RESUMEN
Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease; however, no specific pharmacological therapy has yet been approved for this condition. Plant-derived extracts can be an important source for the development of new drugs. The aim of this study was to investigate the effects of (E)-ß-caryophyllene (BCP), a phytocannabinoid recently found to be beneficial against metabolic diseases, on HepG2 steatotic hepatocytes. Using a fluorescence-based lipid quantification assay and GC-MS analysis, we show that BCP is able to decrease lipid accumulation in steatotic conditions and to change the typical steatotic lipid profile by primarily reducing saturated fatty acids. By employing specific antagonists, we demonstrate that BCP action is mediated by multiple receptors: CB2 cannabinoid receptor, peroxisome proliferator-activated receptor α (PPARα) and γ (PPARγ). Interestingly, BCP was able to counteract the increase in CB2 and the reduction in PPARα receptor expression observed in steatotic conditions. Moreover, through immunofluorescence and confocal microscopy, we demonstrate that CB2 receptors are mainly intracellularly localized and that BCP is internalized in HepG2 cells with a maximum peak at 2 h, suggesting a direct interaction with intracellular receptors. The results obtained with BCP in normal and steatotic hepatocytes encourage future applications in the treatment of NAFLD.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Sesquiterpenos , Humanos , Lípidos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , PPAR alfa/metabolismo , PPAR gamma/metabolismo , Sesquiterpenos/farmacología , Receptor Cannabinoide CB2RESUMEN
The endocannabinoid system (ECS) is involved in the modulation of several basic biological processes, having widespread roles in neurodevelopment, neuromodulation, immune response, energy homeostasis and reproduction. In the adult central nervous system (CNS) the ECS mainly modulates neurotransmitter release, however, a substantial body of evidence has revealed a central role in regulating neurogenesis in developing and adult CNS, also under pathological conditions. Due to the complexity of investigating ECS functions in neural progenitors in vivo, we tested the suitability of the ST14A striatal neural progenitor cell line as a simplified in vitro model to dissect the role and the mechanisms of ECS-regulated neurogenesis, as well as to perform ECS-targeted pharmacological approaches. We report that ST14A cells express various ECS components, supporting the presence of an active ECS. While CB1 and CB2 receptor blockade did not affect ST14A cell number, exogenous administration of the endocannabinoid 2-AG and the synthetic CB2 agonist JWH133 increased ST14A cell proliferation. Phospholipase C (PLC), but not PI3K pharmacological blockade negatively modulated CB2-induced ST14A cell proliferation, suggesting that a PLC pathway is involved in the steps downstream to CB2 activation. On the basis of our results, we propose ST14A neural progenitor cells as a useful in vitro model for studying ECS modulation of neurogenesis, also in prospective in vivo pharmacological studies.
Asunto(s)
Moduladores de Receptores de Cannabinoides/farmacología , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/fisiología , Receptores de Cannabinoides/metabolismo , Animales , Cannabinoides/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Cuerpo Estriado/citología , Estrenos/farmacología , Células-Madre Neurales/fisiología , Neurogénesis/efectos de los fármacos , Pirrolidinonas/farmacología , Ratas , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/antagonistas & inhibidores , Receptor Cannabinoide CB2/genética , Receptores de Cannabinoides/genética , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
Bisphenol A (BPA) is one of the most widely used chemicals worldwide, e.g., as a component of plastic containers for food and water. It is considered to exert an estrogenic effect, by mimicking estradiol (E2) action. Because of this widespread presence, it has attracted the interest and concern of researchers and regulators. Despite the vast amount of related literature, the potential adverse effects of environmentally significant doses of BPA are still object of controversy, and the mechanisms by which it can perturb endocrine functions, and particularly the neuroendocrine axis, are not adequately understood. One of the ways by which endocrine disruptors (EDCs) can exert their effects is the perturbation of calcium signaling mechanisms. In this study, we addressed the issue of the impact of BPA on the neuroendocrine system with an in vitro approach, using a consolidated model of immortalized Gonadotropin-Releasing Hormone (GnRH) expressing neurons, the GT1-7 cell line, focusing on the calcium signals activated by the endocrine disruptor. The investigation was limited to biologically relevant doses (nM-µM range). We found that BPA induced moderate increases in intracellular calcium concentration, comparable with those induced by nanomolar doses of E2, without affecting cell survival and with only a minor effect on proliferation.
Asunto(s)
Compuestos de Bencidrilo/farmacología , Calcio/metabolismo , Disruptores Endocrinos/farmacología , Células Neuroendocrinas/efectos de los fármacos , Fenoles/farmacología , Animales , Compuestos de Bencidrilo/toxicidad , Línea Celular , Disruptores Endocrinos/toxicidad , Hormona Liberadora de Gonadotropina/metabolismo , Transporte Iónico , Ratones , Células Neuroendocrinas/metabolismo , Fenoles/toxicidadRESUMEN
The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron's axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake.
Asunto(s)
Proteína Relacionada con Agouti/metabolismo , Axones/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Ácido Pirrolidona Carboxílico/análogos & derivados , Receptor Cannabinoide CB1/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Benzoxazinas/farmacología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Morfolinas/farmacología , Morfolinos/metabolismo , Naftalenos/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ácido Pirrolidona Carboxílico/metabolismo , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genéticaRESUMEN
Cannabinoids, the bioactive constituents of Cannabis sativa, and endocannabinoids, among which the most important are anandamide and 2-arachidonoylglycerol, control various biological processes by binding to specific G protein-coupled receptors, namely CB1 and CB2 cannabinoid receptors. While a vast amount of information on the mammalian endocannabinoid system does exist, few data have been reported on bony fish. In the goldfish, Carassius auratus, the CB1 receptor has been cloned and its distribution has been analyzed in the retina, brain and gonads, while CB2 had not yet been isolated. In the present paper, we cloned the goldfish CB2 receptor and show that it presents a quite high degree of amino acid identity with zebrafish Danio rerio CB2A and CB2B receptors, while the percentage of identity is lower with the puffer fish Fugu rubripes CB2, as also confirmed by the phylogenetic analysis. The sequence identity becomes much lower when comparing the goldfish and the mammalian CB2 sequences; as for other species, goldfish CB2 and CB1 amino acid sequences share moderate levels of identity. Western-blotting analysis shows the CB2 receptor as two major bands of about 53 and 40 kDa and other faint bands with apparent molecular masses around 70, 57 and 55 kDa. Since the distribution of a receptor could give information on its physiological role, we evaluated and compared CB1 and CB2 mRNA expression in different goldfish organs by means of qReal-Time PCR. Our results show that both CB1 and CB2 receptors are widely expressed in the goldfish, displaying some tissue specificities, thus opening the way for further functional studies on bony fish and other nonmammalian vertebrates.
Asunto(s)
Carpa Dorada/metabolismo , ARN Mensajero/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/aislamiento & purificación , Receptor Cannabinoide CB2/metabolismo , Animales , Secuencia de Bases , Western Blotting/veterinaria , Clonación Molecular , Análisis por Conglomerados , Cartilla de ADN/genética , Perfilación de la Expresión Génica/veterinaria , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB2/genética , Análisis de Secuencia de ADN/veterinaria , Homología de Secuencia , Especificidad de la EspecieRESUMEN
Based on pharmacological, behavioral and neuroanatomical studies, the endocannabinoids appear to be pivotal in some important neuroendocrine regulations of both vertebrates and invertebrates. Interestingly, a well developed endocannabinoid system was recently demonstrated by us in different bonyfish brain areas which control reproduction, energy balance and stress. Fish in particular are very sensitive to different types of stressors which can heavily affect their reproductive activity and negatively reverberate on aquaculture. Since recent new data have been reported on endocrine disruptors (EDs) impact on zebrafish receptor CB1 expression, in the present research we have investigated the response of the endocannabinoid system to acute treatment with an environmental stressor such as the xenoestrogen nonylphenol (4NP) in the brain and peripheral tissues of the goldfish Carassius auratus. First of all the estrogenic effects induced by 4NP were demonstrated by a dose-dependent increase of plasma levels and gene expression of the biomarker vitellogenin, then changes in cannabinoid receptors and anandamide degradative enzyme, the fatty acid amide hydrolase (FAAH), were analysed by means of Real Time PCR. As the exposure to EDs may lead to an activation of estrogen receptors and affects the Aromatase (AROB) transcription, changes in mRNA levels for ER subtypes and AROB were also evaluated. Our results confirm in goldfish the effect of 4NP on ERα and ERß1 receptors and point out a different sensitivity of CB1 and CB2 for this compound, suggesting distinct roles of these cannabinoid receptors in some adaptive processes to contrast stress induced by xenoestrogen exposure.
Asunto(s)
Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Carpa Dorada/metabolismo , Fenoles/toxicidad , Receptores de Estrógenos/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Animales , Aromatasa/genética , Aromatasa/metabolismo , Ensayo de Inmunoadsorción Enzimática , Carpa Dorada/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Cannabinoides/genética , Receptores de Cannabinoides/metabolismo , Receptores de Estrógenos/genética , Vitelogeninas/genética , Vitelogeninas/metabolismoRESUMEN
Even though microplastic (MP) pollution in aquatic environment is nowadays widely studied, a huge gap of knowledge exists on their actual biological effects. In this study we first reported environmental baseline data on the occurrence and characterization of ï¬oating MPs in Italian coastal waters of the Central Adriatic Sea by using a standardized monitoring protocol. Further, we analyzed the concentrations of MP-associated chemicals and evaluated their potential adipogenic effects using 3T3-L1 preadipocytes. MPs were found in each sampling stations showing the highest abundance (1.88 ± 1.78 items/m3) in the sites more distant from the coast with fragments as the most common shape category. All targeted organic pollutants (i.e. polychlorinated biphenyls - PCBs, polycyclic aromatic hydrocarbons -PAHs, organophosphorus - OP, and organochlorine - OC pesticides) have been detected on the surface of the collected MPs. The highest concentrations of PAHs were found on MPs from inshore (i.e. <1.5 NM) surface waters with low-ring PAHs as dominant components. Similarly, MPs from inshore waters had higher ΣPCB concentrations (64.72 ng/g plastic) than those found in offshore (i.e. >6 NM) waters (10.37 ng/g plastic). Among pesticides, all measured OPs were detected in each sample analyzed with pirimiphos-methyl as the most representative compound. For OCs, the sum of all concentrations of congeners was higher in coastal with respect to offshore waters. Moreover, in vitro 3T3-L1 screening of MP extracts indicated potential metabolic effects resulting in both adipogenesis and lipid uptake/storage.
Asunto(s)
Monitoreo del Ambiente , Microplásticos/análisis , Contaminantes Químicos del Agua/análisis , Adipogénesis , Hidrocarburos Clorados/análisis , Italia , Microplásticos/toxicidad , Plaguicidas/análisis , Plásticos , Bifenilos Policlorados/análisis , Hidrocarburos Policíclicos Aromáticos/análisis , Contaminantes Químicos del Agua/toxicidadRESUMEN
The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30-70 % for pre-adipocyte proliferation; this was 40-80 % for triglyceride accumulation induced by pyraclostrobin, 80-100 % for tributyltin, and 80-100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30-70 % active determinations for pyraclostrobin, 30-50 % for tributyltin, and 20-40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes.
Asunto(s)
Adipogénesis/efectos de los fármacos , Compuestos de Bencidrilo/toxicidad , Fenoles/toxicidad , Estrobilurinas/toxicidad , Compuestos de Trialquiltina/toxicidad , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Diferenciación Celular , Proliferación Celular/efectos de los fármacos , Ratones , Reproducibilidad de los Resultados , Rosiglitazona/farmacología , Triglicéridos/metabolismoRESUMEN
(E)-ß-caryophyllene (BCP) is a bicyclic sesquiterpene widely distributed in the plant kingdom, where it contributes a unique aroma to essential oils and has a pivotal role in the survival and evolution of higher plants. Recent studies provided evidence for protective roles of BCP in animal cells, highlighting its possible use as a novel therapeutic tool. Experimental results show the ability of BCP to reduce pro-inflammatory mediators such as tumor necrosis factor-alfa (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), thus ameliorating chronic pathologies characterized by inflammation and oxidative stress, in particular metabolic and neurological diseases. Through the binding to CB2 cannabinoid receptors and the interaction with members of the family of peroxisome proliferator-activated receptors (PPARs), BCP shows beneficial effects on obesity, non-alcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) liver diseases, diabetes, cardiovascular diseases, pain and other nervous system disorders. This review describes the current knowledge on the biosynthesis and natural sources of BCP, and reviews its role and mechanisms of action in different inflammation-related metabolic and neurologic disorders.
Asunto(s)
Antiinflamatorios/farmacología , Enfermedad Crónica/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Extractos Vegetales/farmacología , Sesquiterpenos Policíclicos/farmacología , Animales , Humanos , Mediadores de Inflamación/metabolismo , Estrés Oxidativo/efectos de los fármacos , PPAR gamma/metabolismo , Receptor Cannabinoide CB2/metabolismoRESUMEN
Trans-ß-caryophyllene (BCP) is a natural sesquiterpene hydrocarbon with several important pharmacological activities, including antioxidant, anti-inflammatory, anticancer, and cardioprotective functions. These properties are mainly due to its selective interaction with the peripherally expressed cannabinoid receptor 2. In addition, BCP activates peroxisome proliferated activator receptors α and γ and inhibits the Toll-like receptor signaling pathway. Given the growing scientific interest in BCP, the aim of our study was to investigate the metabolic effects of a black pepper extract (PipeNig®-FL), containing a high standardized content of BCP. In particular our interest was focused on its potential activity on lipid accumulation and glucose uptake. The extract PipeNig®-FL was chemically characterized by gas chromatography-mass spectrometry (GC-MS) and gas chromatography with flame-ionization detection (GC-FID), confirming a high content (814 mg/g) of BCP. Experiments were performed on 3T3-L1 preadipocytes and on C2C12 myotubes. Lipid content following 3T3-L1 adipogenic differentiation was quantified with AdipoRed fluorescence staining. Glucose uptake and GLUT4 membrane translocation were studied in C2C12 myotubes with the fluorescent glucose analog 2-NBDG and by immunofluorescence analysis. Here we show that PipeNig®-FL reduces 3T3-L1 adipocyte differentiation and lipid accumulation. Moreover, acute exposure of C2C12 myotubes to PipeNig®-FL improves glucose uptake activity and GLUT4 migration. Taken together, these results reveal interesting and novel properties of BCP, suggesting potential applications in the prevention of lipid accumulation and in the improvement of glucose uptake.
Asunto(s)
Metabolismo de los Lípidos/efectos de los fármacos , Piper nigrum/química , Extractos Vegetales/farmacología , Sesquiterpenos Policíclicos/farmacología , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cromatografía de Gases y Espectrometría de Masas , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Ratones , Fibras Musculares Esqueléticas/efectos de los fármacos , Extractos Vegetales/químicaRESUMEN
The morphofunctional relationship between the endocannabinoid system and GnRH activity in the regulation of reproduction has poorly been investigated in vertebrates. Due to the anatomical features of lower vertebrate brain, in the present paper, we chose the frog Rana esculenta (anuran amphibian) as a suitable model to better investigate such aspects of the reproductive physiology. By using double-labeling immunofluorescence aided with a laser-scanning confocal microscope, we found a subpopulation of the frog hypothalamic GnRH neurons endowed with CB1 cannabinoid receptors. By means of semiquantitative RT-PCR assay, we have shown that, during the annual sexual cycle, GnRH-I mRNA (formerly known as mammalian GnRH) and CB1 mRNA have opposite expression profiles in the brain. In particular, this occurs in telencephalon and diencephalon, the areas mainly involved in GnRH release and control of the reproduction. Furthermore, we found that the endocannabinoid anandamide is able to inhibit GnRH-I mRNA synthesis; buserelin (a GnRH agonist), in turn, inhibits the synthesis of GnRH-I mRNA and induces an increase of CB1 transcription. Our observations point out the occurrence of a morphofunctional anatomical basis to explain a reciprocal relationship between the endocannabinoid system and GnRH neuronal activity.
Asunto(s)
Moduladores de Receptores de Cannabinoides/fisiología , Endocannabinoides , Hormona Liberadora de Gonadotropina/fisiología , Prosencéfalo/metabolismo , Rana esculenta/fisiología , Receptor Cannabinoide CB1/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Buserelina/farmacología , Moduladores de Receptores de Cannabinoides/metabolismo , Clonación Molecular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hormona Liberadora de Gonadotropina/genética , Hormona Liberadora de Gonadotropina/metabolismo , Masculino , Modelos Biológicos , Datos de Secuencia Molecular , Neuronas/metabolismo , Prosencéfalo/efectos de los fármacos , ARN Mensajero/metabolismo , Rana esculenta/metabolismo , Receptor Cannabinoide CB1/metabolismo , Reproducción/genética , Homología de Secuencia de AminoácidoRESUMEN
Recent studies suggest that exposure to some plasticizers, such as Bisphenol A (BPA), play a role in endocrine/metabolic dispruption and can affect lipid accumulation in adipocytes. Here, we investigated the adipogenic activity and nuclear receptor interactions of four plasticizers approved for the manufacturing of food-contact materials (FCMs) and currently considered safer alternatives. Differentiating 3T3-L1 mouse preadipocytes were exposed to scalar concentrations (0.01-25⯵M) of DiNP (Di-iso-nonyl-phthalate), DiDP (Di-iso-decyl-phthalate), DEGDB (Diethylene glycol dibenzoate), or TMCP (Tri-m-cresyl phosphate). Rosiglitazone, a well-known pro-adipogenic peroxisome proliferator activated receptor gamma (PPARγ) agonist, and the plasticizer BPA were included as reference compounds. All concentrations of plasticizers were able to enhance lipid accumulation, with TMCP being the most effective one. Accordingly, when comparing in silico the ligand binding efficiencies to the nuclear receptors PPARγ and retinoid-X-receptor-alpha (RXRα), TMPC displayed the highest affinity to both receptors. Differently from BPA, the four plasticizers were most effective in enhancing lipid accumulation when added in the mid-late phase of differentiation, thus suggesting the involvement of different intracellular signalling pathways. In line with this, TMCP, DiDP, DiNP and DEGDB were able to activate PPARγ in transient transfection assays, while previous studies demonstrated that BPA acts mainly through other nuclear receptors. qRT-PCR studies showed that all plasticizers were able to increase the expression of CCAAT/enhancer binding protein ß (Cebpß) in the early steps of adipogenesis, and the adipogenesis master gene Pparγ2 in the middle phase, with very similar efficacy to that of Rosiglitazone. In addition, TMCP was able to modulate the expression of both Fatty Acid Binding Protein 4/Adipocyte Protein 2 (Fabp4/Ap2) and Lipoprotein Lipase (Lpl) transcripts in the late phase of adipogenesis. DEGDB increased the expression of Lpl only, while the phthalate DiDP did not change the expression of either late-phase marker genes Fabp4 and Lpl. Taken together, our results suggest that exposure to low, environmentally relevant doses of the plasticizers DiNP, DiDP, DEGDB and TMCP increase lipid accumulation in 3T3-L1 adipocytes, an effect likely mediated through activation of PPARγ and interference at different levels with the transcriptional cascade driving adipogenesis.
Asunto(s)
Adipogénesis/efectos de los fármacos , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Plastificantes/farmacología , Células 3T3-L1 , Animales , Células Hep G2 , Humanos , Ratones , PPAR gamma/metabolismo , Receptor alfa X Retinoide/metabolismo , Transducción de SeñalRESUMEN
Neuroanatomical investigation of the cannabinoid system in a lower vertebrate group such as teleost fishes might improve our understanding of the physiological role of such a signaling system. In the present study, the expression of a CB1 cannabinoid receptor has been demonstrated in the CNS of a teleost fish, the cichlid Pelvicachromis pulcher. Moreover, CB1-like immunoreactivity has been analyzed by using a purified antibody against the CB1 receptor amino-terminus. Immunostained neurons and varicosities were found through the telencephalon as well as in the preoptic area and lateral infundibular lobes of the hypothalamus. Stained cells were observed in the pituitary gland. Several cell bodies and nerve terminals containing an intense CB1-like immunoreactivity were found in the pretectal central nucleus and posterior tuberculum, both lying in a transitional region between diencephalon and mesencephalon. In the brainstem, the CB1 immunopositivity was more restricted than in the prosencephalon, with the exception of some large, intensely immunopositive nerve cells within the dorsal mesencephalic tegmentum, possibly motor neurons of the third cranial nerve. In the cerebellum, among a majority of immunonegative granule cells, a subset of them was immunostained. Some positive Purkinje cells were also observed. In the spinal cord, ventral gray matter, several alpha-motoneurons were stained. Similarities to and discrepancies from the CB1 receptor distributions in other vertebrate CNS are discussed, paying particular attention to the abundant CB1 immunoreactivity observed in the area encompassing the pretectum and glomerular nucleus, which is characterized by a peculiar differentiation in bony fishes.
Asunto(s)
Sistema Nervioso Central/metabolismo , Cíclidos/metabolismo , Regulación de la Expresión Génica/fisiología , Receptor Cannabinoide CB1/biosíntesis , Secuencia de Aminoácidos/fisiología , Animales , Sistema Nervioso Central/química , Cíclidos/genética , Femenino , Masculino , Datos de Secuencia Molecular , Receptor Cannabinoide CB1/análisis , Receptor Cannabinoide CB1/genéticaRESUMEN
Following the discovery of two CB1 genes in the fish Fugu rubripes, investigations on the phylogeny of endocannabinoids have indicated that this system is highly conserved. Our study demonstrated that CB1 receptors are expressed in the CNS and gonads of two teleosts, Carassius auratus and Pelvicachromis pulcher, and they show a high percentage of sequence identity with Fugu rubripes CB(1A) and Danio rerio CB1. By means of immunohistochemistry for CB1, sGnRH, and TH, we found a codistribution of these signaling molecules in the basal telencephalon/preoptic area, which are key centers for gonadotropic regulation. We therefore suggest that endocannabinoids are possibly involved in modulating fish reproduction at both the central and peripheral levels.
Asunto(s)
Cannabinoides/química , Cíclidos/fisiología , Carpa Dorada/fisiología , Receptor Cannabinoide CB1/fisiología , Reproducción/fisiología , Animales , Cannabinoides/metabolismo , Femenino , Peces , Masculino , Reproducción/genéticaRESUMEN
The activator protein 2 (AP-2) transcription factors are essential proteins for oestrogenic repression of the ERBB2 proto-oncogene in breast cancer cells. In the present study, we have examined the possible oestrogenic regulation of AP-2 genes themselves in breast-tumour-derived lines. As early as 1 h after oestrogen treatment, AP-2gamma mRNA was markedly increased, whereas AP-2alpha was down-regulated, but with slower kinetics, and AP-2beta was not affected at all. Addition of anti-oestrogens ablated these effects. Modulation of the protein levels corresponded to changes in the transcript levels, thus suggesting that in oestrogen-treated cells, an inversion of the balance between AP-2alpha and AP-2gamma isoforms occurs. The 5'-untranslated region (5'-UTR) of the human AP-2gamma gene contains one consensus and one degenerate oestrogen-responsive element (ERE). Reporter constructs carrying the AP-2gamma promoter and the 5'-UTR were up-regulated by oestrogens in transient transfection assays. Deletion of the most conserved (but not of the degenerate) ERE from reporter constructs abrogated the oestrogenic response, although both ERE-containing segments were footprinted in DNaseI protection assays. In vitro binding assays demonstrated the ability of oestrogen receptor alpha (ERalpha) to bind to this site, and chromatin immunoprecipitation analysis of the endogenous gene showed that ERalpha occupies this region in response to oestrogens. We conclude that AP-2gamma is a primary oestrogen-responsive gene and suggest that AP-2 proteins may mediate some oestrogenic responses.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Unión al ADN/genética , Estradiol/farmacología , Regulación Neoplásica de la Expresión Génica , Receptores de Estrógenos/metabolismo , Elementos de Respuesta , Factores de Transcripción/genética , Regiones no Traducidas 5' , Secuencia de Bases , Sitios de Unión , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/biosíntesis , Receptor alfa de Estrógeno , Femenino , Humanos , Datos de Secuencia Molecular , Proto-Oncogenes Mas , Factor de Transcripción AP-2 , Factores de Transcripción/biosíntesis , Activación TranscripcionalRESUMEN
In the present research we isolated and characterized Xenopus laevis CB1 cannabinoid receptor mRNA. The CB1 coding sequence shows a high degree of identity with those of other vertebrates, mammals included, confirming that CB1 receptor is conserved over the course of vertebrate evolution. Notably, the similarity between the X. laevis CB1 sequence and that of the urodele amphibian Taricha granulosa is not higher than the similarity existing between Xenopus and mammals, thus supporting phylogenetic distance between anurans and urodeles. By means of in situ hybridization histochemistry, CB1 mRNA expression and distribution was investigated in the X. laevis central nervous system. As revealed, CB1 mRNA-containing neurons are numerous in the prosencephalon, especially in the olfactory bulbs, telencephalic pallium, and hypothalamus. In the midbrain and hindbrain, labeled cells were observed in the mesencephalic tegmentum and dorsolateral romboencephalon. Abundant CB1 mRNA positive neurons are localized throughout the gray matter of the spinal cord, in particular in the dorsal and ventral fields, where labeled motor neurons are also observed. The distribution of CB1 mRNA in the Xenopus CNS is generally consistent with the CB1-like-immunohistochemistry results we have previously obtained, showing in amphibians a well developed cannabinergic system almost comparable to that described in mammals. However, some differences, such as the abundance of CB1 mRNA-containing neurons in the olfactory system and the rich CB1 spinal innervation, are found.
Asunto(s)
Sistema Nervioso Central/metabolismo , Clonación Molecular , ARN Mensajero/metabolismo , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Xenopus laevis/metabolismo , Secuencia de Aminoácidos/genética , Animales , Encéfalo/metabolismo , Femenino , Datos de Secuencia Molecular , Médula Espinal/metabolismo , Distribución Tisular , Vertebrados/metabolismoRESUMEN
Endocannabinoids (eCBs) are natural lipids regulating a large array of physiological functions and behaviors in vertebrates. The eCB system is highly conserved in evolution and comprises several specific receptors (type-1 and type-2 cannabinoid receptors), their endogenous ligands (e.g., anandamide and 2-arachidonoylglycerol), and a number of biosynthetic and degradative enzymes. In the last few years, eCBs have been described as critical signals in the control of male and female reproduction at multiple levels: centrally, by targeting hypothalamic gonadotropin-releasing-hormone-secreting neurons and pituitary, and locally, with direct effects on the gonads. These functions are supported by the extensive localization of cannabinoid receptors and eCB metabolic enzymes at different levels of the hypothalamic-pituitary-gonadal axis in mammals, as well as bonyfish and amphibians. In vivo and in vitro studies indicate that eCBs centrally regulate gonadal functions by modulating the gonadotropin-releasing hormone-gonadotropin-steroid network through direct and indirect mechanisms. Several proofs of local eCB regulation have been found in the testis and male genital tracts, since eCBs control Sertoli and Leydig cells activity, germ cell progression, as well as the acquisition of sperm functions. A comparative approach usually is a key step in the study of physiological events leading to the building of a general model. Thus, in this review, we summarize the action of eCBs at different levels of the male reproductive axis, with special emphasis, where appropriate, on data from non-mammalian vertebrates.
RESUMEN
The endocannabinoid system (ECS) has a well-documented pivotal role in the control of mammalian reproductive functions, by acting at multiple levels, that is, central (CNS) and local (gonads) levels. Since studies performed in animal models other than mammals might provide further insight into the biology of these signalling molecules, in the present paper we review the comparative data pointing toward the endocannabinoid involvement in the reproductive control of non-mammalian vertebrates, focussing in particular on the central regulation of teleost and amphibian reproduction. The morphofunctional distribution of brain cannabinoid receptors will be discussed in relation to other crucial signalling molecules involved in the control of reproductive functions, such as GnRH, dopamine, aromatase, and pituitary gonadotropins.
RESUMEN
The endocannabinoid system has a well-documented pivotal role in the control of mammalian feeding response; nevertheless, some evidence is available regarding a similar role in nonmammalian vertebrates and invertebrates. As in the bonyfish Carassius auratus, CB1 cannabinoid receptors are abundant in brain regions involved in the control of food intake, and fasting affects endocannabinoid levels, in this study the effects of food deprivation and anandamide administration on CB1 expression were evaluated. Fasting led to a time-dependent increase of CB1 mRNA levels in the forebrain, an effect reversed by refeeding. Furthermore, the administration of exogenous anandamide reduced CB1 expression in food-deprived goldfish. Our results support the involvement of CB1 receptors in the control of energy intake in nonmammalian vertebrates.
Asunto(s)
Ácidos Araquidónicos/farmacología , Moduladores de Receptores de Cannabinoides/farmacología , Ayuno/fisiología , Carpa Dorada/metabolismo , Alcamidas Poliinsaturadas/farmacología , Prosencéfalo/fisiología , Receptor Cannabinoide CB1/metabolismo , Actinas/metabolismo , Análisis de Varianza , Animales , Endocannabinoides , Femenino , Expresión Génica/efectos de los fármacos , Carpa Dorada/genética , Masculino , Piperidinas/farmacología , Reacción en Cadena de la Polimerasa , Prosencéfalo/efectos de los fármacos , Pirazoles/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/genética , Rombencéfalo/fisiología , Factores de TiempoRESUMEN
Based on pharmacological, behavioral, and neuroanatomical studies, the endocannabinoid system appears to be pivotal in some neuroendocrine mechanisms, such as modulation of vertebrate reproduction, stress, and food intake. The present study aimed to investigate the involvement of the endocannabinoid system in the control of the feeding response in the goldfish. By means of immunohistochemistry techniques, using anti-CB1 cannabinoid receptor, anti-corticotropin-releasing factor (CRF), and anti-neuropeptide Y (NPY) antisera on brain sections of Carassius auratus, we found a topographical co-distribution of the three signaling molecules through the preoptic area and posterior lobes of the hypothalamus and even a co-localization of CB1 and NPY in the telencephalon. Previous results have shown that food deprivation in goldfish is accompanied by a significant increase of anandamide (AEA) levels in the telencephalon and AEA causes a dose-dependent effect on food intake. We have thus investigated the possible influence of intraperitoneal AEA injections on NPY expression. Our results indicate an interplay between the endocannabinoid system and orexigenic and anorexigenic molecules, such as NPY and, possibly, CRF.