Metabolische Dynamik und Kompartimentierung im Zentralstoffwechsel von Chinese hamster ovary Zellen

The thesis aimed at advancing our knowledge about metabolic compartmentation in the metabolism of mammalian cells. The focus of the study was the central metabolism and its control in Chinese hamster ovary (CHO) cells. An essential part of the work was the development and implementation of innovative methods, e.g. the analysis of extra- and intracellular labeling dynamics, non-stationary 13C metabolic flux analysis, determination of compartment-specific enzyme activities, and elementary mode analysis of the mitochondrial metabolism in selectively permeabilized cells. The results of this thesis increase our understanding of metabolic control at the cytosol-mitochondria interface, including control by mitochondrial transporters. In addition the work highlights aspects in the connection of glycolysis and TCA cycle, microcompartmentation and channeling, metabolite exchange between cells and extracellular medium, as well as metabolic dynamics in general. The comprehensive findings contribute to a deeper understanding of the complexity of mammalian metabolism and the various manifestations of metabolic compartmentation. Understanding the control of metabolism opens up new perspectives for improving biotechnological production processes and designing successful therapies for the treatment of severe diseases.Die Arbeit zielte darauf ab, unser Wissen in Bezug auf metabolische Kompartimentierung im Stoffwechsel von Säugerzellen voranzubringen. Im Mittelpunkt stand die Untersuchung des Zentralmetabolismus und dessen Kontrolle in Chinese hamster ovary (CHO) Zellen. Eine notwendige Voraussetzung für diese Arbeit war die Entwicklung und Umsetzung einer Reihe von innovativen Methoden, u.a. die Analyse von extra- und intrazellulären Markierungsdynamiken, nicht-stationäre 13C metabolische Flussanalyse, Bestimmung von kompartiment-spezifischen Enzymaktivitäten sowie Elementarmodenanalyse des mitochondrialen Stoffwechsels in selektiv permeabilisierten Zellen. Die Ergebnisse dieser Arbeit erweitern unser Verständnis der metabolischen Kontrolle an der Grenzfläche zwischen Zytosol und Mitochondrien, einschließlich der Kontrolle durch mitochondriale Transporter. Darüber hinaus werden Aspekte der Verknüpfung zwischen Glykolyse und TCA Zyklus, Mikrokompartimentierung und Channeling, Metabolitenaustausch zwischen Zellen und extrazellulärem Medium, sowie allgemein die Dynamik des Stoffwechsels beleuchtet. Die umfassenden Erkenntnisse tragen dazu bei, die Komplexität des Säugerstoffwechsels sowie die verschiedenen Spielarten von Stoffwechselkompartimentierung besser zu begreifen. Wissen über die Kontrolle des Stoffwechsels eröffnet neue Perspektiven zur Verbesserung von biotechnologischen Produktionsprozessen sowie zur Behandlung von schweren Krankheiten

Doxorubicin increases oxidative metabolism in HL-1 cardiomyocytes as shown by 13C metabolic flux analysis.

Doxorubicin (DXR), an anticancer drug, is limited in its use due to severe cardiotoxic effects. These effects are partly caused by disturbed myocardial energy metabolism. We analyzed the effects of therapeutically relevant but nontoxic DXR concentrations for their effects on metabolic fluxes, cell respiration, and intracellular ATP. (13)C isotope labeling studies using [U-(13)C(6)]glucose, [1,2-(13)C(2)]glucose, and [U-(13)C(5)]glutamine were carried out on HL-1 cardiomyocytes exposed to 0.01 and 0.02 muM DXR and compared with the untreated control. Metabolic fluxes were calculated by integrating production and uptake rates of extracellular metabolites (glucose, lactate, pyruvate, and amino acids) as well as (13)C-labeling in secreted lactate derived from the respective (13)C-labeled substrates into a metabolic network model. The investigated DXR concentrations (0.01 and 0.02 muM) had no effect on cell viability and beating of the HL-1 cardiomyocytes. Glycolytic fluxes were significantly reduced in treated cells at tested DXR concentrations. Oxidative metabolism was significantly increased (higher glucose oxidation, oxidative decarboxylation, TCA cycle rates, and respiration) suggesting a more efficient use of glucose carbon. These changes were accompanied by decrease of intracellular ATP. We conclude that DXR in nanomolar range significantly changes central carbon metabolism in HL-1 cardiomyocytes, which results in a higher coupling of glycolysis and TCA cycle. The myocytes probably try to compensate for decreased intracellular ATP, which in turn may be the result of a loss of NADH electrons via either formation of reactive oxygen species or electron shunting

Metabolic profiling using HPLC allows classification of drugs according to their mechanisms of action in HL-1 cardiomyocytes.

Along with hepatotoxicity, cardiotoxic side effects remain one of the major reasons for drug withdrawals and boxed warnings. Prediction methods for cardiotoxicity are insufficient. High content screening comprising of not only electrophysiological characterization but also cellular molecular alterations are expected to improve the cardiotoxicity prediction potential. Metabolomic approaches recently have become an important focus of research in pharmacological testing and prediction. In this study, the culture medium supernatants from HL-1 cardiomyocytes after exposure to drugs from different classes (analgesics, antimetabolites, anthracyclines, antihistamines, channel blockers) were analyzed to determine specific metabolic footprints in response to the tested drugs. Since most drugs influence energy metabolism in cardiac cells, the metabolite "sub-profile" consisting of glucose, lactate, pyruvate and amino acids was considered. These metabolites were quantified using HPLC in samples after exposure of cells to test compounds of the respective drug groups. The studied drug concentrations were selected from concentration response curves for each drug. The metabolite profiles were randomly split into training/validation and test set; and then analysed using multivariate statistics (principal component analysis and discriminant analysis). Discriminant analysis resulted in clustering of drugs according to their modes of action. After cross validation and cross model validation, the underlying training data were able to predict 50%-80% of conditions to the correct classification group. We show that HPLC based characterisation of known cell culture medium components is sufficient to predict a drug's potential classification according to its mode of action

Non-stationary 13C metabolic flux analysis of Chinese hamster ovary cells in batch culture using extracellular labeling highlights metabolic reversibility and compartmentation

Mapping the intracellular fluxes for established mammalian cell lines becomes increasingly important for scientific and economic reasons. However, this is being hampered by the high complexity of metabolic networks, particularly concerning compartmentation.Institute of Cell Culture Technology (University Bielefeld, Germany) ; the BMBF (German Federal Ministry of Education and Research