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• Ley-Ngardigal 2022 FEBS J  + (ATP is the most universal and essential en … ATP is the most universal and essential energy molecule in cells. This is due to its ability to store cellular energy in form of high-energy phosphate bonds, which are extremely stable and readily usable by the cell. This energy is key for a variety of biological functions such as cell growth and division, metabolism, and signaling, and for the turnover of biomolecules. Understanding how ATP is produced and hydrolyzed with a spatiotemporal resolution is necessary to understand its functions both in physiological and in pathological contexts. In this review, first we will describe the organization of the electron transport chain and ATP synthase, the main molecular motor for ATP production in mitochondria. Second, we will review the biochemical assays currently available to estimate ATP quantities in cells, and we will compare their readouts, strengths, and weaknesses. Finally, we will explore the palette of genetically encoded biosensors designed for microscopy-based approaches, and show how their spatiotemporal resolution opened up the possibility to follow ATP levels in living cells.lity to follow ATP levels in living cells.)
• Nuskova 2015 Abstract MiPschool London 2015  + (ATP produced by the mitochondrial FoF1-ATP … ATP produced by the mitochondrial FoF1-ATP synthase represents a major source of energy for aerobic organisms. Unsurprisingly, ATP synthase deficiencies are associated with severe pathologic phenotypes. To shed light on the functional consequences of ATP synthase deficiencies, we utilised a model of HEK293 cell line and explored the effect of RNAi mediated knockdown of the three subunits (γ, δ and ε) forming the central stalk of the enzyme connecting Fo and F1 domains.</br></br>For functional evaluations of ATP synthase deficiencies, 10 stable knockdown clones with down-regulated subunits γ (ATP5C1 gene), δ (ATP5D gene), or ε (ATP5E gene) have been selected. The protein content of ATP synthase subunit α among the knockdown clones covers the range of 40–100 % as compared to controls. Further characterization of these clones revealed 2–78 % oligomycin-sensitive ATPase hydrolytic activity that parallels a decrease in the content of fully assembled ATP synthase complex.</br></br>Two aspects of cellular energetics have been examined in detail, specifically respiration and glycolysis, using the Seahorse XFe24 analyser. Our results indicate that the clones with less than 30 % of residual ATPase activity switched their metabolism to enhanced glycolysis. There is a decrease in their basal respiration rate relatively to their respiratory capacity (47 vs 61 % in controls) and in parallel, their basal glycolytic rates utilise by up to 20 % more of their glycolytic capacity. These findings clearly demonstrate metabolic adaptations of these cells. On the other hand, the clones with more than 30 % residual ATPase activity displayed a change neither in the respiration nor in their basal glycolytic rate. </br></br>In the case of ATP synthase deficiency, the mitochondrial membrane potential is expected to rise, which would then stimulate the production of reactive oxygen species (ROS). Indeed, the γ knockdown clones with a very low residual ATPase activity exhibit elevated ROS production. With respect to the role of the oxidative stress in ATP synthase deficiencies, we aim to examine oxidative damage of cell structures and the content of antioxidant enzymes.</br></br>In conclusion, using these model clones, we are planning on investigating the effect of ATP synthase deficiency on the mitochondrial energetics, oxidative stress, energy state, and cell viability and define the threshold residual activity of ATP synthase for the presentation of pathological phenotype. At this moment, our data suggest that the threshold for metabolic remodelling equals to approximately 30 % of ATPase activity. to approximately 30 % of ATPase activity.)
• Cortassa 2022 J Mol Cell Cardiol  + (ATP synthase (F1Fo) is a rotary molecular … ATP synthase (F1Fo) is a rotary molecular engine that harnesses energy from electrochemical-gradients across the inner mitochondrial membrane for ATP synthesis. Despite the accepted tenet that F1Fo transports exclusively H+, our laboratory has demonstrated that, in addition to H+, F1Fo ATP synthase transports a significant fraction of ΔΨm-driven charge as K+ to synthesize ATP. Herein, we utilize a computational modeling approach as a proof of principle of the feasibility of the core mechanism underlying the enhanced ATP synthesis, and to explore its bioenergetic consequences. A minimal model comprising the 'core' mechanism constituted by ATP synthase, driven by both proton (PMF) and potassium motive force (KMF), respiratory chain, adenine nucleotide translocator, Pi carrier, and K+/H+ exchanger (KHEmito) was able to simulate enhanced ATP synthesis and respiratory fluxes determined experimentally with isolated heart mitochondria. This capacity of F1Fo ATP synthase confers mitochondria with a significant energetic advantage compared to K+ transport through a channel not linked to oxidative phosphorylation (OxPhos). The K+-cycling mechanism requires a KHEmito that exchanges matrix K+ for intermembrane space H+, leaving PMF as the overall driving energy of OxPhos, in full agreement with the standard chemiosmotic mechanism. Experimental data of state 4➔3 energetic transitions, mimicking low to high energy demand, could be reproduced by an integrated computational model of mitochondrial function that incorporates the 'core' mechanism. Model simulations display similar behavior compared to the experimentally observed changes in ΔΨm, mitochondrial K+ uptake, matrix volume, respiration, and ATP synthesis during the energetic transitions at physiological pH and K+ concentration. The model also explores the role played by KHEmito in modulating the energetic performance of mitochondria. The results obtained support the available experimental evidence on ATP synthesis driven by K+ and H+ transport through the F1Fo ATP synthase.+ transport through the F1Fo ATP synthase.)
• Juhaszova 2021 Function (Oxf)  + (ATP synthase (F₁F&l … ATP synthase (F₁F_o) synthesizes daily our body's weight in ATP, whose production-rate can be transiently increased several-fold to meet changes in energy utilization. Using purified mammalian F₁F_o-reconstituted proteoliposomes and isolated mitochondria, we show F₁F_o can utilize both Δ''Ψ''_mt-driven H⁺- and K⁺-transport to synthesize ATP under physiological pH = 7.2 and K⁺ = 140 mEq/L conditions. Purely K⁺-driven ATP synthesis from single F₁F_o molecules measured by bioluminescence photon detection could be directly demonstrated along with simultaneous measurements of unitary K⁺ currents by voltage clamp, both blocked by specific F_o inhibitors. In the presence of K⁺, compared to osmotically-matched conditions in which this cation is absent, isolated mitochondria display 3.5-fold higher rates of ATP synthesis, at the expense of 2.6-fold higher rates of oxygen consumption, these fluxes being driven by a 2.7:1 K⁺: H⁺ stoichiometry. The excellent agreement between the functional data obtained from purified F₁F_o single molecule experiments and ATP synthase studied in the intact mitochondrion under unaltered OxPhos coupling by K⁺ presence, is entirely consistent with K⁺ transport through the ATP synthase driving the observed increase in ATP synthesis. Thus, both K⁺ (harnessing Δ''Ψ''_mt) and H⁺ (harnessing its chemical potential energy, Δ''μ''_H) drive ATP generation during normal physiology.istent with K⁺ transport through the ATP synthase driving the observed increase in ATP synthesis. Thus, both K⁺ (harnessing Δ''Ψ''_mt) and H⁺ (harnessing its chemical potential energy, Δ''μ''_H) drive ATP generation during normal physiology.)
• Juhaszova 2019 bioRxiv  + (ATP synthase (F₁F&l … ATP synthase (F₁F_o) synthesizes daily our body’s weight in ATP, whose production-rate can be transiently increased several-fold. Using purified mammalian F₁F_o-reconstituted proteoliposomes and isolated mitochondria, we show that F₁F_o utilizes both H⁺- and K⁺-transport (because of >10⁶-fold K⁺ excess vs H⁺) to drive ATP synthesis with the H⁺:K⁺ permeability of ~10⁶:1. F₁F_o can be upregulated by endogenous survival-related proteins (Bcl-xL, Mcl-1) and synthetic molecules (diazoxide, pinacidil) to increase its chemo-mechanical efficiency via IF₁. Increasing K⁺- and H⁺-driven ATP synthesis enables F₁F_o to operate as a primary mitochondrial K⁺-uniporter regulating energy supply-demand matching, and as the recruitable mitochondrial K_ATP-channel that can limit ischemia-reperfusion injury. Isolated mitochondria in the presence of K⁺ can sustain ~3.5-fold higher ATP-synthesis-flux (vs K⁺ absence) driven by a 2.7:1 K⁺:H⁺ stoichiometry with unaltered OxPhos coupling. Excellent agreement between F₁F_o single-molecule and intact-mitochondria experiments is consistent with K⁺-transport through ATP synthase driving a major fraction of ATP synthesis. (vs K⁺ absence) driven by a 2.7:1 K⁺:H⁺ stoichiometry with unaltered OxPhos coupling. Excellent agreement between F₁F_o single-molecule and intact-mitochondria experiments is consistent with K⁺-transport through ATP synthase driving a major fraction of ATP synthesis.)
• Walker 1994 Curr Opin Struct Biol  + (ATP synthase is regulated so as to prevent … ATP synthase is regulated so as to prevent futile hydrolysis of ATP when the transmembrane proton electrochemical gradient, delta mu H+, falls. Mitochondria and chloroplasts have different mechanisms for inhibition of ATP synthase: by binding an inhibitor protein, and by stabilization of the ADP-inhibited state by making an intramolecular disulphide bond, respectively. The recently determined structure of bovine F1-ATPase is locked in a conformation that probably represents the ADP-inhibited state of the enzyme.nts the ADP-inhibited state of the enzyme.)
• Klusch 2017 Elife  + (ATP synthases produce ATP by rotary cataly … ATP synthases produce ATP by rotary catalysis, powered by the electrochemical proton gradient across the membrane. Understanding this fundamental process requires an atomic model of the proton pathway. We determined the structure of an intact mitochondrial ATP synthase dimer by electron cryo-microscopy at near-atomic resolution. Charged and polar residues of the a-subunit stator define two aqueous channels, each spanning one half of the membrane. Passing through a conserved membrane-intrinsic helix hairpin, the lumenal channel protonates an acidic glutamate in the c-ring rotor. Upon ring rotation, the protonated glutamate encounters the matrix channel and deprotonates. An arginine between the two channels prevents proton leakage. The steep potential gradient over the sub-nm inter-channel distance exerts a force on the deprotonated glutamate, resulting in net directional rotation.te, resulting in net directional rotation.)
• Morciano 2017 Nat Protoc  + (ATP, the energy exchange factor that conne … ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes. In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. Recombinantly expressed wild-type luciferase is exclusively cytosolic; however, adding specific targeting sequences can modify its cellular localization. Using this strategy, we have constructed luciferase chimeras targeted to the mitochondrial matrix and the outer surface of the plasma membrane. Here, we describe optimized protocols for monitoring ATP concentrations in the cytosol, mitochondrial matrix and pericellular space in living cells via an overall procedure that requires an average of 3 d. In addition, we present a detailed protocol for the in vivo detection of extracellular ATP in mice using luciferase-transfected reporter cells. This latter procedure may require up to 25 d to complete.cedure may require up to 25 d to complete.)
• Bayot 2014 Biochimie  + (ATP-dependent proteases are currently emer … ATP-dependent proteases are currently emerging as key regulators of mitochondrial functions. Among these proteolytic systems, Lon protease is involved in the control of selective protein turnover in the mitochondrial matrix. In the absence of Lon, yeast cells have been shown to accumulate electron-dense inclusion bodies in the matrix space, to loose integrity of mitochondrial genome and to be respiratory deficient. In order to address the role of Lon in mitochondrial functionality in human cells, we have set up a HeLa cell line stably transfected with a vector expressing a shRNA under the control of a promoter which is inducible with doxycycline. We have demonstrated that reduction of Lon protease results in a mild phenotype in this cell line in contrast with what have been observed in other cell types such as WI-38 fibroblasts. Nevertheless, deficiency in Lon protease led to an increase in ROS production and to an accumulation of carbonylated protein in the mitochondria. Our study suggests that Lon protease has a wide variety of targets and is likely to play different roles depending of the cell type.ifferent roles depending of the cell type.)
• Kahancova 2018 FEBS Lett  + (ATPase Inhibitory factor 1 (IF1) is an end … ATPase Inhibitory factor 1 (IF1) is an endogenous regulator of mitochondrial ATP synthase, which is involved in cellular metabolism. Although great progress has been made, biological roles of IF1 and molecular mechanisms of its action are still to be elucidated. Here, we show that IF1 is present in pancreatic β-cells, bound to the ATP synthase also under normal physiological conditions. IF1 silencing in model pancreatic β-cells (INS-1E) increases insulin secretion over a range of glucose concentrations. The left-shifted dose-response curve reveals excessive insulin secretion even under low glucose, corresponding to fasting conditions. A parallel increase in cellular respiration and ATP levels is observed. To conclude, our results indicate that IF1 is a negative regulator of insulin secretion involved in pancreatic β-cell glucose sensing.lved in pancreatic β-cell glucose sensing.)
• ATSPB 2023 Hall AT  + (ATSPB 2023, Hall in Tirol, Austria, 2023)
• Gnaiger 2000 Transplant Proc  + (AUTOOXIDATION reactions of highly reduced … AUTOOXIDATION reactions of highly reduced organic compounds are a source of reactive oxygen species that contribute to ischemia/reperfusion injury. Antioxidants such as reduced glutathione (GSH; g-glutamylcysteinylglycine) are added to organ preservation solutions to reduce oxidative stress.^1,2 GSH is unstable, however, in University of Wisconsin (UW) solution in the presence of oxygen.³ To our knowledge, no reports are available on the stability of GSH in other organ preservation solutions, such as histidine-tryptophan-ketoglutarate (HTK) or Celsior solution. The present study reports for the first time a quantitative comparison of autooxidation of GSH in a variety of established preservation solutions, demonstrating in particular the high stability of GSH in HTK solution.nstrating in particular the high stability of GSH in HTK solution.)
• ARVO 2018 Honolulu HI US  + (AVRO - Association for Research in Vision and Ophthalmology, Honolulu, Hawaii, USA, 2018)
• Abcam Mitochondria Meeting 2014  + (Abcam Mitochondria Meeting 2014, London, UK; [http://www.abcam.com/index.html?pageconfig=resource&rid=16185&viapagetrap=mitochondriafeb Abcam Mitochondria Meeting 2014])
• Bricambert 2018 Nat Commun  + (Aberrant histone methylation profile is re … Aberrant histone methylation profile is reported to correlate with the development and progression of NAFLD during obesity. However, the identification of specific epigenetic modifiers involved in this process remains poorly understood. Here, we identify the histone demethylase Plant Homeodomain Finger 2 (Phf2) as a new transcriptional co-activator of the transcription factor Carbohydrate Responsive Element Binding Protein (ChREBP). By specifically erasing H3K9me2 methyl-marks on the promoter of ChREBP-regulated genes, Phf2 facilitates incorporation of metabolic precursors into mono-unsaturated fatty acids, leading to hepatosteatosis development in the absence of inflammation and insulin resistance. Moreover, the Phf2-mediated activation of the transcription factor NF-E2-related factor 2 (Nrf2) further reroutes glucose fluxes toward the pentose phosphate pathway and glutathione biosynthesis, protecting the liver from oxidative stress and fibrogenesis in response to diet-induced obesity. Overall, our findings establish a downstream epigenetic checkpoint, whereby Phf2, through facilitating H3K9me2 demethylation at specific gene promoters, protects liver from the pathogenesis progression of NAFLD.rom the pathogenesis progression of NAFLD.)
• Wu 2018 Adv Sci  + (Aberrant mitochondrial energy transfer und … Aberrant mitochondrial energy transfer underlies prevalent chronic health conditions, including cancer, cardiovascular, and neurodegenerative diseases. Mitochondrial transplantation represents an innovative strategy aimed at restoring favorable metabolic phenotypes in cells with dysfunctional energy metabolism. While promising, significant barriers to ''in vivo'' translation of this approach abound, including limited cellular uptake and recognition of mitochondria as foreign. The objective is to functionalize isolated mitochondria with a biocompatible polymer to enhance cellular transplantation and eventual ''in vivo'' applications. Herein, it is demonstrated that grafting of a polymer conjugate composed of dextran with triphenylphosphonium onto isolated mitochondria protects the organelles and facilitates cellular internalization compared with uncoated mitochondria. Importantly, mitochondrial transplantation into cancer and cardiovascular cells has profound effects on respiration, mediating a shift toward improved oxidative phosphorylation, and reduced glycolysis. These findings represent the first demonstration of polymer functionalization of isolated mitochondria, highlighting a viable strategy for enabling clinical applications of mitochondrial transplantation.ications of mitochondrial transplantation.)
• Scrima 2020 bioRxiv  + (Abnormal hemoglobins can have major conseq … Abnormal hemoglobins can have major consequences for tissue delivery of oxygen. Correct diagnosis of hemoglobinopathies with altered oxygen affinity requires a determination of hemoglobin oxygen dissociation curve (ODC), which relates the hemoglobin oxygen saturation to the partial pressure of oxygen in the blood. Determination of the ODC of human hemoglobin is typically carried out under conditions in which hemoglobin is in equilibrium with O₂ at each partial pressure. However, in the human body due to the fast transit of RBCs through tissues hemoglobin oxygen exchanges occur under non-equilibrium conditions. We describe the determination of non-equilibrium ODC, and show that under these conditions Hb cooperativity has two apparent components in the Adair, Perutz, and MWC models of Hb. The first component, which we call sequential cooperativity, accounts for ∼70% of Hb cooperativity, and emerges from the constraint of sequential binding that is shared by the three models. The second component, which we call conformational cooperativity, accounts for ∼30% of Hb cooperativity, and is due either to a conformational equilibrium between low affinity and high affinity tetramers (as in the MWC model), or to a conformational change from low to high affinity once two of the tetramer sites are occupied (Perutz model).two of the tetramer sites are occupied (Perutz model).)
• Szabo 2020 Int J Mol Sci  + (Abnormal tau protein aggregation in the br … Abnormal tau protein aggregation in the brain is a hallmark of tauopathies, such as frontotemporal lobar degeneration and Alzheimer's disease. Substantial evidence has been linking tau to neurodegeneration, but the underlying mechanisms have yet to be clearly identified. Mitochondria are paramount organelles in neurons, as they provide the main source of energy (adenosine triphosphate) to these highly energetic cells. Mitochondrial dysfunction was identified as an early event of neurodegenerative diseases occurring even before the cognitive deficits. Tau protein was shown to interact with mitochondrial proteins and to impair mitochondrial bioenergetics and dynamics, leading to neurotoxicity. In this review, we discuss in detail the different impacts of disease-associated tau protein on mitochondrial functions, including mitochondrial transport, network dynamics, mitophagy and bioenergetics. We also give new insights about the effects of abnormal tau protein on mitochondrial neurosteroidogenesis, as well as on the endoplasmic reticulum-mitochondria coupling. A better understanding of the pathomechanisms of abnormal tau-induced mitochondrial failure may help to identify new targets for therapeutic interventions.new targets for therapeutic interventions.)
• Valis 2017 Oncotarget  + (Abnormalities in cancer metabolism represe … Abnormalities in cancer metabolism represent potential targets for cancer therapy. We have recently identified a natural compound Quambalarine B (QB), which inhibits proliferation of several leukemic cell lines followed by cell death. We have predicted ubiquinone binding sites of mitochondrial respiratory complexes as potential molecular targets of QB in leukemia cells. Hence, we tracked the effect of QB on leukemia metabolism by applying several omics and biochemical techniques. We have confirmed the inhibition of respiratory complexes by QB and found an increase in the intracellular AMP levels together with respiratory substrates. Inhibition of mitochondrial respiration by QB triggered reprogramming of leukemic cell metabolism involving disproportions in glycolytic flux, inhibition of proteins O-glycosylation, stimulation of glycine synthesis pathway, and pyruvate kinase activity, followed by an increase in pyruvate and a decrease in lactate levels. Inhibition of mitochondrial complex I by QB suppressed folate metabolism as determined by a decrease in formate production. We have also observed an increase in cellular levels of several amino acids except for aspartate, indicating the dependence of Jurkat (T-ALL) cells on aspartate synthesis. These results indicate blockade of mitochondrial complex I and II activity by QB and reduction in aspartate and folate metabolism as therapeutic targets in T-ALL cells. Anti-cancer activity of QB was also confirmed during ''in vivo'' studies, suggesting the therapeutic potential of this natural compound.peutic potential of this natural compound.)
• Roy Chowdhury 2018 Mol Cell Neurosci  + (Abnormalities in mitochondrial function un … Abnormalities in mitochondrial function under diabetic conditions can lead to deficits in function of cortical neurons and their support cells exhibiting a pivotal role in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. We aimed to assess mitochondrial respiration rates and membrane potential or H₂O₂ generation simultaneously and expression of proteins involved in mitochondrial dynamics, ROS scavenging and AMPK/SIRT/PGC-1α pathway activity in cortex under diabetic conditions.</br></br>Cortical mitochondria from streptozotocin (STZ)-induced type 1 diabetic rats or mice, and aged-matched controls were used for simultaneous measurements of mitochondrial respiration rates and mitochondrial membrane potential (mtMP) or H₂O₂ using Oroboros oxygraph. Measurements of enzymatic activities of respiratory complexes were performed using spectophotometry. Protein levels in cortical mitochondria and homogenates were determined by Western blotting.</br></br>Mitochondrial coupled respiration rates and FCCP-induced uncoupled respiration rates were significantly decreased in mitochondria of cortex of STZ-diabetic rats compared to controls. The mtMP in the presence of ADP was significantly depolarized and succinate-dependent respiration rates and H₂O₂ were significantly diminished in cortical mitochondria of diabetic animals compared to controls, accompanied with reduced expression of CuZn- and Mn-superoxide dismutase. The enzymatic activities of Complex I, II, and IV and protein levels of certain components of Complex I and II, mitofusin 2 (Mfn2), dynamin-related protein 1 (DRP1), P-AMPK, SIRT2 and PGC-1α were significantly diminished in diabetic cortex.</br></br>Deficits in mitochondrial function, dynamics, and antioxidant capabilities putatively mediated through sub-optimal AMPK/SIRT/PGC-1α signaling, are involved in the development of early sub-clinical neurodegeneration in the cortex under diabetic conditions.</br></br><small>Copyright © 2018. Published by Elsevier Inc.</small>degeneration in the cortex under diabetic conditions. <small>Copyright © 2018. Published by Elsevier Inc.</small>)
• Jaramillo-Jimenez 2023 Mitochondrion  + (Abnormalities in the Tri-Carboxylic-Acid ( … Abnormalities in the Tri-Carboxylic-Acid (TCA) cycle have been documented in dementia. Through network analysis, TCA cycle metabolites could indirectly reflect known dementia-related abnormalities in biochemical pathways, and key metabolites might be associated with prognosis. This study analyzed TCA cycle metabolites as predictors of cognitive decline in a mild dementia cohort and explored potential interactions with the diagnosis of Lewy Body Dementia (LBD) or Alzheimer's Disease (AD) and APOE-ε4 genotype. We included 145 mild dementia patients (LBD = 59; AD = 86). Serum TCA cycle metabolites were analyzed at baseline, and partial correlation networks were conducted. Cognitive performance was measured annually over 5-years with the Mini-mental State Examination. Longitudinal mixed-effects Tobit models evaluated each baseline metabolite as a predictor of 5-years cognitive decline. APOE-ε4 and diagnosis interactions were explored. Results showed comparable metabolite concentrations in LBD and AD. Multiple testing corrected networks showed larger coefficients for a negative correlation between pyruvate - succinate and positive correlations between fumarate - malate and citrate - Isocitrate in both LBD and AD. In the total sample, adjusted mixed models showed significant associations between baseline citrate concentration and longitudinal MMSE scores. In APOE-ε4 carriers, baseline isocitrate predicted MMSE scores. We conclude that, in mild dementia, serum citrate concentrations could be associated with subsequent cognitive decline, as well as isocitrate concentrations in APOE-ε4 carriers. Downregulation of enzymatic activity in the first half of the TCA cycle (decarboxylating dehydrogenases), with upregulation in the latter half (dehydrogenases only), might be indirectly reflected in serum TCA cycle metabolites' networks. in serum TCA cycle metabolites' networks.)
• Ahluwalia 2017 Sci Rep  + (About two decades ago, West and coworkers … About two decades ago, West and coworkers established a model which predicts that metabolic rate follows a three quarter power relationship with the mass of an organism, based on the premise that tissues are supplied nutrients through a fractal distribution network. Quarter power scaling is widely considered a universal law of biology and it is generally accepted that were in-vitro cultures to obey allometric metabolic scaling, they would have more predictive potential and could, for instance, provide a viable substitute for animals in research. This paper outlines a theoretical and computational framework for establishing quarter power scaling in three-dimensional spherical constructs in-vitro, starting where fractal distribution ends. Allometric scaling in non-vascular spherical tissue constructs was assessed using models of Michaelis Menten oxygen consumption and diffusion. The models demonstrate that physiological scaling is maintained when about 5 to 60% of the construct is exposed to oxygen concentrations less than the Michaelis Menten constant, with a significant concentration gradient in the sphere. The results have important implications for the design of downscaled in-vitro systems with physiological relevance.itro systems with physiological relevance.)
• Skalska 2009 Int J Mol Sci  + (Abstract: The mitochondrial response to ch … Abstract: The mitochondrial response to changes of cytosolic calcium concentration has a strong impact on neuronal cell metabolism and viability. We observed that Ca²⁺ additions to isolated rat brain mitochondria induced in potassium ion containing media a mitochondrial membrane potential depolarization and an accompanying increase of mitochondrial respiration. These Ca²⁺ effects can be blocked by iberiotoxin and charybdotoxin, well known inhibitors of large conductance potassium channel (BKCa channel). Furthermore, NS1619 – a BKCa channel opener – induced potassium ion–specific effects on brain mitochondria similar to those induced by Ca²⁺. These findings suggest the presence of a calcium-activated, large conductance potassium channel (sensitive to charybdotoxin and NS1619), which was confirmed by reconstitution of the mitochondrial inner membrane into planar lipid bilayers. The conductance of the reconstituted channel was 265 pS under gradient (50/450 mM KCl) conditions. Its reversal potential was equal to 50 mV, which proved that the examined channel was cation-selective. We also observed immunoreactivity of anti-β₄ subunit (of the BKCa channel) antibodies with ~26 kDa proteins of rat brain mitochondria. Immunohistochemical analysis confirmed the predominant occurrence of anti-β₄ subunit in neuronal mitochondria. We hypothesize that the mitochondrial BKCa channel represents a calcium sensor, which can contribute to neuronal signal transduction and survival.hannel represents a calcium sensor, which can contribute to neuronal signal transduction and survival.)
• Klein 2018 Int J Digit Libr  + (Academic publishers claim that they add va … Academic publishers claim that they add value to scholarly communications by coordinating reviews and contributing and enhancing text during publication. These contributions come at a considerable cost: US academic libraries paid $1.7 billion for serial subscriptions in 2008 alone. Library budgets, in contrast, are flat and not able to keep pace with serial price inflation. We have investigated the publishers’ value proposition by conducting a comparative study of pre-print papers from two distinct science, technology, and medicine corpora and their final published counterparts. This comparison had two working assumptions: (1) If the publishers’ argument is valid, the text of a pre-print paper should vary measurably from its corresponding final published version, and (2) by applying standard similarity measures, we should be able to detect and quantify such differences. Our analysis revealed that the text contents of the scientific papers generally changed very little from their pre-print to final published versions. These findings contribute empirical indicators to discussions of the added value of commercial publishers and therefore should influence libraries’ economic decisions regarding access to scholarly publications.egarding access to scholarly publications.)
• Teixeira 2022 J Gen Philos Sci  + (Academic publishing is undergoing a highly … Academic publishing is undergoing a highly transformative process, and many established rules and value systems that are in place, such as traditional peer review (TPR) and preprints, are facing unprecedented challenges, including as a result of post-publication peer review. The integrity and validity of the academic literature continue to rely naively on blind trust, while TPR and preprints continue to fail to effectively screen out errors, fraud, and misconduct. Imperfect TPR invariably results in imperfect papers that have passed through varying levels of rigor of screening and validation. If errors or misconduct were not detected during TPR's editorial screening, but are detected at the post-publication stage, an opportunity is created to correct the academic record. Currently, the most common forms of correcting the academic literature are errata, corrigenda, expressions of concern, and retractions or withdrawals. Some additional measures to correct the literature have emerged, including manuscript versioning, amendments, partial retractions and retract and replace. Preprints can also be corrected if their version is updated. This paper discusses the risks, benefits and limitations of these forms of correcting the academic literature.rms of correcting the academic literature.)

• Coelho 2016 Oncol Rep  + (Acceleration of glycolysis is a characteri … Acceleration of glycolysis is a characteristic of neoplasia. Previous studies have shown that a metabolic shift occurs in many tumors and correlates with a negative prognosis. The present study aimed to investigate the glycolytic profile of thyroid carcinoma cell lines. We investigated glycolytic and oxidative parameters of two thyroid carcinoma papillary cell lines (BCPAP and TPC1) and the non-tumor cell line NTHY-ori. All carcinoma cell lines showed higher rates of glycolysis efficiency, when compared to NTHY-ori, as assessed by a higher rate of glucose consumption and lactate production. The BCPAP cell line presented higher rates of growth, as well as elevated intracellular ATP levels, compared to the TPC1 and NTHY-ori cells. We found that glycolysis and activities of pentose phosphate pathway (PPP) regulatory enzymes were significantly different among the carcinoma cell lines, particularly in the mitochondrial hexokinase (HK) activity which was higher in the BCPAP cells than that in the TPC1 cell line which showed a balanced distribution of HK activity between cytoplasmic and mitochondrial subcellular localizations. However, TPC1 had higher levels of glucose‑6-phosphate dehydrogenase activity, suggesting that the PPP is elevated in this cell type. Using high resolution respirometry, we observed that the Warburg effect was present in the BCPAP and TPC1 cells, characterized by low oxygen consumption and high reactive oxygen species production. Overall, these results indicate that both thyroid papillary carcinoma cell lines showed a glycolytic profile. Of note, BCPAP cells presented some relevant differences in cell metabolism compared to TPC1 cells, mainly related to higher mitochondrial-associated HK activity.gher mitochondrial-associated HK activity.)
• Acclimatization and High Altitude Illness - Facts and Myths 2017 Brixen Dolomites IT  + (Acclimatization and High Altitude Illness - Facts and Myths, Brixen Dolomites, IT)
• Arias-Reyes 2023 MitoFit  + (Acclimatization to high altitude relies on … Acclimatization to high altitude relies on adjustments of cellular metabolism that optimize oxygen use and energy production. In tissues with high energy demand and almost exclusive reliance on aerobic metabolism such as the brain, hypoxia is a particularly strong stressor, however, strategies to adjust metabolic pathways for successful high-altitude acclimatization remain poorly understood. Compared to SD rats, FVB mice show successful acclimatization to high altitude, we, therefore, used this model to investigate metabolic adjustments in the retrosplenial cortex (a key area of the brain involved in spatial learning and navigation) in normoxia and during acclimatization to hypoxia (12 % O₂ – 1, 7, and 21 days). We measured in simultaneous the rates of ATP synthesis and O₂ consumption in fresh permeabilized brain samples by coupled high-resolution respirometry and fluorometry. We quantified the citrate synthase (CS) activity as an index of mitochondrial content, the transcriptional regulation of genes involved in mitochondrial dynamics; and the activity of enzymes representative of the glycolytic, aerobic, and anaerobic metabolism. Our findings show that acclimatization to hypoxia significantly increases ATP synthesis in mice and to a lower extent in rats. In mice, this occurs in parallel with a reduction of O₂ consumption, and a three-fold increase in the P»/O ratio. In rats, a six-fold increase in CS activity and altered mitochondrial dynamics gene expression are evident. Finally, activities of glycolytic, aerobic, or anaerobic enzymes remain overall unchanged in both species except for a transient glycolytic and anaerobic peak at day 7 in mice. Altogether, our results show that chronic hypoxia optimizes the efficiency of mitochondrial ATP synthesis in the retrosplenial cortex of mice. Contrastingly, rats sustain the production of ATP only by increasing mitochondrial content and altering mitochondrial dynamics, suggesting drastic mitochondrial malfunctions.<br>ing mitochondrial dynamics, suggesting drastic mitochondrial malfunctions.<br>)
• Schoenfeld 2016 J Cereb Blood Flow Metab  + (According to recent reports, systemic trea … According to recent reports, systemic treatment of rats with methylpalmoxirate (carnitine palmitoyltransferase-1 inhibitor) decreased peroxidation of polyunsaturated fatty acids in brain tissue. This was taken as evidence of mitochondrial β-oxidation in brain, thereby contradicting long-standing paradigms of cerebral metabolism, which claim that β-oxidation of activated fatty acids has minor importance for brain energy homeostasis. We addressed this controversy. Our experiments are the first direct experimental analysis of this question. We fueled isolated brain mitochondria or rat brain astrocytes with octanoic acid, but octanoic acid does not enhance formation of reactive oxygen species, neither in isolated brain mitochondria nor in astrocytes, even at limited hydrogen delivery to mitochondria. Thus, octanoic acid or l-octanoylcarnitine does not stimulate H₂O₂ release from brain mitochondria fueled with malate, in contrast to liver mitochondria (2.25-fold rise). This does obviously not support the possible occurrence of β-oxidation of the fatty acid octanoate in the brain. We conclude that a proposed inhibition of β-oxidation does not seem to be a helpful strategy for therapies aiming at lowering oxidative stress in cerebral tissue. This question is important, since oxidative stress is the cause of neurodegeneration in numerous neurodegenerative or inflammatory disease situations.</br></br>© The Author(s) 2016.erative or inflammatory disease situations. © The Author(s) 2016.)
• Roskams T Falk Workshop Inflammation & Cancer  + (According to the cancer stem cell concept, … According to the cancer stem cell concept, hepatocellular carcinoma (HCC) consists of a hierarchy of cell populations, of which the very small cancer stem cell population is the one that has the growth and metastatic potential of the tumour. The other neoplastic cells are offspring of the cancer stem cells and each can differentiate a little differently, according to the local microenvironment in each part of the tumor, hence explaining the enormous phenotypic heterogeneity of a neoplasm. </br></br>Current therapeutic strategies mostly target rapidly growing differentiated tumour cells. However the results are often unsatisfactory because of the chemoresistance of HCC. New therapies targeting cancer stem cells should therefore be developed. A prerequisite is a good understanding of the mechanisms of activation and differentiation of normal stem/progenitor cells in normal and diseased liver. Hepatocytes and cholangiocytes have stem cell features, but also progenitor cells, located in the smallest branches of the biliary tree. These cells are especially activated in the cirrhotic stage of chronic liver diseases, the stage in which HCC develops. HCC with progenitor cell features, possibly reflecting a progenitor cell origin, have a very bad prognosis and therefore should be recognized and treated accordingly.uld be recognized and treated accordingly.)
• Huetter 2007 Aging Cell  + (According to the free radical theory of ag … According to the free radical theory of aging, reactive oxygen species (ROS) act as a driving force of the aging process, and it is generally believed that mitochondrial dysfunction is a major source of increased oxidative stress in tissues with high content of mitochondria, such as muscle or brain. However, recent experiments in mouse models of premature aging have questioned the role of mitochondrial ROS production in premature aging. To address the role of mitochondrial impairment and ROS production for aging in human muscles, we have analyzed mitochondrial properties in muscle fibres isolated from the vastus lateralis of young and elderly donors. Mitochondrial respiratory functions were addressed by high-resolution respirometry, and ROS production was analyzed by in situ staining with the redox-sensitive dye dihydroethidium. We found that aged human skeletal muscles contain fully functional mitochondria and that the level of ROS production is higher in young compared to aged muscle. Accordingly, we could not find any increase in oxidative modification of proteins in muscle from elderly donors. However, the accumulation of lipofuscin was identified as a robust marker of human muscle aging. The data support a model, where ROS-induced molecular damage is continuously removed, preventing the accumulation of dysfunctional mitochondria despite ongoing ROS production.tochondria despite ongoing ROS production.)
• Huisamen 2015 Abstract MiPschool Cape Town 2015  + (According to the latest statistics, variou … According to the latest statistics, various cardiovascular diseases</br>accounted for 8.3% of natural deaths in South Africa during 2013,</br>ranking the 6th place as cause of mortality. With the efficiency</br>of therapies aimed at decreasing mortality from heart disease, life</br>expectancy increased. As result of this, the focus of recent research</br>changed towards understanding the energy demands of the heart in</br>order to optimize function. Because of its high energetic needs, the</br>human heart utilizes between 3.5 and 6 kg of ATP per day to function.</br>This is produced by its mitochondrial populations which occupy up to</br>50% of the volume of a cardiomyocyte. A close link therefore exists</br>between mitochondrial dysfunction and heart disease. In addition, there</br>is growing recognition that inborn errors of metabolism can influence</br>cardiomyocyte dysfunction [1] and that primary inherited mitochondrial</br>diseases display a full spectrum of cardiac disorders [2].</br>ATM is a 350kDa serine/threonine protein kinase displaying homologies</br>to the large protein family of PI3-Kinases, although it lacks the ability to</br>phosphorylate lipids [3]. It came under scrutiny because of the disease,</br>Ataxia-telangiectasie (A-T), which is an autosomal, recessive disorder</br>that progressively affects multiple organs. This disease is caused by</br>mutations in the Atm gene, resulting in lack or inactivation of the ATM</br>protein [4]. ATM in the cell can be localized to the nucleus, cytoplasm of</br>mitochondria.</br></br>We became interested in myocardial ATM because it was found that</br>skeletal muscle of insulin resistant, obese rats had dramatically reduced</br>levels of the so-called ATM protein, in association with the well-known</br>reduced activation of the insulin/ phosphatidylinositol 3 kinases (PI3-</br>kinase)/PKB/Akt pathway, which is the main mechanism of relaying the</br>metabolic effects of insulin [5]. Foster et al [6] found structural and functional changes in the hearts of ATM KO mice, using echocardiography and</br>Doppler echocardiography.</br></br>The mitochondrial association of ATM protein kinase plays an important</br>role in its integrity and functioning such that ATM deficiency results in</br>defects in mitochondrial respiration [7]. ATM also regulates mitochondrial</br>biogenesis and DNA content [8]. In addition, it was demonstrated that a</br>mitochondria-targeted antioxidant MitoQ, could decrease the features of</br>the metabolic syndrome in ATM+/-ApoE-/- mice [9]. This may be because</br>one of the mechanisms known to activate the ATM protein is increased</br>oxidative stress. Activated ATM initiates an anti-oxidant response based</br>on a metabolic shift while, in fibroblast cell lines, inactivation of ATM</br>is associated with increased ROS levels followed by expression and</br>activation of the transcription factor HIF-1alpha [10]. the transcription factor HIF-1alpha [10].)
• Kozieł 2011 J Invest Dermatol  + (According to the mitochondrial theory of a … According to the mitochondrial theory of aging, reactive oxygen species (ROS) derived primarily from mitochondria cause cumulative oxidative damage to various cellular molecules and thereby contribute to the aging process. On the other hand, a pivotal role of the proteasome, as a main proteolytic system implicated in the degradation of oxidized proteins during aging, is suggested. In this study, we analyzed mitochondrial function in dermal fibroblasts derived from biopsies obtained from healthy young, middle-aged, and old donors. We also determined proteasome activity in these cells, using a degron-destabilized green fluorescent protein (GFP)-based reporter protein. We found a significant decrease in mitochondrial membrane potential in samples from aged donors, accompanied by a significant increase in ROS levels. Respiratory activity was not significantly altered with donor age, probably reflecting genetic variation. Proteasome activity was significantly decreased in fibroblasts from middle-aged donors compared with young donors; fibroblasts derived from the oldest donors displayed a high heterogeneity in this assay. We also found intraindividual coregulation of mitochondrial and proteasomal activities in all human fibroblast strains tested, suggesting that both systems are interdependent. Accordingly, pharmacological inhibition of the proteasome led to decreased mitochondrial function, whereas inhibition of mitochondrial function in turn reduced proteasome activity.ction in turn reduced proteasome activity.)
• Schiffer 2013 Abstract MiP2013  + (According to the rate-of-living and oxidat … According to the rate-of-living and oxidative damage theory of aging, extended lifespan is predicted by low energy metabolism and low reactive oxygen species production rates. Recently, several studies show that dietary inorganic nitrate mainly present in vegetables can reduce oxygen consumption during physical exercise in humans [1] and contribute to attenuated oxidative stress in animal models of disease [2]. Nitrate accumulates in saliva and is bioactivated through reduction to nitrite by oral bacteria. </br></br>We examined the effects of dietary nitrate on basal metabolic rate (BMR) and markers of oxidative stress in man using a double-blind, randomized cross over design. 15 young healthy males volunteered and indirect calorimetry was used to determine basal metabolic rate after three days of dietary intervention with sodium nitrate (NaNO3, 0.1 mmol∙kg-1∙day-1) or placebo (NaCl). The administered amount of nitrate resembles what is found in 100-300 g of nitrate rich vegetables such as beetroot or spinach. </br></br>The intervention reduced BMR by 4.3% after nitrate administration compared with placebo (p<0.02). A strong negative correlation was found between the change in salivary nitrate and the change in BMR (r2=0.72; p<0.002). In addition, nitrate supplementation reduced plasma levels of malondialdehyde, indicating lower oxidative stress as a result of the intervention. Thyroid hormone status was unaffected.</br></br>The cuisines of cultures known for their longevity are usually rich in vegetables and future studies will reveal whether this life span extension is linked to the high nitrate content in this food group. to the high nitrate content in this food group.)
• Nelson 2016 J Fish Biol  + (Accounting for energy use by fishes has be … Accounting for energy use by fishes has been taking place for over 200 years. The original, and continuing gold standard for measuring energy use in terrestrial animals, is to account for the waste heat produced by all reactions of metabolism, a process referred to as direct calorimetry. Direct calorimetry is not easy or convenient in terrestrial animals and is extremely difficult in aquatic animals. Thus, the original and most subsequent measurements of metabolic activity in fishes have been measured via indirect calorimetry. Indirect calorimetry takes advantage of the fact that oxygen is consumed and carbon dioxide is produced during the catabolic conversion of foodstuffs or energy reserves to useful ATP energy. As measuring [CO₂] in water is more challenging than measuring [O₂], most indirect calorimetric studies on fishes have used the rate of O₂ consumption. To relate measurements of O₂ consumption back to actual energy usage requires knowledge of the substrate being oxidized. Many contemporary studies of O₂ consumption by fishes do not attempt to relate this measurement back to actual energy usage. Thus, the rate of oxygen consumption has become a measurement in its own right that is not necessarily synonymous with metabolic rate. Because all extant fishes are obligate aerobes (many fishes engage in substantial net anaerobiosis, but all require oxygen to complete their life cycle), this discrepancy does not appear to be of great concern to the fish biology community, and reports of fish oxygen consumption, without being related to energy, have proliferated. Unfortunately, under some circumstances, these measures can be quite different from one another. A review of the methodological history of the two measurements and a look towards the future are included.view of the methodological history of the two measurements and a look towards the future are included.)
• Fitzgerald 2024 J Cachexia Sarcopenia Muscle  + (Accumulating evidence has demonstrated tha … Accumulating evidence has demonstrated that chronic tobacco smoking directly contributes to skeletal muscle dysfunction independent of its pathological impact to the cardiorespiratory systems. The mechanisms underlying tobacco smoke toxicity in skeletal muscle are not fully resolved. In this study, the role of the aryl hydrocarbon receptor (AHR), a transcription factor known to be activated with tobacco smoke, was investigated.</br></br>AHR related gene (mRNA) expression was quantified in skeletal muscle from adult controls and patients with chronic obstructive pulmonary disease (COPD), as well as mice with and without cigarette smoke exposure. Utilizing both skeletal muscle-specific AHR knockout mice exposed to chronic repeated (5 days per week for 16 weeks) cigarette smoke and skeletal muscle-specific expression of a constitutively active mutant AHR in healthy mice, a battery of assessments interrogating muscle size, contractile function, mitochondrial energetics, and RNA sequencing were employed.</br></br>Skeletal muscle from COPD patients (N = 79, age = 67.0 ± 8.4 years) had higher levels of AHR (P = 0.0451) and CYP1B1 (P < 0.0001) compared to healthy adult controls (N = 16, age = 66.5 ± 6.5 years). Mice exposed to cigarette smoke displayed higher expression of Ahr (P = 0.008), Cyp1b1 (P < 0.0001), and Cyp1a1 (P < 0.0001) in skeletal muscle compared to air controls. Cigarette smoke exposure was found to impair skeletal muscle mitochondrial oxidative phosphorylation by ~50% in littermate controls (Treatment effect, P < 0.001), which was attenuated by deletion of the AHR in muscle in male (P = 0.001), but not female, mice (P = 0.37), indicating there are sex-dependent pathological effects of smoking-induced AHR activation in skeletal muscle. Viral mediated expression of a constitutively active mutant AHR in the muscle of healthy mice recapitulated the effects of cigarette smoking by decreasing muscle mitochondrial oxidative phosphorylation by ~40% (P = 0.003).</br></br>These findings provide evidence linking chronic AHR activation secondary to cigarette smoke exposure to skeletal muscle bioenergetic deficits in male, but not female, mice. AHR activation is a likely contributor to the decline in muscle oxidative capacity observed in smokers and AHR antagonism may provide a therapeutic avenue aimed to improve muscle function in COPD.eutic avenue aimed to improve muscle function in COPD.)
• Ying 2008 Antioxid Redox Signal  + (Accumulating evidence has suggested that N … Accumulating evidence has suggested that NAD (including NAD⁺ and NADH) and NADP (including NADP⁺ and NADPH) could belong to the fundamental common mediators of various biological processes, including energy metabolism, mitochondrial functions, calcium homeostasis, antioxidation/generation of oxidative stress, gene expression, immunological functions, aging, and cell death: First, it is established that NAD mediates energy metabolism and mitochondrial functions; second, NADPH is a key component in cellular antioxidation systems; and NADH-dependent reactive oxygen species (ROS) generation from mitochondria and NADPH oxidase-dependent ROS generation are two critical mechanisms of ROS generation; third, cyclic ADP-ribose and several other molecules that are generated from NAD and NADP could mediate calcium homeostasis; fourth, NAD and NADP modulate multiple key factors in cell death, such as mitochondrial permeability transition, energy state, poly(ADP-ribose) polymerase-1, and apoptosis-inducing factor; and fifth, NAD and NADP profoundly affect aging-influencing factors such as oxidative stress and mitochondrial activities, and NAD-dependent sirtuins also mediate the aging process. Moreover, many recent studies have suggested novel paradigms of NAD and NADP metabolism. Future investigation into the metabolism and biological functions of NAD and NADP may expose fundamental properties of life, and suggest new strategies for treating diseases and slowing the aging process.ew strategies for treating diseases and slowing the aging process.)
• Abu Bakar 2020 Eur J Pharmacol  + (Accumulating evidence indicates that adipo … Accumulating evidence indicates that adipose tissue inflammation and mitochondrial dysfunction in skeletal muscle are inextricably linked to obesity and insulin resistance. Celastrol, a bioactive compound derived from the root of Tripterygium wilfordii exhibits a number of attributive properties to attenuate metabolic dysfunction in various cellular and animal disease models. However, the underlying therapeutic mechanisms of celastrol in the obesogenic environment "in vivo" remain elusive. Therefore, the current study investigated the metabolic effects of celastrol on insulin sensitivity, inflammatory response in adipose tissue and mitochondrial functions in skeletal muscle of the high fat diet (HFD)-induced obese rats. Our study revealed that celastrol supplementation at 3 mg/kg/day for 8 weeks significantly reduced the final body weight and enhanced insulin sensitivity of the HFD-fed rats. Celastrol noticeably improved insulin-stimulated glucose uptake activity and increased expression of plasma membrane GLUT4 protein in skeletal muscle. Moreover, celastrol-treated HFD-fed rats showed attenuated inflammatory responses via decreased NF-κB activity and diminished mRNA expression responsible for classically activated macrophage (M1) polarization in adipose tissues. Significant improvement of muscle mitochondrial functions and enhanced antioxidant defense machinery via restoration of mitochondrial complexes I + III linked activity were effectively exhibited by celastrol treatment. Mechanistically, celastrol stimulated mitochondrial biogenesis attributed by upregulation of the adenosine monophosphate-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) signaling pathways. Together, these results further demonstrate heretofore the conceivable therapeutic mechanisms of celastrol "in vivo" against HFD-induced obesity mediated through attenuation of inflammatory response in adipose tissue and enhanced mitochondrial functions in skeletal muscle.itochondrial functions in skeletal muscle.)
• Arena 2018 Mol Cell  + (Accumulating evidence indicates that the M … Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) ''in vitro'' and ''in vivo'', impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression.y and may contribute to tumor progression.)
• Yang 2018 Cell Death Dis  + (Accumulating evidence revealed that mesenc … Accumulating evidence revealed that mesenchymal stem cells (MSCs) confer cardioprotection against myocardial infarction (MI). However, the poor survival and engraftment rate of the transplanted cells limited their therapeutic efficacy in the heart. The enhanced leptin production associated with hypoxia preconditioning contributed to the improved MSCs survival. Mitochondrial integrity determines the cellular fate. Thus, we aimed to investigate whether leptin can enhance mitochondrial integrity of human MSCs (hMSCs) to protect against various stress. ''In vivo'', transplantation of leptin-overexpressing hMSCs into the infarcted heart resulted in improved cell viability, leading to enhanced angiogenesis and cardiac function. ''In vitro'', pretreatment of hMSCs with recombinant leptin (hMSCs-Lep^pre) displayed improved cell survival against severe ischemic condition (glucose and serum deprivation under hypoxia), which was associated with increased mitochondrial fusion. Subsequently, Optic atrophy 1 (OPA1), a mitochondrial inner membrane protein that regulates fusion and cristae structure, was significantly elevated in the hMSCs-Lep^pre group, and the protection of leptin was abrogated by targeting OPA1 with a selective siRNA. Furthermore, OMA1, a mitochondrial protease that cleaves OPA1, decreased in a leptin-dependent manner. Pretreatment of cells with an inhibitor of the proteasome (MG132), prevented leptin-induced OMA1 degradation, implicating the ubiquitination/proteasome system as a part of the protective leptin pathway. In addition, GSK3 inhibitor (SB216763) was also involved in the degradation of OMA1. In conclusion, in the hostile microenvironment caused by MI, (a) leptin can maintain the mitochondrial integrity and prolong the survival of hMSCs; (b) leptin-mediated mitochondrial integrity requires phosphorylation of GSK3 as a prerequisite for ubiquitination-depended degradation of OMA1 and attenuation of long-OPA1 cleavage. Thus, leptin targeting the GSK3/OMA1/OPA1 signaling pathway can optimize hMSCs therapy for cardiovascular diseases such as MI.can optimize hMSCs therapy for cardiovascular diseases such as MI.)
• Yan 2015 BMC Cancer  + (Accumulating evidence suggests that breast … Accumulating evidence suggests that breast cancer involves tumour-initiating cells (TICs), which play a role in initiation, metastasis, therapeutic resistance and relapse of the disease. Emerging drugs that target TICs are becoming a focus of contemporary research. Mitocans, a group of compounds that induce apoptosis of cancer cells by destabilising their mitochondria, are showing their potential in killing TICs. In this project, we investigated mitochondrially targeted vitamin E succinate (MitoVES), a recently developed mitocan, for its ''in vitro'' and ''in vivo'' efficacy against TICs.</br></br>The mammosphere model of breast TICs was established by culturing murine NeuTL and human MCF7 cells as spheres. This model was verified by stem cell marker expression, tumour initiation capacity and chemotherapeutic resistance. Cell susceptibility to MitoVES was assessed and the cell death pathway investigated. ''In vivo'' efficacy was studied by grafting NeuTL TICs to form syngeneic tumours.</br></br>Mammospheres derived from NeuTL and MCF7 breast cancer cells were enriched in the level of stemness, and the sphere cells featured altered mitochondrial function. Sphere cultures were resistant to several established anti-cancer agents while they were susceptible to MitoVES. Killing of mammospheres was suppressed when the mitochondrial complex II, the molecular target of MitoVES, was knocked down. Importantly, MitoVES inhibited progression of syngeneic HER2(high) tumours derived from breast TICs by inducing apoptosis in tumour cells.</br></br>These results demonstrate that using mammospheres, a plausible model for studying TICs, drugs that target mitochondria efficiently kill breast tumour-initiating cells.ently kill breast tumour-initiating cells.)
• Braganza 2019 Mol Aspects Med  + (Accumulating studies demonstrate that mito … Accumulating studies demonstrate that mitochondrial genetics and function are central to determining the susceptibility to, and prognosis of numerous diseases across all organ systems. Despite this recognition, mitochondrial function remains poorly characterized in humans primarily due to the invasiveness of obtaining viable tissue for mitochondrial studies. Recent studies have begun to test the hypothesis that circulating blood cells, which can be obtained by minimally invasive methodology, can be utilized as a biomarker of systemic bioenergetic function in human populations. Here we present the available methodologies for assessing blood cell bioenergetics and review studies that have applied these techniques to healthy and disease populations. We focus on the validation of this methodology in healthy subjects, as well as studies testing whether blood cell bioenergetics are altered in disease, correlate with clinical parameters, and compare with other methodology for assessing human mitochondrial function. Finally, we present the challenges and goals for the development of this emerging approach into a tool for translational research and personalized medicine.</br></br><small>Copyright © 2019 Elsevier Ltd. All rights reserved.</small> 2019 Elsevier Ltd. All rights reserved.</small>)
• Amaral 2016 J Neurochem  + (Accumulation of 2-methylcitric acid (2MCA) … Accumulation of 2-methylcitric acid (2MCA) is observed in methylmalonic and propionic acidemias, which are clinically characterized by severe neurological symptoms. The exact pathogenetic mechanisms of brain abnormalities in these diseases are poorly established and very little has been reported on the role of 2MCA. In the present work we found that 2MCA markedly inhibited ADP-stimulated and uncoupled respiration in mitochondria supported by glutamate, with a less significant inhibition in pyruvate plus malate-respiring mitochondria. However, no alterations occurred when α-ketoglutarate or succinate was used as respiratory substrates, suggesting a defect on glutamate oxidative metabolism. It was also observed that 2MCA decreased ATP formation in glutamate plus malate or pyruvate plus malate-supported mitochondria. Furthermore, 2MCA inhibited glutamate dehydrogenase (GDH) activity at concentrations as low as 0.5 mM. Kinetic studies revealed that this inhibitory effect was competitive in relation to glutamate. In contrast, assays of osmotic swelling in non-respiring mitochondria suggested that 2MCA did not significantly impair mitochondrial glutamate transport. Finally, 2MCA provoked a significant decrease of mitochondrial membrane potential and induced swelling in Ca²⁺ -loaded mitochondria supported by different substrates. These effects were totally prevented by cyclosporine A plus ADP or ruthenium red, indicating induction of mitochondrial permeability transition (PT). Taken together, our data strongly indicate that 2MCA behaves as a potent inhibitor of glutamate oxidation by inhibiting GDH activity and as a PT inducer, disturbing mitochondrial energy homeostasis. We presume that 2MCA-induced mitochondrial deleterious effects may contribute to the pathogenesis of brain damage in patients affected by methylmalonic and propionic acidemias. This article is protected by copyright. All rights reserved.rticle is protected by copyright. All rights reserved.)
• Liu 2002 Proc Natl Acad Sci U S A  + (Accumulation of oxidative damage to mitoch … Accumulation of oxidative damage to mitochondria, protein, and nucleic acid in the brain may lead to neuronal and cognitive dysfunction. The effects on cognitive function, brain mitochondrial structure, and biomarkers of oxidative damage were studied after feeding old rats two mitochondrial metabolites, acetyl-l-carnitine (ALCAR) [0.5% or 0.2% (wt/vol) in drinking water], and/or R-alpha-lipoic acid (LA) [0.2% or 0.1% (wt/wt) in diet]. Spatial memory was assessed by using the Morris water maze; temporal memory was tested by using the peak procedure (a time-discrimination procedure). Dietary supplementation with ALCAR and/or LA improved memory, the combination being the most effective for two different tests of spatial memory (''P'' < 0.05; ''P'' < 0.01) and for temporal memory (P < 0.05). Immunohistochemical analysis showed that oxidative damage to nucleic acids (8-hydroxyguanosine and 8-hydroxy-2'-deoxyguanosine) increased with age in the hippocampus, a region important for memory. Oxidative damage to nucleic acids occurred predominantly in RNA. Dietary administration of ALCAR and/or LA significantly reduced the extent of oxidized RNA, the combination being the most effective. Electron microscopic studies in the hippocampus showed that ALCAR and/or LA reversed age-associated mitochondrial structural decay. These results suggest that feeding ALCAR and LA to old rats improves performance on memory tasks by lowering oxidative damage and improving mitochondrial function.dative damage and improving mitochondrial function.)
• Schoettl 2015 Endocrinology  + (Accumulation of visceral fat is associated … Accumulation of visceral fat is associated with metabolic risk whereas excessive amounts of peripheral fat are considered less problematic. At the same time, altered white adipocyte mitochondrial bioenergetics has been implicated in the pathogenesis of insulin resistance and type 2 diabetes.</br>We therefore investigated whether the metabolic risk of visceral versus peripheral fat coincides with a difference in mitochondrial capacity of white adipocytes. We assessed bioenergetic parameters of subcutaneous inguinal and visceral epididymal white adipocytes from male C57BL/6N mice employing a comprehensive respirometry setup of intact and permeabilized adipocytes as well as isolated mitochondria. Inguinal adipocytes clearly featured a higher respiratory capacity attributable to increased mitochondrial respiratory chain content as compared to epididymal adipocytes. The lower capacity of mitochondria from epididymal adipocytes was accompanied by an increased generation of reactive oxygen species per oxygen consumed. Feeding a high-fat diet for one week reduced white adipocyte mitochondrial capacity, with stronger effects in epididymal when compared to inguinal adipocytes. This was accompanied by impaired body glucose homeostasis. Therefore, the limited bioenergetic performance combined with the proportionally higher generation of reactive oxygen species of visceral adipocytes could be seen as a candidate mechanism mediating the elevated metabolic risk associated with this fat depot.bolic risk associated with this fat depot.)
• Gnaiger 1990 Thermochim Acta  + (Accurate definitions of efficiency are req … Accurate definitions of efficiency are required to resolve controversies on the significance and comparability of measures of efficiency in biological energetics. This review on concepts of efficiency is arranged into 4 parts. First, some fundamental energy relations of equilibrium and nonequilibrium thermodynamics are defined and placed into a coherent context as relevant for efficiency in biology. The classical expression of the Carnot efficiency of a heat engine obtains a new meaning in terms of flux-force relations of nonequilibrium thermodynamics. Second, within this general thermodynamic frame, the specific treatment of energy transformations of chemical reactions is introduced, with particular emphasis on open systems with internal transformation and external transfer of matter. Third, the chemical transformations in ATP turnover and internal efficiencies of coupled reactions are analyzed in two parts. On the one hand, the enthalpy efficiency is relevant in the context of biological calorimetry in relation to uncoupling and the integration of aerobic and anaerobic metabolism. On the other hand, the molar Gibbs energy efficiency relates to the driving force of coupled reactions and to the control of flux. High metabolic power and maximum efficiency are mutually exclusive. Finally, the discussion of various expressions of efficiency in biological growth requires a careful distinction between energy conservation in transformations (chemical reactions) and energy acquisition in coupled transformation and transfer of energy in the form of externally supplied matter. Better understanding and management of biological resource utilization requires this combined analysis of efficiency in biological energetics.is of efficiency in biological energetics.)
• Lotkova 2009 Acta Vet Brno  + (Acetaminophen (AAP) overdose causes severe … Acetaminophen (AAP) overdose causes severe liver injury and is the leading cause of acute liver injury in humans. The mechanisms participating in its toxic effect are glutathione depletion, oxidative stress and mitochondrial dysfunction. S-adenosylmethionine (SAMe) is the principal biological methyl donor and is also a precursor of glutathione. In our previous studies we have documented a protective action of SAMe against various toxic injuries of rat hepatocytes in primary cultures. The aim of this study was to evaluate a possible protective effect of SAMe against AAP-induced toxic injury of primary rat hepatocytes. Hepatocytes were exposed to AAP (2.5 mM) or AAP together with SAMe at the final concentrations of 5, 25 or 50 mg/l for 24 h. Incubation of hepatocytes with AAP caused a significant increase of the leakage of lactate dehydrogenase (LDH) (p < 0.001) and decline of the activity of cellular dehydrogenases (WST- 1) (p < 0.001). Co-incubation of hepatocytes with SAMe at any dose did not improve these markers of cellular integrity. The functional indicators improved in hepatocytes co-cultured with SAMe - urea production was significantly increased when using the highest dose of SAMe (p < 0.05); albumin synthesis was higher in all cultured hepatocytes exposed to SAMe (p < 0.05). SAMe did not influence AAP-induced decrease of cellular content of glutathione. Mitochondrial respiration of harvested digitonin-permeabilized hepatocytes was measured; Complex II was more sensitive to toxic action of AAP, respiration was decreased by 20%. This decrease was completely abolished by SAMe.y 20%. This decrease was completely abolished by SAMe.)
• Vrbova 2015 Abstract MiPschool London 2015  + (Acetaminophen (APAP) belongs to the most u … Acetaminophen (APAP) belongs to the most used analgetic and antipyretic drugs. APAP overdose causes liver injury and that is why it is the most frequent cause of acute liver injuries in the Western countries. In some cases, it is also associated with renal impairment occurring with frequency 1-2% of patients with acetaminophen overdose [1,2]. Acetaminophen is detoxified by three major pathways, glucuronidation, sulfation and oxidation by cytochrome P450. At therapeutic doses, a small portion of APAP dose is oxidized by cytochrome P450 to a reactive electrophilic molecule (NAPQI). After overdose, APAP is metabolized predominantly through the oxidation pathway and production of the oxidation product is enhanced. NAPQI is considered to be the toxic metabolite causing cell impairment [3]. However, based on our preliminary results, we postulated, that another metabolite could also cause toxicity.</br></br>Our study's aim was to characterize the toxicity of APAP metabolite in the human HK-2 cell line. We used a range of concentrations (10-5 mM) to examine the toxicity in cells. We evaluated the toxicity using the detection of mitochondrial dehydrogenase activity (WST-1 test), lactate dehydrogenase activity assay and detection of intracellular ROS production. </br></br>We observed moderate impairment of cells already after 3 h of treatment based on the finding of decreased mitochondrial dehydrogenase activity in all tested concentrations. After 24 hours, the results showed significant cellular impairment and increased ROS production at all tested concentrations. </br></br>In conclusion, we have proven our hypothesis that APAP metabolites ought to be also concerned in APAP toxicity. The toxic effect is presumably apparent as a decrease in mitochondrial dehydrogenase activity and induction of ROS production. activity and induction of ROS production.)
• Kucera 2016 Drug Chem Toxicol  + (Acetaminophen (APAP) hepatotoxicity is oft … Acetaminophen (APAP) hepatotoxicity is often studied in primary cultures of hepatocytes of various species, but there are only few works comparing interspecies differences in susceptibility of hepatocytes to APAP ''in vitro''.</br></br>The aim of our work was to compare hepatotoxicity of APAP in rat and mouse hepatocytes in primary cultures.</br></br>Hepatocytes isolated from male Wistar rats and C57Bl/6J mice were exposed to APAP for up to 24 h. We determined lactate dehydrogenase (LDH) activity in culture medium, activity of cellular dehydrogenases (WST-1) and activity of caspases 3 in cell lysate as markers of cell damage/death. We assessed content of intracellular reduced glutathione, production of reactive oxygen species (ROS) and malondialdehyde (MDA). Respiration of digitonin-permeabilized hepatocytes was measured by high resolution respirometry and mitochondrial membrane potential (MMP) was visualized (JC-1).</br></br>APAP from concentrations of 2.5 and 0.75 mmol/L induced a decrease in viability of rat (p < 0.001) and mouse (p < 0.001) hepatocytes (WST-1), respectively. In contrast to rat hepatocytes, there was no activation of caspase-3 in mouse hepatocytes after APAP treatment. Earlier damage to plasma membrane and faster depletion of reduced glutathione were detected in mouse hepatocytes. Mouse hepatocytes showed increased glutamate + malate-driven respiration in state 4 and higher susceptibility of the outer mitochondrial membrane (OMM) to APAP-induced injury.</br></br>APAP displayed dose-dependent toxicity in hepatocytes of both species. Mouse hepatocytes in primary culture however had approximately three-fold higher susceptibility to the toxic effect of APAP when compared to rat hepatocytes.effect of APAP when compared to rat hepatocytes.)
• Rousarova 2015 Abstract MiPschool London 2015  + (Acetaminophen (APAP) is a frequently used … Acetaminophen (APAP) is a frequently used analgetic and antipyretic drug. After overdose, it may cause a number of pathophysiological processes that can even lead to acute liver and/or kidney failure. The cause of toxicity can be recognized in its metabolic activation but the entire mechanism of acetaminophen toxicity is still unknown. APAP is metabolized in hepatocytes through various pathways. The most important pathway acting in overdose is oxidation of APAP by cytochrome P450 to a substance, which is detoxified by reaction with glutathione [1,2].</br> </br>We suppose that the metabolite of acetaminophen can also cause toxicity. Thus the main goal of our study was to assess a possible toxic effect of this metabolite in isolated mitochondria using detection of ROS production. We used CM-H2DCFDA molecular probe that is nonfluorescent until oxidized by ROS [3]. We used isolated mitochondria from rat liver and from kidney cells treated with mitochondrial substrates and inhibitors to localize the site of ROS production. </br>We proved that kidney mitochondria and mitochondria from rat liver treated with the acetaminophen metabolite produced ROS in significantly higher extent in comparison with controls. In 5 mM solution, ROS production in mitochondria isolated from rat liver was enhanced 9-fold and 3-fold in presence of glutamate and malate (i.e. complex I-related) and succinate and rotenone (i.e. complex II-related), respectively. Similar results were found in mitochondria isolated from kidney cells. The results support our hypothesis about the possible toxic effect of acetaminophen metabolite likely contributing to the overall toxicity.kely contributing to the overall toxicity.)