Fernando Rodríguez de Fonseca

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.

The endocannabinoid system as a target for therapeutic drugs.

Cannabinoid receptors, the molecular targets of the cannabis constituent Delta9-tetrahydrocannabinol, are present throughout the body and are normally bound by a family of endogenous lipids - the endocannabinoids. Release of endocannabinoids is stimulated in a receptor-dependent manner by neurotransmitters and requires the enzymatic cleavage of phospholipid precursors present in the membranes of neurons and other cells. Once released, the endocannabinoids activate cannabinoid receptors on nearby cells and are rapidly inactivated by transport and subsequent enzymatic hydrolysis. These compounds might act near their site of synthesis to serve a variety of regulatory functions, some of which are now beginning to be understood. Recent advances in the biochemistry and pharmacology of the endocannabinoid system in relation to the opportunities that this system offers for the development of novel therapeutic agents will be discussed.

Colocalization of glucagon-like peptide-1 (GLP-1) receptors, glucose transporter GLUT-2, and glucokinase mRNAs in rat hypothalamic cells : Evidence for a role of GLP-1 receptor agonists as an inhibitory signal for food and water intake

Abstract: This study was designed to determine the possible role of brain glucagon‐like peptide‐1 (GLP‐1) receptors in feeding behavior. In situ hybridization showed colocalization of the mRNAs for GLP‐1 receptors, glucokinase, and GLUT‐2 in the third ventricle wall and adjacent arcuate nucleus, median eminence, and supraoptic nucleus. These brain areas are considered to contain glucose‐sensitive neurons mediating feeding behavior. Because GLP‐1 receptors, GLUT‐2, and glucokinase are proteins involved in the multistep process of glucose sensing in pancreatic β cells, the colocalization of specific GLP‐1 receptors and glucose sensing‐related proteins in hypothalamic neurons supports a role of this peptide in the hypothalamic regulation of macronutrient and water intake. This hypothesis was confirmed by analyzing the effects of both systemic and central administration of GLP‐1 receptor ligands. Acute or subchronic intraperitoneal administration of GLP‐1 (7–36) amide did not modify food and water intake, although a dose‐dependent loss of body weight gain was observed 24 h after acute administration of the higher dose of the peptide. By contrast, the intracerebroventricular (i.c.v.) administration of GLP‐1 (7–36) amide produced a biphasic effect on food intake characterized by an increase in the amount of food intake after acute i.c.v. delivery of 100 ng of the peptide. There was a marked reduction of food ingestion with the 1,000 and 2,000 ng doses of the peptide, which also produced a significant decrease of water intake. These effects seemed to be specific because i.c.v. administration of GLP‐1 (1–37), a peptide with lower biological activity than GLP‐1 (7–36) amide, did not change feeding behavior in food‐deprived animals. Exendin‐4, when given by i.c.v. administration in a broad range of doses (0.2, 1, 5, 25, 100, and 500 ng), proved to be a potent agonist of GLP‐1 (7–36) amide. It decreased, in a dose‐dependent manner, both food and water intake, starting at the dose of 25 ng per injection. Pretreatment with an i.c.v. dose of a GLP‐1 receptor antagonist [exendin (9–39); 2,500 ng] reversed the inhibitory effects of GLP‐1 (7–36) amide (1,000 ng dose) and exendin‐4 (25 ng dose) on food and water ingestion. These findings suggest that GLP‐1 (7–36) amide may modulate both food and drink intake in the rat through a central mechanism.

Long-Term Effects of Mouse Intracytoplasmic Sperm Injection with DNA-Fragmented Sperm on Health and Behavior of Adult Offspring

Abstract Genetic and environmental factors produce different levels of DNA damage in spermatozoa. Usually, DNA-fragmented spermatozoa (DFS) are used with intracytoplasmic sperm injection (ICSI) treatments in human reproduction, and use of DFS is still a matter of concern. The purpose of the present study was to investigate the long-term consequences on development and behavior of mice generated by ICSI with DFS. Using CD1 and B6D2F1 mouse strains, oocytes were injected with fresh spermatozoa or with frozen-thawed spermatozoa without cryoprotector. This treatment increased the percentage of TUNEL-positive spermatozoa, tail length as measured by comet assay, and loss of telomeres as measured by quantitative PCR. The ICSI-generated embryos were cultured for 24 h in KSOM, and 2-cell embryos were transferred into CD1 females. The DFS reduced both the rate of preimplantation embryo development and number of offspring. Immunofluorescence staining with an antibody against 5-methylcytosine showed a delay of 2 h on the active demethylation of male pronucleus in the embryos produced by ICSI. Moreover, ICSI affected gene transcription and methylation of some epigenetically regulated genes like imprinting, X-linked genes, and retrotransposon genes. At 3 and 12 mo of age, ICSI with DFS-produced animals and in vivo-fertilized controls were submitted to behavioral tests: locomotor activity (open field), exploratory/anxiety behavior (elevated plus maze, open field), and spatial memory (free-choice exploration paradigm in Y maze). Females produced by ICSI showed increased anxiety, lack of habituation pattern, deficit in short-term spatial memory, and age-dependent hypolocomotion in the open-field test (P < 0.05). Postnatal weight gain of mice produced by ICSI with fresh or frozen sperm was higher than that of their control counterparts from 16 wk on (P < 0.01). Anatomopathological analysis of animals at 16 mo of age showed some large organs and an increase in pathologies (33% of CD1 females produced with DFS presented some solid tumors in lungs and dermis of back or neck). Moreover, 20% of the B6D2F1 mice generated with DFS died during the first 5 mo of life, with 25% of the surviving animals showing premature aging symptoms, and 70% of the B6D2F1 mice generated with DFS died earlier than controls with different kind of tumors. We propose that depending on the level of DFS, oocytes may partially repair fragmented DNA, producing blastocysts able to implant and produce live offspring. The incomplete repair, however, may lead to long-term pathologies. Our data indicate that use of DFS in ICSI can generate effects that only emerge during later life, such as aberrant growth, premature aging, abnormal behavior, and mesenchymal tumors.

Presence of cannabinoid binding sites in the brain from early postnatal ages.

The present study demonstrates the presence of cannabinoid receptors in the brain from early postnatal ages. Specific and saturable binding was observed in the forebrain and remaining brain from early postnatal ages (2 and 5 days after birth). Female neonate forebrain exhibited a higher receptor density at 2 days after birth than males, but this trend was inverted at 5 days. From postnatal day 10, the receptors could be measured in more defined brain areas, i.e. the striatum, limbic forebrain and ventral mesencephalon. The ontogeny of the receptors in these three areas was relatively similar, exhibiting a progressive increase which maximised on days 30 or 40 and then subsequently decreased to adult values. Subtle sexual dimorphism was found in the striatum and ventral mesencephalon but not the limbic forebrain.

Genetic Impairment of Frontocortical Endocannabinoid Degradation and High Alcohol Preference

Endocannabinoid signaling has recently been implicated in ethanol-seeking behavior. We analyzed the expression of endocannabinoid-related genes in key brain regions of reward and dependence, and compared them between the alcohol-preferring AA (Alko Alcohol) and nonpreferring ANA (Alko Non-Alcohol) rat lines. A decreased expression of fatty acid amidohydrolase (FAAH), the main endocannabinoid-degrading enzyme, was found in prefrontal cortex (PFC) of AA rats, and was accompanied by decreased enzyme activity in this region. Binding of the endocannabinoid-cannabinoid 1 (CB1) receptor ligand 3[H]SR141716A, and [35S]GTPγS incorporation stimulated by the CB1 agonist WIN 55,212-2 were downregulated in the same area. Together, this suggests an overactive endocannabinoid transmission in the PFC of AA animals, and a compensatory downregulation of CB1 signaling. The functional role of impaired FAAH function for alcohol self-administration was validated in two independent ways. The CB1 antagonist SR141716A potently and dose-dependently suppressed self-administration in AA rats when given systemically, or locally into the PFC, but not in the striatum. Conversely, intra-PFC injections of the competitive FAAH inhibitor URB597 increased ethanol self-administration in nonselected Wistar rats. These results show for the first time that impaired FAAH function may confer a phenotype of high voluntary alcohol intake, and point to a FAAH both as a potential susceptibility factor and a therapeutic target.

Chronic (−)-Δ9-tetrahydrocannabinol treatment induces sensitization to the psychomotor effects of amphetamine in rats

Clinical and basic research studies have linked cannabinoid consumption to the onset of psychosis, specially schizophrenia. In the present study we have evaluated the effects of the natural psychoactive constituent of Cannabis (-)-delta9-tetrahydrocannabinol on the acute actions of the psychostimulant, D-amphetamine, on behaviour displayed by male rats on a hole-board, a proposed animal model of amphetamine-induced psychosis. Cannabinoid-amphetamine interactions were studied (1) 30 min after acute injection of (-)-delta9-tetrahydrocannabinol (0.1 or 6.4 mg/kg, i.p.); (2) 30 min after the last injection of 14-daily treatment with (-)-delta9-tetrahydrocannabinol (0.1 or 6.4 mg/kg) and 3) 24 h after the last injection of 14-daily treatment with (-)-delta9-tetrahydrocannabinol (6.4 mg/kg). Acute cannabinoid exposure antagonized the amphetamine-induced dose-dependent increase in locomotion, exploration and the decrease in inactivity. Chronic treatment with (-)-delta9-tetrahydrocannabinol resulted in tolerance to this antagonistic effect on locomotion and inactivity but not on exploration, and potentiated amphetamine-induced stereotypies. Lastly, 24 h of withdrawal after 14 days of cannabinoid treatment resulted in sensitization to the effects of D-amphetamine on locomotion, exploration and stereotypies. Since (-)-delta9-tetrahydrocannabinol is a cannabinoid CB1 receptor agonist, densely present in limbic and basal ganglia circuits, and since amphetamine enhances monoaminergic inputs (i.e., dopamine, serotonin) in these brain areas, the present data support the hypothesis of a role for the cannabinoid CB1 receptor as a regulatory mechanism of monoaminergic neuron-mediated psychomotor activation. These findings may be relevant for the understanding of both cannabinoid-monoamines interactions and Cannabis-associated psychosis.

Elevated circulating levels of anandamide after administration of the transport inhibitor, AM404

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

Expression and Function of CB1 Receptor in the Rat Striatum: Localization and Effects on D1 and D2 Dopamine Receptor-Mediated Motor Behaviors

Cannabinoid CB1 receptors are densely expressed on striatal projection neurons expressing dopamine D1 or D2 receptors. However, the specific neuronal distribution of CB1 receptors within the striatum is not known. Previous research has established that the endocannabinoid system controls facilitation of behavior by dopamine D2 receptors, but it is not clear if endocannabinoids also modulate D1 receptor-mediated motor behavior. In the present study, we show that cannabinoid CB1 receptor mRNA is present in striatonigral neurons expressing substance P and dopamine D1 receptors, as well as in striatopallidal neurons expressing enkephalin and dopamine D2 receptors. We explored the functional relevance of the interaction between dopamine D1 and D2 receptors and cannabinoid CB1 receptors with behavioral pharmacology experiments. Potentiation of endogenous cannabinoid signaling by the uptake blocker AM404 blocked dopamine D1 receptor-mediated grooming and D2 receptor-mediated oral stereotypies. In addition, contralateral turning induced by unilateral intrastriatal infusion of D1 receptor agonists is counteracted by AM404 and potentiated by the cannabinoid antagonist SR141716A. These results indicate that the endocannabinoid system negatively modulates D1 receptor-mediated behaviors in addition to its previously described effect on dopamine D2 receptor-mediated behaviors. The effect of AM404 on grooming behavior was absent in dopamine D1 receptor knockout mice, demonstrating its dependence on D1 receptors. This study indicates that the endocannabinoid system is a relevant negative modulator of both dopamine D1 and D2 receptor-mediated behaviors, a finding that may contribute to our understanding of basal ganglia motor disorders.

Cannabinoid CB1 receptor antagonism reduces conditioned reinstatement of ethanol-seeking behavior in rats

The endocannabinoid system is involved in a variety of effects of drugs of misuse, and blockade of the cannabinoid CB1 receptor by selective antagonists elicits marked reductions in opioid and alcohol self‐administration. The present study was designed to extend our knowledge of the role of the cannabinoid CB1 receptor in the modulation of alcohol misuse vulnerability in rats. Accordingly, using nonselected Wistar rats and genetically selected Marchigian Sardinian alcohol‐preferring (msP) rats, we investigated the effect of the CB1 antagonist SR141716A on operant alcohol self‐administration and on reinstatement of alcohol‐seeking behavior by environmental conditioning factors. In addition, in situ hybridization studies in both strains were performed to measure cannabinoid CB1 receptor mRNA in different brain areas of these animals. Results showed that intraperitoneal administration of SR141716A (0.03, 1.0 and 3.0 mg/kg) markedly inhibits ethanol self‐administration and conditioned reinstatement of ethanol‐seeking behavior in both strains of rats. ED50 analysis showed significantly higher sensitivity (P < 0.05) to the effect of SR141716A in msP rats than in heterogeneous Wistar rats. In situ hybridization studies revealed that, compared with Wistar rats, msP animals have consistently greater cannabinoid CB1 receptor mRNA expression in a number of brain areas, including the frontoparietal cortex, caudate‐putamen and hippocampus (CA1 and dentate gyrus areas). In conclusion, we provide clear evidence that blockade of CB1 receptors reduces both ethanol self‐administration and conditioned reinstatement of alcohol‐seeking behavior in rats. In addition, current pharmacological and neuroanatomical data suggest that an altered function of the CB1 receptor system exists between genetically selected alcohol‐preferring msP rats and a heterogeneous animal population.