Pharmacological Evaluation of Cannabinoid Receptor Modulators Using GRABeCB2.0 Sensor.

Cannabinoid receptors CB1R and CB2R are G-protein coupled receptors acted upon by endocannabinoids (eCBs), namely 2-arachidonoylglycerol (2-AG) and N-arachidonoyl ethanolamine (AEA), with unique pharmacology and modulate disparate physiological processes. A genetically encoded GPCR activation-based sensor that was developed recently-GRABeCB2.0-has been shown to be capable of monitoring real-time changes in eCB levels in cultured cells and preclinical models. However, its responsiveness to exogenous synthetic cannabinoid agents, particularly antagonists and allosteric modulators, has not been extensively characterized. This current study expands upon the pharmacological characteristics of GRABeCB2.0 to enhance the understanding of fluorescent signal alterations in response to various functionally indiscriminate cannabinoid ligands. The results from this study could enhance the utility of the GRABeCB2.0 sensor for in vitro as well as in vivo studies of cannabinoid action and may aid in the development of novel ligands.

Pharmacological blockade of 2-AG degradation ameliorates clinical, neuroinflammatory and synaptic alterations in experimental autoimmune encephalomyelitis.

The endocannabinoid system (ECS) is critically involved in the pathophysiology of Multiple Sclerosis (MS), a neuroinflammatory and neurodegenerative disease of the central nervous system (CNS). Over the past decade, researchers have extensively studied the neuroprotective and anti-inflammatory effects of the ECS. Inhibiting the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) has emerged as a promising strategy to mitigate brain damage in MS. In this study, we investigated the effects of a novel reversible MAGL inhibitor (MAGLi 432) on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We assessed its implications on motor disability, neuroinflammation, and synaptic dysfunction. Systemic in vivo treatment with MAGLi 432 resulted in a less severe EAE disease, accompanied by increased 2-AG levels and decreased levels of arachidonic acid (AA) and prostaglandins (PGs) in the brain. Additionally, MAGLi 432 reduced both astrogliosis and microgliosis, as evidenced by decreased microglia/macrophage density and a less reactive morphology. Flow cytometry analysis further revealed fewer infiltrating CD45+ and CD3+ cells in the brains of MAGLi 432-treated EAE mice. Finally, MAGLi treatment counteracted the striatal synaptic hyperexcitability promoted by EAE neuroinflammation. In conclusion, MAGL inhibition significantly ameliorated EAE clinical disability and striatal inflammatory synaptopathy through potent anti-inflammatory effects. These findings provide new mechanistic insights into the neuroprotective role of the ECS during neuroinflammation and highlight the therapeutic potential of MAGLi-based drugs in mitigating MS-related inflammatory and neurodegenerative brain damage.

Inhibition of fatty acid amide hydrolase reverses aberrant prefrontal gamma oscillations in the sub-chronic PCP model for schizophrenia.

Hypofunctioning of NMDA receptors, and the resulting shift in the balance between excitation and inhibition, is considered a key process in the pathophysiology of schizophrenia. One important manifestation of this phenomenon is changes in neural oscillations, those above 30 Hz (i.e., gamma-band oscillations), in particular. Although both preclinical and clinical studies observed increased gamma activity following acute administration of NMDA receptor antagonists, the relevance of this phenomenon has been recently questioned given the reduced gamma oscillations typically observed during sensory and cognitive tasks in schizophrenia. However, there is emerging, yet contradictory, evidence for increased spontaneous gamma-band activity (i.e., at rest or under baseline conditions). Here, we use the sub-chronic phencyclidine (PCP) rat model for schizophrenia, which has been argued to model the pathophysiology of schizophrenia more closely than acute NMDA antagonism, to investigate gamma oscillations (30-100 Hz) in the medial prefrontal cortex of anesthetized animals. While baseline gamma oscillations were not affected, oscillations induced by train stimulation of the posterior dorsal CA1 (pdCA1) field of the hippocampus were enhanced in PCP-treated animals (5 mg/kg, twice daily for 7 days, followed by a 7-day washout period). This effect was reversed by pharmacological enhancement of endocannabinoid levels via systemic administration of URB597 (0.3 mg/kg), an inhibitor of the catabolic enzyme of the endocannabinoid anandamide. Intriguingly, the pharmacological blockade of CB1 receptors by AM251 unmasked a reduced gamma oscillatory activity in PCP-treated animals. The findings are consistent with the observed effects of URB597 and AM251 on behavioral deficits reminiscent of the symptoms of schizophrenia and further validate the potential for cannabinoid-based drugs as a treatment for schizophrenia.

Resveratrol ameliorates intestinal lipid metabolism through the PPAR signaling pathway in high-fat diet-fed red tilapia (Oreochromis niloticus).

Feeding high-fat (HF) diets has been shown to cause hepatic and intestinal impairment in fish species, but the mode of action, especially the pathways involved in the intestine, has not been determined yet. In this study, the effects of resveratrol (RES) supplementation on the intestinal structure, microbial flora, and fat metabolism in red tilapia (Oreochromis niloticus) were determined. The results showed RES maintained the structural integrity of the intestine and significantly increased the number of goblet cells in the midgut. RES significantly induced interferon (IL)-1ß, IL-6, IL-10, and tumor necrosis factor (TNF)-α, serumal and fecal trimetlylamine oxide (TMAO) and lipopolysaccharides (LPS), intestinal acetic acid levels. However, the concentrations of bound bile acids increased in HF-fed red tilapia. Atp5fa1 and Pafah1b3 significantly increased, Pmt and Acss2 significantly decreased, respectively, with RES supplementation, which was alleviated and retained at the same level in the selisistat (EX527) group. While for transcriptome and proteomics results, RES was found to promote fatty acid ß-oxidation and arachidonic acid metabolism associated with the peroxisome proliferator-activated receptor (PPAR) signaling pathway. The next validation experiment showed some genes related to apoptosis and fatty acid metabolism pathways were altered by RES supplementation. Namely, sn6, loc100702698, new_14481, and prkaa1 were upregulated, while ffrs1, ap3s1, and loc100705861 were downregulated. RES significantly increased Planctomycetes and Verrucomicrobia while decreased Moonvirus, Citrobacter, and Pseudomonas. Akkermansia and Fusobacterium significantly increased and Aeromonas significantly decreased. Thus, unsaturated fatty acid biosynthesis significantly increased and carbohydrate/energy metabolism decreased. To conclude, RES enabled the body to complete fatty acid ß-oxidation and arachidonic acid metabolism, whereas the addition of inhibitors increased the expression of the phagosome transcriptome and reduced fatty acid ß-oxidative metabolism.

Endocannabinoid biosynthetic enzymes regulate pain response via LKB1-AMPK signaling.

Diacylglycerol lipase-beta (DAGLß) serves as a principal 2-arachidonoylglycerol (2-AG) biosynthetic enzyme regulating endocannabinoid and eicosanoid metabolism in immune cells including macrophages and dendritic cells. Genetic or pharmacological inactivation of DAGLß ameliorates inflammation and hyper-nociception in preclinical models of pathogenic pain. These beneficial effects have been assigned principally to reductions in downstream proinflammatory lipid signaling, leaving alternative mechanisms of regulation largely underexplored. Here, we apply quantitative chemical- and phospho-proteomics to find that disruption of DAGLß in primary macrophages leads to LKB1-AMPK signaling activation, resulting in reprogramming of the phosphoproteome and bioenergetics. Notably, AMPK inhibition reversed the antinociceptive effects of DAGLß blockade, thereby directly supporting DAGLß-AMPK crosstalk in vivo. Our findings uncover signaling between endocannabinoid biosynthetic enzymes and ancient energy-sensing kinases to mediate cell biological and pain responses.

Apple consumption affects cecal health by regulating 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HETE) levels through modifying the microbiota in rats.

Apples are rich in many nutrients and functional components. However, the mechanism of the effect of fresh apple consumption on rats remains unclear. In the present study, fresh apples (10 g kg-1) were added to the diet of Wistar rats, and changes in the microbiota and metabolite content of the cecum were analyzed after 28 days of feeding, and changes in the 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HETE) content and indicators related to inflammation, oxidative stress, and apoptosis were detected. Subsequently, a fecal microbiota transplantation (FMT) protocol was designed and carried out to verify the relationship between the microbiota and 12(S)-HETE, the cecal structure, and inflammatory factors. The results show that apple consumption significantly reduced the serum levels of alanine aminotransferase (ALT) and immunoglobulin G (IgG), altered the cecal histomorphology, and significantly upregulated the gene expression of claudin-1 and zonula occludens-1 (ZO-1), which encode tight junction proteins. Apple consumption also changed the structure of the cecal microbiota, increasing the abundance of some species (such as Shuttleworthia) and decreasing the abundance of others (such as Alphaproteobacteria). Metabolomic screening identified 64 significantly different metabolites. The FMT results showed that apple consumption reduced 12(S)-HETE metabolite levels in the cecal contents, improved the intestinal structure, and reduced the levels of proinflammatory factor expression by altering the cecal microbiota. In conclusion, this study provides further insight into the effects of apples on animals using rats as experimental animals. It provides basic data for future exploration of the mechanisms of the effect of apple consumption on humans.

Low colostrum intake results in potential accumulation of peroxisome lipid substrates in vaginal tissue of 3-week-old gilts.

Low colostrum intake relates to poorer health and infertility in swine. We previously connected vaginal lipid profiles at weaning to fertility of sows. We hypothesized vaginal lipidome varied with colostrum intake. Our objective was to determine whether indicators of colostrum intake, immunocrit (IM) and weight gain 24 h postnatal (PN), related to vaginal lipids at d21 PN. Gilts (n=60) were weighed and blood sampled to measure IM. On d21 PN vaginal swabs were taken and lipids measured using multiple reaction monitoring. Abundance of multiple lipids differed (P<0.05) between gilts categorized as high versus low IM and high versus low 24 h gain. The abundance of multiple lipids correlated with IM and 24 h gain. Phosphatidylcholine PC(36:3), PC(36:2), and arachidonic acid (C20:4) positively (P<0.05) correlated with IM. The ether lipid PCo(38:6) and multiple cholesteryl esters negatively (P<0.05) correlated with IM. ROC analysis indicated arachidonic acid and docosanoic acid (C22:0) may serve as excellent biomarkers that distinguish between high and low IM. Similar to gilts found to be infertile, lipid profiles of low colostrum intake animals had greater abundance of very long chain fatty acids, lipids with high levels of unsaturation, and cholesteryl esters, which are metabolized in peroxisomes indicating their potential dysfunction.

Gi/o GPCRs drive the formation of actin-rich tunneling nanotubes in cancer cells via a Gßγ/PKCα/FARP1/Cdc42 axis.

The functional association between stimulation of G-protein-coupled receptors (GPCRs) by eicosanoids and actin cytoskeleton reorganization remains largely unexplored. Using a model of human adrenocortical cancer cells, here we established that activation of the GPCR OXER1 by its natural agonist, the eicosanoid 5-oxo-eicosatetraenoic acid, leads to the formation of filopodia-like elongated projections connecting adjacent cells, known as tunneling nanotube (TNT)-like structures. This effect is reduced by pertussis toxin and GUE1654, a biased antagonist for the Gßγ pathway downstream of OXER1 activation. We also observed pertussis toxin-dependent TNT biogenesis in response to lysophosphatidic acid, indicative of a general response driven by Gi/o-coupled GPCRs. TNT generation by either 5-oxo-eicosatetraenoic acid or lysophosphatidic acid is partially dependent on the transactivation of the epidermal growth factor receptor and impaired by phosphoinositide 3-kinase inhibition. Subsequent signaling analysis reveals a strict requirement of phospholipase C ß3 and its downstream effector protein kinase Cα. Consistent with the established role of Rho small GTPases in the formation of actin-rich projecting structures, we identified the phosphoinositide 3-kinase-regulated guanine nucleotide exchange factor FARP1 as a GPCR effector essential for TNT formation, acting via Cdc42. Altogether, our study pioneers a link between Gi/o-coupled GPCRs and TNT development and sheds light into the intricate signaling pathways governing the generation of specialized actin-rich elongated structures in response to bioactive signaling lipids.

Erythrocyte Membrane Fatty Acid Composition as a Potential Biomarker for Depression.

BACKGROUND: Major depressive disorders is a chronic and severe psychiatric disorder with poor prognosis and quality of life. Abnormal erythrocyte fatty acid (FA) composition in depressed patients were found in our previous study, but the relationship between erythrocyte membrane FA levels and different severity of depressive and anxiety symptoms remains to be explored. METHODS: This cross-sectional study included 139 patients with first-diagnosed, drug-naïve depression and 55 healthy controls whose erythrocyte FA composition was analyzed. Patients with depression were divided into severe depression and mild to moderate depression or depression with severe anxiety and mild to moderate anxiety. Then the differences of FA levels among different groups were analyzed. Finally, the receiver operating characteristic curve analysis was applied to identify potential biomarkers in distinguishing the severity of depressive symptoms. RESULTS: Levels of erythrocyte membrane FAs were elevated among patients with severe depression compared with healthy controls or patients with mild to moderate depression of almost all kinds. While C18:1n9t (elaidic acid), C20:3n6 (eicosatrienoic acid), C20:4n6 (arachidonic acid), C22:5n3 (docosapentaenoic acid), total fatty acids (FAs), and total monounsaturated FAs were elevated in patients with severe anxiety compared with patients with mild to moderate anxiety. Furthermore, the level of arachidonic acid, C22:4n6 (docosatetraenoic acid), elaidic acid, and the combination of all 3 were associated with the severity of depressive symptoms. CONCLUSIONS: The results suggested that erythrocyte membrane FA levels have the potential to be the biological indicator of clinical characteristics for depression, such as depressive symptoms and anxiety. In the future, more research is needed to explore the causal association between FA metabolism and depression.

The impact of subchronic ozone exposure on serum metabolome and the mechanisms of abnormal bile acid and arachidonic acid metabolisms in the liver.

Ambient ozone (O3) pollution can induce respiratory and cardiovascular toxicity. However, its impact on the metabolome and the underlying mechanisms remain unclear. This study first investigated the serum metabolite changes in rats exposed to 0.5 ppm O3 for 3 months using untargeted metabolomic approach. Results showed chronic ozone exposure significantly altered the serum levels of 34 metabolites with potential increased risk of digestive, respiratory and cardiovascular disease. Moreover, bile acid synthesis and secretion, and arachidonic acid (AA) metabolism became the most prominent affected metabolic pathways after O3 exposure. Further studies on the mechanisms found that the elevated serum toxic bile acid was not due to the increased biosynthesis in the liver, but the reduced reuptake from the portal vein to hepatocytes owing to repressed Ntcp and Oatp1a1, and the decreased bile acid efflux in hepatocytes as a results of inhibited Bsep, Ostalpha and Ostbeta. Meanwhile, decreased expressions of detoxification enzyme of SULT2A1 and the important regulators of FXR, PXR and HNF4α also contributed to the abnormal bile acids. In addition, O3 promoted the conversion of AA into thromboxane A2 (TXA2) and 20-hydroxyarachidonic acid (20-HETE) in the liver by up-regulation of Fads2, Cyp4a and Tbxas1 which resulting in decreased AA and linoleic acid (LA), and increased thromboxane B2 (TXB2) and 20-HETE in the serum. Furthermore, apparent hepatic chronic inflammation, fibrosis and abnormal function were found in ozone-exposed rats. These results indicated chronic ozone exposure could alter serum metabolites by interfering their metabolism in the liver, and inducing liver injury to aggravate metabolic disorders.

Inhibition of 2-arachidonoylglycerol degradation enhances glial immunity by single-cell transcriptomic analysis.

BACKGROUND: 2-Arachidonoylglycerol (2-AG) is the most abundant endogenous cannabinoid. Inhibition of 2-AG metabolism by inactivation of monoacylglycerol lipase (MAGL), the primary enzyme that degrades 2-AG in the brain, produces anti-inflammatory and neuroprotective effects in neurodegenerative diseases. However, the molecular mechanisms underlying these beneficial effects are largely unclear. METHODS: Hippocampal and cortical cells were isolated from cell type-specific MAGL knockout (KO) mice. Single-cell RNA sequencing was performed by 10 × Genomics platform. Cell Ranger, Seurat (v3.2) and CellChat (1.1.3) packages were used to carry out data analysis. RESULTS: Using single-cell RNA sequencing analysis, we show here that cell type-specific MAGL KO mice display distinct gene expression profiles in the brain. Inactivation of MAGL results in robust changes in expression of immune- and inflammation-related genes in microglia and astrocytes. Remarkably, upregulated expression of chemokines in microglia is more pronounced in mice lacking MAGL in astrocytes. In addition, expression of genes that regulate other cellular functions and Wnt signaling in astrocytes is altered in MAGL KO mice. CONCLUSIONS: Our results provide transcriptomic evidence that cell type-specific inactivation of MAGL induces differential expression of immune-related genes and other fundamental cellular pathways in microglia and astrocytes. Upregulation of the immune/inflammatory genes suggests that tonic levels of immune/inflammatory vigilance are enhanced in microglia and astrocytes, particularly in microglia, by inhibition of 2-AG metabolism, which likely contribute to anti-inflammatory and neuroprotective effects produced by inactivation of MAGL in neurodegenerative diseases.

Free Fatty Acids Induce Lipid Accumulation, Autophagy, and Apoptosis in Human Sebocytes.

BACKGROUND: A disruption of sebocyte differentiation and lipogenesis has fatal consequences and can cause a wide spectrum of skin diseases, from acne vulgaris to sebaceous carcinoma, however, the relevant molecular mechanisms have not been fully clarified. OBJECTIVES: The induction of autophagy and apoptosis in human sebocytes in response to biologically relevant fatty acids was investigated. METHODS: Free fatty acids (arachidonic acid, linoleic acid, palmitic acid, and palmitoleic acid) and the pan-caspase inhibitor QVD-Oph were added to the supernatant of cultured human SZ95 sebocytes. Individual relevant proteins were analyzed by Western blotting. Apoptosis and cell viability were determined, and typical autophagy structures were detected through electron microscopy. To obtain cell growth curves, cell confluence was continuously monitored by real-time cell analysis. RESULTS: Fatty acids induced the development of intracellular lipid droplets with subsequent apoptosis, whereas arachidonic acid caused the most rapid effect. Cleavage products of caspase-3 were only detected in arachidonic acid-induced apoptosis. The high basal apoptotic rate of cultured SZ95 sebocytes was strongly suppressed by QVD-Oph. Fatty acid-induced apoptosis was also markedly inhibited by QVD-Oph, whereas intracellular lipid droplets further accumulated. While cell viability after incubation with linoleic acid, palmitic acid, or palmitoleic acid and QVD-Oph was comparable with that of non-treated controls, arachidonic acid significantly reduced cell viability and cell density despite the concomitant pan-caspase inhibitor treatment. Using electron microscopy, typical autophagy structures were detected, such as autophagosomes and autolysosomes, at the basal level, which became more pronounced after treatment with fatty acids. CONCLUSIONS: Our findings contribute to a better understanding of the inflammation-associated mechanisms of lipogenesis and cell death induction in human sebocytes and may help to unveil the effects of fatty acid-rich human nutrition.

Radiometric Assay of ABHD2 Activity.

The α,ß-hydrolase fold-containing protein 2 (ABHD2) is a serine hydrolase, responsible for the cleavage of endogenous 2-arachidonoylglycerol (2-AG). ABHD2 is activated by progesterone, thus, it is considered a nonnuclear receptor of this steroid hormone that terminates its biological effects. The products of ABHD2-catalyzed cleavage by the natural substrate 2-AG are glycerol and arachidonic acid; here, instead of 2-AG, the radioactive substrate 2-oleoyl-[3H]glycerol has been used as already done in various acylglycerol lipase activity assays. The amount of [3H]glycerol released allows to measure ABHD2 enzymatic activity.

Integrative Application of Transcriptomics and Metabolomics Provides Insights into Unsynchronized Growth in Sea Cucumber (Stichopus monotuberculatus).

Ever-increasing consumer demand for sea cucumbers mainly leads to huge damage to wild sea cucumber resources, including Stichopus monotuberculatus, which in turn exerts negative impacts on marine environments due to the lack of ecological functions performed by sea cucumbers. Aquaculture of sea cucumbers is an effective way to meet consumer demand and restore their resources. Unsynchronous growth is a prominent problem in the aquaculture of sea cucumbers which has concealed unelucidated molecular mechanisms until now. In this study, we carried out an integrative analysis of transcriptomics and metabolomics on fast-growing (SMF) and slow-growing (SMS) groups of S. monotuberculatus cultured in the same environmental conditions. The results revealed that a total of 2054 significantly differentially expressed genes (DEGs) were identified, which are mainly involved in fat digestion and absorption, histidine metabolism, arachidonic acid metabolism, and glutathione metabolism. 368 differential metabolites (DMs) were screened out between the SMF group and the SMS group; these metabolites are mainly involved in glycerophospholipid metabolism, purine metabolism, biosynthesis of unsaturated fatty acids, pyrimidine metabolism, arachidonic acid metabolism, and other metabolic pathways. The integrative analysis of transcriptomics and metabolomics of S. monotuberculatus suggested that the SMF group had a higher capacity for lipid metabolism and protein synthesis, and had a more frequent occurrence of apoptosis events, which are likely to be related to coping with environmental stresses. The results of this study provide potential values for the aquaculture of sea cucumbers which may promote their resource enhancement.

Epididymal epithelial degeneration and lipid metabolism impairment account for male infertility in occludin knockout mice.

Occludin (OCLN) is a tight junction protein and Ocln deletion mutation causes male infertility in mice. However, the role of OCLN in male reproductive system remains unknown. In this study, we used an interdisciplinary approach to elucidate the underlying mechanism of male infertility in related to OCLN function, including Ocln knockout mice as well as a combined omics analysis and immunofluorescent labelling. Our results showed that the epididymis of Ocln-null mice displayed a phenomenon resembling epididymal sperm granuloma, which occurred especially in the junctional region between caput and corpus epididymidis. Sperm motility and fertilisation capacity were also impaired in these Ocln-null mice, accompanied by enlarged tubules in the proximal regions and degeneration in the distal regions of epididymis. Cellular localization analysis showed that OCLN immunofluorescence was enriched only in the apical junction of epithelial principal cells in the proximal regions of epididymis. Integrative omics analysis revealed the downregulation of gene clusters enriched in acid secretion and fatty acid metabolism in the Ocln-null epididymis, especially the enzymes related to the unsaturated arachidonic acid pathway. The number of proton-pump V-ATPase-expression clear cells, a key player of luminal acidification in the epididymis, declined drastically from prepubertal age before sperm arrival but not in the early postnatal age. This was accompanied by programmed cell death of clear cells and increased pH in the epididymal fluid of OCLN-deficient mice. The lipidomics results showed significantly increased levels of specific DAGs conjugated to unsaturated fatty acids in the Ocln-mutant. Immunofluorescent labelling showed that the arachidonic acid converting enzyme PTGDS and phospholipase PLA2g12a were prominently altered in the principal cells and luminal contents of the Ocln-mutant epididymis. Whereas the carboxylate ester lipase CES1, originally enriched in the WT basal cells, was found upregulated in the Ocln-mutant principal cells. Overall, this study demonstrates that OCLN is essential for maintaining caput-to-corpus epithelial integrity, survival of acid-secreting clear cells, and unsaturated fatty acid catabolism in the mouse epididymis, thereby ensuring sperm maturation and male fertility.

The endocannabinoid anandamide is an airway relaxant in health and disease.

Chronic obstructive airway diseases are a global medical burden that is expected to increase in the near future. However, the underlying mechanistic processes are poorly understood so far. Herein, we show that the endocannabinoid anandamide (AEA) induces prominent airway relaxation in vitro and in vivo. In contrast to 2-arachidonlyglycerol-induced airway relaxation, this is mediated by fatty acid amide hydrolase (FAAH)-dependent metabolites. In particular, we identify mouse and also human epithelial and airway smooth muscle cells as source of AEA-induced prostaglandin E2 production and cAMP as direct mediator of AEA-dependent airway relaxation. Mass spectrometry experiments demonstrate reduced levels of endocannabinoid-like compounds in lungs of ovalbumin-sensitized mice indicating a pathophysiological relevance of endocannabinoid signalling in obstructive airway disease. Importantly, AEA inhalation protects against airway hyper-reactivity after ovalbumin sensitization. Thus, this work highlights the AEA/FAAH axis as a critical regulator of airway tone that could provide therapeutic targets for airway relaxation.

Effects of sub-chronic, in vivo administration of sigma-1 receptor ligands on platelet and aortic arachidonate cascade in streptozotocin-induced diabetic rats.

BACKGROUND: Diabetes mellitus is a chronic metabolic disorder which induces endothelial dysfunction and platelet activation. Eicosanoids produced from arachidonic acid regulate cellular and vascular functions. Sigma-1 receptors (S1R) are expressed in platelets and endothelial cells and S1R expression is protective in diabetes. OBJECTIVES: Our aim was to examine the influence of sub-chronic, in vivo administered S1R ligands PRE-084, (S)-L1 (a new compound) and NE-100 on the ex vivo arachidonic acid metabolism of platelets and aorta in streptozotocin-induced diabetic rats. METHODS: The serum level of the S1R ligands was detected by LC-MS/MS before the ex vivo analysis. Sigma-1 receptor and cyclooxygenase gene expression in platelets were determined by RT-qPCR. The eicosanoid synthesis was examined with a radiolabelled arachidonic acid substrate and ELISA. RESULTS: One month after the onset of STZ-induced diabetes, in vehicle-treated, diabetic rat platelet TxB2 and aortic 6-k-PGF1α production dropped. Sub-chronic in vivo treatment of STZ-induced diabetes in rats for one week with PRE-084 enhanced vasoconstrictor and platelet aggregator and reduced vasodilator and anti-aggregator cyclooxygenase product formation. (S)-L1 reduced the synthesis of vasodilator and anti-aggregator cyclooxygenase metabolites and promoted the recovery of physiological platelet function in diabetic rats. The S1R antagonist NE-100 produced no significant changes in platelet arachidonic acid metabolism. (S)-L1 decreased the synthesis of vasoconstrictor and platelet aggregator cyclooxygenase metabolites, whereas NE-100 increased the quantity of aortic vasodilator and anti-aggregator cyclooxygenase products and promoted the recovery of diabetic endothelial dysfunction in the aorta. The novel S1R ligand, (S)-L1 had similar effects on eicosanoid synthesis in platelets as the agonist PRE-084 and in aortas as the antagonist NE-100. CONCLUSIONS: S1R ligands regulate cellular functions and local blood circulation by influencing arachidonic acid metabolism. In diabetes mellitus, the cell-specific effects of S1R ligands have a compensatory role and aid in restoring physiological balance between the platelet and vessel.

Diversified Effects of COVID-19 as a Consequence of the Differential Metabolism of Phospholipids and Lipid Peroxidation Evaluated in the Plasma of Survivors and Deceased Patients upon Admission to the Hospital.

As a result of SARS-CoV-2 infection, inflammation develops, which promotes oxidative stress, leading to modification of phospholipid metabolism. Therefore, the aim of this study is to compare the effects of COVID-19 on the levels of phospholipid and free polyunsaturated fatty acids (PUFAs) and their metabolites produced in response to reactions with reactive oxygen species (ROS) and enzymes (cyclooxygenases-(COXs) and lipoxygenase-(LOX)) in the plasma of patients who either recovered or passed away within a week of hospitalization. In the plasma of COVID-19 patients, especially of the survivors, the actions of ROS and phospholipase A2 (PLA2) cause a decrease in phospholipid fatty acids level and an increase in free fatty acids (especially arachidonic acid) despite increased COXs and LOX activity. This is accompanied by an increased level in lipid peroxidation products (malondialdehyde and 8-isoprostaglandin F2α) and lipid mediators generated by enzymes. There is also an increase in eicosanoids, both pro-inflammatory as follows: thromboxane B2 and prostaglandin E2, and anti-inflammatory as follows: 15-deoxy-Δ-12,14-prostaglandin J2 and 12-hydroxyeicosatetraenoic acid, as well as endocannabinoids (anandamide-(AEA) and 2-arachidonylglycerol-(2-AG)) observed in the plasma of patients who recovered. Moreover, the expression of tumor necrosis factor α and interleukins (IL-6 and IL-10) is increased in patients who recovered. However, in the group of patients who died, elevated levels of N-oleoylethanolamine and N-palmitoylethanolamine are found. Since lipid mediators may have different functions depending on the onset of pathophysiological processes, a stronger pro-inflammatory response in patients who have recovered may be the result of the defensive response to SARS-CoV-2 in survivors associated with specific changes in the phospholipid metabolism, which could also be considered a prognostic factor.

Integrated analysis of effect of daisaikoto, a traditional Japanese medicine, on the metabolome and gut microbiome in a mouse model of nonalcoholic fatty liver disease.

Dysregulation of lipid metabolism and diabetes are risk factors for nonalcoholic fatty liver disease (NAFLD), and the gut-liver axis and intestinal microbiome are known to be highly associated with the pathogenesis of this disease. In Japan, the traditional medicine daisaikoto (DST) is prescribed for individuals affected by hepatic dysfunction. Herein, we evaluated the therapeutic potential of DST for treating NAFLD through modification of the liver and stool metabolome and microbiome by using STAM mice as a model of NAFLD. STAM mice were fed a high-fat diet with or without 3 % DST for 3 weeks. Plasma and liver of STAM, STAM with DST, and C57BL/6J ("Normal") mice were collected at 9 weeks, and stools at 4, 6, and 9 weeks of age. The liver pathology, metabolome and stool microbiome were analyzed. DST ameliorated the NAFLD activity score of STAM mice and decreased the levels of several liver lipid mediators such as arachidonic acid and its derivatives. In normal mice, nine kinds of family accounted for 94.1 % of microbiome composition; the total percentage of these family was significantly decreased in STAM mice (45.6 %), and DST administration improved this imbalance in microbiome composition (65.2 %). In stool samples, DST increased ursodeoxycholic acid content and altered several amino acids, which were correlated with changes in the gut microbiome and liver metabolites. In summary, DST ameliorates NAFLD by decreasing arachidonic acid metabolism in the liver; this amelioration seems to be associated with crosstalk among components of the liver, intestinal environment, and microbiome.