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Abstract Background A hypoxic environment often persists within solid tumors, including hepatocellular carcinoma (HCC). Hypoxia-inducible factor-1α (HIF-1α) can accelerate cancer malignancy by inducing hypoxia-dependent expression of various genes. Tumor hypoxia can also induce metabolic reprogramming of fatty acid (FA) metabolism, through which HIF-1α plays an essential role in diminishing fatty acid β-oxidation (FAO) in hypoxic cancer cells. Methods We aimed to investigate potential new drug therapy options for targeting hypoxic cancer cells within HCC tumors, specifically through combining HIF-1α inhibition with palmitic acid (PA) + L-carnitine (LC) treatment to effectively induce apoptosis in hypoxic HCC cells. To test this hypothesis, in vitro and in vivo studies were performed. Results We first demonstrated that hypoxia-dependent apoptosis was induced by an overload of PA in two HCC cell lines (HepG2 and Hep3B) via excessive production of reactive oxygen species (ROS). Moreover, this observed PA-induced apoptosis was enhanced by HIF-1α knockdown (KD) in these cells under hypoxia. In addition, the combination of PA with FAO activator LC increased FAO activity and led to stronger cell death than PA alone in hypoxic HIF-1α KD cells, specifically through further ROS generation. To clarify the mechanism of hypoxia-induced FA metabolism reprogramming, expression levels of the genes encoding FAO enzymes CPT1A, ACSL1, MCAD, and LCAD, FA transporter CD36, and FA esterification enzymes DGAT and APGAT were analyzed using HIF-1α KD and scramble control (SC) cells. The results suggested that HIF-1α could repress mRNA expression of the FAO-related enzymes and CD36, while it upregulated FA esterification gene expression. This suggested a central role for HIF-1α in hypoxia-induced reprogramming of FA metabolism in HCC cells. Using a nude mouse model, PA administration was found to induce apoptosis from ROS overproduction in HIF-1α KD tumors compared with SC tumors. Additional LC treatment synergistically enhanced the PA-induced apoptosis in HIF-1α KD tumors. Finally, in vivo therapy composed of HIF-1α inhibitor YC-1 with PA + LC could induce ROS-mediated apoptosis in HepG2 tumors without significant toxicity. Conclusions A combination therapy of YC-1 with PA + LC may be a unique anti-tumor therapy for targeting hypoxic HCC cells, specifically by ROS overproduction leading to forced FAO activation.

Background: A hypoxic environment often persists within solid tumors, including hepatocellular carcinoma (HCC). Hypoxia-inducible factor-1α (HIF-1α) can accelerate cancer malignancy by inducing hypoxia-dependent expression of various genes. Tumor hypoxia can also induce metabolic reprogramming of fatty acid (FA) metabolism, through which HIF-1α plays an essential role in diminishing fatty acid β-oxidation (FAO) in hypoxic cancer cells. Methods: We aimed to investigate potential new drug therapy options for targeting hypoxic cancer cells within HCC tumors, specifically through combining HIF-1α inhibition with palmitic acid (PA) + L-carnitine (LC) treatment to effectively induce apoptosis in hypoxic HCC cells. To test this hypothesis, in vitro and in vivo studies were performed. Results: We first demonstrated that hypoxia-dependent apoptosis was induced by an overload of PA in two HCC cell lines (HepG2 and Hep3B) via excessive production of reactive oxygen species (ROS). Moreover, this observed PA-induced apoptosis was enhanced by HIF-1α knockdown (KD) in these cells under hypoxia. In addition, the combination of PA with FAO activator LC increased FAO activity and led to stronger cell death than PA alone in hypoxic HIF-1α KD cells, specifically through further ROS generation. To clarify the mechanism of hypoxia-induced FA metabolism reprogramming, expression levels of the genes encoding FAO enzymes CPT1A, ACSL1, MCAD, and LCAD, FA transporter CD36, and FA esterification enzymes DGAT and APGAT were analyzed using HIF-1α KD and scramble control (SC) cells. The results suggested that HIF-1α could repress mRNA expression of the FAO-related enzymes and CD36, while it upregulated FA esterification gene expression. This suggested a central role for HIF-1α in hypoxia-induced reprogramming of FA metabolism in HCC cells. Using a nude mouse model, PA administration was found to induce apoptosis from ROS overproduction in HIF-1α KD tumors compared with SC tumors. Additional LC treatment synergistically enhanced the PA-induced apoptosis in HIF-1α KD tumors. Finally, in vivo therapy composed of HIF-1α inhibitor YC-1 with PA + LC could induce ROS-mediated apoptosis in HepG2 tumors without significant toxicity. Conclusions: A combination therapy of YC-1 with PA + LC may be a unique anti-tumor therapy for targeting hypoxic HCC cells, specifically by ROS overproduction leading to forced FAO activation. [ABSTRACT FROM AUTHOR]

RationaleN-acylethanolamine acid amidase (NAAA) is an intracellular cysteine hydrolase that terminates the biological actions of oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), two endogenous lipid-derived agonists of the nuclear receptor, and peroxisome proliferator-activated receptor-α. OEA and PEA are important regulators of energy balance, pain, and inflammation, but recent evidence suggests that they might also contribute to the control of reward-related behaviors.Objectives and methodsIn the present study, we investigated the effects of systemic and intracerebral NAAA inhibition in the two-bottle choice model of voluntary alcohol drinking and on operant alcohol self-administration.ResultsIntraperitoneal injections of the systemically active NAAA inhibitor ARN19702 (3 and 10 mg/kg) lowered voluntary alcohol intake in a dose-dependent manner, achieving ≈ 47% reduction at the 10 mg/kg dose (p

BackgroundPatients with ESRD on maintenance hemodialysis (MHD) are particularly susceptible to dysregulation of energy metabolism, which may manifest as protein energy wasting and cachexia. In recent years, the endocannabinoid system has been shown to play an important role in energy metabolism with potential relevance in ESRD. N-acylethanolamines are a class of fatty acid amides which include the major endocannabinoid ligand, anandamide, and the endogenous peroxisome proliferator-activated receptor-α agonists, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA).MethodsSerum concentrations of OEA and PEA were measured in MHD patients and their correlations with various clinical/laboratory indices were examined. Secondarily, we evaluated the association of circulating PEA and OEA levels with 12-month all-cause mortality.ResultsBoth serum OEA and PEA levels positively correlated with high-density lipoprotein-cholesterol levels and negatively correlated with body fat and body anthropometric measures. Serum OEA levels correlated positively with serum interleukin-6 (IL-6) (rho = 0.19; p = 0.004). Serum PEA and IL-6 showed a similar but nonsignificant trend (rho = 0.12; p = 0.07). Restricted cubic spline analyses showed that increasing serum OEA and PEA both trended toward higher mortality risk, and these associations were statistically significant for PEA (PEA ≥4.7 pmol/mL; reference: PEA

Angiopoietin-like 4 (ANGPTL4) is a potent regulator of TAG metabolism, but knowledge of the mechanisms underlying ANGPTL4 transcription in response to fatty acids is still limited in teleost. In the current study, we explored the molecular characterisation of ANGPTL4 and regulatory mechanisms of ANGPTL4 in response to fatty acids in large yellow croaker (Larimichthys crocea). Here, croaker angptl4 contained a 1416 bp open reading frame encoding a protein of 471 amino acids with highly conserved 12-amino acid consensus motif. Angptl4 was widely expressed in croaker, with the highest expression in the liver. In vitro , oleic and palmitic acids (OA and PA) treatments strongly increased angptl4 mRNA expression in croaker hepatocytes. Moreover, angptl4 expression was positively regulated by PPAR family (PPAR- α , β and γ), and expression of PPAR γ was also significantly increased in response to OA and PA. Moreover, inhibition of PPAR γ abrogated OA- or PA-induced angptl4 mRNA expression. Beyond that, PA might increase angptl4 expression partly via the insulin signalling. Overall, the expression of ANGPTL4 is strongly upregulated by OA and PA via PPAR γ in the liver of croaker, which contributes to improve the understanding of the regulatory mechanisms of ANGPTL4 in fish. [ABSTRACT FROM AUTHOR]

Palmitoylethanolamide is a bioactive lipid that strongly alleviates pain and inflammation in animal models and in humans. Its signaling activity is terminated through degradation by N-acylethanolamine acid amidase (NAAA), a cysteine hydrolase expressed at high levels in immune cells. Pharmacological inhibitors of NAAA activity exert profound analgesic and antiinflammatory effects in rodent models, pointing to this protein as a potential target for therapeutic drug discovery. To facilitate these efforts and to better understand the molecular mechanism of action of NAAA, we determined crystal structures of this enzyme in various activation states and in complex with several ligands, including both a covalent and a reversible inhibitor. Self-proteolysis exposes the otherwise buried active site of NAAA to allow catalysis. Formation of a stable substrate- or inhibitor-binding site appears to be conformationally coupled to the interaction of a pair of hydrophobic helices in the enzyme with lipid membranes, resulting in the creation of a linear hydrophobic cavity near the active site that accommodates the ligand's acyl chain.

Elevated uptake of saturated fatty acid palmitic acid (PA) is associated with tumor metastasis; however, the precise mechanisms remain partially understood, hindering the development of therapy for PA-driven tumor metastasis. The Hippo-Yes-associated protein (Hippo/YAP) pathway is implicated in cancer progression. Here it is shown that a high-palm oil diet potentiates tumor metastasis in murine xenografts in part through YAP. It is found that the palmitoyltransferase ZDHHC15 is a YAP-regulated gene that forms a feedback loop with YAP. Notably, PA drives the ZDHHC15-YAP feedback loop, thus enforces YAP signaling, and hence promotes tumor metastasis in murine xenografts. In addition, it is shown that ZDHHC15 associates with Kidney and brain protein (KIBRA, also known as WW- and C2 domain-containing protein 1, WWC1), an upstream component of Hippo signaling, and mediates its palmitoylation. KIBRA palmitoylation leads to its degradation and regulates its subcellular localization and activity toward the Hippo/YAP pathway. Moreover, PA enhances KIBRA palmitoylation and degradation. It is further shown that combinatorial targeting of YAP and fatty acid synthesis exhibits augmented effects against metastasis formation in mice fed with a Palm diet. Collectively, these findings uncover a ZDHHC15-YAP feedback loop as a previously unrecognized mechanism underlying PA-promoted tumor metastasis and support targeting YAP and fatty acid synthesis as potential therapeutic targets in PA-driven tumor metastasis., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

This study details trends in direct alcohol biomarker concentrations from civil cases within the United Kingdom (UK). Our subject cohort in this study related to family law litigation, where an individual was subject to an alcohol monitoring order by the court. This monitoring was conducted by quantification of alcohol biomarkers Phosphatidlyethanol (PEth) in dried blood spots (DBS) and Ethyl Glucuronide (EtG) and Ethyl Palmitate (EtPa) from hair segments. In total 298 PEth cases predominantly from the South East of England during the period July 2022 to August 2023 were analysed for alcohol biomarkers in DBS and hair. Subjects alcohol intake was classified as abstinence/low alcohol consumption, moderate or excessive alcohol consumption, based on a combination of Society for Hair Testing and PEth Net guidelines. Our results indicate that 33 % of PEth concentrations were consistent with excessive alcohol use (>200 ng/mL DBS), with 36 % consistent with social or moderate alcohol use (20-200 ng/mL DBS). In relation to EtG and EtPa 23 % and 31 % of subjects were classified as excessive alcohol users respectively. This study indicates that DBS sampling of PEth is a more sensitive predictor of alcohol use, in particular, at differentiating between moderate and excessive alcohol use compared to EtG and EtPa testing in hair. The authors suggest that increased frequency in the sampling of PEth in DBS (multiple occasions per month) may provide a more accurate assessment and simplification of the interpretation criteria of alcohol patterns rather than the combined hair testing and DBS sampling that are typically requested by UK courts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

• Induced pluripotent stem cells cultured into 3D spheroids efficiently differentiate into beta like cells. • Spheroid 3D islet-like cells secrete insulin in response to glucose. • Exposure of Spheroid 3D islet-like cells to palmitic acid (PA) perturbs secretory pathways, glucose and lipid metabolisms. • Hippo and FOXO signalings, and in weaker extend Wnt signaling, TGFβ, and Hedgehog signaling, which control endocrine pancreatic development are affected by PA in Spheroid 3D islet-like cells. Type 2 diabetes (T2D) is posing a serious public health concern with a considerable impact on human life and health expenditures worldwide. The disease develops when insulin plasma level is insufficient for coping insulin resistance, caused by the decline of pancreatic β-cell function and mass. In β-cells, the lipotoxicity exerted by saturated free fatty acids in particular palmitate (PA), which is chronically elevated in T2D, plays a major role in β-cell dysfunction and mass. However, there is a lack of human relevant in vitro model to identify the underlying mechanism through which palmitate induces β-cell failure. In this frame, we have previously developed a cutting-edge 3D spheroid model of β-like cells derived from human induced pluripotent stem cells. In the present work, we investigated the signaling pathways modified by palmitate in β-like cells derived spheroids. When compared to the 2D monolayer cultures, the transcriptome analysis (FDR set at 0.1) revealed that the 3D spheroids upregulated the pancreatic markers (such as GCG, IAPP genes), lipids metabolism and transporters (CD36, HMGSC2 genes), glucose transporter (SLC2A6). Then, the 3D spheroids are exposed to PA 0.5 mM for 72 h. The differential analysis demonstrated that 32 transcription factors and 135 target genes were mainly modulated (FDR set at 0.1) including the upregulation of lipid and carbohydrates metabolism (HMGSC2, LDHA, GLUT3), fibrin metabolism (FGG, FGB), apoptosis (CASP7). The pathway analysis using the 135 selected targets extracted the fibrin related biological process and wound healing in 3D PA treated conditions. An overall pathway gene set enrichment analysis, performed on the overall gene set (with pathway significance cutoff at 0.2), highlighted that PA perturbs the citrate cycle, FOXO signaling and Hippo signaling as observed in human islets studies. Additional RT-PCR confirmed induction of inflammatory (IGFBP1, IGFBP3) and cell growth (CCND1, Ki67) pathways by PA. All these changes were associated with unaffected glucose-stimulated insulin secretion (GSIS), suggesting that they precede the defect of insulin secretion and death induced by PA. Overall, we believe that our data demonstrate the potential of our spheroid 3D islet-like cells to investigate the pancreatic-like response to diabetogenic environment. [ABSTRACT FROM AUTHOR]

Transporter-mediated drug-drug interactions (DDIs) are a major cause of drug toxicities. Using published genome-wide association studies (GWAS) of the human metabolome, we identified 20 metabolites associated with genetic variants in organic anion transporter, OATP1B1 (P < 5 × 10-8 ). Of these, 12 metabolites were significantly higher in plasma samples from volunteers dosed with the OATP1B1 inhibitor, cyclosporine (CSA) vs. placebo (q-value < 0.2). Conjugated bile acids and fatty acid dicarboxylates were among the metabolites discovered using both GWAS and CSA administration. In vitro studies confirmed tetradecanedioate (TDA) and hexadecanedioate (HDA) were novel substrates of OATP1B1 as well as OAT1 and OAT3. This study highlights the use of multiple datasets for the discovery of endogenous metabolites that represent potential in vivo biomarkers for transporter-mediated DDIs. Future studies are needed to determine whether these metabolites can serve as qualified biomarkers for organic anion transporters. Quantitative relationships between metabolite levels and modulation of transporters should be established.

Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole-piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders.

This narrative review provides an overview of current knowledge on the impact of nutritional strategies on chronic craniofacial pain associated with temporomandibular disorders (TMDs). Individuals experiencing painful TMDs alter their dietary habits, avoiding certain foods, possibly due to chewing difficulties, which might lead to nutrient deficiencies. Our literature investigation revealed that the causal links between nutritional changes and craniofacial pain remain unclear. However, clinical and preclinical studies suggest that nutraceuticals, including vitamins, minerals, polyphenols, omega-3 fatty acids, isoprenoids, carotenoids, lectins, polysaccharides, glucosamines, and palmitoylethanolamides, could have beneficial effects on managing TMDs. This is described in 12 clinical and 38 preclinical articles since 2000. Clinical articles discussed the roles of vitamins, minerals, glucosamine, and palmitoylethanolamides. The other nutraceuticals were assessed solely in preclinical studies, using TMD models, mostly craniofacial inflammatory rodents, with 36 of the 38 articles published since 2013. Our investigation indicates that current evidence is insufficient to assess the efficacy of these nutraceuticals. However, the existing data suggest potential for therapeutic intervention in TMDs. Further support from longitudinal and randomized controlled studies and well-designed preclinical investigations is necessary to evaluate the efficacy of each nutraceutical intervention and understand their underlying mechanisms in TMDs.

Background: The mental health benefits of cannabidiol (CBD) are promising but can be inconsistent, in part due to challenges in defining an individual's effective dosage. In schizophrenia, alterations in anandamide (AEA) concentrations, an endocannabinoid (eCB) agonist of the eCB system, reflect positively on treatment with CBD. Here, we expanded this assessment to include eCBs alongside AEA congeners, comparing phytocannabinoids and dosage in a clinical setting., Methods: Liquid chromatography-tandem mass spectrometry quantified changes in serum levels of AEA, 2-arachidonoylglycerol (2-AG), alongside AEA-related compounds oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), which were attained from two independent, parallel-designed, clinical trials investigating single, oral CBD (600 or 800 mg), delta-9-tetrahydrocannabinol (Δ 9 -THC, 10 or 20 mg) and combination administration (CBD|800 mg+Δ 9 -THC|20 mg) in healthy volunteers (HVs, n=75). Concentrations were measured at baseline (t=0), 65 and 160 min post administration., Results: CBD-led increases in AEA (1.6-fold), OEA and PEA (1.4-fold) were observed following a single 800 mg (p corr <0.05) but not 600 mg dosage. Declining AEA was observed with Δ 9 -THC at 10 mg (-1.3-fold) and 20 mg (-1.4-fold) but restored to baseline levels by 160 min. CBD+Δ 9 -THC yielded the highest increases in AEA (2.1-fold), OEA (1.9-fold) and PEA (1.8-fold) without reaching a maximal response., Conclusion: CBD-administered effects towards AEA, OEA and PEA are consistent with phase II trials reporting clinical improvement for acute schizophrenia (CBD≥800 mg). Including Δ 9 -THC appears to enhance the CBD-induced response towards AEA and its congeners. Our results warrant further investigations into the potential of these lipid-derived mediators as metabolic measures for CBD dose prescription and co-cannabinoid administration., Competing Interests: Competing interests: FML and DK are shareholders of curantis UG (Ltd.). CR is a shareholder of lero bioscience UG (Ltd.). CR, CB, ID, and AEH are employees of Endosane Pharmaceuticals GmbH. FML received an Investigator Initiated Trial Grant from Endosane Pharmaceuticals GmbH. All other authors have nothing to disclose., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Endocannabinoids and their attending cannabinoid type 1 (CB1) receptor have been implicated in animal models of post-traumatic stress disorder (PTSD). However, their specific role has not been studied in people with PTSD. Herein, we present an in vivo imaging study using positron emission tomography (PET) and the CB1-selective radioligand [(11)C]OMAR in individuals with PTSD, and healthy controls with lifetime histories of trauma (trauma-exposed controls (TC)) and those without such histories (healthy controls (HC)). Untreated individuals with PTSD (N=25) with non-combat trauma histories, and TC (N=12) and HC (N=23) participated in a magnetic resonance imaging scan and a resting PET scan with the CB1 receptor antagonist radiotracer [(11)C]OMAR, which measures the volume of distribution (VT) linearly related to CB1 receptor availability. Peripheral levels of anandamide, 2-arachidonoylglycerol, oleoylethanolamide, palmitoylethanolamide and cortisol were also assessed. In the PTSD group, relative to the HC and TC groups, we found elevated brain-wide [(11)C]OMAR VT values (F(2,53)=7.96, P=0.001; 19.5% and 14.5% higher, respectively), which were most pronounced in women (F(1,53)=5.52, P=0.023). Anandamide concentrations were reduced in the PTSD relative to the TC (53.1% lower) and HC (58.2% lower) groups. Cortisol levels were lower in the PTSD and TC groups relative to the HC group. Three biomarkers examined collectively--OMAR VT, anandamide and cortisol--correctly classified nearly 85% of PTSD cases. These results suggest that abnormal CB1 receptor-mediated anandamide signaling is implicated in the etiology of PTSD, and provide a promising neurobiological model to develop novel, evidence-based pharmacotherapies for this disorder.

Fatty acid ethanolamides (FAEs), which include palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), are endogenous agonists of peroxisome proliferator-activated receptor-α (PPAR-α) and important regulators of the inflammatory response. They are degraded in macrophages by the lysosomal cysteine amidase, N-acylethanolamine acid amidase (NAAA). Previous studies have shown that pharmacological inhibition of NAAA activity suppresses macrophage activation in vitro and causes marked anti-inflammatory effects in vivo, which is suggestive of a role for NAAA in the control of inflammation. It is still unknown, however, whether NAAA-mediated FAE deactivation might regulate pain signaling. The present study examined the effects of ARN077, a potent and selective NAAA inhibitor recently disclosed by our group, in rodent models of hyperalgesia and allodynia caused by inflammation or nerve damage. Topical administration of ARN077 attenuated, in a dose-dependent manner, heat hyperalgesia and mechanical allodynia elicited in mice by carrageenan injection or sciatic nerve ligation. The antinociceptive effects of ARN077 were prevented by the selective PPAR-α antagonist GW6471 and did not occur in PPAR-α-deficient mice. Furthermore, topical ARN077 reversed the allodynia caused by ultraviolet B radiation in rats, and this effect was blocked by pretreatment with GW6471. Sciatic nerve ligation or application of the proinflammatory phorbol ester 12-O-tetradecanoylphorbol 13-acetate decreased FAE levels in sciatic nerve and skin tissue, respectively. ARN077 reversed these biochemical effects. The results identify ARN077 as a potent inhibitor of intracellular NAAA activity, which is active in vivo by topical administration. The findings further suggest that NAAA regulates peripheral pain initiation by interrupting endogenous FAE signaling at PPAR-α.

Objective: This study aimed to evaluate the efficacy and safety of co-micronized palmitoylethanolamide (PEA)/polydatin (PD) in the treatment of abdominal pain symptoms in pediatric patients with irritable bowel syndrome (IBS)., Methods: This was a multicenter trial conducted at three Italian pediatric gastroenterology centers, employing a double-blind, placebo-controlled, parallel-arm design. Participants were ages 10 to 17 y and met Rome IV criteria for pediatric IBS. They were randomly allocated to receive either co-micronized PEA/PD or placebo, administered three times daily in a 1:1 ratio, over a 12-wk period. The study assessed baseline severity using the IBS-Severity Scoring System (IBS-SSS) at enrollment and after 4, 8, and 12 wk of treatment. Abdominal pain frequency was assessed on a scale from 1 to 7 d/wk, while stool consistency was classified using the Bristol Stool Scale (BSS) to categorize various IBS subtypes. The primary outcome was the percentage of patients who achieved complete remission, defined as IBS-SSS score <75 points after 12 wk of therapy., Results: The study involved 70 children with IBS. Of the participants, 34 received co-micronized PEA/PD, and 36 received a placebo. As compared with the placebo group, the co-micronized therapy group had significantly more patients achieving complete remission after 12 wk (P = 0.015), with particular benefit in the IBS-diarrhea subtype (P = 0.01). The treatment group also experienced a significant reduction in abdominal pain intensity and frequency compared with the placebo group. No adverse events were recorded during the study period., Conclusions: Co-micronized PEA/PD is a safe and effective treatment to treat abdominal pain symptoms in pediatric IBS., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Giovanni Di Nardo reports equipment, drugs, or supplies was provided by Epitech group, Milan, Italy. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: N-acylethanolamines (NAEs) include endocannabinoid (EC) and EC-related molecules that impact the anti-obesity and anti-diabetic efficacy of glucagon-like peptide-1 receptor agonists (GLP-1RA) in animal studies. However, the clinical relevance of these findings remains to be determined. Here, we tested whether GLP-1RA treatment affects circulating NAE levels and whether NAEs may predict the efficacy of GLP-1RA treatment in humans with obesity undergoing weight loss maintenance., Materials and Methods: We profiled plasma levels of NAEs in participants with obesity undergoing weight loss maintenance with (n = 23)/or without (n = 20) treatment with the GLP-1RA liraglutide. NAE levels were measured at three different time points: before the start of the study, at the end of the diet-induced weight loss, and after 52-weeks treatment. Linear regression analyses were used to investigate whether pharmacological responses could be predicted by NAEs levels., Results: Liraglutide treatment reduced plasma concentrations of the NAE and oleoyl-ethanolamide (OEA), without altering arachidonoyl-ethanolamide (AEA) levels and palmitoyl-ethanolamide (PEA) levels. High pre-treatment levels of OEA were predictive of superior compound-mediated effects on fasting insulin and triglyceride levels. High pre-treatment PEA and AEA levels were also predictive of superior Liraglutide-mediated effects on triglyceride levels., Conclusions: Our data suggests that specific NAEs such as OEA and AEA are promising biomarkers of GLP-1RA metabolic efficacy in humans with obesity during weight loss maintenance. Plasma profiling of EC-related molecules may be a promising strategy to tailor GLP-1R-based therapies to individual needs in obesity and diabetes management., (© 2023. The Author(s), under exclusive licence to Italian Society of Endocrinology (SIE).)

The amplitude of the depolarization-evoked Ca2+ transient is larger in dorsal root ganglion (DRG) neurons from tumor-bearing mice compared with that of neurons from naive mice, and the change is mimicked by coculturing DRG neurons with the fibrosarcoma cells used to generate the tumors (Khasabova et al., 2007). The effect of palmitoylethanolamide (PEA), a ligand for the peroxisome proliferator-activated receptor α (PPARα), was determined on the evoked-Ca2+ transient in the coculture condition. The level of PEA was reduced in DRG cells from tumor-bearing mice as well as those cocultured with fibrosarcoma cells. Pretreatment with PEA, a synthetic PPARα agonist (GW7647), or ARN077, an inhibitor of the enzyme that hydrolyzes PEA, acutely decreased the amplitude of the evoked Ca2+ transient in small DRG neurons cocultured with fibrosarcoma cells. The PPARα antagonist GW6471 blocked the effect of each. In contrast, the PPARα agonist was without effect in the control condition, but the antagonist increased the amplitude of the Ca2+ transient, suggesting that PPARα receptors are saturated by endogenous ligand under basal conditions. Effects of drugs on mechanical sensitivity in vivo paralleled their effects on DRG neurons in vitro. Local injection of ARN077 decreased mechanical hyperalgesia in tumor-bearing mice, and the effect was blocked by GW6471. These data support the conclusion that the activity of DRG neurons is rapidly modulated by PEA through a PPARα-dependent mechanism. Moreover, agents that increase the activity of PPARα may provide a therapeutic strategy to reduce tumor-evoked pain.

ContextPeripheral and central endocannabinoids and cognate acylethanolamides (AEs) may play important but distinct roles in regulating energy balance.ObjectiveWe hypothesized that in humans central/peripheral endocannabinoids are differently associated with adiposity and energy expenditure and differ by race.DesignWe examined associations of arachindonoylethanolamide, 2-arachidonoylglycerol, palmitoylethanolamide, and oleoylethanolamide (OEA) assayed in plasma and cerebrospinal fluid (CSF) with race, adiposity, and energy expenditure.Setting/participantsIn this monitored clinical inpatient study, CSF was obtained by lumbar puncture in 27 individuals (12 Caucasian, 11 American Indian, and four African-American). Twenty-four hour and sleep energy expenditure were measured by indirect calorimetry in a respiratory chamber.Main outcome measureSamples were analyzed from a previous study originally designed to test a blood-brain barrier leptin transport deficit in human obesity.ResultsCSF (but not peripheral) 2-arachidonoylglycerol was significantly increased in American Indians compared with Caucasians (18.48 ± 6.17 vs. 10.62 ± 4.58 pmol/ml, P < 0.01). In the whole group, peripheral AEs were positively but in CSF negatively associated with adiposity. However, in multivariate models adjusted for the other peripheral and CSF AEs, peripheral arachindonoylethanolamide was the only AE significantly associated with adiposity. Interestingly, CSF OEA concentrations were positively associated with adjusted 24 hour and sleep energy expenditure (r = 0.47, P < 0.05; r = 0.42, P < 0.05), but peripheral OEA was not.ConclusionsThese data indicate a central alteration of the endocannabinoid system in American Indians and furthermore show that AEs in both compartments play an important but distinct role in human energy balance regulation.

Identifying points of control in inflammation is essential to discovering safe and effective antiinflammatory medicines. Palmitoylethanolamide (PEA) is a naturally occurring lipid amide that, when administered as a drug, inhibits inflammatory responses by engaging peroxisome proliferator-activated receptor-alpha (PPAR-alpha). PEA is preferentially hydrolyzed by the cysteine amidase N-acylethanolamine-hydrolyzing acid amidase (NAAA), which is highly expressed in macrophages. Here we report the discovery of a potent and selective NAAA inhibitor, N-[(3S)-2-oxo-3-oxetanyl]-3-phenylpropanamide [(S)-OOPP], and show that this inhibitor increases PEA levels in activated leukocytes and blunts responses induced by inflammatory stimuli both in vitro and in vivo. These effects are stereoselective, mimicked by exogenous PEA, and abolished by PPAR-alpha deletion. (S)-OOPP also attenuates inflammation and tissue damage and improves recovery of motor function in mice subjected to spinal cord trauma. The results suggest that PEA activation of PPAR-alpha in leukocytes serves as an early stop signal that contrasts the progress of inflammation. The PEA-hydrolyzing amidase NAAA may provide a previously undescribed target for antiinflammatory medicines.

Despite the clear roles played by peroxisome proliferators-activated receptor alpha (PPAR-alpha) in lipid metabolism, inflammation and feeding, the effects of its activation in the central nervous system (CNS) are largely unknown. Palmitoylethanolamide (PEA), a member of the fatty-acid ethanolamide family, acts peripherally as an endogenous PPAR-alpha agonist, exerting analgesic and anti-inflammatory effects. Both PPAR-alpha and PEA are present in the CNS, but the specific functions of this lipid and its receptor remain to be clarified. Using the carrageenan-induced paw model of hyperalgesia in mice, we report here that intracerebroventricular administration of PEA (0.1-1 microg) 30 min before carrageenan injection markedly reduced mechanical hyperalgesia up to 24 h following inflammatory insult. This effect was mimicked by GW7647 (1 microg), a synthetic PPAR-alpha agonist. The obligatory role of PPAR-alpha in mediating PEA's actions was confirmed by the lack of anti-hyperalgesic effects in mutant mice lacking PPAR-alpha. PEA significantly reduced the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in sciatic nerves and restored carrageenan-induced reductions of PPAR-alpha in the L4-L6 dorsal root ganglia (DRG). To investigate the mechanism by which PEA attenuated hyperalgesia, we evaluated inhibitory kB-alpha (IkB-alpha) degradation and p65 nuclear factor kB (NF-kappaB) activation in DRG. PEA prevented IkB-alpha degradation and p65 NF-kappaB nuclear translocation, confirming the involvement of this transcriptional factor in the control of peripheral hyperalgesia. These results add further support to the broad-spectrum of biological and pharmacological effects induced by PPAR-alpha agonists, suggesting a centrally mediated component for these drugs in controlling inflammatory pain.

Despite the clear roles played by peroxisome proliferators-activated receptor alpha (PPAR-alpha) in lipid metabolism, inflammation and feeding, the effects of its activation in the central nervous system (CNS) are largely unknown. Palmitoylethanolamide (PEA), a member of the fatty-acid ethanolamide family, acts peripherally as an endogenous PPAR-alpha agonist, exerting analgesic and anti-inflammatory effects. Both PPAR-alpha and PEA are present in the CNS, but the specific functions of this lipid and its receptor remain to be clarified. Using the carrageenan-induced paw model of hyperalgesia in mice, we report here that intracerebroventricular administration of PEA (0.1-1 microg) 30 min before carrageenan injection markedly reduced mechanical hyperalgesia up to 24 h following inflammatory insult. This effect was mimicked by GW7647 (1 microg), a synthetic PPAR-alpha agonist. The obligatory role of PPAR-alpha in mediating PEA's actions was confirmed by the lack of anti-hyperalgesic effects in mutant mice lacking PPAR-alpha. PEA significantly reduced the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in sciatic nerves and restored carrageenan-induced reductions of PPAR-alpha in the L4-L6 dorsal root ganglia (DRG). To investigate the mechanism by which PEA attenuated hyperalgesia, we evaluated inhibitory kB-alpha (IkB-alpha) degradation and p65 nuclear factor kB (NF-kappaB) activation in DRG. PEA prevented IkB-alpha degradation and p65 NF-kappaB nuclear translocation, confirming the involvement of this transcriptional factor in the control of peripheral hyperalgesia. These results add further support to the broad-spectrum of biological and pharmacological effects induced by PPAR-alpha agonists, suggesting a centrally mediated component for these drugs in controlling inflammatory pain.

RationaleAn endocannabinoid signaling system has not been identified in hamsters.ObjectiveWe examined the existence of an endocannabinoid signaling system in Syrian hamsters using neuroanatomical, biochemical, and behavioral pharmacological approaches.Materials and methodsThe distribution of cannabinoid receptors was mapped, and membrane fatty-acid amide hydrolase (FAAH) activity and levels of fatty-acid amides were measured in hamster brain. The impact of cannabinoid CB1 receptor blockade and inhibition of FAAH was evaluated in the elevated plus maze, rota-rod test, and models of unconditioned and conditioned social defeat.ResultsA characteristic heterogeneous distribution of cannabinoid receptors was detected in hamster brain using [3H]CP55,940 binding and autoradiography. The FAAH inhibitor URB597 inhibited FAAH activity (IC50 = 12.8 nM) and elevated levels of fatty-acid amides (N-palmitoyl ethanolamine and N-oleoyl ethanolamine) in hamster brain. Anandamide levels were not reliably altered. The cannabinoid agonist WIN55,212-2 (1- 10 mg/kg i.p.) induced CB1-mediated motor ataxia. Blockade of CB1 with rimonabant (5 mg/kg i.p.) induced anxiogenic-like behavior in the elevated plus maze. URB597 (0.1-0.3 mg/kg i.p.) induced CB1-mediated anxiolytic-like effects in the elevated plus maze, similar to the benzodiazepine diazepam (2 mg/kg i.p.). Diazepam (2-6 mg/kg i.p.) suppressed the expression, but not the acquisition, of conditioned defeat. By contrast, neither URB597 (0.3-3.0 mg/kg i.p.) nor rimonabant (5 mg/kg i.p.) altered unconditioned or conditioned social defeat or rota-rod performance.ConclusionsEndocannabinoids engage functional CB1 receptors in hamster brain to suppress anxiety-like behavior and undergo enzymatic hydrolysis catalyzed by FAAH. Our results further suggest that neither unconditioned nor conditioned social defeat in the Syrian hamster is dependent upon cannabinoid CB1 receptor activation.

Activation of peroxisome proliferator-activated receptor (PPAR)-alpha, a member of the nuclear receptor superfamily, modulates inflammation and tissue injury events associated with spinal cord trauma in mice. Palmitoylethanolamide (PEA), the naturally occurring amide of palmitic acid and ethanolamine, reduces pain and inflammation through a mechanism dependent on PPAR-alpha activation. The aim of the present study was to evaluate the effect of the PEA on secondary damage induced by experimental spinal cord injury (SCI) in mice. SCI was induced by application of vascular clips to the dura mater via a four-level T(5)-T(8) laminectomy. This resulted in severe trauma characterized by edema, neutrophil infiltration, and production of inflammatory mediators, tissue damage, and apoptosis. Repeated PEA administration (10 mg/kg i.p.; 30 min before and 1 and 6 h after SCI) significantly reduced: 1) the degree of spinal cord inflammation and tissue injury, 2) neutrophil infiltration, 3) nitrotyrosine formation, 4) proinflammatory cytokine expression, 5) nuclear transcription factor activation-kappaB activation, 6) inducible nitric-oxide synthase expression, and 6) apoptosis. Moreover, PEA treatment significantly ameliorated the recovery of motor limb function. Together, the results indicate that PEA reduces inflammation and tissue injury associated with SCI and suggest a regulatory role for endogenous PPAR-alpha signaling in the inflammatory response associated with spinal cord trauma.

The N-aryl carbamate URB602 (biphenyl-3-ylcarbamic acid cyclohexyl ester) is an inhibitor of monoacylglycerol lipase (MGL), a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Here, we investigated the mechanism by which URB602 inhibits purified recombinant rat MGL by using a combination of biochemical and structure-activity relationship (SAR) approaches. We found that URB602 weakly inhibits recombinant MGL (IC(50) = 223 +/- 63 microM) through a rapid and noncompetitive mechanism. Dialysis experiments and SAR analyses suggest that URB602 acts through a partially reversible mechanism rather than by irreversible carbamoylation of MGL. Finally, URB602 (100 microM) elevates 2-AG levels in hippocampal slice cultures without affecting levels of other endocannabinoid-related substances. Thus, URB602 may provide a useful tool by which to investigate the physiological roles of 2-AG and explore the potential interest of MGL as a therapeutic target.

A variety of lipophilic 2-oxoamides based on gamma-aminobutyric and delta-aminovaleric analogues were synthesized. 2-oxoamides containing a tetrazole, a thioethyl or a thioacetyl group are weak inhibitors of GIVA cPLA(2), while derivatives containing a methyl tetrazole, a diethyl phosphonate or a thioethyl group are weak inhibitors of GV sPLA(2).

A variety of lipophilic 2-oxoamides based on gamma-aminobutyric and delta-aminovaleric analogues were synthesized. 2-oxoamides containing a tetrazole, a thioethyl or a thioacetyl group are weak inhibitors of GIVA cPLA(2), while derivatives containing a methyl tetrazole, a diethyl phosphonate or a thioethyl group are weak inhibitors of GV sPLA(2).

Peroxisome proliferator-activated receptor (PPAR)-alpha is a nuclear transcription factor. Although the presence of this receptor in different areas of central nervous system (CNS) has been reported, its role remains unclear. Palmitoylethanolamide (PEA), a member of the fatty-acid ethanolamide family, acts peripherally as an endogenous PPAR-alpha ligand, exerting analgesic and anti-inflammatory effects. High levels of PEA in the CNS have been found, but the specific function of this lipid remains to be clarified. Using carrageenan-induced paw edema in mice, we show that i.c.v. administration of PEA may control peripheral inflammation through central PPAR-alpha activation. A single i.c.v. administration of 0.01 to 1 microg of PEA, 30 min before carrageenan injection, reduced edema formation in the mouse carrageenan test. This effect was mimicked by 0.01 to 1 microg of GW7647 [2-[[4-[2-[[(cyclohexylamino)carbonyl](4-cyclohexylbutyl)amino]ethyl]phenyl]thio]-2-methylpropanoic acid], a synthetic PPAR-alpha agonist. Moreover, central PEA administration significantly reduced the expression of the proinflammatory enzymes cyclooxygenase-2 and inducible nitric-oxide synthase, and it significantly restored carrageenan-induced PPAR-alpha reduction in the spinal cord. To investigate the mechanism by which i.c.v. PEA attenuated the development of carrageenan-induced paw edema, we evaluated inhibitor kappaB-alpha (I kappa B-alpha) degradation and nuclear factor-kappaB (NF-kappaB) p65 activation in the cytosolic or nuclear extracts from spinal cord tissue. PEA prevented IkB-alpha degradation and NF-kappaB nuclear translocation, confirming the involvement of this transcriptional factor in the control of peripheral inflammation. The obligatory role of PPAR-alpha in mediating the effects of PEA was confirmed by the lack of the compounds anti-inflammatory effects in mutant mice lacking PPAR-alpha. In conclusion, our data show for the first time that PPAR-alpha activation in the CNS can control peripheral inflammation.

Peroxisome proliferator-activated receptor (PPAR)-alpha is a nuclear transcription factor. Although the presence of this receptor in different areas of central nervous system (CNS) has been reported, its role remains unclear. Palmitoylethanolamide (PEA), a member of the fatty-acid ethanolamide family, acts peripherally as an endogenous PPAR-alpha ligand, exerting analgesic and anti-inflammatory effects. High levels of PEA in the CNS have been found, but the specific function of this lipid remains to be clarified. Using carrageenan-induced paw edema in mice, we show that i.c.v. administration of PEA may control peripheral inflammation through central PPAR-alpha activation. A single i.c.v. administration of 0.01 to 1 microg of PEA, 30 min before carrageenan injection, reduced edema formation in the mouse carrageenan test. This effect was mimicked by 0.01 to 1 microg of GW7647 [2-[[4-[2-[[(cyclohexylamino)carbonyl](4-cyclohexylbutyl)amino]ethyl]phenyl]thio]-2-methylpropanoic acid], a synthetic PPAR-alpha agonist. Moreover, central PEA administration significantly reduced the expression of the proinflammatory enzymes cyclooxygenase-2 and inducible nitric-oxide synthase, and it significantly restored carrageenan-induced PPAR-alpha reduction in the spinal cord. To investigate the mechanism by which i.c.v. PEA attenuated the development of carrageenan-induced paw edema, we evaluated inhibitor kappaB-alpha (I kappa B-alpha) degradation and nuclear factor-kappaB (NF-kappaB) p65 activation in the cytosolic or nuclear extracts from spinal cord tissue. PEA prevented IkB-alpha degradation and NF-kappaB nuclear translocation, confirming the involvement of this transcriptional factor in the control of peripheral inflammation. The obligatory role of PPAR-alpha in mediating the effects of PEA was confirmed by the lack of the compounds anti-inflammatory effects in mutant mice lacking PPAR-alpha. In conclusion, our data show for the first time that PPAR-alpha activation in the CNS can control peripheral inflammation.

We developed a new selective liquid chromatography-electrospray ionization-tandem mass spectrometry method for the identification and quantification of anandamide (AEA), an endogenous cannabinoid receptor ligand, and other bioactive fatty acid ethanolamides (FAEs) in biological samples. Detection limit (0.025 pmol for AEA and 0.1 pmol for palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)) and quantification limit (0.2 pmol for AEA and 0.4 pmol for OEA and PEA) were in the high fmol to low pmol range for all analytes. Linear correlations (r(2)=0.99) were observed in the calibration curves for standard AEA over the range of 0.025-25 pmol and for standard PEA and OEA over the range of 0.1-500 pmol. This method provides a time-saving and sensitive alternative to existing methods for the analysis of FAEs in biological samples.

Palmitoylethanolamide (PEA), the naturally occurring amide of ethanolamine and palmitic acid, is an endogenous lipid that modulates pain and inflammation. Although the anti-inflammatory effects of PEA were first characterized nearly 50 years ago, the identity of the receptor mediating these actions has long remained elusive. We recently identified the ligand-activated transcription factor, peroxisome proliferator-activated receptor-alpha (PPAR-alpha), as the receptor mediating the anti-inflammatory actions of this lipid amide. Here we outline the history of PEA, starting with its initial discovery in the 1950s, and discuss the pharmacological properties of this compound, particularly in regards to its ability to activate PPAR-alpha.

Palmitoylethanolamide (PEA), the naturally occurring amide of palmitic acid and ethanolamine, reduces pain and inflammation through an as-yet-uncharacterized mechanism. Here, we identify the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPAR-alpha) as the molecular target responsible for the anti-inflammatory properties of PEA. PEA selectively activates PPAR-alpha in vitro with an EC(50) value of 3.1 +/- 0.4 microM and induces the expression of PPAR-alpha mRNA when applied topically to mouse skin. In two animal models, carrageenan-induced paw edema and phorbol ester-induced ear edema, PEA attenuates inflammation in wild-type mice but has no effect in mice deficient in PPAR-alpha. The natural PPAR-alpha agonist oleoylethanolamide (OEA) and the synthetic PPAR-alpha agonists GW7647 and Wy-14643 mimic these effects in a PPAR-alpha-dependent manner. These findings indicate that PPAR-alpha mediates the anti-inflammatory effects of PEA and suggest that this fatty-acid ethanolamide may serve, like its analog OEA, as an endogenous ligand of PPAR-alpha.

Palmitoylethanolamide (PEA), the naturally occurring amide of palmitic acid and ethanolamine, reduces pain and inflammation through an as-yet-uncharacterized mechanism. Here, we identify the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPAR-alpha) as the molecular target responsible for the anti-inflammatory properties of PEA. PEA selectively activates PPAR-alpha in vitro with an EC(50) value of 3.1 +/- 0.4 microM and induces the expression of PPAR-alpha mRNA when applied topically to mouse skin. In two animal models, carrageenan-induced paw edema and phorbol ester-induced ear edema, PEA attenuates inflammation in wild-type mice but has no effect in mice deficient in PPAR-alpha. The natural PPAR-alpha agonist oleoylethanolamide (OEA) and the synthetic PPAR-alpha agonists GW7647 and Wy-14643 mimic these effects in a PPAR-alpha-dependent manner. These findings indicate that PPAR-alpha mediates the anti-inflammatory effects of PEA and suggest that this fatty-acid ethanolamide may serve, like its analog OEA, as an endogenous ligand of PPAR-alpha.

The signals involved in axonal trafficking and presynaptic clustering are poorly defined. Here we show that targeting of the gamma-aminobutyric acid-synthesizing enzyme glutamate decarboxylase 65 (GAD65) to presynaptic clusters is mediated by its palmitoylated 60-aa NH(2)-terminal domain and that this region can target other soluble proteins and their associated partners to presynaptic termini. A Golgi localization signal in aa 1-23 followed by a membrane anchoring signal upstream of the palmitoylation motif are required for this process and mediate targeting of GAD65 to the cytosolic leaflet of Golgi membranes, an obligatory first step in axonal sorting. Palmitoylation of a third trafficking signal downstream of the membrane anchoring signal is not required for Golgi targeting. However, palmitoylation of cysteines 30 and 45 is critical for post-Golgi trafficking of GAD65 to presynaptic sites and for its relative dendritic exclusion. Reduction of cellular cholesterol levels resulted in the inhibition of presynaptic clustering of palmitoylated GAD65, suggesting that the selective targeting of the protein to presynaptic termini is dependent on sorting to cholesterol-rich membrane microdomains. The palmitoylated NH(2)-terminal region of GAD65 is the first identified protein region that can target other proteins to presynaptic clusters.

BackgroundExcitotoxic insults such as stroke may induce release of fatty acid ethanolamides (FAEs), contributing to the downstream events in the ischemic cascade. We therefore studied release of FAEs such as anandamide, palmitylethanolamide (PEA), and oleylethanolamide (OEA) in the brain of a patient suffering from malignant hemispheric infarction treated with hypothermia.Case descriptionA patient with life-threatening hemispheric stroke was treated with moderate hypothermia (33 degrees C) that was maintained for 3 days, followed by a 3-day rewarming period. Microdialysis was applied to measure glutamate, lactate, and glycerol by using a microdialysis analyzer. FAEs were measured by microdialysis coupled with high-performance liquid chromatography/mass spectrometry. Release of neuroprotective fatty amides occurred within the first day after ischemia and reached high concentrations for all 3 substances in tissue surrounding the primary ischemic lesion: anandamide up to 42 pmol/mL, PEA up to 120 pmol/mL, and OEA up to 242 pmol/mL. There was a significant correlation with elevation of lactate as early marker for the hypoxic insult.ConclusionsThis is the first report demonstrating release of FAEs in vivo during human stroke and may suggest contribution of the FAE signaling system to the pathophysiological events after ischemia.

The endogenous fatty acid ethanolamide, palmitylethanolamide, alleviated, in a dose-dependent manner, pain behaviors elicited in mice by injections of formalin (5%, intraplantar), acetic acid (0.6%, 0.5 ml per animal, intraperitoneal, i.p.), kaolin (2.5 mg per animal, i.p.), and magnesium sulfate (120 mg per kg, i.p.). The antinociceptive effects of palmitylethanolamide were prevented by the cannabinoid CB2 receptor antagonist SR144528 [N-([1s]-endo-1.3.3-trimethylbicyclo[2.3.1]heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide], not by the cannabinoid CB1 receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide x HCl]. By contrast, palmitylethanolamide had no effect on capsaicin-evoked pain behavior or thermal nociception. The endogenous cannabinoid, anandamide (arachidonylethanolamide), alleviated nociception in all tests (formalin, acetic acid, kaolin, magnesium sulfate, capsaicin and hot plate). These effects were prevented by the cannabinoid CB1 receptor antagonist SR141716A, not the cannabinoid CB2 receptor antagonist SR141716A. Additional fatty acid ethanolamides (oleylethanolamide, myristylethanolamide, palmitoleylethanolamide, palmitelaidylethanolamide) had little or no effect on formalin-evoked pain behavior, and were not investigated in other pain models. These results support the hypothesis that endogenous palmitylethanolamide participates in the intrinsic control of pain initiation. They also suggest that the putative receptor site activated by palmitylethanolamide may provide a novel target for peripherally acting analgesic drugs.

The pathogenesis of renal calcium-oxalate (CaOx) stones is complex and influenced by various metabolic factors. In parallel, palmitic acid (PA) has been identified as an upregulated lipid metabolite in the urine and serum of patients with renal CaOx stones via untargeted metabolomics. Thus, this study aimed to mechanistically assess whether PA is involved in stone formation. Lipidomics analysis of PA-treated renal tubular epithelial cells compared with the control samples revealed that α-linoleic acid and α-linolenic acid were desaturated and elongated, resulting in the formation of downstream polyunsaturated fatty acids (PUFAs). In correlation, the levels of fatty acid desaturase 1 and 2 (FADS1 and FADS2) and peroxisome proliferator-activated receptor α (PPARα) in these cells treated with PA were increased relative to the control levels, suggesting that PA-induced upregulation of PPARα, which in turn upregulated these two enzymes, forming the observed PUFAs. Lipid peroxidation occurred in these downstream PUFAs under oxidative stress and Fenton Reaction. Furthermore, transcriptomics analysis revealed significant changes in the expression levels of ferroptosis-related genes in PA-treated renal tubular epithelial cells, induced by PUFA peroxides. In addition, phosphatidyl ethanolamine binding protein 1 (PEBP1) formed a complex with 15-lipoxygenase (15-LO) to exacerbate PUFA peroxidation under protein kinase C ζ (PKC ζ) phosphorylation, and PKC ζ was activated by phosphatidic acid derived from PA. In conclusion, this study found that the formation of renal CaOx stones is promoted by ferroptosis of renal tubular epithelial cells resulting from PA-induced dysregulation of PUFA and phosphatidic acid metabolism, and PA can promote the renal adhesion and deposition of CaOx crystals by injuring renal tubular epithelial cells, consequently upregulating adhesion molecules. Accordingly, this study provides a new theoretical basis for understanding the correlation between fatty acid metabolism and the formation of renal CaOx stones, offering potential targets for clinical applications., (© 2024. The Author(s).)

Background: Palmitoylethanolamide (PEA) is an endocannabinoid-like lipid mediator which is naturally produced in the body and found in certain foods. The aim of this study was to assess the effect of a bioavailable formulated form of PEA (Levagen+ ® ) on serum BDNF levels and parameters of cognitive function in healthy adults., Methods: A randomised double-blinded placebo-controlled cross-over trial was implemented to measure the effects of a 6-week 700 mg/day course of formulated PEA supplementation versus a placebo. Participants (n = 39) completed pre- and post-assessments of a lab-based cognitive test. Serum samples were collected to measure BDNF concentrations using an immunoassay., Results: A significant increase in serum BDNF levels was found following PEA supplementation compared with the placebo ( p = 0. 0057, d = 0.62). The cognition test battery demonstrated improved memory with PEA supplementation through better first success ( p = 0.142, d = 0.54) and fewer errors ( p = 0.0287; d = -0.47) on the Paired Associates Learning test., Conclusion: This was the first study to report a direct beneficial effect of Levagen+ ® PEA supplementation on memory improvement as well as corresponding increases in circulating neurotrophic marker levels. This suggests that formulated PEA holds promise as an innovative and practical intervention for cognitive health enhancement.

Free fatty acids, like palmitic acid (PA), and xanthophyll pigments, like lutein (LUT) are the natural membrane compounds in plants. To study the effect of PA on LUT and their organization, a model membrane of 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) enriched with 2 mol% PA and 1 mol% LUT was formed. Molecular mechanisms underlying the interaction between these two compounds were examined with application of molecular spectroscopy techniques, e.g., visible spectroscopy, electron paramagnetic resonance and Fourier transform infrared. We determined the monomeric/dimeric organization of LUT in the membrane. We proved that the presence of PA in the lipid phase facilitated and stabilized the formation of LUT structures in the membrane. Lutein with PA did not form strong molecular aggregates like H- and J-structures. We presented the simplified model membrane that could be a suitable representation of the physiological process of de-esterification of PA from LUT appearing in natural biomembranes in humans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: The endocannabinoid (eCB) system and the serotonin (5-HT) are both implicated in the severity of the depression. 5-HT is synthesized from the amino acid tryptophan (Trp), which is also a precursor for kynurenine (Kyn) whose production is increased at the expense of 5-HT in depressed patients. No clinical studies have investigated the crosstalk between the eCB system and the Trp/5-HT/Kyn pathways. Here, we hypothesized that the eCB system is associated with an enhanced Kyn production in relation to the severity of depressive symptoms., Methods: Eighty-two subjects (51 patients with a diagnosis of depressive disorder (DSM-5) and 31 healthy volunteers), were assessed with the Montgomery-Åsberg Depression Rating Scale (MADRS), Beck Depression Scale, and Global Clinical Impression. Serum concentrations of eCBs (N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG)); structurally related fatty acyl compounds 2-oleoylglycerol (2-OG), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA); Trp, Kyn, Kyn/Trp ratio (an index of Trp degradation into Kyn) and 5-HT were also determined., Results: Following a principal component analysis including the severity of depression, Kyn and the Kyn/Trp ratio appear to be directly associated with 2-AG, AEA, and PEA. Interestingly, these biomarkers also permitted to distinguish the population into two main clusters: one of individuals having mild/severe depressive symptoms and the other with an absence of depressive symptoms. Using parametric analysis, higher serum levels of 2-AG, Kyn, and the ratio Kyn/Trp and lower levels of Trp and 5-HT were found in individuals with mild/severe depressive symptoms than in those without depressive symptoms. While in asymptomatic people, PEA was directly associated to Trp, and OEA indirectly linked to 5-HT, in individuals with depressive symptoms, these correlations were lost, and instead, positive correlations between AEA and 2-AG, PEA and AEA, and PEA vs 2-AG and OEA concentrations were found., Conclusions: Parametric and non-parametric analyses suggest a possible association between eCBs, tryptophan/kynurenine biomarkers, and severity of depression, confirming a likely interplay among inflammation, stress, and depression. The enhanced relationships among the biomarkers of the 2-AG and AEA pathways and related lipids seen in individuals with depressive symptoms, but not in asymptomatics, suggest an altered metabolism of the eCB system in depression., (© 2024. The Author(s).)

The biological actions of the endogenous cannabinoid anandamide are terminated by carrier-mediated transport into neurons and astrocytes, followed by enzymatic hydrolysis. Anandamide transport is inhibited by the compound N-(4-hydroxyphenyl)arachidonylamide (AM404). AM404 potentiates several responses elicited by administration of exogenous anandamide, suggesting that it may also protect endogenous anandamide from inactivation. To test this hypothesis, we studied the effects of AM404 on the plasma levels of anandamide using high-performance liquid chromatography/mass spectrometry (HPLC/MS). Systemic administration of AM404 (10 mg kg(-1) intraperitoneal, i.p. ) caused a gradual increase of anandamide in rat plasma, which was significantly different from untreated controls at 60 and 120 min after drug injection. In plasma, both AM404 and anandamide were associated with a plasma protein, which we identified as albumin by non-denaturing polyacrylamide gel electrophoresis. AM404 (10 mg kg(-1), i.p.) caused a time-dependent decrease of motor activity, which was reversed by the cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.hydrochloride (SR141716A, 0.5 mg kg(-1), i.p). These results are consistent with the hypothesis that AM404 inhibits anandamide inactivation in vivo.

Evidence suggests that cannabinoid receptors, the pharmacologcial target of cannabis-derived drugs, and their accompanying system of endogenous activators may be dysfunctional in schizophrenia. To test this hypothesis, we examined whether endogenous cannabinoid concentrations in cerebrospinal fluid of schizophrenic patients are altered compared to nonschizophrenic controls. Endogenous cannabinoids were purified from cerebrospinal fluid of 10 patients with schizophrenia and 11 non-schizophrenic controls by high-performance liquid chromatography, and quantified by isotope dilution gas-chromatography/mass-spectrometry. Cerebrospinal concentrations of two endogenous cannabinoids (anandamide and palmitylethanolamide) were significantly higher in schizophrenic patients than non-schizophrenic controls (p < 0.05). By contrast, levels of 2-arachidonylglycerol, another endogenous cannabinoid lipid, were below detection in both groups. The findings did not seem attributable to gender, age or medication. Elevated anandamide and palmitylethanolamide levels in cerebrospinal fluid of schizophrenic patients may reflect an imbalance in endogenous cannabinoid signaling, which may contribute to the pathogenesis of schizophrenia.

We measured endogenous cannabinoid release in dorsal striatum of freely moving rats by microdialysis and gas chromatography/mass spectrometry. Neural activity stimulated the release of anandamide, but not of other endogenous cannabinoids such as 2-arachidonylglycerol. Moreover, anandamide release was increased eightfold over baseline after local administration of the D2-like (D2, D3, D4) dopamine receptor agonist quinpirole, a response that was prevented by the D2-like receptor antagonist raclopride. Administration of the D1-like (D1, D5) receptor agonist SKF38393 had no such effect. These results suggest that functional interactions between endocannabinoid and dopaminergic systems may contribute to striatal signaling. In agreement with this hypothesis, pretreatment with the cannabinoid antagonist SR141716A enhanced the stimulation of motor behavior elicited by systemic administration of quinpirole. The endocannabinoid system therefore may act as an inhibitory feedback mechanism countering dopamine-induced facilitation of motor activity.

The potent analgesic effects of cannabis-like drugs and the presence of CB1-type cannabinoid receptors in pain-processing areas of the brain and spinal cord indicate that endogenous cannabinoids such as anandamide may contribute to the control of pain transmission within the central nervous system (CNS). Here we show that anandamide attenuates the pain behaviour produced by chemical damage to cutaneous tissue by interacting with CB1-like cannabinoid receptors located outside the CNS. Palmitylethanolamide (PEA), which is released together with anandamide from a common phospholipid precursor, exerts a similar effect by activating peripheral CB2-like receptors. When administered together, the two compounds act synergistically, reducing pain responses 100-fold more potently than does each compound alone. Gas-chromatography/mass-spectrometry measurements indicate that the levels of anandamide and PEA in the skin are enough to cause a tonic activation of local cannabinoid receptors. In agreement with this possibility, the CB1 antagonist SR141716A and the CB2 antagonist SR144528 prolong and enhance the pain behaviour produced by tissue damage. These results indicate that peripheral CB1-like and CB2-like receptors participate in the intrinsic control of pain initiation and that locally generated anandamide and PEA may mediate this effect.

Understanding the mechanisms involved in the biogenesis of N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine is important in view of the possible role of these lipids as endogenous cannabinoid substances. Anandamide (which activates cannabinoid CB1 receptors) and N-palmitoylethanolamine (which activates a CB2-like receptor subtype in mast cells) may both derive from cleavage of precursor phospholipid, N-acylphosphatidylethanolamine (NAPE), catalyzed by Ca(2+)-activated D-type phosphodiesterase activity. We report here that the de novo biosynthesis of NAPE is enhanced in a Ca(2+)-dependent manner when rat cortical neurons are stimulated with the Ca(2+)-ionophore ionomycin or with membrane-depolarizing agents such as veratridine and kainate. This reaction is likely to be mediated by a neuronal N-acyltransferase activity, which catalyzes the transfer of an acyl group from phosphatidylcholine to the ethanolamine moiety of phosphatidylethanolamine. In addition, we show that Ca2+-dependent NAPE biosynthesis is potentiated by agents that increase cAMP levels, including forskolin and vasoactive intestinal peptide. Our results thus indicate that NAPE levels in cortical neurons are controlled by Ca2+ ions and cAMP. Such regulatory effect may participate in maintaining a supply of cannabimimetic N-acylethanolamines during synaptic activity, and prime target neurons for release of these bioactive lipids.