Oleoylethanolamide and human neural responses to food stimuli in obesity.

IMPORTANCE: Obesity has emerged as a leading health threat but its biological basis remains insufficiently known, hampering the search for novel treatments. Here, we study oleoylethanolamide, a naturally occurring lipid that has been clearly implicated in weight regulation in animals. However, its role for weight regulation and obesity in humans is still unclear. OBJECTIVE: To investigate associations between plasma oleoylethanolamide levels and body mass index (BMI, calculated as weight in kilograms divided by height in meters squared) and functional magnetic resonance imaging response to food stimuli in obese patients and matched control participants. DESIGN, SETTING, AND PARTICIPANTS: Case-control study of 21 obese patients and 24 matched control participants. Obesity was defined as having a BMI of at least 30. The mean age of participants was 40.8 years and BMIs ranged from 18.2 to 47.5. MAIN OUTCOMES AND MEASURES: Interactions between plasma oleoylethanolamide levels and obesity on BMI and functional magnetic resonance imaging response to food stimuli. RESULTS: Associations between oleoylethanolamide and BMI differed significantly depending on whether individuals were obese or not (P = .02). In obese individuals, oleoylethanolamide showed a trend toward a positive correlation with BMI (P = .06, ρ = 0.42), while this relationship was inverse for nonobese control participants (P = .07, ρ = -0.34). Similarly, we found significant interactions between oleoylethanolamide levels and obesity on food-related brain activation in cortical areas associated with reward processing and interoceptive signaling (P = .009). Specifically, nonobese individuals with higher oleoylethanolamide levels had higher insular brain activity (P < .001, ρ = 0.70); again, the relationship trended to be inverse for obese patients (P = .11, ρ = -0.36). These effects were not associated with plasma levels of leptin and anandamide, suggesting an independent role of oleoylethanolamide in hunger-associated interoceptive signaling. Analysis of food craving during the functional magnetic resonance imaging task suggested that the identified brain areas may be involved in suppressing food-liking reactions in nonobese individuals. CONCLUSIONS AND RELEVANCE: This study suggests that oleoylethanolamide-mediated signaling plays an important role for hedonic regulation of food craving and obesity in humans and thus may be a valuable target for developing novel antiobesity drugs.

Protein-fatty acid complexes: biochemistry, biophysics and function.

Thirteen years ago, α-lactalbumin (α-LA) was first reported to form a complex with oleic acid (OA). This complex, called HAMLET (human α-lactalbumin made lethal to tumour cells), was found to be cytotoxic to cancer cells. In HAMLET, α-LA assumes a partially unfolded conformation and can bind OA in various stoichiometries. Subsequently, different groups have been able to prepare HAMLET-like cytotoxic complexes in different ways, which all involve the destabilization of α-LA, and a number of different proteins have been able to form similar complexes. This suggests that the ability to form stable complexes with lipids may be a generic feature of the polypeptide chain, although the precise structural and functional details may vary from protein to protein. We review the biophysical and biochemical properties of this class of complexes, focusing on different methods of preparation, complex structure and the role of the protein and the lipid within these complexes. The cellular effects of these complexes are multifaceted and depend on the cell types. There are strong indications that OA has an essential role, whereas the protein component, rather than having a toxic effect on its own, functions as a vehicle for transporting the toxic OA to the cells and keeping the OA in solution. Fatty acids alone can affect numerous cellular signalling and metabolic pathways, in addition to playing important roles in immune responses and inflammatory processes. Further studies will aim to determine how the molecular properties of the different protein-lipid complexes correlate with their biological efficacy.

Antinociceptive effects of the N-acylethanolamine acid amidase inhibitor ARN077 in rodent pain models.

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.

Sympathetic activity controls fat-induced oleoylethanolamide signaling in small intestine.

Ingestion of dietary fat stimulates production of the small-intestinal satiety factors oleoylethanolamide (OEA) and N-palmitoyl-phosphatidylethanolamine (NPPE), which reduce food intake through a combination of local (OEA) and systemic (NPPE) actions. Previous studies have shown that sympathetic innervation of the gut is necessary for duodenal infusions of fat to induce satiety, suggesting that sympathetic activity may engage small-intestinal satiety signals such as OEA and NPPE. In the present study, we show that surgical resection of the sympathetic celiac-superior mesenteric ganglion complex, which sends projections to the upper gut, abolishes feeding-induced OEA production in rat small-intestinal cells. These effects are accounted for by suppression of OEA biosynthesis, and are mimicked by administration of the selective ß2-adrenergic receptor antagonist ICI-118,551. We further show that sympathetic ganglionectomy or pharmacological blockade of ß2-adrenergic receptors prevents NPPE release into the circulation. In addition, sympathetic ganglionectomy increases meal frequency and lowers satiety ratio, and these effects are corrected by pharmacological administration of OEA. The results suggest that sympathetic activity controls fat-induced satiety by enabling the coordinated production of local (OEA) and systemic (NPPE) satiety signals in the small intestine.

Circulating endocannabinoids and N-acyl-ethanolamides in patients with sleep apnea--specific role of oleoylethanolamide.

OBJECTIVE: The endocannabinoid system promotes diverse effects on fat and glucose metabolism as well as on energy balance and sleep regulation. The role of N-acylethanolamides like oleoylethanolamide (OEA) and other endocannabinoids such as anandamide (AEA) and 2-arachidonyl-glycerol (2-AG) has not yet been investigated in patients with sleep apnea. DESIGN AND METHODS: We measured circulating OEA, AEA and 2-AG in patients with sleep apnea (n = 20) and healthy control subjects (n = 57). Respiratory distress index (RDI) as measured by polysomnography was used as a quantitative index of sleep apnea. RESULTS: In patients with sleep apnea OEA serum concentrations were significantly higher than in control subjects (8.4 pmol/ml (95% CI 6.9;9.9) vs. 4.0 (3.5;4.5); p<0.0001, adjusted for body mass index (BMI), fasting insulin, HDL and LDL cholesterol). In contrast, AEA (2.9 (95% CI 1.9;3.9) vs. 1.8 (1.4;2.1), p = 0.09) and 2-AG (20.0 (-14.5;54.5) vs. 32.8 (21.4;44.2), p = 0.56) were not significantly different between patients with sleep apnea and control subjects after adjustment. In the sleep apnea group, OEA serum concentrations were associated with RDI (r (2) = 0.28, p = 0.02) and BMI (r (2) = 0.32, p = 0.01). However, OEA was not associated with BMI in the control group (p = 0.10). CONCLUSIONS: These results indicate that among the three analyzed fatty acid derivatives, OEA plays a specific role in patients with sleep apnea. Together with animal data, the 2-fold elevation of OEA serum concentrations could be interpreted as a neuroprotective mechanism against chronic oxidative stressors and a mechanism to promote wakefulness in patients with nocturnal sleep deprivation and daytime hypersomnolence.

Fat-induced satiety factor oleoylethanolamide enhances memory consolidation.

The ability to remember contexts associated with aversive and rewarding experiences provides a clear adaptive advantage to animals foraging in the wild. The present experiments investigated whether hormonal signals released during feeding might enhance memory of recently experienced contextual information. Oleoylethanolamide (OEA) is an endogenous lipid mediator that is released when dietary fat enters the small intestine. OEA mediates fat-induced satiety by engaging type-alpha peroxisome proliferator-activated receptors (PPAR-alpha) in the gut and recruiting local afferents of the vagus nerve. Here we show that post-training administration of OEA in rats improves retention in the inhibitory avoidance and Morris water maze tasks. These effects are blocked by infusions of lidocaine into the nucleus tractus solitarii (NTS) and by propranolol infused into the basolateral complex of the amygdala (BLA). These findings suggest that the memory-enhancing signal generated by OEA activates the brain via afferent autonomic fibers and stimulates noradrenergic transmission in the BLA. The actions of OEA are mimicked by PPAR-alpha agonists and abolished in mutant mice lacking PPAR-alpha. The results indicate that OEA, acting as a PPAR-alpha agonist, facilitates memory consolidation through noradrenergic activation of the BLA, a mechanism that is also critically involved in memory enhancement induced by emotional arousal.

Nitrooleic acid, an endogenous product of nitrative stress, activates nociceptive sensory nerves via the direct activation of TRPA1.

Transient Receptor Potential A1 (TRPA1) is a nonselective cation channel, preferentially expressed on a subset of nociceptive sensory neurons, that is activated by a variety of reactive irritants via the covalent modification of cysteine residues. Excessive nitric oxide during inflammation (nitrative stress), leads to the nitration of phospholipids, resulting in the formation of highly reactive cysteine modifying agents, such as nitrooleic acid (9-OA-NO(2)). Using calcium imaging and electrophysiology, we have shown that 9-OA-NO(2) activates human TRPA1 channels (EC(50), 1 microM), whereas oleic acid had no effect on TRPA1. 9-OA-NO(2) failed to activate TRPA1 in which the cysteines at positions 619, 639, and 663 and the lysine at 708 had been mutated. TRPA1 activation by 9-OA-NO(2) was not inhibited by the NO scavenger carboxy-PTIO. 9-OA-NO(2) had no effect on another nociceptive-specific ion channel, TRPV1. 9-OA-NO(2) activated a subset of mouse vagal and trigeminal sensory neurons, which also responded to the TRPA1 agonist allyl isothiocyanate and the TRPV1 agonist capsaicin. 9-OA-NO(2) failed to activate neurons derived from TRPA1(-/-) mice. The action of 9-OA-NO(2) at nociceptive nerve terminals was investigated using an ex vivo extracellular recording preparation of individual bronchopulmonary C fibers in the mouse. 9-OA-NO(2) evoked robust action potential discharge from capsaicin-sensitive fibers with slow conduction velocities (0.4-0.7 m/s), which was inhibited by the TRPA1 antagonist AP-18. These data demonstrate that nitrooleic acid, a product of nitrative stress, can induce substantial nociceptive nerve activation through the selective and direct activation of TRPA1 channels.

Milk production and composition from cows fed small amounts of fish oil with extruded soybeans.

Eight Holstein (189 +/- 57 DIM) and 4 Brown Swiss (126 +/- 49 DIM) multiparous cows were used in a replicated 4 x 4 Latin square with 28-d periods to determine the minimal dietary concentration of fish oil necessary to maximize milk conjugated linoleic acid (CLA) and vaccenic acid (VA). Treatments consisted of a control diet with a 50:50 ratio of forage to concentrate (dry matter basis), and 3 diets with 2% added fat consisting of 0.33% fish oil, 0.67% fish oil, and 1% fish oil with extruded soybeans providing the balance of added fat. Dry matter intake (23.1, 22.6, 22.8, and 22.9 kg/d, for control, low, medium, and high fish oil diets, respectively) was similar for all diets. Milk production (21.5, 23.7, 22.7, and 24.2 kg/d) was higher for cows fed the fat-supplemented diets vs. the control. Milk fat (4.42, 3.81, 3.80, and 4.03%) and true protein (3.71, 3.58, 3.54, and 3.55%) concentrations decreased when cows were fed diets containing supplemental fat. Concentration of milk cis-9,trans-11 CLA (0.55, 1.17, 1.03, and 1.19 g/100 g of fatty acids) was increased similarly by all diets containing supplemental fat. Milk VA (1.12, 2.47, 2.13, and 2.63 g/100 g of fatty acids) was increased most in milk from cows fed the low and high fish oil diets. Milk total n-3 fatty acids were increased (0.82, 0.96, 0.92, and 1.01 g/100 g of fatty acids) by all fat-supplemented diets. The low fish oil diet was as effective at increasing VA and CLA in milk as the high fish oil diet, showing that only low concentrations of dietary fish oil are necessary for increasing concentrations of VA and CLA in milk.

[The endocannabinoid system and food intake control]. / Sistema endocannabinoide y control de la ingesta.

Acylethanolamides are endogenous compounds with lipid structure including anandamide (AEA), palmitoilethanolamide, oleylamide and oleylethanolamide (OEA). AEA binds to the cannabinoid receptor CB1, located at the central nervous system, while OEA is an endogenous ligand for the alpha subtype of peroxisome-proliferator activating receptor (PPARalpha). Since AEA acts on the same receptor which binds marihuana active derivatives, this group of compounds were called endocannabinoids. Besides typical central effects of cannabinoids, CB1 receptor activation leads to hyperphagia, whereas its pharmacological blockade is followed by changes in energy metabolism favouring substrate oxidation. OEA has inhibitory effects on food intake by acting on PPARalpha receptors which modulate the autonomous nervous system. Both acylethanolamides, AEA and OEA, have opposite effects suggesting that they form part of a satiety sensor system. Whereas fasting triggers AEA release and inhibits OEA synthesis, eating has the reverse effect. Additionally OEA is also produced by adipocytes ad has some effects on lipid metabolism. All these data suggest a role for acylethanolamides and the endocannabinoid system in the pathophysiology of obesity, diabetes and atherosclerosis.

Metabolism of 1-acyl-2-oleoyl-sn-glycero-3-phosphoethanolamine in castor oil biosynthesis.

We have examined the role of 2-oleoyl-PE (phosphatidylethanolamine) in the biosynthesis of triacylglycerols (TAG) by castor microsomes. In castor microsomal incubation, the label from 14C-oleate of 1-palmitoyl-2-[1-(14)C]oleoyl-sn-glycero-3-phosphoethanolamine is incorporated into TAG containing ricinoleate. The enzyme characteristics, such as optimal pH, and the effect of incubation components of the oleoyl-12-hydroxylase using 2-oleoyl-PE as incubation substrate are similar to those for 2-oleoyl-PC (phosphatidylcholine). However, compared to 2-oleoyl-PC, 2-oleoyl-PE is a less efficient incubation substrate of oleoyl-12-hydroxylase in castor microsomes. Unlike 2-oleoyl-PC, 2-oleoyl-PE is not hydroxylated to 2-ricinoleoyl-PE by oleoyl-12-hydroxylase and is not desaturated to 2-linoleoyl-PE by oleoyl-12-desaturase. We have demonstrated the conversion of 2-oleoyl-PE to 2-oleoyl-PC and vice versa. The incorporation of label from 2-[14C]oleoyl-PE into TAG occurs after its conversion to 2-oleoyl-PC, which can then be hydroxylated or desaturated. We detected neither PE-N-monomethyl nor PE-N,N-dimethyl, the intermediates from PE to PC by N-methylation. The conversion of 2-oleoyl-PE to 2-oleoyl-PC likely occurs via hydrolysis to 1,2-diacyl-sn-glycerol by phospholipase C and then by cholinephosphotransferase. This conversion does not appear to play a key role in driving ricinoleate into TAG.

Fatty acid amide hydrolase expression in rat choroid plexus: possible role in regulation of the sleep-inducing action of oleamide.

The enzyme fatty acid amide hydrolase (FAAH) catalyses hydrolysis of oleamide, a sleep-inducing lipid whose concentration in the cerebrospinal fluid (CSF) is elevated in sleep-deprived mammals. Previous studies have reported expression of FAAH by distinct populations of neurons in the rat brain. Here we demonstrate using immunocytochemical methods that FAAH is also expressed by non-neuronal epithelial cells of the rat choroid plexus. The choroid plexus is formed by invaginations of the pia mater into the ventricle cavities of the brain and an important function of the choroidal epithelium is to regulate production and composition of CSF. Therefore, the role of FAAH in epithelial cells of the choroid plexus may be to control the concentration of oleamide in the CSF and as such FAAH may exert an important regulatory role in shaping the duration and magnitude of the sleep-inducing effect of endogenously or exogenously derived oleamide.

Effect of ileal oleate on interdigestive intestinal motility of the dog.

To determine the effect of ileal oleate on fasting intestinal motility, pairs of duodenal and ileal catheters and bipolar duodenal and jejunal seromuscular electrodes were surgically implanted in six dogs. The ileum was perfused with either normal saline (154 mM NaCl) or oleic acid emulsion (152 mM), while intestinal myoelectric activity was continuously monitored. For transit studies, a bolus of [3H]PEG was injected into the duodenum, and jejunal and ileal alliquots were collected every 15 min for a 6-hr study period. Plasma samples were collected for radioimmunoassays of peptide YY and enteroglucagon. Ileal oleate infusion increased the MMC cycle length and decreased the number of MMCs (P < 0.001) and the myoelectric spike-burst frequency/10 min in the duodenum (P < 0.05). Both duodenal-jejunal (P < 0.05) and duodenal-ileal transit (P < 0.01) were delayed markedly by ileal perfusion with oleic acid emulsion as compared to control studies. Ileal oleate increased plasma levels of peptide YY (P < 0.01) and enteroglucagon (P < 0.01). Ileal perfusion with oleate therefore activated the so-called "ileal brake," diminishing duodenal myoelectric spike bursts and slowing intestinal transit while concurrently increasing plasma levels of peptide YY and enteroglucagon.

Modification of K+ channel properties induced by fatty acids in neuroblastoma cells.

The effects of fatty acids on voltage-dependent potassium (K+) channels in neuroblastoma cells were studied using the whole-cell current recording technique. At a concentration of 5 microM, unsaturated and medium chain length (C10-C14) saturated fatty acids accelerated the apparent inactivation of the K+ current. This effect was reversed by albumin. In the absence of exogenous fatty acids, albumin slowed the inactivation of the K+ current. The acceleration of the K+ current inactivation induced by unsaturated fatty acids was associated with an increase in the sensitivity of K+ channels to 4-amino-pyridine. It is concluded that kinetic and pharmacological properties of K+ channels are, in part, controlled by membrane fatty acids which, in this way, should contribute to an apparent diversity of K+ channels and the modulation of cell excitability.

The stimulatory effect of albumin on luteinizing hormone-stimulated Leydig cell steroid production depends on its fatty acid content and correlates with conformational changes.

The effects of purified albumin species and albumin fragments (0.2-1% w/v) on short-term (4 h) steroid secretion by immature rat Leydig cells, in the presence of a maximally stimulating dose of luteinizing hormone (LH), were investigated. Human albumin and the peptic fragment (comprising residues 1-387) enhanced pregnenolone production in isolated rat Leydig cells, whereas chicken albumin and the tryptic fragment (comprising residues 198-585) were not active. This stimulatory effect of human albumin and the peptic fragment correlated with the potential of these proteins to undergo a pH-dependent neutral-to-base transition as measured by circular dichroism. The tryptic fragment and chicken albumin did not have the potential to undergo such a transition. The pH-dependent conformational changes of albumin and fragments thereof occurred in parallel with a change in the binding affinity for testosterone and pregnenolone. The fatty acid oleic acid and the drug suramin, only when present in a molar ligand-to-albumin ratio equal to or higher than 2, inhibited the albumin-mediated stimulation of steroid production. These data show that the stimulatory effects of albumin species on LH-induced Leydig cell pregnenolone production depend on their fatty acid content and correlate with the potential of these molecules to undergo conformational changes. It is unknown via which mechanisms albumin exerts its stimulatory effect, but the LH action through the cyclic AMP pathway seems not to be affected.

Fatty acids markedly lower the threshold for halothane-induced calcium release from the terminal cisternae in human and porcine normal and malignant hyperthermia susceptible skeletal muscle.

Malignant hyperthermia is caused by an abnormal increase in Ca2+ levels in skeletal muscle in response to anesthetics, including halothane. Since fatty acid production is elevated in skeletal muscle from individuals with malignant hyperthermia, the effects of fatty acids on the threshold of halothane-induced Ca2+ release were examined. In the absence of fatty acids halothane caused Ca2+ release from porcine and human heavy sarcoplasmic reticulum fractions, but only at concentrations above the clinically relevant range. Oleic acid (20 microM), an unsaturated fatty acid, reduced the threshold at which halothane induced Ca2+ release to concentrations used for anesthesia. Stearic acid, a saturated fatty acid had considerably less effect on the threshold of halothane action. The greater sensitivity of malignant hyperthermia muscle to halothane can be explained by elevated fatty acid production.

The effect of non-esterified fatty acids on vascular ADP-degrading enzyme activity.

Following our observations that non-esterified fatty acids (NEFAs) inhibit prostacyclin (PGI2) synthesis and accelerate PGI2 degradation, we have examined the possibility that NEFAs may also affect the activity of vascular ADPase, which converts the platelet pro-aggregatory adenosine diphosphate (ADP) to adenosine, an inhibitor of aggregation and a vasodilator. Incubation, in buffer solutions, of NEFAs with intact rat aortic rings significantly inhibited vascular ADPase activity. This inhibition was more marked at higher NEFA concentrations and with unsaturated fatty acids (linoleic, oleic) than with a saturated fatty acid (stearic). This NEFA-mediated inhibition of vascular ADPase activity could be prevented by the prior addition of fatty acid-free human albumin to the incubate. Similarly, the vascular rings recovered from NEFA-mediated inhibition by washing and further incubation in NEFA-free buffer. Therefore, elevated NEFA concentrations inhibit, reversibly, an enzyme system which is thought to protect the vascular endothelium. The NEFA-mediated inhibition of ADPase activity was also confirmed following incubation of rat aortic rings in human serum enriched with exogenous NEFA. These findings provide further evidence that NEFAs may contribute to the pathogenesis of vascular disease associated with diabetes mellitus and of other conditions where an elevation of serum NEFA concentrations occurs.

Effect of fatty acid modification of cultured hepatoma cells on susceptibility to natural killer cells.

Rat hepatoma (Morris 7777) cells modified with either oleic or linoleic acid exhibited greater susceptibility to normal spleen cell-mediated lysis in a 16-hr 51Cr release assay. At effector:target cell ratios of 300:1, the specific lysis of fatty acid-enriched target cells (cultured for 2 days in fatty acid-supplemented medium) by the normal rat spleen cells was 60% higher than the untreated target cells (P less than 0.01). Prolonging the culture in fatty acid-supplemented medium up to 6 days produced similar effects. Analysis of the fatty acid composition of cellular lipids revealed that an elevation of oleic or linoleic acid was the only significant alteration in the hepatoma cells grown in the oleic or linoleic acid-supplemented medium, respectively. The percentage of the acids was increased in the total cellular phospholipids, the choline, ethanolamine, serine, and inositol phosphoglyceride fractions, and the neutral lipids. In conclusion, we suggest that the elevation of oleic acid and linoleic acid contents in the membranes of the fatty acid-modified hepatoma cells may contribute to the increased susceptibility of these cells to natural killer cell-mediated cytotoxicity.