RESUMO
Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.
Assuntos
Neuroimagem/métodos , Animais , Encéfalo/citologia , Callithrix , Indicadores e Reagentes/química , Camundongos , Microscopia/métodosRESUMO
Mass spectrometry (MS) imaging is a useful tool for direct and simultaneous visualization of specific molecules. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to evaluate the abundance of molecules in tissues using sample homogenates. To date, however, LC-MS/MS has not been utilized as an imaging tool because spatial information is lost during sample preparation. Here we report a new approach for LC-MS/MS imaging using a thermal film-based laser microdissection (LMD) technique. To isolate tissue spots, our LMD system uses a 808-nm near infrared laser, the diameter of which can be freely changed from 2.7 to 500 µm; for imaging purposes in this study, the diameter was fixed at 40 µm, allowing acquisition of LC-MS/MS images at a 40-µm resolution. The isolated spots are arranged on a thermal film at 4.5-mm intervals, corresponding to the well spacing on a 384-well plate. Each tissue spot is handled on the film in such a manner as to maintain its spatial information, allowing it to be extracted separately in its individual well. Using analytical LC-MS/MS in combination with the spatial information of each sample, we can reconstruct LC-MS/MS images. With this imaging technique, we successfully obtained the distributions of pilocarpine, glutamate, γ-aminobutyric acid, acetylcholine, and choline in a cross-section of mouse hippocampus. The protocol we established in this study is applicable to revealing the neurochemistry of pilocarpine model of epilepsy. Our system has a wide range of uses in fields such as biology, pharmacology, pathology, and neuroscience. Graphical abstract Schematic Indication of LMD-LC-MS/MS imaging.
Assuntos
Hipocampo/química , Microdissecção e Captura a Laser/métodos , Neurotransmissores/análise , Espectrometria de Massas em Tandem/métodos , Acetilcolina/análise , Animais , Colina/análise , Cromatografia Líquida/métodos , Epilepsia/diagnóstico , Epilepsia/patologia , Feminino , Ácido Glutâmico/análise , Hipocampo/patologia , Camundongos Endogâmicos C57BL , Pilocarpina/análise , Ácido gama-Aminobutírico/análiseRESUMO
Synaptic plasticity is a cellular mechanism putatively underlying learning and memory. However, it is unclear whether learning induces synaptic modification globally or only in a subset of neurons in associated brain regions. In this study, we genetically identified neurons activated during contextual fear learning and separately recorded synaptic efficacy from recruited and nonrecruited neurons in the mouse basolateral amygdala (BLA). We found that the fear learning induces presynaptic potentiation, which was reflected by an increase in the miniature EPSC frequency and by a decrease in the paired-pulse ratio. Changes occurred only in the cortical synapses targeting the BLA neurons that were recruited into the fear memory trace. Furthermore, we found that fear learning reorganizes the neuronal ensemble responsive to the conditioning context in conjunction with the synaptic plasticity. In particular, the neuronal activity during learning was associated with the neuronal recruitment into the context-responsive ensemble. These findings suggest that synaptic plasticity in a subset of BLA neurons contributes to fear memory expression through ensemble reorganization.
Assuntos
Tonsila do Cerebelo/fisiologia , Condicionamento Operante/fisiologia , Medo/fisiologia , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Transmissão Sináptica/fisiologia , Animais , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Rede Nervosa/fisiologiaRESUMO
This study examined the causal relationships between assertiveness and both internal and external adjustment in children. Elementary school children in grades four through six (N = 284) participated in the study, which used a short-term longitudinal design. The children completed questionnaires twice during a 6-months period. They responded to assertiveness questionnaires that included two components: "self-expression" and "consideration of others". They also completed a self-esteem scale as an index of internal adjustment, and the Class Life Satisfaction scale as an index of external adjustment. There was a positive causative relationship between "self-expression" and internal adjustment and between "consideration for others" and external adjustment. In addition, the effects on adjustment varied according to the type of assertiveness. Cluster analysis and MANOVA indicated that the group with high "self-expression" and "consideration for others" had high internal and external adjustment, while the children with poor assertiveness showed the lowest degree of adaptivity.
Assuntos
Assertividade , Ajustamento Social , Criança , Análise por Conglomerados , Feminino , Humanos , Estudos Longitudinais , Masculino , Inquéritos e Questionários , Fatores de TempoRESUMO
Our objective was to determine whether lipocalin-2 (Lcn2) regulates cardiomyocyte apoptosis, the mechanisms involved, and the functional significance. Emerging evidence suggests that Lcn2 is a proinflammatory adipokine associated with insulin resistance and obesity-related complications, such as heart failure. Here, we used both primary neonatal rat cardiomyocytes and H9c2 cells and demonstrated for the first time that Lcn2 directly induced cardiomyocyte apoptosis, an important component of cardiac remodeling leading to heart failure. This was shown by detection of DNA fragmentation using TUNEL assay, phosphatidylserine exposure using flow cytometry to detect annexin V-positive cells, caspase-3 activity using enzymatic assay and immunofluorescence, and Western blotting for the detection of cleaved caspase-3. We also observed that Lcn2 caused translocation of the proapoptotic protein Bax to mitochondria and disruption of mitochondrial membrane potential. Using transient transfection of GFP-Bax, we confirmed that Lcn2 induced co-localization of Bax with MitoTracker® dye. Importantly, we used the fluorescent probe Phen Green SK to demonstrate an increase in intracellular iron in response to Lcn2, and depleting intracellular iron using an iron chelator prevented Lcn2-induced cardiomyocyte apoptosis. Administration of recombinant Lcn2 to mice for 14 days increased cardiomyocyte apoptosis as well as an acute inflammatory response with compensatory changes in cardiac functional parameters. In conclusion, Lcn2-induced cardiomyocyte apoptosis is of physiological significance and occurs via a mechanism involving elevated intracellular iron levels and Bax translocation.
Assuntos
Proteínas de Fase Aguda/metabolismo , Apoptose/fisiologia , Ferro/metabolismo , Lipocalinas/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas de Fase Aguda/farmacologia , Animais , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Linhagem Celular , Fragmentação do DNA/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , Lipocalina-2 , Lipocalinas/farmacologia , Camundongos , Miócitos Cardíacos/citologia , Proteínas Oncogênicas/farmacologia , Fosfatidilserinas/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Proteína X Associada a bcl-2/metabolismoRESUMO
This study was conducted to examine the influence of acute streptozotocin-induced diabetes on cardiac remodelling and function in mice subjected to myocardial infarction (MI) by coronary artery ligation. Echocardiography analysis indicated that diabetes induced deleterious cardiac functional changes as demonstrated by the negative differences of ejection fraction, fractional shortening, stroke volume, cardiac output and left ventricular volume 24 hrs after MI. Temporal analysis for up to 2 weeks after MI showed higher mortality in diabetic animals because of cardiac wall rupture. To examine extracellular matrix remodelling, we used fluorescent molecular tomography to conduct temporal studies and observed that total matrix metalloproteinase (MMP) activity in hearts was higher in diabetic animals at 7 and 14 days after MI, which correlated well with the degree of collagen deposition in the infarct area visualized by scanning electron microscopy. Gene arrays indicated temporal changes in expression of distinct MMP isoforms after 1 or 2 weeks after MI, particularly in diabetic mice. Temporal changes in cardiac performance were observed, with a trend of exaggerated dysfunction in diabetic mice up to 14 days after MI. Decreased radial and longitudinal systolic and diastolic strain rates were observed over 14 days after MI, and there was a trend towards altered strain rates in diabetic mouse hearts with dyssynchronous wall motion clearly evident. This correlated with increased collagen deposition in remote areas of these infarcted hearts indicated by Masson's trichrome staining. In summary, temporal changes in extracellular matrix remodelling correlated with exaggerated cardiac dysfunction in diabetic mice after MI.
Assuntos
Doenças Cardiovasculares/fisiopatologia , Diabetes Mellitus Experimental/fisiopatologia , Infarto do Miocárdio/fisiopatologia , Remodelação Ventricular , Animais , Colágeno/metabolismo , Diástole , Ecocardiografia , Matriz Extracelular/metabolismo , Fibrose , Reserva Fracionada de Fluxo Miocárdico , Ruptura Cardíaca , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Isoformas de Proteínas/metabolismo , Volume Sistólico , Sístole , Inibidores Teciduais de Metaloproteinases/metabolismo , Função Ventricular EsquerdaRESUMO
Left-right asymmetry of human brain function has been known for a century, although much of molecular and cellular basis of brain laterality remains to be elusive. Recent studies suggest that hippocampal CA3-CA1 excitatory synapses are asymmetrically arranged, however, the functional implication of the asymmetrical circuitry has not been studied at the behavioral level. In order to address the left-right asymmetry of hippocampal function in behaving mice, we analyzed the performance of "split-brain" mice in the Barnes maze. The "split-brain" mice received ventral hippocampal commissure and corpus callosum transection in addition to deprivation of visual input from one eye. In such mice, the hippocampus in the side of visual deprivation receives sensory-driven input. Better spatial task performance was achieved by the mice which were forced to use the right hippocampus than those which were forced to use the left hippocampus. In two-choice spatial maze, forced usage of left hippocampus resulted in a comparable performance to the right counterpart, suggesting that both hippocampal hemispheres are capable of conducting spatial learning. Therefore, the results obtained from the Barnes maze suggest that the usage of the right hippocampus improves the accuracy of spatial memory. Performance of non-spatial yet hippocampus-dependent tasks (e.g. fear conditioning) was not influenced by the laterality of the hippocampus.
Assuntos
Cérebro/fisiologia , Lateralidade Funcional/fisiologia , Memória/fisiologia , Percepção Espacial/fisiologia , Animais , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Procedimento de Encéfalo DivididoRESUMO
The relationship between assertiveness and internal and external adjustment was investigated. Elementary school children in grades four to six (n=207) and their classroom teachers (n=8) participated in the study. Internal and external adjustments were measured by using self-ratings, and self- and other- ratings respectively. The children responded to a questionnaires inquiring about assertiveness that included two components of assessment: "self expression" and "consideration for others". Then, the children were divided into 4 groups according to their scores on these two components of assertiveness. The results indicated that children scoring high on both components of assertiveness had higher self-rating scores than those scoring low on both components. Moreover, children that scored high on "consideration for others" tended to have high external adjustment. Also, boys that scored low on "self expression" had lower external adjustment as indicated by the negative ratings of teachers. Furthermore, girls that scored high on "consideration for others" had high external adjustment as indicated by positive ratings of teachers and same-sexed classmates.
Assuntos
Assertividade , Ajustamento Social , Criança , Feminino , Humanos , Masculino , Fatores SexuaisRESUMO
To better understand the role of equilibrative nucleoside transporters (ENT) in purine nucleoside-dependent physiology of the cardiovascular system, we investigated whether the ENT1-null mouse heart was cardioprotected in response to ischemia (coronary occlusion for 30 min followed by reperfusion for 2 h). We observed that ENT1-null mouse hearts showed significantly less myocardial infarction compared with wild-type littermates. We confirmed that isolated wild-type adult mouse cardiomyocytes express predominantly ENT1, which is primarily responsible for purine nucleoside uptake in these cells. However, ENT1-null cardiomyocytes exhibit severely impaired nucleoside transport and lack ENT1 transcript and protein expression. Adenosine receptor expression profiles and expression levels of ENT2, ENT3, and ENT4 were similar in cardiomyocytes isolated from ENT1-null adult mice compared with cardiomyocytes isolated from wild-type littermates. Moreover, small interfering RNA knockdown of ENT1 in the cardiomyocyte cell line, HL-1, mimics findings in ENT1-null cardiomyocytes. Taken together, our data demonstrate that ENT1 plays an essential role in cardioprotection, most likely due to its effects in modulating purine nucleoside-dependent signaling and that the ENT1-null mouse is a powerful model system for the study of the role of ENTs in the physiology of the cardiomyocyte.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/fisiologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Transportador Equilibrativo 1 de Nucleosídeo/genética , Feminino , Camundongos , Camundongos Transgênicos , Modelos Animais , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Nucleosídeos de Purina/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores Purinérgicos P1/metabolismo , Transdução de Sinais/fisiologiaRESUMO
In vivo monitoring of gene expression using promoter-destabilized fluorescence protein constructs is a powerful method for examining the expression dynamics of immediate-early genes in the brain. However, weak fluorescence signals derived from such constructs have hampered analyses of gene expression over extensive areas of the brain. We succeeded in producing transgenic mice with brains exhibiting high level expression of the reporter gene driven by the Arc gene promoter, which is activated in association with various brain functions (reporter mRNA abundance was near 100-fold greater than endogenous Arc mRNAs). This high expression of the reporter gene enabled us to monitor Arc gene expression dynamics in vivo, over an area that included the whole of the dorsal cerebral cortex. Moreover, we were able to perform three-dimensional analyses of activated regions using paraformaldehyde-fixed brains. In addition to the visual cortex, we found that the cingulate cortex was strongly activated by light stimuli. These mice are extremely useful for the functional analysis of gene expression over extensive areas of the brains in both wild-type mice and mutants with impaired brain function.
Assuntos
Encéfalo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Perfilação da Expressão Gênica/métodos , Camundongos Transgênicos/metabolismo , Microscopia de Fluorescência/métodos , Proteínas do Tecido Nervoso/metabolismo , Espectrometria de Fluorescência/métodos , Animais , Proteínas do Citoesqueleto/genética , Camundongos , Proteínas do Tecido Nervoso/genéticaRESUMO
The incidence of allergic reactions in patients with chronic renal failure during plasma exchange using fresh frozen plasma is not well known. We retrospectively reviewed 62 patients who underwent plasma exchange between January 2013 and May 2018. The most common indication for plasma exchange was desensitization/preconditioning for kidney transplant (61.3%, 38/62). The incidence of allergic reactions was significantly higher in patients with chronic renal failure than patients without (57.1% vs. 25.0%, P = 0.029). Also, the incidence of allergic reactions tended to be higher in peritoneal dialysis patients (75%, 3/4) than in hemodialysis (58.8%, 10/17) and preemptive kidney transplant (58%, 11/19). These results suggested the relationship of chronic renal failure and the incidence of allergic reactions in patients undergoing therapeutic plasma exchange using fresh frozen plasma.
Assuntos
Hipersensibilidade/etiologia , Falência Renal Crônica/terapia , Troca Plasmática/efeitos adversos , Plasma , Adulto , Idoso , Estudos de Coortes , Feminino , Seguimentos , Humanos , Hipersensibilidade/epidemiologia , Hipersensibilidade/fisiopatologia , Incidência , Japão , Falência Renal Crônica/diagnóstico , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Monitorização Fisiológica , Análise Multivariada , Diálise Peritoneal/métodos , Troca Plasmática/métodos , Diálise Renal/métodos , Estudos Retrospectivos , Medição de Risco , Resultado do TratamentoRESUMO
Obesity is a known risk factor for induction of myocardial infarction, but, paradoxically, may also confer a protective effect against subsequent remodeling leading to heart failure. In this study, we investigated the effect of leptin, the product of the obese (ob) gene, on cardiomyocyte apoptosis, a well-characterized component of cardiac remodeling after myocardial infarction. Exposing H9c2 cells to H(2)O(2) decreased cell viability, and this was attenuated by pretreating cells with leptin for 1 h, but not 24 h. Leptin also attenuated the ability of H(2)O(2) to increase phosphatidylserine exposure and annexin V binding. Further investigation of underlying mechanisms of leptin's protective effect demonstrated that the H(2)O(2)-induced decrease in mitochondrial membrane potential (Psi) leading to cytochrome c release was attenuated by leptin pretreatment, and this was associated with reduced translocation of the pro-apoptotic Bax protein to the mitochondrial membrane. Finally, leptin prevented H(2)O(2)-induced increases in caspase-3 cleavage and activity, although again 24 h leptin pretreatment did not confer significant protection. In summary, we have demonstrated that acute leptin pretreatment mediates anti-apoptotic effects in H9c2 rat cardiomyocytes, which may be of significance in clarifying the direct impact of leptin on the heart.
Assuntos
Apoptose , Peróxido de Hidrogênio/antagonistas & inibidores , Leptina/farmacologia , Miócitos Cardíacos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Fosfatidilserinas/análise , Ratos , Proteína X Associada a bcl-2/metabolismoRESUMO
OBJECTIVE: Our aim was to investigate the regulation of glucose and fatty acid metabolism in cardiomyocytes by the globular (gAd) and full-length (fAd) forms of adiponectin. METHODS: We produced fAd (consisting of high, medium and low molecular weight oligomers) in a mammalian expression system and gAd in bacteria. These were used to treat primary neonatal rat cardiomyocytes (up to 48 h), and we employed 3H- or 14C-labeled substrates to monitor glucose uptake and subsequent metabolism via oxidation, glycogen synthesis or lactate production and fatty acid uptake and oxidation. Enzymatic assay for acetyl CoA carboxylase activity was employed, and protein phosphorylation and expression was determined by immunoblotting cell lysates. The role of adiponectin receptor (AdipoR) isoforms was determined via siRNA-mediated knockdown. RESULTS: There was an initial (1 h) increase in glucose uptake and oxidation in response to gAd or fAd. Fatty acid uptake was stimulated by gAd or fAd, and by 24 h a decrease in acetyl CoA carboxylase activity and elevated fatty acid oxidation were observed. After 48 h increased fatty acid oxidation correlated with decreased glucose oxidation and pyruvate dehydrogenase activity, while glycogen synthesis and lactate production increased. Both gAd and fAd elicited phosphorylation of AMP kinase, insulin receptor substrate-1, Akt and glycogen synthase kinase-3beta. Knockdown of AdipoR1 or AdipoR2 attenuated the effect of both gAd and fAd on fatty acid uptake and oxidation. Only AdipoR1 knockdown prevented the ability of gAd (1 h) to increase glucose uptake and oxidation; however, reducing either AdipoR1 or AdipoR2 expression attenuated the long-term (24 h) effects of gAd. CONCLUSIONS: These results clearly demonstrate that gAd and fAd mediate distinct and time-dependent effects on cardiomyocyte energy metabolism via AdipoR1 and AdipoR2.
Assuntos
Adiponectina/farmacologia , Glucose/metabolismo , Miócitos Cardíacos/metabolismo , Palmitatos/metabolismo , Adiponectina/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Antígenos CD36/metabolismo , Células Cultivadas , Proteínas de Transporte de Ácido Graxo/metabolismo , Peso Molecular , Oxirredução , Fosforilação , Estrutura Terciária de Proteína , Interferência de RNA , RNA Mensageiro/análise , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Adiponectina , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Visualization of direct neuronal activity to understand brain function is one of the most important challenges in neuroscience. We have previously demonstrated that in vivo and in vitro gene expression of the ferritin reporter system could be detected by magnetic resonance imaging (MRI). In addition, increased neuronal activity induces Arc, an immediate early gene, and insertion of a destabilized fluorescent reporter dVenus under Arc promoter control has been used for monitoring neuronal activities in the brain by optical imaging. In this study, to visualize Arc promoter-driven neuronal activities directly, we generated transgenic mice and cell lines that express a destabilized fusion reporter ferritin-mKate2 under Arc promoter control. When transgenic mice and cell lines were treated with pilocarpine, a non-selective muscarinic agonist, an increase in T2-weighted image signal was successfully found in neuronal cells. There was a difference in peak time between MRI and fluorescence imaging, which might result from the binding process of iron with ferritin. Visualization of Arc promoter-driven neuronal activity is essential to understand neural mechanisms underlying cognitive processes and complex behaviors, and could be a useful tool for therapeutic approaches in the brain by MRI.
Assuntos
Encéfalo/metabolismo , Expressão Gênica/genética , Imageamento por Ressonância Magnética , Neurônios/metabolismo , Regiões Promotoras Genéticas/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Genes Reporter , Ferro/metabolismo , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Transgênicos , Proteínas Musculares/genética , Proteínas Musculares/metabolismoRESUMO
Obesity is a well-defined risk factor for the development of insulin resistance in target tissues, such as skeletal muscle, and thus type 2 diabetes. This may occur due to endocrine effects mediated by adipokines including leptin, the product of the obese (ob) gene, whose circulating levels positively correlate with body mass index. Induction of suppressor of cytokine-3 (SOCS-3) protein expression has been implicated as a possible mechanism of leptin-induced insulin resistance. Here, we show that treatment of rat skeletal muscle cells with leptin activated the SOCS-3 gene promoter and caused a time-dependent increase in both SOCS-3 mRNA and protein content. Confocal microscopy demonstrated increased co-localization of SOCS-3 with insulin receptor in leptin-treated cells and we confirmed a direct interaction between these two proteins by showing increased coimmunoprecipitation of SOCS-3 and insulin receptor after exposure of cells to leptin. However, the expected functional consequences were not observed, as we saw no change in basal or insulin-stimulated glucose uptake and phosphorylation of GSK3beta, Akt (T308 and S473) or ERK1/2. In summary, leptin induced SOCS-3 expression and its association with the insulin receptor in rat skeletal muscle cells but functional significance of this increase was not apparent upon measuring glucose uptake.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Leptina/farmacologia , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Receptor de Insulina/metabolismo , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glucose/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Imunoprecipitação , Insulina/farmacologia , Camundongos , Músculo Esquelético/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores para Leptina , Transdução de Sinais/efeitos dos fármacos , Proteína 3 Supressora da Sinalização de Citocinas , Fatores de TempoRESUMO
Synapses play a fundamental role in the processing of information in a neuronal network by mediating communication between neurons. To monitor presynaptic and postsynaptic structures simultaneously in living neurons, we developed double transfection method on the basis of calcium phosphate coprecipitation and introduced two fluorescent reporters into cultured neurons. The optimized protocol allowed different populations of neurons to be visualized by different color fluorescences, and presynaptic and postsynaptic structures to be observed in the same field of view of a microscope.
Assuntos
Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neurônios/citologia , Neurônios/fisiologia , Transfecção/métodos , Animais , Células Cultivadas , Hipocampo/citologia , Hipocampo/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Information from the peripheral organs is thought to be transmitted to the brain by humoral factors and neurons such as afferent vagal or spinal nerves. The common hepatic branch of the vagus (CHBV) is one of the main vagus nerve branches, and consists of heterogeneous neuronal fibers that innervate multiple peripheral organs such as the bile duct, portal vein, paraganglia, and gastroduodenal tract. Although, previous studies suggested that the CHBV has a pivotal role in transmitting information on the status of the liver to the brain, the details of its central projections remain unknown. The purpose of the present study was to investigate the brain regions activated by the CHBV. For this purpose, we injected L-arginine or anorexia-associated peptide cholecystokinin-8 (CCK), which are known to increase CHBV electrical activity, into the portal vein of transgenic Arc-dVenus mice expressing the fluorescent protein Venus under control of the activity-regulated cytoskeleton-associated protein (Arc) promotor. The brain slices were prepared from these mice and the number of Venus positive cells in the slices was counted. After that, c-Fos expression in these slices was analyzed by immunohistochemistry using the avidin-biotin-peroxidase complex method. Intraportal administration of L-arginine increased the number of Venus positive or c-Fos positive cells in the insular cortex. This action of L-arginine was not observed in CHBV-vagotomized Arc-dVenus mice. In contrast, intraportal administration of CCK did not increase the number of c-Fos positive or Venus positive cells in the insular cortex. Intraportal CCK induced c-Fos expression in the dorsomedial hypothalamus, while intraportal L-arginine did not. This action of CCK was abolished by CHBV vagotomy. Intraportal L-arginine reduced, while intraportal CCK increased, the number of c-Fos positive cells in the nucleus tractus solitarii in a CHBV-dependent manner. The present results suggest that the CHBV can activate different brain regions depending on the nature of the peripheral stimulus.
RESUMO
Alterations in cardiac glucose and fatty acid metabolism are possible contributors to the pathogenesis of heart failure in obesity. Here we examined the effect of leptin, the product of the obese (ob) gene, on metabolism in murine cardiomyocytes. Neither short-term (1 hour) nor long-term (24 hours) treatment with leptin (60 nmol/L) altered basal or insulin-stimulated glucose uptake and oxidation, glycogen synthesis, insulin receptor substrate 1 tyrosine, Akt, or glycogen synthase kinase 3beta phosphorylation. Extracellular lactate levels were also unaffected by leptin. However, leptin increased basal and insulin-stimulated palmitate uptake at both short and long exposure times and this corresponded with increased cell surface CD36 levels and elevated fatty acid transport protein 1 (FATP1) and CD36 protein content. Whereas short-term leptin treatment increased fatty acid oxidation, there was a decrease in oxidation after 24 hours. The former corresponded with increased acetyl coenzyme A carboxylase phosphorylation and the latter with increased expression of this enzyme. The discrepancy between uptake and oxidation of fatty acids led to a transient decrease in intracellular lipid content with lipid accumulation ensuing after 24 hours. In summary, we demonstrate that leptin did not alter glucose uptake or metabolism in murine cardiomyocytes. However, fatty acid uptake increased while oxidation decreased over time leading to intracellular lipid accumulation, which may lead to lipotoxic damage in heart failure.
Assuntos
Ácidos Graxos/metabolismo , Glucose/metabolismo , Leptina/farmacologia , Miócitos Cardíacos/metabolismo , Animais , Antígenos CD36/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Desoxiglucose/metabolismo , Proteínas de Transporte de Ácido Graxo/metabolismo , Glicogênio/biossíntese , Hipoglicemiantes/farmacologia , Immunoblotting , Insulina/farmacologia , Ácido Láctico/metabolismo , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Oxirredução , Receptores para Leptina , Proteínas Recombinantes/farmacologia , Sais de Tetrazólio , TiazóisRESUMO
Urinary nephrin is a potential non-invasive biomarker of disease. To date, however, most studies of urinary nephrin have been conducted in animal models of diabetic nephropathy, and correlations between urinary nephrin-to-creatinine ratio and other parameters have yet to be evaluated in animal models or patients of kidney disease with podocyte dysfunction. We hypothesized that urinary nephrin-to-creatinine ratio can be up-regulated and is negatively correlated with renal nephrin mRNA levels in animal models of kidney disease, and that increased urinary nephrin-to-creatinine ratio levels are attenuated following administration of glucocorticoids. In the present study, renal nephrin mRNA, urinary nephrin-to-creatinine ratio, urinary protein-to-creatinine ratio, and creatinine clearance ratio were measured in animal models of adriamycin nephropathy, puromycin aminonucleoside nephropathy, anti-glomerular basement membrane glomerulonephritis, and 5/6 nephrectomy. The effects of prednisolone on urinary nephrin-to-creatinine ratio and other parameters in puromycin aminonucleoside (single injection) nephropathy rats were also investigated. In all models tested, urinary nephrin-to-creatinine ratio and urinary protein-to-creatinine ratio increased, while renal nephrin mRNA and creatinine clearance ratio decreased. Urinary nephrin-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA in almost all models, as well as a significant positive correlation with urinary protein-to-creatinine ratio and a significant negative correlation with creatinine clearance ratio. Urinary protein-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA. Following the administration of prednisolone to puromycin aminonucleoside (single injection) nephropathy rats, urinary nephrin-to-creatinine ratio was significantly suppressed and exhibited a significant positive correlation with urinary protein-to-creatinine ratio. In addition, the decrease in number of glomerular Wilms tumor antigen-1-positive cells was attenuated, and urinary nephrin-to-creatinine ratio exhibited a significant negative correlation in these cells. In conclusion, these results suggest that urinary nephrin-to-creatinine ratio level is a useful and reliable biomarker for predicting the amelioration of podocyte dysfunction by candidate drugs in various kidney disease models with podocyte dysfunction. This suggestion will also be validated in a clinical setting in future studies.
Assuntos
Nefropatias/fisiopatologia , Proteínas de Membrana/urina , Podócitos/fisiologia , Animais , Doença Antimembrana Basal Glomerular/fisiopatologia , Doença Antimembrana Basal Glomerular/urina , Biomarcadores/urina , Creatinina/urina , Nefropatias Diabéticas/fisiopatologia , Nefropatias Diabéticas/urina , Doxorrubicina/farmacologia , Nefropatias/induzido quimicamente , Nefropatias/urina , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Puromicina Aminonucleosídeo/farmacologia , Ratos , Ratos WistarRESUMO
Previous studies have shown that many metabolic actions of adiponectin are mediated via the activation of AMP kinase and that adiponectin stimulates GLUT4 translocation and glucose uptake in the muscle. In this study, we demonstrate that adiponectin stimulates actin cytoskeleton remodeling, with increased phosphorylation of cofilin, and that blocking of cytoskeletal remodeling with cytochalasin D prevents adiponectin-stimulated AMPK phosphorylation in L6 myoblasts. LKB1 is an upstream kinase of AMPK, and we observed the colocalization of LKB1 with filamentous actin in response to adiponectin. Adiponectin-stimulated translocation of LKB1 from a nuclear to a cytoplasmic location to activate AMPK was also dependent on actin cytoskeleton remodeling. Cytoskeletal remodeling visualized by rhodamine-phalloidin immunofluorescence indicated that adiponectin-stimulated reorganization resulted in the formation membrane ruffles, which were also clearly visible by scanning electron microscopy in L6-GLUT4(myc) myoblasts. The stimulation of glucose uptake, but not of GLUT4-myc translocation to the cell surface, by adiponectin was also dependent on actin cytoskeleton remodeling. These results suggest that actin remodeling induced by adiponectin is essential for mediating LKB1/AMPK signaling and glucose uptake in skeletal muscle cells.