Erich Gnaiger

Daniel Swarovski Research Laboratory, Department of General and Transplant Surgery, Medical University Innsbruck, Austria; erich.gnaiger@i-med.ac.at

• Mitochondrial Respiratory Control in Textbook on Mitochondrial Physiology

    Oxidative phosphorylation (OXPHOS) is a key element of bioenergetics, extensively studied to resolve mechanisms of energy transduction and respiratory control in the electron transport system (ETS). Electron transport capacity is quantified as oxygen consumption in uncoupled mitochondria or cells (ETS; State E). In contrast, maximum ADP-stimulated respiration is a measure of OXPHOS capacity (P; State 3). P/E ratios yield an index of OXPHOS limitation by the phorsphorylation system.

    Coupled OXPHOS flux was 0.50±0.09 of ETS capacity in permeabilized NIH3T3 fibroblasts respiring on glutamate+malate+succinate (GMS), reflecting control of the phosphorylation system over OXPHOS in this human cell line [1]. Electrons flow to oxygen from Complex I or II with three or two coupling sites. Compared to ETS capacity in intact cells [2], conventional State 3 respiration in permeabilized cells was only 0.38±0.06 with ADP and glutamate+malate. ETS capacities were identical in intact and permeabilized uncoupled cells, however, with convergent electron flow to the Q-junction from glutamate+malate+succinate through Complexes I and II (CI+II e-input [1]). Convergent CI+II e-input provides the relevant basis for quantifying enzymatic thresholds and excess capacities of individual steps of OXPHOS, and for evaluation of mitochondrial defects. Convergent CI+II e-input corresponds to operation of the tricarboxylic acid cycle and mitochondrial substrate supply in vivo and yields novel insights into the physiological diversity of mitochondria from various tissues (compare various OXPHOS control analyses presented at MiPsummer 2008). Multiple substrate-uncoupler-inhibitor titration protocols and advanced OXPHOS flux control analysis extend the diagnostic potential of mitochondrial physiology in health and disease.

     This abstract will be presented at: EBEC 2008 Dublin, Ireland; 19-24 July 2008.

Keywords: Electron transport system, flux control ratio, uncoupling, leak, respiratory control ratio, multiple substrate-uncoupler-inhibitor tirations, Q-junction, fibroblasts.

1. Gnaiger E, ed (2008) Mitochondrial Pathways and Respiratory Control. OROBOROS MiPNet Publications, Innsbruck, 2nd ed: 96 pp.

2. Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley.

Benard G, Rodrigue Rossignol

INSERM U688, Bordeaux, France.- rodrigue.rossignol@phys-mito.u-bordeaux2.fr

    The recently ascertained network and dynamic organization of the mitochondrion, as well as the demonstration of energy proteins and metabolites subcompartmentalization, have led to a reconsideration of the relationships between organellar form and function. In particular, the impact of mitochondrial morphological changes on bioenergetics is inseparable. Several observations indicate that mitochondrial energy production may be controlled by structural rearrangements of the organelle both interiorly and globally, including the remodeling of cristae morphology and elongation or fragmentation of the tubular network organization, respectively. These changes are mediated by fusion or fission reactions in response to physiological signals that remain unidentified. They lead to important changes in the internal diffusion of energy metabolites, the sequestration and conduction of the electric membrane potential (Delta Psi), and possibly the delivery of newly synthesized ATP to various cellular areas. Moreover, the physiological or even pathological context also determines the morphology of the mitochondrion, suggesting a tight and mutual control between mitochondrial form and bioenergetics. In this presentation, we delve into the link between mitochondrial structure and energy metabolism.

Estelle Hirzel¹, Peter Lindinger¹, Peter W Schiller², Alex N Eberle¹

¹University Hospital and Children Hospital Basel, Department of Biomedicine, University of Basel, Switzerland; - estelle.hirzel@unibas.ch; ²Clinical Research Institute of Montreal, Quebec, Canada.

    Overweight and obesity represent a rapidly growing threat to the health in many countries. Obesity-related co-morbidities include coronary heart disease, hypertension and stroke, type 2 diabetes mellitus and dyslipidaemia [1]. The obesity-related elevations of fatty acids cause oxidative stress due to increased mitochondrial uncoupling and b-oxidation, leading to elevated production of reactive oxygen species (ROS) [2]. ROS might cause a decrease in mitochondrial function, thus exacerbating insulin resistance [3]. Szeto-Schiller peptides (SS-peptides) are tetrapeptides which are taken up and concentrated in the inner mitochondrial membrane >1000-fold [4]. They have been shown to reduce intracellular ROS, cell death and mitochondrial depolarization caused by t-butylhydroperoxide (tBHP) in neuronal cells lines [5].

    As a model for fat tissue, primary human bone marrow-derived mesenchymal stem cells (hBM-MSC) will be differentiated into adipocytes in vitro. The influence of different SS-peptides on ATP production, mitochondrial membrane potential, intracellular ROS levels and lipolysis will be tested on cells from healthy, obese and diabetic donors.

    This study will provide insights into the effects of SS-peptides on mitochondrial function and their therapeutic potential in obesity.

1. Obesity (2000): preventing and managing the global epidemic. Report of a WHO consultation. World Health Organ. Tech. Rep. Ser. 894: 1-253.

2. Wojtczak L, Schonfeld P (1993) Effect of fatty acids on energy coupling processes in mitochondria. Biochim. Biophys. Acta 1183: 41-57.

3. Qatanani M, Lazar MA (2007) Mechanisms of obesity-associated insulin resistance: many choices on the menu. Genes Dev. 21: 1443-1455.

4. Zhao K, Zhao GM, Wu D, Soong Y, Birk AV, Schiller PW, Szeto HH (2004) Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. J. Biol. Chem. 279: 34682-34690.

5. Zhao K, Luo G, Giannelli S, Szeto HH (2005) Mitochondria-targeted peptide prevents mitochondrial depolarization and apoptosis induced by tert-butyl hydroperoxide in neuronal cell lines. Biochem. Pharmacol. 70: 1796-1806.

Leandro S. da Costa¹, Nívea Dias Amoêdo¹, Franklin David Runjanek¹,  Antonio Galina¹, Andrea T. Da Poian¹, Tatiana El-Bacha¹

¹Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Brazil. - lcosta@bioqmed.ufrj.br

     In this study we demonstrate for the first time that Sindbis Virus (SV) infection induces important alterations in the respiratory parameters of neuroblastoma cells, Neuro2A. Oxygen consumption was measured in intact cells using high-resolution respirometry (OROBOROS Oxygraph-2k). Our results show that infected cells present a 45 % decrease in ROUTINE respiration (n=5; P<0.05) and a 38 % decrease in FCCP-induced maximum electron transport system capacity (ETS; n=5; P<0.05) when compared to mock-infected cells. Additionally, SV-infected cells show a significant decrease (P<0.05) in oligomycin-inhibited LEAK respiration (mean ± SE; n=5; 18.6 ± 1.3 for SV-infected and 32.4 ± 5.6 for mock-infected cells), and a significantly increase (P<0.05) in respiratory control ratio [(RCR) mean ± SE; n=5; 2.02 ± 0.06 for SV-infected and 1.68 ± 0.13 for mock-infected cells]. The decrease in LEAK respiration and the increase in RCR suggest mitochondrial coupling and a decrease in proton leak induced by SV-infection possibly as a compensatory mechanism for the decrease in ROUTINE respiration and maximum ETS capacity. Since we also found that SV-infection significantly increase by two-fold the K_m of hexokinase for glucose, the mitochondrial coupling found in infected cells may also be important to compensate for a possible decrease in glycolytic flux. We propose that bioenergetic alterations of Neuro2A cells are early signs of cell death and may be involved in the pathophysiology of encephalitis observed in SV-infection.

René G Feichtinger¹, JA Mayr¹, Franz Zimmermann¹, N Jones¹, FH Schilling², P Kogner³, W Sperl¹, B Kofler¹

¹University Hospital Salzburg, Paracelsus Medical University, Müllner- Hauptstrasse 48,  5020 Salzburg, AUSTRIA, ²Olga Hospital, Stuttgart, GERMANY, ³Childhood Cancer Research Unit, Karolinska, SWEDEN. - r.feichtinger@salk.at

    Neuroblastomas (NB) and Wilms tumors (WT) are the two most frequent extra-cranial solid tumors in children. Neuroblastomas originate from the neural crest, whereas Wilms tumors arise from the embryonal kidney. A shift in cellular energy production from oxidative phosphorylation (OXPHOS) to anaerobic glycolysis, called Warburg effect, is a fundamental property of cancer. Succinate dehydrogenase (Complex II) gene mutations are associated with carcinogenesis in pheochromocytoma, another tumor of the neural crest.

    The aim of the present study was to determine specific alterations of OXPHOS in 600g supernatants of human NB (n=15) and WT tissues (n=9) by spectrophotometric measurement of the enzymatic activity of citrate synthase (CS), Complex I, Complex II, Complex III, Complex IV (CI, CII, CIII, CIV) and ATP synthase. Compared to non-malignant tissues the activity of citrate synthase, a pace-maker enzyme of the Krebs cycle, was not altered in NB. The enzymatic activities of control kidneys (K), NB and WT are given as Units / g protein (mean ± SD):  CS:  K: 112±29, NB: 118±29, WT: 75±46; CI: K: 43±9, NB: 10±6, WT 16±11; CII: K: 128±45, NB: 15±16, WT: 24±15; CIII: K: 161±36, NB: 106±36, WT 159±134; CIV: K: 99±34, NB: 28±12, WT: 36±22; ATP synthase: K: 40±17, NB: 9±7, WT: 19±10. 

    When normalized to CS activity and then compared to control values, we found residual activities of CI (22 %), CII (7 %), CIII (69 %), CIV (27 %) and ATP synthase (19 %) in NB tissues, compared to normal kidney cortex tissue. Similar to NB, WT showed a residual enzyme activity of citrate synthase (70 %), CI (31 %), CII (19 %), CIII (78 %), CIV (39 %) and ATP synthase (48 %).

    The reduction of the activity of all complexes of the respiratory chain in NB and WT indicates that in these tumors loss of respiration is not related to a defect of a single enzyme as shown for renal oncocytomas and hereditary pheochromocytomas. Thus an upstream effector (e.g. VHL, HIF-1a), controlling the overall respiration [1,2] seems to cause the downregulation of OXPHOS in NB and WT.

This work was supported by the Salzburger Kinderkrebshilfe.

1. Godinot C et al (2004) A new role for the von Hippel Lindau tumor suppressor protein: stimulation of mitochondrial oxidative phosphorylation complex biogenesis. Carcinogenesis 26: 531-539.

2. Semenza GL (2007) HIF-1 mediates the Warburg effect in clear cell renal carcinoma. J. Bioenerg. Biomembr. 39: 231-234.

Franz A. Zimmermann,¹ Johannes A. Mayr,¹ Rene Feichtinger,¹ Daniel Neureiter,² Beate Alinger,² Nikolaus Schmeller,³ Christian Kögler,⁴ Manfred Ratschek,⁴ Wolfgang Sperl,¹ Barbara Kofler¹

¹Department of Pediatrics, ²Department of Pathology, ³Department of Urology, University Hospital Salzburg, Paracelsus Medical University, Austria; and ⁴Institute of Pathology, Medical University of Graz, Austria.- f.zimmermann@salk.at

   Many solid tumors exhibit a shift in energy metabolism from aerobic oxidation in the mitochondria to anaerobic glycolysis, which was first observed by Otto Warburg more than 80 years ago. Mutations in the mitochondrial enzymes fumarate hydratase and succinate dehydrogenase have been identified in different types of tumors, demonstrating a link between mitochondrial energy metabolism and tumorigenesis. Oncocytomas are tumors characterized by the accumulation of a high number of mitochondria. In order to elucidate the cause of the mitochondrial alterations in oncocytomas, we investigated the activities of respiratory chain enzymes, screened for mitochondrial DNA (mtDNA) mutations and performed by immunohistochemical stainings of oncocytic tumors.

   We showed that enzymatic activity of respiratory complex I was undetectable or extremely reduced in renal oncocytomas (n=15), and assembled complex I was lacking as shown of by Blue Native gel electrophoresis. Sequence analysis of mitochondrially encoded subunits of complex I showed pathogenic mutations in ND1, ND4 and ND5 genes in 10/15 renal oncocytoma samples, most of them were frame-shift mutations.

   Recently, also an association of mtDNA mutations with oncocytic thyroid tumors has been published [1], showing in 26 % of the cases disruptive mutations in mitochondrially encoded complex I genes.

   By immunohistochemical staining, we observed a lack of complex I in renal oncocytomas as well as in oncocytic thyroid tumors, while complex V and porin were considerably up-regulated.

   In summary, renal oncocytomas and oncocytic thyroid tumors seem to be characterized by a complete loss of complex I and a compensatory upregulation of mitochondria. Thus oncocytic tumors can be regarded as a mitochondrial disease, mostly caused by somatic mutations of the mitochondrial DNA.            

   This work was supported by the "Children's Cancer Foundation Salzburg",  and the "Vereinigung zur Förderung der pädiatrischen Forschung und Fortbildung Salzburg".

1. Gasparre G, Porcelli AM, Bonora E, Pennisi LF, Toller M, Iommarini L, Ghelli A, Moretti M, Betts CM, Martinelli GN, Ceroni AR, Curcio F, Carelli V, Rugolo M, Tallini G, Romeo G (2007) Disruptive mitochondrial DNA mutations in complex I subunits are markers of oncocytic phenotype in thyroid tumors. Proc. Natl. Acad. Sci. USA 104: 9001-9006.