RESUMO
Classic galactosemia is an inborn error of metabolism caused by mutations in the GALT gene resulting in the diminished activity of the galactose-1-phosphate uridyltransferase enzyme. This reduced GALT activity leads to the buildup of the toxic intermediate galactose-1-phosphate and a decrease in ATP levels upon exposure to galactose. In this work, we focused our attention on mitochondrial oxidative phosphorylation in the context of this metabolic disorder. We observed that galactose-1-phosphate accumulation reduced respiratory rates in vivo and changed mitochondrial function and morphology in yeast models of galactosemia. These alterations are harmful to yeast cells since the mitochondrial retrograde response is activated as part of the cellular adaptation to galactose toxicity. In addition, we found that galactose-1-phosphate directly impairs cytochrome c oxidase activity of mitochondrial preparations derived from yeast, rat liver, and human cell lines. These results highlight the evolutionary conservation of this biochemical effect. Finally, we discovered that two compounds - oleic acid and dihydrolipoic acid - that can improve the growth of cell models of mitochondrial diseases, were also able to improve galactose tolerance in this model of galactosemia. These results reveal a new molecular mechanism relevant to the pathophysiology of classic galactosemia - galactose-1-phosphate-dependent mitochondrial dysfunction - and suggest that therapies designed to treat mitochondrial diseases may be repurposed to treat galactosemia.
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Glucose and oxygen (O2) are vital to the brain. Glucose metabolism and mitochondria play a pivotal role in this process, culminating in the increase of reactive O2 species. Hexokinase (HK) is a key enzyme on glucose metabolism and is coupled to the brain mitochondrial redox modulation by recycling ADP for oxidative phosphorylation (OXPHOS). GABA shunt is an alternative pathway to GABA metabolism that increases succinate levels, a Krebs cycle intermediate. Although glucose and GABA metabolisms are intrinsically connected, their interplay coordinating mitochondrial function is poorly understood. Here, we hypothesize that the HK and the GABA shunt interact to control mitochondrial metabolism differently in the cortex and the hypothalamus. The GABA shunt stimulated mitochondrial O2 consumption and H2O2 production higher in hypothalamic synaptosomes (HSy) than cortical synaptosomes (CSy). The GABA shunt increased the HK coupled to OXPHOS activity in both population of synaptosomes, but the rate of activation was higher in HSy than CSy. Significantly, malonate and vigabatrin blocked the effects of the GABA shunt in the HK activity coupled to OXPHOS. It indicates that the glucose phosphorylation is linked to GABA and Krebs cycle reactions. Together, these data shed light on the HK and SDH role on the metabolism of each region fed by GABA turnover, which depends on the neurons' metabolic route.
Assuntos
Glucose , Peróxido de Hidrogênio , Glucose/metabolismo , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/metabolismo , Fosforilação , Ácido gama-Aminobutírico/metabolismoRESUMO
Mitochondria are key players of aerobic respiration and the production of adenosine triphosphate and constitute the energetic core of eukaryotic cells. Furthermore, cells rely upon mitochondria homeostasis, the disruption of which is reported in pathological processes such as liver hepatotoxicity, cancer, muscular dystrophy, chronic inflammation, as well as in neurological conditions including Alzheimer's disease, schizophrenia, depression, ischemia and glaucoma. In addition to the well-known spontaneous cell-to-cell transfer of mitochondria, a therapeutic potential of the transplant of isolated, metabolically active mitochondria has been demonstrated in several in vitro and in vivo experimental models of disease. This review explores the striking outcomes achieved by mitotherapy thus far, and the most relevant underlying data regarding isolated mitochondria transplantation, including mechanisms of mitochondria intake, the balance between administration and therapy effectiveness, the relevance of mitochondrial source and purity and the mechanisms by which mitotherapy is gaining ground as a promising therapeutic approach.
Assuntos
Doença de Alzheimer/terapia , Depressão/terapia , Glaucoma/terapia , Hepatite/terapia , Isquemia/terapia , Mitocôndrias/transplante , Distrofias Musculares/terapia , Neoplasias/terapia , Esquizofrenia/terapia , Trifosfato de Adenosina/biossíntese , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Depressão/genética , Depressão/metabolismo , Depressão/patologia , Modelos Animais de Doenças , Glaucoma/genética , Glaucoma/metabolismo , Glaucoma/patologia , Hepatite/genética , Hepatite/metabolismo , Hepatite/patologia , Humanos , Isquemia/genética , Isquemia/metabolismo , Isquemia/patologia , Fígado/metabolismo , Fígado/patologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação Oxidativa , Esquizofrenia/genética , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Resultado do TratamentoRESUMO
The mitochondrial pyruvate carrier (MPC) is an inner-membrane transporter that facilitates pyruvate uptake from the cytoplasm into mitochondria. We previously reported that MPC1 protein levels increase in the hypothalamus of animals during fever induced by lipopolysaccharide (LPS), but how this increase contributes to the LPS responses remains to be studied. Therefore, we investigated the effect of UK 5099, a classical MPC inhibitor, in a rat model of fever, on hypothalamic mitochondrial function and neuroinflammation in LPS-stimulated preoptic area (POA) primary microcultures. Intracerebroventricular administration of UK 5099 reduced the LPS-induced fever. High-resolution respirometry revealed an increase in oxygen consumption and oxygen flux related to ATP synthesis in the hypothalamic homogenate from LPS-treated animals linked to mitochondrial complex I plus II. Preincubation with UK 5099 prevented the LPS-induced increase in oxygen consumption, ATP synthesis and spare capacity only in complex I-linked respiration and reduced mitochondrial H2O2 production. In addition, treatment of rat POA microcultures with UK 5099 reduced the secretion of the proinflammatory and pyrogenic cytokines TNFα and IL-6 as well as the immunoreactivity of inflammatory transcription factors NF-κB and NF-IL6 four hours after LPS stimulation. These results suggest that the regulation of mitochondrial pyruvate metabolism through MPC inhibition may be effective in reducing neuroinflammation and fever.
Assuntos
Peróxido de Hidrogênio , Transportadores de Ácidos Monocarboxílicos , Animais , Febre/induzido quimicamente , Lipopolissacarídeos , Mitocôndrias , Ácido Pirúvico , RatosRESUMO
Hyperglycemia associated with Diabetes Mellitus type 1 (DM1) comorbidity may cause severe complications in several tissues that lead to premature death. These dysfunctions are related, among others, to redox imbalances caused by the uncontrolled cellular levels of reactive oxygen species (ROS). Brain is potentially prone to develop diabetes complications because of its great susceptibility to oxidative stress. In addition to antioxidant enzymes, mitochondria-coupled hexokinase (mt-HK) plays an essential role in maintaining high flux of oxygen and glucose to control the mitochondrial membrane and redox potential in brain. This redox control is critical for healthy conditions in brain and in the pathophysiological progression of DM1. The mitochondrial and mt-HK contribution in this process is essential to understand the relationship between DM1 complications and the management of the cellular redox balance. Using a rat model of one month of hyperglycemia induced by a single administration intraperitoneally of streptozotocin, we showed in the present work that, in rat brain mitochondria, there is a specifically reduction of the mitochondrial complex I (CI) activity and an increase in the activity of the antioxidant enzyme thioredoxin reductase, which are related to decreased hydrogen peroxide generation, oxygen consumption and mt-HK coupled-to-OxPhos activity via mitochondrial CI. Surprisingly, DM1 increases respiratory parameters and mt-HK activity via mitochondrial complex II (CII). This way, for the first time, we provide evidence that early progression of hyperglycemia, in brain tissue, changes the coupling of glucose phosphorylation at the level of mitochondria by rearranging the oxidative machinery of brain mitochondria towards CII dependent electron harvest. In addition, DM1 increased the production of H2O2 by α-ketoglutarate dehydrogenase without causing oxidative stress. Finally, DM1 increased the oxidation status of PTEN and decreased the activation of NF-kB in DM1. These results indicate that this reorganization of glucose-oxygen-ROS axis in mitochondria may impact turnover of glucose, brain amino acids, redox and inflammatory signaling. In addition, this reorganization may be involved in early protection mechanisms against the development of cognitive degeneration and neurodegenerative disease, widely associated to mitochondrial CI deficits.
Assuntos
Diabetes Mellitus Tipo 1 , Hiperglicemia , Doenças Neurodegenerativas , Animais , Encéfalo/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Glucose/metabolismo , Peróxido de Hidrogênio/metabolismo , Hiperglicemia/metabolismo , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/metabolismo , Oxirredução , Estresse Oxidativo , Fosforilação , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mitochondrial dysfunctions are linked to a series of neurodegenerative human conditions, including Parkinson's disease, schizophrenia, optic neuropathies, and glaucoma. Recently, a series of studies have pointed mitotherapy - exogenous mitochondria transplant - as a promising way to attenuate the progression of neurologic disorders; however, the neuroprotective and pro-regenerative potentials of isolated mitochondria in vivo have not yet been elucidated. In this present work, we tested the effects of transplants of active (as well-coupled organelles were named), liver-isolated mitochondria on the survival of retinal ganglion cells and axonal outgrowth after optic nerve crush. Our data show that intravitreally transplanted, full active mitochondria incorporate into the retina, improve its oxidative metabolism and electrophysiological activity at 1 day after transplantation. Moreover, mitotherapy increases cell survival in the ganglion cell layer at 14 days, and leads to a higher number of axons extending beyond the injury site at 28 days; effects that are dependent on the organelles' structural integrity. Together, our findings support mitotherapy as a promising approach for future therapeutic interventions upon central nervous system damage.
Assuntos
Mitocôndrias/transplante , Regeneração Nervosa , Traumatismos do Nervo Óptico/terapia , Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Animais , Fracionamento Celular , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Injeções Intravítreas , Fígado/citologia , Masculino , Traumatismos do Nervo Óptico/patologia , Estresse Oxidativo/fisiologia , RatosRESUMO
Schizophrenia etiology is unknown, nevertheless imbalances occurring in an acute psychotic episode are important to its development, such as alterations in cellular energetic state, REDOX homeostasis and intracellular Ca2+ management, all of which are controlled primarily by mitochondria. However, mitochondrial function was always evaluated singularly, in the presence of specific respiratory substrates, without considering the plurality of the electron transport system. In this study, mitochondrial function was analyzed under conditions of isolated or multiple respiratory substrates using brain mitochondria isolated from MK-801-exposed mice. Results showed a high H2O2 production in the presence of pyruvate/malate, with no change in oxygen consumption. In the condition of multiple substrates, however, this effect is lost. The analysis of Ca2+ retention capacity revealed a significant change in the uptake kinetics of this ion by mitochondria in MK-801-exposed animals. Futhermore, when mitochondria were exposed to calcium, a total loss of oxidative phosphorylation and an impressive increase in H2O2 production were observed in the condition of multiple substrates. There was no alteration in the activity of the antioxidant enzymes analyzed. The data demonstrate for the first time, in an animal model of psychosis, two important aspects (1) mitochondria may compensate deficiencies in a single mitochondrial complex when they oxidize several substrates simultaneously, (2) Ca2+ handling is compromised in MK-801-exposed mice, resulting in a loss of phosphorylative capacity and an increase in H2O2 production. These data favor the hypothesis that disruption of key physiological roles of mitochondria may be a trigger in acute psychosis and, consequently, schizophrenia.
Assuntos
Encéfalo/patologia , Cálcio/efeitos adversos , Mitocôndrias/patologia , Transtornos Psicóticos/complicações , Doença Aguda , Animais , Humanos , Masculino , CamundongosRESUMO
The adult brain is a high-glucose and oxygen-dependent organ, with an extremely organized network of cells and large energy-consuming synapses. To reach this level of organization, early stages in development must include an efficient control of cellular events and regulation of intracellular signaling molecules and ions such as hydrogen peroxide (H2 O2 ) and calcium (Ca2+ ), but in cerebral tissue, these mechanisms of regulation are still poorly understood. Hexokinase (HK) is the first enzyme in the metabolism of glucose and, when bound to mitochondria (mtHK), it has been proposed to have a role in modulation of mitochondrial H2 O2 generation and Ca2+ handling. Here, we have investigated how mtHK modulates these signals in the mitochondrial context during postnatal development of the mouse brain. Using high-resolution respirometry, western blot analysis, spectrometry and resorufin, and Calcium Green fluorescence assays with brain mitochondria purified postnatally from day 1 to day 60, we demonstrate that brain HK increases its coupling to mitochondria and to oxidative phosphorylation to induce a cycle of ADP entry/ATP exit of the mitochondrial matrix that leads to efficient control over H2 O2 generation and Ca2+ uptake during development until reaching plateau at day 21. This contrasts sharply with the antioxidant enzymes, which do not increase as mitochondrial H2 O2 generation escalates. These results suggest that, as its use of glucose increases, the brain couples HK to mitochondria to improve glucose metabolism, redox balance and Ca2+ signaling during development, positioning mitochondria-bound hexokinase as a hub for intracellular signaling control.
Assuntos
Encéfalo/metabolismo , Cálcio/metabolismo , Glucose/metabolismo , Hexoquinase/metabolismo , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Animais , Neurogênese/fisiologia , Fosforilação Oxidativa , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Different exercise protocols lead to long-term adaptations that are related to increased mitochondrial content through the activation of mitochondrial biogenesis. However, immediate mitochondrial response to exercise and energetic substrate utilization is still unknown. We evaluate the mitochondrial physiology of two types rat skeletal muscle fibres immediately after a single session of high intensity interval exercise (HIIE) or aerobic exercise (AER). We found AER was able to reduce the ATP synthesis dependent oxygen flux in the tibialis (TA) when stimulated by complex I and II substrates. On the other hand, there was an increase of the maximum velocity (Vmax) for glycerol-phosphate oxidation and Vmax and affinity (KM) of palmitoyl-carnitine oxidation (PC). The exercise did not affect oxygen flux coupled to ATP synthesis in red gastrocnemius (RG) but, surprisingly, reduced its affinity for PC, decreasing the apparent catalytic efficiency (Vmax/KM) of oxidation for PC. Neither exercise protocol was able to change the electron transfer system capacity of the mitochondria or markers of mitochondrial content. The AER group had increased H2O2 production compared to the SED and HIIE groups, with the mechanism being predominantly the escape of electrons through reverse flux in complex I and other sites in TA, and only through other sites in RG. There were no changes in the activities of antioxidant enzymes. Our results show that mitochondria from different muscles submitted to distinct exercise protocols show alterations in the specific fluxes of substrate utilization and oxygen metabolism, indicating that the dynamics of mitochondria are linked to the metabolic flexibility.
Assuntos
Músculo Esquelético , Oxigênio/metabolismo , Condicionamento Físico Animal , Animais , Antioxidantes/metabolismo , Peróxido de Hidrogênio/metabolismo , Masculino , Mitocôndrias/metabolismo , Consumo de Oxigênio , Fosforilação/fisiologia , Ratos , Ratos Wistar , Especificidade por SubstratoRESUMO
Glucose and oxygen are vital for the brain, as these molecules provide energy and metabolic intermediates that are necessary for cell function. The glycolysis pathway and mitochondria play a pivotal role in cell energy metabolism, which is closely related to reactive oxygen species (ROS) production. Hexokinase (HK) is a key enzyme involved in glucose metabolism that modulates the level of brain mitochondrial ROS by recycling ADP for oxidative phosphorylation (OxPhos). Here, we hypothesize that the control of mitochondrial metabolism by hexokinase differs in distinct areas of the brain, such as the cortex and hypothalamus, in which ROS might function as signaling molecules. Thus, we investigated mitochondrial metabolism of synaptosomes derived from both brain regions. Cortical synaptosomes (CSy) show a predominance of glutamatergic synapses, while in the hypothalamic synaptosomes (HSy), the GABAergic synapses predominate. Significant differences of oxygen consumption and ROS production were related to higher mitochondrial complex II activity (succinate dehydrogenase-SDH) in CSy rather than to mitochondrial number. Mitochondrial HK (mt-HK) activity was higher in CSy than in HSy regardless the substrate added. Mitochondrial O2 consumption related to mt-HK activation by 2-deoxyglucose was also higher in CSy. In the presence of substrate for complex II, the activation of synaptosomal mt-HK promoted depuration of ROS in both HSy and CSy, while ROS depuration did not occur in HSy when substrate for complex I was used. The impact of the mt-HK inhibition by glucose-6-phosphate (G6P) was the same in synaptosomes from both areas. Together, the differences found between CSy and HSy indicate specific roles of mt-HK and SDH on the metabolism of each brain region, what probably depends on the main metabolic route that is used by the neurons.
Assuntos
Córtex Cerebral/enzimologia , Hexoquinase/metabolismo , Peróxido de Hidrogênio/metabolismo , Hipotálamo/enzimologia , Mitocôndrias/metabolismo , Sinaptossomos/enzimologia , Animais , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Glucose-6-Fosfato/farmacologia , Masculino , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacosRESUMO
Reactive oxygen species (ROS) are signaling molecules that regulate plant development and responses to stresses. Mitochondria are the source of most ROS in heterotrophic cells, and mitochondrial complex I and complex III are regarded as the main sites of ROS production in plant mitochondria. Recent studies have demonstrated that succinate dehydrogenase (SDH) also contributes to mitochondrial ROS production. However, the ability of SDH to generate ROS in plants is unclear. The aim of this study was to evaluate the role of SDH in mitochondrial ROS production. Our results demonstrated that SDH is a direct source of ROS in Arabidopsis thaliana and Oryza sativa, and the induction of ROS production by specific SDH inhibitors impaired plant growth. In addition, this effect was accompanied by the down-regulation of cell cycle genes and the up-regulation of stress-related genes. However, the partial inhibition of SDH by a competitive inhibitor decreased ROS production, which was associated with increased shoot and root growth, and prevented the down-regulation of cell cycle genes and the induction of stress-related genes by noncompetitive inhibitors. In conclusion, SDH plays an important role in ROS production, being a direct source of ROS in plant mitochondria and regulating plant development and stress responses.
Assuntos
Arabidopsis/enzimologia , Mitocôndrias/metabolismo , Desenvolvimento Vegetal , Espécies Reativas de Oxigênio/metabolismo , Succinato Desidrogenase/metabolismo , Ciclo Celular , Estresse FisiológicoRESUMO
High intensity interval training (HIIT) is characterized by vigorous exercise with short rest intervals. Hydrogen peroxide (H2O2) plays a key role in muscle adaptation. This study aimed to evaluate whether HIIT promotes similar H2O2 formation via O2 consumption (electron leakage) in three skeletal muscles with different twitch characteristics. Rats were assigned to two groups: sedentary (n=10) and HIIT (n=10, swimming training). We collected the tibialis anterior (TA-fast), gastrocnemius (GAST-fast/slow) and soleus (SOL-slow) muscles. The fibers were analyzed for mitochondrial respiration, H2O2 production and citrate synthase (CS) activity. A multi-substrate (glycerol phosphate (G3P), pyruvate, malate, glutamate and succinate) approach was used to analyze the mitochondria in permeabilized fibers. Compared to the control group, oxygen flow coupled to ATP synthesis, complex I and complex II was higher in the TA of the HIIT group by 1.5-, 3.0- and 2.7-fold, respectively. In contrast, oxygen consumed by mitochondrial glycerol phosphate dehydrogenase (mGPdH) was 30% lower. Surprisingly, the oxygen flow coupled to ATP synthesis was 42% lower after HIIT in the SOL. Moreover, oxygen flow coupled to ATP synthesis and complex II was higher by 1.4- and 2.7-fold in the GAST of the HIIT group. After HIIT, CS activity increased 1.3-fold in the TA, and H2O2 production was 1.3-fold higher in the TA at sites containing mGPdH. No significant differences in H2O2 production were detected in the SOL. Surprisingly, HIIT increased H2O2 production in the GAST via complex II, phosphorylation, oligomycin and antimycin by 1.6-, 1.8-, 2.2-, and 2.2-fold, respectively. Electron leakage was 3.3-fold higher in the TA with G3P and 1.8-fold higher in the GAST with multiple substrates. Unexpectedly, the HIIT protocol induced different respiration and electron leakage responses in different types of muscle.