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Contains fulltext : 153971.pdf (Publisher’s version ) (Closed access) Mitochondrial ATP production is mediated by the oxidative phosphorylation (OXPHOS) system, which consists of four multi-subunit complexes (CI-CIV) and the FoF1-ATP synthase (CV). Mitochondrial disorders including Leigh Syndrome often involve CI dysfunction, the pathophysiological consequences of which still remain incompletely understood. Here we combined experimental and computational strategies to gain mechanistic insight into the energy metabolism of isolated skeletal muscle mitochondria from 5-week-old wild-type (WT) and CI-deficient NDUFS4(-/-) (KO) mice. Enzyme activity measurements in KO mitochondria revealed a reduction of 79% in maximal CI activity (Vmax), which was paralleled by 45-72% increase in Vmax of CII, CIII, CIV and citrate synthase. Mathematical modeling of mitochondrial metabolism predicted that these Vmax changes do not affect the maximal rates of pyruvate (PYR) oxidation and ATP production in KO mitochondria. This prediction was empirically confirmed by flux measurements. In silico analysis further predicted that CI deficiency altered the concentration of intermediate metabolites, modestly increased mitochondrial NADH/NAD(+) ratio and stimulated the lower half of the TCA cycle, including CII. Several of the predicted changes were previously observed in experimental models of CI-deficiency. Interestingly, model predictions further suggested that CI deficiency only has major metabolic consequences when its activity decreases below 90% of normal levels, compatible with a biochemical threshold effect. Taken together, our results suggest that mouse skeletal muscle mitochondria possess a substantial CI overcapacity, which minimizes the effects of CI dysfunction on mitochondrial metabolism in this otherwise early fatal mouse model.

Contains fulltext : 172049.pdf (Publisher’s version ) (Closed access) The acute phase of sepsis is characterized by a strong inflammatory reaction. At later stages in some patients, immunoparalysis may be encountered, which is associated with a poor outcome. By transcriptional and metabolic profiling of human patients with sepsis, we found that a shift from oxidative phosphorylation to aerobic glycolysis was an important component of initial activation of host defense. Blocking metabolic pathways with metformin diminished cytokine production and increased mortality in systemic fungal infection in mice. In contrast, in leukocytes rendered tolerant by exposure to lipopolysaccharide or after isolation from patients with sepsis and immunoparalysis, a generalized metabolic defect at the level of both glycolysis and oxidative metabolism was apparent, which was restored after recovery of the patients. Finally, the immunometabolic defects in humans were partially restored by therapy with recombinant interferon-gamma, which suggested that metabolic processes might represent a therapeutic target in sepsis.

Item does not contain fulltext Complex I (CI) represents a major entry point of electrons in the mitochondrial electron transport chain (ETC). It consists of 45 different subunits, encoded by the mitochondrial (mtDNA) and nuclear DNA (nDNA). In humans, mutations in nDNA-encoded subunits cause severe neurodegenerative disorders like Leigh Syndrome with onset in early childhood. The pathophysiological mechanism of these disorders is still poorly understood. Here we summarize the current knowledge concerning the consequences of nDNA-encoded CI mutations in patient-derived cells, present mouse models for human CI deficiency, and discuss potential treatment strategies for CI deficiency. 01 januari 2012

Contains fulltext : 167305.pdf (Publisher’s version ) (Open Access) We reported before that the minimal alveolar concentration (MAC) of isoflurane is decreased in complex I-deficient mice lacking the NDUFS4 subunit of the respiratory chain (RC) (1.55 and 0.81 % at postnatal (PN) 22-25 days and 1.68 and 0.65 % at PN 31-34 days for wildtype (WT) and CI-deficient KO, respectively). A more severe respiratory depression was caused by 1.0 MAC isoflurane in KO mice (respiratory rate values of 86 and 45 at PN 22-25 days and 69 and 29 at PN 31-34 days for anesthetized WT and KO, respectively). Here, we address the idea that isoflurane anesthesia causes a much larger decrease in brain mitochondrial ATP production in KO mice thus explaining their increased sensitivity to this anesthetic. Brains from WT and KO mice of the above study were removed immediately after MAC determination at PN 31-34 days and a mitochondria-enriched fraction was prepared. Aliquots were used for measurement of maximal ATP production in the presence of pyruvate, malate, ADP and creatine and, after freeze-thawing, the maximal activity of the individual RC complexes in the presence of complex-specific substrates. CI activity was dramatically decreased in KO, whereas ATP production was decreased by only 26 % (p < 0.05). The activities of CII, CIII, and CIV were the same for WT and KO. Isoflurane anesthesia decreased the activity of CI by 30 % (p < 0.001) in WT. In sharp contrast, it increased the activity of CII by 37 % (p < 0.001) and 50 % (p < 0.001) and that of CIII by 37 % (p < 0.001) and 40 % (p < 0.001) in WT and KO, respectively, whereas it tended to increase that of CIV in both WT and KO. Isoflurane anesthesia increased ATP production by 52 and 69 % in WT (p < 0.05) and KO (p < 0.01), respectively. Together these findings indicate that isoflurane anesthesia interferes positively rather than negatively with the ability of CI-deficient mice brain mitochondria to convert their main substrate pyruvate into ATP.

Background: Macrophages are highly specialized immune cells of different developmental origin, which occur in a continuum of diverse functional states in almost all tissues. In order to fulfil their complex role in tissue homeostasis and defence against pathogens they must be able to live with heterogeneous extrinsic nutrient conditions in tissue niches and handle variation in intrinsic metabolic demand that is determined by differentiation state, functional specialization and immune challenge. The purpose of the present study was to gain more insight in how metabolic specialization and versatility in fate and immune effector function of macrophages are coupled. Methods: In vitro phenotypic characteristics of two macrophage cell lineages of profoundly different developmental origin and polarization capacity, RAW 264.7 and Maf-DKO cells, were analysed. By use of biochemical and cell biological approaches and scanning electron microscopy, we studied the metabolic profiles of these two types of macrophages in relation to proliferative capacity, morphological appearance of cell surface and cell shape, and phagocytic activity as index of morphodynamic potential. Results: Comparison of gross features of carbohydrate metabolism, including levels of glycolytic enzymes Hexokinase (HK), Pyruvate Kinase (PK-M2), lactate dehydrogenase and nicotinamide phosphoribosyltransferase (Nampt), glucose and oxygen consumption and lactate production rates, and intracellular concentrations and redox ratios of NAD(P)(H) demonstrated that RAW 264.7 and Maf-DKO cells are conspicuously similar in that they both rely heavily on the use of glycolysis. In this respect they share many characteristics with primary macrophages. Strikingly, this uniform metabolic signature does not translate in behavioral-functional similarities as RAW 264.7 cells have a significantly higher proliferation rate, whereas Maf-DKO cells appear to be morphodynamically more active, form significantly more surface membrane protrusions and phagocytose complement opsonized particles much more efficiently. Conclusion: We conclude that the global rate of glycolysis in intermediary carbohydrate metabolism is similar for the two cell lineages, but that they can make differential use of this important pathway, either to fuel high morphodynamic activity in Maf-DKO cells, or for the sustenance of cell growth in fast proliferating RAW 264.7 cells. Our findings are in keeping with the idea that macrophages may uniformly prefer use of the rapid response time of glycolysis because this pathway provides them with the ability to meet any possible short-timescale energy demand required for immune function.

A BLUEPRINT of immune cell development To determine the epigenetic mechanisms that direct blood cells to develop into the many components of our immune system, the BLUEPRINT consortium examined the regulation of DNA and RNA transcription to dissect the molecular traits that govern blood cell differentiation. By inducing immune responses, Saeed et al. document the epigenetic changes in the genome that underlie immune cell differentiation. Cheng et al. demonstrate that trained monocytes are highly dependent on the breakdown of sugars in the presence of oxygen, which allows cells to produce the energy needed to mount an immune response. Chen et al. examine RNA transcripts and find that specific cell lineages use RNA transcripts of different length and composition (isoforms) to form proteins. Together, the studies reveal how epigenetic effects can drive the development of blood cells involved in the immune system. Science , this issue 10.1126/science.1251086 , 10.1126/science.1250684 , 10.1126/science.1251033

Item does not contain fulltext BACKGROUND: Children with mitochondrial disorders are frequently anesthetized for a wide range of operations. These disorders may interfere with the response to surgery and anesthesia. We examined anesthetic sensitivity to and respiratory effects of isoflurane in the Ndufs4 knockout (KO) mouse model. These mice exhibit an isolated mitochondrial complex I (CI) deficiency of the respiratory chain, and they also display clinical signs and symptoms resembling those of patients with mitochondrial CI disease. METHODS: We investigated seven Ndufs4(-/-) knockout (KO), five Ndufs4(+/-) heterozygous (HZ) and five Ndufs4(+/+) wild type (WT) mice between 22 and 25 days and again between 31 and 34 days post-natal. Animals were placed inside an airtight box, breathing spontaneously while isoflurane was administered in increasing concentrations. Minimum alveolar concentration (MAC) was determined with the bracketing study design, using the response to electrical stimulation to the hind paw. RESULTS: MAC for isoflurane was significantly lower in KO mice than in HZ and WT mice: 0.81 % +/- 0.01 vs 1.55 +/- 0.05 % and 1.55 +/- 0.13 %, respectively, at 22-25 days, and 0.65 +/- 0.05 %, 1.65 +/- 0.08 % and 1.68 +/- 0.08 % at 31-34 days. The KO mice showed severe respiratory depression at lower isoflurane concentrations than the WT and HZ mice. CONCLUSION: We observed an increased isoflurane anesthetic sensitivity and severe respiratory depression in the KO mice. The respiratory depression during anesthesia was strongly progressive with age. Since the pathophysiological consequences from complex I deficiency are mainly reflected in the central nervous system and our mouse model involves progressive encephalopathy, further investigation of isoflurane effects on brain mitochondrial function is warranted.

Contains fulltext : 89702.pdf (Publisher’s version ) (Closed access) Mitochondrial oxidative phosphorylation (OXPHOS) represents the final step in the conversion of nutrients into cellular energy. Genetic defects in the OXPHOS system have an incidence between 1:5,000 and 1:10,000 live births. Inherited isolated deficiency of the first complex (CI) of this system, a multisubunit assembly of 45 different proteins, occurs most frequently and originates from mutations in either the nuclear DNA, encoding 38 structural subunits and several assembly factors, or the mitochondrial DNA, encoding 7 structural subunits. The deficiency is associated with devastating multisystemic disorders, often affecting the brain, with onset in early childhood. There are currently no rational treatment strategies. Here, we present an overview of the genetic origins and cellular consequences of this deficiency and discuss how these insights might aid future development of treatment strategies. 01 juni 2010

Oxygen radical formation in mitochondria is an incompletely understood attribute of eukaryotic cells. Recently, a kinetic model was proposed, in which the ratio between electrons entering the respiratory chain via FADH 2 or NADH determines radical formation. During glucose breakdown, the ratio is low; during fatty acid breakdown, the ratio is high (the ratio increasing—asymptotically—with fatty acid length to 0.5, when compared with 0.2 for glucose). Thus, fatty acid oxidation would generate higher levels of radical formation. As a result, breakdown of fatty acids, performed without generation of extra FADH 2 in mitochondria, could be beneficial for the cell, especially in the case of long and very long chained ones. This possibly has been a major factor in the evolution of peroxisomes. Increased radical formation, as proposed by the model, can also shed light on the lack of neuronal fatty acid oxidation and tells us about hurdles during early eukaryotic evolution. We specifically focus on extending and discussing the model in light of recent publications and findings.

Contains fulltext : 133835.pdf (Publisher’s version ) (Closed access) The mitochondrial oxidative phosphorylation (OXPHOS) system consists of four electron transport chain (ETC) complexes (CI-CIV) and the FoF1-ATP synthase (CV), which sustain ATP generation via chemiosmotic coupling. The latter requires an inward-directed proton-motive force (PMF) across the mitochondrial inner membrane (MIM) consisting of a proton (DeltapH) and electrical charge (Deltapsi) gradient. CI actively participates in sustaining these gradients via trans-MIM proton pumping. Enigmatically, at the cellular level genetic or inhibitor-induced CI dysfunction has been associated with Deltapsi depolarization or hyperpolarization. The cellular mechanism of the latter is still incompletely understood. Here we demonstrate that chronic (24h) CI inhibition in HEK293 cells induces a proton-based Deltapsi hyperpolarization in HEK293 cells without triggering reverse-mode action of CV or the adenine nucleotide translocase (ANT). Hyperpolarization was associated with low levels of CII-driven O2 consumption and prevented by co-inhibition of CII, CIII or CIV activity. In contrast, chronic CIII inhibition triggered CV reverse-mode action and induced Deltapsi depolarization. CI- and CIII-inhibition similarly reduced free matrix ATP levels and increased the cell's dependence on extracellular glucose to maintain cytosolic free ATP. Our findings support a model in which Deltapsi hyperpolarization in CI-inhibited cells results from low activity of CII, CIII and CIV, combined with reduced forward action of CV and ANT.