Basic Science and Clinical Reality

2L-01.    The metabolic syndrome and mitochondrial function - towards an international mtDNA cybrid study group.

               Hong Kyu Lee

Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea. - hkleemd@snu.ac.kr

    Reading:

Morino K, Petersen KF, Shulman GI. Molecular mechanisms of insulin resistance in humans and their potential links with mitochondrial dysfunction. Diabetes. 2006 Dec;55 Suppl 2:S9-S15

Cho YM, Park KS, Lee HK. Genetic factors related to mitochondrial function and risk of diabetes mellitus. Diabetes Res Clin Pract. 2007 Apr 20;

Fuku N, Park KS, Yamada Y, Nishigaki Y, Cho YM, Matsuo H, Segawa T, Watanabe S, Kato K, Yokoi K, Nozawa Y, Lee HK, Tanaka M. Mitochondrial haplogroup N9a confers resistance against type 2 diabetes in Asians. Am J Hum Genet. 2007 Mar;80(3):407-15

Lee HK, Park KS, Cho YM, Lee YY, Pak YK. Mitochondria-based model for fetal origin of adult disease and insulin resistance. Ann N Y Acad Sci. 2005 May;1042:1-18.

West GB, Woodruff WH, Brown JH. Allometric scaling of metabolic rate from molecules and mitochondria to cells and mammals. Proc Natl Acad Sci U S A. 2002 Feb 19;99 Suppl 1:2473-8.

West GB, Brown JH, Enquist BJ. A general model for the origin of allometric scaling laws in biology. Science. 1997 Apr 4;276(5309):122-6.

Lee DH, Lee IK, Jin SH, Steffes M, Jacobs DR Jr. Association between serum concentrations of persistent organic pollutants and insulin resistance among nondiabetic adults: results from the National Health and Nutrition Examination Survey 1999-2002. Diabetes Care. 2007 Mar;30(3):622-8.

Shertzer HG, Genter MB, Shen D, Nebert DW, Chen Y, Dalton TP. TCDD decreases ATP levels and increases reactive oxygen production through changes in mitochondrial F(0)F(1)-ATP synthase and ubiquinone. Toxicol Appl Pharmacol. 2006 Dec 15;217(3):363-74.

2P-01.   Mitochondrial testicular function: Effects of age and diabetes.

               Sandra Amaral, Luís Martins, Paula C Mota, Paulo J Oliveira, João Ramalho-Santos

Center for Neurocience and Cell Biology, Department of Zoology, University of Coimbra, Coimbra, Portugal - scg_amaral@yahoo.com

    Mitochondrial oxidative phosphorylation is the major ATP synthesis pathway in eukaryotes. In this process, oxygen consumption is coupled to ADP phosphorylation with the intermediate generation of a pH and electric gradient across the inner mitochondrial membrane (delta pH and delta psi), respectively. Testicular dysfunction, decreased fertility potential, variations in semen quality and abnormalities in spermatogenesis in male animals have been described in both aging and in chronic diseases linked to oxidative stress, such as Diabetes Mellitus. Also, and most importantly, mitochondrial function is altered during diabetes. Taking into account that sperm motility depends on proper mitochondrial function, we analysed the bioenergetics of isolated rat testicular mitochondria by measuring oxygen consumption and the generation of the mitochondrial delta psi. We examined respiratory parameters and membrane potential with substrates for both complex I (NADH oxidation), and complex II (succinate oxidation) of the membrane respiratory chain. Additionally, we also assessed mitochondrial calcium loading capacity. Changes in mitochondrial function caused by aging were monitored in control rats (2 and 5 months old), while diabetes-induced changes were evaluated in a type I (streptozotocin-induced) diabetic rat model. The respiratory control ratio, an index of mitochondrial coupling between respiration and phosphorylation, was lower in older animals (P≤0.01) regardless of the substrate used, suggesting that mitochondrial functionality is affected in the testis of older males. In accordance with this finding, the maximal oxygen consumption sustained by the respiratory chain (uncoupled respiration) and delta-psi generated was also lower in older animals (P≤0.01), although their calcium loading capacity was increased, suggesting possible adaptations to an age-related increase in oxidative stress. Interestingly, respiratory function was similar in diabetic and control rats, again suggesting adaptations to the deleterious effects of the disease. This idea is reinforced by both the higher uncoupled respiratory rate, and the lower resting oxygen consumption (state 4) in diabetic animals (P≤0.05), the latter of which suggests a decrease in membrane proton leak. Taken together our results suggest that mitochondrial testicular function declines with age in rats. However, in previous work we found that older rats have higher sperm count and motility. Thus, it is reasonable to speculate that when mitochondrial function is affected by age, rodent sperm may switch to alternative metabolic pathways. On the other hand, diabetic animals appear to have some testicular mitochondrial adaptations to overcome the disease process.

     Sandra Amaral is the recipient of a fellowship from FCT, Portugal (SFRH/BD/18734/2004)

2P-02.   Mitochondrial dysfunction and metabolic inflexibility in models of aging and diet-induced obesity.

               Koves TR, Noland RC, Slentz DS, Ilkayeva O, Newgard CB, Muoio DM.

The Depts. of Medicine and Pharmacology & Cancer Biology and the Sarah W. Stedman Nutrition & Metabolism Center, Duke University, Durham, NC USA - koves001@mc.duke.edu

    Obesity, diabetes, and aging are associated with declining skeletal muscle function and are often characterized by accumulation of intramuscular lipids and insulin resistance.  Emerging data suggests that mitochondrial dysfunction may provide a common link among these pathophysiologic states.  In ongoing studies using mass spectrometry-based metabolic profiling, we show that rodent models of aging and diet-induced obesity exhibit muscle accumulation of most short, medium, and long-chain fatty acylcarnitine species, implying a heavy mitochondrial lipid burden.  In addition, muscle levels of long and medium chain acylcarnitines correlated negatively with insulin sensitivity.  Studies in isolated skeletal muscle mitochondria from aged animals fed on a year-long high fat diet displayed low rates of complete fat oxidation but high rates of incomplete oxidation along with “metabolic inflexibility” (impaired substrate switching from fatty acid to a carbohydrate-derived fuel).  We propose that excessive β-oxidation and impaired mitochondrial substrate switching might contribute to skeletal muscle insulin resistance through mechanisms of oxidative stress.  These possibilities, as well as the molecular basis of metabolic inflexibility, are under investigation

2P-03.   Mitochondrial function, -content and -ROS production in rat skeletal muscle: effect of high-fat feeding.

J Hoeks^1,2, JJ Briedé³, J de Vogel^1,2, G Schaart⁴, M Nabben¹, E Moonen-Kornips¹, MKC Hesselink^4,2 , P Schrauwen^1,2

¹Department of Human Biology, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University, Maastricht, The Netherlands.

²TI Food & Nutrition (TIFN), Wageningen, The Netherlands

³Department of Health Risk Analysis and Toxicology, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University, Maastricht, The Netherlands.

⁴Department of Movement Sciences, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University, Maastricht, The Netherlands. - j.hoeks@hb.unimaas.nl

    Aims/hypothesis: The accumulation of fat in skeletal muscle is involved in the development of type 2 diabetes mellitus and a diminished mitochondrial function has been suggested to be a primary cause of this muscular fat accumulation. Although the cause of this mitochondrial dysfunction remains unclear, there are indications that a high intake of dietary fat negatively affects indirect parameters of mitochondrial function. Whether a high intake of dietary fat diminishes intrinsic mitochondrial functioning is however unknown and subject of the present study.

    Methods: Male Wistar rats were subjected to an 8-week low- vs. high-fat dietary intervention after which isolated skeletal muscle mitochondria were analyzed for functional capacity by respirometry and for ROS production by electron spin resonance spectroscopy. In addition, intramyocellular lipids (IMCL) were assessed by Oil Red O staining, peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PPARGC1A) protein and markers for mitochondrial content by western blotting.  

    Results: The high-fat dietary intervention did not affect intrinsic functioning of skeletal muscle mitochondria, neither on a carbohydrate- nor on a lipid substrate. Interestingly, in muscle homogenates PPARGC1A protein increased by ~2-fold upon high-fat feeding and the protein content of structural subunits of 4 different complexes in the respiratory chain tended to increase as well. Protein content of all these subunits correlated tightly with PPARGC1A protein content. IMCL levels increased significantly while mitochondrial ROS production remained unaffected. 

    Conclusions/interpretation: Despite normal (functional) or even improved (content) mitochondrial function, IMCL levels increased upon high fat feeding. This illustrates that a reduced mitochondrial function is not a prerequisite for muscular fat accumulation.

2P-04.   Bile acid signaling, FXR and energy expenditure – novel perspectives for the treatment of metabolic diseases.

Maxi Meissner¹, G Henk Visser^2,3, Frans Stellaard¹, F  Kuipers¹

¹Laboratory of Pediatrics, University Medical Center Groningen, ²Zoological Laboratory, Rijksuniversiteit Groningen and ³Centre for Isotope Research, Groningen, The Netherlands - m.meissner@med.umcg.nl

    The aim of this project is to elucidate the role of bile acid signaling via FXR in the modulation of energy expenditure to potentially allow for novel treatments of metabolic diseases, such as insulin resistance and type 2 diabetes.

    The farnesoid X receptor (FXR) is a nuclear receptor that is predominantly expressed in enterohepatic tissues, as these are exposed to its ligands: bile acids. FXR has applicability in metabolic diseases, for example: Hepatic expression of FXR is reduced in animal models of diabetes.  FXR deficient mice display elevated circulating FFAs, which has been associated with elevated serum glucose levels, impaired glucose and insulin tolerance.  FXR acts to maintain bile acid homeostasis, control glucose and lipo-protein metabolism, and  regulate fuel availability upon fasting / refeeding. Thus FXR is an important metabolic regulator. Furthermore, FXR has recently been shown to be involved in the regulation of energy expenditure. Bile acid signaling appears to have a crucial role herein. However, the precise mechanisms and whether FXR plays a direct or indirect part in the regulation of energy expenditure are not yet understood and need careful investigation.

    Using the doubly labeled water (DLW) technique to measure daily energy expenditure (DEE), 5 Fxr^-/- and WT male mice were injected with DLW in a pilot study. Blood samples were taken via the tail at 25 hours to determine basal energy expenditure and upon a following 24 hour fast to determine fasted energy expenditure. Moreover using the DLW technique, the effect of bile acid sequestration on energy expenditure was studied in ob/ob and db/db mice after a 10 day diet supplemented with a bile acid binding compound.  First results will be presented.

    To further elucidate the role of bile acid signaling and FXR in energy expenditure, research perspective include to investigate mitochondrial function in metabolically active tissues and its relation to energy expenditure in WT and Fxr deficient mice that are on a normal diet, a high fat diet, supplemented with an FXR agonist or a bile acid sequestrant and in a model of cholestasis.

2P-05.   Different approaches to assessing mitochondrial function and their utility and accuracy for pharmaceutical testing

R Böhm¹, L Schild², Th Herdegen¹

¹Dept. of Pharmacology, UK S-H Campus Kiel, Germany

²Klinische Chemie und Pathologische Biochemie, Otto-von-Guericke-Universität Magdeburg, Germany - ruwen.boehm@pharmakologie.uni-kiel.de

    Mitochondria play a pivotal role for energy homoeostasis and apoptosis. Evidence has accumulated that these two primary functions are interacting with each other, e.g. disrupted oxidative phosphorylation might contribute to neurodegeneration in Parkinson’s or Alzheimer’s disease. Traditionally, oxidative phosphorylation is evaluated best using the Clark type electrode. The disadvantage for pharmaceutical testing comes from that substances have to be processed separately in a time consuming manner. Thus optical methods using resazurine or JC-1 were established. We have used these methods to quantify the impact of two c-Jun N-terminal kinases (JNK) inhibitors on mitochondrial function because JNKs are involved in apoptotic processes and cytochrome C release from mitochondria. However, the apparently interesting results turned out to be artifacts caused by the optical activity of the substances or biochemical reactions between resazurine and adjuvants / stabilizing agents like ascorbic acid. Furthermore, results obtained by these methods do not only reflect the oxidative phosphorylation function but also the function of other pathways like the tricarboxylic acid cycle or NADH-depleting reactions. Thus optical methods are inferior to the Clark type electrode concerning quantitative pharmaceutical testing.

2P-06.   Analysis of mitochondria in adipose tissues of bariatric patients: Identification of risk factors and/or predictors for the treatment outcome.

Peter Lindinger¹, Martine Calame¹, Matthias Hoch¹, Alex N Eberle¹, Ralph Peterli², Thomas Peters², Sjouke Hoving³, Hans Voshol³, Ian Fearnley⁴, John E Walker⁴

¹Department of Research, University Hospital and University Children’s Hospital Basel, Switzerland; ²St.Claraspital, Basel, Switzerland; ³Novartis Institutes for Biomedical Research/GPS/Pro, ⁴MRC Dunn Human Nutrition Unit, Cambridge, U.K. - martine.calame@unibas.ch

    At the present bariatric surgery is recognized as the only reliable treatment modality to achieve weight loss in a majority of morbidly obese patients. However, complications and/or absence of weight loss are observed in a number of cases, and this may be related to deficits in metabolic regulation. Recently, impaired mitochondrial activity was shown to be linked with the development of obesity and cardiovascular disease in an animal model (Wisløff et al, 2005). So far there are no predictors for the outcome of bariatric surgery. As a consequence we propose to analyse mitochondria at the sites where energy is stored and produced, i.e. in the human adipose tissue, with a particular focus on protein expression

    This research project focuses on two major aims: (I) Identification of potential predictors for the outcome of bariatric intervention (proteome analysis of patients with vs. without weight loss), and (II) identification of novel mitochondria-based risk factors related to energy metabolism and obesity (comparison of morbidly obese patients before and after weight loss vs. lean control patients).

    Isolated mitochondria populations are subjected to a series of enzymatic assays to characterize functionality of mitochondrial respiratory chain (Citrate synthase, individual complexes I-V of the respiratory chain, ATP production). In addition the mitochondrial count per adipocyte is under investigation using Taqman quantification. The differential proteomic analysis is based on two complementary techniques: (I) 2D Gel-Electrophoresis with isoelectric focusing and (II) pre-fractionation of the proteome by anion-exchange. MS identification will be performed for protein spots of interest.

    In summary this project covers the combination of a carefully characterized patient population and a detailed molecular analysis of human fat tissue. Therefore, this integrated approach considering various important parameters may lead to better criteria for bariatric surgery and eventually pave the basis for a pharmacotherapy of morbid obesity.

2P-07.   h-MitoArray and gene expression analysis in 13 patients with mitochondrial ATP synthase deficiency.

Čížková A^1,2,5, Stránecký V^1,2, Ivánek R^1,2,4, Hartmannová H^1,2, Nosková L², Piherová L^1,2, Tesařová M^1,3, Hansíková H^1,3, Honzík T^1,3, Zeman J^1,3, Divina P⁴, Paul J^1,5, Houštěk J^1,5, Kmoch S^1,2

1 Center for Applied Genomics, 2 Institute of Inherited Metabolic Disorders and 3 Department of Pediatrics, 1st Faculty of Medicine, Charles University, Prague, Czech Republic; 4 Institute of Molecular Genetics, Academy of Science of the Czech Republic, Prague, Czech Republic, 5 Department of Bioenergetics, Institute of Physiology, Academy of Science of the Czech Republic, Prague, Czech Republic - acizk@LF1.cuni.cz

    Mitochondria generate much of the energy of the cell, produce most of the endogenous reactive oxygen species, regulate cellular redox state, cytosolic concentration of calcium and integrate many of the signals for initiating of apoptosis.

    Defects of mitochondrial functions lead in humans to vast array of usually multisystemic pathologies. Several hundreds of diseases resulting from various defects of mitochondrial biogenesis and maintenance, defects of respiratory chain complexes (OXPHOS) or defects of individual mitochondrial proteins are known. To strengthen diagnostic work-up for various mitopathies we designed focused oligonucleotide microarray which allows expression profiling of 1632 human mitochondria-related genes and tested its performance in analysis of genetically heterogeneous group of 13 patients with biochemically proven ATP synthase deficiency. Gene expression data analysis allowed classification of patients into several distinct groups, provided information on subgroup and patient specific gene expression profiles, defined candidate disease causing genes and gave basic information on pathogenic mechanisms associated with ATP synthase deficiency.

2P-08.   Depletion of COX5A subunit in HEK293 cells.

Fornuskova D, Stiburek L, Pejznochova M, Zeman J

Department of Pediatrics, First Faculty of Medicine, Charles University in Prague, Czech

Republic - danielafornuskova@seznam.cz

    COX5A is one of 13 structural subunits of cytochrome c oxidase (COX), the terminal enzyme of respiratory chain. We used RNAi to down-regulate steady-state level of COX5A subunit and analyzed impact of its knock-down on COX assembly. We chose plasmid with short hairpins embedded within a miRNA transcript from the naturally occurring miR30 to mimic a natural microRNA primary transcript. The final transcript contains also GFP and Neomycin Phosphotransferase coding sequences situated tandemly, upstream of the miR30-like hairpins. For success in experiments based on RNAi an appropriate, high-throughput screening method estimating silencing potential of candidate shRNAs is important. We constructed seven derivatives of the plasmid coding for hairpins aimed at different positions of COX5A mRNA.

    A long halflife of COX5A (the level of the protein is at least 48 hours post nucleofection unchanged on Western Blot) contradict the screening on protein level by imunodetection of endogenous COX5A. To circumvent the problem, we introduced COX5A coding sequence into the maxFP-Red – N plasmid to encode fusion protein. The marker plasmid was cotransfected with RNAi-mediating plasmid derivatives (the higher the level of fusion protein the higher the leakage of individual hairpin-mediated RISC systems). A fluorescence of fusion protein was found rapidly lower compared to maxFPRed marker and complicated a setting of FACS measurements, but Western Blot of fusion protein with COX5A antibody gave an acceptable result. To optimize the detection of RNAi-potential through fluorescence by FACS, we re-cloned COX5A coding sequence into 3´UTR of maxFPRed marker. The final transcript contains target sequence for RNAi but leads to translation of merely maxFPRed marker. Fluorescence intensities were comparable with that obtained at empty plasmid.

    Based on the above-mentioned methods, we chose three candidate shRNAs and prepared stable cell lines, where depletion of COX5A level was confirmed to some extent using SDS immunoblotting. Also specific activity of COX was revealed decreased. BN-PAGE showed diminished level of COX holoenzyme and its assembly intermediates.

     Supported by grants GAUK 1/2006/R and GACR 303/03/H065.

2P-09.   Changes in mitochondrial respiration in brain cells of transgenic mice models of Alzheimer’s disease.

V Rhein¹, G Baysang¹, F Meier¹, L Ozmen², H Bluethmann², E Savaskan¹, F Müller-Spahn¹, C Czech², J Götz³, A Eckert¹

¹ Neurobiology Research Laboratory, Psychiatric University Clinic, 4025 Basel, CH

² Hoffmann-La-Roche AG, Pharma Research, Neurosciences, 4070 Basel, Switzerland

³ Alzheimer’s and Parkinson’s Disease Laboratory, Brain and Mind Research Institute, University of Sydney, 2050 Sydney, Australia - virginie.rhein@upkbs.ch

    Alzheimer’s disease (AD) is the most frequent form of dementia among the elderly and is characterized by neuropathological hallmarks of extracellular amyloid plaques and intracellular neurofibrillary tangles in the brain of AD patients. Amyloid plaques are composed of the amyloid-beta (Aβ) protein, derived from its precursor protein APP. Neurofibrillary lesions are formed from paired helical filaments composed of hyperphosphorylated tau protein, a microtubule-associated-protein.

    In addition, mitochondrial dysfunction and energy metabolism deficiencies are recognized as earliest events and correlated with impairments of cognitive abilities in AD. Nevertheless, the specific mechanisms leading to mitochondrial failure in AD are not well understood.

    Interestingly, accruing data indicate that Aβ as well as tau pathology lead to mitochondrial dysfunction and increased levels of reactive oxygen species (ROS). Reduced ATP levels, impairments of mitochondrial membrane potential including reduced cytochrome c oxidase activity in APP transgenic mice and reduced complex I activity in P301L tau transgenic mice have been observed. However, how tau pathology mediates these changes and its role within the amyloid cascade remains unclear.

    To better understand the direct impact of Aβ/tau interplay on mitochondria, we are currently investigating the brains of double (APP (KM670/671NL) / PS2 (N141l)) and triple (APP (KM670/671NL) / PS2 (N141l) / Tau (P301L)) transgenic mice at the age of 7-8, 12 and 16 months. Mitochondrial respiration is studied by the measurement of the oxygen consumption at 37° using an Oxygraph-2k system equipped with two chambers and Datlab software. State 2 respiration is measured after addition of malate/glutamate. Then, ADP is added to measure state 3 respiration. After determining coupled respiration, FCCP is added and respiration is measured in the absence of a proton gradient. To check the integrity of the mitochondrial membrane cytochrome c is added. In order to inhibit complex I and III activities rotenone and antimycine A, respectively are added. Then, ascorbate and TMPD are added and respiration is measured. Finally, sodium azide is added to inhibit complex IV activity. Moreover, enzyme activities of these complexes, mitochondrial membrane potential, ATP levels and ROS production are determined.

    First evidence indicates that the respiration of mitochondria of brain cells from the triple transgenic mice is reduced compared to the double transgenic mice without tau mutation. Based on our preliminary findings, we hypothesize that at the level of mitochondria, the two defining neuropathological AD proteins, tau and Aβ, seem to act in a synergistic or additive way finally leading to/accelerating neurodegenerative mechanisms.

     Supported by SNF grant 310000-108223 and Eli Lilly International Foundation grant to AE

2P-10.   Fibroblasts cell culture as model for apoptosis detection.

O Brantova, J Sladkova, H Hansikova, J Zeman

Department of Pediatrics and Institute for Inherited Metabolic Disorders, Faculty of Medicine, Charles University, Prague, Czech Republic - olbra@seznam.cz

    Introduction: Abnormal apoptosis is one of the primary cause of various diseases such cancer, degenerative diseases, and autoimunne diseases.

Increased susceptibility to apoptosis has been shown in many models of mitochondrial defects but its relevance to human diseases is still discussed. As well, the potential role of apoptotic cell death in the development of the cellular and tissue lessions seen in lysosomal storage diseases, and particularly in neurological diseases is often discussed. The aim of our work was to introduce and to improve the methods for induction and detection of apoptosis in fibroblasts cell culture.

     Material and methods: The study was performed on fibroblasts cell lines. Staurosporine, potent inhibitor of phospholipid/calcium-dependent protein kinase and Actinomycin D, an antineoplastic antibiotic inducing apoptosis were used as chemical apoptogen. Hydrogen peroxid (30uM), UV radiation (15 min.), serum starvation and heat shock (43°C for define time) were used for simulation of  environmental stress.

     Results: Hydrogen peroxid induced apoptosis during 2 hours of cultivation in contrast to staurosporine, which induced first apoptotic changes after 5 hours of cultivation. Serum starvation presents large delay between apotosis induction and appearance of first apoptotic events. Changes in morphology, such a cell shrinkage or membrane blebing under oxidative stress, advert to first apoptotic events and is visible in phase contrast. Mitochondrial membrane potential was measured by MitoTracker ROS or a dual-emission potential-sensitive probe JC-1. Western blot or cytochemistry exploitaging pattern of specific antibodies was used for detection of activated caspase 9 and caspase 3, which cleave cellular substrates in answer to realizing of cytochrome c in cytoplasma. Chromatin condensation and changes in nuclear morphology as apoptotic event was visualized by fluorescent dye Hoechst 33258. Ultrastructural changes in cell during apoptotic events reveals transmission electron microscopy.

     Conclusion: We have verified most of mentioned methods on fibroblast cell culture. In our first approach, we came across some nonspecific difficulties during apoptosis induction such a heterogeneity in apoptotic start in one cell culture under same conditions, divergent sensitivity to one apoptogen in on culture, most of all dependent on localization of cells in cultivation flask. Furthermore fibroblasts present slow growth rate and lower susceptibility to apoptogen in comparison to transformed cell lines and demand very accurate confluence in all tested culture. Despite mentioned disadvantages, we conclude, that under precise defined conditions, fibroblast cell culture represent useful model for apoptosis induction, pursuing of apoptotic kinetics and detection of large scale of apoptotic event on structural, biochemical and ultrastructural level. Consequently, we are going to test suitability of another methods, relating to apoptose, for fibroblast cell culture.

     Supported by GAUK 303/03/H065.

2P-11.   Synergistic effect of glucocorticoids and 2-deoxyglucose - energy metabolism as a new target in the treatment of leukemia.

Karin Eberhart¹, Ireen Ritter¹, Birgit Timischl¹, Reinhard Kofler², Peter Oefner¹, Kathrin Renner¹

¹Institut of Functional Genomics, University Regensburg, Germany, ²Tyrolean Cancer Research Institute, Austria - karin.eberhart@klinik.uni-regensburg.de

    Glucocorticoids (GC) are used in the therapy of acute lymphoblastic leukemia (ALL) in combination with other chemotherapeutics because of their ability to induce cell cycle arrest and apoptosis. Despite a relatively high survival rate, the therapy has severe side effects and resistance develops frequently. The underlying mechanism of GC induced apoptosis is still unclear. Recent gene expression profiles of patients undergoing GC-monotherapy have revealed the enzyme PFKFBP-2 (Schmidt et al. 2006) as promising candidate gene indicating the contribution of metabolic disturbances to GC-induced apoptosis. The glucose analog 2-deoxyglucose (2-DG) that accumulates in the cell as 2 deoxyglucose phosphate (2-DGP) but cannot be further metabolized is often used experimentally to mimic glucose and energy deprivation. Important metabolic pathways, glycolysis and subsequently the citrate acid cycle and oxidative phosphorylation as well as the pentose phosphate pathway can be disturbed by the administration of the glucose analog. It is also reported that through the accumulation of 2-DGP hexokinase II is released from the outer mitochondrial membrane (Krieglstein et al. 1982). Resulting membrane alterations could lead to the release of proapoptotic factors (cytochrome c, Smac/Diablo, AIF), which is the on-set of the intrinsic pathway, leading to apoptosis. Expecting a synergism, we explored the effect of co-administration of 2-DG and the GC dexamethasone in T- and B-ALL cells.

    Co-administration of 2-DG and GC significantly enhanced the kinetic of apoptosis and the sensitivity to dexamethasone in acute lymphoblastic T- and pre B-cells. ATP levels were significant earlier reduced by the co-administration of 2-DG and dexamethasone compared to sole dexamethasone or 2-DG treatment. The two ATP producing pathways glycolysis and oxidative phosphorylation contributed to a different extent to the ATP decline in the two systems. The ATP levels in B-cells were more dependent on changes in the glycolytic flux, whereas in T-ALL cells oxidative phosphorylation was more affected. Although we cannot distinguish between a higher ATP turn-over or less production, it is described that low ATP levels induce apoptosis (Garland et al. 1997).

    Reduced mitochondrial capacity was measured only after digitonin treatment and furthermore single respiratory enzyme activities and a slight cytochrome c release were detected suggesting mitochondrial membrane alterations. These effects were even more pronounced in T-ALL cells, in B-ALL cells mitochondria partially recovered over time. Whether these changes lead to the release of pro-apoptotic factors or are the consequences of the detachment of the hexokinase needs to be clarified. The significantly higher intracellular 2-DGP concentration after co-administration of 2-DG and dexamethasone compared to 2-DG alone supports the latter hypothesis. Disturbances in the energy metabolism seem to play a major role in the earlier on-set of 2-DG and dexamethasone induced apoptosis, since we can abolish the enhanced sensitivity by the administration of pyruvate.

     Supported by GENAU-CHILD, Austrian Ministry of Education, Science and Culture and BayGene Bavarian Ministry of Science, Research and Art

2P-12.   Functional analysis of genes encoding mitochondrial proteins in Trypanosoma brucei.

Zdeněk Paris and Julius Lukeš

Laboratory of Molecular Parasitology , Biology Center, Academy of Sciences of the Czech Republic, Branisovska 31, Ceske Budejovice, 370 05, Czech Republic

    Trypanosoma brucei is a parasitic protist that causes African sleeping sickness in humans and livestock. The unicellular parasite is transmitted by the tse-tse fly. During their life cycle, trypanosomes alternate between the final vertebrate and the intermediate invertebrate hosts. The transmission occurs via the inoculation of flagellates with the tse-tse saliva into the final host.  In order to survive in substantially different environments of both hosts, the parasite’s mitochondrion undergoes dramatic changes. The organelle is strongly downregulated in the vertebrate (bloodstream stage), whereas it is fully active in the insect host (procyclic stage).

    In our lab, we are using RNA interference to analyze the function of genes encoding mitochondrial proteins in procyclic T. brucei. We mainly focus on proteins involved in: i/ mitochondrial mRNA stability and editing ii/ subunits of respiratory complexes, and iii/ iron-sulfur cluster assembly proteins. The ensuing phenotypes are studied by a wide range of methods, by which different mitochondrial functions are followed. To identify novel components of these complex pathways, several approaches are being used, such as homology searches and TAP-tagging of proteins followed by mass spectrometry. Novel approaches for the identification of candidate genes, by forward genetics using RNA interference library of T. brucei, will be discussed.