Antonia Serrano

Oleylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-α

Oleylethanolamide (OEA) is a naturally occurring lipid that regulates satiety and body weight. Although structurally related to the endogenous cannabinoid anandamide, OEA does not bind to cannabinoid receptors and its molecular targets have not been defined. Here we show that OEA binds with high affinity to the peroxisome-proliferator-activated receptor-α (PPAR-α), a nuclear receptor that regulates several aspects of lipid metabolism. Administration of OEA produces satiety and reduces body weight gain in wild-type mice, but not in mice deficient in PPAR-α. Two distinct PPAR-α agonists have similar effects that are also contingent on PPAR-α expression, whereas potent and selective agonists for PPAR-γ and PPAR-β/δ are ineffective. In the small intestine of wild-type but not PPAR-α-null mice, OEA regulates the expression of several PPAR-α target genes: it initiates the transcription of proteins involved in lipid metabolism and represses inducible nitric oxide synthase, an enzyme that may contribute to feeding stimulation. Our results, which show that OEA induces satiety by activating PPAR-α, identify an unexpected role for this nuclear receptor in regulating behaviour, and raise possibilities for the treatment of eating disorders.

Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects

2-Arachidonoylglycerol (2-AG) and anandamide are endocannabinoids that activate cannabinoid receptors CB1 and CB2. Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that, for anandamide, is mediated by fatty acid amide hydrolase (FAAH) and, for 2-AG, is thought to involve monoacylglycerol lipase (MAGL). FAAH inhibitors produce a select subset of the behavioral effects observed with CB1 agonists, intimating a functional segregation of endocannabinoid signaling pathways in vivo. Testing this hypothesis, however, requires specific tools to independently block anandamide and 2-AG metabolism. Here, we report a potent and selective inhibitor of MAGL, JZL184, that, upon administration to mice, raises brain 2-AG by 8-fold without altering anandamide. JZL184-treated mice exhibited a broad array of CB1-dependent behavioral effects, including analgesia, hypothermia, and hypomotility. These data indicate that 2-AG endogenously modulates several behavioral processes classically associated with the pharmacology of cannabinoids and point to overlapping and unique functions for 2-AG and anandamide in vivo.

Antiobesity effects of the novel in vivo neutral cannabinoid receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,4-triazole - LH 21

The present study evaluates the pharmacological profile of the new neutral cannabinoid CB1 receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,4-triazole -LH-21- on feeding behavior and alcohol self-administration in rats, two behaviors inhibited by cannabinoid CB1 receptor antagonists. Administration of LH-21 (0.03, 0.3 and 3 mg/kg) to food-deprived rats resulted in a dose-dependent inhibition of feeding. Subchronic administration of LH-21 reduced food intake and body weight gain in obese Zucker rats. Acute effects on feeding were not associated with anxiety-like behaviors, or induction of complex motor behaviors such as grooming or scratching sequences, usually observed after central administration of cannabinoid receptor blockers with inverse agonist properties. LH-21 did not markedly reduce alcohol self-administration (30% reduction observed only at a high dose of 10 mg/kg). This pharmacological pattern partially overlaps that of the reference cannabinoid CB1 receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide, SR141716A, (0.3, 1 and 3 mg/kg) that reduced feeding and alcohol self-administration with similar efficacy. In vitro analysis of blood-brain barrier permeability using a parallel artificial membrane permeation assay demonstrated that LH-21 has lower permeation through membranes than SR141716A. That was confirmed in vivo by studies showing lower potency of peripherally injected LH-21 when compared to SR141716A to antagonize motor depression induced by intracerebroventricular administration of the CB1 agonist CP55,940. The neutral antagonist profile and the lower penetration into the brain of LH-21 favour this class of antagonists with respect to reference inverse agonists for the treatment of obesity because they potentially will display reduced side effects.

ENDOCANNABINOID INFLUENCE IN DRUG REINFORCEMENT, DEPENDENCE AND ADDICTION-RELATED BEHAVIORS

The endogenous cannabinoid system is an important regulatory system involved in physiological homeostasis. Endocannabinoid signaling is known to modulate neural development, immune function, metabolism, synaptic plasticity and emotional state. Accumulating evidence also implicates brain endocannabinoid signaling in the etiology of drug addiction which is characterized by compulsive drug seeking, loss of control in limiting drug intake, emergence of a negative emotional state in the absence of drug use and a persistent vulnerability toward relapse to drug use during protracted abstinence. In this review we discuss the effects of drug intake on brain endocannabinoid signaling, evidence implicating the endocannabinoid system in the motivation for drug consumption, and drug-induced alterations in endocannabinoid function that may contribute to various aspects of addiction including dysregulated synaptic plasticity, increased stress responsivity, negative affective states, drug craving and relapse to drug taking. Current knowledge of genetic variants in endocannabinoid signaling associated with addiction is also discussed.

Regulation of brain anandamide by acute administration of ethanol

The endogenous cannabinoid acylethanolamide AEA (arachidonoylethanolamide; also known as anandamide) participates in the neuroadaptations associated with chronic ethanol exposure. However, no studies have described the acute actions of ethanol on AEA production and degradation. In the present study, we investigated the time course of the effects of the intraperitoneal administration of ethanol (4 g/kg of body mass) on the endogenous levels of AEA in central and peripheral tissues. Acute ethanol administration decreased AEA in the cerebellum, the hippocampus and the nucleus accumbens of the ventral striatum, as well as in plasma and adipose tissue. Parallel decreases of a second acylethanolamide, PEA (palmitoylethanolamide), were observed in the brain. Effects were observed 45-90 min after ethanol administration. In vivo studies revealed that AEA decreases were associated with a remarkable inhibition of the release of both anandamide and glutamate in the nucleus accumbens. There were no changes in the expression and enzymatic activity of the main enzyme that degrades AEA, the fatty acid amidohydrolase. Acute ethanol administration did not change either the activity of N-acyltransferase, the enzyme that catalyses the synthesis of the AEA precursor, or the expression of NAPE-PLD (N-acylphosphatidylethanolamine-hydrolysing phospholipase D), the enzyme that releases AEA from membrane phospholipid precursors. These results suggest that receptor-mediated release of acylethanolamide is inhibited by the acute administration of ethanol, and that this effect is not derived from increased fatty acid ethanolamide degradation.

Central versus peripheral antagonism of cannabinoid CB1 receptor in obesity: effects of LH-21, a peripherally acting neutral cannabinoid receptor antagonist, in Zucker rats.

The endogenous cannabinoid system plays an important modulatory role in feeding behaviour and metabolism, acting at both central and peripheral levels. Chronic administration of cannabinoid CB1 receptor antagonists has been found to be effective in experimental obesity. However, clinically available cannabinoid receptor antagonists are inverse agonists that can target CB1 receptors located in both central circuits regulating appetite and motivation and in peripheral organs regulating metabolism and energy expenditure. This profile complicates understanding of cannabinoid CB1 receptor blockade as a therapeutic strategy in obesity and metabolic disorders. This review aims to explore the relevance of both inverse agonism and peripheral cannabinoid receptor blockade on the beneficial actions of chronic cannabinoid receptor blockade, by comparing the actions of the reference antagonist/inverse agonist rimonabant and the newly designed drug LH‐21. LH‐21 is a triazol derivative and a neutral cannabinoid receptor antagonist; it has a poor penetration rate into the central nervous system. When given acutely it decreases food intake and enhances the anorectic actions of oleoylethanolamide, a feeding suppressant lipid that acts on peripheral sensory terminals in a similar way as rimonabant. Unlike rimonabant, chronic administration of LH‐21 (3 mg/kg) reduces feeding but does not improve hypertriglyceridaemia or hypercholesterolaemia; nor does it reduce liver fat deposits in Zucker rats. These results suggest that the inverse agonism and/or the antagonism of central cannabinoid CB1 receptors are necessary for the metabolic benefits of cannabinoid CB1 receptor blockade, but not for the appetite reduction.

The cannabinoid CB1 receptor antagonist SR141716A (Rimonabant) enhances the metabolic benefits of long-term treatment with oleoylethanolamide in Zucker rats.

Anandamide and oleoylethanolamide (OEA) are lipid mediators that regulate feeding and lipid metabolism. While anandamide, a cannabinoid CB1 receptor agonist, promotes feeding and lipogenesis, oleoylethanolamide, an endogenous agonist of peroxisome proliferator activated receptor alpha (PPAR-alpha), decreases food intake and activates lipid mobilization and oxidation. The treatment with a cannabinoid CB1 receptor antagonist results in reduction of body weight gain and cholesterol in obese humans and rodents. In the present study, we show the benefits of the treatment of obese Zucker rats with a combination of a cannabinoid CB1 receptor antagonist (Rimonabant) and oleoylethanolamide. This combinational therapy improved the separate effects of Rimonabant and OEA, and resulted in marked decreases on feeding, body weight gain, and plasma cholesterol levels. Additionally, the treatment with both drugs reduced the hepatic steatosis observed in Zucker rats, decreasing liver fat deposits and damage, as revealed by the levels of alanine aminotransferase activity in serum. The combined treatment inhibits the expression of stearoyl coenzyme-A desaturase-1 (SCD-1), a pivotal enzyme in lipid biosynthesis and triglyceride mobilization that is linked to obesity phenotypes. These results support the use of combined therapies with cannabinoid CB1 receptor antagonists and PPAR-alpha agonists for the treatment of obesity associated with dyslipemia.

Anti‐obesity efficacy of LH‐21, a cannabinoid CB1 receptor antagonist with poor brain penetration, in diet‐induced obese rats

BACKGROUND AND PURPOSE Peripheral blockade of cannabinoid CB1 receptors has been proposed as a safe and effective therapy against obesity, putatively devoid of the adverse psychiatric side effects of centrally acting CB1 receptor antagonists. In this study we analysed the effects of LH‐21, a peripherally acting neutral cannabinoid receptor antagonist with poor brain penetration, in an animal model of diet‐induced obesity.

Expression of the cannabinoid system in muscle: effects of a high-fat diet and CB1 receptor blockade

The ECS (endocannabinoid system) plays an important role in the onset of obesity and metabolic disorders, implicating central and peripheral mechanisms predominantly via CB1 (cannabinoid type 1) receptors. CB1 receptor antagonist/inverse agonist treatment improves cardiometabolic risk factors and insulin resistance. However, the relative contribution of peripheral organs to the net beneficial metabolic effects remains unclear. In the present study, we have identified the presence of the endocannabinoid signalling machinery in skeletal muscle and also investigated the impact of an HFD (high-fat diet) on lipid-metabolism-related genes and endocannabinoid-related proteins. Finally, we tested whether administration of the CB1 inverse agonist AM251 restored the alterations induced by the HFD. Rats were fed on either an STD (standard/low-fat diet) or an HFD for 10 weeks and then treated with AM251 (3 mg/kg of body weight per day) for 14 days. The accumulated caloric intake was progressively higher in rats fed on the HFD than the STD, resulting in a divergence in body weight gain. AM251 treatment reduced accumulated food/caloric intake and body weight gain, being more marked in rats fed on the HFD. CB2 (cannabinoid type 2) receptor and PPARα (peroxisome-proliferator-activated receptor α) gene expression was decreased in HFD-fed rats, whereas MAGL (monoglyceride lipase) gene expression was up-regulated. These data suggest an altered endocannabinoid signalling as a result of the HFD. AM251 treatment reduced CB2 receptor, PPARγ and AdipoR1 (adiponectin receptor 1) gene expression in STD-fed rats, but only partially normalized the CB2 receptor in HFD-fed rats. Protein levels corroborated gene expression results, but also showed a decrease in DAGL (diacylglycerol) β and DAGLα after AM251 treatment in STD- and HFD-fed rats respectively. In conclusion, the results of the present study indicate a diet-sensitive ECS in skeletal muscle, suggesting that blockade of CB1 receptors could work towards restoration of the metabolic adaption imposed by diet.

DIFFERENTIAL EFFECTS OF SINGLE VERSUS REPEATED ALCOHOL WITHDRAWAL ON THE EXPRESSION OF ENDOCANNABINOID SYSTEM-RELATED GENES IN THE RAT AMYGDALA

BACKGROUND Endogenous cannabinoids such as anandamide and 2-arachidonoylglycerol (2-AG) exert important regulatory influences on neuronal signaling, participate in short- and long-term forms of neuroplasticity, and modulate stress responses and affective behavior in part through the modulation of neurotransmission in the amygdala. Alcohol consumption alters brain endocannabinoid levels, and alcohol dependence is associated with dysregulated amygdalar function, stress responsivity, and affective control. METHODS The consequence of long-term alcohol consumption on the expression of genes related to endocannabinoid signaling was investigated using quantitative RT-PCR analyses of amygdala tissue. Two groups of ethanol (EtOH)-exposed rats were generated by maintenance on an EtOH liquid diet (10%): the first group received continuous access to EtOH for 15 days, whereas the second group was given intermittent access to the EtOH diet (5 d/wk for 3 weeks). Control subjects were maintained on an isocaloric EtOH-free liquid diet. To provide an initial profile of acute withdrawal, amygdala tissue was harvested following either 6 or 24 hours of EtOH withdrawal. RESULTS Acute EtOH withdrawal was associated with significant changes in mRNA expression for various components of the endogenous cannabinoid system in the amygdala. Specifically, reductions in mRNA expression for the primary clearance routes for anandamide and 2-AG (fatty acid amide hydrolase [FAAH] and monoacylglycerol lipase [MAGL], respectively) were evident, as were reductions in mRNA expression for CB(1) , CB(2) , and GPR55 receptors. Although similar alterations in FAAH mRNA were evident following either continuous or intermittent EtOH exposure, alterations in MAGL and cannabinoid receptor-related mRNA (e.g., CB(1) , CB(2) , GPR55) were more pronounced following intermittent exposure. In general, greater withdrawal-associated deficits in mRNA expression were evident following 24 versus 6 hours of withdrawal. No significant changes in mRNA expression for enzymes involved in 2-AG biosynthesis (e.g., diacylglicerol lipase-α/β) were found in any condition. CONCLUSIONS These findings suggest that EtOH dependence and withdrawal are associated with dysregulated endocannabinoid signaling in the amygdala. These alterations may contribute to withdrawal-related dysregulation of amygdalar neurotransmission.