RESUMO
T cell therapy strategies, from allogeneic stem cell transplantation toward genetically-modified T cells infusion, develop powerful anti-tumor effects but are often accompanied by side effects and their efficacy remains sometimes to be improved. It therefore appears important to provide a flexible and easily reversible gene expression regulation system to control T cells activity. We developed a gene expression regulation technology that exploits the physiological GCN2-ATF4 pathway's ability to induce gene expression in T cells in response to one essential amino acid deficiency. We first demonstrated the functionality of NUTRIREG in human T cells by transient expression of reporter genes. We then validated that NUTRIREG can be used in human T cells to transiently express a therapeutic gene such as IL-10. Overall, our results represent a solid basis for the promising use of NUTRIREG to regulate transgene expression in human T cells in a reversible way, and more generally for numerous preventive or curative therapeutic possibilities in cellular immunotherapy strategies.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante Homólogo , Aminoácidos , Alelos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Linfócitos T , TransgenesRESUMO
Chronic treatment with acetaminophen (APAP) induces cysteine (Cys) and glutathione (GSH) deficiency which leads to adverse metabolic effects including muscle atrophy. Mammalian cells respond to essential amino acid deprivation through the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). Phosphorylated eIF2α leads to the recruitment of activating transcription factor 4 (ATF4) to specific CCAAT/enhancer-binding protein-ATF response element (CARE) located in the promoters of target genes. Our purpose was to study the activation of the eIF2α-ATF4 pathway in response to APAP-induced Cys deficiency, as well as the potential contribution of the eIF2α kinase GCN2 and the effect of dietary supplementation with Cys. Our results showed that chronic treatment with APAP activated both GCN2 and PERK eIF2α kinases and downstream target genes in the liver. Activation of the eIF2α-ATF4 pathway in skeletal muscle was accompanied by muscle atrophy even in the absence of GCN2. The dietary supplementation with cysteine reversed APAP-induced decreases in plasma-free Cys, liver GSH, muscle mass, and muscle GSH. Our new findings demonstrate that dietary Cys supplementation also reversed the APAP-induced activation of GCN2 and PERK and downstream ATF4-target genes in the liver.
Assuntos
Fator 4 Ativador da Transcrição , Fator de Iniciação 2 em Eucariotos , Acetaminofen/efeitos adversos , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Cisteína/metabolismo , Suplementos Nutricionais , Fator de Iniciação 2 em Eucariotos/metabolismo , Glutationa/metabolismo , Mamíferos/metabolismo , Atrofia Muscular/induzido quimicamente , Fosforilação , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Cyclosporin A (CsA) and tacrolimus (FK506) are valuable immunosuppressants for a range of clinical settings, including (but not limited to) organ transplantation and the treatment of autoimmune diseases. They function by inhibiting the activity of the Ca2+/calmodulin-dependent phosphatase calcineurin toward nuclear factor of activated T-cells (NF-AT) in T-lymphocytes. However, use of CsA is associated with more serious side effects and worse clinical outcomes than FK506. Here we show that CsA, but not FK506, causes activation of the integrated stress response (ISR), an event which is normally an acute reaction to various types of intracellular insults, such as nutrient deficiency or endoplasmic reticulum stress. These effects of CsA involve at least two of the stress-activated protein kinases (GCN2 and PERK) that act on the translational machinery to slow down protein synthesis via phosphorylation of the eukaryotic initiation factor (eIF) 2α and thereby induce the ISR. These actions of CsA likely contribute to the adverse effects associated with its clinical application.
Assuntos
Ciclosporina/farmacologia , Imunossupressores/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Tacrolimo/farmacologia , Células A549 , Fator 4 Ativador da Transcrição/metabolismo , Animais , Células Cultivadas , Células HeLa , Humanos , Camundongos , FosforilaçãoRESUMO
The unfolded protein response (UPR) is an endoplasmic reticulum (ER) -related stress conserved pathway that aims to protect cells from being overwhelmed. However, when prolonged, UPR activation converts to a death signal, which relies on its PERK-eIF2α branch. Overactivation of the UPR has been implicated in many neurological diseases, including cerebral ischaemia. Here, by using an in vivo thromboembolic model of stroke on transgenic ER stress-reporter mice and neuronal in vitro models of ischaemia, we demonstrate that ischaemic stress leads to the deleterious activation of the PERK branch of the UPR. Moreover, we show that the serine protease tissue-type plasminogen activator (tPA) can bind to cell surface Grp78 (78 kD glucose-regulated protein), leading to a decrease of the PERK pathway activation, thus a decrease of the deleterious factor CHOP, and finally promotes neuroprotection. Altogether, this work highlights a new role and a therapeutic potential of the chaperone protein Grp78 as a membrane receptor of tPA capable to prevent from ER stress overactivation.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Fibrinolíticos/farmacologia , Camundongos , Neurônios/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tromboembolia/terapia , Ativador de Plasminogênio Tecidual/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
The uncontrolled growth of tumor can lead to the formation of area deprived in nutrients. Due to their high genetic instability, tumor cells can adapt and develop resistance to this pro-apoptotic environment. Among the resistance mechanisms, those involved in the resistance to long-term amino acid restriction are not elucidated. A long-term amino acid restriction is particularly deleterious since nine of them cannot be synthetized by the cells. In order to determine how cancer cells face a long-term amino acid deprivation, we developed a cell model selected for its capacity to resist a long-term amino acid limitation. We exerted a selection pressure on mouse embryonic fibroblast to isolate clones able to survive with low amino acid concentration. The study of several clones revealed an alteration of the eiF2α/ATF4 pathway. Compared to the parental cells, the clones exhibited a decreased expression of the transcription factor ATF4 and its target genes. Likewise, the knock-down of ATF4 in parental cells renders them resistant to amino acid deprivation. Moreover, this association between a low level of ATF4 protein and the resistance to amino acid deprivation was also observed in the cancer cell line BxPC-3. This resistance was abolished when ATF4 was overexpressed. Therefore, decreasing ATF4 expression may be one important mechanism for cancer cells to survive under prolonged amino acid deprivation.
Assuntos
Fator 4 Ativador da Transcrição/genética , Aminoácidos/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Modelos Biológicos , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Transdução de SinaisAssuntos
Aminoácidos Essenciais/deficiência , Alimentos Formulados , Terapia Genética/métodos , Elementos de Resposta , Transgenes , Adulto , Aminoácidos Essenciais/farmacologia , Animais , Proteínas de Ciclo Celular/genética , Galinhas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Ratos , Proteínas Repressoras/genética , Elementos de Resposta/efeitos dos fármacos , Suínos , Timidina Quinase/genética , Ativação Transcricional/efeitos dos fármacos , Transgenes/efeitos dos fármacos , Transgenes/genéticaRESUMO
It is well known that the GCN2 and mTORC1 signaling pathways are regulated by amino acids and share common functions, in particular the control of translation. The regulation of GCN2 activity by amino acid availability relies on the capacity of GCN2 to sense the increased levels of uncharged tRNAs upon amino acid scarcity. In contrast, despite recent progress in the understanding of the regulation of mTORC1 by amino acids, key aspects of this process remain unsolved. In particular, while leucine is well known to be a potent regulator of mTORC1, the mechanisms by which this amino acid is sensed and control mTORC1 activity are not well defined. Our data establish that GCN2 is involved in the inhibition of mTORC1 upon leucine or arginine deprivation. However, the activation of GCN2 alone is not sufficient to inhibit mTORC1 activity, indicating that leucine and arginine exert regulation via additional mechanisms. While the mechanism by which GCN2 contributes to the initial step of mTORC1 inhibition involves the phosphorylation of eIF2α, we show that it is independent of the downstream transcription factor ATF4. These data point to a novel role for GCN2 and phosphorylation of eIF2α in the control of mTORC1 by certain amino acids.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Leucina/deficiência , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Arginina/deficiência , Embrião de Mamíferos/citologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Fibroblastos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Fosforilação , Transdução de SinaisRESUMO
Widespread application of gene therapy will depend on the development of simple methods to regulate the expression of therapeutic genes. Here we harness an endogenous signaling pathway to regulate therapeutic gene expression through diet. The GCN2-eIF2α signaling pathway is specifically activated by deficiencies in any essential amino acid (EAA); EAA deficiency leads to rapid expression of genes regulated by ATF4-binding cis elements. We found that therapeutic genes under the control of optimized amino acid response elements (AAREs) had low basal expression and high induced expression. We applied our system to regulate the expression of TNFSF10 (TRAIL) in the context of glioma therapy and found that intermittent activation of this gene by EEA-deficient meals retained its therapeutic efficacy while abrogating its toxic effects on normal tissue. The GCN2-eIF2α pathway is expressed in many tissues, including the brain, and is highly specific to EAA deficiency. Our system may be particularly well suited for intermittent regulation of therapeutic transgenes over short or long time periods.
Assuntos
Aminoácidos Essenciais/administração & dosagem , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Terapia Genética/métodos , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Administração Oral , Aminoácidos Essenciais/farmacocinética , Animais , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Ingestão de Alimentos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Transgenes/genética , Resultado do TratamentoRESUMO
It has been reported that breast-feeding more than 6 months strongly decreases the risk of allergy, diabetes, obesity, and hypertension in humans. In order to understand the mechanisms responsible for this benefit, it is important to evaluate precisely the composition of maternal milk, especially in response to environmental cues. Mouse models offer a unique opportunity to study the impact of maternal milk composition on the development and health of offspring. Oxytocin injection of the dam is usually used to stimulate milk ejection; however, exogenous oxytocin might have deleterious effects under some experimental conditions by modifying milk content as well as the physiology and behavior of the dam. Taking advantage of the natural stimulation of the mammary gland that occurs after the reunion of a dam that has been separated from her pups, we developed a new procedure to collect mouse milk without the injection of oxytocin. This method is easy to use, low-cost ,and non-invasive. Moreover, it provides a sufficient amount of milk for use in a wide range of biological analyses.
Assuntos
Aleitamento Materno , Glândulas Mamárias Animais/fisiologia , Ejeção Láctea/fisiologia , Leite , Animais , Mama/metabolismo , Mama/fisiologia , Feminino , Humanos , Camundongos , Ocitocina/farmacologiaRESUMO
The eIF2α-ATF4 pathway is involved in cellular adaptation to stress and is dysregulated in numerous diseases. Activation of this pathway leads to phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and the recruitment of the transcription factor ATF4 (activating transcription factor 4) to specific CCAAT/enhancer binding protein (C/EBP)-ATF response elements (CAREs) located in the promoters of target genes. To monitor the spatiotemporal modulation of this pathway in living animals, we generated a novel CARE-driven luciferase mouse model (CARE-LUC). These transgenic mice enable the investigation of the eIF2α-ATF4 pathway activity in the whole organism and at the tissue and cellular levels by combining imaging, luciferase assays, and immunochemistry. Using this mouse line, we showed the tissue-specific activation pattern of this pathway in response to amino acid deficiency or endoplasmic reticulum stress and the hepatic induction of this pathway in a stress-related pathology model of liver fibrosis. The CARE-LUC mouse model represents an innovative tool to investigate the eIF2α-ATF4 axis and to develop drugs targeting this important pathway in the remediation of related pathologies.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Imagem Molecular , Transdução de Sinais , Estresse Fisiológico , Fator 4 Ativador da Transcrição/genética , Animais , Fator de Iniciação 2 em Eucariotos/genética , Camundongos , Camundongos TransgênicosRESUMO
In mammals, metabolic adaptations are required to overcome nutritional deprivation in amino-acids/proteins as well as episodes of malnutrition. GCN2 protein kinase, which phosphorylates the α subunit of the translation initiation factor eIF2, is a sensor of amino-acid(s) deficiencies. On one hand, this review briefly describes the main features of amino-acid metabolism. On the other hand, it describes the role of GCN2 in regulating numerous physiological functions.
Assuntos
Adaptação Biológica/fisiologia , Aminoácidos Essenciais/administração & dosagem , Dieta , Proteínas Serina-Treonina Quinases/fisiologia , Aminoácidos , Aminoácidos Essenciais/deficiência , Animais , Fator de Iniciação 2 em Eucariotos/metabolismo , Homeostase , Humanos , Imunidade , Desnutrição , Plasticidade Neuronal , Estado Nutricional , Fosforilação , Transdução de Sinais/fisiologiaRESUMO
Epidemiological findings indicate that transient environmental influences during perinatal life, especially nutrition, may have deleterious heritable health effects lasting for the entire life. Indeed, the fetal organism develops specific adaptations that permanently change its physiology/metabolism and that persist even in the absence of the stimulus that initiated them. This process is termed "nutritional programming". We previously demonstrated that mothers fed a Low-Protein-Diet (LPD) during gestation and lactation give birth to F1-LPD animals presenting metabolic consequences that are different from those observed when the nutritional stress is applied during gestation only. Compared to control mice, adult F1-LPD animals have a lower body weight and exhibit a higher food intake suggesting that maternal protein under-nutrition during gestation and lactation affects the energy metabolism of F1-LPD offspring. In this study, we investigated the origin of this apparent energy wasting process in F1-LPD and demonstrated that minimal energy expenditure is increased, due to both an increased mitochondrial function in skeletal muscle and an increased mitochondrial density in White Adipose Tissue. Importantly, F1-LPD mice are protected against high-fat-diet-induced obesity. Clearly, different paradigms of exposure to malnutrition may be associated with differences in energy expenditure, food intake, weight and different susceptibilities to various symptoms associated with metabolic syndrome. Taken together these results demonstrate that intra-uterine environment is a major contributor to the future of individuals and disturbance at a critical period of development may compromise their health. Consequently, understanding the molecular mechanisms may give access to useful knowledge regarding the onset of metabolic diseases.
Assuntos
Metabolismo Energético/genética , Lactação/fisiologia , Mitocôndrias/fisiologia , Obesidade/prevenção & controle , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Deficiência de Proteína/fisiopatologia , Tecido Adiposo Branco/metabolismo , Fatores Etários , Animais , Western Blotting , Temperatura Corporal , Peso Corporal , Calorimetria Indireta , Primers do DNA/genética , Dieta Hiperlipídica/efeitos adversos , Ingestão de Alimentos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Obesidade/metabolismo , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and metabolism. It controls many cell functions by integrating nutrient availability and growth factor signals. Amino acids, and in particular leucine, are among the main positive regulators of mTORC1 signaling. The current model for the regulation of mTORC1 by amino acids involves the movement of mTOR to the lysosome mediated by the Rag-GTPases. Here, we have examined the control of mTORC1 signaling and mTOR localization by amino acids and leucine in serum-fed cells, because both serum growth factors (or, e.g., insulin) and amino acids are required for full activation of mTORC1 signaling. We demonstrate that mTORC1 activity does not closely correlate with the lysosomal localization of mTOR. In particular, leucine controls mTORC1 activity without any detectable modification of the lysosomal localization of mTOR, indicating that the signal(s) exerted by leucine is likely distinct from those exerted by other amino acids. In addition, knock-down of the Rag-GTPases attenuated the inhibitory effect of amino acid- or leucine-starvation on the phosphorylation of mTORC1 targets. Furthermore, data from cells where Rag expression has been knocked down revealed that leucine can promote mTORC1 signaling independently of the lysosomal localization of mTOR. Our data complement existing models for the regulation of mTORC1 by amino acids and provide new insights into this important topic.
Assuntos
Aminoácidos/metabolismo , Leucina/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Aminoácidos/farmacologia , Animais , Linhagem Celular , Leucina/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Proteínas Monoméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologiaRESUMO
CHOP encodes a ubiquitous transcription factor that is one of the most important components in the network of stress-inducible transcription. In particular, this factor is known to mediate cell death in response to stress. The focus of this work is to study its pivotal role in the control of cell viability according to the duration of a stress like amino acid starvation. We show that during the first 6h of starvation, CHOP upregulates a number of autophagy genes but is not involved in the first steps of the autophagic process. By contrast, when the amino acid starvation is prolonged (16-48h), we demonstrated that CHOP has a dual role in both inducing apoptosis and limiting autophagy through the transcriptional control of specific target genes. Overall, this study reveals a novel regulatory role for CHOP in the crosstalk between autophagy and apoptosis in response to stress.
Assuntos
Aminoácidos/deficiência , Apoptose/genética , Autofagia/genética , Inanição , Fator de Transcrição CHOP/genética , Animais , Linhagem Celular , Sobrevivência Celular/genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , RNA Mensageiro/biossíntese , Estresse Fisiológico , Proteína Vermelha FluorescenteRESUMO
The reversible phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) is a highly conserved signal implicated in the cellular adaptation to numerous stresses such as the one caused by amino acid limitation. In response to dietary amino acid deficiency, the brain-specific activation of the eIF2α kinase GCN2 leads to food intake inhibition. We report here that GCN2 is rapidly activated in the mediobasal hypothalamus (MBH) after consumption of a leucine-deficient diet. Furthermore, knockdown of GCN2 in this particular area shows that MBH GCN2 activity controls the onset of the aversive response. Importantly, pharmacological experiments demonstrate that the sole phosphorylation of eIF2α in the MBH is sufficient to regulate food intake. eIF2α signaling being at the crossroad of stress pathways activated in several pathological states, our study indicates that hypothalamic eIF2α phosphorylation could play a critical role in the onset of anorexia associated with certain diseases.
Assuntos
Ingestão de Alimentos/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Hipotálamo/metabolismo , Transdução de Sinais , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Técnicas de Silenciamento de Genes , Leucina/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
In response to different environmental stresses, eIF2α phosphorylation represses global translation coincident with preferential translation of ATF4, a master regulator controlling the transcription of key genes essential for adaptative functions. Here, we establish that the eIF2α/ATF4 pathway directs an autophagy gene transcriptional program in response to amino acid starvation or endoplasmic reticulum stress. The eIF2α-kinases GCN2 and PERK and the transcription factors ATF4 and CHOP are also required to increase the transcription of a set of genes implicated in the formation, elongation and function of the autophagosome. We also identify three classes of autophagy genes according to their dependence on ATF4 and CHOP and the binding of these factors to specific promoter cis elements. Furthermore, different combinations of CHOP and ATF4 bindings to target promoters allow the trigger of a differential transcriptional response according to the stress intensity. Overall, this study reveals a novel regulatory role of the eIF2α-ATF4 pathway in the fine-tuning of the autophagy gene transcription program in response to stresses.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Autofagia/genética , Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Ativação Transcricional , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Aminoácidos/metabolismo , Animais , Células Cultivadas , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Elementos de Resposta , Proteína Sequestossoma-1 , Fator de Transcrição CHOP/metabolismo , Regulação para Cima , eIF-2 Quinase/metabolismoRESUMO
PURPOSE: Selection of a balanced diet has a determinant impact on human health. Individual food preferences involve socio-cultural as well as physiological factors and evolve during aging. In mammals, physiological mechanisms governing food choices appear to require the sensing of nutrient concentrations in diet. This is particularly the case for dietary amino acids that are sensed by the protein kinase GCN2. It has been reported that GCN2 is involved in the adaptive response to amino acid imbalanced diets at the level of food intake and lipid metabolism. Here, we hypothesized that GCN2 may play a role in macronutrient selection and its age-related changes. METHODS: Two groups of wild-type and GCN2 knock-out mice were subjected to a food self-selection protocol at ages 6, 12, 18 and 24 months. During each test, mice were allowed to create their own diets by selecting between three separate food sources, each containing either protein, fat or carbohydrates. RESULTS: Our results show that the absence of GCN2 had two main age-related effects. First, it exacerbated fat preference at the expense of carbohydrate consumption. Second, it prevented the increase in protein intake. CONCLUSION: These findings indicate that, in omnivores, the GCN2 ancient pathway participates in the control of food preference.
Assuntos
Envelhecimento/metabolismo , Comportamento Animal , Carboidratos da Dieta/administração & dosagem , Gorduras na Dieta/administração & dosagem , Proteínas Alimentares/administração & dosagem , Preferências Alimentares , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Constitution of oxidative defense systems and, correspondingly, oxidative stress prevention are highly dependent on amino acid supply. In vitro, experiments have demonstrated that amino acid availability participates to the homeostasis of reactive oxygen species. However the molecular mechanisms involved in the maintenance of redox homeostasis responsive to circulating amino acid levels remain unclear. As GCN2 is a protein kinase considered to be an important sensor for amino acids availability and a potential regulator of redox homeostasis, we hypothesized that this kinase can modulate redox homeostasis in vivo, in response to an amino acid-imbalanced diet. We investigated the response of GCN2+/+ and GCN2-/- mice to a long-term (24 weeks) leucine-imbalanced diet (EDΔLeu). In order to evaluate the oxidation level in each group of mice, we determined the degree of protein oxidation in the liver. Interestingly, GCN2-/- mice exhibited an increase in protein carbonylation, a marker of oxidative stress, in response to the EDΔLeu diet. These data correlate with a decrease in hepatic GPX1 expression, a major antioxidant enzyme, and a decrease in total GPX activity in the liver. Our results suggest that GCN2 and its downstream signaling pathway have an important role in the protection against oxidative injuries induced by an amino acid-imbalanced diet, and that it can play a critical role in the prevention of oxidative damage.
Assuntos
Estresse Oxidativo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Dieta , Leucina/deficiência , Fígado/metabolismo , Camundongos , Camundongos Mutantes , Oxirredução , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Transient environmental influences, such as perinatal nutritional stress, may induce deleterious metabolic symptoms that last for the entire life of individuals, implying that epigenetic modifications play an important role in this process. We have investigated, in mice, the consequences of maternal undernutrition during gestation and lactation on DNA methylation and expression of the leptin gene, which plays a major regulatory role in coordinating nutritional state with many aspects of mammalian biology. We show that animals born to mothers fed a low-protein-diet (F1-LPD group) have a lower body weight/adiposity and exhibit a higher food intake than animals born to mothers fed a control diet (F1-CD group). These modifications persisted throughout life and were associated with lower levels of leptin mRNA and protein in starved F1-LPD mice, emphasizing that maternal protein-undernutrition affects the balance between food intake and energy expenditure in adults. Moreover, this nutritional stress resulted in the removal of methyls at CpGs located in the promoter of leptin, causing a permanent specific modification in the dynamics of the expression of leptin, which exhibits a stronger induction in the F1-LPD than in F1-CD mice in response to a meal. This study is an example of a molecular rationale linking transient environmental influences to permanent phenotypic consequences.