Day 1:   Mitochondrial structural organization and respiration

L1-01       Mitochondrial metabolic structure, respiratory capacity and respiratory control.

               Erich Gnaiger

Medical University of Innsbruck, Dept. General and Transplant Surgery, D. Swarovski Research Laboratory, A-6020 Innsbruck, Austria. – erich.gnaiger@i-med.ac.at

    Mitochondrial respiratory physiology continues a tradition of quantitative bioenergetics with a focus on the control of oxidative phosphorylation (OXPHOS) in cells and tissues.  To analyze site-specific H+:e and ADP:O ratios, segments of the electron transport system (ETS) are separated into linear pathways by using either NADH-linked substrates (pyruvate, glutamate, malate; PMG) feeding electrons into Complex I (CI), or succinate (S; with rotenone) for electron input into Complex II (CII).  In living cells, however, the mitochondrial metabolic structure does not conform to electron flow in a linear electron transport chain, but electron flow converges through multiple entry lanes into the Q-junction (Q), including input through CI, CII, glycerophosphate dehydrogenase (GpDH), and electron transferring flavoprotein (ETF) [1,2].  Convergent CI+II e-input corresponds to full operation of the tricarboxylic acid (TCA) cycle in vivo.  This convergent metabolic structure of the electron transport system has fundamental functional consequences:  (1) Convergent CI+II electron input yields an increased respiratory capacity in a wide range of mammalian mitochondria [1,2].  (2) Apparent excess capacities and enzymatic thresholds downstream of Q must be evaluated with respect to convergent electron input into the Q-junction, whereas effective excess capacities (e.g. of Complex IV) are overestimated in relation to any single electron input.  (3) Convergent electron input shifts metabolic flux control towards limitation by the phosphorylation system, particularly in human cells (fibroblasts, HUVEC, skeletal and cardiac muscle, neuronal cells).  In these cases, the apparent excess ETS is high, including CIV.  (4) ROS production of isolated mitochondria or permeabilized cells cannot be evaluated properly in the presence of single electron input through either CI or CII, since fundamentally different patterns emerge with convergent CI+II electron supply [3].  (5) The diversity of mitochondrial respiratory control is largely masked by artifical restriction of electron input, whereas appreciation of the convergent metabolic structure of the electron transport system reveals an unexpected functional diversity, particularly in the comparison of mouse [4] and man [1,5].  (6) Multiple substrate-uncoupler-inhibitor titration protocols and advanced OXPHOS flux control analysis extend the diagnostic potential of mitochondrial physiology in health and disease. 

     ‘.. O2 uptake of intact cells represents the global result of the activity of several respiratory systems.’ [6]. 

     This is a contribution to Mitofood COST Action FAO602. 

1.  Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int. J. Biochem. Cell Biol. 41: 1837-1845. 

2.  Gnaiger E (2009) Mitochondrial Pathways through Complexes I+II: Convergent Electron Transport at the Q-Junction and Additive Effect of Substrate Combinations. In: Mitochondrial Pathways and Respiratory Control. OROBOROS MiPNet Publications 2009, 2nd ed. www.oroboros.at/index.php?compl-12-convergent 

3.  Muller FL, Liu Y, Abdul-Ghani MA, Lustgarten MS, Bhattacharya A, Jang YC, Van Remmen H (2008) High rates of superoxide production in skeletal-muscle mitochondria respiring on both complex I- and complex II-linked substrates. Biochem. J. 409: 491–499. 

4.  Aragonés J, Schneider M, Van Geyte K, Fraisl P, et al (2008) Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism. Nature Genetics 40: 170-180. 

5.  Boushel R, Gnaiger E, Schjerling P, Skovbro M, Kraunsoe R, Flemming D (2007) Patients with Type 2 Diabetes have normal mitochondrial function in skeletal muscle. Diabetologia 50: 790-796. 

6.  Keilin D (1929) Cytochrome and respiratory enzymes. Proc. R. Soc. London Ser. B 104: 206-252.

Day 2:   Mitochondrial transport systems

L2-01         Protein targeting and import into mitochondria: Overview and selected applications.

Steven C Hand

Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, USA. - shand@lsu.edu

    Only approximately 1% of all mitochondrial proteins are encoded by the mitochondrial genome.  Proteome analyses suggest about 800 (yeast) up to 1500 (human) different proteins are associated with mitochondria.  Thus the vast majority of mitochondrial proteins are encoded by the nucleus and must be imported and targeted to one of four locations: outer membrane, intermembrane space, inner membrane or matrix.  Precursor proteins can be separated into two main classes:  (a) preproteins destined for the matrix, and a smaller number heading to the inner membrane and intermembrane space, carry a N-terminal, positively-charged, cleavable presequence; (b) Precursor proteins destined for the outer membrane, and many intermembrane space and inner membrane proteins, carry various internal targeting signals.  These precursors do not have cleavable extensions, and have the same primary sequence as the mature proteins.  

    The translocase of the outer mitochondrial membrane (TOM) is a central entry gate for practically all nuclear-encoded mitochondrial proteins.  After recruitment to and passage through TOM, precursor proteins follow one of three major pathways.  (a) Preproteins with a presequence (those of this type destined for the matrix) are transferred to the translocase of the inner membrane (TIM23 complex; channel forming).  TIM23 cooperates with mt Hsp70, which represents the core of the presequence translocase-associated motor (PAM). (b) Precursors of hydrophobic proteins of the inner membrane (e.g., the ANT) utilize chaperone-like proteins (Tim9, Tim10, Tim12) and the insertion machinery of the inner membrane (TIM22 complex; the ‘carrier protein’ translocase).  c) Precursor proteins with complicated topologies (e.g., porin, Tom40) are first imported via the TOM complex to intermembrane space, then via small Tims are passed on to the SAM Complex (sorting and assembly machinery) for insertion into the outer membrane.  Other smaller proteins with cysteine motifs are aided in their targeting to the intermembrane space by the mitochondrial import and assembly machinery (MIA; main component Mia40), which promotes the formation of intramolecular disulfides and initiates the mature conformation of these proteins.

    Recently, we have discovered a Late Embryogenesis Abundant (LEA) protein in embryos of the brine shrimp A. franciscana that is targeted to the mitochondrion via a N-terminal leader sequence (29 AA).  LEA proteins are known for their ability to stabilize biological structures against stress incurred during drying.  Expressing a fusion protein composed of the leader sequence from this LEA protein and GFP (green fluorescent protein) shows that this chimeric protein is imported into mitochondrial network of human hepatoma cells (HepG2/C3A).  The results indicate the highly conserved nature of the protein import machinery for mitochondria of mammalian and invertebrate cells [perhaps including TIM23, PAM and the mitochondrial processing peptidase (MPP)] and indirectly, the targeting sequence as well.  A potential application for the sugar trehalose and this mitochondrial-targeted LEA protein (AfrLEA3) for stabilization of mitochondria during freezing and drying will be discussed.

     Supported by National Institutes of Health grant 2-RO1-DK046270-14A1, National Science Foundation grant IOS-0920254, and a donation from the William Wright Family Foundation.

P2-02       Differences in oxygen consumption in permeabilized muscle and liver cells following frozen storage and exposure to low pH values

Phung, V. T1., Egelandsdal, B.1*, Sælid, E.1, Volden, J.1 and E. Slinde1,2

1Institute for Chemistry, Biotechnology and Food science, University of Life Science, Postbox 5003, 1432 Ås, Norway; 2 Institute of Marine Research P.O. Box 1870, Nordnes, 5817 Bergen, Norway. *The topic will be presented by Bjørg Egelandsdal: bjorg.egelandsdal@umb.no

    Recently it has been suggested that mitochondria and mitochondrial enzymes are important for meat tenderization and colour stability. Mitochondrial oxygen consumption is vital for keeping myoglobin reduced and thus knowledge of the stability of enzymes participation in the electron transport chain at meat relevant post mortem pH values (most typically pH 5.3-6.2) and storing temperatures (chilled and frozen) is important. 

    Pork liver and muscle (Masseter, fiber type I) were collected just after slaughter, transported to our lab and oxygen consumption measurements were initiated shortly after slaughter (~3hr) on fresh permeabilized samples, samples frozen and stored at -20 °C and samples frozen in liquid nitrogen with a subsequent storage at -80ºC. pH reduction decreased the oxygen consumption of the permeabilized cells following succinate, ADP and cytochrome c addition. Freezing also reduced oxygen consumption as measured after addition of succinate, ADP and cytochrome c. The reduction in oxygen consumption, measured after succinate addition, was most severe if both low pH values (5.5-5.0) and freezing were used for permabilized liver cells; i.e. an interaction effect was observed. This observation was not made for permeabilized muscle cells. Reducing pH still reduced oxygen consumption following succinate, ADP and cytochrome c addition for permeabilized muscle cells, however, freezing increased oxygen consumption following succinate, ADP and cytochrome addition. The interaction effect found between storing temperature and pH, with respect to oxygen consumption upon addition of succinate, ADP and cytochrome c to permeabilized liver cells, was not observed for permeabilized muscle cells. 

P2-03 Molecular partakers of the mitochondrial TIM22 channel

             Patricia Rojo1, Lauro González1, María Luisa Campo1

1Univ. Of Extremadura, Fac. Veterinary Sciences, Dept. Biolochemistry and Molecular Biology, Apt. 643, 10003 Cáceres, SPAIN

    The molecular mechanism by which nucleus encoded proteins are sorted to one of the four compartments enfolded by the two mitochondrial membranes entails import complexes present in both of these membranes. Aqueous channels are key elements of these protein translocases. Previously we have uncovered the conditions to reveal in organello, a channel activity associated to TIM22, the translocase mediating the insertion of multispaning proteins into the inner membrane. Only cargo proteins facing the intermembrane space trigger the activity of this otherwise silent channel. Three membrane proteins partake TIM22: Tim22p, Tim54p and Tim18p. We have performed the molecular dissection of TIM22 present in mitochondria of eight yeast strains with different expression levels of its defined components. A combination of these results with those of the native translocase present in the genetically modified cells, and those obtained patch-clamping their inner mitochondrial membranes, outline the biogenesis of TIM22 and the distinct role played by its components. Our results indicate Tim22p is present in a complex of ~380 KDa also containing Tim18p and Tim54p. Interestingly, the biogenesis of this complex depends on the simultaneous presence of the three membrane proteins. In addition, the detection frequency of the TIM22 channel activity is the greatest, providing the complex is intact, while Tim54p holds together Tim22p and Tim18p. It is noteworthy that over-expression of Tim22p does not correlate with and increase in the channel’s frequency, despite it was described reconstituted Tim22p alone forms pores. 

     Supported by Spanish MCIN BFU2008-475 and Junta de Extremadura PRI07A072

P2-04        Effects of Tim50p on the channel activity of the mitochondrial protein translocase TIM23 

Jorge Bermejo1, María Luisa Campo1

1 Univ. Of Extremadura, Fac. Veterinary Sciences, Dept. Biolochemistry and Molecular Biology, Apt. 643, 10003 Cáceres, SPAIN

    Cytoplasmic synthesized preproteins reach the mitochondrial matrix by means of two translocases, TOM and TIM23, located in the outer and inner membranes of mitochondria respectively. While their components are mostly identified, a precise function for the majority of them has been outlined. TIM23 consists of a core membrane and a peripheral motor, segments. The core segment comprises three essential proteins: Tim17p, Tim23p, and Tim50p. Using patch-clamp techniques we have previously characterized the channel that functions in TIM23. Thus, Tim23p acts as receptor and largely constitutes the preprotein-conducting passageway. Also, Tim17p is required for the twin pore structure of this channel and provides its voltage gate. Recently an interaction Tim23p-Tim50p has been reported that facilitates the transferring of the incoming protein from the TOM to the TIM23 complex and promotes the function of the motor segment of TIM23. In an attempt to determine the structure-function correlations of the feasible channel constituents of TIM23, we are undertaking a comparative study of the biochemical characteristics and electrophysiological properties of mitochondria isolated from Sacharomyces cerevisiae. The strain selected carries a Gal promoter and growing conditions in the absence of galactose rend mitochondria with up to 90% reduced Tim50p. The expressing levels of the remaining TIM23 and TOM components are being determined. The aim of this study is to establish whether Tim50p affects the mechanistic properties of either one of these two channel’s translocases.

     Supported by Spanish MCIN BFU2008-475 and Junta de Extremadura PRI07A072. J.B. is a recipient of a fellowship by Junta de Extremadura.

P2-05        Effects of L-carnitine availability on carnitine palmitoyltransferase I activity in vivo

Janis Kuka1,2, Elina Skapare1,2, Edgars Liepins2, Maija Dambrova2

1Riga Stradins University, Riga, Latvia, 2 Latvian Institute of Organic Synthesis, Riga, Latvia

    Carnitine palmitoyltransferase I (<stockticker>CPT</stockticker>1, EC 2.3.1.21) is a key enzyme involved in the regulation of long-chain fatty acid metabolism. The aim of the present study was to determine the effects of long-term treatment by mildronate, an inhibitor of L-carnitine availability, on different isoforms of <stockticker>CPT</stockticker>1 in heart and liver tissues. 

    Male Wistar rats were orally treated daily for 4, 8 and 12 weeks with mildronate at doses of 100, 200 or 400 mg/kg. The concentration of L-carnitine in blood plasma and tissue homogenates was measured by UPLC-MS/MS. The <stockticker>CPT</stockticker>1 enzyme activity and protein expression was determined in tissue homogenates. Blood plasma and tissue lipid profile measurements were performed. 

    Mildronate treatment induced a significant and dose-dependent 2.5 to 16-fold decrease in the heart and liver tissue L-carnitine concentration. <stockticker>CPT</stockticker>1 activity in heart tissues was significantly increased after 4 weeks in all of the mildronate treated groups by up to 21%. After 8 and 12 weeks of treatment, the <stockticker>CPT</stockticker>1 activity was significantly increased only in the rats treated with a 400 mg/kg dose of mildronate by 25% and 35%, respectively. However, the expression level of <stockticker>CPT</stockticker>1 protein in heart tissues was not changed. In liver tissues, no significant changes in the <stockticker>CPT</stockticker>1 activity were observed.

    These results provide evidence that the lowered L-carnitine concentration induces compensatory increase in <stockticker>CPT</stockticker>1 activity in heart tissues, but not in liver tissues. This observation can be explained by different sensitivity to L-carnitine of different <stockticker>CPT</stockticker>1 isoforms present in liver and heart. 

P2-06        Anthocyanins – potent cytochrome c reducing agents

Kristina Skemienė, Sonata Trumbeckaitė, Daiva Majienė, Vilmante Borutaite, Julius Liobikas

Laboratory of Biochemistry, Institute for Biomedical Research of Kaunas University of Medicine, Eiveniu st. 4, LT-50009 Kaunas, Lithuania; skemiene@yahoo.com

    Cytochrome c functions as electron carrier between cytochrome c reductase and cytochrome c oxidase in mitochondrial respiratory chain, and its heme iron is reversibly oxidized and reduced between the Fe2+ and Fe3+ oxidation states. A study on isolated rat heart and liver mitochondrial functions has indicated that an anthocyanin delphinidin 3-O-glucoside (D3G) by 60% increases cytochrome c activated mitochondrial respiratory rate in the State 4. Generally cytochrome c test is used to evaluate the damage of the outer mitochondrial membrane. However we propose that the observed burst of respiration is due to the ability of anthocyanins to reduce cytochrome c. The aim of the study was to determine the ability of anthocyanins to reduce exogenous cytochrome c in vitro.

    Spectrophotometric measurement of redox state of cytochrome c has revealed that eight tested anthocyanins reduced cytochrome c to a different degree. Three major anthocyanins, found in berries and fruits: D3G, cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside reduced cytochrome c by 80%. Other investigated anthocyanins showed lower reducing power (20–75%). Furthermore we assessed the ability of D3G to reduce exogenous cytochrome c in the presence of isolated rat liver mitochondria. After the suppression of cytochrome reductase with antimycin A all exogenous cytochrome c remained completely oxidized. However, cytochrome c was immediately reduced by added D3G and oxidized over time by mitochondrial cytochrome oxidase complex. When cytochrome oxidase was inhibited, D3G caused fast and complete cytochrome c reduction. Furthermore, D3G had stronger stimulatory effect on rat liver mitochondrial respiration in the presence of antimycin A as compared to ascorbate supported respiration rate.

    These results suggest that anthocyanins are potent cytochrome c reducing agents in vitro and could replace ascorbic acid in respirometric studies. The precise mechanism of proposed stimulatory properties of anthocyanins on mitochondrial respiration in the presence of exogenous cytochrome c is under experimental investigation.

P2-07       Effects of Ginkgo biloba extract on mitochondrial functions: mechanism(s) of action

Baliutyte G, Baniene R, Trumbeckaite S, Borutaite V, Toleikis A.

Institute for Biomedical Research, Kaunas University of Medicine, Eiveniu str. 4, LT-50009, Kaunas, Lithuania

    Though extracts of Ginkgo biloba leaves (GBE) have a wide pharmacological application, little is known about GBE effects on mitochondria. In this work, effects of ethanolic GBE on the respiration of isolated rat heart and liver mitochondria were investigated. We found that GBE stimulates the pyruvate + malate-dependent State 2 respiration of heart mitochondria and decreases mitochondrial membrane potential. Uncoupling effect of GBE was found to be due to its protonophoric action and is likely to be mediated by the ATP/ADP-translocator and uncoupling proteins. The effect of GBE was less in liver than in heart mitochondria. State 3 respiration of heart mitochondria was slightly stimulated at low and depressed at higher GBE concentrations. Inhibition of State 3 respiration of heart mitochondria was not relieved by uncoupler indicating that GBE may inhibit the respiratory chain complexes or the substrate transport. However, Complex IV of the respiratory chain was not inhibited by GBE. H(2)O(2) generation was attenuated by low concentration of GBE probably due to mild uncoupling. The data suggest that mild but not severe uncoupling activity of GBE may be important in providing pharmacological protection of cellular functions in pathological situations.

P2-08        Functional analysis of two novel subunits of FoF1-ATP synthase complex in Trypanosoma brucei

Karolína Šubrtová1,2, Brian Panicucci2, Alena Zíková2

1Faculty of Science, University of South Bohemia, Branišovská 31, 370 05 České Budějovice, Czech Republic; 2Laboratory of Functional Genomics of Protists, Department of Molecular Parasitology, Institute of Parasitology, Biology Centre, ASCR, v.v.i. , Branišovská 31, 370 05 České Budějovice, Czech Republic

    The FoF1-ATP synthase plays an essential role in the protist Trypanosoma brucei, an important pathogen of humans and livestock, which alternates between an insect vector (procyclic stage, PS) and a mammalian host (bloodstream stage, BS). Interestingly, the activities of the respiratory chain complexes in this parasite are altered between the insect and mammalian stages. Most importantly, the function of the FoF1-ATP synthase switches from producing ATP during the PS to consuming ATP in order to maintain its mitochondrial membrane potential in the BS. This ATP hydrolysis activity of the complex is indispensable for the infectious stage of T. brucei.

    My PhD studies will focus on two novel subunits of the T. brucei FoF1-ATP synthase. These two subunits were shown to be essential in PS cells where they have a strong association with the FoF1-ATP synthase, as indicated by their tagged versions pulling down all 22 subunits of the complex. I have generated RNAi knock-down BS cell lines and demonstrated that both subunits are essential for the viability of the parasite. I am now performing more detailed phenotypical analyses (glycerol gradient sedimentation, blue-native electrophoresis, measurement of mitochondrial membrane potential and total ATP) to elucidate if these two proteins are involved in the structural organization of the complex, in its oligomerization and/or in the regulation of the different enzymatic activities found during the two life cycle stages. Moreover, by using a yeast two hybrid analysis, we will determine their specific binding partners within the Fo or F1 moiety of the ATP synthase.

P2-09       Mitochondrial binding Complex I in Trypanosoma brucei: its composition and function in RNA metabolism

Lucie Novotna, Hassan Hashimi, Lucie Hanzalkova and Julius Lukes

Biology Center and University of South Bohemia, Ceske Budejovice, Czech Republic

    Trypanosoma brucei is a member of the protozoan order Kinetoplastida and causes Human African sleeping sickness. It has a very interesting life cycle composed of two main forms – one in the tse-tse fly (procyclic form) and another one in the mammalian host (bloodstream form). The procyclic form of T. brucei contains a single large reticulated mitochondrion with many cristae. The situation is different in the bloodstream form, where the organelle is a small tubular structure with very few cristae. Mitochondrial DNA called kinetoplast DNA is a giant catenated structure composed of dozens of maxicircles and thousands of minicircles. The mitochondrion of trypanosomes is well-known for several unique processes, such as polyadenylation and RNA editing, which is a postranscriptional insertion/deletion of uridines into the mRNAs. I am studying a protein complex termed MRB1 (Mitochondrial Binding Complex 1) that is involved in RNA editing. MRB1 is comprised of 14 proteins, which are associated with the poly(U) binding protein TbRGG1 in a RNA-mediated manner. This complex was shown to have essential functions in RNA metabolism of both procyclic and bloodstream forms of T. brucei. Some of the subunits have domains or motifs involved in RNA metabolism and protein-protein interactions, whereas others have no known motifs. I m particularly interested in the composition and function of MRB1 in cells with down-regulated individual subunits of this complex.

Day 3:   Mitochondria and pathology

L3-01         Mitochondria and apoptosis: cell death, phylogenetic comparisons, and energy limitation

Steven C Hand

Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, USA. - shand@lsu.edu

    Correlations that implicated mitochondrial involvement in apoptosis accumulated for some time, but it was in 1996–1997 when it became clear mitochondria were not just bioenergetic organelles but also controlled life and death decisions in the cell.  Cytochrome c (cyt-c) release by mitochondria in the progression of mammalian apoptosis sparked the realization that mitochondria play a critical gatekeeper role.  We will discuss the apoptotic machinery for the three well-studied systems, those of Caenorhabditis elegans, Drosophila melanogaster and mammalian cells.  At least five factors that reside in the intermembrane space  of mammalian mitochondrion are involved in caspase-dependent and caspase-independent Programmed Cell Death (PCD).  The NADH-oxidase AIF and the endonuclease EndoG translocate to the nucleus where they are involved in chromatin condensation and DNA degradation. Other effectors impact the PCD machinery in a caspase-specific fashion. SMAC/DIABLO releases caspases from inhibition by IAPs (Inhibitor of Apoptosis Proteins). IAPs are intrinsic regulators of the caspase cascade and are the only known endogenous proteins that regulate the activity of both initiator  and effector caspases.  The serine protease Omi contributes to PCD in two ways. Omi neutralizes inhibition of caspases by IAPs, and also contributes to caspase-independent apoptosis through its protease activity.  Within the intermembrane space, cyt-c is essential for oxidative phosphorylation, but after release into the cytoplasm, it initiates the assembly of the apoptosome, i.e. the molecular machinery that activates caspase 9.

    Depending on the nature of the death signal, these factors are released either by permeabilization of the outer mitochondrial membrane, or both inner and outer membranes. Mitochondrial outer membrane permeabilization (MOMP) in mammals involves a complex interplay between the pro- and anti-apoptotic proteins belonging to the Bcl-2 family. Bcl-2 family proteins are divided into three subfamilies: multi-domain anti-apoptotic (e.g. Bcl-2, Bcl-xL), multi-domain pro-apoptotic (e.g. Bax, Bak) and pro-apoptotic BH3-only proteins (e.g. Bid, Bad).  MOMP is mediated by the pore-forming proteins Bak and Bax, whose activation is promoted by BH3-only proteins.  In non-apoptotic cells Bak is tail-anchored to the outer mitochondrial membrane, whereas Bax is mostly cytosolic.  During apoptosis Bax translocates to the mitochondrion where it changes conformation and inserts into the outer membrane.  Bax and Bak undergo conformational changes, oligomerize and form pores in the outer membrane.  Secondly, when mammalian mitochondria are exposed to high Ca2+ in the presence of the co-activator Pi, especially when accompanied by adenine nucleotide depletion and a reduced ∆Ψ, the opening of the mitochondrial permeability transition pore (MPTP) can occur.  Oxidative stress as a result of the generation of ROS is another modulator.  The increase in permeability of the inner membrane to solutes causes swelling of the matrix, rupture of the outer membrane, and release of pro-apoptotic factors.  Exciting discoveries are now linking the MPTP to Alzheimer's disease due to interactions between cyclophilin D and mitochondrial amyloid-β protein.

    Some phenomena like opening of the MPTP are lacking in crustaceans and may represent a vertebrate invention.  Differences in regulation of the intrinsic pathway of crustacean apoptosis might represent a prerequisite for surviving harsh environmental insults in some species. Cellular conditions experienced during energy-limited states – elevated calcium, shifts in cellular adenylate status, compromised ∆Ψ – are precisely those that trigger, at least in mammals, opening of the MPTP.  However, embryos of the brine shrimp, Artemia franciscana, survive extended periods of anoxia and diapause, and evidence indicates that opening of the MPTP and release of cyt-c do not occur. Further, caspase activation in this crustacean is not dependent on cyt-c. Its caspases display regulation by nucleotides that is consistent with ’applying the brakes’ to cell death during energy limitation.  Unraveling the mechanisms by which organisms in extreme environments avoid cell death may suggest possible interventions during disease states and biostabilization of mammalian cells.

     Supported by NIH grant 2-RO1-DK046270-14A1, National Science Foundation grant IOS-0920254, and a donation from the William Wright Family Foundation.

L3-02       Bioenergetics of tumors: role of tumor microenvironment and oncogene activation in the determination of cancer cells metabolic profile.

Caroline Hébert Chatelain1,2, Nadège Bellance1,2, Katarina Smolkova1,2,3, Giovanni Benard1,2 and Rodrigue Rossignol1,2

1INSERM U688, Bordeaux, France; 2Université Victor Segalen Bordeaux 2, Bordeaux, France; 3Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

    Tumor cells exhibit variable metabolic adaptations for rapid growth and survival (for review see [1, 2]). To study this variability requires the analysis of several genetic, metabolic and environmental factors such as the bioenergetic features of pre-cancer cells (cancer stem cell hypothesis), the impact of different oncogenes on energy metabolism and the role of tumor cells microenvironment with regard to oxygen and glucose concentration. Firstly, we analysed human lung cancer cells and bronchopulmonary tumors and observed a reduction of mitochondrial oxidative phosphorylation capacity that was explained by a repression of mitochondrial biogenesis [3]. We concluded that mitochondria are not dysfunctional in lung cancer cells but operate at low capacity. Secondly, we analysed the impact of aglycemia and hypoxia in breast cancer cells [4]. Sustained hypoxia (1% oxygen during 6 days) improved cell respiration in non-cancer cells grown in glucose or glucose-deprived medium (+ 32% and +38%, respectively) while routine respiration was strongly reduced in cancer cells (-36% in glucose medium, -24% in glucose-deprived medium) under these conditions of limited oxygen. These differences in hypoxia and aglycemia tolerance might be caused by oncogene activation in cancer cells. To evaluate this possibility we analysed the bioenergetic impact of different oncogenes as well as their sequence of activation in human fibroblasts. The first results show a considerable effect of G12V-HRAS, HPVE6, HPVE7, SV40 Small T-antigen and TERT on mitochondrial respiration and ATP synthesis. Our work gives a better characterization of cancer cells' metabolic alterations which are essential for growth and survival. They could designate mitochondrial biogenesis and mitochondrial respiratory chain as a possible target for anti-cancer therapy.

1.  Bellance N et al. Frontiers in Biosciences Jan 1 (2009) 4015-4034.

2.  Jezek P et al. Int J Biochem Cell Biol  (2010).

3.  Bellance N et al. Int J Biochem Cell Biol (2009) Dec;41(12):2566-77.

4.  Smolkova K et al. J Bioenerg Biomembr  (2010) Jan 19.

L3-03       Mitochondrial electron & proton leaks in health, disease & aging

Guy Brown

Department of Biochemistry, University of Cambridge, Cambridge, UK

    Up to 1% of electrons flowing down the mitochondrial electron transport chain leak out to react with oxygen and produce reactive oxygen species (ROS), such as superoxide (O2-), hydrogen peroxide (H2O2) and the hydroxyl radical (OH●).  The major sources of ROS are complexes I and III, and ROS production increases when the membrane potential is high and/or the electron transport is reduced.  ROS have signaling roles in the cell, but also damage proteins, membranes and DNA, and are implicated in many diseases and aging. 

    A proportion of the protons pumped out by the mitochondrial proton pumps return across the inner membrane via a ‘proton leak’, which is uncoupled from ATP synthesis, and thus dissipates free energy and generates heat. The nature of the proton leak remains unclear, but may include: i) proton diffusion across the phospholipid bilayer, ii) protons carried by fatty acids, and iii) specific proteins, including the uncoupling proteins.  UCP1 codes for the uncoupling protein thermogenin, found almost exclusively in brown adipose tissue mitochondria, where it uncouples and generates heat.  UCP2 and UCP3 are homologous and expressed in many tissues, but whether they catalyse a significant proton leak remains controversial. 

    There are three main mitochondrial theories of aging. (A) The “mitochondrial free-radical theory of aging” suggests that free radicals produced by mitochondria cause DNA mutations and other cellular damage that eventually causes degenerative diseases and aging.  (B) The “mtDNA theory of aging” suggests that somatic mutations of mtDNA that accumulate with age contribute to aging.  (C) The “bioenergetic decline” theory suggests that mitochondrial function declines with age (either due to mtDNA mutations or free-radical damage or other factors) and this contributes to aging. 

1.  Harper ME, Green K, Brand MD. (2008) The efficiency of cellular energy transduction and its implications for obesity. Annu Rev Nutr. 28:13-33.

2.  Balaban RS, Nemoto S, Finkel T. (2005) Mitochondria, oxidants, and aging. Cell 120:483-95.

3.  Brookes PS (2005) Mitochondrial H+ leak and ROS generation. Free Rad. Biol. Med. 38, 12-23.

4.  Gruber J, Schaffer S, Halliwell B. (2008) The mitochondrial free radical theory of ageing--where do we stand? Front Biosci. 13:6554-79.

5.  Lambert AJ, Brand MD. (2009) Reactive oxygen species production by mitochondria. Methods Mol Biol. 554:165-81.

Day 4:    Mitochondria and neurodegeneration

L4-01         Mitochondria in Inflammatory neurodegeneration

                   Guy Brown

Department of Biochemistry, University of Cambridge, Cambridge, UK

    Inflammation is induced by i) pathogens, or ii) damage, and it promotes survival by killing pathogens and repairing damage.  However, chronic inflammation is thought to contribute to the diseases of aging, including cancer, atherosclerosis, and neurodegeneration.  Inflammatory neurodegeneration is neuronal degeneration due to inflammation, and is thought to contribute to neuronal loss in infectious, ischemic, traumatic and neurodegenerative brain pathologies.  We have identified three mechanisms by which inflamed glia kill neurons: iNOS, PHOX and phagocytosis. 

    A variety of inflammatory mediators induce the expression in microglia and astrocytes of inducible nitric oxide synthase (iNOS), which produces high levels of NO.  NO acutely and potently inhibits mitochondrial respiration at cytochrome oxidase in competition with oxygen, while NO derivatives peroxynitrite and S-nitrosothiols inactivate mitochondrial complex I, resulting in a stimulation of oxidant production by mitochondria. We find that a high level of glial iNOS expression induces neuronal death in synergy with hypoxia, basically by NO inhibition of neuronal respiration resulting in glutamate release and excitotoxicity.  This suggests that the inflamed brain may be more sensitive to hypoxic damage. NO also induces glutamate release from astrocytes, but by a different, calcium-dependent mechanism. NO from nNOS can also synergise with hypoxia to induce neuronal death via inhibition of mitochondrial respiration (if glycolysis is blocked).

    The phagocyte NADPH oxidase (PHOX) is constitutively expressed primarily on the plasma membrane and intracellular vesicles of microglia.  Acute activation of PHOX produces superoxide and hydrogen peroxide, that stimulate microglial production of TNF- and IL-1 and microglial proliferation.  However, if PHOX is activated in glia where iNOS had previously been induced then peroxynitrite is produced, which induces apoptosis in neurons.

    However, we find that apoptosis, as measured by phosphatidyserine (PS) exposure, can be reversible in neurons.  Temporary exposure of neurons to low levels of H2O2, glutamate or peroxynitrite results in reversible PS exposure on neurons.  And such neurons go on to survive, if their phagocytosis by inflamed microglia is prevented at the time of PS exposure.  Inflammatory activation of neuronal-glial co-cultures with LPS, LTA or beta-amyloid (in the absence of pro-inflammatory cytokines) results in progressive loss of neurons (without any apparent cell death) which is accompanied by microglial phagocytosis of neurons, and is prevented by blocking phagocytosis, in culture and in vivo.

1.  Borutaite, V. & Brown, G.C. (2006) S-nitrosothiols induce inhibition of complex I and ROS production by mitochondria. Biochim. Biophys. Acta 1757, 405.

2.  Brown, G. C. (2007) Nitric oxide and mitochondria. Front. Biosci. 12, 1024-33.

3.  Jekabsone, A., Nehrer, J., Borutaite, V. & Brown, G. C. (2007) Nitric oxide from neuronal nitric oxide synthase sensitises neurons to hypoxia-induced death via competitive inhibition of cytochrome oxidase. J. Neurochem. 103, 346-356.

4.  Brown GC, Neher JJ. (2010) Inflammatory neurodegeneration and mechanisms of microglial killing of neurons. Mol Neurobiol. 2010 Jun;41(2-3): 242-7.

L4-02         Mechanisms for regulation of brain mitochondria by extramitochondrial Ca2+ as new targets of neurodegeneration

Zemfira Gizatullina1, Stefan Vielhaber1, Huu Phuc Nguyen3, Sonata Trumbeckaite4, Silke Nuber3, Matthias Jucker5, Frank Striggow2 and Frank N. Gellerich2,

1Neurologische Universitätsklinik der Otto-von-Guericke Universität, Magdeburg, 2KeyNeurotek Pharmaceuticals AG, ZENIT Technology Park, Magdeburg, 3Department für medizinische Genetik, Universität Tübingen, 4Institute for Biomedical Research, Kaunas University of Medicine,5Hertie -Institut für Klinische Hirnforschung, Tübingen

    Due to complex interactions between mitochondria and other cell compartments there are diverse possibilities for involvement of mitochondria in the pathophysiology of neurodegeneration [1]. Recently we detected at isolated muscle mitochondria of R6/2 mice an increased sensitivity of OXPHOS against Ca2+-stress [2]. Therefore we systematically investigated the role of cytosolic Ca2+ (Ca2+cyt) for the regulation of OXPHOS at isolated brain mitochondia of normal and transgenic animals as models of the neurodegenerative diseases Huntington, Parkinson and Alzheimer.

    In contrast to the textbooks we found that Ca2+cyt fully regulates the active respiration of brain mitochondria using the substrates glutamate/malate and also a-glycerophosphate on demand on the actual energy requirements. This occurs via the Ca2+cyt-stimulation of the glutamate aspartate carrier (aralar) and the mitochondrial a-glycerophosphate dehydrogenase. Both enzymes have regulatory Ca2+ binding sites on the mitochondrial surface (in the intermembrane space) and are main constituents of the malat-aspartat-shuttle and the a-glycerophosphate-shuttle, respectively. Activity of both shuttles is reversibly and effectively regulated by Ca2+cyt in the range of physiological Ca2+cyt-concentrations between 50 and 300 nM Ca2+cyt [3-5].

    Brain mitochondria of transgenic animals were investigated with specific respirometric protocols [6]. Active glutamate respiration of brain mitochondria from different transgenic Huntington animals was lower and inhibition by Ca2+-overload started at lower Ca2+cyt compared to the controls. Similar results were obtained with transgenic a-synuclein mice (Parkinson) and also at preliminary measurements of Alzheimer mice with Aß42-pathology.

    Besides aralar and a-glycerophosphate dehydrogenase there are further proteins (porin, MgATP-carrier, Ca2+-uniporter, permeability transition pore) on the mitochondrial surface with assumed or confirmed regulatory Ca2+-binding sites. Therefore, we hypothesize that the cytotoxic proteins (Huntingtinexp, a-Synuclein, Aß42) can interact with these Ca2+-binding sites, disturbing the normal interactions between mitochondria and Ca2+cyt which consequently causes mitochondrial dysfunction, energetic depression mitochondrial cell death and atrophy [1-5].

1. Seppet E et al., Int J Mol Sci 10, 2252-2303.

2. Gizatullina ZZ et al., Ann Neurol 59 (2006) 407-411.

3. Gellerich FN, et al., J Biol Chem 283 (2008) 30715-30724

4. Gellerich FN, et al., PloS one 4 (2009)28181

5. Gellerich FN, et al., Biochim Biophys Acta In press. (2010)

6. Kuznetsov AV, et al., Nature Protocols 3 (2008)965-976

P4-03        Targeting mitochondrial amyloid β through intrabodies: new approach to study pathogenesis of Alzheimer’s disease

Nina Krako1,2*, Giovanni Meli1,2, Antonino Cattaneo1,2

1Scuola Normale Superiore, Pisa, Italy; 2European Brain Research Institute (EBRI), Rome, Italy; * nina.krako@sns.it

    Intracellular antibodies (intrabodies), represent recombinant antibody fragments which are ectopicaly expressed within a cell and target specific intracellular antigen. Concept of using intrabodies is based on induction of phenotipic knockout of relevant target protein. To exert their function, intrabodies have to be directed to the subcellular compartment where the antigen is located. Intrabody-based approaches are now being investigated as the potential treatment of diverse neurodegenerative disorders: Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and prion encephalopaties. In all of these disorders the aggregation of specific misfolded proteins is linked to neurodegeneration of specific brain regions. Our laboratory investigates use of conformational sensitive intrabodies against amyloid β oligomers (AβOs), which are pathologically relevant conformations of amyloid β (Aβ) peptide in Alzheimer’s disease. Functional anti-AβOs intrabodies were selected by using intracellular antibody capture technology (IACT). The specific conformational anti-AβOs intrabodies allows to selectively target AβOs inside different subcellular compartments. Since there are growing evidences for important role of mitochondria in most common neurodegenerative diseases, we are investigating role of mitochondrial AβOs by using intrabody approach. Plasmid vectors containing mitochondrial targeting sequence engineered to code anti-AβOs intrabodies, have been targeted to mitochondria. Functional and biochemical assays are in progress in well established cell models for production and secretion of AβOs (7PA2 cells) in order to study functions of these intrabodies that interfere with mitochondrial Aβ accumulation and cellular toxicity.

P4-04        Effect of amyloid β on rat brain mitochondrial function

P. Cizas

Institute for Biomedical Research, Kaunas University of Medicine, Kaunas Lithuania

    Alzheimer’s disease (AD) is the most prevalent form of progressive neurodegeneration characterized by amyloid-beta (Aβ) plaques, neurofibrillary tangles, and neuronal loss mainly in the cortex and hippocampus. The central event in pathogenesis of Alzheimer’s disease (AD) is thought to be intracellular and extracellular accumulation of polypeptide compounds – so called beta amyloid (Aβ). These molecules tend to aggregate and form complexes of varying size: from small soluble oligomers, bigger protofibrils and finally insoluble fibrils. We have shown that small oligomers of 1-2 nm size are the most cytotoxics species, which induced rapid neuronal necrosis at submicromolar concentrations. A large amount of evidence suggests that mitochondria could intervene in the mechanisms by which Aβ triggers neuronal dysfunction and degradation, and that Aβ may accumulate in mitochondria. In this study we aimed to analyze the direct effect of various Aβ assemblies (monomers, oligomers and fibrils) on respiration rate of mitochondria isolated from rat brain. To study the effect of Aβ on mitochondrial respiration mitochondria were incubated with Aβ1−42 at 1µM concentrations for 15 min at 37 °C.

    We found that all forms of Aβ1-42 (monomers, oligomers and fibrils) did not affect the state 2 and state 3 respiration rates of isolated brain mitochondria with pyruvate and malate as substrate. Therefore, we conclude that A monomers, oligomers and fibrils have no direct effect on respiration of isolated brain mitochondria at low, physiological concentrations.

P4-05        The anti-inflammatory and anti-apoptotic action of humanin-a newly discovered 24-amino acid peptide in LN-18 cells.

Joanna Góralska, Barbara Zapała, Agnieszka Śliwa, Iwona Wybrańska, Aldona Dembińska-Kieć.

Department of Clinical Biochemistry, Collegium Medicum, Jagiellonian University, Kopernika 15a, 31-501 Krakow, Poland

     Background: Humanin (HN) is a newly discovered peptide which suppressed neuronal cell death caused Alzheimer’s disease. It is suggested that HN is part of the physiological mechanisms promoting cell survival under stressful conditions, such as neurodegeneration, inflammation, or energy deficiency.

     Aims: The study of the mitochodria-related metabolic, antiapoptotic mechanisms and neuroprotective effects of humanin isoform Gly14-HN (HNG) against proinflammatory, proapoptotic, metabolic stressors in cell-culture models.

     Methods:  Human brain cell line (LN18) was preincubated with HNG (4uM) for 24 hour, and for the last 4hr exposed to staurosporine (STS;0,025uM) as proapoptotic stressor.  Mitochondrial metabolic potential was observed by measurements of the mitochondrial oxygen consumption rates (OROBOROS® Oxygraph-2k) and ATP generation (Luciferase/Luciferine; ATP Lite Perkin Elmer). Global gene expression changes were measured by using human and mice AFFYMETRIX microarrays.

     Results: Preincubation of cells, exposed to STS, with  humanin increased stimulated mitochondrial respiration rate. This was accompanied by decrease in ATP generation. Moreover the microarray study with the same experimental model demonstrated the influence of HNG on genes expression involved in apoptotic and inflammatory pathway. BAD and CARD4 genes expression was down-regualated in LN18 cells preincubated with HNG and treated with STS. Genes involved in inflammatory pathway like TNFRSF25 and TNF α was also down-regulated with HNG+STS.

     Conclusion: We demonstrated the influence of HN derivative Gly14-HN on metabolic mitochondrial function such as respiration rate and ATP generation in LN18 cells. HNG stimulates the uncoupled respiratory capacity and thus may decrease reactive oxygen species level. Moreover HNG downregulates several genes involved in inflammatory pathway. 

     Supported by Polish-Norwegian Research Grant PNRF-104-AI-1/07 

P4-06       Antiapoptotic activity of humanin is weakly bound to the metabolic changes of HUVEC

Barbara Zapala, Agnieszka Sliwa, <personname productid="Joanna Goralska">Joanna Goralska</personname>, <personname productid="Anna Knapp">Anna Knapp</personname>, Magdalena Awsiuk, <personname productid="Wojciech Dudek">Wojciech Dudek</personname>*,  <personname productid="Aldona Dembinska-Kiec">Aldona Dembinska-Kiec</personname>

Department of Clinical Biochemistry, Collegium Medicum, Jagiellonian University, Kopernika 15a, 31-501 Krakow, Poland, *Department of Gynecology, Province Hospital of Myślenice , Poland

     Background: Humanin (HN) is a newly discovered peptide which is encoded within the mitochondrial and nuclear genomes, with suggested spectrum of cytoprotective, anti-inflammatory and antiapoptotic properties.

     Aim: The study of the metabolic (mitochodria-related)/antiapoptotic mechanisms possible participating in the protective effects of different exogenous humanin-like peptides against proinflammatory, proapoptotic, hypoxic, and metabolic stressors was followed in HUVEC.

     Methods: HUVEC were cultured with humanin peptides: HNG, 13ThrHN10b, 13IleHN10b, and HNM at 4μM for 24h. Mitochondrial metabolic activity was monitored by measurements of the mitochondrial oxygen production rates (OROBOROS® Oxygraph-2k) and ATP generation (Luciferase/Luciferine; ATP Lite Parkin Elmer). The apoptosis- gene expression changes were measured by using the TaqMan Applied microarray system (TLDA) based on Real –time reaction. Measurement of mitochondrial membrane potential (DY) was performed by using BD Bioimager 855 microscopy.

     Results: Mitochondrial metabolic potential, (ATP generation and the mitochondrial oxygen consumption revealed no significant differences after preincubation of HUVEC with  investigated HNs. The microarray results showed that HNG inhibited the FADD gene expression which regulates caspase 8 activities. We observed also the downregulation of APAF1 which is involved in caspase-related apoptotic pathway through regulation of caspase 9.

    Conclusion: The results suggest that  the antiapoptotic activity of humanins is not  connected with the increased mitochondrial oxygen consumption and ATP generation but involves  changes of the  BCL2 protein by activation of BCL2L11.

     This study was supported by Polish-Norwegian grant no. PNRF-104-AI-1/07.

P4-07        Analysis of mitochondrial oxidative metabolism in normal and type 2 diabetic human fibroblasts

Kristina M. Kriauciunas B.A., Stephane Gesta PhD, C.R. Kahn MD

Joslin Diabetes Center, Harvard Medical School, Boston, MA. U.S.A.

    The incidence of diabetes and obesity in the world has increased to an epidemic proportion.  Contributing factors include sedentary life styles and increased adipocity due to aging. Previous studies have shown a decrease in mitochondrial function in the skeletal muscle of Type 2 diabetic patients. Since muscle biopsies are difficult to obtain and skin fibroblasts are more readily available and easy to maintain, we chose to use human skin fibroblasts as a model to explore if there are any differences in the rates of cellular oxidative metabolism between normal and Type 2 diabetic patients, and whether human Type 2 diabetes is associated with defects in mitochondrial metabolism. 

    In the present study we used fibroblasts from five normal and five Type 2  age matched diabetic humans, along with fibroblasts from three normal young and three normal older humans  to generate and compare the bioenergetic profiles of these cells.

    Using Seahorse technology we measured the mitochondrial respiratory capacity and oxidative function of these cells.  Fibroblast cells were seeded at a known density and basal oxygen consumption (OCR) rate was measured to establish a baseline profile. The mitochondria of the cells were then perturbed by using three compounds (oligomycin, FCCP and rotenone) in succession to shift the bioenergetic profile of the cell.

    By measuring the area under the curve of these profiles five metabolic states could be determined; basal respiration, ATP turnover, proton leak, respiratory capacity and non-mitochondrial respiration. Using this analysis we found no statistical significant differences in OCR rates in normal fibroblasts versus age.  In contrast, in the Type 2 diabetic fibroblasts, both basal respiration and proton leak versus age OCR rates were significantly (P=0.0250 and P=0.0396) decreased.

    To access mitochondrial genes which may be involved in these differences we measured mRNA levels by qPCR of CYP7A, COX7A2L, ATP5C1 and ATP5F1. In addition, we measured mRNA levels of some metabolic genes, including the insulin receptor (IR), insulin receptor substrate 1 (IRS-1), insulin receptor substrate 2 (IRS-2), and some aging genes (p16 and p53). We found that the IR, IRS-1, CYP7A, COX7A2L and ATP5F1 decrease with age in both controls and Type 2 diabetics. By contrast, one gene, ATP5C1 showed a two to three fold increase in Type 2 diabetics.

    In summary, we have used Seahorse technology to look at mitochondrial and genetic changes in human fibroblasts from normal and Type 2 diabetic patients.  We found that basal respiration and proton leak in these cells was altered and decreased with age, and that changes in expression of some mitochondrial and metabolic genes may contribute to these differences. These data may help clarify the differences and causes between normal and Type 2 diabetic patients and help identify fibroblasts as a tool to distinguish these differences.

P4-08        Synergistic anti-obesity effects of procyanidins in combination with polyunsaturated fatty acids

Casanova, E., Fernández-Iglesias, A., Quesada, H., Pajuelo. D., Díaz, S., Baselga L., Bladé, C., Arola, L., Salvadó, MJ.

Nutrigenomic Group, Department of Biochemistry and Biotechnology, Rovira i Virgili University, Tarragona, Spain.

    Obesity is a disruption in the balance between fuel intake and energy expenditure which favours energy conservation. A growing body of evidence shows that altered mitochondrial energy production is a major event that can generate a metabolic situation leading to obesity. The data suggest that several classes of bioactive food components may have the potential to modulate mitochondrial function and consequently prevent the risk of developing obesity.

    Experimental plans: The overall aim of the present project is to develop a new strategy to combat obesity by combining natural bioactive compounds and using them as additives to create functional foods.

    We have chosen food ingredients which show special anti-obesity activities: w-3 PUFA (microalgae oil with 40% of omega-3 PUFAs) and GSPE (grape seed procyanidin extract). To study the synergistic/additive effects of the selected combinations of these substances in vivo, we have designed an acute experiment with Wistar rats. There will be 4 groups (6 rats/group): control group, GSPE group, ω-3 group, GSPE + ω-3 group. Doses (250 mg of each compound /kg body weight) will be given five hours before the sacrifice together with 2.5 ml of lard/kg body weight as a high fat vehicle in order to give the maximum effect. During these 5 hours we will measure the respiratory quotient and the energy expenditure using the O2/CO2 analyzer equipment.

    Also, to study adaptive thermogenesis in response to high–fat feeding, we will observe the effects of the compounds tested on the mitochondrial function in mithocondria previously isolated from brown adipose tissue and skeletal muscle samples from each rat.

P4-09        Influence of free fatty acids on metabolic activity of mitochondria during differentiation and dedifferentiation of the human adipose tissue progenitor cells (SVF).

Agnieszka Śliwa, Joanna Góralska, Beata Kieć-Wilk, Magdalena Awsiuk, Urszula Czech, Aldona Dembińska-Kieć

Departament of Clinical Biochemistry Collegium Medicum, Jagiellonian University; Cracow, Poland

     Background: The mechanism of differentiation and dedifferentiation of SVF cells has been extensively studied, but the influence of nutrients on the metabolism, differentiation (angio- vs adipo-) of SVF cells is still poorly understood.

     Aim: The aim of the presented study was to evaluate energy state of the differentiating SVF progenitor cells treated with selected fatty acids (PA, OA, EPA, AA ).

     Methods: The SVF cells were cultured in the adipocyte differentiation medium (MDI) for 48 hours, out which the last 24 hours included incubation with FFA. Cells were investigated after 15-day-long differentiation and than after following 15 days of dedifferentation induced by addition of fetal calf serum or VEGF or L-arginine.

    Changes in mitochondrial membrane potential Dy was assessed by TMRM with BioimagerBD. Metabolic activity of mitochondria were analyzed by ATP generation (luminescence assay kit, ATP LiteTM Perkin Elmer) and by oxygen consumption (high-resolution respirometry, Oxygraph-2k, Oroboros). A phosphorylation control protocol was  used for evaluation of the physiological respiratory control state of the intact SVF cells, the mitochondrial coupling state and uncoupled respiratory capacity.

     Results: Preincubation with some FFA increased ATP level,  mitochondrial coupling state and uncoupled respiratory capacity. After 15 days differentiation we observed tendency to increase Dy, oxygen consumption  and ATP generation by OA, PA and EPA. After the next 15 days dedifferentiation in presence of FSC, OA and AA increased ATP generation, but EPA decreased  oxygen consumption. Mitochondrial activity was reduced in VEGF presence.

     Conclusion: Metabolic status of SVF (EPA, L-arginine) modify their differentiation into adipose tissue.

     Supported by EU FW7 LIPIDOMICNET 202272.

Day 5:    Mitochondrial membrane potential and OXPHOS

<h1 style="TEXT-JUSTIFY: inter-ideograph; MARGIN: 0cm 0cm 0pt 2cm; TEXT-INDENT: -2cm; LINE-HEIGHT: normal; TEXT-ALIGN: justify; tab-stops: 2.0cm">L5-01          Mitochondrial respiratory rates and states – fluxes and forces.

               Erich Gnaiger

Medical University of Innsbruck, Dept. General and Transplant Surgery, D. Swarovski Research Laboratory, A-6020 Innsbruck, Austria. – erich.gnaiger@i-med.ac.at

    Respiratory steady states have been defined by Chance and Williams (1955) according to a protocol for oxygraphic experiments with isolated mitochondria, for studies of mitochondrial respiratory control [1]:  State 2 is induced by addition of ADP to mitochondria incubated without external substrates to assure complete oxidation of endogenous substrates; State 3 is active respiration after addition of substrates; and State 4 is resting respiration after ADP is phosphorylated to a maximum ATP/ADP ratio.  More recently, this termonology became confused, and State 2 was considered to be comparable to State 4 ([2]; ‘State 2’ with added substrate but without ADP).  Clarification is obtained by changing from technical jargon (strictly related to redox states of the cytochrome and corresponding respiratory rates) to an extended concept (including considerations of mt-membrane potential) with functional explanations of coupling states [3,4]:

1. ROX: residual oxygen consumption, in the absence of substrates (the final phase of the original State 2 [1]) or after inhibition of electron transport from substrates to oxygen  (minimum mt-membrane potential).

2. LEAK, L: non-coupled resting respiration, compensating for proton leak, slip and cation cycling in the absence of ADP or inorganic phosphate (maximum mt-membrane potential; ‘static head’).

3. OXPHOS, P: coupled active respiration, with saturating ADP and inorganic substrate (intermittent mt-membrane potential, sufficiently high to generate a high phosphate potential of the ATP system).

4. ETS, E: uncoupled maximum respiration, in the presence of optimum concentrations of uncoupler (low mt-membrane potential).

    Coupling states require complementary definition of substrate states , and sufficient information must be provided on inhibitors, uncouplers, ions and any effectors of mitochondrial respiration.  The term State 3u (ETS, State E) might suggest that the uncoupled state (e.g. in intact cells) provides an alternative for the measurement of OXPHOS capacity (P; State 3).  ETS capacity, however, can be equal to or much higher than OXPHOS capacity, depending on the degree of limitation of OXPHOS by the capacity of the phosphorylation system.

    The most sensitive expressions of mitochondrial quality are flux control ratios, FCR, obtained by normalization of flux for a common metabolic reference state (e.g. ETS with CI+II).  Similarly, coupling control ratios and substrate control ratios provide functional indexes of mitochondrial quality [3,4]:

1. The L/E coupling control ratio provides the appropriate expression of uncoupling, provided that specific limitations of flux by E are considered.  L/E increases with uncoupling from a theoretical minumum of 0.0 for a fully coupled system, to 1.0 for a fully uncoupled system. 

2. The P/E ratio increases from a minimum of L/E if the capacity of the phosphorylation system is zero, to the maximum of 1.0 if the capacity of the phosphorylation system fully matches the ETS capacity (or if the system is fully uncoupled), when there is no limitation of P by the phosphorylation system.  It is important to separate the effect of ADP limitation by ADP concentration (apparent P/E ratio) from limitation by enzymatic capacity at saturating substrate concentrations.

3. The conventional respiratory control ratio, RCR (State 3/State 4 or P/L), strictly is not an index of coupling [3].  The RCR reflects enzymatic limitations of P by the phosphorylation system in addition to coupling.  The RCR increases from 1.0 to infinity from a fully uncoupled to coupled system, but declines independent of coupling as a function of the P/E ratio.  For mathematical reasons, it is more appropriate to use the inverse RCR, which is the L/P ratio with the theoretical boundaries of 0.0 for the fully coupled system to the general maximum of (L/E)/(P/E), which becomes 1.0 in a fully uncoupled system.

    Changes in mitochondrial density constitute a complementary mechanism for alterations of tissue-OXPHOS capacity, which is of primary physiological (training) and pathological (type 2 diabetes; obesity) consequence.  Respiration of permeabilized muscle fibers yields tissue-OXPHOS capacity directly as mass-specific oxygen flux, JO2 [pmol∙s-1∙mg-1 wet weight].  Mass-specific flux must be distinguished from the extensive (per system) quantity of oxygen flow, IO2, obtained in respirometry with cultured cells [pmol∙s-1∙10-6 cells].  Average cell size may change in populations of cultured cells [5,6].  Thus an additional distinction is required between effects on oxygen flow of mitochondrial quality, density and cell size or total mt-amount per cell [5].

1.  Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. III. The steady state. J. Biol. Chem. 217: 409-427.

2.  Nicholls DG, Ferguson SJ (2002) Bioenergetics 3. Academic Press, London. 287 pp.

3.  Gnaiger E (2009) Mitochondrial pathways through Complexes I+II: Convergent electron transport at the Q-junction and additive effect of substrate combinations. In Gnaiger E, ed Mitochondrial Pathways and Respiratory Control. OROBOROS MiPNet Publications, Innsbruck, 2nd ed.

4.  Gnaiger E (2010) MitoPathways: Respiratory States and Coupling Control Ratios. In: Mitochondrial Pathways and Respiratory Control. OROBOROS MiPNet Publications 2007-2010, 2nd ed.: 43-53. www.oroboros.at/index.php?respiratorystates

5.  Renner K, Amberger A, Konwalinka G, Gnaiger E (2003) Changes of mitochondrial respiration, mitochondrial content and cell size after induction of apoptosis in leukemia cells. Biochim. Biophys. Acta 1642: 115-123.

6.  Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem. J. 380: 919-928.

P5-01        The effect of various Aβ1-42 assemblies on viability of neurons

Morkuniene R1,2, Cizas P1,2, Budvytyte R3, Valincius G3, Borutaite V1

1Institute for Biomedical Research, 2 Department of Biochemistry, Kaunas University of Medicine, Kaunas, Lithuania; 3Institute of Biochemistry, Vilnius, Lithuania

    Alzheimer's disease (AD) is the complex, neurodegenerative disease characterized by the impairment of cognitive function in elderly individuals. The central event in the pathogenesis of AD is thought to be intracellular and extracellular accumulation of amyloid-beta (Aß) peptides, however, the contribution of Aß in AD disease progression is still elusive. The inflammatory hypotesis proposes that Aß peptides may promote a damaging inflammation reaction. Aß can also cause mitochondrial dysfunction due to association with mitochondrial membranes and reduction activity of respiration function. Permeabilization of the mitochondrial membranes with release of intermembrane proteins has been strongly implicated in cell death – apoptosis and necrosis. Finally, AD has been related to a disruption of intracellular Ca homeostasis, and elevated intracellular Ca levels are known to trigger cell death. Part of the controversy may relate to the fact that Aß toxicity depends on its assembly state that varies from monomers to small, soluble oligomers and insoluble fibrils. To address these issues we used cultured rat cerebellar granule cells (CGC) to analyze the effect of various Aβ1-42 assemblies (monomeric, oligomeric and fibrillar) on viability of neurons. We found that only Aβ1-42 oligomers with diameter 1-3 nm caused a gradual decrease of neuronal viability in a concentration dependent manner after 24 h incubation. In contrast, the Aβ1-42 fibrils and monomers had no effect on neuronal viability even at higher concentrations. Aβ1-42-induced cell death was mainly necrotic. The percentage of apoptotic cells showing nuclear shrinkage and chromatin condensation was minor with all concentrations of Aβ1-42 oligomers. Moreover, small oligomers of Aβ1-42 (1-2 nm) exhibited propensity to bind to the phospholipid vesicles whereas the bigger aggregates (4-5 nm) did not bind vesicles. These results demonstrate that small oligomers (1-3 nm) at submicromolar concentrations induce rapid neuronal necrosis whereas bigger aggregates did not cause neuronal death.

P5-02        Synergistic antioxidant effects of GSPE and omega-3 PUFAs

Fernández-Iglesias, A., Casanova, E., Quesada, H., Pajuelo, D., Diaz, S., Baselga, L., Blade C., Arola, L., Salvado, MJ.

Nutrigenomic Group, Department of Biochemistry and Biotechnology, Rovira i Virgili, University, Tarragona, Spain

    Proanthocyanidins and omega-3 PUFAs (ω3) are compounds that can act against the oxidative stress underlying such chronic diseases as obesity. The overall objective of this study is to assess the antioxidant effects of grape seed procyanidin extract (GSPE), ω3 (microalgae oil rich in omega-3 PUFAs (40%)) and the possible synergistic effects of these compounds ex vivo. We have designed an acute experiment in which Wistar rats will be orally administered lard oil (2.5ml/kg weight) with or without a high dose of GSPE or ω3 (250 mg compound/kg body weight). There will be four experimental groups: control (vehicle), GSPE, ω3 and GSPE+ω3. The animals will be sacrificed 5h after the treatment and the livers will be collected. As mitochondria are the main intracellular sites of ROS generation, we will focus on the mitochondrial function. First of all, we will isolate viable mitochondria from the liver with a specific protocol. Then, we will determine the oxidative state for each treatment with a 4-hydroxynonenal (4-HNE) protocol in the OROBOROSR Oxygraph and we will compare the NADPH oxidase activity, the major source of ROS, and NADPH oxidase genetic expression for each treatment. With these results, we hope to determine the synergistic antioxidant effect of GSPE and ω3.