RESUMO
Background: Gut microbiota dysbiosis has been linked to obesity-associated chronic inflammation. Microbiota manipulation may therefore affect obesity-related comorbidities. Blueberries are rich in anthocyanins, which have anti-inflammatory properties and may alter the gut microbiota. Objective: We hypothesized that blueberry supplementation would alter the gut microbiota, reduce systemic inflammation, and improve insulin resistance in high-fat (HF)-diet-fed rats. Methods: Twenty-four male Wistar rats (260-270 g; n = 8/group) were fed low-fat (LF; 10% fat), HF (45% fat), or HF with 10% by weight blueberry powder (HF_BB) diets for 8 wk. LF rats were fed ad libitum, whereas HF and HF_BB rats were pair-fed with diets matched for fiber and sugar contents. Glucose tolerance, microbiota composition (16S ribosomal RNA sequencing), intestinal integrity [villus height, gene expression of mucin 2 (Muc2) and ß-defensin 2 (Defb2)], and inflammation (gene expression of proinflammatory cytokines) were assessed. Results: Blueberry altered microbiota composition with an increase in Gammaproteobacteria abundance (P < 0.001) compared with LF and HF rats. HF feeding led to an â¼15% decrease in ileal villus height compared with LF rats (P < 0.05), which was restored by blueberry supplementation. Ileal gene expression of Muc2 was â¼150% higher in HF_BB rats compared with HF rats (P < 0.05), with expression in the LF group not being different from that in either the HF or HF_BB groups. Tumor necrosis factor α (Tnfa) and interleukin 1ß (Il1b) gene expression in visceral fat was increased by HF feeding when compared with the LF group (by 300% and 500%, respectively; P < 0.05) and normalized by blueberry supplementation. Finally, blueberry improved markers of insulin sensitivity. Hepatic insulin receptor substrate 1 (IRS1) phosphorylation at serine 307:IRS1 ratio was â¼35% higher in HF rats compared with LF rats (P < 0.05) and HF_BB rats. Conclusion: In HF-diet-fed male rats, blueberry supplementation led to compositional changes in the gut microbiota associated with improvements in systemic inflammation and insulin signaling.
Assuntos
Mirtilos Azuis (Planta) , Dieta Hiperlipídica/efeitos adversos , Microbioma Gastrointestinal/efeitos dos fármacos , Inflamação/prevenção & controle , Resistência à Insulina/fisiologia , Extratos Vegetais/administração & dosagem , Tecido Adiposo/efeitos dos fármacos , Animais , Antocianinas/administração & dosagem , Bactérias/classificação , DNA Bacteriano/análise , Fibras na Dieta/administração & dosagem , Suplementos Nutricionais , Frutas/química , Microbioma Gastrointestinal/genética , Teste de Tolerância a Glucose , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Análise de Sequência de DNARESUMO
The serine/threonine kinase tumor progression locus 2 (Tpl2, also known as Map3k8/Cot) is a potent inflammatory mediator that drives the production of TNFα, IL-1ß, and IFNγ. We previously demonstrated that Tpl2 regulates T cell receptor (TCR) signaling and modulates T helper cell differentiation. However, very little is known about how Tpl2 modulates the development of regulatory T cells (Tregs). Tregs are a specialized subset of T cells that express FoxP3 and possess immunosuppressive properties to limit excess inflammation. Because of the documented role of Tpl2 in promoting inflammation, we hypothesized that Tpl2 antagonizes Treg development and immunosuppressive function. Here we demonstrate that Tpl2 constrains the development of inducible Tregs. Tpl2(-/-) naïve CD4(+) T cells preferentially develop into FoxP3(+) inducible Tregs in vitro as well as in vivo in a murine model of ovalbumin (OVA)-induced systemic tolerance. Treg biasing of Tpl2(-/-) T cells depended on TCR signal strength and corresponded with reduced activation of the mammalian target of rapamycin (mTOR) pathway. Importantly, Tpl2(-/-) Tregs have basally increased expression of FoxP3 and immunosuppressive molecules, IL-10 and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Furthermore, they were more immunosuppressive in vivo in a T cell transfer model of colitis, as evidenced by reduced effector T cell accumulation, systemic production of inflammatory cytokines, and colonic inflammation. These results demonstrate that Tpl2 promotes inflammation in part by constraining FoxP3 expression and Treg immunosuppressive functions. Overall, these findings suggest that Tpl2 inhibition could be used to preferentially drive Treg induction and thereby limit inflammation in a variety of autoimmune diseases.
Assuntos
Diferenciação Celular/imunologia , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/imunologia , Tolerância Imunológica , MAP Quinase Quinase Quinases/imunologia , Proteínas Proto-Oncogênicas/imunologia , Linfócitos T Reguladores/imunologia , Serina-Treonina Quinases TOR/imunologia , Animais , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Diferenciação Celular/genética , Colite/genética , Colite/imunologia , Colite/patologia , Colite/terapia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Interleucina-10/genética , Interleucina-10/imunologia , MAP Quinase Quinase Quinases/genética , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Linfócitos T Reguladores/patologia , Serina-Treonina Quinases TOR/genéticaRESUMO
Background: The collection of microorganisms, mainly bacteria, which live in the gastrointestinal (GI) tract are collectible known as the gut microbiota. GI bacteria play an active role in regulation of the host's immune system and metabolism, as well as certain pathophysiological processes. Diet is the main factor modulating GI microbiota composition and recent studies have shown that high fat (HF) diets induce detrimental changes, known as dysbiosis, in the GI bacterial makeup. HF diet induced microbiota dysbiosis has been associated with structural and functional changes in gut-brain vagally mediated signaling system, associated with overeating and obesity. Although HF-driven changes in microbiota composition are sufficient to alter vagal signaling, it is unknown if restoring normal microbiota in obesity can improve gut-brain signaling and metabolic outcomes. In this study, we evaluated the effect of lean gut microbiota transfer in obese, vagally compromised, rats on gut-brain communication, food intake, and body weight. Male Sprague-Dawley rats were maintained on regular chow, or 45% HF diet for nine weeks followed by three weeks of microbiota depletion using an antibiotic cocktail. The animals were then divided into four groups (n=10 each): LF - control group on regular chow, LF-LF - chow fed animals that received antibiotics and microbiota from chow fed animals, HF-LF - HF fed animals that received microbiota from chow fed animals, and HF-HF - HF fed animals that received microbiota from HF fed animals. Animals were gavaged with donor microbiota for three consecutive days on week one and once a week thereafter for three more weeks. HF-LF animals received inulin as a prebiotic to aid the establishment of the lean microbiome. Results: We found that transferring a LF microbiota to HF fed animals (HF-LF) reduced caloric intake during the light phase when compared with HF-HF rats and prevented additional excessive weight gain. We did not observe significant changes in the density of vagal afferents terminating in the brainstem among the groups, however, HF-LF animals displayed an increase in postprandial activation of both primary sensory neurons innervating the GI tract and brainstem secondary neurons. Conclusions: We concluded from these data that normalizing microbiota composition in obese rats improves gut-brain communication and restores normal feeding patterns which was associated with a reduction in weight gain.
RESUMO
OBJECTIVE: Our goal is to investigate if microbiota composition modulates reward signaling and assess the role of the vagus in mediating microbiota to brain communication. METHODS: Male germ-free Fisher rats were colonized with gastrointestinal contents from chow (low fat (LF) ConvLF) or HF (ConvHF) fed rats. RESULTS: Following colonization, ConvHF rats consumed significantly more food than ConvLF animals. ConvHF rats displayed lower feeding-induced extracellular DOPAC levels (a metabolite of dopamine) in the Nucleus Accumbens (NAc) as well as reduced motivation for HF foods compared to ConvLF rats. Dopamine receptor 2 (DDR2) expression levels in the NAc were also significantly lower in ConvHF animals. Similar deficits were observed in conventionally raised HF fed rats, showing that diet-driven alteration in reward can be initiated via microbiota. Selective gut to brain deafferentation restored DOPAC levels, DRD2 expression, and motivational drive in ConvHF rats. CONCLUSIONS: We concluded from these data that a HF-type microbiota is sufficient to alter appetitive feeding behavior and that bacteria to reward communication is mediated by the vagus nerve.
Assuntos
Eixo Encéfalo-Intestino , Comportamento Alimentar , Ratos , Masculino , Animais , Ácido 3,4-Di-Hidroxifenilacético , Comportamento Alimentar/fisiologia , Recompensa , BactériasRESUMO
A Bax- and, apparently, mitochondria-dependent increase in superoxide (O(2)(·-)) and other reactive oxygen species (ROS) occurs in apoptotic superior cervical ganglion (SCG) and cerebellar granule (CG) neurons. Here we show that Bax also lies upstream of ROS produced in nonapoptotic neurons and present evidence that caspases partially mediate the pro-oxidant effect of Bax. We used the O(2)(·-)-sensitive dye MitoSOX to monitor O(2)(·-) in neurons expressing different levels of Bax and mitochondrial superoxide dismutase (SOD2). Basal and apoptotic O(2)(·-) levels in both SCG and CG neurons were reduced in SOD2 wild-type (WT) cells having lower Bax concentrations. Apoptotic and nonapoptotic neurons from Bax-WT/SOD2-null but not Bax-null/SOD2-null mice had increased O(2)(·-) levels. A caspase inhibitor inhibited O(2)(·-) in both apoptotic and nonapoptotic SCG neurons. O(2)(·-) production increased when WT, but not Bax-null, SCG neurons were permeabilized and treated with active caspase 3. There was no apoptosis and little increase in O(2)(·-) in SCG neurons from caspase 3-null mice exposed to an apoptotic stimulus. O(2)(·-) levels in nonapoptotic caspase 3-null SCG neurons were lower than in WT cells but not as low as in caspase inhibitor-treated cells. These data indicate that Bax lies upstream of most O(2)(·-) produced in neurons, that caspase 3 is required for increased O(2)(·-) production during neuronal apoptosis, that caspase 3 is partially involved in O(2)(·-) production in nonapoptotic neurons, and that other caspases may also be involved in Bax-dependent O(2)(·-) production in nonapoptotic cells.
Assuntos
Apoptose/fisiologia , Caspase 3/fisiologia , Neurônios/metabolismo , Superóxidos/metabolismo , Proteína X Associada a bcl-2/fisiologia , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mitocondriais/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Vagal afferent neurons (VAN) projecting to the lamina propria of the digestive tract are the primary source of gut-originating signals to the central nervous system (CNS). VAN cell bodies are found in the nodose ganglia (NG). Responsiveness of VAN to gut-originating signals is altered by feeding status with sensitivity to satiety signals such as cholecystokinin (CCK) increasing in the fed state. Chronic high-fat (HF) feeding results in inflammation at the level of the NG associated with a loss of VAN ability to switch phenotype from the fasted to the fed state. HF feeding also leads to compositional changes in the gut microbiota. HF diet consumption notably drives increased Firmicutes to Bacteroidetes phyla ratio and increased members of the Actinobacteria phylum. Firmicutes and Actinobacteria are largely gram positive (GP). In this study, we aimed to determine if byproducts from GP bacteria can induce an inflammatory response in cultured NG and to characterize the mechanism and cell types involved in the response. NG were collected from male Wistar rats and cultured for a total of 72 hours. At 48-68 hours after plating, cultures were treated with neuronal culture media in which Serinicoccus chungangensis had been grown and removed (SUP), lipoteichoic acid (LTA), or meso-diaminopimelic acid (meso-DAP). Some treatments included the glial inhibitors minocycline (MINO) and/or fluorocitrate (FC). The responses were evaluated using immunocytochemistry, qPCR, and electrochemiluminescence. We found that SUP induced an inflammatory response characterized by increased interleukin (IL)-6 staining and increased expression of genes for IL-6, interferon (IFN)γ, and tumor necrosis factor (TNF)α along with genes associated with cell-to-cell communication such as C-C motif chemokine ligand-2 (CCL2). Inclusion of inhibitors attenuated some responses but failed to completely normalize all indications of response, highlighting the role of immunocompetent cellular crosstalk in regulating the inflammatory response. LTA and meso-DAP produced responses that shared characteristics with SUP but were not identical. Our results support a role for HF associated GP bacterial byproducts' ability to contribute to vagal inflammation and to engage signaling from nonneuronal cells.
Assuntos
Microbioma Gastrointestinal , Gânglio Nodoso , Actinobacteria , Animais , Bactérias , Masculino , Neurônios , Ratos , Ratos WistarRESUMO
Circulating levels of bacterial lipopolysaccharide (LPS) or endotoxin are chronically elevated in obesity (metabolic endotoxemia), resulting in low-grade inflammation. Metabolic endotoxemia has been identified as a triggering factor for obesity-associated metabolic complications such as insulin resistance. Furthermore, LPS has been shown to modulate endocannabinoid synthesis and notably to induce cannabinoid receptor type-1 (CB1) ligand synthesis. CB1 activation promotes inflammation, increases food intake and impairs insulin signaling. Therefore, we hypothesized that LPS acts through a CB1-dependent mechanism to aggravate inflammation and promote insulin resistance. Male Wistar rats fed a chow diet were implanted with mini-osmotic pumps delivering a low dose of LPS (nâ¯=â¯20; 12.5⯵g/kg body weight (BW)/hr.) or saline (nâ¯=â¯10) continuously for six weeks. LPS-treated rats were injected daily with a CB1 antagonist (Rimonabant, SR141716A; 3â¯mg/kg, intraperitoneal (ip); LPSâ¯+â¯CB1x; nâ¯=â¯10) or vehicle (1â¯mL/kg, LPS; nâ¯=â¯10). Control and LPS rats' food intake was matched to the LPSâ¯+â¯CB1x group level. Despite no significant differences in body weight among groups, chronic exposure to low-level LPS altered hepatic endocannabinoid signaling, increased inflammation, and impaired insulin sensitivity and insulin clearance (Pâ¯<â¯0.05). CB1 inhibition significantly attenuated LPS signaling (Pâ¯<â¯0.05), which attenuated LPS-induced metabolic alterations. Therefore, we concluded that CB1 contributes to LPS-mediated inflammation and insulin resistance, suggesting that blocking CB1 signaling may have therapeutic benefits in reducing inflammation-induced metabolic abnormalities.
Assuntos
Endotoxemia/metabolismo , Inflamação/metabolismo , Resistência à Insulina/fisiologia , Receptor CB1 de Canabinoide/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Antagonistas de Receptores de Canabinoides/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Endotoxemia/induzido quimicamente , Inflamação/induzido quimicamente , Lipopolissacarídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar , Rimonabanto/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
(1) High-fat (HF) diet leads to gut microbiota dysbiosis which is associated with systemic inflammation. Bacterial-driven inflammation is sufficient to alter vagally mediated satiety and induce hyperphagia. Promoting bacterial fermentation improves gastrointestinal (GI) epithelial barrier function and reduces inflammation. Resistant starch escape digestion and can be fermented by bacteria in the distal gut. Therefore, we hypothesized that potato RS supplementation in HF-fed rats would lead to compositional changes in microbiota composition associated with improved inflammatory status and vagal signaling. (2) Male Wistar rats (n = 8/group) were fed a low-fat chow (LF, 13% fat), HF (45% fat), or an isocaloric HF supplemented with 12% potato RS (HFRS) diet. (3) The HFRS-fed rats consumed significantly less energy than HF animals throughout the experiment. Systemic inflammation and glucose homeostasis were improved in the HFRS compared to HF rats. Cholecystokinin-induced satiety was abolished in HF-fed rats and restored in HFRS rats. HF feeding led to a significant decrease in positive c fiber staining in the brainstem which was averted by RS supplementation. (4) The RS supplementation prevented dysbiosis and systemic inflammation. Additionally, microbiota manipulation via dietary potato RS prevented HF-diet-induced reorganization of vagal afferent fibers, loss in CCK-induced satiety, and hyperphagia.
Assuntos
Bactérias/crescimento & desenvolvimento , Encéfalo/fisiopatologia , Suplementos Nutricionais , Disbiose , Microbioma Gastrointestinal , Inflamação/prevenção & controle , Intestinos/inervação , Intestinos/microbiologia , Obesidade/prevenção & controle , Solanum tuberosum , Amido/administração & dosagem , Nervo Vago/fisiopatologia , Ração Animal , Animais , Bactérias/metabolismo , Encéfalo/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Comportamento Alimentar , Fermentação , Hiperfagia/metabolismo , Hiperfagia/microbiologia , Hiperfagia/fisiopatologia , Hiperfagia/prevenção & controle , Inflamação/metabolismo , Inflamação/microbiologia , Inflamação/fisiopatologia , Masculino , Obesidade/metabolismo , Obesidade/microbiologia , Obesidade/fisiopatologia , Raízes de Plantas , Ratos Wistar , Resposta de Saciedade , Amido/metabolismo , Nervo Vago/metabolismo , Aumento de PesoRESUMO
Development of obesity-associated comorbidities is related to chronic inflammation, which has been linked to gut microbiota dysbiosis. Thus, modulating gut microbiota composition could have positive effects for metabolic disorders, supporting the use of probiotics as potential therapeutics in vivo, which may be enhanced by a microencapsulation technique. Here we investigated the effects of non-encapsulated or pectin-encapsulated probiotic supplementation (Lactobacillus paracasei subsp. paracasei L. casei W8®; L. casei W8) on gut microbiota composition and metabolic profile in high-fat (HF) diet-fed rats. Four male Wistar rat groups (n = 8/group) were fed 10% low-fat, 45% HF, or HF with non-encapsulated or encapsulated L. casei W8 (4 × 107 CFU/g diet) diet for seven weeks. Microbiota composition, intestinal integrity, inflammatory profiles, and glucose tolerance were assessed. Non-encapsulated and pectin-encapsulated probiotic supplementation positively modulated gut microbiota composition in HF-fed male rats. These changes were associated with improvements in gut barrier functions and local and systemic inflammation by non-encapsulated probiotics and improvement in glucose tolerance by encapsulated probiotic treatment. Thus, these findings suggest the potential of using oral non-encapsulated or encapsulated probiotic supplementation to ameliorate obesity-associated metabolic abnormalities.
Assuntos
Glicemia/metabolismo , Dieta Hiperlipídica , Microbioma Gastrointestinal , Intolerância à Glucose/prevenção & controle , Inflamação/prevenção & controle , Intestinos/microbiologia , Lactobacillus/crescimento & desenvolvimento , Probióticos/administração & dosagem , Animais , Modelos Animais de Doenças , Disbiose , Metabolismo Energético , Intolerância à Glucose/sangue , Intolerância à Glucose/etiologia , Intolerância à Glucose/microbiologia , Inflamação/sangue , Inflamação/etiologia , Inflamação/microbiologia , Mediadores da Inflamação/sangue , Insulina/sangue , Masculino , Permeabilidade , Ratos WistarRESUMO
Depriving mouse sympathetic neurons of nerve growth factor (NGF) causes their apoptotic death. A Bax-dependent increase of mitochondrial-derived reactive oxygen species (ROS) begins in these cells soon after NGF withdrawal. We investigated the effects on these ROS of adding NGF to cultures of NGF-deprived neurons. ROS levels were monitored with the fluorescent, redox-sensitive dyes CM-H2DCFDA and MitoSOX Red. The intensity of the former dye increases when it is oxidized by H2O2 and free radicals downstream of H2O2. MitoSOX Red is relatively insensitive to oxidation by H2O2 but is sensitive to oxidation by superoxide (O2*-). Withdrawing NGF increased CM-H2DCFDA intensity, indicating elevated H2O2-associated ROS. Re-exposure of cells deprived of NGF to NGF resulted in rapid suppression of these ROS. Neurons deprived of NGF also had increased MitoSOX Red intensities. Readdition of NGF had no effect on MitoSOX Red fluorescence. The suppression of CM-H2DCFDA-detected ROS by NGF was caused by a rapid activation of glutathione redox cycling. The most likely explanation for these findings is that mitochondria increased O2*- production after NGF withdrawal. The O2*- was converted to H2O2 by dismutation, and the H2O2 was detoxified by accelerated glutathione redox cycling. Our previous work shows that H2O2 induces cytochrome c to be released from mitochondria in NGF-supported sympathetic neurons, whereas antioxidants that detoxify H2O2 block cytochrome c redistribution in NGF-deprived neurons. Readdition of NGF also immediately inhibits cytochrome c release. We present evidence that this inhibition is mediated by the rapid activation of glutathione redox cycling by NGF.
Assuntos
Antioxidantes/metabolismo , Apoptose/fisiologia , Citocromos c/metabolismo , Fator de Crescimento Neural/fisiologia , Neurônios/citologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/fisiologia , Células Cultivadas , Citocromos c/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/metabolismo , Neurônios/metabolismo , Gânglio Cervical Superior/metabolismoRESUMO
Neurites projecting to their target tissues during embryogenesis are subject to many perturbations that could influence their rate of growth. For example, environmental influences such as supply of neurotrophic factor or electrical activity profoundly influence the rate of neuronal protein synthesis. Because accumulation of protein is necessary for outgrowth to proceed normally, a perturbation in protein synthesis could cause a net change in the rate of accumulation of proteins with the result that neurite outgrowth rate increases or decreases. That neurite outgrowth does not normally seem to be subject to such perturbations suggests involvement of a homeostatic system controlling the rate of outgrowth. Consistent with this hypothesis, we show here that the rate of growth of neurites of sympathetic neurons is highly resistant to decreased rates of protein synthesis. Chronic suppression of protein synthesis by 60% had no significant effect on neurite outgrowth over a 2-day period while complete suppression halted it almost immediately. By the 3rd day of exposure, 60% suppression slowed outgrowth. Sustained suppression of protein synthesis rate by 33% had no effect on rate of outgrowth even after 7 days. We show that the ability of the growing neurites to resist protein synthesis suppression appears to be caused, at least in part, by a parallel decrease in the rate of protein degradation. The result of this coupling between degradation and synthesis is that proteins can continue to accumulate even when protein synthesis rate decreases, allowing normal rates of neurite outgrowth.
Assuntos
Neuritos/fisiologia , Neurônios/citologia , Biossíntese de Proteínas/fisiologia , Proteínas/metabolismo , Gânglio Cervical Superior/citologia , Animais , Células Cultivadas , Cicloeximida/farmacologia , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Feminino , Neuritos/efeitos dos fármacos , Gravidez , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Bax is required for the apoptotic death of sympathetic neurons deprived of nerve growth factor (NGF). After NGF withdrawal, Bax translocates from the cytoplasm to the mitochondria of these cells and induces release of the proapoptotic protein cytochrome c. Here, we report that withdrawing NGF from mouse sympathetic neurons caused an increase of mitochondria-derived reactive oxygen species (ROS). Suppressing these ROS inhibited apoptosis. Bax deletion blocked death and prevented the ROS burst. Inducing a pro-oxidant state similar to that in NGF-deprived, wild-type cells caused cytochrome c release even in neurons lacking Bax. A similar ROS burst in cerebellar granule neurons undergoing apoptosis was also blocked by Bax deletion. These findings indicate that Bax lies upstream from increased ROS in NGF-deprived neurons and suggest that the Bax-induced ROS burst is both necessary and sufficient for cytochrome c redistribution in these cells.
Assuntos
Apoptose/fisiologia , Grupo dos Citocromos c/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Transporte de Elétrons/efeitos dos fármacos , Corantes Fluorescentes , Heterozigoto , Homozigoto , Peróxido de Hidrogênio/farmacologia , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fator de Crescimento Neural/deficiência , Fator de Crescimento Neural/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Espécies Reativas de Oxigênio/metabolismo , Gânglio Cervical Superior/citologia , Desacopladores/farmacologia , Proteína X Associada a bcl-2RESUMO
PURPOSE: To identify existing organizations that recognize faculty members' excellence as educators (Academies) in the United States, and describe the organizations' characteristics. METHOD: A 31-item questionnaire inquiring about Academies or equivalent programs was sent to deans of medical education at all 125 U.S. medical schools in February of 2003. Variables examined were general Academy characteristics such as membership selection criteria, goals, benefits of membership, and budget, as well as, estimates of prestige of membership and influence on recruiting new and current faculty to educational activities. RESULTS: Twenty of 97 (21%) respondents reported an implemented Academy or equivalent program (eight begun prior to 2000 and 11 subsequently). Most Academies (75%) did not "cap" membership size, and most (65%) offered lifetime membership. Budgets ranged from 0 dollars to more than 100,000 dollars per year. Full-time faculty status (100%) and involvement in direct undergraduate teaching (95%) affected eligibility the most. Nominations for membership most often came from department or section chairs (89%) and from peers (74%), and learners were involved in the final selection process at 18 of the Academies. Benefits of membership included networking/collaboration, school-wide recognition, and mentoring for educational skills development. The benefit of protected time was offered at only three institutions and was associated with having a larger budget. Respondents believe Academies positively influence faculty participation in educational activities. CONCLUSIONS: Academies are formal organizations recognizing faculty contributions to medical education, and they are increasing in number. They offer important benefits to faculty members and the educational mission of an academic medical center.
Assuntos
Centros Médicos Acadêmicos/normas , Educação de Graduação em Medicina/normas , Avaliação Educacional , Docentes de Medicina , Faculdades de Medicina/normas , Coleta de Dados , Humanos , Inquéritos e Questionários , Gestão da Qualidade Total , Estados UnidosRESUMO
A Bax-dependent increase of reactive oxygen species (ROS) and other reactive species (RS) occurs after withdrawing NGF from mouse sympathetic neurons in cell culture. Possible mechanisms underlying the increased ROS/RS are leakage of electrons from the mitochondrial electron transport chain secondary to caspase cleavage of respiratory complexes or leakage secondary to depletion of cytochrome c from the chain. We previously demonstrated that deletion of Bax or caspase 3 from these cells reduces ROS/RS production to near baseline levels indicating a central role for both Bax and caspase 3 in generating the ROS/RS. Here we depleted cytochrome c to a similar level in neurons from wild type and bax hemizygous or knockout mice by NGF withdrawal or treatment with H2O2. Death was prevented with a caspase inhibitor that caused a partial reduction of ROS/RS levels but did not completely prevent the ROS/RS increase. ROS/RS was highest in bax wild-type cells, lowest in bax knockout cells, and at an intermediate level in the bax hemizygous cells. These and our previous findings indicate that Bax and caspase 3 are necessary for the increased ROS/RS after withdrawing NGF from these cells and that little or none of the increased ROS/RS are secondary to a depletion of cytochrome c from the electron transport chain.
RESUMO
Autoimmune diseases are approaching epidemic levels, estimated to affect 5-8% of the population. A number of autoimmune diseases are believed to be driven by autoreactive T cells, specifically by T helper 1 (Th1) cells and T helper 17 (Th17) cells. One molecule gaining interest as a therapeutic target is the serine-threonine kinase, Tpl2, which promotes expression of proinflammatory mediators. We previously demonstrated that Tpl2 regulates Th1 differentiation, secretion of the inflammatory cytokine IFNγ, and host defense against the intracellular parasite Toxoplasma gondii. The goal of this study was to determine whether Tpl2 also regulates Th1 or Th17 differentiation in vivo in a model of colitis associated with mixed Th1/Th17 pathology. In vitro, Tpl2-/- naïve CD4 T cells were significantly impaired in IL-17A secretion under traditional Th17 inducing conditions. Reduced IL-17A secretion correlated with increased expression of FoxP3, a transcription factor known to antagonize RORγt function. In a murine T cell transfer model of colitis, transfer of Tpl2-/- T cells resulted in reduced proportions of CD4 T cells expressing IFNγ, but not IL-17A, compared to that induced by wild type T cells. Further studies revealed that IL-17A differentiation induced by IL-6 and IL-23, cytokines implicated in driving Th17 differentiation in vivo, was unaffected by Tpl2 deficiency. Collectively, these results implicate Tpl2 in TGF-ß-induced FoxP3 expression. Additionally, they underscore the contribution of Tpl2 to Th1 immunopathology specifically, which suggests that Tpl2 inhibitors may selectively target Th1-based inflammation.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Colite/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/transplante , Diferenciação Celular , Células Cultivadas , Colite/etiologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Interferon gama/genética , Interleucina-17/genética , MAP Quinase Quinase Quinases/genética , Camundongos , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genéticaRESUMO
Half of all neurons produced during embryogenesis undergo apoptotic death shortly before birth or soon thereafter. Two cell culture models have been used extensively to investigate the cellular and molecular mechanisms underlying apoptosis during neuronal development: (a) sympathetic neurons deprived of their required neurotrophic factor, nerve growth factor, and (b) cerebellar granule neurons deprived of serum in low-potassium medium. A dramatic increase in mitochondrial-derived reactive oxygen species (ROS) occurs during the apoptotic death of both of these cell types. These ROS lie downstream from the proapoptotic protein, Bax. Bax normally resides in the cytoplasm, but translocates to the outer mitochondrial membrane during apoptosis. Once associated with mitochondria, Bax causes release of apoptogenic factors from the mitochondria into the cytoplasm, thus inducing or augmenting the apoptotic cascade. Although there is much controversy about the exact mechanism by which Bax causes release of these factors, recent evidence suggests that the Bax-induced ROS are critical for this release to occur in both sympathetic and cerebellar granule neurons. Because Bax is critical for the apoptotic death of many other types of neurons, it is likely that increased ROS is important for the death of these cells as well.
Assuntos
Apoptose/fisiologia , Citocromos c/metabolismo , Fator de Crescimento Neural/fisiologia , Neurônios/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Caspases/farmacologia , Caspases/fisiologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , MAP Quinase Quinase Quinases/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Modelos Biológicos , Neurônios/efeitos dos fármacos , Proteína X Associada a bcl-2RESUMO
An increase of mitochondrial-derived reactive oxygen species (ROS) occurs in nerve growth factor (NGF)-deprived sympathetic neurons undergoing apoptotic death. It has been reported that NGF suppresses increased ROS production by the mitochondria in these cells through a mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein (MAP) kinase pathway because NGF withdrawal inactivates this pathway and the MEK inhibitor, PD98059, increases ROS in the presence of NGF. We show here that treating rat sympathetic neurons in cell culture with PD98059 greatly decreased cellular concentrations of reduced glutathione (GSH), a major cellular antioxidant. Therefore, it is likely that this inhibitor induces a cellular prooxidant state in NGF-maintained sympathetic neurons primarily by decreasing GSH concentration rather than by causing increased mitochondrial ROS production. These data suggest that the MEK/MAP kinase signaling pathway regulates cellular GSH concentration.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fator de Crescimento Neural/deficiência , Neurônios/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Sistema Nervoso Simpático/fisiologia , Acetilcisteína/farmacologia , Animais , Butionina Sulfoximina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Cicloeximida/farmacologia , Feto/inervação , Flavonoides/farmacologia , Glutationa/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Microscopia Confocal , Microscopia de Fluorescência , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Pirazóis/análise , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Gânglio Cervical Superior/citologia , Sistema Nervoso Simpático/citologiaRESUMO
Depriving sympathetic neurons in cell culture of nerve growth factor (NGF) causes their apoptotic death. Bax-induced release of cytochrome c from mitochondria and the subsequent activation of cytosolic caspases are central to this death. A Bax-dependent increase of mitochondrial-derived reactive oxygen species (ROS) that is an important component of the apoptotic cascade in these cells begins soon after NGF withdrawal. Here we report that Bax can also influence mitochondrial production of ROS in non-apoptotic sympathetic neurons. We determined ROS levels by using confocal microscopy to monitor changes in the fluorescence intensity of a redox-sensitive dye loaded into single cells. ROS levels were similar in NGF-replete bax wild-type neurons and neurons from which bax had been deleted. To enhance any effects that Bax might have on ROS levels in NGF-replete cells we exposed cultures to the ATP synthase inhibitor, oligomycin. This treatment hyperpolarizes mitochondrial membrane potential (DeltaPsi(m)), an event that can favor increased ROS production. NGF-replete neurons from mice in which bax had been deleted had much higher levels of mitochondrial-derived ROS when treated with oligomycin than did bax wild-type cells. Oligomycin treatment also caused greater hyperpolarization of DeltaPsi(m) in bax-deleted cells than in wild-type cells. These findings indicate that Bax can affect mitochondrial ROS production in non-apoptotic neurons and may do so by altering DeltaPsi(m).
Assuntos
Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Neurônios/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose , Inibidores Enzimáticos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Knockout , Microscopia Confocal , Mitocôndrias/fisiologia , Membranas Mitocondriais/fisiologia , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Fatores de Crescimento Neural/metabolismo , Neurônios/fisiologia , Oligomicinas/farmacologia , Gânglio Cervical Superior/citologia , Proteína X Associada a bcl-2/deficiênciaRESUMO
It has been suggested that release of cytochrome c (Cyt c) from mitochondria during apoptotic death is through opening of the mitochondrial permeability transition pore followed by swelling-induced rupture of the mitochondrial outer membrane. However, this remains controversial and may vary with cell type and model system. We determined that in mouse cerebellar granule neurons, Cyt c redistribution preceded the loss of mitochondrial membrane potential during the apoptotic process, suggesting that the pore did not open prior to release. Furthermore, when mitochondria were morphologically assessed by electron microscopy, they were not obviously swollen during the period of Cyt c release. This indicates that the pore mechanism of action, if any, is not through mitochondrial outer membrane rupture. While bongkrekic acid, an inhibitor of pore opening, modestly delayed apoptotic death, it also caused a significant (p < 0.05) suppression of protein synthesis. An equivalent suppression of protein synthesis by cycloheximide had a similar delaying effect, suggesting that bongkrekic acid was acting non-specifically. These findings suggest that mitochondrial permeability transition pore is not involved in Cyt c release from mitochondria during the apoptotic death of cerebellar granule neurons.