RESUMO
The present work delves into the enigmatic world of mitochondrial alpha-keto acid dehydrogenase complexes discussing their metabolic significance, enzymatic operation, moonlighting activities, and pathological relevance with links to underlying structural features. This ubiquitous family of related but diverse multienzyme complexes is involved in carbohydrate metabolism (pyruvate dehydrogenase complex), the citric acid cycle (α-ketoglutarate dehydrogenase complex), and amino acid catabolism (branched-chain α-keto acid dehydrogenase complex, α-ketoadipate dehydrogenase complex); the complexes all function at strategic points and also participate in regulation in these metabolic pathways. These systems are among the largest multienzyme complexes with at times more than 100 protein chains and weights ranging up to ~10 million Daltons. Our chapter offers a wealth of up-to-date information on these multienzyme complexes for a comprehensive understanding of their significance in health and disease.
Assuntos
Mitocôndrias , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/enzimologia , Animais , Ciclo do Ácido Cítrico/fisiologia , Complexo Cetoglutarato Desidrogenase/metabolismo , Complexo Cetoglutarato Desidrogenase/químicaRESUMO
KEY POINTS: â¢Bile acids, ethanol and fatty acids affect pancreatic ductal fluid and bicarbonate secretion via mitochondrial damage, ATP depletion and calcium overload. â¢Pancreatitis-inducing factors open the membrane transition pore (mPTP) channel via cyclophilin D activation in acinar cells, causing calcium overload and cell death; genetic or pharmacological inhibition of mPTP improves the outcome of acute pancreatitis in animal models. â¢Here we show that genetic and pharmacological inhibition of mPTP protects mitochondrial homeostasis and cell function evoked by pancreatitis-inducing factors in pancreatic ductal cells. â¢The results also show that the novel cyclosporin A derivative NIM811 protects mitochondrial function in acinar and ductal cells, and it preserves bicarbonate transport mechanisms in pancreatic ductal cells. â¢We found that NIM811 is highly effective in different experimental pancreatitis models and has no side-effects. NIM811 is a highly suitable compound to be tested in clinical trials. ABSTRACT: Mitochondrial dysfunction plays a crucial role in the development of acute pancreatitis (AP); however, no compound is currently available with clinically acceptable effectiveness and safety. In this study, we investigated the effects of a novel mitochondrial transition pore inhibitor, N-methyl-4-isoleucine cyclosporin (NIM811), in AP. Pancreatic ductal and acinar cells were isolated by enzymatic digestion from Bl/6 mice. In vitro measurements were performed by confocal microscopy and microfluorometry. Preventative effects of pharmacological [cylosporin A (2 µm), NIM811 (2 µm)] or genetic (Ppif-/- /Cyp D KO) inhibition of the mitochondrial transition pore (mPTP) during the administration of either bile acids (BA) or ethanol + fatty acids (EtOH+FA) were examined. Toxicity of mPTP inhibition was investigated by detecting apoptosis and necrosis. In vivo effects of the most promising compound, NIM811 (5 or 10 mg kg-1 per os), were checked in three different AP models induced by either caerulein (10 × 50 µg kg-1 ), EtOH+FA (1.75 g kg-1 ethanol and 750 mg kg-1 palmitic acid) or 4% taurocholic acid (2 ml kg-1 ). Both genetic and pharmacological inhibition of Cyp D significantly prevented the toxic effects of BA and EtOH+FA by restoring mitochondrial membrane potential (Δψ) and preventing the loss of mitochondrial mass. In vivo experiments revealed that per os administration of NIM811 has a protective effect in AP by reducing oedema, necrosis, leukocyte infiltration and serum amylase level in AP models. Administration of NIM811 had no toxic effects. The novel mitochondrial transition pore inhibitor NIM811 thus seems to be an exceptionally good candidate compound for clinical trials in AP.
Assuntos
Ciclosporina/uso terapêutico , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Pancreatite/tratamento farmacológico , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Animais , Apoptose , Bicarbonatos/metabolismo , Células Cultivadas , Ciclosporina/efeitos adversos , Ciclosporina/farmacologia , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Ductos Pancreáticos/efeitos dos fármacos , Ductos Pancreáticos/metabolismoRESUMO
Vinpocetine is considered as neuroprotectant drug and used for treatment of brain ischemia and cognitive deficiencies for decades. A number of enzymes, channels and receptors can bind vinpocetine, however the mechanisms of many effects' are still not clear. The present study investigated the effects of vinpocetine from the mitochondrial bioenergetic aspects. In primary brain capillary endothelial cells the purinergic receptor-stimulated mitochondrial Ca2+ uptake and efflux were studied. Vinpocetine exerted a partial inhibition on the mitochondrial calcium efflux. In rodent brain synaptosomes vinpocetine (30 µM) inhibited respiration in uncoupler stimulated synaptosomes and decreased H2O2 release from the nerve terminals in resting and in complex I inhibited conditions, respectively. In isolated rat brain mitochondria using either complex I or complex II substrates leak respiration was stimulated, but ADP-induced respiration was inhibited by vinpocetine. The stimulation of oxidation was associated with a small extent of membrane depolarization. Mitochondrial H2O2 production was inhibited by vinpocetine under all conditions investigated. The most pronounced effects were detected with the complex II substrate succinate. Vinpocetine also mitigated both Ca2+-induced mitochondrial Ca2+-release and Ca2+-induced mitochondrial swelling. It lowered the rate of mitochondrial ATP synthesis, while increasing ATPase activity. These results indicate more than a single mitochondrial target of this vinca alkaloid. The relevance of the affected mitochondrial mechanisms in the anti ischemic effect of vinpocetine is discussed.
Assuntos
Encéfalo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacos , Alcaloides de Vinca/farmacologia , Animais , Encéfalo/metabolismo , Cálcio/metabolismo , Células Endoteliais/metabolismo , Peróxido de Hidrogênio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Ratos Wistar , Sinaptossomos/metabolismoRESUMO
Microglia are highly dynamic cells in the brain. Their functional diversity and phenotypic versatility brought microglial energy metabolism into the focus of research. Although it is known that microenvironmental cues shape microglial phenotype, their bioenergetic response to local nutrient availability remains unclear. In the present study effects of energy substrates on the oxidative and glycolytic metabolism of primary - and BV-2 microglial cells were investigated. Cellular oxygen consumption, glycolytic activity, the levels of intracellular ATP/ADP, autophagy, mTOR phosphorylation, apoptosis and cell viability were measured in the absence of nutrients or in the presence of physiological energy substrates: glutamine, glucose, lactate, pyruvate or ketone bodies. All of the oxidative energy metabolites increased the rate of basal and maximal respiration. However, the addition of glucose decreased microglial oxidative metabolism and glycolytic activity was enhanced. Increased ATP/ADP ratio and cell viability, activation of the mTOR and reduction of autophagic activity were observed in glutamine-supplemented media. Moreover, moderate and transient oxidation of ketone bodies was highly enhanced by glutamine, suggesting that anaplerosis of the TCA-cycle could stimulate ketone body oxidation. It is concluded that microglia show high metabolic plasticity and utilize a wide range of substrates. Among them glutamine is the most efficient metabolite. To our knowledge these data provide the first account of microglial direct metabolic response to nutrients under short-term starvation and demonstrate that microglia exhibit versatile metabolic machinery. Our finding that microglia have a distinct bioenergetic profile provides a critical foundation for specifying microglial contributions to brain energy metabolism.
Assuntos
Metabolismo Energético/fisiologia , Glucose/metabolismo , Glutamina/metabolismo , Lactatos/metabolismo , Microglia/metabolismo , Piruvatos/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Metabolismo Energético/efeitos dos fármacos , Feminino , Glucose/farmacologia , Glutamina/farmacologia , Glicólise/efeitos dos fármacos , Lactatos/farmacologia , Masculino , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Piruvatos/farmacologiaRESUMO
Succinate-driven reverse electron transport (RET) is one of the main sources of mitochondrial reactive oxygen species (mtROS) in ischemia-reperfusion injury. RET is dependent on mitochondrial membrane potential (Δψm) and transmembrane pH difference (ΔpH), components of the proton motive force (pmf); a decrease in Δψm and/or ΔpH inhibits RET. In this study we aimed to determine which component of the pmf displays the more dominant effect on RET-provoked ROS generation in isolated guinea pig brain and heart mitochondria respiring on succinate or α-glycerophosphate (α-GP). Δψm was detected via safranin fluorescence and a TPP+ electrode, the rate of H2O2 formation was measured by Amplex UltraRed, the intramitochondrial pH (pHin) was assessed via BCECF fluorescence. Ionophores were used to dissect the effects of the two components of pmf. The K+/H+ exchanger, nigericin lowered pHin and ΔpH, followed by a compensatory increase in Δψm that led to an augmented H2O2 production. Valinomycin, a K+ ionophore, at low [K+] increased ΔpH and pHin, decreased Δψm, which resulted in a decline in H2O2 formation. It was concluded that Δψm is dominant over ∆pH in modulating the succinate- and α-GP-evoked RET. The elevation of extramitochondrial pH was accompanied by an enhanced H2O2 release and a decreased ∆pH. This phenomenon reveals that from the pH component not ∆pH, but rather absolute value of pH has higher impact on the rate of mtROS formation. Minor decrease of Δψm might be applied as a therapeutic strategy to attenuate RET-driven ROS generation in ischemia-reperfusion injury.
Assuntos
Encéfalo/imunologia , Transporte de Elétrons/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Mitocôndrias Cardíacas/imunologia , Animais , Cobaias , Humanos , Potencial da Membrana MitocondrialRESUMO
Succinate is an important metabolite at the cross-road of several metabolic pathways, also involved in the formation and elimination of reactive oxygen species. However, it is becoming increasingly apparent that its realm extends to epigenetics, tumorigenesis, signal transduction, endo- and paracrine modulation and inflammation. Here we review the pathways encompassing succinate as a metabolite or a signal and how these may interact in normal and pathological conditions.(1).
Assuntos
Carcinogênese/metabolismo , Regulação Neoplásica da Expressão Gênica , Hipóxia/metabolismo , Neoplasias/metabolismo , Succinato Desidrogenase/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Humanos , Hipóxia/genética , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Redes e Vias Metabólicas/genética , Mutação , Neoplasias/genética , Neoplasias/patologia , Comunicação Parácrina/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Succinato Desidrogenase/genética , Ácido Succínico/metabolismoRESUMO
Itaconate is a nonamino organic acid exhibiting antimicrobial effects. It has been recently identified in cells of macrophage lineage as a product of an enzyme encoded by immunoresponsive gene 1 (Irg1), acting on the citric acid cycle intermediate cis-aconitate. In mitochondria, itaconate can be converted by succinate-coenzyme A (CoA) ligase to itaconyl-CoA at the expense of ATP (or GTP), and is also a weak competitive inhibitor of complex II. Here, we investigated specific bioenergetic effects of increased itaconate production mediated by LPS-induced stimulation of Irg1 in murine bone marrow-derived macrophages (BMDM) and RAW-264.7 cells. In rotenone-treated macrophage cells, stimulation by LPS led to impairment in substrate-level phosphorylation (SLP) of in situ mitochondria, deduced by a reversal in the directionality of the adenine nucleotide translocase operation. In RAW-264.7 cells, the LPS-induced impairment in SLP was reversed by short-interfering RNA(siRNA)-but not scrambled siRNA-treatment directed against Irg1. LPS dose-dependently inhibited oxygen consumption rates (61-91%) and elevated glycolysis rates (>21%) in BMDM but not RAW-264.7 cells, studied under various metabolic conditions. In isolated mouse liver mitochondria treated with rotenone, itaconate dose-dependently (0.5-2 mM) reversed the operation of adenine nucleotide translocase, implying impairment in SLP, an effect that was partially mimicked by malonate. However, malonate yielded greater ADP-induced depolarizations (3-19%) than itaconate. We postulate that itaconate abolishes SLP due to 1) a "CoA trap" in the form of itaconyl-CoA that negatively affects the upstream supply of succinyl-CoA from the α-ketoglutarate dehydrogenase complex; 2) depletion of ATP (or GTP), which are required for the thioesterification by succinate-CoA ligase; and 3) inhibition of complex II leading to a buildup of succinate which shifts succinate-CoA ligase equilibrium toward ATP (or GTP) utilization. Our results support the notion that Irg1-expressing cells of macrophage lineage lose the capacity of mitochondrial SLP for producing itaconate during mounting of an immune defense.
Assuntos
Hidroliases/metabolismo , Macrófagos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Succinatos/farmacologia , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Feminino , Glicólise , Hidroliases/genética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Malonatos/farmacologia , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/metabolismo , Fosforilação Oxidativa , Rotenona/farmacologia , Succinato-CoA Ligases/metabolismoRESUMO
Sepsis caused by LPS is characterized by an intense systemic inflammatory response affecting the lungs, causing acute lung injury (ALI). Dysfunction of mitochondria and the role of reactive oxygen (ROS) and nitrogen species produced by mitochondria have already been proposed in the pathogenesis of sepsis; however, the exact molecular mechanism is poorly understood. Oxidative stress induces cyclophilin D (CypD)-dependent mitochondrial permeability transition (mPT), leading to organ failure in sepsis. In previous studies mPT was inhibited by cyclosporine A which, beside CypD, inhibits cyclophilin A, B, C and calcineurin, regulating cell death and inflammatory pathways. The immunomodulatory side effects of cyclosporine A make it unfavorable in inflammatory model systems. To avoid these uncertainties in the molecular mechanism, we studied endotoxemia-induced ALI in CypD(-/-) mice providing unambiguous data for the pathological role of CypD-dependent mPT in ALI. Our key finding is that the loss of this essential protein improves survival rate and it can intensely ameliorate endotoxin-induced lung injury through attenuated proinflammatory cytokine release, down-regulation of redox sensitive cellular pathways such as MAPKs, Akt, and NF-κB and reducing the production of ROS. Functional inhibition of NF-κB was confirmed by decreased expression of NF-κB-mediated proinflammatory genes. We demonstrated that impaired mPT due to the lack of CypD reduces the severity of endotoxemia-induced lung injury suggesting that CypD specific inhibitors might have a great therapeutic potential in sepsis-induced organ failure. Our data highlight a previously unknown regulatory function of mitochondria during inflammatory response.
RESUMO
Although incidence and prevalence of prediabetes are increasing, little is known about its cardiac effects. Therefore, our aim was to investigate the effect of prediabetes on cardiac function and to characterize parameters and pathways associated with deteriorated cardiac performance. Long-Evans rats were fed with either control or high-fat chow for 21 wk and treated with a single low dose (20 mg/kg) of streptozotocin at week 4 High-fat and streptozotocin treatment induced prediabetes as characterized by slightly elevated fasting blood glucose, impaired glucose and insulin tolerance, increased visceral adipose tissue and plasma leptin levels, as well as sensory neuropathy. In prediabetic animals, a mild diastolic dysfunction was observed, the number of myocardial lipid droplets increased, and left ventricular mass and wall thickness were elevated; however, no molecular sign of fibrosis or cardiac hypertrophy was shown. In prediabetes, production of reactive oxygen species was elevated in subsarcolemmal mitochondria. Expression of mitofusin-2 was increased, while the phosphorylation of phospholamban and expression of Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3, a marker of mitophagy) decreased. However, expression of other markers of cardiac auto- and mitophagy, mitochondrial dynamics, inflammation, heat shock proteins, Ca2+/calmodulin-dependent protein kinase II, mammalian target of rapamycin, or apoptotic pathways were unchanged in prediabetes. This is the first comprehensive analysis of cardiac effects of prediabetes indicating that mild diastolic dysfunction and cardiac hypertrophy are multifactorial phenomena that are associated with early changes in mitophagy, cardiac lipid accumulation, and elevated oxidative stress and that prediabetes-induced oxidative stress originates from the subsarcolemmal mitochondria.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Mitocôndrias Cardíacas/metabolismo , Estresse Oxidativo , Estado Pré-Diabético/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Adipocinas/metabolismo , Tecido Adiposo , Animais , Apoptose , Autofagia , Composição Corporal , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Diabetes Mellitus Experimental/fisiopatologia , Neuropatias Diabéticas , Diástole , Dieta Hiperlipídica , Ecocardiografia , GTP Fosfo-Hidrolases , Proteínas de Choque Térmico/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Mitocôndrias Cardíacas/ultraestrutura , Proteínas Mitocondriais/metabolismo , Mitofagia , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Fosforilação , Estado Pré-Diabético/fisiopatologia , Ratos , Ratos Long-Evans , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sarcolema , Serina-Treonina Quinases TOR/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia , Pressão VentricularRESUMO
Herein are reported unique properties of the human 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc), a rate-limiting enzyme in the Krebs (citric acid) cycle. (a) Functionally competent 2-oxoglutarate dehydrogenase (E1o-h) and dihydrolipoyl succinyltransferase components have been expressed according to kinetic and spectroscopic evidence. (b) A stable free radical, consistent with the C2-(C2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (ThDP) cation radical was detected by electron spin resonance upon reaction of the E1o-h with 2-oxoglutarate (OG) by itself or when assembled from individual components into OGDHc. (c) An unusual stability of the E1o-h-bound C2-(2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (the "ThDP-enamine"/C2α-carbanion, the first postdecarboxylation intermediate) was observed, probably stabilized by the 5-carboxyl group of OG, not reported before. (d) The reaction of OG with the E1o-h gave rise to superoxide anion and hydrogen peroxide (reactive oxygen species (ROS)). (e) The relatively stable enzyme-bound enamine is the likely substrate for oxidation by O2, leading to the superoxide anion radical (in d) and the radical (in b). (f) The specific activity assessed for ROS formation compared with the NADH (overall complex) activity, as well as the fraction of radical intermediate occupying active centers of E1o-h are consistent with each other and indicate that radical/ROS formation is an "off-pathway" side reaction comprising less than 1% of the "on-pathway" reactivity. However, the nearly ubiquitous presence of OGDHc in human tissues, including the brain, makes these findings of considerable importance in human metabolism and perhaps disease.
Assuntos
Aminas/metabolismo , Radicais Livres/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Tiamina Pirofosfato/metabolismo , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Ácidos Cetoglutáricos , Cinética , Organofosfonatos/metabolismo , Oxirredução , Ácidos Fosfínicos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Análise Espectral , TiaminaRESUMO
According to recent results, various mitochondrial processes can actively regulate the immune response. In the present report, we studied whether mitochondrial permeability transition (mPT) has such a role. To this end, we compared bacterial lipopolysaccharide (LPS)-induced inflammatory response in cyclophilin D (CypD) knock-out and wild-type mouse resident peritoneal macrophages. CypD is a regulator of mPT; therefore, mPT is damaged in CypD(-/-) cells. We chose this genetic modification-based model because the mPT inhibitor cyclosporine A regulates inflammatory processes by several pathways unrelated to the mitochondria. The LPS increased mitochondrial depolarisation, cellular and mitochondrial reactive oxygen species production, nuclear factor-κB activation, and nitrite- and tumour necrosis factor α accumulation in wild-type cells, but these changes were diminished or absent in the CypD-deficient macrophages. Additionally, LPS enhanced Akt phosphorylation/activation as well as FOXO1 and FOXO3a phosphorylation/inactivation both in wild-type and CypD(-/-) cells. However, Akt and FOXO phosphorylation was significantly more pronounced in CypD-deficient compared to wild-type macrophages. These results provide the first pieces of experimental evidence for the functional regulatory role of mPT in the LPS-induced early inflammatory response of macrophages.
Assuntos
Ciclofilinas/metabolismo , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Animais , Células Cultivadas , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Macrófagos Peritoneais/fisiologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The Warburg effect occurs both in cancer cells and in inflammatory macrophages. The aim of our work was to demonstrate the role of PI3K-Akt-mTOR axis in the Warburg effect in HL-60 derived, rat peritoneal and human blood macrophages and to investigate the potential of selected inhibitors of this pathway to antagonize it. M1 polarization in HL-60-derived and human blood monocyte-derived macrophages was supported by the increased expression of NOS2 and inflammatory cytokines. All M1 polarized and inflammatory macrophages investigated expressed higher levels of HIF-1α and NOS2, which were reduced by selected kinase inhibitors, supporting the role of PI3K-Akt-mTOR axis. Using Seahorse XF plates, we found that in HL-60-derived and human blood-derived macrophages, glucose loading reduced oxygen consumption (OCR) and increased glycolysis (ECAR) in M1 polarization, which was antagonized by selected kinase inhibitors and by dichloroacetate. In rat peritoneal macrophages, the changes in oxidative and glycolytic metabolism were less marked and the NOS2 inhibitor decreased OCR and increased ECAR. Non-mitochondrial oxygen consumption and ROS production were likely due to NADPH oxidase, expressed in each macrophage type, independently of PI3K-Akt-mTOR axis. Our results suggest that inflammation changed the metabolism in each macrophage model, but a clear relationship between polarization and Warburg effect was confirmed only after glucose loading in HL-60 and human blood derived macrophages. The effect of kinase inhibitors on Warburg effect was variable in different cell types, whereas dichloroacetate caused a shift toward oxidative metabolism. Our findings suggest that these originally anti-cancer inhibitors may also be candidates for anti-inflammatory therapy.
RESUMO
We investigated pathogenic mutations relevant in dihydrolipoamide dehydrogenase (LADH; gene: Dld) deficiency, a severe human disease, to elucidate how they alter reactive oxygen species (ROS) generation and associated biophysical characteristics of LADH. Twelve known disease-causing mutants of human LADH have been expressed and purified to homogeneity from E. coli. Detailed biophysical and biochemical characterization of the mutants has been performed applying circular dichroism (CD) spectroscopy, nano-spray mass spectrometry (MS), calibrated gel filtration and flavin adenine dinucleotide-content analysis. Functional analyses revealed that four of the pathogenic mutations significantly stimulated the ROS-generating activity of LADH and also increased its sensitivity to an acidic shift in pH. LADH activity was reduced by variable extents in the mutants exhibiting excessive ROS generation. It is remarkable that in the P453L mutant, enzyme activity was nearly completely lost with a ROS-forming activity becoming dominant, whereas the G194C mutation, common among Ashkenazi Jews, resulted in no alteration in LADH activity but a gain in the ROS-generating activity. There have been neither major conformational alterations nor monomerization of the functional homodimer of LADH associated with the higher ROS-generating capacity as measured by CD spectroscopy and size-exclusion chromatography combined with nano-spray MS, respectively. The excessive ROS generation of selected LADH mutants could be an important factor in the pathology and clinical presentation of human LADH deficiency and raises the possibility of an antioxidant therapy in the treatment of this condition.
Assuntos
Di-Hidrolipoamida Desidrogenase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Cromatografia em Gel , Dicroísmo Circular , Di-Hidrolipoamida Desidrogenase/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Espectrometria de MassasRESUMO
Brain injury is accompanied by neuroinflammation, accumulation of extracellular glutamate and mitochondrial dysfunction, all of which cause neuronal death. The aim of this study was to investigate the impact of these mechanisms on neuronal death. Patients from the neurosurgical intensive care unit suffering aneurysmal subarachnoid hemorrhage (SAH) were recruited retrospectively from a respective database. In vitro experiments were performed in rat cortex homogenate, primary dissociated neuronal cultures, B35 and NG108-15 cell lines. We employed methods including high resolution respirometry, electron spin resonance, fluorescent microscopy, kinetic determination of enzymatic activities and immunocytochemistry. We found that elevated levels of extracellular glutamate and nitric oxide (NO) metabolites correlated with poor clinical outcome in patients with SAH. In experiments using neuronal cultures we showed that the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme of the glutamate-dependent segment of the tricarboxylic acid (TCA) cycle, is more susceptible to the inhibition by NO than mitochondrial respiration. Inhibition of OGDHC by NO or by succinyl phosphonate (SP), a highly specific OGDHC inhibitor, caused accumulation of extracellular glutamate and neuronal death. Extracellular nitrite did not substantially contribute to this NO action. Reactivation of OGDHC by its cofactor thiamine (TH) reduced extracellular glutamate levels, Ca2+ influx into neurons and cell death rate. Salutary effect of TH against glutamate toxicity was confirmed in three different cell lines. Our data suggest that the loss of control over extracellular glutamate, as described here, rather than commonly assumed impaired energy metabolism, is the critical pathological manifestation of insufficient OGDHC activity, leading to neuronal death.
Assuntos
Ácido Glutâmico , Complexo Cetoglutarato Desidrogenase , Ratos , Animais , Ácido Glutâmico/metabolismo , Estudos Retrospectivos , Citoplasma/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Mitocôndrias/metabolismo , Tiamina/metabolismo , Tiamina/farmacologia , Óxido Nítrico/metabolismoRESUMO
AIM: Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) is a modification of two-stage hepatectomy profitable for patients with inoperable hepatic tumors by standard techniques. Unfortunately, initially poor postoperative outcome was associated with ALPPS, in which mitochondrial dysfunction played an essential role. Inhibition of cyclophilins has been already proposed to be efficient as a mitochondrial therapy in liver diseases. To investigate the effect of Cyclophilin D (CypD) depletion on mitochondrial function, biogenesis and liver regeneration following ALPPS a CypD knockout (KO) mice model was created. METHODS: Male wild type (WT) (n = 30) and CypD KO (n = 30) mice underwent ALPPS procedure. Animals were terminated pre-operatively and 24, 48, 72 or 168 h after the operation. Mitochondrial functional studies and proteomic analysis were performed. Regeneration rate and mitotic activity were assessed. RESULTS: The CypD KO group displayed improved mitochondrial function, as both ATP production (P < 0.001) and oxygen consumption (P < 0.05) were increased compared to the WT group. The level of mitochondrial biogenesis coordinator peroxisome proliferator-activated receptor γ co-activator 1-α (PGC1-α) was also elevated in the CypD KO group (P < 0.001), which resulted in the induction of the mitochondrial oxidative phosphorylation system. Liver growth increased in the CypD KO group compared to the WT group (P < 0.001). CONCLUSIONS: Our study demonstrates the beneficial effect of CypD depletion on the mitochondrial vulnerability following ALPPS. Based on our results we propose that CypD inhibition should be further investigated as a possible mitochondrial therapy following ALPPS.
Assuntos
Hepatectomia , Neoplasias Hepáticas , Regeneração Hepática , Mitocôndrias Hepáticas , Peptidil-Prolil Isomerase F , Animais , Peptidil-Prolil Isomerase F/genética , Ciclofilinas/genética , Hepatectomia/métodos , Ligadura/métodos , Fígado/patologia , Fígado/cirurgia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Regeneração Hepática/genética , Regeneração Hepática/fisiologia , Masculino , Camundongos , Mitocôndrias Hepáticas/genética , Mitocôndrias Hepáticas/metabolismo , Veia Porta/cirurgia , ProteômicaRESUMO
α-ketoglutarate dehydrogenase complex (KGDHc), or 2-oxoglutarate dehydrogenase complex (OGDHc) is a rate-limiting enzyme in the tricarboxylic acid cycle, that has been identified in neurodegenerative diseases such as in Alzheimer's disease. The aim of the present study was to establish the role of the KGDHc and its subunits in the bioenergetics and reactive oxygen species (ROS) homeostasis of brain mitochondria. To study the bioenergetic profile of KGDHc, genetically modified mouse strains were used having a heterozygous knock out (KO) either in the dihydrolipoyl succinyltransferase (DLST+/-) or in the dihydrolipoyl dehydrogenase (DLD+/-) subunit. Mitochondrial oxygen consumption, hydrogen peroxide (H2O2) production, and expression of antioxidant enzymes were measured in isolated mouse brain mitochondria. Here, we demonstrate that the ADP-stimulated respiration of mitochondria was partially arrested in the transgenic animals when utilizing α-ketoglutarate (α-KG or 2-OG) as a fuel substrate. Succinate and α-glycerophosphate (α-GP), however, did not show this effect. The H2O2 production in mitochondria energized with α-KG was decreased after inhibiting the adenine nucleotide translocase and Complex I (CI) in the transgenic strains compared to the controls. Similarly, the reverse electron transfer (RET)-evoked H2O2 formation supported by succinate or α-GP were inhibited in mitochondria isolated from the transgenic animals. The decrease of RET-evoked ROS production by DLST+/- or DLD+/- KO-s puts the emphasis of the KGDHc in the pathomechanism of ischemia-reperfusion evoked oxidative stress. Supporting this notion, expression of the antioxidant enzyme glutathione peroxidase was also decreased in the KGDHc transgenic animals suggesting the attenuation of ROS-producing characteristics of KGDHc. These findings confirm the contribution of the KGDHc to the mitochondrial ROS production and in the pathomechanism of ischemia-reperfusion injury.
RESUMO
The effect of Ca2+ applied in high concentrations (50 and 300 microM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H2O2 release from mitochondria was induced by Ca2+ applied in 50 microM, but not in 300 microM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150 sec after exposure to Ca2+. Parallel measurements of membrane potential (DeltaPsim) by safranine fluorescence showed a transient depolarization by Ca2+ followed by the recovery of DeltaPsim to a value, which was more negative than that observed before addition of Ca2+ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca2+ given in 50 microM concentration. In mitochondria having high DeltaPsim in the presence of oligomycin or ATP, the basal rate of release of H2O2 was significantly higher than that observed in a medium containing ADP and Ca2+ no longer increased but rather decreased the rate of H2O2 release. With 300 microM Ca2+ only a loss but no tendency of a recovery of DeltaPsim was detected and H2O2 release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca2+ on mitochondrial ROS release is related to changes in DeltaPsim; in depolarized mitochondria, in the presence of ADP, moderate increase in H2O2 release is induced by calcium, but only in Assuntos
Encéfalo/metabolismo
, Cálcio/farmacologia
, Mitocôndrias/efeitos dos fármacos
, Mitocôndrias/metabolismo
, Espécies Reativas de Oxigênio/metabolismo
, Difosfato de Adenosina/farmacologia
, Animais
, Cobaias
, Peróxido de Hidrogênio/metabolismo
, Técnicas In Vitro
, Potencial da Membrana Mitocondrial/efeitos dos fármacos
, Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores
, Poro de Transição de Permeabilidade Mitocondrial
, Dilatação Mitocondrial/efeitos dos fármacos
, NAD/metabolismo
, NADP/metabolismo
, Oligomicinas/farmacologia
RESUMO
In the present work, the Ca(2+) dependence of mitochondrial H(2) O(2) elimination was investigated. Mitochondria isolated from guinea pig brain were energized by glutamate and malate and incubated with micromolar concentrations of Ca(2+) in the presence of ADP, preventing permeability transition pore formation. After the completion of Ca(2+) uptake, mitochondria were challenged with H(2) O(2) (5 µM), then at various time points residual H(2) O(2) was determined using the Amplex red method and compared with that in mitochondria incubated with H(2) O(2) without Ca(2+) addition. Dose-dependent inhibition of H(2) O(2) elimination by Ca(2+) was detected, which was prevented by the Ca(2+) -uptake inhibitor Ru 360. Stimulation of Ca(2+) release from Ca(2+) -loaded mitochondria by a combined addition of Ru 360 and Na(+) decreased the Ca(2+) -evoked inhibition of H(2) O(2) removal. After Ca(2+) uptake (50 µM), mitochondrial aconitase activity was found to be decreased, which was partially attributable to the impaired elimination of endogenously produced reactive oxygen species. We found that the effects of Ca(2+) and H(2) O(2) on the activity of aconitase were additive. These results confirm that Ca(2+) inhibits elimination of H(2) O(2) in mitochondria and demonstrate that this effect is concentration dependent and reversible. The phenomenon described here can play a role in the modulation of ROS handling under conditions involving excessive cellular Ca(2+) load.
Assuntos
Encéfalo/metabolismo , Cálcio/metabolismo , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Cálcio/farmacologia , Cobaias , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/efeitos dos fármacosRESUMO
Methylene blue (MB) is used in human therapy in various pathological conditions. Its effects in neurodegenerative disease models are promising. MB acts on multiple cellular targets and mechanisms, but many of its potential beneficial effects are ascribed to be mitochondrial. According to the "alternative electron transport" hypothesis, MB is capable of donating electrons to cytochrome c bypassing complex I and III. As a consequence of this, the deleterious effects of the inhibitors of complex I and III can be ameliorated by MB. Recently, the beneficial effects of MB exerted on complex III-inhibited mitochondria were debated. In the present contribution, several pieces of evidence are provided towards that MB is able to reduce cytochrome c and improve bioenergetic parameters, like respiration and membrane potential, in mitochondria treated with complex III inhibitors, either antimycin or myxothiazol. These conclusions were drawn from measurements for mitochondrial oxygen consumption, membrane potential, NAD(P)H steady state, MB uptake and MB-cytochrome c oxidoreduction. In the presence of MB and complex III inhibitors, unusual respiratory reactions, like decreased oxygen consumption as a response to ADP addition as well as stimulation of respiration upon administration of inhibitors of ATP synthase or ANT, were observed. Qualitatively identical results were obtained in three rodent species. The actual metabolic status of mitochondria is well reflected in the distribution of MB amongst various compartments of this organelle.