N-acyl Phosphatidylethanolamine Phospholipase Research Articles

The 16α-hydroxylated Bile Acid, Pythocholic Acid Decreases Food Intake and Increases Oleoylethanolamide in Male Mice.

Modulation of bile acid (BA) structure is a potential strategy for obesity and metabolic disease treatment. BAs act not only as signaling molecules involved in energy expenditure and glucose homeostasis, but also as regulators of food intake. The structure of BAs, particularly the position of the hydroxyl groups of BAs, impacts food intake partly by intestinal effects: (1) modulating the activity of N-acyl phosphatidylethanolamine phospholipase D, which produces the anorexigenic bioactive lipid oleoylethanolamide (OEA) or (2) regulating lipid absorption and the gastric emptying-satiation pathway. We hypothesized that 16α-hydroxylated BAs uniquely regulate food intake because of the long intermeal intervals in snake species in which these BAs are abundant. However, the effects of 16α-hydroxylated BAs in mammals are completely unknown because they are not naturally found in mammals. To test the effect of 16α-hydroxylated BAs on food intake, we isolated the 16α-hydroxylated BA pythocholic acid from ball pythons (Python regius). Pythocholic acid or deoxycholic acid (DCA) was given by oral gavage in mice. DCA is known to increase N-acyl phosphatidylethanolamine phospholipase D activity better than other mammalian BAs. We evaluated food intake, OEA levels, and gastric emptying in mice. We successfully isolated pythocholic acid from ball pythons for experimental use. Pythocholic acid treatment significantly decreased food intake in comparison to DCA treatment, and this was associated with increased jejunal OEA, but resulted in no change in gastric emptying or lipid absorption. The exogenous BA pythocholic acid is a novel regulator of food intake and the satiety signal for OEA in the mouse intestine.

Inhibition of synaptic transmission by anandamide precursor 20:4-NAPE is mediated by TRPV1 receptors under inflammatory conditions.

Transient receptor potential ion channel, vanilloid subfamily, type 1 (TRPV1) cation channel, and cannabinoid receptor 1 (CB1) are essential in the modulation of nociceptive signaling in the spinal cord dorsal horn that underlies different pathological pain states. TRPV1 and CB1 receptors share the endogenous agonist anandamide (AEA), produced from N-arachidonoylphosphatidylethanolamine (20:4-NAPE). We investigated the effect of the anandamide precursor 20:4-NAPE on synaptic activity in naive and inflammatory conditions. Patch-clamp recordings of miniature excitatory postsynaptic currents (mEPSCs) from superficial dorsal horn neurons in rat acute spinal cord slices were used. Peripheral inflammation was induced by subcutaneous injection of carrageenan. Under naive conditions, mEPSCs frequency (0.96 ± 0.11 Hz) was significantly decreased after 20 μM 20:4-NAPE application (55.3 ± 7.4%). This 20:4-NAPE-induced inhibition was blocked by anandamide-synthesizing enzyme N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor LEI-401. In addition, the inhibition was prevented by the CB1 receptor antagonist PF 514273 (0.2 μM) but not by the TRPV1 receptor antagonist SB 366791 (10 μM). Under inflammatory conditions, 20:4-NAPE (20 μM) also exhibited a significant inhibitory effect (74.5 ± 8.9%) on the mEPSCs frequency that was prevented by the TRPV1 receptor antagonist SB 366791 but not by PF 514273 application. Our results show that 20:4-NAPE application has a significant modulatory effect on spinal cord nociceptive signaling that is mediated by both TRPV1 and CB1 presynaptic receptors, whereas peripheral inflammation changes the underlying mechanism. The switch between TRPV1 and CB1 receptor activation by the AEA precursor 20:4-NAPE during inflammation may play an important role in nociceptive processing, hence the development of pathological pain.

NAPE-PLD regulates specific baseline affective behaviors but is dispensable for inflammatory hyperalgesia

N-acyl-ethanolamine (NAEs) serve as key endogenous lipid mediators as revealed by manipulation of fatty acid amide hydrolase (FAAH), the primary enzyme responsible for metabolizing NAEs. Preclinical studies focused on FAAH or NAE receptors indicate an important role for NAE signaling in nociception and affective behaviors. However, there is limited information on the role of NAE biosynthesis in these same behavioral paradigms. Biosynthesis of NAEs has been attributed largely to the enzyme N-acylphosphatidylethanolamine Phospholipase D (NAPE-PLD), one of three pathways capable of producing these bioactive lipids in the brain. In this report, we demonstrate that Nape-pld knockout (KO) mice displayed reduced sucrose preference and consumption, but other baseline anxiety-like or depression-like behaviors were unaltered. Additionally, we observed sex-dependent responses in thermal nociception and other baseline measures in wildtype (WT) mice that were absent in Nape-pld KO mice. In the Complete Freund’s Adjuvant (CFA) model of inflammatory arthritis, WT mice exhibited sex-dependent changes in paw edema that were lost in Nape-pld KO mice. However, there was no effect of Nape-pld deletion on arthritic pain-like behaviors (grip force deficit and tactile allodynia) in either sex, indicating that while NAPE-PLD may alter local inflammation, it does not contribute to pain-like behaviors associated with inflammatory arthritis. Collectively, these findings indicate that chronic and systemic NAPE-PLD inactivation will likely be well-tolerated, warranting further pharmacological evaluation of this target in other disease indications.

Endocannabinoid System Components of the Female Mouse Reproductive Tract Are Modulated during Reproductive Aging.

The endocannabinoid (eCB) system has gained ground as a key modulator of several female fertility-related processes, under physiological/pathological conditions. Nevertheless, its modulation during reproductive aging remains unclear. This study aimed to investigate the expression levels of the main receptors (cannabinoid receptor 1,CB1; cannabinoid receptor 2, CB2; G-protein coupled receptor, GPR55; and transient receptor potential vanilloid type 1 channel, TRPV1) and metabolic enzymes (N-acylphosphatidylethanolamine phospholipase D, NAPE-PLD; fatty acid amide hydrolase, FAAH; monoacylglycerol lipase, MAGL; and diacylglycerol lipase, DAGL-α and -β) of this system in the ovaries, oviducts, and uteri of mice at prepubertal, adult, late reproductive, and post-reproductive stages through quantitative ELISA and immunohistochemistry. The ELISA showed that among the receptors, TRPV1 had the highest expression and significantly increased during aging. Among the enzymes, NAPE-PLD, FAAH, and DAGL-β were the most expressed in these organs at all ages, and increased age-dependently. Immunohistochemistry revealed that, regardless of age, NAPE-PLD and FAAH were mainly found in the epithelial cells facing the lumen of the oviduct and uteri. Moreover, in ovaries, NAPE-PLD was predominant in the granulosa cells, while FAAH was sparse in the stromal compartment. Of note, the age-dependent increase in TRPV1 and DAGL-β could be indicative of increased inflammation, while that of NAPE-PLD and FAAH could suggest the need to tightly control the levels of the eCB anandamide at late reproductive age. These findings offer new insights into the role of the eCB system in female reproduction, with potential for therapeutic exploitation.

Adolescent exposure to low-dose Δ9-tetrahydrocannabinol depletes the ovarian reserve in female mice.

Cannabis use by adolescents is widespread, but its effects on the ovaries remain largely unknown. Δ9-tetrahydrocannabinol (THC) exerts its pharmacological effects by activating, and in some conditions hijacking, cannabinoid receptors (CBRs). We hypothesized that adolescent exposure to THC affects ovarian function in adulthood. Peripubertal female C57BL/6N mice were given THC (5 mg/kg) or its vehicle, once daily by intraperitoneal injection. Some mice received THC from postnatal day (PND) 30-33 and their ovaries were harvested PND34; other mice received THC from PND30-43, and their ovaries were harvested PND70. Adolescent treatment with THC depleted ovarian primordial follicle numbers by 50% at PND70, 4 weeks after the last dose. The treatment produced primordial follicle activation, which persisted until PND70. THC administration also caused DNA damage in primary follicles and increased PUMA protein expression in oocytes of primordial and primary follicles. Both CB1R and CB2R were expressed in oocytes and theca cells of ovarian follicles. Enzymes involved in the formation (N-acylphosphatidylethanolamine phospholipase D) or deactivation (fatty acid amide hydrolase) of the endocannabinoid anandamide were expressed in granulosa cells of ovarian follicles and interstitial cells. Levels of mRNA for CBR1 were significantly increased in ovaries after adolescent THC exposure, and upregulation persisted for at least 4 weeks. Our results support that adolescent exposure to THC may cause aberrant activation of the ovarian endocannabinoid system in female mice, resulting in substantial loss of ovarian reserve in adulthood. Relevance of these findings to women who frequently used cannabis during adolescence warrants investigation.

15P The analgesic compound palmitoylethanolamide reduces inflammation in human cardiomyocytes and vascular endothelial cells exposed to doxorubicin and anti-HER2 monoclonal antibody through PPAR-α and NLRP3-related pathways

Palmitoylethanolamide (PEA) is an endogenous fatty acid mediator that is synthetized from membrane phospholipids by N-acyl phosphatidylethanolamine phospholipase D. It is a new analgesic drug with anti-inflammatory effects through the induction of PPAR-related pathways. We aimed to assess whether palmitoylethanolamide co-incubated during doxorubicin and trastuzumab, reduces anticancer drugs-related cardiotoxicity in cellular models.

N-acylethanolamide metabolizing enzymes are upregulated in human neural progenitor-derived neurons exposed to sub-lethal oxidative stress.

N-acyl amides (NAAs) are a class of lipids that consist of an acyl group N-linked to an amino acid, neurotransmitter, taurine or ethanolamide group (N-acylethanolamines or NAEs) and include some endocannabinoids (eCB) such as anandamide. These lipids are synthesized in a wide variety of organisms and in multiple cell types, including neurons. NAEs are involved in numerous cellular and physiological processes and their concentrations are elevated in response to ischemia and physical trauma to play a role in neuroprotection. The neuroprotective properties of eCB NAEs make the protein targets of these compounds attractive targets for clinical intervention for a variety of conditions. The most promising of these targets include cannabinoid receptor type 1 (CB1), cannabinoid receptor type 2 (CB2), fatty acid amide hydrolase (FAAH), N-acylethanolamine acid amidase (NAAA), and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD). Further characterization of these targets in a more contemporary model system of neurodegeneration and neuroprotection will allow us to fully describe their role and mechanism of action in neuroprotection against oxidative stress leading to better utilization in the clinical setting. Human stem cell-derived or human neural progenitor cell-derived cells, such as ReN cells, have become more utilized for the study of human neuronal development and neurodegenerative diseases. ReN cells can be easily differentiated thereby circumventing the need for using transformed cell lines and primary neurons as cell model systems. In this study, we determined whether ReN cells, a superior cell model system for studying neurodevelopment, differentiation, and neuroprotection, express proteins involved in canonical eCB NAE signaling and whether oxidative stress can induce their expression. We determined that sublethal oxidative stress upregulates the expression of all eCB proteins tested. In addition, we determined that oxidative stress increases the nuclear localization of FAAH, and to a lesser extent, NAAA and NAPE-PLD. This study is a first step toward determining how oxidative stress affects CB1, CB2, FAAH, NAAA, and NAPE-PLD expression and their potential defense against oxidative stress. As such, our data is important for further determining the role of eCB metabolizing proteins and eCB receptors against oxidative stress.

Anandamide Alters Barrier Integrity of Bovine Vascular Endothelial Cells during Endotoxin Challenge.

Vascular endothelial cells are crucial mediators of inflammation during infectious diseases, due to their ability to produce lipid-based inflammatory mediators and facilitate leukocyte migration and translocation to infected tissues. Mastitis is the costliest infectious disease in North America, with over two billion dollars in annual costs due to loss of milk production, medical treatment, and potential loss of the animal. Infections caused by coliform bacteria are particularly deleterious, causing a negative impact on cow well-being and a high mortality rate. Dysfunction and breakdown of the endothelial barrier is a key part of the pathology of coliform mastitis. The endocannabinoid system (ECS), shown to modulate inflammatory responses of vascular endothelial cells in humans and rodents, may be a novel target for inflammatory modulation in dairy cows. The endocannabinoid (EC) arachidonoylethanolamide (AEA) is a potent anti- or pro-inflammatory mediator in endothelial cells, depending on location, timing, and concentration. We hypothesized that elevated AEA during LPS challenge will impair endothelial barrier integrity via increased production of reactive oxygen species (ROS) and activation of apoptotic pathways. Challenge of bovine aortic endothelial cells (BAEC) with 25 ng/mL lipopolysaccharide (LPS) for 8 h induced AEA synthesis, increased expression of cannabinoid receptor 1 and 2 (CB1/2) and the AEA synthesizing enzyme N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), while decreasing gene expression of the AEA degradation enzyme fatty acid amide hydrolase (FAAH). Trans endothelial resistance (TER), measured through electrical resistance across the monolayer, increased 2 h after 0.5 µM AEA treatment and decreased with 5 µM AEA, compared to LPS alone. Addition of AEA to BAEC challenged with LPS induced mitochondrial dysfunction via increased ROS production, cytochrome-C release, and activation of caspase 3/7. Antagonism of CB1 by 1 µM AM251 ameliorated AEA induced ROS production and cytochrome-C release. Addition of AM251 also eliminated 2 h TER increase and improved TER following 5 µM AEA. Doses of 0.5, 1, and 5 µM AEA delayed endothelial barrier recovery, which was eliminated by the addition of AM251. Mitochondrial dysfunction and activation of apoptotic pathways in response to AEA treatment during LPS challenge of BAEC may act to delay inflammatory resolution and contribute to endothelial dysfunction.

The analgesic compound palmitoylethanolamide reduces inflammation in human cardiomyocytes and vascular endothelial cells exposed to doxorubicin and anti-HER2 monoclonal antibody through PPAR-α and NLRP3-related pathways.

e24054 Background: Palmitoylethanolamide (PEA) is an endogenous fatty acid mediator that is synthetized from membrane phospholipids by N-acyl phosphatidylethanolamine phospholipase D. It is a new analgesic drug with anti-inflammatory effects through the induction of PPAR-related pathways. We aimed to assess whether palmitoylethanolamide co-incubated during doxorubicin and trastuzumab, reduces anticancer drugs-related cardiotoxicity in cellular models. Methods: Human vascular endothelial cells and cardiomyocytes were exposed to subclinical concentration of doxorubicin (at 100 and 200 nM) combined to trastuzumab (at 100 and 200 nM) alone or in combination with a formulation composed by palmitoylethanolamide 500 nM for 48h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of peroxisome proliferator-activated receptor-α; mTORC1 Fox01/3a; transcriptional activation of p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6). Results: Palmitoylethanolamide co-incubated with doxorubicin exerts vasculoprotective and cardioprotective effects, enhancing cell viability of 56.3-78.7 % compared to untreated cells (p < 0,001 for all). Notably, PEA reduced significantly the cardiotoxicity through peroxisome proliferator-activated receptor-α –related pathways and NLRP3 inflammasome but without the involvement of calcium homeostasis. Several cytokines and chemokines were also reduced confirming its anti-inflammatory effect. Conclusions: The present study demonstrates that palmitoylethanolamide protects against vasculotoxicity and cardiotoxicity of doxorubicin and trastuzumab by promoting an anti-inflammatory phenotype, representing a new therapeutic approach to resolve doxorubicin-induced vasculo-cardio toxicity and inflammation.

Activation of the endocannabinoid system mediates cardiac hypertrophy induced by rosiglitazone.

Rosiglitazone (RSG) is a synthetic agonist of peroxisome proliferator-activated receptor-γ (PPARγ), which plays a central role in the regulation of metabolism. Meta-analyses have suggested that RSG is associated with increased cardiovascular risk. However, the mechanisms underlying such adverse cardiac effects are still poorly understood. Here, we found that activation of PPARγ by RSG stimulated the endocannabinoid system (ECS), a membrane lipid signaling system, which induced cardiac hypertrophy. In neonatal rat cardiomyocytes, RSG increased the level of anandamide (AEA); upregulated the expression of N-acyl phosphatidylethanolamine phospholipase D (NapePLD), a key enzyme for AEA synthesis; and downregulated the expression of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of AEA. Importantly, PPARγ activation increased the expression of cannabinoid receptor type 1 (CB1) through an identified binding site for PPARγ in the CB1 promoter region. Moreover, both the in vitro and in vivo results showed that inhibition of the ECS by rimonabant, an antagonist of CB1, attenuated RSG-induced cardiac hypertrophy, as indicated by decreased expression of cardiac hypertrophy markers (ANP and BNP), deactivation of the mTOR pathway, and decreased cardiomyocyte size. Thus, these results demonstrated that the ECS functions as a novel target of PPARγ and that the AEA/CB1/mTOR axis mediates RSG-induced cardiac remodeling.

51 Palmitoylethanolamide associated to polydatin reduces inflammation in human endothelial vascular cells exposed to doxorubicin and trastuzumab through PPAR-a and NLRP3-related pathways

Abstract Aims Palmitoylethanolamide is an endogenous fatty acid mediator that is synthetized from membrane phospholipids by N-acyl phosphatidylethanolamine phospholipase D. Polydatin is a nutraceutical agent derived from trans-resveratrol with established anti-inflammatory and anti-atherogenic properties. We aimed to assess whether palmitoylethanolamide combined to polydatin, co-incubated during doxorubicin and trastuzumab, reduces anticancer drugs-related cardiotoxicity in cellular models. Methods Human vascular endothelial cells were exposed to subclinical concentration of doxorubicin (at 100 and 200 nM) combined to trastuzumab (at 100 and 200 nM) alone or in combination with a formulation composed by palmitoylethanolamide and polydatin (500 nM and 50 µM, respectively) for 48 h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of peroxisome proliferator-activated receptor-α; mTORC1 Fox01/3a; transcriptional activation of p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6). Results Palmitoylethanolamide combined to polydatin co-incubated with doxorubicin exerts vasculoprotective effects, enhancing cell viability of 54.7–68.3% compared to untreated cells (P < 0.001 for all). The formulation reduced significantly the cardiotoxicity through peroxisome proliferator-activated receptor-α–related pathways and NLRP3 inflammasome but without the involvement of calcium homeostasis. Conclusion The present study demonstrates that palmitoylethanolamide and polydatin protects against vasculotoxicity of doxorubicin and trastuzumab by promoting an anti-inflammatory phenotype, representing a new therapeutic approach to resolve doxorubicin-induced vasculotoxicity and inflammation.

Structure-Activity Relationship Studies of Pyrimidine-4-Carboxamides as Inhibitors of N-Acylphosphatidylethanolamine Phospholipase D.

N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is regarded as the main enzyme responsible for the biosynthesis of N-acylethanolamines (NAEs), a family of bioactive lipid mediators. Previously, we reported N-(cyclopropylmethyl)-6-((S)-3-hydroxypyrrolidin-1-yl)-2-((S)-3-phenylpiperidin-1-yl)pyrimidine-4-carboxamide (1, LEI-401) as the first potent and selective NAPE-PLD inhibitor that decreased NAEs in the brains of freely moving mice and modulated emotional behavior [MockNat Chem. Biol., 2020, 16, 667−67532393901]. Here, we describe the structure–activity relationship (SAR) of a library of pyrimidine-4-carboxamides as inhibitors of NAPE-PLD that led to the identification of LEI-401. A high-throughput screening hit was modified at three different substituents to optimize its potency and lipophilicity. Conformational restriction of an N-methylphenethylamine group by replacement with an (S)-3-phenylpiperidine increased the inhibitory potency 3-fold. Exchange of a morpholine substituent for an (S)-3-hydroxypyrrolidine reduced the lipophilicity and further increased activity by 10-fold, affording LEI-401 as a nanomolar potent inhibitor with drug-like properties. LEI-401 is a suitable pharmacological tool compound to investigate NAPE-PLD function in vitro and in vivo.

Cannabinoid receptor subtype influence on neuritogenesis in human SH-SY5Y cells

Human SH-SY5Y neuroblastoma cells stably expressing exogenous CB1 (CB1XS) or CB2 (CB2XS) receptors were developed to investigate endocannabinoid signaling in the extension of neuronal projections. Expression of cannabinoid receptors did not alter proliferation rate, viability, or apoptosis relative to parental SH-SY5Y. Transcripts for endogenous cannabinoid system enzymes (diacylglycerol lipase, monoacylglycerol lipase, α/β-hydrolase domain containing proteins 6 and 12, N-acyl phosphatidylethanolamine-phospholipase D, and fatty acid amide hydrolase) were not altered by CB1 or CB2 expression. Endocannabinoid ligands 2-arachidonoylglycerol (2-AG) and anandamide were quantitated in SH-SY5Y cells, and diacylglycerol lipase inhibitor tetrahydrolipstatin decreased 2-AG abundance by 90% but did not alter anandamide abundance. M3 muscarinic agonist oxotremorine M, and inhibitors of monoacylglycerol lipase and α/β hydrolase domain containing proteins 6 &12 increased 2-AG abundance. CB1 receptor expression increased lengths of short (<30 μm) and long (>30 μm) projections, and this effect was significantly reduced by tetrahydrolipstatin, indicative of stimulation by endogenously produced 2-AG. Pertussis toxin, Gβγ inhibitor gallein, and β-arrestin inhibitor barbadin did not significantly alter long projection length in CB1XS, but significantly reduced short projections, with gallein having the greatest inhibition. The rho kinase inhibitor Y27632 increased CB1 receptor-mediated long projection extension, indicative of actin cytoskeleton involvement. CB1 receptor expression increased GAP43 and ST8SIA2 mRNA and decreased ITGA1 mRNA, whereas CB2 receptor expression increased NCAM and SYT mRNA. We propose that basal endogenous production of 2-AG provides autocrine stimulation of CB1 receptor signaling through Gi/o, Gβγ, and β-arrestin mechanisms to promote neuritogenesis, and rho kinase influences process extension.

Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice.

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.

Brain region-dependent changes in the expression of endocannabinoid-metabolizing enzymes in rats following antidepressant drugs.

The endocannabinoid (eCB) system plays a role in the pathophysiology of depression. The aim of this study was to investigate the expression of the eCB synthesizing enzymes (N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) and diacylglycerol lipase α (DAGLα)) and eCB degrading enzymes (fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)) after acute or chronic administration of antidepressant drugs (imipramine (IMI, 15 mg/kg), escitalopram (ESC, 10 mg/kg) and tianeptine (TIA, 10 mg/kg)). eCB metabolizing enzymes were altered in drug-dependent and brain region-specific fashion. After IMI treatment a reduction was noted in the expression of FAAH protein in the dorsal striatum, MAGL in the frontal cortex and DAGLα in the cerebellum. On the other hand, ESC treatment provoked an increase in the MAGL expression in the prefrontal cortex or NAPE-PLD and in DAGLα in the hippocampus and dorsal striatum, while reducing the FAAH expression in the dorsal striatum. TIA administration increased the levels of all enzymes in the prefrontal cortex as well as elevated DAGLα expression in the frontal cortex and dorsal striatum. In conclusion, our results indicate that changes in the eCBs levels after antidepressant drug treatment were related to the expression of their metabolizing enzymes.

A protective role for N-acylphosphatidylethanolamine phospholipase D in 6-OHDA-induced neurodegeneration

N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) catalyzes the cleavage of membrane NAPEs into bioactive fatty-acid ethanolamides (FAEs). Along with this precursor role, NAPEs might also serve autonomous signaling functions. Here, we report that injections of 6-hydroxydopamine (6-OHDA) into the mouse striatum cause a local increase in NAPE and FAE levels, which precedes neuronal cell death. NAPE, but not FAE, accumulation is enhanced in mice lacking NAPE-PLD, which display a substantial reduction in 6-OHDA-induced neurotoxicity, as shown by increased survival of substantia nigra dopamine neurons, integrity of striatal dopaminergic fibers, and striatal dopamine metabolite content. Reduced damage is accompanied by attenuation of the motor response evoked by apomorphine. Furthermore, NAPE-PLD silencing protects cathecolamine-producing SH-SY5Y cells from 6-OHDA-induced reactive oxygen species formation, caspase-3 activation and death. Mechanistic studies in mice suggest the existence of multiple molecular contributors to the neuroprotective effects of NAPE-PLD deletion, including suppression of Rac1 activity and attenuated transcription of several genes (Cadps, Casp9, Egln1, Kcnj6, Spen, and Uchl1) implicated in dopamine neuron survival and/or Parkinson’s disease. The findings point to a previously unrecognized role for NAPE-PLD in the regulation of dopamine neuron function, which may be linked to the control of NAPE homeostasis in membranes.

Bile acids in glucose metabolism and insulin signalling - mechanisms and research needs.

Of all the novel glucoregulatory molecules discovered in the past 20 years, bile acids (BAs) are notable for the fact that they were hiding in plain sight. BAs were well known for their requirement in dietary lipid absorption and biliary cholesterol secretion, due to their micelle-forming properties. However, it was not until 1999 that BAs were discovered to be endogenous ligands for the nuclear receptor FXR. Since that time, BAs have been shown to act through multiple receptors (PXR, VDR, TGR5 and S1PR2), as well as to have receptor-independent mechanisms (membrane dynamics, allosteric modulation of N-acyl phosphatidylethanolamine phospholipase D). We now also have an appreciation of the range of physiological, pathophysiological and therapeutic conditions in which endogenous BAs are altered, raising the possibility that BAs contribute to the effects of these conditions on glycaemia. In this Review, we highlight the mechanisms by which BAs regulate glucose homeostasis and the settings in which endogenous BAs are altered, and provide suggestions for future research.

Cessation of fluoxetine treatment increases alcohol seeking during relapse and dysregulates endocannabinoid and glutamatergic signaling in the central amygdala.

Administration of selective serotonin reuptake inhibitors (SSRIs), typically used as antidepressants, induces long-lasting behavioral changes associated with alcohol use disorder (AUD). However, the contribution of SSRI (fluoxetine)-induced alterations in neurobiological processes underlying alcohol relapse such as endocannabinoid and glutamate signaling in the central amygdala (CeA) remains largely unknown. We utilized an integrative approach to study the effects of repeated fluoxetine administration during abstinence on ethanol drinking. Gene expression and biochemical and electrophysiological studies explored the hypothesis that dysregulation in glutamatergic and endocannabinoid mechanisms in the CeA underlie the susceptibility to alcohol relapse. Cessation of daily treatment with fluoxetine (10 mg/kg) during abstinence resulted in a marked increase in ethanol seeking during re-exposure periods. The increase in ethanol self-administration was associated with (a) reductions in levels of the endocannabinoids N-arachidonoylethanolomine and 2-arachidonoylglycerol in the CeA, (b) increased amygdalar gene expression of cannabinoid type-1 receptor (CB1), N-acyl phosphatidylethanolamine phospholipase D (Nape-pld), fatty acid amid hydrolase (Faah), (c) decreased amygdalar gene expression of ionotropic AMPA (GluA2 and GluA4) and metabotropic (mGlu3) glutamate receptors, and (d) increased glutamatergic receptor function. Overall, our data suggest that the administration of the antidepressant fluoxetine during abstinence dysregulates endocannabinoid signaling and glutamatergic receptor function in the amygdala, facts that likely facilitate alcohol drinking behavior during relapse.

Intestinal epithelial N-acylphosphatidylethanolamine phospholipase D links dietary fat to metabolic adaptations in obesity and steatosis

Variations in N-acylethanolamines (NAE) levels are associated with obesity and metabolic comorbidities. Their role in the gut remains unclear. Therefore, we generated a mouse model of inducible intestinal epithelial cell (IEC)-specific deletion of N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD), a key enzyme involved in NAE biosynthesis (Napepld∆IEC). We discovered that Napepld∆IEC mice are hyperphagic upon first high-fat diet (HFD) exposure, and develop exacerbated obesity and steatosis. These mice display hypothalamic Pomc neurons dysfunctions and alterations in intestinal and plasma NAE and 2-acylglycerols. After long-term HFD, Napepld∆IEC mice present reduced energy expenditure. The increased steatosis is associated with higher gut and liver lipid absorption. Napepld∆IEC mice display altered gut microbiota. Akkermansia muciniphila administration partly counteracts the IEC NAPE-PLD deletion effects. In conclusion, intestinal NAPE-PLD is a key sensor in nutritional adaptation to fat intake, gut-to-brain axis and energy homeostasis and thereby constitutes a novel target to tackle obesity and related disorders.

Palmitoylethanolamide Promotes a Proresolving Macrophage Phenotype and Attenuates Atherosclerotic Plaque Formation.

Objective- Palmitoylethanolamide is an endogenous fatty acid mediator that is synthetized from membrane phospholipids by N-acyl phosphatidylethanolamine phospholipase D. Its biological actions are primarily mediated by PPAR-α (peroxisome proliferator-activated receptors α) and the orphan receptor GPR55. Palmitoylethanolamide exerts potent anti-inflammatory actions but its physiological role and promise as a therapeutic agent in chronic arterial inflammation, such as atherosclerosis remain unexplored. Approach and Results- First, the polarization of mouse primary macrophages towards a proinflammatory phenotype was found to reduce N-acyl phosphatidylethanolamine phospholipase D expression and palmitoylethanolamide bioavailability. N-acyl phosphatidylethanolamine phospholipase D expression was progressively downregulated in the aorta of apolipoprotein E deficient (ApoE-/-) mice during atherogenesis. N-acyl phosphatidylethanolamine phospholipase D mRNA levels were also downregulated in unstable human plaques and they positively associated with smooth muscle cell markers and negatively with macrophage markers. Second, ApoE-/- mice were fed a high-fat diet for 4 or 16 weeks and treated with either vehicle or palmitoylethanolamide (3 mg/kg per day, 4 weeks) to study the effects of palmitoylethanolamide on early established and pre-established atherosclerosis. Palmitoylethanolamide treatment reduced plaque size in early atherosclerosis, whereas in pre-established atherosclerosis, palmitoylethanolamide promoted signs of plaque stability as evidenced by reduced macrophage accumulation and necrotic core size, increased collagen deposition and downregulation of M1-type macrophage markers. Mechanistically, we found that palmitoylethanolamide, by activating GPR55, increases the expression of the phagocytosis receptor MerTK (proto-oncogene tyrosine-protein kinase MER) and enhances macrophage efferocytosis, indicative of proresolving properties. Conclusions- The present study demonstrates that palmitoylethanolamide protects against atherosclerosis by promoting an anti-inflammatory and proresolving phenotype of lesional macrophages, representing a new therapeutic approach to resolve arterial inflammation.