RESUMO
The glucose phosphorylating enzyme glucokinase regulates glucose metabolism in the liver. Glucokinase activity is modulated by a liver-specific competitive inhibitor, the glucokinase regulatory protein (GRP), which mediates sequestration of glucokinase to the nucleus at low glucose concentrations. However, the mechanism of glucokinase nuclear export is not fully understood. In this study we investigated the dynamics of glucose-dependent interaction and translocation of glucokinase and GRP in primary hepatocytes using fluorescence resonance energy transfer, selective photoconversion and fluorescence recovery after photobleaching. The formation of the glucokinase:GRP complex in the nucleus of primary hepatocytes at 5 mmol/l glucose was significantly reduced after a 2 h incubation at 20 mmol/l glucose. The GRP was predominantly localized in the nucleus, but a mobile fraction moved between the nucleus and the cytoplasm. The glucose concentration only marginally affected GRP shuttling. In contrast, the nuclear export rate of glucokinase was significantly higher at 20 than at 5 mmol/l glucose. Thus, glucose was proven to be the driving-force for nuclear export of glucokinase in hepatocytes. Using the FLII2Pglu-700mu-delta6 glucose nanosensor it could be shown that in hepatocytes the kinetics of nuclear glucose influx, metabolism or efflux were significantly faster compared to insulin-secreting cells. The rapid equilibration kinetics of glucose flux into the nucleus facilitates dissociation of the glucokinase:GRP complex and also nuclear glucose metabolism by free glucokinase enzyme. In conclusion, we could show that a rise of glucose in the nucleus of hepatocytes releases active glucokinase from the glucokinase:GRP complex and promotes the subsequent nuclear export of glucokinase.
Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Glucoquinase/metabolismo , Glucose/metabolismo , Hepatócitos/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Citoplasma/metabolismo , Células Secretoras de Insulina/metabolismo , Cinética , Camundongos , Transporte Proteico , RatosRESUMO
The accurate quantification of beta cell mass in humans is one of the key challenges in understanding the role of beta cell loss and dysfunction in the pathogenesis of diabetes mellitus. Autopsy studies indicate that beta cell loss is not only a hallmark of autoimmune diabetes but also plays a pivotal role in type 2 diabetes, owing to the toxic effects of lipids, glucose and cytokines. Thus, there is an urgent need for non-invasive clinical techniques for beta cell mass quantification, which should be optimally integrated into standard diagnostic equipment in hospitals. In this issue of Diabetologia (Brom et al DOI 10.1007/s00125-014-3166-3) it is reported that single photon emission computed tomography (SPECT) data with (111)indium-labelled glucagon-like peptide-1 (GLP-1) receptor agonist exendin-3 correlate with the morphometric analysis of beta cell mass in a rat model of alloxan-induced diabetes. With this validation, the authors were able to demonstrate a significant loss of beta cell mass in C-peptide-negative type 1 diabetic patients. Thus, (111)indium-labelled exendin-3 could serve as a model tracer for future studies of larger cohorts of diabetic patients to monitor the dynamics of beta cell loss and regeneration. Despite the recent progress from SPECT imaging data there remain open questions that await clarification in the near future such as variations in GLP-1 receptor density and physiological variation of beta cell mass in relation to beta cell function. The use of GLP-1-based tracer analysis may open new clinical avenues for non-invasive quantification of beta cell mass in patients with newly diagnosed type 1 diabetes and prediabetic individuals with high titres of autoantibodies.
Assuntos
Diabetes Mellitus Tipo 1/diagnóstico por imagem , Radioisótopos de Índio , Células Secretoras de Insulina/diagnóstico por imagem , Peptídeos , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , MasculinoRESUMO
The Cbl-interacting 85-kDa protein (CIN85) plays an important role as a negative regulator of signaling pathways induced by receptor tyrosine kinases. By assembling multiprotein complexes this versatile adaptor enhances receptor tyrosine kinase-activated clathrin-mediated endocytosis and reduces phosphatidylinositol-3-kinase-induced phosphatidylinositol-3,4,5-trisphosphate production. Here we report the expression of CIN85 in primary splenic B lymphocytes and the B-lymphoma cell lines WEHI 231 and Ba/F3. Cross-linking of the B cell antigen receptor resulted in an increased association of CIN85 with the ubiquitin ligase Cbl. Through a systematic pull-down proteomics approach we identified 51 proteins that interact with CIN85 in B cells, including proteins not shown previously to be CIN85-associated. Among these proteins, the SH2-containing inositol phosphatase 1 (SHIP-1) co-precipitated with both the full-length CIN85 and each of its three SH3 domains. We also showed that this association is constitutive and depends on a region of 79 amino acids near the carboxyl terminus of SHIP-1, a region rich in potential SH3 domain binding sites. Because SHIP-1 is a major negative regulator of the phosphatidylinositol-3-kinase pathway in lymphocytes, we hypothesize that the interaction between SHIP-1 and CIN85 might synergistically facilitate the down-regulation of phosphatidylinositol-3,4,5-trisphosphate levels.
Assuntos
Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linfócitos B/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Inositol Polifosfato 5-Fosfatases , Células Jurkat , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Fosfatos de Fosfatidilinositol/química , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Ligação Proteica , Proteômica , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Baço/citologia , Domínios de Homologia de srcRESUMO
Mitochondria are organelles of all nucleated cells, and variations in mtDNA sequence affect a wide spectrum of human diseases. However, animal models for mtDNA-associated diseases are rare, making it challenging to explore mechanisms underlying the contribution of mitochondria. Here, we identify a polymorphism in the mitochondrial genome, G-to-T at position 7778, which results in an aspartic acid-to-tyrosine (D-Y) substitution in the fifth amino acid of the highly conserved N-terminus of ATP synthase 8 (ATP8). Using a series of conplastic strains we show that this polymorphism increases susceptibility to multiple autoimmune diseases, including collagen-induced arthritis, autoimmune diabetes, nephritis and autoimmune pancreatitis. In addition, it impairs reproductive performance in females, but only in the MRL/MpJ strain. We also demonstrate that the mtAtp8 polymorphism alters mitochondrial performance, increasing H(2)O(2) production and affecting mitochondrial structure. Functional analysis reveals that the polymorphism increase the CD4 T cell adaptive potential to an oxidative phosphorylation impaired condition. Our findings provide direct experimental evidence for the role of mitochondria in autoimmunity and reproduction.
Assuntos
Doenças Autoimunes/genética , DNA Mitocondrial/genética , Infertilidade Feminina/genética , Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/genética , Reprodução/genética , Sequência de Aminoácidos , Animais , Feminino , Genoma Mitocondrial , Peróxido de Hidrogênio/metabolismo , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Polimorfismo GenéticoRESUMO
Galectin-1 (gal-1) triggers T cell death by several distinct intracellular pathways including the activation of the death-receptor pathway. The aim of this study was to investigate whether gal-1 induced activation of the death-receptor pathway in Jurkat T lymphocytes mediates apoptosis via the mitochondrial pathway linked by truncated Bid (tBid). We demonstrate that gal-1 induced proteolytic cleavage of the death agonist Bid, a member of the Bcl-2/Bcl-xL family and a substrate of activated caspase-8, was inhibited by caspase-8 inhibitor II (Z-IETD-FMK). Downstream of Bid, gal-1 stimulated mitochondrial cytochrome c release as well as the activation and proteolytic processing of initiator procaspase-9 were effectively decreased by caspase-8 inhibitor II. Blocking of gal-1 induced cleavage of effector procaspase-3 by caspase-8 inhibitor II as well as by caspase-9 inhibitors I (Z-LEHD-FMK) and III (Ac-LEHD-CMK) indicates that receptor and mitochondrial pathways converged in procaspase-3 activation and contribute to proteolytic processing of effector procaspase-6 and -7. Western blot analyses and immunofluorescence staining revealed that exposure of Jurkat T cells to gal-1 resulted in the cleavage of the DNA-repair enzyme poly (ADP-ribose) polymerase, cytoskeletal alpha-fodrin, and nuclear lamin A as substrates of activated caspases. Our data demonstrate that Bid provides a connection between the death receptor and the mitochondrial pathway of gal-1 induced apoptosis in human Jurkat T lymphocytes.
Assuntos
Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Mitocôndrias/metabolismo , Linfócitos T/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/agonistas , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteínas de Transporte/metabolismo , Inibidores de Caspase , Caspases/metabolismo , Núcleo Celular/metabolismo , Citocromos c/metabolismo , Fragmentação do DNA , Galectina 1/farmacologia , Humanos , Células Jurkat , Lamina Tipo A/metabolismo , Proteínas dos Microfilamentos/metabolismo , Oligopeptídeos/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Linfócitos T/efeitos dos fármacosRESUMO
Mitochondrial Ca2+ flux is crucial for the regulation of cell metabolism. Ca2+ entry to the mitochondrial matrix is mediated by VDAC1 and MCU with its regulatory molecules. We investigated hepatocytes isolated from conplastic C57BL/6NTac-mtNODLtJ mice (mtNOD) that differ from C57BL/6NTac mice (controls) by a point mutation in mitochondrial-encoded subunit 3 of cytochrome c oxidase, resulting in functional and morphological mitochondrial adaptations. Mice of both strains up to 12 months old were compared using mitochondrial GEM-GECO1 and cytosolic CAR-GECO1 expression to gain knowledge of age-dependent alterations of Ca2+ concentrations. In controls we observed a significant increase in glucose-induced cytosolic Ca2+ concentration with ageing, but only a minor elevation in mitochondrial Ca2+ concentration. Conversely, glucose-induced mitochondrial Ca2+ concentration significantly declined with ageing in mtNOD mice, paralleled by a slight decrease in cytosolic Ca2+ concentration. This was consistent with a significant reduction of the MICU1 to MCU expression ratio and a decline in MCUR1. Our results can best be explained in terms of the adaptation of Ca2+ concentrations to the mitochondrial network structure. In the fragmented mitochondrial network of ageing controls there is a need for high cytosolic Ca2+ influx, because only some of the isolated mitochondria are in direct contact with the endoplasmic reticulum. This is not important in the hyper-fused elongated mitochondrial network found in ageing mtNOD mice which facilitates rapid Ca2+ distribution over a large mitochondrial area.
Assuntos
Envelhecimento/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Hepatócitos/metabolismo , Adaptação Biológica , Envelhecimento/genética , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Glucose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutação/genéticaRESUMO
AIM: Mitochondrial DNA (mtDNA) mutations can negatively influence lifespan and organ function. More than 250 pathogenic mtDNA mutations are known, often involving neurological symptoms. Major neurodegenerative diseases share key etiopathogenetic components ie mtDNA mutations, mitochondrial dysfunction and oxidative stress. METHODS: Here, we characterized a conplastic mouse strain (C57BL/6 J-mtNOD) carrying an electron transport chain complex IV mutation that leads to an altered cytochrome c oxidase subunit III. Since this mouse also harbours adenine insertions in the mitochondrial tRNA for arginine, we chose the C57BL/6 J-mtMRL as control strain which also carries a heteroplasmic stretch of adenine repetitions in this tRNA isoform. RESULTS: Using MitoSOX fluorescence, we observed an elevated mitochondrial superoxide production and a reduced gene expression of superoxide dismutase 2 in the 24-month-old mtNOD mouse as compared to control. Together with the decreased expression of the fission-relevant gene Fis1, these data confirmed that the ageing mtNOD mouse had a mitochondrial dysfunctional phenotype. On the functional level, we could not detect significant differences in synaptic long-term potentiation, but found a markedly poor physical constitution to perform the Morris water maze task at the age of 24 months. Moreover, the median lifespan of mtNOD mice was significantly shorter than of control animals. CONCLUSION: Our findings demonstrate that a complex IV mutation leads to mitochondrial dysfunction that translates into survival.
Assuntos
Deficiência de Citocromo-c Oxidase/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Longevidade/genética , Espécies Reativas de Oxigênio/metabolismo , Animais , Encéfalo/metabolismo , Deficiência de Citocromo-c Oxidase/genética , Proteína Glial Fibrilar Ácida/metabolismo , Técnicas In Vitro , Memória/fisiologia , Camundongos Endogâmicos C57BL , Dinâmica Mitocondrial/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxidos/metabolismoRESUMO
Mitochondrial dysfunction affects liver metabolism, but it remains unclear whether this interferes with normal liver aging. We investigated several mitochondrial pathways in hepatocytes and liver tissue from a conplastic mouse strain compared with the control C57BL/6NTac strain over 18 months of life. The C57BL/6NTac-mtNODLtJ mice differed from C57BL/6NTac mice by a point mutation in mitochondrial-encoded subunit 3 of cytochrome c oxidase. Young C57BL/6NTac-mtNODLtJ mice showed reduced mitochondrial metabolism but similar reactive oxygen species (ROS) production to C57BL/6NTac mice. Whereas ROS increased almost equally up to 9 months in both strains, different mitochondrial adaptation strategies resulted in decreasing ROS in advanced age in C57BL/6NTac mice, but persistent ROS production in C57BL/6NTac-mtNODLtJ mice. Only the conplastic strain developed elongated mitochondrial networks with artificial loop structures, depressed autophagy, high mitochondrial respiration and up-regulated antioxidative response. Our results indicate that mtDNA mutations accelerate liver ballooning degeneration and carry a serious risk of premature organ aging.
Assuntos
Envelhecimento/genética , DNA Mitocondrial/genética , Fígado/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Envelhecimento/patologia , Animais , Autofagia/genética , Estágios do Ciclo de Vida/genética , Fígado/patologia , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Estresse Oxidativo/genéticaRESUMO
The glucose sensor enzyme glucokinase plays a pivotal role in the regulation of glucose-induced insulin secretion in pancreatic beta-cells. Activation of glucokinase represents a promising concept for the treatment of type 2 diabetes. Therefore, we analyzed the glucokinase activation through its physiological interaction partner, the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) and the resulting effect on glucose metabolism in insulin-producing cells. In RINm5F-GK-PFK-2/FBPase-2 cells stably overexpressing glucokinase plus islet PFK-2/FBPase-2, colocalization between both enzymes as well as elevation of glucokinase activity were significantly increased at a stimulatory glucose concentration of 10 mmol/liter. RINm5F-GK-PFK-2/FBPase-2 cells showed under this culture condition a significant increase in glucose utilization and in the ATP/ADP ratio compared with RINm5F-GK cells, which only overexpress glucokinase. Also glucose-induced insulin secretion was elevated in RINm5F-GK-PFK-2/FBPase-2 cells in comparison to RINm5F-GK cells. Furthermore, pyruvate accumulation and lactate production in RINm5F-GK-PFK-2/FBPase-2 cells were significantly lower at both 10 and 30 mmol/liter glucose than in RINm5F-GK and RINm5F cells. The significant improvement of glucose metabolism after PFK-2/FBPase-2 overexpression is apparently not exclusively the result of high glucokinase enzyme activity. Stabilization of the closed glucokinase conformation by PFK-2/FBPase-2 may not only activate the enzyme but also improve metabolic channeling in beta-cells.
Assuntos
Glucoquinase/fisiologia , Glucose/metabolismo , Insulina/metabolismo , Fosfofrutoquinase-2/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Glucoquinase/metabolismo , Glucose/farmacologia , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Ácido Láctico/biossíntese , Redes e Vias Metabólicas , Ligação Proteica , Ácido Pirúvico/metabolismo , Ratos , TransfecçãoRESUMO
The glucokinase regulatory protein (GRP) plays a pivotal role in the regulation of metabolic flux in liver by the glucose-phosphorylating enzyme glucokinase. Random peptide phage display library screening for binding partners of GRP allowed the identification of an asparagine-leucine consensus motif. Asparagine-leucine motifs of glucokinase located in the hinge region, as well as in the large domain, were changed by site-directed mutagenesis. The L58R/N204Y and the L309R/N313Y glucokinase mutants showed a significantly reduced interaction with GRP. The L355R/N350Y mutant had a fivefold-higher binding affinity for GRP than wild-type glucokinase. Imaging of glucokinase and GRP fluorescence fusion proteins revealed that the L58R/N204Y glucokinase mutant lacked glucose-dependent translocation by GRP, whereas the L355R/N350Y glucokinase mutant was trapped in the nucleus due to high affinity for GRP. The results indicate that the L58/N204 motif in the hinge region confers binding to GRP, while the L355/N350 motif may modulate the binding affinity for GRP. This latter motif is part of the alpha10 helix of glucokinase and accessible to GRP in the free and complex conformation.
Assuntos
Asparagina , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Glucoquinase/química , Glucoquinase/metabolismo , Leucina , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Células COS , Proteínas de Transporte/genética , Chlorocebus aethiops , Sequência Consenso , Glucoquinase/genética , Proteínas de Fluorescência Verde/genética , Hepatócitos/enzimologia , Humanos , Cinética , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão , Proteínas Recombinantes , Relação Estrutura-Atividade , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
The IDDM (LEW.1AR1/Ztm-iddm) rat is a type 1 diabetic animal model characterized by a rapid apoptotic pancreatic beta-cell destruction. Here we have analyzed the time course of islet infiltration, changes in the cytokine expression pattern, and beta-cell apoptosis in the transition from the pre-diabetic to the diabetic state. Transition from normoglycemia to hyperglycemia occurred when beta-cell loss exceeded 60-70%. At the early stages of islet infiltration, macrophages were the predominant immune cell type in the peripherally infiltrated islets. Progression of beta-cell loss was closely linked to a severe infiltration of the whole islet by CD8+ T-cells. With progressive islet infiltration, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were expressed in immune cells but not in beta-cells. This proinflammatory cytokine expression pattern coincided with the expression of inducible nitric oxide synthase (iNOS) and procaspase 3 in beta-cells and a peak apoptosis rate of 6.7%. Islet infiltration declined after manifestation of clinical diabetes, yielding end-stage islets devoid of beta-cells and immune cells without any sign of cytokine expression. The observed coincidence of IL-1beta and TNF-alpha expression in the immune cells and the induction of iNOS and procaspase 3 mRNA expression in the beta-cells depicts a sequence of pathological changes leading to apoptotic beta-cell death in the IDDM rat. This chain of events provides a mechanistic explanation for the development of the diabetic syndrome in this animal model of human type 1 diabetes.
Assuntos
Linfócitos B/imunologia , Linfócitos B/patologia , Citocinas/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Ilhotas Pancreáticas/imunologia , Animais , Cruzamentos Genéticos , Diabetes Mellitus Tipo 1/genética , Regulação da Expressão Gênica/imunologia , Ilhotas Pancreáticas/patologia , Ratos , Ratos Endogâmicos LewRESUMO
Mitochondrial network functionality is vital for glucose-stimulated insulin secretion in pancreatic beta cells. Altered mitochondrial dynamics in pancreatic beta cells are thought to trigger the development of type 2 diabetes mellitus. Fission protein 1 (Fis1) might be a key player in this process. Thus, the aim of this study was to investigate mitochondrial morphology in dependence of beta cell function, after knockdown and overexpression of Fis1. We demonstrate that glucose-unresponsive cells with impaired glucose-stimulated insulin secretion (INS1-832/2) showed decreased mitochondrial dynamics compared with glucose-responsive cells (INS1-832/13). Accordingly, mitochondrial morphology visualised using MitoTracker staining differed between the two cell lines. INS1-832/2 cells formed elongated and clustered mitochondria, whereas INS1-832/13 cells showed a homogenous mitochondrial network. Fis1 overexpression using lentiviral transduction significantly improved glucose-stimulated insulin secretion and mitochondrial network homogeneity in glucose-unresponsive cells. Conversely, Fis1 downregulation by shRNA, both in primary mouse beta cells and glucose-responsive INS1-832/13 cells, caused unresponsiveness and significantly greater numbers of elongated mitochondria. Overexpression of FIS1 in primary mouse beta cells indicated an upper limit at which higher FIS1 expression reduced glucose-stimulated insulin secretion. Thus, FIS1 was overexpressed stepwise up to a high concentration in RINm5F cells using the RheoSwitch system. Moderate FIS1 expression improved glucose-stimulated insulin secretion, whereas high expression resulted in loss of glucose responsiveness and in mitochondrial artificial loop structures and clustering. Our data confirm that FIS1 is a key regulator in pancreatic beta cells, because both glucose-stimulated insulin secretion and mitochondrial dynamics were clearly adapted to precise expression levels of this fission protein.
Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Linhagem Celular , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , RatosRESUMO
BACKGROUND: Mitochondrial DNA (mtDNA) encodes for the respiratory chain proteins. Genetic alterations in mtDNA have been described during aging and linked to impaired hematopoiesis. MATERIALS AND METHODS: We investigated two novel conplastic mouse strains harboring a mitochondrial nt7778 G/T polymorphism leading to an amino acid exchange in respiratory chain complex V. Effects on reactive oxygen species (ROS) and adenosine triphosphate (ATP) levels, as well as bone marrow composition and peripheral blood counts, were investigated during aging (up to 24 month). RESULTS: The polymorphism correlated with significantly decreased ROS levels in aged mice. Effects on hematopoiesis were marginal and not statistically significant: numbers of erythroid cells in bone marrow, as well as mean corpuscular hemoglobin, tended to decrease over time. CONCLUSION: The investigated polymorphism is associated with decreased ROS levels in aged hematopoietic cells but does not significantly influence hematopoiesis itself.
Assuntos
Senescência Celular/genética , DNA Mitocondrial/genética , Células-Tronco Hematopoéticas/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Polimorfismo de Nucleotídeo Único , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Envelhecimento/genética , Animais , Células Cultivadas , Células Eritroides/citologia , Células Eritroides/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Hemoglobinas/metabolismo , Camundongos Endogâmicos AKR , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Especificidade da EspécieRESUMO
During aging, mitochondrial DNA (mtDNA) can accumulate mutations leading to increasing levels of reactive oxygen species (ROS). Increased ROS were described to activate formerly quiescent hematopoietic stem cells (HSC). Mutations in mtDNA were shown to enhance the risk for myelodysplastic syndrome and leukemia. However, the complex relationship between mtDNA variations, ROS and aging of the hematopoietic system is not fully understood.Herein, three mouse strains with mtDNA polymorphisms in genes of respiratory chain complexes I (ND4), III (CYTB) and IV (COX3) were compared to a reference strain during aging. Analysis focused on ROS and ATP levels, bone marrow composition and blood counts. Additionally, hematopoietic restoration capacity following cytotoxic stress was tested.Mice with polymorphisms in ND4 and CYTB gene had significantly decreasing ROS levels in bone marrow cells during aging, without effecting ATP levels. In addition, the frequency of stem and progenitor cells increased during aging but the amount of lymphocytes in the peripheral blood decreased during aging.In summary, the presence of mtDNA polymorphisms affecting the respiratory chain complexes I, III and IV was associated with altered ROS levels as well as changes in BM and peripheral blood composition during aging.
Assuntos
Envelhecimento/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Genes Mitocondriais , Hematopoese/genética , NADH Desidrogenase/genética , Polimorfismo Genético , Prostaglandina-Endoperóxido Sintases/genética , Trifosfato de Adenosina/metabolismo , Fatores Etários , Envelhecimento/metabolismo , Animais , Contagem de Células Sanguíneas , Células da Medula Óssea/metabolismo , Feminino , Técnicas de Inativação de Genes , Camundongos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Insulin-producing cells are known for their extremely low antioxidant equipment with hydrogen peroxide (H(2)O(2))-inactivating enzymes. Therefore, catalase was stably overexpressed in mitochondria and for comparison in the cytoplasmic compartment of insulin-producing RINm5F cells and analyzed for its protective effect against toxicity of reactive oxygen species (ROS) and proinflammatory cytokines. Only mitochondrial overexpression of catalase provided protection against menadione toxicity, a chemical agent that preferentially generates superoxide radicals intramitochondrially. On the other hand, the cytoplasmic catalase overexpression provided better protection against H(2)O(2) toxicity. Mitochondrial catalase overexpression also preferentially protected against the toxicity of interleukin-1beta (IL-1beta) and a proinflammatory cytokine mixture (IL-1beta, tumor necrosis factor-alpha [TNF-alpha], and gamma-interferon [IFN-gamma]) that is more toxic than IL-1beta alone. Thus, it can be concluded that targeted overexpression of catalase in the mitochondria provides particularly effective protection against cell death in all situations in which ROS are generated intramitochondrially. The observed higher rate of cell death after exposure to a cytokine mixture in comparison with the weaker effect of IL-1beta alone may be due to an additive toxicity of TNF-alpha through ROS formation in mitochondria. The results emphasize the central role of mitochondrially generated ROS in the cytokine-mediated cell destruction of insulin-producing cells.
Assuntos
Catalase/genética , Citocinas/toxicidade , Ilhotas Pancreáticas/enzimologia , Mitocôndrias/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Antifibrinolíticos/toxicidade , Antineoplásicos/toxicidade , Catalase/metabolismo , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica , Glucose/metabolismo , Peróxido de Hidrogênio/toxicidade , Insulina/metabolismo , Insulinoma , Interferon gama/toxicidade , Interleucina-1/toxicidade , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/imunologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Oxidantes/toxicidade , Neoplasias Pancreáticas , Regiões Promotoras Genéticas/fisiologia , Ratos , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Vitamina K 3/toxicidadeRESUMO
The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.
Assuntos
Glucoquinase/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/enzimologia , Vesículas Secretórias/enzimologia , Animais , Anticorpos , Linhagem Celular Tumoral , Glucoquinase/imunologia , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/metabolismo , Secreção de Insulina , Insulinoma , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Camundongos , Neoplasias Pancreáticas , Fosfofrutoquinase-2/metabolismoRESUMO
Proinflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor-alpha [TNF-alpha], and gamma-interferon [IFN-gamma]) initiate a variety of signal cascades in pancreatic beta-cells that affect the expression level of genes involved in both the destruction and the protection of the beta-cell. The generation of nitric oxide (NO) via the inducible NO synthase (iNOS) and oxygen free radicals play a key role in cytokine-mediated beta-cell destruction. Within these signal cascades, the activation of the transcription factor nuclear factor-kappaB (NF-kappaB) is crucial, and many cytokine-sensitive genes contain binding sites for this transcription factor in their promoter regions. The aim of this study was to characterize the cytokine-mediated activation of NF-kappaB and the subsequent expression of iNOS protein in insulin-producing RINm5F cells with an improved antioxidant defense status by overexpression of the cytoprotective enzymes catalase (Cat), glutathione peroxidase (Gpx), and the cytoplasmic Cu/Zn superoxide dismutase (Cu/ZnSOD). RINm5F cells with diverse mitochondrial antioxidative defense status were generated by stable overexpression of MnSOD constructs in sense (MnSOD sense) and antisense orientation (MnSOD antisense). Cytokine-induced (IL-1beta or cytokine mix consisting of IL-1beta + TNF-alpha + IFN-gamma) activation of NF-kappaB in RINm5F cells was reduced by >80% through overexpression of MnSOD. The activity of the iNOS promoter remained at basal levels in cytokine-stimulated MnSOD sense cells. In contrast, the suppression of MnSOD gene expression in cytokine-stimulated MnSOD antisense cells resulted in a threefold higher activation of NF-kappaB and a twofold higher activation of the iNOS promoter as compared with control cells. The iNOS protein expression was significantly reduced after a 6- and 8-h cytokine incubation of MnSOD sense cells. The low activity level of MnSOD in RINm5F MnSOD antisense cells increased the iNOS protein expression in particular during the early phase of cytokine-mediated toxicity. Cat, Gpx, and the cytoplasmic Cu/ZnSOD did not affect the activation of NF-kappaB and the iNOS promoter. In conclusion, the overexpression of MnSOD, which inactivates specifically mitochondrially derived oxygen free radicals, significantly reduced the activation of NF-kappaB in insulin-producing cells. As a consequence of this protective effect in the early cytokine signaling pathways, the induction of iNOS, an important event in the beta-cell destruction process, was also significantly reduced. The results provide evidence that mitochondrially derived reactive oxygen species (ROS) play a critical role in the activation of the cytokine-sensitive transcription factor NF-kappaB. Overexpression of MnSOD may thus be beneficial for beta-cell survival through suppression of oxygen free radical formation, prevention of NF-kappaB activation, and iNOS expression.
Assuntos
Citocinas/fisiologia , Insulina/biossíntese , Insulinoma/metabolismo , Mitocôndrias/enzimologia , NF-kappa B/fisiologia , Oxirredutases/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Catalase/metabolismo , Citocinas/farmacologia , Combinação de Medicamentos , Glutationa Peroxidase/metabolismo , Interleucina-1/farmacologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Regiões Promotoras Genéticas/fisiologia , Superóxido Dismutase/metabolismo , Células Tumorais CultivadasRESUMO
The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was recently identified as a new intracellular binding partner for glucokinase (GK). Therefore, we studied the importance of this interaction for the activity status of GK and glucose metabolism in insulin-producing cells by overexpression of the rat liver and pancreatic islet isoforms of PFK-2/FBPase-2. PFK-2/FBPase-2 overexpression in RINm5F-GK cells significantly increased the GK activity by 78% in cells expressing the islet isoform, by 130% in cells expressing the liver isoform, and by 116% in cells expressing a cAMP-insensitive liver S32A/H258A double mutant isoform. Only in cells overexpressing the wild-type liver PFK-2/FBPase-2 isoform was the increase of GK activity abolished by forskolin, apparently due to the regulatory site for phosphorylation by a cAMP-dependent protein kinase. In cells overexpressing any isoform of the PFK-2/FBPase-2, the increase of the GK enzyme activity was antagonized by treatment with anti-FBPase-2 antibody. Increasing the glucose concentration from 2 to 10 mmol/l had a significant stimulatory effect on the GK activity in RINm5F-GK cells overexpressing any isoform of PFK-2/FBPase-2. The interaction of GK with PFK-2/FBPase-2 takes place at glucose concentrations that are physiologically relevant for the activation of GK and the regulation of glucose-induced insulin secretion. This new mechanism of posttranslational GK regulation may also represent a new site for pharmacotherapeutic intervention in type 2 diabetes treatment.
Assuntos
Glucoquinase/metabolismo , Glucose/metabolismo , Ilhotas Pancreáticas/metabolismo , Fosfofrutoquinase-2/genética , Adenoviridae , Animais , Linhagem Celular , Clonagem Molecular , Colforsina/farmacologia , Vetores Genéticos , Glucoquinase/efeitos dos fármacos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Fígado/enzimologia , Fosfofrutoquinase-2/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The importance of the cellular immune system for the development of T1DM in the LEW.1AR1-iddm rat was investigated by use of polyinosinic-polycytidylic acid (Poly I:C) and by adoptive transfer of concanavalin A (Con A) activated lymphocytes from diabetic LEW.1AR1-iddm rats and the coisogenic LEW.AR1 background strain. Poly I:C treatment induced diabetes, characterized morphologically by a diffuse infiltration of the pancreas, in up to 20% of the animals of the coisogenic LEW.1AR1 background strain. It did not increase the diabetes incidence of 30% of the LEW.1AR1-iddm strain. In contrast Poly I:C treatment induced diabetes in up to 80% of the animals of the Mhc congenic LEW.1WR1 strain. Adoptive transfer of lymphocytes activated by the T-cell mitogen Con A from diabetic donors doubled the incidence of diabetes, characterized morphologically by a focal insulitis, in diabetes prone LEW.1AR1-iddm recipients. In contrast, animals of the LEW.1AR1 background strain did not develop diabetes after adoptive transfer. Moreover, adoptive transfer of Con A activated lymphocytes from LEW.1AR1 rats to LEW.1AR1-iddm rats with 30 or 60% diabetes incidence, significantly decreased the incidence of diabetes in LEW.1AR1-iddm rats with 60% diabetes incidence. The results show that autoreactive lymphocytes induce beta cell destruction in the LEW.1AR1-iddm rat, while the LEW.AR1 background strain apparently contains regulatory potential, which is able to counteract the autoimmune response.
Assuntos
Diabetes Mellitus Experimental/imunologia , Pâncreas/imunologia , Linfócitos T/imunologia , Transferência Adotiva/economia , Animais , Apoptose/imunologia , Glicemia/imunologia , Concanavalina A/farmacologia , Diabetes Mellitus Experimental/patologia , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Ativação Linfocitária/imunologia , Masculino , Pâncreas/patologia , Poli I-C , Ratos , Ratos Endogâmicos Lew , Ratos Nus , Organismos Livres de Patógenos EspecíficosRESUMO
Metabolic disorders, like diabetes and obesity, are pathogenic outcomes of imbalance in glucose metabolism. Nutrient excess and mitochondrial imbalance are implicated in dysfunctional glucose metabolism with age. We used conplastic mouse strains with defined mitochondrial DNA (mtDNA) mutations on a common nuclear genomic background, and administered a high-fat diet up to 18 months of age. The conplastic mouse strain B6-mtFVB, with a mutation in the mt-Atp8 gene, conferred ß-cell dysfunction and impaired glucose tolerance after high-fat diet. To our surprise, despite of this functional deficit, blood glucose levels adapted to perturbations with age. Blood glucose levels were particularly sensitive to perturbations at the early age of 3 to 6 months. Overall the dynamics consisted of a peak between 3-6 months followed by adaptation by 12 months of age. With the help of mathematical modeling we delineate how body weight, insulin and leptin regulate this non-linear blood glucose dynamics. The model predicted a second rise in glucose between 15 and 21 months, which could be experimentally confirmed as a secondary peak. We therefore hypothesize that these two peaks correspond to two sensitive periods of life, where perturbations to the basal metabolism can mark the system for vulnerability to pathologies at later age. Further mathematical modeling may perspectively allow the design of targeted periods for therapeutic interventions and could predict effects on weight loss and insulin levels under conditions of pre-diabetic obesity.