RESUMO
High-calorie diets and chronic stress are major contributors to the development of obesity and metabolic disorders. These two risk factors regulate the activity of the sympathetic nervous system (SNS). The present study showed a key role of the cannabinoid type 1 receptor (CB1) in dopamine ß-hydroxylase (dbh)-expressing cells in the regulation of SNS activity. In a diet-induced obesity model, CB1 deletion from these cells protected mice from diet-induced weight gain by increasing sympathetic drive, resulting in reduced adipogenesis in white adipose tissue and enhanced thermogenesis in brown adipose tissue. The deletion of CB1 from catecholaminergic neurons increased the plasma norepinephrine levels, norepinephrine turnover, and sympathetic activity in the visceral fat, which coincided with lowered neuropeptide Y (NPY) levels in the visceral fat of the mutant mice compared with the controls. Furthermore, the mutant mice showed decreased plasma corticosterone levels. Our study provided new insight into the mechanisms underlying the roles of the endocannabinoid system in regulating energy balance, where the CB1 deletion in dbh-positive cells protected from diet-induced weight gain via multiple mechanisms, such as increased SNS activity, reduced NPY activity, and decreased basal hypothalamic-pituitary-adrenal (HPA) axis activity.
Assuntos
Canabinoides , Neuropeptídeo Y , Camundongos , Animais , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Endocanabinoides/metabolismo , Dopamina beta-Hidroxilase/genética , Dopamina beta-Hidroxilase/metabolismo , Canabinoides/metabolismo , Corticosterona/metabolismo , Obesidade/genética , Obesidade/metabolismo , Dieta Hiperlipídica/efeitos adversos , Neurônios/metabolismo , Norepinefrina/metabolismo , Aumento de PesoRESUMO
The endocannabinoid (eCB) system is widely expressed in many central and peripheral tissues, and is involved in a plethora of physiological processes. Among these, activity of the eCB system promotes energy intake and storage, which, however, under pathophysiological conditions, can favour the development of obesity and obesity-related disorders. It is proposed that eCB signalling is evolutionary beneficial for survival under periods of scarce food resources. Remarkably, eCB signalling is increased both in hunger and in overnutrition conditions, such as obesity and type-2 diabetes. This apparent paradox suggests a role of the eCB system both at initiation and at clinical endpoint of obesity. This review will focus on recent findings about the role of the eCB system controlling whole-body metabolism in mice that are genetically modified selectively in different cell types. The current data in fact support the notion that eCB signalling is not only engaged in the development but also in the maintenance of obesity, whereby specific cell types in central and peripheral tissues are key sites in regulating the entire body's energy homeostasis.
Assuntos
Encéfalo/metabolismo , Endocanabinoides/metabolismo , Metabolismo Energético , Tecido Adiposo/metabolismo , Animais , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismoRESUMO
G protein-coupled receptors (GPCRs) regulate virtually all physiological functions including the release of insulin from pancreatic ß-cells. ß-Cell M3 muscarinic receptors (M3Rs) are known to play an essential role in facilitating insulin release and maintaining proper whole-body glucose homeostasis. As is the case with other GPCRs, M3R activity is regulated by phosphorylation by various kinases, including GPCR kinases and casein kinase 2 (CK2). At present, it remains unknown which of these various kinases are physiologically relevant for the regulation of ß-cell activity. In the present study, we demonstrate that inhibition of CK2 in pancreatic ß-cells, knockdown of CK2α expression, or genetic deletion of CK2α in ß-cells of mutant mice selectively augmented M3R-stimulated insulin release in vitro and in vivo. In vitro studies showed that this effect was associated with an M3R-mediated increase in intracellular calcium levels. Treatment of mouse pancreatic islets with CX4945, a highly selective CK2 inhibitor, greatly reduced agonist-induced phosphorylation of ß-cell M3Rs, indicative of CK2-mediated M3R phosphorylation. We also showed that inhibition of CK2 greatly enhanced M3R-stimulated insulin secretion in human islets. Finally, CX4945 treatment protected mice against diet-induced hyperglycemia and glucose intolerance in an M3R-dependent fashion. Our data demonstrate, for the first time to our knowledge, the physiological relevance of CK2 phosphorylation of a GPCR and suggest the novel concept that kinases acting on ß-cell GPCRs may represent novel therapeutic targets.
Assuntos
Caseína Quinase II/fisiologia , Insulina/metabolismo , Receptor Muscarínico M3/fisiologia , Animais , Células COS , Chlorocebus aethiops , Feminino , Células HEK293 , Humanos , Secreção de Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naftiridinas/farmacologia , FenazinasRESUMO
Spinophilin (SPL), a multidomain scaffolding protein known to modulate the activity of different G-protein-coupled receptors, regulates various central nervous system (CNS) functions. However, little is known about the role of SPL expressed in peripheral cell types including pancreatic ß cells. In this study, we examined the ability of SPL to modulate the activity of ß-cell M(3) muscarinic acetylcholine receptors (M3Rs), which play an important role in facilitating insulin release and maintaining normal blood glucose levels. We demonstrated, by using both in vitro and in vivo approaches (mouse insulinoma cells and SPL-deficient mice), that SPL is a potent negative regulator of M3R-mediated signaling and insulin release. Additional biochemical and biophysical studies, including the use of bioluminescence resonance energy transfer technology, suggested that SPL is able to recruit regulator of G-protein signaling 4 (RGS4) to the M3R signaling complex in an agonist-dependent fashion. Since RGS4 is a member of the RGS family of proteins that act to reduce the lifetime of activated G proteins, these findings support the concept that the inhibitory effects of SPL on M3R activity are mediated by RGS4. These data suggest that SPL or other G-protein-coupled receptor-associated proteins may serve as novel targets for drug therapy aimed at improving ß-cell function for the treatment of type 2 diabetes.
Assuntos
Resistência à Insulina/fisiologia , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptor Muscarínico M3/metabolismo , Animais , Western Blotting , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Resistência à Insulina/genética , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Receptores Acoplados a Proteínas G , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Therapeutic strategies that augment insulin release from pancreatic beta-cells are considered beneficial in the treatment of type 2 diabetes. We previously demonstrated that activation of beta-cell M(3) muscarinic receptors (M3Rs) greatly promotes glucose-stimulated insulin secretion (GSIS), suggesting that strategies aimed at enhancing signaling through beta-cell M3Rs may become therapeutically useful. M3R activation leads to the stimulation of G proteins of the G(q) family, which are under the inhibitory control of proteins known as regulators of G protein signaling (RGS proteins). At present, it remains unknown whether RGS proteins play a role in regulating insulin release. To address this issue, we initially demonstrated that MIN6 insulinoma cells express functional M3Rs and that RGS4 was by far the most abundant RGS protein expressed by these cells. Strikingly, siRNA-mediated knockdown of RGS4 expression in MIN6 cells greatly enhanced M3R-mediated augmentation of GSIS and calcium release. We obtained similar findings using pancreatic islets prepared from RGS4-deficient mice. Interestingly, RGS4 deficiency had little effect on insulin release caused by activation of other beta-cell GPCRs. Finally, treatment of mutant mice selectively lacking RGS4 in pancreatic beta-cells with a muscarinic agonist (bethanechol) led to significantly increased plasma insulin and reduced blood glucose levels, as compared to control littermates. Studies with beta-cell-specific M3R knockout mice showed that these responses were mediated by beta-cell M3Rs. These findings indicate that RGS4 is a potent negative regulator of M3R function in pancreatic beta-cells, suggesting that RGS4 may represent a potential target to promote insulin release for therapeutic purposes.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas RGS/metabolismo , Receptor Muscarínico M3/metabolismo , Análise de Variância , Animais , Betanecol , Glicemia/metabolismo , Western Blotting , Células COS , Cálcio/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Eletroporação , Imunoprecipitação , Secreção de Insulina , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Corticosteroid-mediated stress responses require the activation of complex brain circuits involving mitochondrial activity, but the underlying cellular and molecular mechanisms are scantly known. The endocannabinoid system is implicated in stress coping, and it can directly regulate brain mitochondrial functions via type 1 cannabinoid (CB1) receptors associated with mitochondrial membranes (mtCB1). In this study, we show that the impairing effect of corticosterone in the novel object recognition (NOR) task in mice requires mtCB1 receptors and the regulation of mitochondrial calcium levels in neurons. Different brain circuits are modulated by this mechanism to mediate the impact of corticosterone during specific phases of the task. Thus, whereas corticosterone recruits mtCB1 receptors in noradrenergic neurons to impair NOR consolidation, mtCB1 receptors in local hippocampal GABAergic interneurons are required to inhibit NOR retrieval. These data reveal unforeseen mechanisms mediating the effects of corticosteroids during different phases of NOR, involving mitochondrial calcium alterations in different brain circuits.
Assuntos
Neurônios Adrenérgicos , Corticosterona , Camundongos , Animais , Corticosterona/farmacologia , Receptores de Canabinoides , Cálcio , Mitocôndrias , Endocanabinoides , Receptor CB1 de Canabinoide , Hipocampo/fisiologiaRESUMO
Impaired functioning of pancreatic beta cells is a key hallmark of type 2 diabetes. beta cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific beta cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that beta cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemical-genetic approach that allows for the conditional and selective activation of specific beta cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in beta cells only. Importantly, the two designer receptors differed in their G protein-coupling properties (G(q/11) versus G(s)). They were unable to bind endogenous ligand(s), but could be efficiently activated by an otherwise pharmacologically inert compound (clozapine-N-oxide), leading to the conditional activation of either beta cell G(q/11) or G(s) G proteins. Here we report the findings that conditional and selective activation of beta cell G(q/11) signaling in vivo leads to striking increases in both first- and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated beta cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of beta cell G(s) triggered qualitatively similar in vivo metabolic effects. Thus, this developed chemical-genetic strategy represents a powerful approach to study G protein regulation of beta cell function in vivo.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/fisiologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/anatomia & histologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Animais , Células COS , Chlorocebus aethiops , Clozapina/análogos & derivados , Clozapina/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Teste de Tolerância a Glucose , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Transgênicos , Ensaio Radioligante , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
The endocannabinoid system, with its receptors and ligands, is present in the gut epithelium and enteroendocrine cells, and is able to modulate brain functions, both indirectly through circulating gut-derived factors and directly through the vagus nerve, finally acting on the brain's mechanisms regarding metabolism and behavior. The gut endocannabinoid system also regulates gut motility, permeability, and inflammatory responses. Furthermore, microbiota composition has been shown to influence the activity of the endocannabinoid system. This review examines the interaction between microbiota, intestinal endocannabinoid system, metabolism, and stress responses. We hypothesize that the crosstalk between microbiota and intestinal endocannabinoid system has a prominent role in stress-induced changes in the gut-brain axis affecting metabolic and mental health. Inter-individual differences are commonly observed in stress responses, but mechanisms underlying resilience and vulnerability to stress are far from understood. Both gut microbiota and the endocannabinoid system have been implicated in stress resilience. We also discuss interventions targeting the microbiota and the endocannabinoid system to mitigate metabolic and stress-related disorders.
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Addiction is a chronic relapsing brain disease characterized by compulsive reward-seeking despite harmful consequences. The mechanisms underlying addiction are orchestrated by transcriptional reprogramming in the reward system of vulnerable subjects. This study aims at revealing gene expression alterations across different types of addiction. We analyzed publicly available transcriptome datasets of the prefrontal cortex (PFC) from a palatable food and a cocaine addiction study. We found 56 common genes upregulated in the PFC of addicted mice in these two studies, whereas most of the differentially expressed genes were exclusively linked to either palatable food or cocaine addiction. Gene ontology analysis of shared genes revealed that these genes contribute to learning and memory, dopaminergic synaptic transmission, and histone phosphorylation. Network analysis of shared genes revealed a protein-protein interaction node among the G protein-coupled receptors (Drd2, Drd1, Adora2a, Gpr6, Gpr88) and downstream targets of the cAMP signaling pathway (Ppp1rb1, Rgs9, Pde10a) as a core network in addiction. Upon extending the analysis to a cell-type specific level, some of these common molecular players were selectively expressed in excitatory neurons, oligodendrocytes, and endothelial cells. Overall, computational analysis of publicly available whole transcriptome datasets provides new insights into the molecular basis of addiction-like behaviors in PFC.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/genética , Dependência de Alimentos/genética , Regulação da Expressão Gênica , Córtex Pré-Frontal/fisiologia , Animais , Cocaína/farmacologia , AMP Cíclico/genética , AMP Cíclico/metabolismo , Bases de Dados Factuais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Histonas/genética , Histonas/metabolismo , Memória/fisiologia , Camundongos , Fosforilação , Córtex Pré-Frontal/efeitos dos fármacos , Transmissão Sináptica/genéticaRESUMO
BACKGROUND: Physical activity alleviates chronic stress. The latest research suggests a relationship between resilience and physical fitness. Beneficial adaptations of the hypothalamic-pituitary-adrenal axis, sympathetic nervous system, endocannabinoid system, and tryptophan pathway, which are induced by an active lifestyle, are considered to be conducive to resilience. However, detailed knowledge on the molecular link between the effects of acute and chronic physical exercise and improved resilience to stress in humans is missing. Moreover, the relationship between innate and acquired aerobic capacity and resilience is poorly understood. OBJECTIVE: The aim of this study is to implement a human exercise intervention trial addressing the following main hypotheses: a high innate aerobic capacity is associated with high resilience to stress, and web-based physical exercise training improves aerobic capacity of physically inactive adults, which is accompanied by improved resilience. In this setting, we will analyze the relationship between resilience parameters and innate and acquired aerobic capacity as well as circulating signaling molecules. METHODS: A total of 70 healthy, physically inactive (<150 minutes/week of physical activity) adults (aged 18-45 years) will be randomly assigned to an intervention or control group. Participants in the intervention group will receive weekly training using progressive endurance and interval running adapted individually to their remotely supervised home training performance via web-based coach support. A standardized incremental treadmill exercise test will be performed before and after the intervention period of 8 weeks to determine the innate and acquired aerobic capacity (peak oxygen uptake). Before and after the intervention, psychological tests and questionnaires that characterize parameters implicated in resilience will be applied. Blood and saliva will be sampled for the analysis of cortisol, lactate, endocannabinoids, catecholamines, kynurenic acid, and further circulating signal transducers. Statistical analysis will provide comprehensive knowledge on the relationship between aerobic capacity and resilience, as well as the capacity of peripheral factors to mediate the promoting effects of exercise on resilience. RESULTS: The study was registered in October 2019, and enrollment began in September 2019. Of the 161 participants who were initially screened via a telephone survey, 43 (26.7%) fulfilled the inclusion criteria and were included in the study. Among the 55% (17/31) of participants in the intervention group and 45% (14/31) of participants in the control group who completed the study, no serious adverse incidents were reported. Of 43 participants, 4 (9%) withdrew during the program (for individual reasons) and 8 (19%) have not yet participated in the program; moreover, further study recruitment was paused for an indeterminate amount of time because of the COVID-19 pandemic. CONCLUSIONS: Our study aims to further define the physiological characteristics of human resilience, and it may offer novel approaches for the prevention and therapy of mental disorders via an exercise prescription. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/29712.
RESUMO
Palatable food can promote overfeeding beyond homeostatic requirements, thereby constituting a major risk to obesity. Here, the lack of cannabinoid type 1 receptor (CB1) in dorsal telencephalic glutamatergic neurons (Glu-CB1-KO) abrogated the overconsumption of palatable food and the development of obesity. On low-fat diet, no genotype differences were observed. However, under palatable food conditions, Glu-CB1-KO mice showed decreased body weight and food intake. Notably, Glu-CB1-KO mice were protected from alterations in the reward system after high-fat diet feeding. Interestingly, obese wild-type mice showed a superior olfactory detection as compared to mutant mice, suggesting a link between overconsumption of palatable food and olfactory function. Reconstitution of CB1 expression in olfactory cortex in high-fat diet-fed Glu-CB1-KO mice using viral gene delivery partially reversed the lean phenotype concomitantly with improved odor perception. These findings indicate that CB1 in cortical glutamatergic neurons regulates hedonic feeding, whereby a critical role of the olfactory cortex was uncovered as an underlying mechanism.
Assuntos
Canabinoides , Animais , Dieta Hiperlipídica/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios , Obesidade/genética , Receptor CB1 de Canabinoide/genéticaRESUMO
Stress has a major impact on the modulation of metabolism, as previously evidenced by hyperglycemia following chronic social defeat (CSD) stress in mice. Although CSD-triggered metabolic dysregulation might predispose to pre-diabetic conditions, insulin sensitivity remained intact, and obesity did not develop, when animals were fed with a standard diet (SD). Here, we investigated whether a nutritional challenge, a high-fat diet (HFD), aggravates the metabolic phenotype and whether there are particularly sensitive time windows for the negative consequences of HFD exposure. Chronically stressed male mice and controls (CTRL) were kept under (i) SD-conditions, (ii) with HFD commencing post-CSD, or (iii) provided with HFD lasting throughout and after CSD. Under SD conditions, stress increased glucose levels early post-CSD. Both HFD regimens increased glucose levels in non-stressed mice but not in stressed mice. Nonetheless, when HFD was provided after CSD, stressed mice did not differ from controls in long-term body weight gain, fat tissue mass and plasma insulin, and leptin levels. In contrast, when HFD was continuously available, stressed mice displayed reduced body weight gain, lowered plasma levels of insulin and leptin, and reduced white adipose tissue weights as compared to their HFD-treated non-stressed controls. Interestingly, stress-induced adrenal hyperplasia and hypercortisolemia were observed in mice treated with SD and with HFD after CSD but not in stressed mice exposed to a continuous HFD treatment. The present work demonstrates that CSD can reduce HFD-induced metabolic dysregulation. Hence, HFD during stress may act beneficially, as comfort food, by decreasing stress-induced metabolic demands.
Assuntos
Antígenos de Grupos Sanguíneos/análise , Dieta Hiperlipídica , Estresse Psicológico/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Dieta Hiperlipídica/psicologia , Ingestão de Energia , Teste de Tolerância a Glucose , Insulina/sangue , Leptina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/psicologia , Derrota Social , Estresse Psicológico/sangue , Aumento de PesoRESUMO
Food addiction is linked to obesity and eating disorders and is characterized by a loss of behavioral control and compulsive food intake. Here, using a food addiction mouse model, we report that the lack of cannabinoid type-1 receptor in dorsal telencephalic glutamatergic neurons prevents the development of food addiction-like behavior, which is associated with enhanced synaptic excitatory transmission in the medial prefrontal cortex (mPFC) and in the nucleus accumbens (NAc). In contrast, chemogenetic inhibition of neuronal activity in the mPFC-NAc pathway induces compulsive food seeking. Transcriptomic analysis and genetic manipulation identified that increased dopamine D2 receptor expression in the mPFC-NAc pathway promotes the addiction-like phenotype. Our study unravels a new neurobiological mechanism underlying resilience and vulnerability to the development of food addiction, which could pave the way towards novel and efficient interventions for this disorder.
Assuntos
Dependência de Alimentos/fisiopatologia , Núcleo Accumbens/fisiologia , Córtex Pré-Frontal/fisiologia , Receptores de Dopamina D2/genética , Animais , Modelos Animais de Doenças , Comportamento Alimentar/fisiologia , Dependência de Alimentos/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos Knockout , Vias Neurais/fisiologia , Receptor CB1 de Canabinoide/genética , Transmissão Sináptica , Regulação para CimaRESUMO
BACKGROUND: Obesity is a low-grade inflammation condition that facilitates the development of numerous comorbidities and the dysregulation of brain homeostasis. Additionally, obesity also causes distinct behavioral alterations both in humans and rodents. Here, we investigated the effect of inducible genetic deletion of the cannabinoid type 1 receptor (CB1) in adipocytes (Ati-CB1-KO mice) on obesity-induced memory deficits, depressive-like behavior, neuroinflammation and adult neurogenesis. METHODS: Behavioral, mRNA expression and immunohistochemical studies were performed in Ati-CB1-KO mice and corresponding wild-type controls under standard and high-fat diet. RESULTS: Adipocyte-specific CB1 deletion reversed metabolic disturbances associated with an obese condition confirming previous studies. As compared to obese mice, the metabolic amelioration in Ati-CB1-KO mice was associated with an improvement of mood-related behavior and recognition memory, concomitantly with an increase in cell proliferation in metabolic relevant neurogenic niches in hippocampus and hypothalamus. In mutant mice, these changes were related to an increased neuronal maturation/survival in the hippocampus. Furthermore, CB1 deletion in adipocytes was sufficient to reduce obesity-induced inflammation, gliosis and apoptosis in a brain region-specific manner. CONCLUSIONS: Overall our data provide compelling evidence of the physiological relevance of the adipocyte-brain crosstalk where adipocyte-specific CB1 influences obesity-related cognitive deficits and depression-like behavior, concomitantly with brain remodeling, such as adult neurogenesis and neuroinflammation in the hippocampus and hypothalamus.
Assuntos
Adipócitos/metabolismo , Depressão/genética , Dieta Hiperlipídica/efeitos adversos , Transtornos da Memória/etiologia , Neurite (Inflamação)/genética , Neurogênese/genética , Receptor CB1 de Canabinoide/genética , Células-Tronco Adultas/fisiologia , Animais , Comportamento Animal/fisiologia , Encéfalo/citologia , Encéfalo/fisiologia , Depressão/metabolismo , Deleção de Genes , Masculino , Transtornos da Memória/genética , Transtornos da Memória/metabolismo , Transtornos da Memória/psicologia , Camundongos , Camundongos Knockout , Células-Tronco Neurais/fisiologia , Neurite (Inflamação)/metabolismo , Neurite (Inflamação)/patologia , Especificidade de Órgãos/genética , Receptor CB1 de Canabinoide/deficiência , Receptor CB1 de Canabinoide/metabolismoRESUMO
In the version of this article initially published, Inigo Ruiz de Azua's name was miscategorized. His given name is Inigo and his surname is Ruiz de Azua. This has been corrected in the HTML coding.
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The endocannabinoid (eCB) system, including its receptors, ligands, and their metabolizing enzymes, plays an important role in bone physiology. Skeletal cannabinoid type 1 (CB1) receptor signaling transmits retrograde signals that restrain norepinephrine (NE) release, thus transiently stimulating bone formation following an acute challenge, suggesting a feedback circuit between sympathetic nerve terminals and osteoblasts. To assess the effect of chronic in vivo occurrence of this circuit, we characterized the skeletal phenotype of mice with a conditional deletion of the CB1 receptor in adrenergic/noradrenergic cells, including sympathetic nerves. Whereas the deletion of the CB1 receptor did not affect bone mass accrual in the distal femoral metaphysis and in vertebral bodies of young, 12-week-old mice, it substantially increased bone mass in aged, 35-week-old mutant mice as compared to wild-type controls. Contrary to our expectations, specific deficiency of the CB1 receptor in sympathetic neurons led to a markedly increased bone mass phenotype, associated with an enhanced bone formation rate and reduced osteoclastogenesis. Mechanistically, the reduced skeletal eCB 'tone' in the null mice did not reflect in increased sympathetic tone and reduced bone formation, suggesting that constitutive genetic inactivation of sympathetic CB1 receptor disrupts the negative feedback loop between eCBs and NE signaling in bone.
Assuntos
Envelhecimento/metabolismo , Osteogênese , Receptor CB1 de Canabinoide/metabolismo , Sistema Nervoso Simpático/metabolismo , Animais , Reabsorção Óssea/patologia , Dopamina beta-Hidroxilase/metabolismo , Endocanabinoides/metabolismo , Deleção de Genes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeo Y/metabolismo , Norepinefrina/metabolismo , Receptores Adrenérgicos beta 2/metabolismoRESUMO
Multiple sclerosis (MS) patients exhibit neuropsychological symptoms in early disease despite the immune attack occurring predominantly in white matter and spinal cord. It is unclear why neurodegeneration may start early in the disease and is prominent in later stages. We assessed cortical microcircuit activity by employing spiking-specific two-photon Ca2+ imaging in proteolipid protein-immunized relapsing-remitting SJL/J mice in vivo. We identified the emergence of hyperactive cortical neurons in remission only, independent of direct immune-mediated damage and paralleled by elevated anxiety. High levels of neuronal activity were accompanied by increased caspase-3 expression. Cortical TNFα expression was mainly increased by excitatory neurons in remission; blockade with intraventricular infliximab restored AMPA spontaneous excitatory postsynaptic current frequencies, completely recovered normal neuronal network activity patterns and alleviated elevated anxiety. This suggests a dysregulation of cortical networks attempting to achieve functional compensation by synaptic plasticity mechanisms, indicating a link between immune attack and early start of neurodegeneration.
Assuntos
Córtex Cerebral/fisiopatologia , Encefalomielite Autoimune Experimental/complicações , Encefalomielite Autoimune Experimental/patologia , Hipercinese/etiologia , Recuperação de Função Fisiológica/fisiologia , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Carbazóis/uso terapêutico , Células Cultivadas , Córtex Cerebral/ultraestrutura , Cuprizona/toxicidade , Modelos Animais de Doenças , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacocinética , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/tratamento farmacológico , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Adjuvante de Freund/toxicidade , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Transgênicos , Microglia/patologia , Proteína Proteolipídica de Mielina/toxicidade , Fragmentos de Peptídeos/toxicidade , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Quinoxalinas/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologiaRESUMO
Fluoxetine (FLX) and norfluoxetine (NFLX) racemic mixtures were determined by reversed-phase liquid chromatography with fluorescence detection (lambda(exc)=227 nm, lambda(em)=305 nm). The calibration curves prepared from drug-free plasma and brain were linear in the range of 5-1000 ng ml(-1) and 100-40,000 ng g(-1) for doped samples, with detection limits of 3.2 and 2.1 ng ml(-1) in plasma and 31.5 and 26.1 ng g(-1) in brain tissue for FLX and NFLX, respectively. Enantiomer determination was carried out through normal phase HPLC-FD (lambda(exc)=224 nm, lambda(em)=336 nm) after precolumn chiral derivatization with R-1-(1-naphthyl)ethyl isocyanate. Standard curves also prepared in a drug-free matrix were linear for each enantiomer over the range of 2-1000 ng ml(-1) and 20-7000 ng g(-1) with detection limits for the four compounds ranging between 0.2 and 0.5 ng ml(-1) in plasma and between 3.0 and 8.2 ng g(-1) in brain tissue. In both methods the analytes were isolated from the biological matrix by a new solid-phase extraction procedure with recovery in plasma and brain over 90 and 87%, respectively. The repeatability of this extraction procedure was satisfactory within-day and between-day with CV<9.1%. This study also offered the opportunity to obtain an assessment of the potential relationships between the concentration of individual enantiomers of FLX and NFLX in plasma and brain tissue after chronic treatment with racemic FLX at a dose intended to mimic the human plasma concentration of FLX in standard clinical conditions, and therefore should make for more reliable extrapolation of neurochemical findings in other species.
Assuntos
Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Fluoxetina/análogos & derivados , Fluoxetina/metabolismo , Espectrometria de Fluorescência/métodos , Animais , Fluoxetina/sangue , Ratos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Dysregulated adipocyte physiology leads to imbalanced energy storage, obesity, and associated diseases, imposing a costly burden on current health care. Cannabinoid receptor type-1 (CB1) plays a crucial role in controlling energy metabolism through central and peripheral mechanisms. In this work, adipocyte-specific inducible deletion of the CB1 gene (Ati-CB1-KO) was sufficient to protect adult mice from diet-induced obesity and associated metabolic alterations and to reverse the phenotype in already obese mice. Compared with controls, Ati-CB1-KO mice showed decreased body weight, reduced total adiposity, improved insulin sensitivity, enhanced energy expenditure, and fat depot-specific cellular remodeling toward lowered energy storage capacity and browning of white adipocytes. These changes were associated with an increase in alternatively activated macrophages concomitant with enhanced sympathetic tone in adipose tissue. Remarkably, these alterations preceded the appearance of differences in body weight, highlighting the causal relation between the loss of CB1 and the triggering of metabolic reprogramming in adipose tissues. Finally, the lean phenotype of Ati-CB1-KO mice and the increase in alternatively activated macrophages in adipose tissue were also present at thermoneutral conditions. Our data provide compelling evidence for a crosstalk among adipocytes, immune cells, and the sympathetic nervous system (SNS), wherein CB1 plays a key regulatory role.
Assuntos
Adipócitos/metabolismo , Metabolismo Energético , Macrófagos/fisiologia , Receptor CB1 de Canabinoide/fisiologia , Tecido Adiposo Branco/imunologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Animais , Peso Corporal , Ingestão de Energia , Homeostase , Ativação de Macrófagos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/imunologia , Obesidade/metabolismo , Especificidade de Órgãos , TranscriptomaRESUMO
The levels of expression of G-protein alpha(q/11) (Galpha(q/11)) subunits and PLC-beta(1-4), -gamma, and -delta(1) isoforms were quantified by Western blot analysis in order to establish their contribution to the patterns of PLC functioning reported here. Quantitative measurements of the levels of Galpha(q/11) subunits in each region were obtained by comparison with known amounts of Escherichia coli expressed recombinant Galpha(q) subunits. Quantitative analysis indicated that Galpha(q/11) subunits are abundant polypeptides in human brain, with values ranging from about 1200 ng/mg in cerebral cortex to close to 900 ng/mg of membrane protein in caudate. In cerebral cortical membranes, the PLC-beta(1) isoform was more abundant than in caudate membranes. The highest levels of PLC-beta(2) expression were detected in caudate membranes. PLC-beta(3) was little expressed, and there were no significant differences in the relative values between both brain regions. Finally, the levels of the PLC-beta(4) isoform were significantly lower in caudate than in cortical membranes. It is concluded that although most of these data represent relative, not absolute, measures of protein levels within these regions, they contribute nonetheless to the significant differences observed in signaling capacities through the PLC system in both human brain regions.