RESUMO
BACKGROUND: Several effects of leptin in the immune system rely on its capacity to modulate cytokine expression and apoptosis in the thymus. Surprisingly, some of these effects are dependent on signal transduction through the IRS1/PI3-kinase, but not on the activation of JAK2. Since all the well known effects of leptin in different cell types and tissues seem to be dependent on JAK2 activation, we hypothesized that, at least for the control of thymic function, another, unknown kinase could mediate the transduction of the leptin signal from the ObR towards the IRS1/PI3-kinase signaling cascade. METHODOLOGY/PRINCIPAL FINDINGS: Here, by employing immunoblot, real-time PCR and flow citometry we show that the tyrosine kinase, Fyn, is constitutively associated with the ObR in thymic cells. Following a leptin stimulus, Fyn undergoes an activating tyrosine phosphorylation and a transient association with IRS1. All these effects are independent of JAK2 activation and, upon Fyn inhibition, the signal transduction towards IRS1/PI3-kinase is abolished. In addition, the inhibition of Fyn significantly modifies the effects of leptin on thymic cytokine expression. CONCLUSION/SIGNIFICANCE: Therefore, in the thymus, Fyn acts as a tyrosine kinase that transduces the leptin signal independently of JAK2 activation, and mediates some of the immunomodulatory effects of leptin in this tissue.
Assuntos
Leptina/fisiologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Timo/citologia , Animais , Separação Celular , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Ratos Wistar , Receptores para Leptina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
In diet-induced obesity, hypothalamic and systemic inflammatory factors trigger intracellular mechanisms that lead to resistance to the main adipostatic hormones, leptin and insulin. Tumor necrosis factor-alpha (TNF-alpha) is one of the main inflammatory factors produced during this process and its mechanistic role as an inducer of leptin and insulin resistance has been widely investigated. Most of TNF-alpha inflammatory signals are delivered by TNF receptor 1 (R1); however, the role played by this receptor in the context of obesity-associated inflammation is not completely known. Here, we show that TNFR1 knock-out (TNFR1 KO) mice are protected from diet-induced obesity due to increased thermogenesis. Under standard rodent chow or a high-fat diet, TNFR1 KO gain significantly less body mass despite increased caloric intake. Visceral adiposity and mean adipocyte diameter are reduced and blood concentrations of insulin and leptin are lower. Protection from hypothalamic leptin resistance is evidenced by increased leptin-induced suppression of food intake and preserved activation of leptin signal transduction through JAK2, STAT3, and FOXO1. Under the high-fat diet, TNFR1 KO mice present a significantly increased expression of the thermogenesis-related neurotransmitter, TRH. Further evidence of increased thermogenesis includes increased O(2) consumption in respirometry measurements, increased expressions of UCP1 and UCP3 in brown adipose tissue and skeletal muscle, respectively, and increased O(2) consumption by isolated skeletal muscle fiber mitochondria. This demonstrates that TNF-alpha signaling through TNFR1 is an important mechanism involved in obesity-associated defective thermogenesis.
Assuntos
Obesidade/metabolismo , Consumo de Oxigênio , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Termogênese , Fator de Necrose Tumoral alfa/metabolismo , Gordura Abdominal/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Dieta/efeitos adversos , Gorduras na Dieta/efeitos adversos , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Inflamação/genética , Inflamação/metabolismo , Insulina/metabolismo , Canais Iônicos/metabolismo , Janus Quinase 2/metabolismo , Leptina/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Obesidade/genética , Ratos , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Fator de Transcrição STAT3/metabolismo , Proteína Desacopladora 1 , Proteína Desacopladora 3RESUMO
Infiltrating macrophages play an important role in the production of inflammatory mediators by the adipose tissue of obese subjects. To reach the adipose tissue, peripheral monocytes are recruited by locally produced chemoattractants. However, little is known about the activation of monocytes in the peripheral blood of obese subjects. The objective of this study was to determine reactive oxygen species and endoplasmic reticulum stress as early markers of monocytic commitment with an inflammatory phenotype in the peripheral blood of nondiabetic obese patients. Patients were recruited from an academic general hospital; controls were voluntary students. Seven lean controls and 6 nondiabetic obese patients were included in the study. Monocytes were prepared from peripheral blood. Immunoblot, flow cytometry, and polymerase chain reaction were used to determine reactive oxygen species and endoplasmic reticulum stress. Increased reactive oxygen species and activation of endoplasmic reticulum stress were detected in the monocytes from obese patients. Reducing endoplasmic reticulum stress with a chemical chaperone reversed monocytic activation, as determined by the reduction of reactive oxygen species production. Thus, monocytes from nondiabetic obese patients are already committed with an inflammatory phenotype in peripheral blood; and reducing endoplasmic reticulum stress negatively modulates their activation.
Assuntos
Retículo Endoplasmático/metabolismo , Inflamação/metabolismo , Monócitos/metabolismo , Obesidade/sangue , Estresse Oxidativo , Espécies Reativas de Oxigênio/sangue , Adulto , Cálcio/metabolismo , Catalase/metabolismo , Citosol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Citometria de Fluxo , Humanos , Immunoblotting , Imunoprecipitação , Inflamação/sangue , Masculino , Monócitos/enzimologia , Fenótipo , Reação em Cadeia da Polimerase , Splicing de RNA , RNA Mensageiro/metabolismo , Fatores de Transcrição de Fator Regulador X , Superóxido Dismutase/metabolismo , Fatores de Transcrição/metabolismoRESUMO
In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.
Assuntos
Citocinas/metabolismo , Ácidos Graxos/administração & dosagem , Hipotálamo/metabolismo , Obesidade/induzido quimicamente , Obesidade/patologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Animais , Anticorpos/administração & dosagem , Peso Corporal/efeitos dos fármacos , Citocinas/classificação , Citocinas/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipotálamo/efeitos dos fármacos , Imunoprecipitação , Indóis , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Mutantes , Microglia/efeitos dos fármacos , Mutação , Obesidade/imunologia , Obesidade/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
Uncoupling protein 2 (UCP2) is highly expressed in the hypothalamus; however, little is known about the functions it exerts in this part of the brain. Here, we hypothesized that UCP2 protects hypothalamic cells from oxidative and pro-apoptotic damage generated by inflammatory stimuli. Intracerebroventricular injection of tumor necrosis factor alpha (TNF-alpha)-induced an increase of UCP2 expression in the hypothalamus, which was accompanied by increased expression of markers of oxidative stress and pro-apoptotic proteins. The inhibition of UCP2 expression by an antisense oligonucleotide enhanced the damaging effects of TNF-alpha. Conversely, increasing the hypothalamic expression of UCP2 by cold exposure reversed most of the effects of the cytokine. Thus, UCP2 acts as a protective factor against cellular damage induced by an inflammatory stimulus in the hypothalamus.