RESUMO
OBJECTIVE: The study goal was to analyze the effects of a high-fat diet (HFD) on the histone 3 lysine 27 (H3K27) posttranscriptional modifications and the expression of histone-modifying enzymes in adipose-derived stromal cells (ASCs) from white adipose tissue (WAT). METHODS: Male C57BL/6J mice received control or HFD for 12 weeks. The ASCs were isolated from subcutaneous and visceral (epididymal) WAT, cultivated, and evaluated for expression of H3K27 trimethylation (H3K27me3) and H3K27 acetylation (H3K27ac) by Western blot. The transcription of histone-modifying enzymes was analyzed by real-time polymerase chain reaction. RESULTS: When compared with control, HFD ASCs showed a decrease in H3K27ac enrichment in subcutaneous and visceral WAT and ATP-citrate lyase expression in subcutaneous WAT. Curiously, the expression of CREB-binding protein was increased in visceral ASCs from HFD-fed mice. CONCLUSIONS: These results show that an HFD significantly reduces acetylation of H3K27 in ASCs and the expression of ATP-citrate lyase in subcutaneous ASCs, suggesting that, in this fat depot, the H3K27ac reduction could be partly due to lower acetyl-coenzyme A availability. H3K27ac is an epigenetic mark responsible for increasing the transcription rate and its reduction can have an important impact on ASC proliferation and differentiation potential.
Assuntos
Dieta Hiperlipídica , Histonas , Acetilação , Trifosfato de Adenosina , Animais , Proteína de Ligação a CREB/metabolismo , Coenzima A/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Estromais/metabolismoRESUMO
The molecular targets and mechanisms of propolis ameliorating metabolic syndrome are not fully understood. Here, we report that Brazilian green propolis reduces fasting blood glucose levels in obese mice by disrupting the formation of CREB/CRTC2 transcriptional complex, a key regulator of hepatic gluconeogenesis. Using a mammalian two-hybrid system based on CREB-CRTC2, we identify artepillin C (APC) from propolis as an inhibitor of CREB-CRTC2 interaction. Without apparent toxicity, APC protects mice from high fat diet-induced obesity, decreases fasting glucose levels, enhances insulin sensitivity and reduces lipid levels in the serum and liver by suppressing CREB/CRTC2-mediated both gluconeogenic and SREBP transcriptions. To develop more potential drugs from APC, we designed and found a novel compound, A57 that exhibits higher inhibitory activity on CREB-CRTC2 association and better capability of improving insulin sensitivity in obese animals, as compared with APC. In this work, our results indicate that CREB/CRTC2 is a suitable target for developing anti-metabolic syndrome drugs.
Assuntos
Proteína de Ligação a CREB/metabolismo , Sistemas de Liberação de Medicamentos , Síndrome Metabólica/metabolismo , Camundongos Obesos/metabolismo , Própole/metabolismo , Fatores de Transcrição/metabolismo , Animais , Glicemia , Brasil , Proteína de Ligação a CREB/genética , Desenvolvimento de Medicamentos , Descoberta de Drogas , Gluconeogênese , Resistência à Insulina , Fígado/metabolismo , Síndrome Metabólica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Obesos/genética , Obesidade/metabolismo , Própole/genética , Fatores de Transcrição/genéticaRESUMO
Corticotropin-releasing hormone receptor 1 (CRHR1) activates the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). Both cAMP sources were shown to be required for the phosphorylation of ERK1/2 triggered by activated G protein coupled receptor (GPCR) CRHR1 in neuronal and neuroendocrine contexts. Here, we show that activated CRHR1 promotes growth arrest and neurite elongation in neuronal hippocampal cells (HT22-CRHR1 cells). By characterising CRHR1 signalling mechanisms involved in the neuritogenic effect, we demonstrate that neurite outgrowth in HT22-CRHR1 cells takes place by a sAC-dependent, ERK1/2-independent signalling cascade. Both tmACs and sAC are involved in corticotropin-releasing hormone (CRH)-mediated CREB phosphorylation and c-fos induction, but only sAC-generated cAMP pools are critical for the neuritogenic effect of CRH, further highlighting the engagement of two sources of cAMP downstream of the activation of a GPCR, and reinforcing the notion that restricted cAMP microdomains may regulate independent cellular processes.
Assuntos
Diferenciação Celular , AMP Cíclico/metabolismo , Células Piramidais/citologia , Células Piramidais/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Adenilil Ciclases/sangue , Adenilil Ciclases/metabolismo , Animais , Biomarcadores , Proteína de Ligação a CREB/metabolismo , Pontos de Checagem do Ciclo Celular , Sobrevivência Celular , Células Cultivadas , Hormônio Liberador da Corticotropina/metabolismo , Humanos , CamundongosRESUMO
BACKGROUND: Liposomes are concentric lipid vesicles that allow a sustained release of entrapped substances. GABA (γ-aminobutyric acid) is the most prevalent inhibitory neurotransmitter in the central nervous system. NEW METHOD: Using GABA-containing liposomes (GL) prepared by the freeze-thawing method, we determined the effect of sustained release of GABA on expression of neuronal nitric oxide synthase (nNOS) and GABAA receptor (GABAAR) in an in vitro neuronal model. RESULTS: Neuronal cell line NG108-15 treated with different doses of GL during 24h showed an increase in expression of GABAAR (54 and 50% with 10 and 20ng doses, respectively) and nNOS (138, 157 and 165% with 20, 50 and 100ng doses, respectively) compared with cells treated with empty liposomes (EL). Additionally, cells treated with 50ng of GL showed an increase in GABAAR (23%) after 1h followed by an increase in nNOS (55, 46 and 55%) at 8, 12 and 24h time points, respectively. Immunofluorescence experiments confirmed an increase in nNOS (134%) and basal intracellular levels of nitric oxide (84%) after GL treatment. Further, treatment of cells with GL showed a decrease in expression of a protein inhibitor of nNOS (PIN) (26, 66 and 57% with 20, 50 and 100ng doses respectively) compared with control. COMPARISON WITH EXISTING METHODS: This is first demonstration for the development of GL that allows sustained slow release of this neurotransmitter. CONCLUSION: These results suggest that a slow release of GABA can change the expression of nNOS possibly via alteration in PIN levels in neuronal cells.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lipossomos/administração & dosagem , Óxido Nítrico Sintase Tipo I/metabolismo , Ácido gama-Aminobutírico/administração & dosagem , Animais , Proteína de Ligação a CREB/metabolismo , Linhagem Celular Tumoral , Dineínas do Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Ácido Glutâmico/farmacologia , Lipossomos/farmacologia , Camundongos , Neuroblastoma/patologia , Nitritos/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Receptores de GABA-A/metabolismo , Fatores de Tempo , Ácido gama-Aminobutírico/farmacologiaRESUMO
Phosphorylated-cyclic adenosine monophosphate response element-binding protein (Phospho-CREB) has an important role in the pathogenesis of myocardial ischemia. We isolated the iridoid glycoside cornin from the fruit of Verbena officinalis L, investigated its effects against myocardial ischemia and reperfusion (I/R) injury in vivo, and elucidated its potential mechanism in vitro. Effects of cornin on cell viability, as well as expression of phospho-CREB and phospho-Akt in hypoxic H9c2 cells in vitro, and myocardial I/R injury in vivo, were investigated. Cornin attenuated hypoxia-induced cytotoxicity significantly in H9c2 cells in a concentration-dependent manner. Treatment of H9c2 cells with cornin (10 µM) blocked the reduction of expression of phospho-CREB and phospho-Akt in a hypoxic condition. Treatment of rats with cornin (30 mg/kg, iv) protected them from myocardial I/R injury as indicated by a decrease in infarct volume, improvement in hemodynamics, and reduction of severity of myocardial damage. Cornin treatment also attenuated the reduction of expression of phospho-CREB and phospho-Akt in ischemic myocardial tissue. These data suggest that cornin exerts protective effects due to an increase in expression of phospho-CREB and phospho-Akt.
Assuntos
Proteína de Ligação a CREB/metabolismo , Frutas/química , Glicosídeos Iridoides/farmacologia , Isquemia Miocárdica/tratamento farmacológico , Fitoterapia , Substâncias Protetoras/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Verbena/química , Animais , Western Blotting , Proteína de Ligação a CREB/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Creatina Quinase/sangue , Modelos Animais de Doenças , Hipóxia/tratamento farmacológico , Glicosídeos Iridoides/isolamento & purificação , Masculino , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Ratos Sprague-Dawley , Troponina/sangueRESUMO
Phosphorylated-cyclic adenosine monophosphate response element-binding protein (Phospho-CREB) has an important role in the pathogenesis of myocardial ischemia. We isolated the iridoid glycoside cornin from the fruit of Verbena officinalis L, investigated its effects against myocardial ischemia and reperfusion (I/R) injury in vivo, and elucidated its potential mechanism in vitro. Effects of cornin on cell viability, as well as expression of phospho-CREB and phospho-Akt in hypoxic H9c2 cells in vitro, and myocardial I/R injury in vivo, were investigated. Cornin attenuated hypoxia-induced cytotoxicity significantly in H9c2 cells in a concentration-dependent manner. Treatment of H9c2 cells with cornin (10 µM) blocked the reduction of expression of phospho-CREB and phospho-Akt in a hypoxic condition. Treatment of rats with cornin (30 mg/kg, iv) protected them from myocardial I/R injury as indicated by a decrease in infarct volume, improvement in hemodynamics, and reduction of severity of myocardial damage. Cornin treatment also attenuated the reduction of expression of phospho-CREB and phospho-Akt in ischemic myocardial tissue. These data suggest that cornin exerts protective effects due to an increase in expression of phospho-CREB and phospho-Akt.
Assuntos
Animais , Masculino , Isquemia Miocárdica/tratamento farmacológico , Verbena/química , Proteína de Ligação a CREB/metabolismo , Glicosídeos Iridoides/farmacologia , Frutas/química , Fitoterapia , Troponina/sangue , Linhagem Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Western Blotting , Ratos Sprague-Dawley , Creatina Quinase/sangue , Modelos Animais de Doenças , Proteína de Ligação a CREB/efeitos dos fármacos , Glicosídeos Iridoides/isolamento & purificação , Hipóxia/tratamento farmacológicoRESUMO
Treatment of neuropathic pain is a clinical challenge likely because of the time-dependent changes in many neurotransmitter systems, growth factors, ionic channels, membrane receptors, transcription factors, and recruitment of different cell types. Conversely, an increasing number of reports have shown the ability of extended and regular physical exercise in alleviating neuropathic pain throughout a wide range of mechanisms. In this study, we investigate the effect of swim exercise on molecules associated with initiation and maintenance of nerve injury-induced neuropathic pain. BALB/c mice were submitted to partial ligation of the sciatic nerve followed by a 5-week aerobic exercise program. Physical training reversed mechanical hypersensitivity, which lasted for an additional 4 weeks after exercise interruption. Swim exercise normalized nerve injury-induced nerve growth factor, and brain-derived neurotrophic factor (BDNF) enhanced expression in the dorsal root ganglion, but had no effect on the glial-derived neurotrophic factor. However, only BDNF remained at low levels after exercise interruption. In addition, exercise training significantly reduced the phosphorylation status of PLCγ-1, but not CREB, in the spinal cord dorsal horn in response to nerve injury. Finally, prolonged swim exercise reversed astrocyte and microglia hyperactivity in the dorsal horn after nerve lesion, which remained normalized after training cessation. Together, these results demonstrate that exercise therapy induces long-lasting analgesia through various mechanisms associated with the onset and advanced stages of neuropathy. Moreover, the data support further studies to clarify whether appropriate exercise intensity, volume, and duration can also cause long-lasting pain relief in patients with neuropathic pain.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Terapia por Exercício/métodos , Neuralgia/reabilitação , Neuroglia/metabolismo , Regulação para Cima/fisiologia , Adaptação Fisiológica , Animais , Proteína de Ligação a CREB/metabolismo , Citrato (si)-Sintase , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Coração/fisiopatologia , Hiperalgesia/etiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Músculo Esquelético/fisiopatologia , Neuralgia/complicações , Neuralgia/patologia , Neuroglia/patologia , Medição da Dor , Limiar da Dor/fisiologia , Fosfolipase C gama/metabolismo , Fosforilação , Fatores de TempoRESUMO
Considering that intracellular signaling pathways that modulate brain BDNF are implicated in antidepressant responses, this study investigated whether signaling pathway inhibitors upstream to BDNF might influence the antidepressant-like effect of zinc, a metal that has been shown to display antidepressant properties. To this end, the influence of i.c.v. administration of H-89 (1µg/site, PKA inhibitor), KN-62 (1µg/site, CAMKII inhibitor), chelerythrine (1µg/site, PKC inhibitor), PD98059 (5µg/site, MEK1/2 inhibitor), U0126 (5µg/site, MEK1/2 inhibitor), LY294002 (10nmol/site, PI3K inhibitor) on the reduction of immobility time in the tail suspension test (TST) elicited by ZnCl2 (10mg/kg, p.o.) was investigated. Moreover, the effect of the combination of sub-effective doses of ZnCl2 (1mg/kg, p.o.) and AR-A014418 (0.001µg/site, GSK-3ß inhibitor) was evaluated. The occurrence of changes in CREB phosphorylation and BDNF immunocontent in the hippocampus and prefrontal cortex of mice following ZnCl2 treatment was also investigated. The anti-immobility effect of ZnCl2 in the TST was prevented by treatment with PKA, PKC, CAMKII, MEK1/2 or PI3K inhibitors. Furthermore, ZnCl2 in combination with AR-A014418 caused a synergistic anti-immobility effect in the TST. None of the treatments altered locomotor activity of mice. ZnCl2 treatment caused no alteration in CREB phosphorylation and BDNF immunocontent. The results extend literature data regarding the mechanisms underlying the antidepressant-like action of zinc by indicating that its antidepressant-like effect may be dependent on the activation of PKA, CAMKII, PKC, ERK, and PI3K/GSK-3ß pathways. However, zinc is not able to acutely increase BDNF in the hippocampus and prefrontal cortex.
Assuntos
Antidepressivos/uso terapêutico , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cloretos/uso terapêutico , Depressão/tratamento farmacológico , Transdução de Sinais/fisiologia , Compostos de Zinco/uso terapêutico , Animais , Antidepressivos/farmacologia , Proteína de Ligação a CREB/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Cloretos/farmacologia , Depressão/patologia , Depressão/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Comportamento Exploratório/efeitos dos fármacos , Feminino , Elevação dos Membros Posteriores , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estatísticas não Paramétricas , Compostos de Zinco/farmacologiaRESUMO
BACKGROUND/OBJECTIVES: Early handling alters adult behavioral responses to palatable food and to its withdrawal following a period of chronic exposure. However, the central mechanisms involved in this phenomenon are not known. Since neonatal handling has persistent effects on stress and anxiety responses, we hypothesized that its involvement in the aforementioned association may be associated with differential neuroadaptations in the amygdala during withdrawal periods. METHODS: Litters were randomized into two groups: handled (H, removed from their dam for 10min per day from the first to the tenth postnatal day and placed in an incubator at 32°C) and non-handled (NH). Experiment 1: on PNDs 80-100, females were assigned to receive palatable food+rat chow for 15 or 30 days, and these two groups were compared in terms of palatable food preference, body weight and abdominal fat deposition. In Experiment 2, H and NH rats were exposed to a chronic diet of palatable food+rat chow for 15 days, followed by (a) no withdrawal, (b) 24h withdrawal from palatable food (receiving only rat chow) or (c) 7-day withdrawal from palatable food (receiving only rat chow). Body weight, 10-min rebound palatable food intake, abdominal fat deposition, serum corticosterone as well as TH and pCREB levels in the amygdala were then compared between groups. RESULTS: Experiment 1-chronic exposure to palatable food induces comparable metabolic effects after 15 and 30 days. Experiment 2-neonatal handling is associated with a peculiar response to palatable food withdrawal following chronic exposure for 15 days. Rats exposed to early handling ingested less of this food after a 24h withdrawal period, and displayed increased amygdala TH and pCREB levels. CONCLUSIONS: Variations in the neonatal environment affect both behavioral responses and amygdala neuroadaptation to acute withdrawal from a palatable diet. These findings contribute to the comprehension of the mechanisms that link early life events and altered feeding behavior and related morbidities such as obesity in adulthood.
Assuntos
Encéfalo/metabolismo , Meio Ambiente , Preferências Alimentares , Manobra Psicológica , Estresse Psicológico/enfermagem , Síndrome de Abstinência a Substâncias/patologia , Análise de Variância , Animais , Animais Recém-Nascidos , Proteína de Ligação a CREB/metabolismo , Modelos Animais de Doenças , Ingestão de Alimentos/fisiologia , Comportamento Alimentar/fisiologia , Feminino , Masculino , Gravidez , Ratos , Ratos Wistar , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
To address the role of mixed anxiety/mood disorder on appetitive associative learning, we verify whether previous chronic light deprivation changes ethanol-induced conditioned place preference and its respective expression of c-Fos and pCREB, markers of neuronal activity and plasticity. The experimental group was maintained in light deprivation for 24 h for a period of 4 wk. Subsequently, it was adapted to a standard light-dark cycle for 1 wk. As a control, some mice were maintained in standard cycle for a period of 4 wk (Naïve group). Then, all animals were submitted to behavioral tests to assess emotionality: elevated plus maze; open field; and forced swim. After that, they were submitted to ethanol-induced conditioned place preference. Ninety minutes after the place preference test, they were perfused, and their brains processed for c-Fos and pCREB immunohistochemistry. Light deprivation induced anxiety-like trait (elevated plus maze), despair (forced swim), and hyperlocomotion (open field), common features seen in other animal models of depression. Ethanol-induced conditioned place preference was accompanied by increases on c-Fos and pCREB in the hippocampus, prefrontal cortex and striatum. Interestingly, mice previously submitted to light deprivation did not develop either acquisition and/or expression of ethanol-induced conditioned place preference or increases in c-Fos and pCREB. Therefore, chronic light deprivation mimics several behavioral aspects of other animal models of depression. Furthermore, it could be useful to study the neurochemical mechanisms involved in the dual diagnosis. However, given its likely deleterious effects on appetitive associative memory, it should be used with caution to investigate the cognitive aspects related to the dual diagnosis.