A Cannabinoid Receptor-Mediated Mechanism Participates in the Neuroprotective Effects of Oleamide Against Excitotoxic Damage in Rat Brain Synaptosomes and Cortical Slices.

A number of physiological responses in the central nervous system (CNS) are regulated by the endocannabinoid system (ECS). Inhibition of neuronal excitability via activation of cannabinoid receptors (CBr) constitutes a potential protective response against neurotoxic insults. Oleamide (ODA) is a fatty acid amide with endocannabinoid profile exerting several effects in the CNS, though its neuroprotective properties remain unknown. The tryptophan metabolite quinolinic acid (QUIN) elicits toxic effects via overactivation of N-methyl-D-aspartate receptors (NMDAr) after its accumulation in the CNS under pathological conditions. Here, we investigated the protective properties of ODA against the excitotoxic damage induced by QUIN in rat brain synaptosomes and cortical slices, and whether these effects are linked to the stimulation of the endocannabinoid system via CB1 and/or CB2 receptor activation. ODA (1-50 µM) prevented the QUIN (100 µM)-induced loss of mitochondrial reductive capacity in synaptosomes in a mechanism partially mediated by CB1 receptor, as evidenced by the recovery of mitochondrial dysfunction induced by co-incubation with the CB1 receptor antagonist/inverse agonist AM281 (1 µM). In cortical slices, ODA prevented the short-term QUIN-induced loss of cell viability and the cell damage in a partial CB1 and CB2 receptor-dependent manner. Altogether, these findings demonstrate the neuroprotective and modulatory properties of ODA in biological brain preparations exposed to excitotoxic insults and the partial role that the stimulation of CB1 and CB2 receptors exerts in these effects.

N-Oleoyl-glycine reduces nicotine reward and withdrawal in mice.

Cigarette smokers with brain damage involving the insular cortex display cessation of tobacco smoking, suggesting that this region may contribute to nicotine addiction. In the present study, we speculated that molecules in the insular cortex that are sensitive to experimental traumatic brain injury (TBI) in mice might provide leads to ameliorate nicotine addiction. Using targeted lipidomics, we found that TBI elicited substantial increases of a largely uncharacterized lipid, N-acyl-glycine, N-oleoyl-glycine (OlGly), in the insular cortex of mice. We then evaluated whether intraperitoneal administration of OlGly would alter withdrawal responses in nicotine-dependent mice as well as the rewarding effects of nicotine, as assessed in the conditioned place preference paradigm (CPP). Systemic administration of OlGly reduced mecamylamine-precipitated withdrawal responses in nicotine-dependent mice and prevented nicotine CPP. However, OlGly did not affect morphine CPP, demonstrating a degree of selectivity. Our respective in vitro and in vivo observations that OlGly activated peroxisome proliferator-activated receptor alpha (PPAR-α) and the PPAR-α antagonist GW6471 prevented the OlGly-induced reduction of nicotine CPP in mice suggests that this lipid acts as a functional PPAR-α agonist to attenuate nicotine reward. These findings raise the possibility that the long chain fatty acid amide OlGly may possess efficacy in treating nicotine addiction.

Regulation of GPR119 receptor activity with endocannabinoid-like lipids.

The GPR119 receptor plays an important role in the secretion of incretin hormones in response to nutrient consumption. We have studied the ability of an array of naturally occurring endocannabinoid-like lipids to activate GPR119 and have identified several lipid receptor agonists. The most potent receptor agonists identified were three N-acylethanolamines: oleoylethanolamine (OEA), palmitoleoylethanolamine, and linoleylethanolamine (LEA), all of which displayed similar potency in activating GPR119. Another lipid, 2-oleoylglycerol (2-OG), also activated GPR119 receptor but with significantly lower potency. Endogenous levels of endocannabinoid-like lipids were measured in intestine in fasted and refed mice. Of the lipid GPR119 agonists studied, the intestinal levels of only OEA, LEA, and 2-OG increased significantly upon refeeding. Intestinal levels of OEA and LEA in the fasted mice were low. In the fed state, OEA levels only moderately increased, whereas LEA levels rose drastically. 2-OG was the most abundant of the three GPR119 agonists in intestine, and its levels were radically elevated in fed mice. Our data suggest that, in lean mice, 2-OG and LEA may serve as physiologically relevant endogenous GPR119 agonists that mediate receptor activation upon nutrient uptake.

Cardioprotective effect of a dual acting epoxyeicosatrienoic acid analogue towards ischaemia reperfusion injury.

BACKGROUND AND PURPOSE: Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid that are metabolized into dihydroxyepoxyeicosatrienoic acids (DHET) by soluble epoxide hydrolase (sEH). The current investigations were performed to examine the cardioprotective effects of UA-8 (13-(3-propylureido)tridec-8-enoic acid), a synthetic compound that possesses both EET-mimetic and sEH inhibitory properties, against ischaemia-reperfusion injury. EXPERIMENTAL APPROACH: Hearts from C57BL/6 mice were perfused in Langendorff mode and subjected to ischaemia reperfusion. Mechanistic studies involved co-perfusing hearts with either 14,15-EEZE (a putative EET receptor antagonist), wortmannin or PI-103 (class-I PI3K inhibitor). H9c2 cells were utilized to investigate the protective effects against mitochondrial injury following anoxia reoxygenation. KEY RESULTS: Perfusion of UA-8 significantly improved postischaemic left ventricular developed pressure (LVDP) and reduced infarction following ischaemia reperfusion compared with control and 11,12-EET. UA-7 (13-(2-(butylamino)-2-oxoacetamido)tridec-8(Z)-enoic acid), a compound lacking sEH inhibitory properties, also improved postischaemic LVDP, while co-perfusion with 14,15-EEZE, wortmannin or PI-103 attenuated the improved recovery. UA-8 prevented anoxia-reoxygenation induced loss of mitochondrial membrane potential and cell death in H9c2 cells, which was blocked by co-treatment of PI-103. CONCLUSIONS AND IMPLICATIONS: UA-8 provides significant cardioprotection against ischaemia reperfusion injury. The effects are attributed to EETs mimetic properties, which limits mitochondrial dysfunction via class-I PI3K signalling.

Mono-unsaturated fatty acids protect against beta-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure.

Long-chain saturated fatty acids are cytotoxic to pancreatic beta-cells while shorter-chain saturated and long-chain unsaturated molecules are better tolerated. Mono-unsaturated fatty acids are not, however, inert since they inhibit the pro-apoptotic effects of saturated molecules. In the present work we show that the mono-unsaturates palmitoleate (C16:1) or oleate (C18:1) also cause marked inhibition of apoptosis induced by exposure of clonal BRIN-BD11 beta-cells to serum withdrawal or a combination of interleukin-1beta plus interferon-gamma. This response was dose-dependent and not accompanied by changes in NO formation. Taken together, the results suggest that mono-unsaturated fatty acids regulate a distal step common to several apoptotic pathways in pancreatic beta-cells.

The serotonergic system may be involved in the sleep-inducing action of oleamide in rats.

The mechanism underlying the sleep-inducing effect of oleamide, an endogenous fatty acid amide, was studied in rats. Animals implanted with cerebrocortical and dorsal neck muscle electrodes were monitored continuously by electroencephalograph (EEG) and electromyograph (EMG) for 4 h after i.p. or s.c. injection of drugs. Oleamide induced a dose-dependent increase in slow-wave sleep (SWS), a decrease in wakefulness (W) and sleep latency, but had no effect on rapid-eye-movement sleep (REMS). The oleamide-induced increase in SWS was prevented by 5-HT reuptake inhibitors such as fluoxetine or fenfluramine and by agonists at 5-HT(1A) receptors such as buspirone or 8-hydroxy-2-(di- N-propylamino) tetralin (8-OH-DPAT). Moreover, the selective 5-HT(1A) receptor antagonist WAY100635 markedly antagonized the suppression of the oleamide-induced increase in SWS by 8-OH-DPAT. These data provide the first behavioural evidence that the serotonergic system may be involved in the sleep-inducing action of oleamide in rats.

Protective effect of cobra venom factor on pulmonary injury induced by oleic acid.

The protective effect of cobra venom factor (CVF), separated from the venom of Naja naja atra, on pulmonary injury induced by oleic acid was reported. The blood gas tensions, tidal volume, transthoracic pressure and pulmonary arterial pressure in anaesthetized dogs were continually monitored. The experimental results showed that CVF, given 20 h before the dosage depleting complements exceed 95%, significantly attenuated oleic acid-induced pulmonary dysfunction, including hypoxemia, increasing veno-arterial shunt and P(A-a)O2 and decreasing dynamic compliance and pulmonary blood flow. Histological examination of lung tissues after the experiment also showed improvement of hyaline membrane formation, alveolar haemorrhage and pulmonary arteriolar thrombosis. It was evident that the depletion of serum complements by CVF inhibited the development of lung injury induced by oleic acid. CVF might be a potentially useful drug for the treatment of respiratory distress syndrome in the near future.

Interaction of LY171883 and other peroxisome proliferators with fatty-acid-binding protein isolated from rat liver.

Fatty-acid-binding protein (FABP) is a 14 kDa protein found in hepatic cytosol which binds and transports fatty acids and other hydrophobic ligands throughout the cell. The purpose of this investigation was to determine whether LY171883, a leukotriene D4 antagonist, and other peroxisome proliferators bind to FABP and displace an endogenous fatty acid. [3H]Oleic acid was used to monitor the elution of FABP during chromatographic purification. [14C]LY171883 had a similar elution profile when substituted in the purification, indicating a common interaction with FABP. LY171883 and its structural analogue, LY189585, as well as the hypolipidaemic peroxisome proliferators clofibric acid, ciprofibrate, bezafibrate and WY14,643, displaced [3H]oleic acid binding to FABP. Analogues of LY171883 that do not induce peroxisome proliferation only weakly displaced oleate binding. [3H]Ly171883 bound directly to FABP with a Kd of 10.8 microM, compared with a Kd of 0.96 microM for [3H]oleate. LY171883 binding was inhibited by LY189585, clofibric acid, ciprofibrate and bezafibrate. These findings demonstrate that peroxisome proliferators, presumably due to their structural similarity to fatty acids, are able to bind to FABP and displace an endogenous ligand from its binding site. Interaction of peroxisome proliferators with FABP may be involved in perturbations of fatty acid metabolism caused by these agents as well as in the development of the pleiotropic response of peroxisome proliferation.

Prevention of inhibitory effect of free fatty acids on insulin binding and action in isolated rat hepatocytes by etomoxir.

We recently demonstrated a marked inhibitory effect of high physiological concentrations of free fatty acids (FFAs) on insulin binding, degradation, and action in isolated rat hepatocytes. To elucidate the mechanism, male rats were treated for 3 days with saline (control) or etomoxir (ethyl 2-[6-(p-chlorophenoxy)hexyl]-glycidate), a prodrug, which in vivo is converted to a specific competitive inhibitor of carnitine palmitoyltransferase, and thus, lipid oxidation. Oleic acid (0.4 mM) reduced both 125-I-labeled insulin binding and insulin-stimulated [14C]aminoisobutyric acid transport approximately 40% in cells from control animals. However, this FFA concentration was without effect in cells from etomoxir-treated animals. Etomoxir increased EC50 for the inhibitory effect of oleic acid on insulin binding approximately threefold. The data indicate that the mitochondrial oxidation of fatty acids may be important for their inhibitory effect on insulin binding and action in isolated rat hepatocytes.

Injury induced by fatty acids or bile acid in isolated human colonocytes prevented by calcium.

Measurement of the modulation of the growth fraction of isolated normal colonocytes from adult subjects in primary monolayer culture was used as a sensitive quantitative assay to evaluate toxic effects of several endogenous compounds found within the colon. This assay was used to study the role of CaCl2 in blocking cell injury. When added simultaneously with the injurious agent, 5-10 mM CaCl2 blocked the toxicity of physiological concentrations of deoxycholic acid, oleic acid, palmitic acid and linoleic acid.

Indomethacin and dexamethasone decrease oleic acid-induced pulmonary protein leak in rabbits.

Similarities between oleic acid (OA)-induced pulmonary injury and clinical adult respiratory distress syndrome (ARDS) have resulted in extensive use of this model. Using technetium 99m (Tc-99m)-labeled human serum albumin (Tc-HSA) we examined the effect of indomethacin (a prostaglandin synthetase inhibitor) and dexamethasone (a corticosteroid) alone and in combination on OA-induced pulmonary protein leak. Computer-acquired dynamic gamma camera imaging before (15 min), during, and after (60 min) OA infusion were used to generate time-activity curves for lung and heart regions. A lung:heart activity ratio curve with a positive slope indicates pulmonary capillary protein leak of the labeled substance. Tc-99m labeling of red blood cells followed by OA injury showed no significant change in slope, indicating that lung hemorrhage was not being measured; however, Tc-HSA showed significant protein leakage following OA injury. Pretreatment with indomethacin or dexamethasone did not significantly alter either the preinsult or the postinsult slope. Combined pretreatment with indomethacin and dexamethasone significantly decreased, but did not eliminate, the pulmonary protein leak produced by OA injury. Our results indicate that multiple factors are involved in the production of the pulmonary capillary leak in OA-induced lung injury. In addition to the possible therapeutic efficacy of combined corticosteroids and nonsteroidal antiinflammatory drugs, our results demonstrate that these substances may be useful in defining the pathophysiology involved in permeability pulmonary edema.

Partial purification of fatty-acid binding protein by ammonium sulphate fractionation.

By fractionation of rat liver cytosol with 70% saturation ammonium sulphate, a soluble fraction showing high affinity for oleic acid was obtained. The binding of oleic acid to this fraction was inhibited by flavaspidic acid. The molecular weight of the main protein present in this fraction was 12 000 as determined by SDS-poly-acrylamide-gel electrophoresis. This soluble fraction stimulated the transfer of oleic acid from microsomes to phosphatidylcholine liposomes as demonstrated by a transfer assay in vitro. The behaviour of this fraction is similar to that described for fatty-acid binding protein.

Heptakis(2,6-O-dimethyl)beta-cyclodextrin: a novel growth stimulant for Bordetella pertussis phase I.

The effect of cyclodextrins on the growth of Bordetella pertussis Tohama phase I in synthetic medium was evaluated. The addition of cyclodextrins, especially heptakis(2,6-O-dimethyl)beta-cyclodextrin (Me beta CD), to a complete synthetic medium such as Stainer-Scholte medium gave the same number of individual colonies and growth rates as those on Bordet-Gengou medium. Furthermore, with the addition of Me beta CD, growth inhibition by fatty acids such as oleic or palmitic acid was overcome and normal cell growth was observed. This modified Stainer-Scholte medium, designated as cyclodextrin solid medium (CSM), supported excellent growth of 20 lyophilized clinical isolates. Serotypes of the organisms after 10 passages on this CSM plate were not changed. These results suggest that Me beta CD is a significant growth stimulant and CSM is one of the most suitable synthetic media for culture of B. pertussis phase I.

Reversible effects of fatty acids on respiration, oxidative phosphorylation, and heat production of rat liver mitochondria.

1. Oleic acid at low concentrations (0--70 nmol/mg protein) stimulated mitochondrial state 4 respiration 4-fold, increased the apparent enthalpy change of the respiration per gram atom of oxygen consumed from -112 to -208 kJ/O and completely inhibited ATP synthesis without significant effect on the Mg-ATPase activity of mitochondria. 2. Similar effects on mitochondrial respiratory activities were observed with other fatty acids. 3. Bovine serum albumin (BSA) protected mitochondria from the effects of oleic acid irrespective of the order of addition of oleic acid and BSA to mitochondria. The capacity of BSA to bind oleic acid was calculated to be 3.6--7.1 (mean, 4.9) mol of oleic acid/mol of BSA. 4. The response time of mitochondrial respiration to added oleic acid or BSA was 20--25 s.

[Restoration of glucose utilization in alloxan diabetes by means of inhibiting fatty acid oxidation and gluconeogenesis]. / Vosstanovlenie utilizatsii gliukozy pri alloksanovom diabete putem ingibirovaniia okisleniia zhirkykh kislot i gliukoneogeneza.

Experiments on intact hungry rats showed hydrazine, gluconeongenesis inhibitor, to cause hypoglycemia; it failed to influence glucose utilization and oxidation, and sharply decreased oleate exidation. Hydrazine inhibition of fatty acids (oleate) oxidation and of gluconeogenesis led to practical normalization of blood glucose level, and also to the utilization and oxidation of glucose in alloxan diabetes. It is postulated that agents with an analogous action mechanism could prove to be effective antidiabetic agents.