids were accompanied by concomitant changes in endogenous CYP metabolite profiles (e.g. altering the EET/EEQ/EDP ratio from 87:0:13 to 27:18:55 in the heart). These results demonstrate that CYP enzymes efficiently convert EPA and DHA to novel epoxy and hydroxy metabolites that could mediate some of the beneficial cardiovascular effects of dietary ω-3 fatty acids.     

Emerging mechanisms for growth and protection of the vasculature by cytochrome P450-derived products of arachidonic acid and other eicosanoids

Arachidonic acid (AA) is an essential fatty acid that is metabolized by cyclooxygenase (COX), lipoxygenase (LOX) or cytochrome P450 (CYP) enzymes to generate eicosanoids which in turn mediate a number of biological activities including regulation of angiogenesis. While much information on the effects of COX and LOX products is known, the physiological relevance of the CYP-derived products of AA are less well understood. CYP enzymes are highly expressed in the liver and kidney, but have also been detected at lower levels in the brain, heart and vasculature. A number of these enzymes, including members of the CYP 4 family, predominantly catalyze conversion of AA to 20-hydroxyeicosatetraenoic acid (20-HETE) while the CYP epoxygenases generate mainly epoxyeicosatrienoic acids (EETs). This review will focus on the emerging roles of inhibitors of eicosanoid production with emphasis on the CYP pathways, in the regulation of angiogenesis and tumor growth. We also discuss current observations describing the protective effects of EETs for survival of the endothelium.     

CYP4F3B is induced by PGA1 in human liver cells: a regulation of the 20-HETE synthesis

The regulation of the human liver-specific cytochrome P450 4F3B (CYP4F3B) isoform, a splice variant of the CYP4F3 gene with strong substrate specificity for long chain fatty acids, is yet an unsolved question. This report provides the first evidence that CYP4F3B is uniquely induced by prostaglandin A(1) (PGA(1)) in human hepatocyte-like HepaRG cells and leads to the synthesis of 20-hydroxy-eicosatetraenoic acids (HETEs). Real time PCR, immunoblot analysis with a specific antipeptide antibody, and determination of fatty acid omega-hydroxylase activity demonstrate that PGA(1) treatment strongly increases expression of CYP4F3B. This induction drives the production of 20-HETE (19-fold increase). SiRNA-mediated-silencing of CYP4F3 suppresses both 20-HETE synthesis and PGA(1) induced 20-HETE production. Taken together, these results provide evidence that CYP4F3B is the key enzyme to produce 20-HETE by omega-hydroxylation of arachidonic acid in liver cells. Since 20-HETE is a potent activator of PPARalpha and an important inflammatory mediator, CYP4F3B may exert important functions in lipid homeostasis and in inflammatory diseases.     

CYP-eicosanoids--a new link between omega-3 fatty acids and cardiac disease?

Fish oil omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against arrhythmia and sudden cardiac death by largely unknown mechanisms. Recent in vitro and in vivo studies demonstrate that arachidonic acid (AA) metabolizing cytochrome P450-(CYP) enzymes accept EPA and DHA as efficient alternative substrates. Dietary EPA/DHA supplementation causes a profound shift of the cardiac CYP-eicosanoid profile from AA- to EPA- and DHA-derived epoxy- and hydroxy-metabolites. CYP2J2 and other CYP epoxygenases preferentially epoxidize the ω-3 double bond of EPA and DHA. The corresponding metabolites, 17,18-epoxy-EPA and 19,20-epoxy-DHA, dominate the CYP-eicosanoid profile of the rat heart after EPA/DHA supplementation. The (ω-3)-epoxyeicosanoids show highly potent antiarrhythmic properties in neonatal cardiomyocytes, suggesting that these metabolites may specifically contribute to the cardioprotective effects of omega-3 fatty acids. This hypothesis is discussed in the context of recent findings that revealed CYP-eicosanoid mediated mechanisms in cardiac ischemia-reperfusion injury and maladaptive cardiac hypertrophy.     

Cyclooxygenase-2 dependent metabolism of 20-HETE increases adiposity and adipocyte enlargement in mesenchymal stem cell-derived adipocytes

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a product of the cytochrome P450 (CYP)-catalyzed ω-hydroxylation of arachidonic acid, induces oxidative stress and, in clinical studies, is associated with increased body mass index (BMI) and the metabolic syndrome. This study was designed to examine the effects of exogenous 20-HETE on mesenchymal stem cell (MSC)-derived adipocytes. The expression levels of CYP4A11 and CYP4F2 (major 20-HETE synthases in humans) in MSCs decreased during adipocyte differentiation; however, exogenous administration of 20-HETE (0.1–1 μM) increased adipogenesis in a dose-dependent manner in these cells (P < 0.05). The inability of a 20-HETE analog to reproduce these effects suggested the involvement of a metabolic product of 20-HETE in mediating its pro-adipogenic effects. A cyclooxygenase (COX)-1 selective inhibitor enhanced, whereas a COX-2 selective or a dual COX-1/2 inhibitor attenuated adipogenesis induced by 20-HETE. The COX-derived metabolite of 20-HETE, 20-OH-PGE₂, enhanced adipogenesis and lipid accumulation in MSCs. The pro-adipogenic effects of 20-HETE and 20-OH-PGE₂ resulted in the increased expression of the adipogenic regulators PPARγ and β-catenin in MSC-derived adipocytes. Taken together we show for the first time that 20-HETE-derived COX-2-dependent 20-OH-PGE₂ enhances mature inflamed adipocyte hypertrophy in MSC undergoing adipogenic differentiation.     

The regulation of feeding and metabolism in response to food deprivation in Caenorhabditis elegans

This review considers the factors involved in the regulation of feeding and metabolism in response to food deprivation using Caenorhabditis elegans as a model organism. Some of the sensory neurons and interneurons involved in food intake are described, together with an overview of pharyngeal pumping. A number of chemical transmitters control feeding in C. elegans including 5-hydroxytryptamine (5-HT, serotonin), acetylcholine, glutamate, dopamine, octopamine, and tyramine. The roles of these transmitters are modified by neuropeptides, including FMRFamide-like peptides (FLPs), neuropeptide-like protein (NLPs), and insulin-like peptides. The precise effects of many of these neuropeptides have yet to be elucidated but increasingly they are being shown to play a role in feeding and metabolism in C. elegans. The regulation of fat stores is complex and appears to involve the expression of a large number of genes, many with mammalian homologues, suggesting that fat regulatory signalling is conserved across phyla. Finally, a brief comparison is made between C. elegans and mammals where for both, despite their evolutionary distance, classical transmitters and neuropeptides have anorectic or orexigenic properties. Thus, there is a rationale to support the argument that an understanding of the molecular and genetic basis of feeding and fat regulation in C. elegans may contribute to efforts aimed at the identification of targets for the treatment of conditions associated with abnormal metabolism and obesity.     

Investigation of feeding behaviour in <Emphasis Type="Italic">C. elegans</Emphasis> reveals distinct pharmacological and antibacterial effects of nicotine

Caenorhabditis elegans is an informative model to study the neural basis of feeding. A useful paradigm is one in which adult nematodes feed on a bacterial lawn which has been pre-loaded with pharmacological agents and the effect on pharyngeal pumping rate scored. A crucial aspect of this assay is the availability of good quality bacteria to stimulate pumping to maximal levels. A potential confound is the possibility that the pharmacological agent impacts bacterial viability and indirectly influences feeding rate. Here, the actions of nicotine on pharyngeal pumping of C. elegans and on the Escherichia coli bacterial food source were investigated. Nicotine caused an immediate and concentration-dependent inhibition of C. elegans pharyngeal pumping, IC₅₀ 4 mM (95% CI?=?3.4 mM to 4.8 mM). At concentrations between 5 and 25 mM, nicotine also affected the growth and viability of E. coli lawns. To test whether this food depletion by nicotine caused the reduced pumping, we modified the experimental paradigm. We investigated pharyngeal pumping stimulated by 10 mM 5-HT, a food ‘mimic’, before testing if nicotine still inhibited this behaviour. The IC₅₀ for nicotine in these assays was 2.9 mM (95% CI?=?3.1 mM to 5.1 mM) indicating the depletion of food lawn does not underpin the potency of nicotine at inhibiting feeding. These studies show that the inhibitory effect of nicotine on C. elegans pharyngeal pumping is mediated by a direct effect rather than by its poorly reported bactericidal actions.     

The effect of a selective octopamine antagonist, epinastine, on pharyngeal pumping in Caenorhabditis elegans

This paper investigates the effect of epinastine, a selective octopamine antagonist in invertebrates, in Caenorhabditis elegans. Specifically, its ability to block the inhibitory action of octopamine on C. elegans-isolated pharynx was assayed. Isolated pharynxes were stimulated to pump by the addition of 500 nM 5-hydroxytryptamine (5-HT) (113 ± 2 per 30 s, n = 15). Octopamine inhibited the 5-HT-induced pumping in a concentration-dependent manner (threshold 1–5 μM) with a 61 ± 11% inhibition with 50 μM (n = 5). Epinastine (0.1 μM) antagonized the inhibitory response to octopamine (P < 0.001; n = 15). Tyramine also inhibited pharyngeal pumping induced by 5-HT but was less potent than octopamine. Tyramine, 50 μM to 1 mM, gave a transient inhibition e.g. of 40 ± 5% at 50 μM (n = 5). A higher (10 μM) concentration of epinastine was required to block the tryamine response compared with octopamine. It is concluded that epinastine selectively antagonizes the effect of octopamine on C. elegans pharynx. Further studies are required to test its selectivity for octopamine in other tissues and other nematodes.     

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping^1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.     

Identification of a Feed-Forward Loop Between 15(S)-HETE and PGE2 in Human Amnion at Parturition

Human parturition is associated with massive arachidonic acid (AA) mobilization in the amnion, indicating that large amounts of AA-derived eicosanoids are required for parturition. Prostaglandin E2 (PGE2) synthesized from the cyclooxygenase (COX) pathway is the best characterized AA-derived eicosanoid in the amnion which plays a pivotal role in parturition. The existence of any other pivotal AA-derived eicosanoids involved in parturition remains elusive. Here, we screened such eicosanoids in human amnion tissue with AA-targeted metabolomics and studied their role and synthesis in parturition by using human amnion fibroblasts and a mouse model. We found that lipoxygenase (ALOX) pathway-derived 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and its synthetic enzymes ALOX15 and ALOX15B were significantly increased in human amnion at parturition. Although 15(S)-HETE is ineffective on its own, it potently potentiated the activation of NF-κB by inflammatory mediators including lipopolysaccharide, interleukin-1β, and serum amyloid A1, resulting in the amplification of COX-2 expression and PGE2 production in amnion fibroblasts. In turn, we determined that PGE2 induced ALOX15/15B expression and 15(S)-HETE production through its EP2 receptor-coupled PKA pathway, thereby forming a feed-forward loop between 15(S)-HETE and PGE2 production in the amnion at parturition. Our studies in pregnant mice showed that 15(S)-HETE injection induced preterm birth with increased COX-2 and PGE2 abundance in the fetal membranes and placenta. Conclusively, 15(S)-HETE is identified as another crucial parturition-pertinent AA-derived eicosanoid in the amnion, which may form a feed-forward loop with PGE2 in parturition. Interruption of this feed-forward loop may be of therapeutic value for the treatment of preterm birth.     

The cGMP signaling pathway affects feeding behavior in the necromenic nematode Pristionchus pacificus

Background

The genetic tractability and the species-specific association with beetles make the nematode Pristionchus pacificus an exciting emerging model organism for comparative studies in development and behavior. P. pacificus differs from Caenorhabditis elegans (a bacterial feeder) by its buccal teeth and the lack of pharyngeal grinders, but almost nothing is known about which genes coordinate P. pacificus feeding behaviors, such as pharyngeal pumping rate, locomotion, and fat storage.

Methodology/Principal Findings

We analyzed P. pacificus pharyngeal pumping rate and locomotion behavior on and off food, as well as on different species of bacteria (Escherichia coli, Bacillus subtilis, and Caulobacter crescentus). We found that the cGMP-dependent protein kinase G (PKG) Ppa-EGL-4 in P. pacificus plays an important role in regulating the pumping rate, mouth form dimorphism, the duration of forward locomotion, and the amount of fat stored in intestine. In addition, Ppa-EGL-4 interacts with Ppa-OBI-1, a recently identified protein involved in chemosensation, to influence feeding and locomotion behavior. We also found that C. crescentus NA1000 increased pharyngeal pumping as well as fat storage in P. pacificus.

Conclusions

The PKG EGL-4 has conserved functions in regulating feeding behavior in both C. elegans and P. pacificus nematodes. The Ppa-EGL-4 also has been co-opted during evolution to regulate P. pacificus mouth form dimorphism that indirectly affect pharyngeal pumping rate. Specifically, the lack of Ppa-EGL-4 function increases pharyngeal pumping, time spent in forward locomotion, and fat storage, in part as a result of higher food intake. Ppa-OBI-1 functions upstream or parallel to Ppa-EGL-4. The beetle-associated omnivorous P. pacificus respond differently to changes in food state and food quality compared to the exclusively bacteriovorous C. elegans.     

ALOX5 gene variants affect eicosanoid production and response to fish oil supplementation

The objective of this study was to determine whether 5-lipoxygenase (ALOX5) gene variants associated with cardiovascular disease affect eicosanoid production by monocytes. The study was a randomized, double-masked, parallel intervention trial with fish oil (5.0 g of fish oil daily, containing 2.0 g of eicosapentaenoic acid [EPA] and 1.0 g of docosahexaenoic acid [DHA]) or placebo oil (5.0 g of corn/soy mixture). A total of 116 subjects (68% female, 20-59 years old) of African American ancestry enrolled, and 98 subjects completed the study. Neither ALOX5 protein nor arachidonic acid-derived LTB4, LTD4, and LTE4 varied by genotype, but 5-hydroxyeicosatetraenoate (5-HETE), 6-trans-LTB4, 5-oxo-ETE, 15-HETE, and 5,15-diHETE levels were higher in subjects homozygous for the ALOX5 promoter allele containing five Sp1 element tandem repeats ("55" genotype) than in subjects with one deletion (d) (three or four repeats) and one common ("d5" genotype) allele or with two deletion ("dd") alleles. The EPA-derived metabolites 5-HEPE and 15-HEPE and the DHA-derived metabolite 17-HDoHE had similar associations with genotype and increased with supplementation; 5-HEPE and 15-HEPE increased, and 5-oxo-ETE decreased to a greater degree in the 55 than in the other genotypes. This differential eicosanoid response is consistent with the previously observed interaction of these variants with dietary intake of omega-3 fatty acids in predicting cardiovascular disease risk.     

Mechanotransduction by Membrane Proteins: C. elegans PEZO-1 is a mechanosensitive ion channel involved in food sensation

PIEZO channels are force sensors essential for physiological processes, including baroreception and proprioception. The Caenorhabditis elegans genome encodes an orthologue gene of the Piezo family, pezo-1, which is expressed in several tissues, including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal, whose contraction and relaxation are modulated by mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive ion channel and contributes to pharyngeal function remains unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recording to establish that pezo-1 is expressed in the pharynx, including in a proprioceptive-like neuron, and regulates pharyngeal function. Knockout (KO) and gain-of-function (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, as well as sensing osmolarity and food mechanical properties. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the G-isoform of pezo-1 (among the longest isoforms) demonstrates that pezo-1 encodes a mechanosensitive channel. Our findings reveal that pezo-1 is a mechanosensitive ion channel that regulates food sensation in worms.