Residual Complex I activity and amphidirectional Complex II operation support glutamate catabolism through mtSLP in anoxia

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in the mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in isolated mitochondria in real-time, we demonstrate that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. 13C metabolic tracing or untargeted analysis of metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system showed that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase Complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Complex II operated amphidirectionally during the anoxic event, providing quinones to Complex I and reducing fumarate to succinate. Our results highlight the importance of quinone provision to Complex I oxidizing NADH maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.</p

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Estudio bioenergético mitocondrial en la sepsis experimental y efecto protector de la melatonina

The present work was focused in the study of mitochondrial function during sepsis. Moreover, was evaluated the properties of melatonin against mitochondrial failure during sepsis. The objectives of this research were: 1. To determine the mitochondrial bioenergetics in liver and heart of mice at early and late phases of sepsis. 2. To identify mitochondrial substrate-dependent behavior during septic process in these tissues. 3. To assess the protective effect of melatonin administration to mice on mitochondrial function of liver and heart of septic mice. 4. To evaluate the mechanistics effects of melatonin on mitochondria using different substrate combinations in liver and heart of septic mice.The present thesis reports on the study of mitochondrial function (of 3 mo. C57BL/6 mice) in liver and heart mouse at 8 and 24 h of sepsis induction by cecal ligation and puncture (CLP). Mainly, mitochondrial function was evaluated assessing mitochondrial respiration by high resolution respirometry (HRR). Other bioenergetic parameters including cytochrome a + a3, b, and c + c1 content, mitochondrial mass, and mitochondrial supercomplexes (SC) formation, were analyzed. Regarding to an effective treatment, we assess melatonin, a recognize molecule with important pleiotropic functions.Tesis Univ. Granada. Programa Oficial de Doctorado en: BiomedicinaDurante la realización del siguiente trabajo, la Lda. Carolina Anneliese Doerrier Velasco fue financiada por las siguientes fuentes: RETICEF (2009-2011); Beca del programa de doctorado en Biomedicina, Contrato de técnico de apoyo investigación FIBAO (2011-2013); Proyecto P07-CTS-03135 de la Junta de Andalucíal, Consejería de Innovación, Ciencia y Empresa; Proyecto PI08-1664 de ISCIII

Estudio bioenergético mitocondrial en la sepsis experimental y efecto protector de la melatonina

The present work was focused in the study of mitochondrial function during sepsis. Moreover, was evaluated the properties of melatonin against mitochondrial failure during sepsis. The objectives of this research were: 1. To determine the mitochondrial bioenergetics in liver and heart of mice at early and late phases of sepsis. 2. To identify mitochondrial substrate-dependent behavior during septic process in these tissues. 3. To assess the protective effect of melatonin administration to mice on mitochondrial function of liver and heart of septic mice. 4. To evaluate the mechanistics effects of melatonin on mitochondria using different substrate combinations in liver and heart of septic mice.The present thesis reports on the study of mitochondrial function (of 3 mo. C57BL/6 mice) in liver and heart mouse at 8 and 24 h of sepsis induction by cecal ligation and puncture (CLP). Mainly, mitochondrial function was evaluated assessing mitochondrial respiration by high resolution respirometry (HRR). Other bioenergetic parameters including cytochrome a + a3, b, and c + c1 content, mitochondrial mass, and mitochondrial supercomplexes (SC) formation, were analyzed. Regarding to an effective treatment, we assess melatonin, a recognize molecule with important pleiotropic functions.Tesis Univ. Granada. Programa Oficial de Doctorado en: BiomedicinaDurante la realización del siguiente trabajo, la Lda. Carolina Anneliese Doerrier Velasco fue financiada por las siguientes fuentes: RETICEF (2009-2011); Beca del programa de doctorado en Biomedicina, Contrato de técnico de apoyo investigación FIBAO (2011-2013); Proyecto P07-CTS-03135 de la Junta de Andalucíal, Consejería de Innovación, Ciencia y Empresa; Proyecto PI08-1664 de ISCIII

Proline Oxidation Supports Mitochondrial ATP Production When Complex I Is Inhibited

The oxidation of proline to pyrroline-5-carboxylate (P5C) leads to the transfer of electrons to ubiquinone in mitochondria that express proline dehydrogenase (ProDH). This electron transfer supports Complexes CIII and CIV, thus generating the protonmotive force. Further catabolism of P5C forms glutamate, which fuels the citric acid cycle that yields the reducing equivalents that sustain oxidative phosphorylation. However, P5C and glutamate catabolism depend on CI activity due to NAD+ requirements. NextGen-O2k (Oroboros Instruments) was used to measure proline oxidation in isolated mitochondria of various mouse tissues. Simultaneous measurements of oxygen consumption, membrane potential, NADH, and the ubiquinone redox state were correlated to ProDH activity and F1FO-ATPase directionality. Proline catabolism generated a sufficiently high membrane potential that was able to maintain the F1FO-ATPase operation in the forward mode. This was observed in CI-inhibited mouse liver and kidney mitochondria that exhibited high levels of proline oxidation and ProDH activity. This action was not observed under anoxia or when either CIII or CIV were inhibited. The duroquinone fueling of CIII and CIV partially reproduced the effects of proline. Excess glutamate, however, could not reproduce the proline effect, suggesting that processes upstream of the glutamate conversion from proline were involved. The ProDH inhibitors tetrahydro-2-furoic acid and, to a lesser extent, S-5-oxo-2-tetrahydrofurancarboxylic acid abolished all proline effects. The data show that ProDH-directed proline catabolism could generate sufficient CIII and CIV proton pumping, thus supporting ATP production by the F1FO-ATPase even under CI inhibition

Mitochondrial respirometry reference values from permeabilized mouse soleus muscle fibers.: MitoEAGLE - WG2 pilot study

International audienc

Protective effects of synthetic kynurenines on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in mice

Producción CientíficaMitochondrial complex I inhibition is thought to underlie the neurodegenerative process in Parkinson's disease (PD). Moreover, an overproduction of nitric oxide due to both cytosolic (iNOS) and mitochondrial (i-mtNOS) inducible nitric oxide synthases causes free radicals generation and oxidative/nitrosative stress, contributing to mitochondrial dysfunction and neuronal cell death. Looking for active molecules against mitochondrial dysfunction and inflammatory response in PD, we show here the effects of four synthetic kynurenines in the MPTP model of PD in mice. After MPTP administration, mitochondria from substantia nigra and, in a lesser extent, from striatum showed a significant increase in i-mtNOS activity, nitric oxide production, oxidative stress, and complex I inhibition. The four kynurenines assayed counteracted the effects of MPTP, reducing iNOS/i-mtNOS activity, and restoring the activity of the complex I. Consequently, the cytosolic and mitochondrial oxidative/nitrosative stress returned to control values. The results suggest that the kynurenines here reported represent a family of synthetic compounds with neuroprotective properties against PD, and that they can serve as templates for the design of new drugs able to target the mitochondria

Mis-targeting of the mitochondrial protein LIPT2 leads to apoptotic cell death

<div><p>Lipoyl(Octanoyl) Transferase 2 (LIPT2) is a protein involved in the post-translational modification of key energy metabolism enzymes in humans. Defects of lipoic acid synthesis and transfer start to emerge as causes of fatal or severe early-onset disease. We show that the first 31 amino acids of the N-terminus of LIPT2 represent a mitochondrial targeting sequence and inhibition of the transit of LIPT2 to the mitochondrion results in apoptotic cell death associated with activation of the apoptotic volume decrease (AVD) current in normotonic conditions, as well as over-activation of the swelling-activated chloride current (IClswell), mitochondrial membrane potential collapse, caspase-3 cleavage and nuclear DNA fragmentation. The findings presented here may help elucidate the molecular mechanisms underlying derangements of lipoic acid biosynthesis.</p></div

Commitment to reproducibility in mitochondrial respiration studies with permeabilized muscle fibers.

International audienceThe exponential increase of scientific publications in the mitochondrial field shows the growing interest in mitochondria. However, the lack of methodological consistency in many published projects on mitochondrial respiratory function complicates a quantitative inter/intra-laboratory comparison of datasets. This deficiency manifests the need to improve the quality in science[1]. In this context, the MitoEAGLE COST Action is a powerful framework committed to evaluate and enhance the reproducibility in mitochondrial physiology as a basis to establish a novel mitochondrial database related to Evolution, Age, Gender, Lifestyle and Environment. Permeabilized muscle fibers are widely used to evaluate mitochondrial function in health and disease[2]. Therefore, our main goals are to: 1) compare protocols used in different research laboratories, 2) analyze factors which contribute to experimental variability, 3) define optimal experimental conditions in muscle studies, 4) elaborate guidelines for evaluating mitochondrial function in muscle tissue, 5) establish reference values on mitochondrial respiration, particularly as a test of the skills in preparing high-quality permeabilized muscle fibers, and 6) generate a database. To achieve our aims, two unique studies are currently in progress: 1) 17 international research groups performing independently experiments on respiration in permeabilized fibers of mouse soleus muscle, following the same experimental procedure[3]; 2) a blinded international study measuring simultaneously in the same laboratory respiration of permeabilized human skeletal fibers by high-resolution respirometry and assessing the effect of different experimental conditions[4]. Our results contribute to face the reproducibility crisis and provide the basis for establishing the first database on mitochondrial respiratory parameters in muscle tissues