CD1 protein is involved in diet-induced hypothalamic inflammation in obesity

Orientador: Licio Augusto VellosoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Obesidade é hoje o principal fator de risco para uma série de doenças e o aumento de sua prevalência é alarmante. A ativação de um processo inflamatório subclínico no hipotálamo desempenha papel central como desencadeador dos distúrbios associados ao ganho de peso, como a resistência à insulina. A ativação do processo inflamatório característico da obesidade decorre da complexa associação entre fatores genéticos e ambientais. O consumo de dietas ricas em ácidos graxos saturados é certamente um dos mais importantes fatores ambientais, levando à inflamação através da ativação de receptores TLR4. A participação do sistema imune inato nessa inflamação hipotalâmica tem sido extensivamente estudada. No entanto, o papel da imunidade adaptativa no curso da obesidade é ainda desconhecido. Em doenças neurodegenerativas, a inflamação do sistema nervoso central é responsável pela atração e ativação de linfócitos T, sendo este evento concomitante ao início da doença. Conhecendo o papel dos ácidos graxos nesse cenário imunológico, nós aventamos a hipótese de que o CD1, proteína que apresenta lipídios a células T, teria papel relevante na resposta imune adaptativa no hipotálamo de camundongos obesos. Neste trabalho, mostramos que com poucos dias de dieta, ocorre um aumento da expressão hipotalâmica de CD1 e modificação de sua distribuição anatômica. A redução da sua expressão utilizando um anticorpo imunoneutralizador acarreta em melhora do perfil glicêmico e redução do ganho de peso enquanto sua ativação no hipotálamo exacerba o fenótipo obeso dos animais, inclusive levando animais resistentes à obesidade a apresentarem um perfil de ganho de peso semelhante a animais propensos. A análise por bioinformártica revelou que a expressão de CD1 no hipotálamo se correlaciona a outros marcadores envolvidos no distúrbio. Dessa forma, o CD1 parece exercer papel relevante na inflamação hipotalâmica frente ao consumo de ácidos graxos saturadosAbstract: Obesity is the main risk factor for many comorbidities and its prevalence is rising to astounding numbers. The activation of a subclinic and chronic inflammatory process in the hypothalamus has been shown to be the trigger event leading to several comorbidities, such as insulin resistence and diabetes. This process occurs due to a complex interaction between genetic and envirolmental factors. The consumptions of saturated fatty acid rich diets is one of the most important envirolmental factors, leading to inflammation due to activation of the innate immune receptor, TLR4. The role of innate immune response in the hypothalamic inflammation has been extensively studied. However, the putative role of the adaptive immune response in this scenario is still unknown. In neurodegenative diseases, the inflammation is responsible for the recruitment and activation of T lymphocytes to the brain, which contributes to the emergence of many syptoms of the diseases. As we know that fatty acids trigger an infammatory immune response that disrupts hypothalamic control of food intake and energy homeostases, we hypothesized that CD1, a lipid-presenting protein to lymphocytes, would play a central role in the hypothalamic immune response. In this work, we show that as early as one day of high fat diet, the CD1 expression is higher in the hypothalamus of mice and the distribution in this tissue is also disturbed. The deacrease in CD1 expression leads to improvement of glucose tolerance e reduced weight gain; wheras the increase of CD1 signaling exacerbates the obese phenotype. Bioinformatics analyses showed that the hypothalamic expression of CD1 correlates with other celular mechanisms involved in the pathophysiology of obesity. Therefore, CD1 seems to have a pivotal role in hypothalamic inflammation due to saturated fatty acids consumptionDoutoradoFisiopatologia MédicaDoutora em Ciências2013/25415-9FAPES

Angiopoietin2 is associated with coagulation activation and tissue factor expression in extracellular vesicles in COVID-19

Coagulation activation in immunothrombosis involves various pathways distinct from classical hemostasis, offering potential therapeutic targets to control inflammation-induced hypercoagulability while potentially sparing hemostasis. The Angiopoietin/Tie2 pathway, previously linked to embryonic angiogenesis and sepsis-related endothelial barrier regulation, was recently associated with coagulation activation in sepsis and COVID-19. This study explores the connection between key mediators of the Angiopoietin/Tie2 pathway and coagulation activation. The study included COVID-19 patients with hypoxia and healthy controls. Blood samples were processed to obtain platelet-free plasma, and frozen until analysis. Extracellular vesicles (EVs) in plasma were characterized and quantified using flow cytometry, and their tissue factor (TF) procoagulant activity was measured using a kinetic chromogenic method. Several markers of hemostasis were assessed. Levels of ANGPT1, ANGPT2, and soluble Tie2 correlated with markers of coagulation and platelet activation. EVs from platelets and endothelial cells were increased in COVID-19 patients, and a significant increase in TF+ EVs derived from endothelial cells was observed. In addition, ANGPT2 levels were associated with TF expression and activity in EVs. In conclusion, we provide further evidence for the involvement of the Angiopoietin/Tie2 pathway in the coagulopathy of COVID-19 mediated in part by release of EVs as a potential source of TF activity

A20 deubiquitinase controls PGC-1a expression in the adipose tissue

Orientador: Licio Augusto VellosoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A proteína PGC1? é um co-ativador de transcrição gênica que desempenha papel importante na regulação de uma série de fenômenos metabólicos que compreendem desde o controle da termogênese e mitocondriogênese até a regulação da secreção de insulina e a produção hepática de glicose. Como vários dos fenômenos biológicos controlados direta ou indiretamente pela PGC1? tem importância vital, a regulação dos níveis de PGC1? nos tecidos deve ser finamente ajustada. Nos últimos anos, inúmeros estudos exploraram os mecanismos envolvidos com o controle da expressão gênica e tradução da PGC1?. Entretanto, apenas alguns poucos estudos avaliaram a degradação da mesma. Um dos mais importantes mecanismos envolvidos com a regulação funcional e da meia-vida de proteínas é a ubiquitinação, que pode direcionar proteínas alvo ao proteassoma para degradação ou a outras modificações pós-traducionais. O objetivo do presente estudo foi avaliar a participação de uma proteína com atividade deubiquitinase e ubiquitina ligase, a A20, na manutenção da homeostase do tecido adiposo de animais submetidos à dieta rica em gordura e voluntários humanos magros e obesos antes e após cirurgia de redução de peso. Foram utilizados o tecido adiposo branco visceral e subcutâneo e o tecido adiposo marrom de camundongos Swiss machos submetidos a 16 semanas de dieta hiperlipídica e o tecido adiposo subcutâneo de voluntários magros e obesos antes e após a cirurgia bariátrica. Esses tecidos foram avaliados quanto ao conteúdo protéico e expressão gênica da proteína A20, e sua associação com a PGC1? por imunoprecipitação e imunofluorescência, bem como a ubiquitinação desta última. Os resultados obtidos a partir do tecido adiposo de humanos mostram uma diminuição na expressão da proteína A20 nos pacientes antes e após a cirurgia bariátrica com relação aos voluntários magros. A PGC1? aparece mais ubiquitinada nos pacientes obesos em relação e a associação entre A20 e PGC1? parece aumentar com o ganho de peso na mesma proporção que o conteúdo protéico de PGC1? parece diminuir. No tecido adiposo subcutâneo de camundongos obesos, observamos uma diminuição de PGC1? bem como redução da marcação por cadeias de poliubiquitina desta proteína, associado a um aumento de A20 e aumento da associação de A20 com PGC1?. Camundongos obesos foram também tratados com um oligonucleotídeo antisense (ASO) para A20, resultando na redução de sua expressão gênica. Os animais tratados apresentaram uma piora na tolerância à glicose no teste de GTT o que ocorreu concomitantemente a redução de PGC1?. Nossos resultados indicam que, no tecido adiposo, a A20 se associa a PGC1? e a redução da sua expressão resulta em redução da expressão da PGC1? o que é acompanhando de uma piora no controle homeostático da glicoseAbstract: Peroxisome proliferator-activated receptor ? coactivator 1 alpha (PGC-1?) plays an important role in whole body metabolism and, particularly in glucose homeostasis. Its expression is tightly regulated and, small variations in tissue levels can have a major impact in a number of physiological and pathological conditions. Recent studies have shown that the ubiquitin/proteasome system plays a role in the control of PGC-1? degradation. Here we evaluated the interaction of PGC-1? with the protein A20, which plays a dual-role in the control of the ubiquitin/proteasome system acting as a deubiquitinase and as an E3 ligase. We employed immunoprecipitation, quantitative real-time PCR and immunofluorescence staining to evaluate PGC-1?, A20, PPAR? and ubiquitin in the adipose tissue of humans and mice. Our results show that, in distinct sites of the adipose tissue A20 binds to PGC-1?. At least in the subcutaneous fat of humans and mice the levels of PGC-1? decrease during obesity, while its physical association with A20 increases. The inhibition of A20 leads to a reduction of PGC-1? and PPAR? expression, suggesting that A20 acts as a protective factor against PGC-1? disposal. Thus, we provide evidence that mechanisms regulating PGC-1? ubiquitination are potentially involved in the control of the function of this transcriptional co-activatorMestradoFisiopatologia MédicaMestra em Ciência

Hypothalamic neuronal cellular and subcellular abnormalities in experimental obesity

The characterization of the hypothalamic neuronal network, that controls food intake and energy expenditure, has provided great advances in the understanding of the pathophysiology of obesity. Most of the advances in this field were obtained thanks to the development of a number of genetic and nongenetic animal models that, at least in part, overtook the anatomical constraints that impair the study of the human hypothalamus. Despite the undisputed differences between human and rodent physiology, most seminal studies undertaken in rodents that have unveiled details of the neural regulation of energy homeostasis were eventually confirmed in humans; thus, placing experimental studies in the forefront of obesity research. During the last 15 years, researchers have provided extensive experimental proof that supports the existence of hypothalamic dysfunction, which leads to a progressive whole-body positive energy balance, and thus, to obesity. Here, we review the experimental work that unveiled the mechanisms behind hypothalamic dysfunction in obesity431223612369CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPNão temNão temThe Laboratory of Cell Signaling is supported by funding provided by the Fundação de Amparo a Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico. The Laboratories of Cell Signaling belongs to the Obesity and Comorbidities Research Center and the National Institute of Science and Technology – Neuroimmunomodulatio

Inhibition of hypothalamic leukemia inhibitory factor exacerbates diet-induced obesity phenotype

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOThe consumption of large amounts of dietary fats can trigger an inflammatory response in the hypothalamus and contribute to the dysfunctional control of caloric intake and energy expenditure commonly present in obesity. The objective of this study was to identify chemokine-related transcripts that could be involved in the early stages of diet-induced hypothalamic inflammation. Methods: We used immunoblot, PCR array, real-time PCR, immunofluorescence staining, glucose and insulin tolerance tests, and determination of general metabolic parameters to evaluate markers of inflammation, body mass variation, and glucose tolerance in mice fed a high-fat diet. Results: Using a real-time PCR array, we identified leukemia inhibitory factor as a chemokine/cytokine undergoing a rapid increase in the hypothalamus of obesity-resistant and a rapid decrease in the hypothalamus of obesity-prone mice fed a high-fat diet for 1 day. We hypothesized that the increased hypothalamic expression of leukemia inhibitory factor could contribute to the protective phenotype of obesity-resistant mice. To test this hypothesis, we immunoneutralized hypothalamic leukemia inhibitory factor and evaluated inflammatory and metabolic parameters. The immunoneutralization of leukemia inhibitory factor in the hypothalamus of obesity-resistant mice resulted in increased body mass gain and increased adiposity. Body mass gain was mostly due to increased caloric intake and reduced spontaneous physical activity. This modification in the phenotype was accompanied by increased expression of inflammatory cytokines in the hypothalamus. In addition, the inhibition of hypothalamic leukemia inhibitory factor was accompanied by glucose intolerance and insulin resistance. Conclusion: Hypothalamic expression of leukemia inhibitory factor may protect mice from the development of diet-induced obesity; the inhibition of this protein in the hypothalamus transforms obesity-resistant into obesity-prone mice14FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOsem informaçã

Inhibition of hypothalamic leukemia inhibitory factor exacerbates diet-induced obesity phenotype

Abstract Background The consumption of large amounts of dietary fats can trigger an inflammatory response in the hypothalamus and contribute to the dysfunctional control of caloric intake and energy expenditure commonly present in obesity. The objective of this study was to identify chemokine-related transcripts that could be involved in the early stages of diet-induced hypothalamic inflammation. Methods We used immunoblot, PCR array, real-time PCR, immunofluorescence staining, glucose and insulin tolerance tests, and determination of general metabolic parameters to evaluate markers of inflammation, body mass variation, and glucose tolerance in mice fed a high-fat diet. Results Using a real-time PCR array, we identified leukemia inhibitory factor as a chemokine/cytokine undergoing a rapid increase in the hypothalamus of obesity-resistant and a rapid decrease in the hypothalamus of obesity-prone mice fed a high-fat diet for 1 day. We hypothesized that the increased hypothalamic expression of leukemia inhibitory factor could contribute to the protective phenotype of obesity-resistant mice. To test this hypothesis, we immunoneutralized hypothalamic leukemia inhibitory factor and evaluated inflammatory and metabolic parameters. The immunoneutralization of leukemia inhibitory factor in the hypothalamus of obesity-resistant mice resulted in increased body mass gain and increased adiposity. Body mass gain was mostly due to increased caloric intake and reduced spontaneous physical activity. This modification in the phenotype was accompanied by increased expression of inflammatory cytokines in the hypothalamus. In addition, the inhibition of hypothalamic leukemia inhibitory factor was accompanied by glucose intolerance and insulin resistance. Conclusion Hypothalamic expression of leukemia inhibitory factor may protect mice from the development of diet-induced obesity; the inhibition of this protein in the hypothalamus transforms obesity-resistant into obesity-prone mice

Inhibition of hypothalamic leukemia inhibitory factor exacerbates diet-induced obesity phenotype

The consumption of large amounts of dietary fats can trigger an inflammatory response in the hypothalamus and contribute to the dysfunctional control of caloric intake and energy expenditure commonly present in obesity. The objective of this study was to identify chemokine-related transcripts that could be involved in the early stages of diet-induced hypothalamic inflammation. Methods: We used immunoblot, PCR array, real-time PCR, immunofluorescence staining, glucose and insulin tolerance tests, and determination of general metabolic parameters to evaluate markers of inflammation, body mass variation, and glucose tolerance in mice fed a high-fat diet. Results: Using a real-time PCR array, we identified leukemia inhibitory factor as a chemokine/cytokine undergoing a rapid increase in the hypothalamus of obesity-resistant and a rapid decrease in the hypothalamus of obesity-prone mice fed a high-fat diet for 1 day. We hypothesized that the increased hypothalamic expression of leukemia inhibitory factor could contribute to the protective phenotype of obesity-resistant mice. To test this hypothesis, we immunoneutralized hypothalamic leukemia inhibitory factor and evaluated inflammatory and metabolic parameters. The immunoneutralization of leukemia inhibitory factor in the hypothalamus of obesity-resistant mice resulted in increased body mass gain and increased adiposity. Body mass gain was mostly due to increased caloric intake and reduced spontaneous physical activity. This modification in the phenotype was accompanied by increased expression of inflammatory cytokines in the hypothalamus. In addition, the inhibition of hypothalamic leukemia inhibitory factor was accompanied by glucose intolerance and insulin resistance. Conclusion: Hypothalamic expression of leukemia inhibitory factor may protect mice from the development of diet-induced obesity; the inhibition of this protein in the hypothalamus transforms obesity-resistant into obesity-prone mice1

Additional file 3: Table S3. of Dietary fats promote functional and structural changes in the median eminence blood/spinal fluid interface—the protective role for BDNF

Quantification of immunofluorescence of Fig. 6. (PDF 59 kb